(John R. Whitaker, Alphons G. J. Voragen, Dominic PDF

University of CaliforniaDavis
Davis, California, U.S.A.
Wageningen University
Wageningen, The Netherlands
U.S. Department of Agriculture

Albany, California, U.S.A.
Marcel Dekker, Inc. New York Basel
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Cover gure reprinted with permission from Figure 3, page 643 of Architecture of RNA Polymerase II and Implications for the
Transcription Mechanism, P. Cramer, D. A. Bushnell, J. Fu, A. L. Gnatt, B. Maier-Davis, N. E. Thompson, R. R. Burgess, A. M.
Edwards, P. R. David, and R. D. Kornberg. Science 288(5466): 640649, 2000. Copyright 2000 American Association for the
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Preface
Enzymes are the catalysts of life. They perform the majority of reactions in all living systemsall animals, plants,
and microorganisms. Our lives are absolutely dependent on them. Even inhaling in oxygen and exhaling carbon
dioxide are dependent on enzymes. Our plants take up minerals, water, and air, and combine them with carbon
dioxide and ammonia to provide our fruits and vegetables, via enzymes.
The Handbook of Food Enzymology is a single source that can answer questions about what enzymes are, how
they function catalytically, and how we depend on them and use them every minute of our life. This is not a book
that can be read in a single day. Rather, it is a handbook to be used to nd the answers to various questions: Who
are we biologically and chemically? How do we function biologically? How do our foods, most of our clothing, and
much of our shelter depend on enzymes? What are enzymes? What do they do? How can we manage them in the
most effective way to live a long and healthy life with an abundance of food?
The Handbook is divided into two major parts. The rst 26 chapters deal with general aspects of enzymology.
There are two chapters (1 and 2) on the history of enzymology, which discuss protein structure and the kinetics of
enzyme reactions and emphasize food production, and the trail of knowledge that led to this handbook. The next
two chapters (3 and 4) deal with how enzymes are highly specic and highly efcient catalysts and the environmental
factors that affect their activities. Chapter 5 describes how to inactivate enzymes when their action is no longer
desirable. Chapters 6 and 7 deal with regulatory issues pertaining to using enzymes in our foods to change the taste,
avor, aroma, color, texture, and nutritional quality. Chapter 8 deals with the evolution of enzymes under extreme
conditions of pH, temperature, salinity, toxicity, and pressure, and how these enzymes with special extremophilic
characteristics can be used effectively in foods. Chapters 9 through 16 deal with how enzymes produce the compo-
nents of our foodsthe proteins, lipids, carbohydrates, and nucleic acidsas well as the enzymes that make it
possible to synthesize our food components rapidly, efciently, nutritiously, and in the quantity needed to feed 6
billion persons each day. The last 10 chapters (1726) in Part I deal with management of enzymes postharvest and as
tools for increasing the quality and consumability of our foods.
Part II describes specic prototypic enzymes of the six chemical types of reactions catalyzed by enzymes:
oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. The contributors of these 57 chapters
have followed a common format in which they discuss what the enzyme(s), does, its importance to feed and food
production, its chemical and biological properties, how to measure activity of the enzyme(s) and how to purify it
from raw animal, plant, or microbial sources. Being catalysts, the enzymes are produced in rather small amounts
(0.01 to 1% by weight of the total protein).
The contributors were carefully selected on the basis of their specic knowledge of the importance of enzymology
to food production, food preservation, and food quality. We are very proud and fortunate to have attracted rst-

rate, active scientists worldwide (20 countries are represented) as contributors to the Handbook. We hope the
Handbook will be prominently displayed in all college and university libraries, especially those in which food
production, nutrition, and food quality are important subjects, on the bookshelves of all food manufacturing
companies, and in many faculty ofces at major universities worldwide. When questions arise as to what enzymes
are, what they do, and their importance in food supply and quality, we hope students, educators, and researchers
will nd the answers rapidly and satisfactorily in the Handbook.
We thank all the authors for joining us in this endeavor. Their contributions are excellent, up-to-date, and well
written. Bringing together over 100 rst-rate, active contributors to provide chapters in their selected area of food
enzymology was our rst objective. We wish to thank all the editors and authors who permitted use of copyrighted
material, as well as those who typed the manuscripts. Marcel Dekker, Inc., and its staff were especially helpful as
editors and contributors alike struggled to meet deadlines that extended over four years.
To the reader, may you nd answers to all your questions on food enzymology in this handbook. If you dont,
please ask any of us to help you nd additional material and/or clarication.
John R. Whitaker
Alphons G. J. Voragen
Dominic W. S. Wong

Contents
Preface
PART I. GENERAL ASPECTS
INTRODUCTION
Protein Structure and Kinetics of Enzyme Reactions: A Historical Perspective

John R. Whitaker
History of Enzymology with Emphasis on Food Production

Poul Brge Poulsen and Klaus Buchholz
What Enzymes Do and Why They Are Highly Specic and Efcient Catalysts
John R. Whitaker
Enzyme-Catalyzed Reactions: Experimental Factors that Affect Rates

John R. Whitaker
Inactivation of Enzymes: From Experimental Design to Kinetic Modeling

Ann Van Loey, Indrawati, Chantal Smout, and Marc Hendrickx
REGULATORY ISSUES
Regulatory Issues of Enzymes Used in Foods from the Perspective of the E.U. Market
Danielle P. Praaning
Regulatory Issues of Food Enzymes Used in the United States

John R. Whitaker

EXTREMOPHILIC ENZYMES
Properties of Extremophilic Enzymes and Their Importance in Food Science and Technology
Magnus M. Kristjansson and Bjarni Asgeirsson
THE FOOD CHAIN AND ENZYMES
Nitrogen Fixation and the Enzyme Nitrogenase

William E. Newton
Enzymes in Amylose and Amylopectin Biosynthesis

Bruce P. Wasserman and Ying Yu
Fatty Acid Biosynthesis and Triacylglycerol Assembly

Allan Keith Stobart and Gareth Grifths
Phospholipid Biosynthetic Enzymes in Plants

Ralph E. Dewey and Anthony J. Kinney
Applications of Oxidoreductases in Foods

Colja Laane, Yvonne Bruggeman, and Chris Winkel
Transgenic Plants for Production of Enzymes

Anne S. Ponstein, Rob F. Beudeker, and Jan Pen
Enzymes in Protein Biosynthesis

John R. Whitaker
Nucleic Acid Biosynthesis

Dominic W. S. Wong
ENZYMES AS TOOLS TO MODIFY FOOD COMPONENTS
Protein Modication to Optimize Functionality: Protein Hydrolysates

Ton Kunst
Production and Modication of Acylglycerides

Rob M. M. Diks
ENZYMES IN FOODS POSTHARVEST
Signicance of Indigenous Enzymes in Milk and Dairy Products

Patrick F. Fox
Exogenous Enzymes in Dairy Technology

Patrick F. Fox
Flavor Enhancement in Fruit Juices and Derived Beverages by Exogenous Glycosidases and
Consequences of the Use of Enzyme Preparations
Ziya Gunata

IMMOBILIZED ENZYMES AND THEIR APPLICATIONS
Entrapment of Biocatalysts by Prepolymer Methods

Takuo Kawamoto and Atsuo Tanaka
Genetic Immobilization of Enzymes on Yeast Cell Surface

Mitsuyoshi Ueda, Toshiyuki Murai, and Atsuo Tanaka
Use of Immobilized Enzymes in the Food Industry

Harold E. Swaisgood
ENZYME UTILIZATION AND DEVELOPMENT
Enzymes and Food Analysis

Philip OConnell and George G. Guilbault
Recent Advances in Enzyme Development

Dominic W. S. Wong
PART II. SPECIFIC ENZYMES
OXIDOREDUCTASES
Catalase
Dominic W. S. Wong and John R. Whitaker
Horseradish Peroxidase
Zhong Yi Yuan and Tai Jiao Jiang
Glutathione Peroxidase
Jun-qiu Liu and Gui-min Luo
Glucose Oxidase
A. J. Vroemen
Lactate Dehydrogenase
Hayao Taguchi
Alcohol Dehydrogenase
Sabato DAuria
Sandrine Dallet, Marie Trovaslet, and Marie Dominique Legoy
Amine Oxidase
Akio Ito and Jichun Ma
Prolyl 4-Hydroxylase
Robert B. Rucker, Ana Samimi, and Jerold A. Last

Lysyl Hydroxylase
Ana Samimi, Jerold A. Last, Lucas C. Armstrong, and Robert B. Rucker
Lysyl Oxidase
Robert B. Rucker, Alyson E. Mitchell, Eskouhie Tchaparian, and Jerold A. Last
Superoxide Dismutase
Hirokazu Hara, Tetsuo Adachi, and Kazuyuki Hirano
Polyphenol Oxidase
Edna C. Ramrez, John R. Whitaker, and Victoria M. Virador
Laccase
William H. Flurkey
Mammalian Sulfhydryl Oxidase

Harold E. Swaisgood and Violeta G. Janolino
Xanthine Oxidase
John R. Whitaker
Lipoxygenase and Associated Enzymes

Harold W. Gardner
Sorbitol Oxidase
Kohei Oda and Kazumi Hiraga
TRANSFERASES
Starch Phosphorylases
Ying Yu
Amylosucrase
Volker Buttcher
Dextransucrase
Klaus Buchholz and Pierre F. Monsan
Levansucrase
Ki-Bang Song and Sang-Ki Rhee
Cyclodextrin Glycosyltransferase
Lubbert Dijkhuizen and Bart A. van der Veen
Limonoid Glucosyltransferase
Shin Hasegawa, Masayuki Kita, and Mitsuo Omura
Transglutaminase
Chang-Rak Ha and Ichiro Iuchi

HYDROLASES
Feruloyl Esterases
Craig B. Faulds and Gary Williamson
Lipase
Dominic W. S. Wong
Chlorophyllase
Roger F. McFeeters
Phytase
Onno Misset
-Amylases
Dominic W. S. Wong and George H. Robertson
-Amylases
Glucoamylase
Peter J. Reilly
Limit Dextrinase/Pullulanase
E. Ann MacGregor
Limit Dextrinase
James Hutchison Bryce
Methodologies for Assaying the Hydrolysis of Cellulose by Cellulases

David Johnston
Cellulases in Food Processing

Maija Tenkanen, Marja-Leena Niku-Paavola, Markus Linder, and Liisa Viikari
-Glucosidase
Asim Esen
-D-Fructofuranoside Fructohydrolase
Laura Cantarella, Francesco Alfani, and Maria Cantarella
-Galactosidase
Raymond R. Mahoney
Pectic Polysaccharides
H. A. Schols and Alphons G. J. Voragen
Pectic Enzymes
Jacques A. E. Benen, Alphons G. J. Voragen, and Jaap Visser
Pectic Esterases
Jacques A. E. Benen, Gert-Jan W. M. van Alebeek, Alphons G. J. Voragen, and Jaap Visser

Polygalacturonases
Jacques A. E. Benen and Jaap Visser
Enzymes Releasing L-Arabinose and D-Galactose from the Side Chains of Pectin
Ronald P. de Vries and Jaap Visser
Xylanolytic Enzymes
Peter Biely
Enzymes with Activity Toward Xyloglucan

Jean-Paul Vincken
Enzymes Degrading Rhamnogalacturonan and Xylogalacturonan

Jean-Paul Vincken, Alphons G. J. Voragen, and Gerrit Beldman
Enzymic Hydrolysis of Cereal (1!3, 1!4)--Glucans

Maria Hrmova and Geoffrey Fincher
Enzymology of Endo-1,4--Mannanases
Henrik Stalbrand
Lysozyme
Akio Kato
Ribonucleases
Jaap J. Beintema and Wei Zhao
Proteolytic Enzymes
John R. Whitaker
Thermolysin
Kuniyo Inouye
LYASES
Pectate and Pectin Lyases

Jacques A. E. Benen and Jaap Visser
Alliinases
Edna C. Ramrez
Cystine Lyases in Plants

Edna C. Ramrez
ISOMERASES
Xylose (Glucose) Isomerase

Onno Misset

Contributors
Tetsuo Adachi Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan
Francesco Alfaniy Department of Chemistry, Chemical Engineering and Materials, University of LAquila, LAquila, Italy
Lucas C. Armstrong Division of Pulmonary and Critical Care Medicine, Toxic Substances Research and Training Program,
Department of Internal Medicine, University of California, Davis, Davis, California, U.S.A.
Bjarni Asgeirsson Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, Iceland
Jaap J. Beintema Department of Biochemistry, University of Groningen, Groningen, The Netherlands
Gerrit Beldman Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University,
Jacques A. E. Benen Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University,
Rob F. Beudeker DSM Food Specialties, Delft, The Netherlands
Peter Biely Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia
Yvonne Bruggeman Functional Biomolecules, Unilever Research, Vlaardingen, The Netherlands
James Hutchison Bryce International Center for Brewing and Distilling, Department of Biological Sciences, Heriot-Watt
University, Edinburgh, Scotland
Klaus Buchholz Carbohydrate Technology, Braunschweig Technical University, Braunschweig, Germany
Volker Buttcher Aventis CropScience GmbH, Frankfurt, Germany
y Deceased.

Laura Cantarella Department of Industrial Engineering, University of Cassino, Cassino, Italy
Maria Cantarella Department of Chemistry, Chemical Engineering and Materials, University of LAquila, LAquila, Italy
Sabato DAuria Institute of Protein Biochemistry and Enzymology, Naples, Italy, and Center for Fluorescence Spectroscopy,
University of Maryland, Baltimore, Maryland, U.S.A.
Sandrine Dallet Laboratory of Protein and Cellular Engineering, University of La Rochelle, La Rochelle, France
Ronald P. de Vries Microbiology, Utrecht University, Utrecht, The Netherlands
Ralph E. Dewey Department of Crop Science, North Carolina State University, Raleigh, North Carolina, U.S.A.
Lubbert Dijkhuizen Department of Microbiology, University of Groningen, Haren, The Netherlands
Rob M. M. Diks Unilever Research and Development, Vlaardingen, The Netherlands
Asim Esen Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U.S.A.
Craig B. Faulds Food Materials Science Division, Institute of Food Research, Colney, Norwich, England
Geoffrey Fincher Department of Plant Science, University of Adelaide, Glen Osmond, South Australia, Australia
William H. Flurkey Department of Chemistry, Indiana State University, Terre Haute, Indiana, U.S.A.
Patrick F. Fox Department of Food Chemistry, University College, Cork, Ireland
Harold W. Gardner* National Center for Agriculture Utilization Research, Agricultural Research Service, U.S. Department of
Agriculture, Peoria, Illinois, U.S.A.
Gareth Grifths Horticulture Research International, Wellesbourne, Warwick, England
George G. Guilbault Department of Chemistry, University College, Cork, Ireland
Ziya Gunata Department of Bioengineering and Food Science, University of Montpellier II, Montpellier, France
Chang-Rak Ha Life Science Institute, Sophia University, Tokyo, Japan
Hirokazu Hara Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan
Shin Hasegawa Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany,
California, U.S.A.
Marc Hendrickx Laboratory of Food Technology, Katholieke Universiteit Leuven, Leuven, Belgium
Kazumi Hiraga Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, Japan
Kazuyuki Hirano Department of Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan
Maria Hrmova Department of Plant Science, University of Adelaide, Glen Osmond, South Australia, Australia
*
Retired.

Indrawati Laboratory of Food Technology, Katholieke Universiteit Leuven, Leuven, Belgium
Kuniyo Inouye Laboratory of Enzyme Chemistry, Division of Food Science and Biotechnology, Graduate School of
Agriculture, Kyoto University, Kyoto, Japan
Akio Ito Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan
Ichiro Iuchi Life Science Institute, Sophia University, Tokyo, Japan
Violeta G. Janolino Department of Food Science, North Carolina State University, Raleigh, North Carolina, U.S.A.
Tai Jiao Jiang Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
David Johnston Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
Wyndmoor, Pennsylvania, U.S.A.
Akio Kato Department of Biological Chemistry, Yamaguchi University, Yamaguchi, Japan
Takuo Kawamoto Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto
University, Kyoto, Japan
Anthony J. Kinney DuPont Experimental Station, Wilmington, Delaware, U.S.A.
Masayuki Kita Department of Plant, Cell, and Environment, National Institute of Fruit Tree Science, Tsukuba, Ibaraki, Japan
Magnus M. Kristjansson Department of Food Science, Science Institute, University of Iceland, Reykjavik, Iceland
Ton Kunst Quest International, Naarden, The Netherlands
Colja Laane DSM Food Specialties, Delft, The Netherlands
Jerold A. Last Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California,
Davis, Davis, California, U.S.A.
Marie Dominique Legoy Laboratory of Protein and Cellular Engineering, University of La Rochelle, La Rochelle, France
Markus Linder VTT Biotechnology, Espoo, Finland
Jun-qiu Liu Key Lab of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, China
Gui-min Luo Key Lab of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun,
China
Jichun Ma Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan
E. Ann MacGregor Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada
Raymond R. Mahoney Food Science Department, University of Massachusetts, Amherst, Massachusetts, U.S.A.
Roger F. McFeeters Agricultural Research Service, U.S. Department of Agriculture, and Department of Food Science, North
Carolina State University, Raleigh, North Carolina, U.S.A.
Onno Misset DSM Patents & Trademarks, Delft, The Netherlands

Alyson E. Mitchell Department of Food Science and Technology, University of California, Davis, Davis, California, U.S.A.
Pierre F. Monsan Department of Biochemistry and Food Engineering, National Institute for Applied Sciences, Toulouse,
France
Toshiyuki Murai Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto
William E. Newton Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, Virginia,
U.S.A.
Marja-Leena Niku-Paavola VTT Biotechnology, Espoo, Finland
Philip OConnell Laboratory of Sensor Development, Department of Chemistry, National University of Ireland, Cork, Ireland
Kohei Oda Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, Japan
Mitsuo Omura Department of Citrus Research, National Institute of Fruit Tree Science, Shimizu, Shizuoka, Japan
Jan Pen PlantZyme, Leiden, The Netherlands
Anne S. Ponstein Syngenta Mogen, Leiden, The Netherlands
Poul Brge Poulsen Novozymes A/S, Bagsvaerd, Denmark
Danielle P. Praaning DSM Food Specialties, Delft, The Netherlands
Edna C. Ram rez University of California, Davis, Davis, California, U.S.A.
Peter J. Reilly Department of Chemical Engineering, Iowa State University, Ames, Iowa, U.S.A.
Sang-Ki Rhee Biomolecular Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), and
Real Biotech Co., Ltd., KRIBB, Daejeon, Korea
George H. Robertson Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
Robert B. Rucker Department of Nutrition, University of California, Davis, Davis, California, U.S.A.
Ana Samimi Pharmacology and Toxicology Graduate Program, University of California, Davis, Davis, California, U.S.A.
H. A. Schols Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University,
Chantal Smout Laboratory of Food Technology, Katholieke Universiteit Leuven, Leuven, Belgium
Ki-Bang Song Biomolecular Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), and
Real Biotech Co., Ltd., KRIBB, Daejeon, Korea
Henrik Stalbrand Department of Biochemistry, Lund University, Lund, Sweden
Allan Keith Stobart School of Biological Sciences, University of Bristol, Bristol, England
Harold E. Swaisgood Department of Food Science, North Carolina State University, Raleigh, North Carolina, U.S.A.

Hayao Taguchi Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science,
Noda, Chiba, Japan
Atsuo Tanaka Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto
Eskouhie Tchaparian Agricultural and Environmental Sciences, Department of Nutrition, University of California, Davis,
Davis, California, U.S.A.
Maija Tenkanen VTT Biotechnology, Espoo, Finland
Marie Trovaslet Laboratory of Protein and Cellular Engineering, University of La Rochelle, La Rochelle, France
Mitsuyoshi Ueda Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto
Gert-Jan W. M. van Alebeek Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen
University, Wageningen, The Netherlands
Bart A. van der Veen Department of Microbiology, University of Groningen, Haren, The Netherlands
Ann Van Loey Laboratory of Food Technology, Katholieke Universiteit Leuven, Leuven, Belgium
Liisa Viikari VTT Biotechnology, Espoo, Finland
Jean-Paul Vincken Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen
Victoria M. Virador National Institutes of Health, Bethesda, Maryland, U.S.A.
Jaap Visser Fungal Genetics and Technology Consultancy, Wageningen, The Netherlands
Alphons G. J. Voragen Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen
A. J. Vroemen DSM Food Specialties, Delft, The Netherlands
Bruce P. Wassermany Department of Food Science, Rutgers University, New Brunswick, New Jersey, U.S.A.
John R. Whitaker Department of Food Science and Technology, University of California, Davis, Davis, California, U.S.A.
Gary Williamson Nestle Research Centre, Lausanne, Switzerland
Chris Winkel Department of Enzyme Research, Quest International, Naarden, The Netherlands
Dominic W. S. Wong Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
Ying Yu Department of Food Science, Rutgers University, New Brunswick, New Jersey, U.S.A.
Zhong Yi Yuan Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
Wei Zhao Department of Biochemistry, University of Groningen, Groningen, The Netherlands
y Deceased

1
Protein Structure and Kinetics of Enzyme Reactions

A Historical Perspective
John R. Whitaker
University of California, Davis, Davis, California, U.S.A.
I. INTRODUCTION tively) (1). This was during the time when the word
protein was coined to describe these compounds
This opening chapter focuses primarily on the history that have the compositional formula of carbon, hydro-
of applications of enzymes in food science and technol- gen, oxygen, and nitrogen 16%, with small
ogy. The evolution of knowledge on proteins and food amounts of sulfur and phosphorus. Methionine was
enzymology must also be understood from the funda- discovered in 1922 by Mueller (1) and in 1935 threo-
mental aspects. Enzymes are proteins. Therefore, the nine, the last amino acid, was discovered by Rose. It
history of food enzymology is a composite of advances was very difcult to determine the amino acid composi-
in both understanding the chemistry of protein struc- tion in the earlier days. With the discovery in 1952 by
ture and environmental behavior, as well as the physi- Moore and Stein (2) that the amino acids of protein
cochemical properties (kinetics and thermodynamics) hydrolyzates could be separated and identied readily
of enzyme behavior. within 24 h by ion exchange chromatography, the
This chapter will rst treat the history of food enzy- amino acid composition of many proteins became read-
mology from the evolution of protein chemistry and ily available. Today, the complete amino acid composi-
then the physicochemical behavior of enzymes in relation of a protein is done by automated analysis in 224
tion to environmental conditions, including the effects h using nano- or picogram amounts of hydrolyzed pro-
of enzyme, substrate, cofactor and inhibitor concentra- tein, at a cost of $2535 per total analysis.
tions, and the effects of pH and temperature.
B. Primary Structures of Proteins

II. PROTEIN CHEMISTRY DEVELOPMENT
Real proof of the importance of the peptide bond in
During the period 18201840 crude purications of proteins was when this bond joining individual amino
proteins began to be made by heat coagulation and acids into a longer polymer was discovered in 1903 by
precipitation with organic solvents and high concentra- Emil Fischer, who synthesized the rst peptides (3).
tions of salts, such as ammonium sulfate. Fischer synthesized many peptides during his scientic
career. Hofmeister also reported the peptide bond in
A. Amino Acid Composition 1903 (4). Sanger developed the methodology for deter-
mining the amino acid sequences of proteins in 1955
Glycine and leucine were the rst amino acids to be (5) using insulin (a polypeptide of 5737 daltons).
isolated (from hydrolyzed gelatin and wool, respec- Edman developed a chemical method of sequencing

proteins in 1949 (6); the automated Edman degrada- D. Tertiary Structures of Proteins
tion method, invented in 1967 by Edman and Begg (7),
is used routinely now for sequencing picogram quan- Tertiary structures of proteins, along with secondary
tities of protein within 24 h. The methods are so sensi- structures, were determined by x-ray crystallography
tive these days that one must wear plastic gloves in all of myoglobin by Kendrew et al. in 1960 (9). Even
operations. A typical cost per amino acid residue is though hemoglobin was crystallized in 1848, and the
about $2025. The time required for sequencing law of x-ray diffraction was established by Bragg in the
depends on the size of the protein, since most proteins 1920s, computer capabilities for quantitatively analyz-
must be hydrolyzed to 2550 amino acid residue frag- ing the complex x-ray patterns of proteins were not
ments by two or more highly selective proteases in available until the late 1950s.
order to produce overlapping protein fragments. The primary sequence of amino acids, while deter-
Each of the peptides must then be puried before mining the folding pattern of proteins, is not respon-
sequencing. sible for the folding. The aqueous system is. To be
The fastest, most economical, and most precise more precise, the H-bonded clusters (68 water mole-
method of determining the sequences of proteins, cules) forces the proteins to fold with predominately
since the 1980s, is by determining the nucleotide hydrophobic amino acid residues inside and predomi-
sequence of the gene for a protein. Extremely low nately hydrophilic amino acid residues on the outside,
amounts of DNA or sRNA are needed since PCR along with 2040% (on average) bound water of
(protein chain reaction) catalyzed by S-polymerase hydration around the protein. Folding of proteins is
can be used to make many copies of the gene. an entropy-driven process. The entropic energy from
However, it is important to remember that the amino the hydrogen-bonded clusters of water molecules is
acid sequence determination via gene sequence does used to force the protein to fold so as to occupy its
not include posttranslational modications, including smallest possible surface area, generally a sphere.
proteolysis, that are required to produce the active Proteins, and life, have evolved in an aqueous environ-
mature protein. The relationship between the primary ment. No other solvent could have caused the evolu-
sequence of proteins and gene nucleotide sequence was tion of biological functions as we know them.
rst postulated in the 1950sthe one geneone pro- Aqueous protein solutions contain three types of
tein hypothesis. waterstructural (constitutional) water 0:003 g
water/g protein), interfacial water ( 0:30.5 g water/
C. Secondary Structures of Proteins g protein on the surface of the protein, and free water
(14). The structural water molecules are bound tightly
The -helix secondary element of proteins was rst and are not removed by lyophilization or drying of the
postulated in 1950 by Linus Pauling (8) and was protein or by displacement of water by acetonitrile (15).
found in myoglobin by Kendrew et al. (9) by x-ray In the serine superfamily of enzymes, 20 structural
crystallography in 1960. The determination of water molecules are found in identical locations in all
strands in the primary structures of proteins, subse- (16). The surface of native proteins contains, on the
quently resulting in sheet formation by folding of average, 2040% by weight of bound water molecules.
the polypeptide chain back on itself, was reported in The rst monolayer (vicinal) of water molecules binds
1951 by Pauling and Corey (10). The and turns, primarily to the charged and neutral side chains of the
loops, and bulges were clearly established in proteins surface amino acid residues by hydrogen bonds. Water
by 1987, based on Milner-White and Poets reported molecules surround surface hydrophobic side chains as
observation of reproducible x-ray crystallographic a cage but do not interact with them. The second and
conformation in previously described loops (random subsequent layers (multilayers) of water molecules bind
folding) of a number of proteins (11). The key roles less tightly by dipole interaction with the monolayer
of glycine and proline in forming and turns are water. Bound water and free water can be distinguished
now well established, with subclasses of each of these readily by nuclear magnetic resonance (NMR).
turns. Therefore, the key secondary components of
proteins are -helices (a few other types of helices
also occur in some proteins), strands, and and E. Quaternary Structure
turns and loops. Supersecondary folding patterns
(motifs), involving a minimum of three secondary The importance of subunits in some proteins was
structures, are well known (12, 13). recognized in 1970 by Klotz et al. (17), when it was

discovered that a 0.4 M sodium chloride solution ing quaternary and macromolecular structures. But it
caused hemoglobin to change from 68,000 daltons to is appropriate to mention that many proteins are
34,000 daltons. Later, it was shown that the tetrameric posttranslationally modied in 135 ways (collec-
protein was cleaved to give two identical subunits con- tively among proteins) and that posttranslational
taining two polypeptide chains, and , and that modication plays a very important, sometimes essen-
higher concentrations of salts caused further dissocia- tial role in the folding and stabilizing of a protein in
tion to subunits ( and ) of 17,000 daltons. Perutz et its biologically functional form.
al. (18) determined the x-ray crystallography pattern of Detailed analyses of the data from x-ray diffraction
hemoglobin in 1960. A number of other proteins and of protein crystal structures became available in the
enzymes have subunits (17). Subunits of proteins were 1960s owing to advances in computers. NMR was
found to be devoid of biological activity; in time, a developed sufciently by the 1980s to be used for deter-
molecule of a protein was dened as the smallest unit mining the tertiary structures of proteins. Modern x-
that has biological activity. ray diffraction and new methods of intense beam mag-
nication, using a synchrotron, permit the tertiary
structures to be determined at 1.2 A, sufcient to iden-
F. Macromolecular Structure tify primary amino acid sequence, the secondary struc-
ture, the tertiary structure, and in some cases the
By the late 1960s, it was recognized that the skeletal quaternary structure. In some cases crystallographic
muscles of animals are composed of several different structures of proteins are now being determined at a
proteins, myosin, actin, tropomyosin, and troponin faster pace than the biological function of the proteins.
(three types) that, when associated correctly, had con- The most complex protein in which the tertiary
tractile properties in the presence of the cofactor ATP. structure has been determined recently is that of
Later, it was shown that pyruvate dehydrogenase, the yeast RNA polymerase II (25).
electron transport system, and other systems perform-
ing complex functions require association of several
different proteins to perform the overall biological H. Protein Crystallization
function (19, 20).
Hemoglobin was the rst protein to be crystallized, by
accident, in 1848. Almost anyone can crystallize hemo-
G. Folding of Proteins from the Primary globin from blood, but recognizing that it is a protein
Structure is more difcult. Ovalbumin from chicken egg white
was crystallized in the 1880s. But it was the crystal-
By the 1960s, physical chemists such as Harold lization of urease in 1926 by J.B. Sumner that nally
Scheraga at Cornell University, Phillip Handler at proved that enzymes are proteins (26). Again, seren-
North Carolina State University, Ronald Baldwin at dipity (being at the right place at the right time and
Stanford University, and others began to ask how recognizing it) played an essential role in this discov-
protein folding occurs and how fast it occurs. Dr. ery. As the story goes, Professor Sumner of Cornell
Scheraga, using the hypothesis that every atom of University made a water/alcohol extract of jack
the protein had to nd its proper location by a ran- beans on a Friday, removed the solids, and placed
dom process, and using high-speed computers, pre- the extract on the window sill of the laboratory and
dicted that it would take 1050 years for a 100 went home. On Monday, a solid had formed that,
amino acid residue polypeptide to fold properly. when the liquid/solid was swirled, refracted lighta
About the same time, Hantgan et al. (21) and others biofringence. Recognizing this as unusual, he deter-
(22, 23) began to unfold proteins by heat, urea, pH mined under the microscope that crystals were present.
changes, etc., to remove the unfolding agent and mea- He showed that the crystals were protein with urease
sure the rate of regain of activity. Lu and Whitaker activity, so he suggested that enzymes are proteins.
(24) and others found that denatured proteins could Despite the earlier nding by Willstatter in 1926
regain most of their activities in 610 min at 358C. that highly puried peroxidase was not a protein
Not too long after, sufcient data accumulated to (27), Sumner has been proven correct that all enzymes
show clearly that the primary sequence of amino known to date are proteins. Willstatter made the mis-
acids contained the information needed to permit take that many scientists make at one time or another;
the protein to fold into the native structures, includ- the ability to measure the peroxidase activity was much

more sensitive than the ability to measure the protein amino acid replacement is about as close to the truth as
concentration. one might get of the role of that amino acid.
Protein crystallization is a science these days permit-
ting most proteins, including even glycoproteins and
lipoproteins, to be readily crystallized as demon-
strated, for example, by the crystallization of yeast S- III. DISCOVERY OF ENZYMES AND
RNA polymerase II (25). DEVELOPMENT OF ENZYME
KINETICS
I. Active Sites of Proteins A. Discovery of Enzymes
Active sites of proteins are the locations where the There is some overlap between this section and that of
biological function of a protein resides in the native Chapter 2. This is justied based on the need to present
protein. Study of active sites of proteins became a a complete picture in the book.
major eld of study for enzymologists and protein che-
mists beginning in the 1960s. But as early as 1902, 1. Catalase appears to be the rst enzyme discov-
Brown (28) and Henri (29) independently proposed ered. This occurred in 1812. In reality, the discovery
that enzymes must bind the substrate prior to conver- was that something in blood caused the evolution of
sion to product. Fischer in 1894 (30) proposed that the bubbles, shown to be O2 , when hydrogen peroxide was
active site must t the substrate as stereospecically as added. The reaction observed was
a key ts a lock (lock-and-key analogy) in order to be 2 H2 O2 ! 2 H2 O O 2
bound properly and be converted to product.
Koshland in 1965 (31) introduced the induced t 2. Diastase was discovered in wheat our by Payen
hypothesis to explain how apparently dissimilar mole- and Persoz in 1833 (34), when they reported that the
cules could sometimes serve as substrates for the same enzyme caused the solubilization of starch. In addi-
enzyme. tion, they found the enzyme to be thermolabile and
Initially, study of active sites of enzymes was precipitated by alcohol. They named the substance
approached by use of chemical modication of the diastase because it separated, by solubilization,
side chain groups of amino acid residues of the native the starch from the bran. They further used ase
protein, using loss of biological activity as the measure. in the name to designate it as an enzyme. The reac-
Also, the unfolding and refolding of proteins such as tion observed was
myoglobin and ribonuclease, especially around cofac- Starch H2 O ! dextrins
tors, hematin, etc., gave much information on active
3. Peroxidase in plants was reported by Schoenbein
sites. Much was learned about essential amino acids in
in 1855 (35, 36). Schoenbein used gum guaiac which
the active site. For example, in lysozyme chemical
changes in color from light tan to blue when H2 O2 was
modication established that Glu35, Asn37, Asn44,
added to an extract containing peroxidase. Several
Asp52, Asp59, Try62, Try63, Asp101, and Arg114
other compounds can be used to assay easily for per-
are a part of the active site (32). Asp52 and Glu35
oxidase as shown by the equation:
are the key catalytic general acid/general base residues
(COOH and COO are the catalytic groups). X-ray 4 Guaiacol 8 H2 O2 ! tetraguaiacol 8 H2 O
crystallography was key to determining that His69, colorless brown
Glu72, and His196 (chelates the essential cofactor
Zn(II)), Arg71, Arg145, Tyr198, Tyr248, and Phe279 4. Polyphenol oxidase was discovered by
are in the active site of carboxypeptidase A (33). Zn(II) Schoenbein in plant extracts in 1856 (36). It was
is responsible for catalysis. The other amino acid resi- named polyphenol oxidase because of its ability to
dues are key to binding the substrate properly in the oxidize phenols, cause browning of plant extracts and
active site. bruised plant tissues. The enzyme can use a number of
The best current method of determining the essenti- mono- and polyphenols plus O2 as substrates as shown
ality of amino acid residues in the active site is the use by:
of recombinant biotechnology to replace one amino Phenol 0:5 O2 ! o-dihydroxyphenol
acid at a time, thereby determining its effect on activ-
o-Dihydroxyphenol 0:5 O2 ! o-benzoquinone
ity, is much more certain than the two methods above.
Absence of binding and/or activity based on a single H2 O

5. Invertase was discovered in 1860 by Berthelot in accommodation (exibility) of the active site of the
yeast (37). The enzyme catalyzes the hydrolysis of enzyme in binding the substrate. Further research has
sucrose to glucose and fructose: shown that the induced t concept is an important
factor in the specicity of numerous enzymes and per-
Sucrose + H2 O ! glucose fructose haps less important in more rigid active sites of some
enzymes, such as urease, which converts only urea to
56:58 65:58 (79.28
products.
where the numbers in parentheses are optical rotation.
During this period of enzymology, polarimeters C. Quantition of Rates
were developed that could manually or continuously
follow the changes in optical rotation during the con- The importance of quantitation of rates was intro-
version of sucrose to glucose and fructose by the duced briey in Section III.A above. This section will
change in optical rotation from 56.58 to 23:78 based expand on the topic.
on the difference in optical rotation of the sucrose (the
substrate) to the products (glucose and fructose). This 1. Effect of Temperature on Velocity
ability eventually led to discovery and quantitation of
the effect of pH, temperature, and substrate concentra- In 1889, after a large amount of research on invertase
tion on the kinetics of enzyme reactions. using a polarimeter to study the effect of temperature
The discovery of enzymes continues even today. In on velocity of reaction, Arrhenius developed the
the last listing in 1992 of well-studied enzymes there empirical equation:
were 3196 enzymes listed (38). The types of enzymes so
dlnk=dT AeEa =RT
far discovered based on the chemical reactions cata-
lyzed are oxidoreductases, transferases, hydrolases, which is still in use by most biologists today (39).
lyases, isomerases, and ligases. Many of the individual Physical chemists derived de novo an equation
enzymes have isozymes (same reaction catalyzed but (Gibbs equation; 40) that gives a slightly different
they differ in primary structure of the protein). The numerical value from that of Arrhenius. The difference
nucleotide sequence of the genes that code for the bio- is given by
synthesis of the enzyme can be modied by researchers
to give multiple forms of enzymes via one or more Ea H 6 RT
amino acid changes of the primary sequence.
At 258C Ea is 0.6 kcal/mol larger than H 6 given by
Posttranslational modication of the protein can also
Gibbs equation because a slight temperature effect on
lead to multiple forms.
Ea is removed by using H6 . For most purposes the
two methods of calculation agree within experimental
B. Enzyme Specicity
error.
In the period 1894 to the 1930s Emil Fischer, a
2. Effect of Substrate Concentration on
German chemist, and his associates developed and
Velocity
rened the concept of enzyme specicity and of the
close stereospecicity between an enzyme and its sub- Studies of effect of substrate concentration on velocity
strate(s) (30). Based on their careful studies of enzyme of enzyme-catalyzed reactions advanced primarily
action on numerous compounds his group synthesized, owing to the availability of the polarimeter, permitting
Fischer enunciated the now famous lock-and-key the effect of substrate (sucrose) concentration on the
analogy of enzymesubstrate interaction. Other com- velocity of the invertase-catalyzed reaction to be stu-
pounds, called competitive inhibitors, bind into the died in great detail. The results showed a saturation-
active site in competition with the substrate but they type curve (Fig. 1). At very low substrate concentra-
are not converted to products because they lack the tions, the velocity increased in direct proportion to the
overall keylike t to the active site. increase in substrate concentration. At higher concen-
In 1959, Koshland emphasized the importance of trations, the relationship was curvilinear and at high
the induced t concept of enzyme specicity (31). concentrations, the rate reached a maximum (and even
His detailed research showed that compounds that decreased in some cases) owing to substrate inhibition.
do not have identical shapes can be substrates for the In 1903 Brown (28) and Henri (29) independently
same enzyme and that this is possible because of proposed that the saturation curve for the relation-

ship between velocity and substrate concentration [A]o uous manner (Fig. 2). There are two other methods
could be explained by the equation that give linear plotsthe Augustinsson method (44),
EA!EAEP and the Eadie-Hofstee method (45); however, the
Lineweaver-Burk method continues to be the most
where E A is an obligatory intermediate; i.e., E and A widely used method.
must form a noncovalent complex before conversion of
substrate to products can occur. This concept is now
3. Effect of pH on Enzyme-Catalyzed
universally accepted.
Reactions
In 1913, Michaelis and Menten (41) showed that the
enzymesubstrate saturation curve can be expressed In 1909, Sorensen published a classic paper on the
mathematically by the equation of a right hyperbola effect of pH on invertase activity (46). Not only did
he show that the activity of enzymes is affected by the
vo Vmax Ao =Km Ao
pH of the reaction, he was able to differentiate the
where vo is the observed initial velocity, Vmax is the effect on reaction rates and on stability of the
maximum velocity when the enzyme is fully saturated enzyme.
with substrate, and Km is A at which vo 0:5 Vmax . Detailed studies of pH effects on rates of enzyme-
In 1918, Langmuir (42) showed independently that catalyzed reactions were largely ignored until the 1960s
the same-shape curve is found for adsorption of gases when Bender, Whitaker, and others gave much atten-
onto metal surfaces, soluble solutes onto charcoal, etc. tion to the interpretation of the results from a mechan-
The Langmuir equation is: istic standpoint. See Whitaker (47) for more details of
Fraction of surface covered ns x=ne ns x the studies.
where ns is number of molecules striking 1 cm2 of sur-

face per second, x is the quantity of molecules that 4. Further Renements of Kinetics
binds on the surface, and ne is the number of molecules During 19571963, there were substantial advance-
leaving the surface per second. ments in better understanding of the effect of concen-
In 1934, Lineweaver and Burk (43) showed that a trations of one, two, or multiple substrates on enzyme-
reciprocal plot of vo versus Ao (i.e. 1=vo vs. 1=Ao catalyzed reactions. These advancements were made by
converts the Michaelis-Menten equation to a form in
which Vmax and Km can be determined in an unambig-
Figure 1 Variation of initial velocity with substrate concen- Figure 2 Plot of reciprocal of initial velocity versus recipro-
tration for an enzyme-catalyzed reaction according to the cal of initial substrate concentration by the Lineweaver-Burk
Michaelis-Menten equation. (From Ref. 41.) method. (From Ref. 43.)

Alberty (48), Wong and Haines (49), Cleland (50), and IV. ANALYTICAL TOOLS IN
Segel (51). Clelands descriptive nomenclature (50), ADVANCEMENT OF ENZYMOLOGY
ordered BiBi sequential mechanism, random BiBi
sequential mechanism, and Ping Pong BiBi mechanism Advancement of enzymology, as with all chemical and
terms and methodology are used most frequently. See biological sciences, has been inuenced very much by
Whitaker (47) for a detailed discussion and use of these the development of tools and methodology. Perhaps
methodologies. one of the rst tools was the microscope, which per-
mitted Pasteur to discover the asymmetry of molecules
leading to discovery of isomeric differences among L-,
5. Concept of Initial Velocity D-, and DL-tartaric acid crystals in wine. A specic
enzyme can use only one of the forms as a substrate.
The concept of initial velocity, vo , arose in the 1960s
Another was the development of polarimetry which
and is universally used by those interested in detailed
permitted detailed studies on the effect of pH, tempera-
studies of enzymes. vo is determined as near time zero
ture, and substrate concentration effects on invertase
of the reaction as possible. No more than 5% of the
particularly and other enzymes in general. These stu-
original Ao should be converted to product in deter-
dies led to Sorensens contribution to understanding
mining vo . The method requires that the velocity be
the effect of pH on enzyme activity (46), to
determined on a continuous time basis, generally by
Arrhenius study of temperature effect on rates of
continuous recording of the data.
invertase-catalyzed reactions (the Arrhenius equation
There are several reasons for using vo . It largely
[39], and data on the velocitysubstrate concentration
eliminates problems caused by change in degree of
relationships leading to the Henri (29) and Brown (28)
saturation of the enzyme with substrate during the
hypothesis on essentiality of the EA complex, the
reaction time. It eliminates inuence of the back reac-
Michaelis-Menten equation (41), and the Lineweaver-
tion P E E A. It eliminates effect of product
Burk reciprocal plot (43).
inhibition of the enzyme. It eliminates effect due to
Kinetic analyses are used to determine how much
instability of the enzyme, since the reaction is nor-
enzyme is present in tissues. But their primary use is to
mally done within 5 min. Since it is based on a
determine the mechanism of action of enzymes via a
continuous recording, not on a one-point assay, the
detailed study of the kinetic effect of substrate concen-
substrate concentration Ao at time zero can be
tration, enzyme concentration, pH, temperature, and
used.
inhibitors on the vo of the reaction. Early investiga-
tions in this area were by Koshland and Neet (54)
and by Vallee and Riordan (55). Many articles,
6. Sigmoidal Kinetics books, and treatises of this subject have appeared
Experimentally, not all enzyme-catalyzed reactions fol- over the past 30 years.
low Michaelis-Menten kinetics. In 1965, Monod et al.
(52) published the rst paper conclusively showing that A. Tools for Purication of Enzymes
some enzyme-catalyzed reactions do not follow the
Michaelis-Menten equation. The kinetics are said to Not much was done on the purication of proteins
be sigmoidal or allosteric in nature. The form of the until the 1920s when Willstatter puried horseradish
equation developed to express the results is: peroxidase to the point that no protein could be
detected by the method used, but the peroxidase activ-
vo Vmax Sno =Km Sno ity could be determined, leading to the conclusion that
horseradish peroxidase could not be a protein (27). But
Sigmoidal kinetics are sometimes found when more Sumner (26) crystallized jackbean urease and showed it
than one substrate molecule binds to the enzyme mole- to be a protein. In 1930 Haldane (56) and others
cule. Generally, sigmoidal kinetics may be shown in expressed the view that except for urease, next to noth-
enzymes that have quaternary structure. If binding of ing was known about the chemical nature of proteins.
substrate into the active site of the rst subunit affects, In the 1930s, several enzymes, including pepsin,
positively or negatively, the binding of substrate into trypsin, chymotrypsin, and carboxypeptidase A, were
the active site of the second subunit, sigmoidal beha- crystallized by Northrop, Kunitz, Herriott, and Anson
vior will be seen. Enzymes in metabolic pathways often (57, 58) at the Rockefeller Institute. All were found to
show sigmoidal behavior (53). be proteins. The ultracentrifuge (59) and free boundary

electrophoresis (60) were developed and used to deter- The latest tools to be used in enzymology involve
mine the molecular weight and purity of enzymes. identication of presumed primary amino acid struc-
From 1930 to 1955, use of differential solubility of ture of enzymes by sequencing the genes that encode
proteins at different pHs, as well as differential tem- enzymes, by the replication of enzymes by the polymer-
perature stability, were used to purify proteins. Large ase chain reaction, and the mutagenesis of enzymes by
amounts of protein were needed. In the late 1940s and replacement of one or more nucleotide bases or by
early 1950s Moore and Stein (2) developed ion combinational methods. As noted earlier, signicant
exchange chromatographic methods for separating posttranslational modication of the enzyme can
the amino acids from hydrolysates of proteins, includ- occur after translation of the amino acid sequence.
ing enzymes.
But it was the period beginning in 1956 and extend-
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Muirhead, JC Coppola. Structure of carboxypepti- 12:88118, 1965.
dase A. The 2.0-angstrom resolution studies of the 53. DE Koshland Jr, G Nemethy, D Filmer. Comparison
enzyme and its complex with glycyltyrosine, and of experimental data and theoretical models in pro-
mechanistic deductions. Brookhaven Symp Biol teins containing subunits. Biochemistry 5:365385,
1:2490, 1969. 1966.

54. DE Koshland Jr, KE Neet. The catalytic and regula- 66. EH Cooper, R Turner, JR Webb, H Lindblom, L
tory properties of enzymes. Annu Rev Biochem Fagerstam. Fast protein liquid chromatography
73:359410, 1968. scale-up procedures for the preparation of low-mole-
55. BL Vallee, JF Riordan. Chemical approaches to the cular-weight proteins from urine. J Chromatogr
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Biochem 38:733794, 1969. 67. O Vesterberg. Isoelectric focusing of proteins.
56. JBS Haldane. Enzymes. London: Longmans, Green, Methods Enzymol 22:389-412, 1971.
1930. 68. K Weber, M Osborn. Proteins and sodium dodecyl
57. M Kunitz, JH Northrop. Isolation of a crystalline sulfate: molecular weight determination on polyacry-
protein from pancreas and its conversion into a new lamide gels and related procedures. In: H Neurath,
crystalline proteolytic enzyme by trypsin. Science RL Hill, CL Boeder, eds. The Proteins, Vol 1. 3rd
78:558559, 1933. ed. New York: Academic Press, 1975, pp 179233.
58. M Kunitz, JH Northrup. Isolation from beef pancreas 69. S Moore, WH Stein. Determination of the structures
of crystalline trypsinogen, trypsin, a trypsin inhibitor of proteins. Studies on ribonuclease. Harvey Lect
and an inhibitor-trypsin compound. J Gen Physiol 52:119143, 1958.
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2
History of Enzymology with Emphasis on Food Production
Poul Brge Poulsen

Novozymes A/S, Bagsvaerd, Denmark
Klaus Buchholz
Braunschweig Technical University, Braunschweig, Germany
I. INTRODUCTION tinuous technical development can be traced to the

beginning of the 20th century, where Rohm and the
The scientic development in biocatalysis goes back foundation of the Rohm and Haas company in 1907
to the beginning of the 19th century. The rst enzy- may be seen as a major technical and economic break-
matic actions, notably starch hydrolysis by Diastase, through.
was acknowledged as a catalytic effect by Berzelius in
1835 and utilized industrially following the ndings
and developments of Payen and Persoz on diastase II. THE EARLY PERIOD TO 1880
and its application in the brewing industries about
10 years later. Continuous scientic investigations, Applied biocatalysis has its roots in ancient China
with emphasis on alcoholic fermentation, can be and Japan in the manufacture of food and alcoholic
traced to the 1850s with investigations of Berthelot drinks. Without knowing, mankind utilized microbial
and Bechamp, the school of Pasteur, and the contro- amylases and proteases, in particular for the produc-
versy with the chemical school of Liebig and his inu- tion of soy-derived foods. In Europe also, applied
ence. biocatalysis has a long history. Cheese making has
The rst company based on applied biocatalysis always involved the use of enzymes. As far back as
also dates back to the 19th century, when in 1874, about 400 B.C., Homers Iliad mentions the use of
Christian Hansen started a company in Copenhagen kid stomach for making cheese. It was discovered
named Christian Hansens Laboratory which exists that milk, which was stored in a bag made of a sto-
to this day, producing an enzyme preparation, rennet mach of a recently slaughtered calf, lamb, or kid was
(chymosin), for cheese making. converted into a semisolid substance. Upon pressing
After this early period, research in the subsequent of this substance a drier material was obtained
decades brought about signicant progress with the (namely cheese) which showed preferred properties
important ndings of E. Fischer in 1894 on enzyme over milk, could be easily transported, and gained a
specicity, and E. Buchner in 1897 on the pure chemical avor after some time. These ancient applications
nature of alcohol formation, and the key steps by may best be described as an art and not as a technol-
Sumner and Northrup in crystallizing enzymes in 1926 ogy, a scientically based technique. The context of
and 1930, respectively. These represented turning points biotechnology has been described in a profound ana-
in identifying the nature of the catalytic agent. A con- lysis by Bud (1).

A. Findings and Empirical Results in textbooks on chemical technology within a few
years. Poppes Technology (7) was prescientic insofar
Stahl explored in his book Zymotechnika Fundamentalis as it did not show any chemical formula, working on a
(1697) the nature of fermentation as an important strictly descriptive level. In contrast, Knappes
industrial process, where zymotechnique should be the Chemical Technology (9) gave chemical formulae,
scientic basis (2). About a hundred years later the thus for sugar (not yet fully correct) based on the
liquefaction of meat by gastric juice, as an enzymatic atomic composition of the molecules. Most clearly
activity, yet not recognized as such, was noted by Payens textbook (10, Vol. 1: p. 1) reected the theo-
Spallanzani in 1783 (3). The enzymatic hydrolysis of retical background of chemistry based on atomic and
tannin was described by Scheele in 1786 (4: p. 30). In molecular composition and constitution, differentiat-
1814 Kirchhoff observed that a glutinous component of ing theoretical, applied, and technical chemistry.
wheat was capable of converting starch to sugar and The book by Knapp (9: pp. 300303), about a dec-
dextrin, and Vogel showed in 1817 that an infusion of ade after Poppes (7) view of fermentation, develops a
oats would produce a fermentable sugar from milk rather distinct picture of the action of ferments (mainly
(5: p. 5). referring to diastase and the work by Payen and
Some vital factor, le principe vital, was considered Persoz), and these views are expressed more precisely
an important principle in the chemical processes asso- by Payen (10: pp. 399403). Fermentation is seen as a
ciated with the synthesis of materials isolated from contact (catalytic) process of a degradation (Spaltungs-
living matter. ) or addition process (with water). It can be performed
by two substances or bodies: a nitrogen-containing
All simple bodies in nature are subject to the
organic (nonorganized) substance, such as protein
action of two powers, of which one, that of
material undergoing degradation; or an organized
attraction, tends to unite the molecules of bodies
body, a lower-class plant or an infusorium, such as
one with another, while the other, produced by
with alcoholic fermentation. Probably the type of
caloric, forces them apart. . . . A certain number
effect is the same insofar as the ferment of the second
of these simple bodies in nature are subject to a
class produces a body of the rst class, possibly a big
third force, to that caused by the vital factor,
number of singular ferments. In 1878 Kuhne named
which changes, modies and surpasses the two
the latter class of substances enzymes.
others, and whose limits are not yet understood
A breakthrough occurred with the observations of
(6).
Dubrunfaut and subsequently Payen and Persoz.
In one of the books on technology in the period Dubrunfaut discovered the transformation of starch
preceding Berzelius and Payen, Poppe (7) discussed into sugar and some kind of gum by means of water
mysterious ideas concerning fermentation: and germinated barley, as well as the application of
this nding for technical and economic purposes (11).
Fermentation is seen as aat a time and under
Payen and Persoz in 1833 (12) prepared the acting
circumstances spontaneousoccurring mighty
principle in isolated form, which they named diastase,
movement in a liquid of different compounds. . .,
with the following characteristics: it is a white, amor-
which is due to the fact that several compounds
phous solid insoluble in alcohol and soluble in water.
act in harmony with each other, others in oppo-
For the production of diastase they macerated germi-
sition to each other, so that the rst attract, the
nating barley in cold water, then pressed and ltered it.
latter reject each other. . . . The sugar and
The ltrate was heated to 708C, ltered again, then
a. . .gum extract material act by antagonist forces
precipitated with alcohol. The precipitate was collected
on each other so heavily, that they decompose
on a lter and subsequently, by repeated redissolving
and thus cause the formation of alcohol
and precipitating with alcohol, separated from the
(Weingeist) (7: p. 229).
accompanying residual nitrogenous substances (12).
Gay-Lussac had postulated a generatio spontanea, The dextrin production was performed in a reaction
the hypothesis that a continuing, rather mysterious vessel as shown in Figure 1 (10). The vessel inserted
chain should be the cause of spontaneous generation in a water bath could be heated to 758C, the optimal
(of organisms) (summary in Ref. 8). Pasteur showed temperature for the formation of dextrin by diastase.
that this assumption was without any empirical basis. The reaction progress was controlled by the iodine test
Remarkably, a most signicant change in the theo- and stopped by heating to 1008C. The product solution
retical background, a change in paradigm, took place was subsequently ltered, collected in a reservoir, and

Figure 1 Process for dextrin production with reaction vessel (a), lter (b), reservoir (c), concentration unit (d). (From Ref. 10.)
concentrated by heating with steam. The product Further enzymatic conversions (ferment actions)
rapidly found industrial application. observed in that early period summarized in
Payen and Persoz (12) identied numerous topics Franklands list of soluble ferments (14) and by
for further research, such as the existence of diastase Sumner and Somers (3) are summarized in Table 1.
in plants, its molecular weight and elementary compo- Alcoholic fermentation was a dominating topic of
sition, and the reaction products from different sub- the time in the eld called physiological chemistry
strates. Diastase was the dominating object of research which attracted a good deal of interest and activity
on enzymes throughout the century with 1020% of of leading scientists (5). Trying to identify the active
publications dealing with this topic owing to its eco- principle, Bethelot (15) showed, according to his own
nomic importance. Nevertheless the overall research interpretation, that a peculiar nitrogen-containing sub-
activities remained very weak in this eld, as compared stance, formed by yeast and precipitable by alcohol (he
to chemistry (see below). used casein) and comparable to diastase, could trans-
A few further activities were discovered in subse- form sugar into alcohol. He stated that this substance
quent years. Pectase was found in plants, both in solu- was different from yeast and that there was no produc-
ble and nonsoluble form. It was able to break down tion of yeast cells (8, 15). Thus, he claimed to have
pectose into pectinic acid, and was attributed to have observed the transformation of sugar into alcohol
some similarity to diastase. Neither material could be without the participation of any activity of living
crystallized (10: p. 30). Claude Bernard was the rst to organisms. He was convinced that the formation of
show lipolytic activity in pancreas in 1856 (4: p. 25). alcohol from sugar was a genuine chemical process.
Dobell (13) found that an extract from pancreas However, his experiments were not performed under
hydrolyzed both fat and starch; he gave a procedure sterile conditions, and obviously unknown in part,
for extraction and stabilization and named this pre- anaerobic bacteria (in tests where oxygen was
paration pancreatine. excluded) must have produced scarce amounts of alco-

Table 1 Ferments (Enzyme Activities) Known Up to 1880
Ferment
Enzyme source Catalyzed reaction Author
Protease gastric juice meat liquefaction Spallanzani, 1783a

Cyanogen plant roots substrate: guaiacum Planche, 1810a
Glutin compound wheat starch hydrolysis Kirchhoff, 1814a
Emulsin bitter amygdalin hydrolysis Robiquet and Boutron, 1830a ;
almonds Chalard, Liebig and Wohler, 1837a
Diastatic activity ptyalin starch hydrolysis Leuchs, 1831a
Amylase malt starch hydrolysis Payen and Persoz (1833) (12)
Sinigrinase Faure, 1835a
Pepsin protein hydrolysis Schwann, 1836a
Trypsin protein hydrolysis Corvisart, 1856a
Saccharase yeast sucrose hydrolysis Berthelot, 1860a
Pectase plants pectin hydrolysis Payen (1874) (10)
Pancreas lipolytic activity Bernard, 1856b
Pancreas extract fat and starch hydrolysis Dobell (1869) (13)
Pancreatic ferment pancreas fat hydrolysis Frankland (1885) (14)
a
In (3).
b
In (4).
hol, carbon dioxide, but also hydrogen and acetic and there was about one article per year in the 1860s deal-
butyric acid (16). It was 40 years later when Buchner ing with soluble ferments, meaning enzyme activity,
succeeded in demonstrating cell-free alcohol formation and three or four dealing with fermentation. It was
unequivocally (see below). only in the 1880s that research and publication activ-
Pasteur presented a series of experimental results ities increased signicantly.
showing unequivocally that alcoholic fermentation
proceeded only in the presence of living organisms. B. Technology
And he showed that in all cases where fermentation
could be observed under precisely controlled condi- Work on technological issues was more important and
tions, it was microorganisms (of different kinds, also even dominating over that on basic aspects from the
present in the air) that must be present to initiate fer- beginning of the 19th century, especially for brewing,
mentation. So he presented strong evidence against the wine, bread, and acetic acid production. These topics
assumption of a generatio spontanea postulated by made up important parts of the chemical technology of
Gay-Lussac. He denitely changed, about two decades the time (7, 9, 10, 18). Knapp (9) considered scientic
later, the disciplinary context of fermentation away and practical industrial interests as equally important.
from chemistry, redening the subject to a new auton- Diastase application was a major issue from the
omous disciplinemicrobiology (1). 1830s onward. The diastase presents the best means
The overall research activity on soluble ferments, for production of dextrin and dextrin syrup. These
meaning enzymatic reactions, was scarce in this period. products not only become cheaper, but also more
Thus, in the German Annalen der Pharmacie in the pure and tasty. This latter argument was used in
period from 1833 to 1850, four articles were published favor of the application in food and beverage produc-
on ferments and fermentation, three of them on diation. Thus, it was recommended for the manufacture of
stase. The key paper was essentially a translation from bread, pastries, chocolate, and soups; furthermore, it
Payen and Persoz (12, 17). In the German Journal fur was recommended for cider and fruit wine, but espe-
Praktische Chemie in the period from 1850 to 1860 no cially beer production, where a specic advantage was
paper dealt with soluble ferments (enzymatic activities; that the formation of haze at low temperature could be
eight papers were published on fermentation prevented (12, 17). Also, a sugar could be isolated after
[Gahrung], meaning microbial activity). In the extended action of diastase on starch. A mass balance
Bulletin de la Societe Chimique de Paris, one of the gave, in two experiments where the sugar was subse-
most important of the time for fermentation research, quently fermented by yeast and the amount of carbon

dioxide measured, sugar equivalents of 86.91 and 77.64 the protein nature of enzymes. In 1894 Fischer
parts, with respect to 100 parts of starch. By means of (21:829835) stated that among the compounds that
crystallization the authors obtained a completely pure serve the living cell the proteins are the most impor-
sugar (which) is white, without smell.. . . It was less tant. He was convinced that enzymes were proteins.
sweet than cane sugar. As a chemical formula they However, Willstatter, in 1927, denied that enzymes
presented C12 H28 O14 (12). were proteins (22).
The manufacture of dextrin was established: A few years after Fischers investigations, Eduard
Fouchards dextrin syrup is produced by the factory Buchner published a series of papers (23, 24) which
of Mr. Fouchard at Neuilly as a commercial product signaled a breakthrough in fermentation and enzymol-
(12). It remained so for decades, and was mentioned in ogy. The experiments began in 1893 (24c). In his rst
most of the major books and series on technology and paper on alcoholic fermentation without yeast cells
chemical technology (9:144150, 10:125131, 19, 20a). (23) he stated, in a remarkably short and precise man-
The treatment of starch by acids or diastase yielded a ner, that a separation of the (alcoholic) fermentation
gummy syrup. Notably, French products seemed to from the living yeast cells was not successful up to
have been produced enzymatically, as was obvious by now; in the following year (24) he described a process
their smell of malt (recognized by Knapp [9]). Payen that solved that task. He gave the experimental details
(10) gave a short calculation showing that the applica- for the preparation of a cell-free press juice from yeast
tion of malt is more economical than the use of sulfuric cells with disruption, ltration under high pressure,
acid. and further ltration. He then described the formation
A further application of enzymes was the use of lab of carbon dioxide from carbohydrates (sucrose, glu-
products to produce cheese (9:39, 40). Berzelius was cose, fructose, and maltose). No fermentation was
cited with details stating that one part of lab ferment observed with lactose or mannitol. No microscopic
preparation coagulated 1800 parts of milk, and that organisms were present. Chloroform, an antiseptic
only 0.06 parts of the lab ferment is lost. inhibiting microbial growth, did not inhibit the fer-
The rst company based on applied biocatalysis mentation (the enzymatic reaction). At elevated tem-
dates to the 19th century. In 1874, Christian Hansen perature protein was precipitated and the activity
started a company in Copenhagen. His company, decreased and was nally destroyed. From these
named Christian Hansens Laboratory to this day, results, Buchner derived essential new insights into
was the rst in the industrial market with a standar- the nature of alcoholic fermentation. So the concept
dized enzyme preparation, rennet, for cheese making. of enzyme action as a general principle in biochemical
Rennet, a mixture of chymosin (earlier called rennin) reactions was initiated by Berthelot (16) in the 1850s as
and pepsin, was and still is obtained by salt extraction to priority. The experimental verication, however,
of the fourth stomach of suckling calves. was Buchners work, and he earned the credit for it.
Many more details make up the demarcation of a
breakthrough against an established paradigm which
III. THE PERIOD FROM 1890 TO 1940
taught that processes in living organisms, where alco-
A. Growing Interest holic fermentation was the most important example,
were not of pure chemical nature but required a vis
A pronounced increase in published papers on soluble vitalis. The chemical paradigm, which reduced all reac-
ferments indicated the growing interest, and several tions in physiological (or bio-) chemistry to chemistry
key ndings initiated the enzymology of the 20th cen- without further hidden forces, began to play the domi-
tury. Thus, the numbers of articles on soluble ferments nant role.
summarized in the abstract service Chemisches Further ndings relevant for establishment of the
Zentralblatt rose from 15 per year in the mid- chemical nature of enzymatic catalysis and technical
1880s to 35 in 1895 and to 94 in 1910. application followed within a rather short time.
From about 1894, Emil Fischer investigated in a Croft-Hill performed the rst enzymatic synthesis,
series of experiments the action of different enzymes that of isomaltose, in 1898, by allowing a yeast extract
using several glycosides and oligosaccharides of known (-glycosidase) to act on 40% glucose solutions (3). In
structure; the results revealed specicity as one the key 1900, Kastle and Loevenhart found that the hydrolysis
characteristics of enzymes. Fischer derived therefrom of fat and other esters by lipases was a reversible reac-
his theory on specicity with the famous picture of a tion and that enzymatic synthesis could occur in a
lock and key (21:836844). A second aspect referred to dilute mixture of alcohol and acid (25). This principle

was utilized for the synthesis of numerous glycosides B. Technical Development
by Fischer and coworkers in 1902 and by Bourquelot
and coworkers in 1913 (26). With the exception of Christian Hansens rennet, the
After a long period of 100 years, with mysterious industrial development of enzymes was very slow initi-
hypotheses and several technical applications, where ally. Takamine began isolating bacterial amylases in
one (diastase) was even economically important, the 1890s in what later became known as Miles
research on enzymes obtained a chemically scientic Laboratories (today a part of Genencor). In 1894 he
status. The activity in research increased signicantly obtained a patent for the production of a diastatic
owing to the new direction, and technical development enzyme preparation from molds, which he called
also got a new scientic basis on which to proceed in a Takadiastase. The production was performed in a sur-
rational way. The denitive establishment of the che- face culture with wheat bran as a substrate and inocu-
mical paradigm was the crystallization of urease by lation by spores of Aspergillus oryzae (Fig. 2) (4:401).
Sumner in 1926, and further enzymes (trypsin etc.) Additional early applications and patents on enzyme
by Northrup and Kunitz in 1930/31. In every known application in the food industry have been collected by
case the pure enzyme turned out to be a protein (25). Neidleman (27) (Table 2).
Figure 2 Surface culture with Aspergillus in a cabinet incubator. (From Ref. 4.)

Table 2 Selected Early Enzyme Patents
Inventor Year Enzyme Title
J. Takamine 1894 Amylases Process for making diastatic enzyme

J. Takamine 1906 Amylases Diastatic substance and production procedure
O. Rohm 1908 Trypsin, steapsin Preparation of hides for the manufacture of leather
J. Takamine 1911 Amylases Enzyme
J. Takamine 1911 Amylases Amylolytic enzyme
L. Wallerstein 1911 Malt protease Beer and production procedure
L. Wallerstein 1911 Proteases Preparation of use in brewing
L. Wallerstein 1911 Pepsin Method of treating beer or ale
L. Wallerstein 1911 Papain Method of treating beer or ale
L. Wallerstein 1911 Bromelain Method of treating beer or ale
L. Wallsterstein 1911 Yeast Method of treating beer or ale
O. Rohm 1915 Pancreatin Process for cleaning laundry of all types
S. Frankel 1915 Amylase Manufacture of diastase
I. Pollak 1915 Amylase Diastase preparations and production procedure
I. Pollak 1915 Amylases Malt extract and production procedure
A. Boidin/J. Effront 1917 Amylases Process for treating amylaceous substances
A. Boidin/J. Effront 1917 Amylases Process of manufacturing diastases and toxins by oxidizing
ferments
V.G. Bloede 1918 Amylase Process of manufacturing vegetable glue
H.S. Paine/J. Hamilton 1922 Invertase Process for preparing fondant or chocolate soft cream centers
J. Takamine 1923 Amylases, protease, lipase Enzymatic substance and production procedure
A. Boidin/J. Effront 1924 Amylase, protease, lipase Treatment of textile fabrics or bers
Wallerstein Co. 1931 Amylases, protease, lipase Improvements in process of depilating hides
M. Wallerstein 1932 Amylases or papain Method of making chocolate syrups
R. Douglas 1932 Amylases Process of preparing pectin
L. Wallerstein 1933 Invertase Invertase preparation and production procedure
L. Wallerstein 1937 Proteases Process of chill-proong and stabilizing beers and ales
L. Wallerstein 1937 Proteases Rubber
L. Wallerstein 1938 Proteases Deproteinization of rubber latex
Source: Ref. 27.
At the beginning of the 20th century the production

of plant lipase was performed by mechanical disrup-
tion of the seed of ricinus by procedures of Nicloux
and Hoyer. These seeds had to be stored for several
days with some acid added, which then developed a
much higher activity owing to the activation of lipase.
These were then utilized for the production of fatty
acids from oils and fats. A production vessel is
shown in Figure 3. The vessel A can be heated by
steam and mixed by compressed air from the compres-
sor L and the storage vessel W. The procedure was as
follows: oil or fat was mixed with 35% water, the tem-
Figure 3 Reactor for fatty acid production. Details of reac-
perature held at 20258C, the ferment (enzyme 48%) tion vessel A; water reservoir B; tube for steam c, entrance of
added to the liquid mass, along with manganese sulfate steam a, exit of steam b, compressor L, storage vessel for
for activation of the enzyme. Mixing was performed steam W, tube for air d, valves at positions e and f, M storage
for 15 min, then intermittantly. Eighty percent conver- vessel for medium layer, G storage vessels for glycerol. (From
sion was obtained after 24 h, 90% after 2 days. The Ref. 20b.)

enzyme was inactivated by heating to 80858C. IV. DEVELOPMENTS SINCE 1940
Glycerol was taken from the lower valve e, fatty acid
from that in the higher position f. Details for manu- Even the development of citric acid by fermentation,
facture of 10 tons/week were given. It was pointed out (Pzer) and penicillin (Beecham, Glaxo, Merck, Pzer,
that the reaction is reversible and that an enzymatic Squibb, Bristol-Myers) in the 1940s did not really
synthesis of fat from glycerol and fatty acid was trigger a scale-up of industrial applications of
described by Welter in 1911 (20b). enzymes. We must go forward to around 1955 before
In 1895, Boidin discovered a new process for the the development of enzyme production was gaining
manufacture of alcohol called the amyloprocess. It speed by growing sales of bacterial amylase and pro-
comprised cooking of the cereals, inoculation with a tease. It began in a very modest fashion. As an illus-
mold which formed saccharifying enzymes, and sub- tration, the turnover of the enzyme division of Novo
sequent fermentation with yeast. Together with Industri (now Novozymes), the leading enzyme man-
Effront, working on enzymes for alcohol production ufacturer, did not exceed $1 million (U.S.) annually
since 1900, Boidin founded the Societe Rapidase until 1965. However, with the appearance of the
(today a part of DSM-Gist-brocades) in 1920 (28:6). detergent proteases, the use of enzymes increased.
For chill-proong of beer, proteolytic enzymes (pro- Everybody wanted Biotex, the protease-containing
duced by Wallerstein, later Bayter Travenol) have detergent. At the same time, an acid/enzyme process
been used successfully since 1911 in the United to produce dextrose using glucoamylase was used
States (4:337). So urgent was the demand throughout increasingly in starch processing. By 1969, within
the brewing industry in the United States for a prac- only 4 years, Novos enzyme turnover exceeded $50
tical solution to this problem of sedimentation that in million (U.S.) annually; in 1997 Novozymes enzyme
1909 and again in 1910 the then U.S. Brewmasters division had a turnover of $650 million (U.S.). The
Association offered two cash awards (27). Lintner, as present global market is estimated to be around $1.6
early as 1890, observed that wheat diastase was billion (U.S.) (31).
important in dough making. This effect was exten- One question, which is as old as industrial enzyme,
sively studied, the addition of malt extract came is: Can enzymes be reused? Some of the rst attempts
into practice, and American bakers in 1922 used 30 to reuse enzymes were described by Nelson at the
million pounds of malt extract valued at $2.5 million beginning of the 20th century. But the enzymes
(4:422). The production of pectinases began around adsorbed to charcoal were very unstable. In the
1930 for use in the fruit industry (Schweizerische 1950s, Manecke was the rst really to succeed in mak-
Ferment, now part of Novozymes). ing relatively stable systems; however, he could not
The use of enzymes for the manufacture of leather convince industry of the importance of further devel-
played a major role for the industrial-scale production opment of his systems (30). It became the group of
of enzymes. Wood was the rst in 1898, to show that chemists working with Katchalski-Katzir in Israel
the bating action of the unpleasant dungs used at that who really opened the eyes of industry to the world
time was caused by the enzymes (pepsin, trypsin, of immobilized enzymes. Among Katchalski-Katzirs
lipase) they contained. In the context of Woods inves- coworkers were Klaus Mosbach, Daniel Thomas,
tigations the rst commercial bate, called Erodin, was and Malcolm Lilly. The rst immobilized enzyme pro-
prepared from cultures of Bacillus erodiens, based on a ducts to be scaled up to pilot plant and industrial
German patent granted to Popp and Becker in 1896. application (in 1969) were immobilized amino acid
The bacterial cultures were adsorbed on wood meal acylases (i.e., I. Chibata and colleagues at Tanabe
and mixed with ammonium chloride (4: 491). In Seiyaku Co. in Japan [32]) and penicillin G acylase
1907, Rohm patented the application of a mixture of (M. D. Lilly/University College, London, Beecham
pancreatic extract and ammonium salts as a bating Pharmaceuticals, England, and G. Schmidt-Kastner,
agent (4:488). With this perspective he founded his Bayer, Germany) (33, 34:7).
company in 1907, which successfully entered the mar- The largest immobilized enzyme product still today
ket. In 1913 the company had 22 chemists, 30 other in volume is immobilized glucose isomerase. The rst
employees, and 48 workers (29:101). A systematic commercial enzymatic production of high-fructose
approach toward the interrelation of scientic develop- corn syrups (HFCS) was in Japan in 1969 (Takasaki
ment and engineering aspects, which played a major and coworkers), utilizing heat treated Streptomyces
role in this development, has been published by cells containing glucose isomerase in a batch reactor.
Buchholz (30). In the United States, Clinton Division of Standard

Brands (now ADM) was the rst company using 3. JB Sumner, GF Somers. Chemistry and Methods of
Takasaki immobilized glucose isomerase to make Enzymes. New York: Academic Press, 1953, pp 1316.
industrial quantities of HFCS around 1971 (33). The 4. H Tauber. The Chemistry and Technology of
skyrocking sucrose prices, during 197375, where the Enzymes. New York: Wiley, 1949.
price of sucrose increased from 57/lb to 30/lb 5. SM Roberts, NJ Turner, AJ Willets, MK Turner.
Introduction to Biocatalysis. Cambridge: Cambridge
speeded the interest for HFCS. Thereby, immobilized
University Press, 1995, pp 133.
glucose isomerase increased dramatically in price.
6. MPJ Beral. Notes sur la fermentation. J Pharm Sci
Companies like Novozymes, and later Gist-brocades Access 1:358361, 1815.
developed more stable enzyme products, which were 7. JHM von Poppe. Volks-Gewerbslehre oder allgemeine
easier and cheaper to use. Resources were spent on und besondere Technologie. Stuttgart: Carl
optimizing fermentation for glucose isomerase produc- Hoffmann, 1842.
tion, immobilization processes, and the application 8. Anon. Ueber die Gahrung und die sogenannte gener-
processes for the immobilized glucose isomerases. As atio aequivoca. (summary article). J Prakt Chem
a result, productivities of the commercial immobilized 85:465472, 1862.
glucose isomerase products increased from 500 kg 9. F Knapp. Lehrbuch der chemischen Technologie, Vol
HFCS/kg immobilized enzyme product (1975) to 2. Braunschweig: F. Vieweg und Sohn, 1847.
15,00020,000 kg/kg (1997). 10. A Payen. Handbuch der technischen Chemie. Nach A.
Other immobilized enzyme product successes Payens Chimie industrielle, frei bearbeitet von F.
Stohmann and C. Engler, Vol II. Stuttgart: E.
(where annual production of immobilized enzymes
Schweizerbartsche Verlagsbuchhandlung, 1874,
has surpassed 1 ton/year) comprise: aminoacylase p 127.
(Amano), hydantoinase (Smith Beecham), lactase 11. Guerin-Vary. Wirkung der Diastase auf
(Valio), lipase (Novozymes), penicillin G acylase Kartoffelstarkemehl. Ann Pharm 17:261267, 1836.
(Gist-brocades, Smith Beecham, Rohm), and penicillin 12. A Payen, JF Persoz. Memoire sur la diastase, les prin-
V acylase (Novozymes/Gist-brocades) (33). cipaux produits de ses reactions, et leurs applications
Numerous efforts and papers, as well as standar- aux arts industriels. Ann Chim Physique 2me Ser
dized procedures for the characterization of immobi- 53:7392, 1833.
lized biocatalysts, have been published in recent 13. H Dobell. Action du pancreas sur les graisses et sur
decades. The scientic and technical progress may be lamidon. Bull Soc Chim 506, 1er semestre, 1869.
questioned in the majority of the papers (35). A remark- 14. E Frankland. On chemical changes in their relation to
able issue, however, might be the application of bioca- microorganisms. J Chem Soc 47:159183, 1885.
talysts for synthesis of a broad range of products, most 15. M Berthelot. Remarques sur la note de M. Bechamp
relative a` la fermentation alcoolique. Bull Soc Chim
importantly in the sectors of food, food ingredients,
392393, 1864.
and pharmaceuticals, and recently in glycobiology for
16. M Berthelot. Ueber die geistige Gahrung. J Praktische
making oligosaccharides and glycosides, and in organic Chem 71:321325, 1857.
chemistry, including nonaqueous systems and the 17. Anon. Diastase und Dextrin. Ann Pharm 12:295299,
hydrolysis and modication of fats and products 1834.
derived therefrom, for surfactants (34:141190). 18. R Wagner. Die chemische Technologie. Leipzig: O.
Recombinant techniques have been established dur- Wiegand, 1857.
ing the last two decades for much improved yields of 19. Brockhaus. Diastase, Vol 5, p 255, 1894; Fermente,
enzymes by fermentation, modication of stability, Vol 6, p 679, 1894.
and even selectivity and specicity (36). These techni- 20. F Ullmann. Enzyklopadie der technischen Chemie, (a)
ques have pushed the application, and tend to consid- Vol 3, pp 749773, 1916; (b) Vol 5, p 445, 1914. Berlin:
erably extend the scope of potential applications, of Urban und Schwarzenberg.
enzymes in diverse elds including food technology. 21. E Fischer. Untersuchungen uber Kohlenhydrate und
Fermente. Berlin: J Springer, 1909; Ber D Chem Ges
27:2985, 1894; 27:2031, 1894.
22. JS Fruton. Early theories of protein structure. In: PR
REFERENCES Srinivasan, JS Fruton, JT Edsall, eds. The Origins of
Modern Biochemistry. New York: New York
1. R Bud. The Uses of Life, A History of Biotechnology. Academy of Sciences, 1979, pp 118.
Cambridge: Cambridge University Press, 1993. 23. E Buchner. Alkoholische Gahrung ohne Hefezellen.
2. R Bud. The zymotechnic roots of biotechnology. Br J Ber D Chem Ges 30:117124, 1897.
His Sci 25:127144, 1992. 24. (a) E Buchner. Ueber zellfreie Gahrung. Ber D Chem

Ges 31:568574, 1898. (b) E Buchner, R Rapp. 30. K Buchholz. Reections on the history and scientic
Alkoholische Gahrung ohne Hefezellen. Ber D character of biochemical engineering. Adv Mol Cell
Chem Ges 31:209217, 1898. (c) E Buchner, R Biol 15A:117134, 1996.
Rapp. Alkoholische Gahrung ohne Hefezellen (5). 31. WH Stroh. Industrial enzymes market: growth experi-
Ber D Chem Ges 31:10841090, 1898. (d) E ences from new products and movement into global
Buchner, R Rapp. Alkoholische Gahrung ohne market. Genet Eng News March 1:11, 38, 1998.
Hefezellen (6). Ber D Chem Ges 31:10901094, 1898. 32. T Tosa, T Mori, N Fuse, I Chibata. Studies on con-
(e) E Buchner, R Rapp. Alkoholische Gahrung ohne tinuous enzyme reactions. 6. Enzymatic properties of
Hefezellen (7). Ber D Chem Ges 31:15311533, 1898. DEAE-Sepharose aminoacylase complex. Agr Biol
25. JB Sumner, K Myrback. The Enzymes, Vol 1, Part 1, Chem 33:10471056, 1969.
1950, pp 127. 33. PB Poulsen. Biotechnology and Genetic Engineering
26. K Wallenfels, H Diekmann, Glykosidasen. Hoppe- Review, Vol 1. Newcastle-upon-Tyne: Intercept Inc.,
Seyler/Thierfelder, Handbuch der physiologisch- und 1984.
pathologisch-chemischen Analyse, Vol 6B. Berlin: 34. K Buchholz, V Kasche. Biokatalysatoren und
Springer, pp 11561210. Enzymtechnologie. Weinheim: VCH, 1997, p 711.
27. SL Neidleman. Enzymes in the food industry: a back- 35. K Buchholz. Criteria for the analysis of scientic qual-
ward glance. Food Technol 45(1):8891, 1991. ity. Scientometrics 32:195218, 1995.
28. H Uhlig. Enzyme arbeiten fur uns. Munich: Hanser 36. SB Petersen. Protein engineering. In: AJJ Straathof, P
Verlag, 1991. Adlercreutz, eds. Applied Biocatalysis. Amsterdam:
29. E Trommsdorf. Dr. Otto RohmChemiker und
Harwood Academic Publishers, 2000, p 229270.
Unternehmer. Dusseldorf: Econ, 1976.

3
What Enzymes Do and Why They Are Highly Specic and

Efcient Catalysts
John R. Whitaker
I. INTRODUCTION yellow enzyme so he named it the new yellow

enzyme. When a crude preparation of enzymes that
Enzymes are the key to all life. Therefore, it is not synthesized proteins was rst discovered it was named
surprising that all organisms contain hundreds of dif- the pH 5 extract because maximum enzyme activity
ferent enzymes. It is common to nd isoenzymes, occurred at that pH.
which are multiple protein forms of an enzyme within An International Commission on Enzymes was
the same organ, with qualitatively the same enzymatic established in 1955 by the International Union of
activity. Therefore, each isoenzyme is encoded by a Biochemistry with the charge to consider the classi-
different gene. With so many different organisms in cation and nomenclature of enzymes and coenzymes,
the world, all with hundreds of different enzymes, their units of activity and standard methods of assays,
how is it possible to systematize enzymes and enzyme together with the symbols used in the description of
nomenclature? This was a problem that the biochem- enzyme kinetics. The Commission on Enzymes con-
ical and chemical community faced in 1955. tained one or more members from each of the coun-
As indicated in Chapter 1, the rst enzyme recog- tries that were publishing signicant research on
nized, based only on its activity, was catalase, in 1812. enzymes. The rst Report of the Commission on
It converts hydrogen peroxide to water and oxygen; Enzymes in 1961 contained 712 enzymes. Subsequent
the oxygen bubbles out of the solution. As described updates have been issued. The most recent report in
in Chapter 1, several other enzymes were discovered 1992 contained 3196 enzymes (1). In order for the
over the years. By 1955, more than 600 enzymes were enzyme to appear in the list, the researcher must isolate
known and partially characterized. The naming of the enzyme and characterize it with respect to sub-
enzymes was largely left to the discoverer. Some strate(s) specicity, product(s) formed, nature of the
enzymes, such as catalase, diastase (now amylase), overall reaction, and protein properties, and clearly
polyphenol oxidase, peroxidase, etc., used -ase to demonstrate that it is unique.
indicate that the compound was an enzyme.
However, naming of other enzymes, such as trypsin,
chymotrypsin, cin, and papain, did not use -ase.
II. THE THREE-TIER CLASSIFICATION
Warburg and Christian in 1935 discovered an enzyme
OF ENZYMES
that, when partially puried, was yellow because of the
cofactor riboavin. They named it the yellow
The 1955 Commission on Enzymes recommended that
enzyme. Soon after, Warburg discovered a different
each enzyme have three designations. These are: a

systematic name, a trivial name, and an Enzyme for the nomenclature. The mechanism of the reaction
Commission (EC) number. is not indicated. Therefore, an enzyme cannot be
The systematic name consists of the name(s) of the named systematically until the substrate(s), product(s),
substrate(s). In the case of two or more substrates (or and the chemical nature of the reaction catalyzed are
reactants), the names of the substrates are separated by known.
a colon. The second part of the systematic name, end- The overall reaction catalyzed by a specic enzyme
ing in -ase, is based on one of the six types of chemical is shown in Eq. (1):
reactions catalyzed (see below). When the overall reac-
tion involves two different chemical reactions, such as CH3 CH2 OH NAD CH3 CHO NADH H
oxidative demethylation, the second type of reaction is Ethanol Acetaldehyde
listed in parenthesis following the name of the rst type
1
of reactionfor example, sarcosine:oxygen oxido-
reductase (demethylating). According to the Enzyme Commission, the systematic
The trivial name is one that is generally recognized name of the enzyme is ethanol:NAD oxidoreductase
and is in common usage such as trypsin, chymotrypsin, (more commonly called acohol:NAD+ oxidoreduc-
-amylase, cellulase, peroxidase, or catalase. tase. The substrate (ethanol) that donates the hydro-
The number system derives directly from the clas- gens (becomes oxidized) is listed rst, followed by the
sication scheme. Each enzyme number contains four substrate (NAD ) that accepts the hydrogens
digits, separated by periods, and preceded by EC. The (becomes reduced). According to the number system,
numbers are permanent. Newly discovered enzymes alcohol:NAD oxidoreductase is EC 1.1.1.1). The rst
are placed at the end of a list under appropriate head- digit designates it as an oxidoreductase. The second
ings. When the classication of an enzyme is changed, digit indicates that the donor of the hydrogens is an
the original number remains in the listing, but the alcohol (R2 CHOH). The third digit indicates that
user is directed to the new listing, including the new NAD (or NADP ) is the specic acceptor of the
number of the enzyme. For example, the listing of the hydrogen(s), and the fourth digit indicates that the
proteases has been changed recently. A new arrange- alcohol is ethanol. The trivial name of alcohol:
ment of the carbohydrases will be published soon. NAD oxidoreductase is alcohol dehydrogenase. In a
publication the systematic name would be given at the
A. Rules for Naming of Enzymes rst mention of the enzyme, along with the trivial
name and EC number; i.e., alcohol:NAD oxidoreduc-
1. Classication into Six Groups Based on tase (alcohol dehydrogenase; EC 1.1.1.1). Thereafter in
Type of Chemical Reaction Catalyzed the publication, the trivial name, alcohol dehydrogen-
As noted above, the Enzyme Commission classied the ase, would be used. Table 1 contains the general listing
enzymes into groups based on the type of chemical of enzymes through the second digit as recommended
reaction catalyzed. Together with the name(s) of the by the Enzyme Commission.
substrate(s), this provides the basis for systematically
naming individual enzymes. B. Six Groups of Enzymes
There are six chemical types of reactions catalyzed
by enzymes. These are (with the general group name of The six groups of enzymes, based on the chemical reac-
the enzymes in parentheses): (a) oxidoreduction tion catalyzed, are described in more detail in this sec-
(oxidoreductase), (b) group transfer (transferase), (c) tion.
hydrolysis (hydrolase), (d) formation of double bonds
without hydrolysis (lyase), (e) isomerization (isomer- 1. Oxidoreductases
ase), (f) ligation (ligases).
Oxidoreductases are enzymes that oxidize or reduce
The rst digit in the EC number system is based on
substrates by transfer of hydrogen or electrons or by
the type of chemical reaction catalyzed, with (a)(f)
addition of oxygen. The systematic name is formed as
above being listed as EC 16.
donor:acceptor oxidoreductase. One example for
alcohol dehydrogenase (EC 1.1.1.1) is shown in Eq. (1)
2. The Overall Reaction as Basis of Nomenclature
above, in which the donor substrate is ethanol which
The overall reaction catalyzed, based on the formal contributes two hydrogens to NAD , the acceptor sub-
chemical equation [for example, Eq. (1)] is the basis strate (and cofactor) with the formation of acetal-

dehyde and NADH2 ; NADH2 then loses a proton (H )
based on the pH of the reaction. Another example is the
catalase reaction in which one H2 O2 molecule serves as
the donor substrate and the second H2 O2 molecule
serves as the acceptor substrate to form O2 and H2 O 4
[Eq. (2)]. The systematic name is hydrogen peroxide:-
hydrogen peroxide oxidoreductase (EC 1.11.1.6). When the enzyme specicity is limited to a single group
(of two or more susceptible groups), the name of the
H2 O2 H2 O2 O2 2H2 O 2 group is given as a prex of hydrolase. For example,
enzymatic hydrolysis of a compound such as N-acetyl-
Whitaker (2) has suggested dividing the oxidoreduc- L-alanine ethyl ester [Eq. (4)] can occur at either the
tases into eight subgroups based on mechanistic con- acetyl-nitrogen bond or the ethoxy-oxygen bond,
siderations. But the International Commission on depending on the specicity of the enzyme.
Enzymes considers only the overall reaction in naming In reaction 1 [Eq. (4)], the enzyme is an acyl amino-
enzymes. hydrolase (EC 3.5.) while in reaction 2 [Eq. (4)], the
enzyme is an acyl oxygenhydrolase (esterase, EC 3.1.).
2. Transferases There is no crossreactivity between the two enzymes, as
they are specic for hydrolysis of only one of the scis-
Transferases are enzymes that remove groups (not sile bonds, unlike HCl.
including H, which belongs in group 1, the oxidoreduc-
tases) from substrate(s) and transfers the group to 4. Lyases
acceptor substrates (not including water). The systema-
tic name is formed as donor:acceptor group-trans- Lyases are enzymes that remove groups from their
ferred-transferase. The types of groups transferred substrates (not by hydrolysis) to give a double bond
are given by the second digit in EC 2. of Table 1. A in the product, or which conversely add groups to
specic example of a transferase is shown in Eq. (3). double bonds. The systematic name is formed as sub-
strate prex-lyase. Prexes such as hydro- and
ammonia- are used to indicate the type of reac-
tionfor example, L-malate hydro-lyase (EC
4.2.1.2). Decarboxylases are named as carboxy-
lases. A hyphen is always written before lyase to
avoid confusion with hydrolases, carboxylases, etc. An
example of a lyase is the removal of HOH from malic
3 acid to give fumaric acid [Eq. (5)]:
The systematic name of the enzyme that catalyzes the

reaction in Eq. (3) is ATP:D-glucose 6-phosphotrans-
ferase (EC 2.7.1.2). The trivial name is glucokinase.
The position to which the group (phosphate) is trans-
ferred to the glucose is given in the systematic name
5
when more than one possibility exists, such as D-glu-
cose-1-phosphate. by the enzyme (s)-malate hydro-lyase (fumarate hydra-
tase; EC 4.2.1.2; formerly known as fumarase).
3. Hydrolases
5. Isomerases
Hydrolases are enzymes in which water (H2 O) is the
second substrate. Water participates in the breakage of Isomerases are enzymes that cause rearrangement of
covalent bonds; for example, peptide bonds in pro- one or more groups on substrates without changing
teins, glycosidic bonds in carbohydrates, ester bonds the atomic composition of the product. The general
in lipids, and phosphodiester bonds (and others) in systematic name is formed as substrate prex-isomer-
nucleic acids. The systematic name is formed as sub- ase. The prex indicates the type of isomerization
strate hydrolase. The elements (H and OH) appear in involved, for example, maleate cis-trans-isomerase
the products of the reaction as shown in Eq. (4) (EC 5.2.1.1) [Eq. (6)].

Table 1 Key to Numbering and Classication of Enzymesa
1. Oxidoreductases 3.3 Acting on ether bonds

1.1 Acting on the CHOH group of donors 3.4 Acting on peptide bonds (peptidases)
1.2 Acting on the aldehyde or oxo group of donors 3.5 Acting on carbon-nitrogen bonds, other than
1.3 Acting on the CHCH group of donors peptide bonds
1.4 Acting on the CHNH2 group of donors 3.6 Acting on acid anhydrides
1.5 Acting on the CHNH group of donors 3.7 Acting on carboncarbon bonds
1.6 Acting on NADH or NADPH 3.8 Acting on halide bonds
1.7 Acting on other nitrogenous compounds as donors 3.9 Acting on phosphorusnitrogen bonds
1.8 Acting on a sulfur group of donors 3.10 Acting on sulfurnitrogen bonds
1.9 Acting on a heme group of donors 3.11 Acting on carbon-phosphorus bonds
1.10 Acting on diphenols and related substances as
donors
1.11 Acting on hydrogen peroxide as acceptor 4. Lyases
1.12 Acting on hydrogen as donor 4.1 Carboncarbon lyases
1.13 Acting on single donors with incorporation of 4.2 Carbonoxygen lyases
molecular oxygen (oxygenases) 4.3 Carbonnitrogen lyases
1.14 Acting on paired donors with incorporation of 4.4 Carbonsulfur lyases
molecular oxygen 4.5 Carbonhalide lyases
1.15 Acting on superoxide radicals as acceptor 4.6 Phosphorusoxygen lyases
1.16 Oxidizing metal ions 4.99 Other lyases
1.17 Acting on CH2 groups
1.18 Acting on reduced ferredoxin as donor
1.19 Acting on reduced avodoxin as donor
5. Isomerases
1.97 Other oxidoreductases
5.1 Racemases and epimerases
5.2 cis-trans-isomerases
2. Transferases
5.3 Intramolecular oxidoreductases
2.1 Transferring one-carbon groups
5.4 Intramolecular transferases (mutases)
2.2 Transferring aldehyde or ketone residues
5.5 Intramolecular lyases
2.3 Acyltransferases
5.99 Other isomerases
2.4 Glycosyltransferases
2.5 Transferring alkyl or aryl groups, other than methyl
groups
2.6 Transferring nitrogenous groups 6. Ligases
2.7 Transferring phosphorous-containing groups 6.1 Forming carbonoxygen bonds
2.8 Transferring sulfur-containing groups 6.2 Forming carbonsulfur bonds
6.3 Forming carbonnitrogen bonds
3. Hydrolases 6.4 Forming carboncarbon bonds
3.1 Acting on ester bonds 6.5 Forming phosphoric ester bonds
3.2 Glycosidases
a
The third and fourth levels of classication are given in Ref. 1:vxi.
merization consists of an intramolecular transfer of a

group, such as 2-phospho-D-glycerate to 3-phospho-
6 D-glycerate, the enzyme is called a mutase; a specic
example of a mutase is D-phosphoglycerate 2,3-phos-
phomutase (EC 5.4.2.1). Isomerases that catalyze
inversions of asymmetric groups are termed race-
Enzymes that catalyze an aldose-ketose interconver- mases or epimerases, depending on whether the
sion are known as ketal-isomerases (for example, L- substrate contains one or more than one center of
arabinose keto-isomerase; EC 5.3.1.4). When the iso- asymmetry, respectively.

6. Ligases phoresis, column chromatography, and other separa-
tion methods. Quantitatively, there may be differences
Ligases are enzymes that catalyze the covalent linking
in catalytic efciency of the isoenzymes.
together of two molecules, coupled with the breakage
When isoenzyme forms are present, they contain a
of a pyrophosphate bond as in ATP, UTP, or CTP.
letter (or number) indicating how they behaved by elec-
The systematic name is formed as X:Y ligase (Z),
trophoresis or the order in which they were eluted from
where X and Y are the names of the two molecules
an ion exchange column. For example, the rst isoen-
to be joined together. The compound Z is the product
zyme eluting from a cation exchange column would be
formed from the triphosphate (ATP, UTP, or CTP);
isoenzyme-1, the second would be isoenzyme-2, etc.
they provide energy during the reaction. An example
One might ask whether posttranslational proces-
is:
sing of a nascent protein (see Chapter 1) can give
ATP Lasparate NH3 AMP isoenzymes due to different levels of modication.
7 This is a relatively easy question to answer, but it
+ pyrophosphate + L-asparagine
may be difcult to determine experimentally. An
The systematic enzyme name is L-asparate:ammonia example often observed experimentally is that the
ligase (AMP-forming (aspartate-ammonia ligase, EC extent of glycosylation of a seed protein (for example)
6.3.1.1.) is often different depending on the maturity of the
Proteins, carbohydrates, and triacylglycerols are seed. The glycosyl groups are covalently attached to
synthesized by specic ligases using ATP, UTP, and the protein at the hydroxyl group of seryl residues
CTP, respectively. and -amino groups of asparaginyl residues, as deter-
mined by the genetic code, immediately following
C. Other Considerations in Naming Enzymes translation (and before the protein folds into the
native form; see Chapter 1). The glycosyl groups
1. Organism from Which the Enzyme Is
may then be shortened by cytoplasmic carbohydrases
Isolated
after folding of the protein to the native state (age-
Proteins are specic to an organism. When proteins related processing). Are these isoenzymes? They are
from one organism are injected into a different animal, not products of different genes and so do not qualify
they elicit antibodies, indicating that they differ from as isoenzymes. Native human hemoglobin is glycosy-
the proteins of the recipient animal. They have differ- lated over time at a level depending on its age and the
ences in amino acid sequences and therefore folding concentration of glucose in the blood. Hemoglobin a
patterns. Enzymes, as proteins, are no different. levels are used clinically to determine the severity of
Therefore, the name of the specic organism from diabetes in humans. No one would describe the age-
which an enzyme is derived must be given. related modied hemoglobin as an isoprotein!
Multiple electrophoretic bands (> 30 sometimes)
2. Organ from Which the Enzyme is Isolated occur with peroxidase because of different levels of
glycosylation. The benzoquinones formed due to the
No less important is the organ (of multicellular organ-
activity of polyphenol oxidase action on phenols dur-
isms) from which the enzyme is obtained. -Amylases
ing extraction and purication of proteins (including
from human saliva are different from those produed by
polyphenol oxidase itself) lead to multiple bands since
the human pancreas, for example. Therefore, the organ
the benzoquinones quickly modify the "-amino group
from which the enzyme is obtained must be specied.
of lysyl residues. These different bands are not due to
isoenzymes!
3. Isoenzymes
Isoenzymes are different protein forms of an enzyme
4. In Summary
produced in the same organ by different genes, but
qualitatively they have the same activity. Perhaps mul- In naming an enzyme, one must not only describe it by
tiple gene expression of isoenzymes is protection the overall type of reaction catalyzed (under standard
against failure of a gene. Or they may occur due to conditions), but also by the source of the enzyme
mutation of a nucleotide base in a codon of a gene. (organism, organ) and by isoenzyme number. With
Whatever the reason, the protein expressed is different the excellent tools available today, there is no basis
from one gene to another, even from the same cell. The for publishing quantitative data on the activity of an
proteins have different protein properties by electro- enzyme until the degree of purity of the enzyme has

been established and one can associate the activity with fully grasp the effectiveness of lowering Ea one must
one unique protein catalyst. appreciate that the rate of a reaction, as controlled by
the rate constant, k, is logarithmically related to Ea as
shown in Eq. (8).
III. WHY ARE ENZYMES HIGHLY
k AeEa =RT 8
SPECIFIC AND EFFICIENT
CATALYSTS? where A is the Arrhenius constant, e is the base of the
A. How Efcient Are Enzymes as Catalysts? natural logarithm, Ea is the Arrhenius activation
energy, R is the universal gas constant, and T is
Table 2 provides data on the relative rates of a few Kelvin temperature.
enzyme-catalyzed reactions in relation to the noncata- The effect of temperature on the rate of reactions
lyzed and non-enzyme-catalyzed reactions at 258C. All (including enzyme-catalyzed reactions) is given by the
relative rates are based on a 1 molar concentration of Boltzmann distribution equation:
catalyst for comparison. First, look at the relative rates n0 =n Ea =RT 9
of the reactions at 258C. Catalase is 3:5 108 times
0
more effective in conversion of H2 O2 to H2 O and O2 where n is the number of molecules per unit volume of
than is the noncatalyzed reaction and 1:7 105 times a compound having activation energy Ea or greater
more effective than the I catalyst. Sucrose can be (see Fig. 1) and n is the total number of molecules
hydrolyzed by acids (by the H ) as well as by invertase. per unit volume of a compound. The A factor in Eq.
Invertase is 5:6 1010 times more effective than the (9) is the product of the kinetic energy of the system
H . Carbonic anhydrase, important in our respiratory due to Brownian movement and the probability P
exchange of O2 and CO2 , is 3 106 more effective than that two molecules will collide with proper orientation
no catalyst. In our kidney, urease is important in the of reactive groups. P approaches 1 in enzyme-catalyzed
conversion of urea to ammonia and CO2 . Urease is reactions because the substrates are properly bound
4:3 1011 times more effective than the H . noncovalently in the active site adjacent to the catalytic
Table 2, column 3, gives the Arrhenius activation group(s) required for catalysis (see Ref. 2, pages 314ff).
energy, Ea , required to convert the substrate to Figure 1 shows the change in energy (both activa-
products. Notice that in all cases, the Ea for the tion energy (steps 3, 4, and 6) and thermodynamic
enzyme-catalyzed reaction is signicantly lower than energy (steps 1, 2, 5, and 7) as the enzyme binds with
for the non-enzyme-catalyzed or noncatalyzed reac- substrate and catalyzes conversion of the substrate (A)
tion. Therefore, one can correctly conclude that the to product (P) in the reaction. The rate-limiting step
enzymes are successful in lowering the activation requires the most energy (step 4 in Fig. 1). This energy
energy for conversion of substrate to product. To is the activation energy, Ea , as shown in Eq. (8).
Table 2 Effect of Catalyst on Ea and on Relative Rates of Some Reactions
Ea n0 =na Relative rate

Substrate Catalyst (kcal/mol) (258C) 258C
H2 O 2 None 18.0 5:62 1014 1.00

I 13.5 1:16 1010 2:07 103
Catalase 6.4 1:95 105 3:47 108
Sucrose H 25.6 1:44 1019 1.00
Invertase 11.0 8:04 109 5:58 1010
Carbonic acid None 20.5 8:32 1016 1.00
Carbonic anhydrase 11.7 2:46 109 2:96 106
Urea H 24.5 9:33 1019 1.00
Urease 8.7 3:96 107 4:25 1011
a 0
n n values were calculated by use of Eq. (9) and are approximate only, since differences in Sz for the
comparison reactions are ignored.
Source: Ref. 2.

Figure 1 Change in energy along the pathway of reaction Figure 2 Variation of initial velocity with substrate concen-
during conversion of substrate A to product P in an enzyme- tration for an enzyme-catalyzed reaction according to Eq.
catalyzed reaction. The numbers refer to steps in thermody- (2). (From Ref. 2.)
namic states (ground states); 1, 2, 5, and 7 and transition
states (activated states); steps 3, 4, and 6 in the forward
reaction pathway. (From Ref. 2.) When all of the enzyme is bound to substrate, the
maximum velocity, Vmax , of the reaction is achieved.
Many researchers have veried by other methods that
B. Binding of Substrate by Enzyme formation of the E S complex is essential before con-
version of substrate to product is possible. How does
Very few compounds are substrates for a given this binding contribute to the specicity and efciency
enzyme. In the case of urease, really only urea is a of enzyme-catalyzed reactions? This question is
good substrate. For other enzymes, such as alkaline addressed in the following way.
phosphatase, a number of compounds serve as sub-
strate (phenylphosphate, p-nitrophenyl phosphate, -
1. Nature of the Binding Site
glycerol phosphate, etc.). However, all substrates for
alkaline phosphatase have an ortho-phosphate group. Table 3 lists the characteristics of the active site of an
By competitive inhibition studies with ortho-phos- enzyme. The active site is a small area (with 10
phate, and with different substrates, it is relatively amino acid residues brought together from distant
easy to establish that the major recognition group on
the substrate for the enzyme is the ortho-phosphate
group. This example can be extended to most other Table 3 Active Site Characteristics of an Enzyme
enzymes and their recognition of substrates. Analogs
1. Small area on surface of native protein
of substrates are good inhibitors of enzymes, which
2. Shape can be crevice or hole
tells us that the enzyme must recognize and bind with 3. Active site binds substrate(s) and cofactors
the compounds. a. Stereospecically with at least three points of contact
In 1902, Henri (3) and Brown (4) independently b. Converts substrate to products
proposed that the observed effect of substrate concen- 4. Ionizable groups in active site (side chains of amino acid
tration on the velocity of conversion of substrate to residues)
product (Fig. 2) is due to the essentiality of an inter- a. May be involved in binding
mediate substrateenzyme noncovalent complex prior b. Involved in catalysis
to catalysis [Eq. (10)]. 5. Active site exible (induced t) and can accommodate
substrate(s)
k1 k2 6. Active site may contain cofactor(s)
E S E S!E P 10 7. Active site with substrate bound is hydrophobic
k1

parts of the primary structural chain to the surface of the substrate(s) and cofactors bind in the active site, the
the enzyme). The substrate can diffuse rapidly to the concentration of the catalytic groups of the enzyme in-
active site and binds stereospecically by a minimum crease to 10 M, giving a rate enhancement of 103 104 .
of three noncovalent bonds. In lysozyme, Glu35, In some cases, the scissile bonds of the substrate may
Asn37, Asn44, Asp52, Asp59, Try63, Asp101, Arg be distorted during the bonding process (induced t [8]),
114, and the carbonyl oxygen of the peptide bonds contributing to increase in rate. For example, in the case
between Phe34Glu35, Gln57Leu58, and Ala107 of lysozyme, binding of the sugar substrate to the
Try108 all appear to be in proximity in the active site enzymes active site results in a glucosyl oxocarbonium
(5). In ribonuclease, Lys7, Arg10, His12, Lys41, and ion intermediate with an enzyme-induced distortion of a
His119 are known to be in the active site (6). glucosyl residue at the scissile bond into a half-chair
Carboxypeptidase A and lysozyme form ve and 12 conformation (5). Electrostatic stabilization of the
noncovalent contacts with the substrates benzyloxycar- transition state in an enzyme reaction also plays an
bonyglycyl-L-phenylalanine and hexa-N-acetylchito- important role. For example, the Asn155oxyanion
hexaose, respectively (2). When bound properly in interaction in subtilisin contributes 3.7 kcal/mol activa-
the active site, the substrate is positioned in proximity tion energy to the transition stabilization (9). Bender et
to the catalytic groups that convert it to product(s), al. (10) showed that restriction of rotation of the N-
along with any cofactors that are required. On binding acetyl-L-tryrosyl- (acyl group intermediate) chymotryp-
of substrate into the active site most of the water mole- sin, in comparison with the small acetyl-L-chymotrypsin
cules are forced out so that the reaction takes place (acyl group intermediate) increased Sz from 35:9 to
under hydrophobic conditions. For hydrolases, one 13:4 eu (cal mol1 deg1 ), thereby increasing the rate
water molecule binds stereospecically as a second of deacylation by 3540 times (10). In summation, the
substrate in proximity to the rst substrate. stereospecic binding of the substrate into the active
Table 4 summarizes the factors accounting for the site of the enzyme may increase the rate by 1012 - to
catalytic effectiveness of enzymes. Conversion of the 1026 -fold.
enzyme and substrate (plus any cofactors) from an inter-
molecular reaction to an intramolecular reaction (the E
2. Catalysis
S complex) increases the rate by 104 for one substrate,
109 for two substrates, and 1015 for three substrates. During the catalytic step(s), concerted general acid/
Even ve to six component reactions are possible with general base catalysis or nucleophilic/electrophilic cat-
enzymes, such as the RNA polymerases. There is an alysis increases the overall rate enhancement by a fac-
increase in entropy of the reaction because of the highly tor of 102 103 times. The overall rate enhancement
structured nature of binding of substrate(s) and cofac- predicted for an enzyme-catalyzed reaction is 1018
tors with respect to the catalytic groups, increasing the 1036 . Catalase- and peroxidase-catalyzed reactions
rate by a factor of 103 . Since water is expelled when reach values near 1018 enhancement.
Table 4 Factors Accounting for Catalytic Effectiveness of Enzymes
Rate enhancementa
Factor (approximate)
1. Formation of stereospecic enzymesubstrate complex 104 ; 109 ; 1015;b

(conversion from inter- to intramolecular reaction)
2. Increased entropy of reaction 103
3. Concentration of reactive catalytic groups when substrate binds 103 104
4. Distortion of substrate when bound 102 104
5. General acid/general base catalysis 102 103
6. Nucleophilic/electrophilic catalysis 102 103
7. Use of several step process 102 104
Overall rate enhancement 1018 1036
a
In some cases, these values can be approximated by model experiments. In others, they are the best estimates
available.
b
For one, two, and three substrate reactions, respectively.
Source: Ref. 7.

Proteins of 25,000100,000 molecular weight substrate by nucleophilic/electrophilic catalysis.
200890 amino acid residues per molecule) are Intermediate acyl-enzymes are hydrolyzed in a second
synthesized on the ribosome by polymerases in 26 step with water as the substrate at a rate 109 times
min at 378C! faster than serine esters of similar structures (11).
Figure 3 depicts the active site of ribonuclease with
the substrate, cytidine 20 ; 30 -phosphate bound in the
active site. The catalytic groups of the enzyme are 3. Summary
two imidazole side chains of two histidine residues.
Enzymes are very efcient catalysts because they bind
The distance between reactive groups on the enzyme
substrates (and cofactors) into the active site with the
and the scissile bond is 2 A, nearly covalent bond
scissile bond stereospecically oriented in proximity to
dimensions. The imidazole group on the left side of the
the catalytic groups that carry out the reactions. In
active site functions as a general base to remove a
forming the enzymesubstrate complex, most of the
hydrogen from a water molecule (shown as ROH in
water is removed from the active site. Therefore the
Fig. 3) to form a hydroxide ion (HO ) in proximity to
catalytic reaction is largely in a hydrophobic environ-
the cyclic phosphate of the substrate. The imidazole
ment. Kato et al. (12) tested the effect of excluding
group on the right side of the active site functions as
water from the active site during the catalytic reaction
a general acid by protonating the oxygen adjacent to
by using glutathione synthetase, which requires the two
the PO bond, thereby facilitating the breakage of the
substrates -glutamyl cysteine and glycine with ATP as
cyclic PO bond. Therefore, the hydrolysis by ribonu-
cofactor. In the wild-type enzyme, containing a 17
clease is a general base/general acidconcerted
amino acid loop of the peptide chain that closes the
mechanism. No covalent bonds are formed between
active site to exclude water when the reactants are in
the enzyme and a substrate intermediate.
place, the rate constant, ko , was 151 sec1 . When the
In the nucleophilic/electrophilic mechanism, inter-
lid was removed by genetic engineering, the rate con-
mediate covalent bonds between the enzyme and a por-
stant was 0.163 sec1 , thus decreasing the rate by 930-
tion of the substrate are formed which are then broken
fold by letting some water in.
in a second step. The proteolytic enzymes trypsin, chy-
The role of change in entropy on rates of enzymatic
motrypsin, papain, etc., are known to hydrolyze the
reactions was tested by use of N-acetyl-L-tyrosyl-chy-
motrypsin (A), where the large N-acetyl-L-tyrosyl
group was tightly bound into the binding site, versus
N-acetyl-chymotrypsin (B), where the small N-acetyl
group can rotate freely in the binding site; the relative
rate of A/B was 3540 (10). Sz was 13:4 and 35:9
cal mol1 deg1 for the N-acetyl-L-tyrosyl-chymotryp-
sin and N-acetyl-chymotrypsin, respectively. Most of
the other factors that have been proposed above to
explain why enzymes are so specic and efcient
have been tested by model systems.
Nature is blessed to have such efcient catalysts, the
enzymes, to make life possible! But we must keep our
Figure 3 Proposed orientation of substrate and catalytic

groups in the ribonuclease-catalyzed hydrolysis of the sub-
strate cytidine 20 ;30 -phosphate. Two imidazole groups of his-
tidine of ribonuclease are involved in catalysis, one as a
general base (left) and another as general acid (right). The
arrow at the lower right corner indicates the binding site for
the pyrimidine ring of the substrate which probably involves
several groups on the enzyme protein, and largely determines Figure 4 Ethylene phosphate (A) and dimethyl phosphate
the specicity. (From Ref. 6.) (B)

perspective and respect for very simple chemical exam- 6. AP Mathias, A Deavin, BR Rabin. The active site and
ples of enhanced reaction rates. The relative alkaline mechanism of action of bovine pancreatic ribonu-
hydrolysis rates for ethylene phosphate (A) compared clease. In: TW Goodwin, JI Harris, BS Hartley, eds.
to the structurally similar compound, dimethyl phos- Structure and Activity of Enzymes. New York:
Academic Press, 1964, pp 1930.
phate (B) (Fig. 4) is 1:08 times A=B. This is because
7. JR Whitaker. Enzymes. In: OR Fennema, ed. Food
of the restriction of rotation of the ethylene group in A Chemistry. 3rd ed. New York: Marcel Dekker, 1996,
relative to the free rotation of the methyl groups of B p 473.
(Ref. 2, p. 134). 8. DE Koshland Jr. Conformational changes at the
active site during enzyme action. Fed Proc 23:719
726, 1964.
REFERENCES
9. JA Wells, BC Cunningham, TP Graycar, DA Estell.
Recruitment of substrate-specicity properties from
1. Enzyme Nomenclature. Recommendations of the
one enzyme into a related one by protein engineering.
Nomenclature Committee of the International Union
Proc Natl Acad Sci USA 84:51675171, 1987.
of Biochemistry, published for the International
10. ML Bender, FJ Kezdy, CR Gunter. XXXIII. The
Union of Biochemistry. San Diego: Academic Press,
anatomy of an enzymatic catalysis. -Chymotrypsin.
1992.
J Am Chem Soc 86:37143721, 1964.
2. JR Whitaker. Principles of Enzymology for the Food
11. RL Stein, JP Elrod, RL Schowen. Correlative varia-
Sciences. 2nd ed. New York: Marcel Dekker, 1994.
tions in enzyme-derived structures of catalytic transi-
3. V Henri. Action de quelques sels neutres sur linver-
tion states. Implications for the catalytic strategy of
sion du saccharose par la sucrase. C R Soc Biol
acyl-transfer enzymes. J Am Chem Soc 105:2446
54:353354, 1902.
2452, 1983.
4. AJ Brown. Enzyme action. J Chem Soc 81:373388,
12. H Kato, T Tanake, H Yamaguchi, T Haa, T
1902.
Nishioka, J Oda. Flexible loop that is a novel catalytic
5. LN Johnson, DC Philips, JA Rupley. Activity of lyso-
machinery in a ligase. Atomic structure and function
zyme: an interim review of crystallographic and che-
of the loopless glutathione synthetase. Biochemistry
mical evidence. U.S. Atomic Energy Commission 1968
33:49954999, 1994.
(pub. 1969); BNL 501116, Vol 1:120138, 1969.

4
Enzyme-Catalyzed Reactions
Experimental Factors that Affect Rates
John R. Whitaker
I. INTRODUCTION ciated by Brown (in 1902) and Henri (1903) indepen-

dently (2, 3) when they suggested that the substrate
A. Kinetic vs. Static Processes
saturation effect observed for rates of enzyme-cata-
There are basically two types of determinations in
science. The rst type is to determine the rate of change
of a parameter, whether it be a chemical reaction, the
enlargement of a cell, organ, or an organism, or other
changes. The second determination is primarily a com-
positional one in which chemical or physical analyses
determine one or more compounds, cells, etc., at a
given time. The rst example is followed by change
per unit time, a kinetic process. The second example
is a static process, where change with time is undesir-
able. Enzyme-catalyzed reactions must be investigated
by a rate-of-change process.
Chapter 1 dealt with the history of development of
enzyme kinetics. Undoubtedly, from the beginning
researchers must have noted that the conversion of
substrate to product occurred as a function of time
and that the rate of change over time was not constant
under all conditions as shown in Figure 1, especially
when a signicant amount of substrate was converted
to product.
B. Pathway of Enzymatic Reactions
Before proceeding further with discussion of rates, it is Figure 1 Typical enzyme-catalyzed reaction showing the
important to understand the pathway by which all pre-steady-state (msec), the constant rate, and the declining
enzyme-catalyzed reactions occur. This was rst appre- rate parts of the progress curve. (From Ref. 1)

lyzed reactions as a function of substrate concentra- E EAAo =EA Km Eo Ao
7
tion, A, resulted from an obligatory intermediate, EAAo =EA
the enzymesubstrate complex, EA, prior to product,
P, formation [Eq. (1)]: Rearranging Eq. (7):
k1 k2
Eo Ao Km EA EAAo EAKm Ao
E A EA E P 1 8
k1 k2
Rearranging Eq. (8):
Based on this premise, which has been shown now to
be universal for all enzyme-catalyzed reactions, EA Eo Ao Km Ao 9
Michaelis and Menten in 1913 (4) developed an equa- EA cannot be determined directly in most cases but it
tion to numerically describe the observed relationship can be substituted for since the rate of formation of
between velocity, v, and A. The derivation of the product, P, is
Michaelis-Menten equation is shown below, using the
dP=dt k2 EA vo 10
more modern concepts of initial velocity vo , and the
steady-state assumption. The EA complex has a non- Rearranging Eq. (10):
covalent bond. EA vo =k2 11
Let Eo total enzyme concentration
Ao total substrate concentration, which is Therefore
assumed to be much larger than Eo ; vo k2 Eo Ao =Km Ao 12
therefore, A Ao during initial velo-
city, vo , measurements < 5% A con- The maximum velocity, Vmax , is attained when all
verted to P during time of experiment) active sites of the enzyme are combined with substrate
EA enzymesubstrate concentration, which so that EA Eo . Therefore,
is a noncovalent complex k2 Eo equals Vmax and Eq. (12) gives Eq. (13):
P product concentration (one product is vo vmax Ao =Km Ao 13
formed in the general derivation; more
than one product may be formed and
released in a sequential manner in
C. Concept of Initial Velocity in Enzymology
many examples)
E free enzyme concentration
The importance of determining the initial velocity of
The rate of formation of EA is [see Eq. (1)]:
enzyme-catalyzed reactions was not fully appreciated
dEA=dt k1 EAo 2 and articulated until the early 1960s. Up to that time,
rates of enzyme-catalyzed reactions were frequently
While the rate of disappearance of EA is based on one-point assaysi.e., at a given time of
dEA=dt k1 EA k2 EA 3 the reaction. Also, reactions often were designed so
that many minutes or hours were used in the assays.
When steady-state conditions are reached (within a few Another reason is that the kinetics of non-enzyme-cat-
msec), dEA=dt dEA=dt so that alyzed reactions by chemists are designed to determine
k1 EAo k1 EA k2 EA k1 k2 EA 4 the order of reactions which must be done under con-
Rearranging: ditions of several half-lives of the reaction; i.e., > 90%
of the substrate must be converted to product to give
EAo =EA k1 k2 =k1 Km 5 four half-lives.
The order of chemical reactions is of major impor-
Km , the Michaelis constant, is given by either EAo = tance in determining the mechanisms of reactions.
ES or k1 k2 =k1 . Often the order is not zero, rst or second order, but
The free enzyme concentration, E, cannot be mea- is a fractional order (such as 1.5 for example). Not so
sured experimentally but it is related to Eo by the with enzyme-catalyzed reactions, which are rst, zero,
conservation equation or mixed order, depending on Ao in relation to Km for
E Eo EA 6 reasons explained later in this chapter.
Figure 2 shows the results of determining the velo-
Substitution of Eq. (6) for [E] in Eq (5) gives city of an enzyme-catalyzed reaction at the same pH,

D. Rates of Enzyme-Catalyzed Reactions
As will be shown later in this chapter, the velocities of

enzyme-catalyzed reactions depend on substrate con-
centration, enzyme concentration, cofactor concentra-
tion, inhibitor concentration, pH, and temperature.
We will discuss each of these factors separately. But
at this point, it is useful to provide some data on some
numerical values of the velocity constants shown in Eq.
(1) calculated to 1 molar concentrations of enzymes
and substrates at the pH optimum and standard tem-
perature (usually 25308C).
Velocity constants k1 for formation of the enzy-
mesubstrate complex are in the range of 104 109
M 1 sec1 for the enzymes given in Table 1(5);
these values are typical for many other enzymes.
However, there are exceptions where the formation
of the complex can be the rate-controlling step as
Figure 2 Method of determining initial velocities of reac- for lactate dehydrogenase combining with the cofac-
tion. The solid lines are the experimentally determined tor NAD ( 50 M1 sec1 ).
data; the dashed lines are tangents drawn to the initial The dissociation constant of the EA complex, k1 ,
slope of the experimental data in order to determine the ranges from < 1:4 to 3.1 sec1 for peroxidase and liver
initial velocity, vo . (From Ref. 1.)
alcohol dehydrogenase, respectively, to > 4:5 104
sec1 for fumarase (Table 1). The overall molar cata-
lytic rate constant ko k2 in Eq. (1), often called kcat
temperature, and cofactor concentration (if needed), because more than one step may be involved] ranges
but at different substrate concentrations. At low sub- from 101 sec1 for papain to 107 sec1 for catalase.
strate concentrations the experimental lines (solid) Therefore, one must conclude that k1 , k1 , and k2 dif-
and the initial velocity (vo ) lines (dashed) coincide fer markedly among enzymes. Some enzymes are more
because the concentration of product formed is < 5 efcient than others as measured numerically by the
% of the original substrate concentration. As the sub- ratio of ko =Km , a generally accepted measure since it
strate concentration is increased, and the rate of for- includes all the rate constants of Eq. (1). The good-
mation of product increases (data for Ao3 , Ao4 , and ness of different substrates for the same enzyme is
Ao5 ) the experimental and vo values diverge more also measured by ko =Km .
and more.
There are several reasons why the experimental lines
and the vo lines are not the same and why initial velo- II. EFFECT OF SUBSTRATE
cities, vo , are important in enzymology. Use of vo has CONCENTRATION ON RATES OF
the advantages of (a) not being greatly affected by ENZYME-CATALYZED REACTIONS
instability of the enzyme (the reaction should be
initiated by adding the enzyme last), (b) not being Section I has treated effect of substrate concentration
inuenced by product concentration since this is zero on rates of enzyme-catalyzed reactions in a general
initially (product can inuence the velocity by being a way. Section II will deal with more specic details of
competitive inhibitor or by giving backward reaction), how to determine Km and ko for one- and two-sub-
and (c) the initial substrate concentration changes very strate enzymes, as well as allosteric behavior of
little in the reaction (when < 5% product is formed enzymesubstrate interactions.
during determining of vo .
With modern-day instrumentation, including recor- A. Initial Velocity vs. Substrate Concentration
ders, it is possible to determine vo with excellent pre-
cision using 5 min, or less, overall reaction times. This section will deal only with enzymesubstrate rela-
There are no reasons why data based on single data tionships obeying Michaelis-Menten kinetics. The
point assays should ever be reported in enzymology. expected relation between vo and Ao is shown in

Table 1 Rate Constants for Some Selected Enzymes
ka1 kb1 kc0

Enzyme Substrate (M sec1
1
(sec1 ) (sec1
Fumarase Fumarate > 109 > 4:5 104 103

Acetylcholinesterase Acetylcholine 10a 103
Liver alcohol NAD 5:3 105 74 103
dehydrogenase NADH 1:1 107 3.1
Ethanol > 1:2 104 > 74
Catalase H2 O 2 5 106 107
Peroxidase H2 O 2 9 106 < 1:4 106
Hexokinase Glucose 3:7 106 1:5 103 103
Urease Urea > 5 106 104
Chymotrypsin 102 to 103
Trypsin 102 to 103
Ribonuclease 102
Papain 101
a
Rate constant for formation of enzymesubstrate complex.
b
Rate constant for dissociation of enzymesubstrate complex.
c
Order of magnitude of the turnover number in moles of substrate converted to product per second per mole
of enzyme: k0 kcat is the observed rate constant and may or may not involve a single rate-limiting step.
Source: Ref. 5.
Figure 3. At low substrate concentrations Ao 0:05 dA=dt k dp=dt 15

Km ) the order of the reaction with respect to Ao is rst
order and ts Eq. (14). It is often difcult, if not impossible, to do reactions at
> 100 Km because of (a) insolubility of the substrate,
dA=dt kAo dp=dt 14 (b) cost of the substrate, or (c) the high substrate con-
At very high Ao Ao 100 Km ), the enzyme is > 90% centration becomes inhibitory of the enzyme as two
saturated with substrate. Thus, the rate is independent molecules bind to the enzyme simultaneously, one as
of Ao , and is zero order. This is described by Eq. (15). the substrate and the second as an inhibitor. Such
behavior is observed frequently.
Between Ao 0:05 Km and Ao 100 Km is where
most of the effect of Ao on vo is found and where
most experiments are performed. Over this range of
Ao , the order of the reaction is mixed order; i.e., it
is the sum of rst order and zero order as shown by Eq.
(16), which is the differentiated form of the Michaelis-
Menten equation [see Eq. (12)].
Eo ko t 2:3 Km logAo =Ao P P 16
At Ao < 0:05 Km , the rst-order term, 2.3 Km

logAo =Ao P predominates, while at Ao > 100
Km , the zero-order term P predominates.
At Ao Km the two terms are equally predominate.
1. Determination of Km and Vmax for One-

Substrate Reactions
For reasons discussed above, Km and Vmax cannot be
Figure 3 Variation of initial velocity, vo , with substrate con- precisely determined from a plot of vo vs. Ao , primar-
centration for an enzyme-catalyzed reaction. (From Ref. 1.) ily because Vmax cannot be experimentally determined.

In 1934 Lineweaver and Burk (6) published a mile- enzymes. (For the isomerases see Chapter 3). For the
stone paper showing that by taking the reciprocal of class EC 3 enzymes, the hydrolases are pseudo-rst-
the Michaelis-Menten equation, a straight line rela- order reactions in that the second substrate is water.
tionship between 1=vo and 1=Ao is obtained, which At 55.4 M water concentration compared to 104 102
gives Km and Vmax as shown by Eq. (17) and Figure 4. M for the second substrate, the change in water con-
centration during the reaction is very small and is
1=vo Km =Vmax So 1=Vmax 17 ignored.
In the late 1950s and early 1960s, attention was
The intercept on the y-axis is 1=Vmax and the slope of
given to development of equations for two and more
the line is Km =Vmax . The intercept on the x axis is
substrate enzyme reactions (see Chapter 1 on protein
1=Km . This is one of the most cited references in the
structure and kinetics). Cleland, in a series of papers,
kinetics of enzymology.
provided a clear rationale for dealing with these reac-
Later on, Augustinsson and Eadie and Hofstee (see
tions which follow the general Eq. (18) for two sub-
Ref. 1) rearranged the Lineweaver-Burk equation to
strate/two product reactions.
two other linear transforms of the Michaelis-Menten
equation. For whatever reason, these two methods are ABPQ 18
rarely used, but they can be useful analyses as in more
complex reaction pathways they may give curvilinear He developed equations for when A must bind in the
relationships indicating the reaction may contain addi- active site of the enzyme before B can bind [Ordered
tional intermediates. In 1954, King and Altman (7) Mechanism; Eq. (19)];
published a useful method for determining the equa-
tion for more complex enzyme-catalyzed reactions (see
Ref. 1).
2. Determination of Km and Vmax for Two-

19
Substrate Reactions
There are relatively few one-substrate reactions in when either A or B can bind rst to the enzyme active
enzymology. Most of these occur in the class EC 5 site [Random Mechanism; Eq. (20)];
20
and when A binds rst and is converted to product

before B binds and is converted to product [Ping
Pong Mechanism; Eq. (21)].
21
These are very important concepts and equations
for the serious enzymologist. More information on
this subject is found in the three key articles by
Figure 4 Plot of the reciprocal of initial velocity, vo , versus Cleland (8). A detailed discussion is found in
reciprocal of initial substrate concentration, Ao , according Whitaker (1) on how to experimentally translate
to the Lineweaver-Burk method. (From Ref. 1.) these concepts into practice.

3. Behavior in MultisiteMultisubunit Enzymes where the substrate concentration is raised to power of
n. For linear plotting, Eq. (22) is rearranged to Eq. (23).
Many enzymes have multiple subunits, each with a
substrate binding and catalytic site for substrate(s). log vo =Vmax vo n log Ao log K 23
Some enzymes have two or more domains (indepen- where n is the product of the number of subunits and
dent folding segments on the same polypeptide chain) strength of binding of substrate of interacting sites and
that bind, and may catalyze substrates to products. If K is the average of all the individual Keq values
each of the active sites binds and catalyzes the sub- involved in the various steps of binding and transfor-
strate independently of other active sites, then the mation of substrate to product.
kinetics follow Michaelis-Menten behaving enzymes. Koshland et al. (10) developed an equation to mea-
On the other hand, if binding of substrate into an sure the cooperativity of the interaction based on the
active site affects either binding or catalysis in a second experimental data. This is given in Eq. (24).
active site, then the kinetics do not follow expected
Michaelis-Menten kinetics. Rs Ao at 0:9vo =Vmax =Ao at 0:1vo =Vmax 24
a. Multiple-Subunit Enzymes. As demonstrated For a Michaelis-Menten-behaving enzyme, Rs is 81.
by Monod et al. in 1965 (9), some multiple-subunit For a positive allosteric-behaving enzyme, Rs is < 81
enzymes give the kinetic behavior shown in Figure 5 while for a negative allosteric behaving enzyme Rs is >
for a plot of vo vs. Ao . Note the marked difference 81 (see Fig. 5).
between the curves for the allosteric-behaving enzyme
and the Michaelis-Menten-behaving enzyme. The b. Single Active Site, Multiple-Substrate Binding
allosteric-behaving enzymes follow Eq. (22): Enzymes. Examples of this type of enzyme are tryp-
sin and carboxypeptidase A. A substrate molecule that
vo Vmax Ano =K Ano 22 binds into the active site is converted to product. If a
second substrate molecule binds at a second site and
increases the rate of catalysis at the active site, it is an
activator; if the rate is reduced on binding of the sec-
ond substrate molecule, it is an inhibitor. Generally,
Keq for binding of the second substrate molecule is
about 10 times larger than is Km for the active site
bound substrate molecule.
III. EFFECT OF ENZYME

CONCENTRATION ON RATE OF
ENZYME-CATALYZED REACTIONS
Rates of enzyme-catalyzed reactions are generally

expected to be directly proportional to the enzyme
concentration as shown in Figure 6. This relationship
between vo and Eo holds whatever the Ao is with
respect to Km as long as all other conditions are held
constant and initial velocities are used.
This linear relationship between vo and Eo is very
important from an analytical perspective. Any analysis
where Eo is important to know depends on this rela-
Figure 5 Comparison of effect of substrate concentration,
tionship. In the food, nutritional, and medical elds,
relative to Km , versus initial velocity, vo , relative to Vmax for a
enzyme assays are used to determine how much active
Michaelis-Menten-behaving enzyme (dashed line) versus a
positive allosteric-behaving enzyme (solid line). The vertical enzyme is present. In the frozen food industry, for
and horizontal dashed lines are for calculating Rs Ao at example, polyphenol oxidase, -amylase, and protease
0.9 vo =Vmax =Ao at 0.1 vo =Vmax for the Michaelis-Menten- activities affect the storage stability and storage time of
behaving enzyme and for the allosteric-behaving enzyme. the product. Levels of selected enzyme activities of
(From Ref. 1.) blood are used in diagnostics of diseases and bacterial

increases. As the substrate size decreases the Km may
increase so that the enzyme is less saturated with sub-
strate. Therefore, there may be changes in vo as a func-
tion of Eo .
Proteins are heteropolymers, being composed of 20
different amino acid residues. Proteases hydrolyze pep-
tide bonds selectively based on their specicity for the
amino acid residues on either side of the peptide bond.
Trypsin will be used as a specic example of a protease.
It hydrolyzes peptide bonds only where a lysyl or argi-
nyl residue provides the carboxyl group of the peptide
bond. The nitrogen side of the peptide bond can be any
of the 20 amino acids. However, the relative rate of
hydrolysis of the peptide bond is affected by the spe-
cic nature of the amino acid. Thus, there are a mini-
Figure 6 Expected relationship between initial velocity and mum of 40 different rates of hydrolysis, not counting
enzyme concentration. Substrate concentration, pH, tem- the decreasing size of the peptides. The best peptide
perature, and buffer are kept constant. (From Ref. 1.) bonds are hydrolyzed more rapidly than the less
good bonds. This leads to a decrease in vo vs. Eo as
the enzyme concentration increases.
infections. Purity of enzymes are based on their specic
activities (activity/mg protein) and the absence of
unwanted activities. B. Coupled Enzyme Reactions
But one must not assume that the linear relationship
shown in Figure 6 holds. It is the responsibility of the A coupled enzyme reaction is given by Eq. (25) where
enzyme user to prove this relationship holds under the E1 ;k1 E2 ;k2
conditions used. There are at least ve valid reasons A ! B ! C 25
why the relationship between vo and Eo can be non-
linear (1). These are listed below. More details can be Substrate A is converted to product B by enzyme E1 .
found in Whitaker (1). But B may be hard to determine analytically so E2 is
also added to the reaction to convert product B to
product C, which can be more readily determined
A. Limitations on Substrate Concentration (e.g., by spectrophotometry). To be successful with
the assay, B must be converted to C just as fast as it
1. One of the Substrates is a Gas, Such as
is produced, so that the rate-determining step remains
Oxygen
A to B conversion even as E1 o is increased. Several
Oxygen has limited solubility in aqueous solution. The commercially available enzyme kits for enzyme ana-
solubility of O2 is 0.24 mM at 258C. In such reactions, lyses depend on coupled reactions.
the analyst usually depends on the O2 used to be Similar problems may arise when two coupled
replenished by absorption from the atmosphere to enzyme reactions depend on a common cofactor, such
maintain a constant O2 concentration. As the Eo is as NAD . As the concentration of E1 is increased it
increased, the rate of depletion of O2 is increased. This may limit the concentration of NAD required by E2 .
often leads to a nonlinear relation between vo and Eo .
Only by adequate stirring rate or bubbling 100% O2
C. Presence of Irreversible Inhibitor in Reaction
through the solution can the O2 concentration be kept
System
constant.
Examples of irreversible inhibitors are Pb2 , Hg2 ,
2. The Substrate Is a Polymer
and Ag ions that might be in the water used.
Starch, largely a homopolymer, is used often to mea- Sulfhydryl enzymes (where the sulfhydryl group of
sure - and -amylase activities. As hydrolysis occurs, cysteine is essential for enzyme activity), which are
the product fragments continue to be substrates. 2530% of all enzymes, would be affected.
Initially, the molar concentration of substrates Experimentally, no enzymatic activity would be

found until sufcient enzyme is added to bind all the action effect, and vo will increase. Such effects were
inhibitor. Then, enzyme activity will be seen. This case observed as early as the 1940s and were called dilu-
is easily recognized as the intercept on the x-axis of the tion effects, meaning as one diluted the enzyme con-
vo vs. Eo plot would be on the right side of the zero centration the activity observed was less than that
intercept. expected.
D. Presence of Reversible Inhibitors in the

System IV. KINETIC CONSEQUENCES OF
COFACTORS
The amount of inhibition found would be dependent
A. Nature and Essentially of Cofactors
on the substrate concentration used (the lower the Ao ,
the greater the effect) and the enzyme concentration. In
Cofactors are small, mostly nonpeptide compounds
general, there would be a nonlinear response between
which are required for activity of some enzymes.
vo and Eo , with a downward curvature of the rela-
These include organic compounds, such as NAD
tionship beween vo and Eo .
(nicotine adenine dinucleotide), FAD (avin adenine
dinucleotide), coenzyme B12 , triphosphates of adeno-
E. Need for an Essential Coenzyme (CoE)
sine (ATP), cytidine (CTP), and uridine (UTP), as well
as many others (see Ref. 1 and references cited therein).
A coenzyme is a nonenzyme compound that binds
They also include many inorganic cations and anions
reversibly in the active site of the enzyme and is
as well as metallo derivatives of organic compounds
required for enzyme activity. The B vitamins, for
(coenzyme B12 , metalporphyrin cofactors). See
example, are a part of a coenzyme (see Sec. IV). The
Table 2 for some specic examples.
example is best illustrated by use of Eq. (26).
The cofactors are essential components of the active
E CoEk1 ! E CoEk2 ! E CoE Sk3 site of many enzymes. They may be required for bind-
ing of the substrate into the active site and/or they may
! E CoE P 26
be essential for the chemical conversion of the sub-
At a xed concentration of CoE and a low Eo , not all strate(s) to product(s). Therefore, in most cases they
the enzyme may be bound to CoE to give E CoE, are absolutely essential for activity of the enzyme. Two
which is required for the substrate to bind. As Eo examples, of many, explain this importance. There are
increases, more CoE will be bound owing to mass > 150 enzymes in the human that require Zn2 as a
Table 2 Importance of Phosphate, Ribose, and Purine and Pyrimidine Bases in Cofactors
Enzyme Cofactor Vitamin Phosphate Ribose Base
Oxidoreductases NAD Niacin Adenine

Oxidoreductases NADP Niacin Adenine
Oxidoreductases FMN Riboavin
Oxidoreductases FAD Riboavin Adenine
Ligases ATP a Adenine
Ligases UTP Uridine
Ligases CTP Cytidine
Transferases CoA Pantothenic acid Adenine
Transferases Acetyl phosphate
Transferases Carbamyl phosphate
Tranferases S-Adenosyl methionine Adenine
Adenosine-30 -phosphate-50 -phosphosulfate Adenine
Transferases Pyridoxal phosphate Pyridoxine
and ligases Thiamine pyrophosphate Thiamine
a
, Not present.
Source: Ref. 1.

cofactor. Without adequate Zn2 intake, which can be determine the overall rate of the reaction. The maxi-
compromised by dietary compounds such as phytic mum rate, Vmax , will be obtained when all the enzyme
acid, by pregnancy, etc., > 150 enzymes may not func- is in the E CoE A form.
tion at desirable rates. The second example is for the Glucose oxidase (GO-FAD) is a typical example of
Fe3 prophyrincontaining enzymes catalase and per- an enzyme that requires a prosthetic group for activity.
oxidase. The Fe3 prophyrin alone converts H2 O2 to This enzyme requires FAD (avin adenine dinucleo-
O2 and water at the rate of about 10 sec1 . The protein tide) as the prosthetic group. FAD is bound tightly
portion of the enzyme alone has no activity on H2 O2 . to the enzyme so that it is not lost during purication
The complete enzyme has a catalytic rate of 107 moles or during the reaction as shown by Eqs. (28) and (29).
H2 O2 converted to product per second per mole of k1
catalase (turnover No.). H2 O2 is formed by metabolic Glucose GO-FAD ! -Gluconolactone
28
processes of the cells. It has strong oxidative activity GO-FADH2
on cellular components. It is absolutely essential to
convert H2 O2 to O2 and water as fast as it is formed. k2
GO-FADH2 O2 ! GO-FAD H2 O2 29
B. Unique Features of Cofactors These reactions [Eqs. (28) and (29)] follow a Ping-Pong
BiBi Mechanism, as described by the Cleland nomen-
Only limited examples of unique features of cofactors clature (Eq. 30).
can be given in this chapter. The reader is referred to
Whitaker (1) and specic references therein devoted to
detailed treatment of this very large topic.

1. Two Types of Cofactors
30
Mechanistically, the cofactors can be divided into two
groups, the coenzymes and the prosthetic groups. The Note that the prosthetic group FAD remains bound
coenzymes are loosely bound to the protein and gen- tightly to the enzyme E-FAD indicates covalent
erally act as a substrate in the catalyzed reaction. The bond), the E-FAD is reduced to E-FADH2 by removal
prosthetic groups are more tightly bound, some cova- of the two hydrogens from C1 of glucose and that the
lently, and they are regenerated to the starting form at E-FADH2 is oxidized back to E-FAD by O2 , permit-
the end of the reaction. An example of each type will ting it to be recycled catalytically in the process. The
clarify these differences. reaction takes place in two steps with only one sub-
There are many enzymes that require NAD or strate bound to the enzyme at a time.
NADH as coenzymes. The reaction catalyzed by alco-
hol dehydrogenase is shown in Eq. (27). 2. Same Cofactor, Different Reactions
k1 Earlier in this chapter, it was stated that Zn2 serves as
CH3 CH2 OH NAD CH3 CHO NADH H cofactor for > 150 different enzyme-catalyzed reac-
k1
tions. But the example chosen here to explain this con-
27
cept is the prosthetic group pyridoxal phosphate.
This is a reaction requiring the substrate ethanol and Pyridoxal phosphate is involved in amino acid conver-
the coenzyme NAD . NAD must bind to the enzyme sion to different products, as shown in Figure 7. The
rst, followed by binding of the ethanol. After cataly- prosthetic group binds to the amino acid in the same
sis, the product acetaldehyde dissociates from the way, forming a Schiff base intermediate, as shown by
enzyme, followed by NADH. This is an ordered BiBi the upper central formula. Which of the reactions is
Mechanism (see Sec. II.A.2). The reaction is a two- catalyzed depends on the specicity of the protein
substratethree-product (counting the H ) reaction. (apoenzyme) and the specic nature of the amino
It is a reversible reaction with the backward reaction acid. Therefore, pyridoxal phosphate and apoenzyme
favored. Coenzymes, being loosely bound to the together bind to the amino acid (substrate) to perform
enzyme, are generally lost during purication of the racemization, decarboxylation, deamination, , elim-
enzyme. The researcher must add the coenzyme back ination, , elimination, and addition reactions (to
to the inactive enzyme to restore activity. The concen- indole to form tryptophan) to give a variety of pro-
tration of both the apoenzyme and coenzyme will ducts.

Figure 7 Schematic representation of six enzyme reactions in which pyridoxal phosphate is the cofactor. A common Schiff base
intermediate between serine and pyridoxal phosphate is shown in the upper center. The type of enzyme involved determines the
nature of the reaction. (From Ref. 1.)
V. KINETIC CONSEQUENCES OF
ness of fruits and vegetables. Many pharmaceutical
ENZYME INHIBITORS
compounds are designed to kill microorganisms by
A. Nature of Inhibitors selectively inhibiting their enzyme systems.
Mechanistically, there are two major groups of
Enzyme inhibitors are dened as any compound, except enzyme inhibitors. One group inhibits enzymes by
H ions (see pH effects), which decrease the activity reactions involving covalent bond formation (irrever-
when added to an enzyme reaction. Many enzyme inhi- sible inhibitors); the other group inhibits enzymes by
bitors are known. They are used to kill insects or reversible noncovalent bond formation (reversible inhi-
unwanted plants (herbicides) or animals (rotenone). bitors). Both types are important as they decrease or
They are used to prevent browning or preserve fresh- eliminate enzyme activity.

The rate and extent of inhibition of enzymes by uncompetitive inhibitors (U) bind to the enzyme
irreversible inhibitors will depend on concentration of only after the substrate has bound. The uncompetitive
inhibitor, concentration of the enzyme, and the specic inhibitors can also bind to other intermediate
group modied on the enzyme, as well as pH and tem- enzymeproduct complexes. As shown in Figure 8,
perature. The rate of the inhibition will be relatively at equilibrium the species A, E, U, EAU, and EP0 U
slow (minutes or hours) as a covalent bond is formed. can exist. Actually, the three types of inhibitors are
The reaction cannot be reversed to regain enzyme distinguishable only experimentally where different
activity. effects on the kinetics are shown. The experiments
must be performed in the absence and presence of
E Ik1 !E I 31 the reversible inhibitor.
Figure 9 shows the kinetic effect of a competitive
inhibitor on the rate of an enzyme-catalyzed reaction,
B. Reversible Inhibitors when the data are plotted by the Lineweaver-Burk
method. As noted, there is no effect of inhibitor on
The rate and extent of inhibition of enzymes by rever- the y-axis intercept. Therefore V Vmax is the same
sible inhibitors also depend on the factors listed above. in the presence and absence of the competitive inhi-
However, the rate of inhibition is very rapid (msec) as bitor. But the slope of the line is larger in the pre-
a noncovalent complex is formed. Also, the inhibition sence of the competitive inhibitor, reecting the effect
can be reversed by removing the inhibitor (by dialysis of the inhibitor on K Km for the reaction as
or gel ltration). Our attention in this chapter is shown also by the difference in the x intercept.
focused on the reversible enzyme inhibitors. Dened The numerical difference in slopes and x intercepts
kinetically, a reversible inhibitor is one that reacts in the presence and absence of inhibitors is
with an enzyme in a reversible manner as shown in 1 Io =Ki . Knowing the Io added to the reaction,
Eq. (32), where Ki is an equilibrium (dissociation) con- Ki can be calculated. These data were determined for
stant. Ki EI=E I: a linear competitive inhibitor.
Figure 10 shows the kinetic effect of a noncompe-
Ki titive inhibitor when added to an enzyme-catalyzed
EI EI 32 reaction. Again, data must be obtained for a control
with no inhibitor and one with a xed concentration
The reaction occurs within a few msec and the extent of the inhibitor. Figure 10 shows that both the slope
of inhibition is controlled by the concentration of E and the y-intercept are changed by the term
and I and Ki . Generally, Ki for the reversible inhibitors 1 Io =Ki . There is no effect on the x intercept.
will be in the range of 102 107 M. If the Ki is 108 This is the denition of a simple linear noncompeti-
M, the dissociation rate is so slow that there is not a tive inhibitor.
true equilibrium of the process when perturbed by Figure 11 shows the effect of an uncompetitive inhi-
addition of substrate. bitor when added to an enzyme-catalyzed reaction.
Based on their different effects on the rates of Again, a control without inhibitor must be included.
enzyme-catalyzed reactions, the reversible inhibitors There is no effect of the uncompetitive inhibitor on the
can be divided into three types, as shown in Figure slope of the lines, while there is a marked difference in
8. The three types are the competitive inhibitors (C), the y-intercept (and the x intercept). The difference in
noncompetitive inhibitors (N), and the uncompetitive y-intercept is given by 1 Io =Ki . The example chosen
inhibitors (U). The competitive inhibitors (C) com- is for a linear uncompetitive inhibitor.
pete with the substrate for binding to the active site The examples given in Figures 911 are linear-
of the enzyme. Therefore, the equilibrium involves the behaving inhibitors, as dened in Figure 12. This is
species E, EA, and EC and the free A and C (Fig. 8). determined from a plot of slope (or intercept) vs. Io
The noncompetitive inhibitors (N) affect the rate of where experiments at several different Io are done.
the enzyme-catalyzed reaction by binding outside the In many cases, one nds that the type of inhibition is
active site in a way that permits substrate to bind into linear. Generally, they will be linear for most compe-
the active site but no catalysis of substrate to product titive inhibitors. But for noncompetitive and uncom-
occurs. As shown in Figure 8, the species E, A, EA, petitive inhibitors there are more opportunities for
EA0 , EN, EAN, EA0 N, and N can be present. The observing hyperbolic or parabolic type behavior.

Figure 8 Schematic representation of inhibition of enzyme activity by various types of reversible inhibitors. The model for E
shows only the active site with the binding locus (inner void) and the transforming locus, with catalytic groups A and B. The
symbols for the species involved (in parentheses) are: E, free enzyme; EA, enzymesubstrate complex; EA0 , acylenzyme inter-
mediate; C, competitive inhibitor; N, noncompetitive inhibitor; U, uncompetitive inhibitor; P1 and P2 are products formed from
the substrate, A, and EN, EAN, EA0 N, EC, and EA0 U are complexes with respective enzyme species and inhibitors. All complexes
formed involve noncovalent bonds except EA0 , where a covalent bond is formed with catalytic group A. (From Ref. 1.)
Figure 9 Linear competitive inhibition, plotted by the Figure 10 Simple linear noncompetitive inhibition, plotted
Lineweaver-Burk method. V Vmax in absence of inhibitor. by the Lineweaver-Burk method. See Figure 9 for additional
V 0 Vmax 1 Io =Ki in presence of inhibitor, K Km in explanation. (From Ref. 1.)
absence of inhibitor. K 0 1 Io =Ki in presence of inhibi-
tor. (Ref. 1.)
Figure 13 pH effect on activity of several enzymes. (a)
Pepsin acting on acetyl-L-phenylalanyl-L-diiodotyrosine at
36.78C with 5 104 M pepsin, reaction time 15 min (11).
(b) Ficus glabrata peroxidase acting on 0.03 M guaiacol and
Figure 11 Linear uncompetitive inhibition, plotted by the 0.005 M H2 O2 at 30.08C (12). (c) Trypsin acting on casein
Lineweaver-Burk method. See Figure 9 for additional expla- (13). (d) Hydrolysis of 5 104 M p-nitrophenyl phosphate
nation. (From Ref. 1.) by crude milk alkaline phosphatase at 358C in 0.1 M sodium
glycinate buffer, reaction time 15 min (J.R. Whitaker, unpub-
lished data). (From Ref. 1.)
generally bell-shaped, but the left side of the pH curve

for pepsin and the right side of the pH curve for alka-
line phosphatase are steeper than the other side. We
will return to this point later. Not all pH activity
curves are bell-shaped. They may be sigmoidal or
they may be horizontal lines, as is the case for bovine
catalase over the pH range of 310.
Table 3 lists the pH optima for 24 enzymes. The pH
optima range from 2 to pepsin to 10 for alkaline phos-
phatase. A number of the enzymes have pH optima
near pH 7 (in the range of 68). In the following sec-
tion, the question of why enzymes have pH activity
optima will be addressed. But it should be clear to
Figure 12 Linear, hyperbolic, and parabolic inhibition pat-
terns of enzyme reactions. (From Ref. 1.)
the reader that research must determine the effect of
pH on an enzyme in order to know the range in which
it has activity and at which pH the maximum activity is
found.
VI. EFFECT OF pH ON ENZYME-

CATALYZED REACTIONS B. Why Do Enzyme-Catalyzed Reaactions Have
pH Optima?
A. Some Data on pH Versus Activity for
Enzymes There are two reasons why pH optima are found for
enzyme-catalyzed reactions. The rst is because of pH
Figure 13 shows the effect of pH on activity of porcine instability of the enzymes. The second is because of
pepsin, Ficus glabrata peroxidase, bovine trypsin, and ionization of groups in the active site of enzymes, of
milk alkaline phosphatase. The pH activity curves are the substrates, or of cofactors.

Table 3 pH Activity Optimum of Several Enzymesa Above pH 10, the ammonium groups are nonproto-
nated (NH2 ) and do not form electrostatic bonds.
Enzyme pH optimum
The active site of bovine catalase has no ionizable
Acid phosphatase (prostate gland) 5 carboxyl or amino groups. Below pH 3 and pH 10, the
Alkaline phosphatase (milk) 10 electrostatic bonds are nonexistent and the enzyme
-Amylase (human salivary) 7 loses activity owing to instability. The sharp decrease
-Amylase (sweet potato) 5 in activity of pepsin at pH < 2:0 is because of instabil-
Carboxypeptidase A (bovine) 7.5 ity. The sharp decrease in activity of alkaline phospha-
Catalase (bovine liver) 310 tase at high pH is due to loss of electrostatic bonds to
Cathepsins (liver) 3.55 stabilize the enzyme.
Cellulase (snail) 5
-Chymotrypsin (bovine) 8
Dextransucrase 6.5 2. Effect of pH on the Active Site Essential
(Leuconostoc mesenteroides) Ionizable Groups
Ficin (g) 6.5
Glucose oxidase (Penicillium notatum) 5.6 Table 4 shows six ionizable groups, their ionization,
Lactate dehydrogenase (bovine heart) 7 (forward pKa , and Hion that can be a part of the active site
reaction) of enzymes. Kinetically, ionization of one essential
9 (backward ionizable group gives a sigmoidal pH activity curve,
reaction) while two essential ionizable groups, one in the ionized
Lipase (pancreatic) 7 form and one in the protonated form, give a bell-
Lipoxygenase-1 (soybean) 9 shaped curve. In the case of pepsin there is an aspartic
Lipoxygenase-2 (soybean) 7
acid carboxyl group with pKa of 1 that is responsible
Pectin esterase (higher plants) 7
Pepsin (bovine) 2
for the left side of the pH activity curve. This group
Peroxidase (g) 6 must be as COO for the pepsin to be active. The
Polygalacturonase (tomato) 4 right side of the pH activity curve is due to ionization
Polyphenol oxidase (peach) 6 of another aspartic acid arboxyl group that must be in
Rennin (calf) 3.5 the protonated form (COOH) for pepsin to be active.
Ribonuclease (pancreatic) 7.7 Therefore, the active site of pepsin has two ionizable
Trypsin (bovine) 8 carboxyl groups.
a Trypsin has a pH optimum of 7.8. The left side of
The pH optimum will vary with source and experimental conditions.
These pH values should be taken as approximate values. the activity curve is due to an imidazolium group (pKa
Source: Ref. 1. 5.67.0) in the active site that must be in the unproto-
nated form (Table 4) for activity of the enzyme. The
right side of the bell-shaped curve is due to the ioniza-
tion of the -amino group (pKa 8.08.5) of the N-term-
1. Effect of pH on Instability of Enzymes
inal leucyl residue No. 16 (using the protrypsin
The tertiary and quaternary structures of enzymes are numbering). For activity, the amino group must be
stabilized by noncovalent electrostatic bonds, hydro- in the protonated form.
gen bonds, hydrophobic bonds, and covalent disulde Effect of pH on the observed activity of enzymes
bonds. The electrostatic bonds, formed between nega- can be due to any of the components of the reactions
tive and positive charges on two groups of the enzyme, [see Eq. (1)]. This includes ionization of groups on the
are affected by pH. The two predominant prototropic free enzyme E, the enzymesubstrate complex (EA),
groups in enzymes are the side chain carboxyl groups the substrate or any cofactor involved. To be able to
of aspartic and glutamic acid residues (pKa of 34) and interpret the effect of pH on an enzyme-catalyzed reac-
the side chain ammonium groups (pKa of 910) of tion, the researcher must know the stability of the
the lysine side chain residues and the guanidinium enzyme at different pHs at the temperature to be
groups (pKa 1213) of arginine residues. In the neu- used for activity measurements, the effect of pH on
tral pH range the ionizable side groups are as COO , ionization of group(s) on the substrate and any cofac-
H3 N , and H2 NC( N H2 NH groups, which can tor. Equally important, the researcher must control the
form strong electrostatic bonds (1020 kcal/mol). But conditions of the experiment so that the pH effect can
below pH 3 the carboxyl groups are protonated be on the free enzyme (Ao 0:05 Km ) or on the enzy-
(COOH) and cannot form electrostatic bonds. mesubstrate complex Ao 100 Km ). The reader is

Table 4 Prototropic Groups That May Be Involved in Enzyme Catalysis
a
Located at the end of polypeptide chain. Calories 4.186Joules.
referred to the detailed discussion of Whitaker (1) on

this subject. Properly performed experiments are essen-
tially the only way to determine the essential ionizable
groups in the active site of an enzyme and whether they
are involved in binding of substrate or in catalysis of
substrate to product.
VII. EFFECT OF TEMPERATURE ON

ENZYME-CATALYZED REACTIONS
A. General Discussion
Figure 14 shows (solid line) the effect of different tem-

peratures on the velocity of an enzyme-catalyzed reac-
tion. At 208C, the velocity is constant over the time
period used. At 408C, there is a small deviation from Figure 14 Effect of temperature on rate of product forma-
a constant velocity (compare the solid experimental line tion. The solid lines are for experimental data; the dashed
and the dashed initial velocity line). At 508C there is lines are based on initial velocity, vo . (From Ref. 1)
more deviation from a constant velocity. At 558C and
608C the constant velocity is maintained for only a very plot of initial velocities vs. temperature is curvilinear.
short time of reaction. As expected, vo dp=dt will be As explained below, this is the expected relationship
different depending on the time of the reaction to , t1 , according to the Arrhenius equation. The velocities at
and t2 indicated on the x-axis of Fig. 14). These results time t1 coincide with the expected relationship up to
are plotted in Figure 15 as dp=dt vs. temperature. The 508C. Above 508C, the velocity slows down and then

Eq. (1)]. To eliminate the effect of factors listed in
Section VII.A and B, the enzyme should be saturated
with substrate and performed at the pH optimum for
the enzyme-catalyzed reaction, over a temperature
range where the enzyme is stable.
The rate constant, k2 Vmax =Eo , under the above
conditions is plotted according to the Arrhenius equa-
tion:
k2 AeEa =RT 33
where A is the Arrhenius constant, Ea is the Arrhenius
activation energy, R is the universal gas constant (1.98
cal/mol deg), and T is degrees Kelvin. The plot is
shown in Figure 16. From the right-hand slope, Ea is
obtained. The positive slope (left side of Fig. 16) gives
Ea for denaturation of the enzyme. The relationship
Figure 15 Velocity of formation of product as function of between Ea and H6 from the Absolute Reaction
temperature. Data are from Figure 14, taken at time to , t1 , Rate theory is given by Eq. (34).
and t2 : (From Ref. 1.)
H6 Ea RT 34
6
decreases. At time t2 , the data follow the expected rela- The numerical difference between H and Ea is 0.6
tionship only to 408C, slows at 508C, and decreases at kcal/mol at 258C.
558C and 608C. Therefore, the temperature optima are
608C, 558C, and 508C for the reactions at times to t1 , D. Effect of Catalyst on Ea and Relative Rates
and t2 , respectively. The difference between the three is
due to the effect of temperature on the velocity of dena- Table 5 shows the effect of different catalysts on rela-
turation of the enzyme. Therefore, effect of tempera- tive rates of some reactions. For example, the conver-
ture on velocity of denaturation of the enzyme is an sion of H2 O2 to O2 and H2 O relative to no catalyst is
important factor when one is studying the effect of 2:07 103 times faster for the I and 3:47 108 times
temperature on rates of enzyme-catalyzed reactions.
Two other general factors affect the velocity of
enzyme-catalyzed reactions. The factors are the effect
of temperature on equilibria and on the catalytic ef-
ciency of the enzyme.
B. Effect of Temperature on Equilibria
There are numerous equilibria in an enzyme-catalyzed

reaction. These include: (a) solubility of substrates,
especially gases (O2 solubility is 2:17 103 M at 08C
and 1:12 103 M at 308C); (b) pKa of ionizable
groups on the enzyme (see Hion in Table 4); (c) change
in pH of buffers (Tris buffer pKa changes 0.24 units/
108C; see also Table 4); (d) formation and dissociation
of the enzymesubstrate complex and others (see
Whitaker [1] for a more detailed discussion).
Figure 16 Effect of temperature on rate of an enzyme-cat-
alyzed reaction, plotted according to the Absolute Reaction
C. Effect of Temperature on Catalytic Rates Rate Theory. The positive slope on the left is for rate of
inactivation of the enzyme. The negative slope on the right
The catalytic rate of conversion of the substra- is for effect of temperature on k2 for conversion of EA to
teenzyme complex to product is controlled by k2 [see products. (From Ref. 1.)

Table 5 Effect of Catalyst on Ea and on Relative Rates of Some Reactions
Relative
Ea n0 =n ratesa
Substrate Catalyst (kcal/mol) (258C) (258C)
H 2 O2 None 18.0 5:62 1014 1.00

I 13.5 1:16 1010 2:07 103
Catalase 6.4 1:95 105 3:47 108
Sucrose H 25.6 1:44 1019 1.00
Invertase 11.0 8:04 109 5:58 1010
Carbonic acid None 20.5 8:32 1016 1.00
Carbonic anhydrase 11.7 2:46 109 2:96 106
Urea H 24.5 9:33 1019 1.00
Urease 8.7 3:96 107 4:25 1011
a
Relative rates calculated are approximate since differences in S6 are ignored.
Source: Ref. 1.
faster for catalase. n 0 =n, the fraction of molecules n E A E A6 EA EA EP6

with activation energy equal to Ea or greater, is calcu- EP E P6 E P 36
lated from Eq. (35):
The rate-determining step is number 4 in Figure 17.
n0 =n eEa =RT 35 This is the step that requires the most activation
energy. Activation energy is required to convert a
Equation (1) has been used in this chapter to ground state species to an activated species [Eq. (33)].
describe enzyme-catalyzed reactions. Only ground The thermodynamic energy difference between two
state species are shown. In reality, activated state spe- ground state species is calculated using the Vant
cies should also be included, as shown in Figure 17. Hoff equation [Eq. (37)].
When this is done the complete equation is Eq. (36).
d ln Keq =dT H=RT 2 ; ; ln K2 =K1
37
HT2T1 =T2 T1
E. Factors Accounting for Catalytic Efciency of

Enzymes
Enzymes are superior biological catalysts as shown in

Table 5. The factors that account for their catalytic
effectiveness are listed in Table 6. Factors 14 account
for the rate enhancement due to binding of the sub-
strate stereospecically (at least three points of attach-
ment) into the active site of enzymes. Factor 1
accounts for much of the rate enhancement, especially
for binding of two or more substrates into the active
site simultaneously 109 and 1015 enhancement for two
and three substrates, respectively). The polymerases
Figure 17 Change in energy along the reaction coordinate involved in biosynthesis of proteins bind ve or more
during conversion of A to P in an enzyme-catalyzed reaction substrates and cofactors simultaneously into the active
according to Eq. (36). The numbers refer to steps in the site. Factors 57 account for the rate enhancement
thermodynamic and transition states in the forward direc- during the chemical conversion of the substrate(s) to
tion. (From Ref. 1. product(s).

Table 6 Factors Accounting for Catalytic Effectiveness of Enzymes
Factor Rate enhancementa
1. Formation of stereospecic enzyme-substrate complex 104 ; 109 ; 1015b

(conversion from inter- to intramolecular reaction)
2. Decreased entropy of reaction 103
3. Concentration of reactive catalytic groups 103 104
4. Distortion of substrate 102 104
5. General acid/general base catalysis 102 103
6. Nucleophilic/electrophilic catalysis 102 103
7. Using several steps 102 104
Overall rate enhancement 1018 1036
a
In some cases these values can be approximated by model experiments. In others, they are the
best estimates available.
b
For 1, 2, and 3 substrates, respectively.
Source: Ref. 1.
The overall theoretical rate enhancement shown in 7. EL King, C Altman. A schematic method of deriving
Table 6 is 1018 1036 . The best enzymes, such as catalase the rate laws for enzyme-catalyzed reactions. J Phys
and peroxidase, have rate enhancements 1020 . Chem 60:13751378, 1956.
Therefore the catalytic efciency of enzymes can be 8. WW Cleland. The kinetics of enzyme-catalyzed reac-
tions with two or more substrates or products. I.
explained by well-known chemical principles: enzymes
Nomenclature and rate questions. II. Inhibition:
are not magicians.
nomenclature and theory. III. Prediction of initial
velocity and inhibition by inspection. Biochim
Biophys Acta 67:104137, 173187, 188196, 1963.
REFERENCES 9. J Monod, J Wyman, JP Changeux. On the nature of
allosteric transitions: a plausible model. J Mol Biol
1. JR Whitaker. Principles of Enzymology for the Food 12:88118, 1965.
Sciences. 2nd ed. New York: Marcel Dekker, 1994. 10. DE Koshland Jr, G Nemethy, D Filmer. Comparison
2. AJ Brown. Enzyme action. J Chem Soc 81:373379, of experimental data and theoretical models in pro-
1902. teins containing subunits. Biochemistry 5:365385,
3. V Henri. General Laws of the Action of Diastases. 1966.
Paris: Hermann, 1903. 11. LE Baker. New synthetic substrates for pepsin. J Biol
4. L Michaelis, ML Menten. The kinetics of invertase Chem 193:809819, 1951.
action. Biochem Z 49:333369, 1913. 12. S Kon, JR Whitaker. Separation and partial charac-
5. M Eigen, GG Hammes. Elementary steps in enzyme terization of the peroxidases of Ficus glabrata latex. J
reactions. Adv Enzymol 25:138, 1963. Food Sci 30:977985, 1965.
6. H Lineweaver, D Burk. Determination of enzyme dis- 13. JH Northrop, M Kunitz, RM Herriott. Crystalline
sociation constants. J Am Chem Soc 56:658666, Enzymes. New York: Columbia University Press,
1934. 1948, p 12.

5
Inactivation of Enzymes
From Experimental Design to Kinetic Modeling
Ann Van Loey, Indrawati, Chantal Smout, and Marc Hendrickx

Katholieke Universiteit Leuven, Leuven, Belgium
I. INTRODUCTION by inactivation of the enzymes by physical methods

(e.g., thermal blanching) or by inhibition of the enzy-
Enzymes occur naturally in many biological raw mate- matic reaction using additives, a chemical method.
rials and can, together with processing, affect func- The current chapter will particularly focus on the
tional properties of foods in many ways. Endogenous inactivation of enzymes by the processing factors tem-
enzymes can have benecial or detrimental effects on perature and pressure, discussing the experimental
foods. Some enzymes are positively utilized during design and modeling of kinetic inactivation studies of
food processing for recovery of byproducts, for devel- enzymes, illustrated by some examples. An extended
oping new food products, for achieving higher rates overview on thermal and pressure inactivation kinetics
and levels of extraction, or for improving food quality of enzymes is given by Ludikhuyze et al. (1).
in terms of, e.g., avor, texture. On the other hand,
enzymes might also have detrimental effects. Food
spoilage can be caused by enzymes naturally present II. A GENERAL SCHEME FOR ENZYME
in the food, or by enzymes produced by certain micro- INACTIVATION
organismsfor example, enzymatic browning reac-
tions in fruits and vegetables by polyphenoloxidases, A general scheme for thermal enzyme inactivation was
or rancidity caused by the presence of endogenous proposed by Lumry and Eyring (2): a reversible partial
lipases and lipoxygenases. unfolding, followed by an irreversible reaction step
As a consequence of the benecial and detrimental K kir
N ! U ! I
effects of enzymes, in most food-processing steps con-
trol of enzymatic activity is required. Either one wants where N represents the native, U the reversibly
to promote the benecial effects of the enzyme by unfolded and I the irreversibly inactivated enzyme.
enhancing the enzymatic activity during processing; in K N=U and kir symbolize the unfolding equili-
this case knowledge on enzyme stability under the rele- brium constant and the rate constant of the irreversible
vant processing conditions is required for process reaction step, respectively. In the literature, some evi-
design (e.g., its thermostability, resistance toward acid dence can be found as to an analogous general scheme
environments). On the other hand, in the case of detri- for pressure inactivation of enzymes (3).
mental enzymatic action, one wants to eliminate or If we consider this simple inactivation scheme, the
retard the enzymatic reaction, which is often performed rate of inactivation is given by Eq. (1):

kir U 1 death cans) to minimize heating and cooling lags.
Since the measurement of residual activity involves The samples are immersed in a temperature-controlled
N U U will renature upon cooling), the observed heating bath at constant inactivation temperature for
rate of inactivation kobs is: predetermined time intervals. Immediately upon with-
drawal from the heating bath, the samples are cooled
kir U kobs N U 2 in ice water to stop the thermal inactivation, and the
Because K N=U, assuming rapid equilibrium, the residual enzymatic activity is measured.
experimentally measured rate constant kobs can be For pressure inactivation studies, today only batch
mathematically expressed as Eq. (3). methods are available. For pressure treatments, the
kir enzymic samples should be contained in exible con-
kobs 3 tainers (e.g., microtubes, plastic bags). Because of adia-
1K
batic heating, pressure buildup is inevitably associated
At high temperature or pressure, where the concen- with temperature increase. At the prevailing high pres-
tration of the native form N is much smaller than sure, this temperature increase might provoke a signif-
the concentration of the reversibly unfolded form icant reduction of enzyme activity during pressure
U, the second step becomes rate limiting and the buildup. The temperature reached during pressure
above equation can be simplied to Eq. (4). buildup can seriously be reduced by decreasing the
kobs kir 4 rate of compression. To avoid problems arising from
the temperature increase during pressure buildup, a
i.e., when the inactivation is examined at high inac-
possible approach is to exclude the nonisobaric/non-
tivation temperature or pressure (far above the dena-
isothermal phase from the inactivation data by starting
turation temperature or pressure), the inuence of the
the time course of the experiment (zero point) after
unfoldingrefolding equilibrium is negligible and the
reaching the desired pressure and an additional equili-
inactivation is only determined by the secondary irre-
bration period to allow temperature to evolve to its
versible step.
desired value. At that moment, the pressure vessel is
decompressed and the activity of the corresponding
III. EXPERIMENTAL DESIGN FOR enzyme sample is considered as the blank. The use of
KINETIC ENZYME INACTIVATION this zero-point approach is merely allowed for rst-
STUDIES order reactions (including the special cases of biphasic
and rst-order fractional conversion reactions). After
Enzyme inactivation kinetics can be determined using pressuretemperature treatment, enzyme solutions are
either steady-state or un-steady-state procedures (4; 5), cooled in ice water to stop inactivation and reactiva-
the steady-state procedure being the most straight- tion, and the residual enzymatic activity is measured.
forward approach to study thermal or pressure inacti- To perform kinetic experiments at elevated pressure
vation kinetics. A classical steady-state experiment (up to 1000 MPa), specialized high-pressure equip-
consists of subjecting an enzymic sample to a square ment, consisting of several individual thermostated
wave temperature and/or pressure prole. pressure vessels, is often used (Fig. 1). Such equipment
To study thermal inactivation, batch or ow meth- allows submission of several samples simultaneously to
ods can be used for sample heating and cooling. treatments at the same pressure and temperature for
Whatever method being used, care has to be taken to preset times.
ensure that heating and cooling are quasi-immediate, Upon removal of the denaturing agents (i.e., tem-
or else appropriate compensation for thermal lags has perature or pressure), regeneration of enzyme activity
to be taken into account (e.g., 6, 7), especially when the might occur (see above). Hence, it should be experi-
heating or cooling lag is not sufciently small relative mentally veried whether enzyme reactivation occurs
to the half-life of the reaction. For isothermal batch within a reasonable experimental time after treatment.
treatments, enzyme solutions are usually enclosed in
small, preferably highly conductive vials or tubes
(e.g., glass capillaries, thermal death tubes, thermal IV. MODELING ENZYME INACTIVATION
KINETICS

The half-life of a reaction is the time required to reduce the
concentration (enzyme activity in case of enzyme inactivation Kinetics of the enzyme inactivation process describe its
studies) to half of its initial value. progress in time. The way in which the inactivation

rate constant k not varying with time), Eq. (6) or Eq.
(7) is obtained depending on the reaction order (with
respect to time).
n1 A A0 expkt 6
n 6 1 A1n A01n n 1 kt 7
By trial-and-error procedures an estimate of the

reaction order n (with respect to time) can be obtained
by analyzing graphically the trends and/or deviations
from a linear behavior. However, a complication in
determining the order of a reaction from the method
of integration is that little or no distinction can be
made among zero-, rst-, and second-order reactions
Figure 1 An example of a multivessel high-pressure equip- with respect to time when the response value changes
ment. only little (10). Even when a linear plot is obtained,
conclusions must be drawn cautiously from the data,
especially if the data points correspond to no more
progresses as a function of time is expressed by the than 1020% conversion because many mathematical
mathematical form of the kinetic model. The rate of functions are roughly linear over a sufciently small
inactivation is reected by the numerical values of the range of variables. According to Arabshahi and
kinetic parameter estimates. Lund (7), reactions should be followed, where possible,
Dealing with kinetic studies of thermal or pressure through at least four to ve half-lives (or 12 log
inactivation of enzymes, the rst step of the data ana- reductions). If the method of analysis is not sufcient
lysis procedure involves the identication of an ade- to measure response values as low as this, the longest
quate inactivation rate equation (e.g., rst order, heating times possible should be used (11). According
biphasic, nth order) and the identication of a tem- to Hill and Grieger-Block (8), one should perform at
perature and/or pressure coefcient model (e.g., least experimental runs in which data are taken at
Thermal Death Time model, Arrhenius model, 40%, 50%, or higher conversions.
Eyring model), while the second step is the selection
of a regression method to estimate kinetic parameter 1. First-Order Model
values with the highest probability of being correct.
Thermal or pressure inactivation of enzymes can often
A. Determination of an Inactivation Rate be described by a rst-order reaction [Eq. (6)] (1). This
Equation feature is remarkable since enzyme inactivation is a
complex process involving several events, such as for-
The general rate law to describe the decrease of enzyme mation and/or disruption of different interactions and/
activity A as a function of processing time (t) can be or bonds, decomposition of amino acids, aggregation,
written as and/or dissociation. It is therefore suggested that in
case of apparent rst-order inactivation processes,
dA
kobs An 5 one of these reactions predominates over the others.
dt If several reactions occur at more or less the same
where A is the enzymatic activity at time t; k the reac- rate, complex non-rst-order inactivation kinetics are
tion rate constant, and n the reaction order. The order expected (12).
of reaction can be determined by many different meth-
ods that are briey discussed by Hill and Grieger-
Note that in the context of enzyme inactivation, the concept
Block (8) and Laidler (9). Under simple initial and of reaction order is purely empirical and gives no direct infor-
boundary conditions, the integration of differential mation about the mechanism of the reaction. This procedure
Eq. (5) results in analytical solutions describing the provides a model which describes the kinetics of a reaction,
enzymatic activity A as a function of time. In case but this model should not necessarily be interpreted as repre-
of isobaric-isothermal inactivation experiments (i.e., senting the actual mechanism of the reaction.

Under isothermal-isobaric conditions a rst-order The decimal reduction time at a given inactivation tem-
reaction can be expressed as Eq. (6), which can be perature and pressure can be calculated from the slope
linearized by a logarithmic transformation, yielding of a linear regression analysis of log(A=A0 versus inac-
Eq. (8). tivation time at constant inactivation temperature and
pressure, or alternatively by nonlinear regression ana-
A
ln kt 8 lysis (see below).
A0 The validity of a rst-order inactivating behavior
where A is enzymatic activity at time t; A0 is initial can be examined by plotting residual enzyme activity
enzymatic activity; t is treatment time; and k is the versus treatment time on a semilogarithmic scale and
rst-order inactivation rate constant. As an example, evaluation of the goodness of t by means of, e.g.,
Figure 2 depicts thermal inactivation of tomato pectin lack-of-t test, coefcient of determination R2 , ana-
methylesterase, which can be adequately described by a lysis of the distribution of residuals. Residuals of an
rst-order reaction (adapted from 13). In case of a appropriate t represent ony the experimental error
rst-order inactivation model, the rate constant at a and should therefore be randomly distributed. The
given temperature and pressure can be estimated by existence of trends in residuals (with respect to either
linear regression analysis of lnA=A0 versus inactiva- the independent or dependent variable[s]) suggests that
tion time at constant inactivation temperature and some systematic behavior is present in the data that is
pressure, or alternatively based on Eq. (6) by nonlinear not accounted for by the model (14), in this case by a
regression analysis (see below). rst-order inactivation.
In the area of food science and technology, it is The model for inactivation of enzymes according to
common to characterize rst-order reactions using rst-order kinetics may be applied in many cases.
the Thermal Death Time concept. The decimal reduc- However, often more complex inactivation kinetics
tion time (D-value) is the time, at a given temperature are found, because inactivation may occur by consecu-
and pressure, needed for a 90% reduction of the initial tive or parallel processes, or owing to the presence of
activity. For a rst-order inactivation, D-values and isozymes, enzyme inhibitors, or other food compo-
rate constants are directly related [Eq. (9)]. nents. Several mathematical expressions have been
suggested for modeling a non-rst-order inactivation
ln10 behavior. A biphasic inactivation behavior where a
D 9
k fast inactivation period is followed by a decelerated
decay, eventually leading to an activity plateau, has
Substitution of Eq. (9) into Eq. (8) yields Eq. (10).
been attributed to the occurrence of isozymes with dif-

A t ferent stabilities (distinct isozyme model), to the pre-
log 10 sence of a resistant enzyme fraction (fractional
A0 D
conversion model), or to intermediate (rst-order)
steps in the overall inactivation process (consecutive
step model).
2. Distinct Isozyme Model

Enzymes characterized by several isozymes can often
be subdivided into two (or more) fractions with differ-
ent processing stability, e.g., one more thermal (pres-
sure) resistant than the other and both inactivating
according to a rst-order decay kinetic model. For
constant extrinsic (e.g., pressure, temperature) and
intrinsic factors and assuming that the inactivation of
both fractions is independent of each other, the inacti-
Figure 2 Thermal inactivation curves of tomato pectin vation can be modeled according to Eq. (11).
methylesterase (PME) dissolved in 20 mM Tris-HCl (pH 7)

at 608C ( ), 62:58C ( ), 658C ( ), and 67:58C ( ). Ao and A Residuals are the differences between experimentally
are initial tomato PME activity and activity after thermal observed dependent variable values and those predicted by
treatment, respectively. (From Ref. 13.) the regression equation.

A Al expkl t As expks t 11 applied temperature and pressure. As an example of
where As is the activity of the more stable enzyme fractional conversion, thermal inactivation of the
fraction, Al the activity of the labile enzyme fraction, heat-labile fraction of tomato PG dissolved in 40
k the rst-order inactivation rate constant where the mM Na acetate buffer pH 4.4 is depicted in Figure 4.
subscripts s and l for k denote thermostable and ther- By plotting residual activity after different time
molabile, respectively. As an example of inactivation intervals versus time, the inactivation rate constant
according to a distinct isozyme model, thermal inacti- (k-value) and the remaining activity after prolonged
vation of tomato polygalacturonase (PG) dissolved in treatment A1 -value) can be estimated using nonlinear
40 mM Na acetate buffer pH 4.4 is depicted in Figure 3. regression analysis.
By plotting the residual activity after different time
intervals versus time, the inactivation rate constant of 4. Consecutive Step Model
the labile fraction kl -value), the inactivation rate con-
The consecutive step model is based on a succession of
stant of the stable fraction ks -value), and the activity
(two) irreversible (rst-order) reaction stepsan irre-
of both fractions can be estimated using nonlinear
versible conversion of the native enzyme to an inter-
regression analysis.
mediate with lower specic activity, and the
subsequent irreversible conversion of the intermediate
3. Fractional Conversion Model
to an inactive enzyme form. A consecutive two-step
Fractional conversion refers to a (rst-order) inactiva- model can be mathematically expressed as Eq. (13).
tion process that takes into account a nonzero activity
upon prolonged heating and/or pressurizing ( A1 . A k1
A A1 A2 expk1 t
fractional conversion model can be expressed mathe- k1 k2
13
matically as Eq. (12). k1
A2 expk2 t
A A1 A0 A1 expkt 12 k1 k2
This relation is valid in the temperature and/or pres- By plotting the residual activity after different time
sure domain where only the labile enzyme fraction intervals versus time, the inactivation rate constants k1
inactivates whereas the activity of the stable fraction and k2 and the activity of both fractions can be esti-
A1 -value) does not change with respect to time. This mated using nonlinear, regression analysis. k1 and A1
nonzero activity may or may not be a function of refer to step 1, k2 and A2 refer to step 2.
Figure 3 Thermal inactivation curves of tomato polygalac- Figure 4 Thermal inactivation curves of tomato polygalac-
turonase (PG) dissolved in 40 mM Na acetate buffer (pH 4.4) turonase (PG) dissolved in 40 mM Na acetate buffer (pH 4.4)
at 708C ( ), 758C ( ), 808C ( ), 858C (), and 908C ( ), at 588C ( ), 608C ( ), 628C ( ), and 658C ( ), modeled
modeled using a distinct isozyme model. Ao and A are initial using a fractional conversion model. Ao and A are initial
tomato PG activity and activity after thermal treatment, tomato PG activity and activity after thermal treatment,
respectively. (From Ref. 15.) respectively. (From Ref. 15.)

B. Determination of a Temperature and Pressure reduction time versus temperature. Alternatively, the
Coefcient Model z-value can be estimated directly using nonlinear
regression analysis based on Eq. (15).
1. Temperature Dependence at Constant
Food technologists sometimes use the Q10 -factor to
Pressure
describe the temperature dependence of a reaction rate.
The temperature dependence of the rate constant k is The temperature dependence parameter Q10 is dened
often expressed by an activation energy, Ea , as indi- as the factor by which the reaction rate is increased if
cated in the Arrhenius relationship [Eq. (14)] (16). the temperature is raised by 108C.

Ea 1 1
kobs kobs;refT exp 14 2. Pressure Dependence at Constant
Rg Tref T Temperature
where kobs;refT is the inactivation rate constant at Tref , The pressure dependence of the rate constant is often
Tref the reference temperature, Ea the activation expressed by an activation volume Va , as indicated in
energy, and Rg the universal gas constant Rg 8:314 the Eyring equation [Eq. (16)] (18).
J/Kmol). Equation (14) can be linearized which allows
the activation energy at a certain pressure to be esti- Va
kobs kobs;refP exp P Pref 16
mated based on linear regression analysis of the natural Rg T
logarithm of k versus the reciprocal of the absolute
temperature (Fig. 5). Alternatively, the activation where krefP is the inactivation rate constant at Pref , Pref
energy can be estimated based on the nonlinearized the reference pressure, Va the activation volume at a
Arrhenius relationship [Eq. (14)] using nonlinear certain temperature, T the absolute temperature, and
regression analysis (see below). Rg the universal gas constant Rg 8:314 cm3 MPa/
In the Thermal Death Time model, the temperature Kmol). Also the Eyring equation can be linearized by a
dependence of the D-value is given by the zT -value [Eq. logarithmic transformation. Hence, plotting the nat-
(15)]. The zT -value equals the temperature increase ural logarithm of the rate constant in function of pres-
necessary to obtain a 10-fold decrease of the D-value. sure, the activation volume at a certain temperature
Tref T can be derived from the slope of the regression line
Dobs Dobs;refT 10 zT
15 (Fig. 6). Alternatively, the activation volume can be
estimated based on the nonlinearized Eyring relation-
where Dobs;refT is the inactivation rate constant at Tref ,
ship [Eq. (16)] using nonlinear regression analysis (see
Tref the reference temperature, and zT the z-value.
below).
After linearization of Eq. (15), the z-value at a certain
Analogous to the log-linear relationshp between D-
pressure can be estimated based on linear regression
value and temperature, there is often a log-linear rela-
analysis of the 10-based logarithm of the decimal
Figure 5 Arrhenius plot for thermal inactivation of orange Figure 6 Eyring plot for pressure inactivation at 458C of
pectinmethylesterase, dissolved in distilled water. (From Ref. avocado polyphenoloxidase, dissolved in phosphate buffer
17.) (pH 7, 0.1 M). (From Ref. 19.)

tionship between D-value and pressure [Eq. (17)], G Go Vo P Po So T To
whereby zp is dened as the pressure increase necessary 1
P Po 2 P Po T To
to obtain a 10-fold decrease of the D-value. 2 21

Pref P T
Dobs Dobs;ref 10 zp
17 Cp Tln 1 To
To
where Dobs;ref is the decimal reduction time at Pref , Pref where Cp is the heat capacity change TS=TP ,
the reference pressure, and zP the z-value at a certain the thermal expansibility factor V=TP
temperature. Also the zP -value can be estimated using S=PT , and the compressibility factor
two different regression approaches: plotting the 10- V=PT . This thermodynamic model can be con-
based logarithm of D in function of pressure, the zP - verted into a kinetic model through the transition
value at a certain temperature can be derived from the state theory of Eyring, suggesting that enzyme inacti-
slope of the regression line, or the zP -value can be vation is accompanied by the formation of a meta-
estimated using nonlinear regression analysis (see stable activated state (6 ) which exists in equilibrium
below). with the native enzyme. This conversion is based on
Analogous to the validity evaluation of a rst-order the substitution of Eq. (22) and Eq. (23) in Eq. (21),
kinetic model, the appropriateness of the coefcient obtaining Eq. (24) to describe the combined pressure
models to describe the temperature or pressure depen- temperature dependence of the inactivation rate con-
dency of the inactivation rate constant can be evalu- stant.
ated by determination of the goodness of t of ln k vs. G6 RT lnK 6 22
1=T (Arrhenius model), log D versus T (TDT model),
kh
ln k vs. P (Eyring model), or log D vs. P (PDT model). K 6 23
rkB T
3. Combined PressureTemperature
V06
Dependence lnkobs lnk0 P Po
RT
Based on experimentally determined inactivation rate S06 1 6
constants for an elaborated set of pressuretempera- T To P Po 2
RT 2 RT
ture combinations, an iso-rate contour plot, connect- 24
6
ing pressuretemperature combinations resulting in the P Po T To
same inactivation rate constant can be constructed. RT

Iso-rate contour diagrams for pressuretemperature Cp6 T
Tln 1 To
inactivation of enzymes as well as of microorganisms RT To
are often elliptically shaped. These elliptical pressure The model parameters can be estimated using a non-
temperature kinetic diagrams can be modeled on a linear regression analysis, involving an iterative numer-
thermodynamic basis. ical procedure based on the minimal sum of squares.
The basic thermodynamic equation governing the Figure 7 depicts an iso-rate contour diagram for pres-
behavior of a system during a pressure and a tempera- suretemperature inactivation of soybean lipoxygen-
ture change can be represented as Eq. (18). ase, modeled using Eq. (24).
dG SdT VdP 18
C. Selection of a Regression Model and Method
Since the entropy change S and the volume change for Kinetic Parameter Estimation
V vary with pressure and temperature [Eqs. (19)
and (20), respectively], Eq. (18) can be reformulated Several regression models (linear vs. nonlinear mod-
as Eq. (21) (20, 21). els ) and methods (two-step regression vs. one-step

@S @S
dS dT dP 19 A model is linear if the rst (partial) derivatives of the model
@T P @P T with respect to the parameters are independent of the para-
meters and consequently higher-order derivatives are zero
whereas in a nonlinear model at least one of the derivatives
@V @V
dV dT dP 20 of the model function with respect to the parameters depends
@T P @P T on at least one of the parameters.

parameter values provided by the user, a numerical
algorithm (e.g., Gauss-Newton, Marquardt, DUD) is
used to provide successively better approximations
until the sum of squared residuals do not change
within some specied limit (convergence criterion is
met) so that nally a set of parameter values satisfying
the least-squares criterion is presented. To be more
certain that the minimum found is actually the global
minimum desired and not a local one it is advisable
to start the nonlinear least-squares procedure at several
different starting estimates of the parameters. If the
nal results are independent of the initial values,
there is more condence that the nal parameter values
describe a global minimum.
With the present availability of computing power,
Figure 7 Iso-rate contour diagram k 0:23 min1 ) for ease of parameter estimation is no longer a valid rea-
pressuretemperature inactivation of soybean lipoxygenase
son for linearizing models. It may nevertheless be use-
in Tris HCl buffer (pH 9, 0.01 M). (From Ref. 22.)
ful to perform a nonlinear transformation in order (a)
to produce a linear plot from which the validity of the
model can easily be interpreted graphically, or (b) to be
regression) can be applied to obtain kinetic parameter
able to use a simple linear regression analysis to obtain
estimates. In this paragraph, the selection between a
reasonable starting values for the subsequent nonlinear
linear or a nonlinear kinetic model and between a
regression routine of the untransformed data.
two-step or a one-step regression method will be dis-
cussed.
2. Selection Between Two-Step Versus One-Step
1. Selection Between Linear Versus Nonlinear Regression
Models
It is common practice to estimate kinetic inactivation
Because linear least-squares tting is easier to solve parameters by an individual, or two-step, regression
than nonlinear least-squares tting, an approach fre- method: one estimates at rst inactivation rate con-
quently used is to perform (a) nonlinear transforma- stants (and possibly other kinetic parameters such as
tion(s) of the dependent variable so that a linear model enzyme fraction, reaction order) from inactivation
is obtained from which the least squares estimators of data at constant temperature or pressure by linear or
the linear model parameters can be calculated straight. nonlinear regression analysis. In a second step, one
Typical examples in the context of enzyme inactivation estimates temperature or pressure coefcients from
include linearization of the rst-order rate equation by regression analysis of the obtained inactivation rate
a logarithmic transformation of the inactivation data, constants as a function of temperature and pressure.
or linearization of the temperature and pressure coef- In a global, or one-step, regression approach, one is
cient models by a logarithmic transformation of the considering the inactivation data obtained at different
rate constants. However, one of the assumptions inher- inactivation temperatures or pressures simultaneously.
ent to least-squares tting (for both linear and non- To model a global inactivation data set, the tempera-
linear) is that the experimental errors (in the ture or pressure coefcient model is being incorporated
dependent variables) are normally distributed (23). in the inactivation rate equation. Using nonlinear
Hence, one should not perform any nonlinear trans- regression analysis, one gets estimates of the inactiva-
formation of the dependent variables (such as, e.g., a tion rate constant at reference conditions and a tem-
logarithmic transformation) that will alter the error perature or pressure coefcient.
distribution structure if the original data contain With regard to the choice between an individual or a
Gaussian uncertainties. When the model is nonlinear global approach, in the literature, the effectiveness of
in the parameters no explicit analytical solutions are several least-squares regression methods for the esti-
available for the parameters or the condence inter- mation of kinetic parameters has been assessed mainly
vals, and a solution must be found iteratively by linear based on comparison of the condence region for the
approximation. Starting from an initial estimate of the parameters estimates and on the quality of tting (24,

25). These authors concluded that the two-step regres-
sion approach gives the least accurate estimates prob-
ably because it estimates too many intermediate values,
and does not gain strength in the regression by con-
sidering the data set as a whole. Two-step regression
has the disadvantage of applying regression on regres-
sion parameter estimates. Errors on the rst regression
estimates are not transformed to the second. On the
contrary, they found a global regression procedure a
very good method which yielded unbiased and precise
estimates of the parameters without estimating unne-
cessary ones. Because of several disadvantages inherent
to the two-step approach, one is advised to analyze
kinetic data sets by a global t considering the data
set as a whole. Probably the use of a global t will not Figure 8 90% joint condence contour of the kinetic para-
provide the best individual t at each temperature or meter estimates for thermal inactivation of alkaline phospha-
pressure, but the whole set of results will be better tase in raw bovine milk Tref 608C) (correlation between z
described. and Dref is 0.786). (From Ref. 31).
tic publications. 100(1% joint condence regions

3. Condence Intervals of Parameter Estimates
can be constructed using Eq. (25) (30).
Next to the determination of parameter values with the
p
highest probability of being correct, it is essential to SSQ SSQ 1 Fp; m p; 1
mp
obtain a realistic measure of the statistical condence
of the estimated parameters. In the case of linear mod- 25
els, condence intervals for the parameter estimates are where SSQ represents the error sum of squares asso-
exactly dened and symmetrical and their determina- ciated with the least-squares estimate , p the number
tion is straightforward while in case of nonlinear mod- of parameters estimated simultaneously, m the number
els condence intervals are not symmetric and only of observations, and F the upper 1 quantile for an F
approximate condence intervals are provided (26). distribution with p and mp degrees of freedom. An
The most commonly used method for the evaluation example of a joint condence region for a set of para-
of the condence intervals of parameters estimated in a meter estimates that are highly correlated is given in
nonlinear regression procedure is the use of asymptotic Figure 8. It can be seen from Figure 8 that if the para-
standard errors, computed based on linearizing meters are rather highly correlated no attempt should
assumptions. However, they nearly always provide be made to interpret the individual condence intervals
incorrect estimates of the actual condence limits of simultaneously by constructing a rectangular joint con-
the determined parameters because they neglect the dence interval because a parameter pair may be well
covariances of the simultaneously determined para- within the individual condence interval but is very
meters and assume normal distribution of parameters. unlikely to occur since it is very far outside the joint
According to several authors (23, 2729), asymptotic condence region. Only if the correlation between the
standard errors should not be used in case of nonlinear simultaneously estimated parameters is close to zero
least-squares analysis since there are many other pub- will the rectangular region dened by the individual
lished methods that provide realistic estimates of the condence intervals approximate to the correct joint
condence intervals of parameters determined by non- condence region.
linear least-squares methodse.g., the construction of
joint condence regions for the parameters. Joint con-
dence regions take into account the correlation REFERENCES
between the simultaneously estimated parameters.
Although the use of joint condence regions for simul- 1. L Ludikhuyze, A Van Loey, S Denys, Indrawati, M
taneously estimated parameters is well described in Hendrickx. Effects of combined pressure and tem-
statistical handbooks, it is rarely encountered in scien- perature on enzymes related to quality of fruits and

vegetables: from kinetic information to process engi- 16. S Arrhenius. Uber die reaktionsgeschwindigkeit bei
neering aspects. CRC Crit Rev Food Sci Nutr 2001 der inversion von rohrzucker durch sauren. Z Phys
(submitted). Chem 4:226248, 1889.
2. R Lumry, H Eyring. Conformational changes of pro- 17. I Van den Broeck, L Ludikhuyze, A Van Loey, C
teins. J Phys Chem 58:110120, 1954. Weemaes, M Hendrickx. Thermal and combined pres-
3. A Zipp, W Kauzmann. Pressure denaturation of met- suretemperature inactivation of orange pectinester-
myoglobin. Biochemistry 12:42174228, 1973. ase: inuence of pH and additives. J Agric Food
4. JW Rhim, RV Nunes, VA Jones, KR Swartzel. Chem 47(7):29502958, 1999.
Determination of kinetic parameters using linearly 18. H Eyring, FH Johnson, RL Gensler. Pressure and
increasing temperature. J Food Sci 54(2):446450, reactivity of proteins, with particular interest to inver-
1989. tase. J Phys Chem 50:453464, 1946.
5. RV Nunes, JW Rhim, KR Swartzel. Kinetic para- 19. C Weemaes, L Ludikhuyze, I Van den Broeck, M
meter evaluation with linearly increasing temperature Hendrickx. Kinetics of combined pressuretempera-
proles: integral methods. J Food Sci 56(5):1433 ture inactivation of avocado polyphenoloxidase.
1437, 1991. Biotechnol Bioeng 60(3):292300, 1998.
6. AG Perkin, H Burton, HM Underwood, FL Davies. 20. SA Hawley. Reversible pressuretemperature dena-
Thermal death kinetics of Bacillus stearothermophilus turation of chymotrypsinogen. Biochemistry
spores at ultra high temperatures. II. Effect of heating 10(13):24362442, 1971.
period on experimental results. J Food Technol 21. E Morild. The theory of pressure effects on enzymes.
12:131148, 1977. Adv Prot Chem 34:93166, 1981.
7. A Arabshahi, DB Lund. Considerations in calculating 22. Indrawati, A Van Loey, L Ludikhuyze, M Hendrickx.
kinetic parameters from experimental data. J Food Soybean lipoxygenase inactivation by pressure at sub-
Process Eng 7:239251, 1985. zero and elevated temperatures. J Agric Food Chem
8. CG Hill, RA Grieger-Block. Kinetic data: generation, 47(6):24682474, 1999.
interpretation, and use. Food Technol 34(2):5665, 23. ML Johnson. Review: why, when and how bioche-
1980. mists should use least squares. Anal Biochem
9. KJ Laidler. Chemical Kinetics. New York: Harper 206(2):215225, 1992.
and Row, 1987. 24. E Cohen, I Saguy. Statistical evaluation of Arrhenius
10. MAJS van Boekel, P Walstra. Use of kinetics in study- model and its applicability in prediction of food qual-
ing heat-induced changes in foods. In: PF Fox, ed. ity losses. J Food Proc Preserv 9:273290, 1985.
Monograph on Heat-Induced Changes in Milk. 25. SG Haralampu, I Saguy, M Karel. Estimation of
Bulletin of the International Dairy Federation, Arrhenius model parameters using three least squares
Brussels, 2nd ed. IDF special issue 9501:2250, 1995. methods. J Food Proc Preserv 9:129143, 1985.
11. MK Lenz, DB Lund. Experimental procedures for 26. MAJS van Boekel. Statistical aspects of kinetic mod-
determining destruction kinetics of food components. eling for food science problems. J Food Sci 61(3):477
Food Technol 34(2): 5155, 1980. 485, 489, 1996.
12. RW Lencki, J Arul, RJ Neufeld. Effect of subunit 27. ML Johnson, SG Frasier. Nonlinear least-squares
dissociation, denaturation, aggregation, coagulation analysis. Methods Enzymol 117:301342, 1985.
and decomposition on enzyme inactivation kinetics: 28. HJ Motulsky, LA Ransnas. Fitting curves to data
I. rst-order behavior. Biotechnol Bioeng 40:1421 using nonlinear regression: a practical and nonmathe-
1426, 1992. matical review. FASEB J 1:365374, 1987.
13. I Van den Broeck, L Ludikhuyze, A Van Loey, M 29. ML Johnson, LM Faunt. Parameter estimation by
Hendrickx. Effect of temperature and/or pressure on least-squares methods. Methods Enzymol 210:137,
tomato pectinesterase activity. J Agric Food Chem 1992.
48(2):551558, 2000. 30. NR Draper, H Smith. Applied Regression Analysis.
14. M Straume, ML Johnson. Analysis of residuals: cri- New York: John Wiley & Sons, 1981.
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Enzymol 210:87105, 1992. Inactivation kinetics of alkaline phosphatase and lac-
15. D Fachin, A Van Loey, Indrawati, L Ludikhuyze, M toperoxidase, and denaturation kinetics of -lactoglo-
Hendrickx. Thermal and high pressure inactivation of bulin in raw milk under isothermal and dynamic
tomato polygalacturonase: a kinetic study. J Food Sci temperature conditions. J Dairy Res 68:95107, 2001.
2001 (in press).

6
Regulatory Issues of Enzymes Used in Foods from the

Perspective of the E.U. Market
Danielle P. Praaning
DSM Food Specialties, Delft, The Netherlands
I. INTRODUCTION II. OVERVIEW OF APPLICABLE LAWS
Enzymes were used for food production 2000 years In the European Union, there are so-called horizontal
before the birth of Christ. Good examples are bread, and vertical laws. Horizontal laws are those that cover
cheese, and beer production. Of course, it was all foods. Examples are the laws regarding additives
unknown at the time that it was the enzymes that and those regarding the labeling of foodstuffs.
were doing the job. Nowadays, the production of Vertical laws cover foods with a so-called standard of
food enzymes has become industrialized. European identity, also called compositional regulations or
enzyme manufacturers are market leaders and cater recipe laws. Examples of products for which such
70% of the worlds need for food enzymes, and this standards exist are cheese and bread.
market is still increasing with an annual growth of In both cases, vertical as well as horizontal, there
about 2%. are laws that have been harmonized throughout the
These developments have triggered the creation E.U. and laws that are still subject to national legisla-
of national laws and regulations to ensure both tion. This situation will probably remain so, especially
the safety of the consumer and fair trade. in view of the fact that in the treaty signed in
Although many laws in the European Union Maastricht in February 1992 (1), the European
have been harmonized over the last few decades, Union ofcially laid down the principle of subsidiar-
this has not (yet) happened in the case of ity, which states that the European Commission
enzymes. should not regulate what could be done just as well
The European Association of Manufacturers of or better at the national level.
Fermentation Enzyme Products (AMFEP), which For products regulated solely at the national level,
was founded in 1977, has set as one of its major the principle of mutual recognition applies. If a pro-
aims the achieving of the widest possible harmoniza- duct is legally produced in one E.U. country, it can
tion of regulatory requirements for enzymes in the also be sold in another E.U. country, unless this coun-
European Union (E.U.). To achieve this, AMFEP try has well-founded arguments to reject the product
has initiated discussions with the European on the grounds of protection of consumer health, or
Commission, national authorities, and the food indus- one of the other reasons listed in Article 30 of the
try. This chapter presents an overview of the tangle of Treaty establishing the European Community (2).
present regulations that are applicable for enzymes Other than in this situation, member states are not
when used in food. allowed to create unjustied trade barriers.

If one wants to place a new enzyme on the market, The precise denition of processing aid according to
any of the four cases (horizontal versus vertical, har- the Additives Directive is:
monized versus national) can be applicable, meaning
Processing aid means any substance not con-
that all possibilities have to be assessed. The following
sumed as a food ingredient by itself, intentionally
factors are of importance when establishing how a spe-
used in the processing of raw materials, food or
cic enzyme is regulated.
their ingredients, to fulll a certain technological
1. The purpose of its use. Is the enzyme used only
purpose during treatment or processing and
during processing of the food, or does it still have a
which may result in the unintentional but techni-
specic function in the nal food that is sold to the
cally unavoidable presence of residues of the sub-
consumer?
stance or its derivatives in the nal product,
2. The type of food in which the enzyme is to be
provided that these residues do not present any
used. Does the food in question have a standard of
health risk and do not have any technological
identity?
effect on the nished product.
3. The country in which the enzyme is to be used
in food. Applicable nonharmonized regulations will In the case of enzymes, the distinction between the
differ country by country. use as processing aid or additive (or even ingredient) is
4. The origin or source of the enzyme. Industrial not always easy. The existing denitions in E.U. law
enzymes can be obtained from plant, animal, or micro- are open to interpretation on a number of points. This
bial sources. These may be regulated differently. makes it difcult to decide in certain applications
Moreover, some countries have special requirements whether an enzyme has the status of a processing aid
for enzymes produced by genetically modied organ- or an additive. The lack of consistency in the legisla-
isms (gmos). tion has already led to regulatory uncertainty and dif-
culties surrounding enzymes, which until now had
been regarded as processing aids, suddenly being con-
sidered to be additives.
III. E.U. HARMONIZED LAWS
Recent attempts to bring an increasing number of
A. Horizontal Harmonized Laws enzymes within the scope of the denition of food
additives have principally been based on the argument
The relevant harmonized E.U. Directive is the that it is only possible to speak of a residue when
Council Directive 89/107/EEC Concerning Food there has been a conscious effort made to remove the
Additives Authorized for Use in Foodstuffs Intended enzyme. However, the interpretation of the food indus-
for Human Consumption, also called the Additives try, and most authorities shared this interpretation
Framework Directive (3). until now, has always been that if an enzyme does
According to this directive, additives are everything not have a technological function in the nal food, it
added to food that is not normally used as a characteris a processing aid, even if it remains in the nal food in
istic ingredient (a characteristic ingredient would, for the small quantity (ppm) in which it was added.
instance, be milk in cheese or our in bread). In the If an enzyme does have a technological function in
directive, food additives are divided into those that the nal food, however, it will fall under the Additives
have a technological effect in the nished product Directive. There are at present only two enzymes fall-
and those that do not have a technological effect in ing under the Additives Directive:
the nished product. Additives of the rst category
1. Lysozyme, when used as preservative in ripened
may only be used if they have been authorized and
cheese.
included in a positive list together with their so-called
2. Invertase, when used for soft centered
E-number and, if applicable, permitted applications.
confectionery.
Moreover, their presence in the nal food has to be
declared in the ingredient list of the foodstuff. Confusingly, these very same enzymes may also be
Additives of the second category are exempt from used as processing aids for other food applications!
these obligations; thus they do not have to be author- Thus, in the case of enzymes, one and the same
ized, or labeled on the nal foodstuff. It is rather con- enzyme can fall under both categories, depending on
fusing in practice that only products in the rst its application. If the enzyme has a technological fun-
category are called additives, whereas products in the tion in the nal food, it will fall under the Additives
second category are called processing aids. Directive. If it does not have a function in the nal

food, it is a processing aid and exempt from the 2. Horizontal enzymes regulations. There are three
Additives Directive. E.U. countries having specic horizontal enzyme reg-
Only when use of an enzyme falls under the Additives ulations: Denmark, France and the United Kingdom
Directive does specic approval by the European (see below).
Commission need to be obtained if a company wants 3. Before Spain and Portugal had to implement
to market a new enzyme. This means that the function- the harmonized E.U. Additives Directive, these coun-
ality of the enzyme product has to be proved and its tries regarded all enzymes, whether used as additive or
safety has to be evaluated by a specic group of experts processing aid, as additives. As a consequence, they
called the Scientic Committee on Food (SCF). were mentioned on the positive additive list and had
In 1991 the SCF published guidelines for the pre- to be authorized as such. It may be confusing for these
sentation of data on food enzymes (4). To obtain countries that only very few enzymes are mentioned on
approval, toxicological data need to be submitted. the positive list of the E.U. Additives Directive, while
According to the guidelines, two mutagenicity studies all other enzymes can still be used, albeit only as pro-
and a 90-day oral toxicity study in rat are required as a cessing aids. These countries may see this as a legal
general standard. However, safety data are not vacuum.
required for enzymes originating from edible plants 4. In addition to the above-mentioned regulations,
or animals. Presumably, the SCF did not think about most Member States have vertical laws for cheese,
enzymes obtained from genetically modied plants at our, bread, etc., regulating the use of enzymes.
the time these guidelines were made. These vertical legislations differ from country to coun-
In the case of microbial enzymes, no safety data are try, which may not only create trade barriers but also
needed if the microorganism in question has a long contribute to a very complicated picture for enzyme
history of safe use in food and belongs to a species producers and users alike.
for which documented evidence exists that no toxins Unfortunately, this is also an area where mutual
are produced, and the actual strain used is of well- recognition between Member States does not function
documented origin. To date, the only such microor- properly. If an enzyme is approved for a certain use in
ganism accepted by the SCF is Saccharomyces cerevi- one Member State, this does not automatically mean
siae (bakers yeast). that it is approved for the same use in another Member
State.
B. Vertical Harmonized Laws
A. Horizontal Enzyme Regulations
There are only two vertical harmonized directives,
In the 1970s, the Joint Expert Committee on Food
those on fruit juices and on wines, in which the use
Additives (JECFA) of the FAO/WHO developed tox-
of certain enzymes is expressly provided for. The
icological requirements for enzyme products (7)
Fruit Juice Directive (5) states that only pectolytic,
based on the following classication system: enzymes
proteolytic, and amylolytic enzymes may be used in
from edible tissues of animals; enzymes from edible
fruit juices. The Wine Regulation (6) permits the use
parts of plants; and enzymes from microbial origin,
of pectolytic enzymes and of -glucanase from the
including microorganisms known to be present or
microorganism Trichoderma harzianum only. No
used for food, microorganisms known to be harmless
other enzymes can be used unless specic authoriza-
contaminants in food, and microorganisms unknown
tion, resulting in an amendment of the law, is given.
in food.
The main conclusion from this classication was
that no numerical Acceptable Daily Intake or toxicity
IV. NATIONAL LAWS studies are necessary for enzyme preparations derived
from edible tissues of animals, from edible portions of
National laws covering the use of enzymes are: plants, or from microorganisms that are traditionally
1. The general National Food Safety Laws. Even accepted as constituents of foods or are normally used
when an enzyme is not regulated in a specic Member in the preparation of foods.
State, its use would be subjected to the requirements of Moreover, JECFA also established specications to
General Food Safety Legislation, which stipulates that which enzyme products should comply (8). These speci-
nothing must be added to food which renders it injur- cations cover limits for certain heavy metals, microbial
ious to health. contamination, antibiotic activity, and mycotoxin con-

tent. In their guidelines, the SCF has largely followed cation procedure. Notication is valid only for a spe-
the JECFA safety classication and specications. cic brand or trade name.
Shortly after JECFA development of specications The user or importer of an enzyme preparation is
and toxicological requirements, the United Kingdom, responsible for such notication. It was recently
France, and Denmark started developing guidelines decided that the information to be contained in the
for food enzymes. These guidelines were all largely notication should follow the guidelines laid down
based on the JECFA requirements and specications. by the SCF. Although the term notication is
Only the French and Danish guidelines have been used, there is in practice no real difference between
turned into genuine enzyme legislation. In the United this procedure and an authorization. The authorities
Kingdom, it became a Code of Practice to ask for evaluate the dossier, and the product cannot be mar-
authorization for a new enzyme preparation on the keted until all questions about the dossier have been
basis of the U.K. regulations. satisfactorily answered.
In principle, the French, Danish, and U.K. enzyme
regulations did not mean to differentiate between the
3. National Enzyme Regulation in the United
use of enzymes as additive or processing aid. However,
Kingdom
since the use of an enzyme as additive now falls under
the harmonized E.U. Additives Directive, these In the United Kingdom, the Ministry of Agriculture,
national laws now apply to enzymes used as processing Fisheries and Food published the Food Advisory
aids only. Committees (FAC) Report on the Review of
In all other E.U. Member States, there is no specic Enzyme Preparations in 1982 (11). This document
enzyme legislation. As a result, enzymes when used as was meant to form the basis for a national horizontal
processing aids are merely subjected to the general enzyme legislation.
National Food Safety Laws if the country in question The FAC recommended that food enzymes be
has no horizontal law regulating enzymes and if the approved only when a technical case-of-need had
application of the enzyme is not regulated in a vertical been established and they were considered safe for
way. In such cases, no authorization is needed if a use in food. General specication requirements were
company wants to put a new enzyme on the market. also outlined. Moreover, the FAC established a posi-
tive list based on source and main enzyme activity. In
1. National Enzyme Law in France this list, enzymes are divided into two groups, A and B.
Group A are enzymes of vegetable or animal origin,
The French Enzyme Decree (9) permits the use of all
for which no further specications are necessary.
enzymes mentioned in an Annex to the Decree. This
Group B is a selection of microbial enzymes, which
positive listing of enzymes is by function, enzyme
are temporarily approved for use in food. It was
name, source, foods in which the enzymes are per-
recommended that these enzymes be reviewed within
mitted, and special conditions for use. The list is
2 years of publication of the report.
updated regularly.
As a basis for such a review, the FAC recommended
The French Decree also lays down specications of
the development of a nonspecic screening test to pre-
purity, which are very similar to the specications laid
vent the marketing of microbial enzyme preparations
down by JECFA. Furthermore, permitted preserva-
containing mycotoxins. However, the development of
tives and diluents for enzyme preparations are listed.
the test turned out to be impossible for technical rea-
To obtain approval for a new enzyme, a dossier has
sons.
to be led with the authorities. This dossier should
As an alternative, the U.K. Committee on Toxicity
contain proof of the functionality of the enzyme
(COT) published Guidelines for the Safety Assessment
(case-of-need) and the results of safety studies as
of Microbial Enzyme Preparations in 1993 (12). These
described in the SCF guidelines. In case an already
guidelines are comparable, although somewhat stricter,
approved enzyme is to be marketed for a new food
to those of the SCF. No U.K. guidelines exist for
application, only the case-of-need is required.
enzymes obtained from animals or plants.
Although these COT guidelines have not been con-
2. National Enzyme Law in Denmark
verted into a genuine law, they have the same effect
In contrast to France, the Danish law (10) does not from a practical point of view. Authorization on basis
contain a positive listing system. All enzyme products of the guidelines is required to achieve marketing
and their applications are simply subjected to a noti- approval for a new enzyme preparation.

B. Regulations for Enzymes Produced by be covered by the E.U. Novel Food Regulation. Owing
Genetically Modied Organisms (gmos) to strong opposition of the French authorities, the
Dutch proposal has not been accepted. Instead, the
The SCF guidelines and the national regulations in the European Commission was asked by the Member
United Kingdom, Denmark, and France also cover States to prepare a paper regarding the present legal
specic provisions for enzymes made by gmos. In sim- situation for food enzymes. The European
plied terms this means that the genetic modication Commission transferred this task in October 1998 to
will be evaluated by the authorities before the enzyme a so-called Scientic Cooperation (SCOOP) task force,
can be approved and that the enzyme preparation in which experts from various Member States partici-
should be free of the gmo and recombinant DNA. pated. The four objectives of the SCOOP task force
JECFA has also written down general considera- were to: make a list of food enzymes in use in E.U.;
tions and specications for enzymes from genetically study the way they have been evaluated for their safety;
modied microorganisms (13). This has not been done make overview of the current legal situation; and nd
for enzymes obtained from genetically modied plants criteria for distinguishing between the use of enzymes
or animals. As has been the case for classical enzymes, as additives, processing aids, or ingredients.
these considerations might be used for future E.U. reg- The SCOOP task force nished its work at the end of
ulations. 2000. The report however, has not yet been published at
In the Netherlands, a Novel Food Law was passed the time of this writing. One of the main conclusions the
in July 1993, covering, among others, all additives and SCOOP task force drew was that the present denitions
processing aids made by genetic modication. This law on additives and processing aids do not provide suf-
was issued because of the delays in the adoption of the cient criteria to distinguish between the use of enzymes
harmonized European Novel Food Regulation, but it as an additive or as a processing aid. The problem on
has now been replaced by the latter. how to distinguish between additives and processing
The E.U. Novel Food Regulation was nally aids is also being discussed at international level,
adopted in January 1997 and entered into force in namely within the Codex Committee for Food
May of that year (14). This regulation covers novel Additives and Contaminants. The results of this discus-
foods and food ingredients, but, in contrast to the sion, which may take several years, can potentially have
Dutch Novel Food Law, additives (including proces- a great impact on the way enzymes are regulated in
sing aids) are outside its scope. future in the European Union and elsewhere.
During the prolonged discussions on this regula- In July 2001, the European Commission adopted
tion, the European Parliament obtained a commitment two new proposals for regulating products derived
from the European Commission that an investigation from gmos: (a) a proposal for regulation of genetically
would be carried out as to the need to ll gaps in the modied Food and Feed (15); (b) a proposal for reg-
area regulating the protection of public health, parti- ulation concerning traceability and labeling of gmos
cularly with respect to processing aids (e.g., enzymes), and traceability of food and feed products produced
and that furthermore, where necessary, these gaps from gmos (16). Before becoming law, the proposals
would be lled by proposals for additional legislation. will rst have to be accepted by the European
As a result, the Commission added the following Council and Parliament, a process that may take sev-
statement to the Novel Food Regulation: eral years. The proposals will replace the above-
mentioned E.U. Novel Food Regulation as far as
The Commission conrms that should it appear
products derived from genetic modication are con-
in the light of experience, that there are gaps in the
cerned. Again, enzymes when used as processing aids
system of protection of the public health provided
in food are explicitly exempted from the proposals.
for by the existing legal framework, in particular
Whether this will again lead to discussions within the
in respect of processing aids, it will formulate
European Parliament remains to be seen.
appropriate proposals in order to ll those gaps.
In an attempt to include gmos enzymes when used
V. PROBLEMS
as processing aids into the scope of the E.U. Novel
Food Regulation, the Dutch authorities in July 1997 A. Consequences of Present Regulatory Situation
wrote a proposal to regard enzymes that are not
removed from the nal food as ingredients instead of Since mutual recognition among Member States does
processing aids. In this way, such gmos enzymes would not function properly, the enzyme industry is con-

fronted with an unnecessary burden to obtain and articles have insinuated that enzymes lead to food
approvals for the use of a new enzyme in food in indi- allergy and that enzymes produced using gmos might
vidual Member States. It goes without saying that it lead to an increase in this problem.
will potentially save cost and time if there were only For all these reasons, AMFEP is in favor of a clear,
one approval system. Furthermore, there is not always simple, and more harmonized regulatory regimen for
mutual recognition as regards the type of safety tests to food enzymes in the European Union. Such a regimen
be performed. This may lead to unnecessary use of would not only solve mutual recognition problems, but
laboratory animals. The approval of an enzyme is also ll the perceived gap in the E.U. Novel Foods
thus governed by the toughest standard in any one Regulation. It may also contribute to improving public
country. condence by increasing the transparency of the safety
There is no harmonized system and there are no evaluation of food enzymes.
xed time limits for approvals resulting in uncertainty
about return on R&D investments for the enzyme B. Wishes of the European Enzyme Industry for
industry. the Future
Member States still apply certain national rules on
production within their own territory, which leads to The wish of AMFEP is to have a future regulatory
so-called reverse discrimination. Enzyme and food regimen for food enzymes which:
producers are particularly affected by this mechanism.
1. Leads to only one approval system, thus less
Enzyme producers are in principle able to sell their
burden, less time, less money.
products throughout the European Union, but the
2. Eliminates the present lack of mutual recognition
remaining national rules in some Member States pre-
and thus trade barriers.
vent the food producers, in their own Member State,
3. Simplies and claries the legal situation.
from actually using the food enzyme in question, mean-
4. Removes present legal gaps, giving more legal
ing that they simply will not buy the enzyme!
security to producers and users.
Nevertheless, foodstuffs produced with such
5. Ensures a high level of protection of public
enzymes can be imported into these Member States
health, leading to improved consumer condence.
owing to the open E.U. market. Thus, such Member
6. Ensures that legislation is primarily based on
States discriminate against the production of particular
scientic evidence and risk assessment.
foodstuffs in their own country.
7. Improves the image of producers of food
The reverse discrimination leads to unequal starting
enzymes and its users, the food industry.
points for food producers in the various Member
States and limits their exibility in shifting manufac- Whatever future regulatory situation is created,
turing. AMFEP would like the following ve principles to
The lack of consistency and clarity in the legislation, be fullled:
such as the different interpretations in Member States, 1. Nondiscrimination of gmos enzymes. Gmos
of the terms processing aid, additive and ingredient, enzymes should not end up in a separate legislation,
leads to legal insecurity for the enzyme producers as for instance under the E.U. Novel Food Regulation.
well as the food producers. In some cases, Member 2. Grandfathering mechanism. Enzymes that are
States use the situation as an argument for not giving already on the market should be able to stay on the
approvals: We are waiting for Brussels. In other market.
cases, Member States expressed their desire to develop 3. Processing aid status or a category in their own
their own national enzyme legislation which would right. There should be no more case-by-case discus-
result in an even more fragmented situation. sions about whether a specic enzyme is applied as
In the absence of a harmonized legal situation, the processing aid, additive, or ingredient. They should
food industry creates its own rules, leading to an even all be given the same status. That might be a category
more complicated and costly situation for the enzyme in their own right, as is the case for avors in the E.U.
industry. Political parties, Parliament, and action 4. Application of SCF guidelines for safety testing.
groups are calling for enzyme regulation, especially There should only be one standardized way to do this.
for enzymes obtained from gmos. 5. No fragmentation through different legislation.
Especially in countries without an enzyme legisla- The complicated way enzymes are regulated now
tion, like Germany, the lack of regulation can lead to should be simplied. All food enzymes should be regu-
low consumer condence. Various television programs lated in a uniform way.

VI. SUMMARY use of certain oenological practices provided for in
Council Regulation (EEC) No 822/87. Ofcial
There is no overall E.U. regulatory regimen for Journal No L269, 1995, p 1.
enzymes. Most enzymes are exempted from the harmo- 7. Fifteenth Report of the Joint FAO/WHO Expert
Committee on Food Additives. FAO Nutrition
nized E.U. Food Additives Directive because they are
Meetings Report Series No. 50, 1972.
used as processing aids and not as additives with a 8. Specications for the identity and purity of some
function in the nal food. Enzymes are, however, sub- enzymes and certain other substances. WHO Food
jected to many different national legislations in the Additives Series, No. 2, 1972.
E.U. Member States. The result of these differences 9. Arrete du 5 septembre 1989 relatif a` lemploi de pre-
in legislation is a lack of consistency in the way parations enzymatiques dans la fabrication de cer-
enzymes are regulated throughout the E.U. taines denrees et boissons destinees a` lalimentation
Therefore, for the global enzyme producer, the produc- humaine. Journal Ofciel de la Republique
tion and marketing of an enzyme product is governed Francaise, 1er octobre 1989.
by the toughest standards in any one E.U. country. 10. Bekendtgrelse om tilstningsstoffer til levnedsmi-
The enzyme industry is in favor of a more uniform dler. Sundhedsministeriets bekendtgrelse nr. 1055 af
18 December 1995.
and harmonized regulatory regimen in the European
11. Food Additives and Contaminants Committee
Union.
Review of Remaining Classes of Food Additives
used as Ingredients in Food. Report on the Review
REFERENCES of Enzyme Preparations 1982. FAC/REP/35,
Ministry of Agriculture, Fisheries and Food.
1. Treaty on European Union (signed in Maastricht on 7 12. JM Battershill. Guidelines for the safety assessment of
February 1992), consolidated version. http://eur- microbial enzymes used in food. Food Additives and
ope.eu.int/eur-lex/en/treaties/livre1_c.html Contaminants 10, 5:479488, 1993.
2. Treaty establishing the European Community. http:// 13. Compedium of food additive specications. FAO
europe.eu.int/eur-lex/en/treaties/dat/ec_cons_trea- Food and Nutrition Paper 52, 1988. Annex 1.
ty_en.pdf. General specications for enzyme preparations.
3. Council Directive of 21 December 1988 on the http://apps3.fao.org/jecfa/intro/Annex1.htm.
approximation of the laws of the Member States con- 14. Regulation (EC) No 258/97 of the European
cerning food additives authorized for use in foodstuffs Parliament and of the Council of 27 January 1997
intended for human consumption (89/107/EEC). concerning novel foods and novel food ingredients.
Ofcial Journal No L040 1989, p 27. Ofcial Journal No L043 1997, p 1.
4. Guidelines for the presentation of data on food 15. Proposal for a regulation of the European Parliament
enzymes (opinion as expressed on 11 April 1991). and of the Council on genetically modied food and
Reports of the Scientic Committee for Food feed. Brussels, 2001. Commission Proposal COM
(Twenty-seventh series). EUR 14181 EN, 1992. (2001) 425 nal. http://europa.eu.int/eur-lex/en/com/
5. Council Directive of 21 September 1993 relating to dat/2001/en_501PC0425.html.
fruit juices and certain similar products (93/77/EEC). 16. Proposal for a regulation of the European Parliaments
Ofcial Journal No L244, 1993, p 23. and of the Council concerning traceability and label-
6. Council Regulation of 16 March 1987 on the common ling of genetically modied organisms and traceability
organization of the market in wine (822/87/EEC). of food and feed products produced from genetically
Ofcial Journal No L084, 1987, p 1, and modied organisms and amending Directive 2001/18/
Commission Regulation (EC) No 2624/95 of 10 EC. Brussels, 25.7.2001. Commission Proposal
November 1995 amending Commission Regulation COM (2001) 425 nal. http://europa.eu.int/eur-lex/
(EEC) No 3220/90 laying down conditions for the en/com/dat/2001/en_501PC0182.html.

7
Regulatory Issues of Food Enzymes Used in the United States
John R. Whitaker
I. INTRODUCTION the Food Safety and Inspection Service FSIS), and the
Grain Inspection, Packers, and Stockyard
Thousands of enzymes are found in all of the raw Administration (GIPSA) or by a state department of
materials (plants, animals, microorganisms) produced agriculture. Increases, or decreases, of some enzyme
for foods. In those consumed raw, the enzymes are levels, such as polyphenol oxidase, can be done by stan-
active when ingested but are generally denatured by dard breeding methods. The insertion of a new gene
the acid pH of the stomach. They then become a and its expression to change the sensory, nutrition, cli-
(small) part of our total daily protein requirement. mate tolerance, insect resistance, or storage properties
When foods are processed prior to eating by heating, of the living organism is generally done by genetic engi-
the enzymes are denatured. In dried and freeze-dried neering. The inventor must demonstrate that there
products, the enzymes may be in active form. In fruits are no harmful effects to humans or the environment,
and vegetables, such as apricots, apples, dates, plums, including other types of activities (allergenic, for exam-
potatoes, mushrooms, teas, etc., the enzymes are still ple) of the food produced from such modications. The
active and must be inactivated by blanching ( 858C, federal and/or state departments of agriculture are
or controlled by adding enzyme inhibitors such as responsible for enforcing such modications to living
bisulte, ascorbic acid, or other specic inhibitors potential food sources.
before drying. But these naturally occurring enzymes Additions of enzymes to food materials during pro-
are not regulated. If an enzyme is added (by biotech- cessing are regulated by the Food and Drug
nology) to a plant to increase the level of enzyme, such Administration (FDA) unit of the Department of
as overproduction of 5-enolpyruvylshikimate-3-phos- Health and Human Services. Specically, the Center
phate synthase (EPSPS) or cloning a mutant gene for Food Safety and Applied Nutrition (CFSAN) of
encoding EPSPS that is insensitive to glyphosate to FDA is assigned this responsibility. In carrying out its
prevent killing of the plant by the herbicide glyphosate, responsibility, it may request advice from the National
while the unwanted grasses and weeds are killed, this is Academy of Sciences (NAS) or other expert bodies and
regulated. When an enzyme is added during the pro- scientists. The regulations of FDA become binding
cessing of a food, this is regulated. when published in the Federal Register (published by
Addition of enzymes, whether to increase the level of the Ofce of the Federal Register, National Archives
the enzyme or to include a new enzyme in a live plant or and Records Administration of the U.S. Government
animal or microbe to be used as food, is regulated by Printing Ofce).
the U.S. Department of Agriculture (USDA) via the The regulations are not only published in the
Animal and Plant Health Inspection Service (APHIS), Federal Register, but can be found also on several

electronic Web sites. These web sites include: 13th century. The Sale of Food and Drugs Act of
http://www.fda.gov/; http://vm.cfsan.fda.gov/list.html; the United Kingdom was passed in 1875. The U.K.
http://vm.cfsan.fda.gov/dms/reg-2.html; http://vm Act was amended by the Food and Drug Acts of
.cfsan.fda.gov/dms/eafus.html; http://vm.cfsan.fda 1938 and 1955. A major reason for the amendments
.gov/lrd/biotechm.html; http://vm.cfsan.fda.gov/cgi- was to improve the control of the increasingly com-
bin/ws.cgi?Q. . .+and+food+additives+ (or use OR plex process of food preparation and sale (1). The
in place of AND); http://frwebgate.access.gpo.gov/cgi- U.K. Act prohibited the sale of any food in which
bin/multidb.cgi. substances were added or deleted, or in which the
The EAFUS (Everything Added to Food in the food was processed in a manner that might be unsafe
United States) Web Site (above) is an informational to human health. Other offenses included the sale of
database maintained by the FDA Center for Food misbranded, falsely described foods, and misleading
Safety and Applied Nutrition (CFSAN) under the pro- claims about the nature of the product, its substance,
gram Priority-based Assessment of Food Additives or its quality.
(PAFA). The United Kingdom in concert with other coun-
tries of the European Union (E.U.) have worked to
It contains administrative, chemical and toxico-
standardize the regulations to simplify trade of foods
logical information on over 2000 substances
among the European Union (see Chapter 6 of this
added to food, including substances regulated
book by Praaning).
by the FDA as direct, secondary direct, and
In the United States, the rst Federal Food and
color additives, and Generally Recognized As
Drug Law was established in 1906 (2). The legislation
Safe (GRAS) and prior-sanctioned substances.
was the basis for passage of several food standards of
In addition, the database contains only adminis-
identity, as well as the prohibition of sale of toxic,
trative and chemical information on less than
unsanitary, adulterated, or misbranded foods. The
1000 other substances (without toxicological
law required the federal government to prove that
information). The more than 3000 total sub-
suspect foods were toxic or harmful to human health.
stances together comprise an inventory often
This law remained in effect until 1938, with two
referred to as EAFUS [quoted from EAFUS
minor revisions in 1907 and 1912. In 1938, Congress
database].
enacted the Federal Food, Drug, and Cosmetic Act,
This list of (of some 3000) substances contains following the death of 70 people who took the drug
ingredients added directly to food that FDA Elixir Sulfanilamide. The provisions of the act
has either approved as food additives or listed were: (a) the establishment of the FDA, the primary
or afrmed as GRAS. Nevertheless, it contains functions of which were administrative and provided
only a partial list of all food ingredients that may only for the prohibition of the use of known toxic
in fact be lawfully added to food, because under substances as food additives; (b) the establishment
federal law some ingredients may be added to of standards of identity and food quality, as well as
food under a GRAS determination made inde- (c) mandates to provide for honest labeling and safe
pendently from the FDA. The list therefore con- packaging.
tains many, but not all, of the substances subject In 1958, the Food Additive Amendment was
to independent GRAS determinations. For infor- enacted because of increasing concern by physicians
mation about the GRAS notication program and consumers that chemical additives were a possible
please consult the Inventory of GRAS cause of cancer. Following deliberations of the
Notications [on the EAFUS data base]. Delaney Committee, an amendment was passed
(1960) that required the FDA to consider safety fac-
The EAFUS database is 201 pages in length.
tors which, in the opinion of experts qualied by
scientic training and experience to evaluate the
safety of food additives, are generally recognized as
II. ORIGIN AND HISTORY OF THE U.S.
appropriate for the use of animal experimentation
FOOD AND DRUG ADMINISTRATION
data. This amendment required that the safety of
A. Origin ingredients and products be established before the
consumer was exposed to them. Thus, the onus of
Regulations to prevent adulteration or sale of unt establishing food safety was transferred to the manu-
food in Great Britain have existed since the early facturers, along with the mandated requirement for

animal testing prior to marketing of the food. One Food additives, as dened by the 1958 Food
clause of the amendment, known as the Delaney Additive Amendment are:
clause, caused major scientic and public controversy.
Any substance the intended use of which results
It stated: No food additive shall be deemed to be
or may reasonably be expected to result, directly
safe if it is found to induce cancer when ingested by
or indirectly, in it becoming a component or
man or animals (3). The net result was that all the
otherwise affecting the characteristics of any
compounds classied as GRAS required new petitions
food (including any substance intended for use
to FDA that not only addressed the safety of the
in producing, manufacturing, packing, proces-
food additives but also the reagents used in prepara-
sing, preparing, treating, packaging, transport-
tion and use of the food additives. Since enzymes
ing, or holding food; and including any source
were classied as food additives in 1906, they had
of radiation intended for such use); if such sub-
to be analyzed for safety, activity, and usage level
stance is not generally recognized among experts
in relation to the food in which they were included.
qualied by scientic training and experience to
Nelson (4), Hickman (5), and Roland (6) have written
evaluate its safety as having been adequately
thorough and thoughtful reviews of the issues and
shown through scientic procedures (or, in the
problems caused by this ruling.
case of a substance used in food prior to 1
January 1958, through either scientic proce-
B. Role of the FDA in Regulation of Enzymes in
dures or experiences based on common use in
Food
food) to be safe under the conditions of its
intended use.
Food enzymes are derived from cells of living plants,
animals, and microorganisms. The enzymes, known or The term food additive thus covers any substance
unknown, have been a part of the food of humans, as (i.e., enzyme) directly added to food. Indirect additions
well as animals, as long as humans have been on earth. to food during processing which become a component
The enzymes are an essential part of germination of of the food are also included.
seeds and reproduction of animals and microorgan- The 1958 Food Additive Amendment established
isms, growth of organisms, maturation, storage and three categories of food additives for foods. They were:
utilization in producing the food and in its digestion
1. Food substances grandfathered into the cate-
to the building blocks for proteins, lipids, carbohy-
gory Generally Recognized as Safe (GRAS), or those
drates, and nucleic acids of all animals, including
substances for which prior sanctions had been pre-
humans. From early times enzymes from plants, ani-
viously established.
mals, and microorganisms were used to modify the
2. Substances deemed as automatically unsafe as
taste, aroma, color, texture, and digestibility of agri-
dened by the Delaney clause, no additive shall be
culture products. Many major food industries (baking,
deemed to be safe if it is found to induce cancer
brewing, dairy, meat, and fruits) are dependent on the
when ingested by man or animal, or if it is found,
utilization of enzymes for production of breads, beers,
after tests which are appropriate for the evaluation of
cheese, fruit juices, etc.
the safety of food additives, to induce cancer in man or
Up to 1958, enzymes were considered as food (pro-
animals.
tein) or food additives but safe, since they had been
3. All other food and color additives which need
used in food since humans rst appeared on earth. But
to be tested for safety on an individual basis.
since 1958, the FDA has viewed enzymes as food addi-
tives, not food. However, until 1972 when the FDA In 1972, FDA revoked the GRAS or prior sanction
suspended the generally recognized as safe (GRAS) status of all pre-1958 additives (which to this time were
status, enzymes were considered to be safe, since they cleared under the grandfather clause of long use that
came from natural sources and are proteins, and a part had shown them to be safe, including enzymes) despite
of all raw foods. They also had an excellent safety a lengthy report by the Ad Hoc Enzyme Technical
record for many hundreds of years. Committee (AHETC), established in 1969; membership
The 1958 Food Additive Amendment dened consisted of representatives from most of the rms that
enzymes as food additives, rather than as foods, food- manufactured or marketed enzymes in the United
stuffs, or foreign substances found in very small States. The sole purpose of the AHETC was, and is,
amounts or familiar substances as some other coun- to deal with regulatory issues and requirements invol-
tries did. ving enzymes with the FAO/WHO Joint Expert

Committee on Food Additives (JECFA), a committee or not, the use of higher purity enzymes produced by
of outstanding scientists with experience in food toxi- genetic engineering will alleviate some of the pro-
cology from England, France, Japan, Italy, blems with extraneous materials, including fragments
Switzerland, Holland, Australia, Russia, and the of microbial cells.
United States. After initial review the updated report Nelson (4) has discussed the lengthy time required
was submitted to the FDA and to the Subcommittee on for review of petitions by FDA. The minimum time
Codex Specications, Committee on Food Protection, may be 18 months. But with requirements for toxicol-
National Research Council for inclusion in the Food ogy and mutagenicity studies with animals of up to 2
Chemicals Codex (published in 1974). Despite these years, the time can be 3 years or more, at a cost of
extensive efforts by AHETC and JECFA, the FDA hundreds of thousands of dollars (7). Only enzyme
made it clear that further information would be products with high economic value expectations are
required before any enzyme, even those with a long likely to be submitted for review. Anticipated revenue
history of use, would be conrmed as GRAS. from food enzyme products in no way compares with
At the 1997 Annual Meeting of the Institute of that of enzyme products from drug companies, espe-
Food Technologists, Corbin Miles of the FDAs cially since the latter have to be pure!
Bureau of Foods described the FDA strategy for
enzyme GRAS afrmation. A summary of that report
is found in Nelson (4), which is quoted here. C. GRAS Petitions to FDA
FDA will again review information on enzymes
A GRAS petition for FDA review looks like the fol-
and enzyme sources, particularly microbial-
lowing (6).
derived enzymes. FDA has continued to seek
1. Clearly state the intent of the petition to demon-
such information, including requests published
strate safety, based on one common use in foods for
in the Federal Register. Essentially, FDA will
the enzyme and/or the end product. Alternatively,
hew to the present requirements for food addi-
establish safety through the publication of adequate
tive petitions. Any enzyme which was GRAS
supporting data.
under the 1958 amendment will require signi-
2. Identication of the enzyme: (a) class; (b) spe-
cant safety information before it will be
cies; (c) strain-variety; (d) enzyme source.
included in the present GRAS list. Mr. Miles
3. Method of preparation: (a) complete medium
stressed that the activity of the enzyme and
composition; (b) mechanical units used to produce;
the utilization of such activity in the manufac-
(c) fermentation conditions (aeration, agitation, tem-
ture or modication of foods must be described
perature, pH).
in some detail. Any GRAS regulations on
4. Enzyme specications: (a) single enzyme, multi-
enzymes must include all conditions of use
ple enzymes, or whole cells; (b) impurities, i.e., heavy
which have been evaluated. Mr. Miles stated
metals, solvents; (c) comparative compositional data
that trade secret information (if different than
on a minimum of ve processes of the enzyme.
that publically available or proposed) must be
5. Methods of enzyme recovery: (a) types and
submitted. He suggested that trade secret infor-
levels of solvents employed; (b) types of processing
mation be submitted via an additive petition
aids, i.e., ltration, occulation; (c) other.
which can be exempt from the Freedom of
6. Toxicological data on enzyme or microorgan-
Information Act.
ism: (a) organism must be non pathogenic; (b) organ-
The rst impression of those of us familiar with ism must not produce mycotoxins or other toxic
enzymes (biochemists, members of AHETC and chemicals; (c) organism must not produce antibiotics.
JECFA, and perhaps the E.U. Scientic European Ebert (8), Aunstrup (9) and Grasso (10) have
Committee on enzyme usage in foods) was that this designed formats for typical toxicological evaluations
was a very harsh and unjustied position to take on on animals. Table 1 is from a publication of Dr. Ebert
the clearance of enzymes in food processing and man- (8).
ufacture. But when one recalls that commercial Both FDA and USDA, in reviewing a petition, may
enzyme products are at most 1% of the desired ask advice from the National Academy of Sciences, the
enzyme and 99% of other materials including trace Federation of American Societies for Experimental
amounts of other enzymes, purication materials, Biology, Special Advisory Committees, and individual
etc., the view of FDA can be rationalized. Perhaps, consultants.

Table 1 Typical Toxicological Evaluation of Food Additives
Study Scope
Acute
Oral LD50 Two species (min) range nd for subacute studies.
Dermal
inhalation LD50 As needed (OSHA requirements, etc.).
Subacute
90-day feeding Two species: one nonrodent, dog preferred. Three levels (min) plus
control.
Complete biochemical and histopathologic workup on control,
highest level fed. Other levels if needed. Dene no-effect level.
High level to give effect.
Chronic
2 years Two speciesrat/dog. Carry rat to 25% survival of controls.
Perhaps combine with three-generation reproduction study.
Three levels plus control. Complete biochemical and
histopathologic workup.
Metabolic fate Search for animal model for man. Absorption, distribution kinetics,
organ/tissue concentration (if any) metabolic products dened.
Special Carcinogenicity, teratology, cytogenicity (host mediated, dominant
lethal, chick embryo).
Interaction Special dietary restrictions, drug interactions.
Source: Ref. 8.
III. CURRENT STATUS OF ENZYME A. 170Food Additives and Food Additive

REGULATIONS (11) Petitions
Since the beginning of federal regulations involving 170Food Additives is a general section for all food
enzymes they have been, and continue to be, classied additives (pp 510). 171Food Additive Petitions is a
as food additives. As noted earlier in this chapter, they general section on how to petition FDA for approval
were originally classied as GRAS. But after 1958, of a food additive (pp 2126). Both must be consulted
when the Food Additive Amendment was passed, for the general aspects pertaining to denitions, classi-
safety of the enzyme-containing product had to be cation of foods and ingredients, and general policies
addressed, and in 1972, all GRAS status was revoked. on food additive regulations. The topics covered are
As pointed out in Section II above, these changes given in Table 2.
require extensive data in the petition asking for rein-
statement of GRAS status.
Regulation of food additives is covered in the Code
of Federal Regulation Title 21, 170199 (11). B. 173Secondary Direct Food Additives
Enzymes are covered collectively with other food addi-
tives in Part 170Food Additives, Chapter 1, Food 173Secondary Direct Food Additives Permitted in
and Drug Administration, Department of Health and Food for Human Consumption, Subpart BEnzyme
Human Services Subchapter B, Food for Human Preparations and for a Human Consumption, Subpart
Consumption as to regulation procedures, in 171, BEnzyme Preparations and Microorganisms lists
Food Additive Petitions; in 173, Secondary Direct those enzymes and microorganisms that have been
Food Additives Permitted in Food for Human afrmed by FDA and on what basis they were
Consumption; and in 184, Direct Food Substances approved (Table 3). Note that all the enzymes are
Afrmed as Generally Recognized as Safe. from microorganisms.

Table 2 Code of Federal Registrations 21. Food and Drugs. Part 170Food
Additivesa
Subpart Ageneral provisions

170.3 Denitions
170.6 Opinion letters on food additive status
170.10 Food additives in standardized foods
170.15 Adoption of regulation on initiative of Commissioner
170.17 Exemption for investigational use and procedure for obtaining
authorization to market edible products from experimental animals
170.18 Tolerances for related food additives
170.19 Pesticide chemicals in processed foods
Subpart Bfood additive safety

170.20 General principles for evaluating the safety of food additives
170.22 Safety factors to be considered
170.30 Eligibility for classication as generally recognized as safe (GRAS)
170.35 Afrmation of generally recognized as safe (GRAS) status
170.38 Determination of food additive status
170.39 Threshold of regulation for substances used in food-contact articles
Subpart Cspecic administrative rulings and decisions

170.45 Fluorine-containing compounds
170.50 Glycine (aminoacetic acid) in food for human consumption
170.60 Nitrites and/or nitrates in curing premixes
a
AUTHORITY: 21 U.S.C. 321, 341, 342, 346a, 348, 371.
Source: 42 FR 14483. Mar. 15, 1977, unless otherwise noted.
Table 3 Secondary Direct Enzymes and Microorganisms Permitted in Food for Human Consumptiona
Subsection Enzyme Source
173.110 Amyloglucosidase Rhizopus niveus

173.120 Carbohydrase and cellulase Aspergillus niger
173.130 Carbohydrase Rhizopus oryzae
173.135 Catalase Micrococus lysodeikticus
173.140 Esterase-lipase Mucor miehei
173.145 Alpha-galactosidase Mortierella vinaceae var. rafno seutilizer
173.150 Milk-clotting enzymes microbial (1) Endothia parasitica
(2) Bacillus cereus
(3) Mucor pusillus Lindt
(4) Mucor meihi Cooney et Emerson
(5) Aspergillus oryzae (recombinant modied)
173.160 Several enzymes (citric acid production) Candida guilliermondii
Several enzymes (citric acid production) Candida lipolytica
173.170 Aminoglycoside 30 -phosphotransferase II (Escherichia coli, using klanr gene)
(CAS Reg. No. 58943-39-8)
a
From Code of Federal Regulations 21. Food and Drugs. Part 173, Published in the Federal Register No. 21, pp 111124,
April 1, 2000.

A closer look at the approval use of microbial milk- C. 184Direct Food Substances Afrmed as
clotting enzymes is instructive as to understanding the Generally Recognized as Safe (GRAS)
FDA approval process, including the conditions given
in 173.150. (a) The direct human food ingredients listed in
this part have been reviewed by the Food and
Milk-clotting enzymes produced by pure-culture Drug Administration and determined to be gen-
fermentation process may be safely used in the erally recognized as safe (GRAS) for the pur-
production of cheese in accordance with the fol- poses and under the conditions prescribed. The
lowing prescribed conditions. regulations in this part shall sufciently describe
(a) Milk-clotting enzyme is derived from one each ingredient to identify the characteristics of
of the following organisms by a pure-culture fer- the ingredient that has been afrmed as GRAS
mentation process. and to differentiate it from other possible ver-
(1) Endothia parasitica classied as follows: sions of the ingredient that have not been
class, Ascomycetes; order, Sphaeriales; family, afrmed as GRAS. Ingredients afrmed as
Diaporthacese; genus, Endothia; species, parasi- GRAS in this part are also GRAS as indirect
tica. human food ingredients, subject to any limita-
(2) Bacillus cereus classied as follows: class, tions prescribed in parts 174, 175, 176, 177, 178
Schizomycetes; order, Eubacteriales; family, or Sec. 179.45 of this chapter or in parts 186 of
Bacillaceae; genus, Baccillus; species, cereus this chapter. The purity specications in this part
(Frankland and Frankland). do not apply when the ingredient is used in indir-
(3) Mucor pusillus Lindt classied as follows: ect applications. However, when used in indirect
class, Phycomycetes; subclass, Zygomycetes; applications, the ingredient must be of a purity
order, Mucorales; family, Mucoraceae; genus, suitable for its intended use in accordance with
Mucor; species, pusillus; variety, Lindt. 170.30 (h) (1) of this chapter.
(4) Mucor miehei Cooney et Emerson classied (b) Any ingredient afrmed as GRAS in this
as follows: class, Phycomycetes; subclass, part shall be used in accordance with current
Zygomycetes; order, Mucorales; family, good manufacturing practice. For the purpose
Mucoraceae; genus, Mucor, species, miehei; vari- of this part, current good manufacturing practice
ety, Cooney et Emerson. includes the requirements that a direct human
(5) Aspergillus oryzae modied by recombi- food ingredient be of appropriate food grade;
nant deoxyribonucleic acid (DNA) techniques that it be prepared and handled as a good ingre-
to contain the gene coding for aspartic protei- dient; and that the quantity of the ingredient
nase from Rhizomucor miehei var. Cooney et added to food does not exceed the amount rea-
Emerson as dened in paragraph (a)(4) of this sonably required to accomplish the intended phy-
section, and classied as follows: class, sical, nutrition, or other technical effect in food.
Blastodeuteromycetes (Hyphomycetes); order, (1) If the ingredient is afrmed as GRAS with
Phialidales (Moniliales); genus, Aspergillus; spe- no limitations on its conditions of use other than
cies, oryzae. current good manufacturing practice, it shall be
(b) The strains of organism identied by para- regarded as GRAS if its conditions of use are
graph (a) of this section are nonpathogenic and consistent with the requirements of paragraph
nontoxic in man or other animals. (b), (c), and (d) of this section. When the Food
(c) The additive is produced by a process that and Drug Administration (FDA) determines that
completely removes the generating organism it is appropriate, the agency will describe one or
from the milk-clotting enzyme product. more current good manufacturing practice con-
(d) The additive is used in an amount not in ditions of use in the regulation that afrms the
excess of the minimum required to produce its GRAS status of the ingredient. For example,
intended effect in the production of those when the safety of an ingredient has been evalu-
cheeses for which it is permitted by standards ated on the basis of limited conditions of use, the
of identity established pursuant to section 401 agency will describe in the regulation that afrms
of the Act [42 FR 14526, Mar. 15, 1977; 42 FR the GRAS status of the ingredient, one or more
56728, Oct. 14, 1977, as amended at 62 FR of these limited conditions of use, which may
59284, Nov. 3, 1997]. include the category of food(s), the technical

effect(s) or functional use(s) of the ingredient, come forward with proof of such an applicable
and the level(s) of use. If the ingredient is used prior sanction in response to the proposal will
under conditions that are signicantly different constitute a waiver of the right to assert or rely
from those described in the regulation, that use on such sanction at any later time. The notice will
of the ingredient may not be GRAS. In such a also constitute a proposal to establish a regula-
case, a manufacturer may not rely on the regulation under part 181 of this chapter, incorporating
tion as authorizing that use but shall indepen- the same provisions, in the event that such a reg-
dently establish that that use is GRAS or shall ulation is determined to be appropriate as a
use the ingredient in accordance with a food result of submission of proof of such an applic-
additive regulation. Persons seeking FDA able prior sanction in response to the proposal.
approval of an independent determination that (f) The label and labeling of the ingredient and
a use of an ingredient is GRAS may submit a any intermediate mix of the ingredient for use in
GRAS petition in accordance with 170.35 of nished food shall bear, in addition to the other
this chapter. labeling required by the Act:
(2) If the ingredient is afrmed as GRAS with (1) The name of the ingredient, except where
specic limitation(s), it shall be used in food only exempted from such labeling in part 101 of this
within such limitation(s), including the category chapter.
of food(s), the functional use(s) of the ingredient, (2) A statement of concentration of the ingre-
and the level(s) of use. Any use of such an ingredient in any intermediate mix; or other informa-
dient not in full compliance with each such estab- tion to permit a food processor independently to
lished limitation shall require a food additive determine that use of the ingredients will be in
regulation. accordance with any limitations and good man-
(3) If the ingredient is afrmed as GRAS for a ufacturing practice guidelines prescribed.
specic use, without a general evaluation of use (3) Adequate directions for use to provide a
of the ingredient, other uses may also be GRAS. nal food product that complies with any limita-
(c) The listing of a food ingredient in this part tions prescribed for the ingredient(s) [42 FR
does not authorize the use of such substance in a 14653, Mar. 15, 1977, as amended at 42 FR
manner that may lead to deception of the consu- 55205, Oct. 14, 1977; 48 FR 48457, 48459, Oct.
mer or to any other violation of the Federal 19, 1983; 62 FR 15110, Mar. 31, 1997].
Food, Drug, and Cosmetic Act (the Act).
(d) The listing of more than one ingredient to 1. List of Enzymes That Have Been Afrmed
produce the same technological effect does not as Generally Recognized as Safe
authorize use of a combination of two or more
Table 4 lists the enzymes that have been afrmed as
ingredients to accomplish the same technological
generally recognized as safe (GRAS). Note that the list
effect in any one food at a combined level greater
includes enzymes from animals, plants, and microor-
than the highest level permitted for one of the
ganisms. The afrmation of rennet and bovine rennet
ingredients.
(both animal derived) and chymosin (fermentation
(e) If the Commissioner of Food and Drugs is
derived from microorganisms containing the prochy-
aware of any prior sanction for use of an ingre-
mosin gene) (Section 84.1685) has been chosen to
dient under conditions different from those pro-
indicate (a) requirements for animal rennet,
posed to be afrmed as GRAS, he will
(b) requirements for microbial chymosin, and (c)
concurrently propose a separate regulation cov-
requirements for enzymes produced by genetically
ering such use of the ingredient under part 181 of
engineered microorganisms.
this chapter. If the Commission is unaware of
any such applicable prior sanction, the proposed
2. 184.1685Rennet (Animal Derived) and
regulation will so state and will require any per-
Chymosin (Fermentation Derived)
son who intends to assert or rely on such sanction
Preparation
to submit proof of its existence. Any regulation
promulgated pursuant to this section constitutes (a)(1) Rennet and bovine rennet are commercial
a determination that excluded uses would result extracts containing the active enzyme rennin
in adulteration of the food in violation of section (CAS Ref. No. 9001-98-3), also known as chy-
402, of the Act, and the failure of any person to mosin (International Union of Biochemistry

Table 4 Enzymes Afrmed as Generally Recognized as Safea
Subsection Enzyme (or product) Source (or Substrate)
184.1012 -Amylase Bacillus stearothermophilus

184.1024 Bromelain (peptide hydrolase) Pineapple (Ananas comosus and A. bracteatus L.)
184.1027 Mixed carbohydrase and protease Bacillus licheniformis
184.1034 Catalase Bovine liver
184.1148 -Amylase and -glucanase Bacillus subtilis or B. amyloliquefaciens
184.1150 Subtilisin and neutral proteinase Bacillus subtilis or B. amyloliquefaciens
184.1250 Cellulase Trichoderma longibrachiatum
184.1316 Ficin Tropical g trees
184.1372 Immobilized glucose isomerase Streptomyces rubiginosusb
(high-fructose corn syrup)
184.1387 Lactase Candida pseudotropicalis
184.1388 Lactase Kluyveromyces lactis
184.1415 Lipase From forestomach or pancreas of calves, kids, or lambs
184.1420 Lipase Rhizopus nireus
184.1583 Pancreatin Porcine or bovine pancreas
184.1585 Papain Papaya (Carica papaya L.)
184.1595 Pepsin Hog stomach glandular layer
184.1685 Rennet (animal derived) Fourth stomach (abomasa) of calves, kids, or lambs
Chymosin (fermentation derived) Escherichia coli K-12, Kluyveromyces marxianus var. lactis,
or Aspergillus niger van Teighem var. awamori (all the
microorganisms contain the calf prochymosin gene)
184.1914 Trypsin Bovine pancreas
184.1924 Urease Lactobacillus fermentum
184.1985 Aminopeptidase Lactococcus lactis
184.1063 Phospholipase A2 Gives enzyme-modied lecithin (enzyme from bovine
pancreatin)
184.1287 Lipases from milk or milk powder Fats
184.1857 Acids or enzymes Corn sugar (glucose)
184.1859 Acids or enzymes Invert sugar (glucose + fructose)
184.1865 Acids or enzymes Corn syrup
184.1866 Glucose isomerase High-fructose corn syrup
a
From Code of Federal Regulations 21. Food and Drugs. Part 184, sub part B, published in the Federal Register, No. 21, pp 461
556, April 1, 2000.
b
Other microorganisms approved as a source of glucose isomerase are Actinoplanes missouriensis, S. olivaseus, S. olivochromogenes,
and Bacillus coagulans.
Enzyme Commission (E.C.3.4.23.4). Rennet is luble aggregate that is acid-treated to destroy

the aqueous extract prepared from cleaned, fro- residual cellular material and, after solubiliza-
zen, salted, or dried fourth stomachs (abomasa) tion, is acid-treated to form chymosin. It must
of calves, kids, or lambs. Bovine rennet is the be processed with materials that are generally
product from adults of the animals listed above. recognized as safe, or are food additives that
Both products are called rennet and are clear have been approved by the Food and Drug
amber to dark brown liquid preparations or Administration for this use.
white to tan powders. (3) Chymosin preparation is a clear solution
(2) Chymosin preparation is a clear solution containing the active enzyme chymosin (E.C.
containing the active enzyme chymosin (E.C. 3.4.23.4). It is derived, via fermentation, from a
3.4.23.4). It is derived, via fermentation, from a nonpathogenic and nontoxigenic strain of
nonpathogenic and nontoxigenic strain of Kluyveromyces marxianus variety of lactis, con-
Escherichia coli K-12 containing the prochymo- taining the prochymosin gene. The prochymosin
sin gene. The prochymosin is isolated as an inso- is secreted by cells into fermentation broth and

converted to chymosin by acid treatment. All (d) Prior sanctions for this ingredient different
materials used in the processing and formulating from the uses established in this section do not
of chymosin must be either generally recognized exist or have been waived. [FR 10935, Mar. 23,
as safe (GRAS), or be food additives that have 1990, as amended at 57 FR 6479, Feb. 25, 1992;
been approved by the Food and Drug 58 FR 27202, May 7, 1993].
Administration for this use. It is important to specically point out that FDA
(4) Chymosin preparation is a clear solution has included genetically engineered enzymes as GRAS
containing the active enzyme chymosin (E.C. as food additives, using the same criteria as used for
3.4.23.4). It is derived via fermentation, from a nongenetically engineered sources of enzymes. Under
nonpathogenic and nontoxigenic strain of Part 173, Secondary Direct Food Additives Permitted
Aspergillus niger van Tieghem variety awamori in Food for Human Consumption, Subpart B
Nakazawa); (Al-Musallam synonym A. awamori Enzyme Preparations, FDA permitted the genetically
Nakasawa) containing the prochymosin gene. derived milk-clotting enzyme from Aspergillus oryzae
Chymosin is recovered from the fermentation to be used (173.150).
broth after the acid treatment. All materials
used in the processing and formulating of chymo-
sin preparation must be either generally recog-
nized as safe (GRAS) or be food additives that REFERENCES
have been approved by the Food and Drug
Administration for this use. 1. PP Scott. Attitudes toward regulatory control of car-
(b) Rennet and chymosin preparations meet cinogens in food. Chem Indust 3:8386, 1979.
the general and additional requirements for 2. YH Hui. United States Food Laws, Regulations, and
Standards. New York: John Wiley & Sons, 1979.
enzyme preparations of the Food Chemicals
3. U.S. Congress, House Committee on Appropriations.
Codex, 3d Ed. (1981), pp. 107110, which is
Study of the Delaney and Other Anticancer Clauses.
incorporated by reference in accordance with 5 Washington: U.S. Government Printing Ofce, 1974.
U.S.C. 552(a). Copies are available from the 4. JH Nelson. Enzyme producers, food processors, and
National Academy Press, 2101 Constitution the regulators. In: JP Danehy, B Wolnak, eds.
Avenue NW., Washington, DC 20418, or are Enzymes: The Interface Between Technology and
available for inspection at the Ofce of the Economics. New York: Marcel Dekker, 1980, pp
Federal Register, 800 North Capitol Street, 137147.
NW., Suite 700, Washington, D.C. 5. DH Hickman. The role of government in regulating
(c) In accordance with 184.1(b)(1), the ingre- the commercial use of enzymes. In: JP Danehy, B
dient is used in food with no limitation other Wolnak, eds. Enzymes: The Interface Between
Technology and Economics. New York: Marcel
than current good manufacturing practice. The
Dekker, 1980, pp 149160.
afrmation of this ingredient as generally recog-
6. JF Roland. Regulation of food enzymes. Enzyme
nized as safe as a direct human food ingredient is Microb Technol 3:105110, 1981.
based upon the following current good manufac- 7. General Accounting Ofce Report to Congress. Need
turing practice conditions of use: for more effective regulation of direct additives to
(1) The ingredient is used as an enzyme as food. HRD-80-90, 1980.
dened in 170.03(o)(9) of this chapter; a proces- 8. AG Ebert. Advice to the technologist developing food
sing aid as dened in 170.3(o)(24) of this chap- additives. Food Prod Dev 10(5):40, 44, 49, 1976.
ter; and a stabilizer and thickener as dened in 9. K Aunstrup. Industrial approach to enzyme produc-
170.3(o)(28) of this chapter. tion. In: Z Bohak, N Sharon, eds. Biotechnological
(2) The ingredient is used in the following Applications of Proteins and Enzymes. New York:
foods at levels not to exceed current good man-
10. P Grasso. Methodology in assessing carcinogenic risk.
ufacturing practice: In cheeses as dened in Chem Indust 3:7376, 1979.
170.3(n)(5) of this chapter; frozen dairy desserts 11. Code of Federal Regulations. 21, Food and Drugs.
and mixes as dened in 170.3(n)(20) of this Part 170 to 199. Revised as of April 1, 2000.
chapter; gelatins, puddings, and llings as dened Published by the Ofce of the Federal Register,
in 170.3(n)(22) of this chapter; and milk pro- National Archives and Records Administration,
ducts as dened in 170.3(n)(31) of this chapter. Washington, DC.

8
Properties of Extremophilic Enzymes and Their Importance in

Food Science and Technology
Magnus M. Kristjansson and Bjarni Asgeirsson

University of Iceland, Reykjavik, Iceland
I. INTRODUCTION molecular principles involved in extremophilic adap-

tive strategies could also be of considerable commeri-
Research during the last two decades has increasingly cal interest, as the same principles could be used for
demonstrated that various microorganisms grow and example in designing industrial processes involving
live in environments that previously were considered biocatalysts that have to withstand extreme conditions
unimagineable for the support of life. These habitats with respect to temperature and pH. Given that there
are characterized by extreme conditions with respect to are constantly expanding sources of novel and unu-
temperature, pH, salinity, and pressure. At present the sually stable biocatalysts from extremophiles, the pro-
apparent limits for these biologically relevant physical spects of expanding the limits of biocatalysis in both
parameters in the biosphere are 40 C to 115 C for novel, as well as in more conventional processes, could
temperature, pressures up to 120 MPa (corresponding be realized (3). Enzymes with temperature optima ran-
to hydrostatic pressure in the deep sea), water activity ging from close to the freezing point of water and to
(aw ) of 0.6, (as in salt lakes), and pH values between 1 that in excess of its boiling point have been isolated, as
and 11 (1). In order to survive and to proliferate in well as enzymes that function in extreme salinity, over
such hostile environments, organisms had to adapt a wide range of pHs, pressures, and in essentially non-
their metabolic and other cellular functions to the per- aqueous solvents (3).
sisting extreme condition. Especially important is the High thermal stability is a highly desirable trait in
adaptation of stability and activity of enzymes and many industrial enzymes, as processes involving
other proteins that have to be optimized to function enzymes are frequently carried out at high tempera-
under the extreme conditions. As a result of the tures. There are some distinct advantages in running
requirement for molecular adaption, the properties of biotechnological processes at elevated temperatures. In
proteins, especially enzymes, from extremophiles have fact, most such industrial processes are operated at
been a subject of much interest both for basic studies in temperatures >50 C. Benets of higher operating tem-
biology and chemistry, as well as for potential use as peratures include decreased viscosity, increased diffu-
biocatalysts in industrial applications. sion rates, and solubility of most organic compounds,
Since the discovery of Archaea as a new branch on and thus an increase in reaction rates. Higher tempera-
the phylogenetic tree (2), the number and variety of tures also reduce the risks of microbial contaminations
explored extremophilic organisms have increased enor- in the enzyme reactors (46). For these reasons,
mously, as well as research on the biochemical aspects enzymes from hyperthermophiles have received great
of extremophilic adaptation. An understanding of the attention, as these enzymes are generally found to be

very thermostable. This also makes them ideal experi- packing in the protein core, and the latter arising
mental models for answering fundamental questions mainly by the increase that occurs in conformational
regarding the molecular basis for protein stability. entrophy on unfolding of the polypeptide (11, 12).
Enzymes from cold-adapted organisms, or psychro- As a result of this delicate balance only minor shifts
philes, have gained increasing attention by researchers in the contributions of any of these opposing forces
during the last several years, both as subjects of basic can in principle signicantly alter the stability of a
studies on molecular origins of cold-adaptation, and as protein. The small marginal stability of the native
potential industrial enzymes (7, 8). These enzymes are state, and the fact that these forces are cooperative
usually found to have higher specic activities at low and highly dependent on environmental conditions,
temperatures than their counterparts from meso- and such as temperature and solvent exposure, make the
thermophiles. That property may be benecial in sev- accounting of the contributions of these different
eral industrial applications, such as in the processing of forces to the stability of a protein a highly difcult
sensitive biological materials, including foodstuffs, that task. Partially because of the uncertainty in the magni-
have to be carried out under chilled or refrigerated tude of these different interactions, the free energy of
conditions. Enzymes from cold-adapted organisms stabilization for a protein cannot be calculated, even if
are usually found to be comparatively thermolabile, high-resolution crystal structure for the protein is
which may be benecial in operations when enzyme available (1). Furthermore, such calculations based
treatment has to be terminated rapidly, without exces- on atomic coordinates from crystal structures do not
sive treatment of the raw material (7). consider the dynamics of protein structures, a prere-
The molecular basis for function and stability of quisite for their function (1). Biologically active pro-
proteins from organisms living at the extremes of tem- teins such as enzymes are dynamic molecules and need
perature in the biosphere is still not well understood a certain degree of structural exibility for performing
despite extensive research effort. However, under- their function as biocatalysts (1315).
standing which structural principles direct extreme It appears that during the evolutionary adaptation
temperature adaptation of proteins are fundamental of proteins to different environmental temperatures,
to our general understanding of their structural stabi- preserving function through optimization of exibility
lity and function. Such information on structurefunc- of critical regions of the protein molecules has been a
tion relationships in these enzymes also forms the basis priority. Thus, optimizing a function of an enzyme
for their utilization as industrial biocatalysts under dif- under given temperature conditions is expected to
ferent, and perhaps novel, sets of conditions. involve a compromise between two apparently oppos-
In this chapter we will attempt to give an overview ing structural factors, dynamic exibility and stability
of the current status of knowledge about enzymes that (1, 9, 15). Enzymes that perform the same catalytic
originate from organisms that are adapted to extremes function but are adapted to different habitat tempera-
of temperature. tures, appear to have about the same conformational
exibility at their respective temperature optima (1, 13,
15). This would suggest that temperature-adaptive
II. EFFECT OF TEMPERATURE ON changes of proteins would tend to conserve and opti-
STABILITY AND FUNCTION OF mize the functional state of the proteins, such that they
ENZYMES are in corresponding states, with respect to function-
ally important motions, under the different physical
It is important to realize when considering temperature conditions to which they have adapted (1, 9). This
adaptation that the native conformations of proteins hypothesis suggests that in order to maintain such cor-
are only marginally more stable than their denatured responding states for efcient biological function at
states. Free energies of stabilization of native protein low temperatures, cold-adapted enzymes must have
structures are typically of the order 2080 kJ mol1 , or adopted a higher degree of conformational exibility
what amounts to only a few weak intramolecular inter- (1, 1518).
actions (1, 9, 10). The net stabilization of a native Enzymes from thermophiles, on the other hand,
protein is the result of a delicate balance between a require very rigid and stable conformations that have
relatively large stabilizing and destabilizing forces, to resist excessive thermal motions that lead to their
the former being a combination of the hydrophobic denaturation, but that can maintain optimized exibi-
effect, hydrogen bonding, electrostatic interactions, libity (corresponding states) with respect to function
and van der Waals interactions gained through close at elevated temperatures. That the activity of enzymes

where the temperature-dependent hydrogendeuterium
exchange kinetics of homologous mesophilic and ther-
mophilic pairs of the enzymes 3-isopropylmalate dehy-
drogenase and glyceraldehyde phosphate
dehydrogenase were compared (Fig. 1) (1, 15), and by
theoretical model calculations (20). Hydrogen exchange
experiments with the ultrathermostable protein rubre-
doxin, from the hyperthermophile Pyrococcus furiosus,
however, did not lend support to the hypothesis that
structural rigidity in the native state underlies the
increased thermal stability of the protein (21). The rea-
sons for the apparent contradictions in results obtained
in these studies are not known, but have been commen-
ted on by Jaenicke (22).
On the basis of the proposed (and frequently
observed) inverse correlation between enzyme stability
and activity (14), much research effort has gone into
explaining structural determinants of protein stability
in order to gain insight into the molecular mechanisms
that direct their temperature adaptation. While a glo-
bal structural stabilization is essential for maintaining
functional native states of thermophilic proteins at ele-
vated temperatures, the ne adjustment of adaptive
changes for activity may involve localized reversible
conformational changes that would confer the neces-
sary exibility to critical parts of the protein molecules
for function under given extreme conditions. Such
local structural uctuations within a protein structure
have been dened as the microstability of proteins, as
Figure 1 Hydrogen-deuterium exchange of 3-isopropyl- opposed to macrostability, that describes the ability of
malate dehydrogenase (IPMDH) from E. coli, measured at native conrmations of proteins to resist unfolding
pD 7.15 () and pD 8.15 (&), and from T. thermophilus, at (denaturation) under given set of conditions (15).
pD 7.15 (r) and pD 8.15 (O), plotted as relaxation spectra. Making a distinction between micro- and macrostabil-
For experimental details see (15). (A) Measurements at 25 C
ity of proteins may be necessary when attempting to
for both enzymes. Increasing values of X reect increased
explain the molecular origins of temperature adapta-
rigidity. Curves therefore indicate a more rigid structure for
the thermophilic enzyme. (B) Measurements at the tempera- tion. The noncooperative transitions that determine
ture optima of the enzymes: at 48 C for the E. coli, and at the microstability of a protein may occur on a different
70 C for the T. thermophilus IPMDH. Curves for the two time scale and are not necessarily reected in experi-
enzymes show very similar exibilities. (From Ref. 15. ments that determine the cooperative unfolding/refold-
Copyright 1998 National Academy of Sciences, U.S.A.) ing transitions of protein molecules that dene their
macrostabilities.
In fact, Fields and Somero (18) found no correlation
from thermophiles is greatly diminished at lower tem- between denaturation rates and adaptation tempera-
peratures at which their mesophilic counterparts have tures when comparing these parameters for cold-
optimal activity is a manifestation of the exibility/ adapted orthologs of lactate dehydrogenase A4 from
function relationship, as it would imply that cooling different sh species of Antarctic and South American
would freeze out the molecular uctuations at or origins. They suggested that increased cold activity in
around the active site of the enzyme that are necessary these orthologs involved increased exibility in small
for its activity (19, 20). The role of optimized exibility areas of the molecule that affect mobility (hence micro-
in temperature adaptation and the inverse relationship stability) of adjacent active site structures (18). Recent
between protein stability and function have recently results for mutagenic studies have also shown that the
been supported both by results obtained in experiments stability of enzymes can be considerably increased con-

comitantly with signicantly improved catalytic prop-
erties (2325). This has not been observed, however, in
enzymes from thermophiles, where lower catalytic ef-
ciencies generally accompany higher thermal stabili-
ties, as compared to the corresponding enzymes from
mesophiles. Perhaps, the selective pressure set by the
high-temperature environment imposes more restric-
tion on the thermophilic enzymes to develop more sta-
bility at the expense of function, and are therefore not
catalytically optimized enzymes, but are highly ther-
mostable (19).
For enzymes from psychrophiles the selective pres-
sure is probably more toward optimizing function
which involves maintaining adequate molecular
dynamics in spite of low thermal input from the cold
habitat. This requires lowered microstability of the
cold-adapted enzymes and may accompany, as is Figure 2 Three possible mechanisms for the temperature
most often observed, decreased macrostability of the dependence of free energy of unfolding (Gu ) for a thermo-
proteins, either as a structural consequence, or simply philic protein. Curve M (solid) depicts the relation of Gu
because it is not needed in the low-temperature envir- and temperature (T) for a typical mesophilic protein. In
onment (25). curve 1 (dashed), the thermophilic protein is stabilized across
the temperature range and has a greater maximum stability
(mechanism 1). In curve 2 (dotted), the Gu vs. T relation-
ship is the same as for the mesophilic protein, but is shifted to
III. THERMODYNAMIC ASPECTS OF a higher temperature (mechanism 2). In curve 3 (dash-dot-
TEMPERATURE ADAPTATION dash patterned), the Gu of thermophilic protein depends
less on T, and thus leads to attening of the stability curve,
The thermodynamic stability of a protein (that dena- shifting the melting temperature to higher values (T Tm ,
tures in a reversible, two-state manner) as a function of when Gu 0). (From Ref. 34. Copyright 1999 American
temperature can be described by the modied Gibbs- Chemical Society.)
Helmholtz equation (26):
G H TS Cp T T TlnT=T 1
teins, especially multidomain proteins, do not
where Cp , the difference in heat capacity at constant undergo reversible, two-state thermal denaturation.
pressure, is taken as a constant and is larger than zero. Competing side reactions occur, such as aggregations,
T is any reference temperature (most often the melting that prevent proper refolding at the high temperatures
temperature, Tm ), and S and H are the changes in at which these experiments are carried out (1).
molar entropy and enthalpy evaluated at the reference Recently, however, small, single-domain hyperther-
temperature, respectively. The equation describes a mostable proteins, which alleviate these experimental
parabolic curve for the temperature dependence of problems and fulll the requirements for a two-state,
the free energy of unfolding (Gu ), dened by a reversible transition, have been used as models in such
slope, G=T=-S, and a curvature, 2 G=T 2 =- thermodynamic studies (2735). More than two dec-
Cp =T (26). The curve predicts that proteins have ades ago, Nojima et al. (36, 37) determined the ther-
maximum stabilities (Gmax ) at a temperature Tmax , modynamic parameters for denaturation of
when S=0. The stability decreases on both sides of phosphoglycerate kinase and cytochrome c552 from
the maximum and intersects twice the x-axis (at the thermophile Thermus thermophilus, and compared
G=0), when the proteins undergo cold and heat them to those determined for their counterparts of
denaturation, respectively (Fig. 2). Despite extensive mesophilic origin. From their studies they proposed
research into the causes of thermal stability of (hyper- three possible ways in which thermophilic proteins
)thermophilic proteins, thermodynamic characteriza- could thermodynamically adapt to increase their dena-
tion in the form of protein stability curves are only turation temperatures (Fig. 2). Compared to a meso-
available for relatively few thermophilic proteins. One philic protein (curve M), the free energy of unfolding
major reason for this is that many thermophilic pro- for the thermophilic protein could be increased over

the whole temperature range (curve 1); the stability cially evident with small proteins that are unable to
curve (Tmax ) could be shifted to higher temperatures form a large hydrophobic core, and is reected in
(curve 2); or the curve could be attened (curve 3). In low Cp values (40). The small DNA-binding protein
all cases the result would be to shift the melting tem- Sso7d (64 residues) from the hyperthermophilic
perature (Tm ) of the thermophilic protein to a higher archaeon Sulfolobus solfataricus has its Tmax at 9 C
temperature. with a stabilization free energy of only 7 kcal mol1 ,
In mechanism 1 the higher stability acquired would but still has a Tm of 98.9 C (at pH 5.5) (27, 31). At
require higher absolute values for Hu and Su at Tm 80 C, or close to the optimal growth temperature of S.
than for the mesophilic protein. In the second mechan- solfataricus, the protein has a marginal stability of only
ism the maximal values for G u , Hu , and Su would 2.8 kcal mol1 . Similar stabilities have also been
be of the same magnitude as for the mesophilic protein described for another related small DNA-binding pro-
but would occur at different temperatures: i.e., at tein, Sac7d from S. acidocaldarius (28). These proteins
higher temperatures the thermophilic protein would seem to be thermodynamically stabilized by mechan-
be more stable, whereas the mesophilic protein would ism 3 as discussed above. Since Cp determines the
be more stable at lower temperatures (34). Mechanism curvature of stability curves [Eq. (1)], the small size
3 also predicts that the thermodynamic stability of the of these proteins and the correspondingly low Cp s
thermophilic protein would not be higher than of the (40), have the effect to create shallower protein stabi-
mesophilic protein, but lower temperature dependence lity curves, with broader G maxima, that extend the
of G u for the thermophilic protein, possibly because Tm values to higher temperatures.
of lower entropy of unfolding, would lead to a at- Among cold-adapted proteins only an -amylase
tened stability curve resulting in higher Tm values, as from the psychrophile Alteromonas haloplanktis has
well as lower cold denaturation temperatures. From been characterized with respect to thermodynamic sta-
the relatively few cases available where the thermody- bility (41). The enzyme exhibits a reversible, two-state
namic stability of thermophilic proteins has been char- thermal unfolding with a melting temperature at
acterized, it seems that mechanisms 1 and 3 most often 43.7 C, signicantly lower than its mesophilic counter-
prevail. For phosphoglycerate kinase, both from parts. The measured thermodynamic parameters were
Thermus thermophilus and Thermotoga maritima, used to calculate a protein stability curve according to
mechanisms 1 and 3 both seem to be at work in stabi- Eq. (1) for the enzyme, which is depicted in Figure 3,
lizing the enzymes (36, 38), and the same seems to be and compared to such curves for several small (<18
the case for ribonuclease H from T. thermophilus (35) kDa) proteins from meso- and thermophiles (including
and ferredoxin from T. maritima (29). Sso7d and Sac7d). When the parameters were calcu-
Greater thermodynamic stability according to lated and compared on per mol residue basis, to
mechanism 1 was found to stabilize the catalytic account for the different sizes of the proteins involved,
domain of cellulase E2 from Thermomonospora fusca it is clear that the cold-adapted enzyme is of much
when compared to the analogous domain of cellulase lower stability, characterized by lower absolute values
CenA from the mesophile Cellulomonas mi (34). The for both enthalpy of unfolding and Tm (Fig. 3) (41).
thermostability of the small DNA binding proteins The reverse of mechanism 1 above is apparently in
Sso7d (27, 31) and Sac7d (28) from Sulfolobus spp. effect in the cold adaptation of the enzyme, which is in
appear on the other hand to be enhanced by attened line with the hypothesis that cold-adaptive mechanisms
stability curves in accordance with mechanism 3 involve weakening of intramolecular interactions in
(although data on corresponding mesophilic proteins order to increase exibility (1). A most interesting fea-
were not provided in these studies). A lateral shift in ture of the stability curve for the psychrophilic enzyme,
Tmax values to higher temperatures (mechanism 2), in and what differs from meso- and thermophilic pro-
concomitance with raised Gu values, has also been teins, is that the physiological temperatures lie on dif-
observed in studies on thermal unfolding of cyto- ferent sides of Tmax on the parabolic curve (Fig. 3). As
chrome c552 from T. thermophilus (37) and with both Su and Hu change sign and become negative
archaeal histones (32). at or within few degrees below Tmax , (26), low and high
When considering protein thermal stability, it is temperature adaptation may involve different adjust-
important to observe that depending on the absolute ments in these thermodynamic parameters. As Hu
values for the thermodynamic parameters (H, S, becomes negative on the cold side of the stability
and Cp ), high Tm is not necessarily synonymous curve maximum, destabilization of the native protein
with larger maximum stability (39). This may be espe- occurs for enthalpic reasons, whereas it is stabilized by

IV. MOLECULAR BASIS OF
EXTREMOPHILIC TEMPERATURE
ADAPTATION
It is clear from several studies that proteins from psy-

chro- or thermophilic organisms do not exhibit any
peculiarities with respect to composition, size, or com-
plexities as compared to their counterparts adapted to
moderate temperatures. They are composed of the
same 20 natural amino acids and usually show high
degree of sequence and structural homology with
mesophilic proteins (1, 3, 31). Extremophilic adapta-
tion is therefore an intrinsic property of the proteins,
Figure 3 Protein stability curves for -amylase from the
and the reasons for the striking differences observed
Antarctic psychrophile Alteromonas haloplanktis (heavy both in stability and function at different temperatures
lines), selected mesophilic proteins (continous lines), and of are encoded in their gene sequences. A number of stu-
the thermophilic proteins Sac7d and Sso7d (dashed lines). dies have therefore been carried out attempting to cor-
The Gibbs free energy of denaturation is presented in specic relate different structural factors, at all levels of the
units (per mole residue) in order to account for the different structural hierarchy of the proteins, to differences in
sizes of the proteins compared. By increasing order of Tm , the temperature-adaptive properties (mainly thermal stabi-
curves are for the proteins: psychrophilic -amylase, T4 lyso- lity) in comparative studies of homologous proteins
zyme, barnase, RNase T1, RNase A, spectrin SH3, barstar, from organisms isolated from different temperature
phosphocarrier HPr, chymotrypsin inhibitor CI2, protein G habitats. By far the greatest number of such compara-
IgG binding domain, ovomucoid third domain, thioredoxin,
tive studies have focused on proteins from thermo- and
ubiquitin, and the thermophilic proteins Sac7d and Sso7d.
(From Ref. 41. Copyright 1999 American Chemical Society.)
hyperthermophiles relative to their mesophilic counter-
parts in attempts to elucidate the molecular causes for
their high thermal stability. In the following discussion
the negative TS contribution as the temperature is we will attempt to summarize some of the pertinent
lowered. At higher temperatures, entropic dissipative observations made in such comparative studies on pro-
forces increase faster than favorable enthalpic contri- teins from different temperature habitats.
butions, and hence heat denaturation occurs (11).
As a result of the different thermodynamic forces A. Proteins from (Hyper-)thermophiles
involved, it is apparent that at the molecular level the
causes of destabilization of native proteins to cold and Thermophilic organisms are conventionally classied
heat denaturation may be quite different (42). as those having optimum growth temperatures
Probably the main cause of cold denaturation is the >60 C and hyperthermophiles those with optimal
weakening of the collective waterapolar repulsions growth >80 C (1, 19). While thermophiles are either
that provide the main driving force for maintaining bacteria or Archaea, most hyperthermophiles are
the folded structure under physiological conditions Archaea (45). In earlier comparative studies on ther-
(42). Indeed, cold denaturation has been found to mostabilization, attempts were made to correlate struc-
occur most readily with the most hydrophobic proteins tural factors based on the content of certain amino
(43, 44). An additional cause of cold destabilization of acids and derived parameters such as hydrophobic
proteins is more favorable energetics of interactions of and aliphatic indices (see 4 and reference therein for
water with polar and ionic groups at low temperatures review), to thermal stability of relatively small number
(42). In this respect favorable interactions of water of meso- and thermophilic proteins.
with the polar peptide groups, largely buried in the Argos and coworkers (46) carried out the rst sys-
native structure, but that come into contact with tematic study on sequence comparison of homologous
water on unfolding, seem to play a pivotal role (44). proteins from meso- and thermophiles and identied
Thus, the fact that water increasingly becomes a good certain amino acid exchanges that occurred more fre-
solvent for buried polar and apolar residues at low quently in going from the meso- to thermophilic repre-
temperatures appears to be a major driving force for sentative within the three protein families they
cold denaturation (44). compared. The objective of these and other subsequent

studies, which have included more proteins in their pressure to reduce the number of residues that can
database (47), has been to identify certain trafc cause irreversible thermal denaturation of the proteins
rules used for thermostabilization of proteins in nat- (19, 52).
ure. The effect of the thermostabilizing amino acid Some of the most frequent amino acid replacements
exchanges identied in these studies showed a tendency that lead to a decrease in polar uncharged residues in
toward increasing surface hydrophilicity, decreased the proteins of Methanococcus jannaschii contribute
exibility, and increased hydrophobicity, mainly in - signicantly to increased hydrophobicity (Ser ! Ala,
helical regions of the proteins (46, 47). Critical evalua- Thr ! Ile, Ser ! Pro, Thr ! Val) (53). Moreover,
tion of these early studies, however, showed that the most frequent nonpolar to nonpolar replacements
data set used was too small to make predictions based (e.g., Leu ! Ile, Gly ! Ala, and Met ! Leu) tended
on such compositional comparisons statistically signif- toward bulkier and more hydrophobic residues (53).
icant (48). The hydrophobic interaction has been emphasized as
Several recent studies, including extensive genomic the major stabilizing force for the folded state of pro-
comparisons of meso- and thermophiles, however, teins (11, 12, 57). Strengthening of hydrophobic inter-
have pointed out certain trends in amino acid compo- actions and improved packing in the hydrophobic core
sition and exchanges that have correlated with tem- have indeed been suggested to play an important sta-
perature adaptation, some of which had also been bilizing role in thermophilic proteins (31, 45, 49, 51, 53,
observed in those earlier studies. These include higher 58, 59). In some other comparative studies, however,
content of charged amino acids, especially Arg and no signicant correlations between hydrophobicity and
Glu in hyperthermophiles, often occurring concomi- thermophily have been observed (50, 52). Mutation
tantly with lower content of polar uncharged amino studies have shown that increasing the bulkiness of
acids (4954). In a study where 115 protein sequences buried nonpolar side chains, as discussed above, can
from the genome of the extreme thermophilic archaeon signicantly increase thermal stability of proteins (by
Methanococcus jannaschii were compared with their 1.3 kcal mol1 per CH2 residue buried), by a direct
homologs from several mesophilic Methanococcus hydrophobic effect and by reducing the size of cavities,
spp., the strongest correlation with thermophiles of and hence optimized core packing through, e.g., more
observed amino acid exchanges was the decrease in favorable van der Waals interactions (6064). Indeed,
the content of uncharged polar residues (Ser, Thr, increased residue volume (53) and smaller cavities (54)
Asn, and Gln), besides an increase in charged residues, have been found to correlate well with hyperthermo-
increase in residue hydrophobicity, and increased resi- philic adaptation (see below).
due volume (53). Nearly every other amino acid was The increased number of high-resolution crystal
preferred over polar uncharged amino acids in the structures has allowed more direct structural compar-
thermophilic sequences. isons, where three-dimensional structures of thermo-
A similar trend was observed in sequences deduced philic proteins are being compared to corresponding
from the complete genome of the hyperthermophilic structures of one or more of its mesophilic counter-
bacterium Aquifex aeolicus (50) and in a study where parts (6583). More detailed structural data have
sequences of soluble proteins from complete genomes also facilitated systematic studies where various struc-
of eight thermophiles (including those of M. jannaschii tural parameters have been calculated on the basis of
and A. aeolicus) and 12 mesophiles (51). Moreover, known crystal structures or computer generated
Asn and Gln seem to be more discriminated against homology models from related sequences, and com-
in proteins from hyperthermophiles, as compared to pared within or across protein families (4654, 59,
those from moderate thermophiles, while the decrease 8489). Querol et al. (58) have also surveyed the litera-
in Ser was apparently signicant in moderate thermo- ture on studies reporting thermostabilizing mutations
philes (54). The trend to lower content of Asn and Gln in several different proteins and analyzed the structural
likely reects molecular adaptation in thermophilic consequences of those stabilizing single amino acid
proteins as it would tend to minimize deamidation, a replacements. They created a database comprising
reaction that may limit the thermal stability of the 195 single amino acid exchanges in 164 different posi-
proteins as a result of irreversible denaturation (55, tions in the proteins which had been unambigiously
56). A decrease in Cys, another thermolabile amino related to thermostability enhancement (58). The
acid residue, has also been reported for thermophilic most cited physicochemical reasons for thermostabili-
proteins when compared to their mesophilic counter- zation as observed in this survey are summarized in
parts, which may also be indicating an evolutionary Table 1.

Table 1 Physicochemical Reasons for Enhanced Thermal results as compared to those of Argos and coworkers
Stability Resulting from Single Amino Acid Replacements (49, 85) to some bad-quality or incomplete crystal
in Several Different Enzymes as Cited in the Literature structures (ve of which were mesophilic, but only
Number one thermophilic) that had been included in the data-
of bases used in the former study that introduced a bias in
Type citations the results. If these bad-quality structures had been
omitted from the database used by Argos and cowor-
Better hydrogen bonding 18 kers, the observed difference in the number of hydrogen
Better hydrophobic internal packing 16 bonds between meso- and thermophilic proteins would
Enhanced secondary structure propensity 12 have become insignicant (54).
Helix dipole stabilization 10
Szilagyi and Zavodsky (54) calculated 13 properties
Argos replacements 10
in ve categories (cavities, hydrogen bonds, ion pairs,
Removal of residues sensitive to oxidation or 10
deamidation secondary structure, and polarity of surfaces) from the
Burying hydrophobic accessible area 7 coordinates for each of the proteins in the database,
Improved electrostatic interactions 6 compared them using statistical methods, and corre-
Strengthening of intersubunit association 6 lated with optimal growth temperatures of organisms
Decreased chain strain 5 they originate from. On the basis of the optimal
Salt bridge optimization 4 growth temperatures the proteins were divided into
Better van der Waals interactions 3 two groups; moderately thermophilic (Topt 45
Better afnity for calcium 2 80 C), and extremely thermophilic proteins
Improved H upon substitution 1 (Topt 100 C). The ndings of this comprehensive
Source: Ref. 58. (Copyright 1996 Oxford University Press.) study are summarized in Table 2.
The most signicant difference between mesophilic
and thermophilic proteins was found in the number of
In line with these observations, Argos and cowor- ion pairs (salt bridges), a property that also correlated
kers (49, 85) found, when comparing several structural well with growth temperatures. In fact, several sys-
factors of meso- and thermophilic proteins from 16 tematic comparative studies that have been described
families, that the parameter that most consistently cor- above (49, 51, 52, 59, 85, 88, 89), as well as compar-
related with increased thermal stability was a higher isons of high-resolution three-dimensional structures
number of hydrogen bonds in the thermophilic proof thermophilic and homlogous proteins (69, 72, 73,
teins. An increase in the fractional polar surface area, 7577 79. 81, 90), have pointed to a prominent role
reecting added hydrogen bonding density to water, of ion pairs in their stabilization, especially of
was also found to increase with the thermostability hyperthermophilic proteins. The ion pairs often appear
of the proteins compared (49, 85). In other extensive in the hyperthermophilic structures as clusters or net-
comparative studies of meso- and thermophilic protein works at the surfaces or at interdomain interfaces in
structures, an increase in the number of residues the proteins (Fig. 4). In some studies no correlation has
involved in hydrogen bonding (51) and an increase in been observed between the number of ion pairs, or ion
side chainside chain hydrogen bonds (88) were found pair networks, and stability of hyperthermophilic pro-
to correlate with thermostability. Additional hydrogen teins (80, 88). Indeed, the role of ion pairs in protein
bonds have also been pointed out as a major stabilizing stability has been controversial, as theoretical calcula-
factor from comparisons of known crystal structures tions have indicated that they may actually destabilize
of meso- and thermophiles (70, 72, 77). folded conformations of proteins, at least at room tem-
In a detailed comparative study of high-quality perature (91).
three-dimensional structures of 64 mesophilic and 29 The destabilizing effect of ion pairs or salt bridges
thermophilic proteins (of which ve were hyperthermo- arises primarily from desolvation penalty associated
philic) representing 25 protein families, however, prac- with bringing separated charged groups of the
tically no difference between mesophiles and unfolded protein together to form an ion pair in the
thermophiles was observed in the number of hydrogen fully folded protein (92). The desolvation penalties are
bonds (54). There was, however, a tendency to decrease especially high when burying charged groups in the low
the number of unsatised hydrogen bond acceptors and dielectric environment of the hydrophobic core of pro-
donors as the temperature of adaptation increased. The teins (52, 88, 92). Despite the favorable coulombic
authors attributed the discrepancy observed in their interaction that incurs by bringing oppositely charged

Table 2 A Summary of the Most Signicant Differences Between Structural Parameters of Moderately Thermophilic and
Extremely Thermophilic Proteins When Compared to Their Mesophilic Counterpartsa
Correlation with Change in moderately Change in extremely

Property temperature thermophilic proteins thermophilic proteins
Cavities Number ## 0 ###

Volume # " #
Area # " ##
Hydrogen bonds Number 0 0 0
Unsatised # # #
Ion pairs <4.0 A "" " """
<6.0 A "" "" """
<8.0 A """ """ """
Secondary structure 0 " 0
b " 0 ""
Irregular # # #
Polarity of surfaces Exposed ## """ 0
Buried 0 " "
a
Upward arrows refer to a positive value and downward arrow refer to a negative value for correlation with the structural parameter indicated;
zeros refer to near-zero correlation values. The number of arrows (1, 2, or 3) shows whether the represented correlation is considered insignicant,
moderately signicant, or highly signicant.
Source: Ref. 54. (Copyright 2000 Elsevier Science.)
Figure 4 A schematic representation of an ion pair network cluster in glutamate dehydrogenase from Pyrococcus furiosus. The
twofold axis relating to a CC# dimer is indicated by a diad. Potential ion pair interactions are indicated by dashed lines. The
backbone of the polypeptide chain is shown as a gray ribbon. The individual amino acids are indicated by their one letter code,
followed by their sequence number. (From Ref. 79. Copyright 1998 Blackwell Science Ltd.)
groups together in the folded state, it may not out- less unfavorable, so ion pair formation would become
weigh the unfavorable desolvation (free energy of increasingly favorable at higher temperatures and
hydration) cost. Elcock (92) has recently shown that could provide a signicant stabilization to hyperther-
at higher temperatures, the desolvation effect becomes mophilic proteins at elevated temperatures. It has also

been shown that the location of the ion pair within the and optimized packing of side chains in the interiors,
protein structure, especially with respect to positioning and the absence of cavities have been observed in crys-
of other ion pairs in its vicinity, is a critical determi- tal structures of hyperthermophilic citrate synthase
nant of the stability of these interactions, as they may (73) and lactate dehydrogenase (75).
in a cooperative manner facilitate the formation of ion Increasing structural compactness by decreasing
pair networks (52, 88). cavities and increasing attractive interactions in the
As mentioned earlier, extensive ion pair networks tightly packed protein core reect a trend observed in
have been observed in three-dimensional structures of structures of some hyperthermophilic proteins, i.e., to
several hyperthermophilic proteins. Such cooperative minimize their surface area-to-volume ratio (68, 72).
electrostatic interactions offer simple means of enhan- Minimization of the ratio of surface area to volume
cing stability without disrupting core residues, charac- can stabilize a protein by simultaneously reducing
teristics of different protein families (88). Such unfavorable surface energy and increasing the attrac-
optimally located ion pair networks are therefore tive interior packing interactions (68, 72, 75).
believed to be important stability determinants of pro- Reduction of accessible surface area seems to be part
teins from hyperthermophiles. of the molecular inventory that has been used in the
A signicant observation in the study of Szilagyi high-temperature adaptation of hyperthermophilic
and Zavodsky (54) was a decrease in the number of proteins. At the quaternary structural level this is
internal cavities in the structures of extemely thermo- observed as higher states of subunit associations in
philic proteins, as compared to mesophilic and moder- oligomeric proteins from hyperthermophiles, e.g.,
ately thermophilic proteins (Table 2). A decrease in the triose phosphate isomerase (10, 71), phosphoribosyl
number or the size of cavities leading to improved anthranilate isomerase (76), ornithine carbamoyltrans-
packing in the protein interior has also been observed ferase (78), enolase (10), and in the case of triose phos-
in the structures of hyperthermophilic proteins (31, 67, phate isomerase and phosphoglycerate kinase from
73, 93). However, in a study where the packing density Thermotoga maritima that are expressed as a dimer
of 80 nonhomologous proteins and 24 thermophilic of dimers fusion protein (1, 10, 74). Minimizing
ones was analyzed in context of their possible role in hydrophobic surface area has also been suggested to
thermal stabilization, essentially no difference was be an important stabilizing principle in thermophilic
observed in either partial specic volumes or cavity lactate dehydrogenases (75).
volumes in the two protein groups (86). Cavities cre- Increased compactness of thermophilic proteins
ated in the interior of proteins by use of site-directed may also be accomplished through shortened surface
mutagenesis have been shown to lead to signicant loops (45, 70, 73, 74, 83, 87). Exposed surface loops
destabilization (6062). Creation of a cavity in barnase and turns link successive core elements and are
of the size of a CH2 group (Ile96 !Val) resulted in a believed to be the regions of proteins where their
destabilization of the enzyme of 1.1 kcal mol1 and a unfolding begins (45, 87). Increasing the structural
cavity the size of three such groups (Ile96 ! Ala) by rigidity in such loops, either by shortening them or
4.0 kcal mol1 (60). by stabilizing them by amino acid replacements (e.g.,
In a study of several cavity-creating mutants of T4 by increasing the number of prolines), could therefore
lysozyme, the destablization of a Leu !Ala substitu- signicantly enhance protein stability (45, 87).
tion amounted to a constant term, 2 kcal mol1 , plus In some studies thermophilic proteins have also
a term that increased in proportion to the size of the been found to be smaller than mesophilic counterparts
cavity produced by the substitution (61). The constant (51, 87), reecting truncation of loops, but also of N-
term is approximately equal to the transfer free energy and C-termini as has been observed for some thermo-
of leucine relative to alanine as determined from their philic proteins (77, 80)). It has been pointed out that
partitioning between aqueous and organic solvents the apparently smaller size of thermophilic proteins
(61). These results point to ways to stabilizing proteins may reect a possible thermodynamical adaptive strat-
against high temperatures, i.e., by lling cavities in egy of these proteins (51). As smaller proteins tend to
their core with bulkier, more hydrophobic amino have lower Cp values of unfolding (40), the shape of
acids, leading to enhanced hydrophobic driving force the stability curve could be affected, as its curvature is
and improved van der Waals contacts in the protein determined by Cp =T (see earlier). The net effect
core. As discussed previously, amino acid replacements could therefore be to atten the stability curve (Fig.
of this type have frequently been observed in compara- 2), bringing the denaturation temperature to higher
tive studies of mesophilic and thermophilic proteins, values (51).

B. Proteins from Psychrophiles catalytic efciency under nonsaturating substrate con-
ditions kcat /Km ), since for regulatory purposes most
Many microorganisms and ectothermic animals live intracellular enzymes have Km values approaching
under environmental temperatures that uctuate in the physiological concentrations of their substrates
the range 2 C to 10 C without the opportunity to (95). Then the rate of reaction will vary with oscillation
regulate their cellular temperature (9496). The term in substrate concentration, which gives a way of con-
cold-adaptation refers to the chemical and physiologi- trolling the reaction. Since temperature affects sub-
cal mechanisms that make this possible (97, 98). strate afnity constants, cold-adapted enzymes are
Without adaptation, the chemical enzyme systems of frequently found to have higher Km values than meso-
cold-blooded animals and psychrophilic microorgan- philic counterparts at common assay temperatures of
isms would work too slowly. Enzymes from psychro- 25 C or 37 C.
philic organisms (max. growth temperature at 20 C) or At rst sight, one might therefore expect ortholo-
psychrotrophic organisms (max. growth temperature gous enzymes from organisms with different tempera-
at 40 C) (6) are generally less stable structures than ture optima for their existence to have some obvious
comparable enzymes from warm-blooded or thermo- amino acid substitutions in their active sites. However,
philic organisms (96). This is true for enzymes from active-site residues are usually strictly conserved in psy-
as diverse species as bacteria (16) or sh (7, 98, 100). chrophilic enzymes (18, 102105). This has put the
The need for increased exibility is envisioned to focus for cold adaptation at the molecular level on
call for fewer noncovalent intramolecular interactions the rather loose concept of exibility in protein struc-
as temperatures are lowered (16, 98, 100). This led to ture as a critical factor for improving catalytic ef-
the concept of corresponding states of enzymes at ciency. Indeed, changes in the amino acid sequence
the temperatures in which they nd themselves in the distant from the active site often seem to determine
various living forms on earth (97, 98). In recent years the outcome of temperature adaptation, making
the study of cold adaptation of enzymes has rational design for altered kinetic properties a much
expanded greatly. As more examples accumulate, more difcult proposition than initially hoped for.
however, few generalizations can be made as several Higher exibility of an enzyme molecule should
strategies are used differently in individual cases. Here increase the available number of conformational states,
we will summarize kinetic and stability characteristics and therefore the best conformations for ligand binding
of cold-active enzymes and then give the general con- would become rarer than in a more rigid structure.
cepts used in an attempt to explain temperature adap- Consequently, ligand binding would become poorer,
tations in molecular terms. Several reviews on with higher Km values for substrates and higher Ki
pyschrophilic enzymes have appeared recently (7, 16, values for competitive inhibitors. Early studies estab-
17, 96, 99, 101). lished an inverse relationship within a group of homo-
logous enzymes between the Km value and the working
temperature of individual enzymes (95). Km values for
1. Enzyme Activity at Low Temperatures
enzymes from cold-adapted organisms were higher
As suggested earlier, the selective pressure for evolu- than for mesophiles, when the Km values were deter-
tionary adaptation of enzymes to low temperatures is mined at one common standard temperature (98). This
probably geared more toward optimizing activity than is to be expected, since the evolution of maximal cata-
stability, given that the kinetic properties are somehow lytic rate (high kcat ) can be predicted to be directed
related to the dynamic properties of the protein mole- toward strong binding of the transition state and
cules. It is therefore of interest to examine how cold weak binding of the substrates in ground statesi.e.,
activity is expressed in terms of the kinetic parameters directing the trend toward higher Km values (106).
(Michaelis-Menten) for the enzymes. The nal result of As the goal is often to optimize kcat /Km , kcat values
low temperature adaptation will surely be dependent must increase in psychrophilic enzymes (98, 101). This
on what properties the cold-adapted organism has to would have to occur by improving the rate-limiting
achieve from each enzyme; some extracellular enzymes step of the enzyme-catalyzed reaction under considera-
presumably work at very low substrate concentrations tion, be it binding of substrates, the chemical conver-
and would benet the organism by having low Km sion steps, or the release of products. The most
values, whereas enzymes that work in abundant sub- common situation is a two- to vefold increase in
strate could optimize the kcat rate. Some restrictions kcat , with less prominent changes in Km . There are,
may apply to lowering Km values in order to increase however, notable exceptions, such as in the case of

trypsins from trout, Atlantic cod, Atlantic salmon, and over a wide range of temperatures. The authors pro-
krill (100, 107109). In those cases one isozyme showed posed that adaptation of this class of endocellular
10- to 25-fold improvement in kcat /Km in comparison enzymes to cold environment might only have made
with bovine trypsin, that was to a large extent due to them less stable and not more efcient (114), which is
lowering of the Km value to one-tenth the value mea- a rare outcome. Another interesting group of enzymes
sured for trypsin from the warm-blooded animal. for consideration in the context of temperature adap-
Although small synthetic substrates have mostly been tation are those which have reached the state of diffu-
used in studies of many enzymes with natural poly- sion-controlled reaction rates, such as -lactamase and
meric substrates, being the only choice for Km deter- triose phosphate isomerase (101). Such enzymes have
minations, at least twofold increase in catalytic rate is optimized the activation energy hills throughout the
also seen with natural substrates such as polypeptides reaction pathway to the extent that it is difcult to
(7, 110), or starch (111). improve kcat further when the organisms are chal-
In an early study, the relative importance of the lenged with low temperatures. The higher specic
enthalpic and entropic contributions to the activation activity expected as a result of cold adaptation was
free energy of catalysis (G6 ) was found to differ not observed for beta-lactamase from the Antarctic
between enzymatic reactions of cold-adapted ectother- psychrophilie Psychrobacter immobilis, although it
mic enzymes and homologous enzymes of warm- was very thermolabile in comparison with a mesophile
blooded birds and mammals (112). The psychrophilic (115). As argued from directed evolution experiments
enzymes often have lower enthalpies of activation (see below), there may be no evolutionary pressure for
(H 6 ) and reactions are consequently less temperature thermostability at low temperatures, and this last
driven. Furthermore, the entropic terms (S 6 ) with example would indicate that increasing global heat
psychrophilic enzymes is less positive, which may indi- lability is not always sufcient for improving the cata-
cate looser enzyme-substrate contacts, either to the lytic efciency at low temperatures (101, 115).
ground-state ES complex and/or to the transition- A kinetic comparison of three DNA ligases adapted
state ES* complex (101). As noted before, weaker to different temperatures (Pseudoalteromonas halo-
net binding energy for substrate is one way to increase planktis, E. coli, and Thermus scotoductus) indicated
reaction rates in enzymes; i.e., most of the initial bind- that an increase in kcat is the most important adaptive
ing energy is offset against energetically costly prepara- parameter for enzymatic activity at low temperatures,
tive events, and this avoids a thermodynamic pit in whereas a decrease in Km allowed T. scotoductus DNA
front of the main energy transition curve (106). This ligase to work efciently at high temperatures (116).
includes events such as stopping the entropic move- Km values of psychrophilic enzymes are indeed often
ments of substrates present in solvent, removing hydra- higher compared with a matching mesophilic enzymes
tion shells off substrates, and possibly juxtaposing (101), although a number of exceptions exist. In the
charges of substrates against similar charges in the case of phosphoglycerate kinase from an Antarctic
walls of the active site. Some of the catalytic power Pseudomonas sp., which notably is an intracellular
of enzymes is derived from their abilities to promote metabolic enzyme, both an increase in kcat and a
formation of reactive conformers of substrates that decrease in Km were observed. This double strategy,
precede the transition state (113). Random thermal however, only increased catalytic efciency twofold,
motions of the enzymesubstrate complex would even- compared with a mesophilic enzyme (117). For alka-
tually cause transformation of those conformers into line phosphatases from either a psychrophilic Vibrio
transition states. These dynamic motions might be bacterium or cold-adapted sh, faster product release
initiated at a distance from the active site. Although (lower afnity for the phosphate reaction product) has
correlated motions appear to participate in catalysis, been suggested as the underlying cause for greater cat-
there is still little evidence as to how enzymes use such alytic rates (118, 119). The Km values for the chosen
motions (113); therefore, the promotion of exibility substrate were identical in the former case and lower in
by amino acid substitutions in cold-adapted enzymes the latter case, in comparisons with mesophilic
remains enigmatic. enzymes. From these examples it becomes apparent
kcat values are expected to be higher for an enzyme that various strategies are employed in cold adaptation
isolated from organisms classied as psychrophilic, as reected in Km values, whereas kcat is (nearly)
although there are exceptions. For aspartate amino- always increased. The choice of substrates and/or
transferase from an Antarctic bacterium, kcat was sig- reference mesophile may affect the outcome of such
nicantly lower than determined for the E. coli enzyme in vitro comparisons.

2. Relationship Between Cold Activity and lished (122). The digestive enzyme trypsin is among the
Protein Stability most extensively studied proteins, and in this study 27
trypsin sequences were analyzed from a variety of
For cold-adapted enzymes, increased activity at low organisms to nd unique attributes. The amino acid
temperature is usually reected in low thermal stabili- compositions indicated a high number of charged (pre-
ties. This has been found by comparative studies of dominantly acidic groups) and polar residues, and
natural variants and further corroborated by site- fewer hydrophobic residues in the cold-adapted tryp-
directed mutagenesis experiments. However, it is very sins. All of the psychrophilic trypsins were anionic with
interesting that these two apparently interlinked phe- calculated pIs of 5.56.6 (122). The main unique fea-
nomena can be decoupled (24, 120). Mutations have tures of the cold-adapted trypsins attributable to low-
been produced that increase thermal stability while temperature adaptation seemed to be: reduced hydro-
maintaining low-temperature activity (25). Although phobicity and packing density of the core, mainly
still rare, they bode well for possible development of because of a lower (Ile+Leu)/(Ile+Leu+Val) ratio;
biocatalysts for industral applications having both of reduced stability of the C-terminal helix which cross-
those often desirable properties: high specic activity links to the other (N-terminal) domain; lack of one
and thermal stability. Studies have shown that active- proline residue, which is conserved in temperature-
site residues may not be optimally chosen for stability stable trypsins; and a lack of one proline-plus-tyrosine
as demonstrated by mutagenesis of T4 lysozyme (14), stacking interaction. In addition, there was a difference
but rather have individual needs for movement, as in charge and exibility of loops that extend from the
reected in the observation that inactivation of many substrate binding pocket, and a different conformation
enzymes precedes global conformational changes of of the autolysis loop, which is likely to affect sub-
unfolding (121). Thus, functional amino acids are strate binding (122).
likely optimized for a degree of exibility that is appro- Some of these factors have also been cited as possi-
priate for optimal effectiveness, while the rest of the ble structural determinants for cold adaptation in
protein molecule could maintain stability. other studies. Thus, lower values of calculated hydro-
As in the case of thermophilic adaptation, there phobicity indices have repeatedly been observed in
apparently are no set of trafc rules used for all other studies for cold-adapted enzymes in comparison
cold-adapted proteins that are responsible for their to mesophilic partners (100, 102, 111, 115, 123). For
lower conformational stabilities. By sequence compar- instance, the hydrophobicity index for psychrophilic
isons researchers have attempted to spot the crucial bacterial -amylase in comparison with porcine -amy-
substitutions that differentiate psychrophilic proteins lase was sharply decreased concomitantly with 25 sub-
from their meso- and thermophilic relatives, and lately stitutions out of the 84 residues that make up the
also by tertiary structure comparisons of selected pro- central core in that enzyme (111). Exposure of nonpo-
teins. Reduced-temperature stability may not always lar groups to the solvent may provide entropically dri-
result in improved catalytic properties, as mentioned ven destabilization of the protein, and hence increase
above for aspartate aminotransferase from a psychro- the mobility within the structure. This was cited as a
philic bacterium. In contrast to the observations made factor in a-amylase cold-adaptation (124). As the
with many (extracellular) psychrophilic enzymes, kcat strength of the hydrophobic effect decreases as the
and kcat /Km were signicantly lower than those for the temperature is lowered the cold-adapted proteins
E. coli enzyme over the whole temperature range, may rely less on hydrophobic interactions for stability
although the enzyme showed much reduced thermal than proteins that are adapted to higher temperatures.
stability compared with a similar enzyme from E. coli Reduced hydrophobicity may also protect psychrophi-
(114). The reduction in stability points to the fact that lic proteins against cold denaturations, which may be
stability considerations may be disconnected from caused in part by the diminished strength of hydropho-
functional necessities and only become important as bic interactions at low temperatures (125).
the molecule becomes too unstable for the temperature Comparisons of the relative numbers of hydrogen-
range of its operation. However, increasing the stabi- bond forming residues indicate a lower proportion in
lity of a psychrophilic phosphoglycerate kinase led to some psychrophilic enzymes (102, 126). As an example,
drastically reduced turnover number, an indication of trypsin from an Antarctic sh displayed 20% less
some link between these two properties (117). potential for hydrogen bonds than corresponding
An extensive comparison of trypsins from cold- bovine trypsin (123). In the case of triose phosphate
blooded and warm-blooded animals was recently pub- isomerase from the psychrophilic baterium Vibrio mar-

inus, a single amino acid substitution (Ser238!Ala), temperatures were favorable surface charge distribu-
that eliminates two hydrogen bonds in a loop region of tion for substrate and cofactor binding, and reduced
the molecule, could to a lartge extent account for the intersubunit ion pair interactions that increased rela-
cold-adaptation of the enzyme with respect to catalytic tive exibility of active site residues (131). The crystal
activity and thermal stability (127). structure of dimeric citrate synthase from an Antarctic
Valuable information on the potential structural bacterium revealed that the enzyme had much more
determinants of cold-adaptation has come from a few accessible active site than thermophilic counterparts
recent studies where three-dimensional crystal struc- (105).
tures, or homology models for psychrophilic enzymes, The surface electrostatic potential distribution was
have been compared to the corresponding structures unusual, and an increased relative exibility of the
from organisms adapted to higher temperatures. small domain compared to the large domain of the
Based on such tertiary structural comparison, and cor- protein was noted within each monomer.
roborated with data from sequence comparisons, fewer Furthermore, reduced subunit interface interactions
salt bridges (111, 124, 128, 129), reduction in aro- were found with no intersubunit ion pair networks,
maticaromatic interactions (11, 128, 129), extended and loops were of increased length but carried fewer
surface loops (105, 128130), fewer prolines in such proline residues. Lastly, there was an increase in sol-
loops (105, 115, 124, 129, 130), lower hydrophobicity vent-exposed hydrophobic residues and an increase in
(111, 115), weaker calcium bindings (104, 124, 128), intramolecular ion pairs. In comparison, the thermal
improved solvent interactions through additional sur- denaturation of hyperthermophilic dimeric citrate
face charges (105, 115, 128, 130), increased exposure of synthase appears to be resisted by complex networks
nonpolar groups to the solvent (105, 124), and of ion pairs at the dimer interface, a feature common
reduced intersubunit ion pairs (105, 131) have all to several other hyperthermophilic proteins as dis-
been cited as possible reasons for increased exibility cussed previously. Catalytic efciency of the cold-
and/or decreased thermal stability of proteins from active citrate synthase appears to be achieved by a
psychrophiles. more accessible active site and by an increase in the
The rst x-ray crystal structure of an enzyme from a relative exibility of the small domain compared to the
cold-adapted ectothermic organism was that of salmon large domain in the enzyme (105).
trypsin (102). It was followed by a structure of elastase Molecular modeling of various enzymes has given
from the same species (103) and other trypsin isozymes similar information as x-ray structures. Among fea-
(132). Later, the structures of psychrophilic bacterial tures of a psychrophilic subtilisin S41 that might
enzymes have been determinedthose of -amylase induce a more exible and heat-labile conformation
from Alternomonas haloplanktis that lives in were four extended surface loops, a very hydrophilic
Antarctic seawater (104), and citrate synthase from surface (acidic pI of 5.3), and the lack of several salt
another Antarctic bacterium (105). More recently, a bridges and aromatic interactions (128). The difference
structure was solved for a malate dehydrogenase in the free energy of stabilization between subtilisin
form a psychrophilic bacterium isolate from an S41 and a mesophilic subtilisin was found to lie in
Arctic sediment that grows optimally at 4 C (131). the range 67 kJ/mol, suggesting that the balance of
Superposition of the -amylase structure onto that weak bonds is critical for the enzymes exibility.
of porcine pancreatic -amylase showed that the 24 Detailed model comparison of eight trypsin struc-
residues that form the binding subsites for substrate tures was made to seek explanations for the observed
were strictly conserved. Therefore, the cause for differences in properties seen with mesophilic and cold-
increased catalytic efciency had to be sought outside adapted variants (132). Increased substrate afnity of
the catalytic center. For this enzyme, it was suggested the psychrophilic enzymes was traced to a lower elec-
that reduced number of salt bridges, fewer proline resi- trostatic potential of the S1 substrate binding pocket,
dues in loops, fewer arginine-mediated hydrogen and the lack of ve hydrogen bonds adjacent to the
bonds, and lower hydrophobicity in the core were all catalytic triad. Reduced stability of the cold-adapted
contributing. Also, less interdomain interactions were trypsins might result from fewer interdomain bonds,
proposed as a determining factor for the conforma- including hydrogen bonds and salt bridges, as well as
tional exibility that allows efcient enzyme catalysis poorer packing in the core regions of the two domains.
in cold environments (104). Structural comparison sug- A dimeric 3-isopropylmalate dehydrogenase that
gested that the major factors for the efcient catalytic catalyzes the penultimate step in leucine biosynthesis
activity of A. arcticum malate dehydrogenase at low was cloned from a psychrotrophic Vibrio sp., living in

Arctic seawater (129). The elements that were found to tributing factor to efcient catalysis in cold environ-
be destabilizing in this case were a smaller number of ments (124). Also, this type of adaptation has been
salt bridges, a reduction in aromatic interactions, fewer given consideration in the case of Atlantic salmon
proline residues, and longer surface loops (129). The trypsin, where the active site is divided on both
structural model of a psychrophilic phosphoglycerate sides of two relatively rigid b barrels (102). In contact
kinase indicated the same key determinants of its low regions near the catalytic residues, fewer hydrogen
stability (117). bonds were found in the salmon trypsin, and ion
Some of the examples described above are of pairs might be important in the relative orientation
dimeric enzymes, where psychrophilic variants have of the two domains of the molecule, as well as con-
reduced links between monomers. The formation of tributing to greater stability of the bovine variant
dimers, and higher multimers, has often the sole pur- (102).
pose of increasing stability (133), without linked effects
on kinetic properties. This seems to be the case for
several oligomeric enzymes from thermophiles, as we
discussed earlier. Alkaline phosphatase from cold- V. TEMPERATURE ADAPTATION BY
adapted Vibrio bacteria is apparently monomeric SITE-DIRECTED MUTAGENESIS AND
(118, 134) and may thus be an example of the opposite DIRECTED EVOLUTION
trend, since the well-known enzyme from E. coli,
another Gram-negative species, has a very heat-stable Evolution of a particular trait necessitates that a single
dimeric structure. This may be an example where pres- base change can at times lead to a single amino acid
sure to form dimers was not present. Phosphoglycerate substitution that brings about alterations in a proteins
kinase is a monomeric enzyme, except in extremophilic properties, which is benecial to the host. Site-directed
archaea, where the enzyme has been found as a dimer mutagenesis is therefore often employed in tandem
or a tetramer (135, 136). with rational design ideas in order to understand the
Subtle alterations in structure at dimer interfaces functioning of enzymes. Theories derived from the
can produce enzymes with characteristics of being study of a three-dimensional model of a particular
psychrophilic rather than mesophilic or thermophilic. enzyme are utilized to select particular residues for
Horse liver alcohol dehydrogenase contains two tryp- mutation and predict the possible outcome. Thus,
tophan residues per subunit, one on the surface of the experiments with T4 lysozyme showed that even a sin-
catalytic domain and the second buried in the inter- gle mutation in the hydrophobic core of an enzyme can
face between the subunits of the dimer. After substi- have dramatic effects on stability (138). This may be
tuting the tryptophan at the interface with a leucine, informative, but it does not necessarily cover all the
and making two compensatory mutations that were parts of the molecule that can affect its properties.
required to obtain a stable protein, the turnover num- Attention is often directed at the active-site region
bers for ethanol oxidation, acetaldehyde reduction, and at internal residues, whereas more distant residues
and the dissociation constants of the coenzymes may cause functional alterations, even those that reside
increased by two- to sixfold (137). The three substitu- on the outside surface of the molecule.
tions at the dimer interface apparently activated the Studies using site-directed mutagenesis, however,
enzyme by allowing more rapid conformational have uncovered the fact that thermostability is not
changes that accompany coenzyme binding, probably systematically inversely related to specic activity,
owing to movement of a loop. A subunit interface one example being subtilisin excreted by an Antarctic
was also the site of a single amino acid substitution Bacillus TA39 (139). The enzyme displays the usual
that brought different stabilities of temperatureKm characteristics of cold-active enzymesi.e., a high cat-
relationships to a related group of lactate dehydro- alytic efciency at low temperatures and an increased
genases (95). Furthermore, many enzymes, although thermosensitivity. The afnity for calcium is also
monomeric, can be viewed as aggregates of folding almost 3 orders of magnitude lower than that of meso-
domains. Interdomain interactions could contribute philic subtilisins. An important stabilization of the
to greater movement in cold-active enzymes, allowing molecular structure was achieved through a modica-
more efcient enzyme catalysis in cold environments. tion of one residue acting as a calcium ligand. The
In fact, fewer interdomain interactions were found in thermostability of the mutated product expressed in a
-amylase from a psychrophilic bacterium than in mesophilic Bacillus reached that of mesophilic subtili-
porcine pancreatic amylase, which was likely a con- sin, and this mutation further enhanced the specic

activity by a factor close to 2 when compared to the perature activity, were very rare. The results suggested
wild-type enzyme. that stability at high temperatures was not incompati-
Single-base mutations do not cover a large fraction ble with high catalytic activity at low temperatures
of protein sequence space since they often produce because of perceived mutually exclusive demands on
conservative substitutions. Another approach that enzyme exibility (144).
increases nonconservative substitutions is that of direc- Low-temperature activity can also be generated by
ted evolution, where rapid screening procedures are mutations. It was possible to improve catalytic activity
combined with random mutagensis and in vitro recom- at 37 C of a thermophilic enzyme with a single or
bination. Structural analysis of selected mutants can double amino acid substitution. DNA shufing was
then bring about understanding of the observed phe- used to mutate indoleglycerol phosphate synthase
notype, be it stability or catalytic activity, in terms of from the hyperthermophile Sulfolobus solfataricus
chemical function. The vast majority of possible evolu- (145). The parental enzymes turnover number at
tionary paths lead to poorer enzymes, so for successful room temperature was limited by the dissociation of
directed evolution, the strategic challenge is to choose the enzymeproduct complex, apparently because the
the right path that will eventually improve the desired loops that obstruct the active site were not exible
features (140). enough at low temperatures. In the variants, the bind-
Recent examples can be found where the stability of ing and release of product was much more rapid, and
enzymes from psychrophiles or mesophiles has been this shifted the rate-determining step to a preceding
increased without effects on activity. A moderately chemical step (145). Similarly, multiple random
stable thermolysin-like metalloprotease from Bacillus mutants of b-glucosidase from the hyperthermophile
stearothermophilus was made hyperstable by a limited Pyrococcus furiosus were screened for increased activ-
number of mutations. An eightfold mutant enzyme ity at room temperature (146). Multiple variants were
had a half-life of 2.8h at 100 C, but still displayed identied with up to threefold increased rates of sub-
wild-type-like activity at 37 C (24, 141). Subtilisin E strate hydrolysis. Amino acid substitutions were wide-
from a mesophilic Bacillus subtilis was converted into spread, occurring in the active-site region, at the
an enzyme functionally equivalent to its thermophilic enzyme surface, buried in the interior of the mono-
counterparts by directed evolution (142). Subtilisin E mers, and at subunits interfaces. Interestingly, low-
differs from thermitase at 157 amino acid positions. temperature activity was achieved in different ways,
However, only eight amino acid substitutions were suf- by altering substrate specicity or by overall destabli-
cient to convert subtilisin E into an enzyme equally zation of the enzyme. Single amino acid substitutions
thermostable, a result from ve generations of DNA were sufcient to drastically alter the kinetic proper-
alterations. Interestingly, the eight substitutions were ties, as would be expected, if evolutionary processes are
distributed over the surface of the enzyme. Only two of to work. The enzyme was able to accommodate in its
those are found in thermitase. Also, thermostability interior amino acids with larger or smaller side chains,
could be increased without compromising enzyme and with different properties without affecting thermo-
activity (142). stability. Substrate specicity was also determined by
In another study, thermoresistance was engineered substitutions distant to the active site.
into bacterial b-glucosidase by a directed-evolution The stability of enzymes from psychrophiles can
strategy (143), whereas no signicant alterations in also be increased without reducing activity (25). The
kinetic parameters were observed. The main factors psychrophilic protease subtilisin S41 was subjected to
for increasing the thermostability were a combination two different selection pressures. The evolved subtili-
of one extra salt bridge, replacement of a solvent- sin S41 retained its psychrophilic character as a cata-
exposed asparagine residue, stabilization of the hydro- lyst in spite of its dramatically increased
phobic core, and stabilization of the quaternary struc- thermostability. These results demonstrated that it is
ture. An earlier study used in vitro evolution to probe possible to increase activity at low temperatures and
the relationship between stability and activity in a stability at high temperatures simultaneously. It was
mesophilic esterase. Six generations of random muta- concluded that the fact that enzymes displaying both
genesis, recombination, and screening, stabilized properties are not found in nature, most likely reects
Bacillus subtilis p-nitrobenzyl esterase signicantly the effects of evolution rather than any intrinsic phy-
without compromising its catalytic activity at lower sico-chemical limitations on proteins (25). However,
temperatures. It was also found that mutations that the dependence of slower thermal inactivation on cal-
increased thermostability, while maintaining low-tem- cium concentration indicated that enhanced calcium

binding was largely responsible for the increased sta- enzyme in these applications is the so-called Taq poly-
bility. merase from Thermus aquaticus, but several other
Few studies are to be found where the kinetic char- DNA polymerases from other thermo- or hyperther-
acteristics of a psychrophilic enzyme have been pro- mophilic bacteria and Archaea have been character-
duced by directed evolutionary methodology. A cold- ized and are commercially available (6, 149).
adapted subtilisin has been generated by evolutionary Improved thermal stability is a highly desirable
based sequential methodology. Cold-adaptation was property for the enzymes (-amylase, glucoamylase,
achieved with three mutations, and it was found that pullulanase, glucose isomerase) that are used in the
an increase in substrate afnity (i.e., decreased Km commercial processing of starch to glucose and fruc-
value) was mostly responsible for the observed dou- tose in the production of high-fructose corn syrup. In
bling in catalytic efciency at 10 C (147). this process the starch is typically gelatinized at 105 C
This section has shown that single-residue substitu- for 5 min and then -amylase is used at 95 C and pH
tions can affect stability of enzymes considerably and 66.5 to partially cleave the 1-4-glycosidic linkages in
inuence kinetic properties. However, it may be gen- the interior of the polymer, leading to liquefaction of
erally a fact that more than one substitution is required the starch. For the second (saccharication) step, the
to realize the full potential for temperature adaptation temperature must be lowered to 6062 C and the pH
in an enzyme. to 4.5, to adjust to the stability and the pH optimum of
the exo-acting glucoamylase and pullulanase that
further cleave the chain to smaller saccharides (95
VI. TEMPERATURE EXTREMOPHILES AS
96% glucose in glucose syrups) (6, 19, 150). In the
SOURCES FOR BIOCATALYSTS FOR
nal step the concentrated glucose syrup is again pH
THE FOOD INDUSTRY
adjusted to 78.5, and is passed through a column with
A. Thermophilic Enzymes immobilized glucose isomerase (xylose isomerase) at
5565 C, which converts it preferably to 55% fructose
As mentioned earlier, it is generally advantageous to (150). The limited thermal stability of the currently
run industrial processes at elevated temperatures, as used glucose isomerases determines the moderate tem-
long as sensitive components in the reaction are not peratures used in this process (19).
damaged under such conditions. It is therefore not The use of more thermostable enzymes which are
surprising that many of the biotechnological processes active under similar temperature and pH conditions
involving enzymes are carried out at relatively high could signicantly improve the starch conversion pro-
temperatures, and most of the enzymes used are cess, as it would be possible to run the liquefaction and
quite thermostable, despite them being usually of saccharication process in one step (19, 31). Finding
mesophilic origin (148). A useful industrial enzyme thermostable enzymes with similar pH characteristics
must have several specic properties depending on (activity and stability) would also reduce costs in the
the particular application, but thermostability is process as the use of ion exchanger step could be
usually an absolute necessity (148). Despite the poten- avoided, but that is necessary to carry out to remove
tial benets that may be achieved by using more ther- salts that accumulate as a result of the pH adjustments
mostable enzymes in several of these processes, the between different steps. In this respect hyperthermo-
industrial uses of thermophilic enzymes are still very philic -amylases with lower pH optima are promising
limited (19). In some industrial sectors (including the candidates (6, 151).
food industry), which use bulk industrial enzymes, Studies have also shown that isomerization of glu-
cost is the major issue, as introducing a new enzyme is cose to fructose can be improved considerably by
worthwhile only if the cost improvement provided by running this step at elevated temperatures (150). A
the new enzyme justies the research and development search for more thermostable glucoamylases (or -
costs as well as the required changes to production glucosidases), pullulanases, and glucose isomerases
equipment (19). from hyperthermophilic Archaea and bacteria is
Some successful biotechnological applications of now actively being pursued with the goal of nding
thermophilic enzymes have already been developed enzymes with more desirable properties in the starch
and implemented. At present, by far the most impor- conversion process (see 6, 19, 31, for review).
tant large-scale application of thermophilic enzymes is Furthermore, protein engineering has increasingly
in the use of DNA polymerases in the polymerase been used to improve thermal stability (152, 153),
chain reaction (PCR). The most commonly used or catalytic properties (154) of glucose isomerases.

Mutant enzymes obtained with protein engineering Proteases have increasingly been used in peptide
techniques may prove to be important candidates synthesis, where advantage is taken of their reverse
for future industrial applications, in these and other reaction under modied solvent conditions. Studies
processes. have shown that peptide synthesis is favored under con-
Cyclodextrins are useful in the food industry in spe- trolled conditions at higher temperatures. It has there-
cic separation processes, in avor stabilization, and in fore been suggested that, owing to their optimal activity
controlled release and exclusion of unwanted com- at high temperatures and higher resistance to organic
pounds from the bulk phase (155). They are nonredu- solvents than their mesophilic counterparts, thermophi-
cing cyclic products of six to eight glucose units. At the lic proteases should prove excellent candidates for enzy-
industrial level they are made from starch that rst is matic peptide synthesis (19). Indeed, the use of the
liqueed at high temperature by a thermostable -amy- thermophilic neutral metalloprotease thermolysin for
lase, followed by transglycosylation reaction leading to the synthesis of the artical sweetener aspartame (a
the cyclized product, a reaction catalyzed by cyclodex- dipeptide) has been developed into a large-scale appli-
trin glycosyl transferase (CGTase) (6, 19). The meso- cation, and is currently the only industrial application
philic CTGases that are conventionally used in the that uses a thermophilic protease (19, 148).
process are heat-labile and the second step in the pro-
cess must be run at lower temperatures. Using a ther- B. Psychrophilic Enzymes
mostable CGTase would greatly improve the process,
as liquefaction and cyclization could be carried out in Depsite the fact that stability is a major criterion for
one step (6, 19). the industrial application of enzymes, the use of psy-
Several proteases from thermophiles and hyperther- chrophilic enzymes in selected instances may offer
mophiles have been characterized (19, 48). The major advantages. Examples of the real usage of such
large-scale use of proteases as industrial enzymes is in enzymes in an industrial setting are very rare, although
the detergent industry, as additives in household deter- ideas as to the potential application of cold-active
gents, with alkaline serine proteinases from mesophilic enzymes have been around for the past two decades
Bacillus species (subtilisins) as the main enzymes. (5, 8, 99, 159). The main advantages seen for cold-
These enzymes are generally quite stable at moderately active enzymes are twofold: high activity at low tem-
high temperatures (6065 C) and under alkaline con- peratures, which may reduce energy consumption, and
ditions (pH 911) (148). A number of subtilisin-like easy inactivation through moderate heating or low-pH
serine proteinases from extreme thermophiles have treatment. Low processing temperatures might also
been characterized (see, e.g., 156158) that would be work toward reducing bacterial contamination (5).
expected to have properties more optimized for deter- Potential uses range from the use of cold-active
gent applications, but that have not been used for that enzymes in the detergent industry for washing or dis-
purpose. A major reason is that changing the large infecting at low temperatures (amylases, lipases, and
bulk production lines for the present detergent pro- proteinases), to various applications in food industries.
teases for the more thermophilic enzymes may not be Examples relating to milk processing include lactose
cost-effective at present. hydrolysis by b-galactosidase, or the use of proteases
An advantage of doing proteolysis at high tem- to coagulate milk and accelerate cheese maturing.
peratures is that the protein substrates are denatured, Also, cold-active enzymes may become useful in speci-
which makes them generally more accessible to pro- c biotransformations, such as the production of oils
teolytic attack than when in their native states. This with high polyunsaturated fatty acid content, and in
may be particularly important when hydrolyzing dif- whole organisms for environmental cleaning, where
cult proteinaceous waste materials (148). Because of temperatures are generally low (e.g., removal of
their high thermal activity and stability under condi- heavy metals or toxic materials from aqueous efu-
tions that denature most other proteins, thermophilic ents). More specialized uses include addition to contact
proteases are good candidates for applications aimed lens cleaning uids, and the potential use of ice-nucle-
at utilizing different protein waste materials for mak- ating proteins in the manufacture of ice cream or arti-
ing protein hydrolyzates for different purposes. High- cal snow (5, 99).
temperature treatment of such waste materials would Some other examples relating to food production
also contribute to maintaining aseptic conditions and have been considered in the shing industries of
to prevent the growth of spoilage bacteria and patho- North Atlantic coastal nations, but large-scale use is
gens during processing. still in its infancy (159162). Examples where cold-

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Besides being useful for industrial application,
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1997.

9
Nitrogen Fixation and the Enzyme Nitrogenase
William E. Newton
The Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U.S.A.
genous compounds produced from atmospheric N2

I. INTRODUCTION either commercially or biologically by microbes. In
this way, nitrogen xation assumes signicant impor-
Only a very small proportion of the nitrogen present
tance in agriculture because the availability of xed
on earth is in a usable form at any one time. More than
nitrogen is most often the limiting factor in crop pro-
99.9% of the nitrogen is present as the dinitrogen
duction. Consequently, efforts to grow sufcient crops
molecule (N2 ). Most organisms cannot metabolize
to feed the global population have led to increases in
this abundant, but relatively inert N2 and so they
the application of commercial nitrogen fertilizers.
must assimilate nitrogen in a xed form, such as
However, applying nitrogen fertilizers has its costs.
ammonia (NH3 ) or nitrate (NO 3 ). As part of the geo- These costs, which are directly associated with the
biochemical nitrogen cycle, nitrogen xation drives the
Haber-Bosch process, include: (a) consumption of non-
conversion of atmospheric N2 to ammonia. However,
renewable fossil fuel resources as both feedstock and
the complementary processes of nitrication and
energy source; (b) signicant storage costs for product
denitrication convert ammonia back to dinitrogen,
manufactured year-round but applied seasonally and
which is returned to the atmosphere. This constant
signicant transportation costs due to the production
cycling means that the pool of xed nitrogen within
site usually being remote from the site of use; and (c)
the biosphere must be constantly replenished because
contamination of local water systems through runoff.
ammonia is necessary for the formation of biologically In fact, only about one-third of the applied nitrogen is
essential, nitrogen-containing compounds, such as
assimilated by plants, whereas one-third is leached
amino acids and nucleic acids. Dinitrogen is xed
away (after conversion to nitrate) and one-third is deni-
naturally by both nonbiological processes, like light-
tried and lost to the atmosphere as N2 1. An alter-
ning and combustion, and by biological processes.
native approach to using the Haber-Bosch process for
Natural nonbiological processes contribute 10%
producing commercial nitrogen fertilizer is to exploit
and biological processes contribute about 65% of the
the biological process and considerable effort is cur-
total annual xation rate. Industrial ammonia synth-
rently being directed at understanding the molecular
esis contributes the other 25% (1, 2). Thus, biological mechanism of biological nitrogen xation.
nitrogen xation is a pivotal life-supporting process
that provides the majority of the xed nitrogen source
necessary to sustain life on earth. II. NITROGEN-FIXING ORGANISMS AND
Animals, including man, are directly dependent on CROP PLANTS
plants to supply much of the energy and many of the
nitrogenous compounds required to survive. Plants, in Only prokaryotes, i.e., those living things without an
their turn, are dependent on the availability of nitro- organized nucleus (Eubacteria, Archaea, and actino-

mycetes), can perform biological nitrogen xation. The against O2 occurs through a physical barrier to free O2
ability to x N2 is widely spread among bacterial gen- diffusion provided by the plant-derived, outer cortex
era (3, 4) and, despite a number of claims, no eukar- layer of the nodule. This structure prevents O2 from
yote has been clearly established to x N2 . Although ooding the central core of the nodule where the nitro-
farmers recognized the benets of crop rotations cen- gen-xing bacteria are located. However, some O2 is
turies ago, the source of much of that benet was required for bacterial respiration to provide the
unknown to them. The rst report of nitrogen xation energy and reducing equivalents to drive its nitrogen
in 1838 involved a comparison of the growth and nitro- xation, and this low, but sufcient, concentration is
gen content of cereals with leguminous plants (mainly delivered by an oxygen-transporting protein, called
clover in rotations with wheat and tuber crops) in both leghemoglobin.
greenhouse and eld experiments. The result that Establishment of a symbiotic relationship is a com-
azote (nitrogen) may enter the living frame of the plicated process, which most often involves specicity
plants directly (5, 6) was received with much skepti- between the host plant and its microsymbiont (9). In
cism. It was not until almost 50 years later that the legume symbiosis, initiation of the infection pro-
Boussingaults work was convincingly conrmed by cess occurs when bacteria induce curling of the tip of
Hellriegel and Wilfarth (7), who also solved the per- root hairs (10, 11). These structures then envelop the
plexing question of the source of the xed nitrogen by bacteria and form an infection thread, which pene-
localizing the activity to the bacteria-lled nodules on trates the root cortex and along which the invading
the roots of their pea plants (6, 8). bacteria enter the host plant cells (12). As infection
Most nitrogen-xing microbes are free living and x thread formation occurs, cell division is induced within
nitrogen for their own benet. Except for the cyano- the root cortex, resulting in formation of a nodule
bacteria, the free-living bacteria are generally not agri- primordium (13). When the infection thread contacts
culturally important, contributing only 0:1% of the a newly divided primordial cell, bacteria are released
xed nitrogen of a leguminous association. The most and they enter the cytoplasm of the primordial cell
extensively studied free-living bacterial species are through an endocytotic process, which results in a
Azotobacter vinelandii (an obligate aerobe), plant-derived peribacteroid membrane that surrounds
Clostridium pasteurianum (an obligate anaerobe), the bacteria (14). Following multiple bacterial-cell divi-
Klebsiella pneumoniae (a facultative anaerobe), sion and peribacteroid membrane proliferation, the
Rhodobacter capsulatus (a photosynthetic bacterium), host cell becomes lled with bacteria, which then dif-
and Anabaena sp. 7120 (a heterocyst-forming cyano- ferentiate into bacteroids that are specialized for xing
bacterium). However, some nitrogen-xing microbes nitrogen.
enter into a mutually benecial interaction with other The symbiotic interaction requires coordination
organisms. The most agriculturally important symbio- among the partners at both the developmental and
tic relationship involves leguminous plants and rhizo- metabolic levels. This cooperation is accomplished
bial microsymbionts, but many other nitrogen-xing, through reciprocal communication and control of
symbiotic relationships are known. These include gene expression. Signaling between the partners starts
examples with lichens, bryophytes (mosses and liver- when the regulatory nodulation (nod) genes of the rhi-
worts), pteridophytes (ferns), cycads, and other nonle- zobia detect plant signals present in root exudates.
guminous angiosperms, such as small shrubs and trees, These plant signals are usually avanoids (15). These
like Myrica and alder, and larger trees, like Casuarina avanoids specically induce rhizobial nodD gene
(3). expression (16), which produces a regulatory protein
The leguminous association ensures that the plant (a transcriptional activator) that controls the expres-
receives xed nitrogen directly from the rhizobia bac- sion of all other nod (and nol) genes. The concerted
teria, which are harbored in nodules on its roots. In action of the products of other nod genes results in
return, the bacterium receives a protected environment the synthesis and release of the bacterial signals, called
and a supply of energy from the plant. Another benet Nod factors (17). Each rhizobial Nod factor is species
arising from this association is the provision of an specic. It is chitinlike, being composed of (1-4)-
environment with a low O2 tension within the root linked N-acetylglucosamine residues. The specicity
nodules. This function is vital because the enzyme, of a particular Nod factor depends on both the extent
which catalyzes biological nitrogen xation and is of oligomerization (usually up to ve residues) and the
called nitrogenase, is extremely sensitive to the pre- range of chemical modication of the chitin core, such
sence of O2 . Most often, the rst level of protection as O-acylation or N-acylation at the nonreducing end

and sulfation at the reducing end. The nodABC genes with Klebsiella or ruminants with Clostridium, it is
are called the common nod genes because their pro- unlikely that much is contributed to the nitrogen status
ducts are required for catalyzing formation of the oli- of the animal because its diet likely contains sufcient
gosaccharide core for all Nod factors. The other nod xed nitrogen, which will repress any N2 -xing activity
genes are host specic and their products are respon- by the bacteria. However, with termites and ship-
sible for oligomerization and modication of the oli- worms, the associations are signicant. Citrobacter
gosaccharide core. In some plant strains, isolated Nod infects the intestinal tract of termites and can x N2
factors can initiate root hair curling, infection thread there with the amount xed and the benet gained
development, and nodule formation. depending on the insects diet (26, 27). The N2 -xing,
Root nodules are the usual result of N2 -xing sym- cellulose-decomposing bacterium that inhabits the
bioses involving Rhizobium; however, some tropical Deshayes gland of woodboring shipworms contributes
legumes, like Sesbania, produce stem nodules in asso- signicantly to the mollusks well-being by providing
ciation with Azorhizobium caulinodans (18). In addi- up to 35% of its xed nitrogen requirement (28). The
tion, some stem nodules retain the ability for whole area of animal symbioses is under-researched.
photosynthesis and contain, in the case of
Aeschynomene indica, rhizobia themselves capable of
III. NITROGENASES
photosynthesis (19). This close relationship of photo-
synthesis to nitrogen xation may ease some of the A. Molybdenum Nitrogenase
energy demand of nodules.
Although this tight, nodule-based, symbiotic With one exception (29), all known diazotrophs con-
arrangement is very successful, it does not extend to tain a nitrogenase (or nitrogenases) that is a complex
most of the important food crops, like the cereal grains of two metalloproteins (3033). The individual nitro-
(rice, wheat, and corn) and root and tuber crops. None genase component proteins of the most commonly
of these harbor symbiotic partners in nodules. Hence, occurring nitrogenase, usually referred to as Mo-nitro-
for the productivity of these crops to reach commer- genase, have been designated as the Fe protein (or
cially acceptable levels, extensive augmentation by component 2 or dinitrogenase reductase) and the
commercially xed nitrogen is usually necessary. MoFe protein (or component 1 or dinitrogenase).
However, other, less formal associations occur regu- The trivial names for these proteins are derived from
larly in which some interdependence exists among the composition of their associated metal centers. The
some grasses (family Gramineae) and bacteria. The Fe protein is a homodimer (Mr 64,000, which con-
associations of tropical grasses Paspalum and tains two MgATP binding sites and a single [4Fe4S]
Digitaria with the bacteria Azotobacter paspali and cluster. The MoFe protein is an 2 2 heterotetramer
Azospirillum brasilense, respectively, are good exam- (Mr 230,000), which contains two pairs of two dif-
ples of such a relationship (20). With Paspalum, a ferent metalloclusters, called P-clusters and iron-
mucilaginous sheath forms around the root within molybdenum cofactors (or FeMo cofactors). The
which the bacteria live and x N2 . The bacteria do three-dimensional structures of the Fe protein (34)
not invade the plant tissue. In contrast, the and MoFe protein (35, 36) have been solved, as have
DigitariaAzospirillum association does involve root the structures of their associated metal clusters (34, 37,
invasion but no nodule develops. The extent to which 38). The Fe protein serves as a specic reductant for
the plants benet from these associations is uncertain. the MoFe protein, which contains the site(s) of sub-
Azospirillum lipoferum, which occurs in temperate strate binding and reduction.
zones, associates with certain corn and sorghum culti- Wild-type nitrogenase catalyzes the biological nitro-
vars, but the effect on the plants appears to be small gen-xation reaction, which is usually described as:
(21, 22). The more formalized endophytic associations
N2 8H 8e 16MgATP ! 2NH3 H2
of both Acetobacter diazotrophicus and Herbaspirillum
16MgADP 16Pi
spp. with sugar cane (23) and of Azoarcus spp. with
Kallar grass and possibly rice (24) may supply up to In addition, nitrogenase catalyzes the reduction of
100% of the xed nitrogen required for the host plants many other small molecule substrates, all of which
growth, indicating a signicant agronomic and eco- have the same requirements for catalytic activity,
nomic potential. namely MgATP, a low-potential reductant and an
Nitrogen-xing associations involving animals also anaerobic environment (39, 40). The optimal stoichio-
exist (25). In the case of higher animals, like either man metry for nitrogenase function is four molecules of

MgATP hydrolyzed for each pair of electrons trans- genases are likely to have common structural features
ferred to substrate; this ratio is independent of the and mechanistic similarities. However, the larger com-
substrate reduced. The so-called alternative substrates ponent of these alternative nitrogenases is hexameric
include, for example, acetylene, which is only reduced with three types of subunits, which are encoded by
by two electrons to ethylene, HCN, which is reduced either vnfDGK (51, 53, 54) or anfDGK (52, 55), rather
by six electrons to methane and ammonia and by four than the tetrameric form of the MoFe protein.
electrons to methylamine, and azide, which is reduced There appears to be no logic to the way in which
by two electrons to nitrogen gas and ammonia. Carbon these nitrogenases are distributed among microorgan-
monoxide (CO) is a potent inhibitor of all nitrogenase- isms. For example, Klebsiella pneumoniae has only Mo-
catalyzed substrate reductions with the exception of nitrogenase, whereas Azotobacter chroococcum has
H reduction (41). H2 has a unique involvement with both Mo- and V-nitrogenase, and Rhodobacter capsu-
Mo-nitrogenase and with N2 reduction in particular. It latus has Mo- and Fe-nitrogenase. Moreover, all three
can play any one of four well-documented roles both in nitrogenase types are found in A. vinelandii. The
vivo and in vitro. H2 , via the action of hydrogenase, can expression of each nitrogenase is under hierarchical
be the source of reducing equivalents for nitrogenase; it control through the availability of either Mo or V in
is the sole product in the absence of other reducible the growth medium (56, 57). Whenever Mo is avail-
substrates in an ATP-dependent, CO-insensitive reac- able, expression of the Mo-dependent nitrogenase is
tion (42); it is a specic competitive inhibitor of N2 stimulated and the expression of the others is
reduction, affecting neither the reduction of any repressed. Similarly, when V is available and Mo is
other substrate nor its own evolution (43); and, absent, expression of the V-nitrogenase only occurs.
under an N2 /D2 atmosphere, HD is formed in a CO- If both metals are absent, then only the Fe-nitrogenase
sensitive, MgATP-requiring reaction (4447). These is expressed. Such control by metal availability is phy-
Mo-nitrogenases, from a variety of genera, exhibit a siologically reasonable because Mo-nitrogenase is the
high level of primary sequence identity. The sequence most efcient catalyst for nitrogen reduction, followed
conservation is particularly high in the regions of the by V-nitrogenase, with the Fe-nitrogenase being the
MgATP- and metallocluster-binding sites (48). With least efcient with a large majority of reducing equiva-
the notable exception of C. pasteurianum, the compo- lents being used to evolve H2 rather than to produce
nent proteins from all Mo-based nitrogenases interact ammonia (58, 59). Active heterologous nitrogenase
as heterologous crosses to form catalytically active results from crosses among the two-component pro-
enzymes (49). teins from Mo-nitrogenase and V-nitrogenase, but
crosses involving either component protein from the
B. Alternative Nitrogenases Fe-nitrogenase are ineffective with the complementary
protein from both Mo- and V-nitrogenases (59).
With the single exception noted above and described in
Section III.C below, all nitrogen-xing organisms that C. Streptomyces thermoautotrophicus
have been studied at the level of the enzyme have the Nitrogenase
Mo-containing nitrogenase. There are, however, two
other types of nitrogenases, which are closely related Although this recently discovered (29), unique nitro-
to the Mo-nitrogenase, but neither contains Mo. genase system consists of two component proteins, the
These so-called alternative nitrogenases (5052) consist larger of which contains Mo, Fe, and sulde, that is
of two protein components, including a Fe-protein where the similarities to the classical Mo-nitrogenase
component specic for each alternative nitrogenase. stop. The Fe-protein component of the classical nitro-
The larger of the two component proteins of the rst genase is replaced by a manganese-containing super-
alternative nitrogenase contains a cofactor with a vana- oxide oxidoreductase, which oxidizes superoxide to
dium (V) atom substituting for the Mo atom. This dioxygen and then transfers the electron to a MoFeS-
replacement results in a VFe protein containing a FeV containing protein at which each dinitrogen molecule
cofactor. The second type contains a cofactor where the is reduced by eight electrons to two molecules of
Mo atom is substituted by Fe to produce a FeFe protein ammonium and one molecule of dihydrogen. This
containing a FeFe cofactor. The high level of primary reaction appears to require less MgATP per dinitrogen
sequence identity recognized among the Mo-dependent reduced than that required by the classical Mo-nitro-
nitrogenases also extends to the alternative nitrogenase. In place of avodoxin or ferredoxin as the
genases. This identity strongly suggests that all nitro- electron donor to the Fe protein, a Mo-containing

CO dehydrogenase operates by coupling the oxidation interface of the MoFe protein. This arrangement
of CO to the reduction of dioxygen to produce super- places the Fe proteins [4Fe4S] cluster in close proxi-
oxide. The MoFeS-containing protein is a hetero- mity to the P-cluster of the MoFe protein and also
trimer quite different from the 2 2 composition of the situates the P-cluster between the Fe proteins [4Fe
classical Mo-nitrogenase. Other unique features of this 4S] cluster and the FeMo cofactor. This arrangement
nitrogenase include the insensitivity of its nitrogen xa- suggests that electron transfer occurs from the [4Fe
tion toward dioxygen, CO, and dihydrogen, all of 4S] cluster through the P-cluster to the FeMo cofactor,
which are potent inhibitors of nitrogen xation in the where substrate reduction occurs.
classical system. Finally, this nitrogenase does not cat- One P-cluster is located at each -interface of the
alyze the reduction of acetylene to ethylene. MoFe protein. In its as-isolated form, which is
believed to represent its fully reduced, all-Fe2 state,
D. Mechanism of Nitrogenase Action the [8Fe7S] P-cluster consists of two [4Fe4S] sub-
clusters that share a single, six-coordinated sulde
During catalysis, the Fe protein delivers electrons, one (PN in Fig. 2). Upon oxidation by redox-active dyes,
at a time, to the MoFe protein in a process that couples the P-cluster structurally rearranges through movement
MgATP binding and hydrolysis to the association and of two of its eight Fe atoms to POX , both of which also
dissociation of the two component proteins and con- undergo a change in their ligand environment. This
comitant electron transfer (33, 60). Both component redox-dependent structural change of the P-cluster is
proteins are required for MgATP hydrolysis, and likely to be mechanistically related to its role in accept-
neither component protein alone will reduce substrate. ing electrons from the Fe-protein and coupling their
Based on a substantial amount of kinetic data, a delivery, along with the required protons, to the
numerical model has been developed to describe the FeMo cofactor and substrate (38).
process by which electrons are sequentially delivered The FeMo cofactor is composed of a [Mo7Fe9S]
to the MoFe protein and then to substrate (60). This core plus one molecule of (R)-homocitrate (Fig. 3). This
model treats the MoFe protein as a dimer of dimers basic framework consists of two partial clusters, [Mo
with each -dimer operating independently and being 3Fe3S] and [4Fe3S], bridged by three suldes.
serviced by Fe protein. This approach involves two Homocitrate is coordinated to the Mo atom through
interconnecting processes, which are called the Fe-pro- its 2-hydroxyl and 2-carboxyl groups. Substrate bind-
tein cycle and the MoFe-protein cycle. The Fe-protein ing and reduction at the FeMo cofactor is supported by
cycle describes how the Fe proteins [4Fe4S] cluster several lines of evidence. First, mutant strains, which
cycles between its reduced 1 and its oxidized 2 redox are unable to biosynthesize FeMo cofactor, are also
states as it delivers an electron to the MoFe protein, unable to catalyze nitrogen xation (64) but, when iso-
coupled with MgATP hydrolysis, and is then related FeMo cofactor is added to crude extracts pre-
reduced by another electron transfer protein, usually pared from such mutant strains, the ability to x
either a ferredoxin or a avodoxin. nitrogen in vitro is restored. Second, when citrate
The MoFe-protein cycle is necessarily more com- replaces homocitrate as the organic constituent of an
plex because it involves the progressive reduction of altered FeMo cofactor, the resulting MoFe-protein
the MoFe protein by up to eight electrons for N2 bind- exhibits altered catalytic properties (6466). Third,
ing and reduction, which therefore requires eight turns altered MoFe proteins, which have amino acid substi-
of the Fe-protein cycle. This model is consistent with tutions within the FeMo cofactors polypeptide envir-
N2 becoming bound to the active site only after three onment, also exhibit altered catalytic properties (67,
electrons have been accumulated within the MoFe pro- 68). Although how substrates interact with FeMo
tein. How and where these electrons are stored prior to cofactor during turnover is not known, both the pre-
the binding and reduction of substrate is unknown. sence of six coordinately unsaturated Fe atoms (6973)
However, current biochemical data suggest that and the homocitrate bound to Mo (74) have been sug-
intra-MoFe-protein cluster-to-cluster electron transfer gested to be involved in substrate and inhibitor binding.
occurs (61, 62). In addition, structural data supporting For example, multiple distinct and possibly overlap-
this thesis come from the crystallographic model for ping sites have been proposed involving the FeS fra-
the docked complex of two Fe proteins with one mework (75). In contrast, the carboxylate group, which
MoFe protein (63) (Fig. 1). Docking occurs along the is coordinated to the Mo atom, has been suggested to
Fe proteins twofold symmetric axis, which bisects the serve as a leaving group in a mechanism that activates
single [4Fe4S] cluster, and the pseudosymmetric - Mo to provide a substrate coordination site (76).

Figure 1 Ribbon diagram of the three-dimensional structure of the A. vinelandii Mo-nitrogenase complex, which is stabilized by
using ADP-A1F 4 as an ATP analog. The complex consists of two molecules of the Fe-protein (lightest shading), one at each end
of one molecule of the MoFe-protein a2 b2 tetramer. The twofold symmetry axis of the Fe-protein aligns with the pseudo-twofold
symmetry axis, which bisects the ab-subunit interface of the MoFe-protein. Each half of the MoFe-protein molecule (which may
be visualized by drawing a line through the center from the 1 oclock to the 7 oclock position) represents an b-subunit dimer,
each encompassing one P-cluster and one FeMo-cofactor. The b-subunits (darkest shading) make all contacts among the two ab-
dimers. The a-subunits (medium shading), which do not contact one another, are located in the lower left and upper right (with
the FeMo cofactor clearly visible in both). (Courtesy of Dr. D. C. Rees, California Institute of Technology.)
E. Role of MgATP in Nitrogenase Catalysis formational changes in both nitrogenase proteins that
ensure delivery to and accumulation within the MoFe-
The overall reduction of N2 to yield 2NH3 is thermo- protein of multiple electrons prior to their delivery to
dynamically favorable, although the formation of two- substrate. In support of this concept, primary amino
electron-reduced intermediates, if they exist, is not. It acid sequence and structural comparisons show that
appears, then, that MgATP binding and hydrolysis the Fe-protein is a member of a large class of signal
during nitrogenase catalysis is not a thermodynamic transduction proteins that undergo conformational
requirement but rather it is used to drive electron changes upon MgATP binding and hydrolysis.
transfer toward substrate reduction and to prevent The following sequence of events, which occur dur-
the back ow of electrons to the Fe-protein. This situa- ing a single turn of the Fe-protein cycle and which
tion may be envisioned as the gating of electron ow account for the role of MgATP in nitrogenase cataly-
in which MgATP binding and hydrolysis causes con- sis, is widely accepted (77). First, the reduced Fe-pro-

Figure 3 Ball-and-stick structural model of the FeMo
cofactor of the nitrogenase MoFeprotein. Its iron atoms
are gray and labeled Fe1Fe7; the Mo atom is large and
black; the S atoms are medium-size and black; and all C,
N, and O atoms are small and black. Also shown are the
Figure 2 Ball-and-stick structural models of the two forms, directly bonded entities, which are R-homocitrate and the
POX and PN, of the P-cluster of the nitrogenase MoFe-pro- two a-subunit residues, histidinyl-442 and cysteinyl-275.
tein. Each form is shown in two orientations, which are (Figure courtesy of Dr. J.W. Peters, Utah State University.)
related by a 908 rotation. The iron atoms are gray and
labeled 18; and the S atoms are black with the S atom
that is labeled as S1 being shared among the two subclusters. however, that phosphate release (which is usually
The length of the Fe6S1 bond is longer than the normal Fe
when energy transduction occurs) from the complex
S bond and is shown as a dotted line. (Figure courtesy of Dr.
J.W. Peters, Utah State University.) follows electron transfer and that phosphate release
does not drive the dissociation of this complex into
its component proteins (79). Fourth, the conversion
tein binds two MgATP molecules, which produces a of the Fe-protein from the MgATP-bound state to
conformational change in the Fe-protein that makes it the MgADP-bound state causes complex dissociation
competent for interaction with the MoFe-protein. and this process is the rate-limiting step in nitrogenase
Second, the two proteins form a complex, which catalysis (60). This complex intercommunication
induces changes in the midpoint potentials of the between the two proteins results in the accumulation
metalsulfur clusters such that electron ow toward of electrons within the MoFe-protein, and the whole
FeMo cofactor is energetically favored. In the nitro- process appears to be synchronized by sequential con-
genase complex, the redox potential of the Fe-proteins formational changes induced by MgATP binding, com-
[4Fe4S] cluster is 620 mV, that of the P-cluster is ponent protein interaction, and MgATP hydrolysis.
390 mV, and the redox potential of the FeMo cofac-
tor is 40 mV (78). Third, in addition to electron F. Electron Transport to Nitrogenase
transfer, the docking of the component proteins trig-
gers MgATP hydrolysis, although it is not clear A source of reducing equivalents of sufciently low
whether this occurs shortly before, concomitantly potential is required for regeneration of reduced Fe-
with, or shortly after electron transfer or whether the protein after it has donated one electron to the
timing varies depending on other factors. It is clear, MoFe-protein. Both avodoxins and ferredoxins are

capable of serving this function in vitro. The ultimate genes associated with both of the genetically distinct,
donor of low-potential electrons has not been identi- Mo-independent alternative enzymes have also been
ed for most nitrogenases. In some bacteria, e.g., A identied, including the structural genes for both the
vinelandii, redundancy is apparent with several avo- V-nitrogenase (vnfHDGK) and the Fe-nitrogenase
doxins and ferredoxins capable of supporting nitrogen- (anfHDGK) (82, 83), two nifA-like genes (84), and
ase turnover. However, for K. pneumoniae, two genes, one nifEN-like sequence (85). The functions of the pro-
nifF and nifJ, are involved in coupling the reduction of ducts of these last three genes is described in the next
Fe-protein to intermediary metabolism (80, 81). The two sections.
nifF gene product is a avodoxin that can donate an
electron to the oxidized Fe-protein. In this process, the
reduced hydroquinone form of its avin is oxidized to V. MATURATION OF NITROGENASE
a semiquinone form. The re-reduction of avodoxin is
accomplished through the catalytic activity of the nifJ The primary translation products of the nitrogenase
gene product, which is a pyruvate-avodoxin oxido- structural genes are not active. Many other nif-
reductase. It couples the oxidation of pyruvate, yield- specic genes are required to activate these immature
ing acetyl-CoA and CO2 , to the reduction of the structural components. The function of the products
semiquinone form of the avin to its hydroquinone of these nif-specic maturation genes is to catalyze the
form. The nifJ gene has not been found in other nitro- formation and then insertion of the individual
gen-xing genera, so other means of providing redu- metalloclusters into apo-forms of the Fe-protein and
cing equivalents to nitrogenase must be operating. MoFe-protein. In the case of the Fe-protein, only the
nifM gene product is specically required for its
maturation (86). The nifM gene product has not yet
IV. nif GENES been isolated in an active form, but it appears to be a
member of a family of peptidyl-prolyl isomerases
In addition to the products of the nif genes discussed (87), which assist protein folding by catalyzing the
above, other genes and their products are required for cis/trans isomerization of certain peptidyl-prolyl
the maturation of the nitrogenase component proteins bonds. It is not obvious why such a requirement
and for the regulation of the expression of the nitro- exists for the maturation of the Fe-protein, but it
genase genes. The organism that appears to have the could be involved with the formation of a transient
simplest organization of nitrogen xationspecic (nif) state of the immature Fe-protein necessary for inser-
genes, and which is the one best studied at the mole- tion of the [4Fe4S] cluster.
cular genetic level, is the facultative anaerobe, Maturation of the MoFe-protein, particularly the
Klebsiella pneumoniae. This organism has 20 nif genes formation and insertion of the FeMo cofactor into an
organized into seven transcriptional units. The specic immature form of the MoFe-protein, is much more
designations for individual K. pneumoniae nif genes are complicated. This process involves the products of, at
also used to denote genes whose products have homo- least, the nifH, nifE, nifN, nifB, nifV, and nifQ genes.
logous functions in other organisms. For example, the The nitrogenase Fe-protein, whose subunits are the
nif structural genes from all diazotrophs are designated product of nifH, is required for both formation and
nifH, nifD, and nifK and they encode the Fe-protein insertion of the FeMo cofactor (88, 89). The Fe-pro-
and the MoFe-protein - and -subunits, respectively. teins specic function in these processes is unknown
Complications occur, however, in the genetic nomen- but neither its MgATP-binding and -hydrolysis
clature of nif genes in that some organisms do not have properties nor its electron transfer capability are
homologues of all K. pneumoniae nif genes and others involved (90, 91). Biochemical complementation
have nitrogen xationrelated genes that are not pre- experiments show that the FeMo cofactor is initially
sent in K. pneumoniae. A general convention is that the synthesized elsewhere and then inserted into a FeMo
nif designation is reserved for only those genes that cofactordecient MoFe-protein that contains intact
have functional counterparts in K. pneumoniae, P-clusters (92).
whereas nitrogen xationrelated genes present in A portion of the biosynthetic process occurs within
other organisms without functional counterparts in a complex of the products of the nifEN genes. The
K. pneumoniae are given other designations. For exam- nifEN gene products bear primary sequence similarity
ple, the x designation is used for such genes occur- to the products of the nifDK structural genes (93) and a
ring in the rhizobia. A number of nitrogen xation heterotetrameric complex of the nifEN products likely

serves as a molecular scaffold for FeMo cofactor specic. The requirement for nifB, but not nifEN, is
assembly (93, 94). In A. vinelandii, a second nifEN- surprising because nifB and nifN are fused in
like set of genes, which are called vnfEN, have been Clostridium pasteurianum (110), which expresses a
detected and they appear to have a common function Mo-independent nitrogenase (111). Moreover, the
in both the vnf and anf systems. The vnfEN gene pro- nifV requirement shows that homocitrate is a constitu-
ducts probably work similarly for both the FeV cofac- ent of all three cofactors as are S2 and Fe, which
tor and the putative Anf cofactor (FeFe cofactor) (85). would be provided through the requirement for the
The product of the nifB gene catalyzes the formation of products of the nifUS genes.
a FeMo cofactor precursor called NifB cofactor (95).
NifB cofactor appears to provide the basic FeS
framework necessary for FeMo cofactor construction VI. REGULATION OF NITROGENASE
and becomes accommodated within the NifEN com- EXPRESSION
plex (96).
The nifV gene product catalyzes the condensation of Because both nitrogenase proteins are denatured by
acetyl-CoA and -ketoglutarate to form homocitrate, dioxygen and because nitrogenase turnover requires
the organic constituent of FeMo cofactor (97, 98). The consumption of considerable metabolic energy, it
nifQ gene product has a role in providing the Mo atom would make no sense to express the nif genes when
for the FeMo cofactor, especially under Mo-decient dioxygen is present, when a xed nitrogen source is
conditions, but its exact role is unknown (99). The available, or when cellular growth is limited by the
products of the nifX, nifW, and nifZ genes might also availability of a carbon source. All three factors regu-
have some role in FeMo cofactor biosynthesis (100, late nif gene expression. Moreover, many free-living
101). With K. pneumoniae, a low-molecular-weight nitrogen xers have alternative nitrogenases and so
protein, encoded by nifY, appears to be associated the expression of the nif genes is also controlled by
with the apo-MoFe-protein produced in strains lacking the availability of Mo or V.
either nifB or nifEN activity. The nifY gene product K. pneumoniae has been most thoroughly studied in
may stabilize an apo-MoFe-protein conformation terms of the regulation of nif gene expression. Two of
that is amenable to FeMo cofactor insertion (102, its 20 nif-specic genes are the regulatory genes, nifL
103). A different low-molecular-weight protein, which and nifA (112), and they are contained in one of
is called gamma and is not encoded by nifY, appears to the seven transcriptional units in K. pneumoniae.
serve the same function in A. vinelandii (104). Expression of the nifLA genes is controlled by the glo-
Two other nif gene products, those of nifS and nifU, bal nitrogen regulatory elements consisting of the pro-
catalyze reactions that are involved in the general ducts of the ntrA, ntrB, and ntrC genes. The expression
mobilization of Fe and S for metallocluster assembly of the other six nif transcriptional units is controlled by
(98, 105, 106). The nifS gene product is a cysteine the products of nifL, nifA, and ntrA. The ntrA gene
desulfurase that activates S for FeS cluster formation. product, which is known as either NtrA or 54 , is an
The nifU gene product might have a role in providing alternative sigma factor that controls the expression of
Fe. Homologs to nifU and nifS, whose expression is not only the nif and ntr genes but also that of a wide
not under nif-specic control, are present in many variety of gene families (113). The presence of NtrA
organisms and these may function in general FeS imparts a specicity to RNA polymerase and allows it
cluster formation and repair (107). to recognize a consensus promoter sequence, CTGG-
When the alternative nitrogenases are considered, N8 -TTGCA, which is located 24 to 12 bp before
the situation becomes complicated because the pro- the transcription initiation site. Generally, the normal
ducts of the nifMBVUS genes are essential for func- RNA polymerase, which contains the abundant sigma
tional activity of the V-nitrogenase and Fe-nitrogenase factor called 70 , recognizes a typical prokaryotic pro-
as well as the Mo-nitrogenase (59, 108, 109). The nifM moter sequence, TTGACA-N17 -TATACA, at 35 to
requirement indicates that all three Fe-proteins are so 10 bp before the transcription initiation site.
similar that a single NifM-protein can process them all. Expression of ntrA does not appear to be tightly con-
Because the nifBV gene products are involved with the trolled, and the omnipresence of NtrA apparently
biosynthesis of the FeMo cofactor of Mo-nitrogenase, reserves a portion of the RNA polymerase for specia-
their common requirement suggests similar cofactors lized functions like nitrogen xation.
in all three systems. Further, the nifB requirement sup- The specic regulation of the expression of the
ports the suggestion that its function cannot be Mo nifLA genes is controlled by the products NtrB and

NtrC of the global nitrogen regulatory genes, ntrB and nitrogenase Fe-protein occurs in Rhodospirillum
ntrC. NtrB and NtrC form a two-component regula- rubrum and Azospirillum brasilense. These organisms
tory system in which NtrB is the phosphatase/kinase regulate nitrogenase activity by ADP-ribosylation of
sensor protein that controls the phosphorylation state a specic arginine residue on the docking surface of
of NtrC in response to the ratio of -ketoglutarate the Fe-protein in response to changes in environmental
to ammonia in the cell (114). When this ratio is conditions (121, 122). In strict aerobes, nitrogenase can
high, a condition that signals xed-nitrogen limita- undergo conformational protection to guard against
tion and high energy charge, NtrC is phosphoryl- O2 damage by forming a protein aggregate, which dis-
ated and, when the ratio is low, NtrC is sociates to resume xation when the O2 stress is
dephosphorylated. Under the former condition, phos- relieved (123).
phorylated NtrC initiates a two-tiered regulatory Very different examples of the control of the expres-
cascade that rst leads to activation of the nifLA sion of nitrogenase activity involve the formation of
promoter, which then results in activation of the specialized structures. In many cyanobacteria, for
other nif promoters. Phosphorylated NtrC recognizes example Anabaena 7120, nitrogen xation is restricted
and binds to a consensus DNA sequence that is located to specialized cells called heterocysts (124). The forma-
upstream from the 54 -RNA polymerase-binding site tion of heterocysts, as well as nitrogenase expression, is
of nifLA. Bound, phosphorylated NtrC catalyzes an both temporally and spatially controlled through term-
ATP-dependent conformational isomerization at inal differentiation events involving genomic rearran-
the promoter site, which ultimately results in the initia- gements. The root nodule was discussed earlier, but
tion of transcription of the nifLA transcriptional here another specialized case of the control of nitro-
unit (115). Accumulation of the products of the nifL genase gene expression occurs. Because the function of
and nifA genes then specically controls the other nif the root nodule bacteria (rhizobia) is to produce and
promoters. export xed nitrogen to its host plant, it would defeat
NifA is structurally and functionally similar to its purpose to retain a regulatory mechanism that is
NtrC (116, 117). It also binds to an upstream activator sensitive to the presence of xed nitrogen. However,
sequence, but one with a consensus sequence motif, control of nif gene expression in response to the cellu-
TGT-N10 -ACA, that is different from the NtrC-bind- lar dioxygen concentration is still absolutely required.
ing site. This sequence is located 100 bp before each Similar to the two-component NtrB/NtrC system,
nif promoter except that for nifLA (118). However, dioxygen control of nif gene expression in rhizobia
unlike NtrC, NifA activity is not controlled by phos- occurs through another sensorregulator pair (125).
phorylation/dephosphorylation; rather, its ability to The sensor protein, FixL, is a hemoprotein located
activate nif gene expression is controlled by NifL, within the cell membrane and, like NtrB, it has phos-
which acts as an antiactivator. Although just how phatase/kinase activities. Under microaerobic condi-
NifL complexes with NifA is not known, this com- tions, FixL catalyzes the phosphorylation of FixJ,
plexation occurs when either the dioxygen level or which then activates the expression of nifA similarly
the xed-nitrogen level is sufciently high and the nif to NtrC activation in K. pneumoniae. Rhizobia do
promoters are not activated. NifL is a avoprotein that not contain a NifL analog. Therefore, control of nif
senses the redox status of the cell by conformational gene expression involves no antiactivator; rather, the
changes that are controlled by the oxidation state of its activity of rhizobial NifA proteins appears to be
FAD moiety (119, 120). In A. vinelandii, each of the directly sensitive to the presence of dioxygen (126).
alternative nitrogenase enzyme systems has its own
specic NifA-like protein (either VnfA or AnfA),
which acts as a positive regulator of nif gene expression VII. SUMMARY AND OUTLOOK
(84).
The mechanism by which K. pneumoniae controls nif Considerable progress has been made in understanding
gene expression is not universal. Many other strategies the mechanism of action of nitrogenase, particularly
are used. Very little is known about the regulation of since 1970. In the early 1960s, only crude, cell-free
nif gene expression in the Archaea or clostridia, for preparations were available for investigating the nitro-
example, except that it is different from that described gen xation phenomenon, whereas highly puried,
above. Other control mechanisms, in addition to tran- crystalline preparations of the nitrogenase component
scriptional control, include posttranslational modica- proteins are now available for study. The x-ray derived
tions. For example, the reversible modication of the structures of both protein components and a docked

complex (35, 63) have clearly dened the structural If it were, nature would probably have many N2-xing
goals to add to the obvious reactivity goal for effective plants already in place. Protection from dioxygen and
model building. However, there is still no detailed a sufcient energy supply are also required. These
mechanism that describes biological nitrogen reduction needs could be satised if nitrogenase were relocated
in structural terms, although a very useful numerical to the chloroplasts of leaves where, if properly pro-
model is available (60). Even so, purely chemical sys- tected from the O2 evolved by photosynthesis, it
tems have been devised that bind N2 and, in some could take advantage of directly available reducing
cases, activate it sufciently that reduced nitrogen equivalents produced from sunlight. Finally, at no
compounds, ammonia, and/or hydrazine are produced time soon does it appear likely that the many known
on protonation (127129). These chemical N2 xation associations involving animals will be exploited.
systems, which work under ambient conditions, may in Therefore, man appears destined to remain dependent
the future form the basis of a simple, low-temperature on plants for a supply of xed nitrogen.
and low-pressure system, possibly operated electroche-
mically and driven by a renewable resource, such as
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10
Enzymes in Amylose and Amylopectin Biosynthesis
Bruce P. Wassermany and Ying Yu

Rutgers University, New Brunswick, New Jersey, U.S.A.
I. INTRODUCTION II. THE STARCH BIOSYNTHETIC

PATHWAY
Starch is the major storage carbohydrate of higher
plants. In wild-type maize, starch consists of 25% amy- A current view of the starch biosynthetic pathway in
lose and 75% amylopectin. Starch and its chemically maize endosperm is diagrammed in Fig. 1. The
modied derivatives are of great importance to the immediate precursor of endosperm starch is sucrose.
worlds food supply and economy. Starch-based pro- Sucrose is synthesized by photosynthesis in the leaf,
ducts have long been used as sources for sweeteners and transported through the phloem. By the process
and numerous other food and industrial ingredients, of photoassimilate partitioning, sucrose is taken into
including pharmaceuticals, fat substitutes, textiles, the sink or storage tissue, which in this case is the
packaging materials, and adhesives. The increasing endosperm of the kernel. Sucrose is converted into
agricultural and industrial importance of starch and starch by a series of enzymatic steps (Fig. 1). In the
its derivatives has raised interest in the possibility of cytosol, sucrose synthase and UDP-glucose pyropho-
producing bioengineered starches with improved func- sphorylase convert sucrose into Glc-1-P.
tional properties. The nal three committed steps of starch bio-
Bioengineering of the genes encoding the major synthesis consist of substrate formation, chain
starch biosynthetic enzymes has the potential to modify elongation, and branch point insertion. AGP
starch yield and starch structure. In theory, gene altera- catalyzes the formation of ADP-Glc from Glc-1-P
tion can be used to modify structural features such as and ATP. AGP is widely believed to be the major
the ratio of amylose to amylopectin, chain length and regulatory enzyme of the synthetic pathway (1, 2).
polymer size, degree of branching, the spacing between One reason for this is that AGP is tetrameric and
branch points, and the subsequent attachment of phos- subject to allosteric regulation. A second reason is
phate or sulfate groups. A detailed understanding of that plants carrying mutations in the AGP large or
the enzymes that function within the starch biosyn- small subunits produce markedly decreased levels of
thetic pathway in higher plants is therefore vital. starch (3, 4).
y Deceased.

Abbreviations: ADP-Glc, adenosine diphosphoglucose; AGP, adenosine diphosphoglucose pyrophosphorylase; ATP, adeno-
sine triphosphate; ae, amylose extender mutant; BT2, brittle-2; GBSS, granule-bound starch synthase; Glc-1-P, glucose-1-phos-
phate; SBE, starch branching enzyme; SDBE, starch debranching enzyme; SH2, shunken-2; SS, starch synthase; su-1, sugary-1
mutant.

Figure 1 Starch biosynthetic pathway in maize endosperm.
Chain elongation reactions use ADP-Glc as sub- III. STARCH BIOSYNTHETIC ENZYMES
strate. Starch synthases are glycosyltransferases that AND THEIR ISOFORMS
elongate oligosaccharide chains by successive addition
of (1,4)--linked glucose units to the nonreducing end Many isoforms of AGP, SS, and SBE have been puri-
of growing -glucan chains. There are at least two ed, characterized, and cloned from a variety of plants.
classes of starch synthase. The GBSSI, also known as The occurrence of having multiple isoforms encoded
the Waxy protein, is responsible for the synthesis of by distinct genes may be signicant for several reasons.
amylose. The soluble starch synthases are believed to First, each specic isoform may possess distinct cata-
be involved in chain elongation of amylopectins. lytic properties and therefore determine parameters
Starch chain length is thought to be controlled by spe- such as chain length or the spacing of branch points.
cic starch synthase isoforms. For example, GBSSI is specically responsible for the
The (1,6)--linked branch linkages are introduced synthesis of amylose, and waxy mutant maize which
by isoforms of SBE, which are believed to operate by lacks GBSSI is decient in amylose. In addition, the
a cut-and-past type mechanism (5). SBE is thought to isoforms of SBEI and SBEII transfer chains of differ-
bind to the sites where adjacent starch chains are jux- ent length. Second, the occurrence of multiple isoforms
taposed. The enzyme then cleaves at a (1,4)--linkage, enables differential expression with regard to both tis-
and the newly generated reducing end is then attached sue distribution, and plant growth and differentiation.
at another site to form a (1,6)--branch point. Spacing Therefore, the identication and characterization of
between branch points may be controlled by specic these isoforms is essential to the use of genetic engi-
SBE isoforms. neering to improve starch yield and starch quality.

A. Adenosine Diphosphoglucose 1. Granule-Bound Starch Synthase
Pyrophosphorylase
Based on solubility, the predominant granule-bound
starch synthase is referred to as GBSSI. GBSSI was
AGP is a tetrameric protein composed of two large
identied by studies with the waxy mutant. This
and small subunits known as SH2 and BT2, respec-
mutant lacks amylose, and is also decient in a 60-
tively. In maize, the SH2 and BT2 subunits have mole-
kDa polypeptide known as the Waxy protein. Waxy
cular masses of 54 and 51 kDa, respectively (Table 1)
mutants from numerous plant species are decient in
(3, 6). AGP is the only known tetrameric protein
both the Waxy protein and amylose. In waxy mutants,
within the starch biosynthetic pathway. Most plant
the starch is exclusively amylopectin. On this basis, it
AGPs are subject to allosteric regulation by the acti-
has become widely accepted that the 60-kDa Waxy
vator 3-phosphoglycerate and the inhibitor inorganic
protein is the starch synthase responsible for chain
phosphate (1). The AGP-catalyzed reaction is widely
elongation in amylose (19). Thus, the terms GBSSI
regarded as the rate-limiting step in the pathway. One
and Waxy protein are now used interchangeably. The
body of evidence was obtained by expressing a regula-
Waxy protein is solubilized by gelatinization of starch
tory variant, containing the E. coli AGP gene in
granules in the presence of a detergent such as SDS
tobacco, tomato, and potato. Since the bacterial
(19, 20).
AGP is relatively insensitive to regulatory inhibition
GBSSI was the rst maize starch synthase to be
by Pi, each of the transgenic plants exhibited signi-
cloned (23). Mutations in the waxy locus result in
cant increases in starch content (2).
low GBSS activities, absence of the Waxy protein,
The SH2 and BT2 subunits of AGP have both been
and virtually no amylose in the sink tissue of higher
puried from a variety of plants (3, 711). cDNA (12
plants (19, 2123). When potato plants were trans-
16) and genomic (17) clones have been isolated and
formed to produce antisense RNA so that the expres-
sequenced, and subunit function has been probed by
sion of the waxy gene was transcriptionally inhibited,
site-directed mutagenesis (18).
GBSS activity was greatly reduced and the tubers con-
tained amylose-free starch (24).
B. Starch Synthase
2. Soluble Starch Synthases
Classication of starch synthases has not been straight-
forward because the literature groups classied the The second major starch synthase class, based on solu-
various isoforms according to three different criteria. bility, is synthases which are partially soluble and par-
Particular groupings may be based on solubility con- tially granule bound (Table 1). These starch synthases
siderations, biochemical and catalytic properties, and have been grouped according to the order by which
deduced amino acid sequences derived from cDNA activity peaks from soluble extracts elute from ion
clones. exchange columns. Several soluble starch synthase iso-
Table 1 Starch Biosynthetic Isoforms from Maize
Process Isoforms/subunits Apparent mass (kDa) Reference(s)
Substrate formation AGP large subunit 54 (12, 13, 63)

(SH2)
AGP small subunit 51
(BT2)
Chain elongation GBSSI 60 (19, 23)
SSI 76 (28, 29)
SSII > 180 (30, 31)
Branch insertion SBEI 90 (51)
SBEIIa 89 (55)
SBEIIb 85 (64)
Processing/trimming SDBE (SU1) 79 (58,61)

forms have now been partially puried, characterized, 3. Four Classes of Starch Synthase Genes
and cloned from a diverse range of plants.
Based on the deduced amino acid sequences derived
Ion exchange chromatography of soluble extracts
from cDNA clones, starch synthase genes have been
from whole endosperm revealed the presence of multi-
divided into four classes (33). The extensively charac-
ple starch synthase isoforms (2528). In maize, two
terized GBSSI genes belong to the rst class. The genes
soluble starch synthase isoforms have been documen-
of the second class encode soluble starch synthases
ted. Starch synthase I exhibits maximal activity in the
localized in the endosperms of monocots. Thus far,
presence of citrate (primer independent). On the other
the cDNA clones of two members from this class
hand, starch synthase isoform II is active in the pre-
have been isolated and characterized. These include a
sence of glycogen (primer dependent). The separation
soluble starch synthase from rice (25) and the citrate-
of the two isoforms was also achieved by size exclusion
stimulated starch synthase I from maize (29). A third
chromatography (28). Following additional purica-
group of genes, the SSII class, bears sequence similar-
tion using MonoQ and hydrophobic interaction chro-
ity to the primer-dependent SSII from pea (34) and
matography, starch synthase I activity was correlated
potato (35). In addition to these three classes, starch
with a 76-kDa polypeptide (28).
synthases of the SSIII class, represented by a gene
A cDNA clone encoding the 76-kDa starch
encoding a 139-kDa soluble starch synthase III in
synthase I was subsequently obtained (29). This
potato, are believed to signicantly contribute to
cDNA bears homology to a soluble starch synthase
starch biosynthesis in some species (32, 33). SSIII
cloned from rice (25). Three isoforms of the rice
activity is believed to account for approximately 80%
synthase were isolated by anion exchange chromato-
of the soluble starch synthase activity present in potato
graphy, and their N-terminal amino acid sequences
tuber. The cDNA of potato SSIII exhibits greater simi-
were obtained. From this information, the rice
larity to bacterial glycogen synthases than to members
cDNA phage clone was isolated using oligonucleotide
of the other three classes.
probes prepared according to direct sequences. The
The deduced amino acid sequences of starch
predicted amino acid sequence of the clone contained
synthase clones share several common structural fea-
the KXGG motif which represents the putative ADP-
tures. First, they all contain the Lys-X-Gly-Gly-Leu
Glc binding domain existing in virtually all starch and
(KXGGL) consensus sequence, which is suggested to
glycogen synthases.
be the putative ADP-Glc-binding site. This conserved
The apparent size of SSII is 180 kDa. SSII activity
region was rst identied in E. coli glycogen synthase
has not been aligned with any specic polypeptide
(36). The lysine residue binds to the phosphate moiety
from maize endosperm. However, isolation of a gene
adjacent to the glycosidic linkage of ADP-Glc (37).
encoding a putative polypeptide correlating with SSII
Moreover, starch synthase genes encode transit pep-
activity has been reported (30).
tides ending with a consensus cleavage site motif. It
Moreover, two other starch synthase cDNA clones,
is believed that starch synthases are synthesized in
designated zSSIIa and zSSIIb, have been isolated from
the cytosol and are targeted to the amyloplast by the
a maize endosperm cDNA library (31). The deduced
transit peptides, which are then proteolytically cleaved
amino acid sequences of both clones contain the
upon translocation through the amyloplast envelope
KXGGL consensus sequence. The identity of zSSIIa
into the stroma (38, 39). Finally, several conserved
and zSSIIb as starch synthases has been conrmed by
regions between the KXGGL site and the 3 0 terminus
expression of their activities in E. coli. zSSIIa was
of the coding sequence exist in most of the clones (25).
shown to be predominantly expressed in the endo-
sperm, while zSSIIb transcripts were mainly detected
in the leaf at low abundance. C. Starch Branching Enzymes
In potato, a third kind of soluble starch synthase,
known as SSIII, has been identied. Its cDNA clone The functionality and economic value of starches are
was obtained by using an antibody directed against a largely determined by the relative ratio of amylose and
domain conserved in starch synthases to screen a amylopectin. Since SBE is the enzyme which intro-
tuber-specic expression library. The deduced amino duces -1,6-linked branch points into the -1,4-linked
acid sequence showed that it is a novel type of starch glucan chains of amylopectin, it plays an essential role
synthase. The immature enzyme with its transit peptide in determining starch ne structure. Multiple isoforms
has a mass of 139 kDa (32). of SBE have been identied, puried, and character-

ized from many plants including maize endosperm (40 Immunological evidence and substrate specicity stu-
43), wheat endosperm (44, 45), pea seed (46), and rice dies indicated that wheat BEIA and BEIB are related
endosperm (47, 48). to the SBEI class of maize and rice, while wheat BEII
Different SBE isoforms differ in their specicity for (45) is related to SBEII class (44).
substrate with respect to both chain length and degree
of branching. The SBE isoforms and their clones can D. Starch Debranching Enzyme
be categorized into two groups as initially dened by
the Commission on Plant Gene Nomenclature (1994) An additional putative enzyme of the starch biosyn-
and as later suggested by Burton et al. (49). One family thetic pathway, SDBE, has attracted great recent inter-
(SBEI or class B) includes maize SBEI, rice SBEI, pea est. Ball and others have proposed an additional set of
SBEII, and potato SBE. The second family (SBEII or steps in the biosynthesis of amylopectin consisting of
class A) includes maize SBEII, pea SBEI, and rice formation of a preamylopectin and its subsequent
SBEIII. The establishment of the two groups was trimming to a mature amylopectin (56). SDBE is
based on immunological reactivity, chromatographic believed by some researchers to be involved in convert-
and enzyme kinetic properties, and amino acid compo- ing preamylopectin into mature amylopectin. These
sition (4648, 50). In vitro, maize SBEI preferentially researchers propose that SBE and SDBE work in a
transfers longer chains than SBEII (42). Moreover, concerted fashion to determine the ne structure of
maize SBEI preferentially adds branch points to amy- amylopectin (57).
lose, and its activity toward amylopectin is < 10% Two classes of SDBE have been studied in plant
relative to amylose (41). Conversely, maize SBEII exhi- systems, termed pullulanases and isoamylases. Both
bits a sixfold higher preference for amylopectin than types can directly hydrolyze -(1,6)-branch point lin-
SBEI, but lower rates in branching amylose ( 912% kages. Each class differs in its specicity toward dis-
of that SBEI) (41). tinct substrates. Pullulanases, also referred as R-
In maize, cDNA clones for SBEI (51), SBEIIa (52), enzyme, hydrolyze pullulan at a much higher rate
and SBEIIb (53) have been isolated and their expres- than amylopectin, and do not act on glycogen. In con-
sion patterns characterized (54). Maize SBEI elutes trast, isoamylases act on both amylopectin and glyco-
from ion exchange columns in the pass-through frac- gen, but do not hydrolyze pullulan or, if so, at a very
tion, whereas SBEIIa and SBEIIb require salt for elut- low rate. Pullulanases have been characterized in maize
ing from the column (40). SBEIIa and SBEIIb are very (58), rice (59), and many other plants. Isoamylases,
similar in their immunological reactivities, enzyme however, have only been reported and studied in
kinetic properties, substrate specicity, and amino potato tuber (60) and maize endosperm (58, 61).
acid sequence (1, 41, 42). In maize endosperm, the identication and charac-
For quite some time, there was a controversy as to terization of SDBE, an isoamylase, was achieved using
whether or not SBEIIa and SBEIIb are encoded by a a su1 mutant which carries mutations on the SDBE
single gene. However, the isolation of SBEIIa cDNA gene (61). In maize kernels, these mutations lead to
from ae mutants lacking SBEIIb has claried this ques- increased sucrose levels, reduced amylopectin, and
tion (55). Using an ae mutant in which SBEIIb was accumulation of the highly branched glucopolysac-
absent, Fisher et al. observed signicant SBEIIa charide phytoglycogen (62). The correlation between
enzyme activity and polypeptide levels (53), suggesting the su mutation and the deciency of SDBE was rst
that SBEIIa is encoded by an independent gene distinct reported by Pan and Nelson (58). This study showed
from the Sbe2b locus. The localization of the two that SDBE activities from wild-type maize endosperm
mRNA transcripts in maize tissues also differed. could be separated into three peaks on a hydroxyapa-
Sbe2b is expressed in endosperm, developing embryo tite chromatography column. Their key observation
and tassels (53). Sbe2b mRNA levels were 10-fold was that the su1 endosperm lacked one of the three
higher in embryo than in endosperm tissue, and were activities while the other two were greatly reduced.
much lower than Sbe2b in both tissues (55). They also observed that SDBE activity was propor-
In hexaploid wheat, the situation is more complex. tional to the number of copies of the Su allele, suggest-
Wheat is believed to contain similar classes of SBE as ing that the Su allele was the structural gene for SDBE.
those found in maize and rice. Moreover, each of the This publication classied SDBE as an R-enzyme or
three genomes of wheat may contain a unique set of pullulanase, and assumed that the biochemical lesion
SBE isoforms. The isoforms contained within each in the su1 mutant of maize was induced by mutations
unique set are referred to as allozymes (44). at the structural gene for the R-enzyme. Recently,

James et al. isolated part of the su1 gene locus and a 3. LC Hannah, OE Nelson. Characterization of ADP-
cDNA copy of the su1 transcript using transposon tag- glucose pyrophosphorylase from shrunken-2 and brit-
ging (61). Five new su1 mutations were isolated. The tle-2 mutants of maize. Biochem Genet 14:547560,
cDNA sequence specied a polypeptide of at least 742 1976.
4. CY Tsai, OE Nelson. Starch-decient maize mutant
amino acids bearing high similarity to bacterial isoa-
lacking adenosine diphosphate pyrophosphorylase
mylase. This emerging area is further discussed in a
activity. Science 151:341343, 1966.
recent review paper (57). 5. BP Wasserman, C Harn, C Mu-Forster, R Huang.
Progress toward genetically modied starches. Cereal
IV. CONCLUSION Foods World 40:810817, 1995.
6. DB Dickinson, J Preiss. Presence of ADP-glucose pyr-
A complete understanding of amylose and amylopectin ophosphorylase in shrunken-2 and brittle-2 mutants
synthesis in higher plants requires full knowledge of of maize endosperm. Plant Physiol 44:10581062,
1969.
individual enzymes catalyzing substrate formation,
7. BY Chen, HW Janes. Multiple forms of ADP-glucose
chain elongation, and branch point insertion. The pyrophosphorylase from tomato fruit. Plant Physiol
heightened research efforts have been focused on 113:235241, 1997.
basic biochemistry and molecular biology along the 8. L Copeland, J Preiss. Purication of spinach leaf
following lines: ADP-glucose pyrophosphorylase. Plant Physiol
68:9961001, 1981.
1. Enzyme purication, characterization and poly-
9. LC Hannah, OE Nelson. Characterization of adeno-
peptide identication. sine diphosphate glucose pyrophosphorylase from
2. Isolation and characterization of cDNA and developing maize seeds. Plant Physiol 55:297302,
genomic clones. 1975.
3. Expression of starch biosynthetic enzymes in 10. Y Nakamura, K Kawaguchi. Multiple forms of ADP-
bacteria and transgenic plants. glucose pyrophorylase of rice endosperm. Physiol
Plant 84:336342, 1992.
These efforts will eventually lead to a complete elu- 11. JR Sowokinos, J Preiss. Pyrophosphorylases in
cidation of the molecular mechanisms underlying Solanum tuberosum. III. Purication, physical and cat-
starch biosynthesis and thus enable the bioengineering alytic properties of ADPglucose pyrophosphorylase in
of starch biosynthetic process in cereal plants. potatoes. Plant Physiol 69:14591466, 1982.
12. JM Bae, M Giroux, L Hannah. Cloning and charac-
DEDICATION terization of the Brittle-2 gene of maize. Maydica
35:317322, 1990.
13. MR Bhave, S Lawrence, C Barton, LC Hannah.
This chapter is dedicated to the memory of my mentor,
Identication and molecular characterization of
Bruce P. Wasserman, who passed away on August 26, shrunken-2 cDNA clones of maize. Plant Cell 2:581
1998. 588, 1990.
14. PA Nakata, TW Greene, JM Anderson, BJ Smith-
ACKNOWLEDGMENTS White, TW Okita, J Preiss. Comparison of the pri-
mary sequences of two potato tuber ADP-glucose pyr-
Funding for this research was provided by the U.S. ophosphorylase subunits. Plant Mol Biol 17:1089
1093, 1991.
Department of Agriculture National Research
15. MR Olive, RJ Ellis, WW Schuch. Isolation and
Initiative (95-02531 and 98-01235), and by the New nucleotide sequences of cDNA clones encoding
Jersey Agricultural Experiment Station. ADP-glucose pyrophosphorylase polypeptides from
wheat leaf and endosperm. Plant Mol Biol 12:525
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11
Fatty Acid Biosynthesis and Triacylglycerol Assembly
Allan Keith Stobart

University of Bristol, Bristol, England
Gareth Grifths
Horticulture Research International, Wellesbourne, Warwick, England
I. INTRODUCTION and desirable fatty acids to oil-crop species such as oil-

seed rape, sunower, and soy. The scientic base for
Vegetable oil is an economically important agricultural the production of genetically modied oil-seeds is well
product with an annual production of some 90 million established (3) and it is therefore anticipated that an
metric tons. It is used both as food and in a wide range ever-increasing level of oil and oil products from such
of industrial applications. By using both traditional sources will enter the human diet. Here we describe,
plant breeding methods and genetic engineering tech- therefore, the current understanding of fatty acid bio-
niques, vegetable oils can today be modied for parti- synthesis in plants, oil assembly, and the role of fatty
cular end uses. The production of so-called designer acid oxidation in avor generation. Where appropriate
oils to suit requirements is an ever-increasing prospect, the biotechnological applications of such knowledge
and hence it is anticipated that the world output of will be discussed.
such commodities will increase accordingly (1).
The end use and hence value of an oil are largely
dependent on the fatty acid quality and content. The II. FATTY ACIDS AND COMPLEX LIPIDS
wide range of fatty acids and their relative concentra-
tions, found in oils from many plant species, have Most fatty acids are monocarboxylic and consist of an
made them particularly suited for food production unbranched chain containing an even number of car-
(e.g., confections, ice cream, mayonnaise, margarines, bon atoms (Table 1). The homologous series of satu-
animal foodstuffs, etc.), cooking oils, nutraceuticals, rated fatty acids is represented by the general structure
and pharmaceuticals. Besides being used for human CH3 CH2 n COOH with the carboxy carbon designated
consumption they have many applications in the che- carbon 1. The number of carbon atoms in the fatty
mical industries and particularly for the manufacture acid can vary considerably from medium chain
of detergents and surfactants, polymers, coatings, and (C8C10) to long and very long chain (C12 and up)
specialized lubricants. In the kingdom Plantae perhaps although it is the long-chain ones that tend to predo-
well over 800 different fatty acids have been identied minate. The methyl and carboxy termini of the mole-
(2). Many of these are uncommon and are found in cule are termed the omega (!) and delta () ends,
only a few species. However, this enormous genetic respectively. These fatty acids can be saturated or
resource is of immense commercial value as it becomes unsaturated and in some cases may have additional
feasible to transfer the capacity to synthesize unusual functional groups. A notable characteristic of plants

Table 1 Fatty Acid Structures
General structure (omega [!] terminus)CH3 (CH2)nCOOH (delta [] terminus)
Lauric acid (C12:0) CCCCCCCCCCCC

Palmitic acid (C16:0) CCCCCCCCCCCCCCCC
Hexadecanoic acid
Stearic acid (C18:0) CCCCCCCCCCCCCCCCCC
Octadecanoic acid
Oleic acid (C18:19) CCCCCCCCC
CCCCCCCCC
Octadeca-9-monoenoic acid
Linoleic acid (C18:29,12) CCC
CCCCCC CCCCCCCCC
Octadeca-9,12-dienoic acid
-Linolenic acid (C18:39,12,15) CCC
CCC CCC
CCCCCCCCC
Octadeca-9,12,15-trienoic acid
-Linolenic acid (GLA; C18:36,9,12) CCC
CCCCCC CCC
CCCCCC
Octadeca-6,9,12-trienoic acid
Stearidonic acid (C18:46,9,12,15) CCC
CCC CCC
CCC
CCCCCC
Octadeca-6,9,12,15-tetraenoic acid
Arachidonic acid (C20:45,8,11,14) CCCCCCCCC
CCC
CCC
CC
Eicosa-5,8,11,14-tetraenoic acid CCC
Eicosapentaenoic acid (EPA) CCC CCC
CCC CCC CC
CCC
C20:55,8,11,14,17 CCC
Erucic acid (C22:113) CCCCCCCCCCCCCCCCC

C C C C C
Essential fatty acids are polyunsaturated fatty acids of the omega-6 (n-6) and omega-3 (n-3) families that are essential for life and good health,
and are not produced in man and must be obtained from plants as part of the diet.
is the high incidence of unsaturated and polyunsatu- plant sources for the synthesis of the other n6 and n3
rated fatty acids and particularly those of 18 carbon metabolites. Hence, these are the so-called essential
atoms. fatty acids (EFAs) and are required for the normal
Unsaturation generally describes a four-electron structure of all membranes, for cholesterol transport,
(double bond/olenic) linkage between carbon atoms. for the maintenance of normal permeability barriers
Polyunsaturated fatty acids (the so-called PUFAs) are and as precursors of eicosanoids (prostaglandins,
polyenoic acids having more than one olenic center, thromboxanes, leukotrienes).
the double bonds generally of a cis conguration and A further unsaturated fatty acid terminology, of
methylene interrupted. By convention PUFAs are particular value to biochemists, denes the position
described as being n3 or n6 fatty acids depending on of the double bonds from carbon atom 1 (i.e., the
whether the rst double bond is three or six carbon end of the molecule). The delta system is convenient
atoms from the methyl end (omega carbon), respec- for denoting all double bonds, their position in the
tively. Further double bonds are separated from each carbon chain, and the specic desaturase enzymes
other by a methylene group. Such fatty acids are also which catalyze their introduction into the molecule.
termed omega-3 and omega-6 acids. The omega-3 ser- Hence, linoleic acid and -linolenic acid become
ies are derived from -linolenic acid (C18:3 n3) by C18:29;12 and C18:39;12;15 , respectively. Fatty acid
chain elongation and further desaturation and include structures and trivial and systemic nomenclatures are
eicosapentaenoic acid (EPA; C20:5 n3) and docosa- given in Table 1. Desaturases, which abstract hydrogen
hexaenoic acid (DHA); C22:6 n3). On the other atoms from adjacent CH2 groups to produce double
hand, the omega-6 acids are derived from linoleic bonds, can be specically dened. For example, a 12
acid (C18:2 n6) and include -linolenic acid (GLA; desaturase or a 5 desaturase would introduce double
C18:3 n6) and arachidonic acid (AA; C20:4 n6). bonds at the 12 and 5 carbon atom in the acyl
Linoleic acid and linolenic acid are substances which chain, respectively.
cannot be synthesized de novo in the human body and Triacylglycerols, the major constituents of the oil,
so, like vitamins, must be provided in the diet from consist of a glycerol backbone to which a fatty acid

composition of membrane lipid in leaves and roots,
however, is under strict control and differs little
among plant species whereas the relative abundance
of a fatty acid in the storage triacylglycerol can show
distinct variations among different oil crop species.
III. FATTY ACID BIOSYNTHESIS

A. Carbon Source
Acetyl-CoA is regarded as the direct carbon source for

fatty acid synthesis and can be generated by the pyr-
uvate dehydrogenase (PDC) complex. Despite the fun-
damentally crucial requirements for acetyl-CoA, its
biogenesis in plants is poorly understood. Both chlor-
oplast (cPDC) and mitochondrial (mPDC) forms (4, 5)
have been characterized. There has been speculation
about the supply of pyruvate for acetyl-CoA formation
from cPDC and whether there is sufcient activity of
the glycolytic enzymes to provide pyruvate within this
organelle. In mustard, for example, there appears to be
a sufcient supply of pyruvate to satisfy this require-
ment, although in other tissues important cytosolic
pyruvate may be required to support fatty acid bio-
synthesis (6). Studies with plastids from heterotrophic
tissues indicate that the metabolites utilized for fatty
acid synthesis include glucose, fructose, glucose-6-
phosphate, triose phosphate, malate, pyruvate, and
acetate (7). Of these only acetate has been shown to
be freely permeable to the selective barrier of the plas-
tid envelope. There is considerable variation in the
efciency with which these substrates are utilized by
Figure 1 Glycerolipid structures. plastids, and the basis for this may be determined by
the presence of highly selective transporter proteins on
the plastid envelope.
It has recently been shown that during oil deposi-
acyl group is esteried to each of the three hydroxy tion in oil seed rape, fatty acid synthesis is initially
groups. Each carbon atom in the glycerol molecule is supported by glucose-6-phosphate and then by pyru-
dened by the stereoscopic numbering system and sn1, vate through the corresponding activities of the trans-
sn2, and sn3 describes each carbon, respectively porters for these metabolites (7, 8). In isolated
(Fig. 1). The glycerol carbons of the phospholipids leucoplasts from developing castor seed endosperm,
(membrane polar lipids), important in understanding however, the rate of malate-dependent fatty acid
triacylglycerol biosynthesis, are similarly dened synthesis was threefold higher than that from pyruvate
(Fig. 1). The quality of the oil, and hence its end use, and evidence was presented for a malate/Pi transloca-
is determined by the fatty acids esteried to each of the tor (9). Whether the expression of selective transpor-
glycerol carbon atoms. Most commercial annual oil ters on the plastid envelope determines the ability of
crops have seed oils largely composed of the ve these organelles to accumulate oils or starch requires
major fatty acids, palmitic (C16:0), stearic (C18:0), further research. In this regard it is interesting to note
oleic (C18:19 ), linoleic (C18:29;12 ) and -linolenic the isolation of a novel Arabidopsis mutant producing
(C18:39;12;15 ) acids. These are the housekeeping wrinkled, incompletely lled seeds with an 80% reduc-
fatty acids, so called because they are also found in tion in triacylglycerols. Wild-type and homozygous
the membrane lipids of all plants cells. The fatty acid wri1 mutant plantlets or mature plants were indistin-

guishable. However, developing homozygous wri1 It has now been established, however, that there are
seeds were impaired in the incorporation of sucrose two forms of ACCase in plants (11, 17). In dicotyledo-
and glucose into triacylglycerols but incorporated pyr- nous species there is a low-molecular-weight (LMW)
uvate and acetate at an increased rate (10). Because the form present in the plastid and high-molecular-weight
activities of several glycolytic enzymes, in particular (HMW) one in the cytosol whereas in the Graminaceae
the hexokinase and pyrophosphate-dependent phos- the LMW form has not been detected and HMW
phofructokinase, are reduced in developing homozy- forms are present in both the plastid and cytosol.
gous wri1 seeds, it was suggested that wri1 is This provides an explanation for the differential action
involved in the developmental regulation of carbohy- of the monocotyledon-specic acyloxyphenoxy-pro-
drate metabolism during seed lling. prionate and cyclohexonediose group of herbicides,
whose main target is the HMW ACCase in the plastid.
B. Acetyl-CoA Carboxylase It is currently believed that the HMW ACCase in the
plastid is responsible for de novo fatty acid biosynth-
Acetyl-CoA carboxylase (ACCase) catalyzes the for- esis which is herbicide sensitive and a further HMW
mation of malonyl-CoA from acetyl-CoA and bicarbo- ACCase in the cytoplasm responsible for fatty acid
nate. Plants have two types of acetyl-CoA elongation (see later) which is insensitive (11).
carboxylases; one is the multisubunit complex located Despite the molecular characterization of ACCases,
in plastids, and the other a large multifunctional poly- little progress has been made on the metabolic regula-
peptide localized in the cytosol (11). The plastidic form tion of the enzyme in plants. Unlike the mammalian
consists of four subunits, and in Arabidopsis these are enzyme where regulation by phosphorylation and
encoded by three single copy nuclear genes (cac1, cac2, intermediates of the tricarboxlic acid cycle, particularly
and cac3) and one plastidic gene (accD) (12, 13). The citrate, have been shown to be involved, no evidence
cytosolic ACCase generates malonyl-CoA for the bio- exists for such regulatory mechanisms in plants.
synthesis of a variety of secondary metabolites and for
the elongation of fatty acids. The enzyme is a dimer of C. Fatty Acid Synthase
a 260-kDa subunit and in Arabidopsis is encoded by
two genes (acc1 and acc2) which are in a tandem array The molecular organization of the plant fatty acid
within a 25-kb segment of the genome. In photosyn- synthase is composed of dissociable enzyme activities,
thetic tissue the chloroplast ACCase is modulated by similar to E. coli and termed type II FAS, has been
light/dark. Plants have a light-signal transduction recognized since the early 1980s (Fig. 2). The initial
pathway in chloroplasts which operates when electrons reactions of FAS are catalyzed by transacylases
from photosystem 1 are shuttled through the electron which transfer the acetyl and malonyl units from
transport chain to thioredoxins via ferredoxin and then their CoA esters to the corresponding ACP derivatives
by reducing the disulde bonds of target enzymes (14). (18). The initial condensation reaction in plant fatty
Enzyme activities of the reductive pentose phosphate acid biosynthesis takes place between acetyl (C2)
pathway cycle in chloroplasts are regulated via this ACP and malonyl (C3) ACP, resulting in the forma-
redox cascade. Recent results suggested that a redox tion of acetoacetyl (C4) ACP and carbon dioxide. This
cascade may be responsible for coordination of fatty reaction is catalyzed by ketoacyl ACP synthase (KAS).
acid synthesis with photosynthesis (14). In the next step the acetoacetyl-ACP is reduced to
In mammals the ACCase consists of a single poly- hydroxybutyryl-ACP by -ketoacyl-ACP reductase
peptide chain containing three functional domains, using NAD(P)H. The hydroxybutyryl-ACP is then
while in E. coli the enzyme consists of four separable dehydrated by hydroxyacyl-ACP dehydratase, and
proteins: biotin carboxyl carrier protein (BCCP), bio- the crotonyl-ACP form is converted to butyryl-ACP
tin carboxylase (BC), and carboxyl transferase (CTa by enoyl-ACP reductase using NAD(P)H. This com-
and CTb). Initial attempts to purify the plant pletes the rst round of fatty acid synthesis.
ACCase generated widely differing apparent molecular In the next round the butyryl (C4) ACP is con-
weights (15, 16). These discrepancies were originally densed with malonyl (C3) ACP resulting in the forma-
interpreted as being due to differential rates of proteo- tion of C6 ACP and carbon dioxide which then
lysis in different tissue extracts, and it was therefore undergoes a further two reductions and dehydration
considered that the plant acetyl-CoA carboxylase, step as described earlier to complete the second cycle.
like that from mammals, was a multifunctional pro- Subsequent additions of C2 units from malonyl-ACP
tein. results in the formation of long-chain fatty acids of

Figure 2 The fatty acid synthetase complex. Acetyl-CoA:ACP transacylase (ACAT; reaction 1) and malonyl-CoA:ACP trans-
acylase (MCAT; reaction 2) convert acetyl-CoA and malonyl-CoA to ACP derivatives, respectively. Condensation of these
products by -ketoacyl ACP synthase (KAS I; cerulenin sensitive; reaction 3A) results in the formation of acetoacetyl-ACP (C4
on the gure) and the generation of CO2 . Acetyl-CoA can also be condensed directly with malonyl-ACP in a KAS III mediated
reaction (cerulenin insensitive; reaction 3B). Acetoacyl ACPs are reduced by -ketoacyl reductase (reaction 4) to yield the -
hydroxyacyl-ACP derivatives, which undergo dehydration, catalyzed by a -hydroxyacyl-ACP dehydrase (reaction 5). The
generated ; -unsaturated fatty acyl-ACPs are further reduced by an NADH-specic enoyl reductase (reaction 6), resulting
in the formation of the acyl-ACP product. In subsequent rounds of condensation, malonyl-ACP is used as the primer which
again is followed by reduction (reaction 4), dehydration (reaction 5), and further reduction (reaction 6). In species accumulating
short- to medium-chain fatty acids (e.g., Cuphea), a short chainspecic condensing enzyme has been identied (KAS IV;
reaction 3C). In species not accumulating short-chain fatty acids, KAS I (and/or KAS III up to C6) supports these condensation
reactions. A further three and four rounds of the cycle result in the formation of C8:0 and C10:0 chain length products,
respectively. In species accumulating short-chain fatty acids, these products are hydrolyzed from the ACP by the action of a
specic thioesterase (reaction 7; only C8 illustrated), resulting in the formation of unesteried fatty acids which are subsequently
utilized for storage lipid formation. All species synthesize C16 and C18 chain length fatty acids both for membrane lipid and oil
synthesis. C16:0 is elongated to C18:0 by a specic -ketoacyl-ACP synthetase (KAS II; reaction 3D). These fatty acids are
released from the ACP by thioesterase activity to yield unesteried fatty acid for use in oil assembly.

which palmitate (C16:0) and stearate (C18:0) are the by enoylacyl-ACP reductase (ENR) to form acyl-ACP,
principal end products. The enzyme responsible for the which in developing oilseed rape, is an NADH-depen-
formation of stearate from palmitate has been shown dent enzyme. The Brassica napus ENR is tetrameric
to be an isoform of the KAS I and has been designated (23) and has been cloned and overexpressed in E. coli
KAS II based on reconstitution experiments and on (24) and crystallized (25). Interestingly, the ENR is the
selective inhibitor studies using arsenite and cerulenin target for antibacterial diazaborines and the front-line
(19). antituberculosis drug isoniazid. Recent structural stu-
The transfer of acetyl groups from the CoA track to dies have revealed the chemical basis for its molecular
the ACP track, catalyzed by the acetyl:ACP transacy- binding of drugs which, based on its similarity to other
lase, was considered to be the possible rate-limiting oxidoreductases, may have generic applications in
step in fatty acid synthesis (18). However, in the late other areas of medicinal chemistry (26).
1980s studies with E. coli indicated the presence of a
short-chain condensing enzyme whose activity was not D. Chain Termination
blocked by cerulenin, and short-chain ACPs accumu-
lated. A similar activity was subsequently reported in The principal-product of fatty acid biosynthesis in
plants and it appeared that conversion of acetyl-CoA most plant tissues is palmitoyl-CoA, which can then
to acetyl-ACP could be bypassed and the acetyl-CoA be elongated by a specic -ketoacyl-ACP synthase
and malonyl-ACP condensed directly (20). This activ- type II (KAS II) to generate stearoyl-ACP. However,
ity has been referred to as KAS III. This has subse- since storage lipid accumulation is not under the same
quently brought into question the earlier suggestion functional constraints imposed on membrane lipids,
that the transacylase played a regulatory role and several plant families have evolved mechanisms for
was the rate-limiting step in fatty acid biosynthesis. terminating fatty acid synthesis at the C8C14 chain
There are, however, indications that acetyl ACP:CoA length stage for incorporation into triacylglycerols dur-
transacylase is a partial activity of KAS III, but this ing seed development. The potential applications of
awaits conrmation by characterization of the puried these fatty acids has been a major driving force in
enzyme. Metabolic plasticity has been observed in the search to elucidate the mechanisms by which they
many pathways, and the role of the KAS III and/or are synthesized. A number of potential chain-terminat-
transacylase may yet be another example of this phe- ing mechanisms were hypothesized (see 15) and
nomenon. included the presence of a medium chainspecic
Malonyl-CoA:ACP transacylases (MCAT) have acyl-ACP thioesterase, specic acyl-ACP transferases,
been puried from a number of plant species. The -fatty acid synthetases, and -ketoacyl-ACP synthe-
enzymes usually have molecular weights determined tases. Other possibilities include the availability of the
from standard curves (MW) or by mass spectrometry fatty acid metabolites malonyl-CoA and acetyl-CoA,
(molecular mass) 40 kDa, and tissue-specic iso- and cofactor levels of ACP.
forms have been reported (15). The gene has recently An important role for the medium-chain acyl-ACP
been cloned from Brassica napus using a complementa- thioesterase was rmly established following the
tion approach (21); the protein showed a 47% homol- expression of the Californian bay 12:0-ACP thioester-
ogy to the E. coli MCAT amino acid sequence in the ase in Arabidopsis resulting in the formation of laurate
coding region for the mature protein. The -hydroxya- and the rst genetically engineered rapeseed (27).
cyl dehydratase has also been puried and partially Thioesterases with the potential to confer different
characterized from a number of sources (see 11) and, medium-chain length phenotypes have now been iso-
although the plant enzyme has received little further lated from a number of species, especially from the
molecular characterization, the bacterial dehydratase genus Cuphea (2830). Although acyl-ACP thioes-
has recently been cloned (22). terases with specicities toward medium-chain sub-
The enzymes for the two reductive steps required in strates are requisite enzymes in plants which produce
each round of fatty acid biosynthesis have been puri- C8C14 seed oils, they do not appear to be the sole
ed and shown to have a preference for the ACP- chain length determinants. It has recently emerged that
linked esters over the corresponding CoA derivatives -ketoacyl-ACP synthase also plays an important role
(16). The rst reductive step is catalyzed by -ketoacyl in the production of medium-chain fatty acids and that
ACP reductase, an NADPH-dependent reaction that it is the cooperation of the synthase and thioesterase
produces hydroxyacyl-ACP. Hydroxyacyl-ACP is which determines the phenotype, at least in Cuphea
dehydrated by a dehydratase and then further reduced (28).

cDNA clones encoding the 3-ketoacyl-ACP class, and one from the fatB class. Expression of Garm
synthase have been isolated from Cuphea and overex- FatA1 in Brassica seeds led to the accumulation of
pressed in Brassica (29). Expression of this enzyme in stearate up to 22% in the seed oil. These results suggest
transgenic Brassica seeds which normally do not pro- that Garm FatA1 is at least partially responsible for
duce medium-chain fatty acids did not result in any determining the high stearate composition of mangos-
detectable modication of the fatty acid prole. teen oil and that fatA, as well as fatB, thioesterases
However, coexpression of the Cuphea KAS with the have evolved for specialized roles.
medium chainspecic thioesterase strongly enhanced
the level of C8:0C12:0 fatty acids in the seed oil as E. Unsaturated and Polyunsaturated Fatty Acids
compared with seeds expressing the thioesterase alone.
By contrast, coexpression of the Cuphea KAS along In many oleaceous species the products of fatty acid
with the 18:0/18:1-ACP thioesterase did not result in synthesis in the plastid are palmitic (C16:0), stearic
a modied fatty acid prole. These data indicated that (C18:0), and oleic (C18:1) acids (see above). These
the Cuphea KAS had different acyl chain specicities are made available to the endoplasmic reticulum for
to the previously characterized KAS I, II, and III and triacylglycerol assembly (see below) and the formation
was therefore designated KAS IV, a medium chain of other fatty acids, particularly polyunsaturated fatty
specic condensing enzyme (29). Knowledge of this acids. Unsaturated fatty acids, therefore, are synthe-
enzyme activity and its relationship with the short- sized by the sequential insertion of double bonds into
chain thioesterase may be exploited in the future engi- a more saturated precursor. The rst double bond in
neering of oilseeds to increase yields and medium- the production of C18 unsaturated fatty acids is
chain fatty acids or extend the range of medium- inserted at the 9 position of stearic acid (C18:0)
chain phenotypes. and yields oleic acid (C18:19 ). The 9 desaturase cat-
A few plant species have been identied that accu- alyzing this reaction is a soluble plastidic protein uti-
mulate signicant amounts of stearate in their seed lizing C18:0ACP as substrate. To date the 9
oils; these include cocoa (Theobroma cacao), shea 18:0ACP desaturase is the only desaturase to have its
(Butyrospermum parkii), sal (Shorea robusta), and structure and mode of action fully characterized. A
kokum (Garcinia indica), the oils of which are valuable cDNA encoding this desaturase, isolated from castor
as cocoa butter or cocoa butter substitutes in many bean endosperm and overexpressed in E. coli, gener-
confectionary applications. The level of stearate avail- ated sufcient protein for biophysical characterization
able for triacylglycerol assembly is likely controlled by (34). The desaturase contained a di-iron cluster at the
the combined regulation of the plastid localized active site and in this respect was similar to methane
enzymes -ketoacyl-acyl carrier protein synthase II mono-oxygenase hydroxylase (35). The protein has
(which generates stearoyl-ACP) and stearoyl-ACP been crystallized and its structure determined (36). A
desaturase, acyl-ACP thioesterase, and acyl-ACP acy- mechanism of action for the desaturase proposes that
transferases, all of which use stearoyl-ACP as substrate the removal of two hydrogens results in the formation
(31). Downregulation of stearoyl-ACP desaturase of a transient di-radical that spontaneously recombines
resulted in the signicant accumulation of stearate in to form the 9 double bond (37).
the storage oil of non-stearate-accumulating species Antisense constructs of the 9 stearoyl-ACP desa-
such as Brassica rapa and B. napus (32). turase, with seed-specic promoters inserted into oil-
It should be pointed out that plant acyl-ACP thioes- seed rape, brought about a dramatic increase in stea-
terases fall into two distinct but related classes known rate in the rape oil from the normal < 2% to 40% and
as FatA and FatB (27, 33). The FatA class includes the with a corresponding decrease in oleate (32).
palmitoyl-, stearoyl-, and oleoyl-ACP thioesterases Triacylglycerols with high stearate are of commercial
and these are regarded as ubiquitous. The fatB class interest as cocoa butter substitutes in confectionary fat.
contains all the specialized thioesterases (including the Oleate, from the plastid, is channeled into phospho-
medium-chain thioesterases described above) as well as lipid, mainly phosphatidylcholine of the endoplasmic
an apparently ubiquitous thioesterase with a broader reticulum and there, depending on the plant species,
specicity for longer-chain saturated and unsaturated sequentially desaturated by a 12 and 15 desaturase
substrates. Recently, Hawkins and Kridl (31) have into linoleate and -linolenate (38, 39). Similar desa-
reported the cloning and characterization of three dif- turation steps occur in the plastid galactolipids and are
ferent acyl-ACP thioesterase cDNAs from mangosteen catalyzed by desaturases encoded by genes which are
(Garcinia mangostana) seed RNAtwo from the fatA separate from those encoding the endoplasmic reticu-

lum proteins (40). It is only the endoplasmic reticulum (55). In seed oil, however, -linolenate predominates
desaturases, however, that appear to be responsible for and in borage accounts for some 25% of the triacyl-
the production of the polyunsaturated fatty acids in glycerol fatty acids. The seed -linolenic acid is synthe-
the storage triacylglycerols. The 12 desaturation in sized by a 6 NADH-dependent desaturation of
oil seeds occurs with oleate at both the sn1 and sn2 linoleate esteried to largely microsomal phosphatidyl-
positions of sn-phosphatidylcholine, whereas 15 choline (56). The 6 desaturase has a strong preference
desaturation in linseed exhibits a strong preference for linoleate at position sn2 of phosphatidylcholine. -
for substrate at position sn2. The oil seed endoplasmic Linolenic acid acts as a substrate for the 6 desaturase
reticulum 12 desaturase is cyanide sensitive and and the formation of 18:46;9;12;15 (55).
requires NAD(P)H and molecular oxygen. -Linolenic acid is considered of therapeutic value
Spectrophotometric evidence shows that the safower in alleviating a wide range of clinical and atopic dis-
microsomal 12-desaturation is mediated through the orders (57), and -linolenate-rich oil from a number of
cytochrome b5-cytochrome b5 reductase system (41); species (borage, evening primrose, black currant, and
this was conrmed with antibodies to cytochrome b5, some fungi) (58) is available for this purpose. The
which inhibited desaturase activity (42). Seed microso- inclusion of a 6 desaturase gene in a high linoleate
mal cytochrome b5 has been molecularly characterized oil crop could yield -linolenate oil for medical and
for studies on desaturation and polyunsaturated fatty dietary use and at a competitive price. In borage the
acid synthesis (4346). membrane lipids are also enriched in -linolenate, and
Mutagenesis and conventional plant-breeding pro- the 6 desaturase gene appears to be expressed in the
grams have produced several oil seed varieties with whole plant. It can be assumed, therefore, that its
blocks in both oleate and linoleate desaturase activity, expression in a transgenic plant would not adversely
resulting in dramatic changes in the unsaturation level affect membrane function and this appears to be the
of the seed triacylglycerols (47, 48). The seeds develop case.
normally and are viable. There were no fatty acid Recently a cDNA has been isolated that encodes the
lesions in other parts of the plant, indicating tissue- 6 desaturase from the developing seeds of borage
specic 12 and 15 desaturase genes (4850). Such (59). Its function has been conrmed by ectopic expres-
observations bode well for manipulating the polyunsa- sion in transgenic tobacco plants. The transformants
turation in oil seeds by genetic engineering. The endo- grew well and contained appreciable quantities of -
plasmic reticulum desaturases are integral membrane linolenic acid (13%) and stearidonic acid (10%). The
proteins and highly hydrophobic, and hence little pro- polypeptide encoded by the 6 desaturase cDNA is
gress has been made in their solubilization and puri- unusual and contains diagnostic sequences related to
cation. Much of our understanding of this class of cytochrome b5 at the N-terminus. The microsomal
desaturases has come from Arabidopsis mutants (40, form of borage cytochrome b5 is 32% identical to
51). These mutants, termed FAD mutants, are each the N-terminal extension of the 6 desaturase.
defective in a particular desaturase. For example, Microsomal desaturases (12, 15) that have been
FAD2 and FAD3 genes have been cloned and shown cloned from higher plants do not contain this cyto-
to be responsible for 12 and 15 desaturation in the chrome b5 domain (53), even though the requirement
endoplasmic reticulum, respectively (52, 53). for cytochrome b5 has been demonstrated in vitro (41).
Examination of deduced amino acid sequences show The borage 6 desaturase therefore appears to repre-
that membrane bound desaturases from a number of sent a new class of fatty acid desaturases in higher
sources are characterized by the presence of three plants existing as cytochrome b5 fusion proteins.
regions of conserved histidine residues and that these It is interesting that none of the other plant desa-
are responsible for catalytic activity (37, 54). turases (12; 15) (53) or the closely related hydroxy-
Desaturation reactions involved in the synthesis of a lase from castor (60) have a cytochrome b5 domain
number of other, less common polyunsaturated fatty and use the free microsomal form of the protein. It
acids are also endoplasmic reticulumbound enzymes. is possible that desaturation is more efcient with an
Only -linolenic acid (18:36;9;12 ), however, has been endogenous heme binding domain in the protein. It
studied in detail. Polyunsaturated fatty acids with a 6 may be signicant that the 6 desaturase from borage
double bond are prevalent in the Boraginaceae. Here, differs from the 12 and 15 desaturases in that it
the dominating 6 fatty acid in leaf tissue is an octa- introduces a center of unsaturation between an existing
decatetraenoic acid (stearidonic acid, 18:46;9;12;15 ), double bond and the carboxy carboni.e., a front-end
and this is particularly enriched in the galactolipids desaturase (61), as opposed to double-bond formation

toward the methyl end of the carbon chain. Other- where signicant quantities of VLCFAs are not
front end desaturases have now been cloned and func- found conferred the ability to accumulate these fatty
tionally identied and include the 5 from the lamen- acids. FAE1 thus appears to be the rate-limiting step in
tous fungus, Mortierella alpina (62) and the nematode Arabidopsis seed, and the introduction of extra copies
worm, Caenorhabditis elegans (63); again, these con- resulted in higher levels of VLCFAs. Furthermore, it
tain cytochrome b5 domains. was suggested that the condensing enzyme is the activ-
ity of the elongase that determines the acyl chain
F. Fatty Acid Elongation length of the VLCFAs produced. In contrast, the
other three enzyme activities are found ubiquitously
Very long chain fatty acids (VLCFAs) have chain throughout the plant and are not rate limiting and
lengths from 20 to 30 carbons or greater. For a detailed play little if any role in the control of VLCFA synth-
review of the biochemical and molecular characteriza- esis.
tion of elongase activity, see Postbeittenmiller (64) and Because condensing enzymes are pivotal in the
Domergue et al. (65). Here we will summarize the cur- synthesis of VLCFAs, they may be useful for biotech-
rent understanding and the recent advances in the nology. For example, the accumulation of VLCFAs in
molecular characterization of the process. The major p35S-FAE1 tobacco raises the possibility of producing
site of VLCFA synthesis is the epidermal cells where VLCFAs in plant species that do not normally have
they are used for the formation of waxes including them. The availability of expressed sequence tags
suberin, cutin, and the epicuticular waxes which (ESTs) may allow identication of enzyme activities
cover the aerial parts of the plant (66). The VLCFAs that could synthesize VLCFAs up to C34 in length.
are either directly incorporated into waxes or else act Targeting of these fatty acids to seeds may eventually
as precursors for other aliphatic hydrocarbons which lead to the production of new, agronomically impor-
include alkanes, primary and secondary alcohols, tant VLCFAs. In a broader application, the availabil-
ketones, aldehydes, and acyl esters. VLCFAs also ity of condensing enzymes with different acyl chain
accumulate to high levels in the seed oils of some spe- specicities, together with an ability to manipulate
cies; they are mostly monounsaturated, belonging their expression in epidermal cells, could result in the
chiey to the cis (n-9) series, i.e., higher homologs of generation of plant crops with enhanced performance
oleic acid, e.g., erucic acid, as found in the older vari- physical barriers (waxes) and thereby show increased
eties of oilseed rape. tolerance to environmental stresses and/or resistance
VLCFAs are of particular importance for the oleo- to pathogens.
chemical industries, and the need for renewable raw
materials has led to a signicant interest in this area
IV. TRIACYLGLYCEROL BIOSYNTHESIS
of research, particularly for the characterization of the
so-called fatty acid elongases. The elongase activity is A. Assembly
composed of four activities analogous to those found
in de novo fatty acid synthesis and involves the sequen- Saturated fatty acids up to the levels of palmitic acid
tial additions of C2 moieties from malonyl-CoA to (C16:0), stearic acid (C18:0), and oleic acid (18:19 ) are
preexisting C18 fatty acids exported from the plastid synthesized in the plastid (see above). All further mod-
to the membranes of the endoplasmic reticulum. It is ications of the acyl-chain and the assembly of triacyl-
thought that each cycle of elongation comprises: (a) glycerols are accomplished by membrane-bound
condensation of malonyl-CoA with a long chain acyl- enzymes localized in the endoplasmic reticulum (ER).
CoA; (b) reduction to -hydroxyacyl-CoA; (c) dehy- The oil is released from the endoplasmic reticulum
dration to an enoyl CoA; and (d) reduction of the membranes and stored as discrete oil bodies in the
enoyl-CoA, resulting in an elongated acyl-CoA. cytoplasm. Maximum oil deposition in the seed usually
Recent work with Arabidopsis, which contains occurs over a narrow time window after pollination. In
21% (w/w of total fatty acids) as eicosaenoic acid species such as sunower and safower, > 60% of the
(20:1), has shown that a fatty acid elongation (FAE1) oil is laid down over a period of a few days 15 days
gene encodes a putative seed-specic condensing after owering and when the cells of the cotyledons are
enzyme and catalyzes the rst step in the cycle outlined undergoing cell expansion. At the close of oil accumu-
above (67). lation, the triacylglycerols constitute > 90% of the
Introduction of the FAE1 gene into yeast and total lipid, with the remainder being phospholipids
tobacco and the vegetative tissues of Arabidopsis and glycolipids (68). Seed cotyledons in the active

phase of oil deposition yield microsomal membrane
preparations capable of massive oil assembly in vitro.
Given suitable substrates, microsomal preparations
produce triacylglycerols in mass and these accumulate
as visible oil droplets in the incubation medium (69).
The major pathway for triacylglycerol biosynthesis
in plants is via the so-called Kennedy, or glycerol-3-
phosphate, pathway (70, 71) and is catalyzed by
enzymes in the endoplasmic reticulum. The assembly
of the triacylglycerols proceeds through three sequen-
tial acylation steps each catalyzed by a specic acyl-
transferase (AT; Fig. 3). In the rst reaction, the acyl
moiety from acyl-CoA is transfered to the sn1 position
of sn-glycerol-3-phosphate to yield lysophosphatidic
acid:
sn-glycerol 3-phosphate acyl-CoA ! sn1-acyl-
lyso-phosphatidic acid CoA
The glycerol-3-phosphate acyltransferase (AT1) cata-
lyzing this reaction generally has a broad acyl speci-
city and will accept both saturated and unsaturated
fatty acids (72, 73). In some species, however, which
produce an unusual seed-specic fatty acid, the AT1
may exhibit a greater specicity for the acid than the
enzyme from plants that lack it (74, 75).
The enzyme lysophosphatidic acid acyl transferase
(AT2) catalyzes the acylation of lysophosphatidic acid
at the sn2 position to yield phosphatidic acid:
Figure 3 Glycerol-3-phosphate (G3P) undergoes acylation
sn1-acyl-lyso-phosphatidic acid acyl-CoA ! to yield lysophosphatidic acid (LPA) and phosphatidic acid
(PA) through the action of glycerol phosphate acyltransfer-
phosphatidic acid CoA ase (AT1) and lysophosphatidic acid acyltransferase (AT2),
In most oil seeds, AT2 exhibits a strong selectivity for respectively. A phosphatidic acid phosphohydrolase (PAP)
unsaturated 18-carbon fatty acids (76, 77), and this gives rise to diacylglycerol (DAG) which undergoes acyla-
tion, catalyzed by diacylglycerol acyltransferase (AT3), to
results in a nonrandom distribution of the acyl groups
form triacylglycerol (TAG). Oleic acid (18:1) from the plastid
at the sn1 and sn2 positions of phosphatidic acid and in enters the acyl-CoA pool and can be transferred to the sn2
other lipids derived from this such as phospholipids, position of sn-phosphatidylcholine (PC), through the activity
diacylglycerols, and triacylglycerols (78). As a general of lysophosphatidylcholine acyltransferase (LPCAT), for
rule, therefore, plant triacylglycerols have mainly unsa- desaturation to C-18 polyunsaturated fatty acids. The desa-
turated fatty acids at position sn2. Interesting excep- turated fatty acid products in PC enter the acyl-CoA pool by
tions are plants with triacylglycerols rich in medium- the reverse reaction of LPCAT. During the movement of
chain fatty acids (C8C14) and especially in the genus glycerol backbone towards TAG through the so-called
Cuphea (79). The acyl specicities of the AT2 from Kennedy pathway DAG can freely interconvert with PC
three different Cuphea spp. have been investigated through the forward and reverse reactions of a cholinepho-
and have similar properties. Medium-chain acyl-CoA sphotransferase (CPT). During oil formation DAGs also give
rise to TAG and monoacylglycerol (MAG) through a trans-
substrates are readily utilized. Long-chain acyl-CoA
acylation (TA) reaction, which is freely reversible.
esters, however, were only used if the acyl acceptor,
lysophosphatidic acid, contained a long-chain acyl
group (75, 80). The AT2s from oil seed species which them at a considerably higher rate with long-chain
accumulate triacylglycerols with only long-chain fatty acyl-CoA than medium-chain acyl-CoA (81). The
acids lack these properties. These enzymes poorly uti- rather special properties of the Cuphea AT2 enables
lize medium-chain lysophosphatidates and acylate the cell to synthesize phosphatidic acid in two major

molecular species consisting of medium-chain and diacylglycerol acyl-CoA ! triacylglycerol CoA
long-chain acyl groups, respectively, and with little of
the mixed acyl species. This feature is probably a neces- The acylation of diacylglycerol is the only unique
sity in Cuphea in order to maintain correct membrane step in triacylglycerol assembly. There are many
lipid assembly and function (see below). plant species with unusual fatty acids in their seed
In vitro assays have shown that the AT2 in rape oil. These are often major constituents in the triacyl-
seed has a poor capacity to acylate unusual fatty glycerols and are almost absent in the seed membrane
acids in the sn2 position (81, 82). In line with these lipid. It is probable that the presence of many of these
observations it was found that rape seedaccumulating unusual acids in membrane lipids would severely
erucic acid (C22:1) (82) and transgenic rape seed engi- impair proper membrane function. How particular
neered to accumulate laurate (C12:0) (83, 84) nearly acyl species of diacylglycerols are channeled almost
totally exclude these fatty acids from the sn2 position exclusively to triacylglycerols is not fully understood.
of triacylglycerols. However, this exclusion of unusual Although experimental data suggest that the mechan-
fatty acids from the sn2 position in rape can be sur- isms might differ depending on the type of triacylgly-
passed by introducing other AT2s from other species. cerol-specic fatty acid, it appears that the AT3 must
For example, transgenic laurate-producing rape trans- play an important role in this regulation. It has been
formed with a cDNA encoding coconut AT2 (which shown that AT3 in plants accumulating unusual fatty
acylates medium-chain fatty acids at the sn2 position) acids can exhibit acyl specicity and selectivity which
resulted in rape seeds with 30 mol% of laurate in the enables the preferential channeling of these fatty acids
sn2 position of triacylglycerols (85). When a cDNA into triacylglycerols (92, 93). As yet little progress has
encoding a Limnanthes AT2 was introduced into an been made on the molecular biology of AT3 in plants.
erucate acidproducing rape, it resulted in transgenic Recently, however, a mouse cDNA-encoding AT3 has
seeds with signicant erucic acid at the sn2 position of been isolated and characterized (94). This appears to
the triacylglycerols (86). A thorough review of the be the rst successful identication of an AT3 gene and
molecular biology of AT1 and AT2 has been presented hopefully bodes well for the identication of genes with
by Frentzen and Wolter (87). It is evident that parti- a similar function in plants.
cular cloned AT2 genes can be successfully used to
manipulate fatty acid composition in transgenic
seeds. It also appears that the expression of a yeast B. Desaturation and Its Relationship to Oil
AT2 gene in transgenic rape seed improves oil yield Assembly
(88, 89).
In the further reactions of triacylglycerol assembly Since phospholipids are the prime substrates for the
the phosphatidic acid is converted to diacylglycerols production of linoleic and linolenic acids that are
and inorganic phosphate through the action of the designed for plant triacylglycerol assembly (see
enzyme, phosphatidate phosphohydrolase (PAP): above), there must be efcient mechanisms by which
the oleate is channeled into phospholipids for desa-
phosphatidic acid ! diacylglycerol inorganic turation and the desaturated acyl groups made avail-
phosphate able for triacylglycerol biosynthesis. Among the
phospholipids present in the endoplasmic reticulum,
In oil seeds, the enzyme appears to be present in both it is only phosphatidylcholine that undergoes rapid
soluble and membrane-bound forms and these may turnover and it is this lipid that is important in provid-
have a regulatory function in lipid synthesis (90). ing polyunsaturated fatty acids for triacylglycerol for-
Good purication of this enzyme has recently been mation. An equilibration reaction between acyl groups
achieved and it is currently under investigation (91). in phosphatidylcholine and the acyl-CoA pool occurs
Diacylglycerols are the precursors for the membrane in oil seeds and by this reaction oleate from oleoyl-
lipids, phosphatidylcholine, and phosphatidylethano- CoA enters position sn2 of phosphatidylcholine for
lamine as well as storage triacylglycerols. desaturation. Concomitantly, the products from that
Consequently, diacylglycerol utilization constitutes a position enter the acyl-CoA pool for the acylation of
branch point between membrane and storage lipid all three positions of the glycerol backbone. The reac-
synthesis. The nal step in triacylglycerol biosynthesis tion can be catalyzed by the combined forward and
is the acylation of the diacylglycerols at the sn3 posi- reverse reactions of an acyl-CoA:lysophosphatidyl-
tion catalyzed by diacylglycerol acyltransferase (AT3): choline acyltransferase (LPCAT) (95).

Diacylglycerol may be utilized for phosphatidylcho- glycerols were rapidly converted to di- and triacylgly-
line synthesis by a CDP-choline:diacylglycerol choline- cerols. These interconversions occur in the absence of
phosphotransferase (CPT) catalyzed reaction. In oil acyl-CoA and hence do not involve diacylglycerol acyl-
seeds, this appears to be reversible and thus the phos- transferase activity. The evidence is consistent with the
phatidylcholine can also reconvert with diacylglycerols operation of a diacylglycerol-diacylglycerol transacyla-
(96). In the combined forward and reverse reactions of tion yielding monoacylglycerols and triacylglycerols;
the cholinephosphotransferase, the oleate at both posi- the reaction is freely reversible. A similar transacylase
tions of diacylglycerols can enter phosphatidylcholine reaction has been characterized in rat intestinal micro-
for desaturation and the polyunsaturated products somes where it is involved in the net synthesis of mono-
transferred to the diacylglycerol pool for triacylgly- acylglycerols and triacylglycerols (104). The activity of
cerol synthesis (97). The relative importance of the the transacylase in safower microsomes is of the same
two reactions, the acyl exchange between acyl-CoA order of magnitude as the diacylglycerol acyltransfer-
and phosphatidylcholine and the phosphatidylcho- ase. Hence in any diacylglycerol acyltransferase assays
line-diacylglycerol interconversion, in providing poly- using radioactive diacylglycerols, it would not be pos-
unsaturated fatty acids for triacylglycerol assembly is sible to discriminate triacylglycerol formation via
unclear and might differ among plant species (98, 99). transacylation or direct acylation.
The cholinephosphotransferase has recently been Experimental evidence also shows that the diacyl-
cloned from soybean (100), which should lead to a glycerols, arising directly by the so-called Kennedy
better understanding of the role of this enzyme in inu- pathway, or via transacylation, rapidly interconvert
encing oil quality. with phosphatidylcholine, probably through the rever-
sible action of choline phosphotransferase (96). Any
C. Transacylation oleoyl substrate entering phosphatidylcholine via cho-
line phosphotransferase is readily desaturated and the
Observations suggest that events other than those polyunsaturated fatty acid products become incorpo-
described above may also occur during oil assembly. rated into the triacylglycerol oil. Transacylation, there-
In previous experiments with safower microsomal fore, can account for the further enrichment of
membranes, triacylglycerols appeared to form from preformed triacylglycerols with polyunsaturated fatty
diacylglycerols, but in the absence of acyl-CoA, and acids observed in vivo (101). It cannot be ruled out, of
hence not by the direct acylation through diacylgly- course, that such a desaturation can occur in vivo
cerol acyltransferase activity (97). At that time no while the oil is still associated with the endoplasmic
explanation was offered for this. Also it has recently reticulum, and presumably before stabilization in the
been shown that preformed triacylglycerols in the oil body. In this respect a solubilized 12-desaturase
embryos of sunower at an early stage of development from the choloroplast was found to utilize, to some
can become further enriched with linoleate (101), sug- degree, other substrates (free fatty acids, acyl-CoA,
gesting that fatty acids esteried to triacylglycerols, and the complex lipid monogalactosyldiacylglycerol)
and presumably before stabilization in the oil body, (105). It is clear, however, that the consequence of
are available to the 12-desaturase of the endoplasmic any reaction that gives rise to diacylglycerols will
reticulum. Further, it has been demonstrated that there also bring about the further enrichment of the glycerol
is considerable remodeling of in situsynthesized tria- backbone with C18-polyunsaturated fatty acids.
cylglycerol involving acyl groups from polar lipids in Transacylation could also account for the previous
microsomal preparations from the developing castor observation with microsomal membranes that radio-
bean endosperm (102). activity in diacylglycerols would accumulate in triacyl-
In attempts to reconcile these observations with our glycerols in the absence of acyl-CoA (97), and that in
present understanding of oil assembly, the utilization situsynthesized triacylglycerols are remodeled with
by microsomal membranes of radiolabeled neutral acyl groups from polar lipids (102).
lipid substrates has been investigated. Safower micro- Our current understanding of triacylglycerol assem-
somes were found to catalyze the interconversion of bly and turnover in vitro in developing oil seeds and the
mono-, di-, and triacylglycerols (103). From these stu- relationship to microsomal desaturation is outlined in
dies it was clear that diacylglycerols gave rise to tria- Figure 3. It is also apparent that microsomal mem-
cylglycerols and monoacylglycerols as well as branes catalyze the production of lysophosphatidyl-
phosphatidylcholine. Radioactivity was transferred choline from monoacylglycerols (103). This appears
from triacylglycerols to diacylglycerols and monoacyl- to occur via a phosphocholine transfer between phos-

phatidylcholine and the monoacylglycerol, the pro- oxide formation (107). An important feature of auto-
ducts being diacylglycerols and lysophosphatidylcho- xidation is that it is autocatalytic, the rate of oxidation
line. The lyso derivative can be rapidly reacylated being slow at the start (the induction period) and
from acyl-CoA. Such a combination of reactions increasing as the reaction progresses. In common with
could yield triacylglycerols without the participation other radical chain reactions, autoxidation can be
of diacylglycerol acyltransferase and without any accu- divided into three separate processes: initiation, propa-
mulation of monoacylglycerols. gation, and termination. Initiation is perhaps the pro-
If transacylation occurs in vivo, what might be its cess most difcult to dene because of the low
biological signicance? Clearly, it can provide even concentration of radicals and the likelihood of there
further opportunity for desaturation and the enrich- being more than one process, since a large number of
ment of the oil with C18-polyunsaturated fatty acids. different radicals can abstract hydrogen from RH to

The reactions that channel oleate to phosphatidylcho- form R +H. The initiating species may be, for example,
line for its subsequent desaturation help to concentrate a transition metal ion, a radical generated by photolysis
substrate and give a greater chance(s) for desaturation. or high-energy irradiation, or a radical generated by
It is possible that desaturation of complex lipids, such decomposition of a hydroperoxide. In the propagative
as phosphatidylcholine, is not an efcient process and phase, further reactive species are generated as in the

that a number of mechanisms have evolved, including generation of a radical R from the alkene RH and its
transacylation, which enables the rapid formation of subsequent reaction with oxygen. In the termination
polyunsaturated fatty acids for storage and membrane phase there is an interaction of radicals to produce non-
lipid assembly. One might also speculate that the trans- initiating and nonpropagating products.
acylation may play some role in the assembly of tria- Autoxidation is facilitated by pro-oxidants and
cylglycerols containing > 70% of a particular fatty inhibited by antioxidants. Pro-oxidants, such as metals
acid and particularly those of an unusual nature per- or other radical initiators, operate by promoting the
haps by giving rise to species of diacylglycerols which initiation step or else they may inhibit the activity of
are immediately acylated directly with the required antioxidants. Antioxidants are frequently added to fats
fatty acid. Transacylation, however, could have a and fat-containing foodstuffs to prolong shelf life
more profound biochemical importance. It may help (108). These are often phenolic compounds which act
to regulate the size of an active diacylglycerol pool, a by interferring with the propagation sequence by con-
possible necessity which prevents the detrimental net serving propagating radicals into nonpropagating spe-
movement of phosphatidylcholine to diacylglycerols cies (109, 110). Their effectiveness is often increased by
(via cholinephosphotransferase) and the consumption compounds such as citric acid, ascorbic acid, or phos-
of endoplasmic reticulum structural lipid during the phoric acid (called synergists). In this regard, there is
period of extremely rapid oil deposition that is found considerable evidence for a contributory, antioxidant
in the developing seed. role for vitamins E and C and the carotenoidscon-
stituents of fruit, vegetables, beverages, and grainsin
the maintenance of health and the protection from
V. OXIDIZED FATTY ACIDS
coronary heart disease, cataracts, and certain cancers
A. Autoxidation and Antioxidants (111).
Recent work indicates possible important roles of
Historically, the oxidation of fatty acids has been per- polyphenolic components (the avonoids, phenylpro-
ceived as a deleterious process resulting in the genera- panoids, and phenolic acids) as contributors to antiox-
tion of off-avors in commercial produce. The process idant activities and also as agents of other mechanisms
of oxidation can be considered as either autoxidation, a contributing to cardioprotective and anticarcinogenic
chemical reaction that usually takes place at ambient actions. Epidemiological evidence suggests a negative
temperatures between atmospheric oxygen and an correlation between dietary antioxidants and coronary
organic compound, or as an enzyme-catalyzed reaction. arterial disease. Such an association may involve a
The complexity of the autoxidation reaction has been mechanism whereby antioxidants delay the onset or
well documented (106). The insertion of oxygen is also progression of the disease by reducing oxidative reac-
accompanied by the movement of double bonds result- tions, downregulating thrombic mechanisms, and
ing in the formation of a conjugated diene with a max- attenuating endothelial dysfunction.
imal absorbance at 234 nm. Spectral measurements at A large range of avonoids and phenylpropanoids
this wavelength have been used to monitor hydroper- found in fruit, vegetables, and beverages are more

effective antioxidants in in vitro systems than vitamin 13-hydroperoxides (107, 118). While most LOXs so far
C or vitamin E (111). Examination of the antioxidant characterized are soluble cytosolic enzymes, some are
status of a number of consumables gave a hierarchy of chloroplastic, mitochondrial, or located in the vacuoles
red wine>black tea green tea>red grape juice (118). In soybean, lipoxygenases have been identied
blackcurrant juice>white grape juice orange with involvement in nitrogen and assimilate partition-
juice>white wine apple juice. Indeed, in what has ing and appear to be regulated in response to plant
become known as the French paradox (their rela- nitrogen status in both tissue-specic and developmen-
tively high fat consumption but relatively low coronary tally controlled patterns (120). A key role for some
disease) has been attributed, in part at least, to the LOX isoforms is in the generation of fatty acid hydro-
benecial effects derived from the consumption of peroxides destined for jasmonic acid (JA), which trig-
red wine containing the avonoid constituents derived gers gene activation during wound response in plants
from the black grape skins. (121, 122).
There is also increasing epidemiological evidence The fatty acid hydroperoxides generated by the
for an association between dietary intake of carote- activity of LOX are potentially deleterious to mem-
noid-containing fruit and vegetables and a decreased brane function by causing increased rigidity and
incidence of coronary heart disease and certain can- would not, therefore, be expected to accumulate
cers (111, 112). Recent studies (113) have shown that (123). Hydroperoxides can be metabolized by several
dietary intake of lycopene-rich foods was inversely routes. Glutathione transferases (GSTs) exhibiting glu-
associated with a risk of prostate cancer. These effects tathione peroxidase activity toward phospholipid
have been ascribed in part to the antioxidant activity hydroperoxides have recently been demonstrated in
of carotenoids. In addition to direct radical scaveng- mammalian tissues (124). GSTs in plants perhaps hav-
ing properties of some compounds given above, others ing analogous roles in hydroperoxide detoxication
such as the glucosinolates (114) (present in high could be envisaged (125). Hydroperoxide fatty acids
amounts in vegetables such as brocolli, Brussels can also be metabolized by the peroxygenase cascade
sprouts, and cabbage) or cysteine sulfoxides (115) in (126128). This involves (a) an intramolecular transfer
Allium (garlic and onion) act by stimulating the of one oxygen atom from fatty acid hydroperoxide
animals own enzymic defenses (116, 117). This yielding an epoxyalcohol and/or co-oxidation reac-
appears to operate via the antioxidant responsive tions such as epoxidation of double bonds of unsatu-
element present in the 5 0 -anking region of many rated fatty acids, and (b) an epoxide hydrolase, which
antioxidant enzymes such as glutathione S-trans- preferentially hydrates the epoxides formed by the per-
ferases, glutathione peroxidases, quinone reductase, oxygenase (127). The chloroplast envelope membranes
and superoxide dismutase. appear to be a major site of these metabolic activities
(128); it is suggested that the products of this pathway
B. Oxylipin Biosynthesis (Fig. 4) may play roles in plant defense mechanisms.
During jasmonic acid synthesis upregulation of the
Much of the early work concentrated on characterizing allene oxide synthase (AOS) gene results in the conver-
lipoxygenase (LOX), a nonheme dioxygenase that cat- sion of 13-hydroperoxide to 12,13-epoxylinolenic acid,
alyzes, as a primary reaction, the hydroperoxidation, and is the rst committed step in jasmonic biosynth-
by molecular oxygen, of linoleic acid and other poly- esis. This conversion step has been proposed to be the
unsaturated fatty acids that contain a cis, cis-1,4-pen- rate-limiting step in the octadecanoid pathway (129).
tadiene moiety. Unesteried fatty acids have been AOS is a cytochrome P450 of the CYP74A family, and
regarded as the principal substrates for the enzyme cDNA clones have been obtained from a number of
although LOX isoforms with activity toward complex species (130, 131). The protein contains a peptide lea-
lipid substrates have been reported (118). LOXs are der sequence typical for chloroplast import, indicating
ubiquitous in plants and are encoded by large gene that it is targeted to this organelle. 12,13-
families which may result in the presence in a given Epoxylinolenic acid is very unstable and rapidly
tissue of LOX isoforms differing in substrate prefer- cyclized to racemic 12-oxophytodienoic acid (12-oxo-
ences, kinetic parameters, and stereospecicity of oxy- PDA). In vivo, a stereospecic reaction catalyzed by
genation (119). Linoleic and linolenic acid are the allene oxide cyclase (AOC) occurs specically yielding
major polyunsaturated fatty acids in plant tissues, the 9(S), 13(S)-12-oxo-PDA isomer. The AOC gene
and insertion of the oxygen takes place at either the has not been cloned to date. The 12-oxo-PDA is the
9 or 12 position to generate the corresponding 9- or rst cyclopentenone in the jasmonate pathway and is

Figure 4 Oxylipin biosynthesis. -Linolenic acid (-LNA; C18:39;12;15 ) is converted to 13-hydroperoxylinolenic acid (13-
HPLNA) by a 13-lipoxygenase isoform. The 13-HPNLA can be metabolized by several enzymes of which the formation of
jasmonic acid (JA) and avors volatilities are illustrated. The rst step in JA synthesis is catalyzed by allene oxide synthase (AOS)
forming an unstable allene oxide (AO) which is converted to 12-oxo-phytodienoic acid (12-oxo-PDA) by allene oxide cyclase
(AOC). These reactions are considered plastidic. The 12-oxo-PDA is likely exported to the cytosol and acts as a substrate for 12-
oxo-PDA reductase (12-oxo-PDAR) forming 3-oxo-2 (2 0 -pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0), which yields JA by
-oxidation. When 13-HPNLA is metabolized by hydroperoxide lyase (HPL), short-chain aldehydes and !-oxo acids are
generated. The gure depicts the formation of the C6 aldehyde (cis-3-hexenal) and the 12-oxo-cis-9-dodecenoic acid which in
turn can be metabolized to traumatin. The chain length of the aldehydes and !-oxo acids produced in a particular tissue is
dependent on the cleavage site of the CC bond by HPL. The downstream metabolism of the aldehydes by alcohol dehydro-
genases (ADH) and isomerases generates many of the characteristic avors and aromas of fruits and vegetables.

subsequently converted to jasmonic acid by reduction ized. This, together with biochemical studies, has
of the 10 double bond by 12-oxo-PDA reductase, made it possible to manipulate fatty acid proles in
considered to be a cytosolic enzyme (132) and is fol- oil seeds and even introduce the capacity to synthesize
lowed by three rounds of -oxidation that may occur foreign fatty acids, not normally present, in trans-
in the peroxisomes. Thus jasmonic acid synthesis may formed species. As knowledge and technology
involve the cooperation of three subcellular compart- advance, it will become even more feasible to produce
ments, the chloroplast, cytosol and peroxisomes. The valuable new lipid products in plants to satisfy the
12-oxo-PDA together with its metabolite, jasmonic demands of the food, nutraceutical, and pharmaceu-
acid (collectively termed jasmonates), either directly tical industries.
or indirectly triggers gene activation in a range of
responses of which the defense response has probably
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2000.

12
Phospholipid Biosynthetic Enzymes in Plants
Ralph E. Dewey
North Carolina State University, Raleigh, North Carolina, U.S.A.
Anthony J. Kinney
DuPont Experimental Station, Wilmington, Delaware, U.S.A.
I. PLANT PHOSPHOLIPIDS tions. To achieve this it is necessary to understand the

pathways of phospholipid biosynthesis in plants and
Phospholipids are a minor (0.13%) but important have available the genes that encode phospholipid
component of all edible plant oils. They are removed pathway enzymes.
during rening as a gum fraction but they are not Many of the enzymes involved in plant lipid bio-
discarded. Collectively known as lecithin, they are used synthesis are also similar to their yeast and animal
as high-value emulsiers and wetting agents in food counterparts (2), although there are important differ-
manufacturing (1). The phospholipid fraction of vege- ences in the pathways which predominate. For exam-
table oil, and indeed of all plant tissues, consists mainly ple, in most animal tissues PC is predominantly
of phosphatidylcholine (PC), phosphatidylethanola- synthesized from diacylglycerol and CDP-choline via
mine (PE), phosphatidylserine (PS), phosphatidylino- a phosphoaminoalcohol pathway often referred to as
sitol (PI), phosphatidylglycerol (PG), and the Kennedy pathway (35). In liver tissues and in
diphosphatidylglycerol (DPG). In addition, there are yeast PC is synthesized mainly by the sequential
a number of less abundant phospholipids derived from methylation of PE, catalyzed by a single enzyme in
phosphatidylinositol (2). animals (6) and two separate methylases in yeast (7).
All emulsiers can be characterized by their hydro- In many plants the rst step in committed PC
philiclipophilic balance (HLB). Among plant phos- biosynthesis is the methylation of ethanolamine phos-
pholipids PC has the highest HLB value, PE is phate, followed by the synthesis of phosphatidylmono-
intermediate, and the other phospholipids have a low methylethanolamine and then methylation to PC (see
HLB. Thus, PC has the greatest value as a soluble oil- 2). The enzyme involved in this initial methylation of
in-water emulsier; it is used, for example, in lowering ethanolamine phosphate has recently been identied
the viscosity of cocoa butter during chocolate manu- and its gene cloned from spinach and Arabidopsis (8,
facture. PE is more useful as a wetting agentfor 9). Interestingly, no yeast or animal counterpart has
example, in increasing fat absorption during the pre- been reported.
paration of cake doughnuts (1). For this reason, com- In general, however, the phospholipid pathways of
mercial lecithin preparations are often separated into all eukaryotes may be divided into those in which the
PC and PE fractions using ethanol, and there may be glycerol backbone of the phospholipid molecule
some value in genetically manipulating the ratio of derives from CDP-diacylglycerol and those that it
phospholipids in plant oils for particular food applica- derives from diacylglycerol (DAG). For example,

phosphatidylethanolamine may be made from ethano- source of acyl moieties. PA can then be converted
lamine and diacylglycerol, by the Kennedy pathway, or either to CDP-diacylglycerol or to DAG. The former
by decarboxylation of phosphatidylserine, derived reaction is catalyzed by CDP-diacylglycerol synthase
from CDP-diacylglycerol and free serine (Fig. 1). (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41)
The advent of modern genomics and the rapid and the latter by phosphatidic acid phosphatase (phos-
advances in the understanding of yeast phospholipid phatidate phosphohydrolase, EC 3.1.3.4).
biosynthesis has led to the cloning, heterologous The identication of the yeast gene encoding CDP-
expression, and characterization of many plant phos- diacylglycerol synthase (CDS1) led to the construction
pholipid biosynthetic enzymes that had previously of a null allele at this locus (11). These studies veried
been difcult to purify and characterize. Thus, a that CDP-diacylglycerol synthase activity is essential
great deal of recent understanding of plant phospholi- for cell viability and established CDS1 as the only
pid biosynthetic enzymes has been derived from the gene in yeast that encodes this activity. This latter
molecular approaches described here. The 30 years or observation was somewhat surprising considering
so of classical enzymological studies that laid the that this enzyme is distributed between two very dis-
groundwork for these recent advances are described tinct organellesthe mitochondria and the endoplas-
in earlier reviews (2, 10). mic reticulum.
Studies in animal systems reveal that, in addition to
their involvement in bulk phospholipid synthesis,
II. BIOSYNTHESIS OF CDP- CDP-diacylglycerol synthase are also critical compo-
DIACYLGLYCEROL IN PLANTS nents of the phosphoinositide signal transduction
cycle. A mutation in an eye-specic Drosophila CDP-
Diacylglycerol and CDP-diacylglycerol are both diacylglycerol synthase resulted in the loss of IP3 -
derived from phosphatidic acid (PA), synthesized by mediated light perception in photoreceptor cells (12).
the sequential acylation of glycerol-3-phosphate by This defect was attributed to the inability of these cells
two acyltransferases. These acyltransferasesgly- to adequately regenerate the necessary PI pools
cerol-3-phosphate acyltransferase and 1-monoacylgly- required to maintain the signaling cascade. Multiple
cerol-3-phosphate acyltransferaseuse acyl-CoA as a CDP-diacylglycerol synthase isoforms in animals may
Figure 1 Major pathways of phospholipid synthesis in higher plants.

be produced from independent genetic loci (13), or synthase (glycerophosphate:CDP diacylglycerol phos-
derived from the alternative splicing of a single gene phatidyltransferase, EC 2.7.8.5) and PGP phosphatase
(12). (phosphatidylglycerolphosphate phosphohydrolase,
In plants, CDP-diacylglycerol synthase activity has EC 3.1.3.27). The rst catalyzes the reaction of CDP-
been detected in a number of organelles (2, 14). The diacylglycerol with glycerol phosphate to yield phos-
ability of researchers to identify and isolate plant phatidylglycerolphosphate and CMP, and the second
cDNAs that encode enzymes involved in glycerolipid the dephosphorylation of phosphatidylglycerolphos-
biosynthesis, based on their homology to yeast and phate to PG. In yeast both reactions are localized
animal genes, has been greatly accelerated by the exclusively in the inner membranes of mitochondria
recent proliferation of plant-derived expressed (17) whereas in plants they have been reported in
sequence tagged (EST) databases. Where sequence mitchondria, chloroplasts, and microsomes (10, 18).
information for a given protein and/or gene has been These enzyme activities have been well characterized
elucidated in a nonplant species, computer searchers of in animals and yeast (1921), and a hamster cDNA-
EST databases can rapidly identify candidate plant encoding PGP synthase has been cloned and shown to
cDNAs that are likely to encode the same function complement a PGP-synthase defective mutant in CHO
(15). Additional experimentation, such as expressing cells (22). In plants, however, both of these enzymes
and assaying the candidate cDNA in a heterologous remain substantially uncharacterized and their indivi-
system that lacks the activity in question, is subse- dual activities have not been measured.
quently used to verify function. DPG is synthesized in plant mitochondria as it
Recently, Kopka and coworkers (14) utilized this is in yeast, by reaction of PG with CDP-diacyl-
approach to isolate cDNAs encoding CDP-diacylgly- glycerol (2). The reaction is catalyzed by the enzyme
cerol synthases from plants. Using the CDP-diacylgly- phosphatidylglycerol:CDP diacylglycerol phosphatidyl-
cerol synthase sequence from E. coli as the query transerase, about which very little is known in plants
sequence, a candidate Arabidopsis EST was identied except that the enzyme requires either magnesium, or
and subsequently used as a molecular probe to obtain surprisingly, cobalt, for activity (10).
full-length cDNAs from both Arabidopsis and potato Because of its ubiquitous presence in the mitochon-
gene libraries. The deduced plant proteins had pre- dria of eukaryotes, DPG has long been assumed to
dicted molecular masses of 49 kDa and possessed serve a critical function within the mitochondrial mem-
eight potential membrane-spanning domains. brane. The observation that DGP is required for the in
Although the subcellular location of the encoded vitro activities of several mitochondrial enzymes, and
Arabidopsis and potato proteins was not established, studies indicating a need for this unique phospholipid
the absence of potential mitochondrial or chloroplast for the import of proteins into mitochondria, further
targeting sequences suggested that they represent supported this presumption (23, 24). Doubts were
microsomal CDP-diacylglycerol synthases. To conrm raised concerning the essential nature of DPG when
the function of the putative potato CDP-diacylglycerol a yeast strain possessing no detectable DPG owing to
synthase, a GST/potato cDNA fusion construct was a null mutation at the locus encoding cardiolipin
generated and expressed in E. coli. The puried fusion synthase was shown to be capable of growth on both
protein displayed CDP-diacylglycerol synthase activity fermentable and nonfermentable carbon sources (25).
when assayed in vitro. A more recent report by Jiang and coworkers (26)
demonstrated that although no obvious growth defects
were observed when the null mutant strain was incu-
III. SYNTHESIS OF PHOSPHOLIPIDS
bated at 30 C, no colony formation was observed at
FROM CDP-DIACYLGLYCEROL
37 C. Thus, there does appear to be an absolute
A. Phosphatidylglycerol and requirement for DPG in yeast for growth at elevated
Diphosphatidylglycerol temperatures.
PG in plants has been found in a number of plant B. Phosphatidylinositols

organelles but is mainly associated with chloroplast
thylakoids (16), whereas diphosphatidylglycerol The enzyme PI synthase (CDP-diacylglycerol:myo-ino-
(DPG), also known as cardiolipin, is restricted to the sitolphosphatidyltransferase, EC 2.7.8.11), which cata-
inner mitochondrial membrane (2, 16). In yeast the lyzes the reaction between free inositol and CDP-
synthesis of PG is catalyzed by two enzymes, PGP diacylglycerol, to yield PI and CMP, has been charac-

terized in the membrane fractions of many different polyphosphoinositides (or their breakdown products,
plants (2, 10, 27). DAG and inositol trisphosphate) are involved in the
Identication of an Arabidopsis EST displaying regulation of cell proliferation and membrane trafck-
homology to the yeast PI synthase sequence resulted ing (38, 39). Polyphosphoinositides, and their corre-
in the rst reported characterizations of a plant PI sponding lysophospholipids, have also been detected
synthase cDNA (28, 29). The Arabidopsis cDNA in many plant tissues (2, 35, 40, 41) although PIP
encodes a protein of 227 amino acids with a predicted and PIP2 species represent < 1% each of the total
molecular mass of 25.9 kDa. Because E. coli lacks PI, it inositol lipid. In contrast, PI accounts for 93% of
provides an ideal background for functional verica- the total inositol lipid but < 10% of total lipid phos-
tion of putative PI synthases. Expression of the phorus in plant cells (35).
Arabidopsis cDNA in E. coli resulted in the produc- PI 3-kinase, PI 4-kinase, and PIP 5-kinase activities
tion and accumulation of PI to levels as great as 19% have been measured in plasma membranes isolated
of the total lipid extracted from the bacteria (28). The from a number of plant species (2, 4244). PI 4-kinase
cDNA has also been shown to complement a yeast PI activity has also been characterized in soluble fractions
synthase mutant (29). Studies of the recombinant (45) and in nuclear membranes of plant cells (46). The
enzyme in heterologous systems have conrmed the soluble PI 4-kinases have molecular weights in the 80-
requirement of Mg2 ions for full activity of this kDa range (45) whereas the membrane-associated
enzyme (29). enzymes have reported molecular weights ranging
In yeast and animals PI, in the presence of ATP, is from 65 kDa to 500,000 kDa (42, 47).
converted to PI 4-phosphate [PI(4)P] and AMP, a reac- The existence of both membrane and soluble PI 4-
tion catalyzed by the enzyme PI 4-kinase kinases has been conrmed by molecular studies. The
(ATP:phosphatidylinositol 4-phosphotransferase, EC cloning of PI 4-kinase cDNAs from animals and yeast
2.7.1.67). Alternatively, PI can be phosphorylated at has shown that there are two distinct isoforms that
the D-3 position of the inositol ring by a PI 3-kinase differ in size, subcellular location, and presence or
(ATP:phosphatidylinositol 3-phosphotransferase) to absence of specic motifs (48). The smaller isoforms
give rise to PI(3)P. Among the bisphosphorylated PI correspond to proteins ranging from 90 to 125 kDa
derivatives, there is a vast body of literature document- that are primarily found in the cytosol or associated
ing the cellular functions of PI(4,5)P2, a species most with the Golgi. In contrast, the second class repre-
commonly produced by the ATP-dependent phosphor- sents PI 4-kinases that are considerably larger (200
ylation of PI(4)P by a PIP kinase (ATP:phosphatidy- 230 kDa) and are strictly membrane associated. The
linositol 4-phosphate 5-phosphotransferase, EC plant PI 4-kinase cDNAs that have been reported to
2.7.1.68). Although PI(4,5)P2 is clearly the best char- date are predicted to encode products that generally
acterized of the polyphosphoinositides, PI(3,4)P2 and fall into similar classes. An Arabidopsis cDNA encod-
PI(3,5)P2 species have also been found among a ing a 126-kDa PI 4-kinase was identied that showed
diverse array of eukaryotic species, including plants greatest sequence homology to the smaller PI 4-kinase
(3032). In mammals, PI(3,4)P2 is synthesized primar- isoforms of animals and yeast (49). A partial cDNA
ily through the degradation of PI(3,4,5)P3 by a 5-phos- corresponding to a distinct Arabidopsis PI 4-kinase
phatase, with lesser amounts generated via the 4- isoform has also been identied (50). Western blot
phosphorylation of PI(3)P and 3-phosphorylation of analysis using antibodies raised against the encoded
PI(4)P (33,34). In plants, the 4-phosphorylation of partial protein sequence established the size of the
PI(3)P is the only documented route of PI(3,4)P2 complete polypeptide to be 205 kDa. The 205-kDa
synthesis (30); the 5-dephosphorylation of PI(3,4,5)P3 enzyme is associated with microsomal membranes
to produce PI(3,4)P2 is doubtful, given that and possesses a pleckstrin homology domain, features
PI(3,4,5)P3 has not been detected in any plant species common to the larger PI 4-kinase isoforms of animals
(35). Although the specic route of PI(3,5)P2 synthesis and yeast.
has yet to be conrmed in plants, the 5-phosphoryla- The cDNAs encoding the 126-kDa and 205-kDa PI
tion of PI(3)P has to be considered the most likely, 4-kinase isoforms described above appear to represent
given that both animals and yeast generate PI(3,5)P2 the complete complement of PI 4-kinase genes found in
in this manner (31, 36). Arabidopsis (51). It remains to be determined if the
In animal cells the synthesis and turnover of poly- smaller isoforms puried from plasma membranes
phosphoinositides play an essential role in the response (45, 47) represent the products of new, uncharacterized
of animal cells to various agonists (37), and in yeast plant PI 4-kinase genes, or result from the proteolytic

cleavage or alternative splicing of either of the two In addition, polyphosphoinositides are believed to par-
documented isoforms. ticipate in the regulation of the cytoskeletal architec-
Similar to the PI 4-kinases, animal and yeast PIP 5- ture of the cell and the processes controlling membrane
kinases can also be categorized into two families, desig- trafcking. Some of these functions are believed to be
nated types I and II, based on their structural and mediated by the ability of polyphosphoinositides to
biochemical properties. Type I PIP 5-kinases favor alter the activities of several enzymes, including protein
PI(4)P as the substrate, whereas type II enzymes pre- kinases (57), the plasma membrane ATPase (58), and
ferentially utilize PI(5)P (each yielding PI(4,5)P2 as the certain isoforms of phospholipase D (59). For a com-
end product). Both types also vary according to their prehensive description of the functions of polypho-
ability to recognize D-3 PI substrates (reviewed in 52, sphoinositides in plants, several recent reviews are
53). The Aradidopsis PIP 5-kinase cDNA characterized recommended (35, 40, 41, 51).
by Mikami and coworkers (54) encodes a 638amino
acid product that is equally divergent from both of C. Phosphatidylserine and
these families. Furthermore, the genomic sequences Phosphatidylethanolamine
of additional candidate Arabidopsis PIP 5-kinase
genes (accession Nos. U95973, AF007269, and In yeast PS is synthesized from CDP-diacylglycerol
Y12776) are equally difcult to classify as either type and serine, a reaction catalyzed by the enzyme CDP-
I or type II enzymes based on amino acid sequence diacylglycerol:L-serine O-phosphatidyltransferase (PS
homology. Although these observations suggest that synthase, EC 2.7.8.8). Plant PS synthase activity was
plants may possess a novel class of PI(4)P 5-kinase, originally reported in spinach leaf microsomes (10) and
an analysis of the substrate specicities and subcellular has since been detected in many other plant tissues (2).
localization of individual plant isoforms is clearly war- The recent characterization of a wheat cDNA encod-
ranted in order to establish their specic role within the ing a PS synthase provides denitive evidence that
plant cell. plants are capable of producing PS directly from
Four distinct classes of PI 3-kinases have been char- CDP-diacylglycerol (60). The wheat cDNA encodes a
acterized in animals that differ according to their sub- hydrophobic 239amino acid polypeptide that was
strate specicities and their ability to complex with recovered through a heterologous screening strategy
specic regulatory adaptor proteins (55). Plants and intended to identify cDNAs whose expression in
yeast, in contrast, appear to possess a single type of yeast resulted in enhanced aluminum tolerance.
PI 3-kinase, one that exclusively phosphorylates PI to Although the mechanism by which PS synthase activity
produce PI(3)P. PI(4)P and PI(4,5)P2 are not recog- could serve to increase aluminum tolerance in yeast
nized as substrates. Two soybean cDNAs encoding was not clear, aluminum-stressed wheat roots showed
this class of PI 3-kinase have been characterized (44). increased accumulation of PS synthase transcripts,
Although closely related in size (812 and 814 amino suggesting that the enzyme also plays some role in
acids) and sequence identity (98%), these two genes this phenomenon in its native environment.
were differentially expressed during root module orga- Overexpression of the wheat PS cDNA in transgenic
nogenesis. The modulation of gene expression for one Arabidopsis proved to be very effective in increasing
isoform was coincident with the membrane prolifera- the cellular PS content. Though normally found only
tion during nodulation, lending support to the hypoth- in trace quantities in plants, PS represented > 15% of
esis that, similar to their yeast counterparts, plant PI 3- the labeled phospholipid detected in the transgenic
kinases participate in events associated with membrane Arabidopsis after a 24-h incubation with ortho-32 P.
trafcking. A PI 3-kinase cDNA was also character- Interestingly, overexpression of the wheat cDNA in
ized from Arabidopsis (56). Normal growth and devel- both Arabidopsis and tobacco led to the formation of
opment were severely inhibited in transgenic plants necrotic lesions on leaves. It was hypothesized that
that expressed antisense constructs of this cDNA, overaccumulation of PS could have resulted in
demonstrating the essential nature of this gene in increased PS on the outer surface of these cells, a con-
plants. dition which induces an apoptotic response in animals
Similar to their animal counterparts, a growing (61).
body of literature suggests that polyphosphoinositides In yeast PS is a precursor to both PE and PC (7). PS
and their metabolites function as regulators of a vari- is rst decarboxylated to PE, a reaction catalyzed by
ety of cellular processes in plants. These include PS decarboxylase (EC 4.1.1.65) (62), and then PE is
responses to light, osmotic stress, and pathogen attack. sequentially methylated to PC by the action of two

N-methyltransferases, which utilize S-adenosylmethio- IV. BIOSYNTHESIS OF
nine as the methyl donor. The rst of these enzymes DIACYLGLYCEROL IN PLANTS
PE N-methyltransferase (EC 2.1.1.17) catalyzes the
methylation of PE to phosphatidyl methylethanola- Diacylglycerol, a precursor of PC and PE in plants, is
mine (PME) and the other enzyme phospholipid N- made from the dephosphorylation of PA to form DAG
methyltransferase catalyzes the two methylations and inorganic phosphate. The reaction is catalyzed by
necessary to convert PME to PC. the enzyme phosphatidate phosphohydrolase (EC
PS decarboxylase activity has also been measured in 3.1.3.4). This enzyme has been detected in organelle,
a number of plants (see 2). Thus some plant tissues, membrane, and soluble fractions of a number of plant
like yeast, are capable of synthesizing PE without the tissues (2, 64, 65). A microsomal phosphatidate phos-
participation of DAG. The presence of PE methyl- phohydrolase has been puried to apparent homoge-
transferases in plants would also mean that PC could neity as a 49-kDa species from avocado (66).
be synthesized from PE. This has led to the suggestion Another enzyme that has the potential of inuen-
that all plant phospholipids could be synthesized with- cing the ux of fatty acids through the PA, DAG, and
out the involvement of DAG. CDP-DAG pools is diacylglycerol kinase (EC
The methylation of PE to PC in plants has not 2.7.1.107). Opposing the reaction catalyzed by phos-
been conrmed, however, although plant cDNAs phatidate phosphohydrolase, diacylglycerol kinases
encoding phospholipid N-methyltransferase activities direct the synthesis of PA through the ATP-dependent
have been isolated. Computer-based homology phosphorylation of DAG. Although the inuence of
searches of plant EST databases resulted in the iden- this enzyme is generally not considered to be of
tication of Arabidopsis and soybean cDNAs encod- major consequence in the de novo synthesis of the
ing proteins (162 and 164 amino acids, respectively) major phospholipids or triacylglycerols, in animal sys-
that displayed low but signicant sequence homology tems it is clearly an essential component of inositol
to the yeast phospholipid N-methyltransferase (R.E. lipid signaling pathways. Similar to the CDP-diacylgly-
Dewey, A. J. Kinney, unpublished results). The sub- cerol synthase mutant described above, retinal degen-
strate specicities of the plant enzymes were estab- eration is observed in Drosophila possessing mutations
lished by expressing the Arabidopsis and soybean in an eye-specic diacylglycerol kinase locus due to
cDNAs in yeast mutants lacking either PE methyl- their inability to regenerate essential PI pools (67).
transferase or phospholipid N-methyltransferase Diacylglycerol kinase activities have been documen-
activities (or both), followed by enzymatic analysis. ted in several plant species (6870), and the isolation of
Similar to the yeast phospholipid N-methyl- their corresponding cDNAs from Arabidopsis and
transferase, the plant enzymes were capable of methy- tomato has been reported (71, 72). The identication
lating both PME and PDME. of a calmodulin-binding diacylglycerol kinase isoform
Negligible activity of the plant N-methyltransferases from tomato lends further support to the speculation
was detected using PE as a substrate (R.E. Dewey, A. that the plant enzymes function primarily as compo-
J. Kinney, unpublished results) suggesting that PE is nents of signal transduction pathways (72).
not directly methylated in either soybean or
Arabidopsis. This is supported by the classic radiotra-
cer studies of Datko and Mudd in the 1980s (see refer- V. SYNTHESIS OF
ence 2 for a detailed review of these studies). More PHOSPHATIDYLETHANOLAMINE
recent radiotracer evidence and some very thorough AND PHOSPHATIDYLCHOLINE FROM
computer modeling studies (63) presented clear evi- DIACYLGLYCEROL
dence that the major methylation ux in tobacco is
via phosphoethanolamine and not CDP-ethanolamine Flux modeling experiments in tobacco support the idea
or PE. The recent cloning and characterization of phos- that the ux of ethanolamine into PE is split equally
phoethanolamine N-methyltransferase cDNAs from spi- between PS decarboxylation (or base exchange; see
nach (8) and Arabidopsis (9) provide additional below) and incorporation of phosphoethanolamine
support for this pathway. Thus, while most plant phos- via CDP-ethanolamine and diacylglycerol (63).
pholipids can be synthesized via CDP-diacylglycerol, it Phosphoethanolamine is probably derived from free
appears that the major pathway for synthesis of PC is or phosphorylated serine by decarboxylation (73, 74)
via diacylglycerol. although no enzyme operating on anything other than

phosphatidylserine has been convincingly demon- Two of the cDNAs, designated GmCK1 and
strated. In contrast, it is apparent that choline in plants GmCK2, were full-length encoding proteins of 359
is not synthesized via a free base pathway but by the and 362 amino acids, respectively. Although the third
methylation of phospho- and phosphatidylmono- isoform, GmCK3, was reported to be considerably lar-
ethanolamine (see 2, 74). Furthermore, the bulk of ger than the other two, its nal predicted size was
the ux into choline compounds is likely initiated via unknown because the characterized cDNA was not
the methylation of phosphoethanolamine (63). Free full length. Subsequent work, however, revealed the
choline may then be synthesized by dephosphorylation GmCK3 cDNA to encode a 650amino acid protein,
of phosphocholine. Evidence from many different with most of its increase in size derived from a degen-
plant families supports the idea that PC is synthesized erate 11amino acid sequence that is repeated 23 times
by the methylation of PME derived from methylated at the N-terminus of the protein (D.E. Monks, R.E.
ethanolamine phosphate or by conversion of phospho- Dewey, unpublished results). Expression of the three
choline, also from methylated ethanolamine phos- soybean choline kinase cDNAs in yeast and/or E. coli
phate, to CDP-choline (2, 74). Both pathways utilize demonstrated that each functions strictly as a choline
DAG as the source of glycerol and fatty acids (see Fig. kinase; ethanolamine kinase activity was negligible (78;
1). Free choline is rapidly incorporated into PC (2), R.E. Dewey, D.E. Monks, unpublished results).
suggesting that the plants can also make PC from cho- The discovery of two distinct size classes of soybean
line reserves or exogenous choline. This hypothesis is choline kinases may provide an explanation for discre-
supported by the observation that methylation of pancies in molecular-weight estimations previously
phosphoethanolamine is repressed by free choline in reported in the literature. A smaller, 36-kDa choline
plant tissue cultures (75). Thus, in addition to the kinase from soybean reported by Wharfe and
phosphoethanolamine methyltransferase described Harwood (79) may represent the product of the
above, three types of enzymes are needed to make GmCK1 and/or GmCK2 genes, whereas larger choline
PE and PC using DAG: aminoalcohol kinases, phos- kinase species (58 and 60 kDa) reported by Mellor and
phoaminoalcohol cytidylytransferases, and aminoalco- coworkers (80), also from soybean, may in fact be the
hol phosphotransferases. There is good evidence that product of GmCK3. Although computer searches
all these activities exist in plants for both the ethano- could detect no entries in protein databases that dis-
lamine and the choline pathways. played signicant amino acid sequence homology to
The phosphorylation of ethanolamine is catalyzed the large N-terminal extension found on the GmCK3-
by the enzyme ethanolamine kinase (ATP:ethanolamine encoded protein, the 11-residue repeat structure is remi-
phosphotransferase, EC 2.7.1.82) and the phosphory- niscent of the 11-mer repeats of apolipoproteins and
lation of choline is mediated by an equivalent enzyme, choline phosphate cytidylyltransferase that mediate
choline kinase (ATP:choline phosphotransferase, EC the association of these proteins with membrane phos-
2.7.1.32). The yeast gene for choline kinase (CK1) pholipids (discussed below). Consistent with this
was cloned by complementation of a choline kinase hypothesis, a considerable amount of choline kinase
mutant and was shown to encode a polypeptide of activity was observed in the microsomal fraction
about 66 kDa (76), which corresponded to the native when GmCK3 was expressed in yeast; in contrast no
molecular weight of a partially puried yeast choline membrane-associated choline kinase activity was
kinase (77). However, when the CK1 gene was detected upon expression of GmCK1 or GmCK2 in
expressed in E. coli, both choline kinase and ethanola- yeast (R.E. Dewey, D.E. Monks, unpublished results).
mine kinase activities were observed. Disruption of the The conversion of ethanolamine phosphate and
CK1 gene in wild-type yeast resulted in the loss of both choline phosphate to their respective CDP-amino-
choline and most of the ethanolamine kinase activities alcohols is catalyzed by phosphoaminoalcohol
(76). Thus it is possible that both reactions are cata- cytidylyltransferases. Choline phosphate cytidylyl-
lyzed by the same enzyme in yeast. In contrast, the transferase (CTP:choline phosphate cytidylyltransfer-
bulk of the biochemical evidence favors the hypothesis ase, EC 2.7.7.15) has been extensively characterized
that there are separate ethanolamine and choline in animals (81, 82) and shown to play an important
kinase in plants (see 2 for discussion), and this hypoth- regulatory role in membrane biosynthesis in regulating
esis is supported by recent molecular studies. Using an the ux through the CDP-choline pathway for PC
Arabidopsis EST that displayed homology to yeast and biosynthesis (5). The animal enzyme comprises four
mammalian choline kinases, three distinct choline distinct subdomains: an amino-terminal nuclear local-
kinase isoforms were isolated from soybean (78). ization signal; a highly conserved catalytic core; a

membrane/lipid binding domain; and a carboxyl-term- that different isoforms serve unique cellular functions
inal phosphorylation domain (82). The discovery of is warranted. The newly discovered animal choline
the nuclear targeting signal is consistent with immuno- phosphate cytidylyltransferase differs from the well-
localization studies that unexpectedly revealed the characterized isoform in its subcellular location (cyto-
nucleus, rather than the cytosol, to be the intracellular plasm as opposed to nucleus) and tissue-specic
compartment where the majority of the protein resides. expression prole (88). With the availability of
The lipid/membrane-binding domain consists of three numerous plant choline phosphate cytidylyltransfer-
11-residue repeats that are predicted to form an amphi- ase cDNAs, the tools are now available to dissect
pathic alpha helix. This region is critical not only for and dene the function of specic structural domains
mediating the reversible association of the enzyme with of the protein and test whether different isoforms are
membranes, but also for regulating enzymatic activity functionally distinct.
per se. Current models suggest that in the absence of The enzyme ethanolamine phosphate cytidylyltrans-
activating lipids, the membrane/lipid-binding domain ferase (CTP:ethanolamine phosphate cytidylyltransfer-
inhibits the catalytic activity of the enzyme; lipid bind- ase, EC 2.7.7.14) has not received much attention from
ing increases enzymatic activity > 50-fold by amelior- plant scientists but, like cholinephosphate cytidylyl-
ating the inhibitory effects of this region (82, 83). The transferase, it has also been well studied in yeast and
function of the carboxyl-terminal phosphorylation animals (89, 90). Human, rat, and yeast cDNAs encod-
domain is less clear. Although early studies demon- ing ethanolamine phosphate cytidylyltransferases have
strated that the membrane-associated, enzymatically been isolated and characterized (9193). Not sur-
active choline phosphate cytidylyltransferase was prisingly, signicant homology is observed between
dephosphorylated, and the soluble, inactive form of portions of the ethanolamine phosphate cytidylyltrans-
the enzyme was highly phosphorylated, recent studies ferases and the catalytic domains of choline phosphate
using phosphorylation-decient mutants revealed that cytidylyltransferases. Uniquely found in the ethanola-
the modulation of phosphorylation does not appear to mine phosphate cytidylyltransferase enzymes, how-
be critical either for the reversible translocation of the ever, is a repeat of the rst half of the catalytic
enzyme to membranes or in maintaining cellular domain, located adjacent to the full-length version
growth (82). of this region. Although considerable amino acid
Using an elegant yeast complementation strategy, sequence homology is shared between the catalytic
Nishida and colleagues (84) recovered cDNAs corre- regions of the two aminoalcohol phosphate cytidylyl-
sponding to four distinct choline phosphate cytidylyltransferases, this step of the parallel PE and PC bio-
transferase isoforms from Brassica napus. synthetic pathways is the only one where no functional
Subsequently, choline phosphate cytidylyltransferase redundancy has been reported in any biological system
clones from Arabidopsis and pea were also reported (89). Ethanolamine phosphate cytidylyltransferase was
(85, 86). Although extensive sequence homology rst reported in plants in the membrane fractions of
between the plant enzymes and their animal and mitochondria and ER from castor bean endosperm
yeast counterparts is observed only across the region (94). Curiously, most of the castor bean ethanolamine
dened as the catalytic domain, the presence of a pre- phosphate cytidylyltransferase activity (80%) was in
dicted amphipathic alpha helical motif immediately the mitochondria, whereas the choline phosphate cyti-
following the catalytic domain suggests that plant cho- dylyltransferase is found mainly in the ER and cytosol
line phosphate cytidylyltransferases may possess mem- (94). The mitochondrial ethanolamine phosphate cyti-
brane/lipid binding domains that are functionally dylyltransferase has recently been partially puried
similar to those observed in animals. from postgermination castor bean endosperm (95).
Because of the polyploid nature of Brassica napus, The castor bean enzyme, predicted to have a molecular
the initial observation of multiple choline phosphate mass of 3035 kDa, was modestly activated by the
cytidylyltransferase isoforms in this species was not addition of phospholipids. Membrane association of
considered surprising, as functionally equivalent both the mitochondrial and ER-localized ethanola-
genes could have been derived from different ances- mine phosphate cytidylyltransferases of castor bean
tral parents (84). The presence of multiple isoforms is appears to be much stronger than that observed with
also evident in pea and soybean (86, 87). Given the their mammalian counterparts, as the plant enzymes
recent discovery of a previously unrecognized, distinct remained completely insoluble when treated with salt
choline phosphate cytidylyltransferase isoform in or detergent concentrations that completely solubilize
mammals, however, consideration of the possibility the animal enzymes (95).

PE and PC are synthesized from their cognate CDP- can be detected in vitro suggests that factors altering
aminoalcohol and DAG, reactions catalyzed by the the directionality of this activity could also inuence
aminoalcohol phosphotransferase enzymes cholinephos- the ultimate PC and PE composition of eukaryotic
photransferase (CDP-choline:1,2-diacylglycerol choli- membranes. This may be particularly relevant during
nephosphotransferase, EC 2.7.8.2) and ethanolamine- seed development in oil seeds, where the reverse reac-
phosphotransferase (CDP-ethanolamine: 1,2,diacylgly- tion of choline phosphotransferase is believed to func-
cerol ethanolaminephosphotransferase, EC 2.7.8.1). tion in supplying polyunsaturated fatty acids directly
These enzymes have been measured and characterized into DAG pools destined for triacylglycerol biosynth-
in many different plant tissues (see 2), but they have esis (99). Consistent with previous observations using
remained recalcitrant to solubilization and purication. isolated plant microsomes, the activities of the soybean
The rst cDNA encoding a plant aminoalcohol phos- AAPT1 gene and two Arabidopsis AAPT isoforms
photransferase was obtained from soybean by comple- described above were shown to be readily reversible
mentation of a double-mutant yeast strain defective in when expressed in yeast (97). The ability of CMP to
both CPT1 and EPT1 activities using a colony autora- inhibit the forward reaction and provide a necessary
diography assay (96). The predicted amino acid substrate for the reverse reaction, makes this com-
sequence of the soybean protein showed equal similar- pound a good candidate for regulating the direction
ity to both the yeast CPT1 and EPT1 proteins, and of AAPT-catalyzed reactions in vivo. Further investi-
inhibition studies suggested that the plant enzyme gation of endogenous CMP pools in plants and their
shared equal afnity for both CDP-choline and CDP- inuence on phosphatidylamine metabolism, particu-
ethanolamine. These observations led to the specula- larly during seed development, is warranted.
tion that the enzyme encoded by the soybean cDNA
functions as a general aminoalcohol phosphotransfer-
ase and as a result, was designated AAPT1 (aminoalco- VI. PHOSPHOLIPID MODIFYING
hol phosphotransferase 1). Although this report ENZYMES
presented compelling evidence that plants possess
enzymes that can function as both choline and ethano- There are a number of enzyme activities in plant cells
lamine phosphotransferases, Southern blot data that modify either the polar or lipid portion of phos-
revealed that additional isoforms of the gene were likely pholipid molecules. The best documented polar mod-
to residue in the soybean genome, leaving open the ication enzymes are those which catalyze
possibility that aminoalcohol phosphotransferases phosphoaminoalcohol exchange of preexisting phos-
with more restrictive substrate specicities may exist, pholipids with a similar or different heads. The best
similar to the situation found in yeast. characterized of the head group exchange reactions is
The recent characterization of cDNAs encoding two the synthesis of PS by the reversible exchange of ser-
AAPTs from Arabidopsis provides even further evi- ine with the headgroup of PE by the Ca2 -dependent,
dence that plants utilize broad-specicity aminoalcohol microsomal enzyme PE:L-serine phosphatidyltransfer-
phosphotransferases (97). Southern blot analysis sug- ase (10, 100). No progress has been reported in the
gested that the two Arabidopsis AAPT cDNAs encode characterization of this enzyme or in understanding
the complete complement of AAPT enzymes found in the function of the serine/ethanolamine exchange
that species. Although subtle differences in substrate reaction since an earlier review on the subject (2).
preference, and Ca2 and CMP inhibition proles were This is also true of the PI/inositol base exchange reac-
observed among the two Arabidopsis AAPTs and the tion in castor bean endosperm (2) although a similar
soybean AAPT1 gene product, it was clear that all reaction has been measured in a number of animal
three plant enzymes could effectively utilize either cells (101).
CDP-choline or CDP-ethanolamine to produce PC Only marginally better understood than these
and PE, respectively. An additional AAPT cDNA headgroup modifying enzymes are enzymes that
has been isolated from Chinese cabbage; however, exchange fatty acids with phospholipids, especially
the enzymatic properties of the encoded protein were PC. The activity of an enzyme capable of catalyzing
not reported (98). the exchange of 18:1-CoA with the fatty acid at the
Although the cytidylyltransferase-catalyzed steps sn-2 position of PC (LPCAT EC 2.3.1.23) (acyl-
are generally recognized as the most important in reg- CoA:lysophosphatidylcholine acyltransferase) has been
ulating the net biosynthesis of PC and PE within the assayed in a number of plant tissues (102). This enzyme
cell, the ease with which the reverse AAPT reactions has a potentially important role to play in plant lipid

metabolism since PC is the substrate for fatty acid 5. A Lykidis, S Jackowski. Regulation of mammalian
desaturation of 18:1 to linoleic acid. However, little is cell membrane biosynthesis. Prog Nucleic Acid Res
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ferase: unexpected ndings from curiosity-driven
Rajasekahran (103) used photoafnity labeling of soy-
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7. GM Carman, SA Henry. Phospholipid biosynthesis in
molecular masses of 54 kDa and 114 kDa for putative the yeast Saccharomyces cerevisiae and interrelation-
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Plants also contain a phospholipid:diacylglycerol AD Hanson. cDNA cloning of phosphoethanolamine
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SAM:phosphoethanolamine N-methyltransferase.
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13. S Halford, KS Dulai, SC Daw, J Fitzgibbon, DM
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13
Applications of Oxidoreductases in Foods
Colja Laane
Yvonne Bruggeman
Unilever Research, Vlaardingen, The Netherlands
Chris Winkel
Quest International, Naarden, The Netherlands
I. INTRODUCTION not feasible. In addition, public concerns about the use

of recombinant enzymes in food products is slowing
Oxidative and reductive processes play an important down their market introduction.
role in foods. They inuence not only the taste but also In this chapter the current and potential usage of
the texture, appearance, shelf life, nutritional value, oxidoreductases in controlling the taste, texture,
and process tolerance of food products. Both enzy- appearance (i.e., color), shelf life, and the nutritional
matic and nonenzymatic redox processes are involved. value of food products will be discussed. Increasingly,
In some cases redox processes lead to undesirable redox enzymes are being used for biosensor applica-
effects such as off-avor, reduced shelf life, or textural tions in food systems. This topic will not be discussed
problems. In other cases, they contribute in a positive in this chapter, however. Table 1 lists the most impor-
way to the nal aroma, an improved texture, a more tant redox enzymes and their functions in food sys-
desirable appearance, or an increased shelf life. tems. Most attention will be paid to the market
Controlling the redox behavior in food systems during segments bakery, beverages, and dairy and to oxidases,
all stages of processing and storage is therefore of since relatively little is known about role and applica-
utmost importance. tions of reductases in food systems. Furthermore, the
Up until now redox reactions in foods were con- emphasis will be on added enzymes and not on
trolled mainly by carefully selecting raw materials, by enzymes already present in the food product constitu-
adapting process conditions, by adding chemicals or ents. For detailed information on the properties of the
antioxidants, or by designing air-tight packaging mate- individual oxidoreductases the reader is referred to in
rials. As yet, little attention has been paid to tailor Sec. IIA of this book.
redox reactions in foods by the addition of oxidore-
ductases or by changing the prole or content of oxi-
doreductases in food raw materials by genetic tools. II. TASTE
Presumably, the major bottleneck for the application
of oxidoreductases to foods is that economically ef- Many oxidoreductases play a role by inuencing the
cient enzyme production is, with a few exceptions, still taste prole of food products. They are involved in the
in vivo/in situ biogenesis of desirable aroma compo-

Table 1 Overview of the Main (Potential) Applications of Oxidoreductases in Food Products
Enzymes Taste Texture Appearance Shelf life Nutritional value
Lipoxygenases (LOX) In situ and in vitro Dough extensibility Flour bleaching Destruction vitamin A
(off)-avor and strength
production
Alcohol oxidases and In situ and in vitro AO plus catalase, O2
dehydrogenases avor production removal
(AO, ADH) (ILOX)
Sulfhydryl oxidases Removal of cooked Dough strengthening in
(SOX) avor in UHT-milk combination with
POX
Peroxidases (POX, In vitro avor Crosslinking Assisting PPO in color LPO, antimicrobial agent
LPO) production and biopolymers formation
debittering
(Poly)phenol oxidases Debittering of coffee, (Dis)coloration; in vitro Laccase, O2 removal
(PPO) cacao, and olives production of colors
Carbohydrate oxidases GOX, mild acid Dough strengthening GOX plus catalase, O2
(GOX, HOX, production via H2 O2 ; thickening removal
PYROX) agent
Ascorbic acid oxidase Browning AAO plus catalase, O2 Destruction vitamin C
(AAO) removal
Xanthine oxidases (XO) Antimicrobial agent
Superoxide dismutases Removal reactive O
(SOD) species with catalase
Catalases Removal excess H2 O2
Cholesterol Cholesterol reduction
oxidoreductases

nents, in the in vitro production of avoring topnotes, LOX is also used for the in vitro production of
and in the endogenous formation of off-avors. The several natural topnote avors, which are mainly
main redox enzymes are discussed below. added to beverages and dairy products to tailor the
avor prole (9, 10). Typical examples include the con-
A. Lipoxygenases version of polyunsaturated fatty acids into various
short to medium-chain aldehydes/alcohols (13), or
Lipoxygenase (LOX, EC 1.13.11.12; see Chapter 43, into S(-)--decalactone (butter avor; 12). Well-
for detailed information), formerly known as lipoxi- known fatty acidderived avoring aldehydes/alcohols
dase or carotene oxidase, is an iron-containing include the above mentioned C5 and C6 compounds,
dioxygenase which catalyzes the oxidation of polyun- as well as (E2, E6)-nonadienal (cucumber), 1-octen-3-
saturated fatty acids containing cis, cis-1, 4-pentadiene one (eld mushroom), (Z5)-octadien-3-one (geranium
groups (linoleic, linolenic, and arachidonic acids) to leaves), (E3, E5)-undecatriene (blasamic), and (E3, Z5,
the corresponding conjugated cis, trans dienoic mono- Z8)-undecatetraene (seaweed). Depending on the
hydroperoxides. In addition LOX also accepts a rela- degree of unsaturation and the regioselectivity of the
tively broad range of phenolic compounds as lipoxygenase, different hydroperoxy compounds are
substrates (1) and is capable of oxidizing other sub- formed, from which the above-mentioned compounds
strates than the actual substrate. This process is can be derived by subsequent enzymatic reactions. For
known as co-oxidation and includes compounds like (Z3)-hexenol, linolenic acid is used as a substrate, while
carotenoids and polyphenols (2). for most of the other aldehydes/alcohols higher unsa-
LOX is one of those endogenous enzymes that play turated fatty acids are required. The production of
an important role in both avor generation and off- (Z3)-hexenol has recently been commercialized using
avor formation in virtually all food products derived plant homogenates (e.g., alfalfa sprouts, green pep-
from plant raw materials. Depending on the nal con- pers) which are relatively rich in hydroperoxide lyase,
centration and the type of food product the same a- the enzyme required to split the hydroperoxide fatty
vor component can be a desired aroma component or acid into smaller fragments. The generated (Z3)-hexe-
an off-avor at higher concentrations. For example, nal was converted into (Z3)-hexenol using the reduc-
C6-aldehydes and alcohols derived by the LOX-cata- tive enzymatic power of bakers yeast (13). A very
lyzed oxidation of (poly)unsaturated fatty acids have, elegant approach has been taken recently by
in most cases, a positive effect on the aroma prole Givaudan-Roure. To unify all three enzymes involved
(e.g., wines and juices), but in other beverages, have in the formation of (Z3)-hexenol, they have cloned and
an undesired avor effect (e.g., beer). Likewise, endo- overexpressed both soybean LOX and the hydroper-
genous LOX is known to generate carbonyl com- oxide lyase from banana in bakers yeast. In this way
pounds in dough systems and hence inuences bread they have developed a single-step process which pro-
avor (3). duces (Z3)-hexenol in relatively high yield (14).
The formation of C6 compounds requires the For the production of lactones a different strategy
sequential action of four enzymes of which two are has to be followed after the formation of the linoleic
redox enzymes, namely an acylhydrolase, a lipoxygen- acid hydroperoxide. It involves the fermentative -oxi-
ase, a hydroperoxide lyase, and a yeast-derived alcohol dation of the hydroperoxide intermediate by the yeast
dehydrogenase (4). Typically, off-avor formation is Pichia etchellsii, and the subsequent cyclization of 5-
prevented by using crop variants decient in LOX iso- hydroxydecanoic acid to the corresponding S(-)--dec-
enzymes, either by screening or by genetic tools (5) or alactone (10).
by controlling the oxygen levels during processing (6), As shown by Quest International (1), lipoxygenases
as well as by removing the malodorous oxidation pro- also accept a relatively broad spectrum of phenolic
ducts afterwards. The deodorization of off-avors can compounds as substrates. Of interest to the avor
be achieved by physical techniques such as adsorption, industry are the LOX-catalyzed conversions of isoeu-
or enzymatically by converting the undesired alde- genol and coniferyl benzoate from Siam resin into
hydes (e.g., with aldehyde dehydrogenase/oxidase), or vanillin. At present the commercialization of these bio-
alcohols (e.g., with alcohol oxidase) into their corre- transformations is hampered by the fact that isoeu-
sponding less avorful carboxylic acid. The use of genol is not readily available and that coniferyl
redox enzymes for this purpose has been claimed for benzoate is difcult to handle in a reactor.
several products such as margarine, cream, sh oil, Other well-known reactions of LOX include the co-
noodles, cooked rice, and soybean products (7, 8). oxidation reaction of carotenoids to yield, among

others, -ionone (2, 15). More recently is the genera- C. Sulfhydryl Oxidases
tion of a toasted or cookie-like avor in starchy mate-
rials by a combination of LOX and an -amylase (16). Sulfhydryl oxidase (SOX, no EC number assigned; see
Chapter 41 for more details) catalyzes the formation of
disulde bonds from (protein) thiols. SOX is an Fe/Cu-
B. Alcohol Oxidases and Dehydrogenases containing glycoprotein and has been detected in
bovine, human, goat, pig, rabbit, and rat milks (23).
In general, aldehydes are more potent avor com- The enzyme is capable of oxidizing the sulfhydryl
pounds than their alcoholic counterparts. Hence, alco- groups of cysteine and glutathione, as well as milk pro-
hol oxidases are interesting enzymes for the in vitro teins to their corresponding disuldes using molecular
production of avoring preparations which, among oxygen as electron acceptor (24). In practical applica-
others, can be applied in beverages. Typical examples tions bovine milk SOX may be added to UHT milk to
include the use of methanol oxidase from Pichia, reduce cooked avors. Patents (see Refs. in 24) for this
Hansenula, and Candida (17) for the production of application have been issued. The enzyme has been
natural acetaldehyde from ethanol. This enzyme is immobilized on porous glass, and its effectiveness in
induced during growth on methanol. At the end of ameliorating the cooked avor has been demonstrated
the logarithmic growth phase cells are harvested and on a pilot scale using immobilized enzyme columns.
incubated with ethanol. In this way concentrations of
1:5% natural acetaldehyde can be achieved, which D. Peroxidases
can be concentrated further to the desired application
level. From yeast to yeast the substrate specicity of Peroxidases (POX, EC 1.11.1.7; see Chapters 28 and 29
the alcohol oxidase is different. Hence, this procedure for more details) occur widely in nature and is the
can also be used to convert other alcohols, such as general name for a group of both highly specic and
hexenol and other long-chain alcohols, to their corre- nonspecic enzymes which use hydrogen peroxide
sponding aldehyde (10, 18). instead of oxygen as an electron acceptor.
As alternatives to alcohol oxidases the correspond- Peroxidases, especially the heme-containing ones, can
ing dehydrogenases could in principle be used (19). A catalyze a large number of different reactions including
severe drawback, however, is that these dehydro- sulfoxidation, N-demethylation, oxidation, and hydro-
genases require the expensive cofactor NAD(P) xylation and hence are of potential interest in the pro-
instead of (cheap) oxygen as an electron acceptor. duction of specic avoring topnotes. A recent
Although various sophisticated NAD(P) cofactor example involves the demethylation of methyl N-
regenerating systems have been designed and substan- methylanthranilate (ex Citrus) to monomethylanthra-
tial cost reductions have been realized in this way, it is nilate (25). The latter compound is an important top-
evident that in commercial applications oxidases are note avor in Concord grapes. Soybean, horseradish,
preferred over their dehydrogenase counterparts. The and microperoxidases were found to be convenient cat-
use of dehydrogenases for food purposes seems to be alysts for this reaction. In addition POX gives rise to a
restricted to whole-cell conversions. fresh avor prole when added to tomato paste (26).
Vanillyl alcohol oxidase (VAO) from Penicillium
simplicissimum is a special type of alcohol oxidase. E. Polyphenol Oxidases
Recently, it has been shown that this stable, avin-
containing enzyme has a very broad substrate speci- Polyphenol oxidases (PPO) are a group of several
city and readily converts para-substituted phenols into enzymes. Different activities can be found within this
interesting avor precursors or avoring compounds group: tyrosinase (monophenol monooxygenase, EC
(2022). Apart from natural vanillin and coniferyl 1.14.18.1; see Chapter 39) converting a phenol into a
alcohol, different vinylphenols (e.g., para-vinylguaia- catechol group; 1,2-diphenol oxidase or catechol oxi-
col) and allylphenols can be produced from cheap dase (EC 1.10.3.1) converting catechol into an o-qui-
raw materials and oxygen as an electron acceptor. none, and laccase (EC 1.10.3.2; see Chapter 40)
VAO can also be used for generation of avor building converting a 1,4-diphenol into p-quinone. Usually the
blocks. To that end a natural mix of phenolic com- rst two activities are linked, as catechol is much more
pounds could be treated with VAO to enrich foods readily oxidized than a phenol. Most enzymes that
or avor preparations with a range of vinylic/allylic catalyze 1,4-diphenol oxidation also act on 1,2-diphe-
and aldehydic substances. nols.

The bitter taste in many food products is often the been isolated from seeds of commercial cultivars and
result of the presence of polyphenolic compounds such have been well characterized (31, 32). Recent studies
as guaiacols. PPOs are capable of oxidizing these com- indicate that L2 has the greatest effect among LOX
pounds and hence can be applied to reduce bitterness. isoenzymes on dough extensibility and strength (33)
At present the debittering of coffee beans (27), Adzuki and is also mostly responsible for the production of
beans (28), and cacao beans (29) by PPOs have been undesirable aroma compounds in bread doughs (34;
claimed. Another application involves the use of laccase see Sec. II, Chapter 43). For L3 an increase in foaming
to debittering olives by treating stoned, chopped olives activity has been reported, as well as an overall
in the presence of air at increased temperatures (30). improvement in breadmaking quality of wheat our
(35). The bakery yeast Saccharomyces cerevisiae also
contains LOX. Recently, this enzyme was partially
III. TEXTURE puried (36), but its potential, if any, on breadmaking
remains to be established.
The use of oxidoreductases in texturizing food is based Nowadays, it is feasible to change the prole and
on their ability to crosslink proteins and/or polysac- content of LOX (iso)enzymes in plants either by clas-
charides. This property is of particular interest to sical means (5), or potentially by genetic modication.
improve the texture of dough and paste products (soft- For example, by appropriate crosses, near-isogenic
ness, volume, elasticity, crunchiness) or dairy products soybean seeds have been developed that lack either
(mouth feel, appearance), and of sh and meat pastes. isoenzymes L1 and L3, or isoenzymes L2 and L3.
Basically, enzymatic crosslinking can occur via two These LOX-minus mutants still grow well in the eld
routes: (37). In principle, transgenic plants lacking or overex-
1. Indirect via enzymatically produced hydrogen pressing one or more LOX isoenzymes could be con-
peroxide (e.g., glucose oxidase, hexose oxidase, ascor- structed and tailored to specic applications (38). To
bic acid oxidase), or via the enzymatic production of that end the heterologous expression of one or more
radicals (e.g., peroxidase, lipoxygenase). soybean LOX isoenzymes in wheat could be of interest.
2. Direct via the oxidation of functional groups in
the protein. Examples include the linkage of tyrosine B. Sulfhydryl Oxidases
and ferulic acid residues by tyrosinase and laccases,
linkages of lysine residues by lysyl oxidase, or the for- The action of SOX (see Sec. II, Chapter 41, for general
mation of disulde bridges by sulfhydryl oxidase. information) may be the same as those of chemical
An alternative, nonredox procedure for direct cross- oxidizing agents, provided the formation of disulde
linking is being explored using transglutaminases (see bonds is the primary mode by which these agents func-
Chapter 51). tion. In extensive testing (39, 40) it was found that
These enzymatic routes are being explored to re- SOX alone has no inuence on loaf volume, dough
place chemical oxidizers, such as dehydroascorbic strength, or mixing tolerance. Also, relatively high con-
acid and potassium bromate. Replacement of the non- centrations using recombinant SOX from Aspergillus
specic chemical compounds by redox enzymes could awamori did not have any signicant positive effect
have the benet of more specic and better-controlled (40). One reason could be that SOX has only a limited
oxidation processes. Other disadvantages of the chemi- afnity for thiol groups in gluten proteins and as a
cal substances are related to safety and labeling issues. result its application in the food industry seems to be
One of the major challenges in the baking industry is to limited to the removal of small off-avor molecules
nd a good replacer for potassium bromate, which is a (see Sec. II, Chapter 41).
difcult task since bromate is still more effective and
cheaper than the enzymatic alternative. The most C. Peroxidases
important texturizing redox enzymes will be discussed
below. For detailed information about POX see Section II,
Chapters 28 and 29. Wheat our contains a peroxidase
A. Lipoxygenases that can crosslink phenolic constituents such as ferulic
acid and vanillic acid. However, the pH optimum of
A rich source of LOX is soybeans. Soybean LOX con- the wheat enzyme is 4.5, and in the pH range of 56 of
tains three distinct lipoxygenase isoenzymes, desig- wheat doughs the activity is considerably lower than at
nated as L1, L2, and L3. These isoenzymes have pH 4.5 (41). Although the dough-improving effect of

peroxidase is well documented, the reactions involved gels or viscous media for application as fat or gelatin
in the dough are only poorly understood. replacer, as well as for avor delivery, coating, or glaz-
Model studies with peroxidase, glutathione, and ing.
cysteine indicate that sulfhydryl and/or lysyl groups
may be involved (42). Peroxidase-catalyzed reactions
D. Glucose Oxidases
result in a decreased amount of lysine recovered from
proteins after acid hydrolysis. Peroxidases, or the qui-
Glucose oxidase (GOX, EC 1.1.3.4; see Chapter 30 for
nones formed by the enzyme, oxidatively deaminate
detailed information) catalyzes the conversion of glu-
lysyl residues to form lysylaldehydes, resulting in the
cose into the mild-tasting gluconic acid via glucono--
formation of protein polymers, as was revealed by gel
lactone and hydrogen peroxide. GOX complies with
ltration. These aggregates could not be dissociated by
the FAO/WHO and GRAS requirements for food
detergents, which indicates that covalent bonds were
grade enzymes and is one of the few commercially
formed. In addition, the dough-strengthening effect of
available oxidases from Aspergillus niger and
peroxidases is also ascribed to the crosslinking of car-
Penicillium strains at relatively low costs. Especially,
bohydrates and the coupling of carbohydrates to pro-
the generation of hydrogen peroxide is believed to
teins. The gelation of wheat pentosans is due to the
give the antiweakening effect in bread doughs (40).
oxidative dimerization of ferulic acid moieties, which
In a recent study by Hilhorst et al. (49) the effect of
are covalently linked to pentosans (43).
GOX on Dutch rusk dough was dough stiffening with
Recently, a study has been described showing the
a clear loss in extensibility. This made the overall effect
effect of different peroxidases on the baking perfor-
quite undesirable. For comparison, they also tested
mance of German Kaiser rolls (41). Deliberately
peroxidase (see Sec. III.C above). This enzyme gave a
sticky doughs were prepared in order to demonstrate
dough-stiffening effect without loss in extensibility.
the effects of added peroxidases. Especially the addi-
The authors explain the difference by the fact that
tion of soybean POX led to strengthening of wheat
hydrogen peroxide from the GOX-catalyzed reaction
doughs as determined by the stability of the dough
oxidizes randomly and links the gluten network with
after shaking the dough for 1 min in a laboratory
the arabinoxylan network, whereas the peroxidase only
shaker. This improving effect was observed at both
increases the amount of crosslinks in the arabinoxylan
short and long fermentation times. Dough volume
fraction without affecting the gluten network or the
increased after application of these peroxidases.
coupling between the networks.
Compared with (GOX), the results of peroxidases are
It has been found that synergistic effects occur when
better or at least equal in terms of stability and volume.
using a combination of oxidative enzymes like the
In bread dough, peroxidases seem to act without the
combination of GOX and SOX (50). Furthermore,
extra addition of hydrogen peroxide in spite of their
the dough has an increased stability.
peroxide dependence. This may indicate that peroxide
is present in dough at sufcient amounts, or that it is
generated as a result of the peroxidase reaction. In this E. Hexose Oxidases
way, substrate radicals formed by peroxidase can react
with oxygen to form hydrogen peroxide. When this A newcomer in the eld of redox enzymes for bakery
reaction is occurring, catalytic amounts of peroxide products is hexose oxidase (HOX, EC 1.1.3.5) from red
present in dough are sufcient to get the cycle started, seaweeds. This glycosylated avoprotein is related to
and may explain why no hydrogen peroxide has to be GOX but has a broader substrate specicity toward
added together with a peroxidase. An alternative hexose sugars, including oligomers. Like GOX, it
explanation is that wheat contains endogenous oxi- acts on the C1-position of the sugar moiety (51).
dases, which are active enough to produce some hydro- Recently, the enzyme from Chondrus crispus has been
gen peroxide in situ. Indeed, the addition of hydrogen isolated, cloned, and overexpressed in several recombi-
peroxideproducing enzymes such as GOX has a ben- nant organisms such as Pichia pastoris, Saccharomyces
ecial effect on baking (4446). cerevisiae, and E. coli (51, 52). However, the produc-
In other systems than doughs, POX has been tion levels of active enzyme are still poor at this
claimed as a thickening and stabilizing agent in, for moment and have to be improved. For this reason,
example, ice creams, deserts, sauces, and jams and jel- and because C. crispus has a long standing tradition
lies (47). There is also a recent patent application (48) as an edible organism, efforts have been made to
on the POX-catalyzed gelling of hemicelluloses to form develop a large-scale production method with the use

of carrageenase, which reduces the viscosity of the A. Lipoxygenases
crude extract (51).
For HOX similar applications can be envisaged as It is interesting to note that the use of soybean lipox-
for GOX (see above). In particular, its application in ygenase was described in the 1930s as a means to
bakery products is foreseen (53), since HOX is able to bleach the our in preparation of white bread. More
generate more H2 O2 owing to its broader substrate recent experiments have shown that carotenoids pre-
specicity, and hence should be more effective than sent in wheat our are destroyed by co-oxidation.
GOX in dough strengthening. Likewise, combinations Wheat our itself contains little LOX activity, but
of HOX and H2 O2 -consuming enzymes, such as POX, LOX is abundantly present in, for example, soybeans.
may be envisaged. To that end wheat our is often fortied with up to
Another enzyme with similar properties as HOX, 0.5% enzyme-active soy our (34, 60). Other applica-
isolated from Acremonium strictum T1, has been tions of LOX include the bleaching of noodles, whey
reported in the literature (54). The enzyme is called products, rice, and wheat bran (6164).
an oligosaccharide oxidase and is able to oxidize sev-
eral di- and oligosaccharides at the anomeric site, B. (Poly)phenol Oxidases and Peroxidases
yielding the corresponding di- and oligobionic acids.
A detailed comparison between HOX and oligosac- PPOs (see Sec. II, Chapter 39 for general information)
charide oxidase performance in dough applications play an important role in the browning of fresh fruits
has not been made yet. and vegetables (65, 66), in the coloring and avoring of
tea (67, 68), and in improving the quality of coffee (69).
F. Pyranose Oxidases A major concern in the food industry is to prevent
the development of enzymatic browning prior to the
Pyranose oxidase (PYROX, EC 1.1.3.10; see Refs. processing of fruits and vegetables (70). This is accom-
5557, 59 for more detailed information) catalyzes plished by removing oxygen or by inhibiting PPO
the oxidation of several monosaccharides at the C2- activity using inhibitors such as metal chelating agents,
position and is therefore different from GOX, which inorganic ions (e.g., halide anions), benzoic acid and
oxidizes glucose at the C1-position. While GOX is very some substituted cinnamic acids, reducing agents (e.g.,
specic, PYROX is able to catalyze other substrates L-cysteine, glutathione, sulte, SO2 , ascorbic acid),
such as maltose and pentoses (e.g., xylose). As a result, small natural peptides, and combinations thereof
the PYROX oxidation product of glucose is 2-keto- (68). In contrast, during the fermentation of black
glucose and not gluconic acid. At present, this enzyme tea PPO is used to initiate browning by oxidation of
has been puried from several white rot fungi and a polyphenolic substances such as catechins to theaa-
Bacidiomycetous fungus (5557). PYROX has also vins.
been reported to show signicant activity toward D- The quality of tea, based on sensory evaluation of
glucono-1,5-lactone, which is produced by the GOX- color and bitterness, has been correlated with total
catalyzed oxidation of glucose (58). So if PYROX is theaavin content (71). Theaavins, thearubigins, and
combined with GOX, there will be more substrate caffeine are all essential ingredients in high-quality
available for PYROX, thereby prolonging the activity teas. The addition of microbial laccases and/or perox-
of PYROX and enhancing the total amount of hydro- idases to green tea has shown that no higher theaavin
gen peroxide produced (59). The claimed effects in levels can be obtained than with endogenous PPO.
baking are gluten strengthening, reduced dough sticki- Addition of exogenous laccase/peroxidase to black
ness, and increased volume and crumb structure for tea, however, did yield a very signicant increase in
bread (59). the color intensity of the tea (72).
Polyphenol oxidases also play an important role in
coffee processing (69). The activity of PPO in green
IV. APPEARANCE/COLOR coffee beans has been consistently related to the quality
of the coffee beverage. The exact role of PPO in cocoa
Apart from texture, the appearance of food products is beans is less well understood. There are, however, indi-
determined to a large extent by their color. Several cations that it plays a role in browning during the
redox enzymes are known to inuence the color of curing of the cocoa beans (73).
foodstuffs. The most important ones will be discussed In cereals, PPO is responsible for the darkening of
below. the breadcrumb, particularly in whole-grain and rye

breads. The rate of browning is greatest in sourdough enzyme in bovine milk, where it is found in concentra-
baking. Excessive discoloration can be prevented by tions around 30 mg/L. It is a glycoprotein with a single,
the addition of sodium metabisulte or ascorbic acid covalently bound heme group (78). Lactoperoxidase
(41). Exogenous PPO is also reported to have a posi- requires hydrogen peroxide and thiocyanate (SCN )
tive inuence on the rheological properties of bread for antibacterial activity. All three components are
(74). referred to as the LP system. The growth inhibitory
PPO can also be used for the in vitro production of effect of the LP system is mediated by the generation
colors. For example, Pruidze (75) reported the produc- of SCN oxidation products, mainly hypothiocyanate
tion of a whole range of natural colors by treating ions (OSCN ), which attack sulfhydryl groups of vital
waste streams of beet and tea with PPO. metabolic enzymes of the microorganisms.
Furthermore, it was claimed that safower pigments Mammalian cells are not affected by the LP system.
become darker red when safower petals were sprayed Only 1020 ppm of lactoperoxidase is required for an
with a dilute laccase solution (76). effective system. The cofactor requirements are also
The role of endogenous POX in (dis)coloration is very low: 1025 ppm for thiocyanate and 1015 ppm
not fully understood. It seems that the oxidative role of for H2 O2 . Without the enzyme H2 O2 is also bacterici-
peroxidases is useful in assisting PPOs in oxidizing the dal, but at much higher concentrations: 300900 ppm
polyphenols and ultimately contributing toward the (79). Therefore LPO is often applied in combination
color and avor of tea. The same seems to be true with H2 O2 -generating enzymes. From a toxicological
for coffee and cocoa. point of view, the levels of the cofactors in the LP
system as well as the oxidation products are reported
C. Ascorbic Acid Oxidases to be harmless.
The envisaged applications of the LP system are
Ascorbic acid oxidase (E.C.1.10.3.3; see Ref. 77 for food products (e.g., liquid milk, cheese, meat, sh
detailed information) plays a role in beverages such and poultry products, and functional foods), feed
as lemon and grapefruit juices, where it is responsible and veterinarian products (e.g., milk replacers, and
for the initiation of browning and loss of vitamin C antidiarrhea and antimastitis preparations), and dental
activity during storage (77). The extent of browning products (7880). Typically, applications have been
can be minimized by steam blanching or by the exclu- claimed for Lactobacillus fermented milk products
sion of oxygen. The rate of ascorbic acid oxidation (81), pickled foods (82), sh products (LPO in combi-
increases markedly in the presence of metallic ions, nation with GOX; 83), and white mold cheeses such as
especially copper and iron. Hence, food processed in Camembert (84). Also of interest is the effect of LPO
tin cans and processing equipment should be copper on yogurt. By adding LPO to yogurt the excessive acid
free. While the loss of ascorbic acid cannot be pre- production of lactic acid bacteria in the yogurt is sup-
vented completely, it can be reduced to a minimal pressed (85). These applications are becoming within
level during processing. reach now that it is possible to isolate lactoperoxidase
from milk with high purity on an industrial scale (79).
Currently, LPO is already commercially available at
V. SHELF LIFE relatively low costs.
The application of redox enzymes for improving the B. Xanthine Oxidases

shelf life of food products includes mainly enzymes
which are capable of removing oxygen or reactive oxy- Xanthine oxidase (XO, EC 1.2.3.2; see Chapters 19
gen species (e.g., H2 O2 and superoxide anion), as well and 42 for detailed information) is widely distributed
as enzymes that are able to generate antimicrobial in animals, plants, and microorganisms. It catalyzes
agents. In this way the stability of foods can be the oxidation of hypoxanthine to xanthine and
increased signicantly with respect to taste, appear- xanthine into uric acid. In addition XO is able to oxi-
ance, and microbial spoilage. dize a wide range of purines, aldehydes, and pteridines
with concomitant reduction of O2 to H2 O2 . Under
A. Lactoperoxidases certain conditions XO also produces the highly reac-
tive superoxide anion. Bovine milk is very rich in XO
Lactoperoxidase (LPO; see Ref. 78 and Chapters 19 ( 35 mg=L; 86). XO has been implicated in the oxida-
and 20 for detailed information) is the most prominent tive deterioration of milk and dairy products via the

production of superoxide anion during oxidation of its VI. NUTRITIONAL VALUE
substrates (87). However, Bruder et al. (88) found no
evidence to support a role for native bovine milk XO in Redox enzymes can have both pro- as well as antinu-
lipid oxidation. The results of Weihrauch (89) seem to tritional effects. For example, lipoxygenase, apart from
indicate that in the presence of purines, XO activity all its other functions in food products (see Table 1), is
generates H2 O2 for the lactoperoxidase system in involved in the oxidative destruction of liposoluble
milk, making it a bactericidal or bacteriostatic agent vitamins (provitamin A) and essential fatty acids (3).
in milk (23). Likewise, ascorbic acid oxidase has an antinutritional
effect since it oxidizes vitamin C (97).
Increasingly, redox enzymes are being claimed to
C. Superoxide Dismutases and Catalases improve the healthiness of especially beverages.
For example, peroxidase and catalase have been
Superoxide dismutase (SOD; EC 1.15.1.1) and catalase claimed for the removal of unhealthy hydrogen per-
(EC 1.11.1.6; see Chapters 27 and 38 for detailed infor- oxide in coffee and tea (98). Other examples include
mation) are present in milk and are able to remove the use of cholesterol oxidase, epicholesterol dehydro-
reactive oxygen species generated by other (bio)chem- genase and cholesterol reductase to lower the choles-
ical processes (90). SOD catalyzes the reduction of terol level in foods such as meat, sh, milk, and egg
superoxide anion, as produced, for example, by XO, products (99101).
to H2 O2 and O2 . In turn, catalase is able to neutralize
H2 O2 to water and oxygen. A low level of exogenous
SOD, coupled with catalase, is a very effective antiox- VII. CONCLUDING REMARKS
idant in dairy products (91).
Recently, SOD has been shown to protect beer Compared to the usage of hydrolytic enzymes such as
against free radical damage (92). Obviously, the proteases, carbohydrates, and lipases, the application
commercial feasibility of SOD as an antioxidant of oxidoreductases as a tool to improve the processa-
depends on cost, particularly compared to chemical bility and quality of food products is still in its infancy.
antioxidants, if permitted. As far as is known, SOD is Like hydrolytic enzymes, oxidoreductases are capable
not used commercially as an antioxidant in food of tailoring the taste, texture, appearance, shelf life,
systems. nutritional value, and process tolerance of foods and
Catalase is used for the cold-sterilization of milk in the properties of all major food constituents (e.g., pro-
regions lacking refrigeration and could in principle be teins, carbohydrates, oils, fats, avors; see Table 1). In
applied in developed countries for the treatment of both cases, they can exert a positive as well as a nega-
cheese milk (90). Good sources for catalase are beef, tive effect on the food quality parameters, which can
liver, Aspergillus niger, and sweet potato. There is be reduced or eliminated by careful selection of the raw
also interest in using immobilized catalase reactors materials, by properly controlling the process condi-
for milk pasteurization or for glucose oxidasecata- tion, by the addition of counteracting ingredients,
lase reactions (93). Besides the removal of reactive and by genetic tools. Likewise, hydrolytic and redox-
oxygen species by SOD and catalase, other enzymes active enzymes often have more than one effect. A
can be applied to remove the less but still reactive typical redox example is the multifunctional enzyme
oxygen. Typical examples include glucose oxidase, lipoxygenase, which can be applied to inuence either
D-amino acid oxidase, alcohol oxidase, and ascorbic taste, texture, appearance, and/or nutritional value of
acid oxidase. The disadvantage of these oxidases is food products. The key difference resides in the types
that they produce H2 O2 , which by itself is a powerful of reactions they catalyze. As a result, redox enzymes
oxidant. Catalase can be added to remove H2 O2 , but can be combined with hydrolytic enzymes in a variety
then oxygen is produced again. More recently, poly- of food products to create improved or even new func-
phenol oxidases such as laccase have been proposed tionalities, which cannot be realized by either one of
as a deoxygenation tool for beer (94) and juices (95, them.
96). These enzymes have the advantage that they do At present, major bottlenecks for the large-scale
not produce H2 O2 , and thus the combination with application of oxidoreductases include their limited
catalase is not necessary. As a result PPOs allow a availability, their safety status (not GRAS), their sta-
more efcient oxygen removal. bility, and the fact that they often initiate radical reac-

tions, which propagate via redox-active food constitu- 6. TE Mathiasen. Improving the storage stability of beer
ents such as metal ions and quinones and hence are by fermenting wort into beer and adding a laccase to
difcult to control. In addition, many oxidoreductases the fermented beer. Patent WO9521240.
require expensive cofactors for catalysis. Despite these 7. RL Antrim, JB Taylor. Deodorization of a water-in-
oil emulsion containing sh oil using pure alcohol and
disadvantages over hydrolytic enzymes, certain oxidor-
aldehyde oxidases/dehydrogenase in combinations
eductases are increasingly nding a home in food pro-
with a cofactor. Patent US4961939, 1990.
cessing. The rst generation (see Table 1) includes 8. Y Nomura, N Shiomi. Production of a powder of
enzymes which are cofactor independent and thrive acidic acid bacteria that produce aldehyde oxidase.
on oxygen (e.g., oxidases) and hydrogen peroxide Patent JP10108671, 1998.
(e.g., peroxidases). Owing to advances in recombinant 9. IM Whitehead, BL Muller, C Dean. Industrial use of
DNA technology the low-cost, large-scale production soybean lipoxygenase for the production of natural
of these enzymes in GRAS host microorganisms is green note avour compounds. Cereal Foods World
becoming within reach, and the tools are ready to tai- 40:193197, 1995.
lor redox enzymes to specic needs by site-directed 10. C Laane, I Rietjens, H Haaker, W van Berkel.
mutagenesis and directed evolution. Also feasible will Generation of taste through (redox) biocatalysis. In:
KAD Swift, ed. Flavours and Fragrances.
be the in planta overexpression of desired redox
Proceedings of the 1997 RSC/SCI International
enzymes in food raw materials and the deletion of
Conference on Flavours and Fragrances, Royal
undesirable redox traits. The usage of cofactor-depen- Society of Chemistry, Special Publication No. 214,
dent oxidative enzymes seems tentatively to be con- 1197, pp 137152.
ned to whole-cell systems, in which the cofactor can 11. JC Villettaz, D Dubourdieu. Enzymes in winemaking.
be regenerated, and hence to the in vitro as well as the In: PE Fox, ed. Food Enzymology, Vol 1. London:
in situ production of functional food ingredients such Elsevier Science Publishers, 1991, pp 427454.
as avors. The same seems to be true for reductases, 12. PSJ Cheetham. The use of biotransformations for the
since most if not all require reducing equivalents in the production of avours and fragrances. Tibtech
form of a small protein, hydrogen, and/or NAD(P)H. 11:478488, 1993.
Clearly, the usage of oxidoreductases in food pro- 13. P Brunerie (Pernod Ricard). Synthesis of cis-3-hexenol
from unsaturated aliphatic acidusing combined
cessing is emerging. The benets and limitations are
action of natural enzyme system and yeast. Patent
becoming known, which allows a promising and EP481147, 1991.
focused search for new opportunities. 14. A Muheim, A Hausler, B Schilling, K Lerch. The
impact of recombinant DNA-technology on the a-
vour and fragrance industry. In: KAD Swift, ed.
Flavours and Fragrances. Proceedings of the 1997
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14
Transgenic Plants for Production of Enzymes
Anne S. Ponstein
Syngenta Mogen, Leiden, The Netherlands
Rob F. Beudeker
Jan Pen
PlantZyme, Leiden, The Netherlands
I. INTRODUCTION 38), and biodegradable plastics (2) successfully pro-

duced in plants using this molecular farming
In terms of cost-effectiveness, plants can compete with approach.
any other production system. All that is required is a Plants are gradually earning their place among
suitable plant, sunlight, mineral salts from the soil or other systems for production of proteins and peptides.
fertilizers, and water. The overall costs of production Although research focused initially on the production
in plants is largely determined by the existing and very of high-value pharmaceutical (poly)peptides (2), plants
efcient infrastructure and facilities in agriculture for are equally suited as a competitive source of nonphar-
growing, harvesting, storing, and processing of crops. maceutical enzymes (238). The activity and interest in
Furthermore, traditional breeding has resulted in plant this area are clearly increasing as demonstrated by
varieties optimized for a high yield of harvestable pro- existing collaborations between small plant biotechnol-
duce (1). Crops grown in the eld can yield bulk quan- ogy boutique and established enzyme or agrochemical
tities of biomass. Millions of tons of carbohydrates, companies, such as between Prodigene and Genencor,
lipids, and proteins for the food, feed, or processing Sembiosys and Dow Agrosciences, Zeneca MOGEN
industries are produced annually from the harvested (formerly MOGEN International) and DSM Food
biomass. Specialties (formerly Gist-brocades), Agracetus (now
Biotechnology enables us to produce compounds part of Monsanto), and FinnFeeds (part of Cultor;
of commercial interest in domesticated crops that Cultor was recently acquired by Danisco).
were previously available only from exotic plant spe- This chapter will summarize the method to generate
cies, from other organisms or in limited quantities. transgenic plants, give an overview on what has been
Transgenic plants are an attractive and cost-effective achieved in this area, compare plants with other meth-
alternative for the production of these biomolecules, ods of enzyme production, describe unique application
as demonstrated by the steadily increasing number of methods for enzymes produced in plants, and nally
carbohydrates (2), fatty acids (2), high-value pharma- present an illustrative example of an enzyme, phytase,
ceutical (poly)peptides (24), industrial enzymes (2 applied in animal feed.

II. GENERATION OF TRANSGENIC like the use of promoters, enhancers, leader sequences,
PLANTS EXPRESSING ENZYMES optimizations in codon usage, removal of mRNA-
destabilizing sequences, etc. Other methods to increase
A. Transformation and Regeneration of Plants
the levels of heterologous proteins in plants can be
found in the use of, e.g., integration-independent
In general, expression of (poly)peptides relies on stable
expression, enhancement of correct folding by coex-
integration of the encoding genes in the plant genome.
pression of disulde isomerases or chaperone proteins
An advantage of this approach is that identical off-
and production of polyproteins for stabilization of
spring can be obtained by self-fertilization of the trans-
especially small peptides. Reduction of protein degra-
genic crops and consequently the stable inheritance of
dation by, e.g., expression in tissues, as seeds, where
the trait. An alternative is production by transient
the level of protease inhibitors is high, by expression in
expression in plants via genetically engineered viruses
tissues where the activity of the degrading enzymes is
as CaMV (39), TMV (4042), or CPMV (4345).
low as in seeds owing to the low water content, by
Stable integration of genes generally consists of
targeting the enzyme to the ER or by avoiding the
three phases: (a) introduction of foreign DNA into
presence of susceptible protease-cleavage sites in the
tissue amenable to transformation by methods as
enzyme will also result in an increase of the expressed
PEG-mediated transformation, particle bombardment,
enzyme.
Whiskers, and Agrobacterium tumefaciens-mediated
A unique aspect of expression in higher eukaryotes
transformation; (b) selection of transformed material
is the choice in the species, variety, and the location
in the nontransgenic background by either selecting for
and timing for expression. Firstly, there is a choice in
tolerance to antibiotics, herbicides or another toxic
the plant species to be used for expression. A choice no
chemical and/or by screening for expression of visual
longer severely limited by transformation technology
markers like luciferase, anthocyanin and GFP; (c)
(see above). If the protein content of a crop (tissue)
regeneration of shoots and roots after the successful
would correlate with the accumulation of the transgene
selection of transgenic cells. This last phase may be
protein, crops with higher protein contents would be
quite cumbersome as cells amenable to transformation
more cost-effective for production. However, as far as
(competent cells) may not have the ability to regener-
we are aware, no proof is available that this is indeed
ate.
the case. Moreover, the argument may be turned
Transformation of plants has developed over the
aroundi.e., crops that are already accumulating
last decades into a routine procedure for many crop
high levels of endogenous protein may have little capa-
species, including tobacco, tomato, potato, canola,
city left for the accumulation of heterologous protein.
soybean, alfalfa, sugar beet, lettuce, carrot, cotton,
Secondly, there may even appear to be differences
apple, rubber, rice, corn, wheat, and banana.
among varieties of one and the same species. It is
Preference for Agrobacterium tumefaciensmediated
well known that different germplasms differ in agro-
transformation exists as this generally leads to precise
nomic characteristics as yield, disease resistance, etc. It
integration of only a limited number of copies of the
would therefore not be surprising if they would also
gene of interest into the plant genome. However, cer-
differ in their ability to accumulate heterologous pro-
eals have appeared very recalcitrant to this method.
teins. Thirdly, the enzyme can be expressed in various
Hence, the extensive use in the past of particle bom-
organs or tissues using appropriate promoters.
bardment. With the recent development of a commer-
Fourthly, expression in the plant, tissues, or organs
cial transformation procedure for rice and the initial
can be limited to certain periods of development
successes with the method in corn, wheat, and barley, it
using promoters active only during these stages.
is likely that Agrobacterium tumefaciens will become
Finally, the enzyme can be targeted to specic cellular
the general vehicle for DNA delivery into plant species.
compartments as the vacuoles, the protein bodies (in
seed), the chloroplasts, the mitochondria, and the ER
B. Expression of Enzymes in Plants or to the apoplast using appropriate targeting signals
described in the literature (47).
Even when using an identical expression cassette, the Expression by integration in the plastid genome is
expression levels obtained in plants will differ from another way to express enzymes. This can lead to dra-
gene to gene (7, 28, 46), just as in other production matic increases in expression levels as demonstrated by
systems. However, some general principles apply to the expression of -glucuronidase (GUS) that accumu-
achieve high-level expression of proteins in plants, lated to 2.530% of total soluble protein (20).

Combination of all these possibilities gives an almost D. Exploitation of Plant Characteristics
unlimited choice. As these cellular compartments differ
with regard to pH, ionic strength, and other environ- Various production systems have been developed that
mental factors, these choices in fact allow the choice are based on specic plant characteristics, on the use of
for a location within a plant species or a variety where plant regulatory DNA sequences, or on the exploita-
active enzyme can be accumulated to the highest pos- tion of existing plant processing systems.
sible level. Prodigene (College Station, TX) used a ubiquitin
No study describing the application of a range of promoter that preferentially directs expression of pro-
these possibilities for enhancing the expression level teins to the germ portion of corn seeds. The crude
has been published; there are still ample opportunities protein is present in the waste fraction of the corn
for optimization. Step increases in expression of pro- wet-milling process, and thus essentially produced
teins in plants can be expected, as was the case with free of charge in that process. The only costs involved
microbial systems in their early stages of use. in production are downstream costs for extraction and
Competing systems are much more mature and purication of the protein. A comparison of the costs
improvements in expression levels will therefore be for production in the germ versus the whole seed is
much more modest for the latter expression system. given by Howard (50). Similarly, starch or oil crops
The fact that in its present state plants can already may be interesting as hosts for production, because
be competitive shows that they should have a bright the enzymes may be produced as byproducts in addi-
future as hosts for enzyme production. tion to the primary product (starch or oil), thus redu-
cing the production costs.
A very ingenious way to reduce these downstream
C. Downstream Processing costs was found in the use of plant seed oil bodies as
carriers for proteins and peptides (48). Nonplant
If purication is mandatory (which is not always the (poly)peptides were fused to the structural oil body
case, as shown below), rapid and economical down- protein oleosin and expressed in Brassica napus. The
stream processing of the recombinant protein is essen- lipophilic nature of oleosins forms the basis for a
tial. The benets of the plant-based production should highly efcient purication system for (poly)peptides.
not be offset by increased downstream processing The feasibility of this approach has been demonstrated
costs. Any decrease in these costs will increase the com- with -glucuronidase (GUS; 19, 48) and as a rst com-
petitive advantage of plants. The economic feasibility mercial example the expression and purication of the
of protein purication from plant biomass has not thrombin inhibitor hirudin (51). Alternatively, this sys-
been investigated in any detail, and if at all, mainly tem may be used to immobilize industrial enzymes as
on a lab scale, except for Austin et al. (6), who focus oleosin fusion proteins on the surface of oil bodies
on extraction and purication of industrial enzymes (19).
from alfalfa; by van Rooijen et al. (48; see below), Expression during germination of seeds, using ger-
Wilcox et al. (27), who puried bovine lysozyme mination-specic promoters, has the advantage that a
from transgenic tobacco to 93% homogeneity in an well-established process, available equipment, and fac-
easily scalable process; and by Kusnadi et al. (49), tories of malt production for, e.g., the brewing industry
who described purication of GUS and avidin from can be used. An additional regulatory advantage is
transgenic corn. Downstream processing from plant obtained, because the actual expression takes place in
biomass is generally assumed to be difcult and expen- a contained environment; i.e., seeds are allowed to
sive. One reason for this assumption is the low ratio of germinate in a malting factory (52, 53).
recombinant protein to total biomass. Concentration Using wound-inducible HMG2-derived promoters,
and purication of the protein in the rst step would expression can be effected postharvest by mechanical
reduce this assumed disadvantage considerably. As wounding of the leaves. An advantage is that growth
only few studies have been published, no nal verdict of the plants is separated from the expression of the
is available regarding the cost of downstream proces- protein, which allows expression of proteins that
sing. With step increases in expression level still feasi- would normally have a detrimental effect on the
ble (see above), the economics of production will plant during growth or development. As the proteins
improve. In turn this will make it easier to use plants are expressed in a contained environment postharvest,
for production, even in cases where costly purication this approach could have regulatory advantages as well
is mandatory. (54).

An elegant way to produce and harvest proteins is (14). A cellulase from Thermomonospora fusca was
described by Boffey et al. (55). Proteins are expressed expressed in alfalfa, tobacco, wheat, and maize (10,
in the easily harvestable latex fraction of the rubber 11). In one of these studies (11), the gene was put
tree, constituting a continuous source of the protein. under the control of a chemically inducible promoter
The relatively simple composition of the latex would to prevent detrimental effects of the cellulase.
allow easy purication of the protein from the tapped Production of manganese-dependent lignin peroxidase
latex. A disadvantage may be the long generation time in alfalfa severely affected plant growth and develop-
and consequently the long time to develop this into a ment. Affected plants showed yellowing foliage.
commercially operating system. However, they survived and set seed. In eld trials
Extracellular expression of proteins in the roots of the dry matter content was reduced, as was the plant
hydroponically grown tobacco plants, termed rhizose- height. The enzyme was most likely directed to the ER
cretion, is yet another way to optimize expression, as (6, 8).
well as to facilitate purication (37). GUS (-glucuronidase) is used in molecular biolo-
gical experiments as a reporter for visualizing gene
expression in plants. GUS, produced in transgenic
III. ENZYMES PRODUCED IN PLANTS corn seed and rapeseed (1820), is the rst enzyme
produced in plants that is commercially available
A substantial number of enzymes have meanwhile been from Sigma. The enzyme is produced by Prodigene
produced in plants (see Table 1) (238). Expression of (College Station, TX). Sigma is also selling another
an enzyme in the vicinity of its potential substrate does protein, avidin, produced by Prodigene in corn (57).
not necessarily have a negative effect on the agronomic Alcohol and aldehyde dehydrogenases produced in
characteristics of the plant. Endogenous cell wall corn (5) can nd application in prevention of alcohol
degrading enzymes like glucanases, polygalacturo- toxicity when taken before social drinking. They
nases, and pectin esterases are present at the same should be formulated with NAD to ascertain activity
location as their substrates without a detrimental effect in a pill, paste, or foodstuff.
on the plant. Also, extracellular expression of a vacuo- Although cyclodextrin glycosyl transferase
lar glucanasei.e., an enzyme that normally does not (CGTase) was produced in potato tubers for the in
have access to the glucan substratedid not result in planta production of cyclodextrins (13), these trans-
abberations in growth or morphology (56). Xylanase genic plants could nd application as a source of the
can be expressed in tobacco or Pisum sativum without enzyme as well, provided the enzyme levels can be
an effect on plant phenotype (9, 16, 35). In one pub- increased considerably. Hen egg white and bovine
lication (35), the authors suggest that the high tem- and T-4 bacteriophage lysozymes have been expressed
perature optimum of the enzyme prevents substantial in tobacco (2427). This research was partially driven
degradation at ambient temperatures. As the enzyme by the desire to create plants resistant to bacterial
exhibits considerable residual activity at this tempera- pathogens. However, lysozymes produced in this way
ture, a more likely explanation may be that, for can nd application as preservative in food, feed, cos-
unknown reasons, the apoplastically expressed enzyme metics, and agriculture.
does not, or does only to a very limited degree, degrade Other enzymes expressed in plants include lipase
the xylans in the plant cell wall. (22, 23), -galactosidase (21), -amylase (3, 69), -
Active (1-3,1-4)glucanases can be expressed in amylase (38), and phytase (2834 this chapter).
tobacco without phenotypic alterations despite their
apoplastic location (16). A thermostable (1-3,1-4)glu-
canase, consisting of parts from two Bacillus spp. IV. COMPARISON OF HOSTS FOR
expressed in barley aleurone protoplasts and in trans- ENZYME PRODUCTION
genic barley under control of the germination-specic
high-pI barley -amylase promoter yielded fertile Bacterial and fungal cells selected for high-level expres-
plants (17). In contrast, no viable transgenic tobacco sion have traditionally been used for production of
plants could be obtained after transformation with a industrial enzymes (58). Extraction from these nonre-
endo-(1-4)glucanase gene construct. This was most combinant sources will, for some enzymes, remain the
likely due to degradation of cell wall -glucans, preferred method of production. For the production of
because transient expression in tobacco protoplasts commercially interesting proteins, with the use of
showed that cell wall synthesis was never resumed recombinant DNA techniques, various host organisms

Table 1 Plants as Bioreactors for the Production of Enzymes
Enzyme Origin of gene(s) Application Plant species Ref
Alcohol dehydrogenase Human Prevention alcohol toxicity Corn 5

Aldehyde dehydrogenase Human Prevention alcohol toxicity Corn 5
Alpha-amylase Bacillus licheniformis Liquefaction of starch Tobacco, alfalfa, Vicia narbonensis, 3, 69
Pisum sativum
Beta-amylase Barley Brewing Barley 38
Cellulase Thermomonospora fusca Ethanol production, paper and Alfalfa, tobacco, corn, wheat 10, 11
pulp
Chymosin Calf Dairy industry Tobacco, potato 12
Cyclodextrin glycosyltransferase Klebsiella pneumoniae Production cyclodextrins Potato 13
(CGTase)
Glucanase Trichodema reesei, Hybrid from Brewery, feed Barley cells 1417
two Bacillus spp., Ruminococcus
avefaciens
GUS (-glucuronidase) Escherichia coli Molecular biology tool Corn, canola 1820
Lactase (-galactosidase) Escherichia coli Lactose intolerance Maize, sunower 21
Lipase Dog gastric preduodenal, Food, medical, fatty acid Tobacco, corn, canola, tomato 22, 23
Rhizopus niveus production
Lysozyme Hen egg white, bovine, Food preservation Tobacco, potato 2427
bacteriophage T4
Manganese-dependent lignin Phanerochaete chrysosporium Bleaching and pulping of paper Alfalfa 6, 8
peroxidase
Phytase Aspergillus niger Feed Alfalfa, tobacco, canola, soybean, 2834
soybean cell suspension cultures,
wheat
Xylanase Clostridium thermocellum, Feed, brewing, paper and pulp, Tobacco, Pisum sativum 9, 16, 3537
Cryptococcus albidus, bakery
Ruminococcus avefaciens

are currently used: bacteria (in particular Escherichia from bacterial and fungal origins nd wide application
coli and Bacillus subtilis), lamentous fungi, yeast, in e.g. the starch processing and alcohol industry for
mammalian cells (in particular Chinese hamster liquefaction, in the brewing industry for production of
ovary [CHO] cells), insect cells, transgenic animals low-calorie beer, in bakery for increasing bread
and transgenic plants, as well as a number of other volume, in the juice and wine industries for clarica-
organisms that are less important. The rst tier of bio- tion and in the detergent industry for removal of starch
technology companies have mainly used lamentous stains (6164). The -amylase from B. licheniformis is
fungi, yeast, and bacteria for production of enzymes. the most commonly used enzyme in starch liquefac-
Well-known industrial problems in developing sys- tion, because of its extreme heat stability and its activ-
tems for the production of heterologous proteins ity over a wide pH range (61, 65).
included instability and insolubility of the product, dis-
appointing production rates, instability of selected cell A. Production and Purication from Plants
lines, difculties in scaling up, purication of the (Fig. 1a)
recombinant product, and complicated registration
procedures (59). A specic problem for industrial Bacillus licheniformis -amylase was expressed extra-
enzymes is the production costs, because of the small cellularly in tobacco at a level of maximally 0.5% of
margins on these products. Economic factors inuen- soluble protein in leaves (7, 66). The properties of the
cing the choice of production system are the rate of enzyme as produced in tobacco were very similar to the
biomass production, equipment costs, medium compo- bacterial one. However, the protein is, in obvious con-
sition and costs, processes for protein recovery and trast with the bacterial protein, glycosylated. The
purication, product yields, and the potential hazard enzyme produced in tobacco was active and exhibited
of contamination. A lot of development work is driven extreme thermostability, similar to the Bacillus
by the intended commercial application and by regula- enzyme. No apparent effect of the presence of the
tions set by the FDA and other organizations. enzyme on plant phenotype was observed.
A suitable host for production is selected based on Purication from tobacco has not yet been carried
its ability to fulll all the technical and economic out for bacterial -amylase.
requirements set by the protein of interest and its
application. The production of proteins, by whatever B. Process-Dependent Conversion (Fig. 1b)
system, remains an empirical process, making it impos-
sible to predict the expression level. Such predictability Enzymes produced in crops can be expressed at a
may come with an increase in understanding of the sequestered location in the crop that also harbors its
molecular biology, biochemistry, and physiology of substrate. Alternatively, the enzyme may be expressed
the expression systems and of the expressed proteins. at the same location as the substrate, but in an active
Because expression in each organism is gene depen- form. The conversion thus becomes process dependent.
dent, and this dependency differs from organism to During processing of the crop, enzyme and substrate
organism, some proteins will express well in one system are brought into contact (or the enzyme is activated),
and not well in another. This argues strongly for the resulting in conversion of the substrate into product. In
availability of a multitude of production systems, each this approach, the plant becomes equipped with the
having its own (dis)advantages. Plants will earn their enzyme having optimal characteristics for conversion
place among these systems. A comparison of different of its endogenous substrate. In planta conversion of
hosts for production of enzymes is given in Table 2 the substrate into the desired product (Fig. 1d), by
(60). expression of active enzyme at the same location as
the substrate, will have similar advantages as process-
dependent conversion, but will not be suitable if the
V. UNIQUE APPLICATION METHODS enzymatic activity has a negative effect on the plants
FOR ENZYMES PRODUCED IN phenotype or its agronomic characteristics. The feasi-
PLANTS bility of this concept is exemplied by extracellular
expression of -amylase. Extracellular expression of
The feasibility of different methods for application of -amylase does not have an effect on endogenous
enzymes produced in plants as described below can be starch, as is expected from the separated locations of
nicely demonstrated with -amylase from Bacillus enzyme and substrate. Moreover, the transgenic plants
licheniformis expressed in tobacco (7). Alpha-amylases do not show any abberations in their phenotype.

Table 2 Qualitative Comparison of Hosts for Production of Industrial Enzymes
Transgenic Transgenic
plants animals CHO cells Insect cells Fungi Yeast Bacteria
Research and development

Development time Intermediate Long Intermediate Intermediate Short Short Short
Feasibility improvements (state of the art) Very high High Limited Intermediate Limited Limited Limited
Process characteristics
Reproducibility production
Downstream processing costs ?
Logistics
Crude nonformulated product: Seed Milk Medium Medium Medium Medium Medium
Stability
Storage period Indenite Limited Limited Limited Limited Limited Limited
Upscaling time Fast Fast Intermediate Intermediate Intermediate Intermediate Intermediate
Upscaling cost (capital costs) Low Intermediate High High High High High
Production volume Unlimited Unlimited Limited Limited Limited Limited Limited
Product characteristicsAuthenticity
Glycosylation Yes Yes Yes Yes Yes Yes No
High-mannose Yes Yes Yes Yes Yes Yes No
Complex Yes Yes Yes Yes/No No No No
Human No Yes Yes No No No No
Other modications
Folding capabilities
Safety, regulations, and public acceptance
Food use
Containment level
Presence mammalian pathogens
Public acceptance level
Source: Ref. 60.

Key: : absent; : very low or doubtful; : low; : intermediate; : high

Figure 1 Application methods of industrial enzymes produced in transgenic crops. (a) Production and purication: The enzyme
is puried from the plant, formulated and subsequently applied in the industrial process. (b) Process-dependent conversion;
Expression of the enzyme in the crop sequestered from its substrate. Conversion of the substrate occurs during processing. (c)
Seed-formulated enzymes: The transgenic seeds are directly, without any purication of the enzyme, applied in the industrial
process. (d) In planta conversion: Expression of the enzyme in planta at the same location as its substrate results in conversion of
the substrate and consequently accumulation of product in planta.
Homogenization of transgenic leaf material, followed by in vitro addition of the enzyme. In some cases as
by incubation at 95 C, resulted in conversion of endo- wood pulping, in vitro addition has limited success
genous starch by the transgene product (Table 3). In because the enzymes do not have sufcient access to
starch potatoes, the -amylase levels as obtained in the substrate. Finally, expression in the crop guaran-
tobacco (7, 66) would be sufcient to hydrolyze all tees an optimal dispersion of the enzyme throughout
the starch present in the tuber. Similar applications the biomass; i.e., an optimal homogeneous mixture of
for -amylase and other enzymes may be found in enzyme and substrate is obtained. This is less easily
other industrial processes, like for instance the applica- achieved by in vitro addition of enzymes.
tion of -amylase in the production of bioethanol,
heat-stable -glucanases for the degradation of cell C. Seed-Formulated Enzymes (Fig. 1c)
wall material in the brewing industry, and the produc-
tion of fatty acids in oil plants (23). Another concept for application is the direct use of
In this approach the aim is not producing enzymes engineering plant material in the processing, food, or
in crops per se, but creating either a higher-quality feed industry. The transgenic plant material serves as
input resource or an enabling process through expres- novel formulation for the expressed enzyme. Other
sion of the enzyme inside the plant. This application plant material can be used, depending on the process,
has unique advantages. Firstly, enzyme manufacturing but in general seeds may be the plant organ of choice,
costs will be low, because there is no need for purica- because these create a stable environment for long-
tion and formulation of the enzymes. Secondly, it obvi- term storage of the enzymes. The potential of plants
ates the necessity for separate production of enzymes. as production vehicles for enzymes is enormously
Thirdly, expression in the vicinity of the substrate increased by producing the enzyme in seeds and
assures better access to its substrate than is achieved directly applying these in the industrial process.

Table 3 Process-Dependent Conversion of Leaf Starch by Expressed -Amylase
Incubation time (min)
Sample 0 30 60
Control tobacco
Control tobacco 0:02 T.A.U. B. licheniformis -amylase/mg = =
Control tobacco 0:2 T.A.U. B. licheniformis -amylase/mg =
Control tobacco 2 T.A.U. B. licheniformis-amylase/mg
Transgenic tobacco line MOG227.3
Homogenization of transgenic leaf material containing Bacillus licheniformis -amylase, followed by incubation at 95 C for 30 min hydrolyzed
starch present in the leaves. Addition of 2 T.A.U./mg Bacillus licheniformis -amylase to the nontransgenic control leaf homogenate for 30 min or
of 0.2 T.A.U./mg for 60 min had the same effect, while incubation of nontransgenic control leaf homogenate had no visible effect on the
endogenous starch during the time of the experiment. The presence of starch, as demonstrated by I2 =KI-staining is indicated with , intermediate
staining with = and absence with .
Compared with the production followed by purica- This enzymes-in-seed approach is exemplied by the
tion, this concept has additional advantages over tra- direct application of milled transgenic -amylase seeds
ditional production systems. Firstly, the formulation in liquefaction of corn and potato starch (Fig. 2). Seeds
of the enzyme as obtained by genetic engineering of of a transgenic plant line were milled and used without
the crop is convenient; i.e., it does not require an any further purication in the liquefaction of potato
extra step in the manufacturing process, and it guar- and corn starch. From the nature and the quality of the
antees stable and safe storage of the enzyme. No aller- hydrolysis products obtained from corn and potato
genic problems due to the enzymes are to be expected, starch with the transgenic seeds it can be concluded
because these are contained in the seeds. Secondly, the that the enzyme as produced in tobacco is as suited
manufacturing costs will be lower, because purication for liquefaction of starch as the Bacillus licheniformis
and formulation of the enzyme are no longer required. amylase. As shown in Table 5, the dextrose equivalent
This concept is most easily applied in, but certainly not (DE) values, which were calculated from the HPLC
limited to, cases where enzymes are required for appli- patterns, were found to be similar to those obtained
cation in (processed) food or feed. The enzyme can with the commercial preparations and within the com-
then be expressed in the plants and plant organs that mercially acceptable range (DE-12, preferably DE16;
are the natural component in this animal feed and 67). Seeds containing -amylase maintained full
food. The transgenic plant material containing the enzyme stability for at least a year. When seeds are
enzyme is used as delivery vehicle for the protein the only organs used, seed-specic expression is pre-
with the same advantages as described above. ferred, because it will minimize the chance of effects
The transgenic seeds are a novel specialty enzyme on agronomic characteristics.
product to be added in small quantities into the indus-
trial process, provided the expression level is suf-
ciently high (additive route). This creates
exibility, because various enzymes can be engineered Table 4 Comparison of Bulk and Additive Application of
into plants in the same manner and used and combined Enzymes in Seed
according to the needs of the user. The total premium Additive Bulk
for production, transportation, etc., of transgenic plant
material will be lower than in case the transgene is built Expression level High Low
into the commodity crop. Production, transportation, Research costs Medium Low
and marketing can be strictly and simply controlled, Amount of plant material Low High
preventing the mingling of transgenic with nontrans- Importance host Low High
genic seeds. Alternatively, the enzyme can be expressed Agronomic performance Less critical Critical
Development costs Low High
at low levels in all (or a large part of the) seeds used in
IP production ?
the application (bulk route). A comparison between Level of control High Low
these two approaches in various aspects is given in Maintenance added value High Low
Table 4.

Table 5 Dextrose Equivalent (DE) Values Obtained from
Hydrolysis of Corn and Potato Starch
Potato Corn
DE values starch starch
Transgenic tobacco seeds (MOG227.3) 16 13

Nontransgenic tobacco seeds 0 0
Bacillus licheniformis amylase 18 16
Bacillus amyloliquefaciens amylase 15 18
improve digestibility and utilization of nutrients has

become common practice. Examples are industrial
enzymes such as -glucanase and endoxylanase which
are added to diets containing high contents of barley,
triticale, and wheat to improve growth, quality of eggs,
and feed conversion ratio in poultry (68). Favorable
effects of the inclusion of these enzymes in the diets
of pigs have been reported as well. Another example is
the enzyme phytase which is used to optimize phos-
Figure 2 Liquefaction of potato and corn starch. phorus utilization in monogastric animals such as
Oligosaccharide HPLC patterns obtained from hydrolysis pigs, sh, and poultry.
of corn starch by milled transgenic seeds of plant line
MOG227.3 expressing -amylase (left), Bacillus licheniformis
-amylase (middle), or Bacillus amyloliquefaciens -amylase B. Phytase
(right). The degree of polymerization (DP) of glucose is indi-
cated by the numbers. The products obtained with the trans- Phytases (myoinositol hexakisphosphate phosphohy-
genic seeds and with Bacillus licheniformis amylase are drolase, EC 3.1.3.8) are widely distributed in all classes
virtually identical and clearly different from those obtained of organisms. The enzyme catalyzes the conversion of
with B. amyloliquefaciens amylase. The main difference dis- phytate into myoinositol and phosphorus. The sub-
tinguishing the two specicities is the amount of DP5 (indi- strate of this reaction, phytate, is a salt of phytic acid
cated in black). and divalent cations of calcium, magnesium, iron, or
zinc. In plant seeds up to 80% of the total phosphorus
has been reported to be present in the form of phytate
Plants will often still be a competitive source, (69). During the germination process phytate is rapidly
because of the low-cost production of biomass. The converted into myoinositol and phosphorus for growth
novel concepts of seed-formulated enzymes and pro- and development of the seedlings.
cess-dependent conversion will nd application in var- Seeds of crops like wheat, corn, soybean, and barley
ious markets. For process-dependent conversion the are used in large amounts in animal feed. The amount
increased access to the substrate will for some indus- of phosphorus present in these seeds would be more
trial processes create the required competitive advan- than sufcient to meet the animals requirements for
tage. For seed-formulated enzymes, application will optimal growth. However, monogastric animals, such
depend on the compatibility of the seeds with the as pigs and chickens, are only able to use a small por-
industrial process. A rst commercial example of the tion of the phosphorus present in these seeds, because
latter concept is described below. the remaining part is present in the form of phytate.
These animals cannot convert phytate into myoinositol
and inorganic phosphate. Therefore, inorganic feed
VI. AN EXAMPLE: PHYTASEED
phosphate has to be added to feed. As a consequence
A. Feed Enzymes the manure of the animals contains a huge amount of
phosphate, which, released into the environment,
In livestock farming of pigs and poultry, the supple- causes pollution through eutrophication of surface
mentation of animal feed with industrial enzymes to waters. In many countries, especially those with inten-

sive livestock farming, there is a strong tendency to after transfer of the plants to the greenhouse, was 1.7%
reduce the amount of phosphate. For example, in the of total soluble protein in leaves and 1% of total solu-
Netherlands, farmers are taxed based on the amount of ble protein in seeds. In leaves, phytase accumulated
phosphate released into the environment. In during further maturation of the plants up to a max-
Singapore, pig production is banned because of the imum level of 14.4% of soluble protein 7 weeks after
related environmental problem. The amount of man- transfer to the greenhouse, showing the extreme stabi-
ure secreted into the environment is enormous. Even in lity of phytase in the extracellular space of leaves (33).
as small a country as the Netherlands, 215 million kg The molecular mass of tobacco-derived phytase
of phosphate (P2 O5 ) from animal manure is added to from leaves was 70 kDa and from seeds 68 kDa,
the soil per year. compared to 80 kDa for the Aspergillus enzyme. The
Application of the enzyme phytase will optimize difference in molecular weight between phytase
phosphorus utilization by pigs and poultry (7074). expressed in seeds and leaves and between plant-
Feed phosphate can be substituted by the enzyme phy- derived and Aspergillus phytase was found to be caused
tase, thus reducing the environmental problems caused by differences in glycosylation, which was not unex-
by phosphate considerably. pected because a total of 10 potential asparagine-
The phytase gene has been cloned successfully from linked sites are present in the primary structure and
A. niger var. van Tieghem (75, 76). The enzyme is the Aspergillus niger enzyme is known to be heavily
produced on an industrial scale by means of fermenta- glycosylated (75). Deglycosylation experiments showed
tion of A. niger. The phytase gene is overexpressed by that both leaf- and seed-derived phytase contain high-
placing the gene under control of the A. niger amylo- mannose, as well as complex-type, carbohydrate
glucosidase promoter. Phytase is secreted as a large chains. Tissue-dependent differential glycosylation of
percentage of total protein by this lamentous fungus proteins, as found here for seeds and leaves, has been
during growth in a fermenter. After fermentation, the described before (79). Despite the differences in glyco-
enzyme is separated from the broth through a series of sylation, the specic activity of puried leaf-derived
ltration steps. The nal ultraltrate is either stabilized phytase was found to be identical with that of the
and sold as a liquid product or dried and formulated to puried Aspergillus niger enzyme.
a free-owing product. The unique pH optima at 2 and
5.5 allow application of the enzyme in feed for mono- D. PhytaSeed1
gastric animals because of the optimal compatibility
with the environment in the gastrointestinal tract of For the development of a commercially viable product,
these animals and the crop of poultry. The microbial the fact that plants and plant organs are natural com-
phytase from Aspergillus niger is marketed by the alli- ponents of animal feed was fully exploited. PhytaSeed,
ance of DSM Food Specialties (former Gist-brocades) the enzyme formulated by genetic engineering in
and BASF under the brand name Natuphos for use in canola seed, is used directly without any other post-
animal feed. harvest treatment than milling. In other words, the
seeds are used as the packaging material and the deliv-
C. Expression of Phytase in Tobacco ery vehicle for the protein.
Aspergillus niger phytase was expressed extracellu-
By expressing the phytase gene in plants instead of in larly under control of the seed-specic cruciferin pro-
A. niger, we are substituting an existing production moter in canola (Brassica napus cv. Westar) seeds.
process by a novel and innovative way of producing Canola is commonly used in animal feed, albeit in
industrial enzymes. To demonstrate this concept, low amounts. Therefore, transgenic phytase-contain-
tobacco was transformed with a gene construct (28), ing canola seeds can be simply incorporated.
encoding phytase from Aspergillus niger (75). The con- Transformation of canola is simple and routine, so
struct contains a DNA sequence encoding the tobacco many independent transgenic lines can be obtained.
PR-S signal peptide that will give export of the enzyme As PhytaSeed, canola seeds containing phytase, is envi-
to the extracellular space (77). The cauliower mosaic sioned to be marketed as an enzyme product, and since
virus (CaMV) 35S promoter (78) was used that gives expression levels are high (see below), there is no need
expression of phytase in all parts of the plants. to incorporate the enzyme in seeds of crops that are
A total of 72 independent transgenic tobacco plants major constituents of animal feed like soybean, corn,
were analyzed for phytase expression in leaves and wheat, or barley. Small amounts of canola seed con-
seeds. The highest expression level, measured 3 weeks taining sufcient enzyme for the conversion of the phy-

tate present in the feed mixture will be added. The stored seeds from the previous year. Packaging of the
nutritional value of the seeds will be a bonus to the enzyme in seeds also makes it more resistant to ther-
enzyme formulation. momechanical processing, as for example during feed
A total of 95 independent transgenic canola lines pelleting (83). The effect of addition of milled trans-
were generated and grown in the greenhouse, and genic seeds on the liberation of inorganic phosphate
mature seeds were tested for phytase activity. The aver- from phytate in animal fodder was tested by rst
age phytase expression level was found to be 0.85% of incubating a standard poultry feed sample under con-
total soluble protein. The highest phytase expression ditions simulating chicken crop and stomach con-
level obtained was 9.3%, sufcient for further develop- ditions. Milled transgenic seeds released inorganic
ment into the commercial product PhytaSeed. The phosphate from the fodder, whereas milled control
application experiments described below were carried seeds had no activity (Fig. 4). This experiment shows
out using transgenic tobacco seeds, but similar results that phytase as produced in plant seeds has retained
(to be reported elsewhere) were obtained with the essential characteristics to exert its activity in the
PhytaSeed (8082). gastrointestinal tract and crop of monogastrics ani-
mals.
The effect of transgenic seeds containing phytase on
E. Application of Transgenic Seeds Containing animal growth was tested in vivo by milling and sub-
Phytase sequent addition to the basal diet of broilers. Diets
supplemented with nontransgenic seeds and diets
Phytase packaged in seeds by genetic engineering tech- with and without added phosphate were used as con-
niques was found to be extremely stable. Even milled trols. In addition, the commercial phytase preparation
seeds did not show loss of enzyme activity when stored from Aspergillus niger, Natuphos, was used for com-
at room temperature for a period of a year (Fig. 3). parison. Growth during the 4-week course of the
This characteristic, brought about by the stable seed experiment was used as a measure of the effect of
environment, is commercially very important because each supplementation. Diets supplemented with trans-
it generates a reliable supply to customers, as a reduced genic seeds resulted in signicantly higher growth rates
seed yield in a particular year can be replenished with
Figure 4 In vitro testing of transgenic phytase tobacco seeds

Figure 3 Stability of phytase packaged in tobacco seeds. under conditions simulating the digestive tract of poultry.
Unmilled transgenic tobacco seeds (MOG 413.25) stored at Seeds containing phytase from a transgenic tobacco line
4 C (&) and room temperature ( ), and milled seeds stored (MOG 413.32) and from nontransgenic tobacco were used.
at room temperature (&) were analyzed for phytase activity For comparison, Aspergillus phytase was used in doses of 500
(vertical axis) at different time points during storage (hori- and 750 FTU/kg feed. During the incubation period samples
zontal axis). Milled transgenic seeds stored at 20 C were not were taken and analyzed for the amount of phosphate
analyzed at the rst time point. released.

than those with control seeds or without any addition G. Other Potential Benets of PhytaSeed
(Fig. 5). Growth rates obtained with transgenic seeds
were not signicantly different from those obtained In the processing industries, where seed and/or plant
with Aspergillus phytase as a supplement or those materials are used, seed-formulated enzymes may be
with inorganic phosphate addition. This feeding simply incorporated in existing processes. Phytate pre-
experiment conclusively demonstrates that transgenic sent in corn kernels precipitates during the wet-milling
seeds containing phytase, added to animal feed without process in the concentrated steep water. This causes
prior extraction of the enzymes, can compensate for problems in handling, transportation, and storing of
the lower amount of inorganic phosphorus available the corn steep liquor. These problems may be avoided
to the animals. by using phytase during steeping. The addition of phy-
tase also results in a reduction in steeping time and in
an increase in starch yield (85). An efcient and elegant
F. Low-Phytate Mutants manner to achieve the same goal would be to package
phytase in corn kernels by genetic engineering techni-
Low-phytic acid mutants have been generated in sev- ques.
eral crop species such as corn and barley by Victor The application of transgenic phytase seeds in food
Raboy and coworkers (84). These mutants might be and feed may have other desirable effects. Phytate is
considered to become an alternative to the use of phy- considered to be a nutritional factor, because it che-
tase for optimizing phosphorus utilization from feed lates essential minerals like calcium, iron, and zinc,
components if the agronomic performance is unal- reducing their availability to monogastrics (71, 86).
tered. However, application of phytase in low-phytate An increase in calcium availability by the application
corn-based diets still results in a marked improvement of microbial phytase has been demonstrated in pigs
in the availability of phytate phosphorous from these and poultry, and degradation of phytate in cereals
diets in poultry. It seems likely that there is an oppor- has been shown to increase the availability of iron in
tunity for both technologies to reduce the phosphate vitro. Dietary addition of milled transgenic seeds con-
contents in manure from monogastric animals. taining phytase to feed or food will reduce the amount
of phytate and consequently increase mineral absorp-
tion in animals as well as humans. This is especially
important for infants and elderly people.
Phytate is well known to bind to proteins in general
and to some digestive enzymes such as proteases and
amylases in particular (8789). Application of
PhytaSeed may prevent the formation of phytatepro-
tein complexes and thus improve digestion of feed and
food.
VII. CONCLUSION
This example, as well as the other examples referred to,

clearly demonstrate the advantages of using plants as a
Figure 5 Effect of phytase-containing transgenic tobacco production system for enzymes. As many other appli-
seeds on the growth of broilers. Milled transgenic seeds of cations can be envisioned, plants will certainly earn a
line MOG413.25 was used in a concentration of 295 FTU/kg. prominent place among the various methods for pro-
As controls, diets supplemented with feed phosphate, non-
ducing enzymes.
transgenic seeds, and A. niger phytase (400 FTU/kg) were
included. Growth was measured after 2, and 4 weeks.
Growth observed for diets supplemented with A. niger phy-
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15
Enzymes in Protein Biosynthesis
John R. Whitaker
We shall not unravel the chemistry of life in mole- chromatography by Moore and Stein (4, 5) for separ-
cular detail without knowing at atomic or close to ating, identifying, and quantifying amino acids from
atomic resolution the structure of the biological protein hydrolysates, the Edman method of sequencing
macromolecules especially the proteins (1). proteins (6, 7), and the development of chromato-
graphic (4) and electrophoretic methods (8) for puri-
cation of amino acids and proteins all contributed to
I. INTRODUCTION
Proteins perform many functions in organisms. Some

Table 1 Protein Functions
of these are listed in Table 1. No other polymeric com-
pound can perform so many functions in nature. This Enzymes
is because proteins are so diverse in structure. Enzyme inhibitors
Although only 20 amino acids are used to build the Hormones
primary structures, they can be arranged theoretically Carriers
in 2019 different sequences. They also can fold in many Hemoglobin and myoglobin (O2 and CO2 )
different ways because of their ability to form second- Serum albumin (ions, lipids, hormones)
ary, tertiary, and quaternary structures and combina- Protectors
Antibodies, skin, hair, nails
tion in macromolecular structures with different
Blood clotting
proteins. Table 2 gives a small sample of the diverse
Chaperones
size of proteins. Each molecule of a specic protein has Osmotic regulators (serum albumin)
exactly the same size and amino acid sequence and fold Movement (contractile proteinsmyosin, actin, etc.)
in exactly the same way. Also, we will see later that Connectors
proteins can be posttranslationally modied in at least Muscle to bone (elastin)
135 different ways. Signal transducers (nerve transmission)
The period 19251960 was very active for protein Recognition
chemists and enzymologists. During that period James Cell-to-cell interaction (glycoproteins)
Sumner (2) crystallized urease and showed it to be A, B, AB, O blood types
a protein. In the 1930s, Kunitz and Northrop (3) Elicitors (antigens)
Cell coating, cell glue (adhesion), collagen, others
at Rockefeller Institute learned how to crystallize
Memory (brain: protein or is it DNA?)
trypsin, chymotrypsin, pepsin, and other enzymes,
Taste perception (tongue)
strengthening the argument that all enzymes are likely Vision (opsin, rhodopsin, alcohol dehydrogenase)
to be proteins. The development of ion-exchange

Table 2 Molecular Weights of a Few Selected Proteins
Protein Molecular weight (daltons) Protein Molecular weight (daltons)
Insulin 5,737 Alkaline phosphatase 80,000

Ribonuclease 13,683 Polyphenol oxidase 128,000
Lysozyme 14,100 Fumarase 194,000
Chymotrypsinogen 23,200 Catalase 232,000
-Lactoglobulin 35,000 Aspartate transcarbamylase 310,000
Ovalbumin 45,000 Urease 483,000
Serum albumin 65,000 -Galactosidase 520,000
Hemoglobin 64,500 Glutamate dehydrogenase 2,000,000
rapid advances in understanding the protein nature of The discovery of the chemical nature of the gene
enzymes. resulted from the merging of genetics and biochemistry
During that period, limited thought was given to between 1930 and 1950. In 1909, Archibald Garrod
protein biosynthesis. It was generally assumed that determined that the human disease alkaptonuria was
proteins were formed from activated derivatives of caused by a rare recessive mutation that was inherited
amino acids, probably by use of ATP for activation. according to Mendelian rules (8e). It was noticed that
But no details of the in vivo biosynthesis were known. patients with alkaptonuria had black urine. The dark
In the late 1950s and 1960s a few publications color was determined to be due to a breakdown pro-
appeared, based primarily on studies of microorgan- duct of phenylalanine. In his treatise on Inborn
isms, that began to give evidence that protein biosynth- Errors of Metabolism, Garrod wrote, We may
esis at the molecular level could be more complex than further conceive that the splitting of the benzene ring
originally thought. Especially, studies based on micro- in normal metabolism is the work of a special enzyme,
bial genetics indicated a precise correlation between [and] that in congential alkaptonuria the enzyme is
nucleotide sequences of DNAs and some RNAs and wanted.
the amino acid sequences of proteins. The research of Morgan and his colleagues in the
early 1900s on the inheritance of eye color in
Drosophila (8f), together with that of Beadle and
II. HISTORICAL BACKGROUND Tatum on the bread mold, Neurospora crassa, and its
rare mutant colony development, led Beadle and
There were many important contributions made in the Tatum to conclude that genes somehow control pro-
long journey of understanding protein biosynthesis tein (enzyme) structure (8g). These ndings led to
prior to the 1950s. Genes had been postulated before studies of the chemical structure of genes, as the rst
the turn of the 20th century based on the careful breed- step in elucidating the molecular basis for the genetic
ing studies of Gregor Mendel (8a). August Weismann control of protein synthesis. This occurred in 1944
in 1892 stated that the essence of heredity is the trans- when Avery et al., in studying Streptococcus pneumo-
mission of a nuclear substance of specic molecular niae from patients with pneumonia, showed that DNA
structure (8b). In 1903, Walter Sutton linked chromo- from smooth colonies can transform rough colo-
somes to Mendelian heredity (8c). He observed that all nies into the smooth form (9). They proved DNA was
cell divisions producing germ cells also formed thread- involved. Addition of several different proteases had
like chromosomes, and that a gamete received only one no effect on the transforming activity, eliminating
of each of the chromosomes of different morphologic involvement of proteins. However, small amounts of
types. Through detailed studies of the fruit y, puried deoxyribonuclease (DNAase) immediately
Drosophila melanogaster, Morgan and his colleagues inactivated the transformation.
showed that the genes segregated as four linkage In 1953, Watson and Crick deduced the double-heli-
groups corresponding to the four chromosomes (8d). cal structure of DNA, ushering in the modern era of
Similar observations followed for corn (10 chromo- molecular cell biology (10). At about this same time,
somes, 10 linkage groups) and peas (7 chromosomes, the electron microscope was invented. Light micro-
7 linkage groups). By 1920, the chromosome theory of scopes have a resolving power of 500 nm. Electron
heredity became an accepted fact in genetics. microscopes in the early stage had resolving power of

15 nm; this was eventually reduced to 0.10.5 nm. only when the mRNA is in the cytoplasm. This is the
Therefore, by the beginning of the 1960s the ndings major distinction between prokaryotic and eukaryotic
of genetists, biochemists, and structural cell biologists cells (Fig. 1).
could be brought together, working from the molecu- Eukaryotic RNA synthesis is carried out by three
lar to the subcellular level, and vice versa. Another separate RNA polymerases, while RNA synthesis in
principle that developed about this same time was prokaryotic cells is performed by a single RNA poly-
that the activity of genes is highly regulated. It was merase. The precursor to three of the four eukaryotic
known that genes produced proteins, but the methods rRNAs (the 28S, 5.8S, and 18S rRNAs) is produced by
by which the gene activity was controlled was still RNA polymerase I in the nucleolus of the cell. The
unclear. Perhaps the principle enuciated by Jacob precursors of the mRNAs are made by RNA polymer-
and Monod (11) did the most to clarify gene control. ase II and the small RNAs (tRNAs and the 5S rRNAs)
They proposed that certain genes regulate the activity or their precursors are made by RNA polymerase III.
of other genes. At the molecular level this transforms There are fundamental differences between prokar-
to an understanding that specic sets of proteins are yotes and eukaryotes in chromosome structure due to
produced that build characteristic structures, and some the core of histones wrapped around the DNA of
carry out characteristic enzymatic activities. eukaryotes. With respect to gene control of protein
Further discussion in this chapter will be limited to biosynthesis, prokaryotes are regulated at the tran-
the enzymes involved in in vivo and in vitro protein scription level while many eukaryotes are primarily
biosynthesis. regulated at the translation level.
III. PROTEIN BIOSYNTHESIS B. Steps in Protein Biosynthesis
A. Differences Between Prokaryotic and There are ve steps in the biosynthesis of proteins:
Eukaryotic Protein Biosynthesis transcription phase in which mRNAs, tRNAs, and
rRNAs are produced; translation of mRNAs to give
Being unicellular, formation of bacterial mRNA is polypeptides; termination of translation; posttransla-
always accessible to ribosomes, as well as other com- tional modication of the polypeptides; and folding of
ponents of the protein biosynthesis pathway. Even as the polypeptides into their native structures.
the mRNA is being transcribed from DNA, the specic
tRNAs and tRNA synthetases begin translation of the 1. Transcription Phase
protein and transcription and translation are com-
pleted at almost the same rate and time. Therefore, The overall steps of transcription and translation in
the mRNA nucleotide bases are not modied (by eukaryotes are shown in Eq. (1).
methylation) as they are in eukaryotic cell mRNA. In RNA polymerase
eukaryotes, DNA transcription occurs in the nucleus, DNA 4 NTP ! mRNA, tRNA;
which contains ribosomal precursors undergoing for- transcription
mation (in the nucleolus), but there are no mature amino acids, tRNAs
ribosomes that can perform protein synthesis. In rRNA ! Proteins
ribosomes
eukaryotes, the mRNA is synthesized in the nucleus.
1
The mRNA must then be secreted through the nuclear
membrane into the cytoplasm for translation to occur. During the transcription phase, the mRNAs, tRNAs,
Therefore, transcription and translation are not and rRNAs are produced. The key enzyme is RNA
coupled in eukaryotic cells. polymerase II. The three steps are: initiation signalled
In eukaryotes, the RNA is not a functional mRNA, by the promoter recognized by RNA polymerase (in
tRNA, or rRNA until it is extensively modied in the the case of prokaryotes, it is a subunit of the RNA
nucleus before it is secreted into the cytoplasm where it polymerase (2 0 ; elongation involving a melting
associates with the ribosomes. Types of modication and unwinding of the double helix while sequentially
that occur in the nucleus are: (a) addition of chemical attaching ribonucleotides at the 3 0 end of the growing
groups at both ends of the mRNA; (b) cutting the RNA molecule; termination by the formation of stem-
mRNA into segments; and (c) recombining the seg- loop structures of Rho-dependent terminators.
ments by splicing of original noncontiguous segments The April 28, 2000, issue of Science published a
to produce a very different mRNA. Translation occurs detailed crystallographic study of the protein architec-

Figure 1 The production of functioning mRNA is very different in prokaryotes and eukaryotes. In prokaryotes, the RNA
transcript serves directly as the mRNA, and translation begins before transcription is completed; that is, transcription and
translation are coupled. In eukaryotes, the primary RNA transcript must be modied in the cell nucleus to form mRNA.
Translation takes place only after the completed mRNA is delivered to the cytoplasm. (From Ref. 12.)
ture of a yeast (Saccharomyces cerevisiae) RNA poly-

Table 3 Yeast RNA Polymerase II Subunits
merase II at 3 A resolution (see insert, Fig. 2) in which
10 of the 12 different subunits are shown (Table 3). Amino acid
The yeast RNA polymerase II has a total mass of residues in Identity to
514 kDa. Yeast and human RNA polymerase IIs are Subunit Mass (kDa) sequence human (%)a
considered to be very similar since human genes for 10 Rpb1 191.6 1733 52
of the subunits can be substituted for the yeast genes, (1449)b
and a very similar enzyme is produced. Also, RNA Rpb2 138.8 1224 61
polymerases I and III of prokryotes contain nine of Rpb3 35.3 318 46
the 10 proteins of yeast RNA polymerase II. Rpb4 25.4 221 30
RNA polymerase II is the key of the transcription Rpb5 25.1 215 45
process. Without help of other enzymes it can (see Fig. Rpb6 17.9 155 59
3): (a) recognize the initiation codon, AUG (the regu- Rpb7 19.1 171 61
Rpb8 16.5 146 43
lated initiation complex contains RNA polymerase II,
Rpb9 14.3 122 37
ve transcription factors, and a multiprotein mediator; Rpb10 8.3 70 73
in all there are some 60 proteins involved); (b) recog- Rpb11 13.6 120 50
nize and respond to the operator complex; (c) proof- Rpb12 7.7 70 43
read and unwind the DNA double helix; (d) produce Total 513.6 4565 53
specic mRNA, tRNAs, and rRNAs by catalyzing for- a
mation of phosphodiester bonds among nucleotides Percentage of identical amino acid residues, for Rpb1 excluding the
COOH-terminal domain.
(Str. 1); proofread the nascent mRNA transcript and b
The number in parenthesis corresponds to Rpb1 without the
replace it if necessary; and (f) recognize the termination unstructured COOH-terminal domain (CTD).
point containing the codon AUA, UAG, or UGA. Source: Ref. 13.

Figure 3 Transcription of a double-stranded DNA into a single-stranded RNA by an RNA polymerase. The ve steps are
identied on the left-hand side. Not shown is the termination step. (From Ref. 12.)
Structure 1

Table 4 The Genetic Codea
First position Second position Third position
(5 0 end) U C A G (3 0 end)
U Phe Ser Tyr Cys U

Phe Ser Tyr Cys C
Leu Ser Stop (och) Stop A
Leu Ser Stop (amb) Trp G
C Leu Pro His Arg U
Leu Pro His Arg C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
A Ile Thr Asn Ser U
Ile Thr Asn Ser C
Ile Thr Lys Arg A
Met Thr Lys Arg G
G Val Ala Asp Gly U
Val Ala Asp Gly C
Val Ala Glu Gly A
Val (Met) Ala Glu Gly G
a
Bases are given as ribonucleotides, so U appears in the table instead of T. Stop (och) stands for
the ochre termination triplet, and Stop (amb) for the amber. AUG is the most common initiator
codon; GUG usually codes for valine, but it can also code for methionine to initiate an mRNA
chain.
After synthesis of the three types of mRNAs in mRNA. At the end of the tRNA (rectangle labeled
eukaryotes, they must be secreted from the nucleus amino acid), there is an adeonsyl group to form a
via the cell membrane into the cytoplasm, where trans- covalent binding site for the specic amino acid (Ala
lation takes place. The time to produce the mRNA is shown here). The tRNA synthetase is responsible for
estimated to be about 12 sec, depending on the pro- the reaction leading to the bound aminoacyl-tRNA
moter clearance time, number of RNA polymerase [Eq. 2)].
molecules, and size of the gene.
enzyme
Amino acid ATP tRNA ! aminoacyl-
2
2. Translation Phase tRNA AMP PPi
The mRNA carries the genetic information for protein
biosynthesis that it has transcribed from a gene located Table 5 Degeneracy of the Genetic Code
on DNA via a three-letter code (codon), complemen-
tary to that of the gene. Table 4 lists the genetic codons Number of Amino acid Total number
for each of the 20 amino acids found in proteins. The synonymous of codons
codons
code contains some redundancies, such that 64 codons
(including the stop codons) may be used in protein 6 Leu, Ser, Arg 18
biosynthesis (Table 5). 4 Gly, Pro, Ala, Val, 20
There are tRNAs and tRNA synthetases for each Thr
one of the 20 amino acids found in nascent proteins. 3 Ile 3
The tRNAA1a for alanine is shown in Figure 4. The 2 Phe, Tyr, Cys, His, 18
tRNAs are small ribonucleotides of 7080 residues. Gln, Glu, Asn, Asp,
Some of the nucleotide bases are also modied follow- Lys
ing synthesis, as shown by the shaded circles in Figure 1 Met, Trp 2
Total number of codons for amino acids 61
4.
Number of codons for termination 3
The tRNAA1a contains an anticodon of IGC which Total number of codons in genetic code 64
recognizes and binds to the GCC codon on the

Darnell et al. (12:122123) described the entire pro-
cess in great deal in the following manner (Fig. 7).
a. Initiation. An initiation factor (IF2 in prokar-
yotes and eIF2 in eukaryotes) binds a molecule of GTP
and a molecule of methionyl-tRNAMet i (for prokaryote
E. coli the methionine is replaced with N-formyl-
methionine) to form a ternary complex. This complex
binds to mRNA and the small ribosomal subunit (plus
other initiation factors) to make an initiation complex
(the 30S initiation complex in prokaryotes, and the 40S
complex in eukaryotes). The Met-tRNAMet i is now
positioned correctly at the AUG initiation codon. A
large ribosomal subunit then joins the complex; the
bound GTP is hydrolyzed; and the initiation factors
are detached. The Met-tRNAMet i bearing the rst
amino acid is now bound to the ribosome at the P
site. The initiation complex is ready to begin synthesis
of the peptide chain.
b. Elongation. the met-tRNAMet i is located at the
P (for peptidyl-tRNA) site on the ribosome. The grow-
ing polypeptide is always attached to the tRNA that
brought in the last amino acid. A new aminoacyl-
tRNA (Phe-tRNAPhe here) binds to the ribosome at
the A site (for aminoacyl site). During elongation in
Figure 4 The primary structure of yeast alanine tRNA
(tRNAA1a ). The tRNA is synthesized from the nucleotides
prokaryotes, a protein complex called Tu-Ts catalyzes
A, C, G, and U, but some of the nucleotides are modied the binding of each aminoacyl-tRNA to the ribosome.
after synthesis (shown by labeled shaded circles) (abbrevia- These are two elongation factorsEF-TU and EF-
tions for labels: D dihydrouridine, I inosine, TS. (A protein complex similar in action to Tu-Ts
T thymine, pseudouridine, and m methyl group). exists in eukaryotic cells.) A third elongation factor
The primary structure is a cloverleaf consisting of four EF-Gcontrols the translocation of the A to P sites.
base-paired stems and three loops: the D loop, for dihydrour- An activated Tu-GTP complex binds the TCG. The
idine, a virtually constant constituent of this loop; the anti- Tu-Ts complex probably binds to the TCG loop
codon loop; and the TCG loop, so named because a found in all tRNAs and allows the tRNA to associate
sequence of thymidylate, pseudouridylate, cytidylate, and with the ribosome. GTP is hydrolyzed and the Ts pro-
guanylate is virtually always present in this loop. (From
tein rejoins GDP-Tu and reactivates it. The complex
Ref. 12.)
also binds GTP, whereupon GDP and Ts dissociate
from Tu. The remaining GTP-Tu complex then binds
This chemical step is critical to protein synthesis in to an aminoacyl-tRNA. When the whole elongation
two major ways. The amino acid derivatives are acti- factor complex is bound to the ribosome, the GTP is
vated via the ATP reaction as shown by Eq. (2). The hydrolyzed to GDP, yielding energy to position the
amino acid products are shown in Figure 5. The amino aminoacyl-tRNA in the A site. After the incoming
acid is covalently attached via the 3 0 oxygen of the aminoacyl-tRNA is correctly placed in the A site
ribose diphosphate group of the tRNA. Secondly, the that is, when the codonanticodon pairing is cor-
specicity for binding at the right site on the mRNA is rectthe peptide chain (or, here, the rst methionine)
assured (see above). is transferred to the amino group of the newly arrived
Formation of the primary sequence of a protein is amino-acyl-tRNA. This generates a peptidyl-tRNA
carried out on the ribosomes (Fig. 6). As indicated that has acquired an additional amino acid. (In our
above, the eukaryotic ribosomes are located in the example, the compound is methionyl-phenylalanyl-
cytoplasm of the cells. Protein biosynthesis consists tRNAPhe .) At this stage, the peptidyl-tRNA is bound
of three major stages: initiation, elongation, and termi- to the ribosome at the A site. The ribosome moves one
nation. codon down the mRNA chain. (The mRNA codons

Figure 5 Translation of an mRNA strand into a protein begins with attachment of appropriate amino acids to the appropriate
tRNAs (see Fig. 4). Covalent attachment requires two steps by an aminoacyl-tRNA synthetase that uniquely recognizes and
binds to a specic amino acid and its cognate tRNA (see step 1). In the second step, amino acylation of tRNA occurs by ATP to
attach the COOH group of the amino acid to the 3 0 -hydroxy group of the terminal adenylate of the tRNA. Alternatively, the
initial attachment can be to the 2 0 -OH group, which then rearranges to the 3 0 position.
are illustrated with spaces separating them for conve- IV. POSTTRANSLATIONAL COVALENT
nience; in mRNA there are, of course, no spaces.) The MODIFICATION OF PROTEINS
translocation reaction is catalyzed in bacteria by the
elongation factor G, using energy from the hydrolysis Once the primary structure of a protein is completely
of GTP. With this movement, the empty tRNA is translated by RNA polymerase II, the protein is still
released from the P site, and the peptidyl-tRNA is biologically nonfunctional. It must undergo chemical
shifted to the P site. (In eukaryotes, too, there are modication involving nonhydrolytic and/or hydroly-
special proteins that serve in elongation.) The sequence tic modication of the protein, fold into the tertiary
of events is repeated for every amino acid added to the structure, form quaternary structure (if needed) and
growing chain. Note that two molecules of GTP are macromolecular structure (if needed), and combine
used in the addition of each amino acid. with cofactors (if needed) and often be transported to
the site where it performs its function. In this section,
c. Termination. When the ribosome arrives at the
we deal only with the posttranslational enzymatic
codon UAG, the translation is completed with the aid
modication.
of a terminator factor. Hydrolysis of the peptidyl-
tRNA on the ribosome releases the completed poly-
peptide and the last tRNA, and the two ribosomal A. Nonhydrolytic Posttranslational Enzymatic
subunits separate. At least three TFs are involved in Modication
E. coliRF1, RF2, and RF3. For eukaryotes, one
termination factor eRF recognizes all termination Table 6 lists examples of nonhydrolytic modication of
codons. 11 of the 20 amino acid residues, as well as the carboxy

Figure 6 The composition of prokaryotic and eukaryotic ribosomes is shown, each consisting of a large and a small subunit.
The different subunits contain rRNAs of different lengths and varying numbers and types of proteins (indicated by different
shadings). In addition to the two major rRNA molecules, prokaryotic ribosomes have one small 5S tRNA that is 120 bases
long. Eukaryotic ribosomes have two small rRNAs: a 5S molecule similar to the prokaryotic 5S, and a 5.8S molecule that is 160
bases long. The proteins are named L1, L2, etc., and S1, S2, etc., depending on whether they belong to the large or small subunits.
(From Ref. 12.)
and amino terminal ends of the proteins (1416). There 1 and one pro-2 chains align with each other with
is a highly specic enzyme for each of these modica- the N-terminal amino acid all at one end and the C-
tions. Some modications such as glycosylation and terminal amino acid at the other end. This triple helix
activation of the cascade of pro-proteins in blood clot- forms disulde bonds among the three molecules. At
ting involve a number of enzymes (see below). Most this point the procollagen (MW 115140 kDa for
likely, nonhydrolytic modications occur as soon as each molecule) is secreted through the membrane and
the N-terminal end of the growing protein chain transported to the location where needed. Collagen is
emerges from the ribosomal system. the major protein produced by animals. It forms a
A good example of posttranslational modication coating around all cells, brils, bundles of brils and,
of a protein is that for collagen (Fig. 8). As soon as higher structures, such as the sarcomeres and ber
a part of the nascent primary structure is formed on bundles of muscles.
the ribosome (polysome), hydroxylation of proline and At this point, highly specic and limited enzyme-
lysine (see Chapters 35 and 36) and glycosylation of catalyzed proteolysis occurs. The teleopeptides at the
hydroxylysine are known to occur (17, 18). Two pro-1 N-terminal end of the procollagen are removed by a
and one pro-2 chains are coded for by two different specic procollagen peptidase to give molecules of
mRNAs. While still in the unfolded state, the two pro- 95 kDa. The folded collagen molecules are aligned

Figure 7 Cartoon diagram of the translation of tRNA-acylamino acids and mRNA to form a specic protein. The process
involves: (a) the initiation phase; (b) the elongation phase; and (c) the termination phase. The chemistry involved is shown in
Figure 5 and Eq. (2). (From Ref. 12.)

Table 6 In Vivo Nonhydrolytic Enzyme-Catalyzed Posttranslational Modications of Amino Acid Residues of Proteins
Total Total
Amino acid Typical modications Amino acid Typical modications
(group) modications knowna (group) modications knowna
Arginine methyl-(mono- 6 Serine (hydroxyl) phospho-2 8
(guanidino) and di-)2 b glycosyl-2
ADP-ribosyl-2 methyl-2
citrulline phophopantetheine-2
ornithine ADP-ribosyl2
Lysine (
-NH2 ) glycosyl-2 33 Threonine phospho-2 6
phospho-2 (hydroxyl) glycosyl-2
pyridoxyl-2 methyl-2
biotinyl-2 Cysteine cystine1 7
lipoyl-2 (sulhydryl) glycosyl-2
acetyl-2 dehydroalanyl4
methyl-(mono-, heme
di-, tri-)-2 avin
-hydroxyl-1 seleno-
-glycosyl-2
crosslinks Aspartic acid/ -carboxyl-1 6
2 glutamic acid -phospho-2
Histidine methyl- (1- and 3-) 6 (carboxyl)a methyl-2
(imidazole) phospho- (1- and 3-)2
iodo- Asparagine/ glycosyl2 8
avin- glutamine R-NH-
(amide)a pyrrolidone
Proline 4-hydroxyl-1 5
3-hydroxyl-1 Carboxyl -amide6 11
3,4-dihydroxy-1 terminal residue -amino acid6
4-glycosyloxy-2 (carboxyl)
Phenylalanine -hydroxy-1 2 Amino terminal acetyl-2 19
(benzene ring) -glycosyloxy-2 residue (amino formyl-2
group) glucosyl-2
Tyrosine -hydroxy-1 18 amino acyl-2
(benzene ring/ -glycosyloxy-2 pyruvyl-2
hydroxyl) sulfono-1 -ketobutyryl-2
iodo- (mono-, di-)2 methyl-2
brom- (mono-, di-)2 glycuronyl-2
chloro- (mono-, di-)2 murein2
bis-ester1
adenylyl2 Total 135
uridylyl-2 modications
RNA
a
Including three each for aspartic acid and glutamic acid and four each for asparagine and glutamine.
b
General class of enzymatic reaction: 1 oxidation/reduction; 2 transfer; 4 formation of double bond or addition to double bond; 6 ligation.
Source: Refs. 14, 16.
both end to end in a staggered array and laterally to B. Hydrolytic Posttranslational Enzymatic
give cylindrical brils with diameter range from 50 Modication
2000 A, depending on the tissue and stage of develop-
ment. There are further crosslinkages, especially ins are probably modied by highly specic proteases,
through desmosine formation from four lysyl side since most proteins are secreted from the cell for trans-
chains by lysyl oxidase (see Chapter 37). port to other locations (organs and cells). Secretion

Figure 8 Proposed scheme for in vivo posttranslational enzymatic modications involved in collagen formation. (From Refs.
17, 18.)
from the cell requires a signal peptide targeting the Preproinsulin (Fig. 9) is synthesized as a 100amino
nascent protein for transport. The signal peptide is acid polypeptide. Immediately on synthesis, a highly
required to translocate the protein out of the cell. specic protease in the luminal space of the endoplas-
Table 7 lists a number of physiological systems con- mic reticulum removes the N-terminal signal peptide
trolled by posttranslational proteolysis. Table 8 gives after amino acid 16 by hydrolysis. The remaining 84
some examples of highly specic proteases involved in amino acid proinsulin, in which two disulde bonds
physiological processes. have formed correctly (a posttranslational modica-
In addition to the posttranslational hydrolytic mod- tion), is transported to the Golgi body where a 33
ications to collagen (above), three other examples (of amino acid segment (the C segment in Fig. 9) is hydro-
many) will be given for preproinsulin, chymotrypsino- lyzed from proinsulin by two proteolytic events at two
gen A, and preproproteins in the blood-clotting cas- peptide bonds (Ala30 Arg31 and Arg63 Gly64 of proin-
cade. sulin) to give two peptides (A [21 amino acid residues]

Table 7 Physiological Systems Controlled by Limited
Posttranslation Proteolysis
Physiological system Example
Assembly bacteriophage
virus
membrane
procollagen ! collagen
fibrinogen ! fibrin
Defense reactions blood coagulation
brinolysis
complement reaction
Development maturation of spermatozoa,
release of ova, and fertilization
(proacrosin ! acrosin)
prochitin synthetase ! chitin
synthetase
prococoonase ! cocoonase
Digestion zymogen ! enzyme
Hormone production proinsulin ! insulin
angiotensinogen ! angiotensin
Oncogenic division, growth, migration, and
transformations adhesion
Tissue injury impairment of cell contact
inhibition
prekallikrein ! kallikrein
kininogen ! kinin
Translocation preprotein !protein
Source: Ref. 19. Figure 9 The processing of preproinsulin into proinsulin,

and of proinsulin into insulin. The specic proteolytic clea-
vage of preproinsulin, which occurs immediately after the
synthesis of its chain of 100 amino acids is completed,
remove 16 amino acids termed the signal sequence from the
Table 8 Some Examples of Highly Specic Proteases amino end of the molecule. The remaining 84 amino acids
Involved in Physiological Processes constitute proinsulin, a molecule in which the correct three
disulde bonds have been formed. While the hormone is
Group-specic proteinase of mast cells which inactivates
being packaged for secretion the 33 residues (of the C
PALP-apoenzymes
chain) between the A and the B chains are removed by two
Proteinases of B. megaterium spores which cleave spore
specic proteases to produce insulin. (From Ref. 21.)
protein
RecA gene product from E. coli which cleaves
bacteriophage repressor
The acid proteinase renin, which cleaves angiotensinogen to
angiotensin and B [30 amino acid residues]), held together by two
The carboxydipeptidase that cleaves angiotensin disulde bonds. This is active insulin.
The neutral proteinase enterokinase, which cleaves The proteolytic conversion of chymotrypsinogen A
trypsinogen to trypsin to -chymotrypsin A is shown in Figure 10.
The albumin-degrading light subunit of rat kidney Chymotrypsinogen A is stored in the pancreas as inac-
glutamyl transpeptidase tive protein. Following secretion into the small intes-
The plasminogen-converting streptokinase tine, it is activated by trypsin (another protease).
The proteinase which cleaves the T4 prehead precursor Chymotrypsinogen A is translated on ribosomes as a
protein
single polypeptide chain of 245 amino acids. The key
Calcium-activated protease of myobrils
Methionine aminopeptidase
step in activation of chymotrypsinogen A is hydrolysis
of the Arg15 Ile16 peptide bond by trypsin, thereby
Source: Ref. 20. producing -chymotrypsin. The active -chymotrypsin

Figure 11 A proposed mechanism for blood clotting in
mammalian plasma in the intrinsic system. The factor on
the left side of reaction (proenzyme) is converted to active
enzyme by a highly specic proteolysis. PL, phospholipids.
(From Ref. 23.)
Figure 10 Schematic diagram of the role of several limited

proteolytic steps in the conversion of bovine chymotrypsino- transglutaminase cross-linking of a glutamyl -car-
gen A to - -, and -chymotrypsins. (From Ref. 22.) boxyl group to an "-amino group of a lysyl residue
of brin to form the nal clot. The proenzymes are
always found in the blood but the cascade of events,
then hydrolyzes the Leu13 Ser14 peptide bond to pro- initiated by a cut (for example), takes < 1 min to pro-
duct active -chymotrypsin. -Chymotrypsin can then duce a clot.
split the Tyr145 Thr146 and Asn147 Ala148 peptide After the primary amino acid sequences of proteins
bonds to give -chymotrypsin, a stable enzyme. - are formed, they are posttranslationally modied and
Chymotrypsin can also hydrolyze the Tyr145 Thr146 then folded to give the secondary and tertiary struc-
and Asn147 Ala148 peptide bonds of chymotrypsinogen tures, as dictated by the amino acid sequence. Some
A to form inactive neochymotrypsinogen (two-step proteins, such as hemoglobin, with > 30% hydropho-
process). Proteolytic cleavage forms three large pep- bic amino acid residues on the surface, will form qua-
tides and two dipeptides from the single peptide chy- ternary structures. Figure 12 shows the primary,
motrypsinogen A. The three large peptides are held secondary (-helices) and tertiary structures of myo-
together by four interchain disulde bonds. globin.
Posttranslational modication of the proenzymes in
the blood clotting mechanism is much more complex C. In Vivo Turnover of Proteins
than the two examples given above (Fig. 11). There are
a total of 12 proenzymes in the activation cascade Do proteins last forever, once synthesized? The answer
requiring 12 specic proteolytic events, along with a is no in all except one case. Table 9 shows a few exam-

types of damage caused by metabolic events and to
cofactors of enzymes. Cellular protein turnover occurs
primarily in the lysosomal organelles found in the cyto-
plasm of cells. (See Refs. 26 and 27 for a review of in
vivo protein turnover. This chapter is limited to that of
protein biosynthesis.)
V. COMMERCIAL UTILIZATION OF
KNOWLEDGE FROM IN VIVO
PROTEIN BIOSYNTHESIS
This chapter closes with a brief and incomplete look at

why detailed knowledge of the pathway of protein bio-
synthesis is commercially important.
There is rst, and foremost, the health reason for
being able to potentially x some 600 or so diseases
and defects of humans caused by defective genes. Now
that the entire human genome has been elucidated,
how, and should, the defective genes be repaired in
order to prolong and improve the quality of life? At
what cost, in privacy, dollars, and ethical considera-
Figure 12 Schematic drawing of the primary, secondary, and tions?
tertiary structures of myoglobin as determined by x-ray crys- The opportunity to use nature as an excellent che-
tallography. Only the -carbons are shown. The heme is mist promises many rewards. Organisms, especially
located in the upper center part of the molecule. (From Ref.
microoganisms, can be reprogrammed to concen-
24.)
trate most of their energy in making proteins, enzymes,
and health products needed by humans and other ani-
ples of turnover rates for proteins. Ornithine decarbox- mals. The biological process is less polluting and
ylase has a half-life of 10 min, while lactate dehydro- requires less energy and much less time, and the pro-
genase isoenzyme LDH5 has a half-life of 16 days. ducts are stereospecially those that can be used by
Hemoglobin has a half-life of 40 days. Elastin is other organisms, including humans. DNA shufing
thought to be stable for a lifetime. and enhanced plasmid expression of a gene may lead
Many studies have been done on how a protein to higher yields at faster rates than wild-type organ-
molecule is targeted for replacement. Factors to con- isms can do. The change in one or a few codons may
sider include the equilibrium between native and rever- lead to more product, or a product with better proper-
sible denaturated protein, the state of glycosylation, ties. The Bt gene inserted into corn, cotton, and soy-
beans, for example, eliminates the need for highly toxic
and polluting chemical pesticides.
Table 9 Rates of Intracellular Turnover of Some Selected There are several recombinant gene approaches that
Proteins may be used to modify the properties of enzymes and
Protein Half-life
proteins: (a) plasmid amplication; (b) selective changes
in one or more nucleotides of a gene so as to modify one
Ornithine decarboxylase 10 min or a few amino acid residues in wanted proteins; (c)
-Amino levulinate synthetase 60 min addition of new amino acids at the C- or N-terminal
Catalase 1.4 d end of proteins to increase ease of purication; (d) anti-
LDH5 16 d sense sRNAs to inhibit translation of specic genes in
Hemoglobin 40 d organisms; (e) mRNAprotein fusions to make new or
Mitochondria protein as whole 45 d
modied functions (28); (f) ribosome displays to pro-
Rat protein, 70% 45 d
duce larger libraries for genetic cloning and improve-
Cultured cells 12 h
ment via combinatorial chemical techniques (28); and
Source: Ref. 25. (g) DNA family shufing [molecule breeding (29, 30)].

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Biochem 43:835869, 1974.

16
Nucleic Acid Biosynthesis
Dominic W. S. Wong
U.S. Department of Agriculture, Albany, California, U.S.A.
I. INTRODUCTION the origin of replication consists of a core component

(including an origin recognition element, a DNA-
Biosynthesis of DNA is a biochemical process of high unwinding element, and an A T-rich element) and
complexity, tightly controlled by a wide range of reg- one or more auxillary components anking the ori
ulatory mechanisms. At least 30 proteins are required core, that are binding sites for the activation by tran-
to replicate the chromosome of Escherichia coli. The scription factors (4). The opening of the strands allows
overall pattern of DNA replication is semiconservative several events to occur at the replication fork, each
as unequivocally proved by Meselson and Stahl (1) requires a unique enzyme or enzyme complex: (a)
using 15 N-labeling techniques. At the same time, the DNA helicase for progressively unwinding the parental
biochemistry and enzymology of the process of DNA duplex DNA; (b) primases for the synthesis of primer
replication has become the focus, since Kornberg and RNAs needed for the initiation of polymerization;
colleagues (2) isolated DNA polymerase I from E. coli. (3) DNA polymerase for the continuous 5 0 ! 3 0 synth-
Four decades later, the general picture of DNA repli- esis in the leading strand, and the synthesis of Okazaki
cation is fairly well understood, although many details fragments in the lagging strand in a discontinuous
remain to be lled. This chapter is not an overview of manner.
the replication process, but an attempt to describe the
enzymology of the process, with emphasis on a number
of relatively well-characterized enzymes critical to var- II. UNWINDING
ious stages of DNA replication in prokaryotes.
In simple genomes, DNA replication is initiated by Helicases are a family of enzymes essential to many
the formation of a nucleoprotein complex formed by aspects of DNA metabolism, including DNA replica-
the association of multiple DnaA proteins with the tion, recombination, transcription, and repair and, for
recognition sequence at the replication origin. In E. RNA translation, splicing and ribosomal assembly.
coli, the origin of replication consists of a sequence Comparison of amino acid sequences suggests that
of 245 bp that contains four binding sites to which helicases can be classied into two superfamilies, SF1
DnaA can bind and initiate the stepwise assembly of and SF2, and a smaller family consisting of DnaB-like
all the proteins and enzymes to form a replisome neces- helices (5). In the latter family, E. coli DnaB, RuvB,
sary for replication (3). The binding of multiple dnaA Rho, RepA, and bacteriophage T7 gp4 and T4 gp41
proteins to the replication origin promotes denatura- are helicases with a hexameric ring structure (6). This
tion of the DNA strands in an A T-rich region adja- family of helicases is dened by ve conserved regions.
cent to the nucleoprotein complex. In eukaryotic cells, The E. coli DnaB and the bacteriophage T7 gp4 are the

most extensively studied replicative helicases that cat-
alyze strand separation at the replication fork. Both
are 5 0 3 0 helicases which exhibit a conformational
exibility. The hexameric DnaB has been shown to
exist in two different forms, with a threefold symmetry
(a trimer of dimers), or sixfold symmetry, and a range
of intermediate states (7). The T7 gp4 also likely
involves stable dimer, trimer, and higher oligomer spe-
cies (8). The functional implication of these conforma-
tions is not clear. The T7 gp4 protein, unlike DnaB, is
bifunctional, consisting of a helicase domain and a
primase domain, collectively known as helicase-pri-
mase.
A. Molecular Structure
The primary structure of DnaB helicase, deduced from

the DNA sequence, contains 470 amino acid residues
with a calculated MW of 52,265. In the mature protein,
the N-terminal methionine residue is removed in vivo,
leaving Ala as the N-terminal residue (9). The protein is Figure 1 Ring-shaped hexamer of bacteriophage T7 gp4
proteolytically cleaved in a two-stage process (10). The helicase, with the outer diameter of 120 A and a central
hole diameter of 35 A. The N-terminal helix of each subunit
N-terminal 14 amino acid residues are removed initially,
packs against the neighboring subunit of the hexamer in a
resulting in a 50-kDa polypeptide (Fragment I), which is
swapping arrangement. (Reprinted from Ref. 68, Copyright
subsequently cleaved into two separate segments: a 1999, with permission from Elsevier Science.)
C-terminal 33-kDa Fragment II which contains the
active site for nucleotide binding proteins, single-
stranded DNA binding, DNA-dependent ATPase the transition state during hydrolysis. Arg522 is in con-
activity, and oligomerization to hexameric structure, tact with the phosphate, which is linked to residues in
and an N-terminal 12-kDa Fragment III which is essen- the ssDNA binding site (located in motif H4) via
tial for protein interaction with DnaC and primase in His465. This arrangement may provide a switch cou-
priming DNA replication in the primosome. The crystal pling ATP hydrolysis to DNA binding. Both and
structure of Fragment III consists of a central helix (1) phosphates are coordinated by a Mg2 ion that ligands
wrapped around by ve -helices (11). to Ser319 and Asp424 sidechains.
The crystal structure of the helicase domain of the
bacteriophage T7 gp4 protein has been determined
(68). The T7 helicase domain has a sixfold symmetric B. NTP Binding and ATP Hydrolysis
ring with a central hole of 35 A, that can accommodate
a single-stranded DNA. The structural core is a For the unwinding of double stranded DNA, coopera-
nucleotide-binding fold consisting of a central parallel tive NTP binding and hydrolysis are required to induce
sheet anked by helices (Fig. 1). Four of the ve conformational changes necessary to promote DNA
motifs (H1, H1a, H2, and H3), which are conserved binding and translocation of helicase along the DNA
in the DnaB-like family, are located at the C-terminal (13). Studies on E. coli DnaB suggests that all nucleo-
ends of the strands. The binding of nucleotide lies at tides bind with high afnity to three sites, followed by a
the interface between subunits, involving interactions second phase of low-afnity binding, with a negative
of residues within the four motifs. The subunits are cooperativity (14). For the bacteriophage T7 gp4,
joined with stabilization by hydrophobic and electro- nucleotide binding at the three high-afnity sites exhi-
static interactions over the length of helix A intertwin- bits positive cooperativity (15). The T4 helicase shows a
ing between adjacent domains. Several conserved preference with dTTP, and one to two NTPs per hex-
residues at the NTP binding site have been identied amer is sufcient for the interaction between helicase
for interactions with the phosphates (12). Lys318 inter- and DNA. Crystallographic studies on the T7 gp4 heli-
acts with the phosphate and also serves to stabilize case domain indicate that NTP binding results in the

rotation of the subunits of the ring with a 15 distortion III. PRIMING
from a sixfold symmetry (12). This conformational
change gives rise to the negative cooperativity of NTP DNA polymerases are unable to initiate de novo poly-
binding; binding at one site confers a negative effect on merization, and require the priming of DNA synthesis
the binding of the subsequent site. A model has been by RNA transcription. The process involves replica-
proposed in which the binding sites can exist in either of tion-priming RNA polymerases, called primases, that
two conformational statesR (relaxed) when the site is synthesize short RNA primers to provide a 3 0 -OH
lled by ADP, Pi , and T (tense) when ATP is bound to onto which the rst nucleotide is added. In E. coli,
the site. Conformational changes from R to T and vice the primase, DnaG, is produced separately from the
versa with each cycle of ATP binding and hydrolysis, DnaB helicase, whereas in bacteriophage T7, primase
involves three NTP binding sites at any one stage of the and helicase activities are found in separate domains of
cycle (13). In a two-site sequential model, one catalytic the gp4 protein. The priming step occurs once for the
NTPase site and one site for ADP Pi , alternating leading strand, but multiple times for DNA synthesis
among the six subunits. It is also as likely that active in the lagging strand. The E. coli DnaG participates in
hydrolysis occurs at only one site in any one cycle. proteinprotein interactions with DnaB (helicase), sin-
Nucleotide hydrolysis is mediated by Mg2 coordina- gle-stranded DNA binding protein (SSB), and DNA
tion of the phosphates for nucleophilic attack. The polymerase III holoenzyme, for cooperative synthesis
metal ion also serves to stabilize the developing nega- of DNA. Association of primase with the replication
tive charge of the transition site. The energy released is fork is mediated by proteinprotein interaction with
the driving force for the helicase to unwind DNA the DnaB helicase (22), with the binding site located
duplex and processively translocate along the nucleic at the C-terminal domain of 16 kDa (23). Mutations
acid lattice. on the surfaces of interaction between the two proteins
signicantly reduce primer synthesis (24). The DnaG
C. DNA Binding primase synthesizes primers predominantly 1012
nucleotides in length, starting with a purine (25).
The T7 gp4 helicase, like most hexameric helicases, This (p)ppApPu-rich sequence at the 5 0 end of the
binds ssDNA with a much higher afnity than primer RNAs is structurally similar to some of the
dsDNA. The helicase binds a 10-mer and 30-mer eukaryotes. The primase remains stably bound to the
with a Kd value of 10 nM, and a second strand 50- primer RNA site via contact with the single-strand
fold lower in afnity (16). Similar results have been DNA-binding protein (SSB), and must be displaced
observed for E. coli DnaB (17). Most DNA helicases before the DNA polymerase holoenzyme can attach
show preferences, at least in vitro, for unwinding to the DNA duplexprimer junction. It has been sug-
duplex DNA with ssDNA anking at either 5 0 or 3 0 gested that the subunit of the complex in the
ends. The E. coli DnaB and the T7 gp4 helicase require holoenzyme competitively binds to the SSB to disrupt
a forked DNA, with ssDNA primers attached to both the primaseSSB interactions, resulting in the displace-
5 0 and 3 0 ends of each complementary strand (18). In ment of the primase from the primer (26).
the unwinding process, T7 gp4 helicase encircles the The DnaG gene sequence contains a coding region
lagging strand whereas the 3 0 5 0 ssDNA strand (lead- of 1740 nucleotides, corresponding to 580 amino acids,
ing strand) is excluded from the central cavity (19). In with the rst 14 residues cleaved off in the mature
this model, the helicase translocates unidirectionally protein (27). A sequence comparison of several bacter-
along the single-stranded DNA in the direction of ial primases reveals a conserved zinc-nger motif in the
the DNA duplex, facilitated by ATP hydrolysis. N-terminus where the catalytic domain is located, and
Alternatively, an active mechanism has been proposed a conserved basic region that shares with RNA poly-
for the E. coli Rep protein, in that the helicase binds to merase large subunits (28). That the catalytic function
the dsDNA at the duplexprimer junction, and plays a is located in the N-terminus has been conrmed by a
direct role in destabilizing the base pairs in each cata- study on a partial proteolytic digestion of the protein,
lytic step (20). A recent study showed that T4 helicase and analysis of biochemical properties of the major
can unwind unnatural substrates having the displaced fragments (29). The DnaG primase is a cashew-shaped
DNA strand replaced by a peptide nucleic acid mimic, molecule consisting of three domains: an N-terminal
at similar rates as DNA-DNA substrate, indicating = fold, a central ve-stranded sheet sandwiched
that unwinding is insensitive to the chemical nature by six -helices, and a C-terminal antiparallel, three-
of the displaced strand (21). helix bundle (Fig. 2) (30). The active site is located at a

disassembly and reassembly of the holoenzyme struc-
ture during the synthesis of each Okazaki fragment
(33, 34).
A. DNA Polymerase III
DNA polymerase III holoenzyme is a large enzyme

complex (> 1 MDa) composed of 10 subunits (35),
with four distinct functional components: (a) the poly-
merase core132 kDa, " 27 kDa, and 10 kDa;
(b) the subunit (38 kDa), which acts like a sliding
Figure 2 Structure of Escherichia coli DnaG primase, show- clamp that confers processivity (36); (c) the complex
ing the N-terminal, central, and C-terminal subdomains 2 1 10 1 1 ; 47 kDa which assembles the subunit
from right to left. (Reprinted from Ref. 30, Copyright onto the DNA in an ATP-dependent process (37,
2000, with permission from American Association for the 38); and (d) the linker protein, 71 kDa, which
Advancement of Science.) dimerizes two polymerase cores and one complex.
The core subunits, ", are tightly bound and can
only be dissociated by denaturation. The subunit
shallow, wedge-shaped cleft formed at the interface contains polymerase activity, while the " subunit car-
between the N-terminal and central domains. The ries the editing function of 3 0 5 0 exonuclease activity
invariant carboxylic acids, Glu265 and Asp309, from (39). In contrast, DNA polymerase I consists of both
the side formed by the central domain serve as ligands polymerase and 3 0 5 0 exonuclease activities, located in
for Mg2 coordination. The side covered by the N- the C-terminal Klenow fragment of the enzyme mole-
terminal domain provides an electrostatically positive cule (40).
ridge for surface interaction with the template phos- DNA polymerases are classied into three major
phodiester backbone. familiesA, B, and Cbased on the alignment of
DNA sequences with E. coli polymerases (41).
Family A enzymes are named for their homology to
IV. ELONGATION the product of the polA gene encoding E. coli DNA
polymerase I; Family B enzymes are named for homol-
Two modes of action are introduced in the elongation ogy to polB gene encoding E. coli DNA polymerase II;
process, because of the antiparallel arrangement of the and Family C for their homology to the polC encoding
DNA duplex, and the fact that DNA polymerase can E. coli DNA polymerase II subunit. E. coli DNA
only synthesize DNA in the 5 0 ! 3 0 direction. DNA polymerase III contains 1160 amino acid residues
synthesis in the leading strand occurs in the same direc- with a calculated MW of 129,920 daltons, and a pI
tion as the replication fork progress, and consequently of 4.93 (42). The widely investigated E. coli DNA poly-
only a single priming event is needed and DNA is merase I consists of 928 amino acids with a calculated
synthesized continuously by DNA polymerase III MW of 103,117 and a pI of 5.37 (43, 44). Mild proteo-
holoenzyme. The leading strand synthesis is highly lysis of DNA polymerase I at Thr323-Val324 produces
processive at a rate of 1000 bp/sec, owing to the action a large (Klenow) fragment of 605 amino acid residues
of a sliding clamp ( subunit) that tethers the with a MW of 68,064, and a smaller fragment of 323
enzyme to the DNA template. Processivity (average amino acid residues.
number of bases inserted in a single event of associa-
tion, polymerization, and dissociation of the DNA
polymerase on the DNA template) of 5000 (31) to B. Molecular Structure
even 500,000 (32) has been reported. Elongation of
the lagging strands, however, consists of the formation The crystal structures of several DNA polymerases
of short DNA fragments of 12 kb in length, in a have been published, including E. coli DNA polymer-
discontinuous process requiring repeated RNA primer ase I Klenow fragment (40), HIV-1 reverse transcrip-
synthesis, DNA extensions, primer removal, DNA tase (45), rat DNA polymerase (46), Thermal
repair, and gap lling. The DNA polymerase enzyme aquaticus DNA polymerase (47), bacteriophage T7
complex adapts to these various conditions by partial DNA polymerase (48), bacteriophage RB69 DNA

polymerase (49), Thermococcus gorgonarius DNA as a clamp to position the template primer relative to
polymerase (50), and Archaeon Thermococcus sp. the polymerase active site.
9 N-7 polymerase (51). The small (3 0 5 0 exonuclease) domain contains a
The overall structure of E. coli polymerase I Klenow central mostly parallel sheet with helices on both
fragment consists of a large C-terminal (polymerase) sides (40). A divalent metal ion is coordinated to the
domain that has a handlike shape built by the palm, carboxylate groups of Asp355, Glu357, and Asp501,
ngers, and thumb subdomains, and a smaller globular and to the 5 0 phosphate of dTMP, with a water mole-
N-terminal (3 0 5 0 exonuclease) domain that forms a cule (or hydroxide ion) as a fth ligand. A second
central, mostly parallel -pleated sheet with helices metal ion at site B is observed only when dNMP is
on both sides (Fig. 3) (40). bound to the protein molecule. The meal Mg2 is coor-
The general architecture of the polymerase domain dinated to the carboxylate of Asp355, Asp424, and two
is shared by all known polymerases. The palm subdo- of the 5 0 phosphate oxygens of dNMP, and three water
main contains a six-stranded antiparallel sheet that molecules hydrogen bonded to the carboxyl oxygens of
forms the bottom of the cleft about 2024 A wide and Asp424 and to backbone amides (52).
2535 A deep, which can accommodate 8 bp of The polymerase domain and the exonuclease
duplex DNA. The ngers subdomain contains all domain have separate DNA binding sites and can
helices that form one side of the wall surrounding the function independently. The Klenow fragment, with
cleft. The other side of the wall consists of primarily the polymerase active site lled by a duplex DNA,
two long helices, projecting out and hanging over the can bind and cleave a second DNA substrate at the
cleft like a thumb. The palm subdomain is the most exonuclease active site (53). Based on the crystallo-
conserved structure among all polymerases. This is the graphic study of the Klenow fragment bound to duplex
catalytic center that contains the binding sites for the DNA (54), the duplex DNA (primer strand base-
3 0 terminus of the primer and the substrate dNTP. The paired to the template strand) is in contact with the
active site contains a cluster of three carboxylate side thumb subdomain, the single-stranded template strand
chains of Asp705, Asp882, and Glu883, which are beyond the site of DNA synthesis is in contact with the
ligands for two metal ions. The ngers subdomain ngers subdomain, and the 3 0 end of the primer strand
binds the DNA template strand and directs it in a lies near the carboxylates groups of the three catalytic
productive orientation for interactions with the primer. residues in the active site. In the editing mode, the 3 0
The thumb subdomain functions to control the acces- end of the primer strand is dissociated from the duplex
sibility of the active site to substrate binding and pro- and lies in the active site of the exonuclease domain.
duct release. The thumb helices, together with This particular arrangement of the substrate binding
numerous other proteinDNA interactions, may act allows an interactive mechanism of DNA synthesis and
Figure 3 Schematic illustration of Escherichia coli DNA polymerase I Klenow fragment, showing the N-terminal, central, and
C-terminal subdomains. Arrows indicate sheet; tubes represent helices (From PDB id: 1kfd).

proofreading (or editing) by virtue of allowing the 3 0 delity of polymerization is achieved by a large reduc-
end of the primer strand to shuttle between the two tion in the rate of phosphodiester bond formation for
active sites, when a mismatch at the 3 0 terminus occurs incorrect nucleotides by an induced-t conformational
(55). It involves a competition between these two sites change, in conjunction with editing by the 3 0 5 0 exo-
for synthesis and editing. nuclease (60, 61). DNA polymerases have typical base
In E. coli DNA polymerase III holoenzyme, the substitution errors ranging from 104 to 107 . The
polymerase and 3 0 5 0 exonuclease activities reside in discrimination against a wrong nucleotide insertion
separate subunits, and " respectively, rather than in has been attributed to hydrogen bonding between a
two domains of the same subunit as in the case of DNA template base and a dTNP substrate base, and
DNA polymerase I described above (39). The " subunit more recently, to geometric selection where insertion is
plays a signicant role in replication delity by con- strongly favored by substrates with bond angles and
trolling the editing activity of the holoenzyme. distances that conform to the Watson-Crick model
Proofreading by DNA polymerase III depends on the (62).
cooperative interaction of the polymerase and exonu- Structural and kinetic studies of 3 0 5 0 exonuclease
clease subunits. The efciency and substrate specicity reveal that phosphoryltransfer is mediated by a two-
of the 3 0 5 0 exonuclease activity are markly affected metal ionphosphoryltransfer mechanism (52, 55). One
when the subunit is complexed in the polymerase core. of the metal ions (site A), acting as a Lewis acid, facil-
Compared with that of the free " subunit, the activity itates a nucleophilic attack by a water molecule on the
in an " complex increased 10 to 80-fold, indicating the phosphorous atom of the terminal nucleotide, from a
involvement of the subunit in proofreading and con- position opposite the 3 0 -OH leaving group. Metal ion
tribution to the delity of DNA replication (56). B stabilizes the resulting pentacovalent transition state
and facilitate the leaving of the 3 0 oxyanion. Three
C. Reaction Mechanism carboxylic acid residuesAsp355, Asp501, and
Glu357serve to coordinate the metal ions and,
The chemical reaction at the core of DNA replication together with Tyr497, to position the single-strand pri-
is the DNA polymerase-catalyzed nucleotidyl transfer mer DNA and the H2 O molecule essential for catalysis.
reaction, depicted as follows:
DNA template strand pimer strand dNTP
! DNA template strand primer strand
dNMP PPi;
where dTNP represents one of the four deoxynucleo-
tides and dNMP corresponding deoxyribonucleoside
5 0 -monophosphate. The 3 0 5 0 exonuclease reaction
removes the dNMP from the single-stranded primer
by hydrolysis as follows:
primer strand dNMP H2 O ! primer strand
dNMP:
In the polymerase reaction, at least two carboxylic
acid residues (Asp705 and Asp882 in DNA polymerase
I Klenow fragment) serve to anchor the two metal ions
which play a major role in catalysis. The Mg2 at site
A activates the primers 3 0 -OH with one of the Asp
ligands as the proton acceptor (5759). This facilitates
a nucleophilic attack on the -phosphate of the incom-
ing dNTP which assumes a pentacovalent transition Figure 4 The two-metal ion mechanism of DNA polymer-
state stabilized by both Mg2 ions at sites A and B. ase showing the pentacoordinated transition state. (Adapted
The Mg2 at site B also functions to stabilize the nega- from Ref. 67, Copyright 1994, with permission from
tive charged and phosphates (Fig. 4). The high American Association for the Advancement of Science.)

the primer terminus at the single-strand/double-strand
junction (or nicked duplex DNA). The association of
the clamp increases the processivity of polymerase III
from polymerization of 10 nucleotides to > 50,000
nucleotides per binding event, with 35-fold increase
in the overall rate of polymerization (32). The crystal
structures of the subunit of E:coli DNA polymerase
III holoenzyme, and the corresponding processivity
factors of T4 bacteriophage (gp45, gene 45 protein),
and eukaryotic DNA polymerase (PCNA, proliferat-
ing cell nuclear antigen) have been determined (6365).
The subunit exists as a dimer with a ring structure
and a central cavity that can accommodate a DNA
duplex (63). Each monomer of 366 amino acid residues
consists of three domains of identical topology, each
with a sheet supporting two -helices in a twofold
symmetry (Fig. 5). A dimer formation results in 12 -
helices lining the inner surface of the ring with the
sheets on the outer layer, with an overall structure of a
star-shaped ring. Once loaded by the complex, the
subunit ring can move freely along duplex DNA. The
subunit ring acts as a sliding clamp for the polymerase
core to enable the enzyme for DNA synthesis in a
processive manner. The mechanism regarding how
the dimer ring slips onto the DNA, and eventually
leaves at the end of the template is not fully under-
stood, but some involvement of breaking and closing
of the ring must occur. It has been proposed that the
subunit in the complex plays a major role in binding
the dimer during the loading process (66).
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17
Protein Modication to Optimize Functionality

Protein Hydrolysates
Ton Kunst
Quest International, Naarden, The Netherlands
I. INTRODUCTION mined by the molecular structures and the molecular

interactions of the ingredients; they are affected by the
Proteins are used abundantly in the food industries process conditions.
because of their nutritional value and functional Food research is focused on building up knowledge
properties such as emulsication, foaming, gelation, on the relations between process conditions, structural
hydration, and textural properties. These functional properties, functional properties, and the properties of
properties of proteins are related to their molecular the nal food product. Food product development is
structures and their ability to interact with other often based on a denition of the required properties or
components in the food matrix. The recently published perception of the nal products. The individual compo-
book Functionality of Proteins in Food (1) reviews nents are selected on basis of the required property in
the results of > 50 years extensive research to the nal product and what is (often empirically) known
understand the factors affecting protein functionality about the properties of the individual ingredients. To
and to develop methods to improve functionality. modify the perception of the nal product, normally
Ideally, food proteins should possess a broad range one parameter in the food matrix (e.g., concentration
of functional properties. Most commercial food pro- or pH) or in the process (e.g., temperature) is altered
teins, however, have a limited functionality or have and the effects are studied. If this does not result in the
limitations with respect to their use in food systems. required food product, a new ingredient has to be devel-
A better knowledge about structurefunction relations oped on the basis of a specic functional requirement.
and interactions allows the selection and/or develop- Food scientists can use many methods to broaden
ment of optimal performing ingredients and results in the functionality range and to improve specic func-
the nal food products with improved properties. tional properties of food ingredients. In case of pro-
Figure 1 schematically shows the structurefunction teins, both chemical and enzymatic methods can be
relations in foods. Individual ingredients are combined applied to improve the functionality (25). The present
and are processed into a nal food product. The per- review focuses on the partial enzymatic hydrolysis of
ception of the nal product is determined by its sen- proteins to make peptides with improved solubility and
sory properties, and these are affected by the improved foaming, emulsifying, and nutritional prop-
functional properties of the ingredients in the food erties. Aspects of both the production process and the
matrix. The functional properties in turn are deter- application will be discussed.

Figure 1 Structurefunction relations in foods.
II. PROTEINS, PROTEIN HYDROLYSIS, Neither alkali nor acid hydrolysis is very specic;
AND PROTEIN HYDROLYSATES the rate of hydrolysis of the peptide bond is mainly
determined by the side chain of the amino acid residue
Proteins are linear polymers of amino acids that are of the peptide bond (9). In addition, a number of
covalently linked via a peptide bond. The hydrolysis of amino acids, e.g., cystine, serine, and threonine, are
proteins involves the hydrolytic degradation of this destroyed and undesired components such as lysino-
peptide bond using alkali, acid or enzyme [Eq. (1)]. alanine may be formed (10). Racemerization of
amino acid residues also occurs during alkali hydroly-
sis (4).
Enzymatic hydrolysis is much milder than acid or
alkali hydrolysis and does not lead to the destruction
1 of amino acids. It also is much more specic. By selec-
tion of the proper enzymes the cleavage of specic
Acid hydrolysis has traditionally been used to propeptide bonds is possible. The enzymatic hydrolysis
duce avor products (hydrolyzed vegetable protein or of proteins has extensively been studied by Adler-
HVP). Most of the literature on the production of Nissen (11).
protein hydrolysates with acids is published in the One of the key parameters in protein hydrolysis is
form of patents. A short summary and review is the degree of hydrolysis (DH). This is dened as the
given by Dave et al. (6). Today the use of acid hydro- percentage of peptide bonds cleaved. The DH is
lysis of proteins is limited owing to the risk of the usually approximated by the AN/TN ratio in which
formation of chloropropanols during the hydrolysis. AN is the amount of amino nitrogen and TN is the
Alkali hydrolysis of proteins has long been used to amount of total nitrogen as determined via the
produce foaming agents to substitute egg proteins or to Kjeldahl method. There are different methods to deter-
produce re extinguisher foams. Also, for the alkali mine the number of amino-nitrogen groups in the
hydrolysis most of the literature is published in the hydrolysate. Well-known and often applied methods
form of patents (7, 8). are the Formol titration (12), the determination of

free amino groups by the reaction with o-phthalic pH, the temperature, and the concentrations of sub-
anhydride in the OPA method (13), and the determina- strate and enzyme.
tion of free amino groups by the reaction with 2,4,6- In industrial practice the reaction in general is car-
trinitrobenzene sulfonic acid in the TNBS method (14). ried out in batch form and takes a few hours (16).
The actual properties of a protein hydrolysate are During hydrolysis the degree of hydrolysis increases
determined by the chain length of the peptides. The and phenomena such as enzyme inactivation and pro-
average peptide chain length (PCL), given as the num- duct inhibition start to play a role. After hydrolysis the
ber of amino acids in the peptide, can be approximated enzyme is inactivated (e.g., by heat treatment). The
from the degree of hydrolysis via PCL 100=DH. obtained hydrolysate can be subjected to a number
More important and more informative, however, of posttreatments such as centrifugation or (mem-
than the average peptide chain length is the peptide brane) ltration to remove insoluble components or
chain length distribution. This can be approximated larger peptides. Finally a dried product is obtained
by gel ltration techniques, and can be determined with specic nutritional and functional properties
by methods involving mass spectrometry or by a pro- that are determined by physicochemical properties
cedure involving the Edman degradation technique such as the amino nitrogen (AN), the total nitrogen
(15). (TN), the free and total amino acid prole (FAA and
TAA), and the peptide chain length distribution.
Knowledge about the relations between the nal pro-
III. PRODUCTION OF ENZYMATIC
duct properties and the process conditions allows con-
PROTEIN HYDROLYSATES
trol of the reaction and makes it possible to produce
A. General Aspects hydrolysates that are optimally suited for their appli-
cation.
For the production of protein hydrolysates with
enzymes, a wide variety of endo- and exopeptidases B. The Enzymes
are available as well as a great number of downstream
processing techniques. A schematic overview is shown It will be clear that the selection of a proper enzyme is
in Figure 2. A protein with a certain amino acid com- crucial for the nal results. The choice of the enzyme is
position and sequence is hydrolyzed by one or more determined by parameters such as the required amount
enzymes each with a certain specicity and activity. of free amino acids and the required degree of hydro-
Prior to hydrolysis the protein raw material can be lysis. An enzyme with a broader specicity in general
pretreated. The hydrolysis reaction depends on the results in a higher DH.
Figure 2 Production of protein hydrolysates.

Table 1 Industrially Relevant Endopeptidases Table 2 Examples of Exopeptidases
Enzyme EC number Preferential cleavage Enzyme EC number
Serine proteases 3.4.21 leu-aminopeptidase 3.4.11.1

Chymotrypsin 3.4.21.1 tyr-, trp-, phe-, leu- lys-peptidehydrolase 3.4.11.15
Trypsin 3.4.21.4 arg-, lys- gly-leu dipeptidase 3.4.13.11
Subtilisins 3.4.21.12 mainly hydrophobic- di-peptidyl-peptide hydrolase 3.4.14.4
Cysteine proteases 3.4.22 gly-pro aminopeptidase 3.4.14.5
Cathepsin B 3.4.22.1 arg-, lys-, phe-X- Carboxypeptidase C or Y 3.4.16.1
Papain 3.4.22.2 arg-, lys-, phe-X- Glycine carboxypeptidase 3.4.17.4
Ficin 3.4.22.3 phe-, tyr- Alanine carboxypeptidase 3.4.17.8
Bromelain 3.4.22.4 lys-, arg-, phe-, tyr- Carboxypeptidase S 3.4.17.9
Aspartic proteases 3.4.23
Pepsin 3.4.23.1 aromatic-, leu-, asp-,
glu-
Chymosin 3.4.23.4 cleaves phe105 -met106 how easily it can be hydrolyzed by a given peptidase. It
bond in -casein has been shown that proteins with a higher content of
Metallo proteases 3.4.24 hydrophobic amino acids were more easily hydrolyzed
Thermolysin 3.4.24.27 ile-, leu-, val-, phe- by an enzyme that preferably cleaves after a hydropho-
Neutral proteinase 3.4.24.28 leu-, phe-, and others bic amino acid (11). The structure of the protein raw
material, its solubility, and its state of denaturation all
affect the hydrolysis process.
In its simplied form the hydrolysis of a protein
There are a number of different peptidases, which proceeds according to the scheme originally proposed
can be subdivided into endo- and exopeptidases. There by Linderstrm-Lang (19). It assumes that the native
are four classes of endopeptidases or proteases, which and denatured (reversibly unfolded) state are in equili-
differ in the nature of their catalytic center. Examples brium and that only the (partly) unfolded form can be
of some industrially relevant enzymes and their speci- hydrolyzed by an enzyme into an intermediate form
city are given in Table 1. which then is hydrolyzed to the nal products.
The exopeptidases can be subdivided into a number Basically two types of reactions can be distinguished
of classes. Important exopeptidases are the aminopep- but real hydrolysis processes will be intermediates of
tidases and the carboxypeptidases. The carboxypepti- these two types. In the one-by-one type of reaction the
dases are subdivided according to their catalytic center. overall rate of hydrolysis is determined by the rst step
Exopeptidases are generally not commercially avail- in the process (the reversible unfolding of the mole-
able in puried form but often are used in the form cule). The equilibrium lies strongly on the native side
of a crude extract of microorganisms such as yeast or but once a native protein molecule (partly) unfolds and
lactobacilli or of extracts of gland materials such as a peptide bond can be cleaved, the molecule becomes
pancreatin. In Table 2 some of the more important unstable and further unfolds; more peptide bonds are
exopeptidases are listed. exposed and the intermediate product can be degraded
rapidly into many small peptides. Since the equilibrium
lies strongly on the native side, the protein molecules
C. Protein Raw Materials will be degraded one by one; no signicant amounts of
intermediate products will be present but only native
The protein raw materials commonly used to produce protein and end product. In the zipper type of reaction,
protein hydrolysates on an industrial scale are milk the proteins are rapidly converted into the intermediate
proteins, meat proteins, and a number of vegetable forms. The rate-limiting step is the slow degradation
proteins such as soy and gluten. More recently, rice into the end products.
protein and pea protein (17) are also being used. Fish For those hydrolysis processes that proceed accord-
proteins are also frequently hydrolyzed but their main ing to the one-by-one reaction the denaturation of the
application is animal feed (18). protein (e.g., by heat treatment) will shift the equili-
The amino acid prole of the protein raw material brium of the rst step to the denatured side, and the
not only determines the nutritional properties of the initial reaction rate will increase (11). Extensive dena-
nal hydrolysate, but also affects to what extent and turation, however, impairs the solubility of the protein

owing to molecular aggregation, and this slightly quickly. However, the method only takes solubilized
decreases the hydrolysis rate. peptides into account and consequently the observed
DH may be lower than the actual DH.
D. Reaction Conditions
3. Determination of the Amount of Amino
The reactions conditions such as pH, temperature,
Nitrogen
hydrolysis time, and concentration of enzyme and sub-
strate are selected on basis of the required degree of The increase in the number of amino nitrogen groups
hydrolysis (DH) of the nal product and the pH and can be monitored by taking samples and analyzing
temperature optimum of the enzyme. To control the them as described earlier (OPA, Formol titration,
hydrolysis reaction it is necessary to monitor the DH TNBS). These methods also have the disadvantage of
during the reaction. This can be done with a number of not being continuous in most laboratories; continuous
methods of which the most common are the pH stat, measurements, however, can be done with currently
the measurement of the increase in solubilization, the available automated equipment.
measurement of the osmolarity, and the determination Knowledge and understanding of the relationship
of the amount of amino nitrogen formed. among hydrolysis time, enzyme amount, and degree
of hydrolysis allow the control of the hydrolysis reac-
1. pH Stat tion. Despite many attempts, however, it is still not
possible to give quantitative descriptions of the hydro-
Upon hydrolysis of the peptide bond a free carboxyl
lysis process. This is related to the complexity of the
and a free amino group are formed that are ionized
reactions in terms of specicity and substrate composi-
depending on the pH of the reaction mixture [see Eq.
tion. With 20 different amino acids, 400 different pep-
(1)]. At pH values > 6 the carboxyl group will release a
tide bonds can be formed, for each of which the
H ion and the pH of the reaction mixture will drop.
enzyme will have a different afnity and rate of hydro-
At pH values < 5 the carboxyl group will take up a H
lysis. First the peptide bonds for which the enzyme has
ion and the pH of the reaction mixture will rise. In the
the highest afnity will be cleaved, followed by those
range of 56 the pH will not vary much. Above pH 9,
for which the afnity is lower. In addition, some pep-
H are released from the ammonium group of the
tide bonds are buried inside the tertiary structure and
peptide.
need to be exposed to be hydrolyzed.
When the hydrolysis reaction is carried out at pH
In kinetic studies in general only the rst few min-
values outside the range of pH 56 and is kept constant
utes are taken into account to calculate parameters
during hydrolysis, the amount of alkali or acid
such as Km and Vmax or kcat . The production of protein
required to maintain the pH is a measure of the
hydrolysates on an industrial scale, however, can take
amount of hydrolyzed peptide bonds. This so-called
many hours. Figure 3 shows a typical relation among
pH stat method was originally developed by
process time, enzyme/protein ratio, and DH as mea-
Jacobsen et al. (20). A more detailed description of
sured by the pH stat technique. The steepest increase in
how to use it to determine the DH during hydrolysis
DH takes place in the rst 30 min of the reaction.
is given by Adler-Nissen (11). It is an important tool in
Thereafter the reaction rate slows down. Longer
laboratory-scale experiments. On a production scale it
hydrolysis time and the use of more enzyme result in
has the disadvantages of technical difculties and the
a higher DH. A number of studies are reported in the
extra minerals that are introduced into the reaction
literature in which an empirical model for the hydro-
mixture.
lysis of proteins has been developed. These models are
based either on the amount of solubilized protein (21)
2. Osmometry
or on the degree of hydrolysis (11, 22, 23).
Upon hydrolysis soluble peptides are liberated. As a From the literature data it can be concluded that
consequence the freezing point of the reaction mixture substrate limitation does not play a role in industrial
is lowered. The freezing-point depression can be corre- protein hydrolysis processes. The data in Figure 3
lated with the DH (11). For each set of reactions, how- show that with increasing enzyme concentrations the
ever, the osmometer has to be calibrated by a separate degree of hydrolysis increases. When substrate limita-
determination of the amount of liberated amino nitro- tion is the rate-limiting factor it is expected that the
gen (via TNBS, Formol titration, or OPA). Although degree of hydrolysis at the end of the process is the
it is not a continuous method it can be carried out same for all enzyme concentrations. This is clearly

substrate is present (to prevent autohydrolysis of the
enzyme) and the temperature is not too high (to pre-
vent thermal denaturation).
The control hydrolysis curve in Figure 4 levels off as
expected. In the second experiment, fresh substrate was
added after 135 min and it was found that the rate of
hydrolysis increased. In the third experiment a peptide
cocktail was added at the start of the hydrolysis. This
peptide cocktail was obtained by hydrolyzing the pro-
tein for 20 h, inactivating the enzyme, and removing
the insolubles. It can be seen that the initial rate of
hydrolysis is clearly lower than that of the control.
Similar results were reported by Adler-Nissen (11),
Krause et al. (24), and Haque and Antill (25). Also,
the addition of fresh enzyme was found to result in an
Figure 3 Relation among DH, time, and enzyme concentra- increase in the rate of hydrolysis (21, 23).
tion. Hydrolysis conditions: temperature, 50 C; pH, 8.0; pro-
From these results it can be concluded that product
teinsoy protein, 40 g/L; enzymealcalase 2.4 L; enzyme
inhibition is an important parameter in protein hydro-
concentrations0.2 g/L (^), 0.4 g/L (&), 0.8 gL (~), or
1.2 g/L (*). lysis. The addition of fresh substrate and fresh enzyme
causes a shift in the equilibrium; the addition of peptides
inhibits the reaction. Adler-Nissen (26) measured the
not the case. In industrial protein hydrolysis processes initial hydrolysis rates using hydrolysates with different
the substrate concentrations are high so that substrate DHs as substrates. It was found that the Km was more
saturation most likely prevails throughout most of the or less independent of the DH but that Vmax decreased
hydrolysis reaction (11). with increasing DH. Similar results were obtained by
The data in Figure 4 show the time course of three Snijder and Kunst (27) as shown in Figure 5. From
different hydrolysis experiments in the pH stat. Under these results it is concluded that mixed inhibition or
the hydrolysis conditions as applied, enzyme inactiva- noncompetitive inhibition occurs (28).
tion was found not to be important. In the presence of
10 g/L substrate, 97% of the enzyme was still active
after 48 h at 50 C. In general, enzyme inactivation
does not seem to be very important provided enough
Figure 5 Effect of peptide size on enzyme kinetics.

Hydrolysis conditions: temperature, 50 C; pH, 8.0; sub-
Figure 4 Product inhibition during hydrolysis. Hydrolysis stratepeptides obtained from potato protein. [Intact pro-
conditions: temperature, 50 C; pH, 8.0; proteinpotato protein (^), DH 1.8% (&), DH 4.7% (~), DH 7.5% (*).]
tein, 40 g/L; enzymealcalase 2.4 L, 1.6 g/L. (^) control. Substrate concentrations, 137.6 g/L; enzymealcalase
(*) At t 135 min 20 g/L fresh substrate is added. (&) At 2.4 L; 0.8 g/L; R reaction rate (meq NaOH/L/min);
t 0 min 20 g/L peptide cocktail is added. S substrate concentration (g/L).

E. Downstream Processing G. Immobilized Enzymes
After the actual hydrolysis process the enzyme is inac- The immobilization of enzymes, including proteolytic
tivated via e.g. a heat treatment. The hydrolysate can enzymes, has been extensively studied (see Chapters
be subjected to further processing steps such as separa- 2224 in this book). The advantage of the use of immo-
tion, ltration, or treatments with charcoal or ion bilized enzymes is the possibility to reuse the enzyme
exchange resins (16). Charcoal nonselectively removes (which saves costs) and the possibility to produce
both high- and low-molecular-weight organic com- hydrolysates in a continuous way. The application of
pounds and can be applied to remove off-color mate- immobilized proteolytic enzymes to produce protein
rial and to achieve slight reductions in off-odor and hydrolysates on an industrial scale up to now is lim-
bitterness. Products for patients suffering from phe- ited, however. When the immobilized enzyme is
nylketonuria (PKU) are treated with charcoal and/or applied in a batch reactor it must be separated from
ion exchange resins to remove phenylalanine from the hydrolysate via e.g. a crude ltration step. The
hydrolysates (29, 30). The treatments, however, also costs involved in this isolation can be higher than the
remove tyrosine and reduce the amounts of other savings obtained by reuse of the enzyme.
hydrophobic amino acids. In laboratory experiments immobilized enzymes
Filtration or separation of hydrolysates is generally have been used in columns. Packed columns easily
applied to remove insoluble components in order to get blocked by the insolubles in the raw material or
improve the clarity of the hydrolysate. Membrane by the insolubles formed during hydrolysis (see Sec.
ltration is often used to remove larger peptides and IV.B.3). Expanded bed columns, therefore, have to
undigested protein fragments that are potential anti- be used. With the recently developed beads very
genic materials. This results in a product with a good expanded bed columns can be made that can be
reduced allergenicity. Membrane ltration is also applied at a production scale for adsorption of pro-
applied to reduce the level of endotoxins in the hydro- teins from particulate-containing feedstocks (32).
lysate; this is important when the hydrolysate is These beads can be used also to immobilize enzymes
applied in parenteral formulae or in tissue culture on the surface of the beads. The costs involved are
media. The nal process step usually is drying of the considerable, however, and limit their application as
hydrolysate. carrier for immobilized enzymes.
In addition it is difcult to control the microbiolo-
gical quality of the hydrolysate because microorgan-
isms tend to build up on the carrier material. This
F. Membrane Bioreactors requires operation at elevated temperatures, which
increases the risk of protein denaturation and insolu-
Membrane bioreactors combine the hydrolysis step bilization. The application of immobilized enzymes in
and the membrane ltration step (31). The advantages expanded bed columns thus requires a delicate balance
of a membrane bioreactor are that the enzyme is between microbiological constraints on the one hand
retained (which saves costs), that the peptide size is and protein insolubilization at the other; further
controlled, and that peptides that inhibit the enzyme research is required to establish the best conditions.
are continuously removed after formation.
The disadvantage is that the insoluble fraction
in the hydrolysate is concentrated, which results in IV. APPLICATIONS
ux reduction and reduced productivity. With
prolonged operation times microbiological spoilage Intact, native proteins generally have well-dened
also may become a problem; not only the insoluble three-dimensional structures that affect their func-
protein material is retained, but also all micro- tional properties. Peptides are much smaller, the mole-
organisms. Especially, the more thermophillic cular structure is more random, and a tertiary structure
microorganisms may give rise to uncontrolled is rarely observed (33). For the secondary structure of
hydrolysis reactions. It is for these reasons that mem- proteins, the -helices and the -pleated sheets are the
brane bioreactors are currently not applied on an most important structures. Peptides in general do not
industrial scale for the production of protein hydro- have sufcient length to have enough H bonds to sta-
lysates. Further research is required to optimize the bilize the helix. Upon hydrolysis of proteins the hydro-
process. phobic areas, which are normally buried inside the

folded molecule are exposed and as a consequence the range, which allows them to be used in refreshing
properties of a protein hydrolysate strongly differ from drinks (36).
those of the original protein. Not only is the nutritional value determined by the
Enzymatically hydrolyzed proteins have a wide amino acid composition, but also the peptide chain
range of both food and nonfood applications, which length distribution seems to be an important factor.
relate either to their nutritional or to their functional Some lactic acid bacteria for instance prefer di- and
properties. Larger peptides (25 kDa) are mainly used tripeptides over free amino acids (42, 43). Also, in
as functional ingredients (aeration) or in personal-care the human small intestine most of the dietary proteins
products. Medium-size peptides (12 kDa) are used in are absorbed as small peptides. There are specic car-
clinical nutrition (34) and sports nutrition (35, 36). riers for small peptides that are separate from the car-
Smaller peptides (< 1 kDa) are used in infant food riers for uptake of free amino acids (44). These small
products requiring a reduced allergenicity (15) and peptides are more adequately absorbed in the small
for fermentation and tissue culture media (37). intestine than the equivalent free amino acids. This
allows the formulation of products for enteral nutri-
A. Nutritional Properties tion that gives less intestinal discomfort, as less amino
acids are available for bacterial fermentation in the
1. Nutritional Value
large intestine.
The nutritional value is primarily determined by the
amount of essential amino acids in the protein hydro-
2. Reduced Allergenicity
lysate and mainly depends on the amino acid composi-
tion of the protein raw material. Under some Food allergenicity is a complex topic, and the manifes-
conditions a specic amino acid composition is tations of disease and the diagnostic methods are
required. For example, products for patients suffering highly variable (45). In the majority of allergic
from phenylketonuria (PKU) should be very low in responses a specic immunoglobulin (IgE) mediates
phenylalanine. These products are produced from the immediate hypersensitivity reaction. Immuno-
highly hydrolyzed proteins and are subjected to specic globulins (also called antibodies) are produced by the
downstream processing such as treatments with char- body in response to invasions of foreign compounds
coal or adsorption chromatography resins to remove such as proteins. Materials that elicit antibody produc-
phenylalanine (29, 30). Another example is hydroly- tion (other than IgE) in an organism are called anti-
sates with a high glutamine content. Glutamine is gens. An antigen may contain multiple antigenic
regarded as a conditionally essential amino acid that determinants (epitopes), which are small regions of
is required in periods of physical stress such as severe the antigen molecule that specically elicits the produc-
trauma, surgery, infection, starvation, or heavy train- tion of antibodies to which the antigen binds. In case
ing (38). Peptides rich in glutamine are also increas- an antigen elicits an IgE response, the antigen is called
ingly used for cell nutrition (37). In general such an allergen. In order to initiate a clinical manifestation
products are produced from wheat gluten, which con- of allergenic responses, it is required that the IgE form
tains 27% glutamine. a bridge between two epitopes; the allergen thus should
With respect to sports nutrition it has been shown have at least two epitopes at a certain distance from
that protein hydrolysates stimulate recovery after pro- each other. Destroying the molecular structure of the
longed intensive exercise (39). During high-intensity allergen or antigen forms the basis of the application of
exercise, athletes use up their glycogen reserves, and protein hydrolysates in the control of food allergies.
the time needed to restore their muscle glycogen levels Decreasing the size of the molecules via hydrolysis
determines the time needed to recover from fatigue reduces the number of protein molecules with two epi-
(40). Although the insulin release is stimulated primar- topes (and may even destroy epitopes) and will reduce
ily by carbohydrates, the release of insulin is further the antigenicity (46).
stimulated by proteins and peptides (41), resulting in There is, however, no denite molecular weight
an increased uptake of glucose from the blood, an below which peptides are nonallergenic or above
increased glycogen synthesis rate, and thus an which they are allergenic. In general there appears to
enhanced recovery after prolonged intensive exercise. be a limit for antigenicity for peptides with 1015
Contrary to proteins, peptides are heat stable and can amino acids (47). The rst products, which were mar-
be applied in pasteurized, ready-to-use drinks. keted 40 years ago, comprised > 70% free amino
Furthermore, peptides are soluble over a wide pH acids and peptides up to 8 amino acid residues long.

More recently developed products have < 10% free standing of the physicochemical background of foams
amino acids and peptides up to 15 amino acid residues and emulsions. More detailed information can be
long (15). These developments are based on a better found in recent reviews and textbooks on this subject
understanding of the physiology of the small intestine (1, 55, 56).
(see Sec. IV.A.1).
1. Surface Properties
3. Bioactive Peptides
During the formation of emulsions or foams, energy is
It has long been known that proteins can be the source
applied to disperse, deform, and break up oil droplets
of bioactive peptides. Especially the milk proteins have
or air bubbles in an aqueous phase. To form a good
been extensively studied (48). The main problem is how
foam or a good emulsion it is required that the newly
to isolate them in a commercially attractive manner.
formed air bubbles or oil droplets not (re)coalesce and
As of now only casein phosphopeptides can be manu-
that once the energy input has stopped, the obtained
factured on an industrial scale using a process invol-
foam or emulsion is stable for some time. To meet
ving ion exchange chromatography (49) or a process
these requirements surface-active agents have to be
involving an aggregation step and an ultraltration
used. Surface-active agents are adsorbed at the oil
step (50). Possible applications of casein phosphopep-
water interface (in emulsions) or airwater interface
tides, which relate to their mineral binding capacity,
(in foams) and lower the interfacial tension. Different
are in the treatment of caries (51) or as mineral carriers
aspects, however, play a role in the formation on the
to promote Ca2 uptake (52, 53). Further research is
one hand and stabilization of foams and emulsions on
required to design enzymes that allow the specic lib-
the other hand.
eration of those sequences that are known to have a
biological activity. a. Formation of Foams and Emulsions. During
the formation of emulsions or foams, the oil droplets
B. Functional Properties or air bubbles are subjected to shear stresses and they
break up when the stress exceeds the Laplace pressure
Proteins and peptides are more than just a source of difference. The Laplace pressure difference (p) is
essential nutrients. They also have functional proper- given by p 2=R in which is the surface tension
ties allowing them to contribute to the sensory proper- and R is the radius of the oil droplet or the air bubble.
ties of the nal food product (54). Table 3 shows some From this it follows that for a given energy input smal-
functional and organoleptical properties of protein ler oil droplets or air bubbles are formed when the
hydrolysates. The surface properties of peptides are surface tension is lower. The (re)coalescence is pre-
especially important. To understand the role of protein vented when the surface-active agent in the interface
hydrolysates it is necessary to have a general under- prevents the aggregation of oil droplets or air cells by
electrostatic or steric repulsion.
Table 3 Important Functional and The most important property for surface-active
Organoleptical Properties of Protein agents during the formation of foams and emulsions
Hydrolysates is the ability to quickly reduce the interfacial tension
(57, 58). In the case of a protein or a peptide this
General property Specic property requires that the molecule rapidly adsorb at the inter-
Surface Emulsication face and rapidly unfold to maximize the reduction of
Foaming the interfacial tension.
Hydration Solubility Movement to and adsorption at the interface. Much
Textural Elasticity of the current knowledge on the movement to and
Viscosity adsorption of proteins at interfaces is collected in dif-
Dough relaxation fusion-driven static systems. It is important to realize
Gelation that emulsication and whipping processes are highly
Organoleptic Taste agitated and that in such systems convection is more
Smell important. The time of movement of the protein or
Flavor binding
peptide molecule to a newly formed interface is in the
Color
order of ms. After arrival at the interface, proteins and
Mouth feel
peptides can be adsorbed. The major driving force for

adsorption is the reduction in contact area between The surface elasticity increases when the molecules in
non-polar groups and water. This reduces the free the interface have more intermolecular interactions.
energy of the system and thus the interfacial tension.
The adsorption of proteins at an interface is positively Aggregation or occulation. In this process air
correlated with their surface hydrophobicity (55, 59). bubbles or oil droplets stick together. This process is
retarded when there is more electrostatic repulsion or
Unfolding of the molecule and formation of lms. A more steric repulsion.
protein molecule that is adsorbed at an interface can
unfold. The thermodynamic environment of the inter- Coalescence. After the aggregation of air bubbles
face differs from that of the bulk phase, and unfolding or oil droplets the thin lm separating them may break
results in a reduction of the free energy. The driving and they can ow together. The stability to coalescence
force for unfolding probably is the hydrophobic effect can be improved by increasing the surface viscoelasti-
(56). city by an increase of the intermolecular interactions.
The surface tension of a clean airwater interface is
c. General Requirements. The most important
72 mNm1 , which is sufcient to overcome the activa-
properties for a good emulsifying or foaming agent
tion energy barrier for protein unfolding. The structure
thus are the ability to rapidly reduce the interfacial
will rearrange such that the nonpolar segments are
tension and the ability to form a viscoelastic lm that
predominantly located in the gas phase and the hydro-
is resistant to rupture. These two properties, however,
philic parts in the aqueous phase. The foaming power
are related as can be seen for -lactoglobulin and -
is related to the rate and extent of unfolding and this
casein.
depends on the exibility of the molecule. The exibil-
-Lactoglobulin is a globular protein that slowly
ity of the molecule is determined by the strength of the
adsorbs and unfolds at the interface, but it can form
forces maintaining the structure such as S-S bridges,
strong lms. It performs rather poorly in the formation
electrostatic interactions and repulsions, and the aver-
of emulsions and foams but it has good stabilization
age hydrophobicity (59).
properties. -Casein, on the other hand, is a random
The surface tension of a clean oilwater interface is
coil molecule that quickly adsorbs at the interface; it
only 30 mNm1 , which most probably is not enough
cannot, however, form very strong lms. It has good
to overcome the activation barrier for protein unfold-
emulsication and foaming properties but poor stabi-
ing (55). Consequently for emulsions the surface
lization properties.
hydrophobicity is more important than the average
Modications of (globular) proteins via limited
hydrophobicity (4, 60).
hydrolysis or by rupture of intramolecular disulde
b. Stability of Emulsions and Foams. Once an bridges in general will result in more unfolding of the
emulsion or a foam is formed, its lifetime in part molecule. This will increase the exibility and improve
depends upon the viscoelastic properties of the the ability to quickly reduce the interfacial tension and
adsorbed protein layers and their resistance to rupture. improve the foaming and emulsifying properties, but it
Both foams and emulsions are thermodynamically will reduce the ability to form a coherent lm and
unstable systems and are subjected to various types reduce the stabilization properties.
of instabilities: Figure 6 shows the interfacial properties of intact
protein and protein hydrolysate on an air-water inter-
Drainage (foams) or creaming (emulsions).
face. The intact protein gives a surface pressure of
Drainage and creaming are caused by density differ-
30 mNm1 . The peptides obtained after hydrolysis
ences between the phases. This process can be retarded
gives a signicantly higher surface pressure indicating
by increasing the viscosity of the continuous phase or
a higher foaming capacity. The surface elasticity,
by gelation of the continuous phase.
which is a measure for the amount of interactions in
Disproportionation (Ostwald ripening). This occurs the surface, however, is much larger for the intact pro-
when the dispersed phase is soluble in the continuous tein than for the peptides. This indicates that the pro-
phase; it therefore only plays a role in foams. The tein contributes more to the stability of a foam.
Laplace pressure difference is greater in smaller bub- For good interfacial properties a peptide should
bles, and as a consequence gas diffuses from small bub- have clustered hydrophobic and charged areas and a
bles (which shrink) to large bubbles (which grow). This molecular weight above a certain minimum to allow
process is retarded when the bubbles have a uniform this distribution (62, 63). The distribution of charge
size and when the surface elasticity Ed exceeds =2 (61). and hydrophobicity can be derived from the amino

Figure 7 Hydrophobicity pattern of -lactalbumin. The
hydrophobicity is calculated using the hydrophobicity data
given by Rose and Dworkin (65). The protein parts that have
a hydrophobicity > 0:72 will be buried in the structure.
2. Gelation and Water Binding

The formation of a gel requires large protein molecules
that can form crosslinks in all three dimensions. The
strength and hardness of a gel are related to the size
and shape of the molecule rather than to the amino
acid composition. A very good correlation between
the average molecular weight and the gel hardness
was found (68). Hydrolysis of proteins, which reduces
the size of the molecules, is therefore expected to
reduce the ability to form a gel.
However, it has been shown that the hydrolysis of
whey protein (69) or casein (70) can lead to the forma-
tion of aggregates and the formation of a gel. Electron
Figure 6 Surface properties of proteins and protein hydro- microscopy showed that these gels have a particulate
lysates. The surface properties have been measured in a ring
type of structure. The aggregates in the whey protein
trough at ambient temperature. (a) Surface pressure. (b)
Surface elasticity. Pea protein hydrolysate: ^; intact pea
hydrolysate consist of a number of peptides with mole-
protein: &. cular weights of 20006000 daltons which are held
together by noncovalent interactions. It is hypothe-
sized that a limited hydrolysis causes the protein to
unfold after which exposed hydrophobic areas can
acid sequence of the protein or peptide (64, 65). An interact intramolecularly to form aggregates (71). The
example of the distribution of the hydrophobicity in aggregates from the casein hydrolysates were found to
-lactalbumin is given in Figure 7. It will be clear that have a higher proportion of hydrophobic amino acids.
hydrolysis with different enzymes will give peptides It is likely that the phenomenon of aggregate forma-
with different charge and hydrophobicity distributions, tion is sometimes misinterpreted as a plastein reaction.
and this explains the importance of enzyme selection in At this moment the potential for improvement of the
the production of surface-active peptides. texture of food products is not clear and further
Both foaming properties and emulsifying properties research will be required.
are strongly related to the size of the peptides.
Literature reports that the optimum peptide size gen-
3. Solubility
erally lies in the range of 1535 amino acid residues.
This corresponds to a DH of typically 36% (17, 66). Partial hydrolysis of proteins is often used to improve
Chobert et al. (67) and Turgeon et al. (62) report mini- the solubility of a protein (Fig. 8). For optimal solubi-
mum peptide sizes of 2000 daltons. Also, Caessens lization of soy protein a DH of 8% or higher is
(63) reported that smaller hydrophilic peptides have required (66). A 100% solubility, however, is never
poor foam and emulsion stabilizing properties. achieved; this is related partly to peptidepeptide inter-

Individual peptides will each have a different aver-
age hydrophobicity but also different threshold values
and different bitterness intensity. The conformation of
a peptide and how well it ts in the bitter receptor in
the mouth will affect this. Peptides with a hydrophobic
residue at the C-terminus are reported to be more bit-
ter (73). Hydrophobic peptides are more bitter when
there is another amino acid on both the N- and the C-
side of the hydrophobic residue(s). Bitterness is the
major limitation of the use of protein hydrolysates,
and the reduction, prevention, or removal of the bitter-
ness of protein hydrolysates has been studied exten-
sively.
In an intact globular protein the majority of the
Figure 8 Effect of DH on solubility at various pH values.
Intact protein (*), DH 1.0% (^), DH 3.2 (~), DH 5.1% hydrophobic side chains is hidden in the interior of
(), DH 7.7% (Y), DH 8.3% (*). The hydrolysates were the molecule and cannot interact with the taste recep-
obtained by hydrolyzing soy protein with alcalase 2.4 L. The tors. Upon hydrolysis the protein molecule unfolds
solubility was measured in a 1% protein dispersion in 0.2 M and is degraded into smaller peptides. The hydropho-
NaCl. (From Ref. 66.) bic amino acids now are exposed to the solvent and can
interact with the taste receptors. As the hydrolysis pro-
cess proceeds, more hydrophobic amino acids are
actions and partly to the presence of compact struc- exposed and the bitterness increases. When the hydro-
tures in the protein that cannot be hydrolyzed. Further lysis process proceeds further, the number of hydro-
research will be required to elucidate these limiting phobic amino acids which have another amino acid
factors and to develop methods or new enzyme pre- at both the N- and the C-side decreases and the bitter-
parations that allow the degradation of these compact ness will pass through a maximum. At very high
structures. degrees of hydrolysis, however, a high amount of free
Although peptides as such may be soluble, in a nal amino acid will also be present and this will induce a
application insoluble complexes can be formed due to savory avor. Figure 9 shows the general relation
interactions with other ingredients in the food matrix. among DH, bitterness, and savory avor.
In drinks, for example, peptides may associate with To reduce the bitterness, methods involving specic
polyphenols or caramels, and the formed complexes enzymatic treatments, selective separations, and mask-
can precipitate. In drinks also the concentration of ing can be applied (74). The bitterness of a hydrolysate
the hydrolysate can be important; for soy protein can also be affected via the specicity of the enzyme.
hydrolysates it has surprisingly been found that at Endopeptidases that give peptides with nonpolar
low pH the solubility is better at higher concentrations amino acids at the C-terminus were found to result
(11).
C. Organoleptic Properties
In food applications taste and smell are very important

parameters. When food proteins are hydrolyzed a bit-
ter taste and a savory taste usually develop. In addi-
tion, protein hydrolysates frequently carry the taste
and smell characteristics of the raw material from
which they are derived. The savory taste and smell
are related to the presence of very small peptides and
free amino acids. The bitterness is related to the size
and the average hydrophobicity of the peptides (72).
This can be calculated using the hydrophobicity values
of the individual amino acids as reported in the litera- Figure 9 General relation between DH and organoleptical
ture (11). properties.

in hydrolysates with a relatively high bitterness (75). exopeptidases on the reversed-phase HPLC chromato-
The application of both aminopeptidases (76) and car- gram of the hydrolysates. With increasing elution time,
boxypeptidases (77) was found to reduce the bitterness the hydrophobicity increased. The bitterness was
of hydrolysates. Figure 10 shows the effect of using found to be strongly correlated with the amount of
material eluting after 50 min (78, 79). The neutral pro-
tease hydrolysate contains much hydrophobic material
and is very bitter. When exopeptidases are used, less
hydrophobic material is present and the bitterness is
reduced. It should be realized, however, that the savory
taste will increase owing to increased levels of free
amino acids. Because of this increased level of free
amino acids the osmolarity of the hydrolysate also
increases. When applied in enteral feeding this may
result in an inux of water into the intestine, which
can cause diarrhea.
Another method to reduce the bitterness is the plas-
tein reaction (80). The original idea behind it is that the
molecular weight is increased via a protein resynthesis
but later it was thought that transpeptidation is giving
the effect (81). As a result of the reduction insoluble
components are formed, which are not perceived as
bitter. However, it is also possible that the formation
of aggregates of hydrophobic peptides (see Sec. IV.B.2)
results in a reduction of the number of exposed hydro-
phobic side chains and consequently also results in a
reduced bitterness.
Murray and Baker (82) found that treatment with
activated carbon lowered bitterness. This procedure
suffers from the disadvantage that the protein losses
can be high (83) and that the selective removal of
hydrophobic peptides negatively affects the nutritional
properties (66).
Next to processing and selective separation meth-
ods, several methods to mask the bitter taste of protein
hydrolysates via the addition of specic components
have been reported. Polyphosphates, glycine, gelatin,
gelatinized starch, cyclodextrins, glutamine, and aspar-
agine were found to mask bitterness (74).
V. CONCLUDING REMARKS
Figure 10 Reversed-phase patterns of casein hydrolysates. Enzymatic hydrolysis is a valuable and exible tool to
(Top) Casein hydrolyzed with an endopeptidase (Neutrase, modify and improve the functional properties of pro-
NOVO). (Center) Casein hydrolyzed with an endopeptidase teins. To be able to produce a specic hydrolysate it is
(Neutrase, NOVO) and a carboxypeptidase (obtained from
necessary to have: (a) a detailed knowledge about the
autolysed yeast). (Bottom) Casein hydrolyzed with an endo-
relations between process conditions and the proper-
peptidase (Neutrase, NOVO) and an enzyme mixture con-
taining both carboxypeptidase and aminopeptidase ties of the hydrolysate obtained from a given protein
(BioProtease P23, Quest International). The casein hydroly- and enzyme in combination, and (b) a detailed know-
sates were analyzed on a C18 reversed-phase column using a ledge about the relations between the physicochemical
gradient of 0.1% TFA in water to 0.1% TFA in acetonitrile properties of the hydrolysate and their functional
as eluent. properties.

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18
Production and Modication of Acylglycerides
Rob M. M. Diks
Unilever Research and Development, Vlaardingen, The Netherlands
I. INTRODUCTION surface-active type of acylglycerol. They are widely

applied to stabilize water-in-oil as well as oil-in-water
Acylglycerides are important ingredients of a wide emulsions such as those present in spreads, dressings,
variety of food products, especially those consisting and cosmetic creams (4).
of emulsions of water and oil. Acylglycerides consist In general, the physical behavior of an acylglycer-
of a glycerol backbone with one, two, or three fatty ide, and hence its commercial value, is related to its
acid chains attached to it, thus giving a mono-, di-, or fatty acid composition. Hence the ability to specically
triglyceride molecule. The delicate balance between the modify its composition allows the production of tailor-
apolar hydrophobic fatty acids and the polar OH made acylglycerols. Using conventional organic synth-
groups in the molecule, as well as the type of fatty esis many acylglycerols are being produced on a larger
acid involved, determines the physical behavior of scale. These processes are generally carried out at rela-
the acylglyceride and hence its specic function in the tively high temperatures and pressures, using rather
food product. unspecic catalysts if not fully random catalysts. As
Triglycerides can well be considered the most a result, complex multistep processes are required to
important class of acylglycerides. Being the major obtain the desired product. Moreover, toxic bypro-
component of edible oils and fats, they serve many ducts can be formed as well as off-avor and colors,
functions in daily food products. Apart from being again requiring substantial posttreatment and down-
an energy source, they are nutritionally important for stream processes, especially when food applications
their dietary supply of essential fatty acids, as well as are concerned.
fat-soluble vitamins and antioxidants. Furthermore As opposed to this, enzymes are highly specic and
they signicantly contribute to product characteristics can operate under mild conditions of temperature,
such as spreadability, mouth feel, and the avor release pressure, and pH. In recent years much attention has
during consumption. therefore been focused to the exploitation of enzymes,
Diglycerides are used generally as emulsifers, often especially lipases, for lipid modication.
in a mixture with monoglycerides. However, they are In this chapter various aspects of the use of lipases
also known to act as crystal habit modier in triglycer- for the modication of acylglycerols will be high-
ide mixtures (1, 2), and could act as a building block lighted. Firstly, the general functioning of lipases,
for the organic synthesis of, e.g., drug and phospholi- their specicities and the importance of water in
pids (3). lipase-catalyzed reactions are discussed. Secondly,
Having only one apolar fatty acid chain and two some process engineering aspects of lipase catalyzed
polar hydroxy groups, monoglycerides are the most processes are covered. This comprises the immobiliza-

tion of lipases, reactor design, and modeling as well as dients, this is sometimes an important benet. Apart
the operational stability of the biocatalyst. Finally, from the requirement of food grade solvents, its
examples of glyceride modication processes are absence prevents the need for solvent removal and
reviewed. Though the production of monoglyerides recovery, thus reducing processing costs.
and diglycerides is covered, the main focus in this The ability of lipases to catalyze fatty acid exchange
chapter will be on the modication and processing of reactions on acylglycerols results from two major char-
triglycerides, being the most important processes acteristics. Firstly, the construction of the active site
applied on an industrial scale. of this class of enzymes. It consists of three amino
acids forming a so-called catalytic triad (16). In close
interaction, they cause the lipase to split, e.g., a trigly-
II. LIPASES ceride molecule in a covalent lipasefatty acid complex
and a diglyceride (Fig. 1). In a second reaction step a
A. General Properties water molecule comes in, thereby releasing the fatty
acid and returning the lipase to its native state, thus
Probably the most important class of enzymes studied completing a full hydrolysis cycle (9). This two-sub-
in general are lipases, i.e., glycerol ester hydrolases (EC strate (triglyceride and water) two-step model is gen-
3.1.1.3), especially those that are obtained from micro- erally referred to as the Bi-Bi, Ping-Pong model (59).
bial sources. In living organisms the function of lipases In a similar manner a new ester bond can be estab-
is to hydrolyze oils and fats into free fatty acids, a lished when a new diglyceride molecule reacts with the
mixture of partial glycerides (mono- and di-) and gly- existing lipasefatty acid complex, forming a new tri-
cerol. The latter products are taken up by the organism glyceride molecule. This scheme highlights the impor-
to provide energy and growth intermediates. The tance of diglycerides as intermediate in trans- and
industrial equivalent of this process is widely employed interesterication reactions and hence the necessity of
in detergent systems for laundry cleaning (5). a minimum degree of hydrolysis to start the interester-
In the early 1980s it was shown that under very dry ication reaction.
conditions, such as in organic solvents, lipases remain Secondly, lipases are characterized by a phenom-
active and sometimes even show novel characteristics enon called interfacial activation (10, 11). The entrance
such as improved stability (6). Over the years it has to the active site is covered by a lid or ap when dis-
been shown that lipases in organic media are capable solved in water. However, when in contact with an
of carrying out all major reactions important for interface between water and apolar phase, i.e., oil, or
acylglycerol modication, e.g., hydrolysis, esterica- being dispersed in organic solvent, the ap opens,
tion (Fig. 1), and trans- or interesterication reactions allowing substrates to enter the active site. Thus in a
(Fig. 2). system of a polar and an apolar phase, lipases are
Several extensive reviews on the subject have highly active on substrates present in the apolar
recently been published (7, 8). It should be realized phase, whereas their activity toward water-soluble sub-
that when referring to organic media, this also covers strates is rather low. As a result of the activation in the
liquid oils, i.e., solvent-free systems. For certain appli- presence of an organic phase, lipases, individually xed
cations, especially those aimed at food product ingre- on an inert carrier by adsorption, also show their activ-
ity in organic solvents and vegetable oil. In fact, the
lipases here are located at the interface between the
organic medium and the solid carrier material.
Though lipases are generally considered true cata-
lystssubstances that accelerate the reaction rate but
do not affect the equilibrium compositionthe above-
described phenomenon should be taken into account
when operating in a biphasic system. For example,
using Candida rugosa lipase for triglyceride hydrolysis
> 90% free fatty acids will be formed (12), whereas
90% degree of esterication is obtained when using
fatty acids and long-chain alcohols (> C12 ) (13) or
sterols as substrates (14, 15). The latter result repre-
Figure 1 Reactions on acylglycerides catalyzed by lipases. sents actual thermodynamic equilibrium composition.

Figure 2 Interesterication and acidolysis of triglycerides with 1,3-specic lipases. The rst-order products always comprise two
1,3-isomers.
However, in the former case the synthesis reaction is It should be noted that for the Rhizomucor miehei
blocked by the fact that glycerol is strongly diluted in lipase a surprising phenomenon was reported in that
the aqueous phase and cannot effectively enter the the lipase showed a preference for the sn-1 position
active site of the lipase. when operating under very dry conditions (18). This
property could allow for stereospecic synthesis to be
B. Lipase Specicities carried out. As opposed to this regiospecicity,
lipases, such as that from Candida rugosa and the
Though the catalytic triad principle mentioned above one from Geotrichum candidum, are random and
is true for all lipases, the active site of lipases comes in hence can modify the fatty acids on all positions of
different shapes and sizes (16, 17). This restricts the the acylglycerols.
way in which acylglycerols can bind, or slows down The second type of specicity relates to the type of
and even prohibit certain acylglycerols from binding fatty acid being involved. For example, certain Cuphea
at all. The result is a preference of the lipase to react lipases show a distinct selectivity for short chain fatty
on certain acylglycerols or positions within, e.g., a tri- acids. Another important example is Geotrichum can-
glyceride molecule. This is often addressed as the selec- didum lipase B. Though being non regiospecic, it
tivity or specicity of a lipase. shows a strong selectivity for fatty acids with a cis--
Three major types of specicity can be distinguished 9 double bond (20, 21). Both this lipase and that from
(Table 1). The rst type involves positional or regio- Candida rugosa display a strong selectivity against
specicity, most lipases being either 1,3-specic or fully long-chain polyunsaturated fatty acids. This property
random (nonspecic). The 1,3-specicity is the most is probably related to the tunnel shape of the fatty acid
common and is displayed by, e.g., human gastric binding site of both lipases (22).
lipases, as well as microbial lipases such as from The easiest way of exploiting these fatty acid selec-
Rhizomucor miehei, Thermomyces sp., and various tivities in product purication or fatty acid enrich-
Rhizopus sp. As a result of a trough-shaped active ments is in single-stage reactions such as hydrolysis
site, triglycerides can bind only in specic positions or esterication, in combination with a physical
allowing lipases to modify acylglycerols on the sn-1 means of separating the desired end product and the
and sn-3 positions, both in hydrolysis and inter- or residual unwanted fatty acid fraction (see section on
transesterication. fatty acid enrichment).

Table 1 Exploitation of Lipase Specicities for Acylglycerol Modication
Specicity Lipases Application
Regiospecicity
sn-1, sn-3 specic Rhizomucor mieheia Triglycerides
Rhizopus oryzae interesterication/acidolysis
Rhizopus arrhizus synthesis by esterication
Rhizopus delemar Diglycerides
Rhizopus niveus triglyceride hydrolysis/glycerolysis (directed) fatty acid
Porcine pancreatic lipase esterication
Monoglycerides
triglyceride hydrolysis/glycerolysis fatty acid
esterication
Nonspecic Candida rugosa Fatty acid production by hydrolysis
Chromobacterium viscosum Mono- and diglycerides by directed glycerolysis
Pseudomonas uorescens Fatty acid production by hydrolysis
Pseudomonas cepacia Mono- and diglycerides by directed glycerolysis
Fatty acid specicity
Short-chain fatty acids Cuphea sp. Production of fatty acid concentrates by selective
hydrolysis
Long-chain poly- Geotrichum candidum Production of enriched glycerides by selective hydrolysis
unsaturated acids or enriched fatty acids by esterication
Candida rugosa Production of enriched glycerides by selective hydrolysis
or enriched fatty acids by esterication
Cis-9 unsaturated fatty Geotrichum candidum B Production of enriched glycerides by selective hydrolysis
acids or enriched fatty acids by esterication
Partial glyceride specic
Monoglycerides Potato acylhydrolase (patatin) Monoglycerides by fatty acid esterication
Mono- and diglycerides Penicillium camembertiib Mono- and diglycerides by fatty acid esterication
Penicillium cyclopiumb Mono- and diglycerides by fatty acid esterication
a
sn-1 specic under semi-dry operating conditions (18).
b
The selectivity for monoglycerides over diglycerides varies with the water activity (114, 115).
The third major lipase specicity is that for certain terms of the thermodynamic water activity maintained
types of acylglycerols. Penicillium camembertii lipase is in the reaction medium. It allows comparison of results
known to act upon mono- and diglycerides only, of enzymatic reactions in various organic solvents,
whereas other Penicillium lipases are reported to even though absolute water concentrations are signi-
hydrolyze only triglycerides. Patatin, a potato acyl cantly different (26).
hydrolase, is even more specic, almost exclusively It has been shown that the form of this relationship
producing monoglycerides in synthesis (23). is rather lipase specic. For example, Rhizomucor mie-
Unfortunately, details on its active site are not yet hei lipase has an optimum water activity 0:5 and still
available. retains 40% of its maximum activity at water activities
close to zero (27, 28). On the other hand, the lipases
C. Effect of the Thermodynamic Water Activity from Candida rugosa, Humicola, and Pseudomonas
cepacia not really showed a maximum, their activity
Apart from being a substrate, water also has a vital
function in maintaining the three-dimensional confor-
mation of the enzyme and hence its intrinsic activity
Humicola lanuginosa, currently known as Thermomyces
(24, 25). This effect is most conveniently expressed in lanuginosus.

continuously increasing with the water activity up to III. REACTOR ENGINEERING ASPECTS
saturation level (29). The relationship is rather inde-
A. Efciency of Free Lipases
pendent of the type of reaction catalyzed by the lipase
(30), the carrier material of the lipase (28) as well as the
Using free lipases in organic media, one problem to
type of solvent applied (27). However, in polar solvents
overcome is the low activity of crude enzyme powders.
interactions with the solvent molecule itself may affect
High lipase input obviously reduces the economic via-
the enzyme activity (31), which may explain the low
bility of the system and is hence not feasible. Both
activity of certain lipases in such solvents (32).
physical and chemical modication of free lipases
The existence of an optimum water activity urges
have been developed, describing increased lipase solu-
proper control of the water activity throughout a reac-
bility and activity.
tion. In fact several methods have been developed
In most cases chemical modication involves the
which allow accurate control of the water activity in
covalent coupling of a wide variety of hydrophobic
batch as well as continuous reactor systems: e.g.,
molecules onto lysine amino acid residues on the lipase
vacuum, controlled air sparging (33), membrane per-
(45, 46). This generally results in an enhanced activity
vaporation (34), and equilibration with concentrated
and stability of the catalyst. It was shown that in cer-
salt systems (35, 36). These techniques have been
tain cases these effects are due to an increased porosity
shown adequate to obtain optimum performance of
of the lipase-aggregate structure obtained after lyophi-
biocatalysts in ester synthesis.
lization of the lipase (47). A similar phenomenon may
In some cases water activity control during the pro-
explain the enhanced activities reported after physical
cess is not relevant at all. For example, in the synthesis
modication of lipases. This technique often involves
of esters from hydrophobic fatty acids and hydropho-
drying of a lipase in the presence of hydrophobic com-
bic alcohols degrees of synthesis in the range of 90%
pounds, often lipids (48, 49).
can easily be reached, even in the presence of free water
An alternative modication method, referred to
(13, 14). Moreover, this is independent of the type of
as in situ immobilization, is based on the conversion of
lipase applied (13).
an emulsion of a lipase solution in oil into a dispersion
A more general case is the interesterication or acid-
of solid particles. The latter is achieved by drying the
olysis of oils and fats. Starting with a pure triglyceride
system under reduced pressure. The intensity of mixing
(acylglycerol) mixture, the thermodynamics of the sys-
during the drying stage determined the average particle
tem will cause nearly all water initially present to be
size of the biocatalyst thus obtained (50). Despite these
used for hydrolysis (26). Though this reaction does
developments, dispersion of the dried modied lipase
create the diglyceride intermediates required for the
preparations in hydrophobic media remains essential
interesterication reaction to proceed, it simulta-
but difcult, especially when highly viscous oils are
neously results in a signicant reduction of the water
involved. Moreover, recovery of the suspended lipase
activity. Moreover, the latter may easily drop below
from the reaction medium for reuse may require sig-
the optimum of the lipase applied. Adding extra
nicant catalyst handling and operational cost.
water to the system to increase the aw is no solution
as it will only result in further hydrolysis and hence
decreased product (modied triglyceride) yield (37). B. Immobilization of Lipase on Inert Carriers
Thus, quickly losing water by hydrolysis, the major
part of the interesterication reaction will proceed at a The use of macroscopic inert carrier materials to immo-
very low equilibrium water activity (3842). As a result bilize lipase is important, as it allows easy catalyst
of this drying process, the initial water activity of the handling and reuse after separation of the catalyst by
catalyst itself is of less importance, especially in a con- sedimentation or ltration. A vast amount of literature
tinuous reactor system. Instead, the supply of water via has been published on the immobilization of lipases on
the feed stream to a reactor is essential in order to solid surfaces or inert carrier materials. The methods
establish a low but constant water activity in the sys- described cover covalent coupling, precipitation,
tem, thereby preventing exhaustive drying of the cata- entrapment, and adsorption. Moreover, many different
lyst and providing for fresh intermediates to keep the carrier materials have been used over the years such as
interesterication reaction going. It is therefore gener- hydrophilic powders (celite), ion exchange resins, and
ally accepted that in interesterication reactions, the hydrophobic polymers, either in the form of particles or
water content in the feed steam should be close to as membranes. Extensive overviews on the details of
saturation (41, 43, 44). these techniques can be found elsewhere (8).

The most widely used technique is the adsorption of C. Packed-Bed Reactor Setup
lipases onto porous carrier materials. Such materials
provide a huge internal surface area to immobilize One of the major advantages of immobilization is the
individual lipase molecules from solution with high potential to run a continuous process with maximum
efciency. The carrier materials should fulll a number exploitation but minimum handling of the biocatalyst.
of criteria to deliver a proper biocatalyst (51, 52). The simplest design of a continuous reactor is a
Obviously the material should allow efcient lipase packed-bed reactor, consisting of a cylindrical column
immobilization and an appropriate pore size to allow holding a xed bed of catalyst particles containing
diffusion of the lipase (> 50 nm) (52). Furthermore, immobilized lipase. The reaction medium, being either
particle size is important in order to minimize the liquid oil or organic solvent containing the substrates,
risk of substrate mass transfer limitations (40). is simply pumped through this bed of catalyst particles.
However, to be able to use the biocatalyst in a During its pass through the bed the substrates are con-
packed-bed reactor, a minimum particle size verted into the desired end-products within the pores
(> 500 m) should be accounted for to keep the of the catalyst. The degree of conversion is simply con-
pressure drop over the packed bed within reasonable trolled by the time allowed in the column, i.e., the
limits. residence time. The latter is determined by the height
Carrier materials such as ion exchange resins (e.g., of the xed bed and the ow rate of the reaction med-
Duolite ex Rohm-Haas, Germany (53)) and polypro- ium (see Sec. III.D).
pylene particles (Accurel EP-100, ex Akzo-Nobel, An important design parameter for a packed-bed
Germany) (54, 55) are most widely used for lipase reactor is catalyst particle size, which generally is in
adsorption. Especially the latter material has proven the range of 2001000 m. Too-small particles result
to be a suitable carrier, as the immobilization of a in an unacceptable pressure drop over the packed bed,
wide variety of lipases has been shown to be very ef- whereas too-large particles will introduce mass transfer
cient and reproducible (54). Though easily immobi- limitation effects, thereby decreasing the efciency of
lized on the carrier, it was reported that certain the catalyst. The latter especially holds for high-visc-
lipases gave activities much lower than could be osity systems such as vegetable oils. Applying organic
expected based on their loading. This phenomenon is solvents as the reaction medium, pore diffusion limita-
attributed to conformational changes in the structure tion, i.e., internal mass transfer resistance, can gener-
of the lipase and hence a loss of lipase activity upon ally be neglected (40).
adsorption of lipase molecules onto the carrier surface. External mass transfer generally is not an issue,
Precoating of the carrier with dummy, nonlipase pro- especially when using organic solvents (40, 58). The
tein before adsorption of the lipase has proven a reli- completion of the reaction requires residence times in
able technique to overcome the reduction of the the packed bed from half an hour to several hours,
activity of the immobilized lipase (54, 56). depending on the activity of the biocatalyst applied.
It should be noted that immobilization is men- The oil ow rates applied are generally above the
tioned as a technique to enhance the stability of range in which external limitations become important.
lipases. Fundamental studies have revealed that A detailed study of mass transfer effects in packed-bed
indeed the exibility of enzymes immobilized onto reactors can be found elsewhere (39, 49).
solid surfaces is considerably reduced (24). However,
data actually comparing one and the same type of D. Process Modeling
reaction with both the free and immobilized versions
of one lipase are rather scarce (57). Often research Many models have been developed to describe lipase-
measures the temperature optima of the free and catalyzed reactions. The simplest models generally
immobilized enzymes in different reaction systems; involve Michaelis-Menten kinetics or similar type of
for example, hydrolysis in an emulsion system for rate expressions describing, e.g., substrate or product
the free lipase, and an (inter)esterication in a solvent inhibition. These types of models are often used to
system for the immobilized lipase. However, consider- describe single reactions such as hydrolysis and ester-
ing effects of mass transfer, substrate solubility, and ication (59).
water activity as described above, comparison of Acidolysis (transesterication) and interesterica-
results from different assays can be rather tricky, tion of triglycerides are much more complex. Being
and hence the impact of immobilization itself should multistep and multisubstrate reactions a full-kinetic
be evaluated with care. description is highly complicated. Several models

have been developed which are based on true kinetics reaction, the rst-order model can still describe the
of the multistep process. With varying degrees of change in the composition of the triglyceride fraction.
detail, these models easily contain up to ve or six However, the models fail to describe the change of the
reaction parameters, depending on the reaction composition of the free fatty acid fraction, as well as
mechanism assumed (9, 42, 58). However, to limit the effect of the initial acid/oil ratio on the apparent
the complexity of such a model the number of reacting catalyst activity. In this case a second-order model is
species is generally reduced by assuming only two fatty often applied (42, 60, 62). It should be noted that the
acids to be involved, e.g., the one present in the trigly- latter models can generally only describe the process
cerides before the reaction and the fatty acids to be for a limited number of fatty acids (61), similar to the
incorporated (9). kinetic models referred to above.
A second class of models lacks any mechanistic
background and only describe the change in the com- E. Stability of Immobilized Lipases
position of the triglyceride fraction during acidolysis
and interesterication (39, 60, 61). They neglect the Decisive for the application of any process on indus-
initial phase of the process in which water present in trial scale is variable cost and hence productivity of the
the starting mixture is being used for hydrolysis, biocatalyst. The latter is determined by the activity as
thereby generating free fatty acids and diglycerides. well as the longer-term stability of the biocatalyst. In
This starting point is valid, considering the fact that principle, deactivation is an inherent characteristic of
the rate of hydrolysis is at least an order of magnitude any enzyme in which especially temperature is impor-
faster than the rate of interesterication. As a result the tant. Also the presence of water is important as funda-
hydrolysis phase is short compared to the total time mental studies showed that water is involved in many
required for full triglyceride conversion, and once com- enzyme deactivation reactions. This may explain the
pleted, the overall glyceride composition of the mixture improved stability of enzymes in nonaqueous organic
remains constant (38, 4042). media (63). However, the role of water is much more
The simplest approach is to consider the fatty acid complicated, as it was also reported that some water is
exchange reactions between the triglyceride species as a required to sustain the enzyme activity over time in a
purely statistical process approaching the equilibrium continuous system (41, 125).
end composition (Sec. IV.A) for the triglyceride mix- Another important mechanism causing deactivation
ture concerned. In chemical engineering terms such a is poisoning of the enzyme by a physical or chemical
process is generally referred to as a pseudo-rst-order interaction with impurities in the reaction medium. A
process. Mathematically this is represented by the fol- number of papers have shown that the stability of
lowing one parameter equation: lipases in oils is related to the intrinsic quality of the
X 1 ek 1 feed oil (41, 43, 44, 53). More specically, it has been
shown that primary and secondary products from tri-
in which X is the relative degree of conversion; k is the glyceride auto-oxidation, such as hydroxyperoxides
pseudo-rst-order activity constant of the biocatalyst; and their degradation products, may reduce the activ-
and is the incubation (batch) or average residence ity of the biocatalyst (6466).
time (continuous) in the system. Deactivation is generally described as a rst-order
The degree of conversion is based on the change in process with time (39, 43, 62), given by the following
the composition between the start and the end of the equation:
reaction. The latter is generally characterized by the
absence or presence of certain characteristic substrate k k0 ekd t 3
or product triglycerides:
TAGtt TAGt0 in which k is the catalyst activity at any time; k0 is the
X 2 catalyst activity at the start; kd is the deactivation con-
TAGeq TAGt0
stant [1/time]; and t is the incubation time.
The composition of the product triglycerides at equili- The specic deactivation constant (kd ) is actually a
brium can be calculated from analytical data on the lump parameter, comprising all deactivation mechan-
overall composition of the sn-1,3 and sn-2 positions in isms taking place, such as the thermal deactivation and
the starting triglyceride mixture (see Sec. IV.A). poisoning phenomena mentioned above. This para-
When dealing with the incorporation of specic meter thus is a complex function of the reaction con-
fatty acids in triglycerides, such as in an acidolysis ditions, e.g., temperature and water activity, the

quality of the reaction medium, and the type of acid Y on the 2-positions; and 1; 2 the number of
lipase involved. This complex nature of the problem ways the two and one type of fatty acids, respectively,
generally suggests that it is rather difcult to predict can be distributed over the sn-1 and sn-3 positions in
the performance of a chosen lipase in a certain appli- the triglyceride mixture.
cation and hence substantial testing is required each The overall- and 2-position fatty acid composition
time a new process is being developed. This is con- in a triglyceride mixture can be obtained by conven-
rmed by literature data showing half-life times vary- tional HPLC or GC analysis. The 1,3-position com-
ing between a few days and several months, depending position then follows by calculation. Though the
on specic reaction system studied (7). above presented rules specically refer to interesteri-
cation of triglycerides, they also apply to acid-
olysisi.e., the reaction between triglycerides and
IV. MODIFICATION OF TRIGLYCERIDES fatty acids or single fatty acid esters. However, in
A. Modifying the Fatty Acid Composition this case the overall 1,3-positional fatty acid composi-
tion follows from the 1,3-positional fatty acid
Chemical interesterication is one of the major mod- composition in the triglyceride fraction, the free
ication techniques and basically involves a random fatty acid composition, and the molar ratio of free
rearrangement of the sn-1, sn-2, and sn-3 fatty acids fatty acids to triglycerides.
over the triglycerides present in the mixture (Fig. 1).
A similar result can be obtained when using a random B. Melting Properties of Triglycerides
lipase such as that from Candida rugosa (38, 67),
Pseudomonas uorescens (68), or Pseudomonas cepacia Triglycerides are the basis for the production of
(69). Using a 1,3-specic lipase, only the fatty acids spreads such as margarine and butter, as well as con-
on the outer positions of the triglyceride molecules fectionery fats and chocolate. An important triglycer-
will be randomized. In this case the sn-2 position ide property in this type of product is their melting
remains intact, which implies that a different trigly- behavior and contribution to product texture. The lat-
ceride mixture will result, as compared to the random ter is easily recognized in product properties as soft-
process. ness, spreadability, and mouth feel.
In both cases the rearrangement process can be The melting behavior of oils and fats is the result of
regarded as a statistical process, and hence the nal, complex interactions among large numbers of trigly-
equilibrium composition of the product can be easily cerides, all different in the type and position of the
calculated. For example, assuming only three fatty fatty acids they contain. Thus modication of the
acidsX, Y, and Zpresent in a TAG mixture, the fatty acid composition or distribution will result in a
molar concentration of an individual types of triglycer- change in the melting behavior.
ides follows from: Chemical interesterication using a sodium alkoxide
catalyst is widely being applied for oils and fats mod-
Random process: XYZ = 6 xyz ication (70, 71). However, a growing interest exists
X2 Y = 3 xxy for its enzymatic equivalent. This process runs under
X3 = xxx mild operating conditions of temperature and pressure.
1,3-specic process: XYZ = 2 x1;3 y2 z1;3 Moreover, exploiting the specicity of lipases, more
XYX= x1;3 y2 x1;3 specic triglyceride mixtures are obtained which can-
not be produced by the random, chemical process.
in which x; y; z the molar fractions of X, Y and Z in To date, the main application of enzymatic TAG
overall fatty acid composition of the trigylceride mix- modication is probably the production of cocoa but-
ture; 6; 3; 1 the number of ways in which three, two, terfat equivalents. Cocoa butterfat has a very special
and one type of fatty acids, respectively, can be dis- melting behavior, which is characterized by a rapid
tributed over the sn-1, sn-2, and sn-3 positions; melting within a narrow temperature range. This beha-
x1;3 ; z1;3 the molar fractions of X and Z on the sn- vior is related to its high content of POSt- and StOSt-
1 and sn-3 positions; y2 the molar fraction of fatty type triglycerides (P palmitic; O oleic; St stearic
acid). The latter are not commonly present in vegetable
oils, but can be produced by enzymatic modication of
e.g. palm midfraction (a palm oil fraction rich in POP)

Candida rugosa, formerly referred to as Candida cylindracea. and stearic acid. Using the 1,3-specic lipase from

Aspergillus niger in petroleum ether at 40 C, a POSt- good example of such a process is the production of
and StOSt-rich mixture was thus obtained after 16 Betapol, an additive for infant formulas (81). This pro-
hours (38). Alternatively methylesters (72) or ethylester duct predominantly consists of OPO, which is a major
(73) have been used as stearic donor, using a 1,3-spe- triglyceride in human milk fat (82). Hydrolysis of OPO
cic lipase such as Rhizomucor miehei, Candida antarc- by human pancreatic lipase will result in free oleic acid
tica B (74), or Rhizopus japonicus lipase (60). and 2-monopalmitin, which are both easily absorbed.
Other liquid oils, e.g., olive oil, have also been eval- As opposed to this, vegetable oils in infant formulas
uated for the production of cocoa butter equivalents. generally contain the POO-type triglycerides, the
However, in this case the acidolysis product will also hydrolysis of which results in free palmitic acid. The
contain less functional tri- and di-oleyltriglycerides latter will form insoluble calcium soaps, thereby pre-
such as POO and StOO (75), which requires further venting absorption of both the palmitic acid and the
separation of the POSt and StOSt fraction by conven- calcium (83).
tional fractionation (38). Another example is the MLM triglycerides (M
An improvement of the melting behavior has also medium-chain C6 C12 ; L long-chain), preferably
been reported for butterfat being interesteried with a with an unsaturated fatty acid at the 2-position.
lipase. Using the 1,3-specic Rhizopus arrhizus lipase Because of the improved digestibility of these types
on Accurel interesterication during 96 h at 4060 C of triglycerides (84, 85), they are of interest for indivi-
resulted in signicantly softer products with improved duals suffering from pancreatic deciency and other fat
melting proles, especially when interesterifying but- malabsorption disorders (86, 87).
ter fat with canola oil (7678). It was shown that Starting from liquid oils such as sunower or soy-
chemical randomization yielded even more pro- bean (88) or peanut oil (89), the desired TAGs are
nounced effects on fatty acid composition and spread- obtained by 1,3-specic acidolysis with a medium
ing properties. The latter can also be obtained using a fatty acid donor, e.g., free fatty acids. This reaction
random lipasee.g., that from Pseudomonas uores- can be carried out in batch as well as a packed-bed
cens (68). reactor (90).
Improved melting properties were also reported However, in this one-step acidolysis process, MLM
after the modication of fully vegetable oil blends. is not the only product. As the process is purely statis-
Starting with a 60/40 mixture of palm stearin and soy- tical the nal mixture will also contain MLL- and
bean oil, it was shown that the solid-fat content LLL-type triglycerides. The concentrations of all
dropped after interesterication during 16 h using the TAG types depends on the molar excess of the fatty
1,3-specic Mucor miehei lipase at 70 C (53). Similar acids and the equilibrium composition can be calcu-
results were reported for other fat blends consisting of lated using the principles described above.
palm stearin with coconut oil (43), palm kernel oil, and MLM concentrates were obtained in a two-step
blends of palm with coconut or palm kernel oil (79). In enzymatic process. In the rst step nearly pure 2-
all cases the 1,3-specic rearrangement of the fatty monoglycerides were produced by ethanolysis (reac-
acids resulted in a decrease of the solids content at tion with ethanol) of e.g. triolein or cotton seed oil
all temperatures. using the 1,3-specic Rhizopus delemar lipase (Fig. 3).
This reaction was carried out in organic solvent
C. Structured Triglycerides (ethers) at 40 C and 0.75 water activity. After purica-
tion by crystallization these 2-monoglycerides were
In the last few decades it has become clear that the reesteried with caprylic at 38 C in hexane using
fatty acid composition of dietary fats has important immobilized Rhizomocur miehei lipase. This process
implications for human health. Moreover, in certain yielded 90% MLM (89).
cases also the position of the fatty acids, i.e., the trigly-
ceride structure, is important (80). Much research has
therefore been devoted to the production of so-called D. Fatty Acid Enrichment
structured lipids, which are enriched in TAGs of a very
specic structure and/or in a certain type of fatty acid. Nutritionally interesting triglycerides can also be
It is especially in this area that the selectivities of obtained by enrichment or removal of certain fatty
lipases become of particular interest. acids from a TAG mixture, independent of the TAG
Acidolysis using a 1,3-specic lipase is most com- structure involved. Especially in this area the fatty acid
monly applied to produce structured triglycerides. A selectivity of certain lipases is rather important.

Figure 4 Fatty acid enrichment via selective hydrolysis: the
Figure 3 Two-step synthesis of structured triglycerides. production of a low-saturate oil via selective hydrolysis and
(Adapted from Ref. 89.) nonselective enzymatic reesterication. (From Ref. 91.)
1. Low-Saturate Oils Thus the primary esterication productsi.e., (1(3)-

monoglycerides and 1,3-diglyceridesare converted
The rst example is the production of a low-saturate
to their 2-position isomer. Subsequent esterication
oil. For many decades the lowering of blood choles-
will result in the formation of 1,2-diglycerides and
terol by changing the dietary fat consumption has been
nally triglycerides (95, 96, 103). Research has shown
a hot issue. Nowadays it is recommended to limit the
that the presence of ionic enzyme carriers (94) and the
intake of total fat and especially from saturated fat
use of apolar solvents (97) also promote acyl migra-
(127), and hence there is considerable interest in low-
tion.
saturate fat products.
A two-step enzymatic process was described,
2. Enrichment of Long-Chain Polyunsaturated
exploiting the high selectivity of Geotrichum candidum
Fatty Acids
lipase B for cis--9 unsaturated fatty acids (20, 21). In
the rst step of the process this lipase was used to pre- The second example of fatty acid enrichment is the
ferentially hydrolyze oleic and linoleic acid from sun- production of TAGs enriched in long-chain polyunsa-
ower oil at 40 C. This resulted in > 99% unsaturated turated fatty acids (LPUFA). These fatty acids are now
fatty acids in the free fatty acid fraction (91). After recognized to play an essential role in the human diet
separation of the free fatty acids by conventional dis- and have important biomedial properties. Marine oils
tillation, reesterication with glycerol was carried out are an important source for LPUFAs such as eicosa-
using immobilized Rhizomucor miehei lipase (Fig. 4). pentaenoic acid (C20:5; EPA) and docosahexaenoic
Running the process at 60 C under continuous removal acid (C22:6; DHA). However, their individual levels
of water, > 95% w/w triglycerides were thus obtained are generally rather low and involve a mixture of dif-
with a saturated fatty acid content < 1%. ferent fatty acids. Thus enrichment and separation of
Now Rhizomucor miehei lipase is the most distinct these fatty acids is widely being investigated. Because
1,3-specic lipase, and hence the high yield of triglycer- of the high susceptibility of LPUFAs to oxidation,
ides in this reesterication reaction may seem rather especially in this area low-temperature enzymatic pro-
surprising. However, whenever dealing with partial cessing can be a powerful tool (Fig. 5).
glycerides, acylmigration should be accounted for. At Glycerides enriched in LPUFAs can be produced by
temperatures > 40 C monoglycerides (92) as well as selective hydrolysis using the positionally nonselective
diglycerides (93, 94, 126) undergo acylmigration. but chain lengthspecic lipase from Candida rugosa

chains were hardly hydrolyzed, whereas the rate of
hydrolysis increased with two or only one DHA
chain in the TAG molecule (104). The phenomenon
being less strong for diglycerides and absent for mono-
glycerides, triglycerides will always become the major
compound in the product mixture.
It should be noted that processes similar to those
discussed in this paragraph have been used for the
enrichment of other interesting fatty acidse.g., con-
jugated linoleic acid (CLA) (105), arachidonic acid
(AA) (106), -linoleic acid (GLA) (107109), and
erucic acid (EA) (110).
V. PARTIAL GLYCERIDE PRODUCTION

A. Monoglycerides
Monoglycerides are widely applied as emulsiers or

surfactants and are normally produced by high-tem-
perature glycerolysis using an inorganic catalyst (4).
Figure 5 Production scheme for the enrichment of sh oil. The product is a mixture of mono- and diglycerides
(From Ref. 103.) (1:1 w/w) and residual triglycerides. Moreover, because
of the reaction conditions applied, the crude product is
often dark in color and has a burnt avor. Obviously,
(Table 1). Starting with marine oils such as sardine or this requires extensive purication during further down
cod liver oil (98) or tuna oil (99, 100), partial glycerides stream processing, generally involving molecular distil-
with LPUFA levels between 45% and 65% were lation. Because of the ambient reaction temperatures
obtained. applied, lipase catalyzed processing has widely been
It should be noted that the LPUFAs accumulate in investigated as the more natural process, with
the partial glyceride fraction because of the selectivity fewer byproducts and lower energy consumption.
of the lipase against, i.e. discrimination against, these Producing monoglycerides from oils and fats by
fatty acids. Using Geotrichum candidum lipase similar hydrolysis would be the simplest route. It has been
overall enrichment results were obtained. However, shown that the use of Celite immobilized porcine pan-
this lipase appears even more selective than Candida creas lipase at 40 C in a solvent-free system did result
rugosa, as it is able to discriminate between EPA and in 2-monoglyceride levels as high as 68% w/w in the
DHA (101). residual glyceride fraction (111). This result corre-
Another option for enrichment of oils is selective sponds to 67% of the theoretical molar yield. An
esterication of sh oil free fatty acids, using the con- even better result was obtained in organic solvent
ventional 1,3-specic lipases. Being also selective using Rhizopus arrhizus lipase at 35 C. A molar con-
against LPUFAs but active in the esterication reac- version of > 95% to 2-monoglycerides was obtained
tion, as compared to hydrolysis, reaction with simple (89, 112). Despite this higher molar conversion,
alcohols results in free fatty acids enriched in the obviously the process route implies a low conversion
LPUFAs. These can be puried from the unwanted efciency.
ester fraction by distillation (100, 102). Starting from free fatty acids and glycerol produc-
One interesting feature about the selective hydroly- tion of monoglycerides (predominantly the 1(3)-iso-
sis is the level of triglycerides as high as 75% in the mer) is much more efcient. Using the
residual glyceride mixture, even at degrees of hydroly- monoglyceride-specic enzyme patatin, an acylhydro-
sis as high as 70% (99, 103). This result is probably due lase from potato tubers (113), does offer an opportu-
to the ability of the Candida rugosa lipase to discrimi- nity for a natural process. At temperature < 50 C
nate against triglycerides with a rising number of long- this lipase was shown to produce > 95% monoglycer-
chain fatty acids in their structure. A model study ides in a solvent-free system (23). Though very specic,
revealed that triglycerides containing three DHA the enzyme is not commercially available.

Another option is the use of Penicillium camembertii formed. Optimum diglyceride yield was obtained at a
lipase (lipase G), which is a mono- and diglyceride glycerol/triglyceride molar ratio equivalent to its stoi-
specic lipase and not able to react upon triglycerides. chiometric value for diglyceride production, i.e., 1:2
Moreover, it was shown that the selectivity to produce (119).
monoglycerides over diglycerides is determined by the Directed esterication is another efcient means of
water activity applied during the reaction. The lower diglyceride production. Using the 1,3-specic lipases
the aw , the more monoglyceride-specic the lipase. from Rhizomucor miehei (116, 120) or Rhizopus arrhi-
Thus at a water activity of approximately 0.1, > 90% zus (94, 115), diglyceride yields of 6585% were
pure monoglycerides were produced with 76% degree obtained. Applying continuous removal of water
of conversion (114). (116) during the process, yields > 90% were reported.
Even higher degrees of conversion were obtained by Comparable results were reported using the partial
lowering the reaction temperature, such that the pro- glyceride-specic lipase from Penicillium camembertii.
duct starts to crystallize. This process is generally As described above, at high water activity it loses its
referred to as directed synthesis, referring to the selectivity for monoglyceride production, yielding 60
fact that crystallization shifts the equilibrium towards 85% diglycerides (114, 115).
preferential synthesis of the desired solid product. It should be noted that the directed synthesis
Using this approach it was shown that at 40 C even routes for mono- and diglyceride production prefer
solid palmitic acid could be converted to pure mono- the use of free lipases to minimize the rate of mass
glycerides. Using Penicillium cyclopium lipase, nearly transfer. Still reaction times are rather long.
pure monoglycerides were produced at > 90% degree Moreover, recovery of the free lipase remains rather
of conversion (115). It should be noted that the rate of difcult if the use of solvents is to be avoided.
this solid-to-solid conversion process is determined Allowing solvents in the system, recovery of the bioca-
by the rate of diffusion of the reactants and hence the talyst obviously is much easier. Immobilizing the lipase
process is rather slow. Consequently the process may on nonporous inorganic powders, the catalyst can be
take 1015 days to complete. recovered after the (solvent-free) process by ltration,
Similar results were reported using free lipase from having dissolved the reaction medium in acetone (121).
Pseudomonas uorescens or Chromobacterium visco- Carrying out the whole process in organic solvents,
sum. Monoglyceride yields between 70% and 90% mass transfer is not an issue. For such systems other
were obtained, starting from various oils and fats means of selective product removal also have been
(116, 117). It was shown that the high monoglyceride developed, such as in-line adsorption (122), crystalliza-
yield could be obtained simply by reducing the reaction tion in a separate vessel (123), and extraction (124).
temperature below a certain critical temperature
(118). However, the latter appeared strongly dependent
on the type of triglycerides involved (saturation level). VI. FUTURE PERSPECTIVE
As shown in this chapter, the use of lipases can be an

B. Diglycerides important tool in the synthesis of a wide range of acyl-
glycerides. Maximum production of a desired product
For diglycerides processes have been developed similar can be obtained by carefully selecting the right lipase
to those described above, the only difference being with the right selectivity under the right conditions for
either the optimum composition of the starting mix- the process involved. Both the lipase selectivity and the
ture, the lipase applied, or the processing conditions. relatively low reaction temperatures are important
Again, directed processes, i.e., applying temperature advantages over conventional organic synthesis, result-
programming, have been shown to be an important ing in less byproduct formation and allowing single-
tool for high purity and yield diglyceride production. step syntheses.
Thus stepwise decreasing the reaction temperature Despite these advantages, only a few of the processes
from 62 C to 48 C, glycerolysis of hydrogenated beef described above are actually applied in industry at full
tallow yielded 90% diglycerides using a free scale. This is mainly due to the prohibitively high cost
Pseudomonas lipase (119). of most enzymes, as well as the relatively low opera-
The glycerol/triglyceride ratio was shown to affect tional stability encountered in certain applications.
the product purity, i.e., the level of monoglycerides However, using modern biotechnology the cost of

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18. IC Chandler, PT Quinlan, GP Mcneill. Lipase-cata-
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19
Signicance of Indigenous Enzymes in Milk and Dairy Products
Patrick F. Fox
University College, Cork, Ireland
I. INTRODUCTION Many indigenous milk enzymes are technologically

signicant from ve viewpoints:
About 60 indigenous enzymes have been reported in 1. Deterioration (lipase [potentially the most signif-
normal bovine milk (1); with the exception of -lactal- icant enzyme in milk], proteinase, acid phosphatase
bumin, most of these have no obvious physiological and xanthine oxidase) or preservation (lactoperoxi-
role. The indigenous enzymes are constituents of the dase, sulfhydryl oxidase, superoxide dismutase) of
milk as excreted and arise from three principal sources: milk quality.
(a) the blood via defective mammary cell membranes; 2. As indices of the thermal history of milk; these
(b) secretory cell cytoplasm, some of which is occasion- include alkaline phosphatase, -glutamyl transpepti-
ally entrapped within fat globules by the encircling fat dase, lactoperoxidase, and perhaps others.
globule membrane (MFGM); and (c) the MFGM 3. As indices of mastitic infection; the concentration
itself, the outer layers of which are derived from the of several enzymes increases on mastitic infection,
apical membrane of the secretory cell, which in turn especially catalase, N-acetyl--glucosaminidase and
originates from the Golgi membranes; this is probably acid phosphatase.
the principal source of the enzymes. Thus, most 4. Antimicrobial activity, such as lysozyme and lac-
enzymes enter milk owing to peculiarities of the toperoxidase (which is exploited as a component of the
mechanism by which milk constituents, especially the lactoperoxidaseH2 O2 thiocyanate system for the cold
fat globules, are excreted from the secretory cells. Milk pasteurization of milk).
does not contain substrates for many of the enzymes 5. As commercial source of enzymes; these include
present, while others are inactive in milk owing to ribonuclease and lactoperoxidase.
unsuitable environmental conditions such as pH.
With a few exceptions (e.g., lysozyme and lactoperox-
idase), the indigenous milk enzymes do not have a
The following abbreviations are used: MFGM, milk fat glo- benecial effect on the nutritional or organoleptic
bule membrane; -CN, -casein; s2 -CN, s2 -casein; LPL, attributes of milk, and hence their destruction by
lipoprotein lipase; UHT, ultra high temperature; HTST, heat is one of the objectives of many dairy processes.
high temperature short time; LTLT, low temperature long The technologically signicant indigenous enzymes
time; PE, pasteurization equivalent; HML, human milk lyso-
in milk and their catalytic activities are listed in
zyme; BML, bovine milk lysozyme; EWL, egg white lyso-
zyme; NAGase, N-acetyl--D-glucosaminidase; GGTP, - Table 1. All these enzymes have been isolated and
glutamyl transpeptidase; LPO, lactoperoxidase; XO, well characterized. Other enzymes that have been iso-
xanthine oxidase; SO, sulfhydryl oxidase; SOD, superoxide lated and characterized but which have little or no
dismutase. signicance in milk are listed in Table 2. Enzymatic

Table 1 Technologically Signicant Indigenous Enzymes in Milk
Enzyme Reaction Signicance
Lipase Triglycerides H2 O ! fatty acids partial Off avor in milk; avor development in blue
glycerides glycerol cheese
Proteinase (plasmin) Hydrolysis of peptide bonds, particularly in Reduced storage stability of UHT products;
-casein cheese ripening
Alkaline Hydrolysis of phosphoric acid esters Index of pasteurization
phosphomonoesterase
Acid phosphomonoesterase Hydrolysis of phosphoric acid esters Cheese ripening; reduced heat stability of
milk;
Lysozyme Hydrolysis of mucopolysaccharides Bacteriocidal agent
-Glutamyl transpeptidase Transfer of -glutamyl residues Index of heat treatment
N-Acetylglucosaminidase Hydrolysis of glycoproteins Index of mastitis
Xanthine oxidase Aldehyde H2 O O2 ! Acid H2 O2 Pro-oxidant; cheese ripening
Sulfhydryl oxidase 2RSH O2 ! RSSR H2 O2 Amelioration of cooked avor
Superoxide dimutase 2O
2 2H ! H2 O2 O2 Antioxidant
Catalase 2H2 O2 ! O2 2H2 O Index of mastitis; pro-oxidant
Lactoperoxidase H2 O2 AH2 ! 2H2 O A Index of pasteurization; bactericidal agent;
index of mastitis; pro-oxidant
activities that have been detected in milk but which indices of animal health and of the mechanisms
have not been isolated and have no known signicance involved in the synthesis and secretion of milk. A
in milk are listed in Table 3; it is possible that some of very extensive literature has accumulated. The general
these enzymes are secreted by contaminating bacteria topic has been the subject of several general reviews
in milk. (110); in addition, the literature on the principal tech-
The indigenous enzymes in milk have attracted the nologically signicant enzymes has been reviewed
attention of researchers for > 100 years, mainly separately (see below).
because of their potential to cause defects in milk In this chapter the occurrence, distribution, isola-
and dairy products, especially lipase, and their useful- tion, and characterization of the principal indigenous
ness as indicators of the thermal treatment of milk. enzymes in bovine milk will be discussed, with empha-
More recently, they have assumed importance as sis on their commercial signicance in milk and dairy
Table 2 Other Enzymes That Have Been Isolated From Milk and Partially Characterized but Which Are of No Known
Signicance in Milk
Enzyme Reaction catalyzed Comment
Glutathione peroxidase EC 1.11.1.92 GSH H2 O GSSH Contains Se

Ribonuclease EC 3.1.27.5 Hydrolysis of RNA Milk is a very rich source;
similar to pancreatic
RNase
-Amylase EC 3.2.1.1 Hydrolysis of starch
-Amylase EC 3.2.1.2 Hydrolysis of starch
-Mannosidase EC 3.2.1.24 Hydrolysis of mannan Contains Zn2
-Glucuronidase EC 3.2.1.31 Hydrolysis of glucuronides
5 0 -Nucleotidase EC 3.1.3.5 5 0 - Nucleotides H2 O ribonucleosidesPi Diagnostic test for mastitis
Adenosine triphosphatase EC 3.6.1.3 ATP H2 O ADP Pi
Aldolase EC 4.1.2.13 Fructose, 1,6 diP glyceraldehyde-3-
P dihydroxyacetone-P
Source: Ref. 70.

products. The available information indicates that the A. Plasmin (EC 3.4.21.7)
milks of other species have an enzyme prole similar to
bovine milk, although very considerable interspecies The physiological function of plasmin (brinolysin) is
differences exist in the level of certain enzymes, e.g., to dissolve blood clots. It is part of a complex system
the very high level of lysozyme in human and equine consisting of plasmin, its zymogen (plasminogen),
milks. Human milk and that of other primates contain plasminogen activators, plasmin inhibitors, and inhi-
a bile salts-activated lipase, in addition to the ubiqui- bitors of plasminogen activators (Fig. 1). In milk,
tous lipoprotein lipase, which is not present in the milk there is about four times as much plasminogen as
of other species. The indigenous enzymes in human plasmin, and both, as well as plasminogen activators,
milk have been described by Hamos et al. (11) and are associated with the casein micelles, from which
Hernell and Lonnerdal (12). they dissociate when the pH is decreased to 4.6; the
inhibitors of plasmin and of plasminogen activators
are in the milk serum. It has been reported that there
is a low level of plasmin activity in the milk fat glo-
II. PROTEINASES (EC 3.4..) bule membrane but this appears to be due to the
adsorption of plasmin to casein micelles which are
The presence of an indigenous proteinase in milk was adsorbed on the membrane (15). The concentration
suggested by Babcock and Russel in 1897 but because of plasmin and plasminogen in milk increase with
it occurred at a low concentration or had low activity advancing lactation, mastitic infection, and number
in milk, it was believed until the 1960s that the protei- of lactations. The conversion of plasminogen to plas-
nase in milk might have been of microbial origin. min in milk increases with advancing lactation, and
Recent changes in the dairy industry, e.g., improved there is a positive correlation between plasmin activity
hygiene in milk production, extended storage of milk and the level of plasminogen activator, which itself is
at a low temperature at the farm and/or factory, and positively correlated with somatic cell count (16). The
altered product prolee.g., UHT processing of level of plasmin in milk is also affected by diet and
milkhave increased the signicance of indigenous management practices (16). No activation of plasmi-
milk proteinase which has, consequently, been the nogen to plasmin is reported (17) to occur during
focus of considerable research. storage of milk at 4 C for 6 days; in fact, plasmin
Milk contains at least two proteinases, plasmin and potential plasmin (plasminogen) activity
(alkaline milk proteinase) and cathepsin D (acid milk decreased under these conditions.
proteinase) and possibly several other proteolytic Bovine plasminogen contains 786 amino acids with
enzymes, e.g., two thiol proteinases, thrombin, and a mass of 88.092 kDa. Its primary structure is arranged
an aminopeptidase. In terms of activity and technolo- in ve loops (called kringles), each stabilized by three
gical signicance, plasmin is the most important of the intramolecular disulde bonds. Plasminogen is acti-
indigenous proteolytic enzymes and has been the sub- vated in a two-step process: it is rst cleaved at
ject of most attention. The relevant literature has been Arg557 -Ile558 (bovine) by plasmin (a trace of which
reviewed by Grufferty and Fox 13) and Bastian and occurs in blood) to yield Lys-plasminogen which is
Brown (14). inactive but undergoes a conformational change
Figure 1 Schematic representation of the plasmin system in milk.

Table 3 Partial List of Minor Enzymes in Milk
Distribution
Enzyme Reaction catalyzed Source in milk
EC 1.1.1.1 Alcohol dehydrogenase EthanolNAD acetaldehyde NADH H

EC 1.1.1.14 L-Iditol dehydrogenase L-Iditol NAD L-sorbose NADH H SM
EC 1.1.1.27 Lactate dehydrogenase Lactic acid NAD pyruvate acid NADH H SM
EC 1.1.1.37 Malate dehydrogenase Malate NAD oxaloacetate NADH H Mammary gland SM
EC 1.1.1.40 Malic enzyme Malate NADP pyruvate CO2 NADH H Mammary gland SM
EC 1.1.1.42 Isocitrate dehydrogenase Isocitrate NADP 2-oxoglutarate CO2 Mammary gland SM
NADH H
EC 1.1.1.44 Phosphoglucuronate dehydrogenase 6-Phospho-D-gluconate NADP D-ribose-5- Mammary gland SM
(decarboxylating) P CO2 NADPH H
EC 1.1.1.49 Glucose-6-phosphate dehydrogenase D-Glucose-6-P NADP D-glucono-1,5-lactone-6- Mammary gland SM
P NADPH H
EC 1.4.3.6 Amine oxidase (Cu-containing) RCH2 NH2 H2 O O2 RCHO NH3 H2 O2 SM
Polyamine oxidase Spermine O O2
! spermidine ! putrescine
2
SM
Fucosyltransferase Catalyzes the transfer of fucose from GDP L-fucose to SM
specic oligosaccharides and glycoproteins
EC 1.6.99.3 NADH dehydrogenase NADH acceptor NAD reduced acceptor FGM
EC 1.8.1.4 Dihydrolipoamide dehydrogenase Dihydrolipoamide NAD lipoamide NADH SM/FGM
(diaphorase)
EC 2.4.1.22 Lactose synthetase (A protein: UDP- UDP galactose D-glucose UDP lactose Golgi apparatus SM
galactose: D-glucose, 1-
galactosyltransferase; B protein: -
lactalbumin)
EC 2.4.1.38 Glycoprotein 4--galactosyltransferase UDP galactoseN-acetyl D-glucosaminyl- FGM
glycopeptide UDP 4; -D-galactosyl-N-acetyl-D-
glucosaminyl glycopeptide
EC 2.4.1.90 N-Acetyllactosamine synthase UDP galactoseN-acetyl-D-glucosamine UDP Golgi apparatus
N-acetyllactosamine
EC 2.4.99.6 CMP-N-acetyl-N-acetyl-lactosaminide CMP-N-acetylneuraminate+-D-galactosyl-1,4-N-acetyl SM
-2,3-sialyltransferase D-glucosaminyl glycoprotein CMP
-N-acetylneuraminyl 1-2, 3--D-galactosyl-1,4-N-
acetyl-D-glucosaminyl-glycoprotein

EC 2.5.1.3 Thiamine-phosphate pyrophosphorylase 2-Methyl-4-amino-5-hydroxymethyl/pyrimidine FGM
diphosphate 4-methyl-5-(2-phosphonooxyethyl)-
thiazole pyrophosphate thiamine monophosphate
EC 2.6.1.1 Aspartate aminotransferase L-aspartate 2-oxoglutarate oxaloacetate L- Blood SM
glutamate
EC 2.6.1.2 Alanine aminotransferase L-alanine 2-oxoglutarate pyruvate L-glutamate Blood SM
EC 2.7.7.49 RNA-directed DNA polymerase n Deoxynucleoside triphosphate n
pyrophosphate DNAn
EC 2.8.1.1 Thiosulfate sulfur transferase Thiosulfate cyanide sulfite thiocyanate SM
EC 3.1.1.8 Cholinesterase Acylcholine H2 O choline carboxylic acid anion Blood FGM
EC3.1.3.9 Glucose-6-phosphatase D-Glucose 6-P H2 O D-glucose Pi FGM
EC 3.1.4.1 Phosphodiesterase Phosphodiester H2 O ! phosphomonoester alcohol
EC 3.1.6.1 Arylsulfatase Phenol sulfate H2 O phenol sulfate
EC 3.2.1.21 -Glucosidase Hydrolysis of terminal non-reducing -D-glucose residues Lysosomes FGM
EC 3.2.1.23 -Galactosidase Hydrolysis of terminal nonreducing -D-galactose Lysosomes FGM
residues in -D-galactosides
EC 3.2.1.51 -Fucosidase An -L-fucoside H2 O an alcohol L-fucose Lysosomes
EC 3.4.11.1 Cytosol aminopeptidase (leucine Aminoacyl-peptide H2 O amino acid peptide SM
aminopeptidase)
EC 3.4.11.3 Cystyl-aminopeptidase (Oxytocinase) Cystyl-peptides H2 O amino acid peptide SM
EC 3.4.21.4 Trypsin Hydrolyzes peptide bonds, preferentially Lys-X, Arg-X SM
EC 3.6.1.1 Inorganic pyrophosphatase Pyrophosphate H2 O 2 orthophosphate SM/FGM
EC 3.6.1.9 Nucleotide pyrophosphate A dinucleotide H2 O 2 mononucleotides SM/FGM
EC 4.2.1.1 Carbonate dehydratase H2 CO3 CO2 H2 O SM
EC 5.3.1.9 Glucose-6-phosphate isomerase D-glucose-6-P fructose-6-P SM
EC 6.4.1.2 Acetyl-CoA carboxylase ATP acetyl- FGM
CoA HCO 3 ADP orthophosphate malonyl-
CoA
Source: Ref. 70.

SM skin milk; FGM fat globule membrane; P phosphate:

which exposes the bond Lys77 -Arg78 to hydrolysis by quantied by determining the intensity of uorescence,
urokinase or tissue-type plasminogen activator. with excitation at 380 nm and emission at 460 nm (20).
Hydrolysis of this bond yields a mature enzyme of The chromogenic substrate, Val-Leu-Lys-p-nitroani-
81 kDa, consisting of two polypeptide chains held lide, is also used (16). Plasminogen activity is measured
together by a single disulde bond. The ve kringles by assaying plasmin activity before and after activation
of plasminogen are retained in plasmin; they are of indigenous plasminogen by an excess of added uro-
required for activity and are conserved in plasmins kinase (16). Plasminogen activator activity may be
from different species. Bovine plasmin is also cleaved assayed by the ability of an ultracentrifugal casein
at Arg342 -Met343 to yield midi plasmin (15). Bovine micelle pellet to activate exogenous (added) plasmino-
plasminogen cDNA has been cloned (18). gen (16).
Plasmin is a serine proteinase (inhibited by di-iso-
propyluorophosphate, phenylmethyl sulfonyl uor- 2. Activity of Plasmin on Milk Proteins
ide, and trypsin inhibitors) with a high specicity for
-Casein is the most susceptible milk protein to plas-
peptide bonds to which lysine or arginine supplies the
min action; it is hydrolyzed rapidly at Lys28 -Lys29 ,
carboxyl group. The active site is in the smaller of the
Lys105 -His106 , and Lys107 -Glu108 to yield 1 -CN
two chains and consists of His598 , Asp641 , and Ser736 . It
f29-209), 2 (-CN f106-209), and 3 (-CN f108-
is optimally active at pH 7:5 and 35 C; it exhibits
209) caseins and proteose peptone (PP)5 (-CN f1-
20% of maximum activity at 5 C and is stable over
105/7), and PP8 slowly (-CN f29-105/7) and PP8
the pH range 49.
fast (-CN f1-29). In vitro (in solution), -casein is
Plasmin is usually extracted from casein by washing
also hydrolyzed fairly rapidly at Lys113 -Tyr114 and
with water at pH 3.5 and puried by precipitation with
Lys183 -Asp184 , but it is not known if these bonds are
NH4 2 SO4 and various forms of chromatography,
hydrolyzed in milk. -Caseins normally represent 3
including afnity chromatography. Plasmin is quite
% of total N in milk but can be as high as 10% in late-
heat stable; it is partially inactivated by heating at
lactation milk; as a percent of total N, the value for
72 C for 15 sec, but its activity in milk increases
proteose peptones is about half that of the -caseins.
following HTST pasteurization, probably owing to
s2 -Casein in solution is also hydrolyzed very
inactivation of the indigenous inhibitors of plasmin
rapidly by plasmin at bonds Lys21 -Gln22 , Lys24 -Asn25 ,
or, more likely, inhibitors of plasminogen activators.
Arg114 -Asn115 , Lys149 -Lys150 , Lys150 -Thr151 , Lys181 -
It partly survives UHT sterilization and is inactivated
Thr182 , and Lys188 -Ala189 (see 14), but its hydrolysis
by heating at 80 C for 10 min at pH 6.8; its thermal
in milk has not been characterized. Although less sus-
stability decreases with increasing pH in the range
ceptible than s2 - or -caseins, s1 -casein in solution is
3.59.2.
also readily hydrolyzed by plasmin (14) but it does not
The inactivation of plasmin in milk follows rst-
appear to be hydrolyzed to a signicant extent in milk,
order kinetics. Arrhenius plots show that inactivation
although it has been suggested that -casein is pro-
is not linear with increasing temperature. In the tem-
duced from s1 -casein by plasmin. Although -casein
perature range 63110 C, Ea for inactivation is 52.75
contains several Lys and Arg residues, it appears to
and 74:44 kJ mol1 for low and high somatic cell
be quite resistant to plasmin, presumably owing to a
count (SCC) milk, respectively, while in the range
relatively high level of secondary and tertiary struc-
100130 C, Ea is 22.03 and 24:70 kJ mol1 for low and
tures. The whey proteins are quite resistant to plasmin,
high SCC milk, respectively (19). Plasmin is more heat
probably owing to their compact, globular structures;
stable to low-temperature treatments, e.g., thermization
in fact, -lactoglobulin, especially when denatured,
and HTST pasteurization, in high than in low SCC milk
inhibits plasmin, presumably via sulfhydryl-disulde
but the reverse is true in the UHT range (19). Milk with
interactions which rupture the structurally important
a high SCC has high plasminogen activator activity (19).
kringles.
1. Assay of Plasmin Activity 3. Signicance of Plasmin Activity in Milk

Plasmin activity may be assayed on a wide range of There is sufcient plasmin in milk to cause very exten-
substrates, including proteins, but the most widely sive proteolysis, but this is not realized owing to the
used substrate is the synthetic uorogenic peptide, presence of inhibitors. If the casein micelles are sedi-
N-succinyl-L-Ala-L-Phe-L-Lys-7-amido-4-methyl cou- mented by ultracentrifugation and redispersed in buf-
marin; the liberated 7-amido-4-methyl coumarin is fer, very extensive proteolysis occurs on storage since

the inhibitors are removed in the ultracentrifugal Cheese analogues are usually produced from rennet
serum. According to Guinot-Thomas et al. (17), micro- casein which may contain active plasmin. Hydrolysis
bial proteinases cause more proteolysis in milk than of -casein and undesirable changes in the rheological
plasmin during storage at 4 C for 6 days. properties of cheese analogs have been attributed to
Plasmin and plasminogen accompany the casein plasmin action (14).
micelles on the rennet coagulation of milk and are Plasmin activity may contribute to the age gelation
concentrated in cheese in which plasmin contributes of UHT milk produced from high-quality raw milk
to primary proteolysis of the caseins, especially in (which contains a low level of Pseudomonas protei-
cheeses that are cooked to a high temperature, e.g., nase). The acid precipitability of casein from late-lacta-
Swiss and some Italian varieties, in which the coagu- tion milk is poor, but evidence for the involvement of
lant is totally or extensively inactivated (21). -Casein plasmin is lacking. Reduced yields of cheese and casein
is the principal substrate and even in low-cooked can be expected to result from plasmin action in milk
cheeses (e.g., Cheddar, Gouda), in which the coagulant since the proteose peptones are, by denition, soluble
is the principal primary proteinase, proteolysis of - at pH 4.6 and are not incorporated into acid- or
casein is due mainly to plasmin action; some hydrolysis rennet-produced casein curd.
of s1 -casein by plasmin also occurs.
The level of plasmin activity in cheese varies sub-
4. Proteinase Inhibitors in Milk
stantially with the variety; Emmental, Parmesan, and
Dutch-type cheeses contain about three times as much Milk contains several broad-specicity plasma-derived
plasmin activity as Cheddar-type cheeses (22). This is proteinase inhibitors: 1 -proteinase inhibitor, 2 -anti-
probably due to the greater activation of plasminogen plasmin, C1 inhibitor, antithrombin-III, 2 -macroglo-
in the former as a result of inactivation of inhibitors of bulin, inter--trypsin inhibitor, and two inhibitors
plasminogen activators in high-cooked cheese and analogous to human 1 -antichymotrypsin which pos-
their removal from Dutch-type cheese curd on washing sess inhibitory activity against trypsin and elastase,
(when whey is removed and replaced by water). respectively. Inhibitory activity is highly elevated in
Plasmin activity is decreased in cheeses made from mastitic milk owing to increased leakage of blood pro-
UF-concentrated milk because of the retention of inhi- teins into milk. It has been proposed that trypsin inhi-
bitors of plasmin and plasminogen activators and - bitory activity in milk may be a useful index of mastitis
lactoglobulin in the curd. Proteolysis is less in UF in cows. Bovine colostrum contains colostrum-specic
cheeses than in their conventional counterparts; pre- proteinase inhibitors, including trypsin-inhibitory and
sumably, the lower level of plasmin activity is a con- thiol proteinase-inhibitory activities, which protect
tributory factor. immunoglobulins and other biologically active pro-
It has been suggested that an elevated level of plas- teins and peptides against proteolysis by gastrointest-
min activity in late-lactation milk contributes to its inal enzymes in the newborn (24, 25).
poor cheese-making properties; however, an elevated Apart from the colostrum-specic inhibitors, the
level of somatic cells in milk does not appear to lead to plasma-derived inhibitors are present in milk as a
defects in cheese (13). The casein micelles in bovine result of membrane leakage (mastitis or late lactation)
milk are capable of binding 10 times as much plas- and therefore might be expected to be without signi-
min as occurs naturally in milk. Exogenous plasmin cance. However, they are probably quite signicance:
added to milk is incorporated and uniformly distribu- (a) The level of plasmin in milk is sufcient to cause
ted in the cheese curd, in which it accelerates proteo- very extensive hydrolysis of the caseins, as can be read-
lysis and maturation (23). When other exogenous ily demonstrated by dispersing ultracentrifugally sedi-
proteinases are added to cheese milk, much of the mented casein micelles in buffer (plasmin and
added enzyme is lost in the whey, increasing cost and plasminogen accompany the casein micelles while the
creating potential problems for whey processors. The inhibitors are in the milk serum). Plasmin activity is
yield of cheese may also be decreased owing to early increased by pasteurization apparently because
hydrolysis of casein in the vat. For Cheddar-type proteinase inhibitors are inactivated by heating.
cheese, exogenous proteinases may be added to the (b) The inhibitors are retained in cheese made from
milled curds at salting, but the enzyme is concentrated milk concentrated by ultraltration (UF) whereas
at the surface of the curd chips. Activation of indigen- they are lost in the whey during conventional cheese
ous plasminogen by added urokinase also accelerates making. Consequently, proteolysis is retarded in UF
proteolysis (14, 23a). cheese (14, 25).

Denatured -lactoglobulin also inhibits plasmin, minogen (34), and this may inuence proteolysis in
apparently owing to sulfhydryl-disulde interchange cheese by elevating plasmin levels. Although leucocyte
reactions. Since -lactoglobulin is concentrated by proteinases were more active on -casein at pH 6.6
UF, it probably contributes to the inhibition of pro- than at pH 5.2, their activity at the lower pH was
teolysis in cheese made from UF-concentrated milk such as to suggest that they may be active in cheese
during ripening. during ripening (35). Suzuki and Katoh (36) found two
cysteine proteinases in milk (45 kDa and > 150 kDa).
5. Plasminogen Activators The authors suggested that these proteinases origi-
nated in somatic cells and their level increased during
There are two types of plasminogen activators, uroki-
mastitic infection.
nase-type plasminogen activator (uPA) and tissue-type
Grieve and Kitchen (37) compared the action of
plasminogen activator (tPA). Both types occur in milk;
leucocyte proteinases, plasmin, and some psychotroph
uPA is conned to cells in milk while tPA is associated
proteinases on the caseins. Leucocyte extracts hydro-
with the casein micelles (26, 27). Both activators have
lyzed the caseins in order s1 > . Although these
been characterized and cloned (28). Lu and Nielsen
authors considered that neutral proteinases from leu-
(29) reported that there are ve plasminogen activators
cocytes (isolated from blood) were unlikely to be
in milk, with molecular weights of 93, 57, 42, 35, and
important for proteolysis in milk, other authors have
27 kDa; most were uPA-type activators. PA level
found considerably lower proteolytic activity in leuco-
increases with mastitic infection and advancing lacta-
cytes isolated from blood than from milk (34, 35).
tion (30), which explains the greater conversion of
Kelly et al. (39), who compared proteolysis in
plasminogen to plasmin in such milks.
Gouda cheeses made from milks with the same total
somatic cell count but different levels of polymorpho-
B. Cathepsin D (EC 3.4.23.5) nuclear (PMN) leucocytes, found more rapid produc-
tion of s1 -CN f24-199 and total free amino acids in
It has been known for 30 years that milk also con- cheese made from milk with high PMN levels (s1 -CN
tains an acid proteinase (optimum pH 4:0) which is is s1 -casein).
now known to be cathepsin D, a lysosomal enzyme. It The signicance of minor indigenous proteins dur-
is relatively heat labile (inactivated by 70 C for ing mastitic infections was discussed by Fang and
10 min). Its activity in milk has not been studied exten- Sandholm (40) who investigated the possibility of
sively, and its signicance is unknown. At least some of using specic proteinase inhibitors as therapeutic
the indigenous acid proteinase is incorporated into agents. Although the minor proteinases are probably
cheese curd; its specicity on s1 - and -caseins is less signicant technologically than plasmin, more
quite similar to that of chymosin, but it has very work on the subject is warranted.
poor milk clotting activity (31). It may contribute to
proteolysis in cheese, but its activity is probably nor-
mally overshadowed by chymosin, which is present at a III. LIPASES AND ESTERASES (EC 3.1.1.)
much higher level in cheese.
Lipases catalyze the development of hydrolytic rancid-
C. Other Proteinases ity which is a serious defect in milk and some milk
products, and, consequently, lipases and lipolysis in
The presence of other minor proteolytic enzymes in milk have been studied extensively. Milk contains
milk, including thrombin and a lysine aminopeptidase, three types of esterase: (a) A-type carboxylic ester
has been reported (32). In addition to cathepsin D, hydrolase (arylesterase; EC 3.1.1.2), which hydrolyzes
other proteolytic enzymes from somatic cells are prob- aromatic esters, e.g., phenylacetate. It shows little
ably present in milk. Verdi and Barbano (33), who activity on tributyrin, and is not inhibited by organo-
studied the degradation of caseins in milk by somatic phosphates. (b) B-type esterase (glycerol tricarboxyl
cells or plasmin, found that somatic cell proteinases esterase, aliphatic esterase, lipase; EC 3.1.1.3). Such
and plasmin produced distinctly different peptides, enzymes are most active on aliphatic esters although
and that the plasmin inhibitor 6-aminohexanoic acid they show some activity on aromatic esters; they are
was suitable for studying the action of somatic cell inhibited by organophosphates. (c) C-type esterase
proteinases, without interference from plasmin. (cholinesterase; EC 3.1.1.7; EC 3.1.1.8). These enzymes
Somatic cell proteinases are capable of activating plas- are most active on choline esters but hydrolyze some

aromatic and aliphatic esters slowly; they are inhibited lipoprotein content of the bulk milk to below the
by organophosphates. threshold necessary for lipase activation. Natural var-
In normal milk, the ratio of A : B : C types of ester- iations in the level of free fatty acids in normal milk
ase activity is about 3 : 10 : 1 but the level of A-esterase and the susceptibility of normal milks to induced lipo-
activity increases considerably on mastic infection. A lysis may be due to variations in the level of blood
and C esterases are of little technological signicance serum components in milk.
in milk. Lipases hydrolyze ester bonds in emulsied In addition to LPL, human milk contains a bile
esters, i.e., at a water/oil interface, although some saltsactivated lipase, which probably contributes to
may have limited activity on soluble esters. They are the metabolism of lipids by breastfed babies who
usually activated by blood serum albumin and Ca2 have limited pancreatic lipase activity. Bovine milk
which bind free fatty acids, which are inhibitory. and milks from other dairy animals do not contain
Milk lipase was rst isolated and characterized by this enzyme.
Fox and Tarassuk (41) and Patel et al. (42). The
enzyme was optimally active at pH 9.2 and 37 C and
was found to be a serine enzyme (inactivated by orga- A. Assay of Lipolytic Activity
nophosphates). A lipoprotein lipase (LPL; activated by
lipoprotein cofactors) was demonstrated in milk by Lipase can be assayed by incubating the sample with
Korn in 1962 (42a) and isolated by Egelrud and an emulsied lipid substratee.g., milk fat, olive oil,
Olivecrona (43). The lipase isolated by Fox and tributyrin, etc.extraction of liberated fatty acids with
Tarassuk (41) is, in fact, an LPL which is the principal, diethyl ether-petroleum ether, and titration with etha-
probably the only, indigenous lipase in bovine milk. It nolic KOH. This method is rather tedious and tribu-
has been the focus of considerable research and has tyrin agar diffusion assays may be used when rapid
been characterized at the molecular, genetic, enzy- screening is required. Chromogenic substrates, e.g.,
matic, and physiological levels (44). -naphthyl- or p-nitrophenyl derivatives of fatty
Under optimum conditions, the kcat for milk LPL is acids or uorogenic substrates, e.g., coumarin deriva-
3000 s1 and milk contains sufcient enzymes (1 tives of fatty acids, are very sensitive and satisfactory,
2 mg=L; i.e., 1020 nM) to theoretically cause rancidity especially when the enzyme has been at least partially
in 10 sec. However, this does not occur in practice puried.
because the triglycerides are protected by the MFGM
while the lipase is naturally associated with the casein
B. Signicance of Lipase
micelles. Also, environmental conditions, e.g., pH, are
not optimal. However, if the MFGM is damaged by
Technologically, LPL is, arguably, potentially the most
agitation (e.g., by milking machines, bulk tanks,
signicant indigenous enzyme in milk. Although LPL
pumps, etc.), homogenization or temperature uctua-
may play a positive role in cheese ripening, undoubt-
tions, lipolysis occurs rapidly and rancidity ensues.
edly the most industrially important aspect of milk
Milk LPL appears to be derived from blood plasma
lipase is its role in hydrolytic rancidity which renders
and hence any condition that increases the permeabil-
liquid milk and dairy products unpalatable and even-
ity of mammary cell membranes, e.g., physiological
tually unsaleable. Lipolysis in milk has been reviewed
stress, mastitic infection, or late lactation, increases
extensively (45). It appears to occur mainly at the farm
the level of LPL in milk and hence the risk of lipolysis.
level and the problem may be minimized by good man-
Some individual cows produce milk which becomes
agement practices on the farm:
rancid spontaneously, i.e., without apparent activa-
tion. Apparently, spontaneous rancidity occurs when 1. Proper installation, maintenance, and operation
milk contains a high level of lipoprotein (co-lipase) of milking machines.
from blood serum which activates the LPL. Normal 2. Avoidance of excessive agitation by pumps or
milk will become spontaneously rancid if blood agitators in bulk tanks or risers in milk pipe-
serum is added, suggesting that spontaneous milks lines.
contain a higher than normal level of blood serum. 3. Avoidance of freezing on the walls of bulk
Dilution of spontaneous milk with normal milk pre- tanks.
vents spontaneous rancidity, which, consequently, is 4. Avoidance of cooling and warming cycles in the
not normally a problem with bulk herd milks. bulk tank.
Presumably, dilution with normal milk decreases the 5. Culling of cows with high somatic cell counts.

The rst three factors damage the MFGM, making was shown that the timetemperature combinations
the core triglycerides accessible to lipase. Some casein required for the thermal inactivation of alkaline phos-
micelles probably adsorb on exposed fat surfaces. phatase were slightly more severe than those required to
Pipeline milking machines cause more damage to the kill Mycobacterium tuberculosis, then the target micro-
MFGM than hand milking or bucket milking organism for pasteurization. The enzyme is readily
machines. Suction of air at teat cups (which causes assayed, and a test procedure based on alkaline phos-
foaming), risers, and change of dimensions in the pipe- phatase inactivation was developed as a routine quality
line and the hose connecting the clawpiece to the receiv- control test for the HTST pasteurization of milk.
ing jar are major potential sites for damage.
Homogenization causes total replacement of the nat- A. Assay Methods
ural MFGM by a membrane composed of casein
micelles or submicelles and whey proteins. Unless indi- Several major modications of the original assay have
genous LPL is inactivated by pasteurization before or been developed. The usual substrates are phenyl phos-
immediately after homogenization, rancidity will phate, p-nitrophenyl phosphate, or phenolphthalein
develop very rapidly. Minimal high temperature short phosphate which are hydrolyzed to inorganic phos-
time (HTST; 72 C for 15 sec) pasteurization of milk phate and phenol, p-nitrophenol, or phenolphthalein,
causes extensive inactivation of LPL but pasteurization respectively:
at 80 C for 10 sec is required for complete inactivation.
Freezing of milk on the walls of bulk tanks will
damage the MFGM, inducing lipolysis. Temperature
uctuations, e.g., cooling to 5 C, rewarming to
30 C, and recooling, also activate lipolysis. Such
temperature uctuations may occur if bulk tanks are where XOH phenol, p-nitrophenol, or phenolphtha-
not completely emptied at each milk collection and lein.
warm milk is added at the subsequent milking. The The release of inorganic phosphate may be assayed
mechanism of thermal activation is not clear, but but the other product is usually determined. Phenol is
damage to the MFGM by fat crystals seems likely. colorless but forms a colored complex on reaction with
The propensity of milk to lipolysis increases when one of several reagents, e.g., 2,6-dichloroquinonechlor-
the producing animals are under any type of stress. oimide, with which it forms a blue complex. p-
The mammary cell membranes become more perme- Nitrophenol is yellow while phenolphthalein is red at
able to blood constituents as a result of mastitic infec- the alkaline pH of the assay ( 10) and hence these are
tion in late lactation and as the animal ages. The easily quantied.
number of somatic cells in milk is a good index of A uorogenic aromatic orthophosphoric monoe-
such stress or damage, and upper limits for somatic ster, Fluorophos (Advanced Instruments, Needham
cell count (SCC) are now frequently prescribed by Heights, MA), has been developed and approved for
milk processors. Although the potential for hydrolytic the determination of alkaline phosphatase in milk and
rancidity always exists in raw milk, the problem can be milk products. Hydrolysis of the ester yields a uores-
reduced to insignicant levels by good management cent compound, Fluoroyellow, the concentration of
practices at the farm. which is determined uorometrically (excitation,
439 nm; emission, 560 nm).
IV. ALKALINE PHOSPHATASES (EC 3.1.3.1)
Milk contains several phosphatases, the principal ones

being alkaline and acid phosphomonoesterases, which
are of technological signicance, and ribonuclease,
which has no known function or signicance in milk. Fluorometric methods are about 1001000 times more
The alkaline and acid phosphomonoesterases have sensitive than colorimetric assays. A simple uorom-
been studied extensively (1, 8, 9). eter has been developed for the analysis (Advanced
The occurrence of a phosphatase in milk was rst Instruments). Comparative studies on the uorometric
recognized in 1925. Subsequently characterized as an and the standard colorimetric methods include those of
alkaline phosphatase, it became signicant when it Rocco (46) and Eckner (47).

B. Isolation and Characterization of Alkaline C. Reactivation of Phosphatase
Phosphatase
Much work has been focused on a phenomenon
Alkaline phosphatase is concentrated in the fat globule known as phosphatase reactivation, rst recognized
membrane and hence in cream. The membrane is by Wright and Tramer in 1953, who observed that
released into the buttermilk on phase inversion; conse- UHT-treated milk was phosphatase-negative immedi-
quently, buttermilk is the starting material for most ately after processing but became positive on standing;
published methods for the purication of alkaline microbial phosphatase was shown not to be responsi-
phosphatase. Later methods have used chromatogra- ble. Bulk HTST milk never showed reactivation,
phy on various media to give a homogeneous prepara- although occasional individual cow samples did.
tion; up to 7500-fold purication with a yield of HTST pasteurization after UHT treatment usually pre-
30% have been reported (1). The characteristics of vented reactivation which was never observed in in-
milk alkaline phosphatase are summarized in Table 4. container sterilized milk. Reactivation can occur fol-
The enzyme appears to be similar to the alkaline phos- lowing heating at a temperature as low as 84 C for
phatase of mammary tissue, which is the presumed milk or 74 C for cream. The optimum storage tem-
source of the enzyme in milk. perature for reactivation is 30 C, at which reactivation
The enzyme is a dimer of two identical 85-kDa sub- is detectable after 6 h and may continue for up to 7
units. It contains 4 Zn2 per mole which are required days. The greater reactivation in cream than in milk
for activity; Mg2 are also strong activators, and may be due to protection by fat, but this has not been
1 mM Mg2 is usually added to the assay mixture. substantiated.
The enzyme is strongly but reversibly inhibited by A number of attempts have been made to explain
metal chelators; the apoenzyme is reactivated by the mechanism of reactivation (8). There is evidence
Zn2 , Mg2 , and other metal ions. Reaction of apoen- that the form of the enzyme which becomes reactivated
zyme may in fact be used as a sensitive assay for avail- is membrane bound, and several factors which inu-
able Zn2 in foods. Inorganic orthophosphates are ence reactivation have been established. Mg2 and
strong competitive inhibitors of the hydrolysis of p- Zn2 strongly promote reactivation; Sn2 , Cu2 ,
nitrophenylphosphate. Alkaline milk phosphatase can Co2 and EDTA are inhibitory, while Fe2 has no
dephosphorylate phosphoproteins, including casein, effect. Sulfhydryl (SH) groups appear to be essential
with a pH optimum of 6.57.0; however, it does not for reactivation; perhaps this is why phosphatase
appear to do so in milk, probably owing to inhibition becomes reactivated in UHT milk but not in HTST
by the high level of orthophosphate. milk. The role of -SH groups, supplied by denatured
whey proteins, is considered to be chelation of heavy
metals, which would otherwise bind to -SH groups of
Table 4 Characteristics of Milk Alkaline Phosphatase
the enzyme (also activated on denaturation), thus pre-
Characteristic Conditions venting renaturation. It has been proposed that Mg2
and Zn2 cause a conformational change in the dena-
pH optimum Casein: 6.8 tured enzyme, necessary for renaturation (1).
p-nitrophenylphosphate: 9.9 According to Murthy et al. (48), maximum reactiva-
37 C
Temperature optimum
tion occurs in products heated at 104 C; incubated
Km 0.69 mM on p-
at 34 C, adjusted to pH 6.5, and containing
nitrophenylphosphate
Activators Ca2 , Mn2 ; Zn2 , Co2 , 0:064 M Mg2 ; homogenization of products before
Mg2 heat treatment reduces the extent of reactivation.
Inhibitors Metal chelators (EDTA, There are reports that raw milk contains three isoen-
EGTA, etc.); zymes of alkaline phosphatase and that the zymogram
orthophosphates patterns of raw and reactivated milk or cream are dif-
Native molecular weights 170190 kDa ferent (1, 48); however, these different forms probably
Quaternary structure 2 subunits each of molecular represent free and bound forms of the enzyme. A
weights 85 kDa mechanism for the reactivation of alkaline phospha-
Zn content 4 mol mol1 enzyme tase was proposed by Copius-Peereboom (49); how-
Thermal stability:
ever, this mechanism of reactivation is based on a
D-value at 60 C, pH 9 27.2 min
putative structure of the milk fat globule membrane
63 C, pH 9 8.3 min
which is now known to be incorrect. Linden (50)

proposed that reactivation occurs in stages and full the dephosphorylation of phosphopeptides in cheese is
reactivation requires Zn2 , Mg2 , inorganic phosphate warranted.
(Pi), and substrate (S).
Reactivation of alkaline phosphatase is of consider-
able practical signicance since regulatory tests for V. ACID PHOSPHOMONOESTERASE
pasteurization assume the absence of phosphatase (EC 3.1.3.2)
activity. Methods for distinguishing between renatured
and residual native alkaline phosphatase are based on Milk contains an acid phosphatase which has a pH
the increase in phosphatase activity resulting from optimum at 4.0 and is very heat stable. (LTLT pasteur-
addition of Mg2 to the reaction mixture; various ver- ization causes only 1020% inactivation and 30 min at
sions of the test have been proposed (48, 51, 52). The 88 C is required for full inactivation; when heated in
ofcial AOAC method (53) is based on that of Murthy milk at pH 6.7, the enzyme retains signicant activity
and Peeler (52). However, difculties are experienced following HTST pasteurization but it does not survive
in the interpretation of this test applied to cream or in-bottle sterilization or UHT treatment.) The enzyme
butter (54, 55). is not activated by Mg2 (as is alkaline phosphatase),
but it is slightly activated by Mn2 and is very strongly
inhibited by uoride. The level of acid phosphatase
D. Signicance activity in milk is only 2% that of alkaline phospha-
tase; activity reaches a maximum 56 days postpartum,
Alkaline phosphatase in milk is signicant mainly then decreases and remains at a low level to the end of
because of its use as an index of HTST pasteurization lactation (65).
and is used universally for this purpose. However, the
enzyme may not be the most appropriate for this A. Isolation and Characterization
purpose (56) becomes (a) reactivation of alkaline phos-
phatase under certain conditions complicates inter- Acid phosphatase is found free in skim milk, in mem-
pretation of the test; (b) the enzyme appears to be brane material in skim milk and in the fat globule
fully inactivated by subpasteurization conditions membrane. Kitchen (9) appears to believe that a single
(70 C for 16 sec); and (c) the relationship between enzyme is involved and reported that the membrane-
log10 % initially activity and pasteurization equivalent bound enzyme is strongly attached and is not released
(PE) is less linear than the relationship of lactoperox- by nonionic detergents. The enzyme has been puried
idase or -glutamyl transpeptidase (57). to homogeneity by various forms of chromatography,
Although alkaline phosphatase can dephosphory- including afnity chromatography. Purication factors
late casein under suitable conditions, as far as is of 10,000 to 1 million have been reported (1).
known, it has no direct technological signicance in Adsorption onto Amberlite IRC50 resin is a very effec-
milk. Perhaps its pH optimum is too far removed tive rst step in purication. Andrews (65) stated that
from that of milk, especially acid milk products, all the acid phosphatase activity in skim milk is
although the pH optimum on casein is reported to be adsorbed by Amberlite IRC50, but this is not indicated
7. It is also inhibited by inorganic phosphate. in the original papers. In an unpublished study, N.A.
Proteolysis is a major contributor to the develop- Flynn and P.F. Fox found that only 50% of the
ment of avor and texture of cheese during ripening total acid phosphatase in skim milk was adsorbed by
(58). Most of the small water-soluble peptides in cheese Amberlite IRC50 even after reextracting the skim milk
are from the N-terminal half of s1 - or -casein; many with fresh batches of Amberlite, suggesting that skim
are phosphorylated but show evidence of phosphatase milk may contain two acid phosphatases. About 40%
activity (i.e., they are partially dephosphorylated (59 of the acid phosphatase in skim milk partitioned into
62). In cheese made from pasteurized milk, both indi- the whey on rennet coagulation and this enzyme did
genous acid phosphatase and bacterial phosphatase not adsorb on Amberlite IRC50. The enzyme was
are probably responsible for dephosphorylation partly puried from whey.
(which is the more important is not clear), but in raw Flynn and Fox attempted to purify the alkaline
milk cheese, e.g., Parmigiano Reggiano or Grana phosphatase from MFGM by gel permeation chroma-
Padano, milk alkaline phosphatase appears to be the tography; however, sonication and nonionic detergents
most important (63, 64). Further work on the signi- failed to disassociate the enzyme from the membrane.
cance of indigenous alkaline and acid phosphatases in The MFGM enzyme, which does not adsorb on

Amberlite IRC50, was much less heat stable than the peptides have been isolated from Cheddar and
acid phosphatase isolated from whey or from skim Parmigiano Reggiano and Grana Padano cheeses.
milk by adsorption on Amberlite IRC50. Attempts However, it is not known whether indigenous or bac-
were made to conrm that the MFGM, whey, and terial acid phosphatase is mainly responsible for
Amberlite-adsorbed enzymes are different by studying dephosphorylation in cheese made from pasteurized
the effects of inhibitors, but the results have been equi- milk. It is claimed (6264) that alkaline phosphatase
vocal. Overall, it appears that milk contains more than is mainly responsible for dephosphorylation in raw
one acid phosphatase. Using a zymogram technique, milk cheese. Dephosphorylation may be rate limiting
Andrews and Alichanidis (66) reported that milk from for proteolysis in cheese ripening since most protei-
healthy cows contained one acid phophatase while that nases and peptidases are inactive on phosphoproteins
from mastitic cows contained two additional acid or phosphopeptides. It has been suggested that phos-
phosphatases which were of leucocyte origin. It is unli- phatase activity should be included in the criteria for
kely that the heterogeneity observed by Flynn and Fox starter selection.
was due to a high proportion of mastitic milk. The acid phosphatase activity in milk increases
The acid phosphatase isolated from skim milk by fourfold to 10-fold during mastitic infection. Three
adsorption on Amberlite IRC50 has been well charac- isoenzymes are then present, only one of which is indi-
terized. It is a glycoprotein with a molecular weight of genous milk acid phosphatase, the other two being of
42 kDa and a pI of 7.9. It is inhibited by many heavy leucocyte origin (66). The latter isoenzymes are more
metals, F , oxidizing agents, orthophosphates, and thermolabile than the indigenous enzyme and are inac-
polyphosphates and is activated by thiol-reducing tivated by HTST pasteurization.
agents and ascorbic acid. It is not affected by metal The suitability of acid phosphatase as an indicator
chelators. Its amino acid composition indicates a enzyme for superpasteurization of milk has been
high level of basic amino acids and no methionine (65). assessed (67, 68). It is not as useful for this purpose
The enzyme is quite active on phosphoproteins, as some alternatives, e.g., -glutamyl transpeptidase or
including caseins. It has been suggested that it is a lactoperoxidase.
phosphoprotein phosphatase. Although casein is a
substrate for milk acid phosphatase, the major caseins,
in the order s s1 s2 > > , also act as compe- VI. LYSOZYME (EC 3.2.1.17)
titive inhibitors of the enzyme when assayed on p-
nitrophenylphosphate, probably owing to binding of Lysozyme (muramidase, mucopeptide N-acetyl-mura-
the enzyme to the casein phosphate groups (the effec- myl hydrolase) is a widely distributed enzyme which
tiveness of the caseins as inhibitors is related to their lyses certain bacteria by hydrolyzing the (1-4) linkage
phosphate content). between muramic acid and N-acetylglucosamine of
mucopolysaccharides in the bacterial cell wall.
B. Assay Lysozyme activity is normally assayed by the lysis of
cultures of Micrococcus lysodeikticus measured by a
Acid phosphatase may be assayed, at pH 5, on the decrease in turbidity.
same substrates as used for alkaline phosphatase. If p- Lysozyme was isolated from human milk by Jolles
nitrophenyl phosphate or phenolphthalein phosphate and Jolles (69), who believed that bovine milk was
is used, the pH must be adjusted to > 8 at the end of devoid of lysozyme. Milks of many species, including
the enzymatic reaction in order to induce the color of bovine, have since been shown to contain lysozyme,
the product, i.e., p-nitrophenol or phenolphthalein. and several have been isolated and characterized (70).
Human and equine milks are exceptionally rich
C. Signicance sources, containing 130 mg/L (3000 times the level of
bovine milk) and 800 mg=L, respectively.
Although acid phosphatase is present in milk at a The pH optima of human milk lysozyme (HML),
much lower level than alkaline phosphatase, its greater bovine milk lysozyme (BML), and egg-white lysozyme
heat stability and lower pH optimum may make it (EWL) are 7.9, 6.35, and 6.2, respectively (70). BML
technologically signicant. Dephosporylation of casein has a molecular weight of 18 kDa compared with
reduces its ability to bind Ca2 , to react with -casein, 15 kDa for HML and EWL. The amino acid composi-
to form micelles, and its heat stability. As discussed in tion of BML is considerably different from that of
Section IV.D, several small partially dephosphorylated HML or EWL. The amino acid sequence of BML is

highly homologous with that of -lactalbumin, a whey epithelial cells and, to a lesser extent, from somatic
protein that is an enzyme modier in the biosynthesis cells. Consequently, NAGase activity correlates highly
of lactose. All lysozymes are relatively stable to heat at with the intensity of mastitis. A eld test for mastitis
acid pH values (34) but are relatively labile at pH > 7. based on NAGase activity has been developed, using
More than 75% of the lysozyme activity in bovine milk chromogenic p-nitrophenyl N-actyl--D-glucosamine
survives heating at 75 C for 15 min or 80 C for 15 sec as substrate. Hydrolysis yields p-nitrophenol, which
and is therefore little affected by HTST pasteurization. is yellow at alkaline pH. NAGase activity is also
Low concentrations of reducing agents increase the high in colostrum. NAGase is optimally active at
activity of BML and HML by 330% (70). 50 C and pH 4.2 and is inactivated by HTST pasteur-
Presumably, the physiological role of lysozyme is to ization (7071 C for 1518 sec). Andrews et al. (68)
act as a bacteriocidal agent. In the case of milk, it may proposed that NAGase would be a suitable indicator
simply be a spillover enzyme, or it may have a de- enzyme for assessing heat treatments in the range 65
nite protective role. If the latter is true, then the excep- 75 C for 15 sec. NAGase occurs mainly in the whey
tionally high level of lysozyme in human and equine fraction, from which it has been isolated by various
milk may be signicant. Breastfed babies generally suf- forms of chromatography. Two forms, A and B, differ-
fer fewer enteric problems than bottle-fed babies. ing in molecular weight, 120 and 240 kDa, respectively,
While there are many major compositional and physi- and charge were obtained. Each form is dissociated to
cochemical differences between bovine and human two dissimilar subunits on treatment with 2-mercap-
milks which may be responsible for the observed nutri- toethanol and SDS (9, 70).
tional characteristics, the disparity in lysozyme content
may be signicant. Fortication of bovine milk-based
infant formulae with EWL, especially for premature
babies, has been recommended but feeding studies VIII. -GLUTAMYL TRANSPEPTIDASE
are equivocal on the benets of this practice, and (TRANSFERASE) (EC 2.3.2.2)
recent trials failed to demonstrate any benecial effect
due to inactivation of EWL in the human stomach -Glutamyl transpeptidase (GGTP) catalyzes the
(71, 72). transfer of -glutamyl residues from -glutamyl-con-
One might expect that, owing to this bacteriocidal taining peptides:
effect, indigenous milk lysozyme would have a bene-
cial effect on the shelf life of milk. Such effects do not -glutamyl-peptide X ! peptide -glutamyl-X
appear to have been reported. Exogenous lysozyme 3
may be added to milk for many cheese varieties, e.g.,
where X is an amino acid.
Gouda, Edam, Emmental, and Parmigiano Reggiano,
The enzyme is membrane bound, being found in the
as an alternative to KNO3 to prevent the growth of Cl.
membrane material in skim milk ( 70%) or in the
tyrobutyricum which causes late gas blowing and off-
MFGM, from which it can be dissociated by deter-
avors. At present, lysozyme is not widely used in
gents or solvents. The enzyme, which has been puried
commercial cheesemaking (71, 73). Since indigenous
from the MFGM, has a molecular weight of 80 kDa
milk lysozyme is in the serum phase, very little is incor-
and consists of two subunits of 57 and 25 kDa, both of
porated into cheese.
which are glycoproteins (74, 75). The enzyme is
Addition of lysozyme to milk decreases the heat
optimally active at pH 8.59 and 45 C and has an
stability of milk, but the level of indigenous lysozyme
isoelectric point of 3.85. It is strongly inhibited by di-
is probably too low to contribute to the natural varia-
isopropyluorophosphate, iodoacetamide, and metals,
tions in the heat stability of milk.
e.g., Cu2 and Fe3 (9, 70).
VII. N-ACETYL--D-GLUCOSAMINIDASE A. Assay

(EC 3.2.1.30)
GGTP is usually assayed using -glutamyl-p-nitroani-
N-Acetyl--D-glucosaminidase (NAGase) hydrolyzes lide as substrate; the liberated p-NA can be determined
terminal, nonreducing N-acetyl--D-glucosamine resi- by measuring the absorbance at 410 nm or by reaction
dues from glycoproteins. It is a lysosomal enzyme with naphthylethylenediamine and measuring the
which originates principally from mammary gland absorbance at 540 nm (76).

B. Signicance smaller stabilizing effect on Listeria innocua (79). Thus,
it appears that GGTP is a suitable enzyme for estimat-
GGTP functions in the regulation of cellular glu- ing the intensity of heat treatment in the range
tathione and amino acid transport via the -glutamyl 7277 C for 15 sec to which milk was subjected.
cycle; it may be involved in the biosynthesis of milk GGTP is absorbed from the gastrointestinal tract,
proteins. resulting in high levels of GGTP activity in the blood
From a dairy technologists viewpoint, GGTP is of serum of newborn animals fed colostrum or early
interest mainly because of its heat stability character- breast milk. Since GGTP is inactivated by the heat
istics. As discussed in Section IV.D, alkaline phospha- treatment to which infant formulae are subjected, the
tase is the test enzyme usually used to evaluate the level of GGTP activity in infants can be used to dis-
efciency of HTST pasteurization; however, as distinguish breastfed from formula-fed infants (70).
cussed, reactivation of this enzyme in UHT-treated -Glutamyl peptides have been isolated from Comte
products poses problems with the interpretation of cheese (80). Since casein contains no -glutamyl bonds,
the test. Based on a comparative study on the heat the presence of these peptides in cheese suggests GGTP
stability characteristics of a number of indigenous activity in cheese, but there appear to be no data on
enzymes in milk, Andrews et al. 68) concluded that this.
GGTP was appropriate for monitoring heat treatments
in the range of 7080 C for 16 sec. This conclusion has
been conrmed in pilot-scale studies (77, 78). In whole IX. XANTHINE OXIDASE (EC 1.2.3.2)
or skim milk, GGTP is completely inactivated by heat-
ing at 78 C for 15 sec (77) or 77 C for 16 sec (76). No It has been recognized for 90 years that milk con-
reactivation was found under a variety of conditions, tains an enzyme capable of oxidizing aldehydes and
and little seasonal variation occurs. As little as 0.1% purines with the concomitant reduction of O2 to
raw milk could be detected in skim milk or 0.25% in H2 O2 . The enzyme is now generally referred to as
whole milk (76). xanthine oxidase (XO). Milk is a very good source of
Linear models for the thermal inactivation of XO, at least part of which is transported to the mam-
GGTP and lactoperoxidase (LPO) in a HTST pasteur- mary gland via the bloodstream. A similar enzyme is
izer were developed by McKellar et al. (57). The equa- found in various animal tissues and several bacterial
tion for GGTP was: log10 % initial activity 2:004 species (8, 9, 70).
0:281 PE0:75 . For LPO the equation was: log10 %
initial activity 2:1220:096 PE0:75 . A. Assay

Ea 1 1
t Xanthine oxidase activity can be assayed manometri-
PE e R T T0 dt 4 cally (uptake of O2 ), potentiometrically using a plati-
t0
num electrode, or spectrophotometrically. This last
where Ea activation energy; R 8:314 J=mol K; involves the conversion of the xanthine to uric acid
T0 345 K (72 C reference temperature); T which is quantied by measuring absorbance at
experimental temperature; t0 15 sec (reference hold- 290 nm (75).
ing time); t experimental holding time, sec; and PE
pasteurization equivalent (71:6 C for 15 sec). B. Isolation
These equations indicate that 1 log decrease in
enzyme activity can be achieved with PE values of The enzyme is concentrated in the MFGM, in which it
5.46 and 2.65 for GGTP and LPO, respectively, indi- is one of the principal proteins. Therefore, all isolation
cating that LPO is considerably more heat stable than methods use cream as starting material, using a dis-
GGTP. The assay for LPO (using ABTS; see Sec. sociating agent to liberate XO from membrane lipo-
XIII.A) is subject to greater variation and interference proteins and some form of chromatography for
from milk proteins than the GGTP assay. The relation- purication.
ship between log10 % initial activity and PE was more Milk XO has a molecular weight of 300 kDa and
linear than the relationship for alkaline phosphatase, consists of two identical subunits. The pH optimum is
possibly due to more than one form of alkaline phos- 8:5 and the enzyme requires avin adenine dinucleo-
phatase (56). GGTP was about nine times more stable tide (FAD), Fe and Mo cations, and an acid-labile
in ice cream mix than in whole milk, which had a much sulfur compound as cofactors. Cows decient in Mo

cations have low XO activity. The amino acid compo- 2. Lipid Oxidation
sition of XO has been determined by a number of
XO, which can excite stable triple oxygen (3 O2 ) to sin-
workers; at least ve polymorphic forms have been
gle oxygen (1 O2 , is a pro-oxidant. Some individual-
reported (70).
cow milks, which undergo spontaneous oxidative ran-
XO can be converted to an NAD-dependent dehy-
cidity (i.e., without contamination with metals or expo-
drogenase by treatment with thiol-reducing agents.
sure to light) contain 10 times the normal level of
The enzyme reverts to an oxidase on aerobic storage,
XO, and spontaneous oxidation can be induced in nor-
treatment with sulfhydryl oxidase, or with sulfhydryl
mal milk by the addition of XO to about four times
oxidizing agents.
normal levels. Heat-denatured or avin-free enzyme is
ineffective in oxidation; the susceptibility of unsatu-
C. Activity in Milk
rated fatty acids to oxidation increases with the degree
of unsaturation (8).
XO activity in milk varies substantially2.5-fold (67).
Various processing treatments which damage or alter
3. Atherosclerosis
the MFGM affect the XO activity of milk. Activity is
increased by 100% on storage at 4 C for 24 h, by It has been suggested that XO from homogenized milk
50100% on heating at 70 C for 5 min, and by 60 enters the vascular system and may be involved in
90% on homogenization. These treatments cause the atherosclerosis via oxidation of plasmalogens in cell
release of XO from the MFGM into the aqueous membranes; this aspect of XO attracted considerable
phase, rendering the enzyme more active. The heat attention in the early 1970s (83). However, the experi-
stability of XO is very dependent on whether it is a mental evidence in support of this view is very weak
component of the MFGM or is dissolved in the aqu- and the hypothesis has been disclaimed (8, 70, 84, 85).
eous phase. Aging and homogenization increase heat
susceptibility and explain the inconsistency of early 4. Reduction of Nitrate in Cheese
work in which the history of the sample was unknown
Sodium nitrate is added to milk for Dutch, Swiss, and
or unrecorded. XO is most heat stable in cream and
other cheese varieties to prevent the growth of
least in skim milk. Homogenization of concentrated
Clostridium tyrobutyricum which causes avor defects
milk prepared from heated milk (90:5 C for 15 sec)
and late gas blowing in these cheeses. Xanthine oxidase
partially reactivates XO, which persists on drying the
reduces nitrate to nitrite which is necessary for the
concentrate, but no reactivation occurs following more
bacteriocidal effect of nitrate.
severe heating (93 C for 15 sec). Apparently, homoge-
Various oxidation-reduction reactions occur in milk
nization releases potentially active, undenatured XO
which affect its avor. Perhaps XO is signicant in
from the MFGM. All the major milk proteins can
these reactions, either directly or indirectly.
act as either activators or inhibitors of XO, depending
on their concentration, and may have some signi-
5. Production of H2 O2
cance in the activation, inactivation, and reactivation
of the enzyme. Studies on the heat stability of XO have The H2 O2 produced by the action of xanthine oxidase
been reviewed by Grifths (67), who investigated its on oxidation of xanthine, hypoxanthine, or other sub-
stability in a pilot scale HTST pasteurizer. The enzyme strates can serve as a substrate for lactoperoxidase in
was not completely inactivated after 120 sec at 80 C, its action as a bacteriocidal agent (see Sec. XIII.B).
and a Z-value of 6.8 was calculated.
D. Signicance of Xanthine Oxidase

X. SULFHYDRYL OXIDASE (EC 1.8.3.)
1. As an Index of Heat Treatment
The inactivation of XO parallels the conditions neces- Milk contains an enzyme, sulfhydryl oxidase (SO),
sary for the production of low-heat skim milk powder capable of oxidizing sulfhydryl groups of cysteine, glu-
(82). Andrews et al. (68) considered XO as a suitable tathione, and proteins to the corresponding disulde
indicator of milk heated in the temperature range 80 (for reviews, see 8, 70). The enzyme is an aerobic
90 C but Grifths (67) considered the natural variabil- oxidase which catalyzes the following reaction:
ity in the level of XO activity in milk to be too high for
use as a reliable index of heat treatment. 2RSH O2 ! RSSR H2 O2 5

It undergoes marked self-association and can be has been isolated and characterized. It appears to be
puried readily by chromatography on porous glass. identical to the bovine erythrocyte enzyme. Assay
The enzyme has a molecular weight of 89 kDa, a pH methods for SOD are described by Stauffer (86).
optimum of 6.87.0, and a temperature optimum of SOD inhibits lipid oxidation in model systems. The
35 C. Its amino acid composition, its requirement for level of SOD in milk parallels that of XO (but at a
iron but not for molybdenum and FAD, and the cat- lower level), suggesting that SOD may be excreted in
alytic properties of the enzyme indicate that sulfhydryl milk in an attempt to offset the pro-oxidant effect of
oxidase is a distinct enzyme from xanthine oxidase and XO. Attempts have been made to correlate the stability
thiol oxidase (EC 1.8.3.2). of milk to oxidative rancidity with the SOD activity in
SO is capable of oxidizing reduced ribonuclease and the milk, but these results have been equivocal. Milk
restoring enzymatic activity, suggesting that its physio- contains several pro- and antioxidants, including exo-
logical role may be the nonrandom formation of pro- genous factors such as light, the precise balance of
tein disulde bonds, e.g., during protein biosynthesis. which, rather than any single factor, determines overall
SO immobilized on glass beads has the potential to oxidative stability. The possibility of using exogenous
ameliorate the cooked avor arising from sulfhydryl SOD to retard or inhibit lipid oxidation in dairy pro-
groups exposed by protein denaturation on UHT products has been considered. A marked improvement in
cessing of milk, but the commercial viability of this the oxidative stability of milk with a high level of lino-
system is not known. In any case, sulfhydryl groups leic acid was achieved by adding low levels of SOD.
are effective antioxidants and may serve a benecial SOD is more heat stable in milk than in puried
role in UHT milk. preparations. In milk it is stable at 71 C for 30 min
The production of sulfur compounds is believed to (i.e., it is not affected by HTST pasteurization) but
be very important in avor development in Cheddar loses activity rapidly at even slightly higher tempera-
and other varieties of cheese. Residual sulfhydryl oxi- tures. Slight variations in pasteurization temperature
dase activity may play a role in reoxidizing sulfhydryl are therefore critical to the survival of SOD in heated
groups exposed upon heating cheesemilk; the sulfhy- milk products and may contribute to variations in the
dryl groups thus protected may be reformed during the stability of milk to oxidative rancidity.
ripening process.
XI. SUPEROXIDE DISMUTASE XII. CATALASE (EC 1.11.1.6)

(EC 1.15.1.1)
An indigenous catalase in milk was rst recognized in
Superoxide dismutase (SOD) scavenges superoxide 1907. The catalase activity of whole milk is associated
radicals, O2 , according to the reaction: either with membranes in the skim milk phase or the
MFGM. The pellet obtained from buttermilk on cen-
2O2 2H ! H2 O2 O2 6
trifugation at 10,000 g is a particularly rich source,
The H2 O2 formed may be reduced to H2 O O2 by from which catalase has been highly puried and crys-
catalase, peroxidase or suitable reducing agents. SOD tallized (8, 9, 70).
has been identied in many animals and bacterial cells. Milk catalase is a heme protein with a molecular
Its biological function is to protect tissue against oxy- weight of 200 kDa and an isoelectric pH of 5.5. It is
gen free radicals in anaerobic systems (8, 9, 70). stable between pH 5 and 10 but rapidly loses activity
SOD, isolated from bovine erythrocytes, is a blue- outside the range. Heating at 70 C for 1 h at pH 7.0
green protein due to the presence of copper, removal of causes complete inactivation. Like other catalases, it is
which by treatment with EDTA results in loss of activ- strongly inhibited by Hg2 , Fe2 , Cu2 , Sn2 , CN ,
ity which is restored by adding Cu2 ; it also contains and NO 3 . Analytical methods for catalase are
Zn2 , which does not appear to be at the active site. described by Stauffer (86).
The enzyme, which is very stable in 9 M urea at neutral Catalase activity in milk varies with feed, stage of
pH, consists of two identical subunits of molecular lactation, and especially with mastitic infection, for
weight 16 kDa held together by one or more disulde which it may be used as an index. However, it is not
bonds. usually used for this purpose, since somatic cell count
Milk contains trace amounts of SOD which is pre- and N-acetylglucosaminidase activity are superior
sent exclusively in the skim milk fraction. This enzyme indices of mastitis. Catalase may act as a lipid pro-

oxidant via its heme iron (i.e., nonenzymatically), but A. Assay of Lactoperoxidase
it is probably not very signicant.
There is general agreement that cheese made from The principle generally used in assays for peroxidases
raw milk ripens more quickly and develops a more is the use of a chromogenic or uorogenic reducing
intense (although not always a more desirable) avor agent, AH2 , a number of which have been used (86).
than cheese made from pasteurized milk (87). A highly recommended substrate for lactoperoxidase is
However, for public health reasons and in the interest 2; 2 0 -azinobis(3-ethylbenzylthiazoline-6-sulfonic acid)
of producing a consistent product, pasteurized milk is [ABTS] (91).
now generally used for cheese making. Many cheeses
are still made from raw milk, especially in southern B. Signicance
Europe. Many countries require that if raw milk is
used for cheese making, the cheese must be ripened 1. As for many other indigenous enzymes, the
for at least 60 days during which it was presumed level of LPO in milk increases on mastitic infection
that pathogens die off, a presumption that is now con- and is therefore a possible index of mastitis; however,
sidered to be invalid. Subpasteurized or thermized milk it is not well correlated with somatic cell count, and
(e.g., heated at 6365 C for 16 sec) has been considered superior methods, including enzyme-based methods
as a compromise between raw and pasteurized milk for (Sec. VII) are available to monitor mastitis.
cheese making. The acceptance of such a practice 2. LPO causes nonenzymic oxidation of un-
would require an appropriate validation test, but no saturated lipids, probably due to its heme group.
suitable test is available at present for identifying ther- The heat-denatured enzyme is more active than the
mized milk. The possibility of using the inactivation of native enzyme. Compared with other pro-oxidants,
catalase as an index of thermized milk was investigated LPO is probably not signicant in milk and dairy
by Hirvi and Grifths (88). Although catalase was a products.
useful index of thermization of milk (it was almost 3. LPO has been used in the Storch test for ash
completely inactivated by heating at 65 C for 16 sec), pasteurized milk (67), but this process is not used in
it was unsuitable as an index of cheese made from modern milk processing. However, the pasteurization
thermized milk owing to the production of catalase of milk at a temperature higher than the HTST mini-
in the cheese during ripening, especially by yeasts. mum, i.e., > 72 C for 15 sec, has recently become
The thermal inactivation of catalase was also studied quite common. The assay based on the inactivation
by Hirvi et al. (89). of alkaline phosphatase is not applicable under such
circumstances. Grifths (67), who evaluated the suit-
ability of several indigenous enzymes as indices of the
super-pasteurization of milk, concluded that assay of
XIII. LACTOPEROXIDASE (EC 1.11.1.7) LPO activity was the most promising method for
detecting heat treatments in the order of 76 C for
The occurrence of a peroxidase, lactoperoxidase 15 sec. Andrews et al. (68) reported the results of a
(LPO), in milk was recognized as early as 1881. It is generally similar study, in which LPO was not
one of the most heat-stable enzymes in milk. Its included. They conclude that N-acetylglucosamini-
inactivation was used as an index of ash pasteuriza- dase, -glutamyl transpeptidase (-GGTP), and -
tion (now very rarely used) and is now used as an mannosidase or xanthine oxidase may be the most
index of super-HTST pasteurization, e.g., tempera- suitable indicators of heat treatment of 6575, 70
tures > 75 C for 15 sec. LPO was rst isolated in 80, and 8090 C, respectively. The relative suitability
1943; several isolation procedures have since been of GGTP and LPO were compared by McKellar et al.
published (8, 90). (57). The kinetics of the thermal denaturation of LPO
LPO is a heme protein containing 0:07% Fe, with was reported by Martin-Hernandez et al. (92).
an absorbance peak (Soret band) at 412 nm 4. Milk contains bacteriostatic or bacteriocidal
(A412 =A280 0:9). The pH optimum is 8:0, its substances, referred to as lactenins. One of these is
molecular weight is 77.5 kDa, and it consists of two LPO, which requires H2 O2 and thiocyanate (SCN )
identical subunits. Two principal forms (A and B) to cause inhibition. The nature, mode of action, and
occur, each of which exhibits microheterogeneity with specicity of the LPO-SCN -H2 O2 system have been
regard to amide groups (glutamine and/or asparagine) widely studied. LPO and thiocyanate, which is pro-
and carbohydrate content, giving a total of 10 variants. duced in the rumen by enzymatic hydrolysis of thio-

glycosides from Brassicae plants, occur naturally in LPO
H2 O2 SCN ! OSCN H2 O
milk although at variable and probably suboptimal
levels. Milk does not contain indigenous H2 O2 , thiocyanate anion (hypothiocyanite anion)
which can be generated metabolically by catalase- 7
negative bacteria, or produced in situ through the Microbial membranes have low permeability for
action of exogenous glucose oxidase on glucose
OSCN but are quite permeable to HOSCN
which may be added to milk or produced in situ pKa 5:3). HOSCN or OSCN oxidizes sulfhydryl
from lactose by exogenous -galactosidase or the groups:
action of indigenous XO on added xanthine or
hypoxanthine, or from added sodium percarbonate RSH OSCN!R-S-SCN OH
or it may be added directly. (sulfenyl thiocyanate) 8
Immobilized glucose oxidase has been used to gen-
erate H2 O2 in situ in thiocyanate- and glucose-enriched R-S-SCN H2 O!R-S-OH H SCN
milk or whey. A self-contained LPO-H2 O2 -SCN sys- (sulfonic acid) 9
tem using coupled -galactosidase and glucose oxidase,
immobilized on porous glass beads, to generate H2 O2 Any reaction involving a sulfhydryl group, e.g., thiol
in situ from lactose in milk containing 0.25 mM thio- enzymes, will be inhibited by this oxidation. The effect
cyanate has been developed. may be reversed by thiol compounds such as glu-
The bacteriocidal effect of the LPO-H2 O2 -SCN tathione or cysteine.
system has several applications which have been
reviewed extensively (90, 91, 93, 94): XIV. OTHER ENZYMES
1. Sanitization of immobilized enzyme columns in
which LPO is coimmobilized with the enzyme of inter- In addition to the enzymes described above, several
est. The requisite H2 O2 could be generated by immo- other indigenous enzymes have been isolated and par-
bilized enzymese.g., xanthine oxidase, glucose tially characterized (Table 2) (70). Although a fairly
oxidase, or -galactosidase/glucose oxidase. high level of some of these enzymes occurs in milk,
2. As a bacteriocidal agent in toothpaste. they have no apparent function or signicance in
3. In the therapy of mastitis during the non-lactat- milk which contains no substrate for many of them.
ing period. It is possible that some of these enzymes will assume
4. For the preservation of milk in regions lacking importance in the future as indices of animal health or
refrigeration or pasteurization facilities. of product quality. These enzymes will not be discussed
5. To reduce the incidence of enteritis in calves or further.
piglets fed milk replacers. The indigenous LPO is inac- Nearly 40 other enzymatic activities have been
tivated during the manufacture of these products and detected in milk (Table 3) but have not been isolated,
LPO isolated from milk or whey is added. and only limited information on their molecular and
LPO (and lactoferrin) is cationic at the natural pH biochemical properties in milk is available (70). Some
of milk at which all the principal proteins are anionic. of these enzymes have been evaluated as indices of the
LPO and lactoferrin can therefore be readily isolated heat treatment of milk (67, 68). Perhaps further study
from milk or whey by using cation exchange resins. will identify some important or useful attributes of
The ion exchangers may be used as columns (95), some of these enzymes.
batchwise (96), or as membranes (97). At least some
of these methods are applicable on an industrial scale,
making it possible to isolate LPO for use as a food XV. SUMMARY
ingredient.
Although milk contains 60 indigenous enzymes,
only two, lipoprotein lipase (LPL) and plasmin, are
C. Biochemistry of Lactoperoxidase System really technologically signicant. LPL has the potential
to cause serious problems but these can be avoided
LPO catalyzes the peroxidation of SCN to products through good milking practices on the farm. The
which are nontoxic to mammalian cells but which kill enzyme is relatively heat labile and hence does not
or inhibit the growth of many species of microorgan- cause problems in heat-treated products. Although
isms. The net reaction is: milk contains considerable potential plasmin, relatively

little is expressed owing to the presence of inhibitors. 4. ML Groves. Minor milk proteins and enzymes. In:
The signicance of plasmin is mainly negative but its HA McKenzie, ed. Milk Proteins: Chemistry and
action is probably positive in cheese during ripening. Molecular Biology, Vol. 1. New York: Academic
Acid phosphatase may be signicant in the depho- Press, 1971, pp 367418.
5. KM Shahani, WJ Harper, RJG Jensen, RM Parry Jr,
sphorylation of phosphopeptides in cheese. Several
CA Zittle. Enzymes of bovine milk. J Dairy Sci
oxidoreductasesxanthine oxidase, lactoperoxidase,
56:531543, 1973.
catalase, superoxide dimutase, and sulfhydryl oxi- 6. BK Dwivedi. The role of enzymes in food avors. Part
daseare, at least potentially, signicant in milk sta- I. Dairy products. CRC Crit Rev Food Technol
bility. Lysozyme and lactoperoxidase may have 3:457478, 1973.
antimicrobial effects in the intestine of the consumer. 7. HA Johnson. The composition of milk. In: BH Webb,
Perhaps the most signicant and most useful aspect AH Johnson, JA Alford, eds. Fundamentals of Dairy
of the indigenous enzymes in milk is as indicators of Chemistry, 2nd ed. Westport, CT: AVI Publishing,
mastitis, especially N-acetylglucosaminidase, and of 1974, pp 157.
thermal treatments. The thermal stabilities of the indi- 8. PF Fox, PA Morrissey. Indigenous enzymes of bovine
genous enzymes cover quite a wide range of tempera- milk. In: GG Birch, N Blakeborough, KJ Parker, eds.
Enzymes and Food Processing. London: Applied
tures which make it possible to determine at what
Science, 1981, pp 213238.
temperature milk has been heat-treated in the range
9. BJ Kitchen. Indigenous milk enzymes. In: PF Fox, ed.
6590 C for 15 sec. The principal advantage of Developments in Dairy Chemistry. 3. Lactose and
enzymes compared with other heat-induced changes Minor Constituents. London: Elsevier Applied
in analytical applications is the relative ease with Science, 1985, pp 239279.
which their activity can be quantied. 10. AT Andrews, T Olivecrona, G Bengtsson-Olivecrona,
Most dairy products should undergo no change dur- PF Fox, L Bjorck, NY Farkye. Indigenous enzymes in
ing storage, and hence the indigenous enzymes capable milk. In: PF Fox ed. Food Enzymology. London:
of causing undesirable changes are inactivated by heat- Elsevier Applied Science, 1991, pp 53129.
ing. Cheese is an exception; a very complex series of 11. M Hamos, LM Freed, JB Jones, SE Berkow, J
microbiological, biochemical, and perhaps chemical Bitman, NR Mehta, B Happ, P Hamos. Enzymes in
human milk. In: RG Jensen, MC Neville, eds. Human
reactions occur which lead to desirable characteristic
Lactation: Milk Components and Methodologies.
taste, aroma, and texture of the nished cheese (98,
New York: Plenum Press, 1985, pp 251266.
99). At least four indigenous enzymes contribute to 12. O Hernell, B Lonnerdal. Enzymes in human milk. In:
cheese ripeningplasmin, lipoprotein lipase, acid SA Atkinson, B Lonnerdal, eds. Proteins and Non-
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Bertocchi, I Politis. Changes in plasmin-plasminogen-
plasminogen activator system in milk from Italian
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noglobulin protective proteins in milk: lysozyme,

20
Exogenous Enzymes in Dairy Technology
Patrick F. Fox
I. INTRODUCTION The principal applications of enzymes in dairy tech-

nology will be considered in this review. Earlier reviews
Milk from domesticated animals and products formed include Fox and Grufferty (1), Fox (2), Fox and
therefrom have been components of the human diet Stepaniak (3), Brown (4), and Desmazeaud and
for 10,000 years. Some dairy products are con- Spinnler (5).
sumed in most or all regions of the world and they
are major dietary items in many regions, e.g., Europe,
North America, and South America. Total milk II. PROTEINASES
production is 530 106 metric tons per year, of
which 85% is bovine milk; sheep, goats, water buf- Bovine milk contains 3:5% proteins which can be
falo, camels, and mares are important dairy animals resolved into two groups based on solubility at pH
in some regions. Milk is a very perishable commodity 4.6 and 20 C: the caseins, which are insoluble under
and hence there has been a strong incentive to these conditions and which represent 80% of the
convert it to more stable products, which is facilitated total protein; and the whey (serum) proteins, which
by certain properties of milk. Classically, milk has are soluble. The casein fraction of bovine milk and
been preserved by fermentation, usually with salt that of the other main dairying species comprises
addition (cheese, fermented milks, butter). Newer four proteins, s1 -, s2 -, -, and -caseins, which,
preservation methods include drying, pasteurization/ although they have certain features in common, e.g.,
sterilization, and freezing. Some characteristics of insolubility at pH 4.6, are distinctly different proteins.
milk also render it very amenable to modication The whey protein fraction also comprises four main
by enzymes; that milk is a liquid facilitates enzyme proteins-lactoglobulin, -lactalbumin, blood
addition. Cheese production is probably the oldest, serum albumin, and immunoglobulinsand several
and is still the largest, application of exogenous minor proteins, including 60 indigenous enzymes.
enzymes in food processing. Readers are referred to Fox (6) for a detailed descrip-
Since the principal components of milk are proteins tion of the milk protein system.
( 3:5%), lipids ( 3:6%), and lactose ( 4:8%; a dis- As discussed in Section II.A.1, the colloidal stability
accharide containing galactose and glucose), the prin- of the caseins is extensively changed by limited proteo-
cipal enzymes used in dairy technology are proteinases lysis, leading to gelation of the milk system, which is
and peptidases, lipases, and -galactosidase (lactase). the rst step in the production of many cheese vari-
However, several oxidoreductases have signicant eties. The milk proteins can be easily separated from
applications. the other milk constituents and the caseins and whey

proteins readily separated from each other on an 1. Enzymatic Coagulation of Milk
industrial scale. Both the caseins and whey proteins
The principal gastric enzyme of neonatal ruminants is
have good and diverse functional properties; conse-
chymosin rather than pepsin. Chymosin has low gen-
quently, milk proteins are the preferred functional
eral proteolytic activity but high milk-clotting activity.
food proteins for a wide range of applications (7).
Presumably, it evolved to coagulate milk in the sto-
Some functional properties can be improved and/or
mach and thus delay its discharge into the intestine
modied by limited proteolysis.
and increase the efciency of digestion. Shortly after
Proteolytic enzymes are the most widely used
the domestication of dairy animals ( 8000 B.C.),
enzymes in dairy technology and will be discussed
humans learned to exploit the ability of chymosin
below under three headings: cheese manufacture,
and some other proteinases, collectively referred to as
modication of functional properties, and production
rennets, to coagulate milk for the production of cheese,
of protein hydrolyzates for nutritional and other
which was probably the rst application of enzymes in
applications.
food processing.
The rennet coagulation of milk is a two-stage pro-
A. Cheese Manufacture cess. The rst (primary) phase involves the enzymatic
production of para-casein and TCA-soluble pep-
The manufacture of all cheese varieties essentially tides (glycomacropeptides), while the secondary
involves concentrating the protein and fat of milk six- phase involves the Ca-induced gelation of para-casein
fold to 12-fold, depending on the variety. at a temperature in the range of 3035 C. Proteolysis is
Concentration is achieved by (a) coagulating the prin- essentially complete before the onset of coagulation.
cipal milk proteins, i.e., the caseins (if present, the milk The enzymatic coagulation of milk exploits certain
fat is occluded in the coagulum); (b) cutting or break- properties of the caseins. As discussed above, bovine
ing the coagulum and inducing it to synerese under the casein consists of four proteinss1 -, s2 -, -, and -
inuence of heat and acid; (c) separation of curds and caseinsin the approximate ratio of 40 : 10 : 35 : 12.
whey; and (d) acidication, pressing, and salting of de- These contain 89, 1013, 45, and 12 mol of P per
wheyed curd. mol, respectively. Owing to their high phosphate con-
Coagulation of the casein is induced by one of three tent, s1 -, s2 -, and -caseins bind Ca2 strongly and
methods: precipitate at Ca2 > 6 mM. However, -casein binds
1. Limited proteolysis by a crude proteinase Ca2 weakly and is soluble at high Ca2 . It also reacts
(rennet), which is exploited in the manufacture of all hydrophobically with s1 -, s2 -, and -caseins and can
ripened and some fresh cheeses ( 75% of total pro- stabilize up to 10 times its weight of these Ca2 -sensi-
duction). tive caseins against precipitation by forming colloidal
2. Isoelectric precipitation at pH 4:6, used for aggregates, called micelles.
fresh cheeses, usually by in situ production of lactic In milk, > 95% of the casein exists as micelles,
acid by a culture (starter) of lactic acid bacteria and which consist, on a dry weight basis, of 94% protein
less frequently by direct acidication with preformed and 6% of other species, mainly Ca2 and PO3 4 with
acid, usually HCl, or acidogen, usually gluconic acid some Mg2 and citrate, collectively called colloidal cal-
-lactone. cium phosphate (CCP). The micelles are spherical, 50
3. Acid plus heat, i.e., acidication to pH 5:2 with 600 nm (mean, 120 nm) in diameter, with particle
acid whey, acid milk, citrus juice, vinegar, or acetic weights of 108 daltons; i.e., a typical micelle contains
acid at 8090 C; this method is used to produce a 5000 monomers (Mr 2024 kDa). The micelles
small number of relatively minor varieties, e.g., typically bind 2 g H2 O=g protein.
Ricotta. Within the micelles, the caseins are held together by
Concentration of the total colloidal phase of milk CCP bridges, hydrophobic interactions, and hydrogen
(i.e., fat and total protein) to the level present in cheese, bonds. There is a widely held view that the casein
i.e., to a pre-cheese, by ultraltration is now used monomers are organized as submicelles (spherical par-
commercially for the manufacture of several cheese ticles, Mr 5 106 daltons). The micelles dissociate
varieties. Additions of rennet and starter to the when CCP is removed (e.g. by Ca2 chelators or acid-
concentrate are still necessary for texture and avor ication/dialysis), or when the pH is increased above
development. 9, or on addition of detergents (e.g., sodium dodecyl

sulfate) or urea. In most models of the casein micelle, it parent pentapeptide, -CN f103107, with a kcat =KM
is envisaged that the Ca2 -sensitive s1 - s2 -, and - of 2 M1 sec1 .
caseins interact hydrophobically to form the core of The two residues, Phe-Met, are not intrinsically
the micelles, with -casein located predominantly on essential for chymosin action. Replacement of Phe by
the surface. The N-terminal two-thirds of -casein is PheNO2 or cyclohexylalanine decreases kcat =KM about
hydrophobic and reacts hydrophobically with the core threefold and 50-fold, respectively. Oxidation of
proteins, leaving the hydrophilic C-terminal region Met106 decreases kcat =KM 10-fold but substitution
projecting into the surrounding environment. It has of Ile for Met increases this ratio about threefold. In
been proposed that submicelles contain variable fact, the chymosin-susceptible bond in porcine or
amounts of surface -casein and aggregate such that human -caseins is Phe-Ile, which is readily hydrolyzed
-casein-rich submicelles predominate at the surface of by calf chymosin. Thus, the sequence around the Phe-
the micelles with the -casein-decient submicelles bur- Met bond, rather than the bond itself, contains the
ied within. The micelles are stabilized by a zeta poten- important determinants of hydrolysis. The sequence
tial of 20 mV and by steric factors caused by the Leu103 -Ser-Phe-Met-Ala-Ile108 of -casein, which may
protruding C-terminal segments of -casein which exist as a -structure, ts into the active site cleft of
form a hairy layer, 710 nm thick, on the surface acid proteinases. The hydrophobic residues, Leu103 ,
of the micelles, preventing close approach. Phe105 , Met106 , and Ile108 , probably interact with
hydrophobic residues along the active site cleft while
a. Primary Phase of Rennet Action. During the
the hydroxyl group of Ser104 forms a hydrogen bridge
primary phase of rennet action, -casein is the only
with a counterpart on the enzyme. Residues 98102
protein hydrolyzed to a signicant extent. It is cleaved
and 109111 probably form -turns around the edges
by chymosin, and for most of the other proteinases
of the active site cleft in the enzyme substrate com-
used as rennets, at the bond Phe105 -Met106 , which is
plex; this conformation is stabilized by Pro residues at
many times more susceptible to hydrolysis by acid pro-
positions 99, 101, 109, and 110. One or more of the
teinases (which include all commercial rennets) than
three His residues, 98, 100, 102, and Lys111 , are prob-
any other bond in the milk protein system. -Casein
ably involved in electrostatic bonding.
f1105, which is referred to as para--casein, remains
Pepsins and most other acid proteinases used as
attached to the rennet-altered micelle but the hydro-
rennets hydrolyze -casein at Phe105 -Met106 but the
philic peptide, -CN f106169, referred to as the case-
acid proteinase of Cryphonectria parasitica hydrolyzes
ino(glyco)macropeptide, diffuses into the whey and
Ser104 -Phe105 . Although the specicity of cathepsin D
consequently the micelle-stabilizing properties of -
on s1 - and -caseins is generally similar to that of
casein are lost.
chymosin, it has very poor milk-clotting activity. The
A number of attempts have been made to explain
aggregation characteristics of micelles vary with the
the unique sensitivity of the Phe-Met bond. Di-, tri-, or
rennet used, suggesting differences in the extent and/
tetrapeptides containing a Phe-Met bond are not
or specicity of the hydrolysis of -casein or perhaps of
hydrolyzed. However, the Phe-Met bond is hydrolyzed
the other caseins. Furthermore, the commonly used
in the pentapeptide, H.Leu-Ser-Phe-Met-Ala-OMe,
rennets have markedly different specic activities on
i.e., a derivative of -CN f103107. The length of the
synthetic -casein-related peptides.
peptide and the sequence around the cleavage site are
Reviews on the enzymatic coagulation of milk
important determinants of enzyme-substrate interac-
include Dalgleish (8, 9), Fox (1012), Fox and
tion. Ser104 is particularly important and its replace-
Mulvihill (13), and Fox and McSweeney (14).
ment by Gly or Ala in the above pentapeptide
renders the Phe-Met bond very resistant to hydrolysis b. Rennets. The rennets used to coagulate milk
by chymosin but not by pepsins. Even substituting D- are crude preparations of selected proteinases. Many
Ser for L-Ser markedly reduces the sensitivity of the proteinases can coagulate milk but most are too pro-
adjacent Phe-Met bond. Extension of the above penta- teolytic relative to their milk clotting activity and
peptide from the N- and/or C-terminal to reproduce hydrolyze the coagulum too quickly, causing reduced
the sequence of -casein increases the efciency of cheese yield and/or defective, e.g., bitter cheese.
hydrolysis of the Phe-Met bond by chymosin; the tet- Traditionally, rennets were prepared from calves,
radecapeptide, -CN f98111, is hydrolyzed as ef- kids, or lambs stomachs; the principal proteinase in
ciently as intact -casein and 66,000 faster than the such rennets is chymosin. The molecular and enzy-

matic properties of chymosins have been studied exten- which lacks three residues, Asp244 -Phe246 .
sively (1517). Commercially available microbial recombinant chy-
Owing to increasing world production of cheese mosins contain only chymosin A or B; it is not
( 3% per year over the past 30 years), concomitant known if the different forms of chymosin differ in spe-
with a reduced supply of calf vells, the supply of veal cicity, but it is claimed by rennet manufacturers that
rennet has been inadequate for many years, which has the cheesemaking properties of chymosin A and B are
led to a search for rennet substitutes. Although many not equivalent.
proteinases can coagulate milk, only six have been The primary, and probably higher, structures of
found to be more or less acceptable as rennets: bovine, commercially available microbial recombinant chymo-
porcine, and chicken pepsins and the acid proteinases sins are identical to that of calf chymosin. However,
from Rhizomucor miehei, R. pusillus, and C. parasitica. several modied chymosins have been produced from
Chicken pepsin is the least suitable of these and is used genetically engineered microorganisms. At present, the
only in special circumstances. Bovine pepsin gives gen- objective of these investigations is to study the mechan-
erally satisfactory results with respect to cheese yield ism of chymosin action at the molecular level, but it is
and quality; many commercial calf rennets contain a probable that chymosin with improved cheese-making
substantial proportion of bovine pepsin. Although the properties will emerge from such studies, e.g., enzymes
proteolytic specicity of the three commonly used fun- with increased activity on certain bonds shown to pro-
gal rennets on s1 - and -caseins is considerably differ- duce cheese with improved quality or reduced activity
ent from that of calf chymosin, they generally yield on other bonds, cleavage of which results in avor or
acceptable cheese and were widely used in the United textural defects. It should be remembered that chymo-
States before the introduction of microbial recombi- sin evolved to coagulate milk in the neonatal stomach
nant chymosin. Acid proteinases from owers of the (to improve the efciency of digestion) and not to pro-
genus Cynara are used to coagulate sheeps milk for duce cheese. It is fortuitous that chymosin is the best
some artisinal cheeses in Portugal and Spain, especially proteinase for cheese production, not just for milk coa-
Serra dEstrala cheese; these proteinases are not gen- gulation, but it is highly probable that it can be
erally suitable as rennets. The extensive literature on improved. For reference on genetically engineered chy-
rennet substitutes has been reviewed (1821). mosins, see (14, 17).
The gene for calf chymosin has been cloned in Most (7090%) of the rennet added to cheese milk
selected bacteria, yeasts, and molds. Chymosin from is lost in the whey. Therefore, the possibility of immo-
genetically engineered Kluyveromyces marxianus var. bilizing rennet has been investigated as a means of
lactis (Gist-brocades), Escherichia coli (Pzer), and extending its working life. Several rennets have been
Aspergillus nidulans (Hansens) is commercially avail- immobilized but their efcacy as milk coagulants has
able and used extensively, with excellent results; how- been questioned. There is widespread support for the
ever, these products are not yet permitted in all view that properly immobilized enzymes can not coa-
countries. Reviews on microbial recombinant chymo- gulate milk owing to inaccessibility of the Phe-Met
sin include Teuber (22) and IDF (23). bond of -casein and that the apparent coagulating
Microbial recombinant chymosin preparations con- activity of immobilized rennets is due to leaching of
tain no pepsin whereas 550% of the milk-clotting enzyme from the support. Even if immobilized rennets
activity of calf rennets may be due to pepsin; hence, could hydrolyze micellar -casein, operational difcul-
some minor differences in the pattern of proteolysis in ties would exist at the cheese factory level.
cheese made with microbial recombinant chymosin or Furthermore, as discussed in Section II.A.2.d, the resi-
calf rennet are observed, most notably the formation dual rennet in cheese curd plays an essential role in
of the peptide s1 -CN f110199 in cheese made using cheese ripening and it would be necessary to add
calf rennet (owing to the action of pepsin). For those some chymosin or similar enzyme to the curd after
wishing to simulate the action of calf rennet more clo- coagulation, which would be difcult or impossible
sely, blends of microbial recombinant chymosin and (see 8, 9, 24 for reviews).
bovine pepsin are commercially available.
Calf chymosin contains three isoenzymesA, B, c. Factors Affecting the Hydrolysis of -
and C. A and B are gene products that differ from Casein. The pH optimum for chymosin and bovine
each other by one amino acid; A has Asp at position pepsin is 4:7 on small Phe-Met-containing peptides
243 while B has Gly at this position. Chymosin C and 5.35.5 on -CN f98111 or on whole -casein.
appears to be a degradation product of chymosin A The pH optimum for the rst stage of rennet action

in milk is 6:0. Milk for most cheese varieties is -lactoglobulin and/or -lactalbumin is primarily
renneted at about pH 6.5. responsible for the increased rennet coagulation time
Increasing ionic strength (0.010.11) decreases the of heated milk; both the primary phase and especially
rate of hydrolysis of -CN f98112, especially if the the secondary phase are adversely affected. The
pH is also increased. At 1 mM, NaCl, CaCl2 , and adverse effects of heating can be reversed by acidica-
MgCl2 stimulate the hydrolysis of -casein in isolated tion before or after heating or by addition of CaCl2 .
form or in sodium caseinate. Rennet-coagulated milk gels are relatively stable if
The optimum temperature for the coagulation of left undisturbed but synerese strongly if cut or broken.
milk by calf rennet at pH 6.6 is 45 C. The tempera- The rate and extent of syneresis are promoted by redu-
ture coefcient ( rate/10 C) for the hydrolysis of - cing the pH, increasing the temperature, and applying
casein in solutions of Na-caseinate is 1:8, the Ea is pressure, e.g., agitation. By controlling the extent of
10,000 cal mol1 and the activation entropy is syneresis, the cheese maker can control the moisture
39 cal deg1 mol1 ; generally similar values have content of cheese which is a major factor affecting the
been reported for the hydrolysis of isolated -casein. rate and pattern of ripening and the stability of cheese.
The efcacy of -casein as a substrate for chymosin Differences in moisture content are, in fact, a major
decreases as its level of glycosylation increases. At pH factor responsible for the diversity of cheese avor and
6.6, kcat decreases from 43 sec1 for carbohydrate- texture.
free -casein to 25 sec1 for -casein containing 6
moles N-acetyl neuraminic acid (NANA) per mol. 2. Proteolysis During Cheese Ripening
However, Km is lowest for the -casein component
Acid-coagulated cheeses are usually consumed fresh,
containing 3 moles NANA per mol. Polymerization
but the vast majority of rennet-coagulated cheeses
(aggregation) markedly increases Km with little effect
are ripened (matured) for a period ranging from 3
on kcat .
weeks to > 2 years; the rate of ripening is directly
d. Secondary (Nonenzymatic) Phase of Rennet related to the moisture content of the cheese. During
Coagulation. Hydrolysis of -casein removes its ripening, numerous microbiological, biochemical, and
highly charged, hydrophilic C-terminal segment from chemical events occur, as a result of which the princi-
the surface of the casein micelles, thereby reducing pal constituents of the cheesethe proteins, lipids, and
their zeta potential from 20 mV to 7 mV and lactoseare transformed to primary, and later to sec-
removing the steric stabilizing layer. When 85% of ondary, products. Among the principal avor com-
the total -casein has been hydrolyzed, the casein pounds present in most cheese varieties are: peptides,
micelles begin to aggregate and eventually form a gel. amino acids, amines, acids, thiols, and thioesters
Reducing the pH or increasing the temperature from (derived from proteins); fatty acids, methyl ketones,
the normal ( 6:6 and 31 C, respectively) induces lactones, esters, and thioesters (derived from lipids);
coagulation at a lower degree of -casein hydrolysis. organic acids (lactic, acetic, and propionic); carbon
The mechanism involved in the coagulation of dioxide; esters; and alcohols (derived from lactose).
rennet-altered micelles is not known precisely. At appropriate concentrations and combinations,
Coagulation is dependent on Ca2 and on colloidal these compounds are responsible for the characteristic
calcium phosphate, which are exchangeable to a cer- avor of the various cheese varieties.
tain extent. Ca2 may function by neutralizing nega- The biochemistry of cheese ripening has been
tive charges on the caseins. Coagulation is highly reviewed by Fox et al. (25, 26) and Fox and Wallace
temperature dependent; rennet-altered micelles do not (27); only proteolysis is discussed here.
coagulate below 20 C, above which the Q10 C for a. Signicance of Proteolysis. Proteolysis is
coagulation is 16. The high temperature dependence essential in all rennet-coagulated cheese varieties, espe-
of coagulation suggests that hydrophobic bonds may cially internal- and surface-bacterially ripened cheeses
be involved or that multiple bonds are formed. in which it is probably the principal biochemical event
The rennet coagulability of milk is adversely during ripening. Proteolysis contributes to cheese
affected by heat treatments at temperatures > 65 C ripening in at least four ways: (a) makes a direct con-
and is prevented by very severe heat treatments tribution to avor, or off-avor, e.g., bitterness, or
(> 90 C for 10 min). Although changes in the equili- indirectly since free amino acids are catabolized to
bria of milk salts, especially calcium phosphate, are amines, acids, thiols, thioesters, etc.; (b) facilitates the
contributory factors, complexation of -casein with release of sapid compounds during mastication; (c) the

production of NH3 from amino acids released by pro- making, pH (low pH favors retention of chymosin
teolysis affects avor and texture; (d) changes in tex- but not microbial rennets and reduces denaturation,
ture due to breakdown of the protein network, increase e.g., of pepsins), and cooking temperature, e.g., little,
in pH, and greater water binding by the newly formed if any, coagulant survives the cooking conditions used
amino and carboxyl groups. There is a good correla- for Swiss-type cheeses. About 6% of the added chymo-
tion between the intensity of Cheddar cheese avor sin is retained in Cheddar cheese and up to 2030% in
and the extent and depth of proteolysis. high-moisture, low-cook, low-pH cheeses, such as
Considerable information is available on the level Camembert.
and type of proteolysis in the principal cheese groups The proteolytic specicity of calf chymosin on s1 -,
(14, 25, 26, 2833). s2 -, and -caseins in solution has been established
and these ndings can, largely, be extended to cheese
b. Proteolytic Agents in Cheese. Four, and in
(14, 33).
some varieties ve, agents contribute to proteolysis in
The principal chymosin cleavage sites on s1 -casein
cheese during ripening: rennet or rennet substitute;
in cheese are: Phe23 -Phe24 , which is hydrolyzed rapidly
indigenous milk enzymes, especially plasmin; starter
and completely in cheese (e.g., within 3 months in
bacteria and their enzymes, released on cell lysis; non-
Cheddar), and Leu101 -Lys102 , which is cleaved fairly
starter bacteria, which either survive pasteurization of
extensively; Phe33 -Gly34 and Leu98 -Leu99 are also
the cheese milk or gain access to the pasteurized milk
cleaved to some extent in cheese. Surprisingly, the
or curd during manufacture; and secondary inocula,
Trp164 -Tyr165 bond, which is the second most suscep-
e.g., propionic acid bacteria, Brevibacterium linens,
tible bond in s1 casein in solution, does not appear to
yeasts, and molds (Penicillium roqueforti and
be hydrolyzed in cheese. The small peptide, s1 -CN f1
Geotricum candidum), and their enzymes are of major
23, does not accumulate in cheese but is hydrolyzed
importance in some varieties. Techniques have been
rapidly by the cell envelope-associated lactococcal pro-
developed which permit quantitation of the contribu-
teinase, with a specicity dependent on the strain (34).
tion of each of these ve agents to the primary aspects
Some peptide bonds in -casein in solution are
of cheese ripening (25, 31).
hydrolyzed quite rapidly by chymosin in the order:
Studies using these techniques have shown that pri-
Leu192 -Tyr193 , Ala189 -Phe190 , Leu163 -Ser164 , and
mary proteolysis, as determined by gel electrophoresis
Leu139 -Leu140 . In cheese, these bonds are hydrolyzed
or the formation of water- or pH 4.6-soluble N, is due
to a very limited extent or not at all, probably because
mainly to the coagulant except in those varieties, e.g.,
the C-terminal region of -casein is very hydrophobic
Emmental, Parmesan, and mozzarella, cooked to a
and undergoes hydrophobically driven interactions in
high temperature (5255 C) in which the coagulant is
cheese. These interactions appear to be accentuated by
extensively or completely denatured. In these latter
NaCl; even in solution, the hydrolysis of -casein is
varieties, primary proteolysis is relatively limited and
strongly inhibited by 5% NaCl, which is the typical
is due mainly to plasmin, which also contributes to
NaCl concentration in the aqueous phase of many
proteolysis, especially of -casein, in low-cooked
cheese varieties. Inhibition of the hydrolysis of -casein
cheese. The peptides produced by the coagulant and
in cheese is desirable since the peptide -CN f193209
plasmin are further hydrolyzed by the proteinases and
and fragments thereof are very bitter.
peptidases of starter and nonstarter bacteria, leading
Although s2 -casein in solution is fairly readily
to the formation of many small peptides and free
hydrolyzed by chymosin, its fate in cheese is not
amino acids. Proteolysis varies widely among varieties,
clear, and para--casein (-CN f1105) is very resistant
from very limited in short-ripened cheeses such as moz-
to chymosin (and to other proteinases in cheese).
zarella, to very extensive in extramature Cheddar,
The specicity of pepsins is generally similar to that
Parmesan, or blue cheeses. Proteolysis in cheddar
of chymosin but has not been established precisely.
cheese has been well characterized and considerable
Bovine pepsin cleaves the Leu109 -Glu110 bond of s1 -
information is also available on Gouda, Emmental,
casein quite rapidly, a bond which is cleaved very
and Parmesan cheeses (33).
slowly by chymosin. The specicity of the fungal
c. Contribution of Coagulant to Proteolysis in rennet substitutes is quite different from that of chy-
Cheese. Most of the rennet added to cheese milk is mosin (35). The principal cleavage sites of R. miehei
either denatured or lost in the whey. The amount proteinase in s1 -casein in solution are Phe23 -Phe24 ,
retained is inuenced by the type of rennet; e.g., por- Met123 -Lys124 , and Tyr165 -Tyr166 while those in -
cine pepsin is extensively denatured during cheese casein are Glu31 -Lys32 , Val58 -Val59 , Met93 -Gly94 , and

Phe190 -Leu191 (35). C. parasitica proteinase is much dehydration, low Eh , and salting. Chemical and
more active on -casein in cheese than chymosin, biochemical changes do occur during storage but
pepsin, or Rhizomucor proteinases, but its specicity stability was the prime objective. While still important,
on the caseins has not been determined; since it does stability is no longer the primary objective of cheese
not cause signicant bitterness in cheese, its primary manufacture, and since ripening is expensive, its
cleavage sites in -casein may be in the N-terminal acceleration, especially in low-moisture, slow-ripening
rather than in the C-terminal region (the N-terminal varieties, is desirable, at least under certain circum-
region of -casein is quite hydrophilic, and small stances, provided the whole process can be maintained
peptides produced from it would be expected to be in balance.
nonbitter). Some high-moisture cheeses develop an intense a-
vor through a very active secondary ora, e.g., internal
d. Signicance of Secondary Coagulant
blue mold, external white mold, or a bacterial surface
Proteolysis. The proteolytic activity of the coagulant
smear; these cheeses ripen quickly, e.g., 416 weeks.
in cheese inuences quality in four ways:
High-moisture internal bacterially ripened cheeses
1. Some rennet-produced peptides may have a posi-
also mature rapidly but develop a low avor intensity;
tive inuence on avor but excessive or unbalanced
if the ripening of such cheeses is extended, they will
proteolysis, e.g., too much or excessively proteolytic
probably develop off-avors. It is possible to develop
rennet or unsuitable environmental conditions, e.g.,
an intense avor in internal bacterially ripened cheeses
too much moisture or too little NaCl, leads to bitter-
only if the moisture content is low and they are ripened
ness. The peptide -CN f193209 derived from the C-
for a long period, e.g., Parmesan, extramature
terminal of -casein and fragments thereof are particu-
Cheddar, or extramature Gouda, which are ripened
larly bitter.
for 23 years. Owing to the high cost of ripening facil-
2. Rennet-produced peptides serve as substrates for
ities and stocks, the ripening of extramature cheeses is
microbial proteinases and peptidases which produce
expensive. Consequently, there is commercial interest
small peptides and amino acids. These contribute at
in accelerating the ripening of these cheeses, provided
least to background avor and perhaps to bitterness
quality can be maintained. It might also be possible to
if the activity of such enzymes is excessive. Catabolism
apply similar techniques to medium-moisture, med-
of amino acids by microbial enzymes and perhaps
ium-avor cheeses, e.g., regular Cheddar and Gouda,
alterations via chemical mechanisms leads to a range
with the objective of accentuating their avor. Most of
of sapid compoundsamines, acids, NH3 , thiols
the work on accelerating cheese ripening has in fact
which are major contributors to characteristic cheese
been on Cheddar. Techniques for accelerating ripening
avors.
may also be applicable to reduced-fat cheeses, which
3. Alterations in cheese texture appear to inuence
tend to ripen slowly. A substantial literature on
the release of avorful and aromatic compounds, aris-
attempts to accelerate cheese ripening has accumulated
ing from proteolysis, lipolysis, glycolysis, and second-
and has been reviewed regularly (36).
ary metabolic changes, from cheese during
Glycolysis is rapid in all cheeses and does not
mastication, and this may be the most signicant con-
require acceleration. Lipolysis is limited in most
tribution of proteolysis to cheese avor.
cheeses and excessive lipolysis is undesirable.
4. Texture is an important attribute of all cheeses
Consequently, most studies on accelerated ripening
and is critical in some varieties. Protein forms a con-
of cheese have focused on proteolysis, which contri-
tinuous solid matrix in cheese, and its hydrolysis leads
butes to avor and is mainly responsible for changes
to a softening of the texture. Chymosin is primarily
in texture.
responsible for textural changes during the early stages
Methods for accelerating cheese ripening fall into
of ripening. The functionality (stretchability and melt-
six categories: elevated ripening temperature, exogen-
ability) of mozzarella is strongly inuenced by proteo-
ous enzymes, chemically or physically modied cells,
lysis; a low level of proteolysis improves functionality,
genetically modied starters, adjunct starters, and
but quality deteriorates on further proteolysis.
enzyme-modied cheeses. These methods either seek
to make the conditions under which indigenous
3. Acceleration of Cheese Ripening
enzymes function more favorable (i.e., elevated tem-
The original objective of cheese manufacture was perature) or to increase the level of certain key enzymes
conservation of the principal nutrients in milk (i.e., which are considered to be particularly important in
lipids and proteins) by a combination of acidication, cheese ripening.

A complex cascade of enzymes is involved in cheese A simpler and probably more effective approach is
ripening, and most key enzymes have not been identi- the use of lactose-negative strains of Lactococcus,
ed. Not surprisingly, the use of single enzymes, e.g., which cannot grow in milk or cheese curd but serve
additional coagulant (which is mainly responsible for as a balanced natural source of enzymes important
primary proteolysis), plasmin (for which some benets in cheese ripening. Such cultures are available commer-
are claimed), or Neutrase (from B. subtitis), while accel- cially and are claimed to give satisfactory results.
erating proteolysis, does not accelerate avor develop- Cells of selected non-lactic-acid bacteria, which do
ment and may cause off-avors. It has been claimed not grow in a particular cheese, either because they are
that a combination of exogenous proteinases and lacto- aerobic, e.g., Pseudomonas spp. or Brevibacterim spp.,
coccal cell-free extracts (rich in peptidases) accelerate or because they have a high growth temperature, e.g.,
ripening, but the results are equivocal. Uniform incor- Propionibacterium, might also serve as suitable
poration of the enzyme preparation into cheese curd packages of enzymes for addition to cheese curd;
poses problems. For Cheddar, the enzyme preparation, however, such cultures do not appear to be used com-
diluted with salt, may be added to milled curd, but this mercially at present.
method is not applicable to most varieties. Addition of It is probable that certain enzymes are rate limiting
microencapsulated enzyme(s) to cheese milk is tech- in cheese ripening. At present, the key limiting enzymes
nically feasible, but microencapsulation techniques are unknown, but studies are in hand with the objec-
currently available are not very efcient. At present, tive of identifying these enzymes using decient or
exogenous enzymes are not being used commercially overproducing mutants. It is possible to genetically
to accelerate the ripening of natural cheese. modify Lactococcus to overproduce certain desirable
Exogenous lipase, traditionally pregastric esterase, enzymes, to delete undesirable genes, or to introduce
is added to certain hard Italian cheeses, e.g., Romano foreign genes for putitively important enzymes. It is
and provolone. It has been claimed that inclusion of highly probable that genetically modied bacteria
selected lipases in the blend of exogenous enzymes with the ability to accelerate ripening and improve
accelerates the ripening of other cheeses, e.g., cheese quality will become available in due course for
Cheddar and Ras. use as primary or secondary cultures.
Most of the enzymes involved in cheese ripening are All cheese acquires an adventitious nonstarter
produced by microorganisms that grow in or on the microora, predominantly mesophilic lactobacilli,
cheese. By increasing the number of these organisms it which grow from low numbers initially (e.g.,
should be possible to accelerate ripening. The use of < 103 cfu=g to 107 108 cfu=g and which dominate
whole cells should have two advantages over isolated the microora of cheese after 3 months owing to
enzymes: they contain the natural cocktail of the death of the primary starter. These nonstarter lac-
enzymes found in cheese and they are cheaper to pro- tic acid bacteria (NSLAB) probably affect cheese
duce. Three approaches have been considered in the ripening, and there is considerable interest in inoculat-
use of bacterial cells to accelerate cheese ripening: atte- ing cheese milk with selected strains of NSLAB to
nuated starter cells, genetically modied cultures, accelerate or modify cheese avor development (37).
adjunct cultures. An extreme form of accelerated ripening is practiced
The starter, in addition to its essential role in curd in the production of enzyme-modied cheese (EMC);
acidication, is also essential for secondary proteolysis the subject has been reviewed by Kilcawley et al. (38).
and avor development. Therefore, it might be EMCs are produced by adding a cocktail of enzymes
expected that increasing the number of starter cells in (proteinases, peptidases, lipases) and perhaps bacterial
cheese would accelerate ripening. However, increasing cultures to homogenized, pasteurized fresh curd or
the level of active starter added to the cheese milk young cheese. The mixture is incubated for a requisite
causes an excessively rapid rate of acid production period and repasteurized to terminate the microbiolo-
which has undesirable effects, e.g., a crumbly texture gical and enzymatic reactions. The preparation may be
and overacid avor. The acid-producing ability of star- spray-dried or commercialized as a paste.
ter cells may be attenuated or destroyed by heat-shock- Although the avor of EMCs does not approximate
ing, freeze-shocking, or solvent treatment with very that of natural cheese, they have the ability to potenti-
little effect on their enzyme activities. These attenuated ate cheeselike avor in various food products, e.g.,
cells are in effect packages of enzymes and their use has processed cheese, cheese analogs, cheese sauces, and
been reported to accelerate the ripening of a number of dips, and products containing cheese, e.g., crackers,
cheese varieties. crisps, etc. For such applications, EMCs may replace

2050 times their weight of natural cheese and are cells, and inhibition of acid secretion in the stomach
cheaper. Cheddar EMCs are the most important com- (47, 48). Methods have been developed for the indus-
mercially, but EMCs that stimulate several varieties trial production of CMP from whey (48, 49).
have been developed, e.g., blue, Swiss, and Romano. There is considerable interest in the fortication of
performance-enhancing drinks with protein hydroly-
sates; bitterness is a problem here, and whey protein
B. Other Applications of Proteinases
hydrolysates are preferred to casein hydrolysates.
In comparison with their use in cheese making, the
2. Physiologically Active Peptides from Milk
other applications of proteinases in dairy technology
Proteins
are quite small but some have considerable growth
potential. The more important of these are discussed Peptides with various physiological activities have been
below. isolated from milk protein hydrolysates; at least some
of these peptides are produced in vivo and may play a
1. Dietary Products physiological role (50). The best-studied of these are
the -caseinomorphins, a family of peptides containing
Protein hydrolyzates for use in soups, gravies, avor-
47 amino acids (representing -CN f6063/7) with
ings, and dietetic foods are generally prepared from
opioid activity. These peptides are produced in the
soy proteins, gluten, milk proteins, meat, or sh pro-
intestine in vivo but it is still unclear whether or not
tein by acid hydrolysis. Neutralization results in a high
they reach the brain. Peptides with opiate properties
salt content which is acceptable for certain applications
have also been isolated from hydrolysates of s1 - and
but may be unsuitable for dietetic foods and food sup-
-caseins, lactotransferrin, -lactalbumin, and -lacto-
plements. Furthermore, acid hydrolysis causes total or
globulin.
partial destruction of some amino acids. Enzymatic
Other biologically active peptides that have been
hydrolysis is a viable alternative (39), but bitterness
isolated from hydrolysates of milk proteins include:
due to hydrophobic peptides is frequently encountered.
immunomodulating peptides, an inhibitor of angioten-
Caseins are strongly hydrophobic and yield very bitter
sin-converting enzyme, blood platelet modiers, and
hydrolyzates, but bitterness may be eliminated, or at
stimulators of DNA synthesis (50). Whether any of
least reduced, by one of several treatments (39, 40).
these peptides are active in vivo remain to be estab-
There is increasing interest in the production of
lished, but their formation has led to casein being
casein-derived peptides with special nutritional or phy-
referred to as a pro-hormone (51).
siological properties; some of the possibilities have
The release of biologically active peptides requires
been reviewed (41, 42). Apart from the interest in
precise hydrolysis of the parent molecules at specic
casein hydrolyzates for the nutrition of patients with
bonds. If the application of these peptides develops
digestive problems, interest has been focused recently
as predicted, very interesting applications for protei-
on phosphopeptides derived from casein which it is
nases in the dairy industry will emerge.
claimed stimulate the absorption of calcium and iron,
but views on this are not unanimous (43). Methods for
3. Modication of Protein Functionality
the production of caseinophosphopeptides for nutri-
tional and/or medical applications have been devel- Milk proteins are among the principal functional pro-
oped (4446). teins used in food products (52). In general, milk pro-
The casein(glyco)macropeptide (CMP), -CN f106 teins possess very good functional properties but suffer
169, produced from -casein during the enzymatic coa- some limitations, notably the insolubility of casein in
gulation of milk for cheese or rennet casein, is lost into the pH range 3.05.5. The functional properties of milk
the whey. CMP is devoid of aromatic amino acids and proteins may be improved by limited proteolysis (1, 40,
hence is a suitable nutrient for patients suffering from 53, 54). An acid-soluble casein, free from off-avor
phenylketonuria. Several biological activities have been and suitable for incorporation into beverages and
attributed to the CMP, including inhibition of adhesion other acid foods, has been prepared by limited proteo-
of oral actinomyces and streptococci to erythrocytes, lysis. The antigenicity of casein is destroyed by proteo-
effects on gastrointestinal motility, growth factors for lysis, and the hydrolysate is suitable for use in milk
Bidobacterium spp., inhibition of the binding of cho- protein-based foods for infants allergic to cows milk.
lera toxin, inhibition of inuenza virus hemagglutinin, Controlled proteolysis improves the meltability of
stimulation of cholecystokinin release from intestinal directly acidied cheese but excessive proteolysis

causes bitterness. Casein solutions are very viscous at lipase(s) in the rennet paste traditionally used in their
concentrations > 20%, w/v, which increases the cost of manufacture.
drying caseinates. Viscosity may be reduced by limited Rennet pastes are prepared from the stomachs of
proteolysis, but only a small increase in solids is pos- calves, lambs, or kids slaughtered after suckling; the
sible owing to bitterness after even moderate levels of stomachs and contents are held for a considerable per-
proteolysis. iod prior to maceration. Because of possible risks to
The surface activity of sodium caseinate can be public health, the use of rennet pastes, which have
increased considerably by the treatment with plasmin, proteolytic and lipolytic activities, is prohibited in
apparently owing to the formation of 2 - and 3 -case- some countries. The lipase in rennet paste is of oral
ins (-CN f106209, -CN f108209) which are very origin and its secretion is stimulated by suckling: the
surface active. Generally, the emulsifying and foaming secreted lipase is washed into the stomach with the
properties of small peptides are poor, since they form ingested milk. Oral (lingual) lipase, commonly referred
very thin interfacial layers. to as a pregastric esterase (PGE), is secreted by several
Limited proteolysis of lactalbumin (heat-denatured species and probably makes a signicant contribution
whey protein), which is insoluble and has very poor to the digestion of lipids by the neonate in which the
functional properties, yields a product with greatly activity of pancreatic lipase is limited. The consider-
improved solubility and functionality. Limited proteo- able literature on PGE has been comprehensively
lysis of whey protein concentrate (WPC) reduces its reviewed (3, 56). PGE shows a high specicity for
emulsifying capacity and increases its specic foam short chain fatty acids, especially butyric, esteried
volume but reduces foam stability. The heat stability on the Sn-3 position of glycerol, although some inter-
of WPC may be improved considerably by limited species differences in specicity have been reported.
hydrolysis without concomitant impairment of other They are maximally active at 3242 C, pH 4.85.5,
functional properties or off-avor development. and in the presence of 0.5 M NaCl. Calf, kid, and
The plastein reaction has been proposed as a lamb PGEs have been partially puried from commer-
mechanism by which the functional and nutritional cial preparations (57). Calf PGE has been isolated
properties of proteins may be improved. The reaction from oral tissue and characterized with respect to pI
is of little relevance to milk proteins because they (7.0), molecular weight ( 49 kDa), and amino acid
already have good functional and nutritional proper- composition (58, 59). The secondary structures of rat
ties and yields of plastein are low. lingual lipase and pancreatic lipase were studied and
One of the principal food applications of whey pro- compared (60). The gene for rat lingual lipase has been
tein (WP) and whey protein isolates (WPI) is in the cloned and sequenced, and the amino acid sequence of
production of thermo-set gels. The gelation of WPCs the enzyme has been deduced (61).
at low temperatures has been achieved by limited pro- PGE-containing extracts from calf, kid, or lamb tis-
teolysis by certain enzymes (55); perhaps gelation sue are commercially available as alternatives for
occurs through the plastein reaction. rennet pastes. Slight interspecies differences in speci-
city render one or another more suitable for particular
applications. Particularly large differences in the abil-
III. LIPASES ity of lipases to release 4-methyloctanoic acid, which
exhibits a goat-muttony aroma, have been found (62).
Lipases have a number of relatively low-volume appli- Such differences in specicity permit the generation of
cations in the dairy industry. Some of these applica- a range of avors in cheese products. The propensity of
tions are traditional and essential for the manufacture PGE to synthesize triglycerides is increased in the aw
of particular products: others are emerging but hold range 0.750.90 or by the addition of ethanol to the
considerable potential. reaction mixture (63); this property may be exploited
to modify cheese avors.
A. Lipases in Cheese Production Although PGE extract is now widely used in the
manufacture of hard Italian cheese varieties, connois-
The principal application of lipases in dairy technology seurs of Italian cheese claim that rennet paste gives
is in cheese manufacture, particularly, some Italian superior results. Perhaps rennet paste contains
varieties, e.g., Romano and provolone. The character- enzymes in addition to chymosin and PGE. A second
istic piquant avor of these cheeses is due primarily lipase, termed gastric lipase, was identied in an
to short-chain fatty acids resulting from the action of extract of cleaned gastric tissue and partially charac-

terized. A combination of calf gastric lipase and goat ably more lipolysis occurs in cheeses made from raw
PGE gave Cheddar and provolone of superior quality milk than in pasteurized milk cheeses, possibly owing
to cheese made with PGE alone (64). However, Nelson to the indigenous milk lipoprotein lipase and/or a more
et al. (56) expressed reservations on the occurrence of a diverse nonstarter microora in the former. It has been
gastric lipase distinct from PGE. Traditional rennet claimed that the avor of many cheeses can be intensi-
paste would be expected to contain both PGE and ed or their ripening accelerated by incorporating PGE
gastric lipase. or fungal lipases, although their use appears to be very
R. miehei secretes a lipase that is reported to give limited (13). Relatively extensive lipolysis occurs in
satisfactory results in Italian cheese manufacture (65). Parmigiano-Reggiano, which can probably be attribu-
The enzyme has been characterized (66) and is com- ted to the use of raw milk (which contains an indigen-
mercially available as Piccantase. The lipases secreted ous lipase) and the long ripening time (2 years).
by selected strains of Penicillium roqueforti, P. candi- Lipases, probably mainly of fungal origin, are used in
dum, or A. niger are considered to be potentially useful the production of some enzyme-modied cheeses (38).
for the manufacture of Romano, provolone, and other
cheese varieties (67, 68). Unlike PGE, the fungal B. Other Applications of Lipases
lipases do not preferentially release short-chain fatty
acids. The use of different lipase preparations or blends Lipases are used to hydrolyze milk fat for a variety of
of lipases opens possibilities for producing Italian uses in the confectionery, candy, chocolate, sauce, and
cheeses with different degrees of sharpness. snack food industries. The partially hydrolyzed fat
A. oryzae secretes a lipase which has as exception- imparts a greater intensity of butterlike avor to the
ally high specicity for C6 C8 acids (69). Another products and delays stalling, presumably as a result of
interesting characteristic of this enzyme is that it the emulsifying effect of di- and monoglycerides (56,
forms micelles, 0:2 m in diameter, in aqueous 71, 74).
media, as a result of which 94% of the enzyme An important new application of lipases is in the
added to milk is recovered in cheese curds. The forma- trans/interesterication of fatty acids on triglycerides
tion of short-chain fatty acids was reported to parallel (75). This approach can be used to modify the melting
avor intensity in Cheddar cheese containing this point of triglycerides, and hence their rheological prop-
enzyme. In contrast to the FFA prole caused by erties. An important application of this technology is
calf PGE, which liberated high concentrations of in the production of cocoa butter substitutes for cho-
C4:0 , the FFA prole in the cheese containing A. oryzae colate manufacture. Mono- or polyunsaturated fatty
lipase was similar to that in the control cheese, but the acids may also be introduced to relatively saturated
level of FFA was much higher. milk lipids to improve their nutritional qualities.
Extensive lipolysis also occurs in blue cheese vari- Immobilized lipase systems have been developed for
eties, and in addition to making a direct contribution these applications (74).
to avor, the free fatty acids serve as substrates for
fungal enzyme systems in the biosynthesis of methyl
ketones, which are the principal contributors to the IV. -GALACTOSIDASE
typical avor of blue cheese (70). P. roqueforti lipase
predominates the ripening of blue cheeses. However, Lactose is a reducing disaccharide containing galactose
blue cheese ripening may be accelerated and quality and glucose linked by a -1-4 O-glycosidic bond (O--
improved by the addition of exogenous lipases (71, 72). D-galactopyranosyl-(1-4)-- or -D-glucopyranose, -
Blue cheese is a popular ingredient for salad dres- and -lactose, respectively). Lactose is by far the domi-
sings and cheese dip. High-quality natural cheese is not nant carbohydrate in milks which are, in turn, the only
normally required for these applications and there is signicant natural sources of lactose. The concentra-
considerable interest in the production of cheaper sub- tion of lactose in milk ranges from 0% in the milk of
stitutes. Various methods have been developed for the marine mammals to 10% in milk from some species
production of blue cheese avor concentrates; most of of monkey; bovine and human milk contain 4:8%
these methods involved the use of fungal lipases and and 7% lactose, respectively. Lactose is an important
usually P. roqueforti spores (1, 2, 73). source of energy for the newborn mammal, but when a
The low level of lipolysis that occurs in most other very energy-dense milk is required (mammals in aqua-
varieties is catalyzed by lipases/esterases derived from tic or polar environments), the lipid rather than the
the starter or nonstarter lactic acid bacteria. Consider- lactose content is increased. In fact, there is a fairly

good inverse relationship between the level of fat and and as a reducing sugar in products in which Maillard
that of lactose. browning is desirable as a source of color and avor.
Among sugars, lactose possesses many fairly unique The chemistry, properties, problems, modications,
properties: low solubility ( 18 g=100 mL H2 O at and applications of lactose and lactose derivatives
20 C); a marked tendency to form supersaturated solu- have been reviewed (7685).
tions which are difcult to crystallize; when crystalliza- It is claimed that lactose promotes the intestinal
tion does occur, the crystals are hard and sharp and, absorption of calcium and phosphorus and hence
unless kept to dimensions < 20 m, cause a sandy tex- should be nutritionally benecial, especially in infant
ture in foods; crystallization is complicated by its nutrition. However, lactose is involved in two enzyme
mutarotation characteristics since - and -lactose dif- deciency diseases: lactose intolerance and galactose-
fer considerably in solubility and degree of hydration; mia. There are in fact two forms of galactosemia, both
it has low sweetness (16% as sweet as sucrose at 1%, arising from the congenital deciency of an enzyme in
w/v); owing to its crystallization and mutarotation their Leloir pathway for galactose metabolism (86).
characteristics, it is hygroscopic and may cause cak- Classical galactosemia is due to a deciency of galac-
ing of dairy powders; it has a strong tendency to tose-1-phosphate:uridyl transferase. Ingested galactose
absorb avors and odors. (from lactose or other source) is phosphorylated to
At 20 C, -lactose is considerably less soluble galactose-1-phosphate which is not metabolized
( 7 g=100 g H2 O) than -lactose ( 50 g=100 g H2 O). further, leading to the accumulation of galactose and
However, the solubility of the -anomer increases galactose-1-phosphate. Galactosemic infants appear
more sharply with increasing temperature than that normal at birth but develop various symptoms, includ-
of the -anomer and the solubility curves intersect at ing mental retardation, unless put on a galactose-free
93:5 C; therefore, when lactose is crystallized at a diet within 23 months. The second form, galactoki-
temperature < 93:5 C, -lactose is obtained. - nase-decient galactosemia, results in the failure to
Lactose crystallizes as a monohydrate while -lactose phosphorylate galactose, some of which is metabolized
crystals are anhydrous. to galactitol which accumulates in the eye, causing cat-
When milk is spray-dried, there is insufcient time aracts.
for lactose to crystallize and an amorphous lactose Disaccharides must be hydrolyzed to monosacchar-
glass is formed. If the moisture content of the powder ides in the intestine prior to absorption, in the case of
is low, the glass is stable, but if the moisture content lactose by -galactosidase. The vast majority of infants
increases, the glass become hygroscopic, and lactose secrete adequate levels of -galactosidase in the brush
crystallizes as -lactose monohydrate, leading to cak- border of the small intestine to hydrolyze ingested lac-
ing. In practice, the problem is solved by precrystalliz- tose; however, a small minority of infants secrete an
ing the lactose, usually induced by seeding with inadequate level of -galactosidase. The level of intest-
powdered lactose crystals. inal -galactosidase reaches a maximum shortly after
When milk is frozen, the lactose usually does not birth and declines thereafter to a low level. With the
have time to crystallize and the concentration of inor- exception of northwestern Europeans and a few
ganic solutes in the liquid aqueous phase increases. On African tribes, the level of intestinal -galactosidase
holding in the frozen state, calcium phosphate crystal- becomes so low within 68 years as to render the sub-
lizes as Ca3 PO4 2 , releasing H and reducing the pH ject incapable of hydrolyzing ingested lactose at an
to 5:8, and lactose crystallizes as -lactose monohy- adequate rate, leading to lactose intolerance. The
drate, reducing the amount of solvent water, further unhydrolyzed lactose passes to the large intestine
increasing the concentration of inorganic solutes. The where it leads to atulence, cramps, diarrhea, and pos-
combination of low pH and high Ca2 destabilizes the sibly death. Thus, only a minority of the worlds popu-
casein micelles which aggregate when the milk is lation can consume large quantities of lactose-
thawed. Unless properly controlled, these characteris- containing foods with impunity. The subject of lactose
tics of lactose may cause defects in concentrated, dehy- intolerance has been reviewed extensively (87, 88).
drated, and frozen dairy products. However, some of The third feature of the lactose problem is the devel-
the same characteristics may be exploited to make lac- opment of economic outlets for the large quantities
tose an interesting and useful food additivee.g., as a ( 5 109 kg=year) available from cheese and casein
free-owing agent, an agglomerating (instantizing) wheys. The technology for lactose production is well
agent, an additive to stabilize color, avor, and texture, developed but < 10% of the potentially available lac-
especially when concomitant sweetness is undesirable, tose is recovered as such; although lactose has a num-

ber of special food applications, the market appears to The principal applications of -galactosidase in
be limited. Increasing concern about environmental dairy technology are: (a) production of low-lactose
pollution has focused attention on more complete milk and dairy products for -galactosidase-decient
utilization of whey for which there are many options patients; (b) modication of dairy products for use in
(7685). ice cream, baked goods, yogurt, etc.; (c) production of
The dairy industry has developed methods for con- syrups and sweeteners for food applications; and (d)
trolling the physicochemical problems posed by lac- pretreatment of milk for freezing.
tose, and lactose intolerance may not be too serious In spite of the widespread incidence of -galactosi-
if lactose-containing products are introduced gradually dase deciency among non-Caucasians, such subjects
to the diet. However, all the various problems posed by adjust to lactose-containing diets if lactose is intro-
lactose (technological, nutritional, utilization) may be duced gradually and the response to lactose is moder-
solved via hydrolysis by -galactosidase (lactase) to the ated by other components in the diet. Researchers are
less problematic sugars, glucose and galactose. divided as to the desirability of including milk in the
-Galactosidase (-D-galactoside galactohydrolase; diets of lactose-intolerant subjects (87, 88). However, it
EC 3.2.1.23) catalyzes the hydrolysis of lactose to its appears to be generally agreed that treatment with -
component monosaccharides, glucose and galactose. galactosidase would enhance the nutritional value of
The use of -galactosidase in dairy technology has dairy products in such cases and render protein-rich
been considered as one of the most promising appli- dairy products suitable for supplementation of nutri-
cations of exogenous enzymes in food processing. tionally decient diets. Lactose-hydrolyzed milk is
However, although many interesting applications commercially available but is more expensive than nor-
have been demonstrated, the use of -galactosidase mal milk. -Galactosidase is also available in powder
has not yet become commercially successful for or liquid form for home use (89, 90). Direct addition of
economic reasons. The voluminous literature on the -galactosidase to milk at mealtime (enzyme replace-
preparation, properties, and uses of -galactosidases ment therapy) has also given satisfactory results. A
has been the subject of several reviews (76, 81, 83, promising method for reducing treatment cost is the
89, 90). addition of a very low level of soluble -galactosidase
Although -galactosidase is widely distributed in to UHT milk which, during prolonged storage, induces
plant, animal, and microbial sources, only the enzymes extensive hydrolysis. This approach has been commer-
from Aspergillus niger, A. oryzae, Kluyveromyces marx- cialized by the Tetra-Pak Company (Lund, Sweden).
ianus var. lactis, K. fragilis, Bacillus stearothermophilus, The increased sweetness of lactose-hydrolyzed milk
and Escherichia coli are commercially available. - does not appear to be a problem and may actually be
Galactosidases from several sources have been isolated preferred by some individuals.
and characterized; the important properties have been -Galactosidase can act as a transferase resulting in
summarized by Mahoney (89, 90). In general, -galac- the synthesis of several oligosaccharides (galacto-oligo-
tosidases produced by molds have an acid pH opti- saccharides), the range and concentration of which
mum (2.54.5) and are therefore best suited for use appear to vary with the source of the -galactosidase
in acid wheys, while yeast and bacterial -galactosi- and the duration of treatment. Many of the oligosac-
dases, with a pH optimum in the range 67.5, are charides are -(1 ! 6 galactosides which are not
more suitable for use in milk or rennet wheys. - hydrolyzed in the small intestine and pass into the
Galactosidases with high thermal stability have been large intestine where they are acted on by bacteria
isolated and are attractive because they can be used at leading to intestinal disturbances, mainly atulence.
high temperatures at which microbial growth is slow or However, galacto-oligosaccharides stimulate the
absent. The heat stability of -galactosidase from K. growth of Bidobacterium spp. in the lower intestine,
lactis and Streptococcus thermophilus is considerably which is believed to be benecial. A product (oligo-
greater in milk than in buffer systems owing to the nate, 6 0 -galactosyl lactose) is produced commercially
combined effects of casein and lactose. by the Yokult Company in Japan for addition to infant
-Galactosidases from several sources have been formulae. Some galacto-oligosaccharides have interest-
immobilized by encapsulation; entrapment in bers, ing functional properties and may nd commercial
gels, or semipermeable membranes; adsorption or applications (91, 92).
covalent attachment by a variety of techniques to var- Hydrolysis of lactose in milk for yogurt has been
ious supports, e.g., porous glass, collagen, cellulose suggested as a means of increasing the sweetness of
derivatives, and various resins (76, 83, 89, 90). yogurt without a concomitant increase in calories; it

is reported to reduce fermentation time (89, 90). Some whey appears to be suitable for incorporation into ice
authors have reported that lactose-hydrolyzed yogurt cream in which up to 25% of the skim milk solids may
has superior texture and consistency, with less wheying be replaced by hydrolyzed whey syrup without adverse
off, than controls. Off-avors have been reported in effects on quality; 50% of the sucrose may also be
some lactose-hydrolyzed yogurts, perhaps owing to replaced by whey syrup (89, 90, 97). Lactose-hydro-
the action of contaminating proteinases. Yogurt and lyzed whey may be fed in larger amounts than normal
other cultured milks are well tolerated by lactose-intol- whey to animals, especially pigs.
erant subjects, apparently owing to the secretion of - Probably the principal commercial interest in -
galactosidase by the thermophilic culture used or to galactosidase is for the production of syrups as a prof-
slower gastric emptying (88). itable outlet for lactose. Glucose-galactose syrups are
It is also claimed that the manufacturing time for 70% as sweet as sucrose and about four times
Cheddar and other cheeses is reduced by pretreatment sweeter than lactose. It has been known for a long
of cheese milk with -galactosidase, and, possibly more time that lactose can be hydrolyzed to glucose and
signicantly, the quality of the cheese is improved and galactose by strong mineral acids, and there has been
ripening accelerated. However, a proteinase present in renewed commercial interest in the process, using free
the commercial -galactosidase preparations used, acid or ion exchange resins, as a means of producing
rather than to the action of -galactosidase, was prob- glucose-galactose syrups. Production costs are
ably responsible for the accelerated ripening. reported to be lower than for enzymatic hydrolysis,
Hydrolysis of lactose improves the functionality of but acid hydrolysis is applicable only to puried lac-
milk powder in bakery products. The glucose moiety is tose solutions or perhaps ultraltration permeates.
fermentable by bakers yeast (Saccharomyces cerevi- Numerous applications have been reported for glu-
siae), leading to increased loaf volume, while the non- cose-galactose syrups (in addition to the use of lactose-
fermentable galactose contributes to avor and crust hydrolyzed whey) (83, 89, 90). Although effective sys-
color through Maillard browning. Prehydrolysis of lac- tems for the production of glucose-galactose syrups
tose using -galactosidase renders milk stable to freez- and other lactose-hydrolyzed products are available
ing (89, 90, 93). and continued research will undoubtedly lead to
Dulce de Leche, a sweetened concentrated dairy improved systems, the cost of such syrups vis-a`-vis
product, is popular in Latin America as a dessert or alternatives, mainly glucose syrups from starch,
spread. Lactose crystallization is a major problem in remains a problem.
this highly concentrated product. Growth of K. marx- The sweetness of glucose-galactose syrups may be
ianus var. lactis in milk for the preparation of Dulce de increased by isomerizing the glucose to fructose (which
Leche has been recommended for the control of lactose is about twice as sweet as glucose) using glucose iso-
crystallization (94). About 50% of the lactose was merase, which is now widely used in the commercial
hydrolyzed in 12 h (at 5:2 107 cells/mL) or 100% in production of high-fructose syrups from starch. Such a
24 h. When the delactosed milk was mixed in propor- system has been patented and a glucose-fructose-galac-
tions of 1 : 2 with normal milk and concentrated, no tose-lactose syrup with a sweetness equal to sucrose
lactose crystallization and no signicant changes in (both at 10% solution) has been prepared by treating
avor were noted. The use of permeabilized K. marx- a glucose-fortied lactose hydrolyzate with glucose
ianus var. lactis cells for the same application was isomerase. Conversion of lactose to lactulose offers
found to be very satisfactory (95). Presumably, isolated another avenue for the production of novel sugar mix-
-galactosidase could be used successfully in the pre- tures. Lactulose is a disaccharide consisting of galac-
paration of Dulce de Leche. tose and fructose, which can be produced from lactose
Lactose crystallization may also be a problem in by mild alkaline treatment. At least some -galactosi-
conventional sweetened condensed milk, although the dases can hydrolyze lactulose, although more slowly
problem can be controlled by appropriate manufactur- than lactose.
ing steps. Prehydrolysis with -galactosidase offers an It will be apparent that -galactosidase has numer-
alternative solution. ous applications in the dairy industry. However, in
Treatment with -galactosidase prevented lactose spite of very considerable research and demonstrated
crystallization in a whey retentatebuttermilk powder technological feasibility in pilot-scale experiments, -
spread (96). Acid (mold) -galactosidase was prefer- galactosidase is not yet exploited commercially on a
able to neutral (yeast) enzyme, and hydrolysis of signicant scale, except for lactose-hydrolyzed pasteur-
30% of the lactose was sufcient. Lactose-hydrolyzed ized and UHT milks. Zadow (98) concluded that the

markets for lactose-hydrolyzed syrups are likely to late blowing, the level of lysozyme already per-
remain limited for economic reasons. mitted by regulatory authorities in a number of
countries appears to be satisfactory until further
evidence is obtained. Lysozyme appears to be of
V. LYSOZYME
value in the control of late blowing in countries
which prohibit nitrate. Published information
Lysozyme (muramidase, EC 3.2.1.17), an enzyme
indicates that in some cheese types which are
which causes lysis of certain bacteria by hydrolyzing
very sensitive to late blowing, such as Gouda,
cell wall polysaccharides, is widely distributed in ani-
lysozyme used at the current normal addition
mal tissues and secretions. Some bacteria and bacter-
under normal manufacturing and storage condi-
iophage secrete similar enzymes, lysins. Egg white is a
tions is less effective than the usual amount of
particularly rich source of lysozyme, which is the best
nitrate. In this case, lysozyme can not be consid-
characterized of these enzymes. The molecular and
ered a suitable alternative to nitrate at present.
biological properties of lysozyme and its use in food
More information will become available for the
preservation and as a pharmaceutical have been com-
various cheese types about the critical number of
prehensively reviewed by Proctor and Cunningham
spores in the raw milk to cause defects when
(99) and Cunningham et al. (100).
lysozyme is used. Combinations of lysozyme
The milks of most species contain an indigenous
addition and other control measures can then
lysozyme; human and equine milks are particularly
be evaluated further.
rich in this enzyme. Various aspects of indigenous
milk lysozyme were reviewed by Farkye (101, 102). Lysozyme appears to be quite effective against
In view of its antibacterial activity, the large differ- Listeria monocytogenes and other bacteria involved in
ence in lysozyme content between human and bovine foodborne diseases and food spoilage (105).
milks may have signicance in infant nutrition. It is Considering the widespread attention now focused on
claimed that supplementation of baby food formulae Listeria in dairy products, especially cheeses, it is likely
based on cows milk with egg-white lysozyme gives that this application of lysozyme will be the subject of
benecial results, especially with premature babies; further research. The preservative effects of lysozyme
however, results are equivocal (1). in other foods were reviewed by Proctor and
Lysozyme has some other minor applications in Cunningham (99) and Cunningham et al. (100). It is
dairy technology, but most current interest is focused possible that some of these may be applicable to dairy
on its use in Dutch, Swiss, Italian, and other cheese products.
varieties to prevent late gas blowing and/or off-avors
caused by the growth of Clostridium tyrobutyricum. VI. GLUCOSE OXIDASE
Contamination of cheese milk with Clostridium spp.
can be reduced by good hygienic practices, and popu- Glucose oxidase (GO) catalyzes the oxidation of glu-
lations may be further reduced by bactofugation or cose to gluconic acid (via gluconic acid--lactone)
microltration. However, in most countries it is nor- according to the following reactions (106):
mal practice to add sodium nitrate as a further precau- GO-FAD GO- 1
tion. The use of nitrate in foods is suspect because it
leads to nitrosamine formation, and many countries
have reduced permitted levels or prohibited its use.
Lysozyme is effective in killing Clostridium cells and GO-
preventing the outgrowth of their spores. It has been
shown to be an effective alternative to nitrate in pre-
venting the butyric acid fermentation and late gas @-
blowing in several cheese varieties (1, 2, 99, 100, 103,
The hydrogen peroxide formed is normally reduced by
104). It was concluded (103, 104) that
catalase present as a contaminant in commercial pre-
the published information indicates that for some parations of GO (from P. notatum, P. glaucum, or A.
cheese types, lysozyme is a suitable substance for niger) or added separately. GO, which has a pH opti-
the control of late blowing, provided the number mum 5:5, is highly specic for D-glucose and is used
of clostridial spores is low. For these cheese to assay specically for D-glucose in the presence of
types, which may be considered less sensitive to other sugars, blood, urine, etc.

In the food industry, GO has four principal application of gluconic acid from added glucose or from glu-
tions, which are not commercially very signicant, cose produced in situ from lactose by -galactosidase
especially in the dairy industry (1, 106, 107): or from added sucrose by invertase has been proposed
1. Removal of residual, trace levels of glucose. This (108). It is possible to produce gluconic acid from glu-
application, which is particularly useful for the treat- cose using immobilized glucose oxidase. However, it is
ment of egg white prior to dehydration (although an doubtful whether immobilized glucose oxidase could
alterative procedure using yeast fermentation is more be applied to the acidication of milk because of the
commonly used), is of little if any signicance in dairy high probability of fouling by precipitated protein even
technology. if low temperatures at which less extensive casein pre-
2. Removal of trace levels of oxygen. Traces of oxy- cipitation occurs were used.
gen in wines and fruit juices cause discoloration and/or
oxidation of ascorbic acid. Chemical reducing agents
may be used to scavenge oxygen, but enzymatic treat- VII. SUPEROXIDE DIMUTASE
ment with GO may be preferred. The GO system has
been proposed as an antioxidant for high-fat products, Superoxide dismutase (SOD) catalyzes the reduction of
such as mayonnaise, butter, and whole milk powder, superoxide anions, O2 , according to the following:
but it does not appear to be used commercially for this
2O2 2H !H2 O2 O2 2
purpose, probably because of cost vis-a`-vis chemical
antioxidants (if permitted) and the relative effective- The hydrogen peroxide formed may be reduced by
ness of inert gas ushing of canned milk powder. catalase, peroxidase, or a suitable reducing agent.
3. Generation of hydrogen peroxide in situ. The SOD occurs widely in tissues where it plays a major
hydrogen peroxide generated by glucose oxidase has antioxidant role in scavenging superoxide radicals
a direct bacteriocidal effect (which is a useful side effect which are produced through the action of several
of GO applied to egg products), but its bacteriocidal enzymes, e.g., XO and peroxidases.
properties can be much more effectively exploited as a Milk contains a low level of indigenous SOD which
component of the lactoperoxidase/hydrogen peroxide/ has been isolated and characterized (101, 102); the milk
thiocyanate system (see Sec. X). Two components of enzyme appears to be identical to SOD from bovine
this system occur naturally in milk: lactoperoxidase is erythrocytes. The level of indigenous SOD in milk,
present at 30 mg/L, and thiocyanate, produced in which is entirely in the skim phase, varies considerably
the rumen by hydrolysis of thioglucosides from mem- among individual cows, with stage of lactation and
bers of the Brassicae family, varies from 0.017 to mastitic infection. It may play a role in the oxidative
0.26 mM. Hydrogen peroxide does not occur naturally stability of milk, but attempts to correlate stability
in bacteria-free milk, but it can be generated metabo- with SOD activity have been inconclusive, presumably
lically by catalase-negative bacteria, added directly owing to the interaction of various pro- and antioxi-
(which is usually preferred) or produced in situ from dants. The tendency of fat in milk to oxidize is directly
sodium percarbonate or by the action of xanthine oxi- correlated with increases in XO and negatively with
dase on added hypoxanthine or by the action of glu- increases in SOD activity. A low level of exogenous
cose oxidase on glucose, either added or produced in SOD, together with catalase, is a very effective antiox-
situ from lactose by -galactosidase. Activation of the idant in dairy products, and it has been suggested that
lactoperoxidasehydrogen peroxidethiocyanate sys- treatment of milk with SOD may be effective in pre-
tem suppresses the growth of psychrotrophs in milk serving the avor of UHT milk which is prone to lipid
stored at 5 C and has given promising results as a oxidation (1). It has been reported that a combination
milk preservative in tropical regions where refrigera- of SOD and catalase is a more effective antioxidant
tion is lacking. It would appear that in such applica- than butylated hydroxyanisole. However, it appears
tions the use of exogenous hydrogen peroxide is the to be ineffective as an antioxidant in the presence of
simplest and most appropriate. 0:1 ppm Cu2 , apparently because Cu2 effectively
4. Production of acid in situ. Direct acidication of competes with SOD for O2 and converts it to lipid-
dairy products, particularly cottage, feta-type, and reactive species such as
OH. The commercial feasi-
mozzarella cheeses, is now fairly common. bility of using SOD as an antioxidant depends on
Acidication is normally performed by addition of cost, particularly vis-a`-vis chemical antioxidants, if
acid or acidogen (usually gluconic acid--lactone) or permitted. As far as is known, SOD is not used
by a combination of acid and acidogen. In situ produc- commercially as an antioxidant in the dairy industry.

VIII. SULFHYDRYL OXIDASE low concentrations of H2 O2 are required (see Sec. X).
However, at 400 mg/kg, H2 O2 alone is a more effective
Milk contains an enzyme capable of oxidizing sulfhy- long-term bactericidal agent than 8.5 mg/kg H2 O2 in
dryl groups to disuldes: the lactoperoxidase system. There was no signicant
2RSH O2 !RSSR H2 O 3 difference in the quality of cultured milk made from
either lactoperoxidase-activated or H2 O2 -treated milk.
The enzyme, which has molecular and catalytic proper- The activity of lactic starter was signicantly lower in
ties distinctly different from thioloxidase (EC 1.8.3.2), the former, but the rennet coagulability of the latter
glutathione:protein disulde oxidoreductase (EC was extended.
1.8.4.2), and protein disulde isomerase (EC 5.3.4.1), There is interest in using immobilized catalase reac-
has been isolated and well characterized (101, 102). tors for milk pasteurization or for glucose oxidase-cat-
Sulfhydryl oxidase (EC 1.8.3.) also has been demon- alase reactors. Although catalase may be readily
strated in human milk. immobilized, it is inactivated rapidly on exposure to
It has been proposed that sulfhydryl oxidase has a hydrogen peroxide. Hydrogen peroxide appears to be
physiological role in the formation of disulde bonds an effective agent for inactivation of aatoxin M1 . As
in vivo to give proteins the correct three dimensional discussed above, catalase, usually as a contaminant, is
structure. Industrially, the potential of the enzyme lies necessary for many of the applications of glucose oxi-
in its ability to ameliorate the cooked avor of UHT- dase and also to optimize the antioxidative properties
treated milk (1, 109). The milk enzyme occurs exclu- of SOD.
sively in the serum (whey) from which it may be easily
isolated by exploiting its marked tendency to aggre-
gate, thus facilitating its isolation by chromatography X. LACTOPEROXIDASE
on porous glass. The enzyme has been immobilized on
porous glass and titanium oxide, and its effectiveness
in ameliorating the cooked avor of UHT-treated milk H2 O2 2AH!2H2 O 2A 5
has been demonstrated on a pilot scale using immobi- Bovine milk is rich in lactoperoxidase ( 30 g=mL),
lized enzyme columns. For industrial-scale use, an ade- which is distinct from the myloperoxidase of leucocytes
quate supply of the enzyme from whey may be a and salivary peroxidase. Lactoperoxidase has been
limitation but production of the enzyme by genetically puried by several investigators and well characterized
engineered microorganisms might be feasible. (109112).
The most important physiological and technological
feature of lactoperoxidase is its ability, in the presence
IX. CATALASE of H2 O2 and thiocyanate, to inhibit the growth of sev-
eral bacteria. Since lactoperoxidase is relatively heat
resistant, there is adequate lactoperoxidase activity
2H2 O2 !2H2 O O2 4
even in milk that has been moderately to severely
Hydrogen peroxide is used for the cold-sterilization of heat-treated. Thiocyanate occurs in many animal tis-
milk in regions lacking refrigeration and perhaps in sues and uids. It is produced endogenously during the
some developed countries also; e.g., it may be used to detoxication of thiosulfates and metabolic products
treat cheese milk in the United States (1, 107). In of sulfur amino acids and cyanide and from foods con-
underdeveloped regions, treatment of milk with H2 O2 taining thioglucosides. Cows on pastures containing
is performed at ambient temperature and excess H2 O2 clover (rich in RCN) and nongrasses (e.g., Cruciferae
is reduced by indigenous milk catalase or by chemical containing thioglucosides) yield milk containing higher
interaction with milk proteins in which it causes some concentrations of thiocyanide than cows on winter feed
physicochemical changes, principally oxidation of or lay pastures. Saliva contains high levels of thiocya-
methionine, with adverse effects on cheese quality. nate which is also secreted by gastric mucosal cells.
Side effects can be decreased by short exposure to Milk does not contain indigenous H2 O2 but it may
H2 O2 at 65 C, after which the residual H2 O2 is be added or produced in situ (see Sec. VI). Thus,
reduced by added catalase, usually from beef liver or bovine milk possesses an effective antibacterial
Aspergillus niger. Most of the recent interest in the use system which probably affects the intestinal micro-
of H2 O2 as a preservative for milk has focused on the ora of calves (and presumably other species).
lactoperoxidase-H2 O2 -thiocyanate system in which Lactoperoxidase also appears to protect the mammary

gland against mastitic infection, especially during the including blood clotting, wound healing, keratinization
dry period. The antibacterial signicance of lactoper- of epidermal tissue, and stiffening of cell membranes.
oxidase in vivo has been reviewed (113, 114). Human An enzyme with a similar activity has been found in
milk appears to contain a low level of lactoperoxidase the bacterium Streptoverticillium mobaraense; this is
(< 0:1 g=mL) as well as myloperoxidase and eosino- referred to as MTGase (M microbial. However,
phil peroxidase. The lactoperoxidase system may be TGase and MTGase differ in a number of respects,
exploited in vitro to extend the shelf life of milk most notably in molecular weight ( 77 and
under conditions where refrigeration and pasteuriza- 38 kDa, respectively) and dependence on Ca2 ;
tion facilities are lacking (111114). TGase requires Ca2 , MTGase does not.
While most interest in lactoperoxidase has focused The activity of (M)TGase suggests several possible
on the indigenous enzyme, it may also acquire signi- applications in the food industry:
cance as an exogenous enzyme. Techniques have been
1. Gelation of proteins by crosslinking
developed for the commercial-scale purication of lac-
2. Formation of restructured meat and sh pro-
toperoxidase from milk (42). Large-scale trials have
ducts from smaller pieces or comminuted meats
shown that addition of puried lactoperoxidase to
3. Stabilization of protein-stabilized foams or
calf milk replacers markedly reduces the incidence of
emulsions (by crosslinking proteins in the stabi-
diarrhea and increases weight gain. Although the anti-
lizing layer)
bacterial properties of human milk do not depend on
4. Modication of the functionality, e.g., solubility
lactoperoxidase, its addition to bovine milk-based
or water binding, of proteins by converting glu-
infant formulae may have benecial effects (111114).
tamine to glutamic acid residues or binding of
amino sugars or phospholipids
XI. TRANSGLUTAMINASE 5. Modication of the nutritional value of foods
by enzymatically attaching essential amino
Transglutaminase (TGase; protein-glutamine -gluta- acids to proteins
myltransferase; EC 2.3.2.13) catalyzes an acyl transfer- 6. Conjugation of the same or different proteins
ase reaction between the -carboxyamide group of 7. Conjugation of amino sugars with proteins
peptide-bound glutamine residues (acyl donors) and a 8. Conjugation of phospholipids with proteins
variety of primary amines (acyl acceptors), including 9. Immobilization of enzymes
amino acids, and the "-amino group of lysine residues The feasibility of these applications has been demon-
in certain proteins. In the absence of an amine, TGase strated in laboratory studies, but the limited availabil-
catalyzes the deamination of glutamine residues, with ity and cost of the enzyme to date have restricted
water molecules acting as acyl acceptors. Thus, TGase larger-scale trials (guinea pig liver is the usual source
can modify proteins by incorporation of (a) an amine, of TGase). However, the gene for TGase has been
(b) crosslinking, or (c) deamination: inserted into microorganisms (115) which would be
expected to increase the availability and reduce the
cost of the enzyme.
Various aspects of the chemistry of TGase and its
applications in foods have been reviewed by Motoki
and Seguro (116) and Dickinson (117).
XII. -LACTAMASE
The amino group might be from a simple aliphatic or

aromatic amine, an amino acid, an amino sugar, or a
phospholipid (-aminoethanol moiety). 9
TGase is present in several animal tissues and body Residues of penicillin in milk, resulting from treatment
uids and is involved in several biological phenomena, of mastitis-infected cows with antibiotics, inhibit or

prevent the growth of lactic acid bacteria used in the bial recombinant chymosins will be improved through
production of cheese and fermented milks. They may genetic engineering. The production of cheese is
also evoke an allergic response in susceptible consu- expanding worldwide, and the market for rennets will
mers and create conditions for the selection of penicil- therefore continue to increase. Development of cheese-
lin-resistant pathogens. The usual approach taken to like products as food ingredients is a growth area, and
prevent contamination of the milk supply with antibio- it is likely that the application of proteinases and
tics is to withhold milk from treated cows for an ade- lipases in the production of such products will increase.
quate period after treatment; obviously, this results in The use of proteinases and peptidases to produce pro-
economic losses to farmers. tein hydrolysates with specic functional, nutritional,
Procedures developed for the removal of -lactam- or physiological properties appears to be very promis-
type antibiotics from milk include columns of activated ing and has been attracting increasing attention.
charcoal, ultraltration or dialtration (1). While these Lipases have some traditional and novel applica-
methods may be suitable under certain circumstances, tions in cheese technology, but the growth potential
they have obvious limitations. An alternative approach of these applications is probably limited. However,
is the inactivation of penicillin by -lactamase [Eq. (9)]. new and more effective lipases may be identied.
Korycka-Dahl et al. (118) demonstrated the ability Perhaps the greatest potential application of lipases is
of -lactamase from Bacillus cereus to inactivate peni- in the production of tailor-made lipids with superior
cillin G such that Cheddar or Swiss cheeses of normal nutritional or functional properties.
quality could be produced from the decontaminated Although -galactosidase has applications in dairy
milk. The enzyme was active at 4 C so that penicillin technology, most of these are not commercially viable
in milk could be inactivated during cold storage on the at present, mainly for economic reasons. Niche mar-
farm or at the factory. The enzyme lost only 50% of its kets exist for lactose-hydrolyzed milks and these may
activity following heating at 63 C for 30 min and hence expand. Exploitation of the transferase activity of -
would retain considerable activity in pasteurized milk, galactosidase in the production of new oligosacchar-
which may be illegal. These problems may be solved by ides with novel functional and/or nutritional properties
using immobilized -lactamase (119). This procedure appears to hold potential.
appears promising and awaits further development. Transglutaminase should have several interesting
An alternative approach to solving the problems applications in dairy technology. The availability of a
caused by penicillin in fermented products is the selec- cheaper enzyme should encourage work on the appli-
tion of -lactamase-producing mutants of lactic acid cation of this enzyme.
bacteria (120). With the possible exception of lactoperoxidase and
lysozyme, the other enzymes discussed in this chapter
appear to have very limited application in dairy tech-
XIII. PERSPECTIVES nology, at least under present circumstances.
Owing to the physicochemical properties of its consti-

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applications of the natural antimicrobial agents of

21
Flavor Enhancement in Fruit Juices and Derived Beverages by

Exogenous Glycosidases and Consequences of the Use of
Enzyme Preparations
Ziya Gunata
University of Montpellier II, Montpellier, France
I. INTRODUCTION the properties and current use of these enzymes for

avor enhancement in fruit juices and derived bev-
Research over the past two decades has revealed that erages (mainly wines) and future developments.
in a great number of fruit and plant tissues important Flavor enhancement by glycosidases has been the sub-
avor compounds are glycosylated and accumulate as ject of a review by the author and colleagues in 1993
nonvolatile and avorless glycoconjugates (16). (10), and brief summaries are also found in the reviews
Although results in the literature had long suggested of Williams (11), Vasserot et al. (9), and Winterhalter
the occurrence of glycosidically bound avor and Skouroumounis (6).
compounds in plants, the rst clear evidence was
found in 1969 by Francis and Allcock in rose owers
(7). The work of Cordonnier and Bayonove in 1974, II. STRUCTURE AND OCCURRENCE OF
suggesting the occurrence in grapes of important GLYCOCONJUGATED FLAVOR
avor compounds (monoterpenes) as glycoconju- COMPOUNDS
gates on the basis of enzymatic work (8), was later
conrmed by Williams et al. in 1982 by the identica- The publication of two techniques in 1982 (12) and
tion of glycosides (1). These ndings opened a new 1985 (2), which allow the selective retention of glyco-
eld of intensive research on the chemistry of sides on hydrophobic adsorbants (C18 reversed-phase
glycoconjugated avor compounds to exploit this and Amberlite XAD-2) from the complex matrix of
important avor source present in both fruit and fruit and plant materials, replaced tedious classical
plant tissues. The expanding literature on the chemis- techniques and greatly contributed to progress in the
try and occurrence of glycosidically bound volatiles in eld of glycosidic avor precursors. Once the glycosi-
the plant kingdom has been recently reviewed by dic extract is obtained, different liquid chromatogra-
Vasserot et al. (9), Stahl-Biskup (4), and Winterhalter phy techniques are applied to isolate and purify
and Skouroumounis (6). glycosides. Countercurrent chromatography, which is
This chapter briey presents the diversity and struc- a liquidliquid partition chromatography, improved
ture of glycosides found in fruits and plants. Attention separations especially by eliminating or decreasing
will focus on those glycosidases involved in avor the formation of artifacts observed on solid adsorbents
release from glycoconjugated precursors, examining (13). Various combined chromatographic and spectral

techniques (GC-MS , GC-FTIR, HPLC-MS/MS, (33), and strawberry (34). The amount of glycosidically
FAB-MS/MS) are used for identication. A recent bound volatiles is typically two to eight times greater
review (6) gives general approaches to the analysis of than that of their free counterparts (2, 3, 19).
glycosides with abundant references related to the Moreover, most norisoprenoids in fruit, some of
subject. which are precursors of very potent avor compounds,
Glycosidically bound volatiles identied in fruits have been detected mainly in glycosidic forms. This,
and plants are highly complex and diverse, especially together with the low aroma threshold and sensory
with regard to the aglycone moiety. The sugar parts properties of aglycones, makes the glycosidic com-
consist of -D-glucopyranosides and different digly- pounds an important potential source of avor vola-
cosides: 6-O--L-arabinofuranosyl--D-gluco- tiles during fruit juice processing.
pyranosides; 6-O--L-arabinopyranosyl--D-gluco- The glycosidically bound volatiles can be released
pyranosides (vicianosides); 6-O--L -rhamno- by either acid or enzyme hydrolysis (1, 2, 36). Slow
pyranosyl--D-glucopyranosides (rutinosides); 6-O-- acid hydrolysis takes place at fruit juice pH (35) and
D-glucopyranosyl--D-glucopyranosides (gentiobio- can be accelerated by thermal treatment (3638). This
sides); 6-O--D-apiofuranosyl--D-glucopyranosides; treatment, however, can reduce the sensory quality of
and 6-O--D-xylpyranosyl--D-glucopyranosides (pri- the products (38).
meverosides) (Fig. 1) (1, 2, 47, 1419). In rare cases Some aglycones are already odorous when released
trisaccharide glycoconjugates were isolated (6). As far from glycosides. They can therefore contribute to the
as we are aware, only (1 ! 6) intersugar linkages have oral aroma of some wines (36, 39), grapes (10, 36, 39),
been detected in plant avor glycoconugates, while apricots (40), peaches (41), and tea (5, 7, 16, 17). This is
1 ! 2, 1 ! 3, and 1 ! 4 linkages together with the case, for example, of monoterpenes such as gera-
1 ! 6 linkages were observed for diglycosides of a- niol, nerol and linalol which possess mainly oral attri-
vonoids (20). Therefore, different glycosyl transferases butes and low odor thresholds (100400 ppb in water)
seem to exist in plants. Recently, -D-glucopyrano- (39).
sides of some volatiles of fruit and plant tissues have In contrast, polyhydroxylated monoterpenes (poly-
been shown to be acylated at the C-6 hydroxyl group ols) and the majority of norisoprenoids are odorless,
of the sugar moiety with malonic acid (21, 22). but can generate potent aroma compounds due to
Malonylated glycoconjugates of avonoids are often their reactivity in the acidic medium (pH 2.83.8) of
detected in plants (20). fruit juices (36, 37, 42). For example, some hydroxy-
A common feature of glycosidic avor precursors is linalool derivatives can give rise at ambient tempera-
that the aglycone moiety is always linked to -D-glu- ture and grape juice pH (3.03.6), to the formation of
copyranose. To date 200 aglycones have been iden- odorous monoterpenes, such as hotrienol, nerol
tied in 150 plant species (4, 6). The aglycone moiety is oxide, and linalool oxides (37). The review by
predominantly mevalonic acid (monoterpenes, C13 - Strauss et al. discusses the acid catalyzed conversions
norisoprenoids, sesquiterpenes), and shikimic acid of monoterpenes and role of these compounds in
derived secondary metabolites. Medium-chain alka- grape and wine avor (36).
nols and alkenols have also been detected. Some abun- Potent C13 -norisoprenoid avorants in fruit- and
dant and important aglycones for avor are given in plant-derived products, such as theaspiranes, viti-
Figure 1. Further information can be obtained from spiranes, edulans, and -damascenone, are similarly
the reviews (4, 6). generated from relevant progenitors by acid-catalyzed
Glycoconjugates of avor compounds are present in reactions. The major pathways leading to these
several fruits, such as grape (1, 2, 19), apricot (3, 23), avor compounds are discussed in the review by
peach (3), yellow plum (3), quince (24), sour cherry Winterhalter (42). For example, -damascenone,
(25), passion fruit (15, 26), kiwi 927), papaya (28, which is among the most potent avoring substances
29), pineapple (30), mango (31), lulo (32), raspberry known to date (odor threshold 2 ppt in water; oral,
fruity, and iononelike odor) (43), can be generated
from multiple glycosylated precursors detected in

Abbreviations: GC, gas chromatography; MS, mass spectro- fruits and plants (42).
metry; FTIR, Fourier transform infrared spectroscopy; Glycosylated volatile compounds in plants are often
HPLC, high-performance liquid chromatography; FAB, considered as physiologically inactive plant storage
fast atom bombardment; TLC, thin-layer chromatography; and transport forms of secondary metabolites.
pNP, p-nitrophenyl. However, their exact role is far from understood (4).

Figure 1 Structure of glycoconjugated aroma compounds from plants.
III. ENZYMES INVOLVED IN THE will be later discussed, the release of the aglycone moi-
RELEASE OF FLAVOR COMPOUNDS ety may occur through two-step (sequential hydrolysis)
FROM GLYCOSIDES or one-step (endoglycosidase) enzyme-catalyzed reac-
tions. The term endoglycosidase is used in this chap-
The elucidation of the enzymes responsible for the ter for glycosidases involved in avor release as a
release of avor compounds from glycosides is the function of their catalytic activities.
rst step in the development of technological applica-
tions. Glycosidases catalyze hydrolysis of the glycosidic A. Sequential Hydrolysis
bond of their carbohydrate substrates by two general
mechanisms, leading to either the retention or inversion Preliminary work carried out on the hydrolysis of grape
of the anomeric conguration at the cleavage point (44). glycosides, by using fungal enzyme preparations (pecti-
The mechanism of hydrolysis is presumed to proceed in nases, hemicellulases, and glucanases), has shown that
the same way as the acid-catalyzed cleavage of glycosicic the release of avor compounds depends on the pre-
bond, via a carbocation intermediate. Glycosidases dis- sence of glycosidases as side activities in the enzyme
cussed here refer to those enzymes which catalyze the preparations (47). With the aid of suitable analytical
hydrolysis of O-glycosyl compounds. techniques (TLC, HPLC, GC/MS), a study using pur-
As had been suggested as early as 1913 (45) and ied glycosidases and synthesized substrates was able to
1924 (46), volatile compounds (monoterpenes, vanillin) elucidate the sequential reaction involved in the hydro-
can be liberated from -D-glucosides through the lysis of disaccharidic avor precursors (Fig. 2) (48).
action of -glucosidase (EC 3.2.1.21). The involvement First, the inter-sugar linkage is cleaved by exoglyco-
of enzymes in the release of volatiles from disacchar- sidases; either an -arabinofuranosidase (EC 3.2.1.55),
ides, however, was shown only recently because the an -rhamnopyranosidase (EC 3.2.1.40), or a -apio-
relevant substrates were rst reported in 1982 (1). As furanosidase depending on the disaccharidic moiety.

Figure 2 Sequential enzymatic hydrolysis of disaccharidic avor precursors.
This releases terminal sugars and -D-glucosides. demonstrated. With regard to microbial enzymes,
Subsequently, -glucosidase cleaves the aglyconic link- mainly those originating from GRAS (generally
age of -D-glucosides to liberate volatiles. This hydro- recommended as safe) will be discussed.
lysis mechanism has been patented (49). Such two-step
reactions, involving exoglycosidases (5, 6, 50, 51), are A. Plant-Originated Glycosidases
also possible in the hydrolysis of diglycosides of fruit
or plant volatiles, which possess -L-arabinopyrano- Most work has been devoted to grape glycosidases
syl, -D-xylopyranosyl or -D-glucopyranosyl as because the rst evidence of multiple forms of glycosi-
terminal sugars. dic avor precursors was found in this fruit. -
Glucosidase (55, 56, 69, 67, 68), -arabinofuranosidase
B. One-Step Hydrolysis of Disaccharide (56, 59), -arabinopyranosidase (19), -rhamnopyra-
Glycosides (Endoglycosidase) nosidase (56, 69), and -xylosidase (51) activities
were detected in grapes of various cultivars. The pre-
This one-step reaction occurs through the cleavage of sence of -apiosidase in grapes has not yet been con-
the aglyconic linkage which yields a disaccharide and rmed. On the basis of glycosidase activity
aglycone, the identity of which were conrmed by determinations with pNP-glycosylated substrates, -
HPLC and GC/MS analysis (Fig. 3) (5, 51). The glucosidase activity is considered the most important.
enzymes catalyzing this reaction were isolated from tea Multiple forms of -glucosidase exist in grape berries
leaves (5) and grapes (51). The enzyme from tea is called (51, 55, 59) and are also found in almond emulsin (69)
primeverosidase, after primeverosides, which are the but not in papaya fruit (54). The enzyme from papaya
most abundant glycoconjugated avors in this plant. fruit, unlike that from grapes, is readily soluble and
does not require the use of detergent for extraction.
Glycosidases are found predominantly in grape
IV. OCCURRENCE OF GLYCOSIDASES skins (67, 68), although contradictory results may
arise owing to the enzyme extraction method employed
Exoglycosidases involved in avor release are widely (59). -Glucosidase, -arabinofuranosidase, and -
distributed among plants (10, 5260) and microorgan- rhamnosidase activities have been shown to increase
isms (6166). The enzymes presented here mainly con- during the ripening of grape berries (56, 59, 67), unlike
cern those found in fruits or plants where the -glucosidase activity from papaya fruit which is unaf-
occurrence of glycoconjugated avors has been fected by the ripening stage of the fruit (54).

Figure 3 One-step hydrolysis of disaccharidic avor conjugates by grape endoglycosidase.
The presence of -glucosidase activity has also been 1. Botrytis cinerea

reported in vine leaves (59, 67) where avor glycocon-
The ability of B. cinerea to produce -glucosidase (64,
jugates are abundant (70). It should be noted that gly-
65) and -arabinofuranosidase (62) when cultured in a
cosidases substrates other than glycosidic avor
suitable medium was shown. Grape berries
conjugates are found in plants. Indeed, many of the
contaminated by fungi such as B. cinerea were found
secondary metabolites synthesized are glycosylated,
to contain higher levels of -arabinofuranosidase and
and > 1500 avonoid glycosides have been character-
-rhamnosidase activities compared to uncon-
ized (71).
taminated grapes (56). This fungus has been shown
Endoglycosidase, active on disaccharidic avor pre-
to exhibit these same activities when developed on
cursors in fruits, has been recently isolated and par-
grape juice (56). In these conditions, -glucosidase is
tially characterized from grape skins (51). The
also produced but its activity is strongly inhibited by
extraction was only possible with the use of a deter-
glucono--lactone which is produced from glucose
gent, the type of which greatly inuenced the yield. In
through glucose oxidase activity of the fungus
contrast, the extraction of tea enzyme (primeverosi-
(10, 56). The presence of gluconolactone in grapes is
dase) was possible without the use of detergent (5,
one of the major limitations on avor release by
72). This suggests that the structure of grape endogly-
glycosidases and will be discussed later.
cosidase differs from that obtained from tea and/or
The development of B. cinerea on grapes has a very
could be a membrane-bound protein. Moreover,
detrimental effect not only on the avor of juice and
although needing conrmation, grape endoglycosidase
wine, through the degradation of monoterpenes (73
also seems to possess -glucosidase activity (51).
75) and fatty acid esters (76), but also on the overall
quality due to the action of several enzymes produced
by the fungus (7779).
B. Fungal-Derived Glycosidases
2. Yeast
These can be present in fruit due to the development of
fungi such as Botrytis cinerea. Other lamentous fungi, In wine making the alcoholic fermentation is essen-
for example Aspergillus sp., and yeast are also able to tially performed by yeast Saccharomyces cerevisiae. -
produce glycosidases. Arabinofuranosidase (80), -rhamnosidase (80), and

-glucosidase (80, 81) activities were only detected at localized at the cell membrane and in the cytoplasm
low levels in juices and in cells during fermentation. (63, 87, 93, 96).
-Glucosidase activity was the most abundant, fol-
lowed by -arabinofuranosidase activity. The activ-
ities reached their maximum levels during
3. Filamentous Fungi
exponential growth of the yeast population, then
dropped dramatically. No differences have been Filamentous fungi, particularly Aspergillus sp., are
observed among enological yeast (S. cerevisiae) good producers of exoglycosidases and used in indus-
strains in the production of glycosidases during fer- try to obtain pectic and hemicellulase enzyme prepara-
mentation (80). tions (61, 97, 98). These preparations are widely used
Owing to the low level of glycosidases production in fruit juice processing and winemaking, mainly to
by S. cerevisiae, a recombinant yeast strain, improve juice clarication and yield. In addition to
expressing -glucosidase from C. molischiana (82) their main activities, they contain several other enzyme
and -arabinofuranosidase from A. niger (83), has side activities including glycosidases. -Glucosidase
been recently constructed. The relevant proteins and -arabinofuranosidase are often the most abun-
were detected during grape juice fermentation, dant, followed by -rhamnosidase, while -apiosidase
although poor secretion of the C. molischiana protein is rarely detected (10) (Table 1). The production of -
was observed. There is a lack of data on the apiosidase by A. niger appears to be inducible because,
contribution of these recombinant yeast strains to unlike -glucosidase, which is a constitutive enzyme in
avor release during fermentation. -Glucosidases fungi, it requires the presence of a carbon source indu-
from Kluyveromyces fragilis (84), A. niger (6), and cer such as apiin (a trihydroxyavone-apiosyl (1 ! 2
Bacillus polymyxa (85) have been expressed in glucoside) (99). Enzyme preparations shown in Table 1
S. cerevisiae in order to obtain strains able to ferment differ notably in their glycosidase activity levels.
disaccharides (cellobiose and lactose). Furthermore, these levels vary greatly from one
The yeast species from the genera Saccharomyces batch to another, as current pectinase enzyme prepara-
(63, 86), Dekkera (87), Debaryomyces (88, 89), tions are formulated by producers as a function of
Kloeckera (90), Hansenula (91), and Candida (9295) their main activities.
are able to synthesize -glucosidase when cultured on The production by fungi of endoglycosidase, cap-
carbon sources such as glucose, cellobiose, xylose, and able of hydrolyzing disaccharidic conjugates of avor
alkyl thioglucosides. The lowest enzyme synthesis is compounds, has to date been the subject of only one
generally found in the Saccharomyces spp. Except for paper. An A. niger strain was shown to produce such
Candida and Debaryomyces spp., yeast seldom secretes as enzyme (an endo--glucosidase) when cultured on
-glucosidase into a growth medium. The enzyme is rutin as the sole carbon source (100).
Table 1 Glycosidase Activities (nkat/mg Product) in Some Enzyme Preparations
-Apiosidase -Glucosidase -Arabinofuranosidase -Rhamnosidase
Cellulase A a 6.1 0.6 0.007

Hermicellulase 7.1 7.0 0.9
Pectinol VR 0.2 0.1
Rohament CW 3.3 0.7 0.4
Pectinol D5S 0.5 0.7
Pektolase 3PA 0.3 1.5 3.8 0.04
Ultrazym 100 0.03 0.5 0.1
Pectinase 263 0.2 7.2 1.4 0.3
AR 2000 1.08 5.7 14.7 0.3
Novoferm 12 0.15 8.4 0.5 0.05
Source: Ref. 10.

Commercial sources: Gist Brocades (CellulaseA, Hemicellulase, Pectinase 263, AR 2000), Rohm (Pectinol VR, Rohament CW,
Pectinol D5S), Ciba-Geigy (Ultrazym 100), Grindsted (Pektolase 3PA), Novo (Novoferm 12).
a
Undetected.

V. ENZYME ASSAY METHODS A. Effect of pH on Activity and Stability
The substrates used may be composed either of a mix- The optimum pH activity of plant -glucosidase (55,
ture of glycosides isolated from fruits and plants by 59) and endoglycosidase (51) is similar (5.06.0) to that
selective retention on hydrophobic adsorbents (2, 12), of intracellular yeast -glucosidase (63, 87, 93, 105).
or of puried and synthesized natural glycosides (6, 35, This value is generally lower (4.05.0) for extracellular
101, 102). Alternatively, articial pNP-glycoconju- yeast (63, 92, 93) and Aspergillus enzymes (10, 61, 106).
gated substrates are often used. The optimum pH of an extracellular endo--glucosi-
Glycosidase activities can be determined by two dase from A. niger was found to be unusually low (3.4)
techniques: (100). Most -glucosidases exhibit only 515% of their
1. Direct analysis of glycosides by chromatographic maximum activity in the pH range of fruit juices (2.8
techniques (TLC, HPLC, GC) (2, 5, 6, 19, 35, 48, 51). 3.8).
Before GC analysis, a derivatization step, generally A. niger -arabinofuranosidase (62, 107) and -
involving the silylation and triuoroacetylation of gly- rhamnosidase (47, 108) possess similar pH optima to
cosides, is carried out in order to obtain their volatile that of extracellular -glucosidase from the same fun-
forms. gus, while the value for A. niger -apiosidase is higher
2. Analysis by colorimetric or chromatographic (5.06.0). (10, 99, 106). Both -arabinofuranosidase and
(TLC, HPLC, CPG) techniques of aglycone or sugar -rhamnosidase exhibit > 50% of their maximum
moieties (monosaccharides or disaccharides) released activities in the pH range of fruit juices.
from glycosides (2, 5, 48, 51, 103). In the case of agly- An important parameter that must be considered
cone liberated by enzymatic hydrolysis from natural for technological applications is the stability of glyco-
glycosides, the volatiles are recovered from the enzyme sidases in the acidic pH of fruit juices. Glycosidases
assay before GC analysis, by either liquidliquid differ notably in this respect (Fig. 4) (10).
extraction with a solvent or selective retention on -Glucosidases from grapes, S. cerevisiae, and C.
hydrophobic adsorbents (2, 6, 12). A colorimetric wickerhamii, are not very stable in these conditions.
assay of monoterpenes (aglycones) liberated from For example, C. wickerhamii -glucosidase was able
sugars has also been proposed (104). to hydrolyse -D-glucosides of monoterpenes in a
pNP-glycoconjugated substrates are widely used as wine only when the initial pH (3.0) was adjusted to
the standard for assaying glycosidase activities using 3.5 or higher (109). Only 10% of initial -glucosidase
mainly colorimetric techniques owing to their ready activity from S. cerevisiae was observed after 90 min
availability (except for certain disaccharidic substrates) incubation (208C) in a buffer at pH 3.0. (10). This may
and the simplicity of the analysis. Often, this analysis explain the large decrease in S. cerevisiae -glucosidase
does not reect the catalytic characteristics of enzymes, activity during grape juice fermentation. In fact the
owing to the presence of multiple forms of glycosidases optimum pH stability of the enzyme corresponds to
and, in some cases, the narrow aglycone specicity (see the pH of yeast cells (6.0). This decrease is also
Sec. VI.C). observed for -rhamnosidase and -arabinofuranosi-
dase activities from S. cerevisiae during juice
fermentation (10, 80). A -glucosiase from C. peltata
was fairly stable at pH 3.56.0 (95). The enzyme from a
mutant strain of C. molischiana was shown to be more
VI. FOOD-PROCESSING PROPERTIES OF stable at acidic pH than an enzyme from a wild-type
GLYCOSIDASES strain (110,111). This difference could be explained by
the difference in glycosylation of the relevant proteins.
This section will examine the properties of enzymes, In contrast, -glucosidase, -arabinofuranosidase,
focusing on the specic conditions encountered in the and -rhamnosidase from A. niger are remarkably
processing of fruit juice and derived beverages. In par- stable over a pH range of 2.57.0 (Fig. 4) (10, 62,
ticular, three important parameters inuencing avor 106). The enzymes exhibit > 80% of their maximum
release will be discussed: the effect of pH; aglycone and activity after 24 h of incubation in a buffer at pH
sugar specicity of glycosidases; and inhibition of 3.0. However, A. niger -apiosidase was less stable at
activity by sugar and sugar analogues. A summary of a pH < 4:0 (10, 106). The same trends were observed
the properties of -glucosidase from some plants and when the stability of A. niger glycosidases were mon-
microorganisms is given in Table 2. itored during grape juice fermentation at 208C (pH 3.0)

Table 2 Properties of -Glucosidases from Some Plants and Microorganisms
Origin pH Temperature (8C) Glucose inhibition Gluconolactone inhibition Aglycone alcohol
a
Optimum Stability Optimum Stability Ki (mM) % Ki (mM) % substrates References
Plant
Grape 5.0 6.07.0 45 50 170 66% at 100 mM 0.22 88% at 10 mM Primary 2, 10, 19, 55, 59
Almond 5.5 n.d. 50 65 210 12% at 100 mM 0.49 n.d. Primary 10, 52, 59, 69, 115
Papaya 5.0 n.d. 50 n.d. n.d. 50% at 10 mM n.d. n.d. n.d. 11, 54
Yeast
S. cerevisiae (i) 6.06.8 5.07.0 45 45 6.7 13% at 100 mM n.d. 42% at 10 mM Primary 10, 80, 105
D. intermedia (i) 5.0 n.d. 55 50 3.0 n.d. n.d. n.d. n.d. 87
C. molischiana (e) 4.04.5 4.05.0 5060 60 7.0 n.d. n.d. 68% at 10 mM Primary tertiary 92, 116
C. wickerhamii (e) 4.5 4.05.0 50 50 230 18% at 100 mM n.d. 100% at 10 mM Primary tertiary 93, 94, 116
(i) 6.0 n.d. 50 50 9 n.d. n.d. 100% at 10 mM n.d. 93
C. peltata (e) 5.0 3.56.0 4550 50 1400 n.d. n.d. n.d. n.d. 95
D. hansenii (e) 4.05.0 n.d. 40 n.d. n.d. 10% at 100 mM n.d. n.d. Primary tertiary 89, 164
Fungus
A. niger (e) 4.06.0 2.57.0 6065 6065 3.03.8 90% at 100 mM n.d. 100% at 1 mM Primary tertiary 10, 61, 106, 113, 115
3.4 n.d. 65 n.d. 40 81% at 250 mM n.d. n.d. Primary tertiary 100, 161
4.06.0 4.07.0 55 60 543 n.d. n.d. n.d. n.d. 131
A. oryzae (e) 4.56.0 3.07.0 n.d. n.d. 958 10% at 100 mM n.d. 80% at 50 mM Primary tertiary 13, 129
B. cinerea (i) 6.57.0 n.d. 50 n.d. 5.5 n.d. 0.02 n.d. n.d. 64
(e) 3.0 4.010.0 60 n.d. n.d. n.d. n.d. n.d. n.d. 65
a
Temperature at which rapid inactivation occurs.
n.d., not determined; (i), intracellular; (e), extracellular.

Figure 4 Effect of pH (phosphate-citrate buffer) on stability of glycosidases of A. niger (24 h, 308C), C. wickerhamii (1 h, 308C),
S. cerevisiae (90 min, 208C), and grape (24 h, 308C). (From Ref. 10.)
(106). -Glucosidase and -arabinofuranosidase activ- not very surprising since 1 H and 13 C NMR spectra of
ities remained practically unchanged after 7 days, while disaccharide glycosides show that the orientation of
only 70% of initial -rhamnosidase and 50% of - the terminal sugar is unaltered whether the monoter-
apiosidase activities were observed. This remarkable penyl moiety is a primary or tertiary alcohol (35, 101).
stability of extracellular A. niger enzymes is interesting The hydrolysis of monoterpenyl -D-glucosides by
for fruit juice processing and could be explained by the plant (grape and almond; 2, 19, 55, 59, 115), fungal
glycosylation of proteins (110, 112). (yeast, A. niger; 10, 89, 115, 116), and bacterial (B.
polymyxa; 117) -glucosidases is, however, greatly
B. Effect of Temperature inuenced by the structure of the aglycone. Plant -
glucosidases showed a pronounced specicity and
The optimum temperature activities of plant (54, 55) acted on -D-glucosides of primary alcohols (e.g., ger-
and yeast -glucosidases (63, 89, 93, 95) is generally aniol, nerol, and citronellol) but not on those of ter-
lower (45508C) than that (50608C) of A. niger tiary alcohols (e.g., linalool, -terpineol, linalool
enzymes (61, 106, 113). At the normal temperature oxides) which, when hydrolyzed, can give rise to potent
(208C) of grape juice fermentation, for example, glyco- volatils. This shows the limitations in avor recovery
sidases exhibit only 1020% of their maximum activity. with plant endogenous -glucosidases in fruit juice
Rapid inactivation of glycosidases occurs at tem- processing.
peratures > 508C, (55, 63, 99, 113), although some la- The aglycone specicity of -glucosidases from S.
mentous fungal -glucosidases are only inactivated at cerevisiae and B. polymyxa resembled grape and
temperatures > 658C (61). The pH of the meidum plays almond -glucosidases more than microbial enzymes
an important role in the thermal denaturation of gly- from Aspergillus, Candida, Debaryomyces spp. The lat-
cosidases. In general, glycosidases from lamentous ter were able to catalyze the hydrolysis of both mono-
fungi are more heat stable than those from yeasts terpene primary and tertiary alcohol -D-glucosides.
and plants (61, 63). The rate of hydrolysis of -D-glucosides of mono-
terpene primary alcohols by various -glucosidases
C. Aglycone and Sugar Specicity of was found to be higher than that of -D-glucosides
Glycosidases of tertiary alcohols (115, 116). These results are in gen-
eral agreement with the catalytic activity of almond
The hydrolysis of monoterpenyl diglycosides by exo- emulsin -glucosidase on other primary and tertiary
glycosidases-arabinofuranosidase (48, 107), -ara- alkyl--D-glucosides (118). It is interesting to note
binopyranosidase (50, 114), -rhamnosidase (48), and that the opposite results were obtained when the
-apiofuranosidase (99, 106)is not signicantly inu- acid-catalyzed hydrolysis of these substrates was per-
enced by the structure of the aglycone moiety. This is formed (119).

Furthermore, the conguration of monoterpenes sides. An enzyme from Viburnum furcatum was found
has an effect on the rate of hydrolysis since diastereoi- to be very specic to the apiosyl (1 ! 6 glucoside unit
someric monoterpenyl -D-glucosides (linalool, -ter- (122). Glycosides possessing rutinose and apiosyl 1 !
pineol, and citronellol) and isomeric monoterpenyl - 2 glucoside as sugar moiety were not substrates. In
D-glucosides (geraniol and nerol) are hydrolyzed at contrast, the latter was substrate for an enzyme from
different rates by enzymes (115, 116). For example, Cicer arietinum L. (123). An endoglycosidase from
geranyl--D-glucoside was often hydrolyzed at a Rhamnus (124) and from Fagopyrum esculentum
higher rate than neryl--D-glucoside. (125), however, showed, a broad specicity for avo-
The 1 H and 13 C NMR spectra of -D-glucosides of noid glycosides. Further work is needed to determine
monoterpene primary and tertiary alcohols differ in whether plants possess multiple forms of endoglycosi-
the orientation of the -D-glucopyranosidic linkage dase differing in their catalytic activities towards gly-
(35, 101), which may partly explain differences cosidic avor precursors.
observed in enzymatic hydrolysis. The conclusion The broad substrate specicity of tea and grape
reached from these results is that the source of the - endoglycosidases provides new challenges for the use
glucosidase can inuence the pattern of liberated agly- of such enzymes through genetic engineering for avor
cones and thereby the sensory properties of products. enrichment of fruit juices and other plant-derived pro-
Little work has been devoted to the determination ducts.
of the afnity of -glucosidases for glucosidic avor With regard to fungal endoglycosidase, an enzyme
precursors. Among the substrates tested (geranyl, from A. niger was able to hydrolyze geranyl--rutino-
neryl, and citronellyl -D-glucosides), grape enzyme side (100). We have observed in our laboratory (data
showed the highest afnity for geranyl--D-glucoside not published) that Aspergillus sp. can produce an
(55). Additionally, differences were observed between endoglycosidase when specic carbon souce inducers
two -glucosidases isolated from grapes with regard to were present in the culture medium, in similar condi-
their afnity for monoterpenyl -D-glucosides. Two tions to those described by Westlake et al. (126).
-D-glucosidases isolated from A. niger differed also However, the enzyme showed a distinct specicity for
signicantly in their afnity toward geranyl -D-gluco- both the sugar and aglycone moiety of substrates. It
side and malvidin -D-glucoside (participating in the was active on avonoid rutinosides, such a rutin and
red color; see Sec. VII.B) (103) and monoterpene ter- hesperidin. Neither naringin, a avonoid glycoside
tiary alcohol -D-glucosides (115). It is worthwhile to with rhamnosyl 1 ! 2 glucoside as sugar moiety,
note that often multiple forms of glycosidases were nor monoterpenyl, 2-phenylethyl, or benzyl rutinosides
detected in plants (53, 55, 120) and microorganisms was the substrate.
such as fungi (61, 103, 106). This emphasizes the
importance of the use of natural substrates to deter- D. Inhibition of Glycosidase Activities
mine the catalytic properties of enzymes, as suggested
1. Glucose Inhibition
by Hosel and Conn (120), instead of commonly used
articial substrates (pNP-glycosides). An important constraint for avor release in fruit juice
With regard to endoglycosidase, the enzyme from processing is the inhibition of -glucosidases by glu-
grapes shows a broad specicity toward the sugar moi- cose. This inhibition is often of a competitive type and
ety of disaccharides (51). The enzyme is active against a common characteristic of these enzymes (10, 16, 127,
arabinofuranosyl, rhamnopyranosyl, and apiofurano- 128). In contrast, -glucosidase activity is not inu-
syl (1 ! 6 glucosides from grapes. Arabinopyranosyl enced by other sugars such as fructose, galactose, ara-
and xylopyranosyl (1 ! 6 glucosides moieties from binose, mannose, lactose, and sucrose at the
passion fruit (15) and tea leaves (5), respectively, are concentrations occurring in food products (95, 100).
also substrates for the enzyme. Inhibition of -glucosidase activity by glucose has
-Primeverosidase from tea leaves also shows fairly long been known to be a major problem for enzymatic
broad specicity toward disaccharide glycosides. debittering of citrus juices by hydrolysis of naringin
Xylopyranosyl (1 ! 6 glucosides and apiofuranosyl (108) and for efcient enzymatic saccharication of
(1 ! 6 glucosides are better substrates than arabino- cellulosic material (61). Cellobiose, produced by the
pyranosyl and arabinofuranosyl (1 ! 6 glucosides consecutive action of cellulases (exo and endogluca-
(121). nases) on cellulose, can accumulate in the medium as
Work carried out on other plant endoglycosides has the consequence of end-product (glucose) inhibition of
mainly focused on the hydrolysis of avonoid glyco- -glucosidase, which catalyzes the hydrolysis of cello-

biose. This leads to the inhibition of cellulase activities occurring at 0.2 M glucose (132). That from another
by cellobiose. Much attention has therefore been strain was activated only up to 0.2 M glucose concen-
focused on the search for glucose tolerant -glucosi- tration (133). The activity of both enzymes decreased
dase. at > 0:4 M glucose but the extent of this decline dif-
Intracellular -glucosidases from yeast are generally fered among the strains. No convincing explanations
very sensitive to glucose inhibition. Inhibition con- have emerged to account for this unusual behavior of
stants vary from 3.0 to 23 mM (63, 87, 93). During the enzymes.
grape juice fermentation, both excreted and cell -glu- The activities of fungal exoglycosidases are not sig-
cosidase of S. cerevisiae were found to be less sensitive nicantly affected at the usual glucose concentrations
to glucose inhibition (80, 81). Puried -glucosidase found in fruit juices (47, 135). The enzymes -arabino-
from S. cerevisiae showed, however, a low value of furanosidase and -apiofuranosidase were more resis-
K1 (6.7 mM) (105). tant to glucose inhibition than -rhamnosidase (10).
Some strains belonging to the genera Candida (92 Glucose has been demonstrated to be a competitive
95) and Debaryomyces (89) are able to produce glu- inhibitor Ki 120 and 350 mM) of fungal -rhamno-
cose-tolerant extracellular -glucosidase. C. molischi- sidase (66, 136).
ana extracellular enzyme (92) is strongly inhibited by Among plant -glucosidases, those from grape (55)
glucose Ki 7 mM), while that from C. wickerhamii and almond (52) were resistant to glucose inhibition,
Ki 230 mM) (93) and particularly from C. peltata with a Ki of 170 mM and 210 mM, respectively, while
(Ki 1400 mM) (95) were distinguished by high resis- that from papaya fruit was more sensitive (11).
tance to glucose inhibition. Rosi et al. (89), after With respect to endoglycosidases, the enzyme from
screening 317 yeast strains from 20 species, selected a grapes showed a high Ki value (212 mM) (51). Glucose
D. hansenii strain producing a highly glucose-tolerant did not inuence the activity of an endoglycosidase
-glucosidase. produced from Aspergillus sp. in our laboratory (data
Bacterial and lamentous fungal -glucosidases are not published), which was found to be specic to a-
generally strongly inhibited by glucose, the inhibition vonoid rutinosides as discussed previously. However,
constant being in the range of 0.610 mM (95, 113, an A. niger endo--glucosidase able to hydrolyze ger-
127129). However, we recently demonstrated that anyl -rutinoside was found to be sensitive to glucose
the production of highly glucose-tolerant extracellular inhibition (Ki 40 mM) (100). Furthermore, the com-
-glucosidase from A. niger and A. oryzae is possible mon inhibition of glycosidases by their end product
(129, 130). This involves an adaptive enzyme whose (sugar) was observed for fungal -rhamnosidase activ-
production was induced by using uncommon carbon ity (66, 136). The inhibition with rhamnose was com-
sources in the culture medium. The highest production petitive, with a Ki of 1.2 and 1.75 M.
was obtained with quercetin (a pentahydroxyavone)
as the sole carbon souce. In these conditions, two -
2. Glucono--Lactone
glucosidases were produced. The major -glucosidase
was strongly inhibitied by glucose Ki 3:5 mM) as is This is one of the most powerful inhibitors of -gluco-
the case for Aspergillus enzymes (113, 127, 128). The sidases. Plant (52, 55) and microbiol -glucosidases
minor -glucosidase, however, was highly resistant to (63, 92, 93, 113, 137) are more strongly inhibited by
glucose inhibition Ki 953 mM). The molecular this compound than by glucose. The high afnity of
weight of this enzyme is quite low (30 kDa) compared the compound for the enzyme has been explained by its
to that of most Aspergillus enzymes. To date only one structural analogy with an intermediate product in the
strain of A. niger has been reported to produce (MW enzymatic cleavage of -D-glucosides (138). The inhi-
98 kDa) a constitutive enzyme which is not very sensi- bition is usually of the competitive type with Ki values
tive to glucose inhibition Ki 543 mM) (131). often < 1 mM and applies equally to -glucosidase
Activity enhancement by glucose was observed for from Streptomyces sp. (Ki 0:44 mM), the activity
an intracellular -glucosidase from Streptomyces sp. of which was enhanced by glucose (133).
(132, 133) and from Microbispora bispora (134). Gluconolactone concentration may reach levels of
Multiple forms of -glucosidases have been observed 510 mM in grape juice and wine obtained from grapes
in Streptomyces sp., but only one of these forms was contaminated by fungi, such as B. cinerea (56, 139). At
found to display this particular behavior. - these levels, grape (55), S. cerevisiae (80), C. wickerha-
Glucosidase activity from one strain was stimulated mii (93), and A. niger (113) -glucosidases are strongly
by up to 0.4 M glucose, with the maximum activation inhibited. However, glucose-tolerant -glucosidase

from Aspergillus strains, the production of which was on the source of the enzyme (55, 63, 99). The strongest
induced with quercetin, displays 30% of its activity at 5 inhibition was often observed with Ag , Hg2 , and
mM and 20% at 50 mM of gluconolactone (129, 130). Cu2 .
At high concentration glucunolactone is also an
inhibitor of A. niger -rhamnosidase and -apiosidase
activities. For example, the former was inhibited by VII. USE OF GLYCOSIDASES FOR
50% and the latter by 85% with 100 mM gluconolac- AROMA ENRICHMENT
tone (10). A. niger -arabinofuranosidase was unaf-
fected at this concentration. The use of glycosidases to release avor compounds
from glycosidic precursors, was initially examined in
3. Miscellaneous Compounds wines. Two major reasons for this are: (i) important
avor compounds in wines of Vitis vinifera cultivars
a. Ethanol. Generally, fungal -glucosidase (80,
are accumulated in grapes as avorless glycoconju-
93, 100), -arabinofuranosidase (47, 135), -arabino-
gates, and especially (ii) the glucose inhibition of -
pyranosidase (47), and -apiosidase (99) are weakly
glucosidase in available enzyme preparations, limits
inhibited by 10% (v/v) ethanol which is the average
the use of glycosidases to media, like wine, containing
value in wines. Inhibition often only occurs above
trace levels of glucose (< 1 g/L). Nevertheless, because
15% ethanol. However, A. niger -rhamnosidase was
of the occurrence of avor glycoconjugates in several
found to be more sensitive to ethanol, with a loss of
fruits, enzyme applications have been found for fruit
2040% of activity at a 10% ethanol concentration,
juices.
depending on the enzyme preparation (47).
An enhancement of particularly microbiol -gluco-
A. Wine Flavor Enhancement
sidase activity has been observed when pNP--gluco-
side was used as substrate, which may be due to the
The amount of glycosidic avor precursors decreases
glucosyltransferase activity of the enzyme (47, 87, 140).
only slightly during grape juice fermentation (145).
Conversely, the hydrolysis of pNP--glucoside by
This phenomenon may be explained by the low levels
grape (55, 59) and almond (59) -glucosidases clearly
of glycosidase activities exhibited by grapes (55, 56)
decreased in the presence of ethanol. Grape -glucosi-
and enological yeast strains (80), as discussed pre-
dase activity declined by 60% in 10% ethanol (55).
viously. Consequently, attention has been focused on
b. Sulfur Dioxide. At the usual levels (50200 the use of commercially available fungal enzyme pre-
ppm) used in fruit juice processing and wine making parations (pectinases, hemicellulases) containing glyco-
to prevent oxidation, this compound was not found to sidase activities derived mainly from Aspergillus spp.
cause an inhibitory effect on yeast -glucosidase (80, Various enzyme preparations have been tested for a-
82) or A. niger endo--glucosidase (100). vor enhancement of wines from different cultivars by
incorporating the enzymes either into the juice during
c. Phenols and Polyphenols. Phenols (141) and
fermentation or into the wine. Current regulations,
polyphenols (142) display an inhibitory effect on
however, do not allow the addition of enzymes into
plant -glucosidase. The afnity of phenolic inhibitor
wine.
for the enzyme increases with increasing molecular size
Muscat cultivars were often used as a model to test
(142). Catechin, dimeric procyanidins, and especially
the effect of the enzyme treatment because of the abun-
yellow oxidation products of catechin are powerful
dance of monoterpenyl glycosides. The hydrolysis of
inhibitors of sweet almond -glucosidase (143).
these glycosides liberates highly odorous alcohols
Similarly, apple -galactosidase is also inhibited by
which can be detected by sensory analysis and easily
catechin, quercetin, and glycosides of quercetin (144).
analyzed by GC/MS. In general, the addition of
These polyphenols are either present in intact fruits or
enzyme preparations possessing glycosidase activities
produced during fruit juice processing by oxidative
during wine making results in a signicant increase in
reactions. To our knowledge, the effect of polyphenols
the concentration of monoterpenes, C13 -norisopre-
on fungal glycosidase activities has not yet been stu-
noids, and shikimate derived compounds.
died.
The total free monoterpene concentration in the
d. Cations. Ag , Hg2 , Cu2 , Mg2 , Ca2 , Fe2 , treated wines increased by several fold compared to
and Fe3 were found to be inhibitors of plant and control wines (Fig. 5) (10, 109, 146152). The concen-
fungal glycosidases, the extent of inhibition depending tration of free monoterpenes often exceeded their

Figure 5 Total free monoterpene and C13 -norisoprenoid concentrations in control and glycosidase treated wines.
aroma threshold in wines made from varieties, such as It is difcult to attribute the increase in concentra-
Muscat and Gewurztraminer, in which large amounts tion of each monoterpene directly to the enzymatic
of monoterpenes occur as avorless glycoconjugates release from glycoconjugate precursors. The acid-cata-
(2, 19, 109). These treated wines developed a more lyzed rearrangement of released monoterpenes occurs
intense oral note owing to the abundance of mono- in grape juice and wine (Fig. 6) (3638, 157), and some
terpenes. In nonoral varieties, such as Sauvignon, monoterpenes, as mentioned above, may be involved
Semillon, Shiraz, Greanche, Carignan, and Trajadura in yeast metabolism. The ideal way to determine the
(10, 109, 148152), the increase in monoterpenes is effect of enzymatic release from glycoconjugate precur-
below the aroma threshold of these compounds.
However, there is no information available about the
effect of this increase on the avor nuances that are
sometimes detected in treated wines.
Among the monoterpenes, an increase was observed
for geraniol, nerol, citronellol, linalool, furan, and
pyran forms of linalool oxides, -terpineol, and mono-
terpene polyols (e.g., 8-hydroxylinalools, p-menthene-
7,8-diol, exo-2-hydroxy-8-cineol). The highest increase
often occurred for geraniol and nerol, which impart
oral attributes to the wines (10, 146, 147, 151, 152).
The concentration of linalool, and linalool oxides,
and some polyols often increased to a lesser extent
because of the low activity of fungal -glucosidase
toward the relevant glycoconjugates. However,
mono- and diglycosides of these tertiary alcohols are
more prone to acidic hydrolysis in fruit juice than those
of primary alcohols (35).
Citronellol concentration increased greatly in some
enzymatically treated wines (Muscat). This could not
be the consequence of the enzymatic hydrolysis of
related glycosidic precursors since the glycosidic con-
jugates of this terpenol have not been detected in
grapes (153). The increase was explained by the action
of yeast (S. cerevisiae) reductase on geraniol and nerol,
which were more abundant in the enzymatically trea-
ted wines (154156). The production of citronellol was
inuenced by the yeast strain (155). The high amounts
of this compound resulted in the perception of citrus Figure 6 Acid-catalyzed rearrangement of monoterpenes.
attributes in the wines. (From Ref. 157.)

sors is by direct quantitative analysis of monoterpenyl alcohol glucosides which are generated from diglyco-
glycosides. However, this analysis requires reference sides by the action of exoglycosidases.
glycosides which are not yet commercially available, Fermentation trials with various Muscat grape
such as certain monoterpenes. Furthermore, their juices, in the presence of different levels of glycosidase
synthesis, mainly for diglycosidic conjugates, is quite activities, showed that several hundred nanokatals of
complex and leads generally to low yields (35, 101, -glucosidase, -arabinofuranosidase, -apiosidase,
102). and -rhamnosidase activities per liter of grape juice
In our laboratory several monoterpenyl glycosides are needed to efciently hydrolyze monoterpenyl
were synthesized (35, 101), which allowed us to quan- glycosides (10, 109). These activities are much greater
tify them in control and enzymatically treated wines than those found in grape berries (55, 56) or
(10, 109, 147). An enzyme preparation, rich in glycosi- produced by S. cerevisiae (80) during alcoholic
dase activities, was added to grape juices from different fermentation.
cultivars before alcoholic fermentation. The glycosides Several C13 -norisoprenoids that were barely detect-
hydrolysis was measured 1 month after the comple- able in control wines were nonetheless detected in the
tion of fermentation. the concentration of monoterpe- treated wines (Fig. 5) (10, 109, 147). This is not surpris-
nyl arabinofuranosyl and rhamnopyranosyl glucosides ing as they occur mainly in glycoconjugated forms in
decreased notably in treated wines, while apiosylgluco- grapes. These compounds are not often analyzed in
sides decline less sharply (Fig. 7). This was due to the glycosidase-treated wines, mainly because the refer-
low levels of -apiosidase activity compared to - ences compounds are not commercially available.
rhamnosidase and -arabinofuranosidase activities in They are not specic to any grape variety, as was
the enzyme preparation. With regard to monogluco- found for monoterpenes in Muscat cultivars, although
sides, primary alcohol (geraniol and nerol) -D-gluco- some cultivars, such as Chardonnay (158, 159) and
sides were almost totally hydrolyzed by exogenous Shiraz (10, 109), do seem to contain larger amounts
enzymes (Fig. 8) (10, 109). In contrast, the level of of C13 -compounds.
tertiary alcohol (linalool) glucoside increased signi- In the enzymatically treated wines, the most abun-
cantly in wines containing high levels of relevant dis- dant C13 -norisoprenoids were often 3-hydroxy--
accharidic conjugates. This accumulation is the result damascone, 3-oxo--ionol, and vomifoliol (10, 109,
of the low activity of fungal -glucosidase on tertiary 147), as was also observed in the enzymatic hydrolysis
Figure 7 Monoterpenyl diglycoside concentrations in control and glycosidase-treated wines.

Figure 8 Monoterpenyl glucoside concentrations in control and glycosidase-treated wines.
of grape glycosidic extracts. These molecules are odor- cally treated white wine from a nonoral grape
less and are considered not to generate odor-active variety (150) contained high levels of vanillin, which
compounds by further reactions (42, 158). In contrast, is a potent avorant usually derived from wood used in
3-hydroxy-7,8-dehydro--ionol, an acetylenic alcohol, wine aging.
detected in treated wines (10, 109) can generate a
highly esteemed, avorant, -damascenone, along
with a major product, 3-hydroxy--damascone, at
wine pH during storage through acid catalyzed reac-
tions (Fig. 9) (42, 70, 158). A high concentration of -
damascenone, exceeding several fold its odor thresh-
old, has been detected in an enzymatically treated wine
1 month after the end of fermentation (147). This can
be explained by the fact that glycosidase treatment
may have increased the concentration of an allenic
triol which is known to rearrange much faster than
aforementioned acetylenic alcohol to generate -
damascenone (158, 160).
-Damascenone, like other norisoprenoid avor-
ants, is generally produced in tiny amounts (in the
range of ppt) (42, 158, 160) that are difcult to quan-
tify by conventional GC/MS techniques. Nowadays,
stable isotope dilution techniques are being used
more and more because of their sensitivity and accu-
racy in quantitative analysis of trace avorants.
Among the shikimate-derived volatiles, benzyl alco-
hol, 2-phenyethanol, vanillin, eugenol, and zingerone Figure 9 Products issued from 3-hydroxy-7,8-dehydro--
were often detected at higher concentrations in enzy- ionol by acid-catalyzed reactions in fruit juice and wine.
matically treated wines (10, 109, 150). An enzymati- (From Ref. 158.)

The increase in avor compounds, using the same chemistry of aglycones (6, 36), very few data are avail-
wine and at the same dosage of enzyme preparation, is able on their effect on the formation of odor-active
strongly inuenced by the enzyme preparations used molecules in normal wine storage conditions.
(usually from 1 to 3 g/HL) (10, 151, 152). This can The role of aglycones, derived from grape glycosidic
be explained by the different glycosidase activities of precursors, in the generation of important wine aroma
enzyme preparations, as mentioned previously. descriptors has been shown in mimetic conditions
Furthermore, the vintage had an inuence on the (165). Indeed, some important key aroma descriptors
aroma enrichment of wine, which could be explained (honey, tea, lime) of white wines were detected when a
by differences in the biosynthesis of glycosidic precur- glycosidic extract from nonoral varieties
sors. It has also been found that enzyme preparations (Chardonnay, Semillon, Sauvignon) was subjected to
were more effective when added into wine instead of an acid hydrolysis (pH 3.2) at elevated temperature
grape juice (10, 109, 151). (508C, 4 weeks, under nitrogen) in order to accelerate
In addition to commercial pectinase preparations, the acid-catalyzed conversions that slowly occur dur-
-glucosidase preparations have been used to enhance ing wine conservation. The contribution of glycosidic
the varietal avor of Muscat wines, including an precursors to the characteristic aroma of an important
immobilized endo--glucosidase from A. niger (161), red wine cultivar (Grenache) has been recently
an immobilized -glucosidase from C. molischiana observed in our laboratory under similar conditions.
(162, 163), and a -glucosidase from D. hasenii (164). The volatiles responsible for the key aroma descrip-
Monoterpenes (geraniol, nerol, linalol, linalool oxi- tors have not been identied yet, most probably due to
des), benzyl alcohol, and 2-phenylethanol concentra- the presence of trace quantities of important avor-
tions all increased in the treated wines. Since the ants.
authors only analyzed volatiles after enzymatic treat- The rapid liberation of aglycones from glycoconju-
ment, it is difcult to judge the extent of glycoside gates, through the use of exogenous glycosidases, can
hydrolysis. Furthermore, the hydrolysis of disacchari- thereby accelerate the formation of odor-active vola-
dic glycosides was probably quite limited owing to the tiles during wine storage. In fact the glycosidic avor
use of an unique activity (-glucosidase). precursors do undergo slow hydrolysis at wine pH dur-
Finally, an important aspect in the use of glycosi- ing storage (35, 109, 145). For example, several Muscat
dases is the prevention of any contamination of the wines prepared without glycosidase addition and ana-
grapes or fruits by molds. As previously mentioned, lyzed after 18 months contained from 1.2 to 1.9 times
such contamination may lead to the inhibition of - more monoterpenes in glycosylated form than in the
glucosidase activity by gluconolactone. This was free fraction (145). Bound forms of primary alcohols
observed when glycosidases were added to a wine (geraniol and nerol) were 610 times more abundant
made from mold infected grapes (personal communi- than their free counterparts.
cation). The diglycosides were effectively hydrolyzed
by exoglycosidases in the treated wine, while the result- B. Fruit Juices
ing monoglucosides were not hydrolyzed owing to glu-
conolactone, of which the presence in the wine was Owing to the inhibition by glucose of the key
conrmed by analysis. enzyme -glucosidase in the available fungal derived
Sensory difference tests often distinguished between enzyme preparations, very little work has been devoted
the control and enzyme treated wines (10, 109, 147, to avor enhancement in fruit juices.
152, 161, 162). The difference was clearly observed in Treating mango and passion fruit juices with a yeast
wines whose avor is monoterpene dependent (like -glucosidase from Candida cacaoi resulted in an
Muscat and Gewurztraminer) even after 2 or 3 weeks increase in monoterpene concentrations (166).
of enzyme application, due to the enhanced oral attri- However, the increase was much lower than that due
butes. For nonoral varieties, which constitute the to acid hydrolysis. The immobilized enzyme (cova-
greatest volume of world wine production, the effect lently bonded to sodium alginate beads) liberated
of glycosidases can be perceived usually after several fewer terpenols than the free enzyme. The addition of
months or more. Most of the liberated aglycones, espe- -glucosidase from C. molischiana (immobilized onto
cially those corresponding to the C13 -norisoprenoids Duolite A-568 resin) to apricot, mango, strawberry,
and monoterpene polyols, are avorless but can gen- peach, and apple juices yielded an increase in concen-
erate odorous molecules during wine conservation. In tration of monoterpene alcohols (linalool, -terpineol,
contrast to the advances in our understanding of the geraniol), benzyl alcohol, and 2-phenylethanoal (162).

No differences were observed in the avor released raspberry (167), while in grapes they are esteried
using free and immobilized -glucosidase from C. with tartaric acid (167169).
molischiana. The immobilized enzyme was found to A large number of pectinase and hemicellulase pre-
be quite stable under fruit juice conditions. parations derived from A. niger used in fruit juice pro-
An A. niger endo--glucosidase, immobilized onto cessing exhibit an cinnamate esterase as side activity
acrylic beads or cellulose, increased the concentration which leads to the enrichment of the medium in free
of volatiles (linalool, benzyl alcohol and benzaldehyde) caffeic, p-coumaric, and ferulic acids (Fig. 10) (170
in passion fruit juice (161). In general, an aroma 173). Free p-coumaric and ferulic acids can be con-
enhancement in the treated juice was observed (161, verted to volatile high-avorant p-vinylphenols, 4-
162). vinylphenol, and 4-vinylguaiacol, respectively, by
Although the volatile levels in fruit juices increased either nonenzymatic thermal (174) or enzymatic
after the applications of -glucosidase, two important (yeast and bacteria) decarboxylations (175179) (Fig.
parameters may have limited the efciency of the pro- 10). These vinylphenols possess low detection thresh-
cess: (a) many avor compounds occur in fruits as olds (< 100 ppb in water) (174, 179) and spicy and
diglycosides which fungal -glucosidases are, in gen- medicinal odors, and they contribute positively only
eral, unable to hydrolyze; (b) the fungal -glucosidases at low levels to the avor of food products (180
tested are inhibited by glucose, which could therefore 182). Accordingly, the increase in the concentration
limit their action in fruit juices. of hydroxycinnamic acids precursors of these volatiles
Because the authors only analyzed the volatile con- due to enzyme treatments can have an important con-
tent of fruit juices and not the glycosides, the yield of sequence to the avor of the products.
hydrolysis cannot be evaluated. However, a paper 4-Vinylguaiacol (or p-vinylguaiacol) generated from
reports how the amount of glycosides changes in a ferulic acid through nonenzymatic decarboxylation
sweet wine (containing 30 g/L glucose) when fungal during orange and grapefruit juices processing and sto-
glycosidases were used in the wine-making process rage contributes to the development of the major detri-
(147). The level of monoterpene diglycosides decreased mental off-avor (old fruit, rotten avor) in these
notably in the treated wine, which may be explained by products (174). It has been shown that the rate of 4-
the action of exoglycosidases. The monoterpenyl--D- vinylguaiacol formation during orange juice storage
glucosides produced were not hydrolyzed and there- was closely dependent on the amount of free ferulic
fore accumulated in the wine. This clearly indicates acid (183).
the inhibition of fungal -glucosidase activity by glu- A phenolic off-avor is perceived in fermented pro-
cose, which greatly limited the liberation of volatiles as ducts (beer, cider, wine) when aforementioned volatile
conrmed by their analysis. In contrast, the hydrolysis phenols are present at high levels (171173, 181, 184).
of monoglucosides was very efcient when a dry wine The wines made from grape juice, that was initially
was prepared using the same grape juice, enzyme spiked with some pectinase preparations rich in glyco-
dosage, and conditions (147). sidase activities contained exceedingly high levels
(> 770 ppb) of vinylphenols, masking the pleasant fru-
ity and oral odors which were generated by hydrolysis
VIII. POTENTIAL ADVERSE EFFECTS OF of glycosidic avor precursors (109, 171, 172). It was
ENZYME PREPARATIONS demonstrated recently that the high concentration of
volatile phenols detected sometimes in wines was due
Certain side activities of fungal enzyme preparations to the consecutive action of cinnamate esterase activity
can be detrimental to fruit juice and wine quality from enzyme preparation and decarboxylase activity
owing to the formation of off-avors and the decolor- from yeast (S. derevisiae) (171173) present in the
ization. majority of enological strains (175). Consequently,
particular attention is now being directed to develop
A. Off-avor Formation an enzyme preparation without cinnamate esterase
activity and to select yeast strains with low activity
Hydroxycinnamic acids, such as p-coumaric and feru- toward hydroxycinnamic acids (185). Since the 1970s,
lic acids, occur mainly in bound forms in fruits. Quinic pectic enzyme preparations have been widely used in
acid and/or glucosidic esters of these hydroxycinnamic wine making mainly to improve clarication and juice
acids have been detected in numerous fruits: apple, yield, and the problem of off-avors has been some-
pear, apricot, peach, orange, grapefruit, strawberry, times observed without explanation (186).

Figure 10 Some volatile phenols formation pathways during processing and storage. (Courtesy of C. Flanzy, Oenologie:
fondements scientiques et techniques, Tec-Doc, Paris, 1998.)

The formation of vinylphenols is found to be lower (197, 198), and -galactosidase (197, 199) from com-
in red than white wines, and is explained by the inhibi- mercial enzyme preparations may potentially degrade
tion of yeast decarboxylase activity by red wine poly- anthocyanins. The decolorization effect of these
phenols (187). enzymes has been reported for raspberry (200), straw-
The high levels of vinylphenol and vinylguaiacol in berry (201), blackberry (202), and grape anthocyanins
wines dropped notably after 1 year of storage, while (197, 203, 204). Certain molds, particularly B. cinerea,
there was a simultaneous decrease of unpleasant odor which develop on fruits are also able to degrade antho-
(188). In the acidic pH of wine, these compounds cyanins as has been observed in strawberry juice (205),
were partly transformed into their corresponding raspberry juice (206), and red wines (207, 208).
ethoxyethylphenols by addition of ethanol (Fig. 10). Only one in 26 enzyme preparations, used in fruit
Ethoxyethylphenols, at the levels found in wines, prob- juice processing within the mean recommended dosage,
ably have no direct inuence on avor owing to their degraded the anthocyanin--D-glucoside (cyanidin-3-
odor threshold (188). glucoside) and di- and trisaccharides of cyanidin
Ethylphenols, 4-ethylphenol (woody, phenolic (209). Six enzyme preparations acted on disaccharidic
odor), and 4-ethylguaiacol (clovelike, smoky odor), conjugates only at high doses.
which impart undesirable avors to wine, beer, and Two of four pectic enzyme preparations employed
cider are produced from 4-vinylphenol and 4-vinyl- in wine making caused a signicant color loss in red
guaiacol, respectively, through reductase activity wine making (204). The major anthocyanin (malvidin-
from bacteria (176, 181) and more frequently from 3-glucoside) and its acylated forms (malvidin-3-gluco-
spoilage yeast, Brettanomyces (184, 189). The produc- sylacetate and malvidin-3-glucosylcoumarate) both
tion of ethylphenols from hydroxycinnamic acids is decreased signicantly. The degradation of acylated
possible by another pathway by Lactobacillus: rstly anthocyanins requires the consecutive action of two
the double bonds of hydroxycinnamic acids were enzymesan esterase and -glucosidase (170).
reduced, and subsequently the resultant phenyl propio- Accordingly, the choice of fungal enzyme prepara-
nic acids were decarboxylated (177, 178). tions for avor enhancement in fruit juices and derived
In addition to this off-avor formation, fungal beverages should be made carefully. Glycosidases that
enzyme preparations cause the oxidation of C13 -nori- are highly active on anthocyanins may have specic
soprenoids belonging to the structure of 3-hydroxyme- applications for the decolorization of some orange
gastigmanes when they are used in assays for the juices or wines containing low levels of anthocyanin
determination of the aglycone moiety of glycosides pigments.
(190). High enzyme concentrations are employed to An anthocyanin--glycosidase from A. niger has
ensure the complete hydrolysis of glycosides. This oxi- been partially puried and characterized (201). Two
dation, by altering the structure of polyol aglycones, -glucosidases that were isolated and characterized
prevents the generation of desired aroma compounds, from an A. niger pectinase enzyme preparation differ
such as -damascenone (6). However, at the dosage mainly in their catalytic activity (103). The major one
used in wine making for avor release, the fungal- was highly active on cellobiose and geranyl-glucoside
derived enzyme preparations do not provoke such oxi- but only slowly degraded malvidin-3-glucoside. In con-
dations (10, 109). trast, the minor one was highly active in degrading mal-
vidin-3-glucoside. Both enzymes were strongly
B. Decolorization Effect inhibited by glucose. These two -glucosidases were
also observed in another pectinase enzyme preparation.
Anthocyanins are among the most widespread natural In rare cases, an oxidation of white wine color was
pigments and responsible for the red, blue, and purple observed in the enzyme-treated sample. This might be
color of fruits (191, 192). They are composed of antho- due to polyphenoloxidase activity from enzyme pre-
cyanidins glycosylated with mono- (usually glucose), parations or to the chemical or enzymatic oxidation of
di- or trisaccharides (193). Anthocyanidins become avonoids released through the action of glycosidases.
unstable and are readily converted to brown or color- Finally, as positive effect of glycosidases as biocata-
less compounds when released from their sugar moiety lysts, is their role in the detoxication of certain foods
(194, 195). through the hydolysis of cyanogenetic glycosides (210)
The hydrolytic activity of fungal enzyme prepara- and the debittering of citrus juices by hydrolysis of
tions toward anthocyanins was reported as early as narigin (211). These topics are not of the subject of
1955 (194). -Glucosidase (196, 197), -arabinosidase this chapter.

IX. CONCLUSION AND FUTURE tions of some grape aroma components. J
PROSPECTS Chromatogr 331:8390, 1985.
3. G Krammer, P Winterhalter, M Schwab, P Schreier.
Major progress has been made during the past two Glycosidically bound aroma compounds in the fruits
of Prunus species: apricot (P. armeniaca, L.), peach (P.
decades in our understanding of the chemical composi-
persica, L.), yellow plum (P. domestica, L. ssp.
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Syriaca). J Agric Food Chem 39:778781, 1991.
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strated. S Dong, Q Tong, K Sakata. Purication of a -prime-
Several approaches, through genetic engineering verosidase concerned with alcoholic aroma formation
techniques, will doubtless be used in the near future in tea leaves (Cv. Shuixian) to be processed to oolong
to overcome, by using recently discovered glycosidases, tea. J Agric Food Chem 45:877882, 1997.
the limitations (glucose inhibition, substrate specicity, 6. P Winterhalter, GK Skouroumounis. Glycoconjugated
aroma compounds: occurrence, role and biotechnolo-
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inhibition constant for glucose, together with highly Phytochemistry 8:13391347, 1969.
glucose-tolerant -glucosidases from Aspergillus sp. 8. R Cordonnier, C Bayonove. Mise en evidence dans la
and yeasts (Candida and Debaryomyces spp.), repre- baie de raisin, variete Muscat dAlexandrie, de mono-
sent important biotechnological challenges in avor terpe`nes lies, revelables par une ou plusieurs enzymes
release processes. du fruit. C R Acad Sci Paris 278:33873390, 1974.
The construction of fungal strains such as S. cere- 9. Y Vasserot, A Arnaud, P Galzy. Monoterpenol glyco-
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glycosidases without undesirable activities (such as vour potential from grape glycosides in winemaking.
esterase, anthocyanase, and oxydase). This would In: P Schreier, P Winterhalter, eds. Progress in
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Sensible Principles and Techniques. Washington:
ACKNOWLEDGMENTS American Chemical Society, 1983, pp 287308.
12. PJ Williams, CR Strauss, B Wilson, RA Massy-
The author thanks his colleagues identied in the lit- Westropp. Use of C18 reversed-phase liquid chroma-
erature, and Professor Christian Ambid for his helpful tography for the isolation of monoterpene glycosides
discussion and advice. and nor-isoprenoid precursors from grape juice and
wines. J Chromatogr 235:471480, 1982.
13. P Winterhalter. Application of countercurrent
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22
Entrapment of Biocatalysts by Prepolymer Methods
Takuo Kawamoto and Atsuo Tanaka

Kyoto University, Kyoto, Japan
I. INTRODUCTION There are various immobilization methods at pres-

ent. In general, the immobilization methods can be
Immobilization of biocatalysts which include enzymes, classied as follows:
cellular organelles, microbial cells, plant cells, and ani- (a) carrier binding via covalent linkage, ionic bonds,
mal cells is attracting worldwide attention. physical adsorption, or biospecic binding; (b) cross-
Immobilized biocatalysts are, in general, easier to han- linking by using bi- or multifunctional reagents; (c)
dle and more stable than native counterparts, and they entrapment in gel matrices, microcapsules, liposomes
can be used either repeatedly in a long-term series of or hollow bers; and (d) combination of the above-
batchwise reaction or continuously in ow systems, mentioned methods (1, 2).
which are useful and important features for practical Of these immobilization methods developed
applications of biocatalysts. At present, immobilized hitherto, entrapment methods are the most promising
biocatalysts have contributed to a wide variety of elds because this method is applicable to the immobiliza-
including: tion of not only single enzymes but also multiple
enzymes, cellular organelles, microbial cells, plant
1. Production of useful compounds by stereospe-
cells, and animal cells.
cic and/or regiospecic bioconversions.
The disadvantages of this method are: (a) high-
2. Production of energy and reducing equivalent
molecular-weight substrates may not be able to access
by biological processes.
the entrapped biocatalysts, and (b) renewal of the car-
3. Selective degradation or removal of pollutants.
rier is difcult.
4. Analyses of various compounds with high sen-
Among the entrapment methods, entrapment in gel
sitivity and specicity.
matrices is the most widely used. Various types of gel
5. Application to foods, such as hydrolysis of lac-
materials, such as polysaccharides, proteins, and syn-
tose of milk, alcohol fermentation, production
thetic polymers, are now used to entrap biocatalysts.
of glucose from starch, and production of high-
Because applications of immobilized biocatalysts are
fructose corn syrup.
aimed toward a wide variety of bioreactions, including
6. Utilization in medical elds, such as new types
synthesis, transformation, degradation, or assay of
of drugs and articial organs.
various compounds having different chemical proper-
These processes require immobilization not only of ties (3), it is desirable to entrap biocatalysts in gels of
single enzymes that catalyze simple reactions but also adequate physicochemical properties to satisfy the dif-
of multienzyme systems that mediate more compli- ferent demands in such a variety of applications. It is,
cated reactions. however, difcult to select suitable gels for each pur-

pose among those prepared from natural polymers. ties of the several prepolymers are summarized in
Furthermore, it is not easy to modify natural polymers Table 1.
for the preparation of proper gel materials. As for Polyethylene glycol dimethacrylate (PEGM) was
synthetic materials, acrylamide and its analogs have synthesized with polyethylene glycol and methacry-
been used widely as starting materials for gels. These late. ENT and ENTP were prepared with hydro-
monomers are, however, liable to inactivate biocata- xyethylacrylate, isophoronediisocyanate, and
lysts in the polymerization and immobilization pro- polyethylene glycol or polypropylene glycol, respec-
cesses. tively. Each prepolymer has a linear skeleton of
For these reasons, continuous efforts have been optional length, at both the ends of which are
devoted to development of prepolymer methods attached the photosensitive functional groups, acry-
which are new entrapment methods using synthetic loyl, methacryloyl, etc. PEGM (46) and ENT (7, 8)
prepolymers as starting materials for gels. containing polyethylene glycol as the main skeleton
are water-soluble and form hydrophilic gels, while
ENTP (9) with polypropylene glycol as the main skel-
eton is water-insoluble and gives hydrophobic gels.
II. PREPOLYMER METHODS
By using polyethylene glycol or polypropylene glycol
of different molecular weights, prepolymers of differ-
In the case of prepolymer methods, synthetic resin pre-
ent chain length, PEGM-1000 to PEGM-4000, ENT-
polymers are used as gel-forming starting materials for
1000 to ENT-6000, and ENTP-1000 to ENTP-4000,
entrapment of biocatalysts. Specic features and
can be prepared. Chain length of the prepolymers
advantages of prepolymer methods can be summarized
correlates with the size of network of the gels formed
as follows:
from these prepolymers. Prepolymers having ionic
1. Entrapment procedures are very simple and pro-
properties can also be prepared by introducing ionic
ceed under mild conditions.
functional group(s) to the main skeleton of prepoly-
2. Prepolymers do not contain monomers which
mers. An emulsion-type photocrosslinkable resin pre-
may have a negative inuence on the biocatalysts to
polymer, ENTE-1, was prepared in the following
be entrapped.
way. Hydrophobic polyvinyl acetate was coated with
3. The network structure and strength of gels can be
hydrophilic polyvinyl alcohol to be easily dispersed in
regulated by the chain length and the content of the
an aqueous solution. Photosensitivity was introduced
reactive functional groups of the prepolymers.
by the reaction with N-hydroxymethyl acrylamide in
4. The physicochemical properties of gels, such as
the presence of an acid catalyst. After neutralization,
hydrophilicityhydrophobicity balance and ionic na-
an emulsion-type photocrosslinkable resin prepolymer
ture, can be controlled by selecting suitable prepoly-
mers which are synthesized chemically in advance in
the absence of biocatalysts.
Several examples of the prepolymer methods are
described below. Table 1 Properties of Several Photocrosslinkable Resin
Prepolymers
A. Photocrosslinkable Resin Prepolymer Method Prepolymer MW of main chain Property
1. Principle PEGM-1000 1000 Hydrophilic
In the case of photocrosslinkable resin prepolymers, PEGM-2000 2000
the mixture of the prepolymer and the biocatalyst is PEGM-4000 4000
ENT-1000 1000 Hydrophilic
gelled by radical polymerization of the prepolymer
ENT-2000 2000
initiated by illumination with near-ultraviolet light
ENT-4000 4000
for several min in the presence of a proper photosensi- ENTP-1000 1000 Hydrophobic
tizer (initiator) such as benzoin ethyl ether or benzoin ENTP-2000 2000
isobutyl ether. ENTP-3000 3000
ENTP-4000 4000
2. Prepolymers ENTE-1 Emulsion-type
ENTA-1 2200 Anionic
The structures of some typical photocrosslinkable ENTC-1 940 Cationic
resin prepolymers are shown in Figure 1. The proper-

Figure 1 Structures of typical photocrosslinkable resin prepolymers.
was obtained. ENTE-1 is useful when swelling of the maximum intensity at 360 nm) for several minutes.
gels is to be avoided (10). Photocrosslinkable resin The gel lm formed is cut into small pieces and
prepolymers can be autoclaved at 1208C and stored used as the entrapped biocatalyst.
in the dark. Bead-type biocatalysts entrapped with photo-
Among these photocrosslinkable resin prepolymers, crosslinkable resin prepolymers can also be obtained
ENT and ENTP prepolymers are commercially avail- (11). Ten parts (by weight) of prepolymer are mixed
able from Kansai Paint Co., Tokyo, Japan. with 0.08 parts of the initiator, 2 parts of 3% sodium
alginate, 2 parts of distilled water, and 2 parts of the
biocatalyst. The mixture obtained is dropped into
3. Procedures 1.5% aqueous calcium chloride solution to form the
Typical procedures for entrapment with photocross- beads. The beads thus formed are irradiated by near-
linkable resin prepolymers are as follows: 1 g of ultraviolet light. After irradiating for 5 min, the
ENT-4000 or mixture of ENT-4000 and ENTP-4000 entrapped biocatalyst beads obtained are washed
(ENT-4000 content is above 70%) is mixed with with sterile water. For practical applications, the
10 mg of a photosensitizer such as benzoin ethyl bead-type is often preferable to the cube-type or lm-
ether. The mixture is melted by warming at 608C. type.
An adequate buffer, such as 50 mM potassium phos- Tube-type biocatalysts can be easily prepared by
phate buffer (0.5 mL) is added to the molten mixture using ENTE-1. The inner surface of glass tubes
and mixed at 608C. The molten mixture is cooled to (ID 2 mm) is silanized with vinyltriethoxysilane (10).
48C, and 2 mL of solution or suspension of biocata- ENTE-1 is mixed with photosensitizer and the
lysts is added to the mixture. The obtained mixture is solution or suspension of biocatalyst. This viscous
layered on a sheet of transparent polyester lm mixture is injected at the top of each silanized glass
framed (70 cm2) with a plastic tape (thickness tube to form a thin layer of biocatalyst-containing
0.5 mm). The layer is covered with a transparent prepolymer on the inner surface of the tube. The
thin lm to eliminate air and then illuminated with tube is illuminated with near ultraviolet light for
near-ultraviolet light (wavelength range 300400 nm; several minutes under an atmosphere of nitrogen to

complete the photocrosslinking of the prepolymer. Urethane resin prepolymers can be sterilized at
The resulting tube-type biocatalysts can be used in 1208C under anhydrous conditions and stored in a
continuous-ow systems to assay various com- desiccator.
pounds. Although these urethane resin prepolymers are not
currently available commercially, Hypol having a simi-
B. Urethane Resin Prepolymer Method lar structure can be obtained from W. R. Grace Co.,
Lexington, MA.
1. Principle
3. Procedures
The urethane resin prepolymer method is very simple
and convenient, because isocyanate functional groups Typical entrapment procedures with urethane resin
at both terminal ends of the urethane prepolymer (Fig. prepolymers are as follows. The urethane prepolymer
2) react with each other only in the presence of water, (1 g) melted by incubation at 508C (if necessary) is
forming a urea linkage and liberating carbon dioxide cooled to 48C and mixed quickly and well with 2 mL
(12). Consequently, when the prepolymers are mixed of aqueous solution or suspension of biocatalyst in a
with an aqueous solution or an aqueous suspension of small beaker. When gelation starts after a few minutes
biocatalyst, gels are easily formed within a few minutes of mixing, the mixture is kept at 48C for 3060 min to
and gelation is complete in 3060 min. complete the gelation. The gel thus formed is cut into
small pieces, washed thoroughly with the buffer, and
2. Prepolymers used as the entrapped biocatalyst.
If the mixture, quickly and well stirred, is immedi-
Structures of typical urethane resin prepolymers are ately layered on a glass plate framed with adhesive tape
shown in Figure 2, and the properties of the pre- with the proper dimensions before the gelation starts, a
polymers are summarized in Table 2. Urethane resin lm of entrapped biocatalyst can be obtained (15).
prepolymers (PU) are synthesized from toluene
diisocyanate and polyether diols composed of poly-
ethylene glycol and polypropylene glycol or polyethyl- C. Miscellaneous
ene glycol alone. Prepolymers with different
hydrophilic or hydrophobic properties can be Owing to the advantages of prepolymer methods men-
obtained by changing the ratio of the polyethylene tioned above, continuous efforts have been devoted to
glycol and polypropylene glycol in the polyether develop new prepolymer methods.
diol moiety of the prepolymers. For example, PU-2 Photosensitive resin prepolymers (PVA-SbQ) were
to PU-4 with a high content of polypropylene glycol also developed by Ichimura (16). Photosensitive resin
give relatively hydrophobic gels, while PU-1 and prepolymers are derivatives of polyvinyl alcohol having
PU-5 to PU-11 with a high content of polyethylene styrylpyrydinium groups as photosensitive functional
glycol give hydrophilic gels, although all the prepoly- sites and polymerized by photodimerization with irra-
mers are water miscible (13, 14). The chain length diation of visible or ultraviolet light. Hydrophilicity of
and the content of isocyanate group of the prepoly- the prepolymers can be controlled by changing the
mers, which correlate to the size of network of the saponication degree of polyvinyl acetate.
gels formed from these prepolymers, can also be Several prepolymers, such as polyvinyl alcohol (17),
changed. polyethylene glycol diacrylate (18), and polyethylene
Figure 2 General formula of urethane resin prepolymers.

Table 2 Properties of Urethane Resin Prepolymers
Polyethylene glycol
MW of NCO content in content in Property of gel
Prepolymer polyether diol prepolymer (%) polyether diol (%) formed
PU-1 1486 4.0 100 Hydrophilic

PU-2 2529 3.1 57a Hydrophobic
PU-3 2529 4.2 57a
PU-4 2529 5.6 57a
PU-5 2627 2.7 91a Hydrophilic
PU-6 2627 4.0 91a
PU-7 2627 5.6 91a
PU-8 2616 2.7 100 Hydrophilic
PU-9 2616 4.0 100
PU-10 2616 5.6 100
PU-11 4285 4.0 73a Hydrophilic
a
The remaining percentage (100X) is polypropylene glycol.
glycol dimethacrylate (18, 19), are also useful gel mate- developed for the entrapment of different kinds of bio-
rials for entrapment by radiation. In the case of 10% catalysts.
polyvinyl alcohol, 57 Mrad of radiation is sufcient
to obtain rigid gels (17). The glass-forming prepoly- A. Enzymes
mers, such as polyethylene glycol diacrylate and poly-
ethylene glycol dimethacrylate, are used for Examples of enzymes entrapped with prepolymers are
entrapment through radiation polymerization at low listed in Table 3. Some enzymes listed in Table 3 have
temperature. Entrapment can be carried out at -248C been employed for development or appraisal of new
with 10% prepolymer and a radiation of 1 Mrad (19). prepolymer methods. Not many reports are available
A mixture of polyvinyl alcohol and polyethylene glycol concerning the applications of enzymes entrapped by
dimethacrylate is also applicable (18). This radiation prepolymer methods.
method has several advantages such as possible appli- Lipases entrapped with hydrophobic photocross-
cation of a wide variety of prepolymers as gel materials linkable resin prepolymers (ENTP-2000 and ENTP-
and entrapment at low temperature. However, utiliza- 4000) have been applied to the hydrolysis and ester
tion of this method is limited because radiation appa- exchange reaction of triacylglycerides. Production of
ratus is required. cacao butter-like fat from olive oil and stearic acid or
Water-soluble linear polyacrylamide (MW 15104 palmitic acid by enzymatic ester exchange reaction has
to 17 104 partially substituted with the acyl- been successfully achieved with ENTP-entrapped
hydrazide group was synthesized (20). This pre- Rhizopus delemar lipase in an organic solvent system
polymer can be gelled and used for entrapment of (21, 22). Entrapment markedly enhanced the opera-
biocatalysts in the presence of a crosslinking agent tional stability of the enzyme. In the case of the hydro-
such as glyoxal, glutaraldehyde, or periodate-oxidized lysis of olive oil to glycerol and fatty acids, Candida
polyvinyl alcohol. cylindracea lipase entrapped with ENTP showed high
activity (23). Entrapment also made it possible to use
the enzyme repeatedly or continuously. PU-entrapped
Candida cylindracea lipase was used for optical resolu-
III. BIOCATALYSTS ENTRAPPED BY tion of dl-menthol through enantioselective esterica-
PREPOLYMER METHODS tion in an organic solvent (24).
Enzymes entrapped with photocrosslinkable resin
As described above, various kinds of prepolymers hav- prepolymers were applied to the analyses of various
ing different physicochemical properties have been compounds. Tubes entrapping glutamate decarboxyl-

Table 3 Examples of Enzymes Entrapped by Prepolymer Methods
Enzyme Polymerization method (prepolymer) Ref.
Amino acylase Photocrosslinking (ENT) 13

Invertase Photocrosslinking (PEGM, etc.) 4, 5, 37
Urea linkage (PU) 12
Lipase Photocrosslinking (ENTP) 21, 22, 38
Urea linkage (PU) 23, 38
Glucose isomerase Photocrosslinking (ENT) 13
Catalase Photocrosslinking (ENT, etc.) 8, 37
Hydrogenase Photocrosslinking (ENT) 39
Choline oxidase Photocrosslinking (ENT and ENTA) 13
Glucose oxidase Photocrosslinking (ENT) 13
Uricase Photocrosslinking (ENT) 13
Lactate oxidase Photocrosslinking (ENT) 13
Pyruvate oxidase Photocrosslinking (ENT) 13
Glycerophosphate oxidase Photocrosslinking (ENT) 13
Glutamate decarboxylase Photocrosslinking (ENTE-1) 10
Lysine decarboxylase Photocrosslinking (ENTE-1) 25
ase (10) and lysine decarboxylase (25) were prepared 2. The cell wall and/or cell membrane of intact cells
with the emulsion-type photocrosslinkable resin prepo- often prevents substrates, products, and other reaction
lymer ENTE-1. These tubes were used in continuous- components from permeating into and out of cells. In
ow systems to measure the concentration of L-gluta- this case, appropriate treatment of cells before or after
mate and L-lysine, respectively, in fermentation broths. immobilization is required to eliminate the barriers to
Thin enzyme lms, in which the enzymes were permeability.
entrapped with photocrosslinkable resin prepolymers, The immobilized cells can be kept in a growing state
were mounted on an oxygen electrode and used as by appropriate supply of suitable nutrients. This type
enzyme electrodes (13). Several oxygen-consuming oxi- of immobilized cells (immobilized growing cells) is very
dases, such as choline oxidase and glucose oxidase, advantageous. Immobilized growing cells serve as
were employed for this purpose. These enzyme electro- self-proliferating and self-regenerating biocatalysts.
des were found to be stable. However, immobilized growing cells also have some
disadvantages as follows:
1. Various nutrients and energy sources are required
B. Cells to maintain the cells in living or growing state(s), and
yield of wanted products may be lowered by the con-
Applications of immobilized cells are very attractive sumption of substrates and/or products as nutrients or
and important because cells possess multienzyme sys- energy sources.
tems which mediate complex reactions (3). In the case 2. Products are liable to be contaminated by leakage
of immobilization of cells, it is not necessary to extract of cells from carriers.
the enzymes from the cells. This avoids inactivation of In spite of these disadvantages, cell immobilization
the enzymes during the time-consuming and expensive is a key technology to construct an efcient bioprocess
purication procedures, and makes it possible to use because immobilized cells have many attractive char-
the enzymes under more stable conditions. However, acteristics, as described above. For immobilization of
immobilized cells have some disadvantages that should cells, entrapment methods have been mainly used.
be considered for practical application: Especially, other immobilization methods, such as car-
1. Byproducts may be synthesized because the cells rier binding and crosslinking methods, are difcult to
contain many enzymes, which may catalyze undesir- use for immobilization of living or growing cells. For
able reactions. Selection of a strain, mutation, or entrapment of cells, prepolymer methods have been
proper treatment of cells, or genetic improvement of also widely used. Various types of cells have been
a cell line helps to overcome this problem. entrapped by prepolymer methods.

1. Microbial Cells ENT- or PU-entrapped cells of Rhodotorula minuta
var. texensis were successfully employed for the enan-
Microbial cells are thought to be excellent biocatalysts
tioselective hydrolysis of a dl-menthyl ester, giving
due to high growth rate, insensitivity to shear force,
optically pure l-menthol (28).
and a relatively low cost for cultivation. Moreover,
Entrapment in suitable gels renders microbial cells
microbial cells can synthesize and secrete not only
signicantly tolerant against unfavorable effects of
primary metabolites but also various secondary metab-
organic solvents. Applications of such entrapped
olites. Thus, various conditions of microbial cells
microbial cells to bioconversion of hydrophobic or
dried, untreated, resting, living, growing, etc.have
water-insoluble compounds are very important from
been entrapped by prepolymer methods and employed
a practical point of view. In these cases, introduction
widely. Table 4 lists examples of microbial cells
of organic solvents into reaction systems is essential to
entrapped by prepolymer methods. Typical applica-
dissolve substrates and/or products. Various reactions
tions of entrapped microbial cells are described below.
have been successfully carried out by using microbial
Generation of ATP by using the energy of glycolysis
cells entrapped with prepolymers in waterorganic
is very important for the production of useful
cosolvent systems and in organic solvent systems (28,
compounds by biocatalysts. Dried yeast cells
29). However, it is very difcult for the entrapped cells
entrapped by photocrosslinkable resin prepolymers
to live in organic solvent. Recently, we have demon-
were employed for the phosphorylation of adenosine
strated that doubly entrapped bakers yeast, i.e., the
with glucose as an energy source (26). Such
yeast cells entrapped with urethane resin prepolymer
ATP-generating systems can be combined with an
(PU-6) after entrapment in calcium alginate, can main-
ATP-requiring system in the same cells.
tain a living state during long-term stereoselective oxi-
By using growing Saccharomyces cells entrapped
doreduction in an organic solvent (30).
with photocrosslinkable resin prepolymers, pilot-scale
production of ethanol has been successfully carried out
(27).
Table 4 Examples of Microbial Cells Entrapped by Prepolymer Methods
Microorganism (condition) Polymerization method (prepolymer) Ref.
Alcaligenes eutrapus (freeze-dried or untreated) Urea linkage (PU) 39

Brevibacterium ammoniagenes (freeze) -Radiation (PEGM+PVA) 18
Brevibacterium ammoniagenes (acetone-dried) Photocrosslinking (ENT) 40
Citrobacter freundii (acetone-dried) Photocrosslinking (ENTE) 41
Enterobacter aerogenes (freeze-thawed) Photocrosslinking (ENT and ENTP) and urea linkage (PU) 36
Escherichia coli (untreated) Urea linkage (PU) 42
Escherichia coli (acetone-dried) Photocrosslinking (ENTE) 43
Hansenura jadinii (dried) Photocrosslinking (ENT) 44
Arthrobacter simplex (acetone-dried) Photocrosslinking (ENT and ENTP) 9, 37
Arthrobacter simplex (acetone-dried) Urea linkage (PU) 14, 45
Corynebacterium sp. (living) Photocrosslinking (ENT and ENTP) 46
Norcardia rhodocrous (freeze-thawed) Photocrosslinking (ENTP, etc.) and urea linkage (PU) 35
Nocardia rhodocrous (freeze-thawed) Photocrosslinking (ENT and ENTP) 37, 47
Propionibacterium sp. (growing) Photocrosslinking (ENT) and urea linkage (PU) 48
Streptomyces clavuligerus (resting) Crosslinking with dialdehyde (water-soluble polyacrylamide 20
having acylhydrazide group)
Streptomyces peucetius (growing) Photodimerization (PVA-SbQ) 49
Streptomyces rimosus (growing) Urea linkage (PU) 50
Streptomyces roseochromogenes (untreated) Photocrosslinking (PEGM) 6
Curvularia lunata (living) Photocrosslinking (ENT) 34, 51
Rhizopus stolonifer (living) Photocrosslinking (ENT) 52
Bakers yeast (dried) Photocrosslinking (ENT) 26
Rhodotorula minuta var. texensis (freeze-thawed) Photocrosslinking (ENT) and urea linkage (PU) 28
Saccharomyces sp. (growing) Photocrosslinking (ENT, etc.) 27

2. Plant and Animal Cells sized chemically in advance in the absence of
biocatalysts.
Higher plants are important sources of numerous use-
ful natural compounds such as pharmaceuticals, a-
vorings, and coloring materials, which have
A. Chain Length
complicated structures and cannot be synthesized by
microbial cells. Animal cells are also attractive bioca-
The chain length of prepolymers can be changed.
talysts for the production of vaccines and other bioac-
For example, the chain length of ENT-
tive peptides. For practical application of these cells,
1000-containing polyethylene glycol-1000 (average
immobilization, especially, entrapment has received
MW1000) is 10 nm, and that of ENT-2000
increasing interest as one of the most promising tech-
containing polyethylene glycol-2000 (average MW
niques. Entrapment of these cells has been carried out
2000) is 20 nm.
mostly with natural polymers such as alginate. As for
In general, a looser network of gels prepared from
prepolymer methods, cultured plant cells of Lavandura
longer-chain prepolymers increases the rate of diffu-
vera were entrapped with photosensitive resin prepoly-
sion of substrates and products through the gels.
mer (PVA-SbQ) and used for production of blue pig-
However, leakage of biocatalysts, especially enzymes
ments (31).
with low molecular weights, sometimes becomes a sig-
nicant problem with the gels which have a loose net-
C. Cellular Organelles
work (13). When invertase was entrapped with PEGM-
1000 to PEGM-4000 (4) or PU of MW 20005000 (12),
Cellular organelles have their own organized functions
the apparent enzyme activity was highest in the case of
for metabolizing cellular substances. This means that
the longest-chain prepolymer. A similar result was
these organelles are useful biocatalysts for carrying out
obtained when catalase was entrapped with ENT-
well-organized multistep reactions when they are iso-
1000 to ENT-6000 (8).
lated intact and immobilized without the loss of their
The loose network of gels is generally favorable to
native characteristics. Prepolymer methods have
growth if the entrapped cells are allowed to grow
proved to be suitable for the immobilization of organ-
inside gel matrices. When fungal spores (Curvularia
elles because the immobilization can be achieved by
lunata) were entrapped with photocrosslinkable resin
entrapment under mild conditions (32, 33). Only a
prepolymers (ENT) and allowed to germinate and
few reports have been published; the kinds of orga-
grow inside gel matrices, development of mycelia
nelles immobilized with prepolymer methods are sum-
was abundant in gels formed from prepolymers of
marized in Table 5.
longer chain length, while the growth was inhibited
in gels of tight network (34). Leakage of mycelia was
often observed with gels prepared from longerchain
IV. EFFECTS OF THE PROPERTIES OF length prepolymer.
PREPOLYMERS As described above, the chain length of prepolymers
is an important factor affecting the activity of biocata-
One of the advantages of prepolymer methods is lysts entrapped within the prepolymers. Prepolymer
easy preparation of gels having different properties methods offer an easy selection of adequate chain
by selecting suitable prepolymers which are synthe- length.
Table 5 Examples of Cellular Organelles Entrapped by Prepolymer Methods
Organelle (source) Polymerization method (prepolymer) Ref.
Chromatophores (Rhodopseudomonas capsulata) Photocrosslinking (ENT) and urea linkage (PU) 53

Thylakoids (lettuce) Photocrosslinking (ENT) and urea linkage (PU) 54
Chloroplasts (spinach) -Radiation (polyethylene glycol diacrylate + PEGM) 19
Mitochondria (acetate-grown Candida tropicalis) Photocrosslinking (ENT) and urea linkage (PU) 55
Peroxisomes or microbodies (methanol-grown Photocrosslinking (ENT) 7
Kloeckera sp.) Urea linkage (PU) 45

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Microbiol 27:505509, 1981.
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10. N Itoh, N Hagi, T Iida, A Tanaka, S Fukui. Photo-
Anionic or cationic property of gels has a signicant crosslinkable prepolymer method for immobilization
of biocatalysts. Preparation of immobilized glutamate
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1980. Continuous CoA production with immobilized
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28. T Omata, N Iwamoto, K Kimura, A Tanaka, S polyurethane and its application for L-aspartic acid
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nate by gel-entrapped Rhodotorula minuta var. texen- 43. K Watanabe, N Itoh, A Tanaka, S Fukui. Application
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44. A Kimura, Y Tatsutomi, N Mizushima, A Tanaka, R lized growing Streptomyces rimosus cells. Appl
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16, 1978. various gels, and steroid 11-hydroxylation by the
45. A Tanaka, IN Jin, S Kawamoto, S Fukui. entrapped mycelia. J Ferment Technol 59:465-469,
Entrapment of microbial cells and organelles with 1981.
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Microbiol Biotechnol 7:351-354, 1979. Hydroxylation of progesterone by gel-entrapped
46. K Sonomoto, N Usui, A Tanaka, S Fukui. 9- Rhizopus stolonifer mycelia. Eur J Appl Microbiol
Hydroxylation of 4-androstene-3,17-dione by gel- Biotechnol 16:57-62, 1982.
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Microbiol Biotechnol 17:203-210, 1983. Thomas. Comparative stabilization of biological
47. T Yamane, H Nakatani, E Sada, T Omata, A Tanaka, photosystems by several immobilization procedures.
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Continuous production of oxytetracycline by immobi-

23
Genetic Immobilization of Enzymes on Yeast Cell Surface
Mitsuyoshi Ueda, Toshiyuki Murai, and Atsuo Tanaka

Kyoto University, Kyoto, Japan
I. INTRODUCTION whole-cell biocatalysts, because surface-immobilized

proteins are covalently linked to glucan in the cell
Extensive studies have been conducted on various wall, rendering them resistant to extraction. Second,
methods of immobilization of enzymes. Conventional as S. cerevisiae is generally regarded as safe (GRAS)
methods for immobilization of enzymes through for oral use, it can be used in food and pharmaceu-
covalent bonds have some merits; the enzymes tical products.
are immobilized through strong bondage, and dissocia- The immobilized enzyme displayed on the cell sur-
tion is low in substrate or salt solution even at high face is also regarded as a kind of a self-immobilized
concentrations. However, change in structure of enzyme on the cell surface; this phenomenon is passed
the immobilized protein or in their characteristics on to daughter cells as long as the plasmid or the inte-
often occurs owing to severe treatment and there grated gene is retained by the cells. This immobiliza-
are many difculties in the determination of the tion system could turn or remake S. cerevisiae into a
immobilization reaction conditions. On the other novel and attractive microorganism as a whole-cell
hand, immobilization through ionic bonds does not biocatalyst by surface expression of various enzymes,
share these disadvantages, but the enzymes are easily especially when target substrates are not able to be
dissociated. taken up by the cells.
The genetic engineering method, combined with
the immobilization method using cell surface as a
carrier for immobilization of enzymes, overcomes II. PRINCIPLES OF GENETIC
the disadvantages of the above methods; enzymes IMMOBILIZATION OF ENZYME ON
are immobilized by a strong covalent bondage. They YEAST CELL SURFACE
can be regenerated according to the activation of
the promoter, and they are naturally immobilized To immobilize protein on the cell surface of S. cerevisiae,
on the cell surface. Immobilization of enzymes on molecular information of the native cell wall-anchored
cell surface saves tedious purication process of protein, -agglutinin, was utilized. -Agglutinin (1)
enzymes to be used in conventional immobilization. is a mannoprotein involved in the sexual adhesion of
Expression of proteins on the cell surface of S. cere- mating-type S. cerevisiae cells with mating-type a
visiae would offer more advantages than other micro- S. cerevisiae cells. -Agglutinin has a glycosylphospha-
bial cells. First, since S. cerevisiae is widely used in tidylinositol (GPI) anchor attachment signal (Fig. 1;
industrial production of proteins and chemicals, Table 1), which is involved in anchoring cell wall
enzyme-coated yeast cells could be used as novel proteins. This anchoring signal was combined with the

Figure 1 Transportation system of cell surface proteins in S. cerevisiae and GPI anchor structure. ER, endoplasmic reticulum;
PI-PLC, phosphatidylinositol-specic phospholipase C; GPI, glycosylphosphatidylinositol; Man, mannose; GlcNH2 , glucosamine.
signal of the secreted enzymes using genetic engineering S. cerevisiae itself is unable to utilize starch. A
techniques. Figure 2 shows the general structure of the number of strategies have been adopted for the
gene for cell surface immobilization of an enzyme (2). 30 - construction of starch-utilizing systems, which include
Half of -agglutinin contains a glycosylphosphatidyl- the addition of amylolytic enzymes in culture broth
inositol (GPI) anchor attachment signal at the C-term- and the introduction of heterologous genes encoding
inal end, like other cell surface proteins. amylolytic enzymes into yeast cells for secretive
production of the enzymes. As one of strategies, gluco-
amylase (EC 3.2.1.3) from Rhizopus oryzae (3), an
III. GENETIC IMMOBILIZATION OF exo-type amylolytic enzyme cleaving -1,4-linked
AMYLOLYTIC ENZYMES ON YEAST and -1,6-linked glucose effectively from the non-
CELL SURFACE reducing end and/or -amylase (EC 3.2.1.1) from
Bacillus stearothermophilus CU21 (BSTA) (4), an
Although starchy materials are available in abun- endo-type amylolytic enzyme cleaving -1,4-glycosidic
dance as carbon sources for cultivation, wild-type linkage, were displayed on the yeast cell surface
Table 1 GPI Anchor Attachment Signal in S. cerevisiaea
Cell surface protein GPI anchor attachment signal
Ag1 TSTSLMISTYEG KASIFFSAELGSIIFLLLSYLLF

Aga1 TSSMVTISQYMG SGSQTRLPLGKLVFAIMAVACNVIFS
Flo1 STASLEISTYAG SANSLLAGSGLSVFIASLLLAII
Cwp1 QAPNTVYEQTEN AGAKAAVGMGAGALAVAAAYLL
Cwp2 SSTETISQQTEN GAAKAAVGMGAGALAAAMLL
Tip1 VETASNAGQRVN AGAASFGAVVAGAAALLL
Sed1 SASSHSVVINSN GANVVVPGALGLAGVAMLFL
Tir1/Srp1 ATKAVSEQTENG AAKAFVGMGAGVVAAAAMLL
"
a
The predicted cleavage sites are indicated by an arrow. The predicted ! sites are underlined.

Figure 2 General strategy for cell surface immobilization of an enzyme.
(Fig. 3). The constructed strains immobilizing gluco- mid for expression of the glucoamylase/-agglutinin
amylase or glucoamylase and -amylase should be fusion gene containing the secretion signal sequence
able to saccharify starch on its cell wall and assimilate of the glucoamylase under the control of the
the released glucose to proliferate and ferment. GAPDH promoter (2). The plasmid pGA11 was con-
structed as follows (Fig. 4): an XhoI site was generated
A. Immobilization of Glucoamylase on Yeast at the end of the glucoamylase coding region on the
Cell Surface Using a Multicopy Plasmid plasmid pYGA2270 (5) by site-directed mutagenesis
(U.S.E. Mutagenesis Kit, Pharmacia Biotech. Co.,
1. Construction of a Multicopy Plasmid Uppsala, Sweden) with the primers 50 -GCACCTG
A plasmid pGA11 to immobilize glucoamylase on the CCGCTGGCTCGAGAAATTTAAATGC-30 and 50 -
yeast cell surface was constructed as a multicopy plas- CTGTGACTGGTGACGCGTCAACCAAGTC-30 as
mutation and selection primers, respectively. A DNA
fragment containing glucoamylase coding region was
isolated from the mutated plasmid by EcoRI-XhoI
digestion. A DNA fragment of the -agglutinin gene
(AG1), containing 30 -half of the coding region encod-
ing 320 amino acids of the -agglutinin and 446 bp of
the 30 -anking region, was prepared by PCR (primers,
50 -GTACCTCGAGCGCCAAAAGCTCTTTTATC-
30 and 50 -GCGGTACCTTTGATTATGTTCTTCTTT
CTAT-30 ) with genomic DNA from S. cerevisiae MT8-
1 (MATa ade his3 leu2 trp1 ura3). (6) as a template,
followed by digestion by XhoI and KpnI. These two
fragments were substituted for the EcoRI-KpnI section
between the GAPDH promoter and the GAPDH ter-
minator of the yeast expression cassette vector
pYE22m. The resulting plasmid was named pGA11.
2. Detection of Glucoamylase Activity

Figure 3 Model of cell surfaceengineered yeast assimilat- The constructed plasmid was introduced into S. cere-
ing starch or cellulose. E, amylolytic or cellulolytic visiae strain MT8-1 as a host cell. Yeast was preculti-
enzyme(s). vated in YPD medium (1% yeast extract, 2% peptone,

Figure 4 Construction of the multicopy plasmid pGA11 for immobilization of glucoamylase on the yeast cell surface.
2% glucose). After cells were cultivated aerobically at The cell wall of S. cerevisiae mainly consists of glu-
308C in modied Burkholders medium (containing can and mannoproteins. Glucan is composed of -1,3-
0.002% adenine sulfate, 0.002% L-histidineHCl, and -1,6-linked glucose. Some mannoproteins seemed
0.003% L-leucine, 0.002% uracil, and 1% casamino to be covalently linked with glucan, because they can
acids) to which 2% glucose was added as a carbon be extracted by -1,3- or -1,6-glucanase. The localiza-
source, culture medium and cell pellets were isolated tion of the glucoamylase protein and its association
by centrifugation to measure the glucoamylase activ- with cell wall was determined. Cells were harvested
ities in both fractions. Cell growth in the culture broth by centrifugation at 3000g and washed in cooled
was measured by absorbance at 600 nm. For measure- (08C buffer (10 mM Tris-HCl [pH 7.8], 1 mM phenyl-
ment of glucoamylase activity (2), the substrate for methylsulfonyl uoride [PMSF]). The cells, the buffer,
glucoamylase reaction was prepared by adding soluble and glass beads (diameter 0.450.50 mm) were mixed
starch to boiling 20 mM sodium acetate buffer (pH in a ratio of 1:2:1 wet-w/v/w in a glass tube and agitated
4.6) to give a concentration of 0.5%. After keeping vigorously, using a benchtop vortex mixer, for 5 min at
0.9 mL of the solution at 308C for 5 min, 0.1 mL of 08C. The cell wall fraction was recovered by centrifu-
enzyme solution was added and the mixture was incu- gation of the homogenate at 1000g for 5 min and was
bated at the same temperature for 15 min. The reaction washed with the same buffer. Glucoamylase was
was stopped by boiling the mixture for 10 min and the extracted from this fraction in a two-step procedure.
concentration of glucose was determined using F-kit First, noncovalently bound proteins, and proteins
for glucose (Boehringer Mannheim, Mannheim, bound through disulde bridges, could be extracted
Germany). One unit of glucoamylase was dened as from cell wall with hot sodium dodecyl sulfate
the amount of enzyme required to release 1 mmol glu- (SDS). Following this, the SDS-treated cell wall was
cose/min from starch. As a result, the cells harboring further digested with laminarinase (-1,3-glucanase).
the plasmid pGA11 had only the cell-associated glu- To quantify the amount of the SDS-extracted and glu-
coamylase activity without secretion of the active canase-extracted glucoamylase protein, intensities of
enzyme (2). signals from each fraction on the lter were measured

using antiglucoamylase IgG and horseradish peroxi- bon source at 308C for 24 h, and the supernatant of the
dase protein A (2). The enzyme was almost extracted culture broth was used as the source of free glucoamy-
by glucanase. lase. Free glucoamylase solution was prepared by dia-
The amylolytic activity was also detected by halo lysis of the culture supernatant in 20 mM sodium
formation on an agar plate. Cells harboring the plas- acetate buffer (pH 4.6) at 48C. The glucoamylase activ-
mid pGA11 or pYE22m, as a control, were inoculated ities in the cell wall suspension and the dialyzed super-
on a plate of modied Burkholders medium contain- natant were measured. The properties of the anchored
ing 2% glucose and 1% soluble starch. After incuba- glucoamylase examined were similar to those of the
tion for 3 days at 308C, the plate was stained with free glucoamylase, suggesting that the enzymatic func-
iodine vapor. Figure 5 shows that the cells harboring tion of the anchored glucoamylase was comparable to
the plamid pGA11 hydrolyzed starch and produced a that of the free enzyme.
halo strictly around the colony, while no halo forma-
tion was observed around the cells harboring the plas-
mid pYE22m. This indicates that the former cells 3. Microscopic Observation
obtained amylolytic activity owing to the expression For immunouorescence microscopy, immunostaining
of the glucoamylase/-agglutinin fusion gene on the was performed as follows. As the primary antibody,
cell surface. the antibody against R. oryzae glucoamylase at the
To know the properties of the anchored enzyme, dilution rate of 1:1000 was used. Cells and the anti-
thermal stability, optimal temperature, and optimal body were incubated at room temperature for 1.5 h.
pH of the glucoamylase anchored on the cell surface After washing of cells, the second antibody, uores-
were compared with those of the secreted free gluco- cein isothiocyanate (FITC)-conjugated goat anti-rabbit
amylase (7). The cell wall fraction of the cells harbor- IgG diluted 1:300, was reacted with the cells at room
ing the plasmid pGA11 was suspended in 20 mM temperature for 1 h. After washing, the image of cells
sodium acetate buffer (pH 4.6). The cells harboring was observed. Cells expressing the glucoamylase/-
the plasmid for expression of the glucoamylase without agglutinin fusion gene were uniformly labeled,
being fused to -agglutinin were cultivated in modied although not all the cells were equally intensively
Burkholders medium containing 2% glucose as a car- labeled. This is probably due to differences in expres-
sion levels among the cells. The cells harboring the
control plasmid were hardly labeled (Fig. 6). For
immunoelectron microscopy, yeast cells were xed
with 4% paraformaldehyde, and immunostaining was
done as follows. The antibody against glucoamylase
diluted 1:100, as the primary antibody, was incubated
with cells at 48C for 2 h. The second antibody, goat
anti-rabbit IgG conjugating 5 nm gold diluted 1:40,
was reacted with the cells at 48C overnight, followed
by xation with 1% glutaraldehyde. Thereafter,
embedding and microscopic observation were per-
formed. Gold particles were detected on the surface
of the cell wall of the cells harboring the plasmid
pGA11. Few gold particles were detected in the case
of the control cells (Fig. 7).
4. Assimilation of Starch
When cells were cultivated aerobically with 1%
Figure 5 Plate assay for detection of amylolytic activity. 1, soluble starch as the sole carbon source, the cells
S. cerevisiae with cell surface-immobilized glucoamylase harboring the plasmid pGA11 utilized starch and
(S. cerevisiae MT8-1/pGA11); 2, S. cerevisiae secreting glu- proliferated to reach an absorbance of 10, which
coamylase; 3, S. cerevisiae MT8-1 as the control (S. cerevisiae was the same level as in the case of the culture on
MT8-1/pYE22m). 1% glucose.

Figure 6 Immunouorescent labeling of transformed cells. Normarsky differential interference micrographs (a, c, e, g) and
immunouorescence micrographs (b, d, f, h). a, b, e, f: S. cerevisiae MT8-1 harboring pYE22m (control); c, d: S. cerevisiae MT8-
1 harboring pGA11; g, h: S. cerevisiae MT8-1 harboring pCMC11.

Figure 7 Immunoelectron micrographs of S. cerevisiae MT8-1 harboring pGA11 (a) and pCMC (b). Cells were immunogold-
labeled with the respective antibodies. CW, cell wall; M, mitochondrion. Arrowheads, gold particles (diameter, 5 nm).
B. Immobilization of Glucoamylase on Yeast ment containing the glucoamylase/-agglutinin fusion

Cell Surface Using an Integrative Plasmid gene excised from the plasmid pGA11 was inserted into
the HindIII site of the self-ligated plasmid. The result-
For the stable expression of enzymes on the yeast cell ing plasmid was named pIGA11 as the integrative plas-
surface, a gene to be integrated into the chromosome mid. The plasmid pIGA11 prepared was cut by ApaI
of S. cerevisiae was constructed using the fusion gene and introduced into S. cerevisiae MT8-1 strain, and the
of glucoamylase and C-terminal half of -agglutinin transformant obtained was named IGA strain.
genes (8). Integration of the fusion gene was conrmed by
Southern blot hybridization analysis using a full-length
fragment of URA3 as a probe.
1. Construction of an Integrative Plasmid
An integrative plasmid for integration of the glucoamy-
2. Stability of the Strain Harboring the
lase gene to the chromosome was constructed as fol-
Integrative Plasmid
lows (Fig. 8a): The yeast integration vector pRS406 (9)
was digested with KpnI and SalI, treated with T4 DNA Mitotic stability of the integrated exogenous DNA
polymerase, and then self-ligated. The HindIII frag- sequence after 120 h cultivation was estimated by com-

Figure 8 Construction of integrative plasmids for cell surface immobilization of glucoamylase (a, pIGA11) and -amylase
(b, pIAA11).
paring the number of colonies (cells corresponding to bp of the 30 -anking region, was prepared by the same
A600 10 grown on a nonselective YPD plate with method as the case of glucoamylase. These two frag-
that grown on a selective SD (synthetic [S] medium ments and BamHI-XhoI fragment of the GAPDH pro-
[0.67% yeast nitrogen base without amino acid with moter were inserted into the plasmid pRS404 at its
appropriate supplements] containing 2% glucose) BamHI-KpnI site.
plate using the replica-plate method. As for IGA On the constructed plasmid, introduction of the
strain, the number of colonies on SD plate without XbaI site changed its corresponding amino acid
uracil was 97% of that on YPD plate. When cultiva- sequence Lys-Arg to Ser-Arg, where Lys-Arg is the
tion was repeated three times, the number of colonies recognition sequence of the Kex2 endopeptidase to
of uracil nonauxotrophs was still 90%. cleave -factor prosequence. Therefore, the site-direc-
ted mutagenesis was performed by PCR-based techni-
C. Immobilization of -Amylase on Yeast Cell que to change Ser-Arg to Lys-Arg. The resulting
Surface Using an Integrative Plasmid plasmid was named PIAA11. When the IAA strain
harboring pIAA11 was cultivated in modied
1. Construction of an Integrative Plasmid
Burkholders medium containing 2% glucose at 308C
To express the -amylase gene from B. stearothermo- for 24 h, 60% of the total -amylase activity was
philus CU21 (BSTA in S. crevisiae and display the detected in the culture supernatant. This solubilization
synthesized protein on its cell surface (8), the following seemed to be caused by the proteolytic processing of
DNA fragments were prepared (Fig. 8b). The prepro the fusion protein.
leader sequence of the -factor precursor (MF1) was The deduced amino acid sequence of the BSTA gene
rst fused to the -amylase gene. The prepro secretion contains one possible processing site for the Kex2
signal sequence of the MF1 was amplied from the endopeptidase, Val-Pro-Arg sequence, at amino acid
plasmid pLS01 by PCR, using the primers 50 - residues No. 481483. The Kex2 enzyme exhibits the
GCAGCTCGAGATGAGATTTCCTTCAATTTTT- substrate specicity toward each carboxyl site of
ACTGC-30 and 50 -TCTCTAGAATCCAAAGATAC- Lys-Arg, Arg-Arg, and Pro-Arg sequences (11). Then,
CCCTTC-30 . pLS01 has a 1.7-kbp EcoRI-EcoRI frag- a Kex2-resistant BSTA/-agglutinin fusion protein was
ment of the MF1 inserted at EcoRI site of pUC13 constructed by eliminating 33 residues from the
(10). The amplied fragment was cut with XhoI and C-terminus of the BSTA. The C-terminal truncated
XbaI, then ligated at the XbaI site with the -amylase BSTA/-agglutinin fusion gene was introduced into
gene fragment, which was amplied from pBR2.0A (4) S. cerevisiae (Fig. 9), using the integration plasmid
by PCR using the primers 50 -CCTCTAGAGCCGCA- pIAA12. For construction of the plasmid pIAA12,
CCGTTTAACGGCAC-30 and 50 -GCCTCGAGCCA- PCR was performed with primers 50 -GCAGCT-
GGCCATGCCACCAACCG-30 and digested with CGAGATGAGAT-TTCCTTCAATTTTTACTGC-30
XbaI and XhoI. A DNA fragment of the -agglutinin and 50 -TCCTCGA-GCCAGGAACCCAAACCGAA-
gene (AG1), containing 30 -half of the coding region ACCG-30 and pIAA11 as a template, followed by
encoding 320 amino acids of the -agglutinin and 446 XhoI digestion. The XhoI fragment of the plasmid

Figure 9 Strategy for the construction of the C-terminal Figure 10 Model of cell surfaceengineered yeast coim-
truncated BSTA/-agglutinin fusion protein. mobilized with glucoamylase and -amylase.
pIAA11 was substituted for this newly synthesized IAA strain on a starch-containing plate, unlike that
fragment to produce the plasmid pIAA12 to be immo- of the IGA/IAA strain, formed a small halo strictly
bilized without cleavage by proteases. Southern blot around the colony as observed in the case of the
hybridization analysis was carried out to conrm the IAA and IGA strains, showing the expression of
integration into chromosomal DNA. The obtained both glucoamylase and -amylase on the cell surface.
strain was named IAA strain. The IAA strain These results demonstrated that glucoamylase and -
exhibited -amylase activity only in the cell pellet frac- amylase were coexpressed and coimmobilized on the
tion. This indicated that the genetic immobilization of cell surface of the same cell.
-amylase without leaking any detectable activity to the
culture medium was performed by the elimination of 2. Assimilation of Starch
the possible Kex2 enzyme processing site.
The transformants were cultivated aerobically at 308C
in modied Burkholders medium containing 1% solu-
D. Coimmobilization of Glucoamylase and - ble starch as the sole carbon source. The IGA and
Amylase on Yeast Cell Surface IGA/IAA strains grew on starch and reached an
absorbance of 10 monitored at 600 nm, which was
Coexpression of glucoamylase and -amylase on the the same level as in the case of the culture on 1%
cell surface was expected to be effective for efcient glucose, although the growth on glucose of these
assimilation of starch (Fig. 10). An S. cerevisiae strains was more rapid. No growth on starch was
strain was constructed in which glucoamylase/- observed with the MT8-1 and IAA strains. The strain
agglutinin gene and C-terminal truncated -amylase/ codisplaying glucoamylase and -amylase, IGA/
-agglutinin gene were integrated into its chromo- IAA, grew faster than the glucoamylase-displaying
somes (8). The plasmid pIAA12 was cut by strain, IGA (Fig. 11). This result demonstrated that
HindIII and introduced into the IGA strain harbor- the yeast strain armed with -amylase in addition to
ing pIGA11. The cell of uracil and tryptophan non- glucoamylase had the enhanced ability to degrade
auxotrophy was picked up and named IGA/IAA starch by sequential hydrolytic reaction on the cell sur-
strain. Integration of the fusion genes was conrmed face.
by Southern blot hybridization analysis using URA3
and TRP1 as probes.
IV. GENETIC IMMOBILIZATION OF
CELLULOLYTIC ENZYMES ON
1. Detection of Glucoamylase and -Amylase
YEAST CELL SURFACE
The strain IGA/IAA exhibited both glucoamylase
and -amylase activities only in the cell pellet fraction. Cellulose, consisting of glucose units linked together
Immunouorescent labeling and immunoelectron by -1,4-glycosidic bonds, is the most abundant carbo-
microscopy conrmed the location of both enzymes hydrate in the biosphere. An estimated synthesis rate
on the yeast cell surface. The colony of the IGA/ of cellulose is 4 107 tons/year. For a long-range

promoter from S. cerevisiae. The multicopy plasmid
pCMC11 to immobilize CMCase was constructed as
follows (Fig. 12): An XhoI site was generated at the end
of the CMCase coding region on the plasmid
YEpM93R (14) by the PCR with the primers 50 -
AGTACTCGAGCCCTGTACGCTGGCAGACCA-
GT-30 and 50 -CGGTAGGTATTGATTGTAATTCT-
G-30 . A DNA fragment containing CMCase coding
region was isolated by EcoRI-XhoI digestion. A
DNA fragment of the -agglutinin gene (AG1), con-
taining the 30 -half of the coding region encoding 320
amino acids of the -agglutinin and 446 bp of the 30 -
Figure 11 Time courses of cell growth (absorbance at 600 anking region, was prepared by the previously
nm) during aerobic cultivation in the medium containing described method. These two fragments were substi-
starch as the sole source of carbon and energy. Symbols for
tuted for the EcoRI-KpnI section between the
strains: *, MT8-1; &, IGA; ~, IAA; *, IGA/IAA.
GAPDH promoter and the GAPDH terminator of
the yeast expression cassette vector pYE22m. The
resulting plasmid was named pCMC11.
solution to resource problems of energy, chemicals,
2. Detection of CMCase
and food, cellulose is the most promising renewable
carbon source that is available in large quantity. Cells were cultivated in SD medium (Sec. III.B.2) at
However, the yeast S. cerevisiae is unable to utilize 308C for 24 h to the stationary phase. Culture medium
cellulosic materials in spite of its versatility in indus- and cell pellets were isolated by centrifugation to mea-
trial fermentation. The cell surface engineering system sure the CMCase activities in both fractions. The cells
might be applicable. The genetic immobilization on the harboring the plasmid pCMC11 had the cell-associated
cell surface of yeast of carboxymethylcellulase CMCase activity without secretion of the active
(CMCase) from Aspergillus aculeatus, classied as enzyme. The CMCase activity was stably maintained
endo-1,4--D-glucan glucohydrolase (endoglucanase, for 120 h at least. SDS and -1,3-glucanase treatment
EC 3.2.1.4), which cleaves the -1,4-glycosidic linkage indicated that the CMCase was covalently attached to
of cellulose (12) or/and -glucosidase (1,4--D-gluco- the cell wall and immobilized by the genetic engineer-
side glucohydrolase, EC 3.2.1.21), an exo-type enzyme ing techniques. A plate assay was carried out using
(13) from the same microorganism, was carried out. It Congo Red staining (15). The cells harboring the plas-
was predicted that short-chain cello-oligosaccharides mid pCMC11 hydrolyzed carboxymethylcellulose and
formed by the endo-action of CMCase would be con- produced a halo strictly around the colony, while no
verted quickly to glucose by -glucosidase. However, halo formation was observed around the cells harbor-
S. crevisiae lacks -glucosidase activity and conse- ing the plasmid pYE22m (Fig. 13). Immunouorescent
quently is unable to utilize cellobiose as the carbon labeling and immunoelectron microscopy conrmed
source. Thus, to construct an S. cerevisiae strain the localization of CMCase/-agglutinin fusion protein
which is able to utilize cellulosic materials, it is neces- on the yeast cell surface (Figs. 6, 7).
sary to endow it with -glucosidase activity.
B. Immobilization of -Glucosidase on Yeast
A. Immobilization of CMCase on Yeast Cell Cell Surface
Surface
1. Construction of a Multicopy Plasmid
1. Construction of a Multicopy Plasmid
The DNA fragment composed of the GAPDH promo-
A cDNA encoding Fl-CMCase of the fungus A. acu- ter and the secretion signal sequence of glucoamylase
leatus with its secretion signal peptide was fused with gene from R. oryzae was prepared by PCR (primers
the gene encoding the C-terminal half (320 amino acid 50 -CCGAGCTCACCAGTTCTCACACGGAACA-30
residues from the C-terminus) of yeast -agglutinin and 50 -GCCCGCGGCAGAAACGAGCAAAGAAA-
(12). The constructed plasmid containing this fusion A-30 ) with the plasmid pYGA2269 (3) as a template,
gene was expressed under the control of the GAPDH and then cut with SacI and SacII. The resulting SacI-

Figure 12 Construction of the multicopy plasmid pCMC11 for cell surface immobilization of CMCase.
Figure 13 Plate assay for detection of CMCase activity. 1, S. cerevisiae with cell surfaceimmobilized CMCase (S. cerevisiae
MT8-1/pCMC11); 2, S. cerevisiae secreting CMCase; 3, S. cerevisiae MT8-1 as the control (S. cerevisiae MT8-1/pYE22m).

SacII fragment, the SacII-XhoI fragment created by cellooligosaccharides was added as the sole carbon
annealing two oligonucleotides 50 -GGAGATCTCCA- source. The cello-oligosaccharides contain 11%
TGGC-30 and 50 -TCGAGCCATGGAGATCTCCGC- (w/w) cellohexaose, 29% (w/w) cellopentaose, 33%
3, and the XhoI-KpnI fragment containing the 30 -half (w/w) cellotetraose, 17% (w/w) cellotriose, 4%
of the coding region encoding 320 amino acids of - (w/w) cellobiose, and < 1% (w/w) glucose.
agglutinin and 446 bp of the 30 -anking region excised -Glucosidase activity was measured as follows (16):
from the plasmid pGA11, were inserted into the The reaction mixture was composed of 0.1 mL of
sections of SacI-SacII, SacII-XhoI, and XhoI-KpnI, 1.7 mM p-nitrophenyl--D-glucoside in 0.1 M sodium
respectively, of the plasmid pRS404 (9). The resulting acetate buffer, pH 5.0, and 0.1 mL of enzyme
plasmid was named pICAS1. The plasmid pBG211 solution. After incubation at 378C for 10 min, p-nitro-
to immobilize -glucosidase on the yeast cell phenol released was measured spectrophotometrically
surface was constructed as follows. The yeast high- as an increase of absorbance at 400 nm (molecular
copy vector pMH1 (15) was constructed by intro- extinction coefcient, 17,700 M1 cm1 ). One unit
ducing the 2.14-kbp EcoRI-EcoRI fragment of of the enzyme activity was dened as the amount
PMT34(3) (4) containing a part of 2 m DNA into of enzyme that released 1 mol of p-nitrophenol
the AatII site of the plasmid pRS403 (9). The BglII- from the substrate per minute. Immuno-
XhoI fragment of the cDNA encoding -glucosidase 1 uorescence microscopy was performed the same as
(BGL1) from A. aculeatus was generated by PCR (50 - in other cases.
GTCGAGATCTCTGATGAACTGGCGTTCTCT-30
and 50 -TTCACTCGAGCCTTGCACCTTCGGGAG-
CGCCG-30 ) with the plasmid pABG7 as the template, 3. Analysis of Saccharides
and substituted for the BglII-XhoI section of pICAS1,
from which the BssHII-BssHII fragment was For thin-layer chromatography of cello-oligosaccha-
transferred to the plasmid pMH1 at its BssHII- rides, aliquots (1 L) of the culture supernatants
BssHII section. The resulting plasmid was named were spotted on a silica gel 60F254 TLC plate, and
pBG211 (Fig. 14) (14). developed twice with 1-butanol-pyridine-water
(70:15:15, v/v). Sugars were detected by the diphenyl-
amine-aniline method; the detection reagent, consist-
2. Detection of -Glucosidase
ing of 4 mL aniline, 4 g diphenylamine, 200 mL
Yeast harboring pBG211 was precultivated in YPD acetone, and 30 mL 85% phosphoric acid, was
medium and cultivated aerobically in the S-medium sprayed on the plate, which was then heated at
to which 2% glucose, 1% cellobiose, or 0.5% 1058C for 30 min.
Figure 14 Construction of the multicopy plasmid pBG211 for cell surface immobilization of -glucosidase.

C. Coimmobilization of CMCase and -Glucosidase source. The strains MT8-1/pBG211 and MT8-1/
on Yeast Cell Surface (PCMC11, pBG211) grew on cellobiose and reached
an absorbance of 2, which was a little lower than
1. Construction of Cells for Coimmobilization
that in the case of the culture on 1% glucose. No
of CMCase and -Glucosidase
growth on cellobiose was observed with strains
The plasmids pBG211 and pCMC11 described above MT8-1 and MT8-1/pCMC11. No difference was
were introduced stepwise into S. cerevisiae MT8-1. The observed between the growth curves of the strains
resulting strain was named MT8-1/(pCMC11, MT8-1/pBG211 and MT8-1/(pCMC11, pBG211).
pBG211) (13). These results showed that the -glucosidase geneti-
cally immobilized on the cell surface endowed the
2. Detection of CMCase and -Glucosidase yeast with the ability of cellobiose degradation and
utilization. The transformants were precultivated in
To determine the location of CMCase and -glucosi-
YPD medium and cultivated aerobically in S-medium
dase, yeast strains were cultivated in the SD medium
supplemented with cello-oligosaccharides as the sole
(Sec. III.B.2) at 308C for 24 h. The cells harboring the
carbon source, and the cell growth was monitored by
plasmid pCMC11, i.e., strain MT8-1/pCMC11 and
counting colonies that appeared on YPD plates on
strain MT8-1/(pCMC11, pBG211), had the cell-asso-
which aliquots of the culture broth were spread.
ciated CMCase activity, and the cells harboring the
The strains MT8-1/pBG211 and MT8-1/(pCMC11,
plasmid pBG211, i.e., strain MT8-1/pBG211 and
pBG211) grew on cello-oligosaccharides employed
strain MT8-1/(pCMC11, pBG211), exhibited the cell-
(Fig. 16).
associated -glucosidase activity. Moreover, the cells
-Glucosidase was capable of degrading cello-oligo-
harboring the plasmids pCMC11 and pBG211 showed
saccharides with glucose units of 2 to 6; the breakdown
both CMCase and -glucosidase activities. Strain
of cello-oligosaccharides by -glucosidase seemed to be
MT8-1 itself exhibited neither of the activities. These
sufcient to sustain the growth of the yeast displaying
results suggested that CMCase and -glucosidase pro-
this enzyme. The strain MT8-1/(pCMC11, pBG211),
teins were efciently synthesized and codisplayed on
however, grew a little more rapidly than the strain
the cell surface in their active forms. MT8-1/
(pCMC11, pBG211) cells were labeled by uorescence
with both anti-CMCase IgG and anti--glucosidase
IgG, conrming again that these cells coimmobilized
CMCase and -glucosidase proteins on their cell sur-
face (Fig. 15).
3. Assimilation of Cello-oligosaccharides
The transformants were precultivated in YPD me-
dium and cultivated aerobically in S-medium (Sec.
III.B.2) containing cellobiose as the sole carbon
Figure 16 Time courses of the cell growth during aerobic

cultivation in the medium containing cello-oligosaccharides
as the sole source of carbon and energy. Symbols for strains:
*, MT8-1; ~, MT8-1/pCMC11; &, MT8-1/pBG211; *,
MT8-1/ (pBG211, pCMC11). Cell growth was monitored
Figure 15 Model of cell surfaceengineered yeast coim- by counting colonies that appeared on YPD plates on
mobilized with CMCase and -glucosidase. which aliquots of the culture broths were spread.

MT8-1/pBG211, indicating that the yeast strain codis- Our cell surface engineering system has a possibility
playing CMCase and -glucosidase had the enhanced of being used to construct new functional yeast strains.
ability to degrade cello-oligosaccharides. The degrada- This may contribute to resolution of many problems
tion and assimilation of cello-oligosaccharides by the concerning environmental pollution and biomass con-
transformants were conrmed by thin-layer chromato- version at the global level.
graphy. The cello-oligosaccharides dissolved in the cul-
ture supernatants were gradually degraded and
assimilated by the strains MT8-1/pBG211 and MT8- VI. SUMMARY
1/(pCMC11, pBG211).
The strain MT8-1/(pCMC11, pBG211) degraded We established the genetic system to immobilize
cello-oligosaccharides more rapidly, especially cello- enzymes as their active forms on the cell surface of
pentaose (G5) and cellohexaose (G6), than the strain yeast, S. cerevisiae. As a principle, a cDNA encoding
MT8-1/pBG211. This is probably due to the activity of enzyme with a secretion signal peptide was fused with
CMCase displayed on the cell surface of the strain the gene encoding the C-terminal half (320 amino acid
MT8-1/(pCMC11, pBG211). CMCase puried from residues from the C-terminus) of yeast -agglutinin, a
A. aculeatus showed activity against G5 and G6 (17). protein involved in mating and covalently anchored to
Surely, it was conrmed that the strain MT8-1/ the cell wall. The constructed plasmid containing this
pCMC11 degraded G5 and G6 among the cello-oligo- fusion gene was introduced into S. cerevisiae and
saccharides employed, while the strain MT8-1 could expressed under the control of the glyceraldehyde-3-
not degrade any of the cello-oligosaccharides. These phosphate dehydrogenase (GAPDH) promoter from
results gave evidence that the strain MT8-1/ S. cerevisiae. The activity and localization of the
(pCMC11, pBG211) hydrolyzed the cello-oligosac- enzyme on the cell surface was biochemically demon-
charides with synergistic actions of CMCase and - strated by subcellular fractionation and glucanase
glucosidase, the produced glucose being assimilated treatment. Appearance of the fused protein expressed
for proliferation. on the cell surface was further conrmed by halo for-
mation, unlike that of secretive enzyme, and immuno-
uorescence and immunoelectron microscopies.
Arming cells displaying glucoamylase and -amylase,
V. PERSPECTIVE or carboxymethylcellulase and -glucosidase, could
assimilate starch or cello-oligosaccharides as the sole
The immobilization of amylolytic and cellulolytic carbon source, although S. cerevisiae cannot intrinsi-
enzymes on the yeast cell surface may facilitate the cally assimilate them. This technique endowed the cell
utilization of starch and cellulose by yeast. In this sys- with novel functions.
tem, the cell surface of yeast was used as a carrier for
immobilization of enzymes, and the living whole cells
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24
Use of Immobilized Enzymes in the Food Industry
Harold E. Swaisgood
1. INTRODUCTION the soluble enzyme are listed in Table 1 (11). Because

the same enzyme is used continuously, the amount of
Most of the literature on immobilized enzymes has product obtained per unit of enzyme is much greater.
resulted from research and development during the For example, in the current commercial use of immo-
past 40 years. A number of reviews have appeared bilized glucose isomerase 12,00015,000 kg of dry pro-
(111) that discuss the preparation, characteristics, duct is typically obtained per kilogram of biocatalyst
and uses of immobilized enzymes. Actual commercial- during its operational lifetime (J. Shetty, personal com-
ization of immobilized enzyme bioprocesses has not munication, 1999).
occurred at a rapid pace in the food industry, perhaps Precise control of the extent of reaction is a key
owing to a natural reluctance to replace existing solu- advantage for many processes where a limited extent
ble enzyme processes and the fact that most of the of reaction is desirable. Use of the immobilized form
industry involves low value-added products with eliminates the need for a downstream enzyme inactiva-
small prot margins. Nevertheless, immobilized tion step that could be harmful to the product. These
enzyme technology has gained acceptance in several considerations are important for processes involving
areas. The most successful example is the production protein modication or avor generation. Even if pre-
of high-fructose corn syrup using immobilized glucose cise control of the extent of reaction is not required, in
isomerase, which is a unique product produced only many cases a downstream enzyme inactivation step is
with the immobilized enzyme. necessary because the commercial enzyme preparation
may contain contaminating activities, e.g., proteinases,
that over long periods can cause product deterioration.
II. WHY USE AN IMMOBILIZED When the immobilized preparation is used, the short
ENZYME? exposure time may not be detrimental.
Use of the immobilized enzyme in a bioreactor, for
The commercial success of an immobilized enzyme example, a xed-bed or uidized-bed reactor, allows it
process requires the cost per unit of product to be to be incorporated into a continuous automated bio-
less than that for a comparable soluble enzyme process process. This feature greatly reduces the materials
or that a unique product can be produced with the handling and, consequently, the labor costs. For exam-
immobilized enzyme that cannot be obtained with ple, Tanabe-Seiyaku Co. in Japan experienced a three-
the soluble enzyme. Some possible advantages of an fold reduction in labor costs when they introduced the
immobilized enzyme process as compared with use of use of immobilized aminoacylase for L-amino acid

Table 1 Some Possible Advantages of an Immobilized ports have a polyol type structure that is known to
Enzyme Process as Compared with Use of the Soluble stabilize proteins (13).
Enzyme Finally, it may be possible to produce a unique pro-
1. Greater productivity per unit of enzyme duct using an immobilized enzyme. Such is the case for
2. Precise control of the extent of reaction production of high-fructose corn syrup using immobi-
3. Continuous operation and automation of the process lized glucose isomerase. Recently, the potential to
is possible design functionality by limited proteolysis has been
4. Materials handling is minimized demonstrated (14, 15). However, this requires precise
5. Product does not contain the biocatalyst control of the extent of hydrolysis, but a downstream
6. Enzyme activity may be stabilized heat inactivaction of the enzyme would also denature
7. A unique product may be produced the polypeptides formed and thus destroy functional-
ity. Use of immobilized proteinases would eliminate
the required downsteam enzyme inactivation; conse-
quently, functional polypeptides could be produced
production (4). Another example is the Valio process that have unique properties in foods.
for producing hydrolyzed whey syrup using immobi-
lized b-galactosidase. One of the largest whey-proces-
sing plants in the world, operated by Dairy Crest in III. ECONOMIC CONSIDERATIONS FOR
northern Wales and using the Valio process, is fully AN IMMOBILIZED ENZYME
automated and controlled by a microprocessor, thus BIOPROCESS
requiring only two operators to be present at any one
time (M. Harju, personal communication, 1999). The commercial success of an enzyme process depends
Immobilization can also result in stabilization of the on its cost per unit of product balanced against the
enzyme activity. Such stabilization is especially true for value added. The major factors that determine the
proteinases because autolysis is prevented (12). cost of an enzyme process are listed in Table 2
Moreover, in many food processes, in order to limit (6, 11). For a soluble enzyme process, a major cost is
microbial growth it is desirable to operate at tempera- that associated with its purication. The degree of
tures where structural stability of proteins is marginal purication must be balanced against the desirability
and thus most susceptible to proteolysis. In addition, of a high specic activity. Usually, costs escalate when
the surface microenvironment of the support may sta- additional purication is sought in the higher range of
bilize the enzyme structure; for example, many sup- purity; however, higher specic activities mean less of
Table 2 Major Factors That Determine the Cost of an Enzyme Process
Purication costs Required specic activity

Required removal of contaminating activities
Immobilization costs Characteristics of the immobilization methodology
Amount of enzyme loading
Operation stability Enzyme stability
Amount of enzyme leaching
Resistance of the support to attrition
Regenerative capability of the biocatalyst Number of times the activity can be regenerated
Complexity of the regeneration procedure
Capability of support regeneration
Upstream processing requirements Complexity of the process
Costs of substances added
Sanitation requirements Frequency of required cleaning
Complexity of the cleaning procedure

the enzyme preparation will be required in a process. tions for removal of constituents adsorbed from the
The same considerations apply to an immobilized stock and for sanitizing the bioreactor.
enzyme process. Preparations with higher specic
activities will result in greater loading of active enzyme
and higher activities of the biocatalyst; thus, a smaller IV. IMMOBILIZED ENZYME
bioreactor will be required for the same production PROCESSES THAT HAVE
rate. BEEN COMMERCIALIZED
Characteristics of the immobilization procedure IN THE FOOD INDUSTRY
are very important. In general, the fewer number
of steps required, the lower the costs. However, a
number of factors come into play, including the A. Production of High-Fructose Corn Syrup
effect of the procedure on enzyme activity, the Using Immobilized Glucose Isomerase
efciency of immobilization (percent of the original
activity that becomes immobilized), and the cost of This industry has grown from the initial commercial
the support. production in 1970 by Clinton Corn Pocessing to
The productivity of the biocatalyst, i.e., the amount become the largest industrial use of an immobilized
of product per unit of biocatalyst per half-life, is a key enzyme process in the world (16, 17). Most recent
economic parameter. Both the specic activity and the data indicate that the global annual production of
operational stability of the biocatalyst contribute to high fructose corn syrup is 10 million metric tons dry
this factor. The higher specic activities of immobilized substance(dsb) which is produced with 1500 metric
glucose isomerase biocatalyst currently in use have a tons of immobilized enzyme (J. Shetty, personal com-
much greater (10-fold) productivity than the orginal munication, 1999). Currently, the major producers of
biocatalysts that were simply crosslinked whole cells immobilized enzyme are Novo Industry A/S and
(16; J. Shetty, personal communication, 1999). Most Genencor. The Genencor immobilized enzyme is pro-
commercial immobilized enzymes have half-lives of duced by adsorption of substantially puried enzyme
months or even years and typically are operated on ion exchange resins, crosslinked with polyethylenei-
through several half-lives. Some factors that contribute mine and glutaraldehyde, and granulated by extrusion.
to the operational stability of the biocatalyst are the This immobilized enzyme typically has a productivity
stability of the enzyme, the amount of enzyme leaching of 12,00015,000 kg dsb/kg and a half-life of 80150
(desorption), and the resistance of the support to attri- days (19203600 h) (J. Shetty, personal communica-
tion. Enzymes immobilized by adsorption, such as on tion, 1999). The immobilized enzyme produced by
ion exchange supports, will exhibit a nite desorption Novo (Sweetzyme T) is prepared by crosslinking
rate that depends on environmental conditions, includ- whole cell material from Streptomyces murinus with
ing enzyme characteristics, ionic strength, and tem- glutaraldehyde followed by extrusion (18). A typical
perature. Covalently immobilized enzymes will not bioprocess uses 1.5 m 5 m xed-bed bioreactors
leach from the support but the biocatalyst cannot be operated at 60658C to produce a 42% fructose iso-
regenerated. The operational stability of the bioreactor syrup (16, 17). More than half of this isosyrup is con-
is also affected by the presence of proteinases, either verted to 55% fructose using fractionation technology.
those in the feed stock or those produced by microbial Future development of a thermostable enzyme may
growth. Thus, sanitation of the bioreactor is very allow direct production of a 55% fructose isosyrup
important. For example, immobilized glucose isomer- by performing the isomerization at 958C because the
ase bioreactors are operated at 608C to control micro- equilibrium is shifted in favor of fructose.
bial growth.
Capability of regeneration of the biocatalyst can B. Production of Hydrolyzed Whey Syrups
signicantly lower the cost of the bioprocess per unit Using Immobilized b-Galactosidase
of product. Thus, if the support can be regenerated
many times, even an expensive support can be econom- The current commercial process, Valio Hydrolysis
ical. This feature would favor the use of an adsorption Process, was initiated in Finland in 1980 by Valio
immobilization procedure over the use of a covalent Ltd. (19). The biocatalyst, Valio IML, consists of a
bond. Nevertheless, a strong adsorption interaction is mold b-galactosidase (Aspergillus oryzae) adsorbed
most desirable because less leaching would occur and and crosslinked on a food grade resin (19, 20; M.
the biocatalyst could be cleansed with stronger solu- Harju, personal communication, 1999). The major

product produced by Valio is a demineralized hydro- sized acyl-D,L-amino acids are asymmetrically hydro-
lyzed whey syrup containing 60% solids with 72% lyzed to produce the L-amino acid and acyl-D-amino
hydrolysis of the lactose, Valio Hydroval 80 (M. acid that are easily separated by differences in solubi-
Harju, personal communication, 1999). Hydrolyzed lity. The acyl-D-amino acid is then racemized and
whey syrups without demineralization and with 50% passed back through the bioreactor for resolution.
demineralization are also produced. Whey from Bioreactors are prepared by immobilization of the
cheese-making operations is received in a liquid form enzyme from Aspergillus oryzae by adsorption on
(6% total solids), pasteurized, passed through xed- DEAE-Sephadex. A typical half-life of a bioreactor is
bed immobilized enzyme bioreactors for lactose hydro- 65 days at 508C. Operating as a xed-bed bioreactor,
lysis, demineralized (if desired), and concentrated to the productivity ranges between 13 and 46 kg amino
60% total solids. The half-life and productivity of a acid/L/half-life, depending on the amino acid being
typical bioreactor are 20 months and 2000 kg dry mat- produced. According to Uhlig (17), the annual produc-
ter/kg enzyme, respectively. The annual production of tion in 1988 was 1000 metic tons of Phe, Met, Tyr, and
hydrolyzed whey syrups by this process in Finland, the Val. The overall cost of amino acid production using
United Kingdom and Norway is 50006000 metric the immobilized enzyme was 60% of that for use of the
tons. soluble form (24).
Bioreactors containing b-galactosidase immobilized
covalently on porous silica were developed at Corning
Glass Co. (21) and used commercially (6, 22) in D. Production of Specic Amino Acids
England (Specialist Dairy Ingredient Co.), the U. S.
(Nutrisearch), and France (Union Laitie`re L-Aspartic acid has been produced from ammonium
Normande). The Nutisearch plant used the hydrolyzed fumarate commercially in Japan since 1973 using
whey for production of bakers yeast; however, this immobilized L-aspartate ammonia lyase (aspartase)
plant is no longer in operation. The only plant still in (EC 4.3.1.1.) prepared by entrapment of Escherichia
operation is the SDI plant in the UK owned by Dairy coli cells in k-carrageenan (27). In 1988, 1000 metric
Crest; however, they currently use Valio IML as the tons of L-aspartate was produced (17). The production
biocatalyst. of L-alanine from ammonium fumarate using two
In addition to production of whey hydrolysates, bioreactors with different immobilized cell prepara-
immobilized enzyme also has been used commercially tions was successfully commercialized in 1983 in
to hydrolyze lactose in milk. The yeast (Kluyveromyces Japan by Tanabe Seiyaku Co. (28). The rst bioreactor
lactis) enzyme, with a pH optimum near 7, was contained E. coli cells that were pH-treated to inacti-
entrapped in cellulose triacetate bers in a process vate alanine racemase and fumarase activities and
developed by SNAM Progetti for production of hydro- entrapped in carageenan, and the second bioreactor
lyzed lactose milk by a plant in Milan, Italy (6, 11, 17, contained pH-treated and glutaraldehyde-crosslinked
23). This plant, Centrale del Latte, has a minimum Pseudomonas dacunhae also entrapped in carrageenan.
capacity of 8000 L/day (23). Snow Brand Milk Thus, the rst bioreactor provided the aspartase activ-
Products in Japan also developed an industrial process ity while the second provided L-aspartate b-decarbox-
for production of hydrolyzed lactose milk using the ylase activity. This bioprocess represents the rst
SNAM Progetti immobilized enzyme (23). The industrial use of sequential bioreactors.
Japanese scientists also developed a bioreactor sanita- Because of its demand as a percursor in the produc-
tion process, involving immersion in glycerol at 108C, tion of aspartame (-L-aspartyl-L-phenylalanine
that allowed them to use 60 processing cycles without methyl ester), various methods for enzymatic produc-
an increase in microbial load. tion of L-phenylalanine have been developed (29).
Purication Engineering (Rhone Poulenc) (11,29)
C. Production of L-Amino Acids Using developed an immobilized enzyme bioprocess using
Immobilized Aminoacylase phenylpyruvate as the starting material. A mutant of
E. coli having exceptionally high activities of transami-
This process for enzymatic resolution of D, L-amino nases with the required specicities was immobilized by
acids was developed by Chibata and co-workers at entrapment in a polyazetidine layer on Amberlite IRA-
Tanabe-Seiyaku Co. in Japan, and its commercializa- 938 porous beads. Bioreactors typically exhibited a
tion in 1969 represented the rst industrial use of an half-life>8 months. A 600 metric ton per year plant
immobilized enzyme (6, 2426). Chemically synthe- was constructed (29).

Industrial processes for production of aspartame cess developed by Cheetham (34) in England, Erwinia
using immobilized enzymes also have been developed rhapontici cells were entrapped in calcium alginate gels
(10, 11, 30, 31). The process developed in Japan by and formed into pellets by extrusion. The enzyme
Toya Soda Co. (30) utilized immobilized Thermoase expressed by these cells was specic for sucrose and
(thermolysin) in a biphasic system to stereospecically followed an unusual intramolecular mechanism in
couple N-protected aspartate with D, L-phenylalanine which both the glucose and fructose moieties remain
methyl ester to give -aspartame after hydrogenolysis enzyme bound (34). Operating as a xed-bed bioreac-
to remove the protecting group. The enzymatic process tor, the immobilized enzyme exhibited a half-life of 1
eliminates the need to protect and deprotect the b-car- year and had a productivity of 1500 times its own
boxyl group of aspartate to prevent formation of the weight in crystalline isomaltulose.
bitter isomer b-aspartame and allows use of the less In another process developed at the South Germany
expensive phenylalanine methyl ester racemate. The Sugar Co. Protaminobacter rubrum cells were occu-
technology has been licensed to Holland Sweetener lated and crosslinked with glutaraldehyde (34, 35).
Co., a joint venture between Tosoh Corp. and DSM. Palatinit AG, a subsidiary of South Germany Sugar
More recently, another industrial process has been Co. and Bayer AG, used this process for commercial
reported that uses enzymes in immobilized cells to production of isomaltulose.
make phenylalanine from cinnamic acid and ammonia Using a similar procedure, except that the cells
followed by direct coupling to aspartate (31). entrapped in calcium alginate gels were crosslinked
with polyethyleneimine and glutaraldehyde, Mitsui
E. Production of 5 0 -Ribonucleotides Sugar Co. in Japan commercialized a process using
Serratia plymuthica or P. rubrum cells (34, 36). The
A market exists for 5 0 -ribonucleotides as avor productivity of a xed-bed bioreactor containing the
enhancers in foods and as precursors for pharmaceu- immobilized P. rubrum enzyme was 260 kg/L of bioca-
ticals. A process was developed in Germany by Keller talyst/half-life. The company initiated production with
and co-workers at Hoechst Aktiengesellschaft for a 600 metric ton per year plant in Okayama, Japan.
hydrolysis of RNA using 5 0 -phosphodiesterase immo-
bilized on oxirane acrylic beads (Eupergit C from G. Production of Invert Sugar (Hydrolysis of
Rohm Pharma) (32). Elimination of the necessity of Sucrose)
fractionation of RNA and DNA was accomplished
because DNA could not penetrate the matrix and Invertase immobilized by adsorption on ion exchan-
thus was not hydrolyzed. In a pilot plant operation gers (31) or covalently bound to a macroporous
using crude nucleic acid feed stocks at 608C, the immo- Plexiglas (17), Plexazyme IN (Rohm GmbH), has
bilized enzyme was used continuously for 500 days been commercialized in Europe for hydrolysis of
with no detectable loss of activity and produced 10 sucrose obtained from sugar beets or cane sugar. The
metric tons per year (32). Earlier work in Japan also productivity of the Plexazyme IN bioreactor is 6000 kg
resulted in a process for production of 5 0 -ribonucleo- dry weight/kg biocatalyst when producing a syrup with
tides, including IMP, using a mixed bed of 5 0 -phospho- 90% conversion (17). Enzymatic hydrolysis of sucrose
diesterase and 5 0 -adenylate deaminase immobilized on is less expensive than the HCl chemical process because
porous silica (33). of side reactions in the latter that lower the yield and
decrease product quality. The annual production of
F. Production of Isomaltulose invert sugar using immobilized invertase was 1000
metric tons in 1988 (17).
Isomaltulose (6-O--D-glucopyranosyl-D-fructofura-
nose) is a natural component of honey with a sweetness H. Production of Modied Triacyglycerols (Fats)
one-third that of sucrose. However, it appears to be
noncariogenic and it encourages the growth of Chemically catalyzed interesterication results in ran-
Bidobacter. Several processes for its commercial pro- dom interchange of fatty acids among the three posi-
duction using immobilized enzymes have been devel- tions of the glycerol backbone. However, enzyme
oped (3436). Although only one enzyme, isomaltulose catalysis using a 1,3-specic lipase connes acyl migra-
synthase, is required for conversion of sucrose to iso- tion to the 1- and 3-positions yielding a mixture of
maltulose, normally whole cells were immobilized triacylglycerols not obtainable by any other means.
rather than incur the cost of purication. In the pro- Several commercial processes have been reported for

immobilization of lipase and its use for fat modica- This may be the case for production of modied pro-
tion (3740). Workers at Unilever (37, 39) immobilized teins designed for specic functionality in a foodfor
lipases from Aspergillus, Mucor and Rhizopus spp. by example, modication of the emulsifying, gelling, or
adsorption from acetone, methanol, or ethanol solu- foaming properties of a protein ingredient by limited
tions on inorganic matrices such as diatomaceous proteolysis (14, 15, 44, 45). If the proteolysis is carefully
earth, hydroxylapatite, or alumina and used these pre- controlled, large oligopeptides with native-like struc-
parations in xed-bed bioreactors to interesterify shea ture can be released that have more desirable function-
oil or the mid fraction of palm oil. The substrates were ality than the parent protein (14, 15). Use of an
dissolved in petroleum ether or hexane and passed immobilized proteinase allows precise control of the
through the bioreactor under carefully controlled extent of proteolysis without a downstream enzyme
water concentrations to maximize inter- or transester- inactivation step such as a heat treatment. Because
ication. Transesterication of the midfraction of palm excellent functionality is destroyed by heat treatments
oil with myristic acid produced a cocoa butter substi- that cause unfolding and aggregation, such a down-
tute from these less expensive starting materials (37). stream process is often counterproductive. Another
Scientists at Fuji Oil Co. Ltd, in Japan also have devel- potential example is the alteration of the structural
oped an immobilized lipase process for production of a stability or gelling characteristics of a protein by limited
cocoa butterlike fat using the palm midfraction as a crosslinking using transglutaminase as a catalyst (46,
starting material (41). Similarly, the triacylglycerols in 47). As in the case with proteinases, use of immobilized
shea oil or shea oleine have been modied by interes- enzyme avoids the need for a downstream enzyme
terication using these adsorbed lipases in two-phase inactivation step to prevent eventual gel formation
systems (11, 38). and to stop reaction at the desired level of crosslinking.
Tsukishima Kikai Co. (17, 42) developed a unique Another major economic factor is the cost of
countercurrent two-phase multistage bioreactor for tri- enzyme purication and immobilization. Designing
glyceride hydrolysis using an immobilized thermo- the enzyme for bioselective adsorption can minimize
stable lipase from a Pseudomonas sp. for production this cost. In the future, many enzymes will most likely
of glycerin and fatty acids. Workers at AKZO be recombinant proteins designed with consideration
Chemicals (17, 43) also produced glycerin and fatty of specic catalytic requirements such as specicity,
acids using a lipase from Candida sp. immobilized by thermal stability, pH optima, and cofactor require-
adsorption on high-density polyethylene. The produc- ments. In addition, it would not be difcult to design
tivity of the CSTR bioreactor was 1100 kg of fatty structures allowing ease of purication and immobili-
acid/kg biocatalyst resulting in a cost that was compe- zation. A number of fusion proteins have been geneti-
titive with the cost of steam splitting, and the product cally designed for this purpose. The cellulose-binding
was of superior quality (43). domain of Cellulomonas mi exoglucanase has been
Eigtved (40), at Novo Industri A/S, developed an fused to the gene for b-glucosidase allowing this fusion
immobilized lipase bioreactor that could be used in a protein to bind to cellulose (48). Similarly, the
solvent-free system. Lipase from Mucor miehei was starch-binding domain of Aspergillus glucoamylase
adsorbed from aqueous solution onto macroporous was fused to b-galactosidase, resulting in a fusion
phenolformaldehyde resin and dried to a water content protein that bound to starch (49). The streptavidin-
of 220%. The support has a relatively large particle biotin interaction represents an even stronger bio-
size, allowing it to be employed with melted triglycer- specic interaction with a dissociation constant of the
ides in a xed-bed bioreactor without excessive pres- order of 10-15 M-1. Genetic constructs have been
sure drop. Operating to interesterify liquid fat (olive prepared that allow expression of streptavidin-b-galac-
oil) at 608C, the bioreactor exhibited a half-life of tosidase (50), streptavidin-lipase (51), streptavidin-
3100 h and a productivity of 6.5 metric tons of mod- transglutaminase (52), and trypsin-streptavidin (53).
ied triglyceride/kg of biocatalyst. Biotin can be covalently attached to a variety of sup-
ports using commercially available biotinylation
reagents. Streptavidin-enzyme fusion proteins were
V. FUTURE OPPORTUNITIES puried and immobilized in one-step from crude
recombinant cell lysates (5053). Furthermore, the bio-
A major factor driving the commercialization of an tinylated supports can be regenerated and repeatedly
immobilized enzyme process is the ability to produce used by desorption of spent enzyme and readsorption
a product that cannot be produced by other means. of fresh recombinant fusion enzyme (54).

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enzymatic transesterication. In: A Tanaka, T Tosa, T and characterization of the immobilized fusion pro-
Kobayashi, eds. Industrial Application of tein. Enzyme Microb Technol 22:246-254, 1998.
Immobilized Biocatalysts. New York: Marcel 52. VW Valentine, DA Clare, HE Swaisgood.
Dekker, 1993, pp 337-352. Engineering a protein recombinase: construction and
42. Tsukishima Kikai Co., Ltd Bioprocessing Technol characterization of heterologous fusions of streptavi-
9(4):3, 1987. din and transglutaminase. Am Soc Biochem Mol Biol,
43. C Brady, L Metcalf, D Slaboszewski, D Frank. Lipase Annual Meeting (Abstr B 394), 1998.
immobilized on a hydrophobic, microporous support 53. DA Clare, VW Valentine, GL Catignani, HE
for the hydrolysis of fats. J Am Oil Chem Soc 65:917- Swaisgood. Molecular design, expression, and afnity
921, 1988. immobilization of a trypsin-streptavidin fusion pro-
44. HE Swiasgood, XL Huang, GL Catignani. tein. Enzyme Microb Technol 28:483491, 2001.
Characteristics of the products of limited proteolysis 54. XL Huang, MK Walsh, HE Swaisgood. Simultaneous
of b-lactoglobulin. In: N Parris, A Kato, LK Creamer, isolation and immobilization of streptavidin-b-lacto-
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Interfacial properties of tryptic peptides of b-lactoglo-
bulin. J Agric Food Chem 40:669-675, 1992.

25
Enzymes and Food Analysis
Philip OConnell
National University of Ireland, Cork, Ireland
George G. Guilbault
I. ADVANTAGES AND DISADVANTAGES and enhances the thermal stability of the enzymes.
Limited dynamic ranges can be extended through the
The advantages of enzyme analyses are numerous, as use of diffusion limiting membranes (5). Gibson et al.
are the disadvantages. The advantages of using (6) have employed polyelectrolyte backbones along
enzymes must therefore be weighed against the disad- with sugar alcohols to stabilize enzymes for extended
vantages for each application. periods of time, even at room temperature. Glucose
The benets of using enzyme analyses include rapid oxidase was shown to have an operational stability
assay times (relative to most analytical assays), low for 5 months (7) when modied. With so much effort
cost, inherent selectivity, and simplistic systems. The being focused on improving enzymatic analyses, the
disadvantages include pH and temperature depen- applications of the technology are likely to expand
dence, a limitation to primarily aqueous analysis, lim- throughout the coming decade.
ited dynamic ranges, and relatively low stability. Thus,
enzymatic analyses are conned to those areas where
the advantages outweigh the disadvantages.
In recent years much work has been focused on II. METHODOLOGIES AND TOOLS
overcoming the shortcomings mentioned above and AVAILABLE
hence widening the applicability of enzymes in ana-
lyses. It has been shown that pH and temperature In science, one of the main methodologies is standar-
dependence of enzymes is signicantly reduced when dizationto be able to compare different techniques
immobilized; this is probably due to the enforced struc- or products to a standard. However, in a relatively new
ture induced by immobilization (1). Thus, the use of area such as enzymatic analyses, such standards are
immobilization can increase the ruggedness of most rare. McAteer et al. (8) have proposed a model for
enzymatic assays. Several groups (24) have focused shelf life prediction of stabilized commercial enzyme
on the use of enzyme sensors in organic media. They assays. Being able to place a well-dened shelf life on
found that when using organic solvents their sensors a product would make it much more viable in the
could still work well, but that the enzyme response was commercial marketplace. Boujtita et al. (9) have con-
dependent on both the concentration and type of sol- centrated on making biosensors applicable to indus-
vent. Such technology allows the detection of enzy- trial needs. They compared different modes of
matic substrates sparingly soluble in aqueous media production of renewable carbon paste electrodes and

found that they can produce high analytical quality (20) and sol-gels. Other methods include encapsulation
sensors. of the enzyme within a growing polymer (21), entrap-
Enzymes can be coupled to a myriad of detection ment behind membranes (22), encapsulation within
techniques; however, some are more appropriate to gels (23), entrapment within carbon pastes (24), and
food analysis than others. Commonly used tech- passive adsorption of the enzyme on a suitable surface
niques include visible spectrophotometry, lumine- (25). Sol-gels have become important owing to the ease
scence, uorescence, amperometry, and potentiometry. with which they are prepared and because they have
Thus, development of the tools of these assay tech- controllable porosity, are chemically inert and physi-
niques has taken place. Examples of each system cally stable, and can be produced at low temperatures
used in conjunction with food analysis are shown in under mild conditions (26). Another important prop-
Table 1. erty is that they can be produced as a bulk-modied
For most food samples, some pretreatment would electrode (27), providing simple renewal of surface by
have to take place. Solid samples would generally be polishing, or as a coating of electrodes (28), where
dissolved or the analyte extracted from them. This is rapid responses are seen and a high enzyme loading
particularly the case for optical detection systems can be achieved. This makes sol-gel-based enzymatic
where solid particles can seriously disturb the response. assays very versatile.
The importance of pretreatment cannot be underem-
phasized, even in an area such as enzyme analysis
where the need for pretreatment is minimized. The B. Covalent Binding
strong pH, ionic strength, and temperature dependence
of most enzymes means that any pretreatment must be The use of glutaraldehyde as a crosslinking agent is
carefully considered before being applied to enzymatic perhaps the most common procedure in this area.
analysis. This is due to the simplicity of the procedure. The
In electrochemical analysis, a major challenge has three-dimensional structure produced allows a high
been immobilization, to make the sensors reusable. enzymatic loading. Coupling to activated surfaces
Several techniques have been developed for immobili- (29) produces a more reproducible surface coverage
zation, but generally speaking they can be classied but at the expense of decreased enzyme loading.
into two major groups. Other methods for covalent immobilization of enzymes
include silanization (30) and multipoint covalent
immobilization on epoxy-activated supports (31).
A. Adsorption or Physical Entrapment Each of these immobilization methods succeeds in
making the associated assays reusable, but most
This method has become more important in the last decrease the activity of the enzymes and change their
decade owing mainly to the development of hydrogels analytical performance such as pH optima, selectivity,
Table 1 Direct Enzyme Assays Using Various Transduction Techniques
Analyte Detection method Enzyme Dynamic range Ref.
Cyanogenic potential Absorbance at 510 nm/linamarin Linamarase 0.540 mg/kg of HCN 10

Asparagine Absorbance/pH indicator Asparaginase 0.22.3 mM 11
Glucose Fluorimetry/FAD GOxa 0.52 mM 12
Lactate Fluorimetry/FMN LMO 1.0410 mM 13
Mannitol Fluorimetry/NADPH MDH/LDH 501500 mM 14
Lactate Luminescence/luminol-H2 O2 LOx 0.15 nM0.3 mM 15
Sulte Luminescence/luminol-H2 O2 SOx/HRP 3.2320 mM 16
Biogenic amines Amperometry/H2 O2 DAO 150 mM 17
Fructose Amperometry/O2 redox polymer FDH 0.220 mM 18
Asparagine Potentiometry/H Asparaginase 0.14 mM 11
Formaldehyde Potentiometry/H AOx 5200 mM 19
a
Glucose oxidase (GOx); lactate monooxygenase (LMO); lactate dehydrogenase (LDH); malate dehydrogenase (MDH); lactate oxidase (LOx);
sulte oxidase (SOx); horseradish peroxidase (HRP); diamine oxidase (DAO); fructose dehydrogenase (FDH); alcohol oxidase (AOx).

and temperature dependence (1). Enzyme kits, of III. TYPES OF ANALYSIS POSSIBLE
which there are a wide variety (32), ignore this problem
and use enzymes directly in solution. However, this can Enzymes are already used in a variety of assays of
make the assay expensive if the enzyme cannot be foods. These can be generally classied into three
produced cheaply enough. Kits are available for a main areasdirect, indirect, and inhibitionas
wide variety of analytes, but most require optical shown in Figure 1.
detection by eye or through a spectrophotometer. Direct enzyme assays use the specicity of an
Thus, only colorless and fully soluble samples can be enzyme toward the analyte to determine the concentra-
assayed with this technique, which makes food analysis tion of the analyte or enzyme. An example of this is a
difcult. glucose assay, using glucose oxidase to oxidize the glu-
Figure 1 Schematics of direct, indirect, and inhibition enzyme assays. Example system 1 (a) is a direct enzyme assay; system 2
(b) is an ELISA, an indirect assay; and example 3 (c) is an inhibition assay.

Table 2 Summary of Sugar Analysis Through Enzymatic Methods
Analyte Detection method Enzyme Application Ref.
Glucose Spectrophotometric GOxa Fruit juices, soft drinks, fermentation processes 33

Glucose Amperometry (0.7 V) GOx Fruit juices, soft drinks 34
Galactose Amperometry (0.1 V) GaOxa/HRP Dairy products 35
Fructose Amperometry (0.1 V) FDH Honey 18
Sucrose Amperometry Invertase/GOx Juices 36
mutarotase
Lactose Potentiometry (CO2 ) Lactase/S. cerevisiae Milk 37
a
Galactose oxidase (GaOx).
cose in a sample and produce a measurable response. A. Direct Enzyme Analysis

Table 2 shows a brief list of direct enzyme assays.
Indirect enzyme assays use enzymes as a label. This Direct enzyme analysis is the simplest of the three
means that the assay uses only the catalytic properties areas. It can only be used to quantify substrates and
of an enzyme to produce an easily measured response, enzymes, and is only practical when the products are
achieving specicity through another molecule. An readily measurable. Many modications have been
example of this type of assay is ELISA, where speci- made to direct enzyme analysis, some to expand the
city is provided by antibodies but the nally measured applicability of the system, and some to overcome pro-
signal is produced by an attached enzyme. blems in the analysis.
Inhibition assays use the inhibitory effects of certain
molecules or ions on enzymes to quantify their pre-
1. Simple Substrates Analysis
sence in samples. Heavy metals are known to inhibit
most enzymes but to different extents depending on the The main applications of this system are the assays of
enzyme and metal ion involved. Organophosphorous chemical components of foods, such as sugars (Table
and carbamate pesticides inhibit certain cholines- 2) and organic acids (Table 3). This is because there are
terases. Depending on the pesticide and enzyme cho- many enzymes available for these metabolic com-
sen, the extent of inhibition varies. pounds. Given the inherent instability of most
Table 3 Summary of Organic Acid Analysis Through Enzymatic Methods
Lactate Amperometry LOxa/HRP Wine and yogurt 38

(0 V)
Pyruvic acid FOB LDH Wine, beer, and milk 39
Formate FOB FDH/LDH Fermentation of milk 14
Malate Amperometry MDH/Diaphorase Wine 40
(0.3 V)
Glutamate Amperometry GluOx Flavor enhancers 41
(0.65 V)
Ascorbate Amperometry (0.4 V) AscOx Fruit juices and pharmaceutical 42
preparations
Uric acid Amperometry (0.1 V) UOx Fish freshness 43
Inosine monophosphate Amperometry IMPDH/NOx Flavor enhancers 41
(0.65 V)
a
Lactate oxidase (LOx); glutamate oxidase (GluOx); ascorbate oxidase (AscOx); urate oxidase (UOx); inosine monophosphate dehydrogenase
(IMPDH); NADH oxidase (NOx).

enzymes, these analyses are performed with a reference bioprocess. This is achieved by selecting the key ana-
of known analyte concentration. Such assays are rarely lytes in a given system. To dene the quality of wine,
applicable to analytes not commonly found in meta- Miertus et al. (47) developed a multianalyte sensor for
bolic processes. An example of such a rare assay is the glucose, fructose and ethanol and for lactate, malate,
organophosphate pesticide sensor. Using organopho- and sulte. The sensors were grouped in this way to
sphorus hydrolase, Mulchandani et al. (44) developed allow different fermentation processes to be analyzed.
a biosensor for the determination of organophosphate The rst group would allow monitoring of any alco-
pesticides with no crossreactivity with carbamates or holic fermentation process, while the second could be
other neurotoxins. They achieved a detection limit of 2 used to monitor the malolactic fermentation processes
M for several common pesticides. in red wine.
2. Enzyme Sequence Reactions 4. Simple Enzyme Analysis

Systems employing a sequence of enzyme reactions can It is possible to detect the presence of an enzyme by
be used to increase the ease with which certain enzy- exposure to its substrate. This system is employed in
matic products can be assayed. Peroxidase is fre- metabolic detection, where the metabolic action of
quently employed to detect the peroxide product of bacterial enzymes indicates their presence in a sample.
oxidase reactions at low applied potentials (45), thus It is especially useful to determine the efciency of
avoiding electrochemical interferences that may be pre- sterilization procedures such as pasteurization. Swain
sent in a sample. Difculties with these systems include et al. (48) used this type of assay to detect foodborne
the possibilities that the optima for performance of the pathogenic bacteria.
enzymes in the sequence may not be the same and that
multiple enzymes decrease specicity. Such systems are 5. Substrate Recycling
therefore only used for important analytes such as
Another modication of the direct analysis method is
sucrose (36) and aspartame (46) or to overcome
substrate recycling (enzyme amplication). This pro-
major problems, such as electrochemical interference
cess can dramatically enhance the sensitivity of enzyme
(45). Sucrose is hydrolyzed with the enzyme invertase,
assays but, as it uses multiple enzymes, it suffers from
where the resultant glucose can be detected with glu-
the same limitations as the enzyme sequence reactions
cose oxidase. Mutarotase is commonly added to such
described above. One example of this system is the
systems to speed up the conversion of -D-glucose to
detection of malate (49). In the presence of malate
-D-glucose.
(analyte) and oxygen, lactate mono-oxygenase pro-
duces oxaloacetate. Malate dehydrogenase then con-
3. Multianalyte Systems
verts this back to malate, where the sequence starts
With direct enzyme sensors it is relatively easy to pro- again (Fig. 2). While this simple procedure amplies
duce multianalyte assay systems, which can detect a the response to malate, it also makes the assay strongly
range of analytes simultaneously. The main advantage susceptible to interference from lactate. Table 4
of this is that the analyst can build up an accurate describes examples of assays for miscellaneous food
picture of the current state of the food product or analytes using direct analysis.
Figure 2 Schematic of a simple enzymatic amplication system for malate using an oxygen sensor.

Table 4 Summary of General Food Analytes Through Enzymatic Methods
Ethanol Amperometry (0.7 V) ADHa Alcoholic beverages 50

Polyphenols Amperometry for O2 (0:7 V) Laccase Catechols in tea 51
Glycerol Amperometry (0.65 V) GK/GPO Alcoholic fermentation 52
Lysine Amperometry Lysine oxidase Milk and pasta 53
Lecithin Amperometry for O2 (0:7 V) PL/COx Soybean our and chocolate 54
Asparagine Spectrophotometric Asparaginase Fruits and vegetables 11
Aspartame Amperometry for O2 (0:7 V) CT/AOx Articial sweeteners 46
a
Alcohol dehydrogenase (ADH); glycerokinase (GK); glycerol-3-phosphate oxidase (GPO); phospholipase D (PL); choline oxidase (COx);
a-chymotrypsin (CT)
B. Indirect Enzyme Analysis Is Composed of limit of detection (LOD) of 100 cells/mL. Ivnitski et al.
Many Approaches (63) have reviewed the different techniques used for
bacteriological detection using immunosensors.
1. Immunoassays
It can be difcult to detect the presence of pesticides
The most commonly used indirect technique is ELISA, using immunoassays. Owing to their small size, the
or enzyme-linked immunosorbant assay. An ELISA antibodies are raised against pesticides conjugated to
system can be designed to detect any molecule that immunogenic carriers and may thus be of low afnity/
can produce an immunogenic response, and many selectivity. Keay and McNeil (64) used an ampero-
molecules that dont produce one on their own can metric immunosensor to detect atrazine.
be modied to generate a response. This makes the Polychlorinated biphenyls (PCB) are found through-
technique very versatile. While other biorecognition out the food chain (sh extracts, clams, milk), and
molecules are available, such as DNA, they are not Bender and Sadik (65) produced an electrochemical
generally coupled to enzymes. The enzyme labels are immunosensor to detect for them. Dankwardt and
selected very carefully to fulll dened parameters. The Hock (66) reviewed the use of immunoassays to detect
enzyme must be cheap, highly puried, easy to conju- pesticides in foods and discussed the use of immuno-
gate to antibodies, and have colorless substrates with a sensors.
highly colored product. The main enzymes employed Marine toxins are an area of increasing interest, as
are horseradish peroxidase and alkaline phosphatase their incidence of outbreaks has been rising in recent
(55). They both meet the above requirements, but to years. Kreuzer et al. (67) developed an assay for oka-
different degrees. Given the versatility of ELISA, it is daic acid in shellsh. This diarrhetic toxin was detect-
not surprising that a large number of food assays have able down to a concentration of 6 1013 M in a rapid
been based on it. Primarily these have been focused time of 90 min, far better than any HPLC system for
upon the pathogenic bacteria and pesticides in foods, okadaic acid. Also, a rising issue in recent times has
which can be difcult to detect using traditional tech- been the use of genetically modied (GM) foods. Brett
niques. et al. (68) have concluded that antibody-based assays
Bacteria analyzed by ELISA include Listeria mono- are the natural detector for the presence of novel pro-
cytogenes (56), Salmonella sp. (57), Escherichia coli teins from GMs. Van Duijin et al. (69) have used
(58), Staphylococcus aureus (59), and Bacillus cereus immunoblotting to detect the presence of CP4 synthase
(60). The popularity of the method is due to the sensi- in GM soya. Thus, ELISA and immunosensors could
tivity of the technique, which can yield results much become powerful tools in the conrmation of GM pro-
faster than conventional techniques. Crowley et al. (61) ducts in our foods.
developed a rapid immunosensor based on ampero-
metric detection for Listeria monocytogenes in milk
2. Induced Bioluminescence
samples. The assay was performed in 3.5 h, which is
an improvement over the 24 h needed to complete even Another type of indirect enzyme analysis is the use of
the most rapid of traditional analysis. Abdel-Hamid et luciferase-encoded bacteriophage. The bacteriophage
al. (62) developed a sensor for E. coli O157:H7 with a infect specic bacteria present in the sample and

cause them to bioluminesce by forcing them to produce this medium and the low solubility of the enzyme,
luciferase. A major advantage of this system, unlike thus increasing the ease of immobilization.
ELISA, is that it distinguishes between viable and non-
viable cells. Salmonella sp. (70) and Listeria were 2. Heavy Metal Analysis
assayed (71). Loessner et al. (72) produced a system
Fennouth et al. (78) compared the effectiveness of
for the detection of a variety of Listeria strains.
heavy metal salts at inhibiting the activity of lactate
dehydrogenase. They found that they could detect
C. Inhibition Enzyme Assays the presence of heavy metals below the European
norms, with the exception of lead acetate. They con-
There are two main groups of inhibition enzyme cluded that such technology could be applicable to
assays, pesticides and heavy metals. metal contamination in foods. Bertocchi et al. (79)
found that they could detect mercury levels in the
1. Pesticide Analysis 1060 ppb range using enzyme inhibition. Volotovsky
et al. (80) showed that additions of NaI to the sample
Since pesticides are not normally present in the food
improved the selectivity of their urease-based mercury
chain, there are few specic enzymes that can be used
sensor by decreasing the effects of silver.
for their determination, although nonspecic inhibi-
tion of cholinesterase is commonly used (73). The
determination of pesticides in food and water is a par- IV. ONLINE ANALYSIS
ticular area of interest for biosensors. This is due to the
function of pesticides to inhibit cholinesterases in pests The use of enzymatic methods for continual and quasi-
(and possibly mammals). Organophosphates have been continual analysis is a challenge. Continual measure-
particularly well examined as they are toxic to mam- ments require the enzymatic performance to be con-
mals. Carbamates are less toxic to mammals but are stant over a set period of time. Most enzyme assays
not very soluble in water and thus accumulate in foods cannot meet this challenge. A more applicable use
if incorrectly used. Cholinesterase inhibition is used for would be quasi-continuous analysis, where samples
the determination of both these pesticides. Nunes et al. are sent to an enzymatic detector at set time intervals.
(74) used a sensor based on a low charge of cholines- These intervals can be set by the need of the produc-
terase to determine carbamates in food samples. As tion process, but they are limited by the baseline recov-
with all sensors of this type, the sample solutions ery of the sensor. Flow injection analysis (FIA)
must be exposed to the sensor prior to the analysis. provides a rapid means of sending a sample plug to a
This slows down the analysis time. Their sensor is sensor, but after each analysis there is a recovery per-
usable only once (74). Overcoming this problem iod when excess sample is washed away and the
would reduce the cost per assay. Jeanty and Martin response returns to baseline (81). A simple ow injec-
(75) used a reactivation sequence in their pesticide tion system is shown in Figure 3. Such systems have
assay system. Based on ow analysis, their system two major classeswhere the enzyme is contained in a
showed good operational stability of 3 weeks and 6 reactor and the product is detected downstream at a
months storage stability. transducer, or where the enzyme is immobilized near
The inhibition of cholinesterases by both organo- the surface of the transducer. The needs of the fermen-
phosphates, carbamates, and other neurotoxins causes tation industry have led to the development of ow
problems for those who wish to measure just one class injection sensors for sugars (82) and ethanol (83).
of pesticides. To achieve class specicity Mulchandani The versatility of this technique led to its widespread
et al. (44) used organophosphorus hydrolase as use in enzymatic analysis (84, 85).
described in the direct enzymatic assay section. Wang
et al. (76) showed that a tyrosinase-based sensor could
be inhibited by carbamates as well as other phenolic V. QUALITY CONTROL
compounds. They also showed that tyrosinase did not
require the lengthy exposure periods that characterize Production processes require quality control. The
most enzyme inhibition assays. Perez Pita et al. (77) quality of food going to the consumer must always
used this system in conjunction with a reversed micelle be assured. Traditionally this was performed by taking
as the working medium. The advantages of this system a sample and sending it to a remote laboratory. While
include the increased solubility of the carbamates in this is successful for most samples, this was not always

Figure 3 Schematics of a simple ow injection analysis system for use with an enzyme reactor or enzyme coated sensor
(biosensor)
the case. Having remote laboratories means that the of protein is of primary importance in determining
results are not instantaneous. It also means that nutritional quality. Hsu et al. (88) developed a multi-
unstable samples may have changed by the time they enzyme technique for estimating protein digestibility.
are analyzed. Enzyme-based systems, especially those
incorporated within ow injection analyzers, overcome
this problem. As the systems are small and of low cost
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26
Recent Advances in Enzyme Development
Dominic W. S. Wong
I. INTRODUCTION and substantiates the potential of their future roles in

enzyme technology.
The important role of enzymes in food and food sys-
tems is aptly underscored by the description of some 60
enzymes in this handbook. These encompass all major
II. EXTREMOPHILIC ENZYMES
six classes of enzymesoxidoreductases, transferases,
hydrolases, lyases, isomerases, and ligasesthat cata- A. Search of Enzymes from Exotic Environments
lyze various reactions contributing to the characterisitc
features and quality attributes of a particular food. A majority of the currently known 4000 enzymes
Food scientists investigate the fundamentals of enzy- have been isolated from mesophilic organisms. In
mology, extract the information obtained from bio- terms of microbial diversity, < 1% of the world
chemical studies, and apply them to the use and microbial species have been identied and cultured
control of enzymes for preserving and producing better in laboratories. These enzymes have evolved to func-
foods. Of the 4000 known enzymes, only a handful tion under a narrow range of pH, temperature, ionic
of them are currently used in industry, 45% are for strength, pressure, and chemical concentration, and
food processing (Table 1). The future of enzyme tech- are not optimized for performance under industrial
nology foresees an increasing use of recombinant DNA conditions. Considerable efforts have been devoted
technology and biotechnological processes for the dis- to harvest organisms, mostly in the Eubacterial and
covery and development of novel enzymes and for Archaeal phylogenetic domains (1), that thrive in
better utilization of our food resources. Four research extreme environments for enzymes with industrial
areas of emerging importance are described in this compatible properties. Major types of extremophiles
chapter: The study of extremophilic enzymes will include: thermophiles (found in hot spring, deep-sea
change the landscape of our current knowledge on thermal vents); psychrophiles (Arctic water, Antarctic
the use of enzymes; the rapid advances in developing Circle); acidophiles (geothermal sulfur-rich springs,
novel techniques in protein engineering provide an ever coal deposits); alkalophiles (alkaline lakes, sewage
increasing powerful tool for tailoring enzyme struc- sludge); halophiles (salt lakes, mines); and barophiles
tural and functional properties; the ability to mani- (deep-sea oor). Crystal structures are available for a
pulate metabolic enzymes in biosynthetic pathways number of thermophilic and psychrophilic enzymes,
provides new opportunities for in vivo modication and some of these results provide the current infor-
of food components; and the recent development of mation on factors that might stabilize them at
innovative approaches in the construction of articial extreme temperatures, compared with their mesophilic
enzymes results in much improved catalytic activities, counterparts.

Table 1 A List of Important Industrial Enzymes and Their Applications
-Amylase
Conversion of starch to dextrins (liquefaction) in the production of corn syrup
Supplement to our types low in -amylase to ensure a continuous supply of fermentable sugar for yeast growth and gas
production in dough making
Solubilization of adjuncts (nonmalt carbohydrate materials from barley and other cereal grains) used in brewing
Glucoamylase
Conversion of dextrins to glucose (saccharication) in the production of corn syrup
Conversion of residual dextrins to fermentable sugar in brewing for the production of light beer
-Amylase
Production of high-maltose syrup
Xylose (glucose) isomerase
Isomerization of glucose to fructose in the production of high-fructose corn syrup
-Glucanase
Breakdown of -glucans in malt and other raw materials to aid ltration of wort after mashing in brewing
Lipase
Enhancing avor development and shortening the time for cheese ripening
Production of specialty fats with improved qualities
Production of enzyme-modied cheese/butter from cheese curd or butterfat
Papain
Used as meat tenderizer
Used in brewing to prevent chill-haze formation by digesting proteins that can otherwise react with tannic substances to
form insoluble colloid particles.
Chymosin
Curding of milk by specic proteolytic action on -caseins in cheese making
Microbial proteases
Processing of raw plant and animal proteins
Production of sh meals, meat extracts, texturized proteins, and meat extenders
Pectinase
Treatment of fruit pulp to facilitate juice extraction and for clarication and ltration of juice
Lactase
Additive for dairy products for individuals lacking lactase
Breakdown of lactose in whey products for manufacturing polyactide
Acetolactate decarboxylase
Reduction of maturation time in wine making by converting acetolactate to acetoin
(In the absence of the enzyme, acetolactate is oxidized to diacetyl that requires secondary fermentation to reduce it to
acetoin)
Lysozyme
Antimicrobial preservative
Glucose oxidase
Conversion of glucose to gluconic acid to prevent Maillard reaction in products caused by high heat used in dehydration
Potential use to remove O2 in food packaging for protection against oxidative deterioration
Cellulase
Conversion of cellulose wastes to fermentable feedstock for ethanol or single-cell protein production
Source: Ref. 24.
Protein stability can be expressed in terms of the the folded state. Macrounfolding represents the
free energy, associated with the micro- and macrostability of the macroscopic state of the protein
unfolding of the protein. Microunfolding represents molecules and the energy required for the protein
qualitatively characteristics of the rigidity of the changing from the native to the unfolded macroscopic
protein structure, and is associated with local, state (2). Adjustment of conformational exibility is a
reversible, noncooperative unfolding reactions within key step in the thermal adaptation of proteins.

Zavodszky et al. (3) compared both the macrostability nica) (13), and triosephosphate isomerase (psychro-
(DSC and CD measurements) and microstability philic bacterium Vibrio marinus) (14). The Antarctic
(hydrogen H/D exchange and FT-IR) of Thermus bacteirum A. haloplanctis secretes a Ca2 and Cl -
thermophilus (a hyperthermophilic microrganism) and dependent -amylase composed of 453 amino acids
E. coli 3-isopropylmalate dehydrogenase (IPMDH), with a predicted Mr of 49,340 and a pI of 5.5 (15).
and conrmed the correlation between enzyme activity The enzyme is synthesized as a preproenzyme com-
and conformational exibility. The thermophilic prised of a 24-residue N-terminal signal peptide, and
IPMDH has increased conformational rigidity as a 192-residue C-terminal propeptide which contains
compared to the mesophilic counterpart, so as to features common to -pleated transmembrane proteins
stabilize the protein against heat denaturation. (16). The amino acid sequence displays 66% similarity
However, the two enzymes show similar conforma- with procine pancreatic -amylase. It is characterized
tional exibility at their corresponding physiological by a seven fold higher kcat and kcat =Km values at 48C
temperature optima. This thermometer effect has and a lower conformation stability with the difference
been previously reported by Wrba et al. (4) on the in free energy of 1.9 kcal mol1 (17). Homology mod-
study of D-glyceraldehyde-3-phosphate dehydrogenase eling reveals several features responsible for the more
isolated from Thermotoga maritima. Maes et al. (5) exible, heat-labile conformation of the psychrophilic
compared the crystal structure of triosephosphate -amylase: the lack of 12 surface salt bridges by repla-
isomeraes (TIM) from Thermotoga maritima with a cement with Gln or Asn residues; substitution of 25
series of nine known TIM from a wide variety of residues in the hydrophobic clusters of the porcine
organisms, and did not nd a common theme in the enzyme, causing a sharp decrease of the hydro-
adaptive mechanism for the temperature scale. phobicity and increase in exibility; and deletion or
Current structural investigations indicate that meso- substitution of 12 proline residues in loops or turns.
philic and corresponding thermophilic proteins are These ndings seem to substantiate the concept of
virtually identical in structure with the difference in local exibility as an adaptation to thermal stability
free energy of stabilization in the order of 515 kcal by extremophiles. The treatment of thermodynamic
mol1 (6). This small energy difference is equivalent to parameters of activation as a structurefunction
only a few hydrogen bonds, salt bridges, or hydro- model accounting for local exibility for acquiring
phobic interactions (7). Proteins may have achieved high activity at low temperature has been described
increased thermostability by various combinations of by Lonhienne et al. (18).
these additional interactions. Delboni et al. (8)
compared the crystal structures of six known thermo- B. Design Enzyme Stability
philic and mesophilic TIM enzymes, and found that
1. Immobilization
hydrophobic stabilization contributes to the thermo-
stability of the Bacillus stearothermophilus TIM. The Until the advent of genetic modication, immobiliza-
enzyme shows increased hydrophobic packing with the tion was routinely used for improving the stability of
smallest number of cavities and the total cavity surface enzymes in industrial processing. Immobilization
area. The enzyme also has the largest hydrophobic anchors the enzyme onto a suitable carrier such as
surface area (314534 A) buried in the dimer formation agarose, alginate, gelatin, or polyacrylamide either by
compared with other TIM structures, with an energy covalent coupling, adsorption, matrix entrapment, or
gain of 713 kcal mol1 . It was also shown that the B. encapsulation. The primary advantage of immobiliza-
stearothermophilus TIM contains 13 prolines, exceed- tion is to increase the stability of the enzyme, as well as
ing all other TIM enzymes, suggesting that the large convenience in separation and recovery, and reuse of
number of Pro residues are responsible for thermo- the enzyme from the product mixture. In the manufac-
stability by decreasing the entropy of the unfolded ture of high-fructose corn syrup, glucose isomerase is
state. Similar effects have been achieved by introducing immobilized by direct crosslinking of lysed cells with
disulde bridges, for example, into subtilisin (9) and glutaraldehyde and extruded with structural support
lysozyme (10). substances, such as gelatin and polyamine (19).
The crystal structure of several psychrophilic Although not directly relevant to food applications,
enzymes has been elucidated, including that of -amy- immobilization of enzymes has been shown to have
lase (Antarctic Alternomonas haloplanctis) (11), subti- higher stability in reactions carried out in water-
lisin (Antarctic Bacillus TA39 and TA41) (12), trypsin miscible organic solvents (20). Apart from the conven-
(Atlantic salmon, Antarctic Paranotothenia magella- tional approaches, Stempfer et al. (21) engineered a

fusion protein of -glucosidase containing a hexa-argi- mutations to increase the rigidity of the molecule,
nine polypeptide stretch at the C-terminal end. The including Ala69!Pro and Ser65!Pro. The resulting
cationic peptide extension provides a strong, noncova- mutant enzyme shows a dramatic increase in stability;
lent attachment of the fusion protein to a polyanion it is 340 times more stable than the wild type in terms
matrix. Ureda and Tanaka (22) have constructed a of the half-life time at 1008C. All these results suggest
genetic system to immobilize enzymes in their active the importance of considering the balance between
and functional forms on the cell surface of favorable and unfavorable factors involved in engi-
Saccharomyces cerevisiae. The display of both CM- neering structural changes.
cellulase and -glucosidase on the surface of S. cere-
visiae results in digestion of cello-oligosaccharides as
the sole carbon source. Likewise, coimmobilization of III. CREATING NOVEL ENZYMES BY
glucoamylase and -amylase on cell surfaces has been DIRECTED EVOLUTION
shown to digest soluble starch as the sole source of
carbon for yeast growth (23). In the use of rational design to engineer a proteins
structural and functional properties, the change in
the amino acid sequence is preconceived based on a
2. Protein Engineering
prior knowledge of the structural and mechanistic
With the advent of recombinant DNA technology, details of the protein. In spite of some remarkable suc-
enzymes have been tailored by manipulating the cesses, prediction of the outcome of a particular amino
genetic code to target specic amino acid substitutions. acid substitution remains largely empirical, and expla-
Site-directed or cassette mutagenesis is rapidly becom- nations for the effects of site-directed mutagenesis are
ing a routine approach in many investigations. done in hindsight, simply because there is an enormous
Subtilisin, lysozyme, trypsin, and amylases are gap in our ability to correlate sequence structure and
among some of the most extensively studied enzymes function. A novel approach based on the concept of
as models for enhancing various enzyme properties combinatorial chemistry has revolutionized the way to
(24). Considerable work has been conducted on the engineering protein molecules. Initially, the concept of
contribution of disulde bridges to the stability of pro- directed evolution was utilized in generating RNA
teins by decreasing the conformational entropy in the molecules with novel binding and catalytic activities
unfolded state. In T4 lysozyme, a single change of (31). Libraries of RNA can be generated by in vitro
Ile3 !Cys creates a disulde bridge between the new transcription of DNA by PCR with random priming.
Cys3 and the native Cys97, resulting in enhancement A selection step is applied to the RNA pool to isolate
of stability against irreversible inactivation (25) as well variants possessing the targeted function/property. The
as reversible thermal unfolding (26). Matsumura et al. few selected RNA molecules are subjected to iterative
(10) have shown that mutants containing three con- cycles of amplication, mutation, and selection until
structed disulde bonds (397, 9164, 21142) exhibit the best-t molecule is identied. The use of this con-
an additive effect with Tm of 23.48C higher than the cept for evolving proteins has been realized with the
wild type. However, mutants containing Cys90-Cys122 development of the DNA shufing technique (32) and
and Cys127-Cys154 are less stable due to the introduc- subsequent rening and expansion of this technique.
tion of strains by the new bonds. The method involves random digestion of a pool of
It has been known that salt bridges play an impor- DNA sequences (typically amplied error-prone PCR
tant role in protein stability. The introduction of products of a gene of interest) with DNaseI to a mix-
His31-Asp30 in T4 lysozyme stabilizes the protein by ture of small DNA fragments. These fragments are
35 kcal mol1 (27). Engineering Ser38!Asp and reassembled by DNA polymerase, relying on self-prim-
Asn144!Asp at the N-terminal end of the helices B ing of homologous end sequences, and in the process,
and J increases stability with G of 1.6 kcal mol1 recombination or crossover occcurs by template
(28). Some investigations indicate that the contribution switching. This DNA shufing step is followed by
to stability from an engineered salt bridge is very small selecting the recombinant libraries for the best combi-
( 0:10.25 kcal mol1 ), which is largely offset by the nation of mutations yielding the targeted properties
entropic cost of localizing the ion pair on the surface of (33).
the enzyme (29). Van den Burg et al. (30) constructed a Modication and expansion of the original DNA
thermolysin-like protease mutant with a disulde shufing concept have been continuously developed
bridge (Asn60!Cys with Gly8!Cys), and several and reported. One of the adaptations, called family

shufing, allows two or more related genes to be used as expression system, while yeast is the choice for working
the starting genetic materials (34). This approach is with eukaryotic genes. Various techniques have been
based on the assumption that naturally occurring developed to allow the generation of a library of pro-
genes in a family of species are already enriched for tein molecules to be displayed (anchored) on the cell
functional diversity by selection in the natural evolu- surface of bacteria (42), phage (43), or yeast (44) as a
tion process. A simplied method for in vitro recombi- fusion protein. Currently, there are two cell-free sys-
nation, called staggered extension process (StEP) tems developed for in vitro selection and evolution of
recombination, eliminates the steps of DNaseI diges- proteins. Tawk and Grifth (45) showed that in vitro
tion and reassembly of DNA fragments (35). The key transcription/translation apparatuses can be incorpo-
to this method is to employ very short extensions in the rated with single genes into reverse micelles, a cell-like
PCR reaction, so that in the following cycle, denatura- compartment that links genotype to phenotype. In
tion will free the growing fragments to anneal to differ- robosome display, in vitro transcription and transla-
ent templates. Kikuchi et al. (36) developed an effective tion are performed on ribosomes, with a number of
family shufing method using single-stranded DNA of modication steps to retain the expressed protein on
two or more related genes to avoid the problem of the ribosome after translation (46, 47). The ribosome-
homoduplex formation caused by the annealing of bound proteins are screened and reverse transcription
DNA fragments derived from the same gene. and PCR of the mRNA produce the DNA sequence of
In a greater deviation from the Stemmers shufing the corresponding protein. Mutations in sequence can
method, Coco et al. (37) developed a method called be introduced during amplication to generate a new
random chimeragenesis on transient templates, which population in the next cycle of protein synthesis and
involves separating the gene homologs into single- selection.
stranded DNA, digesting one strand into fragments Directed evolution has been successfully employed
which will align with the other strand as the template. in numerous investigations to engineer various aspects
Unhybridized aps are removed, and gaps are lled to of structurefunction properties of proteins and
generate a full-length heteroduplexed gene. The enzymes (48). However, the focus has been primarily
parental template is destroyed by uracil-DNA-glycosy- on the improvement or alteration of existing proper-
lase treatment, and is replaced by a homoduplex strand ties. In a recent study, Alan R. Fershts group created
during PCR. Ostermeier et al. (38) described a metho- totally new catalytic functions in the transmutation of
dology, incremental truncation for the creation of indole glycerol phosphate synthase (IGPS) to phos-
hybrid enzymes, that creates hybrid gene libraries phoribozyl anthranilate isomerase (PRAI) (49). This
that are independent of sequence homology. Two was achieved by modication of the loops of IGPS
parent genes are truncated incrementally and the that are differed from those in PRAI, followed by
fragments are fused pairwise to generate single cross- directed evolution. The nal evolved enzyme loses its
overs. A more rened method, called sequence homol- original IGPS activity, but acquires PRAI activity with
ogy-independent protein recombination (39), allows kcat =Km value of 4:8 107 sec1 M1 which is sixfold
the proper sequence alignment maintained between that displayed by the wild-type enzyme. This study
the parent genes in the hybrid sequence. A more recent exemplies the remarkable power of combining
development involves exon shufing, which is based on rational design and directed evolution in achieving
the fact, that in nature, genetic recombination occurs novel structural and functional properties.
by crossover in the intron regions to produce various
structure genes (40). This approach had already been
successfully utilized earlier by Kumagai et al. (41) to IV. METABOLIC ENGINEERING
introduce lysozyme activity to -lactalbumin by engi-
neering a hybrid protein by articial exon shufing. Recombinant DNA technology has progressed rapidly
Directed evolution as described above contains an so that foreign genes can be introduced or endogenous
in vivo step of expressing the gene variants in micro- genes can be manipulated to redesign metabolic path-
organisms. It requires an appropriate system for the ways in microbial, plant, and animal cells. For the eld
transcription and translation of the DNA sequences of agriculture and food, this means the ability of in
to proteins for in vivo selection, while in the following vivo manipulation of enzymes to tailor the chemical
step, the benecial mutations are combined and sub- composition and physical properties of various food
jected to in vitro mutagenesis and amplication for the components, to enhance characteristics suitable for
next cycle. E. coli has been frequently used as a suitable food formulation and processing, to improve nutri-

tional values, to remove or reduce the level of antienzyme complex catalyzes repeated Claisen condensa-
nutrients, or even to produce value-added nonfood tion between acylthioesters (ketosynthase), followed by
products. The following description illustrates the repeated reductive cycles comprising a ketoreduction
application of metabolic engineering to change the (ketoreductase), dehydration (ketodehydratase), and
quality characteristics of starch in cereal plants. The enoylreduction (enol-reductase) on the -keto group
composition and molecular structure of starch have a of the growing polyketide chain. After the chain has
direct impact on the rheological properties, such as elongated through a xed number of cycles, the poly-
gelatinization, swelling, viscosity, and retrodegrada- ketide product is released via the action of a thioester-
tion. With the current trend of developing more pro- ase. Structural variability is introduced by variations of
cessed and formulated foods, the demand for substitutions in the starter units, the extent of reduc-
specialty starches is increasing. The ratio of amylose tion, and the location of intramolecular ring closure
to amylopectin has a signicant impact on the physi- (57). This natural process can be manipulated to pro-
cochemical properties of the starch, and for some duce designer polyketides by combinatorially expres-
applications enriched fractions of either one is desir- sing PKS subunits in modules, and a large repertoire of
able. Currently, most waxy mutants that produce polyketides can be generated for screening for specic
starches with little or no amylose are created by selec- structurefunction, including antibiotics and anti-
tive breeding in maize. However, controlling the cancer agents (58).
degree of branching or the ratio of amylose to Recently, a wide range of structurally different car-
amylopectin can be realized only through the use of otenoids have been successfully synthesized by manip-
metabolic engineering. Introduction and expression of ulating terpenoid pathways in bacteria and yeasts. To
Escherichia coli glycogen branching enzymes into direct carotenoid biosynthesis in Escherichia coli, the
potato tubers showed an increase in branching degree terpenoid pathways need to be extended with expres-
of the amylopectin (50, 51). Lloyd et al. (52) intro- sing a number of genes, including phytoene synthase
duced a chimeric antisense construct of two starch (for forming the C40 carotenoid phytoene), isopente-
synthase isoforms (SSII and SSIII) into potato tubers, nyl pyrophosphate isomerase (for the isomerization of
which led to accumulation of amylopectin with an isopentenyl pyrophosphate to dimethylallyl pyropho-
increase in short-chain branching. Antisense inhibi- sphate which is the substrate for elongation), and
tion of starch branching enzyme (isoform SBE A) in geranylgeranyl pyrophosphate synthase (for forming
potato increases the average chain length of amylo- geranylgeranyl pyrophosphate which is the substrate
pectin and the level of phosphorylation (53). for phytoene synthase). A combination of these and
Suppression of both SBE A and B results in very several other genes for specic modications of the
high ( 2:5 times) amylose potato starch containing phytoene chain were cloned into E. coli plasmids,
insignicant levels of highly branched amylopectin, reuslting in the synthesis of eight different hydroxy
and sixfold increase in phosphorus content (54). derivatives of carotenoids, including 1-hydroxyly-
Other targets for genetic manipulating of starch copene, 1; 10 -dihydroxylycopene, dimethylspheroidene,
biosynthesis include (a) increased production or 3-hydroxy--zeacarotene, 7,8-dihydrozeaxanthin, 3- or
alteration of storage proteins, (b) changes in size and 30 -hydroxy-7,8-dihydro--carotene, and 10 -hydroxy--
ratio of A and B starch granules, and (c) specialty carotene (59). Using a similar approach, but combin-
carbohydrate products, such as cyclodextrins utilizing ing it with directed evolution of phytoene desaturase
starch as substrate (55). All these can be achieved, as and lycopene cyclase, Schmidt-Dannert et al. (60) were
future investigations continue to reveal the complex able to introduce six double bonds into phytoene for
interactions and mode of action of the various enzymes the production of the fully conjugated carotenoid,
involved in starch synthesis. 3,4,30 ,40 -tetradehydrolycopene, and a unique cyclic car-
Metabolic pathways often employ enzyme com- otenoid torulene.
plexes to achieve multifunctional steps of catalysis in
the formation of the nal product via a number of
intermediates. The concept of combinatorial chemistry V. ARTIFICIAL ENZYMES
can be applied to biological systems by combining,
changing, or shufing the genes in a biosynthetic path- The idea of replacing enzymes with synthetic biocata-
way, resulting in a whole range of novel molecules and lysts has long been an intellectual challenge. Miniature
their variants (56). For example, in polyketide bio- enzymes, devoid of polypeptide backbones, offer an
synthesis, the multifunctional polyketide synthase attractive alternative for use in processing because

they are less prone to the effects of high temperature, of the antigen-binding site may be involved in nucleo-
extreme pH, and ionic strength existing in industrial philic catalysis (74). Applying directed evolution tech-
applications. Cyclodextrins have proved to be the niques to evolve a hydrolytic abzyme generated by the
most popular host matrix for constructing enzyme conventional method with immunization of the transi-
mimics, usually with the attachment of one or more tion state analog, Takahashi et al. (75) produced a var-
reactive groups, such as a pyridoxamine to the hydro- iant with six- to two-fold increases in the kcat value.
phobic binding cavity to produce selectivity for trans- An alternative approach of generating catalytic
amination of ketoacids bound into the cavity (61). antibodies has been proposed by Briboulet et al. (76)
Examples also include the grafting of an imidazolyl using the properties of anti-idiotypic antibodies to gen-
benzoic acid moiety to achieve a chymotrypsin-like erate an internal image of an enzyme-binding site. This
activity (62, 63). Another synthetic host that has approach follows the concept of idiotypic mimicry (77)
been used quite extensively includes porphyrin or in that antienzyme antibodies generated by animal
porphyrin-like structures as well as various macrocyc- immunization are screened for those that recognize
lic compounds with built-in functions. For example, (idiotypic) the enzyme active site. The selected antien-
FeIII and MnIII (porphyrinato) complexes have been zyme antibodies are used as immunogens to induce the
studied by several groups as superoxide dismutase production of anti-idiotypic antibodies which carry the
mimics (64). Salvemini et al. (65) developed a Mn(II) imprint of the enzyme active site and are subject to
bis(cyclohexylpyridine) macrocyclic complex that enzyme activity screening. Crystallographic studies of
mimics superoxide dismutase catalytically and acts antihen egg white lysozyme antibody complexed with
exclusively on superoxide, without interfering with an anti-idiotopic antibody suggest that the mimicking
other reactive species, such as nitric oxide, hydrogen is functional, involving similar binding interactions
peroxide, or peroxynitrite. An example of mimicking rather than exact topological images (78).
not only the important structural features but also the Another technique known as molecular imprinting
same chemistry as an enzyme was reported for the deals with a very similar concept in biological recogni-
development of models of galactose oxidase, which tion. The overall approach is to allow a mixture of
oxidizes benzylic or allylic alcohols with O2 to yield functional monomers (of either acrylate-, styrene-, or
an aldehyde and H2 O2 (66). In the model, the deriva- silica-based materials) to prearrange around the
tized Schiff base diiminediphenolate ligands mimics selected ligand by noncovalent interactions (79, 80).
the copper coordination in the natural enzyme invol- The reslting complexation is immobilized by polymer-
ving two tyrosine phenolates, two histidine imidazoles, ization of the monomers, and subsequent extraction
and exogenous H2 O ligand. (or dissolution) of ligand results in the development
Catalytic antibodies are currently the best-known of an imprint within the polymeric structure.
enzyme mimics. These so-called abzymes are generated Plastic enzyme mimics could be produced by using
by screening a huge library of antibodies raised against template-containing analogs of substrates, transition
transition state analogs for catalysts in a particular state, or product of an enzyme reaction. Several studies
reaction of interest (67, 68). The many different reac- used phosphonate derivatives to mimic the transition
tions developed for catalytic antibodies include amide of carboxylic ester hydrolysis, and the results have
hydrolysis, ester hydrolysis, peptide cleavage, transes- been very moderate. In one study, phosphonic acid
terication, -elimination, cationic cyclization, Diels- monoesters were imprinted using a polymerizable ami-
Alder reaction, Claisen rearrangement, lactonization, dine derivative, and the catalysts were screened for
and various chemical transformations. Esterolytic activity (81). The hydrolysis reaction follows a typical
antibodies are the best characterized of the catalytic MichaelisMenten curve with saturation kinetics.
antibodies probably due to the fact that excellent tran-
sition state analogs are readily available. Several crystal
structures of esterase-like antibodies have been pub-
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27
Catalase
Dominic W. S. Wong
John R. Whitaker
I. INTRODUCTION First, it is not possible to saturate the enzyme with

substrate even as high as 5 M H2 O2 . Second, there is
A. Reactions Catalyzed: Behavioral Anomalies
a rapid inactivation of catalase at H2 O2 concentrations
above 0.1 M, because the active enzyme-H2 O2 com-
Catalase (hydrogen peroxide : hydrogen peroxide oxi-
pound I is converted to the inactive compounds II
doreductase; EC 1.11.1.6) catalyzes two different reac-
and III (Fig. 1) (4). Therefore, one cannot experimen-
tions and shows unusual features kinetically (1, 2). The
tally determine Vmax by saturating catalase with H2 O2 .
rst and best-known reaction is the decomposition of
As a result, the reactions must be performed at 10
H2 O2 to give H2 O and O2 . This is called catalatic
mM H2 O2 Successful and reproducible assays must be
activity [Eq. (1)]. But catalase also oxidizes several
performed at catalase concentrations high enough so
H donors, such as methanol, ethanol, formic acid,
that the half-time (t1=2 ) for decomposition of the H2 O2
and phenols with the consumption of 1 mole of hydro-
is 30 sec or less (4). Temperature changes have a low
gen peroxide. This is called peroxidatic-like activity
effect on rate of reaction (Q10 1:051.12; about the
[Eq. (2)].
same effect as on diffusion rates). Therefore, the acti-
catalase
2H2 O2 !2H2 O O2 1 vation energy, Ea , for conversion of H2 O2 to H2 O and
catalase O2 is 0:601.7 kcal mol1 . Conversion of H2 O2 to
ROOH AH2 !H2 O ROH A 2 products initially (030 sec) follows a linear reaction
Which reaction predominates depends on the con- (rst order with respect to H2 O2 concentration)
centration of H donor and the steady-state concentra- between 0.01 and 0.05 M H2 O2 .
tion of H2 O2 in the system. In both reactions the active The rate constant (k) for the overall reaction is given
catalase-H2 O2 compound I is formed rst. The decom- by Eq. (3).
position step of H2 O2 , in which H2 O2 serves as H
k 2:3=t logS1 =S2 sec1 3
donor for compound I proceeds very rapidly
(k 107 M1 sec1 , while peroxidatic-like reactions where t t2 t1 near the beginning of reaction (see
[Eq. (2)] proceed much slower (k 102 above), [S1 ] and [S2 ] are H2 O2 concentrations at t1 and
3 1 1
10 M sec ) (3). t2 , respectively. In studies with puried enzyme pre-
Another anomaly of catalase-catalyzed reactions is parations the specic activity (k10 ) is given by Eq. (4).
that catalase does not obey the expected order of reac-
tion (Michaelis-Menten) shown by most enzymes. k10 k=E M 1 sec1 4

Figure 1 Reactions of catalase with hydrogen donors. The Ds refer to the donors: D1 is ascorbate or ferrocyanide; D2 is
phenols; D3 is alcohols or formic acid; D4 is nitrite; D5 is azide or hydroxamine; and D6 is hydrogen peroxide. (From Ref. 4.)
The k10 for pure catalase from human erythrocytes is iron despite his partial purication of catalase.
3:4 107 M1 sec1 . This value permits estimation of Willstatter (13) also doubted the function of iron in
the concentration of catalase in blood and tissues. peroxidase.
There are some problems in estimating the concen- In 1930, Zeile and Hellstrom (14) prepared purer
tration of catalase because of molecular heterogeneity catalase from liver and demonstrated the hematin nat-
due to genetic origin (not all isozymes have the same ure of the prosthetic group. The analogies between
specicity), conformational alterations due to -SH catalase and other hematin compounds were soon
group oxidation or dissociation of the active tetramer recognized (1517). Bray and Gorin (18) postulated
(catalatic activity) into the dimer which has peroxidatic the reaction with iron of the hematin prosthetic
activity but not catalatic activity (68). group was probably that shown in Eq. (5). This was
the rst formulation of a catalytic role for quadrivalent
(ferryl) iron.
B. Historical Aspects
Fe2 H2 O2 ! FeO2 H2 O 5
Catalase appears to be the rst potential enzyme
Earlier, Manchot and Lehmann (19) reported that a
recorded (about 1812), based on the appearance of
pentavalent iron Fe2 O5 could perform a catalatic
rapid bubble evolution when biological extracts were
reaction [Eq. (6)].
mixed with H2 O2 . The catalatic activity was regarded
originally as an attribute of protoplasm, a term Fe2 O5 2H2 O2 2H ! 2FeOH2 2O2 H2 O
shared with other general catalysts such as platinum. 6
In 1901, Loew introduced the specic name catalase
(9). But the true nature of catalase was overlooked by But in enzymes, the theory of the enzyme-substrate
the more prevalent discussion of the actions of metals complex formation was an alternate explanation to
in biological oxidation. Wieland (10) considered the the free-radical hypothesis required by Eq. (6). Von
initial oxidation reaction to be the transfer of hydrogen Euler and Josephsons data (20) supported the idea
to oxygen to form peroxide, and the role of the metal that catalase formed a Michaelis complex with H2 O2 ,
was to remove the peroxide so formed. Warburg (11) with Km of 0.025 M. Bonnichsen et al. (21) proved that
considered that hydrogen peroxide was not formed the reaction velocity could be explained by the collision
during biological oxidation, but that the iron of the theory of reaction rates and strongly argued against
cells activated O2 to react directly with substrates. the assumptions of chain mechanisms postulated ear-
Therefore, he did not consider special catalatic or per- lier. At the same time Chance (22) discovered the pri-
oxidative roles for the heavy metals in contrast to mary compound (FeOOH2 ) formed between catalase
Wieland (10). Hennichs in 1926 (12) also failed to and H2 O2 (compound I; [Eq. (7)], which then reacts
recognize that the prosthetic group of catalase contains with a second H2 O2 [Eq. (7)].

FeOH2 H2 O2 !FeOOH2 H2 O Administration some concern about whether catalase
7 was the best enzyme to use. In 1949, Joslyn (29), based
FeOOH2 H2 O2 !FeOH2 H2 O O2 on his research from 1933 to 1945, concluded that loss
Chances demonstration of the similarity between of peroxidase activity paralleled the loss of enzymes
the catalatic and peroxidatic behaviors of the catalase responsible for off-avor development in blanched fro-
compound I required a reexamination of the hypoth- zen foods more closely than the loss of catalase activ-
esis that the biological role of catalase is in fact a per- ity. Neither catalase nor peroxidase has been shown to
oxidatic one (23). be directly involved in quality deterioration of frozen
blanched foods. But it is easy to determine their activ-
ities and demonstrate the conditions required to inac-
II. IMPORTANCE OF CATALASE IN tivate them in the blanching process.
FOOD SCIENCE AND TECHNOLOGY In the dairy industry, catalase activity is a sensitive
and easily detected indicator of contamination of milk
Catalase is widely distributed in nature. It is found in by neutrophil granucytes. In biomedical research and
all aerobic microorganisms, and in all plant and animal medical clinics, catalase in liver, leukocytes, and ery-
cells. The catalase activity of mammalian cells varies. It throcytes has received substantial attention as to its
is highest in liver and kidney and low in connective role in oxidative metabolism and its protective role
tissues. Catalase has been used in the food processing as a H2 O2 scavenger. In maize genetics, investigation
industry to determine the adequacy of blanching (70 of catalase has been extensively studied in relation to
105 C) of vegetables and fruits to destroy microorgan- human acatalasemia. Severe cases of acatalasemia in
isms and enzymes to preserve color, texture, avor, humans indicate that the catalase activity is near zero,
taste, and aroma of the products when stored frozen raising the question of how such individuals deal with
up to 2 years. Early in the application of blanching for the metabolic destruction of H2 O2 . Catalase activity
stabilization of frozen foods catalase was used espe- has been used as a diagnostic predictor in some hae-
cially for Birdseye peas (2426), the best on the market. matological disorders and in determining urinary tract
But catalase is much less heat stable than peroxidase infections.
(Fig. 2) (27), causing the U.S. Federal Drug
Figure 2 Rates of inactivation of catalase, peroxidase, lipoxygenase, and lipase in English green pea homogenates incubated at
60 C. (From Ref. 27.)

Following cold pasteurization of milk by H2 O2 , the with a prosthetic group. In the homotetrameric mole-
residual H2 O2 is removed by addition of catalase. cule, the four Cys residues in each subunit do not form
Catalase has been widely used in removing O2 from disuldes (31). BLC belongs to a family that may be
the head space of several foods, including dried egg referred to as true catalases (or heme catalases), corre-
powder, fruit juices, and wines, to prevent enzymatic sponding to homotetrameric, heme-containing
browning by polyphenol oxidase. It is also used in enzymes with an fold, ubiquitously found in
analytical determinations of O2 and H2 O2 concentra- eukaryotes and also in many prokaryotes (32). These
tion in foods (30). heme catalases have molecular weights (MW) in the
range of 200330 kDa. The other family of catalases
consists of Mn-catalases that are nonheme hexameric
III. MOLECULAR STRUCTURE enzymes with an all -fold, and are present only in
prokaryotes (33).
A large number of amino acid sequences of catalases An interesting group of bifunctional catalase-perox-
are known, primarily deduced from the gene idases have been identied in Escherichia coli,
sequences. These include (based on entries listed in Mycobacterium tuberculosis, and Synechococcus sp.
Swiss-Prot): Aspergillus fumigatus (Sartorya fumi- These KatG enzymes are phylogenetically related to
gata), A. niger, Arabidopsis thaliana (mouse ear eukaryotic ascorbate peroxidases and yeast cyto-
cress), Bacillus subtilis, B. rmus, B. stearothermophi- chrome peroxidase forming class I of the plant perox-
lus, Bacteroides fragilis, Bordetella pertussis, Bos taurus idase superfamily (34). The E. coli KatG enzyme (or
(bovine), Botrytis cinerea (Botryotinia fuckeliana), hydroperoxide I, or HPI) is a tetramer of 84-kDa sub-
Brucella abortus, Caenor habditis elegans, units and 2 protoheme IX groups, with a primary
Campylobacter jejuni, Candida albicans, C. tropicalis, structure of 726 amino acid residues (35).
Cavia porcellus (guinea pig), Cucurbita pepo (summer E. coli also produces hydroperoxidase II (HPII)
squash), Deinococcus radiodurans, Desulfovibrio vul- which has been characterized as a monofunctional cat-
garis (strain Miyazaki), Drosophila melanogaster alase containing a modied heme imparting a charac-
(fruit y), Emericella nidulans (Aspergillus nidulans), teristic green color to the protein (36). It is a tetramer
Glycine max (soybean), Gossypium hirsutum (upland of identical 84-kDa subunits, each encoded by the katE
cotton), Haemophilus inuenzae, Helianthus annuus gene, with a predicted sequence of 753 amino acid
(common sunower), Helicobacter pylori residues (37). The Cys residues in HPII do not have
(Campylobacter pylon), Homo sapiens (human), a catalytic role. Moreover, the highly conserved
Hordeum vulgare (barley), Ipomoea batatas (sweet Cys438 is blocked with a unique hemithioacetal struc-
potato), Lactobacillus sake, Listeria seeligeri, ture originated from a pyruvate modication (38).
Lycopersicon esculentum (tomato), Micrococcus luteus Crystal structures of several catalases have been elu-
(M. lysodeikticus), Mus musculus (mouse), cidated, including bovine liver, human erythrocyte,
Mycobacterium avium, M. bovis, M. intracellulare, M. Penicillium vitale, E. coli, Proteus mirabilis, and
tuberculosis, Neisseria gonorrhoeae, Nicotiana plumba- Saccharomyces cerevisiae. The beef liver catalase sub-
ginifolia (leadwort-leaved tobacco), N. tabacum (com- unit consists of four domains: an N-terminal section of
mon tobacco), Onchocerca volvulus endobacterium), 70 residues forms an arm extending from the globulin
Oryza sativa (rice), Penicillium janthinellum (P. vitale), region and intersects with the neighboring subunits;
Phaseolus aurens (mung bean), Pichia angusta the second domain (residues 76320) forms a -barrel
(Hansenula polymorpha), Pisum sativum (garden consisting of two topologically similar, four-stranded
pea), proteus mirabilis, Pseudomonas aeruginosa, Ps. antiparallel sheets; the third domain (residues 321
putida, Ps. syringe, Rattus norvegicus (rat), Rhizobium 436), also known as the wrapping domain, is a long
meliloti (Sinorhizobium meliloti), Rhodobacter capsula- stretch of polypeptide chain forming an outer layer;
tus (Rhodopseudomonas capsulata), Ricinus communis and the C-terminal domain (residues 437506) consists
(castor bean), Saccharomyces cerevisiae), Salmonella of four -helices on the external part of the molecule
typhimurium, Schizosaccharomyces pombe (ssion (32, 3941) (Fig. 3A, B). In the tetrameric structure,
yeast), Secale cereale (rye), Solanum melongena (egg- most of the intersubunit interactions are conned to
plant), Solanum tuberosum (potato), Triticum aestivum the N-terminal arm and the wrapping domain. The -
(wheat), Vibrio scheri, and Zea mays (maize). barrel and the C-terminal domain form a hydrophobic
The primary structure of bovine liver catalase channel leading to the heme pocket which is 20 A
(BLC) subunit consists of 506 amino acid residues below the surface. Heme protein interactions involve

Figure 3 Stereo view of beef liver catalase (A) subunit (PDB id 7cat) and (B) tetramer (PDB id 4b1c).
mostly van der Waals contacts between the heme vinyl from the heme iron, respectively. The ligand 6 site on
and methyl groups and nonpolar side chains, and a few the distal side of the heme is unoccupied.
charge interactions between the heme propionyl The tetrameric enzyme from human and bovine
groups and Arg and carboxylic acid residues. At the contains four molecules of tightly bound NADPH
proximal side of the heme, Tyr357 provides a protein located toward the carboxyl ends of 5 and 10 helices
ligand (as ligand 5) with the Fe-phenolic oxygen dis- (42, 43). However, the binding of NADPH is not
tance of 1.9 A. Ionization of the Tyr-OH is assisted by essential for activity, but may function to prevent the
interactions with the guanidinium group of Arg353. formation of compound II, an inactive intermediate in
On the distal side of the heme are the essential His74 the catalatic cycle (see below). The binding site shows
and Asn147, with their N atoms at 4.3 A and 6.2 A relative afnities in the order of: NADPH > NADH >

NADP > NAD . The crystal structure of the fungal Step. 1. Two-electron oxidation of native ferric
Penicillium vitale catalase is similar to that of the beef enzyme to catalase compound I, with the substrate
liver enzyme, except that there is an additional C-term- H2 O2 reduced to H2 O:
inal avodoxin-like domain (residues 520670) (44,
45). This domain has an = structure with a central En-FeIII H2 O2 ! En-FeIV O H2 O
-sheet of ve parallel strands and four helices around
Step 2. Two-electron reduction of compound I to
it, with a topology similar to that of avodoxin (46).
native ferric enzyme, with a second molecule of H2 O2
Escherichia coli catalase HPII, encoded by the katE
oxidized to O2 .
gene, shows a very close structural relationship with
other catalases. It differs from BLC in that the C-term- En-Fe(IV)=O H2 O2 ! En-FeIII O2
inal domain of 150 residues is structurally avo-
doxin-like (47). In addition, the N-terminal 60 amino
acids increase the contact area between subunits. The
A. Formation of Compound I
crystal structures of Proteus mirabilis PR catalase and
Saccharomyces cervisiae catalase-A show that the
Compound I contains a -cation radical of the heme
bound NADPH has the same conformation as in
structure of O FeIVporphyrin similar to that of
BLC, and lacks a C-terminal domain equivalent to
compound I formed by horseradish peroxidase (HRP)
that found in Penicillium vitale (48, 49).
(53). The rate constant for the formation of compound
Escherichia coli catalase HPII exhibits an unusual
I with H2 O2 as substrate in the catalatic reaction is 6
absorption spectrum distinctly different from that of
106 M1 sec1 (54). Other hydroperoxide substrates,
other catalases containing heme b (protoporphyrin
such as alkyl or acyl peroxides, can also be reduced
IX). The heme prosthetic group of HPII is an unusual
to corresponding alcohols, albeit with much lower
green chromophore derived from a member of the
rate constants (Table 1).
family of d-type hemes, which is also found in
Upon the binding of a peroxide molecule, two water
Penicillium vitale catalase. The structure has been char-
molecules in the heme cavity are displaced for the sub-
acterized as having a conguration of a cis-hydroxy-
strate to form interactions with the enzyme. The per-
chloin -spirolactone at the saturated pyrrole ring III
oxide oxygen interacts with the heme Fe(III) via
of the macrocycle in the C-6 position, which may arise
electrostatic interactions, and with His74-N
through
from heme b by a self-catalyzed epoxide formation on
hydrogen bonding (Fig. 4) (40). The net result is
ring III followed by hydrolysis to the cis diol (5052).
increasing acidity of the -proton and the weakening
of the peroxide O H bond. The O O bond is further
polarized by the formation of hydrogen bonding
IV. REACTION MECHANISM between the oxygen and Asn147. Finally, a charge-
relay system formed between His74-N and Ser113-O ,
The overall reaction catalyzed by catalases involves the and the propionic carbonylic group of pyrrole III facil-
conversion of two moles of H2 O2 to yield water and itates the transfer of the -proton from O to O of the
oxygen as the nal products. The substrate H2 O2 par- peroxide. The deprotonation of O in turn enhances the
ticipates in both oxidizing and reducing activities, with interaction between O and heme (Fe(III), delocaliza-
a two-electron transfer in each process. tion of the negative charge on O , and development of a
Table 1 Rate Constants of Catalase in the Enzymatic Cycle
Substrate Formation of Compound I Substrate Reduction of Compound I

k1 M1 sec1 k4 app M1 sec1
HOOH 6 106 HOH 1:8 107

CH3 OOH 8:5 105 CH3 OH 830
CH3 CH2 OOH 2 104 CH3 CH2 OH 1020
CH3 CH2 2 OOH 5:5 103 CH3 CH2 2 OH 6.5
HOCH2 OOH 3 104 HOCH2 OH 1200
Source: Ref. 54.

Figure 4 Reaction mechanism of catalase showing the formation and reduction of compound I. (From Ref. 70; adapted from
Ref. 40.)
positive charge of the heme group, allowing the nal C. Formation of Compound II
transfer of the O to the heme iron.
Catalase can slowly form compound II by a two-step
one-electron reduction of compound I under a steady
B. Reduction of Compound I low concentration of H2 O2 or with other special sub-
strates, such as ferrocyanide and ascorbate. Catalase
In the second half of the cycle, catalase compound I compound II is an inactive form in the catalatic mode
reacts with a second molecules of H2 O2 to yield the of the enzyme. This is in contrast to HRP compound
native enzyme with generation of O2 and H2 O. II, which is an active species produced by two sequen-
However, compound I reacts poorly with other hydro- tial one-electron reductions of compound I with most
gen donors, such as alcohol, hydroxylamine, or formic of its substrates. The difference in the reactivities of
acid. Reduction of compound I occurs via a hydride compound II species of catalase and HRP is probably
transfer by abstracting the -hydrogen by the heme due to protein control of the accessibility of substrates
ferryl oxygen (55). Deprotonation of the substrates to the heme group (57). In addition, the phenoxylate
-proton is unlikely to occur because the His74 is oxygen of the proximal Tyr residue provides more elec-
less nucleophilic as the heme in compound I is posi- tron density to the heme iron than the His residues of
tively charged. The -hydrogen abstraction is stereo- HRP, and favors a two-electron transfer instead of a
specic for the pro-R hydrogen in the oxidation of sequential addition of electrons (58).
ethanol substrate (56). A hydride transfer facilitates The slow inactivation of catalase by its own sub-
the development of a double-bond character at O strate can be prevented by experimentally limiting the
O , and the nal transfer of the proton to the exposure to H2 O2 , or reducing the concentration of
heme ferryl oxygen (Fig. 2). compound I. However, the primary inhibition of the

formation of compound II is provided by the enzymes volumes of reagents can be prepared by keeping the
cofactor, NADPH, which has been shown to prevent concentrations constant as given.)
and reverse the process (59). It has been suggested that
d. Sample of Catalase. Prepared in the 50 mM,
the initial formation of compound II involves a com-
pH 7.0, phosphate buffer.
pound II radical intermediate, and NADPH reduces
both the iron and the free radical by a two-electron
3. Stability of Solutions
transfer (60, 61).
The phosphate buffers are stable as long as bacterial
contamination is avoided; store at 2 C. Prepare the
V. MEASUREMENT OF CATALASE
buffered H2 O2 solution fresh each day.
ACTIVITY
4. Reaction
Catalase activity can be measured spectophotometri-
cally, manometrically, polargraphically, and titrimetri-
a. Blank. Place 1.00 mL of 50 mM phosphate
cally. The best methods are the spectrophotometric
buffer into a quartz cuvette. Add 2.00 mL catalase
and titrimetric methods. They will be presented below.
sample.
A. Spectrophotometric Method (1, 2, 62) b. Catalase Reaction. Place 2.00 mL catalase
sample into a quartz cuvette. Add 1.00 mL of H2 O2
Hydrogen peroxide has a molar extinction coefcient solution (above). Mix rapidly ( 5 sec) with a plastic
(
m ) of 52 M1 cm1 at 235 nm and its disappearance paddle or by inversion three times using paralm.
can be followed by continuous spectrophotometry
c. Place in Spectrophotometer. Record continu-
when puried catalase preparations are used. As
ously for 3060 sec. If a recorder is not available,
noted in paragraph 3 of Section I, 10 mM H2 O2 should
take readings at 5- to 10-sec intervals. The temperature
be used in the catalase assay so as to avoid formation
should be at 2025 C. Abs/15 sec should be no larger
of inactive compounds II and III. Therefore, the time
than 0.100 and not smaller than 0.020. If necessary,
zero absorbance reading will be 0.520. This method is
adjust concentration of enzyme solution to be within
the easiest to perform, is sensitive, and is the best when
these limits and repeat the experiment.
puried catalase is used.
5. Calculation of Results
1. Equipment Needed
As noted earlier in Section I.A, the reaction approxi-
UV spectrophotometer suitable for accurate measure-
mates a rst-order reaction. Therefore, the following
ments at 235 nm, connected to a high-speed recorder.
relations can be used for a 15-sec or other interval of
Quartz cuvettes (10 mm light path).
reaction time.
2. Reagents and Solutions k 2:3=15 sec logA1 =A2
8
a. Purity of Reagents. Glassware, buffer, and 0:153 log A1 =A2 sec1
substrate should be free from heavy metals, as they
will cause decomposition of H2 O2 . Potential problems To calculate the catalytic concentration b of the
caused by metal impurities can be determined by a sample (k/L) or catalytic content Zc =ms of the sample
control containing an equivalent volume of phosphate (unit k/g Hb) use:
buffer, pH 7.0, in place of the enzyme.
b V=v2:3=15 log A1 =A2 k=L 9
b. Phosphate Buffer (50 mM, pH 7.0). (1)
Dissolve 6.81 g KH2 PO4 in water and make to 1 L. b 3=22:3=15 log A1 =A2 k=L
(2) Dissolve 8.90 g Na2 HPO4 2 H2 O in water and
0:23 log A1 =A2 k=L 10
make to 1 L. Mix solutions (1) and (2) in the propor-
tion of 1 : 1.5 (V/V), pH 7.0.
where A1 is A235 at time 0; A2 is A235 at t 15 sec; V
c. Hydrogen Peroxide (30 mM). Dilute 0.34 mL is total assay volume; v is enzyme sample volume in the
of 30% hydrogen peroxide (freshly opened) with phos- assay mixture. The 3/2 is total reaction volume/enzyme
phate buffer, pH 7.0 (above) to 100 mL. (Smaller sample volume (see Sec. V.4 above).

Zc =ms 2:3=15 Hb log A1 =A2 3. Equipment
0:153 log A1 =A2 sec1 g Hb Zc =g Hb Thermostatic water bath (20 C) with rotating shaking
11 attachment.
where is the quotient of weight concentration of

hemoglobin (Hb; unit g/L) in blood or erythrocyte 4. Reagents and Solutions
sediment, and in the cuvette.
For a difference in absorbance of 0.450 (t0 0:400 a. Purity of Reagents. See remarks in Section
t15 sec log A1 =A2 0:05115, the following relation- V.A.3 under spectrophotometric method.
ship holds:
b. Preparation of Solutions. Use repuried water.
1
k 2:3=t log A1 =A2 0:1175=t sec 12 Phosphate buffer (50 mM, pH 7.0). See Section
V.A.2.b under spectrophotometric method.
6. Precision, Accuracy, and Sensitivity Sodium perborate (100 mM, pH 7.0). Dissolve
With values around 300 k/g Hb, a precision of 10 k=g 7.694 g NaBO3 4 H2 O n ca. 20 ml of 2.4 M HCl,
Hb was found. The coefcient of variation is 23%. add ca. 400 ml of water, adjust pH to 7.0 with addi-
tional 2.4 M HCl and make to 500 ml with water.
7. Detection Limits Potassium permanganate (0.01 M). Dissolve 1.58 g
The limits of this method are set by the turbidity (of KMnO4 in water and make 1 liter.
tissue homogenates) or by the hemoglobin content (of Stability of solutions. The phosphate buffer is stable
the hemolysate). Whenever low catalase activity of a as long as bacterial contamination is avoided; store at
sample does not permit dilution of at least 1 : 100 (i.e., 2 C. The perborate solution is stable for several days at
activity in blood is < 20 k=g Hb), use of the alternative 2 C; check titer with permanganate in each series of
titration technique (below) is recommended. measurements.
B. Titrimetric Method (62)

5. Procedure
1. Method Design
The conversion of H2 O2 to products is measured by a. Assay Conditions. Incubation at 20 C; nal
determining the H2 O2 left in the reaction after a certain reaction volume 5.00 mL.
time by back-titration with permanganate. Portions of
b. Measurement. The blank contains 1.00 mL
the assay mixture are taken after 0, 10, 20, and 30 sec,
phosphate buffer and 3.00 mL perborate solution.
and reactions are stopped immediately by addition of 1
After preincubation for 10 min at 20 C, add 1.00 mL
M H2 SO4 .
water; mix. Stop the reaction after 30 sec by addition
of 3.00 mL of 1 M H2 SO4 . Mix.
2. Optimized Conditions for Measurement
The sample contains 1.00 mL phosphate buffer and
See spectrophotometric method above (Sec. V.A). 3.00 mL of perborate solution. After preincubation at
Titrimetric methods with incubation times > 30 sec 20 C for 10 min, 1.00 mL catalase solution is added;
give only comparative values. Preparation of rate mixed. Stop the reaction after 30 sec by adding 3.00
curves require time and effort, with a large series of mL of 1 M H2 SO4 . Mix.
measurements. Perborate, a stabler substrate than The remaining perborate in the blank and sample is
H2 O2 , is best for routine studies. The inactivation of determined by titrating with permanganate solution.
the enzyme is somewhat slower than with H2 O2 . The To construct a rate curve, prepare several assay mix-
results obtained with H2 O2 and perborate are of the tures and stop the reactions at 10, 20, and 30 sec,
same order of magnitude. The method of Feinstein respectively. The concentration of the enzyme solution
(63) is suitable, insofar as the assay conditions are should be such that after 30 sec not > 50% and not <
modied according to the recommended procedure of 10% of the perborate is converted to product. If the
Bonnichsen (64) (i.e., 30 sec incubation time at pH 7.0 catalase activity in the sample is low, the incubation
and 20 C). time should be increased to 1 min, or even to 5 min.

6. Calculations horse and pig blood (64), bacteria [Micrococcus lyso-
deikticus (67), and spinach leaves (68).
The rate constant (k) for the overall reaction is given
The purication procedure for spinach leaf catalase
by:
will be described here, as being more appropriate for
titration vol for blank food enzymology (69). Freshly harvested spinach
k 2:3=t log sec1 leaves are washed in tap water and stored in a cold
titration vol for sample
13 room at 24 C. The leaves are transferred to large
stainless steel vats, covered with commercial acetone
The specic activity (k10 )
is obtained by dividing k by previously chilled to 15 C, and let sit for at least
the molar concentration of enzyme [E]. 2 h at this temperature. The cold, partially dehydrated
leaves are very brittle and are easily reduced to small
k10 k=E M 1 sec1 14 pieces with a large Waring-type blender. The blending
is complete in 3060 sec and the slurry is gravity-l-
Owing to the progressive inactivation of the catalase tered in the cold; the ltrate is discarded. The residue is
the calculated k values for the various time intervals washed several times with portions of cold acetone
must be plotted and extrapolated to t 0. The values (4 C), and the resulting gray-green material is dried
obtained in this way for the rate constant k are some- overnight in a ventilated hood.
what lower than nonextrapolated values. The acetone powder is extracted repeatedly with ice-
For comparative purposes the results can also be cold 0.1 M Na2 HPO4 , yielding a ltrate of Kat.f: 50
expressed in arbitrarily dened unitse.g., the num- and an inactive residue, which is discarded. The ltrate
ber of millimoles of perborate decomposed under the is half-saturated with solid NH4 2 SO4 and left over-
standard experimental conditions related to mg wet night in the cold room (4 C). The precipitate, obtained
weight of tissue (or g hemoglobin, etc.) (perborate by centrifugation at 0 C and containing all the activity,
units) (62, 63). is redissolved in a small volume of 0.1 M Na2 HPO4 .
This preparation, after clarication by ltration or cen-
7. Validation of Method trifugation, has a Kat.f. of 180. Three successive
fractionations with 20%, 7%, and 20% saturated
According to the type of sample, the relative standard
(NH4 2 SO4 are carried out, the precipitates being
deviation is 35%. The source of errors includes the
saved in each instance. The product has a Kat.f. of
titration of concentrated homogenates or blood sam-
920, shows a faint absorption band at 630 nm, and
ples (because of their low catalase activity). The end-
gives absorption peaks at 275, 330, 405, and 625 nm in
point is not sharp since the protein in the sample
a Beckman spectrophotometer.
slowly reacts with permanganate. If the titration is
The catalase solution is adjusted to pH 6.5 and
carried out rapidly and always to the same endpoint
made 0.1 M with respect to Na acetate by addition
(relative excess of KMnO4 ), the resulting error is small.
of the solid salt. Cautious fractionation with saturated
A correction can be applied by titration of an addi-
NH4 2 SO4 , also adjusted to pH 6.5, yields several pre-
tional control mixture containing no perborate or by
cipitates varying in Kat.f. from 1200 to 10,000. The
deproteinization of the incubation reaction mixture
most active preparations are dialyzed for 2 days in
with trichloroacetic acid. Oxidizable compounds (e.g.,
the cold against pH 7.14 phosphate buffer of ionic
Tris) must not be used as components of buffers if
strength 0.1 and then subjected to preparative free
catalase activity is determined by the titrimetric
boundary electrophoresis (18 mA, 330 volts, 4 h),
method.
yielding ve fractionsthree on the anodic side, one
in the bottom cell, and one from the cathodic side.
(Note: polyacrylamide gel electrophoresis should be
VI. PURIFICATION OF CATALASE used today.) The Kat.f. and spectrophotometric data
for these fractions, as well as Kat.f.s and all the other
As reported in Section III, the amino acid sequence has fractions in the preparatory scheme, are shown in
been determined for 63 catalases, largely by gene Table 2.
sequencing. Individual researchers have developed The cathodic fraction of Kat.f. 23,600 gives needle-
their own methods for purication. In Methods in like crystals after concentration, dialysis against 0.1 M
Enzymology, Vol II, 1955, detailed methods are Na2 HPO4 , and addition of solid NH4 2 SO4 to
given for purifying catalase from beef liver (65, 66), 12% saturation. These needles, when redissolved in

Table 2 Kat.f. Values and Spectrophotometric
Characteristics of Various Fractions of the Spinach Leaf
Catalase Preparation
Fraction Kat.f.a;b E280 nm

E405 nm
1. Na2 HPO4 extract of acetone powder 50

2. Ppt. of Fraction 1 insol. in 50%
satd. NH4 2 SO4 redissolved in
0.1 M Na2 HPO4 180
3. Ppt. of Fraction 2 insol. in 20%
0.1 M Na2 HPO4 , sol. in 7% satd.
NH4 2 SO4 425
4. Ppt of Fraction 3 insol. in 20%
0.1 M Na2 HPO4 920
5. Ppts. obtained from adjusting Figure 5 Absorption spectrum of electrophoretically pre-
Fraction 4 to pH 6.5, making pared spinach leaf catalase. Kat:f: 23,600. Concentration
0.1 M with respect to Na acetate, of the enzyme was 0:5 mg=mL. (From Ref. 69.)
then cautiously fractionating with
solid NH4 2 SO4 10,100
6. Electrophoretic fractions of REFERENCES
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Anodic (top cell) 2,620 2.54 peroxidases. In: D Glick, ed. Methods of
Biochemical Analysis, Vol 1. New York: Interscience
a
k 1=t log10 A0 =A0 x, where k 1st-order rate constant, t
time in min, A0 mL permanganate used at zero time and A0 x
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b
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4. D Keilin, P Nicholls. Reactions of catalase with
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28
Horseradish Peroxidase
Zhong Yi Yuan and Tai Jiao Jiang

Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
I. INTRODUCTION lapthifola, Cochlearia armracia L., etc.); the highest

activity is in the roots. The content of peroxidase not
Horseradish peroxidase (HRP; donor:hydrogen-perox- only varies with location but also with maturity and
ide oxidoreductase; EC 1.11.1.7) catalyzes the reaction: season. However, little is known about its cellular loca-
Donor +H2 O2 ! oxidized donor + 2H2 O 1 tion, microenvironment, or specic participation of
HRP in physiological pathways. Plant peroxidases
The enzyme utilizes hydrogen peroxide, some organic play very important physiological roles:
peroxides, or peroxy acids of the general formula 1. Degrading and synthesizing lignin in cell wall
ROOH as hydrogen acceptors to catalyze the oxida- damage and repair. It is also important in the elonga-
tion of a variety of organic and inorganic hydrogen tion of plants (13).
donors, such as phenols, aromatic amines, and 2. Providing defense against pathogens and stress
others. (14).
Horseradish peroxidase occupies an important posi- 3. Catalyzing the catabolism of phyto-growth
tion in the history of enzymology. Because of its sta- hormones, e.g., indole acetic acid (IAA) and auxin to
bility and availability, the puried enzyme is often used regulate plant growth (15).
as a typical model for the family of peroxidases. Since 4. Controlling respiration (16).
the 1950s, research on the reaction mechanism, struc- 5. Promoting formation of highly reactive species,
ture, and function of this enzyme has drawn much i.e., free radicals and electronically excited states.
attention (16). The practical applications of HRP in These species may participate in cell damage or be of
clinical, food, and industrial analyses (ELISA, etc.) benet against pathogens (17).
(79) and catalysis in nonaqueous media (producing 6. Catalyzing removal of hydrogen peroxide (18).
pharmaceuticals, synthesizing polymers, etc.) (10, 11)
have been exploited. Horseradish peroxidases are
glycoproteins, containing one polypeptide chain, one
protoporphyrin IX as prosthetic group, three to eight III. PEROXIDASE FAMILIES AND
carbohydrate chains, and two Ca2 (1, 12). SUPERFAMILIES
Besides being found in horseradish, peroxidases are

II. OCCURRENCE AND PHYSIOLOGY found also in other plants, microorganisms, and ani-
FUNCTION mals including humans. On the basis of sequence
homologies, Welinder (3) proposed classication of
Peroxidases are widely distributed in the leaf, stem, peroxidases by families and superfamilies as shown in
and root of horseradish (Armoracia rusticana, A. Table 1.

Table 1 Superfamilies and Families of Peroxidases B. Primary, Secondary, and Tertiary Structures
of HRP
Superfamily Family
Plant (a) Peroxidases of prokaryotic origin: Determination of the complete amino acid sequence of
peroxidase yeast cytochrome C peroxidase, HRP-C was largely contributed by Welinder (21).
chloroplast and cytosolic ascorbate HRP-C consists of 308 amino acid residues, a hemin
peroxidases, gene duplicated bacterial group, and eight neutral carbohydrate side chains
peroxidases. attached to asparagine residues. The amino acid
(b) Secreted fungal peroxidases: sequence and carbohydrate attachment points are
lignin peroxidase (Phanechaete shown in Figure 1. The molecular weight was reported
chryosporium), manganase peroxidase
to be between 40,000 and 45,000, and the carbohydrate
(P. chrysoporium), ink cap mushroom
moiety constitutes 18%. HRP-C is the most exten-
peroxidase (Coprinus sinereus),
chloroperoxidase (Caldariomyces sively investigated isoenzyme in the plant peroxidase
fumago), cytochrome C peroxidase superfamily. Within the superfamily, HRP-C belongs
(Pseudomonas aeruginosa). to Class III of the plant extracellular peroxidases (22).
(c) Classical plant peroxidases: Peroxidases from owering plants have considerable
HRP isoenzyme C is the most identity of amino acid sequences (4064%). In particu-
intensively studied. lar, those sequences in the regions of the two His resi-
Animal Myeloperoxidase, verdoperoxidase, dues (the distal His42 and proximal His170 in HRP)
peroxidases eosinophil peroxidase, prostaglandin H and Cys residues forming disulde bonds are con-
synthase, glutathione peroxidase, served (12, 22).
lactoperoxidase, thyroid peroxidase.
Gajhede et al. (23) published a 2.15 A resolution
Catalases They are found in plants and animals.
structure of HRP-C, which gave complete information
These are tetrameric proteins.
on overall structure and its comparison to other per-
oxidases. HRP-C has the same overall fold typical of
the peroxidase superfamily, with the secondary struc-
ture elements (especially the 10 prominent helices AJ),
IV. PROPERTIES AS PROTEINS
heme group, and calcium-binding sites in similar posi-
A. HRP Isoenzymes tions. The secondary and the tertiary structures of
HRP-C are summarized in Figure 2.
Over 40 isoenzymes of peroxidase have been found in More recently, the synthetic and native HRP-C
the horseradish plant. By cloning techniques, some of have been cloned in various microorganismsE. coli,
the isoenzymes come from different genes and others Baculovirus, and Pichia pastoris, etc. (2426).
may be produced by posttranslational modication,
especially by degree of glycosylation. There are differ-
V. PROPERTIES AS AN ENZYME
ences in amino acid composition and sequences among
the isoenzymes (19). Based on the isoelectric points, the A. Specic Mechanism of Action
isoenzymes can be classied as shown in Table 2 (20).
The normal peroxidase cycle is shown in Figure 3. The
reaction of HRP with H2 O2 produces a two-electron
oxidized species known as Compound I in which the
ferric iron is oxidized to a ferryl (FeIV
O) species and
Table 2 Horseradish Peroxidase Isoenzymes
the porphyrin to a radical cation. Stepwise reduction of
FORM pI pHopt Carbohydrate (%) Compound I by two substrate-derived electrons pro-
duces Compound II, in which the porphyrin radical
HRP-A 4 Acid Higher cation has been quenched, and subsequently the resting
HRP-B, -C 5.75, 9.63 Neutrala, Low ferric state form. The second-order rate constants
slightly for the reaction HRP and H2 O2 are: k1 , (1:58 0:05
alkaline
107 M1 s1 ; k2 , (9:97 0:28 106 M1 sec1 ; and k3 ,
HRP-D, -E 10.6, > 12 Strongly Low
alkaline (3:64 0:01 102 M1 sec1 (1, 27, 28).
Generally, k3 is always 1020 times slower than k2 ,
a
Neutral HRP-C is the main isoenzyme in the plant. and under most steady-state conditions is rate limiting.

Figure 1 The amino acid sequence of horseradish peroxidase (carb site of carbohydrate attachment). Disulde bridges: 1191,
4449, 97301, 177209. (From Ref. 21.)
The values of k2 and k3 depend on the nature of the play a role in mediating the reactions with a general
substrate. acid/base mechanism. The aromatic donor molecule
Both competitive binding (1) and spectroscopic interacts with a substrate accessible channel contribu-
studies (29) have revealed that the peroxide- and ted by Phe residues (Phe68, Phe142, and Phe179) and
substrate-binding sites are not the same. Specic-site the heme C18 methyl group.
mutation analyses (30, 31) and the high-resolution
crystallographic structure of HRP-C (23) have B. Substrate Specicity
provided detailed information on structural basis of
HRP catalysis. Peroxide binding, activation, and Peroxidase is specic to the hydrogen acceptors. Only
Compound I formation occur in the distal heme hydrogen peroxide, methylhydroperoxide, and ethyl-
pocket, where the key residues (His42 and Arg38) hydroperoxide can be used as hydrogen acceptors.

Figure 2 (a) Stereo C trace of HRP-C with the heme C20 edge in the foreground. Every 10th residue is labeled. This gure was
prepared with program SETOR. (b) The relative positions of the secondary structural elements (-helices and -sheets) in
HRP-C. The helices are labeled with their conventional names. (From Ref. 23.)
The enzyme is not specic to the hydrogen donor. A 2. Inhibitors

variety of phenols, aminophenols, diamines, indophe-
Horseradish peroxidase is inhibited reversibly by cya-
nols, leucodyes, ascorbate, and several amino acids
nide (36, 37), sulde, and dithionite (104 106 M)
are hydrogen donors. In HRP kinetic studies and
(36). Azide uoride, hydroxylamine, diethyldithiocar-
activity assays, guaiacol and 2; 20 -azino-bis(3-ethyl-
bamate, cyclopropanone hydrate, and borohydride at
benzthiazoline-6-sulfonic acid) (ABTS) are the most
103 M inhibit HRP (36, 38). Vanadate and hydro-
commonly used hydrogen donors as shown in Table
xymethylhydroperoxide inhibit the enzyme irreversibly
3 (32, 33). The kinetic parameters of other substrates
(39, 40). As HRP inhibitor, 1,10-phenanthroline is
(including hydrogen acceptors) are also listed in Table
used in immunoperoxidase procedures (41).
4 (3436).
3. Stability
C. Effect of Environmental Factors Horseradish peroxidase is very stable. The commercial
HRP, as a lyophilized powder, may be stored at 48C
1. pH and Temperature Optima
for several years without apparent loss of activity. A
pH optimum is 7.0 for the commercial HRP prepara- solution of puried HRP (1 mg/mL) is stable for 1 year
tions. As seen from Table 2, each isoenzyme has a at 48C.
different optimal pH.
Temperature optimum is 40528C, depending on
the type of isoenzyme and the substrates (1). VI. DETERMINATION OF HRP ACTIVITY
Based on chromogenesis and chemiluminescence of

products, numerous methods have been developed
for determining the activity of HRP. The hydrogen
donors in common use are guaiacol, pyrogallol,
ABTS, 4-methoxy--naphthol, and phenol plus amino-
antipyrine. Among them, guaiacol is easily available
and simple to operate and is used very often, although
Figure 3 The catalytic cycle of horseradish peroxidase. its product is not stable. ABTS and phenol plus amino-
(From Ref. 1.) antipyrine are widely used in microanalysis, enzyme-

Table 3 The Steady-State Kinetic Parameters for the Peroxidation of Guaiacol and ABTS by HRP
Specic activity
Substrate Enzyme (nM) Km M kcat (s1 ) kcat =Km s1 m1 (units/mg)
ABTSa 0.2 8:0 102 4:1 103 5:1 0:2 1:15 103
Guaiacolb 2 5:8 103 4:2 102 7:2 1:1 102 c

a
pH 4.6, 10 mM H2 O2 .
b
pH 6.0, 1 mM H2 O2 .
c
Unveried.
linked immunosorbent assay (ELISA), and histochem- 2. Solutions

ical tracer techniques. Both methods are rather sensi-
All solutions are prepared with deionized water and
tive, and the chromogenic products are relatively
kept at 48C.
stable. On the other hand, aromatic amines, such as
o-dianisidine, o-phenylenediamine, and mesidine are a. ABTS. 1:5 102 M as a stock solution.
less used now, because the oxidized products are car- Dissolve 0.077 g ABTS (diammonium salt) in 10 mL
cinogenic. water.
b. H2O2. Dilute the 10 M commercial solution
to a stock solution of 0.01 M. Titrate the stock solution
A. Determination of HRP Activity Using ABTS with a 0.02 M KMnO4 solution. The concentration
(42, 43) of the stock solution can be checked by its
240
1. Reaction 4:36 104 M1 cm1 .
The reaction catalyzed is shown in equation (2). c. HRP. Dissolve 1.00 g lyophilized HRP in 1
mL deionized water, and dilute to 0.01 g/mL just
HRP before use. The concentration of HRP is determined
H2 O2 AH2 ! 2H2 O A 2 by the Soret absorbance at 403 nm using
403
1:02 105 M1 cm1 .
AH2 ABTS d. Sodium Acetate Buffer. 0.2 M, pH 5.0.
ABTS (2,20 -azinobis(3-ethylbenzthiazolin-6-sulfonate) 3. Procedure
has an absorption maximum at 340 nm (pH 4.4) and

340 3:97 104 M1 cm1 . It is oxidized by HRP to a The method is according to Chailds and Bardsley (42)
radical cation that has an adsorption at pH 5.0 with with some modication. Ten microliters of HRP sam-

414 3:6 104 M1 cm1 . ple with the appropriate dilution is added to a cuvette
containing 50 L of ABTS, and 0.1 M sodium acetate
buffer in a nal volume of 1.9 mL. After the mixture is
Table 4 Kinetic Parameters of HRP with Different preincubated at 208C, 100 L of H2 O2 (0.5 mM) is
Substrates added to initiate the reaction and formation of the
ABTS radical cation is monitored at 414 nm for the
Substrate Rates constants (31) rst 5 min against a blank in which the HRP sample is
(a) Hydrogen acceptors k1 [M1 sec1 ] replaced by deionized water. One unit of enzymatic
H2 O 2 9 108 activity is dened as the amount of HRP which causes
Methyl hydroperoxide 1:5 106 oxidation of one mol of ABTS per min at 258C, pH 5.
Ethyl hydroperoxide 3:6 106 Molar absorption coefcient
414 3:6 104 M1
(b) Hydrogen donors Km (mM) (32, 33) cm1 .
KI 181.7
o-dianisidine 0.48 4. Calculation
4-Aminophenazone 83.33
Pyrogallol 23.80 Specic activity (U/mg) A414 =3:6 104 :dtE,
where d is light path 1 cm, t is time of reaction
Source: Refs. 3133. in min, and [E] is enzyme concentration in mg/mL.

B. Determination of HRP Activity Using 2. Solutions
Guaiacol (35, 44, 45)
a. Phosphate Buffer. 0.2 M sodium phosphate
1. Reaction buffer, pH 7.0.
b. H2O2. Dilute 1 mL of 30% H2 O2 to 100 mL
H2 O2 AH2 ! 2H2 O A 3 with redistilled water. Further dilute 1 mL of this solu-
AH2 Guaiacol tion to 50 mL with 0.2 M phosphate buffer to obtain
1.7 mM H2 O2 . Prepare fresh daily.
2. Solutions c. 4-Aminoantipyrine and Phenol. Dissolve 810

a. Phosphate Buffer. 0.2 M sodium phosphate mg phenol and 25 mg 4-aminoantipyrine in 40 mL
buffer, pH 7.0. redistilled water and add more water to a nal volume
of 50 mL.
b. Guaiacol Solution. 18 mM (245 mg of guaiacol
in 100 mL water). 3. Procedure
c. H2O2 Solution. 8 mM (dilute 0.1 mL of 30% Pipette 1.4 mL of phenol/4-aminoantipyrine solution
H2 O2 solution with redistilled water to read A240 and 1.5 mL of 1.7 mM H2 O2 into each cuvette, and
0:400; light path 1 cm. preincubate at 258C for 34 min. Add 0.1 mL of the
diluted enzyme and measure the A510 for 45 min.
3. Procedure One unit of the enzyme is dened as the amount which
The appropriately diluted HRP sample is mixed with brings about 1 mol of hydrogen peroxide to result in
0.3 mM guaiacol in 0.1 M phosphate buffer in a total A510 increase per min at 258C and pH 7.0 under the
volume of 3.0 mL and preincubated at 258C. Addition specied conditions.
of 0.03 mL 8 mM H2 O2 initiates the oxidation reac-
tion; the oxidation of guaiacol is monitored at 470 nm 4. Calculation
(
470 2:75 103 M1 cm1 ) for the rst 5 min. One Specic activity (U/mg) A510 /min/(6:58 mg
unit of HRP activity is the amount of enzyme that enzyme/mL reaction mixture).
results in the oxidation of 1 mol of guaiacol per The RZ (Reinheitzahl) number, the absorbance
min at 258C, pH 7.0, under the above-described con- ratio A403 =A275 , is used as an evaluation of enzyme
ditions. purity. As an example, partially puried HRP has an
RZ of 0.81.0 with a specic activity of 85200 U/mg.
4. Calculation The chromatographically puried HRP has an RZ of
Specic activity 3:03 A470 min1 =2:75 103 M1 3.0 with a specic activity of 650900 U/mg. However,
cm1 d E where 3.03 is the total reaction volume the RZ values for the isozymes have been found to
in mL, d is the light path 1 cm, [E] is the enzyme vary from 2.50 to 4.19 (47); RZ is also pH dependent.
concentration in mg/mL, d is the light path and Therefore, both the specic activity and RZ value have
A=min is the change of absorbance at 470 nm/min. been given together as the criterion of purity.
C. Determination of HRP Activity Using Phenol

Plus 4-Aminoantipyrine (46) VII. PREPARATION OF PEROXIDASE
FROM HORSERADISH
1. Reaction
Since the 1950s, a variety of isolation and purication
2H2 O2 Phenol 4-Aminoantipyrine methods have been developed. Preparation of HRP is
4 generally composed of (a) initial extraction, (b) isola-
!4H2 O Quinol Red
tion and partial purication, and (c) renement.
When phenol plus 4-aminoantipyrine is used as hydro-
gen donor, the HRP-catalyzed reaction is monitored A. Initial Extraction
by measuring the increase in absorbance at 510 nm due
to formation of quinol in red color with extinction Fresh roots of horseradish plant are cut, minced,
coefcient
510 6:58 M1 cm1 . extracted with water, and ltered through cheesecloth.

The extract may contain > 40 HRP isoenzymes as cation-exchange column to remove all the basic iso-
harvested (47, 48). enzymes and contaminant proteins. The efuent
contained neutral HRP. After being desalted and lyo-
philized, a puried HRP with a specic activity 500
B. Traditional Purication of HRP Umg1 and RZ 3:0 was obtained (Zhong-Yu Ji,
personal communication, 1995).
In the traditional processes of HRP production pio-
neered by Theorell (1945) and Keilin and Hartree
(1951), the common biochemical methods involved C. Reverse Micellar Purication of HRP
ammonium sulfate fractionation, organic solvent (acet-
one, ethanol, etc.) different precipitation, calcium As an alternative to the above methods, reverse
phosphate gel adsorption, and corresponding dialysis micelles were recently utilized in isolation and purica-
and crystallization selected to achieve the HRP with tion of the enzyme from the root extract (51). The
RZ 2:33.04 (4648). reverse micellar isolation involved two stages.
HRP isoenzyme C was successfully separated from The horseradish root extract with a specic activity
isoenzyme B by using a modied elution gradient from of 1.1 Umg1 was dialtrated using 10 kDA MWCO
CM-cellulose column, and further puried using a pre- membranes against deionized water. In the rst for-
parative isoelectric focusing to achieve HRP C as a ward transfer, the anionic surfactant AOT (aerosol-
single component (49). However, this process was OT [sodium-bis(2-ethylhexl)]sulfosuccinate]) in isooc-
time-consuming and expensive with respect to ampho- tane (110 mM) was mixed with the dialyzed extract
lyte needed. at pH 4.15 and 0.15 M NaCl (Vaq/Vorg 5). In this
Ion-exchange column chromatography was also case, HRP protein solubilized in the aqueous phase,
used in isolating ve major neutral isoenzymes of and most contaminant proteins ( 49% protein)
HRP from a partially puried preparation were removed into the organic phase. After centrifuga-
(RZ 0.8). Three columns of SP-Sephadex C-50 com- tion, the organic phase was separated and regenerated
bined with an improved elution system in which a step- by backtransfer. In the second stage, the forward
wise increase of pH of elution buffer at a xed low transfer of the HRP micelles was carried out at pH
concentration was used instead of increasing the con- 3.5, 0.15 M NaCl by mixing with AOT in isooctane
centration of the buffer at a xed pH (50). (Vaq/Vorg 10). Finally an HRP of specic activity of
In recent years, a cation-exchange column was com- 86 guaiacol Umg1 was obtained with 80-fold puri-
bined with an anion-exchange column to form a simple cation and 46% yield.
and effective approach to purify HRP. The partially Comparison between the two-stage reverse micellar
puried preparation of HRP owed in an anion- extraction and traditional methods (two ammonium
exchange column. The acidic HRP isoenzymes and sulfate fractionations plus two differential ethanol
other contaminant proteins were adsorbed on the precipitations with the corresponding dialysis steps)
resin, whereas the neutral and basic HRP passed gave a good efciency of the former (see Table 5).
through. Then the efuent was applied on the second Other techniques, such as afnity chromatography
Table 5 Peroxidase Purication Using the Traditional Biochemical Methods in Combination with
Reverse-Micelle Extraction (Horseradish Peel only; Method C)a
Specic activity Purication

Purication stage Protein (mg)b Activity (U)c (U mg1 ) factor Yield (%)
Crude extract 5500 5300 1.0 1 100

Crude preparation 100 4100 41 40 76
Two-stage reverse-micelle
extraction 16 2400 150 150 45
Source: Ref. 51.

a
Values are the mean of three replicates with SE within 5% of the mean.
b
Total protein was measured using the BCA method (32).
c
Activity measured by the guaiacol assay (30).

and hydrophobic chromatography, were also used for 14. CP Vance, TK Kirk, RT Sherwood. Lignication as a
peroxidase purication (52, 53). mechanism of disease resistance. Annu Rev
Phytopathol 18:259288, 1980.
15. J Ricard, D Job. Reaction mechanism of indole-3-
acetate degradation by peroxidase. A stopped ow
and low-temperature spectroscopic study. Eur J
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198990.

29
Glutathione Peroxidase
Jun-qiu Liu and Gui-min Luo

Jilin University, Changchun, China
I. INTRODUCTION and addition reactions with endogenous and xenobio-

tic electrophiles as well as the reduction of organic
Glutathione peroxidase (GPX; EC 1.11.1.9) is a sele- hydroperoxides, but not hydrogen peroxide. The
noenzyme that protects biomembranes and other cel- enzyme has subsequently been demonstrated to be glu-
lular components against oxidative damage (13). Its tathione S-transferase (11). Among the GPX isozymes,
activity was discovered by Mills in 1957 (1). In 1973, cGPX, pGPX, and giGPX are tetramers and have
the stoichiometric amount of selenium was found in similar substrate specicity. They catalyze the reduc-
this peroxidase in the effort of searching for the pros- tion of H2 O2 , tert-butyl hydroperoxide, cumene hydro-
thetic group of GPX and the essentiality of selenium as peroxide, and linoleic acid hydroperoxide with
the trace element (4, 4a). The enzyme catalyzes the glutathione, but reduce cholesterol hydroperoxide
reduction of a variety of hydroperoxides including and phospholipid hydroperoxide poorly (913). The
hydrogen peroxide, organic hydroperoxides, and lipid giGPX displays most of the enzymatic properties of
hydroperoxides using glutathione (GSH) as the redu- the classical cellular GPX, but shows little activity
cing equivalent [Eq. (1)]: toward lipid hydroperoxides (9). Plasma glutathione
peroxidase is an extracellular enzyme, which is differ-
2GSH ROOH ! GSSG ROH H2 O 1
ent from other GPX enzymes in structural and enzy-
where ROOH is the hydroperoxide such as H2 O2 , tert- matic properties (7, 8). PHGPX is a membrane-bound
butyl hydroperoxide, cumene hydroperoxides, etc. enzyme, a monomer, and has different substrate speci-
(Table 1), GSSG is the oxidized form of glutathione city compared to cGPX, pGPX, and giGPX. It cata-
which is reverted to GSH by glutathione reductase, lyzes the reduction of phospholipid hydroperoxide,
and ROH is the corresponding alcohol or H2 O in the linoleic acid hydroperoxide, and cholesterol hydroper-
case of H2 O2 acting as substrate. oxide in biological membranes, and also shows activity
Four types of GPX have been discovered: (a) the on H2 O2 and organic hydroperoxides (5, 14).
classical cellular glutathione peroxidase, cGPX (1);
(b) the phospholipid hydroperoxide glutathione perox-
idase, PHGPX (5); (c) the plasma glutathione peroxi- II. BIOLOGICAL DISTRIBUTION OF GPX
dase, pGPX (68); and (d) the gastrointestinal
glutathione peroxidase, giGPX (9). Another type of Glutathione peroxidase has been found in most
enzyme that contains GPX activity but does not animals but not in plants (1517). A low level of the
depend on selenium for catalysis has been previously enzyme has been demonstrated in some insects (18),
classied as selenium-independent GPX (10). It cata- and GPX activity has been detected in yeast,
lyzes glutathione-dependent nucleophilic substitution Hansenula mraki (19). The enzyme is found chiey in

Table 1 Substrates and Their Structures of Glutathione Peroxidase
Chemical
Substrate structure Examples
Glutathione g-Glu-Cys-Gly
Hydroperoxides R-O-O-H H2O2
Organic hydroperoxidase: ethyl, cumene, tert-butyl, 5-phenyl-4-pentenyl,
linoleic, linoleic acid hydroperoxides, and their methyl esters: 13-hydroperoxy-
9, 11-octacadienoic acid, cholesterol 7b-hydroperoxide; allopregnanolone 17a-
hydroperoxide; thymine hydroperoxide.
Lipid hydroperoxide: phosphatidylethanolamine, phosphatidyl-choline,
phosphatidylserine, cardiolipin and phosphatidic acid hydroperoxides: mono-
and dihydroperoxydilinoleoylphosphatidyl cholines
respiratory tissues (blood vessels, lung, heart), diges- III. UTILITY OF GPX
tive tissues (liver, kidneys), and brain tissues where
oxygen is required. In rats, a high level of the enzyme The biological damage such as peroxidation of lipids
is found in the liver with lower concentrations detected and nucleic acid damage can be caused by reactive
in red cells, heart, lung, and kidney. In hepatocytes oxygen species. Antioxidant defenses consist of a num-
70% of the GPX activity is from the cytosol and ber of antioxidant enzymes protecting cells and mem-
30% is localized within the mitochondrial matrix (20). branes against the reactive oxygen. The rst line of
Classical GPX is present abundantly in erythro- defense seems to be governed by superoxide dismutase

cytes, kidney, and liver (16). The lens of different ani- which acts by converting the superoxide ion O2: into
mal species contain remarkably high GPX activity hydrogen peroxide. Secondly, catalase, and GPX share
(21, 22). PHGPX has been identied in all tissues in the task of metabolizing H2 O2 to water. In the last line
which it was searched for, namely, rat kidney, heart, of defense, the GPX is the sole enzymatic system cap-
lung, muscle, and brain; bull spermatozoa; and sh able of preventing lipid peroxidation. The enzyme can
liver (23). A high level of GPX activity has been reduce a wide variety of hydroperoxides, and acts as an
observed in rat testis (24). pGPX is detected in plasma, important part of the antioxidant defense of biology
milk, and lung (6, 7). giGPX has been found in the body.
cytosol of human and rat gastrointestinal tracts (9). Many studies have shown that low selenium in the
It was detected in human liver and colon, but not diet can result in low levels of GPX activity leading to
other human tissues including kidney, heart, lung, oxidative damage and several medical disorders such
placenta, and uterus. In rodent tissues, giGPX is only as Keshan disease, Kashin-Beck disease, cancer, heart
detected in the gastrointestinal tract, not in other disease, premature aging, and diabetes. The impor-
tissues. In rat, the highest activity of the enzyme is tance of this enzyme as an oxidant defense enzyme
detected in stomach followed by decreasing activities has led to its proposed strong possibilities to be used
in esophagus, colon, and intestine. in treatment for these diseases (2932).
The study by Godin and Ganett (25) showed that
the distribution of GPX in mammalian tissues is
IV. PROPERTIES AS PROTEIN
strictly functionally related to other antioxidant
enzymes such as superoxide dismutase (SOD; EC A. Molecular Weight and Physical Properties
1.15.1.1) and catalase (EC 1.11.1.6). It should be
kept in mind that the level of GPX is subject to numer- cGPX, pGPX, and giGPX are tetrameric proteins of
ous inuences, including dietary selenium, age and sex, four identical subunits, with each subunit containing
and estrous cycle, and other environmental factors one atom of selenium in the form of selenocysteine
(2628).It has been documented that GPX activity is residue. The average molecular weight is 1925 kDa
subject to large species differences in all organs (20). (33, 34, 42). PHGPX is a monomeric protein of 23

kDa containing a single selenocysteine residue (5, 13, N-terminus. The other two, 3 and 4 , are antiparallel.
35). The properties of GPXs from different sources are The -helices, 1 , 2 , and 4 , are located on the side of
listed in Table 2. the central -sheets and another one, 3 , is on the other
side. The 1 and 4 are nearly parallel and their axes
B. Protein Structure seem to parallel with -sheets. The active selenocystine
residue 45 is located at the N-terminal end of helix 1
Multiple forms of GPX exist in different animal tis- which forms a substructure. The structure
sues. In the cGPX family, the bovine erythrocyte may be important for catalysis and substrate binding.
cGPX was well investigated. It consists of four identi- The structure of the other GPX, human pGPX, was
cal subunits each of 198 amino acids. The catalytic proved to be similar to that of cGPX, although they
active selenocysteine is localized at residue 45 with belong to different species and have 42% sequence
four other cysteine residues present at positions 74, identity. No signicant deviation of secondary struc-
91, 111, and 152 (36). The primary structures of several ture was found between the two enzymes (34). The
GPXs have been determined from their cDNA main differences in two enzyme subunits are an
sequences (Fig. 1) (9, 3742). extended N-terminus and possible existence of a disul-
Within each GPX class high-sequence homology is de bridge in the pGPX.
found. The similarity between any two cGPXs ranges cGPX exists as a rather at, tetrahedral quaternary
from 80% to 90% (43). The mouse pGPX protein structure with point symmetry 222. Each asymmetric
sequence is 98% identical to rat, 89% identical to unit consists of two identical subunits in the dimer.
human, and 88% identical to bovine pGPX protein The contact regions are extensive along two of the
sequences (44); the homology between different classes perpendicular twofold symmetry axes, a local axis
is much lower. The sequence homology of human relating the monomer of an asymmetric unit, and a
pGPX is 44% identical with the human cGPX, 34% crystallographic axis producing the tetrameric struc-
with the human giGPX, and 25% with the human ture from the dimer. The interactions at each inter-
PHGPX (9, 41). The homology of mouse pGPX is face contain hydrogen bonds formed across the local
< 52% identical to mouse cellular cGPX (44). axis by residues Glu77, Arg86, Arg286, and Glu227;
However, the homology is much higher in the active- hydrophobic actions; and the main-chain hydrogen
site domain with the selenocysteine residue and in bonds (45). The active-site selenocysteine residues
some regions containing residues 6174, 93108, 119 are located on two opposite surfaces of the tetramer.
138, and 181192 (41, 44). The active center of GPXs is found in a at depres-
In the GPX family, only two crystal structures have sion on the tetramer surface surrounded by aromatic
been determinedthe bovine erythrocyte GPX (45) amino acid residues, with selenocysteine located at the
and the human plasma GPX (34). Bovine cGPX exists N-terminal ends of -helices forming structures
as a tetramer with each asymmetric unit consisting of together with adjacent parallel -sheets. Three resi-
two identical subunits. The secondary structure within dues, Arg40, Glu130, and Arg167 are involved in
each subunit contains four -sheets, four -helices, 12 binding of substrate glutathione (GSH). Arg167 and
clearly dened -turns, and three one-turn 310 helices. Arg40 form salt bridges with the carboxyl groups of
Two -turns, 1 and 2 , are parallel, and close to the GSH, and Glu130 can form a hydrogen bond with
Table 2 Properties of GPXs from Different Sources
Subunits
Subcellular molecular Activiation energy
Enzyme location weight (kDa) Subunits Isoelectric (kcal/mol1 ) Ref.
Bovine cGPX Cytosolic 21 4 5.66.0 4, 56

Human cGPX Cytosolic 23 4 4.9 8.2 60
Hamster liver cGPX Cytosolic 23 4 5.1 3 58
Rat lunk cGPX Cytosolic 20 4 67
Pig heart PHGPX Membrane 22 1 5
Human pGPX Plasma 21.5 4 64

Figure 1 Amino acid sequences of GPXs: bovine erythrocyte cGPX (37), mouse erythrocyte cGPX (38), human cGPX (39),
human giGPX (9), pig PHGPX (40), human pGPX, bovine pGPX (42). The selenocysteine is indicated by an asterisk.
GSH. A proposed model of glutathione binding was exons; exons 3 and 5 show homology with exons of
given by Epp et al. (45). cGPX, indicating that this gene, although related to
Compared with cGPX, the three residues for cGPX, is distinct from it (40, 46). Multiple isoforms
binding GSH are mutated or deleted in pGPX. The of PHGPX have been detected in human, rat, pig, and
surface of pGPX is more negatively charged, especially bovine tissues (5, 23, 24, 4951). Another intercellular
close to the active site. These differences may affect GPX is giGPX. The gene expression was found in
substrate binding and lead to different enzyme human liver and colon and also detected in mouse
activities (34, 41). and rat gastrointestinal tract (9). giGPX has been
Although there are no data on the crystal structure regarded as the most closely related offspring of the
of PHGPX, a model has been developed by molecular cGPX family (43).
dynamics calculation (43). According to this model, An articial GPX mimic, selenosubtilisin, was cre-
the shape of the core of these enzyme families compris- ated by converting the active serine residue of the cat-
ing the pleated sheets and three of the helices is largely alytic site of subtilisin into selenocysteine. It displays
identical to that of cGPX. However, the deletion of GPX activity but not as efcient as cGPX (52, 53).
several amino acids in the PHGPX sequence causes a Another articial selenium-containing enzyme has
substantial short-cut of the subunit interaction loop been generated by monoclonal technique and chemical
and an elimination of a small helix. In the PHGPX modication (54). The abzyme displays a higher GPX
model, the active site appears more freely accessible. activity compared with rabbit liver cGPX.
This allows the enzyme to attack more complex hydro- All the cDNA sequences of these GPX contain a
peroxy lipids. UGA codon coding for selenocysteine (9, 41, 38).
The UGA codon, usually functioning as a stop
codon, was demonstrated to encode the amino acid
C. Isoforms residue selenocysteine, which could be incorporated
directly during protein synthesis (46, 38).
The existence of multiple forms of GPX is due to the
expression of different genes (41, 46). Many mamma-
lian cGPX genes display highly conserved sequences
V. ENZYMETIC PROPERTIES
around the site of selenium (47). The gene for human
pGPX has been found at chromosome 5q32, and con- A. Kinetics of GPX
tains ve exons rather than two for cGPX. Rat and
bovine pGPX genes have been identied similar to The steady-state kinetics of bovine erythrocyte GPX
human pGPX (48). The gene for PHGPX has seven was studied by Flohe et al. (56). The enzyme system

follows tert uni Ping-Pong mechanism with indenite tion, suggesting that pGPX is a very effective hydro-
Michaelis constants and indenite maximum velocities peroxide scavenger at low concentration of plasma
(56, 57). The double-reciprocal plots of GPX reaction GSH.
with hydroperoxides in various concentrations of both
GPX and hydroperoxide lead to parallel lines. The rate B. Specic Mechanism of Action
equation was described in Eq. (2).
E0 = 1 =ROOH2 =GSH 2 In the catalytic cycle of the enzyme reaction with a tert
uni Ping-Pong mechanism, the selenol form (E-SeH) of
In this mechanism, [E0 , [ROOH], and [GSH] are the enzyme is rst oxidized by hydroperoxide to a sele-
the concentrations of GPX, ROOH, and GSH, respec- nenic acid (E-SeOH) which is reduced back by two
tively, is initial rate of the enzyme reaction, and 1 GSH through an intermediate selenenyl sulde form
and 2 are Dalziel coefcients, examples of which are (E-SeSG) (5, 56).
presented in Table 3. In double-reciprocal plot of
E0 = vs 1/[ROOH], the slope is identical to 1 and the k1
ROOH ESeH ! ESeOH ROH 5
intercepts equal 2 =GSH. The Michaelis constant for
one substrate depends on the other and is expressed as k2
ESeOH GSH ! ESeSG H2 O 6
follows:
k3
Km;ROOH 1 =2 GSH 3 ESeSG GSH ! ESeH GSSG 7
Km;GSH 2 =1 ROOH 4 A proposed catalytic cycle containing selenenic acid,

selenenyl sulde, and selenol has been suggested (Fig.
cGPX from bovine and ovine erythrocytes and 2), which is consistent with Ping-Pong kinetics (45, 56,
hamster liver show no saturation for GSH, and true 60). The seleninic acid (E-SeOOH) is not involved in
Michaelis constants for GSH cannot be measured (56, the main catalytic cycle but may become important at
58). However, the apparent Michaelis constant for high concentrations of hydroperoxide.
ROOH and GSH can be calculated according to Eqs.
(3) and (4).
The reaction kinetics of cGPX and PHGPX is in C. Substrate Specicity
agreement with a tert uni Ping-Pong mechanism (5).
The Km values and other kinetic parameters for The enzyme has a broad substrate specicity. The
cGPX and PHGPX are listed in Tables 4 and 5. relative substrate specicity of different GPXs are
The reaction mechanism of pGPX also displays presented in Table 6. All GPXs have specicity
Ping-Pong and is similar to those for cGPX and toward hydroperoxides (ROOH) but not on perox-
PHGPX. However, the pGPX shows saturation ides (ROOR). GSH is the only physiological donor
kinetics on both substrates (8, 60). The true Km values substrate although GPX exhibits activities for some
can be obtained from kinetic plots. Some kinetic con- sulfhydryl compounds such as mercaptoethanol (5,
stants for human cGPX and pGPX are listed in Table 62). cGPX, PHGPX, pGPX and giGPX display
4 and Table 5. The value of kcat =Km are very close to substrate specicity for H2 O2 and organic hydroper-
the diffusion controlled limit for a bimolecular reac- oxides. Although it shows lower hydroperoxide
Table 3 Dalziel Coefcients for cGPX and PHGPX
cGPX PHGPX
Substrate f1 108 msec f2 106 msec f1 106 msec f2 104 msec
H2O2 0.56 1.27 5.5 1.3
tert-Butyl hydroperoxide 13.5 2.24 1.4 31
Cumene hydroperoxide 7.8 2.24 9.1 2.5
Linoleic acid hydroperoxide 5.5 2.3
Phosphatidyl choline hydroperoxide 1.2 1.6
Source: Refs. 5, 20, 56.

Table 4 Kinetic Constants for Human pGPX, Human cGPX, and Ovine cGPX
Enzyme Hydroperoxide Km mM 1mM GSH kcat 103 min1 kcat =Km 107 M1 min1
Human H2O2 3.3 18.4 2.3

pGPX 13-hydroperoxy-9,11- 0.66 9.20 5.6
octadecadienoic
15-hydroperoxy-5,8,11,
13-eicosatetrenoic 2.1 13.2 4.8
5-phenyl-4-pentenyl
hydroperoxide 2.6 20.0 3.0
Cumene hydroperoxide
t-butyl hydroperoxide
Ovine 80
cGPX H2O2 120
5-phenyl-4-pentenyl
hydroperoxide
Human 12
cGPX 10
activities for phospholipid hydroperoxides than

organic hydroperoxides, pGPX possesses the ability
to reduce phospholipid hydroperoxides, whereas
giGPX and cGPX have no activities for lipid hydro-
peroxides (Table 6).
Phospholipid hydroperoxides are the primary sub-
strates for PHGPX, which is also active on H2 O2 and
organic hydroperoxides such as tert-butyl hydroperox-
ide, cumene hydroperoxide and linoleic acid hydroper-
oxide (5). pGPX is about one-tenth less reactive Figure 2 The proposed catalytic mechanism of GPX for
against organic and hydrogen peroxides than cGPX reduction of ROOH by GSH.
(63, 64). giGPX displays lower activity than pGPX
against organic and hydrogen peroxides.
The second-order constants for the reduction of small and unhindered substrate such as H2 O2 and
the hydroperoxides show that H2 O2 is a poorer ethyl hydroperoxide, this effect can be negligible (63).
substrate for PHGPX than for cGPX, while linoleic
acid hydroperoxide is reduced faster by PHGPX.
PHGPX is highly active on lipid hydroperoxides D. Effect of Environmental Factors
(Table 7). 1. pH Effects
In cGPX, the hydrophobic effect of a bulky sub-
strate seems important, the more hydrophobic, the GPX is relatively stable in the range of pH 710 (20).
faster a bulky substrate is reduced by cGPX. For Enzymes from different sources have similar pH opti-
mum > 8 (Table 8). The enzyme alone can form oxi-
dized state (seleninic acid) in the air, but can be
reactivated by incubation with GSH (45, 65).
Table 5 Km Values for Human pGPX and cGPX is very stable at pH 7.0 at 48C. The enzyme
Human cGPX lost only 1520% activity when stored at 48C at pH 7.0
for 4 months. However, it is very unstable at low pH;
Enzyme Kmt,BuOOH(mM) KmGSH(mM)
At pH 4.0, the enzyme lost all its activity in 20 min
pGPX 570 5.3 (65). The GPX from rat lung was found to have very
cGPX 52 4.1 little activity at physiological pH 7.0, < 20% of the
activity at the pH optimum of 8.8 (67).
Source: Ref. 8, 65.

Table 6 Activity of GPX Toward Different Hydroperoxides
Specic activity (U/ mg enzyme)

a
Substrate cGPX pGPXb PHGPXc giGPXd
H2O2 216 31.3 135 2.1
tert-Butyl hydroperoxide 224 26.6 13.5 0.6
Cumene hydroperoxide 199 25.0 8.7 0.9
Linoleic acid hydroperoxide 187 1.2
Phosphatidyl choline hydroperoxide 0 8.41 226 0.004
The specic activities are dened as mmol NADPH oxidized/min/mg protein.
Data shown for GPXs: ahuman erythrocyte cGPX (63); bhuman plasma pGPX (63); cpig heart
PHGPX(5); dhuman carcinoma MCF-7 cells giGPX (9).
2. Inhibitors VI. DETERMINATION OF ACTIVITY

Divalent cations, sulfhydryl reagents, and iodoace- A. Methods for Determining the GPX Activity
tate inhibit the enzyme activity. The enzymes from
human erythrocyte and human plasma exhibit simi- Three methods are used for determining the GPX
lar inhibitory spectrum by divalent cations (8, 66). activity. The most commonly used assay is the GSH
Cu2 and Hg2 completely inhibit the two enzyme reductaseNADPH coupled assay (33, 70, 71). The
activities. Zn2 completely inhibits human pGPX method takes advantage of the capability of glu-
but partly inhibits human cGPX. Ni2 , Co2 , tathione reductase to regenerate GSH from GSSG.
Ca2 , and Mg2 also inhibit the two enzymes. The loss of NADPH is monitored continuously by
Most enzymes from various sources are inhibited absorbance spectrophotometry. The other two meth-
by iodoacetate in the presence of GSH or a sulfhy- ods involve measurement of the GSH removal by the
dryl reagent such as mercaptoethanol or dithioer- formation of thionitrobenzoate from Ellmans reagent,
thritol (5, 8, 66). The active selenocysteine residue 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) (72) and by
of the reduced enzyme can be alkylated by iodoa- polarographic GSH analysis (20).
cetate, but the other alkyling reagent, iodoaceta-
mide, has no inhibitory effect. Human erythrocyte
1. Method 1: Coupled Enzymatic Assay
GPX was reported to be inactivated by N-ethylmal-
eimide (66, 68). In an inhibition study for hamster a. Reagents. 100 mM potassium phosphate buf-
liver GPX, a number of mercaptocarboxylic acids fer, pH 7.0, containing 1 mM sodium azide and 1 mM
and tertiary mercaptans have been found to be EDTA; 2.4 U/mL glutathione reductase (from yeast),
strong and specic inhibitors (69). prepared daily; 10 mM GSH (AR) in distilled water;
2.5 mM NADPH in 0.1% NaHCO3 , prepared daily;
12 mM hydroperoxide (AR) in water.
Table 7 Second-Order Rate Constants (k1) for the
Reaction Between cGPX and PHGPX with Hydroperoxidic
Substrates (pH 7.6)
cGPX PHGPX Table 8 Optimum pH of GPXs

Substrate k1105 (mM1 min1 )
Enzyme Optimum pH Ref.
H2O2 2.98 1.8
tert-Butyl hydroperoxide 7.1 0.71 Bovine erythrocyte cGPX 8.8 56
Cumene hydroperoxide 10.4 1.1 Human erythrocyte cGPX 8.5 66
Linoleic acid hydroperoxide 23.6 1.8 Hamster liver cGPX 8.0 58
Phosphatidyl choline 0 8.6 Rat lung cGPX 8.8 57
hydroperoxide Human pGPX 8.9 64

b. Procedure. 500 L of 100 mM phosphate 4. Denition of Units
buffer (pH 7.0), 100 L of enzyme sample, 100 L
In the above three methods, 1 unit of the enzyme activ-
of glutathione reductase (0.24 U), 100 L of 10 mM
ity is dened as the amount of enzyme oxidizing 1
GSH, and 100 L of 2.5 mM NADPH are added into a
mol of NADPH per min at 378C. The specic activity
1-mL curette. The reaction mixture is preincubated for
expresses units/mg of protein or units/mol of protein.
10 min at 378C. The reaction is started by adding 100
Some GPX specic activities with different peroxide
L of hydroperoxide solution. The linear decrease of
substrates are listed in Table 6.
NADPH absorption is monitored at 340 (or 366) nm
for about 5 min. The control is assessed by replacing the
B. Factors Affecting Activity Determination
enzyme with phosphate buffer.
The coupled enzyme method is suitable for activity
determination of both puried and crude enzymes.
2. Method 2: Assay of GSH Removal by
In working with crude biological materials (e.g.,
DTNB
liver and kidney), it is necessary to use H2 O2 as
a. Reagents. 50 mM potassium phosphate buf- the substrate in order to differentiate GPX from
fer, pH 7.0; 10 mM GSH (AR) in distilled water; 1.5 glutathione S-transferase which has similar activity
mM DTNB in potassium phosphate buffer, pH 7.0; 12 on organic hydroperoxides but is unreactive toward
mM hydroperoxide (AR) in water. H2 O2 . In this case, sodium azide should also be
added to inhibit catalase. For blood red cells, the
b. Procedure. 700 L of potassium phosphate
conversion of hemoglobin into cyanomethemoglobin
buffer, 100 L of 10 mM GSH, 100 L of enzyme
prior to assay is necessary because of the interference
solution are added into a 1-mL cuvette and preincu-
of methemoglobin (20). PHGPX activity is measured
bated for 10 min at 378C. The reaction is started by
by this method with the phospholipid hydroperoxide
adding 100 L of hydroperoxide solution. Over 10 min
substrate dispersed in mixed micellar form using
50 L of the reaction mixture is taken at 1-min inter-
Triton X-100 (5).
vals. The solution is transferred into a cuvette contain-
The method for assay of GSH removal is suitable
ing 1.5 mM DTNB. The amount of GSH remaining in
for determining only puried enzymes. Inorganic
the reaction is determined by measuring the absor-
halides interfere with polarographic determination.
bance at 412 nm with an extinction of coefcient 13.0
Other sulfhydryl compounds such as mercaptoethanol
mM1 cm1 .
and cysteine will interfere with both polarography and
DTNB methods.
In all assay systems, the assay mixture should be
3. Method 3: Assay of GSH Removal by
preincubated with GSH. The inactive state of the
Polarography
enzyme (E-SeOOH) can be activated by GSH to
a. Reagents. 100 mM potassium phosphate buf- the active E-Se-SG form. High concentrations of poly-
fer, pH 7.0, containing 1 mM sodium azide and 2 mM valent cations (e.g., Hg2 , Cu2 , and Zn2 ) can inhibit
EDTA; 4 mM GSH (AR) in distilled water; 5 mM GPX activity.
H2 O2 in distilled water prepared from a commercial
solution (30%); 0.7 M HClO4 .
VII. PURIFICATION
b. Procedure. 1 mL of 100 mM phosphate buffer
is incubated with 0.5 mL GSH solution for 10 min at Several GPXs have been puried from various sources
378C. The reaction is started by addition of 0.5 mL of 5 (1, 514, 21, 63, 66, 67). A currently used procedure for
mM H2 O2 and stopped after various times by addition cGPX isolation has been described by Wendel (20).
of 2 mL of 0.7 M HClO4 . The acidied assay mixture is This strategy includes dialysis of the hemolysate,
cooled to room temperature and bubbled with nitrogen organic solvent precipitation, ion-exchange chromato-
for 3 min. Polarogram is recorded between 0 and 0:5 graphy on DEAE-Sephadex, hydrophobic chromato-
V with 5 A full scale, versus a mercuric sulfate graphy on phenyl-Sepharose, gel ltration on Biogel
reference electrode. The calibration curve is prepared P-100, or hydroxyapatite chromatography.
by adding HClO4 to a series of different GSH concen- Step 1. Hemolysate. Eight liters of bovine blood is
trations. The control is measured without enzyme centrifuged. The erythrocytes are washed twice with
addition. 0.9% NaCl and are hemolyzed by adding water to

form 8 L of hemolysate. Six liters of 0.6 M potassium maximum power, for 5 min in 3 volumes of ice-cold
phosphate at pH 6.6 is added and the mixture is stored 0.1 M Tris-HC1 (pH 7.4), 5 mM mercaptoethanol.
in the cold for 212 h. The soluble fraction is prepared by two centrifuga-
Step 2. Organic solvent precipitation. A mixture of tions of the homogenate: 30 min at 10,000 g and
chloroform/ethanol (3:5, v/v), 4 L, cooled to 208C, 45 min at 100,000 g. Before the second centrifugation,
are added dropwise to the vigorously stirred the supernatant is ltered through cheesecloth to
hemolysate. The dark-brown precipitate is removed eliminate uffy material. The supernatant is dialyzed
by centrifugation at 2000 g for 30 min at 08C. The exhaustively against 10 mM potassium phosphate
yellowish supernatant liquid is ltered and immedi- buffer (pH 7), 5 mM mercaptoethanol. During
ately concentrated to 1 L with a membrane with dialysis some proteins precipitate and are eliminated
exclusion limits of 50 kDa. by centrifugation.
Step 3. Phosphate precipitation. One liter of concen- Step 2. The supernatant is applied to a DEAE-
trated enzyme solution is mixed with 2 L of 3.65 M Sepharose 6B column (135.5 cm) equilibrated with
potassium phosphate at pH 7.0. The oating precipi- 10 mM potassium phosphate buffer (pH 7), 5 mM
tate is collected by ltration and dissolved in 0.7 M mercaptoethanol. The column is washed with 2 L of
potassium phosphate at pH 8.0. the equilibration buffer and then eluted with 0.5 L of
Step 4. Phenyl-Sepharose. The enzyme is adsorbed 0.2 M potassium phosphate buffer (pH 7), 5 mM mer-
in a batch step to phenyl-Sepharose equilibrated captoethanol. The active fractions are collected and
with 0.7 M potassium phosphate at pH 8.0. The pooled, and 10% (v/v) (nal concentration) glycerol
adsorbed gel is added to a column (230 cm) pre- is added.
viously prepared with 5 mL of fresh equilibrated gel. Step 3. The above fraction is loaded onto a
The column is washed with 300 mL of 0.7 M phos- Sepharose-bromosulfophthalein glutathione (BSP)
phate buffer and then with 300 mL of 0.5 M afnity column. The column is equilibrated with 25
phosphate buffer with the same pH. A linear, mM Tris-HCI (pH 7.2), 5 mM mercaptoethanol, and
300-mL gradient, from 100 to 10 mM potassium 10% (v/v) glycerol. After the sample is loaded, the
phosphate, pH 8.0, is used to elute the enzyme at a column is washed with 300 mL of 25 mM Tris-HCl
ow rate of 6 mL/h. (pH 7.2), 100 mM KSCN, 5 mM mercaptoethanol,
Step 5. DEAE-Sephadex. The pooled fraction is aand 10% (v/v) glycerol. The elution is carried out
diluted to a nal buffer concentration of 5 mM phos- with 25 mM Tris-HCl (pH 7.6), 300 mM KSCN, 5
phate, pH 8.0, and applied to a column of DEAE- mM mercaptoethanol, and 10% (v/v) glycerol. The
Sephadex equilibrated by the same buffer. After wash- active fractions are pooled and concentrated to 510
ing with 1 L of starting buffer, the enzyme is eluted mL using an Amicon ultraltration apparatus with a
with a linear gradient of 580 mM potassium phos- YM10 membrane.
phate containing 1 mM GSH at pH 8.0. Step 4. The above fractions are applied onto a
Step 6. S-300 Sephacryl. The active fractions are Sephadex G-50 column (1405.5 cm) equilibrated
concentrated by a Diao cell to 15 mL and charged with 25 mM Tris-HCl (pH 7.4), 5 mM mercaptoetha-
onto a S-300 Sephacryl column (2.0180 cm), equili- nol, and 10% (v/v) glycerol. The active fractions are
brated with 20 mM potassium phosphate, pH 8.0, con- pooled and concentrated with the same ultraltration
taining 0.5 M NaC1, with a rate of 12 mL/h. The apparatus as before. This fraction is dialyzed against
enzyme is eluted as a single protein that has an appar- potassium phosphate buffer (pH 6.5), 0.1 M KCl, and
ent molecular weight of 100 kDa. 5 mM mercaptoethanol.
Step. 7. The enzyme is dialyzed against 5 mM potas- Step 5. The nal purication step is carried out by
sium phosphate at pH 7.4, adsorbed onto a hydroxy- HPLC using a weak cation-exchange column, TSK
apatite column (1100 cm), equilibrated with the same CM. The chromatographic conditions are as follows:
buffer, and eluted with a gradient up to 80 mM phos- Buffer A: 10 mM potassium phosphate buffer, 100
phate. mM KCl, 5 mM mercaptoethanol (pH 6,5); buffer B:
Purication of PHGPX from pig heart has been 10 mM potassium phosphate buffer, 300 mM KCl, 5
described by Ursinbi et al. (49). This procedure can mM mercaptoethanol (pH 6.5). The gradient from 0
also be used for other tissues. 100% buffer B is developed in 25 min after 3 min in
Step 1. A total of 500 g of ventricular muscle isocratic conditions. Injection volume is < 0.2 mL.
is prepared from pig heart and is minced and Peaks isolated from several runs are pooled, and
thoroughly homogenized in a Polytron mixer, set at 10% glycerol is added.

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30
Glucose Oxidase
A. J. Vroemen
I. INTRODUCTION acid with simultaneous production of hydrogen perox-

ide. The decrease of the pH caused by the gluconic acid
Glucose oxidase (GOX) is produced predominantly by as well as the antibacterial effect of the hydrogen per-
fungi as part of an enzyme complex also containing oxide produced, are favorable for the fungus. In addi-
catalase. The best-studied microorganisms are species tion, A. niger is able to consume the gluconic acid
of Aspergillus (13) and Penicillium (4, 5). In produced, which is not the case for most of the com-
Aspergillus the enzymatic complex is located in cell peting microorganisms. Bacteria like Gluconobacter are
compartments called peroxisomes (6), which results also capable of converting glucose into gluconic acid,
in the need to open the fungal cell walls before the but these bacteria use a different mechanism.
enzyme can be released and puried. The enzyme com- Gluconobacter oxidans contains two types of glucose
plex of Penicillium is secreted into the extracellular dehydrogenases, one PQQ and one NADP dependent,
environment (7). Almost all GOX preparations avail- which convert glucose to gluconic acid without forma-
able on the market are produced by Aspergillus niger. tion of hydrogen peroxide. The PQQ-dependent
These preparations are either highly or somewhat or enzyme seems to be the most important (8). This oxi-
completely exempt of catalase (see Sec. II.A.1). This dation process yields energy for the bacteria con-
chapter will focus mainly on GOX of Aspergillus niger. cerned. It is notable that these bacteria are rather
The question can be raised why fungi like acid tolerant.
Aspergillus niger invest a high amount of nutrients
and energy in the synthesis of the enzymatic complex. II. REACTION CATALYZED
As we will see later, the catalyzed reaction of glucose to
gluconic acid does not yield energy for the microorgan- As substrates GOX uses b(D)-glucose and oxygen.
ism. Most probably the enzymatic complex gives the Two hydrogen atoms are rst transferred from glucose
microorganiism an ecological advantage in the soil. In to the coenzyme FAD during formation of d-glucono-
nature, GOX is produced by the fungus only when lactone (9). Subsequently the enzyme transfers the two
glucose is present: It is an inducible enzyme. When hydrogen atoms directly to molecular oxygen during
glucose is liberated from, for instance, starch or cellu- formation of hydrogen peroxide. The d-gluconolactone
lose, the synthesis of the enzymatic complex starts and is hydrolyzed to gluconic acid spontaneously or by the
in the presence of air, glucose is oxidized to gluconic enzyme gluconolactonase.

C6H12O6 + E-FAD C6H10O6 + E-FADH2 (1) cglu cO 2
v kcat E
Kmglu cglu KmO cO2
glucose gluconolactone 2
The Km values as determined by Gibson et al. (11)

C6H10O6 + H2O ! C6H12O7 (2)
Kmglu 110 mM; KmO 0:48 mM
gluconolactone gluconic acid 2
(both determined at 278C.)

E-FADH2 + O2 ! E-FAD + H2O2 (3)
Here the molarity of glucose is dened as total glucose,
not as b-glucose.
C6H12O6 + H2O + O2 C6H12O7 + H2O2 The value of 110 mM mentioned by Gibson seems
rather high. Other values mentioned in the literature
glucose gluconic acid hydrogen
are 37 mM (12), which is more in agreement with GOX
peroxide
from other microorganisms. The Penicillium amagasa-
kiense enzyme has a higher afnity for glucose: a Km
The fact that GOX uses only b-glucose as substrate
value of 5.2 mM is reported (13). The problem in the
does not mean that glucose can be only partially oxi-
determination of the Michaelis-Menten constant for
dized; during the process a-glucose spontaneously
glucose is that precautions must be taken to ensure
mutarotates to a-,b-glucose, making all the glucose
that the concentration of dissolved oxygen, which
available as substrate. GOX is rather specic for glu-
is consumed during the reaction, remains constant.
cose. There is < 1% activity on related sugars like
Technically it seems possible to maintain the dissolved
xylose and galactose. Also, the activity on other nat-
oxygen concentration constant during the reaction in
ural mono- and disaccharides is very low. In a systema-
for instance a fermenter, enabling the determination of
tic study Whitaker showed that in the deoxyglucoses,
correct velocity values at different glucose concentra-
replacement of the hydroxyl group by a hydrogen
tions. However, to the authors best knowledge such an
atom at successively the 6, 4, 2, 3 and 5 position
approach has never been published so far. Initial velo-
resulted in respectively 10%, 4%, 3%, 1% and 0%
city, vo, can also be used to obtain correct data.
remaining activity (10).
These Km values mentioned above show that the
The hydrogen peroxide formed causes the reaction
afnities of GOX for the two substrates are rather
to stop since it inactivates the GOX rapidly and irre-
poor. This is especially true for the substrate oxygen
versibly. In principle, hydrogen peroxide can be hydro-
when we compare the Km for oxygen with the solubility
lyzed by the enzyme catalase, or the active oxygen
of oxygen in water. At 1 bar, 258C, and in the presence
can be transferred spontaneously or enzymatically to
of air, the maximum solubility of oxygen is 6 mg/L or
another molecule.
0.2 mM, equaling < 50% of the Km. Even with pure
As mentioned before, the fungi concerned also pro-
oxygen the maximum solubility of 30 mg/L is only
duce catalase, which oxidizes/reduces the hydrogen
twice the Km. With glucose concentrations far below
peroxide produced into water and oxygen. The overall
the Km value, which is between 7 to 20 g/L for the
reaction of the enzyme complex becomes:
Aspergillus enzyme, the velocity of the reaction will
be low. As a consequence it can be concluded that
2C6H12O6 + 2H2O + 2O2 2C6H12O7
for the elimination of low quantities of glucose or oxy-
+ 2H2O2 (4)
gen from certain foods, high quantities of enzyme and/
2H2O2 ! 2H2O + O2 (5) or long incubation times are necessary even under opti-
mal conditions. The afnity of the enzyme catalase for
Overall reaction: its substrate hydrogen peroxide is extremely low: Km
2C6H12O6 + O2 ! C6H12O7 (6) = 1.1 M or 37.4 g/L. This aspect is very important in
certain applications.
B. Determination of GOX Activity

III. ENZYME CHARACTERISTICS
A. Michaelis-Menten Constants From a theoretical point of view, the analysis of GOX
is rather complicated. The enzyme has two substrates:
The enzyme has two substrates: glucose and molecular glucose and oxygen. In principle high glucose concen-
oxygen. Simple Michaelis-Menten kinetics results in trations can be chosen, far above the Km, so that the

velocity of the reaction is no longer dependent on the tionship between IU and Sarrett units is determined
glucose concentration. This is more difcult for the as 1 IU=1.12 SU (16, 18) or 1.1 SU(17). (See Annex
second substrate oxygen. To obtain oxygen concentra- 1 for a detailed description of a method with o-dia-
tions in solution far above the Km, very high oxygen nisidine.) Methods based on the oxidation of a chro-
pressures have to be chosen, which is not practical. The mogen by hydrogen peroxide are now generally used.
concentration of oxygen is below the Km if the reaction The relationship between IU and Sarrett units can be
is performed under normal conditions, and as a result different for glucose oxidation from different organ-
the velocity of the reaction is below the maximum isms. Finally, it should be mentioned that comparison
velocity. of units in literature is extremely difcult owing to
An extra complication is that the reaction consumes different incubation conditions, including saturation
oxygen. When there is insufcient supply to maintain levels of oxygen, and different unit denitions. The
the oxygen concentration at a saturation level, there best thing to do is to work with an internal standard
will not be a linear production of gluconic acid in of GOX of known activity.
time at a xed enzyme concentration or a linear rela-
tionship between the amount of gluconic acid pro-
duced within a xed time and amount of enzyme. C. Specic Activity
Such linear relationships are desirable for an analytical
method. Therefore, an analytical method for GOX is By measuring the oxygen consumption of a pure, cat-
preferably based on a system in which only very small alase-free GOX preparation of A. niger, Tsuge et al.
quantities of oxygen are consumed. A sensitive method (21) found a specic activity of 172 IU/mg protein at
is not realized when it is based on the consumption of 308C and pH 5.6. About the same value has been
glucose or the formation of gluconic acid. If the ana- reported by Hayashi and Nakamura (22). Several
lysis is based on the consumption of oxygen, the result research groups have puried the A. niger enzyme by
is inuenced by the presence of catalase. In this type of ion-exchange chromatography and obtained pure pre-
method high quantities of GOX-free catalase have to parations with activities from 200 to 250 Sarrett units/
be added. This is the principle underlying a method mg protein, equaling 180225 IU/mg protein. Pure cat-
described in 1953 by Scott (14) and in 1957 by alase-free preparations can be obtained from compa-
Underkoer (15). In a Warburg equipment the amount
of oxygen consumed under optimal conditions was
determined. The unit most frequently used is the
Annex 1 Analysis of Glucose Oxidase
Sarrett unit, which is dened as the amount of enzyme
which catalyzes the uptake of 10L oxygen/ min at The method used by Whittington (26) is mentioned here:
308C, pH 5.9, and a glucose concentration of 3%. Glucose solution A: 2 g/L in 0.2 M Tris phosphate buffer,
Working with a Warburg is rather laborious, and pH 7.0.
therefore Underkoer also describes a method using o-Dianisidine solution B: 2 g/L in 0.2 M Tris phosphate
a sodium hydroxide solution neutralizing the gluconic buffer pH 7.0.
acid produced. Horseradish peroxidase solution C: 60 units/mL in 0.2 M
Tris phosphate buffer pH 7.0.
A very sensitive and accurate method is based on
Glycerol.
the production of hydrogen peroxide. The amount of
Enzyme solution standard D: 10 IU glucose oxidase/mL.
hydrogen peroxide is determined indirectly by the oxi- Hydrochloric acid solution E: 5 M.
dation of a chromogen catalyzed by a peroxidase. All reagents Sigma quality.
Owing to the very high afnity of this peroxidase Preincubate all solutions at least 10 min at 308C.
for hydrogen peroxide compared to catalase, the pre- Add to 1 mL A, 0.1 mL B, 0.1 mL C, and 0.8 mL glycerol.
sence of this last enzyme in GOX preparations does Mix carefully.
not inuence the analytical result. As chromogen, o- Add 1020 L of an enzyme sample containing 010 IU/
dianisidine, o-toluidine, and others are used, the mL.
developed color is measured in a spectrophotometer, Incubate 30 min at 308C.
and by using internal standards the amount of oxi- Stop reaction by adding 2 mL of 5 M hydrochloric acid
solution. Mix carefully.
dized glucose can be determined. The activity is often
Oxidized o-dianisidine is measured in a spectrophotometer
expressed as IU (international units), dened as
at 525 nm.
micromolar glucose oxidized per min under optimal Compare with internal standard of 010 IU/mL enzyme.
conditions. For the Aspergillus niger enzyme the rela-

nies selling research chemicals, or by growing a trans- of two identical subunits with 1 molecule FAD per
genic yeast harboring a GOX gene. subunit as coenzyme. Because of this, the color of the
The specic activity of the puried Penicillium ama- oxidized protein is yellow with absorption maxima at
gasakiense enzyme is 60% higher than that of the A. 377 and 455 nm. Under anaerobic conditions in the
niger enzyme (22). presence of glucose, the enzyme molecule is reduced
and the color disappears. When oxygen is admitted to
D. Turnover Number the system the color reappears. The FAD is not cova-
lently linked and can easily be removed by acid, urea, or
Gibson et al. (11) found a turnover number of 16,200/ guanidine. But without these reagents, the FAD is
min. Whittington et al. (27) cloned the A. niger enzyme bound to the enzyme. The apoenzyme is not active
in yeast and found a value of 17,00020,000 for the but activity is restored upon incubation with FAD (15).
yeast-derived enzyme. Vmax values of 235/sec for the GOX is a glycoprotein. The amount of carbohydrate
wild-type enzyme and 500 for the yeast-derived enzyme in different preparations from A. niger can vary from
are found, but it remains unclear whether these differ- 10% to 18% (22, 23). Mannose is by far the most
ences are really signicant. important sugar with amounts of 7080%. Galactose
contributes 5% and glucosamine 15% to the total
E. pH Activity Prole carbohydrate content. In the Penicillium enzyme Kalisz
(13) found 95 residues of mannose, 12 residues of glu-
The pH activity curves for both the Penicillium and cosamine, and ve residues of galactose per molecule of
Aspergillus enzymes show a horizontal prole between enzyme, and a total carbohydrate content of 13%.
pH 4.5 and 7.5, with a sharp decline on both sides (10, In the A. niger enzyme the sugar molecules are N
20). There is almost no activity at < pH 3.0 or > pH and O linked. By selective elimination of the O-linked
8.5. GOX is stabilized by its substrate glucose (32), sugars Takegawa (12) showed that predominantly
explaining why in certain applications with a pH mannose monomers are linked to serine and threonine,
between 2.5 and 3.0 and with a high concentration of but the exact place in the molecule is not yet known.
glucose the enzyme is still active. Also at > pH 8.0 the The N-linked sugar moiety can be selectively removed
stability of the enzyme is improved by adding the by an enzyme from Flavobacterium, resulting in the
substrate. liberation of 30% of the total carbohydrates. The
partly deglycosylated enzyme has the same kinetic
F. Temperature Activity Prole and biochemical properties and resistance to proteases
as the native enzyme except that the native enzyme
In determination of the temperature activity prole of precipitates at higher concentrations of ammonium
GOX, some specic complications are encountered. sulfate or TCA, and the deglycosylated enzyme is less
GOX is sensitive to the hydrogen peroxide formed espe- stable in relation to pH and temperature. The same
cially at higher temperatures. This means that when a results were obtained with the Penicillium enzyme by
method is applied in which the hydrogen peroxide is not Kalisz et al. (13). In this case 95% of the carbohydrate
removed immediately and completely, the optimum was split off by the enzymatic treatment. Losses in pH
temperature and the maximum temperature will be and temperature stability were observed as well.
low as compared to a method where this is done prop- The enzyme from Phanerochaete chrysosporium (24)
erly (10). Moreover, long incubation times give a lower was found to be a avoprotein with a molecular weight
optimum. In the analytical method using perosidase of 160 kDa and containing 2 mol FAD per mol pro-
and a chromogen, the hydrogen peroxide reacts imme- tein, but it does not seem to contain any carbohydrate
diately with the chromogen. This method gives an activ- and its specicity is lower. Although glucose is the
ity prole which is rather constant with time between main substrate, considerable activity is still found on
308C and 608C. Above 608C activity goes down slowly sorbose, xylose, and maltose.
with still 10% activity at 708C but no activity at 808C. The gene of the A. niger enzyme has been isolated
and the sequence analyzed by various research groups
G. Protein Properties with identical results (2527). This sequence is now
available in data bases. The gene codes for a signal
The molecular weights of the enzymes from both peptide of 22 amino acids (122), followed by the
Aspergillus and Penicillium have been determined at 583 amino acid subunit itself (23605). The molecular
between 140 and 160 kDa (1921). The enzyme consists weight of the subunit plus signal peptide is calculated

to be 65,638, and without signal peptide it is 63,250. strate-binding site explains the high specicity of GOX
With 16% sugars and two molecules of FAD the described above. Part of the entrance to the pocket is
total molecular weight can be calculated to be 152 at the interface to the second subunit and is formed by
kDa. When the gene is expressed in yeast, an enzyme is a 20-residue lid. The carbohydrate moiety attached to
obtained with a higher glycolysation level and an Asn89 at the top of this lid forms a link between the
improved thermostability. This is in line with the subunits of the dimer. In total, there are four N-glyco-
observed lower thermostability the deglycosylated sylation sites, with an extended carbohydrate moiety at
enzymes. The kinetic parameters of the yeast-derived Asn89. Starting from the 3D structure of the
enzyme and the original one are not signicantly dif- Pencillium amagasakiense enzyme, it could be shown
ferent. The yeast expression system can be used to by mutation of key conserved active-site residues that
obtain GOX completely free of catalase. The trans- Arg516 is involved in the binding to the 3-OH group of
genic yeast must not been grown on glucose since the the glucose molecule (31). Replacement of this arginine
hydrogen peroxide formed will inactivate the enzyme by another amino acid lowers the afnity for the sub-
and stop growth. Instead it can be grown on sacchar- strate. Aromatic residues on other locations like 73,
ose, since this sugar is hydrolyzed by membrane-bound 418, and 430 are important for the correct orientation
invertase in the cell to fructose and glucose, which and maximum velocity of glucose oxidation.
are immediately metabolized without formation of glu-
conic acid and hydrogen peroxide.
IV. APPLICATIONS OF GOX IN FOOD
H. Genetic Aspects
Looking at the reaction catalyzed by the enzyme, the
A comparison of the mature GOX sequence with applications are linked to four different aspects:
sequences present in data bases shows 26% homology
1. Removal of glucose
with the alcohol oxidase from Hansenula polymorpha.
2. Removal of oxygen
The GOXs from Aspergillus and Penicillium show
3. Production of gluconic acid
66% identity and 79% similarity. The Penicillium
4. Production of hydrogen peroxide
enzyme consists of 587 amino acids. The two enzymes
are highly conserved in the FAD-binding and sub- A separate subject is the application of glucose oxidase
strate-binding domain, in their secondary structures as an analytical agent.
and in regions at the subunit interface. The highest
similarity with other oxidoreductases is observed in A. Removal of glucose (10)
the FAD-binding domain. Aryloxidase, a FAD-depen-
dent enzyme involved in lignin degradation, has been Glucose is a reducing sugar that can react with amino
cloned from Pleurotus eryngii. The enzyme is com- groups in, for instance, proteins, to form colored
posed of 593 amino acids, 27 of which form a signal Maillard components that are often undesirable in
peptide. It shows 33% sequence identity with GOX food products. Additions of GOX to the food system
from Aspergillus niger. The predicted secondary struc- in the presence of air or an oxygen donor like hydrogen
tures of the two enzymes are very similar (28). peroxide will result in the conversion of glucose to the
non-amine-reactive gluconic acid and thus prevent the
I. Three-Dimensional Structure formation of these Maillard components. The best
known application is the prevention of nonenzymatic
The three-dimensional structures of the two enzymes browning in egg white powder. A detailed description
of Aspergillus and Penicillium have been determined by of this application is given by Scott (10). Treatment
x-ray crystallography at 2.3 Angstrom resolution (29) can be done up to 508C and in the pH range from 4
and later improved to 1.9 A resolution (30). The FAD- to 7. Egg white is neutral at the time of laying, but the
binding domain is very similar to other FAD-binding pH rises quickly as carbon dioxide is lost, such that it is
proteins, and 11 amino acid residues from different generally close to 9 when the egg white is processed.
parts of the molecule are involved in this binding. The pH is brought below 7 by adding citric acid; addi-
The same is true for substrate binding. The substrate tion of hydrogen peroxide as oxygen donor is advan-
enters a deep pocket and is stabilized by 12 hydrogen tageous. Owing to the low afnity of GOX for glucose,
bonds and hydrophobic contacts to three aromatic high quantities of enzyme and/or long incubation times
residues and to FAD. A detailed analysis of this sub- are necessary to remove the glucose almost completely,

making this application less competitive than other into glucose and galactose, with GOX and hydrogen
systems like addition of active yeast. Canadian peroxide (40). Owing to the pH drop, the milk coagu-
researchers have shown that GOX is also efcient in lates. In principle, the same can be done in cheese
the reduction of nonenzymatic browning in potato manufacture, where direct acidication is fairly com-
products like chips and French fries (33, 34). mon. However, in industrial practice, the addition of
However, complete removal of glucose is also not an acid or gluconolactone, which under the conditions
realized in this process. It is clear that in this type of of cheese manufacturing slowly hydrolyzes into glu-
application only glucose is converted to a nonreducing conic acid, is preferred.
component; other sugars, such as maltose and lactose, Until, now production of gluconic acid on an indus-
maintain their reducing characteristics. A solution can trial scale is done by fermentation with selected strains
be the application of an oxidase active on a broad of Aspergillus niger and Gluconobacter oxydans.
range of reducing sugars. Some enzyme candidates Several attempts have been made toward production
are under investigation now. of gluconic acid using an enzymatic system. Until
recently complete bioconversion was only obtained
B. Removal of Oxygen with glucose solutions of 10% and lower, which is
not interesting from an economical point of view.
During storage of food, oxygen can have a detrimental Beverini and Vroemen (41) have now shown that glu-
effect on quality. As an example, oxidation of unsatu- cose concentrations as high as 4050% can be comple-
rated fatty acids can lead to rancidity of vegetable oils. tely converted in a fermenter at pH 56 and up to
Oxidation of colored components will change the color 308C, using low quantities of GOX rich in catalase in
of beverages or wine. In addition, oxygen inuences a relatively short time. The high catalase content
the taste of beer in a negative way during storage. needed is necessary owing to the low afnity of this
The majority of foods contain certain quantitites of enzyme for hydrogen peroxide. Under the conditions
glucose. In closed systems, the quantity of oxygen to mentioned, the concentration of hydrogen peroxide
be removed, in order to prevent oxidation, is generally remains low such that the two enzymes GOX and cat-
rather low. Therefore GOX can be used to remove alase are not completely inactivated before the end of
oxygen. If necessary a small quantity of glucose is the bioconversion. The advantages of this enzymatic
added. An important aspect is the stability of the process are that no fermentation is necessary (steriliza-
enzyme under application conditions. Fruit juices and tion, nutrients, time), the yield is up to 100%, and the
wine have a very low pH of 2.53, conditions in which purication of the nal product is much easier.
the enzyme is neither active nor stable. However, the
high glucose concentrations in fruit juices appear to
D. Production of Hydrogen Peroxide
have a stabilizing effect on the enzyme.
A large number of publications describe the positive
Hydrogen peroxide is a potent oxidant, which can be
effect on quality of adding GOX to food. A good
used as an active antimicrobial agent. Based on this
review is given by Scott (10). Other publications con-
property of hydrogen peroxide, several applications
cern: prevention of rancidity in oils, fats and sh (35
of GOX have been developed of which some are
37); and prevention of off-avors and color changes in
applied on a large scale. For these applications the
fruit juices, fruit concentrates, white wine, and beer
GOX preparations have to be exempt or poor in cat-
(10, 38, 39). Despite all these positive results, no appli-
alase. Unfortunately, the GOX itself risks also being
cations of adding GOX to food has been realized on an
inactivated by the hydrogen peroxide formed.
industrial scale until now. The main reason seems to be
the existence of competing technologies to prevent oxi-
1. Toothpaste
dation; for example, ushing out of oxygen by carbon
dioxide or nitrogen in beverages, addition of antioxi- In the Netherlands, toothpaste containing enzymes has
dants as BHA, ascorbic acid, sulte, etc. been developed (42). If sufcient glucose is present, the
GOX generates hydrogen peroxide, which kills plaque-
C. Production of Gluconic Acid forming bacteria in the mouth. To obtain sufcient
glucose, amyloglucosidase can be added, which
Milk can be directly acidied by adding glucose, GOX, together with the amylase present in saliva, hydrolyzes
and hydrogen peroxide as oxygen donor. A variant is starch into glucose. In addition, it is claimed that GOX
combining lactase, which hydrolyzes the milk sugar stimulates the lactoperoxidase system in the mouth.

2. Milk and Milk Products combination of GOX and sulfhydryl oxidase. The lat-
ter enzyme catalyzes the selective oxidation of sulfhy-
GOX can generate hydrogen peroxide for the lactoper-
dryl groups to disuldes by oxygen.
oxidase (LPO) system naturally present in milk. This
combined GOX-LPO system has been studied exten- 2 RSH O2 RSSR H2 O2 7
sively by a number of research groups to solve severe This leads to interprotein disulde bonds. The role of
contamination problems in milk and cheese produc- GOX is not yet completely understood. It clearly par-
tion (43, 44). Especially in cheese made from raw non- ticipates in the formation of disulde bonds between
pasteurized milk, there is an urgent need for a natural gluten proteins, but most probably it also participates
antibacterial system. Although a strong effect could be in the formation of other bonds like oxidative gelation.
shown on a high number of pathogens, introduction This is the coupling of two ferulic acid residues of
of the GOX-LPO system has not yet taken place. One neighboring arabinoxylan chains by the hydrogen-
of the reasons is that it has not given an absolute peroxide formed by glucose oxidase.
guarantee against all pathogens. The second patent (47) concerns the synergistic
effect of GOX and hemicellulases, like xylanases. It is
3. Baking believed that GOX makes stronger doughs, permitting
An important aspect of baking is the strength or weak- the addition of higher amounts of hemicellulases.
ness of the dough. Flours with a low protein content Addition of such amounts of hemicellulases alone
are characterized as weak, and the gluten is very exten- often results in softer and sometimes sticky doughs.
sible under stress but does not return to its original Especially, the latter combination of GOX and hemi-
dimensions when the stress is released. Bakers gener- cellulases has obtained a high market acceptance. This
ally prefer strong doughs because of their better rheo- Hemilox preparation, produced and commercialized
logical and handling properties, which result in a better by DSM Baking Ingredients under license of Cultor,
form and texture of the nal bread. Bakers have used permits production of clean-label breads of high qual-
dough conditioners to strengthen the dough. These ity without addition of nonspecic oxidants or emulsi-
conditioners are mostly nonspecic oxidants like bro- ers (48).
mates, iodates, and ascorbic acid. In North America
and Western Europe, public opinion and legislation 4. Glucose Oxidase as an Analytical Agent
are more and more opposed to the use of chemicals GOX is the most widely employed enzyme as an ana-
in bread. Moreover, these nonspecic oxidants can lytical agent, particularly for the determination of glu-
have a negative inuence on the bread aroma. In the cose in clinical laboratories, fermentation media, food,
United Kingdom bromates are no longer allowed since feed, etc. Different systems have been developed using
1990, and in France no addition of chemicals is per- glucose oxidase in soluble form, but also in immobi-
mitted in the production of traditional bread (pain a` lized form as glucose electrodes or sticks for the deter-
tradition francaise). Therefore, a need exists to replace mination of glucose in urine, as practiced by every
the nonspecic oxidants by a natural alternative. physician. For a good detailed review see Raba and
Enzymes are judged as natural and in agreement with Mottola (49).
clean label. Already in 1957, a patent appeared (45)
which describes the addition of GOX to our to
improve the dough quality and the baking properties. ACKNOWLEDGMENTS
As a particular advantage, the combination with ascor-
bic acid is mentioned. This is already an indication that The author wants to thank his son Casper W. Vroemen
GOX added gives a better result than ascorbic acid or of Wageningen University for valuable comments on
other nonspecic oxidants alone, which has been con- the manuscript.
rmed in many tests. This, together with the fact that
in that era the acceptance of chemicals was still high,
resulted in a low market penetration of the addition of REFERENCES
GOX to our.
The big breakthrough came in the beginning of the 1. B Drews, H Smalla. Brantweinwirtschaft 22, 1969.
1990s, when researchers of the Finnish company 2. K Zetelaki, K Vas. Biotech Bioeng 10:45, 1968.
Cultor discovered the synergistic effect of combining 3. W Franke, L Mochel, K Haye. Arch J Mikrobiol
several enzymes. The rst patent (46) concerns the 51:323, 1965.

4. T Yoshimura, T Isemura. J Biochem Tokyo 1971 25. KR Frederick, S Chakraborty, et al. J Biol Chem
69:839. 265:3793, 1990.
4a. S Nakamura, S Hayashi. FEBS Lett 41:327, 1974. 26. H Whittington et al. Curr Genet 18:531536, 1990.
5. JP van Dijken, M Veenhuis. Eur J Appl Microbiol, 27. E Varela, MJ Martinet, AT Martinez. Biochim
9:275, 1980. Biophys Acta 1481:202208, 2000.
6. T Yoshimura, T Isemura. J Biochem (Tokyo) 69:839, 28. HJ Hecht et al. J Mol Biol 229:153172, 1993.
1971. 29. G Wohlfahrt et al. Acta Cryst D55:969977, 1999.
7. JT Pronk, PR Levering, W Olijve, JP van Dijken. 30. S Witt, G Wohlfahrt, D Schomburg, HJ Hecht, H M
Enzyme Microb Technol, 11:160, 1989. Kalisz. Biochem 347:553559, 2000.
8. D Keilin, EF Hartree. J Biochem 42:221229, 1948. 31. P Vaha-Vahe. Food Technol Int Eur, p 139, 1994.
9. D Scott. Enzymes in Food Processing. New York: 32. Z Jiang, B Ooraikul. J Food Prod Preserv 13:175186,
Academic Press, 1975, p 228. 1989.
10. Q H Gibson, BEP Swoboda, V Massey. J Biol Chem 33. N Low et al. J Food Sci 54:118121, 1989.
239:3927, 1964. 34. CA Kannt et al. J Food Sci 58:104107, 1993.
11. K Takegawa, K Fujiwara, S Wahora. Biochem Cell 35. YH Lin. Dissertation, University of Rhode Island,
Biol 67:460, 1989. 1987.
12. HM Kalisz, J Hendle, RD Schmid. Appl Microbiol 36. M Dedek, J Hanus, M Vedlich. Int Dairy Cong
Biotechnol 47:502507, 1997. Sydney, 1970, p 225.
13. D Scott, J Agric Food Chem, 1:727, 1953. 37. Ough, Mcleod. Am J Enol Viticult 26:3036, 1975.
14. LA Underkoer. Proc Int Symp on Enzyme 38. U Schobinger, P Durr, R Waldvogel. Fluss Obst
Chemistry, Tokyo and Kyoto 1957. London: 59:586588, 1992.
Pergamon Press, 1957, p 586. 39. AGJ Rand. J Food Sci 37:698701, 1972.
15. G Tholey, B Wurtz. Soc Biol 159:2512, 1965. 40. M Beverini, AJ Vroemen. European Patent.
16. Catalog PL Biochemicals 1974. 41. Technical Documentation Zendium. Akzo Dental
17. Catalog Serva Biochemicals 1975. Research now Sara Lee, Veenendaal, Netherlands.
18. T Yoshimura, T Isemura. J Biochem (Tokyo) 69:839, 42. M Sandholm et al. J Vet Med B 35:346352, 1988.
1971. 43. M Desmazeaud et al. Food Ingredients Europe. Conf
19. S Nakamura, S Fujiki. J Biochem (Tokyo) 63:51, Proc Maarssen, Netherlands, 1989, pp 96103.
1968. 44. -. Luther, U.S. Patent 2783150, 1957.
20. H Tsuge, O Natsuaki, K Ohashi. J Biochem (Tokyo) 45. S Haarasilta, S Vaisanen, D Scott. U.S. Patent
78:835, 1975. 136003, 1987.
21. S Hayashi, S Nakamura. Biochim Biophys Acta 46. S Haarasilta, T Pullinen, S Vaisanen, I Tammersalo-
438:3748, 1976. Karsten. U.S. Patent 4990343, 1991.
22. S Hayashi, S Nakamura. Biochim Biophys Acta 47. DSM-Gist, P.O. Box 1, 2600 MA Delft, Netherlands.
657:4051, 1981. Technical Documentation Hemilox.
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24. M Kriechbaum et al. FEBS Lett 255:6366, 1989. 1995.

31
Lactate Dehydrogenase
Hayao Taguchi
Tokyo University of Science, Noda, Chiba, Japan
I. INTRODUCTION catalytic properties in different species and tissues.

The structures, properties, and relationships of various
This chapter will focus on the NAD-dependent lactate LDHs have been described in great detail by some
dehydrogenases (LDHs), although there are NAD- other reviews (14). This chapter outlines these aspects
independent LDHs that are usually linked to avo- of LDHs, with a few recent advances.
proteins. There are two forms of NAD-dependent
LDHs with distinct stereospecicities for lactate:
L-LDHs (L-lactate:NAD oxidoreductases, EC 1.1.1.27) II. IMPORTANCE TO QUALITY OF FOOD
and D-LDHs (D-lactate:NAD oxidoreductases, EC
1.1.1.28). This chapter will mainly concern L-LDHs, LDHs play key roles in the production of lactic acid,
D-LDHs being concomitantly mentioned. L-LDHs cat- which gives food a sour taste, by stimulating glycolysis
alyze the following overall reaction: through the consumption of pyruvate and the recycling
of NAD+ under anaerobic conditions. It is known that
muscles of exhausted animals are acidic and taste sour
because of the lactic acid produced. Glycolysis from
glycogen also proceeds in the meat after the death of
the animal, producing ATP and lactic acid. While the
ATP produced is mostly consumed, lactic acid is accu-
L mulated and reduces the pH of the meat, usually to 5.5
(ultimate pH). The pH decrease in the meat mostly
D-LDHs catalyze virtually the same reaction as L- results from the lactic acid produced, and the rate differs
LDH beside the distinct chirality of the lactic acid sub- with animal species and storage conditions. The rapid
strates (products). The physiological reaction of both pH decrease, before cooling of the meat, tends to occur
types of LDHs generally comprises pyruvate reduction particularly in the case of pork, and causes the dena-
(NAD oxidation), the reverse reaction, in which the turation of some proteins in the meat, leading to the
equilibrium is much more favorable than that of the decline of meat quality. Since the isoelectric points of
forward reaction under physiological (neutral) pH con- many meat proteins are around pH 5, the pH decrease
ditions (see Sec. IV). Therefore, LDHs act in the last also releases water molecules from hydrated proteins of
step of the glycolytic pathway, converting pyruvate meat and thus makes the meat watery.
and NADH into lactate and NAD+. It should, how- In processed foods such as dairy products, lactic
ever, be noted that LDHs are highly diverged enzymes acid is produced through lactic acid fermentation by
during evolution, and exhibit a multiplicity in their some bacteria, in particular, lactic bacteria.

Homofermentative lactic bacteria usually produces pounds (7). In particular, the pyruvate content can
> 85% lactate as an end product, and heterofermen- be simply determined through measurement of the
tative bacteria form mixed end products containing reduction of NADH by the direct LDH reaction.
acetic acid or ethanol besides lactic acid. Lactic bac- The decrease in NADH, which can be monitored as
teria ferment D- or L-lactic acid, or both, depending on the change in absorbance at 340 nm, is equal to the
the growth conditions, such as the oxygen concentra- reduction in the pyruvate content. The equilibrium
tion, temperature, pH, and sugars or other catabolites constant (Keq) of this reaction is shown in the following
supplied, and also on the nature of the LDHs con- equation:
tained in bacterial strains. D- and L-LDHs play key pyruvate NADH
roles in the fermentation of D- and L-lactic acids in Keq
lactate NAD
lactic bacteria, respectively, and the amount and prop-
erties of each LDH greatly affect the fermentation pat- 2:76 106 pH 7:0; 258C
terns of lactic bacteria. Bacteria that possess L-LDHs The favorable equilibrium allows pyruvate to be quan-
activated by fructose-1,6-bisphosphate (Fru-1,6-P2) titatively converted into lactate under conventional
(see below) produce mainly L-lactic acid when Fru- LDH assay conditions. The contents of some other
1,6-P2 is accumulated in the cells, but under other catabolites in glycolysis, such as phosphoenolpyruvate
growth conditions the bacteria may ferment a large and D-glycerate 2-phosphate, can also be quantitatively
amount of D-lactic acid, or some other end products, determined by means of the LDH reaction, coupling
rather than L-lactic acid (3). with enzyme(s), such as pyruvate kinase (EC 2.7.1.40)
and/or enolase (EC 4.2.1.11). Therefore, LDHs are
III. LOCATION OF LDH also useful as coupling enzymes for the determination
of enolase and pyruvate kinase activities. In addition,
L-LDHs are widely distributed in animals, plants, and some common LDHs exhibit low but sufcient cataly-
bacteria. Vertebrates have L-LDH in isoforms, depend- tic activity on certain pyruvate analog such as
ing on the tissues and organs, such as the A (or M), B hydroxypyruvate, and these substrates can also be
(or H), and X types of the enzymes, which are mainly assayed using the enzyme.
located in skeletal and heart muscles, and testes, On the other hand, the L- and D-lactate contents in
respectively. These isozyme subunits can also be hybri- food can be individually determined by the use of L-
dized, and form heterotetramers such as A4, A3B, and D-LDHs, respectively. To remove the pyruvate
A2B2, AB3, and B4. On the other hand, D-LDHs are produced, appropriate coupling reagents are required
widely found in invertebrates, fungi, and bacteria, and for quantitative determination because of the unfavor-
the tissue-specic izozymes of D-LDHs are found in able equilibrium. For example, L-alanine aminotrans-
invertebrates. It has been reported that invertebrates ferase (EC 2.6.1.2) is used to convert pyruvate into L-
possess either L- or D-LDH, depending on the species alanine in the presence of L-glutamate. Assay kits for
(5). For example, only D-LDHs are found in horseshoe D- or L-lactate including LDHs and aminotransferase
crab, arachnids, and gastropods, but insects or crusta- are available commercially (such as F-kits for L- and
ceans only possess L-LDHs. On the other hand, some D/L-lactate from Boehringer Mannheim).
lactic bacteria contain D- or L-LDHs, or both, and L-LDHs from many sources can be purchased from
therefore ferment D-, or L-, or both lactic acids (3, 6). commercial source, such as the muscle, heart, and liver
Activity staining with a tetrazolium salt (see below) isozymes from rabbit, cow, and pig, and the enzymes
is often used for the histological analysis of the iso- of lobster and some bacteria. The D-LDHs of lactic
zymes, or phylogenic or taxonomic analysis of LDHs bacteria, for example, Lactobacillus leichmannii and
in lactic bacteria. LDHs are usually separated by elec- Leuconostoc mesenteroides enzymes, are also available
trophoresis of a native protein sample, and distinct from commercial sources.
bands on the electrophoresis gel are visualized by stain-
ing with D- or L-lactate, or both.
V. STRUCTURES AND STRUCTURE
FUNCTION RELATIONSHIP
IV. UTILIZATION OF LDH IN FOODS
The three-dimensional structures of LDHs were rst
LDHs are used for the quantitative or qualitative determined for vertebrate L-LDHs in apo and ligand
determination of pyruvate, lactate, and related com- complex forms (2, 4), and then extensively analyzed for

the bacterial L- (810) and D- (11) LDHs. Most L- L-LDHs. The N-terminus of vertebrate L-LDHs is
LDHs are tetrameric enzymes composed of identical usually acetylated, while bacterial L-LDHs have a
polypeptides (subunits) with three twofold symmetric free amino group at the N-terminus. In addition, ver-
axes, P- Q-, and R-axes, although some L-LDHs, such tebrate L-LDHs possess a long additional amino acid
as the Alcaligence eutrophus enzyme (12), are homo- sequence (R-arm) at the N-terminus, which is followed
dimers. Vertebrate L-LDHs can form heterotetramers by the -sheet strand of the NAD-binding domain.
through association among the different subunits of The R-arm (20 amino acids) extends from the
the isozymes both in vivo and in vitro, and some NAD-binding domain to another subunit across the
bacterial enzymes may also associate in vitro, if they R-axis subunit interface, and is responsible for inter-
are closely related to each other (13). Subunits of L- subunit interactions on the R-axis subunit interface in
LDHs, as well as those of D-LDHs, are usually com- vertebrate L-LDHs. On the other hand, bacterial L-
posed of 310350 amino acids (molecular weights LDHs, in general, exhibit no or much weaker interac-
34,00040,000). tions on the R-axis subunit interface, since they largely
The subunits of L-LDHs comprise two similar sized or completely lack the R-arm sequence and are
domains: NAD-binding and catalytic domains. The thought to form tetramers mostly through P- and Q-
NAD-binding domain is located in the N-terminal axis interactions (9). The lack of an R-arm may be
half of the L-LDH primary structure and is folded important in allosteric types of L-LDHs, which are
into a typical open a=b structure (Rossmann fold), widely distributed in bacterial cells and are usually
which is a highly conserved domain structure among activated by fructose 1,6-bisphosphate (see below).
NAD-dependent dehydrogenases. The structure of the The 3D structures of the active and inactive forms of
catalytic domain in the C-terminal half appears unique the Bidobacterium longum enzyme indicate that the
in L-LDHs among these dehydrogenases, except for L- quaternary structure markedly changes through altera-
malate dehydrogenases (MDH), the catalytic domain tion of the intersubunit interactions in the allosteric
of which quite resembles that of L-LDHs as well as the transition (9). The lack of an R-arm possibly provides
NAD-binding domain. Although MDHs are usually the enzyme sufcient exibility for the structural
dimeric enzymes with a symmetric axis, their quatern- change in the allosteric transition.
ary structures are quite homologous to that of the Q- Unlike L-LDHs, many D-LDHs have dimeric struc-
axis dimer of L-LDHs. The homology of the tertiary tures comprising identical subunits and only one two-
and quaternary structures indicates that MDHs and L- fold symmetric axis in the quaternary structure, which
LDHs appear to have recently diverged during enzyme is not homologous to the P-, Q-, or R-axis dimer of
evolution, although there is little similiarity in their L-LDHs. But some D-LDHs, such as the Haemophilus
primary structures. It is known that only one amino inuenzae enzyme, the molecular weight of which was
acid substitution in L-LDH (Arg102 to Gln) estimated to be 35,000 and 135,000 by SDS-PAGE and
sufciently alters the substrate specicity of L-LDHs gel lration, respectively (18), are possible tetrameric
to that of MDHs (14). forms. D-LDHs have been distantly separated from L-
In the case of L-LDHs, the amino acid sequences LDHs in the enzyme evolution (19) in spite of the quite
have been systematically aligned with a standard align- homologous catalytic functions, which possibly
ment system (N-system) based on the primary struc- resulted from convergent evolution. D-LDHs exhibit
ture of a vertebrate isozyme (15). Although all known little sequence similarity with L-LDHs except for only
L-LDHs have essentially the same overall protein a small locus such as a Gly-X-Gly-X-X-Gly motif (X is
structures, however, the primary structures of L- unconserved amino acid) in the NAD-binding domain,
LDHs are highly divergent in different species and tis- and the 3D structure of the enzymes is very distinct
sues. The vertebrate isozymes usually exhibit more from that of L-LDHs. It is known that D-LDHs consti-
than 70% amino acid identity with one another, but tute a dehydrogenase family distinct from that of L-
bacterial L-LDHs share considerably less common LDH, together with other NAD-dependent 2-D-hydro-
amino acids with one another, depending on the spe- xyacid dehydrogenases such as 2-D-glycerate dehydro-
cies, and with the vertebrate enzymes up to < 40%. genase, 3-D-phosphoglycerate dehydrogenase, 2-D-
Evolutionary trees have been proposed for L-LDHs on hydroxyisocaproate dehydrogenase, and interestingly,
the basis of the divergence of their primary structures also some NAD-dependent formate dehydrogenases,
(16, 17). since these enzymes exhibit homologous primary and
There are, in particular, marked differences in the 3D structures (2023). Recently, furthermore, it has
N-terminal region between vertebrate and bacterial been shown that L-alanine dehydrogenases also

resemble D-LDH in 3D structure, in spite of their poor and the protonated imidazole of His195 onto the 2-
amino acid sequence identity with D-LDH (24). The carbon and the carbonyl oxygen of the substrate,
primary structure of D-LDHs appear to be also diver- respectively, while NAD+ and the unprotonated
gent among species, since even the enzymes from His195 act as acceptors for hydride and proton hydro-
Lactobacillus pentosus and L. bulgaricus strains of the gens from lactate on lactate oxidation. The carboxyl
same genus have < 50% identical amino acids (25). group of Asp168, which is located close to the imida-
The subunits of most D-LDHs are composed essen- zole of His195, is thought to promote the catalytic
tially of two domains: NAD-binding and catalytic function of the imidazole, through stabilizing the pro-
domains, which are connected by a hinge containing tonated form of the imidazole (26). Two guanidino
two peptides (11). The NAD-binding domain of the groups of the enzyme are known to play an essential
enzyme is folded into a typical Rossmann fold, which role in promoting enzyme catalysis. The guanidino
is quite homologous to the NAD-binding domain of group of Arg171 undergoes strong interactions with
the L-LDH, but is located at a different position in the the substrate carboxyl group, and allows the substrate
primary structure from that of L-LDH, i.e., in the mid- to be correctly orientated in the catalytic site of the
dle of the primary structure near the C-terminus (resi- enzyme (27). The other guanidino group of Arg109 is
dues 101 to 299 in Lactobacillus pentosus D-LDH [total located on a loop polypeptide (residues 98110) over
332 amino acids]) (11). the active site of the enzyme. The substrate binding
On the other hand, the structure of the catalytic induces marked structural rearrangement in the L-
domain is composed of a large N-terminal and a LDH active site, in which the loop closes over the
small C-terminal part (residues 1100 and 300332, active site. Arg109 moves toward the active site and
respectively, in Lactobacillus pentosus D-LDH), and is comes near the carbonyl oxygen of pyruvate through
folded in to an a=b structure, which resembles the the rearrangement, and promotes the hydrogen trans-
NAD-binding domain structure but greatly differs fer reaction by polarizing the substrate carbonyl group
from the L-LDH catalytic domain structure. Two of (28). The structural rearrangement, like the hydrogen
the NAD-binding domains are tightly bound to each transfer, is one of the slow steps in the L-LDH catalytic
other with no covalent bond, and are responsible for process. It has been indicated that the rearrangement
not only the main interactions for the subunit assembly step is fully rate-determining in the pyruvate of Bacillus
but also the rigid base that allows the catalytic stearothermophilus L-LDH, which exhibits a kcat value
domains to exibly move during catalysis through of 250 sec1 for pyruvate reduction, due to the primary
the hinge motion of the two connecting peptides. isotope effect of an NADH deutrium derivative on the
This motion of D-LDH in catalysis is also different enzyme transition state kinetics, while the hydrogen
from the loop motion of L-LDH (see below). transfer step is rate limiting in lactate oxidation (4).
In the case of D-LDHs, the catalytic site of the
enzyme exists in a cleft between the NAD-binding
VI. CATALYTIC PROPERTIES AND and catalytic domains, and the substrate binding
MECHANISMS allows the cleft to be closed through the hinge motion
of the catalytic domain. In the active site of D-LDH,
The kinetics of L-LDH are explained by an ordered bi- the His296Glu264 pair plays essentially the same role
bi mechanism, in which coenzyme NADH (or NAD+) as the His195Asp168 pair does, and Arg235 possibly
is bound rst, and subsequently the substrate, pyruvate fullls the roles of both Arg102 and Arg171 (11, 29,
(or L-lactate), is bound to the enzyme active site. In the 30). In spite of their distinct substrate stereospecici-
L-LDH catalysis, the imidazole group of the His195 ties, L- and D-LDHs exhibit common stereospecicity
(the numbering is based on the vertebrate enzyme for the NADH hydrogen, which is located on the A-
according to Eventof et al. [15]) residue acts as an side (re-side) at the C-4 position (pro-R hydrogen) of
essential acid/base catalyst, which mediates proton the nicotinamide ring (31).
transfer between the substrate and the solvent. The Both L- and D-LDHs are sensitive to chemical mod-
catalytic reaction occurs through the concerted attack ication reagents for His imidazole and Arg guanidino
by the nicotinamide ring of NAD and the His195 imi- groups, such as diethylpyrocarbonate and 2,3-butan-
dazole toward the 2-carbonyl (or hydroxymethyl) dione, respectively, and are protected from in-
group of pyruvate (or lactate). In the case of pyruvate activation by coenzymes and subtrates (analogues),
reduction, hydride and proton hydrogens are trans- because the enzymes possess essential His and Arg
ferred from NADH (4-H of the nicotinamide ring) residues in their catalytic sites. Sulfhydryl reagents,

such as p-hydroxymercuribenzoate and N-ethylmalei- Fru-1,6-P2 as a specic activator for their catalytic
mide, also inactivate vertebrate L-LDHs by the mod- activities. The regulations of these enzymes greatly
ication of Cys165, which is highly conserved in differ, depending on the bacterial species (3,39).
vertebrate L-LDHs, but not in other L-LDHs. Some of the bacterial enzymes absolutely require
Although the D-LDHs of invertebrates (31) and Fru-1,6-P2 and exhibit virtually no catalytic activity
Haemophilus inuenzae (18) are also inactiviated by unless Fru-1,6-P2 is present. On the other hand, some
sulfhydryl reagents, some bacterial D-LDHs have no of the enzymes exhibit signicant activity even without
cysteine residue in their primary structures (19). Fru-1,6-P2, but much more enhanced activity in the
The pH prole of LDH activity differs markedly in presence of Fru-1,6-P2, which usually improves the
the forward (from lactate to pyruvate) and reverse substrate binding. In the case of the B. stearothermo-
(from pyruvate to lactate) reactions, depending on philus L-LDH, for example, the pyruvate Km is reduced
the pK of the catalytic His residue, which is 7.0 in from 2 mM to 0.04 mM by 5 mM Fru-1,6-P2 at pH 6.0
most LDHs (6.7 in apo and binary complex L-LDHs of (28). On the other hand, the L-LDHs of L. casei,
mammals) (2, 26), since the unprotonated and proto- Thermus sp., and Bifdobacterium longum have
nated forms of imidazole are required for the lactate positive cooperative effects on pyruvate binding, in
and pyruvate binding, respectively. Therefore, the pyr- the absence of Fru-1,6-P2, shown by the sigmoidal
uvate Km is virtually constant at < pH 6.0, and pyruvate saturation curve, which changes into a hyper-
increases to > pH 8.0 in a reverse proportion to the bolic one in the presence of Fru-1,6-P2. Besides Fru-
H+ concentration in the solvent, in usual cases, while 1,6-P2, the L-LDHs of L. casei and curvatus (35)
the lactate Km is constant at > pH 8.0, and increases and Enterococcus faecalis (40), previously called
to < pH 6.0. In contrast, the kcat values and dissocia- Streptococcus faecalis, require certain divalent metal
tion constants of NAD are usually much less sensitive ions such as Mn2+ under physiological pH conditions,
to pH conditions. In the LDH reaction, thus, the opti- and thus constitute a subclass of divalent metal ion-
mal pH of the pyruvate reduction is usually lower than dependent allosteric L-LDHs (35). The metal ions
that of the lactate reduction. improve the activation function of Fru-1,6-P2 by
The A- and B-types of the bovine and chicken increasing its afnity to these enzymes. In the case of
5
L-LDH isozymes exhibit pyruvate Kms of 10 the L. casei enzyme, 10 mM Mn2+ reduces the
4 3 2
10 M, and L-lactate Kms of 10 10 M, dissociation constant for the enzyme and Fru-1,6-P2
and the B-type of L-LDH isozymes shows several- from 15 mM to 0.2 mM at pH 7.0 (41).
fold lower substrate Km values than the A-type iso- It has been demonstrated that the Arg173 and
zymes (32). Most L-LDHs, including all known His188 residues, which are located on the P-axis sub-
vertebrate L-LDHs, are signicantly inhibited by a unit interface of L-LDHs, are directly involved in the
high concentration of the substrate, pyruvate (32), Fru-1,6-P2-binding in common allosteric L-LDHs,
although the B-type isozyme generally shows more such as the enzymes of L. casei (42), B. stearothermo-
marked substrate inhibition than the A-type isozymes. philus (8, 43), Thermus caldophilus (44, 45), and B.
It has been indicated that the substrate inhibition longum (9, 46). The side chains of these amino acids
mostly results from the formation of an abortive face each other across the P-axis subunit interface, and
enzymeNAD+pyruvate ternary complex (1). Everse constitute the Fru-1,6-P2-binding site with a highly
and Kaplan (1) and Holbrook et al. (2) reviewed the positive charge. The Fru-1,6-P2 molecule neutralizes
steady-state and transition kinetics of vertebrate L- the positive charges and thereby reduces the static
LDHs in detail. repulsions, which stabilizes the inactive form of the
Vertebrate L-LDHs exhibit no apparent cooperative enzyme in the absence of Fru-1,6-P2. Thus, Fru-1,6-
effects on substrate binding, and require no particular P2 binding is one of the triggers for the change in the
factors for their enzyme activity. On the other hand, quaternary structure of the enzyme, which is closely
bacterial L-LDHs show great variety in their catalytic coordinated with the structural change of the sub-
properties, and often allosteric regulation by certain strate-binding site (9) and the coenzyme in the enzyme
factors, as reviewed by Garvie (3) in detail. Some (47). The T. caldophilus enzyme is converted into the
bacterial L-LDHs, such as the enzymes of Lacto- fully active form that is independent of Fru-1,6-P2
bacillus casei (3335), Thermus spp. (36, 37), and through chemical modications of the Arg residues,
Bidobacterium longum (38), show positive cooperative mainly Arg173 (44) and/or Arg216 (48), which are
effects on substrate binding. It was, in addition, also located on the P-axis interface of the enzyme,
particularly noted that many bacterial L-LDHs require with protection of the catalytic site by NADH and

oxamate (49, 50). Reduction of positive charge, that is regulated by Fru-1,6-P2. Nevertheless, some D-
through the replacement of Arg173 or Arg216 by LDHs, such as the enzymes of Butyribacterium rettgeri
site-directed mutagenesis, also leads to partial activa- (55) and Escherichia coli B (56), show positive coop-
tion of the enzyme (44, 48). erativity in the substrate binding, as shown by the sig-
The x-ray structures revealed that one Fru-1,6-P2 moidal pyruvate saturation curves. The enzyme from
molecule, which possesses a pseudosymmetric struc- Pythium devaryanum, a homolactate-fermenting fun-
ture, is bound per one P-axis dimer in the B. stearother- gus, is unique in that it also shows cooperativity in
mophilus (8) and B. longum (9) L-LDHs (two Fru-1,6- the NADH and D-lactate binding, but not in NAD+
P2 molecules per tetramer), although equilibrium dia- or pyruvate binding (57). In addition, GTP has an
lysis of the L. casei L-LDH indicates that one Fru-1,6- allosteric inhibition effect on the enzyme by increasing
P2 molecule is bound per one of the subunits (four Fru- the cooperative effects on the NADH and D-lactate
1,6-P2 molecules per tetramer) (41). The pH conditions binding, while ATP inhibits the enzyme reaction by
greatly inuence the regulation by Fru-1,6-P2, in the only competing with coenzyme NAD+.
cases of usual allosteric L-LDHs, since enzyme activa- Adenine nucleotides such as AMP, ADP, and ATP
tion highly depends on the interaction between the generally inhibit the catalytic reactions of LDHs
protonated His188 (the numbering is based on the ver- through competition with coenzymes at the NAD-
tebrate enzyme according to Eventof et al. [15]) and the binding site of the enzymes, and exhibit K1 values of
phosphate moiety of Fru-1,6-P2 (both pKs are 6. 103 M in human L-LDH A- and B-type isozymes.
For example, the L. casei enzyme shows a dissociation On the other hand, oxamate and oxalate, the structures
constant of 1 mM with Fru-1,6-P2 at pH 5.4, but one of of which are analogous to those of pyruvate and lac-
15 mM at pH 7.0 (41). Usually allosteric L-LDHs are tate, respectively, are representative substrate-relative
markedly stabilized, or protected from the dissocia- inhibitors that inhibit LDH reactions by competing
tions of subunits, in the presence of allosteric ligands with substrates, and exhibit particularly strong inhibi-
such as Fru-1,6-P2, or divalent cations in some cases, tory effects on pyruvate reduction and lactate oxida-
as well as coenzymes, although the E. faecalis enzyme tion, respectively. In the case of the bovine A-type
is unique in that it is rather labile in the presence of isozyme, for example, oxamate and oxalate show dis-
these ligands (51). sociation constants of 1.1 105 and 5.5 103 M with
Nonallosteric L-LDHs of vertebrates possess a cor- the enzyme-NADH binary complex, and 1.7 104
responding binding site at the same locus of the Fru- and 2.1 106 M with the enzyme-NAD+ complex,
1,6-P2-binding site, called the anion-binding site, where respectively (58, 59). Nevertheless, oxamate also some-
citrate ions were found to be bound on x-ray crystal- times exhibits an apparent activation effect on the
lographic analysis (2). In vertebrate enzymes, the active LDH reaction. In the case of LDHs that show positive
forms are sufciently stabilized in the absence of Fru- cooperativity in the substrate binding, a low concen-
1,6-P2, through some intrinsic interactions, such as the tration of oxamate usually stimulates the enzyme reac-
R-axis interaction that is missing in bacterial enzymes. tion when the substrate concentration is sufciently
It is nevertheless thought that the anion-binding site is low through reduction of the cooperativity of substrate
not involved in the regulation of the enzyme activity, binding.
but in the stabilization of the enzyme structure through In the case of Fru-1,6-P2-activated L-LDHs, inor-
binding certain organic or inorganic anions. Both ganic phosphate usually competes with Fru-1,6-P2 at
Arg173 and His188 are also highly conserved in most the Fru-1,6-P2-binding site, and has a strong inhibitory
known nonallosteric L-LDHs including vertebrate effect on the enzyme reaction, besides the coenzyme-
enzymes, except for the enzymes of L. pentosus (19, and substrate-related inhibitors. It is nevertheless
52), L. plantarum (53), and Pediococcus acidilactici known that the Enterococcus faecalis L-LDH is not
(54), in which His188 is substituted by Asp. The markedly sensitive to inorganic phosphate (40).
mutant L. pentosus enzyme, in which Asp188 is
replaced with His by site-directed mutagenesis, is not
signicantly regulated, but markedly stabilized by Fru- VII. QUALITATIVE AND QUANTITATIVE
1,6-P2 (52). DETERMINATION OF ACTIVITY
D-LDHs of invertebrates and bacteria generally dis-
play hyperbolic saturation curves for substrates, and The enzyme activity is usually assayed based on pyr-
exhibit high activities independent of certain allosteric uvate reduction, in which the catalytic efciency of
factors. Unlike L-LDHs, no D-LDH has been found most LDHs together with the equilibrium are much

more favorable than those of lactate oxidation. The semiquantitative or qualitative determination of the
rate of decrease of the NADH is quantitatively deter- enzyme activity, and can be applied to histological
mined by monitoring absorbance at 340 nm (e of staining or phylogenic or taxonomic differentiation
NADH 6,220 M1 cm1 at 338 nm, pH 7.5). of distinct LDHs or isozymes. For example, LDHs
Pyruvate concentrations of 2 and 0.3 mM are usually are separated on a native electrophoresis gel and give
appropriate for the A4 and B4 isozymes of mammals, distinct colored bands on staining of the gel with lac-
respectively, at pH 7.2 and 258C. In general, however, tate, NADH, PMS, and tetrazolium salt (35). If an
the pyruvate concentrations must be chosen or enzyme hardly catalyzes the lactate oxidation, the
searched for carefully, together with pH and tempera- reverse reaction (pyruvate reduction) is also available
ture conditions, in particular for new or unknown for the assay (negative staining). In the case of the
LDHs, because the pyruvate Km and inhibitory con- reverse reaction, the sample gel is primarily treated
centrations of pyruvate change greatly depending on with NADH and pyruvate (and Fru-1,6-P2 if neces-
the pH and temperature. It should be noted that the sary) for the LDH reaction to proceed in the gel, and
suitable concentrations of pyruvate markedly differ subsequently with PMS and a tetrazolium salt. Since
with individual enzymes, depending on the species LDHs consume NADH during the pretreatment, the
and tissues containing the enzymes, in particular in tetrazolium gives clear bands for the LDHs in a blue-
bacterial L-LDHs, which often exhibit sigmoidal colored background.
saturation curves for pyruvate or require Fru-1,6-P2
for enzyme activities. The concentration of NADH
which is more favorable for the reaction than VIII. PURIFICATION
NADPH in LDHs in general, and does not critically
affect the enzyme activity in the usual LDH assay com- Since LDHs are usually localized in the cytoplasmic
pared with the pyruvate concentration, because the fraction of cells, the rst step of purication is the
dissociation constants between LDH and NADH are preparation of cell-free extract through disruption of
sufciently small (107 106 M for vertebrate A and the cells with a homogenizer, or through osmotic
B L-LDH isozymes) under usual assay conditions, in shock or sonication. The conventional methods for
which the NADH concentration is 0.1 mM (A340 is protein purication, such as ammonium sulfate pre-
0.62). In addition, the NADH binding is usually less cipitation, and ion exchange, or gel ltration chroma-
sensitive to the pH condition, and the excess concen- tography, are generally available for both L- and D-
tration of NADH inhibits the enzyme reaction less LDH purication. The excess dilution or repeated
than in the case of pyruvate. freezing and thawing of an enzyme sample is usually
The concentration of Fru-1,6-P2 must also be care- undesirable during the purication or storage of the
fully chosen for the assay of Fru-1,6-P2-activated L- enzyme, since these promote the dissociation of the
LDHs, since it highly depends on the bacterial strain enzyme subunits. Purication of LDHs is usually per-
and pH. Phosphate buffer is usually unsuitable, since formed in conventional buffers such as phosphate and
inorganic phosphate reduces the activation effects of Tris-HC1 buffers, around neutral pH (between 6.0
Fru-1,6-P2 on the enzymes. and 7.5), where the enzymes are relatively stable.
Lactate oxidation is also useful for the assay of However, acetate buffers of pH 5.5 are often used
LDHs in some cases, and can also be determined by in the case of Lactobacillus LDHs. The L. casei and
following the rate of the change in the absorbance at L. curvatus allosteric L-LDHs tend to dissociate into
340 nm. However, it is recommended that this enzyme dimers or monomers under neutral or slightly alkaline
reaction be performed in the presence of much higher conditions (60).
coenzyme and substrate concentrations and/or under a Heat-treatment is sometimes used for the purica-
more alkaline pH (pH >9) than those in the pyruvate tion of certain bacterial L-LDHs, although the thermo-
reduction, since the equilibrium is unfavorable at neu- stability of the enzymes greatly differs with the original
tral pH, and LDHs generally show higher optimal pHs source of the enzyme (3). The L. casei and L. curvatus
for lactate oxidation than pyruvate reduction. The allosteric enzymes have been three to vefold puried
NADH resulting from lactate oxidation can also be from cell-free extract by heat treatment at 608C and
detected using a tetrazolium salt with a coupling pH 5.5, at which the enzymes are stable in the presence
reagent, phenazine methosulfate (PMS). Since the of Mn2+ as compared with other cell proteins (35). In
reduced tetrazolium salt displays a blue color as lactate the case of L. plantarum cells, which contain both L-
oxidation proceeds, this assay is also useful for the and D-LDHs, the former is much more thermostable

than the latter, and it alone can retain activity after
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32
Sabato DAuria
Institute of Protein Biochemistry and Enzymology, Naples, Italy, and University of Maryland, Baltimore,
Maryland, U.S.A.
I. INTRODUCTION water. Conversion rates in the range of 1020% are

typically obtained, and in a continuous batch reactor
Of the estimated 25,000 enzymes present in nature, system the concentration of acetaldehyde is 2:5 g=L
2800 have been classied and 400 have been com- after a period of 9 h (2).
mercialized. On a larger industrial scale, in particular Recently it has been shown that genetic manipula-
for detergent and food processing, only 50 enzymes tion of alcohol dehydrogenase levels in ripening
are used. The main impetus for the use of enzymes is the tomato fruit affected the balance of some avor
continuous growth in the demand for enantiomerically aldehydes and alcohols. In particular, modied
pure compounds. This trend plays an important role in alcohol dehydrogenase levels in the ripening fruit
the use of chiral drugs, but is also essential in the avor inuenced the balance between some of the aldehydes
industry where, besides microbial fermentation, and the corresponding alcohols associated with avor
enzyme technology is an alternative for the biotechno- production. Hexanol and Z-3-hexenol levels were
logical production of avor compounds (1). For exam- increased in fruit with increased alcohol dehydrogen-
ple, acetaldehyde is an important low-molecular-weight ase activity and reduced in fruit with low alcohol
avor compound that plays a signicant role in the dehydrogenase activity. In some taste trials, fruits
avor of yogurt and certain fruits, such as orange. with elevated alcohol dehydrogenase activity and
Figure 1 shows the conversion of ethanol to acetalde- higher levels of alcohols were identied as having a
hyde using alcohol dehydrogenase. This process suffers more intense ripe fruit avor (3). Moreover, the
from the same drawbacks as all enzymatic oxidation involvement of alcohol dehydrogenases in the bio-
reactions that rely on cofactor regeneration. genesis of six carbon alcohol constituents of the
Specically, the process involves the use of alcohol aroma of olive oil has been recently discussed (4).
dehydrogenase and its cofactor NAD , which during In yeast, ethanol is produced from acetaldehyde dur-
the reaction is reduced and subsequently regenerated by ing anaerobic fermentation of sugar and this could
light-catalyzed oxidation with avin mononucleotide represent an important process in the production of
(FMN). The reduced avin mononucleotide alcohol-containing beverages.
(FMNH2) is reconverted to FMN by oxidation with A survey of the actual use of enzymes in synthetic
molecular oxygen. The byproduct of this reaction is organic chemistry shows that 65% of all reported
hydrogen peroxide, which is in turn decomposed by applications fall into the classes of hydrolytic and
the action of the enzyme catalase to oxygen and dehydrogenase reactions (1).

Figure 1 Enzymatic oxidation of ethanol to acetaldehyde. (From Ref. 2.)
II. PROPERTIES AS PROTEIN alcohol formation is favored. ADHs can be very effec-
tive for the conversion of the readily available alde-
Alcohol dehydrogenase (ADH) (EC 1.1.1.1) is an oxi- hydes citronellal and citral for the production of ()-
doreductase enzyme that catalyzes the oxidation of citronellol and geraniol, compounds with a much
alcohols and the reduction of carbonyl compound higher avor impact than the corresponding aldehydes
such as aldehydes and ketones. The enzyme displays or ketones (8). Thus, the potential applications of
a broad substrate specicity, being active on a variety ADHs in industry are numerous. With mesophilic
of primary, secondary, branched, and cyclic alcohols. enzymes, however, limitations due to narrow speci-
Alcohol dehydrogenases are widely distributed city, instability to heat and organic solvents, and loss
enzymes and have been found in many microorgan- of activity on immobilization have been incurred. The
isms, fungi, and animal cells (5). Most bacterial use of these enzymes from thermophilic sources in
ADHs are so-called soluble enzymes and are located immobilized and continuous reactor systems has also
in the cytoplasmic, but some have been shown to be been proposed for the regeneration of NAD(P)H
membrane bound. The intracellular compartmentation needed for such reactors, the lifetime of which would
of eukaryotes gives rise to greater possibilities of var- be extended with stable enzymes. The ADH from
iation in location. Thus, Saccharomyces cerevisiae con- Thermoanaerobium brockii has been shown to reduce
tains three ADHs of which two are cytoplasmic and aliphatic acyclic ketones asymmetrically with an opti-
the third is mitochondrial (6). Some of these enzymes mal purity. Furthermore, this enzyme has been used to
(e.g., ADHs from horse liver and yeast) have been obtain optically pure cyclic ethers, which are con-
extensively studied, because they can provide new stituents of civet used as a xative in the perfume
data on the relationship between structure and func- industry (9).
tion, being oligomeric, coenzyme dependent, and con- ADHs have been subdivided into different families
taining structural and functional metal atoms (5). according to the polypeptide chain lengths: the short-
ADH requires a nicotinamide cofactor for the cat- chain family consisting of non-metalloenzymes with
alytic activity, and it is capable of distinguishing the subunits of 250 residues (10); the medium-chain
diastereotopic methylene hydrogens at the dihydropyr- family with subunits of 350375 residues, often
idine C-4 position of NADH or NADPH, transferring containing zinc (11); and the long-chain family with
the hydride to the substrate stereospecically (7). This subunits of > 700 residues (12). Moreover, ADHs
interesting enzymatic feature suggests a potential use lacking zinc as well as alcohol dehydrogenases requir-
of ADHs for the production of alcohols with a 100% ing iron for activation have been described (13). A
optical yield (7). monomeric ADH was also isolated from
ADHs catalyze the interconversion of alcohols to Saccharomyces cerevisiae, but its metal content is yet
aldehydes/ketones: unknown (14).
ADHs have been classied as = proteins with high
Alcohol NADP Aldehyde=ketone amounts of -helices and -structures. Figure 2 shows
NADPH H the far-UV circular dichroism spectrum of B. acidocal-
darius ADH. The deconvolution of the spectrum
This reaction is highly reversible and the direction of resulted in 22% -helix and 45% -structure, sug-
the reaction is inuenced by pH of the reaction gesting that ADHs from thermophilic microorganisms
medium. At pH 9 the equilibrium of this reaction is could also have a secondary structural organization
directed toward aldehyde formation, while at pH 7 similar to that described for mesophilic ADHs (15).

that in almost all the zinc containing ADHs studied
to date, three zinc-binding amino acids (Cys46, His67,
and Cys174) are highly conserved (20). However, in
ADH from Thermoanaerobacter brockii only Cys37
and His59, which align with Cys46 and His67 of
HLADH, are conserved. Although the residue corre-
sponding to Cys174 of HLADH is absent in
Thermoanaerobacter brockii ADH, it has been sug-
gested that Asp150 could serve as the third Cys residue
in the latter enzyme. Moreover, the absence in
Thermoanaerobacter brockii ADH of all four amino
acid residues found in HLADH to coordinate the struc-
tural zinc ions (Cys97, Cys100, Cys103, Cys111) sug-
gests that the Cys37 is involved in the catalytic activity
of T. brockii enzyme (21). The ADHs isolated from
Bacillus acidocaldarius and Sulfolobus solfataricus have
two zinc atoms per enzyme subunit, one with a catalytic
role and the other with a structural role (15, 22).
Figure 2 Far-UV circular dichroism spectrum of Bacillus The thiol groups of cysteines are important for pro-
acidocaldarius ADH. Protein concentration 0.2 mg/mL. tein structure and folding (23). As in other proteins,
Temperature 25 C. chemical modication of reactive cysteine residues has
been used extensively to investigate the importance of
Moreover, conformational variations have been cysteine residues in the structure and function of the
reported in many ADHs on binding to coenzyme or ADHs. Figure 3 shows the structure of the E-subunit
substrate. One of the many techniques utilized to of HLADH resolved at 2.2 A. In the same gure the
detect conformational changes in protein structure is cysteine residues are shown as spacell.
uorescence spectroscopy. Horse liver ADH In zinc-containing ADHs, cysteine serves as binding
(HLADH) contains two tryptophan residues: Trp15 site for the catalytic zinc atom (24). In most eukaryotic
located at an outer edge of the catalytic domain and and prokaryotic ADHs, cysteine serves as binding site
accessible to solvent, and Trp314 located in the apolar for a structural zinc atom as well (25). The role of other
intersubunit region of the catalytic domain, inaccessi- cysteine residues present in the ADHs has not been
ble to the solvent. In a detailed uorescence study determined. Of the three catalytic zinc atoms that
Lakowicz and coworkers showed that these two tryp- have been identied in the 3D structure of HLADH,
tophan residues displayed different emission spectra the only enzyme for which a detailed 3D structure of
and time decay by using one- or two-photon excita- an ADH with its coenzyme is available (24), two resi-
tion. The results suggest that the intrinsic uorescence dues, Cys46 and Cys174, can be selectively alkylated
of HLADH can be used to probe small changes in the by -halo acids in an afnity-labeling type of reaction
active site of the enzyme (16). (28). Whereas carboxymethylation of Cys46 and
Zinc-dependent alcohol dehydrogenases have a Cys174 of HLADH, or the putative active site zinc
dimeric or tetrameric structure. Dimeric enzymes are ligand cysteines of human ADH, sheep liver sorbitol
the ADHs from mammals (11) and from higher plants dehdyrogenase, and human liver aldehyde dehydro-
(17). Tetrameric enzymes are the ADHs from yeasts genase, abrogates enzymatic activity (27), similar mod-
and bacteria. Recently, a detailed characterization of ication of the ADH from S. solfataricus signicantly
the ADH from S. solfataricus has ascertained the pre- increases the oxidation rate of alcohols (28). Recently
sence of both dimeric and tetrameric forms, with the it has been shown that Cys283 and Cys295 of the ADH
dimeric conformation showing a lower level of cataly- from Thermoanaereobacter brockii, although they are
tic efciency than the tetrameric one (18). Moreover, buried within the protein core and are not accessible
mammalian ADHs have been divided into three major for chemical modication, are oxidized to cystine when
classes (I, II, and III) based on their electrophoretic the enzyme is heated at 75 C, forming a disulde
mobility and kinetic properties (19). bridge that was not present in the native enzyme, with-
Multiple sequence alignment of > 60 ADHs from out affecting either enzymatic activity or thermal sta-
different sources with that of HLADH has shown bility (21).

Figure 3 Structure of E-subunit of horse liver ADH solved at 2.4 A. The cysteine residues are shown as spacell.
Figure 4 shows the 22 amino acid residues strictly isolated recently from thermophilic bacteria utilize
conserved in 16 ADHs from different sources (11). It is NADP as coenzyme (29, 30), while the ADHs from
worth noting that His ! Arg substitution at the posi- the eubacteria Bacillus acidicaldarius and Bacillus
tion 47 (HLADH numbering) has been reported to stearothermophilus require NAD as coenzyme (15,
affect both the afnity for the coenzyme and pH opti- 31). Moreover, ADHs containing pyrroloquinolin qui-
mum for the activity in human ADH isoenzymes, the none (Fig. 5) as a cofactor have been isolated from
native enzyme displaying a lower optimum and higher various Gram-negative bacteria. Based on the cofactor
Km and Vmax values than the mutant (18, 25). requirement two classes of ADHs can be distinguished:
Table 1 shows some biochemical features for some quinoprotein alcohol dehydrogenase (containing PQR
ADHs with His or Arg at position 47. and Ca2 ) and quinohemoprotein alcohol dehydrogen-
ase (containing PQQ, Ca2 , and heme c). The rst class
includes the methanol dehydrogenases from methylo-
III. PROPERTIES AS ENZYMES
tropic bacteria and alcohol dehdyrogenases occurring
in Pseudomonas (32).
According to their source ADHs differ in substrate and
HLADH is a well-characterized zinc-containing
coenzyme specicity (5). It is commonly believed that
ADH. Each subunit of the dimeric enzyme contains
ADHs from eukaryotes are mainly NAD dependent,
one catalytic and one structural zinc atom (5) and
while those from prokaryotic organisms are either
binds one ligand of NAD(P)H.
NAD or NADP dependent. However, two ADHs
Figure 4 The 22 amino acid residues strictly conserved in several ADHs (11). The amino acids are numbered according to
HLADH sequence.

Table 1 Optimal pH and Km Values Related to Amino Acid 47 of Horse Liver (HL), Yeast
(Y), 1 1 Human (1 1 h), 2 2 Human (2 2 h), and Bacillus acidocaldarius (Ba) ADHs
HL ADH Y ADH 1 1 h ADH 2 2 h ADH Ba ADH
Amino acid Arginine Histidine Arginine Histidine Not

residue 47 determined
Optimal pH 10 8.6 10 8.5 10
activity
Km (M) 53 160 7.4 180 1600
(NAD )
The most widely accepted reaction mechanism for The stereospecicity of the ADH isolated from the
alcohol dehydrogenase is indicated in Figure 6 (33) and thermoacidophilic archaeon Sulfolobus solfataricus
includes: binding of NAD ; binding of alcohol sub- (36) toward an asymmetric ketone was examined by
strate by replacing a zinc-bound water molecule; using 3-methyl-butan-2-one as a model compound.
deprotonation of the alcohol; hydride transfer from The hydride attack, using the enzyme immobilized on
the alkoxide ion to NAD , yielding NADH and a Eupergit C was on re-face of the ketone, producing the
zinc-bound aldehyde; dissociation of NADH. In corresponding (s)-3-methyl-butan-2-ol with a 100%
applicable steps (79), the zinc-bound water molecule optical yield. Moreover, the potentiality of the use of
equilibrates with a zinc-bound hydroxyl ion, which this enzyme for the preparation of chiral compounds
gives rise to a dead-end complex. The catalytic zinc was also pointed out by the use of resting cells of S.
in HLADH is bound to Cys46, His67, and Cys174. solfataricus to reduce a series of acyclic, cyclic, and
The fourth ligand is a water molecule that is linked aromatic ketones (37). Another interesting aspect of
to a hydrogen-bonding network involving Ser48 and stereospecicity of ADH from S. solfataricus was dis-
His51 (34). The four cysteine residues coordinating the covered by studying the oxidation of secondary alco-
noncatalytic zinc (Cys97, 100, 103, and 111) and the hol enantiomers (38): (s)-enantiomers are far better
binding amino acids of the catalytic zinc are arranged substrates than (r)-enantiomers. For example, (s)-2-
in a distorted tetrahedral geometry (35). butanol is oxidized about 60 times more efciently
The nicotinamide ring system is redox active, than (r)-2-butanol, and (r)-sec-phenethyl alcohol is
accepting a hydride of two electrons and a proton to not oxidized at all. In a detailed study Keinan and
form 1,4-dihydronicotinamide derivatives of NADH coworkers showed that the ADH isolated from
or NADPH. The reversible hydride transfer from a Thermoanaerobium brockii exhibited a high stereospe-
reduced substrate to NAD , and that from NADH cicity depending on the enzyme assay temperature
to an oxidized substrate, is stereoselective and charac- (39). However, the nicotinamide cofactors are too
teristic of individual enzyme. Each enzyme is able to expensive to be used as stoichiometric reagents in
transfer stereoselectively one of the diastereotopic large-scale synthesis. Thus, recycling of the cofactors
methylene hydrogens at C-4 of NADH to a substrate is needed if enzymes requiring nicotinamide cofactors
carbonyl group or an equivalent sp2 center with high are to be used in a preparative scale. In spite of the use
enantiofacial or diastereofacial selectivity. of HLADH with its broad substrate acceptance, nar-
row stereoselectivity and bidirectional functionality,
there is continuing research for novel reductases, in
particular for alcohol dehydrogenases puried from
thermophilic microorganisms, that are stable and
active at high temperature and in the presence of
organic solvents. Enzymes isolated from thermophilic
sources display high levels of enzyme activity at high
temperatures, being barely active at room temperature.
Figure 7 shows the thermophilic behavior of two
ADHs: B. acidocaldarius ADH (moderate thermophilic
microorganism), and S. solfataricus ADH (hyperther-
mophilic microorganism). Several research groups
Figure 5 Structure of pyrroloquinolin quinone. have related these functional features to a high struc-

Figure 6 Reaction mechanism of ADH according to Petterson (33).
tural rigidity at room temperature (40) that could dehydrogenase activity by monitoring the decrease in
prevent the utilization of these enzymes in biotechno- absorbance at 340 nm, as a consequence of the oxida-
logical applications. tion of the NADH (NADPH) molecules.
The alcohol dehydrogenase from yeast exhibits the
maximal enzymatic activity in the range of pH 8.69.0
IV. ENZYMATIC ASSAY at 25 C. The typical assay for measuring it is the fol-
lowing: 50 mM Tris-HCl (pH 8.6), 10 mM ethanol, 1.0
Usually alcohol dehydrogenase activity is assayed by mM NAD , and 1:0 g enzyme/mL nal volume, at
following the increase in absorbance at 340 nm owing 20 C. The ADH activity is determined by monitoring
to the reduction of the NAD NADP molecules. On the increase of absorbance at 340 nm. One unit of
the contrary, it is also possible to assay alcohol enzyme activity is dened as the amount of the enzyme
that catalyzes the reduction of 1:0 M of NAD at
25 C.
For assaying enzymatic activity of thermophilic
alcohol dehydrogenases it is of fundamental impor-
tance to use a sealed cuvette, in order to avoid the
evaporation of the assay mixture. The alcohol dehy-
drogenase from the thermophilic microorganism
Bacillus acidocaldarius shows the maximal enzyme
activity at 80 C. However, Bacillus acidocaldarius
ADH activity is usually assayed spectrophotometri-
cally at 65 C by measuring the increase in the absor-
bance at 340 nm in a sealed quartz cuvette. The
reaction mixture contains 50 mM glycine-NaOH (pH
10.0) and 1:0 g protein/1.0 mL nal volume. The con-
centrations of cofactors and substrate are 5.0 mM
NAD and 3.0 mM ethanol for alcohol oxidation,
and 0.12 mM NADH and 3.0 mM p-anisaldehyde
for aldehyde reduction, respectively. One unit of
enzyme activity is dened as the amount of the enzyme
that catalyzes the reduction of 1:0 M of NAD at
65 C, using ethanol as substrate.
It is also possible to monitor ADH activity by uor-
escence spectroscopy. In this case, we follow the
increase of the uorescence emission at 450 nm
(upon excitation at 340 nm) due to NADH formation.
The uorescence assay is more sensitive than the spec-
Figure 7 Thermophilic behavior of Bacillus acidocaldarius trophotometer one, and it is utilized when it needs the
ADH(A) and Sulfolobus solfataricus ADH (B). detection of low ADH activity levels.

V. PURIFICATION buffer, at a ow rate of 20 mL/h. The column was
washed with 150 mL of Buffer B, and the homoge-
Several purication strategies have been described to neous enzyme was eluted with a step of 2 mM
isolate ADHs from different sources. However, the NAD in the same buffer. The results of the purica-
utilization of an afnity column has been reported as tion are summarized in Table 2.
the main purication step in almost all described pur-
ication procedures. Specically, an afnity chromato- Bacillus acidocaldarius ADH was puried to homo-
graphy based on the interaction of ADH coenzyme- geneity by ve steps. The crucial step in the purica-
site-blue dye molecule was used for large-scale puri- tion procedure was the specic elution of the enzyme
cation of horse liver ADH. Moreover, it has recently by NAD at pH 7.4 from the afnity Blue A column.
been shown that the afnity chromatography on The presence of DTT in the buffers used in Bacillus
Matrex Gel Red A was the essential purication step acidocaldarius ADH purication was necessary in
for the isolation of the ADH from a novel strain of order to avoid the complete inactivation of the enzyme.
Bacillus sterotheromphilus (31). On the other hand, no signicant enzyme stabilization
Here we report the typical procedure of purication was observed utilizing buffers containing divalent ions
of the ADH from Bacillus acidocaldarius (15). (Zn2 , etc.).
However, it is worth noting that a similar method However, it is worth stating that the expression of
can be also used for the purication of alcohol dehy- thermophilic ADHs in mesophilic hosts (e.g., E. coli)
drogenases from different sources. makes the enzyme purication procedure very easy; in
fact, it needs a thermoprecipitation step in order to
1. Preparation of cell extract. One hundred and separate the thermostable recombinant enzyme from
twenty grams of wet cell pellet was suspended in 360 the native proteins of the host. Moreover, the cloning
mL of 10 mM Tris/HCl, 0.5 mM dithiothreitol, pH 9.0 and expression of thermostable ADHs in organisms
(Buffer A), and broken by ve 1-min cycles of sonica- generally recognized as safe (GRAS) from the U.S.
tion at regular intervals, utilizing an MSE Soniprep Food and Drug Administration (e.g., Saccharomyces
150. Cell debris was removed by centrifugation at cerevisiae) give the opportunity to obtain large
160,000 g for 90 min (at 4 C). The supernatant repre- amounts of stable ADHs to use in the food industry.
sented the crude extract. In conclusion, ADHs have been isolated from dif-
2. Column chromatography steps. The crude ferent organisms and they differ according to their
extract was applied to a DEAE-Sepharose Fast Flow sources, in substrate and coenzyme specicity, as well
column, previously equilibrated in Buffer A. The col- as in their protein structural organization. Their wide
umn was washed with 1000 mL of Buffer A, and eluted substrate specicity makes this class of enzymes good
with a linear gradient from 0.0 to 0.3 M NaCl in Buffer biocatalysts in biotechnological applications, as well as
A, at a ow rate of 60 mL/h. The alcohol dehydrogen- interesting proteins in the study of evolution.
ase activity was eluted at 0:1 M NaCl. The fractions
containing the enzymatic activity were pooled and
Table 2 Purication of Alcohol Dehydrogenase from
loaded on a Phenyl-Superose column, at a ow rate
Bacillus acidocaldarius
of 30 mL/h, after addition of 1.0 M ammonium sulfate.
After washing with 100 mL of Buffer A, the enzymatic Protein Specic Fold Yield
activity was eluted with a gradient of ammonium sul- Purication step (mg/mL) activity purication (%)
fate from 1.0 M to 0.0 M in Buffer A. Fractions con-
taining the ADH activity were eluted at 0.3 M Cell extract 12,200 0.3 1.0 100
(homogenate)
ammonium sulfate, pooled, and dialyzed overnight
DEAE-Sepharose FF 1,100 3.3 11 99
against 10 L of Buffer A, at 4 C. The active pool Phenyl-Superose 500 6.1 20 83
was loaded at a ow rate of 30 mL/h on a Mono Q Mono-Q Sepharose 150 16 53 65
FPLC column previously equilibrated with the same Blue Sepharose 15 150 500 61
buffer. The column was washed with a linear gradient CL-6B
from 0.0 M to 0.5 M NaCl in Buffer A, at a ow rate of
60 mL/h. The enzymatic activity, eluted at 100 mM Source: Ref. 15.
The alcohol dehydrogenase activity was assayed spectrophotometri-
NaCl, was dialyzed overnight against 5 L of 10 mM cally by measuring the change in absorbance at 340 nm. The reaction
Tris/HCl, pH 7.4 (Buffer B), and applied onto a Blue A mixture contained 50 mM glycine-NaOH, pH 10, 5.0 mM NAD ,
afnity column, previously equilibrated with the same and 3.0 mM ethanol.

ACKNOWLEDGMENTS 11. H Jornvall, B Persson, J Jeffery. Characteristics of
alcohol-polyol dehydrogenases. The zinc-containing
I thank Professor Joseph R Lakowicz (University of long-chain alcohol dehydrogenases. Eur J Biochem
Maryland), Professor Mose Rossi (University of 167:195201, 1987.
12. PE Goodlove, PR Cunningham, J Parker, DP Clark.
Naples), and Professor Carlo Fini (University of
Cloning and sequence analysis of the fermentation
Perugia) for their stimulating scientic discussions; alcohol dehydrogenase-encoding gene of Escherichia
and Mr. Carlo Vaccaro (Italian National Research coli. Gene 85:209214, 1989.
Council) for his technical assistance. Skillful assistance 13. RK Scopes. An iron-activated alcohol dehydrogenase.
in the preparation of the gures by Ms. Krystyna FEBS Lett 158:303306, 1983.
Gryczynska is gratefully acknowledged. This work 14. MR Wales, CA Fewson. NADP-dependent alcohol
was supported by a grant from the Ministry for dehydrogenase in bacteria and yeast: purication
University and for Technological and Scientic and partial characterization of enzymes from
Research (M.U.R.S.T.) 60%, and EU contract Acinetobacter calcoaceticus and Saccharomyces cerevi-
Extremophiles, and the National Center for siae. Microbiology 140:173183, 1994.
Research Resources, NIH RR-08119. 15. S DAuria, F La Cara, F Nazzaro, N Vespa, M Rossi.
A thermophilic alcohol dehydrogenase from Bacillus
This chapter is dedicated to Cristina, Leandro
acidocaldarius not reactive toward ketones. J Biochem
Maria, and Gerard Joseph. 120:498504, 1996.
16. JR Lakowicz, B Kierdaszuk, I Gryczynski, H Malak.
Fluorescence of horse liver alcohol dehydrogenase
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33
Sandrine Dallet, Marie Trovaslet, and Marie Dominique Legoy

University of La Rochelle, La Rochelle, France
I. INTRODUCTION were renamed as the medium-chain family after the

still longer ADHs were discovered (3).
NAD -dependent alcohol dehydrogenases (AHDs) Many different type I ADHs have been character-
(alcohol : NAD ) oxidoreductase E.C.1.1.1.1) are ized as subclasses based on their dimeric and tetra-
enzymes that occur widely in living organisms where meric forms such as horse liver alcohol
they are important for the detoxication and metabo- dehydrogenase (HLADH) and yeast Saccharomyces
lism of ethanol and other alcohols (1). ADHs catalyze cerevisiae alcohol dehydrogenase (SADH or YADH),
the reversible oxidation of various alcohols to the cor- respectively. Primary structures of most ADHs are
responding aldehydes and ketones, with the concomi- known but only the tertiary structure of HLADH is
tant reduction of NAD (2). Depending on the known at present (6,7). Crystallization of HLADH
biological source, ADHs show different substrate spe- with or without NAD has been achieved in order to
cicity (short-long-chain alcohols, aliphatic/aromatic understand the binding mechanism of different sub-
alcohols, and branched alcohols). strates and the kinetic mechanism (8, 9). The three-
Even though ADHs show some divergence in dimensional modeling of other medium chain ADHs
amino acid sequences, isoenzymes, and substrate is based on the structure of HLADH. Comparison of
specicity, they have certain structural and func- structure between HLADH and the other ADHs has
tional similarities. Three structurally and catalyti- shown that the three-dimensional structures of these
cally different types of ADHs are currently known enzymes must be very similar in the catalytic domains.
(3). The medium-chain ADHs (containing 350 Although the general overall sequence homology is
residues per subunit) are named type I. They are < 40%, all the structurally important residues are
zinc dependent and are characterized by a prefer- either homologous or conservatively substituted (Fig.
ence for primary alcohol. The short-chain ADHs 1) (4). Since HLADH and YADH are the most studied
(containing 250 residues per subunit), named ADHs, we will present below only the state of the
type II, are zinc independent and they display a science concerning these two enzymes.
better afnity toward secondary alcohols. Iron-acti-
vated long-chain ADHs, named type III, have an
average subunit size of 385 residues. Among II. SOME UTILIZATION OF ADH IN
these NAD -dependent ADHs, type I ADHs are FOOD TECHNOLOGY
the most studied with respect to structure and
kinetic mechanism (4, 5). Originally, the ADHs of The applications of ADHs as industrial catalysts are
type I had been termed as the long-chain ADHs but limited by the expense of the cofactors NAD(H). Many

Figure 1 Alignment of the amino acid sequences of alcohol dehydrogenases from horse liver, chicken, and yeast. Top row, horse
liver enzyme (6). Middle row, chicken enzyme (10). Bottom row, yeast enzyme (11). All residues numbering is based on alignment
to HLADH, homologous residues conserved; * residue involved directly or not directly in zinc binding; bold: homologous
residues involved in NAD binding; underlined: homologous residues involved in substrate binding pocket.
complex methodologies have been studied for recycling tion. Pasteur showed that living yeast cells caused fer-
the redox coenzyme, as for instance the coupling of mentation in the absence of air, converting sugar into
ADH with another enzyme together with a coupled ethanol and carbon dioxide. Research later in the 19th
substrate in order to regenerate the coenzyme (12). century showed that fermentation resulted from the
An alternative approach consists in using a coenzyme action of substances contained within the yeast cells.
mimic, bearing functional similarity to NAD (13). Later, one of the major discoveries of fermentation
Independently of the cofactor recycling, ADHs microbiology was made by Hansen at the Carlsberg
obviously have been used for the production of alco- center in Copenhagen while working on wild yeast.
hols during the alcoholic fermentation or the produc- These wild yeast were known to give problems during
tion of aldehydes which represent a major source of beer fermentation and, by isolating pure cultures of
numerous avors. yeast, which he then used in the brewing process,
In the case of alcoholic beverage production, it is Hansen initiated the use of pure cultures in beer pro-
not the enzyme that is used but the cell. The produc- duction. Alcoholic beverages are produced by the alco-
tion of beverages by alcoholic fermentation is one of holic fermentation of the sugar-containing material to
the oldest fermentations known. Beer and wine were ethanol and carbon dioxide (see Chapter 2 for other
probably the two earliest alcoholic drinks produced. A details).
brief history of the knowledge in alcoholic fermenta- Fermentation is carried out by species of the yeast
tion could be summarized as follows. Until Pasteurs Saccharomyces. In some cases, sugar is present natu-
work in the late 19th century, little was known of the rally, such as in grapes used in wine making; in others,
actual processes and mechanism of alcohol produc- sugars are produced from starches in cereals, as in beer

production. Free sugar is essential for alcoholic fer- Lys366) that are different are all located in different
mentation by Saccharomyces as the species is not regions from the catalytic domain (4).
able to hydrolyze polysaccharide material. Each subunit is divided into two domains separated
Production of ethanol from glucose occurs via the by a crevice that contains a wide and deep hydropho-
Embden-Meyerhof-Parnas pathway. In the last reac- bic pocket (active site). One of these domains, called
tion of this alcoholic fermentation, yeast ADHs cata- the coenzyme-binding domain, binds the coenzyme.
lyze the reduction of acetaldehyde to ethanol (14). The two zinc atoms are bound within the second
The yeast used in the manufacture of alcoholic bev- domain, called the catalytic domain. The two domains
erages are strains of Saccharomyces cerevisiae or S. are unequal in size. The catalytic domain is larger and
carlsbergensis. The denitive difference between these comprises 231 residues, whereas the coenzyme-binding
yeasts is that S. carlsbergensis ferments rafnose com- domain contains 143 residues. The primary and sec-
pletely, whereas S. cerevisiae does not (14). ondary structures in the subunit of HLADH are illu-
strated in Figure 2 (6).
The two subunits of the dimeric enzyme are joined
III. PROPERTIES AS PROTEIN together mainly through noncovalent interactions
within the coenzyme-binding domains. These domains
A. Horse Liver Alcohol Dehydrogenase
thus form a core in the middle of the molecule by
homologous interactions within corresponding regions
Horse liver alcohol dehydrogenase (HLADH) is gen-
of the coenzyme binding to enzyme. The catalytic
erally considered to be a symmetrical dimer, composed
domains are located on the exterior of the molecule,
of two identical chains of 40 kDa but with difference in
and the catalytic sites are found in the junction
amino acid sequence at six positions. One cannot
between the two domains and the core. The whole
ignore the different forms observed in several experi-
molecule has an approximate helical content of 29%
ments owing to dissociation-reassociation or in other
of the residues while 34% are in pleated-sheet regions.
studies of the puried isoenzymes. It is known that
three main isoenzymes are formed by the dimeric com-
bination of the two different types of subunit chains, 2. Coenzyme-Binding Domain
which are based on the difference in substrate speci-
The coenzyme-binding domain of the subunit comprises
city. There is a subunit called E for its ethanol activity
residues 176318. The amount of secondary structure in
and another subunit S for its steroid activity, therefore,
this domain is considerable; 45% of the residues are
the main isoenzymes are EE, ES, and SS. The three
helical (mainly -helices), 32% are in pleated-sheet
isoenzymes differ in that SS also has ethanol activity
structure, and 13% in reverse bends. Thus only 10%
but lower than ES and still lower than EE and that EE
of the residues have no regular secondary structure. The
has some activity toward certain steroid side chain
domain is built of six parallel strands of pleated sheet
hydroxyl groups (4).
(AF) anked by ve helices (AE) in a regular
pattern. There are three hydrophobic regions in this
1. Primary, Secondary, Tertiary, and
domain. One is involved in subunit interactions; the
Quaternary Structures
F strands of each subunit run in opposite directions
Studies of primary structure of E- and S-subunits have perpendicular to the twofold axis and are joined together
shown that each subunit is composed of 374 residues by hydrogen bonds forming two strands of antiparallel
plus two zinc atoms. The crystal structure analysis of structure. The other two hydrophobic regions are impli-
the apoenzyme helped to determine the position of cated in the folding of the domain and form hydropho-
each residue in the polypeptide chains and showed bic cores between helices and the parallel pleated sheet.
that the principal difference between these subunits is The unique fold of the coenzyme-binding domain cre-
a 6 amino acid difference at positions Glu17, Thr94, ates a large cleft region between the two domains of
Arg101, Phe110, Asp115, and Glu366 (residues listed HLADH for binding of a coenzyme molecule (4, 6, 15).
for E-subunit). Three of the six amino acid differences Two coenzyme molecules are bound per enzyme
observed between the E- and S-chains may be respon- molecule independently of each other. The coenzyme
sible for the difference in substrate specicity. These binds to the enzyme in the central region of the car-
are Phe110, Asp115, and Thr94 in the E-subunit, and boxyl end of the parallel pleated sheet. The coenzyme
Leu110, Ser115, or a gap and Ile94, respectively, in the binding induces different conformational changes: the
S-chain. The other three residues (Gln17, Ser101, and active site is more shielded from the solution; the active

Figure 2 Primary structure and schematic diagram of the regions of secondary structure in the subunit of HLADH.
Helices are called with a numeral for those that appear in the catalytic domain and a letter for those that are in the
coenzyme-binding domain. Strands of pleated sheet are called with a similar nomenclature as for the helices. Reverse
turns are denoted by the following symbol ( ). : indication of length of helix or strand. (From Ref. 6.)
site is then in a more hydrophobic environment in the 3, and 4); 35% are in pleated-sheet regions (I,
holoenzyme than in the apoenzyme. All water mole- II, III), and 14% are in reverse bends. Thus, a
cules that are located in the active site in the apoen- large number of residues, 32%, have no regular sec-
zyme are displaced in the ternary complex, and the ondary structure. An important region in the domain,
catalytic reaction of the enzyme takes place in a com- necessary for the structural stability of the enzyme, is
pletely water-free environment (15). The increase of the lobe comprising residues 95113 that binds the
hydrophobicity around the nicotinamide group in the structural zinc atom by four sulfur atoms from cysteine
ternary enzyme-NAD -alcohol complex facilitates the residues 97, 100, 103, and 111. Although it is bound
reaction (8). near the surface of the molecule, this zinc atom is com-
pletely surrounded by the protein and is not accessible
3. Catalytic Domain in the native conformation of the enzyme (6).
The catalytic domain comprises residues 1175 and
319374 plus two zinc atoms. Both ends of the poly-
4. Substrate-Binding Site
peptide chain are within this region. The two zinc
atoms of the subunit are bound to ligands from the The substrate-binding site, lined almost exclusively by
domain; these bindings are illustrated in Fig. 2. Only hydrophobic residues, is located in a cleft between the
the zinc atom participates in the catalytic activity; the coenzyme-binding core and the catalytic domain. This
second zinc atom is remote from the active site and cleft is open in the apoenzyme form, which is well
may help in stabilizing protein folding (a structural suited for the initial coenzyme binding and does not
zinc). Only 19% of the residues are helical (1; 2; hinder its entrance.

The catalytic zinc atom is situated at the bottom of Table 1 Residues at Positions that Participate in
this deep pocket, with a distance of 2025 A from Enzymatic Functions of the HLADH
the exterior surface of the protein. The active-site zinc
Function Horse livera Yeast
performs several functions associated with the catalytic
reaction. Three of the four coordination ligands to the Adenine-binding pocket interior Phe198 Ser198
catalytic zinc belong to the protein; two sulfur atoms Val222 Ile222
from Cys46 and Cys174 and one nitrogen atom from Ile224 Gly224
His67. In the apoenzyme, the fourth ligand is an ioniz- Pro243 Phe243
able water molecule that is hydrogen-bonded to the Ile250 Val250
hydroxyl group of Ser48. Following the binding of Thr274 Ala274
Thr277 Ala277
the coenzyme, the water is displaced by the substrate.
Surface Arg271 Ser271
This is facilitated by a coenzyme-induced shift in equi-
Asp273 Ala273
librium from an open/closed conformation of the Adenosine ribose binding Asp223 Asp223
enzyme. Gly199 Gly199
Ile269 Ser269
B. Alcohol Dehydrogenase from Yeast (YADH) Asn225 Gly225
Lys228 Lys228
YADH has a molecular weight of 150 kDa and con- Pyrophosphate binding Arg47 His47
tains four identical subunits of 3637 kDa. Just like Ile269 Ser296
in HLADH, each subunit contains two zinc atoms. Nicotinamide ribose binding Gly293 Gly293
They have structural and catalytic roles and are Nicotinamide binding Thr178 Thr178
Substrate-binding pocket Leu57 Trp57
bound tetrahedrally to four sulfur atoms all close to
Phe93 Trp93
one another in the primary sequence (structural zinc Phe110 Asn110
atom) or to three protein ligands (two sulfur atoms and Leu116 Leu116
one nitrogen atom) and a water molecule or hydroxyl Phe140 Tyr140
ion, depending on the pH (catalytic zinc atom) (16). Leu141 Thr141
The primary structure of YADH has been compared to Pro296 Ala296
the known tertiary structure of the corresponding Ile318 Ile318
HLADH after proper alignment of the two proteins Acid-base system Ser48 Thr48
(17). The results show that YADH and HLADH are His51 His51
distantly homologous with many insertions/deletions Ligands to active site zinc atom Cys46 Cys46
and with only 25% of all residues conserved, but all His67 His67
Cys174 Cys174
functionally essential residues are similar or identical.
Moreover, the difference in the tertiary structures of Source: Ref. 17.
a
YADH and HLADH are less than the large differences Numbers refer to the amino acid sequence of the horse protein.
in the primary structure of these proteins (Fig. 1) (17).
This comparison shows that the general subunit con-
formations and enzymatic mechanisms of both tially expressed, at least partly related to the require-
enzymes are probably similar (Table 1). The residues ment for fermentation or for oxidation of alcohols.
in HLADH that participate in coenzyme binding, sub- The three chromosomally encoded isoenzymes are dif-
strate binding and the catalysis are listed in Table 1, ferentially expressed. Although YADH I and YADH
together with the equivalent residues in YADH. II perform separate metabolic roles, they do not differ
Quaternary structure of YADH and residues greatly in their Vmax values for both forward and
involved in subunit contacts are not yet well known. reverse reactions, but they do differ in their afnity
But, in 1995, De Bolle et al. (18) suggested that a part for ethanol as a substrate. YADH I is the fermentative
of the subunit contacts observed in HLADH are cytoplasmic enzyme that, when the yeasts are grown
located at homologous positions in YADH. The on glucose under anaerobic conditions, can account
main difference in quaternary structure is likely to be for up to 1% of cell protein. An opposite physiological
due to surface changes of the subunits. role of ethanol oxidation is carried out by YADH II
Four alcohol dehydrogenase isoenzymes are found which converts ethanol accumulated in anaerobic
in the yeast Saccharomyces cerevisiae. These enzymes growth to acetaldehyde and is expressed when ethanol
have different substrate specicities and are differen- is used as a carbon source. These two isoenzymes have

94% sequence homology (11). YADH III is mito- dehydrogenase has more restricted specicity than
chondrially located and has 79% and 78% amino horse liver alcohol dehydrogenase. The activity of
acid identity with YADH I and YADH II, respectively YADH decreases as the chain length of the primary
(11). The function of YADH III has not been identi- alcohols increases (19). YADH and HLADH also have
ed, although its mitochondrial location would indi- quite different activities and stereospecicities on sec-
cate that it may have had a role in respiration. Cells ondary and branched alcohols. The difference in sub-
lacking YADH III isoenzyme can survive aerobically strate specicity between HLADH and YADH could
or anaerobically (11). The fourth ADH is an iron acti- be due to a difference in the size of the substrate bind-
vated enzyme. ing pocket. The main differences in the substrate bind-
ing pocket are the changes from Phe93 and Ser48 in
HLADH to Trp and Thr, respectively, in YADH. As a
IV. PROPERTIES AS ENZYMES
consequence YADH has a smaller substrate binding
A. Substrate Specicity and Activity pocket (20).
The activities of HLADH and YADH on primary,
Alcohol dehydrogenases catalyze the reversible inter- secondary, and branched alcohols and the kinetic con-
conversion of a wide variety of aldehyde/alcohol sub- stants (kcat [turnover number], KM , and kcat =KM [cat-
strates. The specicity of ADHs for their substrates is alytic efciency]) characteristic for HLADH and
not the same for all enzymes. In fact, yeast alcohol YADH are listed in Section V (see Tables 24).
Table 2 Kinetics Parameters for Oxidation of Alcohol by NAD Catalyzed by HLADH
KM M KM (mM) kcat =KMsubstrate

Substrates kcat sec1 coenzyme substrate M1 sec1 )
Primary alcohols
Ethanol (a) 1.14 16.3 0.460 2,480
Propanol (b) 8.20 0.390 21,000
Butanol (b) 5.20 0.270 19,000
Pentanol (b) 4.30 0.150 29,000
Hexanol (b) 2.80 0.076 37,000
Heptanol (b) 2.50 0.020 125,000
Octanol (b) 2.30 0.040 58,000
Secondary and branched alcohols
Isopropanol (c) 0.58 9.0 64
2-methyl-propanol (b) 5.30 0.4 13,000
(R)-2-butanol (c) 2.00 7.5 270
(S)-2-butanol (c) 1.00 1.35 740
2-methyl-1-butanol (S) (b) 5.70 0.31 18,000
2-methyl-1-butanol (RS) (b) 5.30 0.52 10,000
3-methyl-1-butanol (b) 2.80 0.15 19,000
(S)-2-pentanol (c) 0.87 0.73 1,200
(R)-2-pentanol (c) 1.01 31.7 32
3-pentanol (c) 2.26 1.6 1,400
(S)-2-octanol (c) 1.52 0.18 8,400
(R)-2-octanol (c) 0.053 1.92 29
Source: Refs. 20, 33, 34.

Measurements were made (a) at 25 C, in a 47 mM phosphate, 0.25 mM EDTA buffer, pH 8. Kinetics were measured
with NAD concentration varied from 5 to 55 M and ethanol concentration varied from 1 to 10 mM; (b) at 30 C
in a 83 mM potassium phosphate, 40 mM KCl, 0.25 mM EDTA buffer, pH 7.3. [NAD ] was xed at 2 mM.
Concentration of substrates were varied over a 10-fold range around the corresponding KM values; (c) at 25 C, in a
0.1 M Taps buffer, pH 8.5. Kinetics were measured with 0.5 mM NAD and a substrate concentration ranging from
0.5 to 5 times the KM .

Table 3 Kinetics Parameters for Reduction of Aldehydes Catalyzed by HLADH

Acetaldehyde (a) 125.00 14.37 0.46 271800

Acetone (b) 0.33 135.00 2
2-butanone (b) 0.12 15.00 8
2-pentanone (b) 0.073 14.20 5.1
3-pentanone (b) 0.36 75.00 5
Cyclohexanone (c) 26.00 5.30 4900
(3R)-3-methylcyclohexanone (c) 0.64 5.0 128
Benzaldehyde (d) 110.00 9.90 0.12 916700

Measurements were made (a) at 23:5 C, in a sodium phosphate buffer, pH 6. Range of concentrations of coenzyme
and substrates were: [NADH] 0.520 M, [acetaldehyde] 291440 M; (b) at 25 C, in a 0.1 M Taps buffer, pH 8.5,
with a coenzyme concentration of 0.2 mM and a substrate concentration range of 0.5 to ve times the KM ; (c) at
30 C, in a 33 mM sodium phosphate buffer, pH 8, containing 0.25 mM EDTA. Range of concentrations of sub-
strates were: [cyclohexanone] 0.28 mM, [(3R)-3-methylcyclohexanone] 19 mM, [()-3-methylcyclohexanone] 0.28
mM; (d) at 30 C, in a 50 mM sodium TES and 0.25 mM EDTA buffer, pH 7.
Table 4 Kinetics Parameters for Oxidation of Alcohol and Reduction of Aldehydes Catalyzed by
YADH

Oxidation of alcohols by NAD

Primary alcohols
Ethanol (a) 340 17.0 20,000
Propanol (a) 120 27.0 4,400
Butanol (a) 51 55.0 930
Pentanol (a) 29 37.0 780
Hexanol (a) 16 10.0 1,700
Heptanol (a) 14 7.8 1,800
Octanol (a) 17 5.3 3,300
Nonanol (a) 8.6 1.7 5,100
Secondary and branched alcohols
2-propanol (a) 4.8 190 25
2-butanol (R) (a) 0.05 61 0.8
2-butanol (S) (a) 1.0 55 18
2-methyl-1-propanol (a) 0.19 25 8
2-methyl-1-butanol (S) (a) NAa NA NA
2-methyl-1-butanol (RS) (a) NA NA NA
3-methyl-1-butanol (a) NA NA NA
Benzyl alcohol (a) NA NA NA
Cyclohexanol (a) NA NA NA
Reduction of aldehydes by NADH
Acetaldehyde (b) 3850 96 0.93 4,139,800
Butyraldehyde (b) 3450 97 27.5 125,450

Measurements were made (a) at 30 C in a 83 mM potassium phosphate, 40 mM KCl, 0.25 mM EDTA buffer,
pH 7.3. [NAD ] was xed at 2 M. Concentration of substrates were varied over a 10-fold range around the
corresponding KM values; (b) at 25 C, in a sodium phosphate buffer, pH 7.05, containing bovine serum albumin.
Range of coenzyme and substrate concentrations were: [NADH] 3330 M, [acetaldehyde] 0.0275.5 mM,
[butyraldehyde] 1.937 mM.
a
NA, no measurable activity.

B. Effects of Environmental Factors the optimal pH is 9 in the forward direction and 6 in
the backward direction. The kinetic constants obtained
ADHs, like most enzymes, may be affected structurally versus pH are listed in Table 5. The Keq (equilibrium
and catalytically by environmental factors such as pH, constant of enzyme-coenzyme complex with sub-
temperature, and pressure. strates) decreases when pH increases. The approximate
values are of 2, 1, and 0.1 nM at pH 7, 8, and 9,
1. pH respectively (22).
Alcohol dehydrogenases catalyze a reversible reaction,

and forward and backward reactions occur simulta- 2. Effect of Temperature on Activities of
neously. The reaction of NAD acidies an unbuffered YADH and HLADH
reaction medium by producing a proton while the oxi- The optimal temperature for YADH is 25 C. The opti-
dation of NADH consumes a proton (21). For YADH mal temperature for HLADH is 53 C. The behavior of
and HLADH, a double pH dependence for the for- YADH and HLADH versus temperature is opposite in
ward and the backward directions has been estab- direction. Whereas YADH activity decreases when
lished. For YADH, the optimal pH in the forward temperature increases above 25 C, HLADH activity
reaction (oxidation of alcohol) is 8.3 and in the back- increases when temperature increases up to 53 C.
ward direction 6.0 (reduction of aldehyde). These pH The half-life of YADH is 3.5 h when a solution of
optima have been obtained under standard conditions YADH at 0.1 mg/mL is stored at 25 C in 50 mM Tris-
(absence of buffer at high substrate concentration (100 HCl buffer, pH 8.5 (23). Concerning HLADH, the
mM), 5 mM NAD or 0.5 mM NADH and zero initial half-life at 53 C is 8.5 h and it is 14.3 h at 25 C.
product concentration). The distance between the two These studies have been performed in a solution of
optimal pH values (the pH optimal distance) corre- HLADH at 0.045 mg/mL, in 50 mM Tris-HCl buffer,
sponds to two pH units. Other substrates (2-propa- pH 8 (24).
nol/propanal) shift the optimal pH of the forward
and the backward directions by about two pH units
3. Effect of Pressure on Activities of YADH
toward higher values. Moreover, whatever the sub-
and HLADH
strates, the forward and the backward directions
have different maximum rates, even at their corre- In studying the effect of pressure on YADH activity at
sponding optimal pH (20). In the case of HLADH, 25 C, it has been shown that pressurization of YADH
Table 5 Effect of pH on Kinetic Constants
Oxidation of ethanola
pH kcat (sec1 ) KM NAD M KM substrate (M) kcat =KM M1 sec1 b
6 1.6 4.0 452 3,600

7.1 2.7 3.0 178 15,000
8 3.2 2.9 161 20,000
9 3.8 4.6 396 9,600
Reduction of aldehyde
pH kcat sec1 ) KM NADH (M) KM substrate (M) kcat =KM (M1 sec1 b
6 125.0 14.4 463 270,000
7.1 125.0 12.5 413 303,000
8 47.6 6.3 186 256,000
9 7.6 1.6 97.7 78,000
Source: Ref. 32.

a
The values have been determined in the following conditions: T 23:5 C, in 100 mM sodium phosphate buffers
(for experiments at pH 5.358) or in 36 mM glycine-NaOH buffer, pH 9, with 1.8500 M NAD /0.18.1 mM ethanol
or 0.9819:6 M NADH/0.0271.0 mM acetaldehyde.
b
KM for substrate.

resulted in a decrease of catalytic efciencies. This phe- analog to NAD in which the nicotinamide 0 ring is
nomenon became more pronounced for pressure > 50 linked to the sugar via a C-glycosyl (C5C1 ) bond
MPa. This loss in catalytic efciency is mainly due to a and 5--D-ribofuranosylnicotinamide adenosine dinu-
decrease of kcat (70% loss from 0.1 to 175 MPa). On cleotide (CPAD ) is another one. The inhibition con-
the contrary, KM for ethanol seems to be less sensitive stant of CNAD with respect to NAD is 4 nM; this is
to pressure (20% less in the same range of pressure) signicantly smaller than the Ki for NADH (0:4 M).
(25). YADH activity inhibition is therefore not due to Further, CNAD competes with both cofactor and
a dissociation of the tetrameric enzyme because only substrate binding (Ki 2 nM). The other isosteric ana-
the tetrameric form binds ethanol. log of NAD , CPAD, shows competitive inhibition of
In contrast to YADH, HLADH activity increases NADH with respect to NAD and does not compete
with pressure up to 225 MPa at 53 C. From atmo- with substrate binding (27).
spheric pressure to 225 MPa kcat increases by a factor
of 13. But pressure affects the afnity of HLADH for 5. Some Inhibitors for YADH (4)
its substrates. At the same conditions KM for NAD
Some coenzyme competitive inhibitors include N-alkyl
increases by a factor of 50 and KM for ethanol
nicotinamide derivatives (the inhibitory ability
increases by a factor of 45, but the inhibition by etha-
increases with increase in length of the alkyl chain),
nol observed at atmospheric pressure is not observed
N-alkyl substituted ammonium chlorides and uores-
above 150 MPa (24).
cent dyes, and drugs such as chloroquine and propa-
nol. Other inhibitors include chelating agents such as
4. Some Inhibitors of ADH 1,10-phenanthroline (inhibition is considerably smaller
with YADH than with HLADH), and nonchelating
There are two general classes of ADH inhibitors. One
analogs such as 1,5-phenanthroline and 2,9-dimethyl-
class includes analogs of alcohols and aldehydes. These
1,10-phenanthroline. With pyrazole, the inhibition is
compounds bind in the substrate pocket to the cataly-
competitive with ethanol and uncompetitive with coen-
tic zinc atom displacing the zinc-bound water molecule
zyme and acetaldehyde and with 4-substituted pyrazole
(ex: pyrazole; Ki 0:22 M [4]). Amides and forma-
amide derivatives (weaker inhibitors than pyrazole).
mides are analogs of aldehydes and potent inhibitors
Thiocyanate is an inhibitor that is competitive with
of HLADH. These inhibitors bind preferentially to the
ethanol and noncompetitive with NAD . It is reversi-
enzyme NADH complex. The amides inhibit alcohol
ble or irreversible depending on the concentration.
metabolism and might be useful therapeutic agents
Urea at low concentrations inhibits the enzyme non-
for example, to prevent oxidation of methanol or ethy-
competitively with respect to NAD , NADH, alcohol,
lene glycol to their toxic acids. The amides are uncom-
and aldehyde, and at higher concentrations inhibits the
petitive inhibitors of ethanol and competitive of
enzyme irreversibly. Thiourea, guanidium salts, pheny-
acetaldehyde (N-cyclohexylformamide; Ki 8:7 M
lisopropyl hydrazine, and canavanine are also inhibi-
[26]). The amides may bind to free enzyme or the
tors.
enzyme NAD complex (26). The chelating agents
such as 2,2-bipyridine (Ki 400 M [4]) or salicylate
(Ki 1:25 M [4]) are competitive inhibitors with C. Reaction Mechanism of HLADH
NAD and NADH and partially competitive with
alcohol. These inhibitors bind to the catalytic zinc The enzyme binds coenzyme and substrate to form a
and displace the zinc-bound water molecule at the pre- ternary complex which, in general, can be assumed to
sumed substrate binding site. Heterocyclic nitrogen be ordered with coenzyme binding preceding substrate
bases such as imidazole form both binary and ternary binding and product release preceding coenzyme
complexes with HLADH. Imidazole binds to the cat- release.
alytic zinc in the same way as chelating agents (4). Aldehyde formation during the catalytic action of
The second type of ADH inhibitors consists of ana- the enzyme requires a net removal of two hydrogen
logs of the coenzymes NAD and NADH, as well as atoms from an alcohol substrate. This dehydrogena-
structurally related compounds. These inhibitors bind tion process is known to proceed by a mechanism of
in the coenzyme-binding cleft generally mimicking the combined proton and hydride ion transfer. It is well
binding of the adenosine portion of the cofactor. For established that transfer of the hydride ion occurs
instance, 5--D-ribofuranosylnicotinamide adenosine directly between substrate and coenzyme in the pro-
dinucleotide (CNAD ) is an isosteric and isomeric ductive ternary complex formed. The rst step is an

initial binding of NAD . The E NAD complex iso- reaction is spectrophotometrically silent because there
merizes (i.e., changes conformation) with a forward is no net synthesis of NADH. The Vmax for dismuta-
rate constant of 620 sec1 at pH 8 and 1100 sec1 at tion is higher than that for oxidation of the corre-
pH 7.6 (28). By isomerization of the enzyme NAD sponding alcohol. At high concentrations of NAD ,
complex, the pKa of the zinc bound water molecule is the enzyme catalyzes oxidation of aldehydes to acids
shifted from 9:6 to 7:6 (29). This isomerization is with net NADH production with a Vmax much less
facilitated in the presence of alcohol in which case the than that for alcohol oxidation (30).
pKa drops to < 4:5. As a result of this isomerization, a
hydroxide ion remains bound to the zinc atom, and a
proton is released. It has been suggested that this pro-
ton is released through the system of hydrogen bonds D. Reaction Mechanism for YADH
involving Ser48 and His51 (28). The electric eld cre-
ated by the protein framework facilitates this proton The main features of the reaction mechanisms for
tunneling along the hydrogen bonds. The presence of YADH are in all probability essentially the same as
NAD and zinc with a small positive charge further in HLADH (because of structural similarities of the
enhances this effect. It is similar to a proton pump catalytic domain of the two enzymes). Finer details
which pumps protons from the interior hydrophobic of the reaction mechanism, however, are different
pocket to the surface of the molecule. The transfer of between the two enzymes. There are differences in sub-
the proton from the alcohol to the enzyme active site strate specicity, in the rate of catalysis, and in the
occurs simultaneously with the hydride transfer from requirements for ordered events during the catalytic
the substrate to the coenzyme. Figure 3 presents the mechanism which may be random or ordered depend-
ordered ternary complex mechanism during HLADH ing on substrate (31). The step of dissociation of
catalysis. NADH from the enzyme could be the rate-limiting
The rate of the conformational change (isomeriza- step in the oxidation of alcohol.
tion) could limit the transient rate of oxidation of alco-
hol. The isomerization of the E NAD complex is
partially, but not fully, rate limiting for the oxidation
of longer-chain alcohols. The rate of interconversion of
V. QUALITATIVE AND QUANTITATIVE
ternary complexes can be limited by the rate of transfer
DETERMINATION OF ACTIVITY
of hydride ion from alcohol to NAD , the release of a
proton to solvent, or other unimolecular steps depend- A. Specic Activity with Assay Conditions
ing on the substrate (28).
When NAD concentrations are approximately ADH activity assays for alcohol oxidation or aldehyde
stoichiometric with enzyme, HLADH catalyzes the dis- reduction were followed by an increase or a decrease of
mutation of different aldehydes to equimolar quanti- NADH absorption at 340 nm, respectively. As the
ties of the corresponding alcohol and acid (30). This molar absorptivity of NADH at 340 nm is dependent
on the conditions of pH, ionic strength, buffer, tem-
perature, and pressure, it must be determined for each
kinetics condition (25, 32). However, some character-
istics of HLADH must be known before using this
enzyme. Its isoelectric point is 6.8, its optimal pH is
9, its optimal temperature is 53 C. In Tables 2 and 3
some kinetic parameters obtained for oxidation of
alcohol or reduction of aldehydes catalyzed by
HLADH under different assay conditions are pre-
sented.
Figure 3 The ordered ternary complex mechanism usually
As for HLADH, some characteristics of YADH
considered for evaluation of the kinetics of HLADH cataly-
sis. E, E NADH, E NAD , E NAD RCH2 OH, and must be known before using this enzyme. Its isoelectric
E NAD RCH2 OH denote enzyme, coenzyme-enzyme point is 5.4, its optimum pH is 8:6 (for the alcohol
complex, coenzyme-enzyme complex isomerized, enzyme- oxidation), its optimal temperature is 25 C. The speci-
coenzyme-alcohol complex, and enzyme-coenzyme-alcohol c substrates and the corresponding kinetic constants
complex isomerized, respectively. (From Ref. 28.) are listed in Table 4.

B. Enzyme Stability Table 6 Characterization of Commercially Available
Lyophilized preparations of YADH stored at 20 C
Yeast ADH Horse liver ADH
are stable for 612 months. Crystalline suspensions in
ammonium sulfate are stable for 6 months at 28 C. Classication medium-chain medium-chain
However, preserving an active enzyme solution dehydrogenase dehydrogenase
remains a problem. A solution of enzyme (0.1 mg/ Required coenzyme NAD(H) NAD(H)
mL in 50 mM Tris-HCl buffer, pH 8.5) stored at 4 C Specic activity (U/mg) 300a 12a
is not stable; its half-life (t1=2 ) is 4.5 h. When stored at Enzyme costs 2 330
20 C, its half-life is 56 h. In order to increase the ($/500 units)
(Sigma catalog 1998)
stability of the enzyme solution, it has been shown
Stability sensitive to O2 stable
that storing the enzyme with NAD (1.5 mM) or
Limitations low stability
with NAD =sorbitol (1.5 mM/1 M), enhances the sta-
bility. The t1=2 (25 C) was 28 h with NAD and 16 h a
oxidation of 1 mol ethanol/min.
for NAD /sorbitol. The t1=2 (4 C) increases to 82 and
68 h in the presence of NAD and NAD /sorbitol,
respectively (23).
Both lyophilized and crystalline suspensions in buf-
fer preparations are stable for 36 months when Figure 4 shows an example of purication of
HLADH is stored at 20 C. In solution, HLADH human liver alcohol dehydrogenase (which is very
was more stable than YADH; at the same storage con- similar to HLADH except in regard to specicity)
ditions of buffer and temperature, the t1=2 (25 C) is 680 (37). Human liver alcohol dehydrogenase has been
h and the t1=2 (4 C) is 755 h (24). See above for stability puried by a procedure designed to process 10- to
of YADH under these conditions. To stabilize the 20-kg quantities of liver. The results are summarized
enzymes, it is possible to add bovine serum albumin in Table 7.
at 1 g/L.
VI. PURIFICATION OF ADH
The enzyme has been puried, and the EE and SS

isoenzymes separated by afnity chromatography on
an immobilized AMP analog. Commercially available
preparations contain the EE isoenzyme and small
amounts of the ES isoenzyme (4). The more studied
YADH is a cytoplasmic protein, commercially avail-
able and usually obtained from bakers yeast. Several
purication methods have been reported including
toluene plasmolysis; DEAE-cellulose chromatography;
fractionation with protamine sulfate and calcium phos-
phate gel; and purication without autolysis, heat
denaturation, or use of solvents. Separation of
YADH from enzyme mixtures by afnity chromato-
graphy on immobilized NAD or AMP analogs has
also been reported (4). It is now not necessary to purify
HLADH and YADH in ones laboratory because of
their commercial availability at low price and high Figure 4 Example of purication: human liver alcohol
purity. Table 6 presents the characteristics of commer- dehydrogenase (which is very similar to HLADH except in
cially available HLADH and YADH. regard to specicity). (From Ref. 37.)

Table 7 Large-Scale Purication of Human Liver Alcohol Dehydrogenase
Act. Protein Specic act.

Fraction Vol (L) (A340 nm=min (mg) (A340 nm=min=mg
Crude extract 27 24,000 760 0.03

Dialyzed extract 32 23,300 770 0.03
DEAE-cellulose-treated extract 41 19,200 36 0.53
Concentrated CM-cellulose eluates
Peak I 0.16 1,000 0.78 1.1
Peak II 0.19 2,700 2.6 1.0
Peak III 0.21 1,920 2.4 0.8
Source: Ref. 37.

The total activity recovered from CM-cellulose column chromatography, which represents 6070% of that present in the DEAE-cellulose-treated
extract, exceeds that exhibited by the selected fractions from peaks IIII which were combined and concentrated.
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enzyme. Primary structure, cDNA-cloning, and rela-
1. L Ribas de Pouplana, S Atrian, R Gonzalez-Duarte, tionships to other alcohol dehydrogenases. Eur J
L-A Fothergill-Gilmore, S-M Kelly, N-C Price. Biochem 194:593602, 1990.
Structural properties of long and short chain alcohol 11. MF Reid, CA Fewson. Molecular characterization of
dehydrogenases. Biochem J 276:433438, 1991. microbial alcohol dehydrogenases. Crit Rev Microbiol
2. H Jornvall, B Persson, J Jeffery. Characteristics of 20:1356, 1994.
alcohol/polyol dehydrogenases. Eur J Biochem 12. CR Lowe. The application of coenzyme-dependent
167:195201, 1987. enzymes in biotechnology. Phil Trans R Soc Lond
3. W Hummel. New alcohol dehydrogenases for the 300:335353, 1983.
synthesis of chiral compounds. Adv Biochem Eng 13. S Dilmaghanian, C Vivian Stead, RJ Ansell, CR
Biotechnol 58:145184, 1997. Lowe. Synthesis and properties of a naphthalene-con-
4. C-I Branden, H Jornvall, H Eklund, B Furugren. taining articial redox coenzyme. Enzyme Microbiol
Alcohol dehydrogenases. In: PD Boyer, ed. The Technol 20:165173, 1997.
Enzymes, Vol XI. New York: Academic Press, 1975, 14. GA Beech, MA Melvin, J Taggart. Food, drink and
pp 103190. biotechnology. In: IJ Higgins, DJ Best and J Jones,
5. J Jornvall, B Persson, J Jeffery. Alcohol and polyol eds. Biotechnology: Principles and Applications.
dehydrogenases are both divided into two types and London: Blackwell Scientic Publications, 1985, pp.
structural properties cross-relate the different enzyme 73110.
activities within each type. Proc Natl Acad Sci USA 15. Eklund, BV Plapp, JP Samana, C-I Branden. Binding
78:42264230, 1981. of substrate in a ternary complex of horse liver alcohol
6. H Eklund, B Nordstrom, E Zeppezauer, G Soderlund, dehydrogenase. J Biol Chem 257:1434914358, 1982.
I Ohlsson, T Boiwe, B-O Soderberg, O Tapia, C-I 16. BL Vallee, DS Auld. Zinc coordination, function, and
Branden. Three dimensional structure of horse liver structure of zinc enzymes and other proteins.
alcohol dehydrogenase. J Mol Biol 102:2759, 1976. Biochemistry 29:56475659, 1990.
7. H Eklund, B Nordstrom, E Zepperzauer, G 17. H Jornvall, H Eklund, C-I Branden. Subunit confor-
Soderlund, I Ohlsson, T Boiwe, C-I Branden. The mation of yeast alcohol dehydrogenase. J Biol Chem
structure of horse liver alcohol dehydrogenase. 253:84148419, 1978.
FEBS Lett 44:200204, 1974. 18. X De Bolle, C Vinals, D Prozzi, JY Paquet, R Leplae,
8. H Eklund, JP Samana, L Wallen, C-I Branden, A E Depriereux, J Vandenhaute, E Feytmans.
Akenon, TA Jones. Structure of a triclinic complex Identication of residues potentially involved in the
of horse liver alcohol dehydrogenase at 2.9 A resolu- interactions between subunits in yeast alcohol dehy-
tion. J Mol Biol 146:561587, 1981. drogenases. Eur J Biochem 231:214219, 1995.
9. R Rella, CA Raia, M Pensa, FM Pisani, A 19. FM Dickinson, GP Monger. A study of the kinetics
Gambacorta, M De Rosa, M Rossi. A novel archae- and mechanism of yeast alcohol dehydrogenase with a
bacterial NAD -dependent alcohol dehydrogenase. variety of substrates. Biochem J 131:261270, 1973.
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10. M Estonus, C Karlsson, EA Fox, J-O Hoeoeg, B strate specicity of yeast alcohol dehydrogenase. J
Holmouist, BL Vallee, WS Davidson, H Jornvall. Biol Chem 268:77927798, 1993.

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dehydrogenase from Thermoanaerobium brockii. PhD dehydrogenases. J Biol Chem 262:37543761, 1987.
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24. M Trovaslet. Inuence of temperature and pressure ity of NADH. Clin Chem 22:141150, 1976.
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29. J Kvassman, G Pettersson. Unied mechanism for
proton-transfer reactions affecting the catalytic activ-

34
Amine Oxidase
Akio Ito and Jichun Ma

Kyushu University, Fukuoka, Japan
I. INTRODUCTION and dopamine, and the synthetic tertiary amine 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Amine oxidase catalyzes the reaction RCH2 NH2 O2 has been found to be oxidized by both forms of
H2 O ! RCHO H2 O2 NH3 with mono-, di-, or MAO, which suggests that MAO may oxidize MPTP
polyamine as substrate. Amine-oxidizing enzymes are analogs and generate toxic effects on the central ner-
classied by their prosthetic group(s), FAD or copper vous system. These features make MAO an extremely
plus topaquinone, or by their sensitivity to inhibitors, important enzyme related to neuronal dysfunction in
2-chlorocyclopropylamine or semicarbazide. The schizophrenia, Parkinsons disease, Downs syndrome,
FAD-containing amine oxidases have monoamine-oxi- and some other neurodegenerative diseases and psy-
dizing activity, and most of the copper-containing chological disorders in human, and vast numbers of
enzymes belong to the diamine-oxidase type. its inhibitor have been developed as medicines.
II. MONOAMINE OXIDASE (FAD- A. Properties as Protein

CONTAINING)
The study of MAO has a long history. However, it was
Monoamine oxidase (EC 1.4.3.4; MAO) is a avin- at the end of 1980s that the cDNA cloning of MAOA
containing enzyme. It catalyzes the oxidation of ali- and MAOB ended the notion that MAOA and MAOB
phatic and aromatic primary, secondary, and tertiary were the same protein with different catalyzing sites or
amines. that they had different functions at different environ-
Two forms of MAO, MAOA and MAOB, are ments, and proved that they were different proteins.
dened by their substrate and inhibitor afnities. Essentially, all higher eukaryotic organisms express
MAOA has a higher afnity for catecholamines and MAO (3). Both forms of MAO are localized in the
5-hydroxytryptamine (5-HT or serotonin) and is more outer mitochondrial membrane. They are expressed
sensitive to inhibition by clorgyline, whereas MAOB at different levels in different cell types. For example,
has a higher afnity for dietary amines such as phenyl- the highest expressions of both forms are found in the
ethylamine and is selectively inhibited by deprenyl liver for most species. Placenta expresses predomi-
(1, 2). nantly MAOA whereas platelet contains primarily
In human and animals, the enzyme plays an impor- MAOB. Most of the other tissue cells express both
tant role for the metabolism of biogenic monoamines forms of MAO.
in the central nervous system and peripheral tissues. MAOA and MAOB are encoded by two very simi-
MAO oxidizes neurotransmitters such as serotonin lar but different genes. In human and probably in

other mammals, the two genes are closely linked on the substrate specicity and inhibition from different
X-chromosome (Xp11.2311.4) (4), suggesting that the laboratories publications show divergence. Since the
two genes may have arisen from duplication of a com- Km value is a ratio of rate constants, it is easily varied
mon ancestral gene. Amino acid sequences of MOAO with the purity and the lipid content of the prepara-
and MOAB are deduced from cDNAs for human, tion. Lipophilic substrates may be concentrated
bovine, and rat enzymes (58). MAOA and MAOB around the mitochondrial membrane to which the
contain 527 and 520 amino acid residues (rat MAOA MAO is located, leading to a lower apparent Km
lacks one amino acid residue at the carboxy-terminus), when using mitochondria than when using puried
respectively. Molecular mass of human MAOA and MAO for the assay (13). The same is true in the
MAOB are 59.7 and 58.8 kDa, respectively. assay for reversible inhibition. Table 1 summarizes
Approximately 90% of the amino acid residues are some results on the specicity of MAOA and MAOB
identical among the same forms of MAO from differ- for a number of substrates. Some are from assays using
ent species, while there is only 70% homology between puried MAO, others from that using mitochondria or
MAOA and MAOB from the same species. One highly whole cell extract.
conserved region, residues 389460 for MAOA and Since the discovery that MAO inhibitors are
380451 for MAOB, contains the cysteine (residue antidepressants, large numbers of inhibitors of
406 for MAOA and residue 397 for MAOB) to MAOA and MAOB have been synthesized. The early
which the FAD covalently binds as 8--S-cysteinyl- MAO inhibitors were hydrazine or cyclopropylamine
FAD. The N-terminus shows signicant similarity to derivatives that showed little selectivity to the two
several avoproteins. This region fullls the criteria for forms of MAO. The acetylenic MAO inhibitors, clogy-
a predictive ngerprint for a avin-binding domain line and ()-deprenyl, however, exhibit a high degree
and contains the FAD- and ADP-binding fold. of selectivity to MAOA and MAOB, respectively. They
This structure has been suggested to act as a nuclea- have been found to act as mechanism-based irreversi-
tion center. That is, during biosynthesis this domain ble inhibitors of MAO. These compounds rst form a
will form rst, and the rest of the protein structure noncovalent complex with the active site of the
forms around it. The C-termini of both forms of enzyme, followed by the normal catalytic process to
MAO are responsible for anchoring the enzyme to generate a reactive species, which reacts with the
the outer membrane of mitochondria (9). enzyme to form an irreversibly inhibited species.
Another type of inhibitor is a selectively reversible
B. Properties as Enzyme inhibitor, which can sometimes be derived from the
substitution of the hydrogen at -carbon of MAO sub-
Since the crystal structures of both forms of MAO strate by a methyl group. The afnity of MAO to these
have not been established, the actual mechanism of inhibitors is often higher than it is for its substrates,
catalysis has yet to be elucidated. A conserved region with a Ki value of 105 to 109 M vs a Km value of 104
in the central parts of the proteins (residues 187230 to 105 M. This may be a reection of the fact that Km
for MAOA and 178221 for MAOB) is postulated as is not a simple dissociation constant for the initial
part of the active site. Chimeric and site-directed muta- noncovalent complex but includes steps that may
tion studies suggest that the region between about resi- orient the -C-H so as to optimize orbital overlap for
dues 120220 for MAOA and 111211 for MAOB are both the electron transfer and proton abstraction
responsible for substrate specicity (10, 11) and the processes (14). IC50 s of some inhibitors are summar-
phenylalanine of residue 208 for MAOA and isoleucine ized in Table 2.
of residue 199 for MAOB are found to be the key
amino acids responsible for substrate selectivity.
C. Determination of Activity
Exchange of these two residues by site-directed muta-
tion leads to an exchange of the enzyme properties;
The widely used methods for determination of activity
that is, F208I-MAOA shows properties of MAOB,
of MAO are the kynuramine oxidation method and
and I199F-MAOB shows properties of MAOA (12).
radiochemical method.
MAO catalyzes many compounds. The typical sub-
strate is 5-HT for MAOA and phenylethylamine
1. Kynuramine Oxidation Method
(PEA) and benzylamine for MAOB. MPTP is found
to be a substrate of MAOB, although it is oxidized by Kynuramine can be oxidized by both MAOA and
MAOA at a lower rate. The kinetic parameters for MAOB to produce 4-hydroxylquinoline, which has

Table 1 Kinetic Parameters of MAOs for Some Substrates
Turnover number (sec1 ) kcat sec1 Km M
Substrate MAOA MAOB MAOA MAOB MAOA MOAB Note Ref.
Kynuramine 146 170 puried human MAO 17

11.63 121.5 human MAO expressed in 11
HEK293 cell
Serotonin 119.3 human MAO expressed in 18
Cos cell
310 rat MAO expressed in yeast 10
cell
Phenylethylamine 3.07 2.57 154.4 2.4 human MAO expressed in 11
HEK293 cell
4.6 human MAO expressed in 18
Cos cell
240 8 rat MAO expressed in yeast 10
cell
MTPT 20 204 140 390 puried human MAO 17
the highest absorption at 315 nm and uorescences at length of 380 nm with an excitation wavelength of
380 nm. According to the absorbance or the intensity 315 nm.
of uorescence, the concentration of the product can
be quantitatively determined. The following protocol
2. Radiochemical Method
is a modication of Krajls method (15). In a test
tube, mix 0.25 mL of 200 mM potassium phosphate This method provides more sensitive determination
buffer, pH 7.5, and suitable volume of the enzyme but needs special devices and laboratory for using
sample. Add distilled water to a nal volume of radioisotopes. Many 14 C-labeled substrates for MAO
0.90 mL. Incubate the test tube at 30 C for 5 min, are commercially available. For example, DuPont
then add 0.10 mL 1 mM kynuramine preincubated at NEN(r) provides hydroxytrypamine binoxalate,
30 C, mix immediately, and incubate at 30 C with 5-[2-14 C[tryptamine bisuccinate, [side chain-2-
14
shaking for 20 min. Terminate the reaction by adding C]tyramine hydrochloride, [1-14 C]phenylethylamine
0.4 mL of 20% TCA. Precipitate the protein by hydrochloride, etc. MAO can oxidize these compounds
centrifugation at 10,000 g for 10 min. Take 0.5 mL and produce radioactive products. The enzyme activity
of the supernatant and add 1.5 mL of 1 N NaOH, can be estimated by measuring the radioactivity of the
and measure the intensity of uorescence at a wave- products.
Table 2 IC50 of Some Inhibitors for MAOs
IC50 (M)
Substrate Inhibitor MAOA MAOB Note Ref.
Serotonin Deprenyl 1:5 106 5:2 105 human MAO expressed in Cos cell 18
Clorgyline 7:0 1010 human MAO expressed in Cos cell 18
Ro41-1049 3:6 108 human MAO expressed in HEK293 cell 11
Phenylethylamine Deprenyl 2:7 109 human MAO expressed in Cos cell 18
6
5:0 10 1:3 107 rat MAO expressed in yeast cell 10
Clorgyline 4:0 107 human MAO expressed in Cos cell 18
2:5 108 7:9 105 rat MAO expressed in yeast cell 10
Lazabemide 1:2 104 1:1 108 human MAO expressed in HEK293 cell 11

Hot and cold substrates are mixed to a nal con- hydroxyapatite chromatography is not encouraged
centration of 5 mM (usually, the hot accounts for 1/50 because it leads to obvious inactivation, although this
by concentration when using 1:5 GBq/mmol hot can produce almost homogeneous MAOA.
substrate) to make an application solution of sub- MAOB can be puried essentially by the same
strate. In each Eppendorf tube, add 10 L of 300 method, but use Ficol, dextran, and PEG at the poly-
mM potassium phosphate buffer, pH 7.5, and certain mer partition step. MAOB will go to the interface
volume of sample (e.g., 50 g of rat liver mitochondrial between the upper (Ficol-PEG) and lower (dextran)
protein), and add sample buffer to make a nal volume layer, mainly in the solid material. See Salach (19)
of 54 L. Incubate the tube at 30 C for 1 min, and for details.
start the reaction by adding 6 L of application sub-
strate. Incubate at 30 C for 20 min and stop the reac-
tion by adding 40 L of 2 M HCl. Add 300 L water- III. AMINE OXIDASE (COPPER-
saturated ethyl acetate-toluene (1 : 1 v/v) and vortex to CONTAINING)
extract the product. Centrifuge at 10,000 g for 1 min
and transfer 200 L into a new tube. Add same volume The copper-containing amine oxidase (EC 1.4.3.6
of 0.5% PPQ; measure the radioactivity in a liquid amine:oxygen oxidoreductase [deaminating]) is obtain-
scintillation spectrometer. Blank should be made by able from bacteria, fungi, higher plants, blood plasma,
adding 2 N HCl before adding the substrate. and various mammalian organs. Most of this type of
enzymes contain a cofactor possessing one or more
D. Purication carbonyl groups, making them sensitive to inhibition
by carbonyl reagents such as semicarbazide and are
The Welter and Salachs procedure (16) is widely used classied as semicarbazide-sensitive amine oxidase
for purication of MAOA. Briey, mitochondrial frac- (SSAO). The enzyme is relatively abundant comprising
tion containing 140150 units of enzyme activity is at least 0.1% total soluble protein in etiolated pea
homogenized with glass/Teon homogenizer in 0.1 M seedling, but its function(s) remain unclear despite
triethanolamine HCl buffer, pH 7.2, to give a nal extensive study. Amine oxidases in Lens and Pisum
concentration of 20 mg protein/mL. The homogenate are absent in ungerminated seeds, and appear during
is digested with 1 mg and 670 units of phospholipases the early period of germination. The enzyme activity is
C and A, respectively, per 500 mg of the total protein greatest in the growing parts of the plant and decreases
in the presence of 25 mM CaCl2 at 25 C for 1 h. After with maturity and senescence.
centrifugation of the mixture at 43,000 g for 15 min at Plasma copper-containing amine oxidase is soluble,
15 C, the pellet is resuspended in the same buffer to while in most tissues the enzymes appear to be mem-
give a concentration of 15 mg protein/mL, solubilized brane bound, as plasmalemmal enzymes, which may be
with Triton X-100 at a nal concentration of 1 mg capable of metabolizing extracellular amines. A possi-
detergent/3 mg protein, and centrifuged at 43,000 g ble role for the cellular amine oxidase is the regulation
for 15 min at 15 C. For each 8 mL of resulting super- of histamine and polyamine levels, whereas the serum
natant, add 0.5 g of dextran (average Mr 500,00) and proteins have been speculated to control the level of
0.4 g of PEG (average Mr 8000) and centrifuge at circulating biogenic amines such as dopamine and phe-
9500 g for 20 min. nylethylamine. For review articles, see references
This produces a two-phase system. The enzyme in (2027).
the upper layer is precipitated by addition of PEG to
25%, resuspended in 20 mM sodium phosphate buffer, A. Properties as Protein
pH 7.2, solubilized by octylglucoside at 2 mg/mg pro-
tein and subjected to DEAE-Sepharose CL-6B column Copper amine oxidases are homodimers of 7095 kDa
chromatography. The enzyme is eluted with a linear subunits depending on the source (Table 3) (2833).
gradient of 20250 mM potassium phosphate buffer, Each subunit contains one Cu(II) and a quinone cofac-
pH 7.0, containing 20% glycerol (w/v), 0.8% octylglu- tor. The enzyme-bound copper is removable by treat-
coside, 3 mM 2-mercaptoethanol, and 1 mM D- ment with diethyldithiocarbamate at acidic pH.
amphetamine. The buffer should rst be made anaero- Inactive apoprotein recovers its original activity on
bic and anaerobiosis should be maintained in the gra- addition of cupric ion. A quinone cofactor was identi-
dient chambers during the elution. This will result in ed as 2,4,5-trihydroxyphenylalanine quinone (TPQ or
partially puried MAOA. Further purication by TOPA quinone) rst in the bovine serum enzyme.

Table 3 Molecular Properties of Some Cu-Containing Amine Oxidases
Molecular Ref. for Ref. for

weight Ref. cDNA crystal
Enzyme source (subunit) Cu/mol Absorption maxima Yield starting material No. cloning structure
Arthrobacter 167,900 2 480 nm 92 mg/84 g wet weight 28 44 61

(82,250)a A480=280 0:0137 47
Asp. niger 150,000 1 490 (e 5300 M1 cm1 ) 427 mg/105 L culture 29 29
(75,000)
Pea seedling 113,000 2 525 nm (e 1200 M1 cm1 0.68 mg/2 kg seedling 30 48 60
(77,000)
Bovine plasma 180,000 2 480 nm 36 mg/5 L blood 31 46
(90,000)
Bovine lung 400,000 1.84 425 nm (nitrophenyl 0.9 mg/1.5 g 32
(membrane bound) (100,000) hydrazine neutral pH) microsomal protein
Porcine aorta 200,000 456 nm (nitrophenyl 33
(95,000, hydrazine neutral pH)
102,000)
a
Subunit mw.
Subsequently TPQ has been identied biochemically Crystal structures have recently been reported for
and its presence inferred from amino acid sequence several amine oxidases (5862).
homology in copper-containing amine oxidases from
various sources (3440). B. Properties as Enzyme
Amine oxidase cDNA clones have been obtained
from various sources, and the primary structure of The enzyme catalyzes the oxidation by molecular oxy-
the enzyme was deduced from the cDNAs (4152). gen of the primary amino group of di- and polyamines
Sequence alignment of representative amino acid to corresponding amino aldehydes, hydrogen peroxide,
sequences of bacterial, yeast, plant, and mammalian and ammonia. The amino aldehyde products from
amine oxidases suggests separate protein domains putrescine, cadaverine, and spermidine spontaneously
with a carboxyl-terminal domain encoding the cofactor cyclize to 1-pyrroline, 1-piperidine, and 1,5-diaza-
consensus sequence and putative copper-binding sites, bicyclononane, respectively.
and a more divergent amino-terminal domain accom- Substrate specicity varies widely among the dif-
modating differing physiological functions and sub- ferent enzymes (Table 4) (2032, 63, 64). The best
strate specicities of these proteins. There are 33 substrates for all plant enzymes are putrescine and
completely conserved residues mainly within two cadaverine. The relative reaction rates with some sub-
regions: residues 286459 in the pea enzyme, approxi- strates are shown in Table 4. Km values for putrescine
mately centered on Tyr387, and the C-terminal end. seem to be of the same order of magnitude for all
In this enzyme the amino acid sequence of the phe- enzymes investigated (0.1 mM). The pH optimum is
nylhydrazine-labeled peptide was determined to be found around pH 7.0 with putrescine or cadaverine as
VGNXDNVID, in which the X corresponds to substrate. Plasma and tissue amine oxidases also
Tyr387. TOPA quinone is posttranslationally gener- show considerable species-related variations in sub-
ated by oxidation of a specic tyrosine precursor strate specicity. Some endogenously occurring aro-
occurring in the consensus sequence of NYD/E in matic amines such as tyramine and tryptamine are
the active site of amine oxidases (5357). The oxida- metabolized well by copper-containing amine oxidase
tion to TPQ is a spontaneous reaction mediated by in homogenates of rat blood vessels, and also in vitro
the bound copper ion. Numerous studies point inhibition of the enzyme can potentiate vasoconstric-
toward the presence of three histidines as ligands to tor actions of these amines in rat vascular prepara-
copper in the enzyme. One HXH motif 4050 resi- tions. These amines are poor substrates for human
dues toward the carboxyl terminus from the cofactor amine oxidase, thus complicating attempts to general-
consensus sequence is conserved in all alignments. ize possible physiological roles for these enzymes. The

Table 4 Substrate Specicity of Some Cu-Containing Amine Oxidases
Kinetic parameters
Enzyme source Substrate V or kcat Km (mM) Compound not oxidized Ref.
Arthrobactor methylamine 68 (mole/min 0.20 tyramine, spermine, putrescine 28

ethylamine 85 0.15
benzylamine 32 3.76
Yeast (Candida methylamine 2.41 (mmole/min/mg) 0.227 benzylamine 63
boidinii, methylamine ethylamine 5.05 0.77
oxidase) phenylethylamine 0.815 0.11
Asp. niger n-hexylamine 100 (relative rate) spermidine 29
benzylamine 36.0
histamine 42.0
tyramine 109.8
putrescine 6.3
Pea seedling putrescine 165 (sec1 ) 0.19 cyclohexylamine (competitively 30
cadaverine 498 0.065 inhibit, Ki 0:045 mM),
spermidine 193 5.9 diethylamine
Bovine plasma butylamine 0.1 (relative rate) ethylamine, tyramine, tryptamine, 64
amylamine 2.5 serotonin
heptylamine 5.4
benzylamine 6.1 0.025
spermine 10.0
kynuramine 1.0
Bovine lung benzyl amine 170 (sec1 ) 0.05 32
phenylethylamine 62 1.19
methylamine 580 0.21
histamine 110 1.11
endogenously occurring aliphatic amines methylamine preparations of the enzyme (2833). The extinction
and aminoacetone are metabolized in vitro to formal- coefcient at 500 nm is 4100 M1 cm1 and at 278
dehyde and methyglyoxal, respectively, by amine oxi- nm is 246,000 M1 cm1 for highly puried Lens
dase in some animal (including human) tissues enzyme. The aerobic addition of phenylhydrazine
suggesting the possibility of toxicological conse- and semicarbazide to the Lens enzyme is followed by
quences of cellular function (2123, 2527). the formation of a strong absorption band at 445 nm
Inhibitors are common to all these enzymes. Metal (e 64,000 M1 cm), 492 nm (e 8,600 M1 cm1 ),
chelators such as diethyldithiocarbamate, cuprizone, o- and 360 nm (e 34,800 M1 cm1 ), respectively, con-
phenanthroline, 2,2 0 -bipyridil, 8-hydroxyquinoline, comitant with the disappearance of the absorption
and CN compounds inhibit Cu-containing amine oxi- band at 500 nm. In anaerobiosis a stable yellow inter-
dases noncompetitively with different sensitivities. mediate absorbing at 346, 432, and 462 nm is observed
Carbonyl group reagents irreversibly form adducts in the presence of substrates. Under this condition a
with the enzymes with concomitant loss of catalytic Cu(I)-semiquinone state is generated by substrate
activity. The most effective agents are hydrazines reduction, and this intermediate may react directly
(phenylhydrazine and benzylhydrazine), hydrazides with oxygen. The interaction of the Lens enzyme
(semicarbazide, thiosemicarbazide, and phenylsemicar- with primary amines gives rise to a covalent amino
bazide), hydroxylamine, isoniazide, and iproniazid group quinone compound, a quinoketimine, which is
(2027). converted to quinoaldimine. This releases the aldehyde
In addition to the protein absorbance maximum at product and forms a species containing Cu(II) and a
278 nm, the visible spectrum of the enzyme is charac- reduced form of TPQ, aminocatechol, which coexists
terized by a broad absorption peak centered around with the radical species containing Cu(I) and TPQ
500 nm which confers a typical pink color to puried semiquinone. Finally, the semiquinone reacts with

oxygen and restores the oxidized enzyme, liberating homogenate is subjected to controlled heat treatment
ammonia and hydrogen peroxide (24, 65). (48 C, 10 min) followed by centrifugation. To the
supernatant ammonium sulfate is added to 70%
C. Determination of Activity saturation and centrifuged. The precipitate is dissolved
in water, dialyzed, and loaded onto a DEAE-cellulose
The enzyme activity of amine oxidase is measured by column equilibrated with 15 mM potassium phosphate
various methods, including radiochemical, spectropho- buffer, pH 7, and eluted with the same buffer. The
tometrical, and polarographical techniques. When eluate containing the activity is diluted with an equal
necessary, MAO activity is inhibited by preincubation volume of water and applied to an AH-Sepharose 4B
with 1 mM clorgyline or deprenyl. column. The enzyme is eluted with 50 mM potassium
In a radiochemical method (32, 66), the reaction is phosphate buffer, pH 7, with 43% recovery and 880-
carried out at 37 C in a nal volume of 225 L of 50 fold purication; 0.25 mg of the enzyme is obtained
mM potassium phosphate buffer, pH 7.2, with radio- from 900 g of lentil seedling.
labeled substrates (e.g., 20 M [14 C]benzylamine Membrane-bound amine oxidase has recently been
[3 mC/mmol], 100 M [14 C]2-phenylethylamine [2.5 puried from bovine lung microsomes (32). The
mCi/mmol], etc.) and is stopped by the addition of enzyme is solubilized with Triton X-100 and purica-
100 L of 2 M citric acid. Radioactively labeled tion is achieved, in the presence of detergent, by
products are extracted with toluene/ethyl acetate chromatography with Cibacron Blue 3GA-agarose,
(1:1, v/v) containing 0.6% (w/v) 2,5-diphenyloxazole hydroxylapatite, Lens culinaris agarose, Resource Q-
before liquid scintillation counting. In the assay FPLC, and gel ltration on Superdex 200 HR-FPLC.
for the activity toward methylamine (100 M The enzyme was puried 2320-fold from the micro-
[14 C]methylamine [1 mCi/mmol]) as substrate, the reac- somal membranes with an overall yield of 15%.
tion is stopped by cooling the tube in an ice bath and
the reaction product ([14 C]formaldehyde) is separated
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35
Prolyl 4-Hydroxylase
Robert B. Rucker, Ana Samimi, and Jerold A. Last

I. INTRODUCTION substrates are (Pro-Pro-Gly)5 or (Pro-Pro-Gly)10 . In

noncollagenous proteins, such as C1q complement,
A. Chemical Reactions Catalyzed
4-hydroxyproline is found in -Ala-X-4Hyp-Gly- and
-Gly-X-4-Hyp-Ala- sequences. In nonvertebrate col-
Prolyl 4-hydroxylase catalyzes the formation of 4-
lagens, Pro in the sequence, -Pro-Y-Gly-, is the pre-
hydroxyproline residues in collagens and a number of
ferred site of hydroxylation (1), and in plants, Ser-
other proteins in both plants and animals, e.g., acetyl-
Pro-Pro-Pro-Pro- is the preferred sequence, in con-
choline esterase, C1q component of complement, and
trast to the -X-Pro-Gly- sequences in animals (4, 5).
elastin. Equation (1) shows the conversion of L-proline
Polyproline-enriched sequences are common to plant
to L-hydroxyproline.
structural proteins, e.g., the hydroxyproline-rich gly-
coproteins (4). The plant enzyme also recognizes and
B. Chemical Structure of Substrates
hydroxylates substrates with distinguishable second-
ary and tertiary structure (5).
In vertebrate animals, an -X-Pro-Gly- triplet fullls the
minimum sequence requirement for interaction with
prolyl 4-hydroxylase, wherein the Pro residue is hydro- C. Enzyme Commission Designation
xylated (13). For example, the Pro residue in the cen-
tral position of -Gly-Pro-Pro- or -Pro-Gly-Pro is not The systematic name and number for prolyl 4-hydro-
hydroxylated. Moreover, in vertebrate animals, single xylase are 4-proline:2-oxo-glutarate dioxygenase and
polypeptide chains serve as substrates. Useful synthetic EC 1.14.11.2, respectively.
(1)

II. PHYSIOLOGICAL IMPORTANCE molecular weight of 240,000. The cDNA encoding
the enzyme has been characterized from chicken,
The formation of hydroxyproline is necessary for the human, and rat (13). The -subunit from mammalian
intracellular assembly of procollagen polypeptide sources is 64,000 daltons. The -subunit contains the
chains into helical structures. When collagen chains active site, but does not have detectable prolyl hydro-
are underhydroxylated, helical formation is impaired xylase activity unless assembled as the heterotetramer.
and collagen is retained within the cell. As a conse- The -subunit has a molecular weight of 60,000 dal-
quence, underhydroxylation of collagen leads to tons. Although in most animals both the - and -
decreased collagen production (13, 6). In plants, subunits are necessary for activity, prolyl 4-hydroxy-
increased hydroxyproline-rich glycoprotein synthesis lase activity in algae and some plants is structurally
is associated with wound healing and resistance to related to only the -subunit (7).
fungal infections. Impaired hydroxylation impairs the The -subunit is identical to the enzyme protein
ability to respond to infections and wounds, particu- disulde isomerase (811). The -subunits also provide
larly situations wherein encapsulation of a foreign sub- retention and docking signals for prolyl hydroxylase
stance is a requisite (5). within the endoplasmic reticulum (2). In addition,
Wilson et al. (9) have provided data that the -subunit
acts as a chaperone for procollagen chain assembly. Of
III. LOCATION IN TISSUES interest, there is also partial sequence homology
between the -subunit and thyroid hormone-binding
Prolyl 4-hydroxylase is found in the rough endoplas- protein, phospholipase C, thioredoxin, and the estro-
mic reticulum in loose association with the cistern gen-binding domain of the estrogen receptor (2, 3).
inner membrane. Prolyl hydroxylation takes place
while the polypeptide chain is growing on the ribo- B. Genes and Posttranslational Modications
some. In plants, hydroxylation also occurs within the
cistern of the rough endoplasmic reticulum. The The cDNA for prolyl hydroxylase has been cloned and
enzyme is ubiquitous, wherever collagen is found in the active enzyme expressed in insect cells using a bacu-
animals or hydroxyproline-rich glycoproteins are lovirus vector (12). When the - and -subunit cDNAs
found in plants (15). are expressed separately in baculovirus, neither has
prolyl 4-hydroxylase activity, but when they are
IV. COMMERCIAL APPLICATIONS expressed together, prolyl 4-hydroxylase activity is
observed (12). The gene coding for the -subunit
There are few commercial applications for prolyl sequence has been mapped to the human chromosome
hydroxylase per se. However, inhibitors of prolyl 10 (q21.323.1 region), while the -subunit maps to
hydroxylase are considered valuable if they can effec- human chromosome 17 q25, the same chromosome
tively modulate wound healing or abnormal collagen that contains the gene for the type I collagen -chains
deposition. (13, 14, 15).
With regard to postranslational processing, the -
subunit contains 16 residues of mannose, one residue
V. PHYSICAL AND CHEMICAL of galactose, and two residues of N-acetylglucosamine.
PROPERTIES The -subunit contains two residues of mannose and
three residues of galactose (2, 3). Using a cell-free sys-
The descriptions that follow apply mostly to vertebrate tem, John and Bulleid (12) have shown that enzyme
prolyl 4-hydroxylase. For additional descriptions assembly is redox sensitive and ATP dependent. The
regarding plant prolyl 4-hydroxylase or prolyl 3- assembled subunits are intramolecularly crosslinked by
hydroxylase, the reader should refer to references 35 disulde bonds.
and 7, respectively.
VI. ENZYMATIC PROPERTIES
A. Molecular Weight and Structural
Characteristics A. Substrates
The enzyme from human placenta and chick embryos As noted above, the peptides (Pro-Pro-Gly)5 or
exists as a heterotetramer (an 2 2 tetramer) with a (Pro-Pro-Gly)10 are excellent substrates for detection

of prolyl 4-hydroxylase activity. The Km s for these VII. DETERMINATION OF ACTIVITY
substrates are in the 110 M range.
The standard prolyl 4-hydroxylase assay is based on
B. Effect of Environmental Factors the release of tritium from underhydroxylated procol-
lagen -chains (1, 27). Although tedious to prepare and
Mammalian prolyl 4-hydroxylase is often assayed at sometimes variable in consistency, tritiated procolla-
the physiological pH and temperature corresponding gen substrate can be used in assays that are reliable
to that of the source. Activity is lost upon freeze/thaw- in providing good relative values for enzyme activity
ing and concentration. The enzyme is best stored at when appropriately standardized. The procedure for
20 C in ethylene glycol. substrate preparation is largely adapted from
Peterkofsky and DiBlasio (28), Kivirikko and
C. Cofactors and Proposed Mechanism Myllyla (1) and Kivirikko et al. (2). The method may
be used to prepare a [3 H]-L-proline labeled collagen
The reaction mechanism involves the following pro- substrate of high specic activity.
posed sequence of scheme:
A. Substrate Preparation
Phase I Step 1. Chick embryo calvaria (15 days old) are incu-
bated in the presence of [2,3,4,5-3 H]proline (16 Ci/
E Fe2 ! E Fe2 2
mmol [New England Nuclear, MA] in the proline-
E Fe 2
2-OG ! E Fe 2
2-OG 3 free basal medium (Dulbeccos modied Eagles med-
ium [DME], GIBCO, Long Island, NY) with - 0 -
E Fe 2
2-OG O2 ! E Fe 2
2-OG O2 4 dipyridyl to inhibit endogenous hydroxylation of pro-
collagen chains. First, a basal medium is prepared con-
E Fe 2
2-OG O2 Peptide !
taining DME with NaHCO3 (3.7 g/L), penicillin (100
E Fe2 2-OG O2 Peptide 5 units/mL), streptomycin (100 g/mL), and sodium
ascorbate (50 g/mL) added. This medium is adjusted
Phase II to pH 7.6.
E Fe2 2-OG O2 Peptide ! Step 2. From 100 to 200 calvaria should be used.
The calvaria (one per 0.5 mL) are rst incubated in
E Fe2 Succ CO2 Peptide-OH 6 the basal DME medium without isotope addition at
3940 C for 1 h.
E Fe2 Succ CO2 ! EFe2 Succ CO2 7 Step 3. Next, the calvaria are transferred to basal
E Fe2 ! E Fe2 8 DME, but with the following additions: sodium pyru-
vate (1.0 mM), ; 0 -dipyridyl (0:2 M), and L-
[2,3,4,5-3 H]proline at 5 Ci/mL. Culture asks are

Ascorbic acid is essential in keeping both iron and the
ushed with 95% oxygen 5% CO2 , capped, and the
enzyme in a reduced state. Enzyme activity is lost after incubation is continued for 1224 h.
four to six catalytic turns if a strong reducing agent, such Step 4. The calvaria are harvested by decanting
as ascorbic acid, is not present. The Km values for Fe2 , 2- the medium. The decanted medium is saved, centri-
OG, and ascorbate are 2, 5, and 100 M, respectively. 2-OG, fuged (2000 g for 10 min) to remove particulate mate-
2-oxoglutarate; Succ, succinate. rial, and dialyzed against distilled water to remove
unincorporated [3 H]-L-proline. Following dialysis,
the medium is stored (20 C) and saved for Step 5.
Step 5. The decanted calvaria are homogenized in
D. Inhibitors 0.5 M acetic acid (1:5 w/v) using a Polytron (Brinkman
Instruments) and extracted twice (12 h each with agita-
Several classes of inhibitors for prolyl hydroxylase tion at 4 C). Since the dialyzed medium contains col-
have been described, including proline analogs, agents lagen, it is added to the acetic acid extracts. The
that interfere with 2-oxoglutarate or cofactor binding, combination is exhaustively dialyzed against 0.05 M
and peptides containing 5-oxoproline (13). Table 1 Tris, pH 7.4, until < 100 dpm of [3 H]-L-proline is
lists various inhibitors and their modes of action. detected per mL dialysate.

Table 1 Examples of Prolyl 4-Hydroxylase Inhibitors
Inhibitor Description Ref.
Active-site modication
Diethyl pyrocarbonate Modication of active site histidine residues, Ki 10 M 7
Substrate binding
Mimosine IC50 100 M 16, 17
S4682 Ki 150 nM 18
Iron chelation
Pirfenidone (methyl-1-phenyl-2-[1 H]- Can be used in vivo (0.5% of diet, w/w) 19
pyridone)
HOE 077 (2,4-pyridine dicarboxylic Can be used in vivo (200 g/g dry food) 20
acid bis[2-methoxyethyl] amide)
; 0 -dipyridyl and related compounds Classical inhibitor of prolyl and other iron-requiring 21, 22
hydroxylases,
Ki 1 M
2,2 0 -bisbipyridine-5,5 0 -dicarboxylic acid Most potent inhibitor of its type, Ki 50100 nM. Note: 23
2,2 0 -bipyridines lacking a 5-carboxylate are poor inhibitors
2-Oxo-glutarate binding
2,7,8-trihydroxyanthraquinone Competes with 2-oxo-glutarate, Ki 40 nM 24
Oxalylalanine and oxalylglycine Structural analogs of 2-oxoglutarate in which the CH2 25, 26
moiety is replaced by -NH-, Ki 100200 nM
Step 6. Following dialysis, the material is ali- 13 100 mm glass culture tubes, and the reaction is
quoted and frozen until use. Note, however, that the stopped by freezing.
storage of tritiated samples in the frozen state can Step 2. The tritiated water that is released (Step 1)
result in some tritium exchange with water. is collected by vacuum distillation (60 C) and radio-
Additional dialysis prior to use or passage through a activity determined (1). Hughes (27) has described a
Sephadex G-10 column equilibrated in sample buffer simple method for measuring release of tritium that
to remove tritiated water is often necessary. As a qual- may also be considered. A useful variant of this
ity control measure, the collagenous nature of the sub- assay is to use ion-exchange chromatography to sepa-
strate may be assessed by digestion overnight with rate the tritiated substrate from titriated water (28),
puried bacterial collagenase (1). The digested material allowing one to omit the vacuum distillation step and
is divided into two portions: one that was dialyzed the need for specialized equipment (see chapter on
against 0.05 M Tris buffer at pH 7.8 and one that Lysyl Oxidase).
was not dialyzed. Loss of > 80% of the tritium follow- Step 3. Data are expressed as tritium released per
ing collagenase digestion and dialysis indicates that mg protein and tissue weight of the extract or DNA in
most of the labeled material is collagen. the sample. This assay is reliable when at least 10 ng of
prolyl 4-hydroxylase is present in samples. The assay is
also useful for semiquantication, if standardized by
B. Assay for Prolyl Hydroxylase using puried or partially puried prolyl 4-hydroxylase
as a reference. The assay is linear over the range of 5-50
ng of enzyme.
Step 1. The assay reaction mixture (0.5 mL) should
contain 80 M FeSO4 , 0.5 mM 2-oxo-glutarate, 0.1 The same procedures may be used to prepare [14 C]-
mM dithiothreitol, 2.0 mM sodium ascorbate, 1 mg L-proline-labeled collagen as substrate. About 40,000
bovine serum albumin, and 300 units of catalase dis- 50,000 dpm of [14 C]-L-proline-labeled collagen is
solved in 0.05 M Tris-HCl buffer, pH 7.8. The sub- needed per assay. This substrate may be stored for
strate should contain at least 100,000 dpm of tritium. long periods (1016 months), in contrast to the tri-
Prior to its addition, the substrate should be boiled for tiated substrate. [14 C]-L-hydroxyproline formed during
810 min to denature the collagen. Reaction mixtures the enzymatic reaction can be measured following
are incubated at 37 C for 1 h on a rotating platform in hydrolysis (6 N HCl, 1224 h, 98 C) by any procedure

that allows precise and reliable measurement of [14 C] dithiothreitol, and 0.01 M Tris-HCl buffer, pH
as hydroxyproline. adjusted to 7.8).
Prolyl 4-hydroxylase activity can also be assayed by Step 3. A crude enzyme extract is prepared by
the decarboxylation of 2-oxo-[1-14 C]glutarate. In this homogenizing from 200 to 1000 g of tissue in enzyme
case, the assay mixture can be similar to that described buffer (1 M NaC1, 0.1 M glycine, 10 M dithiothrei-
above, except 1 mg/mL (Pro-Pro-Gly)10 is used as sub- tol, and 0.01 M Tris-HCL buffer, pH adjusted to 7.8
strate (1). The reaction is followed by measuring the containing 0.1% Triton X-100). The extract should be
release of 14 CO2 (for additional details see Ref. 29). 1:4 w/v. The homogenization is usually carried out
using a Waring blender. For tendon, skin, aortae,
we have found that rst powdering small pieces
of the tissue (35 g each) in a commercial blender
VIII. PROLYL 4-HYDROXYLASE
(appropriately vented) or with a mortar and pestle
PURIFICATION
in the presence of liquid nitrogen increases the yield
of enzyme.
For purication of prolyl 4-hydroxylase, we use a pro-
Step 4. The homogenate is allowed to stand with
cedure adapted from Kivirikko and Myllyla (1). The
occasional stirring for 15 min and then centrifuged at
procedure is simple and based on the afnity of the
15,000 g for 30 min. This and subsequent steps are
enzyme for poly-L-proline linked to agarose, elution
carried out at 48 C unless stated otherwise. This
with a low-molecular-weight poly-L-proline peptide,
step may be repeated.
and gel ltration. Using this method, prolyl 4-hydro-
Step 5. Solid NH4 2 SO4 is slowly stirred into the
xylase has been isolated from a number of sources, e.g.,
supernatant fraction from Step 4 to a nal concentra-
chick embryos, cultured broblasts, and skin. Steps in
tion of 25%. After centrifugation at 15,000 g for 20
purication are as follows.
min, the pellet is discarded and solid NH4 2 SO4 is
Step 1. Poly-L-proline (MW 50020,000) may be slowly stirred into the supernatant fraction to a nal
obtained from a number of commercial sources. Poly- concentration of 70% saturation. Following centrifu-
L-proline is coupled to agarose by cyanogen bromide gation (15,000 g for 20 min), this pellet is dissolved into
activation. Commercially available forms of activated buffer (see above) and aliquots (corresponding to 50 g
agarose are also available from a number of venders, tissue) are dialyzed to remove (NH4 2 SO4 and then
which simplies this process. Briey, 100 mL of 4% stored at 20 C.
agarose is used (Sepharose 4B, Pharmacia, washed Step 6. When enzyme is needed, an aliquot of the
twice with deionized water). Cyanogen bromide (25 NH4 2 SO4 fraction is thawed and adjusted with buffer
g) is added, and the reaction is allowed to proceed to yield 10 mg of soluble protein/mL. The sample is
for 1520 min at 48 C with stirring. The pH is main- passed through the afnity column at a ow rate of
tained at 11.0 by addition of a saturated solution of about 4060 mL/h, and the column is washed with
NaOH. The mixture is next washed on a Buchner fun- enzyme buffer until the absorbance of the eluate (at
nel using a solution containing 0.14 M NaCl and 0.1 M 230 nm) is 0.05 or less. The enzyme is then eluted
NaHCO3 , pH 9.0. When the pH reaches 9.99.5, the with 20 mL enzyme buffer containing 3 mg poly-L-
coupling reaction is carried out by stirring into the gel proline/mL (MW < 6000. The column may be regen-
1 g of poly-L-proline (MW 30,000). The reaction is erated by washing with 6 M urea and re-equilibration
allowed to proceed overnight and the nal gel product with enzyme buffer.
is washed extensively with water, followed by buffer Step 7. The pooled fractions containing activity
containing 0.1 M NaCl, 0.1 M glycine, 10 mM dithio- are concentrated to 2 mL using an Amicon ultral-
threitol, and 0.01 M Tris-HCl adjusted to pH 7.8. The tration cell.
efciency of coupling can be estimated by taking an Step 8. For further purication, this sample is cen-
aliquot of the gel, subjecting the aliquot to hydrolysis trifuged at 20,000 g for 10 min, and the clear super-
in 6 M HCl (98 C for 16 h), and measuring the total natant is applied to an 8% agarose gel column
proline content by standard procedures for amino acid (BioGel A-1.5m, 200400 mesh, 1:5 90 cm, Bio-
analysis. The poly-L-proline content should be 35 Rad, Richmond, CA). Prior to application of the sam-
mg/mL gel. ple, the column is equilibrated with enzyme buffer.
Step 2. Next, the gel is poured into a column with Following application of the sample, the column is
a bed volume of 4060 mL and equilibrated with eluated with enzyme buffer. Fractions are collected
enzyme buffer (1 M NaCl, 0.1 M glycine, 10 M and their absorbance is monitored (230 nm). The frac-

tions containing activity are pooled and concentrated 8. R Myllyla, V Gunzler, KI Kivirikko, DD Kaska.
by ultraltration. Since poly-L-proline eventually Modication of vertebrate and algal prolyl 4-hydro-
adsorbs to the column, after three or four uses the xylases and vertebrate lysyl hydroxylase by diethyl
column packing should be discarded. pyrocarbonate. Evidence for histidine residues in the
catalytic site of 2-oxoglutarate-coupled dioxygenases.
This procedure is highly specic for prolyl hydroxy- Biochem J 286:923927, 1992.
lase and usually results in a several thousand-fold 9. R Wilson, JF Lees, NJ Bulleid. Protein disulde iso-
purication. merase acts as a molecular chaperone during the
assembly of procollagen. J Biol Chem 273:9637
Note added in proof: Since submission of this
9643, 1998.
chapter, several relevant articles have appeared. 10. RB Freedman, NJ Bulleid, HC Hawkins, JL Paver.
Briey, isoforms of prolyl hydroxylase generated by Role of protein disulde-isomerase in the expression
alternative splicing have been identied in both of native proteins. Biochem Soc Symp 55:167192,
human and mouse (30). Prolyl hydroxylase (I) gene 1989.
is inducible by hypoxia (31), providing an explanation 11. AR Walmsley, MR Batten, U Lad, NJ Bulleid.
for observations made more than 20 years ago that Intracellular retention of procollagen within the endo-
hypoxia increases its enzymatic activity. plasmic reticulum is mediated by prolyl 4-hydroxylase.
J Biol Chem 274:1488414892, 1999.
12. DCA John, NJ Bulleid. Intracellular dissociation and
reassembly of prolyl 4-hydroxylase: the -subunits
REFERENCES associate with the immunoglobulin-heavy-chain bind-
ing protein (BiP) allowing reassembly with the -sub-
1. KI Kivirikko, R Myllyla. Posttranslational enzymes in unit. Biochem J 317:659665, 1996.
the biosynthesis of collagen: intracellular enzymes. 13. T Helaakoski, P Annunen, K Vuori, IA MacNeil, T
Methods Enzymol 82:245304, 1982. Pihlajaniemi, KI Kivirikko. Cloning, baculovirus
2. KI Kivirikko, R Myllyla, T Pihlajaniemi. expression, and characterization of a second mouse
Hydroxylation of proline and lysine residues in col- prolyl 4-hydroxylase alpha-subunit isoform: forma-
lagens and other animal and plant proteins. In: JJ tion of an alpha 2, beta 2 tetramer with the protein
Harding, and MJC Crabbe, eds. Post-Translational disulde-isomerase/beta subunit. Proc Natl Acad Sci
Modications of Proteins. Boca Raton: CRC Press, USA 92:44274431, 1995.
1992, pp 151. 14. L Pajunen, R Myllyla, T Helaakoski, T Pihlajaniemi,
3. NA Guzman, GC Fuller, JE Dixon. Hydroxyproline K Tasanen, M Hoyhtya, K Tryggvason, E Solomon,
containing proteins and their hydroxylations by KI Kivirikko. Assignment of the gene coding for
genetically distinct prolyl hydroxylases. In: WS both the beta-subunit of prolyl 4-hydroxylase and
Adair, RP Mecham, eds. Organization and the enzyme disulde isomerase to human chromo-
Assembly of Plant and Animal Extracellular Matrix. some region 17p11. Cytogen Cell Gen 47:3741,
San Diego: Academic Press, 1990, pp 302348. 1989.
4. AM Showalter, D Rumeau. Molecular biology of the 15. L Pajunen, TA Jones, T Helaakoski, T Pihlajaniemi, E
plant wall hydroxyproline-rich glycoproteins. In: WS Solomon, D Sheer, KI Kivirikko. Assignment of the
Adair, RP Mecham, eds. Organization and Assembly gene coding for the alpha-subunit of prolyl 4-hydro-
of Plant and Animal Extracellular Matrix. San Diego: xylase to human chromosome region 10q21.3-23.1.
Academic Press, 1990, pp 247282. Am J Hum Gen 45:829834, 1989.
5. DG Robinson, M Andreae, Blankenstein. Plant prolyl 16. H Ju, J Hao, S Zhao, IMC Dixon. Antiproliferative
hydroxylase. In WS Adair, RP Mecham, eds. and antibrotic effects of mimosine on adult car-
Organization and Assembly of Plant and Animal diac broblasts. Biochim Biophys Acta 1448:5160,
Extracellular Matrix. San Diego: Academic Press, 1998.
1990, pp 283301. 17. TA McCaffrey, KB Pomerantz, TA Sanborn, AM
6. EG Eleftheriades, AG Ferguson, ML Spragia, AM Spokojny, B Du, MH Park, JE Folk, A Lamberg,
Samarel. Prolyl hydroxylation regulates intracellular KI Kivirikko, DJ Falcone. Specic inhibition of eIF-
procollagen degradation in cultured rat cardiac bro- 5A and collagen hydroxylation by a single agent.
blasts. J Mol Cell Card 27:14591473, 1995. Antiproliferative and brosuppressive effects on
7. K Tryggvason, K Majamaa, J Risteli, KI Kivirikko. smooth muscle cells from human coronary arteries. J
Partial purication and characterization of chick- Clin Invest 95:446455, 1995.
embryo polyl 3-hydroxylase. Biochem J 286:923927, 18. M Bickel, KH Baringhaus, M Gerl, V Gunzler, J
1992. Kanta, L Schmidts, M Stapf, G Tschank, K

Weidmann, U Werner. Selective inhibition of hepatic 25. CJ Cunliffe, TJ Franklin, NJ Hales, GB Hill. Novel
collagen accumulation in experimental liver brosis in inhibitors of prolyl-4-hydroxylase. 3. Inhibition by the
rats by a new prolyl 4-hydroxylase inhibitor. substrate analogue N-oxaloglycine and its derivatives.
Hepatology 28:404411, 1998. J Med Chem 35:26522658, 1992.
19. SN Iyer, SB Margolin, DM Hyde, SN Giri. Lung 26. E Baader, G Tschank, K-H Baringhaus, H Burghard,
brosis is ameliorated by perfenidone fed in diet V Guenzler. Inhibition of prolyl 4-hydroxylase by
after the second dose in a three-dose bleomycin-ham- oxalyl amino acid derivatives in vitro, in isolated
ster model. Exp Lung Res 24:119132, 1998. microsomes and in embryonic chicken tissues.
20. Y Matsumura, I Sakaida, K Uchida, T Kimura, T Biochem J 300:525530, 1994.
Ishihara, K Okita. Prolyl 4-hydroxylase inhibitor 27. WL Hughes. A simple fast micromethod for measur-
(HOE 077) inhibits pig seruminduced rat liver bro- ing enzyme activities which release tritium as tritium
sis by preventing stellate cell activation. J Hepatol water. Anal Biochem 161:529532, 1987.
27:185192, 1997. 28. B Peterkofsky, R DiBlasio. Modication of the tri-
21. RI Dowell, EM Hadley. Novel inhibitors of prolyl-4- tium-release assays for prolyl and lysyl hydroxylases
hydroxylase. J Med Chem 35:800804, 1992. using Dowex-50 columns. Anal Biochem 66:279286,
22. YR Kim, B Peterkofsky. Differential effects of ascor- 1975.
bate depletion and alpha, alpha 0 -dipyridyl treatment 29. P Annumen, P Koivunen, KI Kiviriko. Cloning of the
on the stability, but not on the secretion, of type IV alpha-subunit of prolyl 4-hydroxylase from
collagen in differentiated F9 cells. J Cell Biochem Drosophila and expression and characterization of
67:338352, 1997. the corresponding enzyme tetramer with some unique
23. NJ Hales, JF Beattie. Novel inhibitors of prolyl 4- properties. J Biol Chem 274:67906796, 1999.
hydroxylase. 5. The intriguing structureactivity rela- 30. M Nokelainen, R Nissi, L Kukkola, T Helaakoski,
tionships seen with 2,2 0 -bipyridine and its 5,5 0 -dicar- J Myllyharju. Characterization of the human and
boxylic acid derivatives. J Med Chem 36:38533858, mouse genes for the subunit of type II prolyl
1993. hydroxylase. Eur J Biochem 268:53005309, 2001.
24. TJ Franklin, M Hitchen. Inhibition of collagen 31. Y Takahashi, S Takahashi, Y Shiga, T Yoshimi,
hydroxylation by 2,7,8-trihydroxyanthraquinone in T Miura. Hypoxic induction of prolyl 4-hydroxylase
embryonic-chick tendon cells. Biochem J 261:127 (I) in cultured cells. J Biol Chem 275:1413914146,
130, 1989. 2000.

36
Lysyl Hydroxylase
Ana Samimi, Jerold A. Last, Lucas C. Armstrong, and Robert B. Rucker

I. INTRODUCTION hydroxylase. The tripeptide Lys-Gly-Pro is not hydro-

xylated whereas a tripeptide Ile-Lys-Gly is hydroxy-
lated by lysyl hydroxylase. This indicates a minimum
structural requirement of an X-Lys-Gly triplet for this
Lysyl hydroxylase catalyzes the oxidation of specic
enzyme.
lysine residues in collagen to hydroxylysine. The reac-
Studies with synthetic peptides have shown that the
tion sequence is given in Eq. (1)
amino acid sequences, as well as the peptide chain
R-CH2 -CH2 NH2 ! R-CHOH-CH2 NH2 length and peptide conformations, are critical for
O2 2-oxoglutarate succinate CO2 determining their specicity as substrates. Ile-Lys-
Gly, (Ile-Lys-Gly)2 , and (Ile-Lys-Gly)5 -Phe have been
Lysine (in collagen) Hydroxylysine
shown to have, respectively, Lys Km s of 4, 1, and 0:14
(in collagen) M (2). The conformation of the peptide may regulate
1 the extent of hydroxylation of the peptide. It has been
suggested that a -bend involving the lysine to be
where R HOOC-CH2 2 -NH2 hydroxylated is required for lysyl hydroxylase recogni-
tion (3). Some amino acid sequences around the Y
B. Chemical Structure of Substrates positioned lysine can in effect prevent hydroxylation
of the lysine by exerting a conformational change not
The enzyme recognizes the sequence X-Lys-Gly. In inherently recognizable to the enzyme.
some types of collagen, hydroxylation of lysine resi-
dues occurs at the end of -chains in the short non- C. Enzyme Commission Designation
triple-helical region where there is recognition of tri-
plet sequences, e.g., X-Lys-Ser and X-Lys-Ala. In addi- Lysyl hydroxylase, classied as a member of the keto-
tion to collagens, the lysine in sequences X-Lys-Ser, X- glutarate-dependent dioxygenase family, has the
Lys-Ala, and X-Lys in arginine-rich histone proteins ofcial name of lysine:2-oxoglutarate 5-dioxygenase
can also be hydroxylated (1). Evidence now points to and an EC number of 1.14.11.4.
the possibility that different isozymes of lysyl hydro-
xylase are responsible for catalyzing these varied sub-
strate recognition sequences. Lysine vasopressin, which II. PHYSIOLOGICAL IMPORTANCE
has the structure Cys-Thr-Phe-Gln-Asn-Cys-Pro-Lys-
Gly-NH2 , and lysine-rich histone, which contains X- The conversion of collagen-bound lysine to hydroxy-
Lys-Gly sequences, also act as substrates of lysyl lysine alters the properties of the collagen crosslinks,

e.g., instead of Schiff-base products and simple aldi- and may thereby affect the tensile strength of the
mines, ketoamine derivatives also become possibilities. collagen brils in the tissues involved (14, 15).
Further, hydroxylation provides a site for the O-glyco-
sylation of collagen (4). Both functions are important
determinants of the nal structure and properties of III. LOCATION IN TISSUES
the mature collagen bres. The O-glycosylation results
in the addition of either galactose as a monosaccharide Lysyl hydroxylase is an intracellular enzyme found
or glucosylgalactose as a disaccharide linked to the within the lumen of the rough endoplasmic reticulum,
hydroxylysyl residue(s). where it comes in contact with nascent collagen chains
Nascent collagen -chains, not triple helical col- prior to their folding. It can hydroxylate lysyl residues
lagen, are the substrates for lysyl hydroxylase. on unfolded nascent collagen chains, but has no
Generally nonhelical lysyl residues are preferred sites demonstrable activity on triple helical collagen. This
of hydroxylation over residues occurring in the helical enzyme is ubiquitous, wherever collagen is found;
regions of the collagen chain. However, this property is that is, all organs of the body contain some level of
related to the rate of folding of the collagen molecule; this enzyme. In Northern blot analysis of total RNA or
overhydroxylation of helical lysyl residues occurs poly A mRNA from various adult rat tissues, the
under conditions of delayed or impaired assembly of rat lysyl hydroxylase cDNA probe (the so-called type I
the triple helical form of collagen (57). Lysyl residues lysyl hydroxylase; see below) hybridizes to a 3.2-kb
in denatured (nonhelical) collagen may also be mRNA in all tissues, with highest levels of expression
hydroxylated by the enzyme (8). There are differential in liver, lung, heart, and cartilage.
hydroxylation levels in different collagens; type IV is
relatively heavily hydroxylated as compared, for exam-
ple, with types I or III collagens. In addition, lysyl
residues in the same collagen type may be differentially IV. COMMERCIAL APPLICATIONS
hydroxylated (and glycosylated) in different tissues.
Bone collagen (type I) contains relatively more hydro- There are none at this time. The enzyme has been
xylysine than type I collagen from lung, skin, or liver, notoriously difcult to purify, is sparingly soluble in
and hypertrophied tendon contains relatively more aqueous buffers, and is fairly labile (16). Thus, pure
hydroxylysine than does normal tendon (9). There enzyme is not easily prepared, and has mainly been
are also apparent age-related variations in the level produced by recombinant DNA techniques. Since the
of lysine hydroxylation in collagen (10). Finally, enzyme is a glycoprotein, its cognate DNA has to be
hydroxylysine also occurs in noncollagenous proteins expressed in an eukaryotic vector such as a baculovirus
that have a triple helical collagenlike domain in their system (17, 18) to prepare active enzyme. Inhibitors of
structure, such as C1q (complement component), lysyl hydroxylase such as minoxidil (19) may have
acetyl cholinesterase, and other related proteins (1). future biomedical or clinical applications as preventive
Evidence for the biological importance of col- agents for various brotic disorders, but none has yet
lagen lysine hydroxylation catalyzed by lysyl hydro- been approved for this indication because of their toxi-
xylase comes from patients with Ehlers Danlos city.
Syndrome Type VI, who exhibit thin skin, loose
joints, and ocular disruption. These patients are
decient in collagen hydroxylysine and lysyl hydro- V. PHYSICAL AND CHEMICAL
xylase activity. In several such patients, defects in PROPERTIES
the lysyl hydroxylase gene have been identied (6, 7,
11). Increases in collagen lysine hydroxylation and/ The descriptions that follow apply to vertebrate lysyl
or lysyl hydroxylase activity have been described in hydroxylase.
a number of pathological conditions, including
dimethylnitrosamine-induced liver brosis (12) and A. Molecular Weight
bleomycin-induced pulmonary brosis (13).
Abnormal hydroxylation of lysyl residues causes The enzyme has been puried from human placenta
changes in the ratios of hydroxylysinoleucine and chick embryos, and it chromatographs as a homo-
(HLNL) and dihydroxylysinoleucine (DHLNL), dimer of two identical 85,000-MW -subunits on a gel
two lysine- and/or hydroxylysine-derived crosslinks, ltration column (20).

B. Structural Characteristics human and the chicken lysyl hydroxylases.
Interestingly, Asn163 in the human lysyl hydroxylase
The cDNA encoding the enzyme has been character- has been replaced with a serine at the corresponding
ized from chicken, human, and rat (17, 21, 22). residue 164 in the rat, while Ser176 in the human
Although the sequence does not closely resemble any enzyme has been converted to a consensus glycosyla-
other known protein, there is much homology in the C- tion site asparagine at residue 177 in the rat (1719). It
terminal region between various species. The C-term- is not known which of these sites is glycosylated in the
inal region also contains two histidyl residues that may mature protein.
be conserved between different members of the -keto-
glutarate-dependent dioxygenase family (23).
The lysyl hydroxylase cDNA sequence has little
similarity to the prolyl 4-hydroxylase - or -subunit VI. ENZYMATIC PROPERTIES
cDNA sequences (21, 22). The rat cDNA encodes a
A. Substrates
protein of 728 amino acids, and has an overall homol-
ogy at the protein level of 91% and 77% to the human
Several synthetic peptides have been shown to be good
and chicken lysyl hydroxylases, respectively.
substrates for determining lysyl hydroxylase activity.
Two histidyl residues in the chicken lysyl hydroxy-
In addition to lysine vasopressin and (Ile-Lys-Gly)2
lase and other members of the ketoglutarate-depen-
(as mentioned earlier), Ala-Arg-Gly-Ile-Lys-Gly-Ile-
dent dioxygenase family have been identied as
Arg-Gly-Phe-Ser-Gly, Ala-Arg-Gly-Met-Lys-Gly-His-
possible catalytic sites (11). These histidyl residues
Arg-Gly-(Pro-Pro-Gly)4 and (Pro-Pro-Gly)4 -Ala-Arg-
are conserved in the rat lysyl hydroxylase, and indeed
Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Gly)4 can be
the sequence around these histidyl residues is also per-
used in enzymatic assays. Their respective Km s are
fectly conserved between human and rat. It has been
1.1, 2.0, 0.4, 0.2, and 0:2 M (2).
proposed that two histidyl residues, located 3050
Underhydroxylated protocollagen substrate can
amino acids apart in the primary sequence which are
also be prepared from chick calvaria or chick embryo
conserved in different members of the -ketoglutarate-
tendon (20).
dependent dioxygenase family, may be involved in
cofactor binding (23). Conservation of the histidyl resi-
dues and surrounding residues in the rat lysyl hydro-
xylase supports this hypothesis. Although these B. Effects of Environmental Factors
enzymes can be inactivated by diethylpyrocarbonate,
a histidine reactive reagent, the direct involvement of Vertebrate lysyl hydroxylase is assayed under physio-
these particular residues in cofactor binding has not logical conditions of 37 C and pH 7.8. Activity is lost
been demonstrated (23). by 50% after one freeze-thaw cycle or several days at
4 C. In ethylene glycol, the enzyme is fairly stable and
1. Genes can be scored at 20 C for several months.
There are three known isoforms (from DNA

sequences) arising from different, although closely
C. Proposed Mechanism
related, genes (18, 24). Isoform-1 in the human gene
(PLOD) has been mapped to chromosome 1p36.3-36.2
Lysyl hydroxylase and prolyl-4-hydroxylase are very
(22). Isoform-2 has been mapped in human gene
similar in their substrate and cofactor requirements.
(PLOD2) at 3q23-q24 (25), and isoform-3 has been
Both enzymes are therefore classied as -ketogluta-
mapped to the human gene (PLOD3) at chromosome
rate-dependent dioxygenases, which couple hydroxyla-
7q36 (24).
tion to the conversion of oxygen and -ketoglutarate
to succinate and CO2 . These enzymes also require Fe2
2. Posttranslational Processing
and ascorbate as cofactors. Furthermore, both prolyl
Lysyl hydroxylase is a glycoprotein wherein the aspar- 4-hydroxylase and lysyl hydroxylase require that the
agine-linked carbohydrate units are critical for the hydroxylated amino acid be in the Y position of the
enzymes activity. Four consensus sites for N-linked (X-Y-Gly)n collagen motif.
glycosylation are found in the rat lysyl hydroxylase. The following reaction sequence is involved in the
Three of these are in the same location as in the hydroxylation of lysyl residues by lysyl hydroxylase:

E Fe2 ! E Fe2 2-OG ! E Fe2 2-OG hydroxylase is digested with 18 units of type VII col-
O2 ! E Fe 3
2-OG O2 Peptide lagenase (Sigma, St. Louis, MO) for 24 h at 37 C. The
reaction mixture is fractionated by passage through a
E Fe 2-OG O2 Peptide ! E Fe2
3
Centricon 10 ultraltration unit (Amicon, Beverly,
Succ CO2 Peptide-OH MA). The separated fractions are hydrolyzed in 6 N
E Fe2 Succ CO2 ! E Fe2 Succ CO2 HCl at 110 C overnight. HPLC separation of hydro-
E Fe2 Succ ! E Fe2 Succ xylysine and lysine in hydrolysates is performed by
isocratic reversed-phase HPLC. A Beckman C18
E Fe2 ! E Fe2
Ultrasphere column (0:46 25 cm) is equilibrated
If the enzyme is oxidized it can be reduced by ascor- and run with 23% 1-propanol, 0.3% sodium dodecyl
bic acid and is therefore regenerated to undergo further sulfate, 0.01 M phosphate, pH 2.84, as the elution sol-
catalytic reaction cycles (see reaction below). The Km vent (28). Fractions from the HPLC are collected and
values for Fe2 , 2-OG, and ascorbate using protocol- mixed with an appropriate scintillation cocktail and
lagen as a substrate are: 2, 70, and 200 M, respec- radioactivity determined.
tively (Table 1).
E Fe2 ! E Fe3 Ascorbate ! E Fe2 B. Decarboxylation of 2-Oxo[1-14 C]Glutarate
Dehydroascorbate ! E Fe2
This assay is based on the determination of the stoi-
Dehydroascorbate chiometric release of 14 CO2 from 2-oxo[1-14 C]glutarate
during the enzymatic assay. Synthetic peptides can be
utilized as substrates instead of natural procollagen
D. Inhibitors substrates, which allow for a more rapid and simple
assay (20).
Inhibitors of lysyl hydroxylase can be grouped as given The procedure involves sequential addition of sub-
in Table 2 (1, 4). strate, enzyme, and cofactors (ferrous iron, ascorbate,
and 2-oxo[1-14 C]glutarate), as well as supplements
bovine serum albumin, catalase, and dithiothreitol to
VII. DETERMINATION OF ACTIVITY
the assay mixture, which is maintained at a pH of 7.8
A. Determination of Tritriated Water Release in 0.05 M Tris-HCl buffer. The assay mixture is kept
on ice until the nal compound is added, after which it
Determination of lysyl hydroxylase activity by tritiated is immediately sealed with a rubber stopper containing
water release is conveniently performed by a modica- a 1:5 3:0 cm Whatman lter paper saturated with
tion of the method of Peterkofsky and DiBlasio (27), NCS hung from a wire hook. NCS is a quaternary
as described in detail elsewhere (13). For direct deter- amine that is sold as a proprietary product of
mination of tritiated procollagen substrate in assay Amersham Pharmacia Biotech. Incubation is at 37 C
mixtures, the trichloroacetic acidprecipitable material in a shaker water bath for 3040 min. The vials are
from the enzymatic reaction after addition of lysyl then transferred to an ice bath where they are acidied
Table 1 Recombinant and Native Lysyl Hydroxylase Kinetics
Km Specic activity
-Ketoglutarate O2 Fe2 Procollagen (cpm 3 H/h/

Ascorbate M M M pM mg 105 )
Recombinant
lysyl hydroxylase
(rat) 122 106 ND ND 100 60
Lysyl hydroxylase
(chick embryo) 220 70 ND 2 10 20.2

Table 2 Lysyl Hydroxylase Inhibitors
Inhibitor Description
Interference with Fe2 binding

Zn2 Competes with Fe2 ; 5 m
, 0 -dipyridyl Chelates Fe2
Interference with O2 binding
Zn, Cu superoxide dismutase Dismutation of activated oxygen; 30 m
Nitroblue tetrazolium Scavenges activated oxygen
Interference with 2-oxoglutarate binding
Pyridine 2,4-dicarboxylate Competes with 2-OG; 50 M
Pyridine 2,5-dicarboxylate Competes with 2-OG; 150 M
Some TCA cycle intermediates Compete with 2-OG
Coumalic acid Suicide substrate, 2-OG analog; > 2 mM
Active site interference
Diethyl pyrocarbonate A histidine reactive reagent, > 50% inhibition at 50 M
Mechanism unclear
Minoxidil > 50% inhibition at > 50 M
by injection of an equal volume of KH2 PO4 , pH 5.0, buffer in preparation for collagenagarose afnity
buffer. The reaction is allowed to incubate at room chromatography. The collagenagarose afnity col-
temperature for 30 min, after which the lters are umn is prepared by coupling citrate-soluble calf skin
placed in a toluene-based scintillation cocktail (20). collagen to cyanogen bromide-activated agarose (20).
The entire dialysate is applied to the collagenagarose
column. The enzyme is eluted from the column with
VIII. LYSYL HYDROXYLASE enzyme buffer containing 60% (v/v) ethylene glycol.
PURIFICATION As a nal step, the enzyme fractions containing
activity are concentrated to 23 mL by ultraltration
All steps are carried out at 4 C. Chick embryos (14 using an Amicon PM30 membrane. The concentrated
days old; 50100 embryos) are homogenized (1:4) in sample is centrifuged at 20,000 g for 10 min and the
an enzyme buffer (0.2 M NaCl, 0.1 M glycine, 10 M supernatant fraction is loaded onto an 8% agarose gel
dithiothreitol, 0.02 M Tris-HCl buffer, pH 7.5, con- column (Bio-Gel A 1.5 m, 2004000 mesh, Bio-Rad,
taining 0.1% Triton X 100 and 60% (v/v) glycerol). 1 90 cm). The nal puried enzyme is subsequently
This homogenate is centrifuged at 15,000g for 30 min eluted with enzyme buffer. This process usually results
and then subjected to ammonium sulfate precipitation. in a 5000 to 10,000-fold increase in specic activity
The supernatant is saturated to 17% with solid with a recovery of 26%. The enzyme fractions in ethy-
NH4 2 SO4 and centrifuged at 15,000 g for 20 min, lene glycol are the most stable. There is often a large
the pellet is discarded, and the supernatant is once loss of activity during the gel ltration step (20).
again saturated to 55% with NH4 2 SO4 and centri- The puried enzyme has a molecular weight of 180
fuged at 15,000 g for 20 min. This pellet is dissolved kDa determined by gel ltration column chromatogra-
in the enzyme buffer and dialyzed against two 16-L phy under nondenaturing conditions, while it migrates
volumes of the same buffer supplemented with 0.003 at 85 kDa in polyacrylamide gels in the presence of
M MnCl2 overnight. The dialyzed fraction is adjusted SDS. As noted above, the enzyme is a homodimer
to 20 mg protein/mL and next passed (ow rate, 40 (16). Since the protein expressed by insect cells infected
50 mL/h) through a concanavalin A-agarose afnity with baculovirus containing the lysyl hydroxylase
column (4050 mL of packed gel) equilibrated with cDNA sequence from rat is active with the addition
the dialysis buffer. Lysyl hydroxylase is then competi- of cofactors such as ascorbate, -ketoglutarate, and
tively eluted with methyl -D-glucoside (1 M) dis- ferrous iron (17), it is likely that no other cofactors
solved in the enzyme buffer and ethylene glycol remain to be discovered. That insect cells infected
(40%:60%, v/v). with baculovirus containing the lysyl hydroxylase
The efuent from chromatography on concanavalin cDNA express lysyl hydroxylase with a similar specic
A-agarose is next dialyzed overnight against enzyme activity to that of the chick embryo lysyl hydroxylase

conrms that lysyl hydroxylase does not require Danlos syndrome in two siblings. Genomics 15:399
protein disulde isomerase or any other mammalian 404, 1993.
protein for its activity (Table 2) (17). 7. VT Ha, MK Marshall, LJ Elsas, SR Pinnell, HN
Note added in proof: Since submission of this Yeowell. A patient with Ehlers-Danlos syndrome
type VI is a compound heterozygote for mutations
chapter, several relevant articles have appeared.
in the lysyl hydroxylase gene. J Clin Invest 93:1716
Briey, the lysyl hydroxylase 3 isoform not only cata- 1721, 1994.
lyzes the hydroxylation of lysyl residues on collagen, 8. L Ryhanen, KI Kivirikko. Hydroxylation of lysyl resi-
but the pure enzyme also is a collagen glucosyl- dues in native and denatured protocollagen by proto-
transferase, catalyzing the addition of glucose to collagen lysyl hydroxylase in vitro. Biochim Biophys
galactosyl residues on collagen hydroxylysines (29). Acta 343:129137, 1974.
Thus, LH3 is also galactosylhydroxylysyl glucosyl- 9. JE Gerriets, SL Curwin, JA Last. Tendon hyper-
transferase, EC 2.4.1.66. Neither LH1 nor LH2 possess trophy is associated with increased hydroxylation of
this additional activity. A specic 40 amino acid-long nonhelical lysine residues at two specic cross-linking
sequence in the C-terminal end of lysyl hydroxylase has sites in type I collagen. J Biol Chem 268:2555325560,
been identied that is responsible for its membrane 1993.
10. MJ Barnes, BJ Constable, LF Morton, PM Royce.
binding and localization in the endoplasmic reticulum
Age-related variations in hydroxylation of lysine and
(30). An important role for hydroxylysine and its proline in collagen. Biochem J 139:461468, 1994.
glycosylated forms in collagen may be regulation of 11. J Hyland, L Ala-Kokko, P Royce, B Steinmann, KI
the rate of brillogenesis and determination of the Kivirikko, and R Myllyla. A homozygous stop codon
morphology of the bers produced (31). in the lysyl hydroxylase gene in two siblings with
Ehlers-Danlos syndrome type VI. Nat Genet 2:228
12. J Risteli, KI Kivirikko. Intracellular enzymes of col-
1. KI Kivirikko, R Myllyla, T Pihlajaniemi. lagen biosynthesis in rat liver as a function of age and
Hydroxylation of proline and lysine residues in col- hepatic injury induced by diemthylnitrosamine.
lagens and other animal and plant proteins. In: JJ Biochem J 158:361367, 1976.
Harding, and MJC Crabbe, eds. Post-Translational 13. JA Last, JE Gerriets, LC Armstrong, TR Gelzleichter,
Modications of Proteins. Boca Raton: CRC Press, KM Reiser. Hydroxylation of collagen by lungs of
1992, pp 151. rats administered bleomycin. Am J Respir Cell Mol
2. KI Kivirikko, K Shudo, S Sakakibara, DJ Prockop. Biol 2:543548, 1990.
Studies on protocollagen lysine hydroxylase. 14. KM Reiser, JA Last. Collagen crosslinking in lungs of
Hydroxylation of synthetic peptides and the rats with experimental silicosis. Collagen Rel Res
stoichiometric decarboxylation of -ketoglutarate. 6:313324, 1986.
Biochemistry 11:122129, 1972. 15. KM Reiser, AF Tryka, RC Lindenschmidt, JA Last,
3. K Takahashi, YK Kang, G Nemethy, HA Scheraga. HR Witschi. Changes in collagen cross-linking in
Low-energy conformations of two lysine-containing bleomycin-induced pulmonary brosis. J Biochem
tetrapeptides of collagen: implications for post-trans- Toxicol 1:8391, 1986.
lational lysine hydroxylation. Biopolymers 26:1781 16. TM Turpeenniemi-Hujanen, U Puistola, KI Kivirikko.
1788, 1987. Isolation of lysyl hydroxylase, an enzyme of collagen
4. KI Kivirikko, R Myllyla. The hydroxylation of prolyl synthesis from chick embryos as a homogeneous
and lysyl residues. In: RB Fredman, HC Hawkins, protein. Biochem J 189:247253, 1980.
eds. The Enzymology of Post-Translational 17. LC Armstrong, JA Last. Rat lysyl hydroxylase:
Modication of Proteins, Vol 1. London: Academic molecular cloning, messenger-RNA distribution and
Press, 1980, pp 53104. expression in a baculovirus system. Biochim Biophys
5. A Ihme, T Krieg, A Nerlich, U Feldmann, J Acta 1264:93102, 1995.
Rauterberg, RW Glanville, G Edel, PK Muller. 18. K Passoja, K Rautavuoma, L Ala-Kokko, T
Ehlers-Danlos syndrome type VI: Collagen type Kosonen, KI Kivirikko. Cloning and characterization
specicity of defective lysyl hydroxylation in various of a third human lysyl hydroxylase isoform. Proc Natl
tissues. J Invest Dermatol 83:161165, 1984. Acad Sci USA 95:1048210486, 1998.
6. T Hautala, J Heikkinen, KI Kivirikko, R Myllyla. 19. S Murad, SR Pinnell. Suppression of broblast pro-
A large duplication in the gene for lysyl hydroxy- liferation and lysyl hydroxylase activity by minoxydil.
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the biosynthesis of collagen: intracellular enzymes. FASEB J 3:16091617, 1989.
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hydroxylation: Prolyl 4-hydroxylase, an enzyme with

37
Lysyl Oxidase
Robert B. Rucker, Alyson E. Mitchell, Eskouhie Tchaparian, and Jerold A. Last

I. INTRODUCTION and extracellular forms of the enzyme exist. Although

the mechanism(s) of action remains unclear, there is
A Chemical Reactions Catalyzed
loss of expression of lysyl oxidase in malignant cells
(6). It is speculated that the intracellular form(s) of
Lysyl oxidase is a copper-containing enzyme that cat-
lysyl oxidase targets oncogenic and growth-promoting
alyzes the oxidative deamination of amine substrates
transcription factors as substrates. Two examples are
according to Eq. (1)
the RAS oncogene and IRF-1 transcription factor (7
R-CH2 NH2 O2 H2 O ! R-CHO NH3 9). Moreover, lysyl oxidase is also found in the cell
1 nucleus (10).
H2 O2
Within the extracellular matrix (ECM), lysyl oxi-
dase catalyzes the crosslinking of the collagens and
B. Chemical Structure of Substrates elastin. Developmental processes beyond gastrulation
are blocked when lysyl oxidase is inhibited (1113).
Substrates include simple monoamines, diamines, and/ For the food chemist, a general knowledge of lysyl
or the
-amine function of lysyl and/or hydroxylysyl oxidase function is essential. In general, the location
residues in oligopeptides and proteins that range from and types of cross links in collagen and elastin have
histone (1) to collagen and elastin (2). For polypep- profound inuence on their tensile and elastic proper-
tides, the location and chemical properties of vicinal ties. For example, an increase in the amount, or a
dicarboxylic amino acid residues adjacent to poten- change in the distribution, of crosslinked collagen in
tially oxidizable lysyl residues inuence the efciency edible meats affects rheological properties (4, 14). In
of lysyl oxidation (3). addition, crosslinking renders collagen and elastin less
susceptible to proteolysis (15). Therefore, a decrease in
C. Enzyme Commission Designation lysyl oxidase activity is often associated with increased
proteolysis of collagens and elastin, or altered tensile
Lysyl oxidase is an amine oxidase with the numerical and elastic properties.
designation of EC 1.4.3.13.
III. LOCATION IN TISSUES

II. PHYSIOLOGICAL IMPORTANCE
Lysyl oxidase is found in highest concentrations in
Lysyl oxidase is a key enzyme in the growth and devel- tissues enriched in collagen and elastin, ranging from
opment of all higher animals (4, 5). Both intracellular 20 to 400 g=g tissue (16). For cellular localization

studies, poly- or monoclonal antibodies have been used C. Genes and Posttranslational Processing
against lysyl oxidase or synthetic peptide sequences for
1. Genes
a given form of lysyl oxidase. Methods for detection
range from indirect immunouorescence (17) to immu- The human lysyl oxidase gene resides on chromosome
nocytochemical approaches employing confocal micro- 5 while the mouse homologue is on chromosome 18
scopy (10). Four separate genes have been identied to (22, 23). Three additional lysyl oxidaselike (LoxL)
date that may produce a distinct family of lysyl oxidase genes have also been identied. LoxL 1 is localized
proteins (see below). Most of the comments herein on chromosome 15 in humans (24). Its complete
apply to the forms of lysyl oxidase designated as 1 or sequence is encoded by seven exons distributed
2 (also known as lysyl oxidaselike protein, LoxL). throughout 25kb of genomic DNA. Exons 26 encode
Lysyl oxidase 1 is usually found in tissues containing the region of greatest homology to lysyl oxidase. LoxL
brillar collagens or elastin, whereas lysyl oxidaselike 2 maps to chromosome 8 (19). LoxL 2 (21) shares some
protein is often found in tissues enriched in nonbrillar sequence similarity to lysyl oxidase 1, e.g., 48% in pro-
collagens, i.e., laminar or basement membrane col- tein domains transcribed and translated from exons 2
lagens (2, 18). 6. LoxL 3 is an 87-kDa protein and maps to human
Recently, lysyl oxidaselike 2 and 3 have also been chromosome 2. This form of lysyl oxidase is also novel,
identied as separate genes. LoxL 2 appears to be one since it contains four scavenger receptor cysteine-rich
of the lysyl oxidases that is involved in tumor suppres- domains, which are not present in other known forms
sion and is present in reproductive tissue (1820). of lysyl oxidase.
LoxL 3 has yet to be fully characterized, but it is Northern blot analysis of smooth muscle or bro-
expressed in response to cell senescence (21). blast mRNA often results in two to four mRNA spe-
cies of 5.5, 4.3, 2.4, and 2 kb in size (2) corresponding
to lysyl oxidase and the lysyl oxidaselike proteins.
IV. COMMERCIAL APPLICATIONS
2. Posttranslational Processing
There are no commercial applications for the enzyme
The pre part of pre-prolysyl oxidase is removed by a
at this time. A potential long-range use of genetically
protease that cleaves at the peptide bond between
engineered lysyl oxidase might be crosslinking of col-
Cys21 and Ala22 (Fig. 1), giving prolysyl oxidase.
lagens for gelling purposes.
Prolysyl oxidase is N-glycosylated at two sites (residues
9193 and 138140 in the rat enzyme) in the region of
the propeptide (Fig. 1; Table 1). These residues are lost
V. PHYSICAL AND CHEMICAL
when the propeptide is cleaved by a prolysyl-oxidase
PROPERTIES
peptidase located on the external cell surface (2). For
activation of lysyl oxidase, oxidation of a tyrosyl resi-
The descriptions that follow apply mostly to extracel-
due (Table 1) within the active site of the enzyme to
lular forms of lysyl oxidase.
peptidyl trihydroxyphenylalanine (TOPA) and even-
tually to the quinone, topaquinone (TPQ), or alterna-
A. Molecular Weight
tively dihydroxyphenylalanine (DOPA), is essential.
This cofactor is generated after copper is incorporated
Human lysyl oxidase is rst expressed as a polypeptide
into the apoenzyme. In subsequent steps, a lysine deri-
of 417 amino acid residues (MW 48,000), including a
vative is formedi.e., lysyl tyrosine quinone (Fig. 1)
signal peptide of 21 amino acids (Table 1; Fig. 1). The
(25). This product is formed by the addition of the lysyl
active form of the enzyme has a MW of 30,000.
amine to topaquinone with the lost of water or by the
direct addition to DOPA.
B. Structural Characteristics
The translated primary amino acid sequences from

VI. ENZYMATIC PROPERTIES
human genomic, human, rat, and mouse lysyl oxidase
cDNAs indicate a highly conserved primary structure. A. Substrates
Likewise, computer-predicted secondary structure
appears similar for lysyl oxidases from different When synthetic oligopeptides are used as substrates,
animals (Fig. 1; Table 1). e.g. acetyl-(Gly)n -Lys-(Gly)n -CONH2 (n 15), the

Table 1 Rat Lysyl Oxidase: Amino Acid Sequence and Sites for Potential Posttranslational Modications
1 NH2 -Met-Arg-Phe-Ala-Trp-Thr-Val-Leu-Phe-Leu-Gly-Gln-Leu-Gln-Phe-Cys-Pro-Leu-Leu-Arg-
21 Cys-Als-Pro-Gln-Ala-Pro-Arg-Glu-Pro-Pro-Ala-Ala-Pro-Gly-Ala-Trp-Arg-Gln-Thr-Ile-
41 Gln-Trp-Glu-Asn-Asn-Gly-Gln-Val-Phe-Ser-Leu-Leu-Ser-Leu-Gly-Ala-Gln-Tyr-Gln-Pro-
61 Gln-Arg-Arg-Arg-Asp-Ser-Ser-Ala-Thr-Ala-Pro-Arg-Ala-Asp-Gly-Asn-Ala-Ala-Ala-Gln-
81 Pro-Arg-Thr-Pro-Ile-Leu-Leu-Leu-Arg-Asp-Asn-Arg-Thr-Ala-Ser-Ala-Arg-Ala-Arg-Thr-
101 Pro-Ser-Pro-Ser-Gly-Val-Ala-Ala-Gly-Arg-Pro-Arg-Pro-Ala-Ala-Arg-His-Trp-Phe-Gln-
121 Val-Gly-Phe-Ser-Pro-Ser-Gly-Ala-Gly-Asp-Gly-Ala-Ser-Arg-Arg-Ala-Ala-Asn-Arg-Thr-
141 Ala-Ser-Pro-Gln-Pro-Pro-Gln-Leu-Ser-Asn-Leu-Arg-Pro-Pro-Ser-His-Val-Asp-Arg-Met-
161 Val-Gly-Asp-Asp-Pro-Try-Asn-Pro-Tyr-Lys-Try-Ser-Asp-Asp-Asn-Pro-Tyr-Tyr-Asn-
181 Tyr-Asp-Thr-Tyr-Glu-Arg-Pro-Arg-Ser-Gly-Ser-Arg-His-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-
201 Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-
221 Tyr-Val-Gln-Lys-Met-Ser-Met-Tyr-Asn-Leu-Arg-Cys-Ala-Ala-Glu-Glu-Asn-Cys-Leu-Ala-
241 Ser-Ser-Ala-Tyr-Arg-Ala-Asp-Val-Arg-Asp-Tyr-Asp-His-Arg-Val-Leu-Leu-Arg-Phe-Pro-
261 Gln-Arg-Val-Lys-Asn-Gln-Gly-Thr-Ser-Asp-Phe-Leu-Pro-Ser-Arg-Pro-Arg-Tyr-Ser-Trp-
281 Glu-Trp-His-Ser-Cys-His-Gln-His-Tyr-His-Ser-Met-Asp-Glu-Phe-Ser-His-Tyr-Asp-Leu-
301 Leu-Asp-Ala-Ser-Thr-Gln-Arg-Arg-Val-Ala-Glu-Gly-His-Lys-Ala-Ser-Phe-Cys-Leu-Glu-
321 Asp-Thr-Ser-Cys-Asp-Tyr-Gly-Tyr-His-Arg-Arg-Phe-Ala-Cys-Thr-Ala-His-Thr-Gln-Gly-
341 Leu-Ser-Pro-Gly-Cys-Tyr-Asp-Thr-Tyr-Ala-Ala-Asp-Ile-Asp-Cys-Gln-Trp-Ile-Asp-Ile-
361 Thr-Asp-Val-Gln-Pro-Gly-Asn-Tyr-Ile-Leu-Lys-Val-Ser-Val-Asn-Pro-Ser-Tyr-Leu-Val-
381 Pro-Glu-Ser-Asp-Tyr-Ser-Asn-Asn-Van-Val-Arg-Cys-Glu-Ile-Arg-Tyr-Thr-Gly-His-His-
401 Ala-Tyr-Ala-Scr-Gly-Cys-Thr-Ilc-Scr-Pro-Tyr-COOH
Underlined and highlighted are sites associated with: leader sequence cleavage (Cys-Ala), glycosylations
(Ans-Arg-Thr), prolysyl oxidase peptide cleavage to lysyl oxidase (Gly-Asp-Asp), potential trypsin-like
proteinase cleavage sites (Arg-Arg and Arg-Arg-Arg), the proposed copper binding region
(Trp-His-Ser-Cys-His-Gln-His-Tyr-His-Ser), and the location of the Lys and Tyr associated with lysine
tyrosylquinone formation (39, 40, and Refs. cited).
kcat =Km increases with increasing peptide length. recombinant tropoelastin (MW 70,000, 56 residues
Insertion of a Glu residue immediately preceding the of Lys per 1000 total residues) is 5 M (29).
Lys residue causes an increase in the kcat =Km 10-fold
over that for -Lys-Glu- and about vefold over B. Effect of Environmental Factors
sequences containing only Lys. Replacement of Glu
with Gln increases the Km and lowers kcat =Km . Lysyl oxidase is optimally active at pH 7.88.6 and 50
Changes in kcat =Km also respond to the carbon chain 60 C. No externally added cofactors are required, since
length of the vicinal amino acid at this position. In copper and TPQ are tightly associated with the
particular, decreasing the carbon chain length enzyme.
decreases kcat =Km (26, 27). For simple aliphatic
mono- and particularly diamines, as the carbon chain
C. Proposed Mechanism
length decreases, such compounds become poorer sub-
strates; e.g., ethylenediamine is an irreversible inhibitor
Lysyl oxidase carries out oxidative deaminations by a
of lysyl oxidase (28). The Km s for simple amine sub-
classical Ping-Pong mechanism [Eqs. (2) and (3)]
strates are in the 100 M to mM range (see Ref. 2 and
Refs. cited within). The Km for the oxidation of A RCH2 NH2 BO2 !P RCHO
2
Q H2 O2 and NH3
(3)

Figure 1 Lysyl oxidase processing. (A) Lysyl oxidase is rst synthesized as a 46-kDa pre-proenzyme. In steps associated with
Golgi and post-Golgi processing. N-linked glycosylation, copper binding, and quinone cofactor formation occur (5). A part of
this process is the packaging of lysyl oxidase into vesicles for eventual secretion. At the cell surface, prolysyl oxidase is cleaved to
lysyl oxidase by the same enzyme that carries out procollagen cleavage to collagen. (B) The gure insert shows the structure of
lysyl tyrosine quinone. This cofactor is formed as a product of posttranslational modication following the insertion of copper
into lysyl oxidase. (C) The diagram corresponds to lysyl oxidase mRNA. The size of the coding, 3 0 - and 5 0 -untranslated regions,
and the approximate location of modications that occur in pre-prolysyl oxidase are depicted. Lysyl oxidase mRNA arises from
a gene that is 15 kb.
Shah et al. (30) have studied selected aspects of this al. (30) noted, such stereospecicity and proton
mechanism. To iterate, substrate efciency often exchange uniquely differentiates lysyl oxidase from
decreases with decreased carbon chain length. most of the other semicarbazide-sensitive amine oxi-
Regarding the two half reactions, enzyme reoxidiza- dases.
tion is the most rate-limiting step. 1 H NMR spectro-
scopy of the alcohol that is reductively derived D. Inhibitors
(nonenzymatically) from the aldehyde product of the
lysyl oxidasecatalyzed oxidation of deuterated tyra- The most potent inhibitors are derivatives of semicar-
mine indicates that the pro-S, but not the pro-R, bazides and aminoallylnitriles, e.g., -aminopropioni-
alpha-deuteron is catalytically abstracted. As Shah et trile (BAPN). BAPN administration in vivo promotes

degeneration of arteries, increased friability of the skin, M of -aminopropionitrile/mL assay buffer.
and bone fragility. In direct assays of lysyl oxidase Released tritium is recovered by distillation.
activity, BAPN has a Ki of 1020 M. Other com- Alternatively, the reactions may be stopped by the
pounds that act as pseudo-substrates and/or potential addition of 0.2 mL of 3 M trichloroacetic acid.
inhibitors are the aminoalkylaziridines, homocysteine Released tritium is determined after centrifugation of
thiolactone, selenohomocysteine lactone, and homo- individual samples and passage of the supernatant
serine lactone. Liu et al. (31) showed that the activities fraction through a column of AG50W-X8(H) resin
of plasma amine oxidase and diamine oxidase are only (1 4 cm). After a wash with two to three column
partially inhibited at concentrations of the sulfur or volumes of distilled water, an aliquot of the combined
selenium lactones that fully inhibit lysyl oxidase. fractions is assayed for radioactivity by liquid scintilla-
Homocysteine thiolactone, selenohomocysteine lac- tion spectrometry (33). The data are expressed as
tone, and homoserine lactone are found to be compe- radioactivity released per unit time per weight of tissue
titive, irreversible inhibitors of lysyl oxidase, with Ki or unit of protein or DNA.
values of 20, 8, and 420 M, respectively. The rst-
order rate constants for inactivation (k2 ) of the enzyme
vary from 0.12 to 0.18 to 0.28 min1 for the Se-, thio-, B. Simple Amine Substrates
and O-lactones, respectively.
The assay described by Trackman et al. (34) based on
cadaverine oxidation is a good method for routine
assays. However, the lysyl oxidase preparation must
VII. DETERMINATION OF ACTIVITY
be free of endogenous inhibitors, high levels of cata-
A. Natural Substrates lase, and natural substrates (5). The assay also requires
the equivalent of 12 g of enzyme per assay. The oxi-
The classical substrate for assessing lysyl oxidase activ- dation of cadaverine is measured in a coupled assay
ity is prepared using aortae or calveria from 10- to 16- system utilizing peroxidase to measure the H2 O2
day-old chick embryos (32). Briey, the aortae or cal- formed [see Eq. (1)]. In typical assays, samples (equiva-
veria are incubated in Eagles medium devoid of L- lent to 50200 mg of tissue homogenate) are fragmen-
lysine, but containing 3 H-6-L-lysine or 3 H-4,5-L-lysine ted and ground in liquid nitrogen using a metal mortar
in amounts sufcient to cause 200,000400,000 dpm and pestle. Next, readily soluble protein is extracted
( 36 kBq) of tritium to be incorporated per aorta into 23 mL of phosphate-buffered (10 mM, pH 7.6)
or calveria. Standard procedures are used for the tissue saline for 12 h. The tissue is recovered by centrifuga-
culture (95/5% O2 =CO2 , 3941 C for 1824 h) employ- tion (10,000 g for 30 min), and the pellet is rehomogen-
ing vessels that allow convenient recovery of the tissue. ized into 3 mL of 6 M urea buffered with 100 mM
BAPN is added at 50 g/mL to inhibit endogenous sodium borate at pH 8.2. This homogenate is extracted
lysyl oxidase activity. Usually 35 g fresh tissue (100 at least two times (4 C for 812 h) with agitation, and
200 aortae) is cultured to prepare a single lot of sub- the supernatant fraction is collected (10,000 g for 30
strate. Following incubation, the tissue is homogenized min) and combined.
in saline containing 1 mM L-lysine. The tissue is re- Assays (3 mL total volume) must contain 0.25 mg of
covered by centrifugation (10,000g, 30 min) and sodium homovanillate, 40 g horseradish peroxidase,
repeatedly washed to remove unincorporated radio- urea extract (equivalent to 1 g of lysyl oxidase;
chemically labeled L-lysine. Following a nal wash in usually 0.5 mL urea extract), 3.33 mM cadaverine,
0.01 M HCl, the tissue residue is lyophilized and stored and sodium borate buffer (50 mM, pH 8.2). The uor-
at room temperature in a desiccator. escence resulting from homovanillate oxidation is
In typical assays, tissue extracts containing enzyme monitored continuously at 315 nm excitation and 425
(equivalent of 250 ng or more of lysyl oxidase) are nm emission for 1020 min at 45 C. Production of
added to assay mixtures that contain at least one H2 O2 is usually linear for 2040 min. Lysine-rich pep-
aorta or calveria equivalent of substrate suspended in tides and proteins can also be used as substrates, if nM
0.1 M sodium borate buffer containing 0.15 M NaCl, amounts of potentially oxidizable amine functions are
pH 8.0. The samples are next incubated at 45 C for 2, present in the assay mixture. For example, if resources
4, 6, and 12 h (assay volume 1.52.0 mL). Assays are are available to prepare recombinant proteins, recom-
performed with substrate alone, with enzyme, or with binant tropoelastin could be considered as a substrate
enzyme preincubated for 30 min in the presence of 50 (29).

Although aldehyde production and NH3 can also be phosphate-buffered (10 mM, pH 7.6) saline at 1 : 5 w/v.
measured, the low sensitivity, inconvenience, and This step is repeated two or three times using the tissue
potential of interfering substances, e.g., NH3 asso- pellet recovered following centrifugation (10,000g, 30
ciated with the use of urea (see Sec. VIII), compromise min at 4 C). At the onset of purication, it is impor-
their use for routine measurements or as endpoints for tant to remove endogenous inhibitors and native sub-
kinetic assays. strates that inhibit lysyl oxidase activity. Little lysyl
oxidase activity is lost in these initial washes, since
lysyl oxidase is tightly bound to the ECM (16 and
VIII. PURIFICATION Refs. cited within).
Step 2. The tissue is next homogenized in buffered
The methodological steps that follow may be applied 6 M urea (sodium borate or phosphate, 50 mM, pH
to sources of lysyl oxidase containing > 50 g enzyme/ 7.88.0). Three extractions (1 : 4 w/v) are repeated with
g tissue. In general, lysyl oxidase may be puried to constant agitation or stirring and are usually sufcient
homogeneity by differential extraction procedures and to extract all measurable activity. The enzyme is most
sequential chromatography using columns of collagen, easily concentrated by precipitation using ammonium
elastin-hexylamine-Sepharose, DEAE-cellulose, and/ sulfate. NH4 SO4 is added to the combined urea
or Sephacryl S-200. The end product is usually the extracts to 50% saturation. The resulting precipitate
activated or fully processed form of lysyl oxidase is collected by centrifugation (10,000g, 30 min).
( 30 kDA). Lysyl oxidase from some tissues, e.g., Step 3. The product from Step 2 is next suspended
skin, may copurify with an associated protein, a tyro- in buffered 0.5 M urea to give 20 mg protein per mL.
sine-rich associated matrix protein (35). NH4 2 SO4 is removed (by dialysis against 0.5 M urea
in 0.05 M sodium phosphate or borate buffer, pH 7.8
A. Purication Steps (Table 2) 8.0) or chromatography on columns of Sephadex G-10
(Pharmacia Co., Uppsala, Sweden). Note, however,
Step 1. To prepare connective tissue for subsequent that the presence of some urea is essential in all sub-
homogenization, the tissue is frozen and pulverized in sequent steps to maintain lysyl oxidase in a nonaggre-
liquid nitrogen using a mortar and pestle or a blender gated state. If aggregated, there is loss of activity.
vented to allow the escape of nitrogen. Next, the pul- Step 4. Depending on the goal for the purication,
verized tissue (tendon, skin, aorta) is homogenized in either afnity chromatography or ion-exchange chro-
Table 2 Lysyl Oxidase Puricationa
Volume Total Protein Specic Fold Recovery

Steps (mL) (mg) activityb purication (%)
1. PBS extractionb 150 30006000c NAc

2. Urea extraction 120 500600d 0.80.9 1 100d
3. Concentration (NH4 2 SO4 10 300400 1.11.5 1:3 100
4. Dialysis 25 300400 0.91.1 1:1 100
5. Sephacryl S-200/elastine;f 10 6 6570 7080 9095
or
6. DEAE-cellulose chromatographye;f 50 4060 100150 125190 5060
7. HPLCg 1 23 200 200 4070
a
The summary is based on using 10 g of chick tendon as the enzyme source, which normally contains 200 g=g or more lysyl oxidase.
b
Greater than 8590% of the total protein in tendon is collagen. This amount reects the total protein homogenized into phosphate buffered
saline, pH 7.0 (PBS). All of the extracted protein at this step is discarded.
c
One unit of activity is the production of one nanomol of H2 O2 /min/mg protein.
d
About 15% of the total initial protein is extracted into urea and > 90% of the total enzyme (16). For purposes of estimating indices of
purication and recovery, this step is set at onefold and 100%, respectively.
e
Afnity chromatography often results in > 50-fold purication in a single step and enzyme that is > 20% pure based on assessment using
polyacrylamide gels. DEAE-cellulose chromatography may be useful in separating specic forms of isoforms of lysyl oxidase (see Fig. 2).
f
High-performance liquid chromatography yields a product (following Step 5 or 6) that is suitable for amino acid sequencing.
g
From Ref. 38.

Figure 2 Lysyl oxidase purication. (A) Lysyl oxidase binds to Sephacryl S-200. An elution prole for lysyl oxidase indicates
that the enzyme is released only upon addition of 6 M urea. Further purication can be achieved by reversed-phase HPLC
chromatography using conventional protocols for protein and peptide elution (small insert). (B) Separation of lysyl oxidase
isoforms may be achieved using ion exchange chromatography. The elution of lysyl oxidase from skin, tendon, lung, or aorta
from DEAE-cellulose. Columns result in four distinct isoforms, which are immunologically indistinguishable. The protein is
applied in starting buffer (0.025 M sodium phosphate, pH 7.6, containing 6 M urea). Elution is achieved with a linear gradient of
NaCl (equivalent to increasing the conductivity from 0 to 10 mmho).

matography is useful as a next step. At urea concen- 4. L Knott, AJ Bailey. Collagen cross-links in mineraliz-
trations of < 1:0 M, lysyl oxidase binds tenaciously to ing tissues: a review of their chemistry, function, and
columns of Sephacryl S-200, and elution can only be clinical relevance. Bone 22:181187, 1998.
achieved using buffers containing 46 M urea (see Ref. 5. RB Rucker, T Kosonen, MS Clegg, AE Mitchell, BR
Rucker, JY Uriu-Hare, CL Keen. Copper, lysyl oxi-
36 for additional details). The choice of column size
dase, and extracellular matrix protein cross-linking.
depends on the amount of protein applied. Am J Clin Nutr 67:996S1002S, 1998.
Approximately 3 mL of hydrated gel/1015 mg protein 6. K Kenyon, S Contente, PC Trackman, J Tang, HM
is adequate. Columns composed of Sephacryl S-200 Kagan, RM Friedman. Lysyl oxidase and rrg messen-
and insoluble elastin (mixed 3 : 1 w/v) further improve ger RNA. Science 253:802804, 1991.
resolution and separation from other proteins (Fig. 2). 7. K Kenyon, WS Modi, S Contente, RM Friedman. A
The dialyzed urea extract is loaded onto a Sephacryl S- novel human cDNA with a predicted protein similar
200/insoluble elastin column. The column is washed to lysyl oxidase maps to chromosome 15q24-q25. J
with 35 column volumes of buffered 0.5 M urea Biol Chem 268:1843518437, 1993.
(Step 3). Lysyl oxidase is then eluted with 6 M buffered 8. C Ren, G Yang, TL Timme, TM Wheeler, TC
urea. Columns of collagen and hexylamine-Sepharose, Thompson. Reduced lysyl oxidase messenger RNA
levels in experimental and human prostate cancer.
which are available commercially (Elastin Products, St.
Cancer Res 58:12851290, 1998.
Louis, MO), can also be used instead of Sephacryl. At 9. RS Tan, T Taniguchi, H Harada. Identication of the
this point the enzyme is usually sufciently pure for lysyl oxidase gene as target of the antioncogenic tran-
enzymatic studies, such as dening inhibitor proles. scription factor, IRF-1, and its possible role in tumor
Step 5. If further purication is required, DEAE- suppression. Cancer Res 56:24172421, 1996.
cellulose chromatography or reversed-phase HPLC is 10. W Li, K Nellaiappan, T Strassmaier, T Graham, KM
an option. Prior to or following Sephacryl chromato- Thomas, HM Kagan. Localization and activity of
graphy, selected fractions may be pumped onto col- lysyl oxidase within nuclei of brogenic cells. Proc
umns of DEAE-cellulose (see Fig. 2). Lysyl oxidase Natl Acad Sci USA 94:1281712822, 1997.
often elutes as four chromatographically different iso- 11. E Butler, J Hardin, S Benson. The role of lysyl oxidase
forms, although distinguishing features that give rise to and collagen crosslinking during sea urchin develop-
ment. Exp Cell Res 173:174182, 1987.
such forms have not been fully assessed (37). Reversed-
12. G Wessel, D McClay. Gastrulation in the sea urchin
phase HPLC (Fig. 2) can yield products that are sui- embryo requires the deposition of crosslinked collagen
table for amino acid sequencing (38). with the extracellular matrix. Dev Biol 121:149165,
Step 6. The enzyme is best stored at 4 C in 24 M 1987.
urea solutions at concentrations of 2040 g/mL. 13. A Di Donato, JC Lacal, M Di Duca, M Giampuzzi, G
Freezing or concentration, which results in aggrega- Ghigger, R Gusmano. Microinjection of recombinant
tion, causes rapid loss of activity, i.e., in hours to a lysyl oxidase blocks oncogenic p21-Ha-Ras and pro-
few days (16). gesterone effects on Xenopus laevis oocyte maturation.
FEBS Lett 419:6368, 1997.
14. K Reiser, RJ McCormick, RB Rucker. The enzymatic
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38
Superoxide Dismutase
Hirokazu Hara, Tetsuo Adachi, and Kazuyuki Hirano

Gifu Pharmaceutical University, Gifu, Japan
I. INTRODUCTION SOD has been found in many kinds of animals (verte-

brates and invertebrates), fungi and slime molds, and
Superoxide (O2 ), a free radical generated during the plants, and EC-SOD has been found in mammals and
monovalent metabolic reduction of oxygen, is toxic in pine trees. Fe-SOD and Mn-SOD are found in pro-
to biological systems. The major enzymatic protector karyotes and in eukaryotes. Mn-SOD has been found
against superoxide in the body is superoxide dismutase in mammals, while Fe-SOD has not.
(SOD; EC 1.15.1.1) which disproportionates the super- The cellular localization of Cu,Zn-SOD is different
oxide to hydrogen peroxide (H2 O2 ) and oxygen as fol- from that of EC-SOD. Cu,Zn-SOD is located in the
lows: cytosol and the stroma of chloroplasts, while EC-
SOD SOD is mostly located in the extracellular matrix,
O2 O2 2H ! H2 O2 O2 since it is a secretory protein and has an afnity for
Mitochondria and endoplasmic reticulum have been heparin and heparan sulfate. Mn-SOD is located in
shown to produce superoxide as a consequence of the mitochondrial matrix and the thylakoids of chlor-
autooxidation of electron transport chain components. oplasts. Fe-SOD is found in the cytosol of bacteria and
Other sources of superoxide are cytosolic enzymes such the stroma of chloroplasts.
as xanthine oxidase, and activated neutrophils. Under The contents of Cu,Zn-SOD, Mn-SOD, and EC-
physiological conditions, the nonenzymatic dismuta- SOD were determined in tissues from mammalian spe-
tion of superoxide proceeds with a rate constant of cies (2). Various tissues contain the Cu,Zn-SOD activ-
approximately k 105 M1 sec1 , and the reaction is ity, and Cu,Zn-SOD activities are especially high in
accelerated by a factor of 104 in the presence of SOD. metabolically active organs, such as liver and kidney.
The Mn-SOD content is high in organs with high
respiration, such as liver, kidney, and heart. The tissue
II. DISTRIBUTION OF SOD distribution of EC-SOD is different from those of
other isoforms, and it is present at high levels in
SODs are proteins with metal ions at the active site. lung, thyroid gland, and uterus. EC-SOD is the least
Four isoforms, copper,zinc-SOD (Cu,Zn-SOD), extra- abundant SOD isoform in tissues, and 9099% of the
cellular-SOD (EC-SOD), manganese-SOD (Mn-SOD), EC-SOD in the body is located in the interstitial spaces
and iron-SOD (Fe-SOD), have been isolated so far of tissues. EC-SOD is a major SOD form in extra-
from many species, and the cellular distribution pat- cellular uids such as plasma, lymph, and synovial
tern of these isoforms has been determined (1). Cu,Zn- uid. Recently, it was reported that the blood vessel
SOD and EC-SOD, which are known to contain cop- walls, particularly the walls of the arteries, contain
per and zinc atoms, are found in eukaryotes. Cu,Zn- large amounts of EC-SOD (3).

III. PROPERTIES OF SOD PROTEINS Table 1 Comparison of Human SOD Isoforms
Cu,Zn-SOD Mn-SOD EC-SOD

The characteristics of three SOD isoformsCu,Zn-
SOD, Mn-SOD, and EC-SODare presented in Chromosomal location 21q22.1 6q25.2 4p16.3-q21
Table 1. A comparison of the primary structures of Amino acid length 153 198 222
several Cu,Zn-SODs are presented in Figure 1, and Predicted MW 32 kDa 80 kDa 135 kDa
the sequences of human Cu,Zn-SOD and EC-SOD Subunit structure dimer tetramer tetramer
are shown in Figure 2. Glycosylation
Cu,Zn-SOD is a homodimer in which each subunit Heparin afnity
has one copper and one zinc atom. The molecular
weight of Cu,Zn-SOD is 32,000. The amino acid
sequences of Cu,Zn-SODs have been determined in -120 in human) with an uneven tetrahedral distortion
various species. Human Cu,Zn-SOD consists of 153 from square planar geometry. The geometry of Zn
amino acid residues. The amino acid sequences of var- atom ligands, His61, -69, -78, and Asp81 of bovine
ious mammalian Cu,Zn-SOD show > 80% identity enzymes (His63, -71, -80, and Asp83 in human), is tet-
but are signicantly different from those in yeast rahedral. The cysteine residues forming an intrasubunit
( 50% identity) and spinach ( 50% identity) (Fig. disulde bond Cys55-Cys144 of bovine enzyme (Cys57-
1) (4). The amino acid residues at the active site Cys146 in human) are conserved in the primary struc-
responsible for binding Cu and Zn, and the cysteine ture of Cu,Zn-SODs (Fig. 1).
residues of a disulde bond, are conserved in various Recently a cDNA of EC-SOD was isolated from a
species. The crystal structure analysis of bovine Cu,Zn- human placenta cDNA library and the primary struc-
SOD at 2 A resolutions was reported, and the disulde ture of the enzyme was determined (6). Human EC-
bond, the metal atoms, and the amino acid residues to SOD is a tetrametric glycoprotein containing one cop-
which they are bound were identied (5). Each subunit per and one zinc atom per one subunit. The molecular
of Cu,Zn-SOD is composed primarily of eight antipar- weight of EC-SOD is 135,000. EC-SOD has a puta-
allel -strands that form a attened cylinder, plus three tive signal sequence (18 amino acid residues), and the
external loops. The Cu atom is bound by His44, -46, - mature enzyme of EC-SOD consists of 222 amino acid
61, and -118 of bovine enzymes (His46, -48, -63, and residues. The presence of the signal sequence indicates
Figure 1 Comparison of amino acid sequences of human, rat, bovine, horse, yeast, and spinach Cu,Zn-SODs. Asterisks indicate
identical amino acid residues.

Figure 2 Comparison of amino acid sequences of human Cu,Zn-SOD and human EC-SOD. Asterisks indicate identical amino
acid residues. The bold letter indicates the N-glycosylation site. Cu and Zn indicate amino acid residues binding the metals.
Cysteine residues forming a disulde bond are Cys-57-Cys-146 in Cu,Zn-SOD and Cys-107-Cys-189 in EC-SOD.
that EC-SOD is a secreted protein. The N-terminal Since the central core active site portions of Cu,
region (amino acid residues 195) of the mature Zn-SOD and EC-SOD are conserved, the enzymatic
enzyme shows no homology with other proteins, but properties of the two enzymes are very similar.
the region corresponding to amino acid residues 96 However, the properties of Mn-SOD, which has no
193 shows 50% homology with the carboxyl-term- structural homology of active site with that of Cu,Zn-
inal two-thirds of the sequences of eukaryotic Cu,Zn- SOD and EC-SOD, are signicantly different from
SODs. The amino acid residues binding the Cu and Zn those of the other two enzymes. Both Cu,Zn-SOD
atoms (Cu atom ligands His96, -98, -113, and His163; and EC-SOD are very sensitive to cyanide. The values
Zn atom ligands His113, -121, -124, and Asp127) and of the IC50 of cyanide for human Cu,Zn-SOD and
the cysteines forming the intrasubunit disulde bridge human EC-SOD are 10 M and 3 M, respectively
(Cys107-Cys189) in EC-SOD can all be identied with (10). In contrast, Mn-SOD is insensitive to cyanide
corresponding residues in Cu,Zn-SOD (Fig. 2). The and the enzymatic activity of Mn-SOD is not inhib-
carboxyl-terminal region (194222), including nine ited at 10 mM cyanide (11). All SOD isoforms are
amino acids with a positive charge, confers the afnity inhibited by azide. The values of the IC50 of azide for
of EC-SOD for heparin and heparan sulfate. There is Cu,Zn-SOD, Mn-SOD, and EC-SOD are 21, 20, and
one deduced N-glycosylation site (Asn89). 6.5 mM, respectively (10, 12). Cu,Zn-SOD and EC-
The primary structure and three-dimensional struc- SOD are sensitive to H2 O2 , while Mn-SOD is resis-
ture of Mn-SOD show no homologies with those of tant to it (10, 11). Cu,Zn-SOD and EC-SOD are inac-
Cu,Zn-SOD or EC-SOD (7). tivated at 1 mM H2 O2 with half-times of 9.3 and 6.2
7.1 min, respectively.
IV. PROPERTIES OF ENZYMES

The rate constants of the catalytic reactions of all of DETERMINATION
the SOD isoforms are very similar, with values of
1 109 M1 sec1 1. The enzymatic activities of Several methods for assaying SOD, such as the cyto-
Cu,Zn-SOD and EC-SOD are stable over a wide pH chrome C assay (13), nitro blue tetrazolium (NBT)
range, from pH 5 to 11 (8, 9). On the other hand, Mn- assay (14, 15), nitrite assay (16), KO2 assay (17), and
SOD has a lower pH resistance than other isoforms enzyme-linked immunosorbent assay (ELISA) (18)
(8). SOD is a heat-stable enzyme, and Cu,Zn-SOD have been reported. There are three SOD isozymes in
and EC-SOD are especially stable, up to 708C, while eukaryotes and they have similar specic activity. The
Mn-SOD is stable up to 608C (8, 9). methods for assaying SOD activity (1317) show total

SOD activity. When we want to know the activity of The nal concentrations of the above reagents are 1
one of the SOD isozymes, it is necessary to separate mM DETAPAC, 1 unit/mL catalase, 56 M NBT, and
them with reagents such as NaCN (for Mn-SOD) or 100 M xanthine.
pretreatment of samples with concanavalin A
Sepharose column (for EC-SOD) and/or immobilized 2. Procedure
antibodies (2). It is possible to assay one of the SOD
isozymes exclusively with ELISA, because there are no 1. Mix the following solution for a 20-sample
immunological cross-reactivities among three SOD iso- assay: 13 mL solution A, 0.5 mL catalase solu-
zymes. ELISA is not a method for the assay of SOD tion, 0.5 mL NBT solution, 1.1 mL xanthine
activity, but rather for SOD protein measurement, solution, and 0.9 mL 50 mM potassium phos-
while the other methods (1317) are for the assay of phate buffer, pH 7.8 (to measure total SOD
activity. We describe here the procedure for the NBT activity), or 0.1 M NaCN in the buffer (to mea-
assay and ELISA. sure Mn-SOD activity).
2. Add 800 L of this solution to a cuvette.
3. Add 100 L of standard SOD or sample to the
cuvette.
A. NBT Assay
4. Add 100 L xanthine oxidase solution to the
cuvette.
This assay was originally reported by Beauchamp and
5. Mix and monitor at 560 nm by spectrophot-
Fridovich (14). SOD catalyzes the dismutation reac-
ometer for several minutes.
tion of superoxide generated by the xanthine-xanthine
oxidase reaction. However, since superoxide cannot
easily be detected directly by conventional analytical 3. Comments
tools, this assay utilizes NBT as an indicator of super- Dilute xanthine oxidase with solution A until the
oxide. The SOD activity is dened as the ability to change of the absorbance rate without SOD
inhibit the reduction of NBT caused by superoxide. (blank) is between 0.015 and 0.025/min at 560
One unit of SOD activity is dened as the amount of nm.
protein which gives half-maximal inhibition. The SOD The change of the absorbance rate is recorded for at
activity is usually expressed as units per milligram pro- least 2 min after a good straight line is rst
tein. The following NBT assay was reported by obtained.
Oberley and Spitz (15). Diethylenetriaminepentaacetic To measure the Mn-SOD activity, the xanthine oxi-
acid (DETAPAC) is used as chelator in this assay to dase solution is added after the reaction mixture
suppress interference by metal ions. Catalase is also is incubated in the presence of 5 mM NaCN
necessary for protection of SODs against inactivation (nal concentration) for at least 30 min and no
by H2 O2 formed in the reaction. more than 2 h.
It is necessary that NBT reductase activity and
xanthine oxidase inhibitory activity in tissues
1. Reagents are checked when SOD activities in extracts of
tissues are measured.
50 mM potassium phosphate buffer, pH 7.8
Solution A: 50 mM potassium phosphate buffer, pH
7.8, containing 1.33 mM DETAPAC B. Enzyme-Linked Immunosorbent Assay
Xanthine solution (prepare fresh every week): 1.8
mM xanthine in 50 mM potassium phosphate ELISA is a convenient and sensitive assay for the mea-
buffer, pH 7.8. surement of SODs. ELISAs for each of the SOD iso-
NBT solution (keep in brown bottle): 2.24 mM forms have been developed by various investigators.
NBT in 50 mM potassium phosphate buffer, We describe here the procedure of ELISA for human
pH 7.8 Cu,Zn-SOD in our laboratory (19).
Catalase solution: 40 units/mL catalase in 50 mM
potassium phosphate buffer, pH 7.8
1. Reagents
Xanthine oxidase solution (diluted only at time of
assay): about 102 units/mL xanthine oxidase in Coating buffer: 50 mM sodium carbonate buffer,
solution A pH 9.5, containing 0.02% sodium azide

Washing buffer: 10 mM sodium phosphate buffer, SOD preparation from spinach leaves reported by
pH 7.4, containing 150 mM NaCl, 0.05% Tween Kitagawa et al. (22). All procedures are carried out
20, and 0.02% sodium azide at 4 C. Spinach leaves are homogenized with 0.1 M
Blocking buffer: 10 mM sodium phosphate buffer, potassium phosphate buffer, pH 7.8, using a Waring
pH 7.4, containing 150 mM NaCl, 0.05% Tween blender. The homogenate is forced through cotton
20, 0.02% sodium azide, and 1% bovine serum cloth and centrifuged at 24,000 g by a continuous cen-
albumin (BSA) trifugation technique. The supernatant is precipitated
Substrate solution: 0.1 M diethanolamine hydro- with 4080% saturated ammonium sulfate. The preci-
chloride, pH 9.8, containing 0.5 mM MgCl2 , pitate is dissolved and dialyzed against 10 mM potas-
0.02% sodium azide, and 2.7 mM p-nitrophenyl- sium phosphate buffer, pH 7.8. The dialyzed solution
phosphate is applied to a column of DEAE-Sepharose CL-6B.
5 N NaOH The column is eluted with a linear gradient (1060
Antibody against Cu,Zn-SOD mM) of potassium phosphate, pH 7.8. Fractions with
Alkaline phosphatase-labeled antibody against SOD activity are collected, concentrated, and applied
Cu,Zn-SOD to a column of Sephacryl S-200. The column is eluted
with 20 mM potassium phosphate buffer. Active frac-
2. Procedure tions are collected and concentrated.
1. Add 80 L of 50 g/mL Cu,Zn-SOD antibody
dissolved in coating buffer to each well of a 96- REFERENCES
well immunoplate, and leave the plate to stand
overnight at 4 C. 1. K Asada, S Kanematsu, S Okada, T Hayakawa.
2. Wash each well with washing buffer. Phylogenic distribution of three types of superoxide
3. Add 300 L blocking buffer to block the dismutase in organisms and in cell organelles. In: JV
remaining protein-binding sites, and leave the Bannister, HAO Hill, eds. Chemical and Biochemical
plate to stand at 4 C until use. Remove the Aspects of Superoxide and Superoxide Dismutase.
blocking buffer before use. Amsterdam: Elsevier North-Holland, 1980, pp 136
4. Add 70 L sample or standard diluted with 153.
blocking buffer to the wells, and incubate the 2. SL Marklund. Extracellular superoxide dismutase and
other superoxide dismutase isoenzymes in tissues from
plate for 2 h at room temperature.
nine mammalian species. Biochem J 222:649655,
5. Wash three times with washing buffer.
1984.
6. Add 80 L alkaline phosphatase-labeled 3. P Stralin, K Karlsson, BO Johansson, SL Marklund.
Cu,Zn-SOD antibody diluted with blocking The interstitium of the human arterial wall contains
buffer to each well, and incubate the plate very large amounts of extracellular superoxide dismu-
for 2 h at room temperature. tase. Arterioscler Thromb Vasc Biol 15:20322036,
7. Wash three times with washing buffer. 1995.
8. Add 150 L substrate solution to each well, 4. http://www.genome.ad.jp/
and incubate the plate for 30 min at room 5. JA Tainer, ED Getzoff, KM Beem, JS Richardson,
temperature. DC Richardson. Determination and analysis of the
9. Stop the enzyme reaction by the addition of 2 A structure of copper,zinc superoxide dismutase. J
Mol Biol 160:181217, 1982.
50 L of 5 N NaOH.
6. K Hjalmarsson, SL Marklund, A Engstrom, T
10. Measure the absorbance at 415 nm.
Edlund. Isolation and sequence of complementary
DNA encoding human extracellular superoxide dis-
VI. PURIFICATION mutase. Proc Natl Acad Sci USA 84:63406344, 1987.
7. GEO Borgstahl, HE Parge, MJ Hickey, WF Beyer Jr.,
RA Hallewell, JA Tainer. The structure of human
Since Cu,Zn-SOD was rst isolated from bovine ery-
mitochondrial manganese superoxide dismutase
throcytes in 1969 (13), Cu,Zn-SOD and other isoforms reveals a novel tetrametric interface to two 4-helix
have been puried from various species (2022). bundles. Cell 71:107118, 1992.
Generally, the purication of Cu,Zn-SOD was carried 8. M Sugiura, T Adachi, H Inoue, Y Ito, K Hirano.
out by means of extraction with organic solvent, Purication and properties of two superoxide dismu-
anion-exchange column chromatography, and gel l- tases from human placenta. J Pharm Dyn 4:235244,
tration. We briey describe a procedure for Cu,Zn- 1981.

9. L Tibell, R Aasa, SL Marklund. Spectral and physical 17. SL Marklund. Direct assay with potassium superox-
properties of human extracellular superoxide dismu- ide. In: RA Greenwall, ed. CRC Handbook of
tase: a comparison with CuZn superoxide dismutase. Methods for Oxygen Radical Research. Boca Raton:
Arch Biochem Biophys 304:429433, 1993. CRC Press, 1985, pp 249255.
10. SL Marklund. Properties of extracellular superoxide 18. T Adachi, H Ohta, H Yamada, A Futenma, K Kato,
dismutase from human lung. Biochem J 220:269272, K Hirano. Quantitative analysis of extracellular-
1984. superoxide dismutase in serum and urine by ELISA
11. K Asada, K Yoshikawa, M Takahashi, Y Maeda, K with monoclonal antibody. Clin Chim Acta 212:89
Enmanji. Superoxide dismutase from a blue-green 102, 1992.
alga, Plectonema boryanum. J Biol Chem 250:2801 19. T Adachi, Y Usami, T Kishi, K Hirano, K Hayashi.
2807, 1975. An enzyme immunoassay for cuprozinc superoxide
12. HP Misra, I Fridovich. Inhibition of superoxide dis- dismutase using monoclonal antibody: application
mutase by azide. Arch Biochem Biophys 189:317322, for pharmacokinetic study. J Immunol Methods
1978. 109:93101, 1988.
13. JM McCord, I Fridovich. Superoxide dismutase. J 20. JR Jabusch, DL Farb, DA Kerschensteiner, HF
Biol Chem 244:60496055, 1969. Deutsch. Some sulfhydryl properties and primary
14. C Beauchamp, I Fridovich. Superoxide dismutase: structure of human erythrocyte superoxide dismutase.
improved assays and an assay applicable to acryla- Biochemistry 19:23102316, 1980.
mide gels. Anal Biochem 44:276287, 1971. 21. SL Marklund. Human copper-containing superoxide
15. LW Oberley, DR Spitz. Assay of superoxide dismu- dismutase of high molecular weight. Proc Natl Acad
tase activity in tumor tissue. In: L Packer, ed. Sci USA 79:76347638, 1982.
Methods in Enzymology 105. New York: Academic 22. Y Kitagawa, S Tsunasawa, N Tanaka, Y Katsube, F
Press, 1984, pp 457464. Sakiyama, K Asada. Amino acid sequence of copper,
16. EF Elstner, A Heupel. Inhibition of nitrite formation zinc-superoxide dismutase from spinach leaves. J
from hydroxylammonium chloride: a simple assay for Biochem 99:12891298, 1986.
superoxide dismutase. Anal Biochem 70:616620,
1976.

39
Polyphenol Oxidase
Edna C. Ramrez and John R. Whitaker

Victoria M. Virador
National Institutes of Health, Bethesda, Maryland, U.S.A.
I. INTRODUCTION
Polyphenol oxidase, also known as tyrosinase, pheno- (2)

lase, catechol oxidase, catecholase, o-diphenol oxidase,
monophenol oxidase, and cresolase, was discovered in
mushrooms in 1856 by Schoenbein (1). The problem
with naming the enzyme is that it may act on two
general types of substrates, a monohydroxyphenol
as an o-diphenol oxidase. The monophenol oxidases
(such as p-cresol) to hydroxylate it in the o-position
generally also act as o-diphenol oxidases, often at a
with respect to the original hydroxyl group (monophe-
faster rate (3). Therefore, they are sometimes classied
nol, L-dopa:oxygen oxidoreductase; EC 1.14.18.1 (2)
either as monophenol oxidases, o-diphenol oxidases, or
[Eq. (1)]), and on o-dihydroxyphenols, such as cate-
both depending on substrates used. But not all o-
chol, oxidizing them by removal of the hydrogens of
diphenol oxidases can act as monophenol oxidases
the hydroxyl groups, forming benzoquinones (1,2-ben-
(4, 5). This view is not shared by all researchers in
zenediol:oxygen oxidoreductase; EC 1.10.3.1 (2) [Eq.
this eld. The benzoquinones formed by o-diphenol
(2)]).
oxidases are very reactive nonenzymatically with O2 ,
sulfhydryl compounds, amines, amino acids, and pro-
teins, so a variety of compounds, including melanin of
the skin, are formed with colors including yellow, red,
(1) brown, and black.
A third type of polyphenol oxidase reaction
occurs with the enzyme laccase (benzenediol:oxygen
oxidoreductase EC 1.10.3.2 (2) [Eq. (3)]), acting on p-
dihydroxy compounds, but not exclusively (Chapter
40), to give colored compounds. The laccases are also
In this chapter, the rst enzyme activity will be copper-containing enzymes, but the mechanistic oxida-
referred to as a monophenol oxidase and the second tion pathway differs from that of the o-diphenol oxi-

dases. The two types of enzymes can be distinguished and other leafy vegetables. Black spot development in
by use of p-phenylene diamine or syringaldazine (these shrimp is a major economic problem.
are substrates for laccase only). Heat processing to inactivate polyphenol oxidase,
Salicylhydroxamic acid and cinnamic acid inhibit especially in juices, is standard practice with certain
only o-diphenol oxidases (6), while p-diphenol oxidases fruits and vegetables. Some acceptable compounds
are selectively inhibited by quaternary ammonium such as ascorbic acid, thiol compounds, and sultes
compounds such as cetyl tetra-ammonium bromide may be added to prevent browning of cut fruits and
(CTAB) (7). See also Flurkey et al. (8) and Chapter vegetables (11). Cinnamic, p-coumaric, and ferulic
40 on laccase in this handbook. acids can be added to apple juice (sometimes at
Polyphenol oxidases are found in many plant tissues < 0:01%) to prevent browning due to polyphenol oxi-
(9, 10); in some fungi, especially those that produce dase (20). 4-Hexyl-resorcinol is a safe and effective
brown laments including edible mushrooms (11); inhibitor of enzymatic browning, is used in shrimp
and in many animals, including insects (12), mice processing (21, 22), and prevents apple slice browning
(13), and humans (14). There are numerous genes in (23) among other fruits and vegetables. Browning of
humans that affect pigmentation (15, 16). According to foods can also be prevented by removal of O2 , acidi-
Witkop (14), there are at least 40 clinical manifesta- cation (if acceptability of the food permits), and
tions of hypopigmentation and 27 of hyperpigment- removal of polyphenols by complexing with cyclodex-
ation in humans, some leading to cancerous skin trins and polyvinylpyrrolidone (2426). Polyphenol
melanoma. oxidase levels in fruits and vegetables can be decreased
by breeding and through biotechnology. An example
of the use of biotechnology is the use of a specic
II. IMPORTANCE TO FOOD QUALITY antisense RNA to turn off expression of polyphenolox-
AND FOOD PROCESSING idase in grape tissues (27).
Exclusion or decrease of O2 and separation of poly-
Polyphenol oxidases are very important enzymes in phenol oxidase and substrate (phenols) provide excel-
determining the quality and economics of fruit and lent prevention from browning. Fruits and vegetables
vegetable harvesting, storage, and processing (1719). have skins (waxes and other O2 impermeable com-
Bruises, cuts, and other mechanical damage during pounds) on the surface that prevent O2 absorption. As
harvest, storage, and processing that allow O2 penetra- long as this protective skin is intact and the cellular
tion result in rapid browning in many fruits and vege- tissue is undamaged, browning does not occur. In
tables. Up to one-half of some tropical fruits are lost food processing and packaging O2 can be excluded
for consumer consumption owing to browning, since or reduced by use of gaseous N2 , use of O2 -imperme-
the off-color, off-taste, and loss of nutritional quality able coatings and lms, and controlled atmospheric
are unacceptable to consumers. Apricots, apples, storage (O2 reduced to the minimum needed to main-
peaches, grapes, strawberries, and bananas (among tain cellular integrity at reduced temperature ( 5 C)).
others) and several tropical fruits and juices therefrom Color development due to polyphenol oxidase activ-
become brown, as do Irish potatoes and some lettuces ity is desirable in the processing of tea, coffee, cocoa,

apple cider, prunes, black raisins, Black Mission gs, The amino acid sequence homologies among the
and zapote. nine polyphenol oxidases (Fig. 2) are much higher
when compared only in the active-site regions A and
B (28). In active-site region A, of 26 amino acids, there
III. PROPERTIES OF POLYPHENOL is a range from 100% to 31% homology (Table 2). In
OXIDASES AS PROTEINS active-site region B of 56 amino acids, the homology
A. Primary Sequences ranges from 96% to 34% (Table 3).
Several amino acid sequences of propolyphenol oxi- B. Molecular Weights

dases and polyphenol oxidases are known, primarily
from sequencing the gene. Table 1 shows the similari- The presumed mature molecular weights of the 12
ties of nine plant PPO sequences. Another group of polyphenol oxidases listed in Table 4 range from 30.7
plant PPO sequences are depicted by a dendrogram kDa for S. antibioticus polyphenol oxidase to 128.0
(Fig. 1). kDa for mushroom polyphenol oxidase (with four sub-
Whitaker (28) has compared the amino acid units). The potato, tomato, and broad bean polyphe-
sequence relationships among polyphenol oxidases of nol oxidases have molecular weights in the range of
higher plants, fungi, and higher animals known to 56.558.1 kDa while grape polyphenol oxidases have
1995. The homologies (relative to potato 1) among molecular weights of 40.7 kDa. Three of the fungi
the higher plant polyphenol oxidase gene sequences polyphenol oxidases have molecular weights of 30.9
from potato 1, potato 2, tomato, and broad bean 46.0 kDa, while mushroom polyphenol oxidase is 128.0
were 96.6% (potato 1 and 2), 92.2% (potato 1 and kDa. The human and mouse polyphenol oxidases are
tomato), and 38.1% (potato 1 and broad bean). 62.6 and 57.8 kDa, respectively. With the exception of
Among the fungi S. glaucescens, S. antibioticus, and the polyphenol oxidases from N. crassa, S. glaucescens,
N. crassa, the homolgies were 87.5% (S. glaucescens and edible mushroom (Agaricus bisporus) (29, 30), the
with S. antibioticus) and 17.0% (S. glaucescens and molecular weights are based on the gene sequence
N. crassa). Comparison between human and mouse translated to amino acid sequence. Nothing is known
polyphenol oxidases showed 41.0% homology. about posttranslational modications.
Overall, when potato 1 sequence was compared with
those of S. glaucescens, S. antibioticus, N. crassa, and C. Secondary, Tertiary, and Quaternary
human and mouse polyphenol oxidases, there was Structure
19.4%, 18.8%, 13.0%, 10.4%, and 10.9% homology,
respectively. Only the primary sequences of N. crassa X-ray crystallography data have been published only
and S. glaucescens polyphenol oxidases were deter- for the sweet potato polyphenol oxidase (31, 32).
mined by the Edman degradation method on the There are two isozymes of polyphenol oxidase in
mature protein. sweet potatoes of molecular masses 39.0 and 40.0
Table 1 Similarity of the Aligned Plant PPO Sequences
Grenache Sultana
Tomato Tobacco grape grape Apple Bean Pokeweed Spinach Sugarcane
Tomato 100 90.48 59.52 60.03 56.97 56.8 55.2 53.74 43.2
Tobacco 100 58.01 58.52 56.49 56.83 53.83 51.43 43.68
Grenache grape 100 99.18 67.17 64.42 64.22 57.83 43.16
Sultana grape 100 67.34 64.42 64.4 57.73 43.09
Apple 100 69.7 63.03 54.88 45.29
Bean 100 62.69 56.84 43
Pokeweed 100 57.58 43.44
Spinach 100 40.42
Sugarcane 100
The GCG program OldDistances was used to calculate percent similarity among the plant PPOs from a complete PileUp alignment; part of this
alignment is depicted in Figure 1. Genbank accession numbers are as follows: tomato, Z12837 S61013; tobacco, Y12501; Grenache grape,
U83274; Sultana grape, Z27411; apple, D87670; bean, Z11702 S45506; pokeweed, D45385; spinach, X90869; sugarcane, U46014.

protein. There are two disulde bridges (Cys11Cys28
and Cys27Cys89). Each of the two active-site coppers
is coordinated to three histidine residues on -helices.
Copper of site A is coordinated to His88 of helix 2
and His109 and His118 of helix 3. The copper of site
B is coordinated to His240 and His244 of helix 6 and
His274 of helix 7.
As noted above, mushroom tyrosinase is thought to
have quaternary structure (29, 30), probably composed
of four subunits. The similarity of the subunits is
uncertain. Based on gene sequences, there appear to
be at least two isozymes of tyrosinase in mushrooms
(33, 34).
IV. ENZYMATIC PROPERTIES OF

POLYPHENOL OXIDASES
Some polyphenol oxidases oxidize both monophenols

such as p-cresol, tyrosine etc. (monophenols), and
diphenols such as catechol and o-dihydroxyphenylala-
nine [Eqs. (1) and (2)]. Lerch and Ettlinger (3) investi-
gated the activity of pure Streptomyces glaucescans
polyphenol oxidase (tyrosinase) on a large number of
mono- and o-diphenols. In all cases, the o-diphenolase
activity was greater than the monophenolase activity.
The ratio of kcat for activity on o-diphenol to that of
kcat for activity on the analogous monophenol ranged
from 222 for homocatechol/p-cresol to 2.89 for
3,4-dihydroxyphenylacetic acid/p-hydroxyphenylacetic
acid. The Km values for the analogous o-diphenol to
monophenol ranged from 1.00 to 7.88. Therefore, Km
and Kcat are both responsible for the large differences
in activity on mono- and o-diphenols.
Figure 1 Dendrogram illustrating the degree of similarity Table 5 shows some activity results of plant poly-
between the protein sequences of reported plant PPO phenol oxidases on a limited number of mono- and o-
genes. The Genbank accession numbers are apple (L29450), diphenols. Polyphenol oxidase from peaches was not
bean (Z11702), grape (Z27411), potato-1 (M95196), potato-2 able to hydroxylate p-cresol or p-coumaric acid, and
(U22921), tomato-a (Z12833), tomato-b (Z12834), tomato-c polyphenol oxidase from pears was not able to hydro-
(Z12834), tomato-d (Z12836), tomato-e (Z12837), tomato-f xylate p-coumaric acid. The highest activities on mono-
(Z12838), pokeweed (D34385), spinach (Z6655), and sugar- phenols were the 5.5% and 4% on p-cresol compared
cane (U846014). Sequences were aligned using the GCG pro- to that of catechol for potato and broad bean, respec-
grams PILEUP and FIGURE.
tively. Note also the variable relative activities on o-
diphenols compared to that on catechol.
kDa (determined by MALDI-MS) (20). The sweet There is some disagreement by researchers on
potato polyphenol oxidase molecular weight of 39.0 whether or not all polyphenol oxidases can perform
kDa has 345 amino acid residues, is a monomeric, the hydroxylation step [Eq. (1)]. As shown in Eq. (1)
ellipsoid molecule with dimensions of 55 45 45A. a reducing compound, BH2 , is required in the reaction.
The secondary structure is primarily -helical in nat- BH2 is an o-diphenol such as catechol. If no BH2 is
ure, with seven -helices and probably four short - present, some polyphenol oxidases appear to be able to
sheets with - and - turns and random coils in the slowly produce BH2 during a lag period in the initial
structure (Fig. 3). It appears to be an globular reaction with a monophenol (Fig. 4). The in vivo nat-

Figure 2 Amino acid sequence relationships in and near the active sites of potato (Pot 1 and Pot 2), tomato (Tom A and Tom
B), broad bean (BB), S. glaucescens (S.g.), S. antibioticus (S.a.), Neurospora crass (N.c.), Homo sapiens (H.s.), Mus musculus
(M.m.), putative mouse transcript 4 (pMT 4) polyphenol oxidases, hemocyanin E (spider, Eurypelma californicum) and hemo-
cyanin D (spider, E. californicum). Shown are regions A and B containing the presumed copper-binding histidine residues. The
Cu indicates histidines that ligand to copper in N. crassa polyphenol oxidase, and presumably in the other polyphenol oxidases
and hemocyanins. The - indicates that the sequence is the same as for POT1. (From Ref. 28.)
ure of BH2 is not known. Lerch and Ettlinger (3) Kinetically, the mechanism of peach polyphenol
reported lag periods of 0.3216.0 min for S. glauces- oxidase (5) and S. glaucescens polyphenol oxidase (3)
cens polyphenol oxidase acting on N-acetyl-L-tyrosine followed an ordered BiBi mechanism (Fig. 5). The O2
hydrazide and p-hydroxyphenylacetic acid, respec- must bind rst to the deoxy form (Cu(I) state) of the
tively. Whitaker (unpublished data) showed that 1 enzyme followed by phenol. The ordered BiBi mechan-
107 M 4-methylcatechol added to mushroom poly- ism assumes that the products come off in the order of
phenol oxidase, along with p-cresol, was able to elim- the benzoquinone followed by the hydrogen peroxide.
inate the 9-min lag period entirely. Equation (4) shows This author does not know of published experimental
that the BH2 acts on the Cu(II) met form to reduce the data to support this assumption.
two coppers to Cu(I) (deoxy form), which can then The overall proposed mechanism of action of poly-
bind O2 and oxidize the monophenol substrate to the phenol oxidase is shown in Figure 6. The top part
o-diphenol. Would similar experiments with other shows the pathway for oxidation of o-diphenols. The
polyphenol oxidases show that all polyphenol oxidases met form (at the No. 1 position in the A cycle) is
can do both types of reactions? thought to be the resting form of the enzyme when

Table 2 Amino Acid Sequence Homologies Among Nine Table 4 Mature Protein Molecular
Polyphenol Oxidases in Active Site Region A Weights of Some Polyphenol Oxidases
AA sequence Identical % Source MW (kDa)

Source region AA Homology
Pot 1 56.5
Pot 1 110135 26a 100 Pot 2 56.6
Pot 2 110135 26 100 Tom 56.5
Tom B 110135 26 100 BB 58.1
BB 110135 25 96 GPO1 40.7
S.g. 5476 13 50 GPO2 40.7 (29.0)a
S.a. 5477 11 42 S.g. 30.9
N.c. 96121 11 42 S.a. 30.7
H.s. 190215 8 31 N.c. 46.0
M.m. 191216 8 31 Mushroom 128.0
a
H.s. 62.6
The number 26 amino acid residues includes amino acid M.m. 57.9
residues spaces for alignment. (Taken from Figure 2.)
a
There are two peaks of polyphenol oxidase
activity from a Sephadex G-100 column
no substrate is present. Addition of o-diphenol (cate- (40.7 and 29.0 MW). The 40.7-kDa peak
chol; BH2 ) binds to the met Cu(II) form (#2) produ- has the most activity.
cing the deoxy [Cu(I)] form of the enzyme (and o- Source: Whitaker, 1998, unpublished data.
benzoquinone). The deoxy form binds O2 (#3) to give
the oxy form [Cu(II)] which then binds a molecule of o- hydroxylamine, dithionite) and by H2 O2 in the pre-
diphenol (#4) to give the enzyme CuII O2 diphenol sence of O2 (39). The overall mechanism indicates
ternary complex. Two hydrogens are removed to give there are three different steps in Figure 6 leading to
the benzoquinone (#5) and the met form of the enzyme oxidation of o-diphenols to benzoquinone.
in a complete cycle. Polyphenol oxidases are irreversibly inactivated
Depending on substrate available, the oxy form of during the oxidation of substrate to product. Golan-
the enzyme (produced as above from the met form) can Goldhirsh and Whitaker (40) calculated that inactiva-
bind a monophenol (Pathway B) (#1 0 ) which is oxi- tion of mushroom polyphenol oxidase occurs at the
dized to the o-diphenol (#20 ) and recycles through the rate of approximately one in 5000 turnovers of the
B pathway via the diphenol intermediate to the deoxy substrate to product (an efciency of 0:02%). But
form (#40 ) which can bind O2 to form the oxy form complete inactivation of the enzyme can occur in 23
(#50 ), etc. The met form, reduced to the deoxy form by min reaction with substrate (with no added reductant
an o-diphenol (Fig. 6), can also be reduced to the deoxy such as ascorbic acid). The inactivation is due to a free
form by other reducing compounds (ascorbic acid, radical-catalyzed fragmentation of one or more of the
six histidine residues that bind the two coppers at the
active site. The fragmentation leads to loss of histidine
Table 3 Amino Acid Sequence Homologies Among Nine and release of copper (4042). Reaction inactivation of
Polyphenol Oxidases in Active Site Region B N. crassa polyphenol oxidase is due to loss of His306 in
the active site (42).
AA sequence Identical %
Source region No. Homology Golan-Goldhirsh et al. (43) later showed that ascor-
bic acid, copper, and O2 mimic the above reaction not
Pot 1 241296 56a 100 only with polyphenol oxidase but also with ovalbumin,
Pot 2 241296 54 96 Kunitz trypsin inhibitor, bovine serum albumin, and
Tom B 241296 54 96 small histidine-containing peptides, leading to loss of
BB 241295 35 62 most of the histidine residues in 24 h at 25 C. In the
S.g. 189229 20 36 case of mushroom polyphenol oxidase, the histidine
S.a. 189229 19 34
residues of the protein are converted by the free radical
N.c. 277320 20 36
process in the absence of mono- or diphenols, but
H.s. 350390 19 34
M.m. 352393 21 38 require O2 , to several products including aspartic
acid (major product), glycine, alanine, and urea (stoi-
a
The No. 56 includes amino acid residues spaces for alignment. chiometric with the aspartic acid formation). Free radi-

Figure 3 Ribbon drawing of sweet potato catechol oxidase, showing the front view of 39.0-kDa single polypeptide enzyme.
(From Ref. 32.)
Table 5 Relative Substrate Specicities of Four Polyphenol Oxidases
Activity relative to catechol
Broad bean
Substrate Potatoa Peachb leafc Peard
Di- or triphenolic compounds

Catechol 100 100 100 100
4-Methylcatechol 51.5 200225 72.3
d-Catechin 31.8 7.79
Chlorogenic acid 140 22.2 8 71.8
Caffeic acid 76.5 0 12.5 4.41
Protocatechuic acid 16.3 0.11
3,4-Dihydroxy-L-phenylalanine 54.3 40.5 50
Dopamine 45.6 15.6
Gallic acid 25.7 0.22
Pyrogallol 8595
Monophenolic compounds
p-Cresol 5.5 0 4
p-Coumaric acid nil 0 0.05 0
a
Ref. 35; pH 7.0.
b
Ref. 4. For isozyme A of clingstone peach at pH 6.8 and 30 C.
c
Ref. 36.
d
Ref. 5; 35 C and pH 6.2; isozyme B.

Figure 4 Theoretical graph for the velocity of product for-
mation from oxidation of monophenols by polyphenol oxi-
dases in the absence of BH2 . There is an initial lag period
followed by maximum velocity and then slowing of the velo- Figure 5 (Top) Effect of O2 and chlorogenic acid concen-
city as the enzyme undergoes reaction inactivation. The nega- trations on initial velocity, v0 , of pear polyphenol oxidase-B-
tive velocity phase is due to melanin formation, which catalyzed reactions. The reactions were followed with an oxy-
absorbs at a different wavelength. gen electrode at pH 4.0 and 30:0 C, permitting initial v0 to be
determined. (Bottom) Kinetic mechanism for pear polyphe-
nol oxidase-B catalysis, diagrammed according to the
Cleland nomenclature for an Ordered Sequential Bi-Bi
cals due to semiquinone formation of the substrate can Mechanism. P is an o-diphenol; B is an o-benzoquinone;
be detected during polyphenol oxidasecatalyzed reac- E O2 is a binary enzyme O2 complex; E O2 P is a ternary
tions (44, 45; Sugumara et al., cited in 46) in insect enzyme O2 o-diphenol complex, and E H2 O B is a ternary
cuticle formation by polyphenol oxidase. Free radical enzyme H2 Oo-benzoquinone complex. (From Ref. 5.)
formation (11) requires O2 , a reducing agent (we used
ascorbic acid only), a histidine residue (histidine in
small peptides are also fragmented), and Cu(II) (we
could not detect Cu(I) formation by bathocuproine
disulfonate in the reaction). The specic free radical V. MEASUREMENT OF POLYPHENOL
formed has not been identied yet, to the authors OXIDASE ACTIVITY
knowledge.
The two main methods of measuring polyphenol oxi-
dase activity are spectrophotometry and polarography.
There are strong advocates of each method, including
statements that both methods are unreliable (47, 48).
The basis of the unreliability is caused by the further
nonenzymatic oxidation of the benzoquinone formed.
As shown by Mayer et al. (47), results from the two are
identical very early on in the reaction (Table 6), but by
54 sec the difference is 28%. Note that O2 consump-
(4) tion measurements contribute primarily to the differ-
ence because of the continued nonenzymatic oxidation
of benzoquinone and other intermediate products. For
example, the oxidation of 4-methyl catechol to 4-
methyl-2,3-benzoquinone requires 1.0 equivalent of
O2 , while further reactions (generally nonenzymatic)
require 1.4 additional equivalents of O2 . But, as
shown by Whitaker (28), there is also a decrease in

Figure 6 Proposed kinetic scheme depicting the mechanisms of oxidation of o-diphenol (catechol [A]) and monophenol (phenol
[B]) by Neurospora crassa polyphenol oxidase. (From Refs. 37, 38.)
absorbance after 2 min as the benzoquinone is con- In the measurement of monophenol activities, the
verted to other products that do not absorb maximally rate of conversion of the intermediate product (an o-
at the wavelength of benzoquinones. Therefore, it is diphenol) to the benzoquinone is generally measured.
critical to obtain initial velocity (v0 ) early on in the But in some cases, kcat values for the monophenol and
reaction. As shown by Eqs. (1) and (2), the oxidation product o-diphenol can be very similar (3). Examples
of monophenols to benzoquinone plus H2 O requires are shown in Table 7.
two equivalents of oxygen, while oxidation of o-di- The monophenol oxidation can be measured
phenols to benzoquinone requires one equivalent of uniquely by use of 18 O-labeled O2 or tritium-labeled
oxygen. substrate.
In reactions involving monophenols, a lag phase is
generally observed (see Fig. 4). As shown in Eq. (1) A. Measurement of Monophenolase Activity
and Figure 6, BH2 is required to reduce the met form
1. Use of Tritium-Labeled Substrate
of polyphenol oxidase to the deoxy form that can bind
O2 . Addition of as little as 1 107 M catechol to the As shown in Eq. (1), one atom of oxygen (from O2 ) is
reaction can eliminate the lag period. covalently attached to the 2 position of p-cresol, along

Table 6 Comparison of the Spectrophotometric and label. The unincorporated 18 O2 can be ushed from the
Polarographic (O2 electrode) Methods of Measuring Apple solution by N2 , or the water can be distilled from the
Catechol Oxidase Using 4-Methyl Catechol as Substrate solids, and the water collected to measure the increase
(1)a (2)b in 18 O level of the water, or the 4-methyl catechol, in
Time Quinone Formed O2 consumption Ratioc the presence of a reducing agent such as ascorbic acid,
(sec) (mol) (mol) (2)/(1) can be obtained. The increase in 18 O in the product will
give a measure of the velocity of the monophenol oxi-
18 0.256 0.261 1.02 dase reaction.
54 0.512 0.653 1.28
93.6 0.768 1.133 1.48
198 1.024 1.568 1.53 3. Spectrophometric Method at 280 nm
480 1.408 2.464 1.75
An easier method than those in 1 and 2 above is to
a
Measured spectrophotometrically. measure the increase in absorbance at 280 nm due to
b
Measured polarographically.
c the formation of 4-methyl catechol from p-cresol.
o-Benzoquinone formation from 4-methylcatechol consumes 1
gram-atom of O2 per mole of catechol, whereas conversion of 4- Again, the measurements must be made within the
methylcatechol to melanin consumes 2.4 gram-atoms O2 per mole rst 3090 sec of beginning of the reaction.
of catechol oxidized.
Source: Ref. 47.
4. Use of Diphenol Conversion Rates to
with a H to give 4-methyl catechol. By the use of the o-Benzoquinone
2-tritiated p-cresol, tritium will be released into the
Km values for monophenols are in the range of 4:5
aqueous phase. During the initial phase of the reac-
105 M (N-chloroacetyl-L-tyrosine ester ester) to 5:34
tion (between 0 and 60 sec), the rate of release of
103 M (glycyl-L-tyrosine) for the S. glaucescens
tritium should give a good measure of the initial velo-
polyphenol oxidase (3) and 2:62 104 M and 4:62
city of the hydroxylation step. The remaining titriated
104 M for L-tyrosine methyl ester for the - and -
p-cresol must be removed by chromatography or by
isozymes of mushroom polyphenol oxidase (30).
an adsorption method.
When the Km and kcat values of the monophenol and
18 o-diphenol substrates are similar, the observed rates of
2. Use of O-Labeled O2
the reactions of benzoquinone formation will be con-
As shown by Eq. (1), one atom of oxygen (from 18 O2 ) tributed by both rate constants (v0 k2 E0 S0 =
will be covalently attached to the 2 position of p-cresol, k2 k3 =k2 k3 S0 .
along with a H to give 4-methyl catechol with an 18 O See discussion below for methodology to use.
Table 7 Ratios of kcat =Km and kcat =kcat , in Parentheses, for Some Monophenol/o-Diphenol
Substrate Pairs
Substrate pairs
Monophenol Diphenol (1)a (2)b
L-Tyrosine 3,4-Dihydroxy-L-phenylalanine 7.81 (110)

p-Cresol Homocatechol 25.7 (222)
p-Hydroxyphenylpropionic acid 3,4-Dihydroxyphenylpropionic acid 6.31 (13.6)
p-Hydroxyphenylacetic acid 3,4-Dihydroxyphenylacetic acid 2.89 (2.89)
L-Tyrosine methyl ester 3,4-Dihydroxy-L-phenylalanine 1.77 (6.17)
L-Tyrosine methyl ester ()c 3,4-Dihydroxyl-L-phenylalanine ()a 3.44 (2.62)
L-Tyrosine methyl ester ()c 3,4-Dihydroxy-L-phenylalanine ()a 3.41 (5.20)
a
(kcat =Km ) diphenol/(kcat =Km ) monophenol.
b
kcat , diphenol/kcat , monophenol.
c
Based on kinetic constants for - and -isozymes of mushroom polyphenol oxidase, using a molecular weight
of 32,400.
Source: Refs. 2830.

B. Measurement of o-Diphenol Oxidase Activity
Reports of Km values for S. glaucescens enzyme activ-

ity on o-diphenols range from 3:72 104 M for
caffeic acid to 1:50 102 M for 3,4-dihydroxy-D-phe-
nylalanine (3). For mushroom isozymes and , Km
values are 3:75 104 M and 4:00 104 M, respec-
tively (30). Other reports of Km include those of
Wong et al. (4) where Km values for catechol and
peach polyphenol oxidase were 6.6, 4.2, 7.0, and 36.0
mM for isozymes A, B, C, and D, respectively, and of
Rivas and Whitaker (5) with pear isozymes A and B,
where Kms were 20.9 and 33.9 mM for pyrocatechol,
8.0 and 5.8 mM for 4-methyl catechol, 16.1 and 11.9
mM for chlorogenic acid, and 3.1 and 1.6 mM for d-
catechin, respectively. These Km values permit one to
use an appropriate concentration of substrate for
measuring activities. Figure 7 Relationship between enzyme concentration and
L O2 consumed by oxidation of 4-methyl catechol by
1. Spectrophotometric Method apple catechol oxidase, using an O2 electrode. The reaction
was performed in phosphate-citrate buffer, pH 5.1 with 5
In the authors laboratory, o-diphenol oxidase activity 103 M 4-methyl catechol and stock solution of 50 g enzyme
is generally determined using catechol or 4-methyl protein/mL. (From Ref. 47.)
catechol (prepared fresh daily) by mixing 4.00 mL of
0.1 M sodium phosphate buffer, pH 7.0, 0.50 mL of
5:00 102 M 4-methylcatechol (nal concentration
5:00 103 M) and adding 0.50 mL of polyphenol
oxidase of a concentration that gives a change in absor-
bance of 0:100/min. The wavelength used is 395 nm;
the
m for 4-methylbenzo-2,3-quinone is 1:350 103
M1 cm1 at 395 nm.
Absorbance should be measured with a recording
spectrophotometer so that v0 can be determined pre-
cisely at the very early stage of the reaction for reasons
discussed above. The temperature and pH should also
be controlled.
2. Polarographic Method
The polarographic method, also called the O2 -elec-
trode method, determines the rate of O2 uptake. As
shown in Figures 7 and 8 and Table 6, the O2 electrode
and spectrophotometric methods give linear relation-
ships over a wide range of enzyme concentrations. The
chronometric method measures the lag time before
browning occurs or ascorbic acid is exhausted, and
the manometric method (O2 uptake) does not give
linear relationships between enzyme concentration Figure 8 Relationship between enzyme concentration and
and O2 uptake. L O2 consumed by oxidation of 4-methyl catechol by
The concentration of enzyme, substrate, buffers, and apple catechol oxidase using spectrophotometric, chrono-
pH used may be the same as described for the spectrometric, and Warburg techniques. The reaction was per-
photometric method (above). Specic conditions are formed under the experimental conditions given in Figure
also given in the legends of Figures 7 and 8 (47). 7. (From Ref. 47.)

VI. PURIFICATION OF POLYPHENOL also successfully used ascorbic acid and Sephadex
OXIDASES G-25 to bind the phenols. This is done at 0 C.
The following method (E. Ram rez and J.R.
Purication of polyphenol oxidase must be done with- Whitaker, unpublished data) was used to purify
out any development of browning. Otherwise, the grape polyphenol oxidase to homogeneity for crystal-
polyphenol oxidase will be modied by reaction of lization for x-ray structure determinations. Acceptable
the
-amino group of lysyl residues with the o-benzo- extraction yields were obtained only when a maximum
quinone. Modication leads articially to several of 250 g of grapes per batch were used as starting mate-
bands with activity as shown by gel electrophoresis rial. Attempts to extract PPO from larger batches were
or by column chromatography (E. Ram rez and J.R. unsuccessful, owing to insufcient mechanical agita-
Whitaker, unpublished data). tion during the second extraction step because of
Several methods have been used by the authors to high viscosity of the solution. All steps were done at
prevent browning. The tissue was frozen in dry ice or 4 C. In a typical extraction, 100200 g of frozen whole
Freon (4, 5), lyophilized, and the powder extracted Grenache grapes were blended in a Waring blender for
repeatedly with cold (5 to 10 C) acetone to remove 5 min at low speed with 2 volumes (w/v) of buffer A
the phenolic compounds. This method of removing (100 mM sodium phosphate, 0.03 M ascorbic acid, pH
phenols with acetone, while used widely in the 1950s 6.5) and 10% PVPP. Pulp was separated by centrifu-
to 1970s, has been shown to modify proteins under gation (23,000 g, 30 min) and the supernatant dis-
certain conditions. We have found Freon to be a carded. It contained no PPO activity. The precipitate
good method for rapidly freezing the tissue (5). More was transferred to a beaker with 1 volume of buffer B
recently, we have used 0.03 M ascorbic acid and inso- (100 mM sodium phosphate, 0.1% Triton X-100, pH
luble polyvinyl polypyrrolidone with frozen whole 6.5) and 2.5% PVPP. This suspension was mechani-
grapes to prevent browning by binding the phenols. cally stirred for 30 min. After centrifugation
This gave an extract that did not brown. We have (23,000 g, 30 min) the precipitate was discarded and
Figure 9 Mono Q [R-CH2 -N CH3 3 HR 5/5 column chromatography of grape polyphenol oxidase. Protein was eluted
initially with a sodium chloride linear gradient (00.3 M), held for 2 mL at 0.3 M sodium chloride, and then stepped up to 1
M (dashed line). Activity of polyphenol oxidase is indicated by closed circles (and shaded area). Protein is indicated by open
squares. The small peak of activity at Fraction No. 30 is that designated as Mono Q II in the text.

Table 8 Purication of Grenache Grape Leaf Polyphenol Oxidase
Activity Spec. activity Total

(units/mL) Protein (units/mg) Volume protein Total activity Yield Purication
Sample 103 (g/mL) 103 (mL) (mg) (units 103 ) (%) (fold)
Extraction 3.64 53.1 68.5 2267 120 8250 100 1.00

40% AS (sum) 13.8 26.9 513 480 12.9 6620 80.0 7.47
95% AS (sum) 90.2 272 332 22 5.98 1980 24.0 4.82
Mono QI 164 260 631 3 0.78 492 6.75 9.19
Mono QII 33.3 105 317 14 1.47 466 5.64 4.63
the supernatant was concentrated by ultraltration substrate and measuring the initial change in absor-
(Amicon microconcentrator, Diao PM10 membrane). bance at 420 mM (pH 6.5, 25 C). One unit of activity
The concentrated solution was brought to 40% is dened as an increase in absorbance of 0.001/min.
saturation with ammonium sulfate, let stand for 30 The Mr of the mature PPO was determined on gel
min, centrifuged, and the precipitate discarded. The ltration columns. Mr was 41:2 2 kDa (replications
supernatant was further brought to 95% saturation 4) on Sephadex G-75 (ne), 34.6 kDa on Superose
with ammonium sulfate, let stand for 30 min, centri- 12 column (50), 28.0 kDa on TSKgel G3000 SW, and
fuged, and the supernatant separated. The supernatant 22.9 kDa on TSKgel 2000 SW. Superose 12 columns
still contained 50% of total activity; owing to very low are known to give Mr values that are too low (51).
protein concentration, some PPO remained in solution. Note that the Mr values on TSK gels are even lower
The precipitate was stored at 4 C (or frozen) until all than those on Superose 12. All standard proteins were
extractions were done. the same (bovine serum albumin, ovalbumin, -lacto-
The precipitates from all extractions were com- globulin [dimer], chymotrypsinogen, and ribonuclease
bined, dissolved in a minimal amount of buffer (5 A). Based on our use of Sephadex columns for Mr
mM sodium phosphate buffer, pH 8.0), and dialyzed determination for 37 years, we suggest the correct Mr
with 200 volumes of the same buffer. The sample was is near 41.2 kDa.
applied to a Mono Q [(R-CH2 -N CH3 3 HR 5/5 col-
umn linked to an FPLC system and the polyphenol
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fruit juices by cinnamic acids. J Food Technol 11:341 oxidase in Agaricus bisporus. Accession No. X85113,
345, 1976. March 6, 1995.
21. I Iyengar, A McEvily. Anti-browning agents: alterna- 34. C Ebbelaar, H Wichers, T Van den Bosch, C Sarzier, J
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1998. Detection of semiquinone radicals of N-acyldopa-
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1968. 45. W Korytowski, T Sarna, B Kalyanaraman, RC Sealy.
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inhibitors of the activated tyrosinase of broad bean catechol (amines): a kinetic electron spin resonance
(Vicia faba L.). Phytochemistry 5:665675, 1966. investigation using spin-stabilization and spin label
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1975. ing the principle of protein-dye binding. Anal Chem
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40
Laccase
William H. Flurkey
Indiana State University, Terre Haute, Indiana, U.S.A.
I. INTRODUCTION Fungal laccases have also been found to convert syr-

ingylglycol guaiacol ethers into a variety of products
Laccase was rst discovered in the latex of Rhus verni- containing aldehyde, carboxylic acid, ester, or quinone
cifera, Japanese lacquer tree, by Yoshida in 1883 (1) groups (Fig. 1f). Depending on the type of substrate
where it was observed to catalyze the oxidation of and the type of laccase, a variety of products can be
urushiol. The enzyme was rediscovered and named formed.
some 10 years later (18941896) by Bertrand (2), who Chemical structures of some of the articial sub-
found the enzyme in the lac trees and mushrooms. strates used to monitor laccase are shown in Figure
Laborde (3) also reported the existence of the enzyme 2. This list includes di- and tri-phenols, methoxy and
in mushrooms in 1896. Laccase is a multicopper amino phenols, phenylamine and phenylamine-like
enzyme member of the blue-colored oxidase family compounds and heterocyclic compounds. 2,2 0 -azino-
(for reviews see 415). Laccase catalyzes the four-elec- bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) is
tron reduction of molecular oxygen to water. A variety currently one of the more common laccase substrates.
of compounds, including substituted monophenols, o- Laccase is classied as an oxidoreductase (benzene-
and p- diphenols, methoxyphenols, aminophenols, aryl diol:oxygen oxidoreductase; EC 1.10.3.2). However,
amines, and even some inorganic ions (i.e., potassium there is still apparent confusion over its Enzyme
ferrocyanide), can serve as the electron donors in the Commission (EC) classication number since there
reaction and are oxidized in the process (Table 1). are recent publications about laccase using an EC
In general, laccases can oxidize a variety of p-diphe- number of 1.10.3.1 (p-diphenol:oxygen oxidoreduc-
nols, resulting in the production of p-diquinones (Fig. tase) and 1.14.18.1 (p-diphenol:oxygen oxidoreductase)
1a). Oxidation of syringaldazine, a common laccase in the current literature.
substrate, also proceeds through a free radical inter-
mediate followed by formation of a quinone (Fig. 1b).
The oxidation and formation of free radicals as semi- II. IMPORTANCE TO QUALITY OF FOOD
quinones also occurs when laccase acts as a dimethyox-
yphenol oxidase (Fig. 1c). With some substrates, this The importance of laccase to the quality of food is
free radical formation leads to coupling reactions and directly related to its proposed biological function.
or generation of quinone products. For example, Rhus Laccases are found in a variety of plants, fungi, and
laccase can act upon urushiol to yield coupled products even some bacteria. In higher plants, laccases are
(Fig. 1d), and plant laccases have been found to oxi- thought to be involved in lignication and detoxica-
dize monolignols into dimers and trimers (Fig. 1e). tion/protection. In fungi, laccases produce colored pig-

Table 1 Partial List of Natural and Articial Substrates Used to Monitor Laccase Activity
t-butylcatechol 4-methoxy--naphthol o- and p-aminophenols

catechol 4-methylcatechol o- and p-anisidines
caffeic acid -naphthol 4-amino-N,N-diethylaniline
chlorogenic acid protocatechuic acid 2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)
coniferyl alcohol phloroglucinol benzidine
p-coumaric acid pyrogallol 3,3 0 -diaminobenzidine
coumaryl alcohol resveratrol 2,7-diaminouorene
p-cresol resorcinol N,N-dimethylaniline
2,6-dimethoxyphenol quercetin 3,3 0 -dimethylbenzidine
dopa toluqinol p-phenylenediamine
gallic acid urushiol 5,6-dihydroxyindole
hydroquinone guaiacol 1,8-dihydroxynaphthalene
ferulic acid vanillic acid 6-hydroxydopamine
hydroxycinnamic acid sinapyl alcohol phenylhydrazine
veratryl alcohol sinapic acid syringaldazine
quinol eugenol o-tolidine
ferrocyanide ascorbic acid 1,2,4,5-tetramethoxybenzene
ments during chestnut blight pathogenic infections. products formed during enzymatic oxidations. Some of
Although laccases are different from lignin peroxi- these tests have been used to distinguish white rot fungi
dases, Mn peroxidases, and other peroxidases, they from brown rot fungi and have been used for taxo-
can utilize some of the same substrates, but they are nomic classication. Immunocytochemical location of
proposed to be a less strong oxidant than these perox- the fungal laccase from Rigidoporous lignosus grown
idases. Because laccases can oxidize a variety of phe- on wood was carried out using antilaccase polyclonal
nolic and phenolic-like compounds, they can also sera and immunogold labeling (17). These studies
cause browning-like reactions in fruits and vegetables. found laccase to be located in fungal cytoplasm, in
The browning reactions decrease consumer product vesicle-like structures at the plasmalemma, in the cell
appeal, nutritional value, and marketability. The asso- wall, and in the extracellular slime layer connecting
ciation of fungal laccases with many plant and fruit fungal cells to wood. In liquid-grown cultures of the
products poses other potential problems related to fungus Coprinus congregatus, laccase located in the
browning and crosslinking of polymeric material, and hyphal tips was suggested to be involved with sclero-
could affect product characteristics and properties dur- tia/primordia formation (18).
ing storage and processing. In plants, cytochemical localization of laccase has
relied upon the use of syringaldazine, 2,7-diamino-
uorene (DAF), 2,2 0 -azinobis(3-ethylbenzthiazoline-
III. LOCATION OF LACCASE
6-sulfonate; ABTS), and 4-methylcatechol as sub-
A. Tissue Localization strates. Laccase was localized to active lignifying
zones of xylem cells in loblolly pine sections using
In plants, laccase has been located in lignifying xylem DAF as a substrate (19). Similarly, cytochemical loca-
cells where it is associated with cell walls. Extracellular lization of laccase in tobacco stem cells was found in
laccase is secreted by plant cell cultures. In these cul- the outermost lignifying zones in xylem tissue (20).
ture conditions, laccase was not found in the cyto- Driouch et al. (21) found laccase excreted in the
plasm, but it was associated with cell walls. An extracellular medium and associated with cell walls in
epidermal location of laccase was indicated in stem sycamore cell cultures by cytochemical and immuno-
tissue. cytochemical methods. In contrast, De Marco and
Roubelakis-Angelakis (22) observed laccase in areas
B. Methods for Determining Location where lignication was thought to occur in tobacco
protoplasts.
Spot tests, either of liquid culture samples or cultures Tissue printing of stem cross sections using 4-methyl
grown on solid media, have relied on the use of colored catechol and syringaldazine as cytochemical substrates

Figure 1 Types of reactions catalyzed by laccases. Reactions appearing in (a)(c) were taken from Sanchez-Amat and Solano
(16) and reactions appearing in (d)(f) were taken from Solomon et al. (6).

from apple juice (25) and in must and wine (26). Lante
et al. (27) also reported the use of laccase as a stabilizer
during must and wine processing. Soft rot in some crop
plants and gray rot in grapes are a result of Botrytis
cinerea infection. The extracellular laccases produced
by B. cinerea are apparently involved in pathogenesis
(28). Thus, laccase may have detrimental effects on
infected food products.
Laccase is extremely important for lignin degrada-
tion in providing compost material and nutrients dur-
ing mushroom cultivation. Constitutively produced
laccase increases during growth of Agaricus bisporus
on compost and declines when fruit formation occurs
(29). Laccase seems to be associated with vegetative
growth. Extracellular laccases (ECLs) have also been
used to dene distinct species/strains in Agaricus bis-
porus and can be used as phylogenetic markers (30).
Recently, Figueroa-Espinoza and Rouau (31)
reported the use of laccase to crosslink wheat arabi-
noxylans in model systems. This crosslinking affected
the structure and properties of nonstarch polysacchar-
ides, including gelation properties. The potential
effects on food functionality related to this observation
have yet to be determined.
Figure 2 Chemical structures of substrates used to monitor Other commercial uses for laccases involve lignin
laccase activity. degradation, bioadhesives, biosensors, bioremediation,
removal of phenolic compounds from waste water, use
coupled with immunolabeling located laccase in the in enzyme assays and immunosensors, and applica-
epidermal tissue (21). In petiole tissues, laccase was tions in organic synthesis (4).
located in lignifying cell walls of xylem and epidermal
cells. All cytochemical methods must employ some
V. MOLECULAR PROPERTIES OF
method to distinguish peroxidase and tyrosinase (cate-
LACCASE
chol oxidase, phenol oxidase) from laccase. Peroxidase
can be distinguished from laccase by samples treated A. Molecular Weight
with and without hydrogen peroxide and by including
catalase in the incubation medium to remove hydrogen Fungal and plant laccases vary considerably in size.
peroxide. A variety of tyrosinase inhibitors (i.e., tropo- Depending on the method of size estimation (ultracen-
lone, SHAM (salicylhydroxamic acid), 4-hexylresorci- trifugation, size exclusion, electrophoresis, predicted
nol) can be included in the incubation medium to block size from gene sequence), the molecular size of laccase
tyrosinase activity without affecting laccase activity. ranges from 40 to 100 kDa. The more common size
range is from 60 to 80 kDa, however. Most laccases are
active as monomeric subunits, but dimeric and other
IV. UTILIZATION OF LACCASE IN FOOD multimeric combinations of the monomeric subunits
have been reported.
Besides its use as a bleaching agent in the wood pulp
and paper industry, laccase has been used in dechlor- B. Structure
ination and for removal of phenolic compounds from
wastewater. In relation to the food industry, laccase Laccases sequenced by protein or DNA methods have
has been used in assays for phenols in natural juices been shown to be composed of a single polypeptide
(23). It has also been used as a biosensor for determi- containing 500580 amino acids (4, 5, and references
nation of polyphenols/catechols in tea (24). therein). They are synthesized with an N-terminal lea-
Immobilized laccase has been used to remove phenols der peptide that is cleaved after translation. The

mature polypeptide contains N- and O-linked glycosy- multiple genes coding for the enzyme (33). Because
lation sites, four copper atoms, and two or three dis- many laccases are glycoproteins, isoform differences
ulde bonds. Because laccase has not been crystallized can arise from variations in carbohydrate content, lin-
and analyzed by x-ray crystallography, the secondary kages between carbohydrate units, and location of lin-
and tertiary structures are unknown. However, lac- kages to the polypeptide backbone. Heterogeneity due
cases do show some amino acid homology and simi- to carbohydrate content/structure can arise posttran-
larity to ascorbate oxidase and ceruloplasmin, slationally and after excretion. Other isoforms and
especially at the copper-binding sites. Because of this enzyme inactivation can arise from limited proteolysis
homology, the hypothetical secondary and tertiary (34). Because laccases are often associated with pheno-
structures are thought to be very similar to ascorbate lic material, the potential for artifactual protein mod-
oxidase. ication during synthesis, excretion, culture
Using this homology and similarity as model, three conditions, and isolation also exists. Support for this
domains found in ascorbate oxidase are postulated to comes from nding a yellow laccase that apparently
be present in laccase (4, 9). These domains in ascorbate is a modied form of the original blue laccase
oxidase are composed of -barrel/-strand motifs. By because of modication of a copper center by lignin
analogy to ascorbate oxidase, domain 1 should contain phenolics (35).
pleated sheets forming a -sandwich motif. Domain
2 should contain -pleated sheets and short stretches of
VI. ENZYME PROPERTIES
-helices. Domain 3 should contain two ve-stranded
-pleated sheets forming a -barrel and short -helix A. Substrate Specicity
stretches. Support of this comparative analogy comes
from differentially scanning calorimetry, which has Although laccase can utilize a variety of substrates as
suggested the presence of three domains in laccase (32). electron donors, few recent reports have examined sub-
Predictive structural features would suggest that strate specicity in terms of kcat =Km . In general, plant
domain 1 would be connected to domain 3 by disulde and fungal laccases have low substrate specicity (high
bonds. Overall, predictions suggest there would appear Km ) in the mM range for organic substrates. Other
to be very little -helix, a large percentage of -sheets, studies have shown that the kcat =Km is correlated
and several types of turns. The C-terminal end may be with the substrate oxidation potential (6). These obser-
blocked, buried, and/or susceptible to limited proteo- vations have suggested that laccase has no phenolic
lysis. In general, laccase is predicted to show more substrate binding pocket and oxidation takes place
homology to ascorbate oxidase than ceruloplasmin, by direct transfer of an electron(s) through an outer
plastocyanin, or azurin (4, 5, and references therein). sphere mechanism to the T1 Cu-binding site (6).
Based on amino acid sequence homology, laccase has Physiological substrates for plant laccases include p-
homology to ascorbate oxidase in the Cu-binding coumaryl, coniferyl, and sinapyl alcohols in which lac-
ligands, linkage of domains 1 and 3, the trinuclear case can cause oxidative coupling of the substrates (6,
Cu center, and the C-terminus. See Table 2 for a com- 19). Physiological substrates for fungal laccases depend
parison of the amino acids in the copper binding sites on the role laccase plays. A list of natural and articial
for 10 laccases and three ascorbate oxidases. substrates for laccase appears in Table 1.
Comparisons of the blue oxidases suggest gene dupli-
cation from an ancestral blue copper protein. Even B. Effect of Environmental Factors
though laccase and tyrosinase both contain a type 3
copper center, this center is distinctly different in lac- Many laccases have pH optima in the acidic pH range
case (6). There is apparently little to no homology of (pH 36), although some alkaline pH optima have
laccase to the copper-binding regions of tyrosinase. been reported (Table 3). In general, fungal laccases
Complete sequence information for cloned or deter- usually have lower pH optima than plant laccases;
mined sequences is available (4, 5). however, the pH optimum is often dependent upon
which substrate is chosen to assay laccase. The type
C. Isoforms of product formed also depends on the pH and the
substrate. For example, at pH 3.5 two products were
Isoforms of laccase have been found in both fungi and identied from oxidation of syringic acid by laccase
plants. These isoforms differ in their size, charge, and while at higher pH values four different products
kinetic properties. Some of these isoforms are due to were observed (7). The pH stability of the laccase iso-

Table 2 Comparison of Amino Acids in the Copper Centers of Laccase and Ascorbate Oxidase
177 178 179 238 239 240 718 719 720 721 722 723 803 804 805 806 807 808 809 810 811 812 813 814
Laccase
Aspergillus nidulans H W H H S H H P I H K H H C H I A S H Q M G G M
Phebia radiata H W H H S H H P F H L H H C H I D W H L E A A L
Coriolus hirsutus H W H H S H H P F H L H H C H I D F H L E A G F
Neurospora crassa H W H H S H H P I H L H H C H I A W H V S G G L
Cryphonectria parasitica H W H H S H H P I H L H H C H I A W H V S A G L
Filobasidiella neoformans H W H H S H H P Y H L H H C H I G W H L T E G K
Pleurotus ostreatus H W H H S H H P F H L H H C H I D W H L E I G L
Agaricus bisporus H W H H A H H P F H L H H C H I D W H L E A G L
Basidiomycete PM1 H W H H S H H P F H L H H C H I D F H L E A G F
Acer pseudoplatanus H W H H A H H P M H L H H C H F E R H T T W G M
Ascorbate oxidase
Cucumber H W H H G H H P W H L H H C H I E P H L H M G M
Pumpkin H W H H G H H P W H L H H C H I E P H L H M G M
Zucchini H W H H G H H P W H L H H C H I E P H L H M G M
2 3 3 3 1 2 3 3 1 3 1 1
Numbers at the top of the columns represent alignment of amino acid residue numbers in ascorbate oxidase. Numbers at the bottom of the columns represent amino acid residues
associated with the type 1, 2, or 3 copper centers of both enzymes.

Table 3 pH and Temperature Properties of Laccases
Temperature Temperature
Laccase source pH optimum pH stability optimum ( C) stability ( C)
Fungal
Coriolus hirsutus 3.54.5
Schizophyllum commune 5.46.0
Rhizoctonia practicola 67
Coriolus versicolor 45
Agaricus bisporus 3.6, 5.6 t1=2 40 min @ 60
Pleurotus eryngii I 4 57 65 t1=2 30 min @ 50
Pleurotus eryngii II 3.5 812 55 t1=2 25 min @ 55
Basidiomycete PM1 4.5 39 80 60 min @ 60
Trametes villosa 1,2,3 55.5
Pleurotus ostreatus POXC t1=2 30 min @ pH 3 5060 t1=2 30 min @ 60
POXA1 t1=2 24 h @ pH 3 4565 t1=2 200 min @ 60
POXA2 t1=2 2 h @ pH 3 2535 t1=2 10 min @ 60
Monocillium indicum 3.0, 5.0 26 60 t1=2 10 min @ 70
Cerrena unicolor 5.05.6 68 60
Plant
Schinus molle 6.2
Acer pseudoplatanus 6, 6.8 410
forms varies considerably. Some are stable over a wide D. Mechanism of Action
pH range (pH 37) while others are more pH sensitive.
Many laccases are relatively heat stable, but specic Laccases contain three copper centers. The type 1 cop-
thermal stability properties are associated with each per center (T1) is responsible for the blue color of the
laccase isoform (Table 3). Half-lives of 30 min at 50 protein because of the intense absorption at 600 nm.
65 C for laccase isoforms are not uncommon, while T1 centers also show small hyperne splitting in an
temperature optima are generally > 50 C. electron paramagnetic spectrum (EPR). Type 1 centers
are proposed to involve the 1e oxidation of the sub-
C. Inhibitors strate. Type 2 (T2), or normal copper centers, have no
absorption to the visible spectrum and show EPR spec-
Laccases are inhibited by a variety of compounds trum characteristic of tetragonal Cu complexes. Type 3
that can be classied into groups such as Cu centers (T3) contain two copper ions and are termed
chelators, reducing agents, detergents, and free coupled binuclear copper centers. They have no EPR
radical scavengers. Cu chelators include EDTA, signals but do absorb in the UV spectrum (330 nm). In
CN , sodium azide, and diethyldithiocarbamic acid. laccases, T2 and T3 centers form a trinuclear copper
Cetyltrimethylammonium bromide (CTAB), a catio- cluster. This cluster is responsible for the 4e reduction
nic detergent, seems to inhibit some laccases in a com- of molecular oxygen to water.
petitive or noncompetitive manner (36). Short-chain The exact mechanism for laccase catalysis has not
carboxylic acids have also been reported to inhibit been elucidated. A proposed mechanism appears in the
some laccases (37). Desferal inhibits laccase by deacti- work by Solomon et al. (6). In this mechanism an elec-
vating phenoxy radicals (38). Tiron, a superoxide ion tron from the substrate is transferred to the T1 copper
and hydroxyl ion quinone scavenger, also decreased site. The site transfers an e to the T2T3 sites.
the activity of laccase-like oxidases in tobacco xylem Reduction of the trinuclear cluster is thought to
(39). Halides, especially F , are potent inhibitors of occur by either sequential reduction of each copper
laccase (40). N-Hydroxyglycine has been reported to in the trinuclear cluster or by reduction of the T3 cop-
be a specic inhibitor of laccase by Murao et al. (41) per pair by T1 and T2. Because there are a total of 4
and later by others (4244). However, recent work in Cu ions, a variety of intermediates with different Cu
our laboratory suggests this may be an artifact (data oxidation states in the copper sites have been postu-
not shown). lated. A somewhat different mechanistic scheme is

presented in the review by Yarpolov (8). In all of G. Unique Properties
the proposed mechanisms, all e pass through the T1
site rst. Laccase catalyzes a variety of reactions including oxi-
dation, coupling of free radical intermediates, carbon
E. K m and kcat Values carbon bond cleavage, polymerization, and crosslink-
ing. Oxidative and nonoxidative decarboxylation of
Km values for organic laccase substrates are generally phenolic compounds have also been reported.
in the 0.110 mM range for a variety of phenolic and Hydroxyindole derivatives are also acted upon by
phenolic amine-like compounds (6, 8, 40). Some plant and fungal laccases. Laccase oxidizes ascorbic
hydroxyindole derivatives, such as 5,6-dihydroxyin- acid in the absence of a phenol-like substrate. More
dole, have Km values < 0:1 mM (45). For molecular recently, laccase contamination in commercial tyrosi-
oxygen as the second substrate, Km values are in the nase preparations was responsible for the novel con-
order of 105 M and Vmax values are 50 to version of dimethoxy allyl phenol to its corresponding
300 M1 sec1 (8). kcat =Km values versus electron quinone methide instead of tyrosinase (46).
reduction potentials at the T1 site show a positive cor-
relation (6). As a result, comparisons between different
VII. QUALITATIVE AND QUANTITATIVE
laccases and different substrates correspond to redox
potentials between the T1 site and the electron donor
substrates. kcat values range from 70 to 2000 sec1 (8) A. Assay Conditions
or from 100 to 3600 min1 (40). According to Xu, kcat
=Km differences in a variety of substituted phenolic In general, most laccases are assayed in buffers of pH
substrates were associated with changes in kcat primar- 38 with variations in buffer concentrations from 10 to
ily (40). Turnover numbers (expressed as kcat ) are gen- 100 mM. Changes in phenolic substrates can be mon-
erally larger than individual intramolecular electron itored spectrophotometrically using continuous, xed
transfer rate constants (8). end points, and scanning absorption methods. Assay
volumes vary from 1 to 3 mL for spectrophotometric
F. Inhibitors and Other Physicochemical Data assays and 34 mL for oxygen consumption assays.
There is no one best method for determining laccase
Surprisingly, there appear to be few data for Eact , activity because of the differences in substrate and pH
Hz , Gz , or Sz with regard to laccase. More preference for each laccase isoform. A suitable sub-
data are available relating to electron reduction poten- strate, pH, and buffer must be determined experimen-
tial differences in native and modied laccases. Most tally for each laccase.
data for inhibition of laccases are reported in terms of Colorimetric assays have employed four different
percent inhibition relative to some control/standard. A substrates that give uniquely colored products2,6-
few reports have determined I50 values for selected dimethoxyphenol (DMP; brown), ABTS (blue-green),
inhibitors. Because there are few if any specic inhibi- syringaldazine (SYR; pink), o-tolidine (TOL; blue).
tors of laccase, few data are available for Ki values. For example, 3 mL of 5 mM DMP, freshly prepared
Walker and McCallion (36) examined the effect of in buffer, can be monitored at 468 nm (
14,800 M1
CTAB, a cationic detergent, on several p-diphenol oxi- cm1 ) after the addition of 550 L enzyme. Likewise,
dases. Using CTAB, Ki values ranged from 0.25 to 23 3 mL of 0.5 mM ABTS, prepared fresh in buffer, can
mM depending on the substrate and source of laccase. be monitored at 420 nm (
36,800 M1 cm1 ). With
Desferal has a high I50 for laccase (38), while N-hydro- the third substrate, stock solutions of 15 mM syrin-
xyglycine was shown to have a low I50 for Coriolus galdazine are dissolved in 95% ethanol. Because of the
vesicolor laccase of 0:1 M (41). Faure et al. (43) also limited solubility of syringaldazine, assays for laccase
determined the I50 for N-hydroxyglycine using usually contain 10% ethanol. Even then, syringalda-
Pyricularia oryzae and A. lipoferum laccases and o- zine precipitates out of solution with time. The assay
aminophenol, p-aminophenol, and syringaldazine as solution, usually 1 mL, contains 2050 M concentra-
substrates. These I50 values ranged from 50 to tions of syringaldazine dissolved in buffer and is mon-
800 M. Caution must be used when interpreting itored at 525 nm (
65,000 M1 cm1 ). Because of
data with N-hydroxyglycine because it may not inhibit turbidity at this wavelength, blank assays in a reference
the enzyme but might interact with the products of the cell should contain all assay components except
reaction (51). enzyme. With the fourth substrate, tolidine stock solu-

tions (540 mM) are also dissolved in 95% ethanol. monolignols is usually monitored using high-perfor-
Assay solutions, usually 3 mL, contain 15 mM toli- mance liquid chromatography size exclusion chroma-
dine when added to buffer so that the ethanol concen- tography (HPLC-SEC). The following method, in
tration remains below 10% (v/v). Five to 50 L of which production of DHPs were made from coniferyl
enzyme is added to initiate the reaction, and the pro- alcohol, is a summary from Sterjiades et al. (47, 48).
duct is monitored at either 366 nm (UV) or 630 nm Coniferyl alcohol (10 mM) in 20 mM phosphate buffer
(VIS). Because the
is not known for tolidine, units of (pH 6.8) was incubated for 24 h with laccase ( 5 g)
enzyme activity are expressed in absorbance changes isolated from cultured sycamore maple cells. DHPs
per min. For substrates with known
values, enzyme were recovered from the reaction by centrifugation at
activity is expressed in either changes in absorbance 48,000 g for 30 min. The precipitates were resuspended
per min or mol product formed per min. in water and centrifuged again. This resuspensioncen-
Oxygen uptake assays can be determined using trifugation cycle was repeated for a total of three times.
Clark-type oxygen electrodes by YSL, Hansatech, or DHPs were dissolved in dimethylformamide and
Rankin Brothers. Air-saturated buffers are commonly diluted to a concentration of 10 g/mL for analysis
used as the oxygen source and are 240280 M in by HPLC-SEC. DHPs were separated on an 8 300
oxygen. Reactions using any of the methods are nor- mm Showdex KD-802 Millipore HPLC column (styr-
mally carried out at ambient temperatures, although ene divinylbenzene gels) equilibrated in dimethylfor-
some reactions have been carried out at 30 C. mamide and 100 mM LiBr. Separations were carried
Oxygen uptake assays must have the temperature con- out at 55 C with a ow rate of 1 mL/min. Column
trolled more precisely, because oxygen solubility in eluants were monitored at 280 nm. Several peaks of
aqueous solutions is temperature dependent and the polymerized monomeric material larger in size than
enzyme reaction rate is affected by temperature. coniferyl alcohol were observed. To monitor the oxida-
Water-insoluble substrates or those with limited aqu- tion of coniferyl alcohol during this process, the reac-
eous solubility are sometimes dissolved in a polar sol- tions were scanned from 230 to 400 nm at various time
vent (ethanol, methanol, dimethylsulfoxide) to make intervals. In comparison to control reaction, mixtures
stock solutions. Oxygen has a greater solubility in that contained no enzyme, a decline in absorbance at
some polar solvents than aqueous buffer solutions; 264 nm occurs. Depending on the choice of initial sub-
therefore the amount of dissolved oxygen may increase strates, new absorption peaks sometimes appear at
with the addition of substrates dissolved in these sol- lower wavelengths.
vents. Most laccase assays can tolerate at least 10% Other substrates used to monitor laccase activity are
ethanol, but this depends on the source of laccase listed in Table 1. Spot tests for laccase have used
and its stability. ABTS, gum guaiac, -naphthol, syringaldazine, and
Using oxygen uptake assays for example, a 4-mL o-tolidine as substrates. ABTS, o- and p-phenylenedia-
assay volume of 0.5 mM ABTS is pre-equilibrated mine, o-dianisidine, syringaldazine, 4-methoxy--
until the percent oxygen reaches a steady value. naphthol, tetramethylphenylenediamine, and tetra-
Enzyme is added (525 L) and the oxygen uptake is methyl benzidine have been used for histochemical
followed for 110 min, depending on the substrate and localization of laccase in plants and fungi.
enzyme concentration. Units of enzyme are expressed
in % O2 consumed per min or L of O2 consumed per C. Unique Factors That Affect Enzyme Activity
min. If the exact concentration of O2 can be deter-
mined in the air-saturated buffer, then units may be Several factors affect the ability to obtain meaningful
expressed in mol O2 consumed per min. assays for laccase. One factor is halide inhibition.
Although F is a very potent inhibitor of laccase (I50
B. Natural and Synthetic Substrates of 0.020.5 mM), I50 values for Cl range from 0.05 to
600 mM for plant and fungal laccases (39). Chloride
The natural substrate for fungal laccase is presumably ions are often found in buffers and may have some
lignin. This lignin can originate from pulp Kraft or effect on the enzyme during purication and in buffer
sulte-type processed softwoods and hardwoods. preparation. Short-chain carboxylic acids, including
Plant laccases, on the other hand, use monolignols acetic acid, also inhibit laccase. Since acetic acid/acet-
(coniferyl, sinapyl, and p-coumaryl alcohols) as sub- ate is present in some buffers used for the assay of
strates to produce water-insoluble dehydrogenation laccase, they may also affect the enzyme. The pH
polymers (DHPs). Oxidation and oligomerization of affects the type of products produced (8) and kinetic

parameters (Km , Vmax , kcat ). Inhibition of the enzyme tion after IEC and SEC. When necessary, further
also increases as the pH is increased and when the ionic purication methods have employed hydrophobic
strength is increased. interaction chromatography (HIC) on phenyl
Peroxidases can utilize many of the same substrates Sepharose using initial buffers containing high
as laccase (i.e., syringaldazine, ABTS, tolidine, guaia- ammonium sulfate concentration. Enzyme is eluted
col, etc.). If care is not taken to eliminate or distinguish by decreasing the salt concentration stepwise or by
peroxidase from laccase, estimation of laccase activity gradient elution.
or reactions attributed to laccase can be misinter- The following method is a summary of the purica-
preted. The presence of peroxidase activity is usually tion of laccases from Trametes villosa by Yaver et al.
monitored with a suitable electron donor in the pre- (49). The fungus was grown in a dened medium at
sence or absence of hydrogen peroxide. Catalase can be room temperature. The dened medium depends on
added to laccase assays to remove any hydrogen per- the type of fungi cultured. After 4 days, 1.3 mM
oxide formed, thus eliminating reactions which might (nal concentration) 2,5-xylidine was added to induce
arise from peroxidase. laccase production. Cultures were allowed to grow
Tyrosinase can also use many of the same substrates another 24 h before ltering, high-speed centrifuga-
as laccase (i.e., catechol, dopa, phenylenediamine, pyr- tion, and concentrating the ltrate using an Amicon
ogallol). This activity can be eliminated by using spe- S1Y100 ultraltration membrane. After dialysis, the
cic tyrosinase inhibitors (tropolone, salicylhydroxamic sample was subjected to IEC on a Q-Sepharose
acid, 4-hexylresorcinol) in assays for laccase (44). (Pharmacia) column equilibrated with 10 mM Tris
However, these inhibitors may react with products dur- (pH 7.7).
ing the oxidation process and can possibly interfere Some laccase activity was not adsorbed onto the IEC
with laccase estimations. Catalase can be added to resin and was eluted with buffer. Two other peaks of
remove endogenous hydrogen peroxide; however, oxi- laccase activity were adsorbed onto the column and
dation of some phenolic substrates has been attributed separated by a linear 00.5 M NaCl gradient elution.
to peroxidatic activity of catalase. The three laccase peaks were pooled separately, con-
centrated by Centricon-10 concentrators (Amicon),
and subjected to SEC on a Superdex 75 column
VIII. PURIFICATION OF LACCASES
(Pharmacia). SEC columns were equilibrated in 10
A. Methods mM Tris (pH 8) containing 150 mM NaCl. Active frac-
tions were pooled, resulting in three different laccase
There is no best method for the purication of lac- fractions, identied as neutral (laccase 3), neutral-acidic
case because of the varied number of isoforms and (laccase 2), and acidic (laccase 1) forms (Table 4).
different characteristics. Laccases have been puried All three laccases showed the same apparent MW of
most often from extracellular uids from fungal and 6070 kDa after SDS-PAGE. SEC suggested a native
plant cell cultures. In general, fungal laccase puri- size of 130 kDa. Laccase 1 consisted of a single isoform
cations employ some method for removal of cells with a pI of 3.5. Laccase 2, apparently homogeneous
(ltration, centrifugation) followed by concentration by SDS-PAGE, consisted of ve isoforms identied by
of the extracellular uid using ultraltration or pre- isoelectric focusing with pIs of 5, 6, 6.2, 6.5, and 6.8.
cipitation methods (ethanol, ammonium sulfate). Laccase 3, apparently homogeneous by SDS-PAGE,
The dialyzed and concentrated extract has been sub- contained three isoforms with pIs of 6.2, 6.5, and 6.8.
jected to ion-exchange chromatography (IEC). IEC While forms 1 and 3 were typical of other blue oxi-
methods have utilized chromatography on DEAE- dases, laccase 2 showed a reduced absorbance at 605
cellulose, QAE and mono Q columns at low salt nm. Laccase 1 had a different N-terminal sequence
concentrations near physiological pH. Laccase iso- from laccases 2 and 3, which had similar N-terminal
forms are eluted from the exchangers using increas- sequences. All forms showed differences in substrate
ing linear salt gradients (i.e., 00.5 M NaCl) preference and pH stability characteristics.
depending on how tightly the enzyme binds to the Extracellular plant laccases have been isolated by
exchanger. IEC can be preceded or followed by size concentrating the extracellular uid followed by HIC
exclusion chromatography (SEC). Typical SEC on phenyl Sepharose (2:6 30 cm column; 2 M potas-
methods have used Superose-12, Superdex 75, sium phosphate buffer, pH 6.7). Enzymes are typically
Sephadex G-100, and Sephacryl S-200. In many eluted from HIC columns with a decreasing salt
cases, the enzyme is highly puried after concentra- gradient.

Table 4 Purication of Laccases from T. villosa Culture Filtrates
Volume Total Total Specic

(mL) protein activity activity Purication Yield
Sample (mg) (mol min1 ) (mol min1 =mg protein (fold) (%)
Culture ltrate 1500 11,000 1800 0.16 1 100

conc. culture ltrate 280 460 1500 3.3 21 83
Q-Sepharose
neutral form 0.40 5.1 270 53 330 15
neutral-acidic form 0.42 9.6 108 11 69 6
acidic form 0.2 14.0 430 31 190 24
Superdex-75
neutral form 4.0 1.7 122 72 450 6.8
neutral-acidic form 7.0 4.1 117 29 180 6.5
acidic form 4.5 2.0 131 66 410 7.3
Source: Dr. D. Yaver and Dr. F. Xu at Novo Nordisk Biotech Inc., Davis, CA.
Because fungal and plant laccases are glycoproteins, activity and levels. The distribution of expressed lac-
some purication methods have employed lectin af- case isoforms is often dependent on the type of culture
nity chromatography on concanavalin A (Con A) conditions (shake vs. stationary), culture medium, and
Sepharose. Following is a summary of the purication phenolic inducer used. Many of the puried laccases
used by Drouich et al. (21) to isolate laccase from have small fold purication factors (based on the spe-
sycamore cell cultures. Proteins in the extracellular cic activity of starting material compared to puried
uid were precipitated with 5 M ammonium sulfate samples), indicating the laccase is a major protein in
at 4 C. After centrifugation of the precipitated protein, the extracellular uid or that there is a signicant loss
the pellet was dissolved in 10 mM Tris buffer (pH 7.4) in activity during purication. Puried laccases are
containing 1 mM MnCl2 and 1 mM CaCl2 . The sample typically blue in color when concentrated, but the
was applied to a Con A Sepharose column Agaricus bisporus laccase was reported to be yellow
(1:5 30 cm) equilibrated in the above buffer. in color (34) and a white laccase (50) has also been
Laccase was eluted from the column with buffer con- reported. Yellow laccases, modied by reactive pro-
taining 50 mM methylmannoside. Laccase was preci- ducts of lignin degradation, may be generated from
pitated from the solution with 5 M ammonium sulfate, blue laccases during the isolation of laccases (35).
washed with cold acetone, and dissolved in a small Puried laccases are usually stable for several years
volume of 10 mM Tris buffer (pH 8.8), containing when stored frozen. Rhus vernicifera laccase is com-
10% glycerol. The sample was subjected to preparative mercially available from Sigma Chemical Co.; a
native gel electrophoresis using the Laemmli system. Coriolus versicolor laccase is available from Mercian
After locating the laccase activity by staining the gel Corp., Tokyo.
briey with catechol, areas of active enzyme were
excised from the gel and subjected to electroelution.
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Microbiol 61:11441146, 1995.

41
Mammalian Sulfhydryl Oxidase
Harold E. Swaisgood and Violeta G. Janolino

I. INTRODUCTION but does not exhibit much activity with proteins such
as reduced RNase, whereas the mammalian enzyme
Most of the characteristics of sulfhydryl oxidase (EC exhibits no activity with DTT, contains iron, and
1.8.3.) reported in this chapter are those of the enzyme rapidly oxidizes reduced proteins (7). A mammalian
isolated from the skim milk membrane vesicles avoprotein sulfhydryl oxidase that oxidizes DTT
obtained from bovine milk (13). The enzyme is an has been isolated from the male reproductive tract
integral membrane iron-containing glycoprotein (810).
found in mammary tissue, kidney, and pancreas (4).
Immunouorescent staining also indicated its location
in the endothelial cells lining the capillaries of the II. IMPORTANCE TO FOOD QUALITY
kidney, heart, and small intestine (4). The enzyme cat- AND UTILIZATION IN FOOD
alyzes oxidation of sulfhydryl groups of cysteine, PROCESSING
cysteine-containing peptides, and proteins to disuldes
with molecular oxygen as the electron acceptor accord- Heating of milk causes protein denaturation resulting
ing to the stoichiometry given below (1, 5): in exposure of the sulfhydryl group, Cys121, in -
lactoglobulin and other chemical reactions of the sul-
2RSH O2 ! RSSR H2 O2
fhydryl and disulde residues of proteins yielding vola-
This enzyme differs from thiol oxidase (EC 1.8.3.2) tile sulfur compounds resulting in formation of a
not only by its substrate specicity and protein char- cooked avor. Ultra-high-temperature (UHT) steri-
acteristics but also in the fact that water is a product of lized and aseptically packaged milk, typically processed
the thiol oxidasecatalyzed oxidation (6), whereas, at 140150 C for 24 sec, initially has strong cooked
hydrogen peroxide is the product of sulfhydryl oxi- avor that slowly dissipates with storage (11). The
dasecatalyzed reactions. Also, sulfhydryl oxidase dif- intensity of the avor is objectionable to most North
fers from glutathione oxidase (EC 1.8.3.3) because of Americans and northern Europeans who are accus-
its broader substrate specicity and it does not contain tomed to high-quality refrigerated pasteurized milk.
FAD (6). Hence, the enzyme is a sulfhydryl:oxygen UHT treatment causes extensive denaturation of the
oxidoreductase (EC 1.8.3. ), but it has not been globular whey proteins and complete loss of sulfhydryl
assigned a serial number as yet. oxidase activity. Dissipation of the cooked avor
The mammalian enzyme is also distinct from micro- occurs concomitantly with the oxidation of sulfhydryl
bial sulfhydryl oxidase. The microbial enzyme from groups. Furthermore, sulfhydryl oxidasecatalyzed
Aspergillus niger is a soluble avoprotein that oxidizes oxidation of the exposed sulfhydryls also results in
small thiol compounds such as dithiothreitol (DTT) elimination of cooked avor (1116).

A number of studies using various preparations and could detect the cooked avor and the normalized resi-
forms of sulfhydryl oxidase have shown that the dence time in the bioreactor (Fig. 2) (16). Two patents
cooked avor of UHT milk can be immediately elimi- have been issued describing preparation of the enzyme
nated by treatment with the enzyme (1216). For and treatment of UHT milk with the enzyme for
example, bioreactors for treatment of UHT milk removal of cooked avor (17, 18).
have been prepared by covalent immobilization of Some evidence suggests that enzymatic oxidation of
the enzyme on succinamidopropyl porous glass beads heat-liberated sulfhydryl groups may provide longer
(1215) or by direct adsorption from whey on avor stability. A major defect of UHT milk that
Spherosil QA (16). The concentration of free sulfhy- develops during the long-term storage arises from
dryl groups in freshly processed UHT milk was rapidly avors derived from oxidative reactions (19).
depleted by treatment with sulfhydryl oxidase bioreac- Autoxidation of sulfhydryls produce superoxide
tors operating in either a xed-bed or a uidized-bed anion that can lead to other activated oxygen species
conguration. Activities of the immobilized enzyme that have a pro-oxidative effect on milk lipids (20).
were routinely assayed using reduced glutathione However, sulfhydryl oxidasecatalyzed oxidation of
(GSH) as the substrate. These data allowed a correla- sulfhydryls does not produce superoxide anions (5).
tion to be established between the percent oxidation of Comparison of untreated UHT milk with UHT milk
GSH and the normalized residence time which is the treated with soluble lter-sterilized sulfhydryl oxidase
ratio of units of activity/the ow rate through the bio- after a 10-month storage at ambient temperature indi-
reactor (Fig. 1) (12, 15, 16). The avor of the enzyme- cated that the untreated milk had undergone extensive
treated UHT milk was evaluated by a trained taste browning and was undrinkable because of its strong
panel. Most signicantly, a direct correlation was oxidized avors, whereas the treated milk retained
observed between the percent of the judges that its original color and the avor was acceptable (un-
published observations).
Figure 2 Relationship between the degree of cooked avor

and the extent of enzyme treatment of UHT milk. The nor-
malized residence time in a bioreactor represents the units of
Figure 1 Conversion of GSH to GSSG as a function of enzyme activity in the bioreactor divided by the ow rate
normalized residence time. Column assays (*); recirculation through the bioreactor. The panel consisted of trained milk
assays (&); batch assays (~). (From Ref. 12.) judges. (From Ref. 16.)

III. PROPERTIES OF THE PROTEIN shown by resolution of the proteins by transient cova-
lent chromatography on cysteinylsuccinamidopropyl
The molecular weight of sulfhydryl oxidase deter- glass and by specic immunoprecipitation of sulfhy-
mined with SDS-PAGE is 89 kDa (1). A similar dryl oxidase activity (25, 26). Furthermore, the activ-
value was obtained from light-scattering studies of ities of sulfhydryl oxidase, -glutamyltransferase,
a substantially puried preparation in 5 M guanidi- xanthine oxidase, and alkaline phosphatase in deter-
nium chloride (21, 22). Like other membrane pro- gent-solubilized skim milk membranes were resolved
teins, preparations of the enzyme in the absence of by isoelectric focusing (3).
dissociating agents are highly aggregated; thus, the
size of the active protein as it exists in the mem-
brane has not been established. The enzyme is pre- IV. ENZYMATIC PROPERTIES
sent in skim milk in lipid vesicles > 0:3 m in
diameter as indicated by their exclusion from Cysteine, all peptides containing cysteine, and proteins
Nucleopore lters (21) and their limited penetration with free sulfhydryl groups are all excellent substrates
of 0:3-m-diameter controlled-pore glass (3, 23). for mammalian membrane sulfhydryl oxidase (Table
Active enzyme can be solubilized with nonionic deter- 1). Unlike the soluble avoprotein microbial enzymes,
gents. Polyoxyethylene-9-lauryl ether (C12 E9 ) and - this enzyme is not active with other simple thiol com-
octyl-D-glucoside are best suited for this purpose (2). pounds such as DTT. Activity is completely lost upon
More than 80% of the activity is solubilized in 1% treatment with EDTA (1) or reagents that react with
C12 E9 ; however, the complex is still quite large having sulfhydryl groups such as iodoacetate (24). Substrate
limited ability to penetrate 7.5% polyacrylamide gels inhibition is observed at concentrations > 510 Km (1,
(2). Nevertheless, all of the solubilized enzyme was 5, 28). With GSH as the substrate, a pH optimum of
able to penetrate 0:1-m-diameter controlled-pore 6.87.0 and a temperature optimum of 35 C have been
glass (3). Dissociation of the aggregated enzyme with established (1). The stoichiometry of sulfhydryl oxi-
either nonionic detergent or 1 M guanidinium chloride dasecatalyzed oxidation of GSH has been conrmed
results in increased activity (3, 21). by quantitation of the disappearance of GSH and O2
Analysis of a substantially puried form of the and the appearance of H2 O2 and GSSG (1, 5, 25). A
enzyme indicated that it contains 11% carbohydrate number of observations, including kinetic studies (5),
and 0.5 g-atoms of Fe/89,000. Moreover, treatment have indicated that catalysis follows a Bi Uni Uni Uni
with EDTA caused removal of the Fe and complete Ping-Pong mechanism as shown by the Cleland
loss of activity (1). Subsequent dialysis of the apopro- diagram below.
tein against dilute solutions of Fe2 restored 70% of In addition to analysis of initial rate data (5), a
the original activity. However, treatment with other substituted enzyme mechanism is supported by the
divalent metals did not restore any activity, with the specic covalent interaction with cysteinylsuccinami-
exception of Cu2 which yielded 30% of the dopropyl glass and its release by reducing agents
original activity (1). The active enzyme contains two such as GSH or DTT (25). Binding of GSH prior to
DTNB-reactive sulfhydryl groups/89,000, whereas the release of H2 O2 is supported by the requirement of a
denatured protein contains three reactive groups (1, sulfhydryl oxidase substrate for the observed oxidation
24). Kinetics of activity loss due to carboxymethylation of horseradish peroxidase (HRP) in the presence of the
with iodoacetate indicated that modication of one enzyme (29). Enhancement of sulfhydryl oxidasecat-
sulfhydryl group/89,000 caused complete inactivation alyzed oxidation of GSH in the presence of HRP is
(24). thought to occur by direct transfer of peroxide from
That sulfhydryl oxidase activity and -glutamyl- sulfhydryl oxidase to HRP, thus speeding up the rate-
transferase activity arise from distinct proteins was limiting dissociation of peroxide from the enzyme (29).

Table 1 Kinetic Characteristics of Several Sulfhydryl Oxidase Substrates
Relative pseudo-rst-order
Substrates Km (mM) rate constant (Vmax =KM )
Reduced glutathione 0.1a , 0.3b , 0.6c 1.0

Gly-Gly-L-Cys 2.4d 1d
L-Cysteine 0.8c 0.62c
D-Cysteine 1.33b , 0.97c 0.62c
N-Acetyl-L-cysteine 3.85b , 1.13c 0.34c
Cysteamine 30b , 1.3c 0.68c
5-Nitro-5-thiobenzoic acid 100b
Reduced ribonuclease 0.14c
No activity with (5, 22):
Dithiothreitol Mercaptoethanol Mercaptopopionic acid
Dithioerythritol Mercaptoacetic acid Lipoic acid
a
Janolino and Swaisgood (1).
b
Swaisgood and Horton (22).
c
Sliwskowski et al. (5).
d
Schmelzer et al. (27).
e
Janolino and Swaisgood (28). This value was calculated on the basis of the sulfhydryl group concentration.
As indicated by the kinetic parameters in Table 1, activity is restored within minutes in the presence of
reduced proteins are excellent substrates. Fully sulfhydryl oxidase, whereas > 15 h is required in its
unfolded and reduced RNase A (1, 28, 30) and chymo- absence. Restoration of biological activity proves
trypsinogen A (31) are rapidly oxidized with nearly that native disulde bonds are formed. Comparison
complete restoration of biological activity (Figs. 3, of the intermediates formed during enzyme-catalyzed
4). Thus, sulfhydryl groups are oxidized and biological oxidation and air or GSH/GSSG-catalyzed oxidation
Figure 3 Comparison of the rates of sulfhydryl oxidase Figure 4 Comparison of the rates of sulfhydryl oxidase
catalyzed oxidation (*) and activity regeneration (*) with catalyzed oxidation (*) and restoration of trypsin-activata-
that observed in air (~, ~) for reduced ribonuclease. Also ble chymotryptic activity *) with that observed in air (~,
included are the data (&) obtained with the microbial ~) for immobilized reduced chymotrypsinogen A. Activity
enzyme from A. niger (7). Activity data are plotted as data are plotted as (Y1 Y)/(Y1 Y0 ) where Y1 and Y0 are
(Y1 Y)/(Y1 Y0 ) where Y1 and Y0 are the activities at in- the activities at innite and 0 time, respectively. Nearly 50%
nite and 0 time, respectively. Nearly 100% of the activity was of the original biological activity was restored. (From Ref.
restored. (From Refs. 1 and 7.) 31.)

of reduced RNase A (28) and bovine pancreatic trypsin where 1 mol SH oxidized/min is dened as 1 unit of
inhibitor (32) suggests that the same disulde bond activity.
formation pathway is followed in all cases.
Consequently, the folding pathway of the reduced pro-
tein is determined by its structure, with the enzyme VI. PURIFICATION PROCEDURES
acting as a true catalyst affecting only the rate of the
reaction and not the pathway. The procedures described have been used to obtain the
Another reaction observed to be catalyzed in vitro enzyme from bovine milk. Because more of the enzyme
by sulfhydryl oxidase that may have biological signi- exists in skim milk membranes than in the milkfat glo-
cance is the conversion of xanthine dehydrogenase to bule membranes, initially fresh raw skim milk is pre-
xanthine oxidase (33). The sulfhydryl groups of pared (1). It is very important to remove all of the
xanthine dehydrogenase are rapidly oxidized in the milkfat globules because they apparently interfere
presence of the enzyme with the concomitant appear- with later isolation procedures (unpublished observa-
ance of xanthine oxidase activity. Sulfhydryl oxidase is tions). Fresh milk is centrifuged at 4100 g for 30 min at
absent in liver where xanthine dehydrogenase is found, 30 C and the skim milk is carefully removed from
whereas in bovine milk xanthine oxidase is present. under the fat layer. Caseins are then coagulated by
addition of four units of chymosin (one unit will coa-
gulate 10 mL of skim milk in 1 min at 30 C)/100 mL
skim milk, followed by incubation at 30 C for 30 min
V. MEASUREMENT OF SULFHYDRYL (34). Whey is prepared by centrifugal removal of the
OXIDASE ACTIVITY clotted caseins at 16,300 g for 45 min at 4 C. The next
step is to prepare the skim milk membrane vesicles.
Typically, the enzyme is assayed with 0.8 mM GSH in Several procedures have been developed for this isola-
0.047 M sodium phosphate buffer, pH 7.0, at 35 C (1, tion (1, 25, 35) as described in Table 2. Preparations of
5, 25, 34). To 1.0 mL of the 0.8 mM GSH solution the membrane vesicles by chromatography on 300 nm
equilibrated at 35 C, 0.1 mL of enzyme solution is pore diameter controlled-pore glass (CPG-3000) or by
added. A blank solution is prepared using 0.1 mL of membrane fractionation using a 100,000-dalton exclu-
boiled enzyme solution. At 1-min intervals, 0.1 mL is sion limit membrane are more homogeneous than that
removed and added to 5.0 mL of 0.1 mM DTNB solu- obtained by ammonium sulfate fractionation.
tion in 0.1 M sodium phosphate buffer at pH 8.0, Typically, puried vesicles represent a 200- to 300-
containing 10 mM EDTA. The absorbance of each fold increase in specic activity compared to whey
of these solutions is read at 412 nm after a 2-min reac- (35, 36).
tion. Sufcient enzyme activity must be added to Three procedures have been developed for purica-
complete the assay within 10 min to avoid effects of tion of the enzyme from membrane vesicles. The initial
autoxidation. The initial slope of the absorbance procedure developed involved a differential centrifuga-
change versus time is used to calculate the activity. tion taking advantage of the concentration-dependent
Enzyme activity per milliliter of enzyme solution is aggregation of the enzyme (1). Thus, the ammonium
calculated from the relationship sulfate precipitate is made to 0.15% protein in 0.047
mol SH=min=mL 561 A412 =min= mM phosphate buffer, pH 7.0, and centrifuged at
2000 g for 30 min at 4 C. The supernatant is concen-
13:6 mM1 cm1 trated to 3% protein and recentrifuged under the same
Table 2 Three Methods for Preparing Skim Milk Membrane Vesicles from Whey
NH4 2 SO4 Size exclusion chromatography Membrane fractionation

fractionation (1) (36) (25)
1. Adjust the whey to 50% 1. Apply to a CPG-3000 column 1. Dialter the whey against six volumes
saturation with NH4 2 SO4 at 4 C (300 nm pore diameter) equilibrated of 0.047 mM phosphate buffer,
2. Incubate overnight (with 0.047 mM phosphate buffer, pH 7.0, using a 100,000-dalton
3. Collect precipitate by centrifuging pH 7.0. exclusion limit membrane.
at 16,500 g for 60 min at 4 C. 2. Membrane vesicles are obtained in 2. Concentrate the retentate containing
Represents crude vesicles. the void volume fraction. the vesicles 10-fold.

Table 3 Two Methods for Purication of Sulfhydryl Oxidase from Detergent-Solubilized Membrane Vesicles
Transient covalent chromatography on Bioselective adsorption on monomeric

cysteinylsuccinamidopropyl glass (25, 37) avidin (35)
1. Recirculate 150 mL solubilized vesicles prepared from 1. The enzyme is biotinylated using 0.25 mL of biotin-
1 L whey through 60 mL of the adsorbent at 30 C HPDPa /mL of solubilized membrane vesicles. Incubate for
for 4 h. 90 min at room temperature.
2. Remove adsorbed materials by washing with 1 L 2. Recirculate the biotinylated enzyme in 47 mM phosphate
phosphate buffer, pH 7, containing the detergent. buffer, pH 7, through monomeric avidin beads for 20 min
3. Reductively release the enzyme by recirculating 100 mL at room temperature. Add 0:5 mg total protein/mL of
of 2 mM GSH until no thiol groups can be detected adsorbent.
with DTNB. 3. Wash the matrix with ve volumes of 47 mM phosphate
4. Regenerate the adsorbent by recirculating 50 mL buffer, pH 7, containing 1% C12 E9 .
DTT in 8 M urea/1 M NaCl, followed by 0.1 M 4. Release the enzyme by recirculating 0.30.5 mL 50 mM
acetic acid. DTT/mL matrix in 47 mM phosphate buffer for 20 min at
room temperature
a
Biotin-HPDP is N-[6-(biotinamido)hexyl]-3 0 (2 0 -pyridyldithio)propionamide.
conditions, yielding sulfhydryl oxidase activity in the prepared by reaction of cysteine with 1-ethyl-3-(3-
pellet. Finally, the pellet is solubilized in twice the dimethylaminopropyl)carbodiimide-activated succina-
volume of the previous solution and again centrifuged midopropyl glass as described by Sliwkowski et al.
under the same conditions, yielding the puried (25). A typical purication using this procedure results
enzyme in the pellet. in a 4800-fold increase in the specic activity compared
The other two methods of purication are based on to that in whey (Table 4).
the catalytic mechanism or the active-site sulfhydryl Bioselective adsorption on monomeric avidin is
group (Table 3). First, the membrane vesicles are solu- based on selective biotinylation of the active site
bilized by adjustment to 1% C12 E9 in phosphate buf- sulfhydryl group of the enzyme with N-[6-(biotin-
fer, pH 7, and centrifuged at 100,000g to remove any amido)hexyl]-3 0 (2 0 -pyridyldithio)propionamide (bio-
insoluble material. Transient covalent chromatogra- tin-HPDP) (35). The resulting mixed disulde,
phy on cysteinylsuccinanidopropyl glass based on the containing a biotinyl residue, strongly binds to avidin
fact that sulfhydryl oxidase follows a substituted but can be released by reduction of the disulde bond
enzyme mechanism. Thus, a mixed disulde is formed (35). The puried enzyme obtained by this procedure
between the enzyme and the immobilized cysteinyl resi- exhibits a 3100-fold increase in specic activity com-
due which cannot be reduced without the addition of pared to that of whey (Table 5).
free thiols (34). Cysteinylsuccinamidopropyl glass is
Table 4 Data from a Typical Purication Using Cysteinylsuccinamidopropyl Glass
Total Total units Specic

Volume protein of activity Yield Purication
Fraction (mL) (mg) activitya (units/mg) (%) (fold)
Whey 910 8190 40 0.005 100 1

Solubilized skim 156 565 17 0.031 47 6.3
milk membranesb
Puried enzyme 99 0.545 13 24.2 33 4830
a
Assay mixtures contained 0.8 mM GSH in 50 mM sodium phosphate, pH 7.0. Activity was calculated from
the rate of disappearance of sulfhydryl groups due to enzyme-catalyzed oxidation at 35 C.
b
Prepared by membrane fractionation (see Table 2). The dialtered whey was solubilized with 1% C12 E9 .
Source: Ref. 25.

Table 5 Purication of Sulfhydryl Oxidase by Avidin-Biotin Afnity Chromatography
Total units Specic

Total protein of activitya activity Recovery Purication
Fraction (mg/L whey) (U/L whey) (U/mg enz) (%) (fold)
Whey 6510 53 0.008 100 1

Skim milk membranesb 23.6 51 2.2 96 275
Puried enzyme 0.84 21 25 39 3125
a
Assay mixtures contained 0.8 mM GSH in 50 mM sodium phosphate, pH 7.0. Activity was calculated from the
rate of disappearance of sulfhydryl groups due to enzyme-catalyzed oxidation at 35 C.
b
Dialtered whey was fractionated with CPG-3000 yielding the puried skim milk membrane vesicles in the void
volume fraction.
Source: Ref. 35.
REFERENCES 12. HE Swaisgood, VG Janolino, MX Sliwkowski. An

enzymatic approach to avor enhancement.
1. VG Janolino, HE Swaisgood. Isolation and character- Proceedings of International Symposium on UHT
ization of sulfhydryl oxidase from bovine milk. J Biol Processing and Aseptic Packaging of Milk and Milk
Chem 250:25322538, 1975. Products, Raleigh, NC, 1980, pp 6776.
2. MB Sliwkowski, HE Swaisgood, HR Horton. 13. HE Swaisgood, VG Janolino, HR Horton.
Solubilization of sulfhydryl oxidase, a bovine skim Immobilized sulfhydryl oxidase. AIChE Symposium
milk membrane enzyme. J Dairy Sci 65:16811687, Series, Vol 74, No. 172, Am Institute Chem Engr,
1982. 1978, pp 2530.
3. VG Janolino, HE Swaisgood. Isolation, solubiliza- 14. HE Swaisgood. Sulphydryl oxidase: properties and
tion, fractionation by electrofocusing,and immobiliza- applications. Enzyme Microb Technol 2:265272,
tion of skim milk membrane. J Dairy Sci 67:1161 1980.
1168, 1984. 15. HE Swaisgood, MX Sliwkowski, PJ Skudder, VG
4. DA Clare, HR Horton, TH Stabel, HE Swaisgood, Janolino. Sulfhydryl oxidase: characterization and
JG Lecce. Tissue distribution of mammalian sulfhy- application for avor modication of UHT milk. In:
dryl oxidase. Arch Biochem Biophys 230:138145, P Dupuy, ed. Utilisation des Enzymes en Technologie
1984. Alimentaire. Paris: Lavoisier, 1982, pp 229235.
5. MX Sliwkowski, HE Swaisgood, DA Clare, HR 16. HE Swaisgood, VG Janolino, PJ Skudder.
Horton. Kinetic mechanism and specicity of bovine Continuous treatment of ultrahigh-temperature steri-
milk sulfhydryl oxidase. Biochem J 220:5155, 1984. lized milk using immobilized sulfhydryl oxidase.
6. Nomenclature Committee of the International Union Methods Enzymol 136:423431, 1987.
of Biochemistry and Molecular Biology. Enzyme 17. HE Swaisgood. Process for removing cooked avor
Nomenclature. San Diego, CA: Academic Press, from milk. U.S. patent No. 4,053,644 (1977).
1992, p 110. 18. HE Swaisgood. Purication and immobilization of
7. VG Janolino, HE Swaisgood. A comparison of sulf- sulfhydryl oxidase. U.S. patent No. 4,068,328 (1978).
hydryl oxidases from bovine milk and from 19. S Rerkrai, IJ Jeon, R Bassette. Effect of various direct
Aspergillus niger. Milchwissenschaft 47:143146, 1992. ultra-high temperature treatments on avor of com-
8. TSK Chang, B Morton. Epididymal sulfhydryl oxi- mercially prepared milks. J Dairy Sci 70:20462054,
dase: a sperm-protective enzyme from the male repro- 1987.
ductive tract. Biochem Biophys Res Commun 66:309 20. JJ Lee, WF Shipe. Effects of sulfhydryl compounds on
315, 1974. lipid oxidations catalyzed by copper and heme. J
9. MC Otrowski, WS Kistler. Properties of a avopro- Dairy Sci 65:14141420, 1982.
tein sulfhydryl oxidase from rat seminal vesicle secre- 21. VG Janolino, CS Barnes, HE Swaisgood. Dissociation
tion. Biochemistry 19:26392645, 1980. and unfolding of sulfhydryl oxidase in solutions of
10. MC Otrowski, WS Kistler, HG Williams-Ashman. A guanidinium chloride. J Dairy Sci 63:19691974, 1980.
avoprotein responsible for the intense sulfhydryl oxi- 22. HE Swaisgood, HR Horton. Sulphydryl oxidase: oxi-
dase activity of rat seminal vesicle secretion. Biochem dation of sulphydryl groups and the formation of
Biophys Res Commun 87:171176, 1979. three-dimensional structure in proteins. Ciba
11. HE Swaisgood. New developments in UHT sterilized Foundation Symposium No. 72: Sulphur in Biology,
milk. Dairy Ice Cream Field 160:4860, 1977. Excerpta Medica, 1980, pp 205222.

23. VG Janolino, HE Swaisgood. Effect of support size on proteins associated with a surface? J Appl Biochem
activity of immobilized sulfhydryl oxidase. J Dairy Sci 7:3337, 1985.
61:393399, 1978. 31. VG Janolino, MX Sliwkowski, HE Swaisgood, HR
24. KB Song, HE Swaisgood, HR Horton. Requirement Horton. Catalytic effect of sulfhydryl oxidase on the
for a sulfhydryl group for sulfhydryl oxidase activity. formation of three-dimensional structure in chymo-
J Dairy Sci 69:25892592, 1986. trypsinogen A. Arch Biophys 191:269277, 1978.
25. MX Sliwkowski, MB Sliwkowski, HR Horton, HE 32. DC Chen. Coupled use of immobilized enzymes with
Swaisgood. Resolution of sulphydryl oxidase from mass spectrometry for peptide analysis including loca-
-glutamyltransferase in bovine milk by covalent tion of disulde bonds and sulfhydryl oxidase-cata-
chromatography on cysteinylsuccinamidopropyl- lyzed formation of disulde bonds in bovine
glass. Biochem J 209:731739, 1983. pancreatic trypsin inhibitor. Ph.D. dissertation,
26. CH Schmelzer, HE Swaisgood, HR Horton. North Carolina State University, Raleigh, NC, 1993.
Resolution of renal sulfhydryl oxidase from -gluta- 33. DA Clare, BA Blakistone, HE Swaisgood, HR
myltransferase by covalent chromatography on cystei- Horton. Sulfhydryl oxidasecatalyzed conversion of
nylsuccinamidopropyl-glass. Biochem Biophys Res xanthine dehydrogenase to xanthine oxidase. Arch
Commun 107:196201, 1982. Biochem Biophys 211:4447, 1981.
27. CH Schmelzer, HE Swaisgood, HR Horton. 34. HE Swaisgood, HR Horton. Sulfhydryl oxidase from
Glycylglycyl-L-cysteine at a substrate for renal sulfhy- milk. Methods Enzymol 143:504510, 1987.
dryl oxidase (Glutathione oxidase). Biochim Biophys 35. VG Janolino, HE Swaisgood. Purication of reactive
Acta 827:140143, 1985. sulfhydryl enzymes by bioselective adsorption on
28. VG Janolino, HE Swaisgood. Sulfhydryl oxidasecat- monomeric avidin: purication of sulfhydryl oxidase.
alyzed formation of disulde bonds in reduced ribo- J Food Biochem 16:389399, 1993.
nuclease. Arch Biochem Biophys 258:265271, 1987. 36. VG Janolino, DA Clare, HE Swaisgood.
29. GW Koszalka, HE Swaisgood, HR Horton. Characteristics of sulfhydryl oxidase isolated by a sim-
Enzymatic enhancement of the catalytic rate of sulf- ple chromatographic procedure. Biochim Biophys
hydryl oxidase. Biochim Biophys Acta 915:315329, Acta 658:406409, 1981.
1987. 37. VG Janolino, HE Swaisgood. Homogeneity of sulfhy-
30. VG Janolino, HE Swaisgood, HR Horton. dryl oxidase preparations obtained by transient cova-
Renaturation of soluble and immobilized ribonu- lent afnity chromatography. J Dairy Sci 73:308313,
clease: are the polypeptide folding pathways for struc- 1990.
ture formation the same for soluble proteins and for

42
Xanthine Oxidase
John R. Whitaker
I. INTRODUCTION neously isomerizes to some keto form of xanthine

that can be catalyzed by XO to the enol form of uric
The enzyme described in this chapter is called acid, which then spontaneously isomerizes to some
xanthine oxidase, xanthine dehydrogenase, or keto form of uric acid. The isomeric forms of the sub-
xanthine oxidoreductase by various authors. strates make interpretation of the kinetic results dif-
Except for the oxidation state of two sulfhydryl cult, as the enol form of xanthine and the keto form of
groups in the active site of the protein, they are the uric acid may be competitive inhibitors of XO. Other
same. When the two sulfhydryl groups are present, purines and adenine are oxidized more slowly (Table
the enzyme performs primarily as a dehydrogenase 1). A number of aldehydes (Table 1) are oxidized also
but still has some oxidase activity. When the to the corresponding carboxylic acids by XO.
two sulfhydryl groups (SH) are oxidized to give a Xanthine dehydrogenase (XDH; EC 1.1.1.204)
disulde group (SS), or two sulfonic acid groups reduces NAD to NADH (7), as well as O2 to H2 O2
3 , the enzyme functions as an oxidase (15).
(SO2 (8) with xanthine as the second substrate [Eqs. (2) and
Reduction of the SS bond converts the oxidase (3)].
back to a dehydrogenase, with a signicant conforma-
tional transition in the avin binding site (3). Xanthine NAD ! uric acid NADH H 2
Xanthine O2 ! uric acid H2 O2 3
A. Reactions Catalyzed
Steady-state kinetic rate constants of xanthine=O2 and
Xanthine oxidase (XO; EC 1.1.3.22) catalyzes the oxi- xanthine=NAD reactions catalyzed by XDH are 2:1
dation of hypoxanthine and xanthine to uric acid 0:1 sec1 (7) and 6:3 sec1 (8) at 25 C and pH 7.5,
(Structure 1), respectively. For comparison, the oxidation of
NADH=O2 is 2:5 0:9 sec1 under the same condi-
Hypoxanthine H2 O O2 ! uric acid H2 O2 tions. Therefore, XDH has a signicant and intrinsic
some O2 xanthine oxidase activity on some substrates (7, 8).
Xanthine dehydrogenase has an NAD binding site
1
and a substantially lower reduction potential for the
with O2 being the second substrate (oxidant). The FADH=FADH2 couple relative to XO (9, 10). In the
heavy arrows in Structure 1 show the reactions cata- case of the milk enzyme, the difference is 180 mV.
lyzed by XO; the dashed arrows show the net result of Xanthine oxidase has low specicity for both the
the reactions. Note that oxidation of hypoxanthine substrates and the electron acceptor. More than 100
gives the enol form of xanthine, which then sponta- compounds are known to serve as substrates, and a

Structure 1
large number of electron acceptors are known, includ- kinetic studies on the rate of reduction of Methylene
ing O2 , NAD , methylene blue, 2,6-dichlorophenol- Blue. Dixon (14) showed that aldehydes are substrates
indophenol, triphenyltetrazolium chloride, phenazine for XO. In 1939, Ball (15) and Corran et al. (16)
methosulfate, cytochrome c, and ferricyanide. obtained nearly pure XO, with a golden brown color
due to intrinsic avin (FAD), plus another unknown
chromophore (now known to be molybdenum) (17,
B. Historical Aspects 18). In 1954, Richert and Westerfeld (19) reported
that addition of ferric chloride in the diet of rats sub-
In 1902, Schardinger (11) determined that fresh milk stantially increased the activity of XO in rat liver; they
decolorized Methylene Blue. In 1922, Morgan et al. found iron, FAD, and molybdenum to be present in
(12) reported that bovine milk converted hypoxanthine the ratio of 8 : 2 : 1 in XO. In 1955, Avis et al. (20)
and xanthine into uric acid under both aerobic and developed an isolation procedure for XO from cows
anaerobic (when Methylene Blue was included) condi- milk which gave crystalline XO with A280 =A450 of 5.0
tions. They found the same activity in liver, spleen, and 5.2, a level of purity that has not been increased to the
lungs of rats and cows. The activity, which they named current time. The ratio of iron, molybdenum, and
xanthine oxidase, was destroyed by boiling. In 1924, FAD was 8.1 : 1.4 : 2.0. Later, the iron, labile sulde,
Dixon and Thurlow (13) partially puried XO and did molybdenum, and FAD were found to be in the ratio
Table 1 Substrate Specicity of Xanthine Oxidase from Milk
Relative Relative
Substrate rates Substrate rates
Xanthine 140 Acetaldehyde 0.72

Hypoxanthine 100 Cinnamaldehyde 0.41
8-Hydroxypurine 6 Vanillin 0.01
6-Amino-2-hydroxypurine 4.5 Glyceraldehyde 0.014
6-Amino-8-hydroxypurine 7 p-Phthalaldehyde 0.013
Adenine 6 Decanaldehyde 0.0080
2,8-Dihydroxypurine 0.6 n-butyraldehyde 0.0060
p-Hydroxybenzaldehyde 1.3 Octaldehyde 0.0002
Benzaldehyde 0.8
Source: Ref. 6.

attention was given to this concern by several research-
ers. In particular, Dr. Clifford at U.C. Davis carefully
researched this area and determined that no intact XO
(active or inactive) was found in the blood of rabbits
(27). His results are not surprising when one considers
the size of the XO (290 kDa).
Harrison (25) reported that puried human breast
milk XO has only 23% the specic activity of cows
milk. His data indicate that XO activity is very low in
most human tissues, except for liver and intestinal tis-
Structure 2 sues.
II. MECHANISM OF XANTHINE OXIDASE

CATALYSIS
of 2 : 2 : 1 : 1 per subunit (21). The molecular weight of
XO was determined by ultracentrifugal sedimentation A. Protein Nature of Xanthine Oxidase
and diffusion data to be 290 kDa (22), with two iden-
tical subunits. The complete amino acid sequences of XOs from var-
The mechanistic aspects will be presented in Section ious sources have been deduced by sequencing the
II. respective cDNAs (genes). They are highly homolo-
gous consisting of, for example, 1332 amino acid resi-
dues for bovine milk XO and 1333 residues for human
C. Importance of Xanthine Oxidase to Food liver XO, with 90% sequence identity (2830).
Science, Nutrition, and Medicine The x-ray crystallographic structure of bovine milk
XO has been published (9). As reported by other meth-
During metabolism of nucleic acids, animals convert ods, XO has a molecular weight of 290 kDa. It is com-
the purine bases adenine and guanine to uric acid. Uric posed of two identical subunits, each containing an
acid then forms calcium urate which is deposited in active site. In addition to the several amino acid resi-
joints. As a result, XO has been implicated in a number dues in the active sites there are two molecules of iron
of diseases ranging from rheumatoid pathology, ische- [Fe(III)], two molecules of labile sulde, one molecule
mia-reperfusion injury, and in reactive oxidant- of molybdenum, one molecule of FAD per subunit
mediated signal transduction. The bases for these (21), and one molecule of molybdopterin, a cofactor
implications are due to known metabolic free radical that binds the molybdenum (31) (see Structure I). In
generation by XO and correlation of XO concentra- XO/XDH from prokaryotes, the dinucleotides of gua-
tions with certain diseases. As pointed out in Section nine, cytosine, adenine, or hydroxyxanthine are found.
I.A, the oxidation of hypoxanthine and xanthine and For example, in the gram-positive bacterium
of NADH=O2 by XO leads to the formation of H2 O2 Desulfouibrio gigas, the cofactor is a molybdenum
and O2 (superoxide ion). In experimental animals, pri- molybdopterin cytosine dinucleotide.
marily rats and mice, there are good relationships The dimensions of the active dimeric (two subunits)
between XO activity and the development of the enzyme are 155 A90 A70 A (Fig. 1) (9). It is butter-
pathological problems listed above. Evidence for this y shaped with the dimer interface on the smaller side
is well presented by the recent reviews of Nishino et al. of the vertically elongated subunits. Each subunit con-
(5), Wright and Repine (23), Bulkley (24), Harrison sists of three domains. The small N-terminal domain
(25), and Blake et al. (26). In particular, the review (residues 1165) contains the two iron/sulfur cofactors.
by Blake et al. (26) details the current experimental It is connected to the FAD-binding domain (residues
evidence for the possible role of XO in rheumatoid 226531) by a long segment of amino acid residues
pathology. (166225). The FAD domain is connected to the
In 1977, major consumer concerns arose as to how third domain (residues 5901332) by the amino acid
milk was processed, in particular raw milk and lightly residues 532589. This large third domain contains
pasteurized milk. There was concern that active XO the Mo-pterin cofactor (Structure 2). The three
might pass through the small intestinal wall into the domains are folded in proximity to each other so as
blood and lead to major problems in tissues. Special to form the active site of the enzyme containing the

Figure 1 (A) Molecular structure of xanthine dehydrogenase (XDH) as determined by x-ray crystallography. (B) For clarity, the
cofactors and salicylate (added to stabilize XDH) are shown in the same positions as found in the protein molecule. (From Ref.
9.)
two iron/sulfur cofactors, the FAD cofactor and the Cleland Nomenclature (35) in Eq. (4) where X is
Mo-pterin cofactor. Each subunit functions indepen- xanthine and U is uric acid.
dently of the other. The reader is referred to the pub-
X U O2 H2 O2
lication by Enroth et al. (9) for a color-coded diagram # " # "
of the XO molecule. E-FAD E-FADX E-FADH2 E-FAD2 O2 E-FAD
E-FADH2 O E-FADH2 O2
The tertiary structure of aldehyde oxidoreductase, a
member of the xanthine oxidase family, is also known 4
for Desulfouibrio gigas (32, 33); it is very similar to that
In 1964, Bray et al.(36) used electron paramagnetic
of XO.
resonance spectroscopy to investigate the rates of elec-
tron transfer to and decay from the several cofactors of
B. Mechanism of Action of Xanthine Oxidase the enzyme-substrate system. The XO and xanthine
were mixed very rapidly (< 1 msec) and frozen very
When xanthine and O2 are added to XO, the reaction rapidly (< 1 msec) in liquid N2 . The Mo- signal,
pathway was shown by Gutfreund and Sturtevant (34)
to be that described by Fig. 2. The rate constants k1 ,
k2 , k3 , and k4 for the reactions were determined by
rapid reaction techniques to be 5:0 105 M1 sec1 ,
10:5 sec1 , > 4:0 105 M1 sec1 , and 21:5 sec1 ,
respectively. The rate constants k1 and k3 are for for-
mation of the Michaelis-Menten complexes of
xanthine E FAD and O2 E-FADH2 , respectively,
while k2 and k4 are the rate constants for oxidation of
xanthine to uric acid and reduction of O2 to H2 O2 ,
respectively.
When the 1/(turnover numbers) are plotted versus
1/[xanthine] at three different concentrations of O2 : air
saturated (0.24 mM), 60% O2 saturated enzyme and
the results of the two are extrapolated to 1O2 concen-
tration, all three lines are parallel (Fig. 3) (21), indicat-
ing that the reaction follows a Ping-Pong Bi-Bi Figure 2 Mechanism of action of xanthine oxidase as deter-
mechanism. This is diagrammed according to the mined by fast reaction kinetics. (From Ref. 34.)

6
7
While Eqs. (6) and (7) give the same nal products, the
Figure 3 Effect of xanthine and O2 concentrations on the mechanisms of the reactions are fundamentally differ-
rate of xanthine oxidation by xanthine oxidase. T.N. is the ent.
turnover number measured at pH 8.3 and 25 C. (From Ref. In the rst proposed mechanism [Eq. (6)], the pro-
21.) duct of hydroxylation of the substrate 2-hydroxy-6-
methylpurine (B; in the enolate tautomeric form) is
suggested to be coordinated to the molybdenum via
believed to be conversion of Mo(VI) to Mo(V), the induced oxygen atom from the OH group of the
reached a maximum in 15 msec. The Mo-, molybdenum cofactor (A) of the enzyme to form the
FADH FAD ! FADH, and Fe(II) FeIII ! bracketed intermediate/transition state compound (C).
FeII signals were maximum at 40, 45, and 100 The intermediate/transition state compound is pro-
msec, respectively (Fig. 4). Therefore, Bray et al. (36) posed to be formed in a base-assisted (B: group in
suggested that the sequential steps in the reductive the active site of the enzyme) nucleophilic attack of
reaction are: xanthine ! Mo ! FAD ! Fe ! O2 . the MoVI OH group on the C-8 of the substrate with
More recently, the reaction order has been shown to a concomitant hydride transfer to the Mo S group
IV
be: xanthine ! Mo ! 2Fe=2S ! FAD ! O2 (21). (bracketed structure) to give Mo SH (D). The very
Therefore, the overall reaction involving the ow of rapid species is formed by a one-electron oxidation
electrons is best described by Eq. (5). and deprotonation to give the EPR-detectable
MoV OS(OR) species (D).
In the second proposed mechanism [Eq. (7)], the
purine substrate (B0 ) is complexed (noncovalently) to
the molybdenum cofactor (A 0 ) of the enzyme as the
keto tautomer permitting insertion of the C8H-bond
The order in which electrons and water are trans-

ferred from xanthine and oxygen to produce uric
acid and hydrogen peroxide (H2 O2 ) is generally
agreed to be that shown in Eq. (5). However, the
mechanistic pathway of the Mo-pterin cofactor and
substrate in the rst step is still in dispute. The key
difference among scientists in the eld is primarily
whether the rst chemical event following formation
of the noncovalent enzyme-substrate complex
involves an Mo-C or a Mo-O-C covalent linkage Figure 4 Electron paramagnetic resonance signals gener-
between the Mo-cofactor and substrate as shown in ated by xanthine oxidase in a single turnover experiment
Eqs. (6) and (7) (37). with xanthine as substrate. (From Ref. 36.)

across the MoVI S bond to give a species with a Mo 1. Although XO had been partially puried by
C(SH) bond (C 0 ). D 0 is formed by removal of a proton other researchers, Avis et al. (20) were the rst to
from the MoOH group to provide an O-bridge obtain a homogeneous XO preparation and to crystal-
between the Mo and C-8 of the purine. Subsequent lize it.
proton (H ) and electron (e ) transfers lead to the 2. Researchers have shown this method to be
very rapid species (E 0 ), with some resemblance to the best one, even after 45 years. Some modica-
E in Eq. (6) (both have MoV OS(OR) structure) tions of the method have been published, but all
which is EPR detectable. report the same maximum specic activities as Avis
et al. (20).
3. Fraction M7 contained FAD, iron, and molyb-
III. DETERMINATION OF XANTHINE denum in the ratio of 2.0 : 8.1 : 1.4 per unit of protein;
OXIDASE ACTIVITIES recent methods give nearly the same values.
A. Xanthine Oxidase Activity 4. QO2 is 2300 units activity using xanthine as sub-
strate at 23:5 C, indicating purity. The steps involved
A number of methods are available to determine XO in purication are described below.
activity. Using xanthine as the substrate, the rate of
oxidation of xanthine to uric acid can be followed by 1. M1
the rate of Methylene Blue reduction (under anaerobic Cream separated from fresh milk. The milk was cooled
conditions), by cytochrome c reduction (aerobically), by
to 5 C overnight, skimmed, churned, and passed
oxygen uptake (oxygen electrode use), or by the rate of
through a cream separator to remove additional fat.
uric acid formation (increase in absorbance at 290 nm). Approximately 50% of the original activity was
The easiest, and perhaps best assay is to follow the retained; volume was 25.1 L. The buttermilk was
rate of uric acid formation from xanthine, in the prewarmed to 34 C in a pressure controlled cast-iron ves-
sence of O2 , in a recording spectrophotometer at 290 sel. Calcium chloride (0.5 M: 250 mL) and pancreatin
nm, pH 8.3, 25 C (8). The molar extinction coefcient, (40 g) were added. After 15 min a dense precipitate of

m ; 9:6 103 M1 cm1 . casein formed and was removed by straining through
xanthine O2 ! uric acid H2 O2 8 nylon net to give 25 L of M2.
Only initial rates should be used (< 5% conversion of
2. M2
xanthine to uric acid) in analyses, as H2 O2 slowly inac-
tivates the enzyme. Catalase can be added to remove Filtration was performed on type PF 40 lters and 13
the H2 O2 when reaction times need to be extended sterimats precoated with Hyo Super Cel (650 g) at
(when low concentrations of XO are present). 5 lb=in:2 Hyo Super Cel (325 g was mixed with
The rate of reaction [Eq. (8)] can also be followed M1). Starting pressure was 7 lb/in.2 using a nitrogen
with cytochrome c under aerobic conditions. The rate cylinder. The rst 3 L of colorless ltrate was dis-
of reduction of cytochrome c is determined by increase carded. Filtration rate decreased after 10 L was col-
in absorbance at 550 nm (reduced band of cytochrome lected and a nal pressure of 28 lb/in.2 was required.
c). Total volume of M3 was 21 L.
B. Xanthine Dehydrogenase Activity 3. M3
See Section IV.B.1. The cloudy ltrate was stored for 16 h at 5 C, then
reltered through PF 40 and 11 sterimats of grade
GS to give 20 L of clear yellowish brown liquid
IV. PURIFICATION OF XANTHINE (M4).
OXIDASE
4. M4
A. Xanthine Oxidase from Cows Milk
M4 was chromatographed on a 6 in.12 in. Pyrex
An abbreviated description of the method of Avis et al. column packed with calcium phosphate gel/Celite 545
(20) for the purication of xanthine oxidase from cows (1 : 1) (see Ref. 38 for preparation). M4 was cooled to
milk, published in 1955, is given below. This method is 5 C, adjusted to pH 6.2 with 0.1 N acetic acid, and
chosen for several reasons: passed through the column at 4 lb/in.2 pressure. The

brown enzyme bound to the calcium phosphate gel chloride at 5 C. The solution was diluted to 50 mL
column. The column was washed with 0.02 M phos- (M7).
phate buffer, pH 6.2, until the washes gave a negative A second crop of crystals was obtained from centri-
protein test by trichloroacetic acid addition (ppt.). The fugation of the mother liquor after 28 days at 1 C.
enzyme was eluted with 1 M phosphate buffer, pH 5.8, The crystals were collected by centrifugation and dis-
to give M5. solved in 0.2 M sodium chloride (15 mL) (M7a).
Part of the M7 solution (10 mL), after storage at 2
5. M5 C for 28 days, was recrystallized by the following
method to give M8. The enzyme was precipitated by
M5 was precipitated with an equal volume of 4 M
adding 70% (v/v) ethanol (5 mL), then centrifuged,
phosphate buffer (pH 9) and the precipitate col-
and the precipitate dissolved in water to give 10 mL
lected on a sintered-glass funnel with aid of 10 g
of solution. Phosphate buffer (1 M; 0.7 mL; pH 5.8)
Hyo Super Cel. The precipitate was dissolved in a
and 50% ethanol (1.5 mL) were added. The immediate
minimum amount of water and ltered using Hyo
precipitate formed at 10 min was removed by centrifu-
Super Cel, which was washed with small amounts of
gation at 1 C and the supernatant liquid was seeded
water to give 400 mL enzyme solution. The solution
and left at 1 C for 16 h. The crystals formed were
was then dialyzed in a Visking cellulose casing (size 32/
collected by centrifugation and dissolved in 0.2 M aqu-
32) against 40 L of 0.01 M acetate buffer, pH 5.1, at
eous sodium chloride (nal volume of 10 mL) (M8).
5 C. This treatment gave a copious, colored precipitate
Results of the purication are summarized in Table 2.
which was removed by centrifugation and discarded.
The 450 mL of supernatant is M6.
B. Xanthine Dehydrogenase from Cows Milk
6. M6
Hunt and Massey (8) puried cows milk XO in a
M6 was concentrated from 450 to 320 mL by pereva-
dehydrogenase form (XDH) in the presence of 2.5
poration (air current produced by fan) in a Visking
mM dithiolthreitol. While XO reacts rapidly only
cellulose casing for 16 h at 5 C. The protein concen-
with O2 (and xanthine) and not with NAD , the
tration was 1% as measured by absorbance at 280 nm.
XDH form of the enzyme reacts rapidly with NAD .
The solution was cooled to 0 C; 160 mL of 70% (v/v)
XDH has a turnover number for the NAD -dependent
ethanol, precooled in a carbon dioxideacetone bath,
conversion of xanthine to urate of 380 mol/NAD /
was added to the solution from a burette tted with a
min/mol of XDH at pH 7.5 and 25 C, with a Km <
ne capillary, with sufcient stirring to ensure rapid
1 M for xanthine and a Km of 7M for NAD , but
mixing but without frothing. The solution, which
very little O2 -dependent activity. XDH can be con-
began to form a small amount of precipitate, was
verted to the XO form by addition of three to four
held for 16 h at 6 C. The precipitate was collected
equivalents of the disulde-forming reagent 4, 4 0 -
in a refrigerated (6 C) centrifuge at 1300g for 40 min.
dithiodipyridine, suggesting that in the XDH form of
The precipitate was dissolved in water (88 mL), giving
the enzyme disulde bonds are reduced to SH groups.
a total volume of 100 mL. Phosphate buffer (1 M; 1
This may create a binding site for NAD that changes
mL; pH 5.8) and 10 mL of 50% (v/v) ethanol were
the protein structure near the avin (8).
added slowly to give a nal ethanol concentration of
8%. The solution was held at 1 C for 0.5 h and
1. Enzyme Assays
centrifuged. The precipitate was discarded. The super-
natant liquid was seeded with crystals from a previous Xanthine oxidase activity was measured using 165 M
batch and held at 1 C. Crystals appeared after 12 xanthine in 0:1 M sodium pyrophosphate buffer con-
h. In other preparations crystals appeared within 36 h taining 0.3 mM EDTA, pH 8.5, and 25 C in an air-
without seeding. equilibrated solution, by monitoring production of
After 4 days at 1 C, the crystals were collected by urate at 295 nm (
M 9,500 M 1 cm1 ) (8).
centrifugation at 1 C and washed twice with phos- Xanthine dehydrogenase activity was measured
phate buffer containing 7% ethanol (0.01 M phos- using the same assay mixture as above, plus 530 M
phate, pH 6.2, 1 C), using 70 mL of the ethanolic NAD and determining the NADH production at 340
buffer each time. The crystals were dissolved in aqu- nm (
M 6:22 103 M 1 cm1 ) (39). Activities are
eous 0.2 M sodium chloride ( 45 mL) and the solu- expressed in turnover numbers, as moles of substrate
tion was dialyzed for 16 h against 1 L of 0.2 M sodium catalyzed to product/min/mol enzyme-bound avin.

Table 2 Summary of Purication of Xanthine Oxidase
Fraction Volume Total units Spec. Act.a Spec. Act.b

No. (L) (XO) (units/L/E280 ) (units/L/E450 ) E280 =E450 c Step
M1 25 277 0.14d Buttermilk

M2 25 275 Pancreatin treatment
M3 21 96 1st ltrate
M4 20 85 0.53 35 65 2nd ltrate (whey)
M5 1.22 96e 7.4 70 9.4 Eluate
M6 0.45 43 11.1 88 8.0 Starting concentrate
M7 0.050 17 15.2 79 5.2 Crystalline
M7a 0.015 3 10.4 66 6.3 Crystalline
M8 14.2f 71c 5.0c Crystalline
a
Equivalent to total units activity/L/total protein.
b
Equivalent to total units activity/L/total protein.
c
Equivalent to total protein/total avin content.
d
Approx. value due to turbidity of solution.
e
Apparent increase in activity probably due to experimental error.
f
M7 solution before 2nd crystallization gave units/L/E450 of 71, units/L/E280 of 13.3, and E280 =E450 of 5.3.
Source: Ref. 20.
Since XO/XDH have two avins per mol, the turnover 6. Titrate the liquid portion from above using 30
number is per active site. The XDH/XO ratio is calcu- mL enzyme preparation/mL calcium phosphate gel
lated on the basis of these specic assays. (38). Typically, 1 L of calcium phosphate gel is stirred
well with 470 mL of enzyme solution. Centrifuge for 10
2. Enzyme Purication min at 4,000g. Discard supernatant.
7. Resuspend the precipitate in phosphate buffer
Purication of XO in the reduced XDH form (8).
(see 5 above) and centrifuge to collect insoluble mate-
1. To 15 L fresh unpasteurized cows milk, add 50 rial.
mL of 0.75 M dithiothreitol (DTT), 15 mL of 0.1 M 8. Resuspend the precipitate in 500 mL of 10%
phenylmethanesulfonyl uoride (to inhibit serine pro- ammonium sulfate. Centrifuge. Repeat the washing
teases), 15 mL of 1.0 M sodium salicylate, 240 g of of the precipitate three more times, saving the washes.
sodium bicarbonate, and 2840 g of ammonium sulfate. 9. To the combined washes, add ammonium sul-
Stir rapidly for 1 h at 4 C (all steps below are done at fate to increase its concentration to 50%. Centrifuge at
4 C). 16,000g for 30 min. Save the precipitate.
2. Add 2.5 L chilled 1-butanol and stir for 30 min. 10. Resuspend the precipitate in a mixture of 80%
3. Centrifuge the suspension in 800-mL aliquots of 0.05 M Tris, 0.3 mM EDTA, pH 7.8, buffer and
for 15 min at 4000g. Remove the top butanol layer 20% of 0.1 M sodium pyrophosphate, 0.3 mM EDTA,
by suction. Remove the solid lipid layer. Discard both. pH 8.5, with 2.5 mM DTT (Buffer A). Dialyze against
4. To the 15 L of liquid, add 160 g/L of ammo- 2 L of the same buffer overnight.
nium sulfate and stir for 30 min. Then let stand for 2 h 11. A 100-mL portion is removed from the dialysate
to permit the precipitate to rise to the top. Skim off the and loaded onto a folate afnity column, prepared
brown precipitate (in 1:7 L); centrifuge for 1 h at according to Nishino et al. (40), and equilibrated in
16,000g. Remove the liquid and discard. Buffer A. The column is washed with 500 mL of
5. Resuspend the solid precipitate in 500 mL of Buffer A until the 280 nm absorbance reaches a mini-
0.05 M potassium phosphate buffer containing 0.3 mum, then elution is begun with Buffer A containing
mM EDTA, 1 mM salicylate, 2.5 mM DTT, pH 6.0. 10 mM salicylate. XDH moves gradually down the
Dialyze twice, using 6 L of the same buffer solution column and elutes as a single dark band.
each time. Centrifuge the dialysate ( 470 mL) at 12. The dark band fractions from step 11 above are
16,000g for 1 h. Decant the liquid portion and save. pooled, concentrated, and dialyzed in 0.1 M sodium
Discard precipitate. pyrophosphate, 0.3 mM EDTA, pH 7.5, buffer. The

Table 3 Purication of Milk Xanthine Dehydrogenase
XDH/XO
specic XDH/XO Dehydrogenase:
Purication Volume XDH/XO Protein activity purication oxidase activity
step (mL) (total units) (mg/mL) (units/mg) (fold) (ratio)
Fresh milk 15,000 747/870 8 0.0062/0.0072 1/1 1.3

After (NH4 2 SO4 =butanol 470 800/260 7 0.24/0.79 38/11 3.1
fractionation and dialysis
After calcium phosphate gel 2,000 810/250 0.4 1.0/0.31 161/43 3.2
Before folate column 100 620/85 4.7 1.3/0.18 210/25 7.3
After folate column 24 450/46 12 1.56/0.16 252/22 9.7
Source: Ref. 8.
liquid fractions from step 1 are stored at 4 C in the 7. CM Harris, V Massey. The reaction of reduced
buffer above with 1 mM salicylate and 2.5 mM DDT xanthine dehydrogenase with molecular oxygen. J
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21485, 1992.
Table 3 summarizes the results of the purication of 9. C Enroth, BT Eger, K Okamoto, T Nishino, T
Nishino, EF Pai. Crystal structures of bovine milk
milk XDH.
xanthine dehydrogenase and xanthine oxidase: struc-
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43
Lipoxygenase and Associated Enzymes
Harold W. Gardner
U.S. Department of Agriculture, Peoria, Illinois, U.S.A.
I. INTRODUCTION tions, oxidations of C:20 fatty acids occur either at !6

(C-15), !9 (C-12), or !16 (C-5) position with (S)-
A. Reactions Catalyzed
stereospecicity (7). As discussed below, the list of sub-
strates include additional natural and synthetic sub-
Lipoxygenase (LOX), a nonheme iron protein, is
strates; many of the latter give reduced activity.
undoubtedly universally distributed in plants and ani-
Generally, plant LOXs having high pH optima oxidize
mals. Fungal LOXs are known that give unique pro-
at the !6 position if incubated at high pH, and those
ducts and some of these LOXs apparently operate by
with optima at neutral or low pH mainly afford !10
different mechanisms (1), including a LOX with a man-
oxidation or both types of oxidation. The spatial rela-
ganese active site (2). A number of recent publications
tionship of removal of the bis-allylic hydrogen, and an
review various aspects of LOX (315). Although many
S-oxidation at !6 versus !10 requires an alignment
LOXs have been thoroughly characterized, much of
of the substrate into the active site of LOX in reverse
the literature focuses on one soybean seed isozyme,
orientationsthat is, head rst or tail rst. This
LOX-1. In this review a detailed account of soybean
can be deduced by reference to Figure 1. Since redu-
LOX-1 is unavoidable because the literature is too
cing the pH of the incubation has been observed to
extensive to realistically review the eld in its entirety.
lower the ratio of !6 to !10 oxidation of linoleic
LOXs catalyze the dioxygenation of methylene-
acid by soybean LOX-1 (16) and also lowers the
interrupted pentadiene fatty acids to afford conjugated
ratio of !6 to !16 oxidation of arachidonic acid by
hydroperoxydiene fatty acids. With few exceptions,
rice LOX-2 (17), this suggested that either a head-
plant LOXs oxidize linoleic and linolenic acids regios-
rst or tail-rst alignment at the active site was depen-
pecically at either the !6 (C-13) or !10 (C-9) position
dent on the charge of the carboxylate/carboxylic acid
with (S)-stereospecicity (Fig. 1). In the animal king-
group. It is probably because of reverse orientation
dom LOXs oxidize linoleic and linolenic acids by
phenomenon that arachidonic acid is all-S doubly
mechanisms similar to those in the plant kingdom,
dioxygenated at both ends of the polyene structure
but many of the important oxidations of physiological
(17, 18).
signicance utilize arachidonic acid and other C:20
The rate-limiting step is an H removal from the bis-
polyenoic acids as substrates. Although plant LOXs
allylic methylene of the pentadiene on the opposite side
are capable of oxidizing C:20 polyenoic acids, these
of O2 insertion (19, 20). The immense size of an
substrates are unnatural in plants. With some excep-
observed kinetic isotope effect with deuterated linoleic
acid indicates that hydrogen tunneling occurs in the

Retired. LOX reaction (21). As shown in Figure 2, the iron

Figure 2 The iron redox cycle of the LOX active site shows
the reaction of linoleic acid with O2 under conditions of
Figure 1 Oxidation of either linoleic (18:2) or linolenic adequate aeration, as well as an aberrant reaction with lino-
(18:3) acid by plant LOXs is preceded by stereospecic leic acid hydroperoxide under O2 -starved conditions. Shown
removal of hydrogen followed by placement of oxygen on in bold are the entry of linoleic acid (RH) and O2 into the
the opposite side of the molecule from hydrogen removal. cycle and products initially released from the active sitethat
Plant LOXs usually produce the 13(S)- and/or 9S-hydro- is, linoleic acid hydroperoxide (ROOH), the alkoxyl radical
peroxides of linoleic or linolenic acid as shown. of linoleic acid hydroperoxide (RO), and the linoleic acid
pentadienyl radical (R). The alkoxyl radical (RO) often
active site cycles electrons by a redox mechanism from rearranges into an epoxyallylic carbon radical which reacts
H removal (Fe3 to Fe2 transition) to conversion of with O2 to form epoxyhydroperoxyoctadecenoic acid
the fatty acid peroxyl radical to the hydroperoxide (HOORO), leading to epoxyhydroxyoctadecenoic acid.
anion (Fe2 to Fe3 transition) (15, 22, 23). Also ROR represents dimers of epoxyoctadecenoic acid with the
shown in Figure 2 is an O2 -starved cycle of LOX conjugated diene of linoleic acid. RR represents conjugated
(22, 23). When LOXs become decient in O2 the linoleic acid dimers, and rac-ROOH signies racemic hydro-
enzyme undergoes an aberrant reaction whereby the peroxides formed by nonenzymic reaction of O2 with the
pentadienyl radical of linoleic acid. Refs. 15, 22, and 23.
product hydroperoxide fatty acid (ROOH) replaces
ROO as an oxidant of the active site. This results in
formation of a fatty acid alkoxyl radical (RO) from and phosphoglycerides (29, 30) have been described,
the hydroperoxide, as well as unoxidized pentadienyl but the activity is often reduced compared to free poly-
radicals from substrate. From linoleic acid and its 13- unsaturated fatty acids. Recently, it has been found
hydroperoxide, a number of compounds are formed by that one of the olenic double bonds can be replaced
free radical chemistry, among which are pentane, 13- by a carbonyl double bond, such as a ketone (31) or
oxo-9,11-octadecadienoic acid, 13-oxo-9,11-tridecadie- aldehyde moiety (32). Many unnatural substrates have
noic acid (24), dimers (25), and racemic fatty acid been synthesized to study enzyme mechanisms, among
hydroperoxides (26). Similarly, from linolenic acid which two detailed studies are cited here as examples of
and its 13-hydroperoxide, one obtains isomeric pente- the substrate structural requirements other than those
nols, 13-oxo-9,11-tridecadienoic acid, and dimers (27). found in usual substrate fatty acids (33, 34).
It is relatively easy to obtain O2 -starved conditions,
such as by using excess concentrations of LOX and
substrate, and by low O2 solubility due to increased C. Classication According to Enzyme
temperatures of incubation. For good yields of hydro- Commission
peroxides, one should optimize O2 dispersal/solubility,
and use optimal enzyme concentrations (6). According to the IUBMB (35), LOX is classied as a
linoleate:oxygen 13-oxidoreductase (EC 1.13.11.12).
However, many LOX isozymes that have been thor-
B. Structure of Substrates oughly characterized by sequence have been shown to
catalyze other oxidations, such as linoleate:oxygen 9-
Natural fatty acid substrates were discussed above. oxidoreductase; thus, it would appear that classica-
Other natural substrates, such as glycerides (28, 29) tion of LOX should be amended.

II. IMPORTANCE TO QUALITY OF FOOD aldehydes and a C-12 oxoacid, the former giving ran-
cid-green (hexanal) and grassy (3Z-hexenal) odors. 9-
LOX is known to have several detrimental effects in Hydroperoxides cleaves into C-9 aldehydes (cucumber-
foods (see reviews: 36, 37), and these effects can be like odors) and C-9 oxoacid (see review: 46). The alde-
controlled by various methods of enzyme denatura- hydes are then subject to further transformation by
tion. By the process of cooxidation by free radicals, enzymes, such as reduction to alcohols. The alcohols,
LOX activity is known to cause bleaching of carote- which generally have similar odor proles as the alde-
noids, chlorophyll (38, 39, and Refs. therein), and oxi- hydes, can also be transformed into other avor vola-
dation of ascorbic acid (40). LOX probably accounts tiles, such as the intense apple avor hexyl acetate. The
for a large portion of pigment bleaching and loss of 3(Z)-alkenals produced by hydroperoxide lyase are
quality observed in unblanched frozen vegetables. In susceptible to transformation by isomerase into 2(E)-
addition, products of LOX action, fatty acid hydroper- alkenals (47) or oxidation to 4-hydroperoxy-2(E)-alke-
oxides, are cleaved both enzymically and nonenzymi- nals by LOX itself and possibly other enzymes (32).
cally into odorous shorter-chain aldehydes, alcohols, Most of the volatile aldehydes and alcohols are con-
and alkanes. The nonenzymic formation of odors is sidered very desirable fresh vegetable odors, and con-
very detrimental leading to complex free radical pro- trast with the complex mixture of odors obtained by
ducts broadly dened as rancidity (see review: 41). the free radical process of rancidity development.
LOX reactions can even occur in dry substrates at However, in soybean hexanal is considered to be an
the lowest relative humidities examined (52%) (42). It off-avor, and, as described below, mutants have
was also demonstrated that LOX action in dry sub- been developed to eliminate LOX isozymes from the
strates initiated free radical autoxidation of polyunsa- seed. Also, it has been reported that a LOX/hydroper-
turated fatty acids. oxide lyase sequence is responsible for the odor of fresh
Although LOX is often considered to be detrimental sh (48).
to foods, there are examples of positive uses. LOX has In the multistep biosynthesis of an important jas-
been used in replacing bromates to improve dough mine odor, methyl 7-iso-jasmonate (49), the enzyme
elasticity for bread making presumably by increasing allene oxide synthase catalyzes the inaugural step of
disulde bonds through oxidation (see review: 43). In 12-oxo-10,15-phytodienoic acid formation from 13-
addition, LOX bleaches carotenoid through cooxida- hydroperoxide of linolenic acid. It is of interest to
tion in doughs increasing the whiteness of bread. As note that methyl jasmonate, the racemate of methyl
discussed below, the LOX/hydroperoxide lyase 7-iso-jasmonate, is odorless (49). Allene oxide
sequence, also involving other enzymes such as alcohol synthase, a cytochrome P450 designated as CYP74A
dehydrogenase and double-bond isomerase, is impor- (50), has been cloned and sequenced from a number of
tant in imparting odors and avors to fresh fruit and sources (e.g., 50). When allene oxide synthase is active,
vegetables. These avors are important enough to per- it also has the practical function of removing rancidity-
suade Firmenich to develop an enzymic procedure for causing hydroperoxide fatty acids from foods.
their biosynthesis (44). Another plant hemoprotein, hydroperoxide peroxy-
genase (not yet sequenced), utilizes one of the hydro-
peroxide oxygens to epoxidize a double bond intra- or
III. ENZYMES FOUND IN RAW FOODS
intermolecularly, and in the process the hydroperoxide
A. Enzymes Acting on Hydroperoxide Products itself is reduced to a hydroxide (51). Hydrolysis of the
of LOX epoxide by epoxide hydrolase furnishes the corre-
sponding diol. The resulting hydroxyl/dihydroxy/trihy-
Generally, in raw foods LOX often operates in concert droxy fatty acids have been implicated in imparting
with other enzymes by sequential reactions. In plants, bitter tastes to certain foods (52). Other hydroperox-
some of the cytochrome P450 enzymes, usually mem- ide-metabolizing enzymes, like divinyl ether synthase
brane bound, are of considerable importance to food (53 and Refs. therein), have little known effects on
quality. Each raw food has its unique blend of hydro- foods.
peroxide-utilizing enzymes. One of the most important
of these enzymes is hydroperoxide lyase, which has B. Location of LOXs
been cloned, sequenced, and identied as a cytochrome
P450 (CYP74B) (45). Hydroperoxide lyase cleaves 13- In plants LOXs are found in all tissues with a few
hydroperoxides of 18:2 and 18:3 fatty acids into C-6 exceptions. For example, soybean mutants have been

found devoid of all three LOX isozymes normally and mammalian LOXs of 2127%, among plants of
found in the seed (54), but it is likely that the absence 4386%, and among mammals of 3993% (68).
of LOX in these mutants is only seed specic. Coral LOX, which catalyzes an unusual 8R-specic
LOX is generally regarded as a soluble enzyme oxidation of arachidonic acid, warrants special men-
located in the cytoplasm, but recent research demon- tion as it exists as a fusion protein with allene oxide
strated a far more complex organization. Using tech- synthase (69). The allene oxide synthase portion of the
niques such as immunolocalization, LOX isozymes in protein is not a cytochrome P450, like that found in
soybean seed are generally found in the cytoplasm plants, but it shares homology with catalase.
around the protein bodies of storage parenchyma,
but LOX is also associated with aberrant protein B. Isozymes
bodies in the hypodermis and vascular bundle sheaths
(55, 56). In soybean leaves, LOX isozymes also func- Known LOX isozymes are too numerous to review
tion as vegetative storage proteins being regulated by here. However, soybean can serve as an example. As
source-sink status. These isozymes differentially locate discussed above, soybean seeds normally have three
in vacuoles, in the cytosol of paravienal mesophyll and isozymesLOX-1, -2, and -3 (54, 70). The process of
in the bundle sheath along with other storage proteins germination gave expression of an additional three iso-
(57). Also, organ-specic expression of LOX isozymes zymes in seedlings, LOX-4, -5, and -6 (70). Multiple
has been observed, such as that found in potato (58). nonseed LOX isozymes have been identied in the soy-
It seems certain that membrane-associated LOXs bean leaves (71). Ten isozymes of the soybean plant
are important. Although there are a number of reports have now been identied (see review: 4).
showing LOX to be associated with microsomes and Posttranslational modication of soybean seed iso-
plasma membranes, there is little evidence to show zymes has been studied. It has been shown that LOX-1
specic targeting to these membranes; that is, such underwent transformation with incubation at room
associations may be nonspecic binding. Numerous temperature to furnish ve separable forms with dif-
reports of the localization of LOXs in chloroplasts of ferent specic activity (72). Cleavage of LOX-1 and -3
a variety of plants (e.g., 59) have been authenticated in by trypsin or chymotrypsin afforded two domains
some cases by showing the presence of a chloroplast 60 and 40 kDa without inactivation (73 and Refs.
transit peptide (60, 61). Chloroplast LOXs have been therein). The two domains were tightly associated,
reported to be induced by wounding, pathogenesis, because the separated fragments did not possess activ-
and methyl jasmonate. LOXs presence in chloroplasts ity (74).
presumably functions to initiate a cascade of reactions
through chloroplast-bound hydroperoxide-utilizing
V. PROPERTIES AS ENZYME
enzymes (62). There are several reports of a specic
LOX being localized in lipid bodies (e.g., 63), and A. Specic Mechanism of Action
this type of LOX has been reported to preferentially
metabolize esteried fatty acids (64). As shown by Figure 2, the mechanism of action is an
iron-catalyzed one-electron redox cycle that causes free
radical oxidation of polyunsaturated fatty acids. X-ray
crystallography revealed many clues of the details of
A. Protein Structure catalysis. Not obvious in the structural depiction of the
LOX protein shown in Figure 3 is a conical hydropho-
Except for some LOXs from fungi, all are nonheme bic cavity, or tunnel, that connects the bottom of the
iron enzymes ranging in size from 90 to 100 kDa. enzyme with the iron active site, which is believed to be
Soybean seed LOX-1, with a molecular weight of 94 a path for O2 . A substrate channel was identied by a
kDa, serves as a model for several reasons, not the long, narrow hydrophobic cavity with two bends lying
least of which are its abundance and priority of isola- to the right of the iron (75). As discussed above, the
tion in crystalline form (65). LOX-1 has been cloned iron active site serves to cycle electrons. Iron is com-
and sequenced (66). As shown in Figure 3, the three- plexed by three histidines and one isoleucine. Higher-
dimensional structure has been determined by x-ray resolution x-ray crystallographic analysis showed that
crystallography (67). Numerous LOXs from plants iron was additionally liganded with one water and
and animals have now been cloned and sequenced, weakly with asparagine694 (76), making a total of six
affording a pairwise sequence identity between plant coordinates (Fig. 4). It has been proposed that the

water ligand exists as a hydroxyl anion/water transi-
tion that accepts hydrogen from the substrate (see
review: 15). Asparagine694 (or its equivalent in sequen-
tial position) is mostly conserved in various LOXs, but
not completely (15); replacement by histidine, as
observed in certain LOXs of mammalian origin,
reduced kcat signicantly (77).
B. Kinetics, K m , kcat , Activation Energy
A number of factors must be considered prior to deter-

mining LOX kinetics. Kinetics should be determined
below the critical micelle concentration (CMC), which
is dependent principally on the pH, and the presence of
Figure 3 The three-dimensional structure of soybean lipox- other hydrophobic substances, including surfactants.
ygenase-1 determined by x-ray crystallography. The iron The CMC for linoleic acid has been reported for Na
active site is shown as a sphere, -helices by cylinders, strands borate buffer (0.1 M) at 167, 60, and 21 M at pH 10,
in -sheets by arrows, coils by narrow rods. (From Ref. 75.)
9, and 8, respectively (78). Ca2 has been reported as a
cofactor of LOX, but this ion is ineffective below the
CMC, indicating that it affects CMC by calcium salt
formation (79). However, Ca2 may not be completely
without effect in vivo. It has been recently reported
that Ca2 stimulated the membrane binding of soy-
bean LOX-1, implying a regulatory mechanism for
Ca2 (80).
An initial lag in the kinetics of LOX must also be
considered. The lag can be overcome simply by adding
a small quantity of product hydroperoxide (81). This
can be interpreted as an initial oxidation of native
Fe2 -LOX by hydroperoxide product to give Fe3 -
LOX to prime the catalytic cycle, but detailed kinetic
analysis suggested a somewhat more complex interpre-
tation (23).
The turnover rate, kcat , and Km for soybean LOX-1
with linoleic acid substrate has been variously reported
at 280350 sec1 and 1225 M, respectively (15, 81,
and Refs. therein).
The activation energy (Ea ) for soybean LOX-1 and
linoleic acid was determined to be 22 kcal/mol, and
H6 21 kcal/mol, S6 20 e.u., and G6 15
kcal/mol (82, 83).
A kinetic scheme has been developed (23 and Refs.
therein) and was recently modied by Solomon et al.
(15) to account for recent ndings of the active site.
Figure 4 The six-coordinate ligands of the iron active site of Figure 2 essentially illustrates this scheme modied to
soybean LOX-1 showing ve amino acids with their num-
clearly show the aerobic and O2 -starved aspects of the
bered positions in the sequence; the sixth ligand (H2 O) is
active site redox cycle, as well as the products obtained.
thought to abstract hydrogen from substrate (OH to H2 O
transition) as shown. The position of the three histidines and
isoleucine was determined by x-ray crystallography (67), and C. Effect of pH, Inhibitors, and Surfactants
high-resolution x-ray analysis showed the presence of H2 O
and asparagine as ligands (76). The OH to H2 O transition Soybean LOX-1 is generally stable over a wide range
has been proposed by a number of investigators (e.g., see 15). of pHs. Its activity is optimum between pH 9 and 10.

Activity is negligible at pH 6.5, but it is stable at pHs as efciencies are obtained with combinations of heat and
low as 4.5. Adjustment to pH 4.5 has been used with other treatments, such as ionizing irradiation (97),
crude preparations to remove unwanted protein in alcohol (96), sonication (98), and pressure (99). As dis-
LOX purication (84). However, pH 3.0 and below cussed above, low pH alone inactivates LOX (85), but
causes irreversible inactivation of soybean LOX activ- pH also plays a role in inactivation by heat (97) and
ity (85). combined treatments (98, 99).
Although many inhibitors of LOX have been inves-
tigated, only representative examples can be cited here.
VI. QUALITATIVE AND QUANTITATIVE
First, it should be emphasized that soybean LOX cat-
alyzes its own destruction during oxidation of sub-
strate. The destruction is greater with substrates of a A. Class Action with Assay Conditions
higher degree of unsaturation (86). Several types of
inhibitors mainly can be categorized as follows (for Axelrod et al. (84) outlined spectrophotometric meth-
citations also see 86 and Refs. therein): (a) substrate ods for assaying all three soybean seed isozymes. For
suicide inhibitors, such as acetylenic fatty acids (irre- assay of LOX-1, the cuvette contains 2.975 mL of 0.2
versible) (87); (b) chain-breaking antioxidants (usually M Na borate buffer, pH 9, containing LOX-1, and
competitive and reversible) (88); (c) iron chelators then 25 L substrate solution is added, stirred and
(often reversible) (89); (d) disrupters of the active site monitored at 234 nm at 25 C. The substrate solution
(90); (e) reductants of the active-site iron (91); (f) free was Na linoleate (10 mM) containing a weight of
radical reactions with LOX, such as with hydroxyl Tween 20 equal to the weight of linoleic acid used;
radicals produced from H2 O2 (presumably irreversible) nal concentration of Na linoleate in the reaction
(92); and (g) substrate mimics (usually competitive) was 0.083 mM. The rate is the straight-line portion
(93). In regard to substrate mimics, such as various observed after the initial lag period. Activity is dened
fatty acids, potential errors are possible because of in units (mol of product formed/min). Specic activ-
their disruption of CMC stability (94). That is, some ity is dened as units/mg or g protein. The mol of
fatty acids actually may not be inhibitors, and exert product is calculated from the molar extinction coef-
their effect by changing the CMC of the substrate. cient of the conjugated hydroperoxydiene measured at
Nonionic surfactants, such as Tween 20 and Triton 234 nm. Like most investigators, Axelrod et al. (84)
X-100, are often used by researchers to clarify sub- used a molar extinction coefcient of 25,000 M1
strate solutions. However, it has been found that sur- cm1 , but this value is probably too low. Our lab
factants actually decrease activity, except when uses a higher value of 26,800 M1 cm1 (6).
concentrations of substrate and surfactant are high Several comments should be made regarding the
and low, respectively (95). Kinetic data suggested spectrophotometric method. The method is very satis-
that surfactant sequestered substrate in micelles, factory for highly puried LOXs with high pH optima.
whereas the actual substrate was indicated to be sol- However, assay of crude enzyme preparations by this
vated monomers. That is, substrate could be oxidized method is hazardous because protein and other UV-
only after escaping micelles in equilibrium with the absorbing materials may absorb all or most of the light
aqueous phase. The rate increase at high substrate at 234 nm, and hydroperoxide-utilizing enzymes, if
and low surfactant concentrations was interpreted as present, generally destroy the conjugated diene chro-
alleviating substrate inhibition by sequestering sub- mophore as it is being formed. Also, when LOX con-
strate in micelles. Besides surfactants, ethanol or centrations are extremely high, the absorbance could
methanol is often used to conveniently disperse fatty increase to a maximum before the assay could be
acid substrates, but alcohols also inhibited the activity started; this could mislead the investigator to the
of LOX, increased the Km and decreased the Vmax (96). false conclusion that no LOX activity is present.
The alcohol effect was found to be reversible. Third, inhibitory surfactants are included to clarify
the solutions to avoid scatter of UV light. In our
D. Control Methods in Foods laboratory we have found that pHs below 7.5 result
in artifactual absorbance changes due to imperceptible
Heat is a simple, commonly used method to inactivate changes in micelles, even in the presence of surfactant.
enzymes, including LOX. The inactivation rate con- For these reasons, the use of O2 -electrode measure-
stant of soybean LOX activity at 65 C was found to ment of O2 uptake can be a reliable and sensitive
be 8 104 sec1 at pH 57 (97). Greater inactivation method to assay LOX activity. The method described

Table 1 Purication of LOX-1 from 200 g Defatted Soybean Meal
Total Specic
Volume Protein activity activity Fold
Purication step (mL) (mg) (units)a (units/mg) purication
Crude extract (0.2 M Na acetate, pH 4.5) 1,160 20,800 95,900 4.72 1b

NH4 2 SO4 (0.30.6 saturation, pH 6.8) 170 3,424 73,400 21.4 4.5
DEAE-Sephadex chromatography 172 229 24,900 101 21
Rechromatography on DEAE-Sephadex 106 81 14,600 180 38
a
A unit is dened as 1 mol product formed/min.
b
The rst step of extraction at pH 4.5 gave some purication from unwanted protein that would normally be extracted at higher pH. This
inherent purication is not reected in the fold purication of 1.
Source: Ref. 84.
above can be utilized, except the substrate is linoleic Triton X-100 was removed from the supernatant
acid dissolved in a minimum of methanol to aid dis- after centrifugation with Amberlite XDA-2. An initial
persal. This avoids the use of inhibitory surfactants, purication was achieved by a DEAE-Toyopearl col-
but alcohols are also inhibitory at high concentrations umn, and nal separation was accomplished with
(96). Methanol can be kept to a minimum to give neg- Mono Q. One isozyme that did not bind to DEAE-
ligible inhibition (e.g., 25 L substrate dissolved in Toyopearl required a different purication with CM-
methanol injected into 2.5 mL buffered enzyme solu- Toyopearl and Mono S columns.
tion). With the advent of cDNA technology, LOX is
increasingly puried from E. coli; however, it has
B. Assays for Screening Purposes been found necessary to cultivate E. coli at low tem-
peratures in order to obtain active LOX (105, 106).
In instances where seed or other material is being
screened for absence or presence of LOX activity,
there is need for rapid assay methods. A number of REFERENCES
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44
Sorbitol Oxidase
Kohei Oda and Kazumi Hiraga

Kyoto Institute of Technology, Kyoto, Japan
I. INTRODUCTION SOX gene and succeeded in expression of the recombi-

nant SOX with covalently bound FAD in E. coli with
Various pathways for the use of sorbitol have been total activity about 240-fold higher than that of
described. Sorbitol dehydrogenase (SDH; EC Streptomyces sp. H-7775 (5).
1.1.1.14) is an enzyme of the polyol pathway in a Sorbitol oxidase (SOX) from Streptomyces sp. H-
wide variety of species (Fig. 1) (1-3). SDH acts on 7775 converts D-sorbitol to glucose in the absence of
polyols such as D-sorbitol and D-xylitol but has no exogenous cofactors, and the other reaction product is
activity toward primary alcohols. SDH catalyzes the hydrogen peroxide. SOX catalyzes the following reac-
oxidation of D-sorbitol to form D-fructose with tion (4).
NAD as a cofactor.
D-Sorbitol O2 ! Glucose H2 O2
In the course of screening for glycerol oxidase-pro-
ducing microorganisms, we found an enzyme that oxi-
CH2 OH
dized D-sorbitol in the cell-free extract of a strain
j
isolated from soil. The enzyme is capable of oxidizing
HCOH
D-sorbitol to produce hydrogen peroxide and glucose
j
without the requirement of exogenous cofactors such
HOCH
as NAD , NADP , and FAD. Apparently, this type
j
of sorbitol-oxidizing enzyme has a different reaction
HCOH
mechanism from the SDHs described above. Hence,
j
according to the reaction mechanism, we named the
HCOH
enzyme sorbitol oxidase (EC 1.1.3.) (SOX). This
j
enzyme contains covalently bound FAD. Moreover,
CH2 OH
when D-sorbitol is used as a substrate, the SOX does
not act on the reaction product, glucose. These proper-
D-Sorbitol
ties of the SOX will provide a precise and routine
determination of D-sorbitol concentration even in a Based on the chemical reaction catalyzed by SOX
crude sample and may be suitable for technical appli- as described above, SOX is classied in a subclass of
cations (4). oxidoreductases (subclass 1), within six main types of
The expression level of SOX in Streptomyces sp. H- enzymes. SOX can act on the CHOH group of donors
7775 was very low (10 units/L culture). To obtain a (subclass 1.1). This can be further classied in a
large amount of SOX from H-7775, we cloned the subclass according to the acceptor, which is an

V. PROPERTIES AS PROTEIN
The molecular weight of SOX puried from the cell-

free extract is 45,000 daltons. The prosthetic group is a
covalently bound FAD. The absorption spectrum of
SOX puried from Streptomyces sp. H-7775 has a typi-
Figure 1 The polyol pathway.
cal avoprotein spectrum with the absorption maxima
at 276, 358, and 455 nm and a shoulder at 480 nm
(Fig. 2A,B). A hypsochromic shift of the second
oxygen. According to Enzyme Nomenclature (6), absorption band to 358 nm relative to that of ribo-
SOX from Streptomyces sp. H-7775 is classied as avin at 372 nm has also been observed. The spectrum
EC 1.1.3. is similar to those of avoproteins with covalently
bound avin (1114). With the addition of D-sorbitol,
the peaks at 358 nm and 455 nm decrease owing to the
reduction of avin (Fig. 2C). This indicates that the
II. IMPORTANCE TO QUALITY OF FOOD
avin component is functionally involved in the oxida-
DURING GROWTH, MATURATION,
tion of D-sorbitol. The uorescence intensity of the
STORAGE, AND PROCESSING
puried SOX is pH dependent and is similar to that
of FAD as in the case for choline oxidase, but different
Applications of SOX from Streptomyces sp. H-7775 to
from that of FMN and riboavin (14). The avin
quality of food during growth, maturation, storage,
prosthetic group could not be liberated from the
and processing have not been investigated. The regula-
puried SOX protein by (a) acidication with 5%
tion of the metabolism and transport of sorbitol in
trichloroacetic acid, (b) boiling for 5 min, (c) treatment
fruit may have an important inuence on the accumu-
with 1% SDS, or (d) dialysis against 3 M KBr in 1 mM
lation of photosynthates in fruit, and consequently on
EDTA for 2 days at 4 C (15). It was calculated that 0.9
their yield and quality (79). Sorbitol-oxidizing activity
mol of FAD is bound to 1 mol of SOX, assuming the
was detected in apple leaves (10).
molecular absorption coefcient for FAD at 460 nm to
be 11,300 M1 cm1 (16).
Recombinant SOX has been expressed in E. coli and
III. RAW FOODS IN WHICH ENZYMES puried. The SOX was homogeneous by SDS-PAGE
ARE FOUND showing a strong yellowish uorescence under UV
light ( 265 nm) and acidic condition. The failure
SOX from Streptomyces sp. H-7775 is localized in
mycelia. The subcellular location of SOX was deter-
mined semiquantitatively in the cell-free extract, cell
debris, and culture supernatant after disruption of
the mycelia by ultrasonication using the method
described in Section VIII. SOX activity was detected
only in the cell-free extract.
IV. UTILITY/UTILIZATION OF ENZYMES

IN FOODS (INCLUDING EXOGENOUS
APPLICATION)
SOX from Streptomyces sp. H-7775 may be used for

determination of D-sorbitol and D-xylitol concentra- Figure 2 Absorption spectra of puried SOX. Absorption
tion added as additives in foods. As SOX contains spectrum of puried SOX (A) in 20 mM potassium phos-
covalently bound FAD, the enzyme activity is not phate buffer, pH 6.0, at the nal concentration of 0.64 mg/
inuenced by exogenous cofactors such as NAD , mL. The insert shows the enlarged spectrum before (B) and
NADP , or FAD in food. after (C) addition of 50 mM D-sorbitol.

to dissociate the cofactor by the same methods The deduced amino acid sequence of the SOX gene
described above has led to the conclusion that FAD has 25.3% identity and 68.1% similarity to that of rat
is covalently bound to the SOX even when it is L-gulonolactone oxidase (EC 1.1.3.8) (10) (Fig. 3).
expressed in E. coli (5). SOX does not oxidize L-gulonolactone. One of the
The molecular weight of SOX from Streptomyces sp. features of rat L-gulonolactone oxidase is that the
H-7775 was estimated to be 45,000 daltons by both enzyme contains a FAD covalently bound to histidine
SDS-PAGE and Sephadex G-75 column chromatogra- (18, 19) and has no nucleotide-binding motif (Gx
phy. This has the same value of the molecular weight of GxxG) often found at the amino terminal region
the puried recombinant SOX expressed in E. coli. in a number of dehydrogenases (20-26). Streptomyces
The SOX gene from Streptomyces sp. H-7775 was SOX also contains no nucleotide-binding motif. The
cloned and sequenced (accession number for cofactor that may be involved in the SOX is covalently
GenBank, EMBL, and DDBJ Nucleotide Sequence bound FAD. Until now, ve types of covalently bound
Database is AB000519). It consists of an open reading avins are known: 8--[N(1)-histidyl]-FAD, 8--[N(3)-
frame of 1260 bp encoding a protein of 420 amino histidyl]-FAD, 8--S-cysteinyl-FAD, 6-S-cysteinyl-
acids with a molecular weight of 45,148 daltons (5). FMN, and 8--O-tyrosyl-FAD (27). Thus, His, Cys,
This value is in good agreement with that of an authen- and Tyr residues are suggested to constitute the site
tic SOX puried from Streptomyces sp. H-7775. for the covalently bound FAD. The SOX molecule
Figure 3 Comparison of the deduced amino acid sequence of SOX with that of the rat L-gulonolactone oxidase. The deduced
amino acid sequence of the SOX gene (upper, I) is compared with that of rat L-gulonolactone oxidase (lower, II). Asterisks and
dots indicate identical and related amino acids, respectively. Closed circles indicate the histidine residue that is possible for
covalent attachment of FAD.

has 12 His, two Cys, and seven Tyr residues. The simi- tol were oxidized most rapidly. D-Xylitol, D-mannitol,
larity between the SOX and rat L-gulonolactone oxi- and D-arabitol were oxidized at a rate of 93.5%, 55%,
dase shows that the similarities are mainly found and 39%, respectively, of that for D-sorbitol. Glycerol,
around His residues (His45 and -374). These His resi- 1,3-propanediol, 1,3-butanediol, and 1,4-butanediol
dues occupy similar positions in the alignment as were oxidized at low rates, while D-glyceraldehyde,
marked by a closed circle in Fig. 3. The His residue the dihydroxyacetone, methanol, ethanol, 1-propanol,
that has been suggested to be responsible for covalent and 1-butanol were not attacked at all at various sub-
attachment of FAD of the rat L-gulonolactone oxidase strate concentrations from 1 to 100 mM (Table 1).
and mitomycin C resistance protein is located at the N- SOX did not react with glucose, a reaction product
terminal region according to the sequence similarity of D-sorbitol.
(28). However, in the case of SOX, amino acid residues Enzyme characteristics of SOX are summarized in
around His374 located at the C-terminal showed Table 2. Optimum pH is between 6.5 and 7.5 (Fig. 4A)
higher similarity than those of His45 located at the N Optimum temperature is 50 C in 10 min incubation at
terminal. Comparison of the amino acid sequences pH 7.5. SOX is stable below 55 C in 15 min incubation
around Cys and Tyr residues between SOX and rat at pH 7.5 (Fig. 4B). The enzyme activity is completely
L-gulonolactone oxidase shows no possible site contri- lost after incubation for 15 min at pH 7.5 at 80 C.
buting to the binding of avin. Moreover, Cys- and SOX is stable between pH 7.5 and 10.0 after 24 h
Tyr-binding regions of other avoproteins have no incubation at 30 C. The enzyme activity is inhibited
sequence similarity to SOX (29, 30). From these by 1 mM monoiodoacetate (IAA) and 1 mM N-ethyl-
results, it is suggested that the His374 is responsible maleimide (NEM) by 53.8% and 54.8%, respectively.
for the binding of FAD. The enzyme activity is inhibited by 1 mM of Zn2 and
There are no isoforms of SOX from different genes. Hg2 by 96.8 and 90.7%, respectively (Table 2).
There are no data about the possible formation of Sugar alcohols that have the R conguration on C3,
isozymes from posttranslational modication. such as D-sorbitol, D-xylitol, D-mannitol (C2 epimer
of D-sorbitol), and D-arabitol (C4 epimer of D-xylitol),
are good substrates of SOX. However, ribitol (C3 epi-
VI. PROPERTIES AS ENZYME mer of D-xylitol) and D-threitol, which have S cong-
uration at C3 are poor substrates of SOX. The results
Substrate specicity of SOX from Streptomyces sp. H- suggest that the stereochemical requirement at position
7775 is summarized in Table 1. D-Sorbitol and D-xyli- C3 of sugar alcohol is important for SOX activity.
Figure 4 Optimum pH (A) and heat stability (B) of SOX. (A) SOX activity was measured at various pHs for 10 min at 37 C.
Buffers used were 40 mM citrate-Na2 HPO4 (pH 4.07.5, open circles), 40 mM Tris-HCl (pH 7.58.5, closed circles), 40 mM
glycine-NaOH (pH 8.510.0, open triangles). (B) SOX was incubated at various temperatures for 15 min in 40 mM Tris-HCl
buffer, pH 7.5, and the remaining activity was measured at 37 C.

Table 1 Substrate Specicity of Sorbitol Oxidasea
Relative
activity Relative activity
Compound (%) Compound (%)
D-Sorbitol 100 (Km 0.26 mM) D-Fructose 0
D-Xylitol 93.5 (Km 0.38 mM) Saccharose 0.30
D-Mannitol 55.0 (Km 7.38 mM) Maltose 1.30
D-Arabitol 39.0 (Km 7.73 mM) Lactose 0.10
Ribitol 4.10 Methanol 0
meso-Erythritol 3.80 Ethanol 0
D-Threitol 0 1-Butanol 0
Inositol 3.30 1-Propanol 0
Glycerol 3.70 2-Propanol 0
D-Glyceraldehyde 0 1,2-Propanediol 0.80
Dihydroxyacetone 0 1,3-Propanediol 2.70
Dihydroxyacetone phosphate 0 1,2-Butanediol 0.80
D-Arabinose 0 1,3-Butanediol 3.20
D-Glucose 2,3-Butanediol 0
D-Galactose 3.50 1,4-Butanediol 7.40
D-Mannose 1.00 Ethylene glycol 0.20
L-Sorbose 0.70 PVAb 0.10
a
The enzyme assay was carried out at 50 mM concentration of each compound.
b
PVA; Polyvinyl alcohol 2000.
Table 2 Characteristics of Sorbitol Oxidase from Streptomyces sp. H-7775
Localization Intracellular Substrate specicitya
Inducibility No D-Sorbitol 100 (Km 0.26 mM)

D-Xylitol 93.5 (Km 0.38 mM)
Molecular weight D-Mannitol 55.0 (Km 7.38 mM)
SDS-PAGE 45 kDa D-Arabitol 39.0 (Km 7.73 mM)
Glycerol 3.70
Gel ltration 45 kDa D-Glucose 0
D-Fructose 0
Optimum pH 6.57.5 D-Mannose 1.00
pH stability 7.510.0 Inhibitorb

(for 24 h at 30 C) Zn2 96.8
Hg2 90.7
Optimum temp 50 C IAA 53.8
(pH 7.5, 10 min) NEM 54.8
Heat stability < 55 C Cofactor FAD

(pH 7.5, 15 min
incubation) Reaction products Glucose H2 O2 c
a
Relative activity, %.
b
Inhibition, %.
c
Substrate; D-sorbitol.

SOX has a Km value of 0.26 mM for D-sorbitol, D-xylitol are used as sweetening agents. SOX may be
0.38 mM for D-xylitol, 7.38 mM for D-mannitol, used to quantitatively determine these sugar alcohols
and 7.73 mM for D-arabitol. in foods. A histochemical approach elucidates the loca-
A SOX-like activity that converts sorbitol to glucose tion of accumulated D-sorbitol (or D-xylitol) in vivo.
was reported in an apple leaf (10). The partially pur- A microtiter plate assay is an easy and fast way for
ied enzyme converts sorbitol to glucose in the absence detection of D-sorbitol and D-xylitol in a large number
of NAD or NADP . This enzyme is reported to cat- of samples. At rst, purple color is detected visually by
alyze the following reaction: the microtiter plate assay; a precise concentration of
Sorbitol 1=2O2 ! Glucose H2 O the sugar alcohols is determined spectrophotometri-
cally.
One of the reaction products of this enzyme is not In order to detect a reaction product directly, ana-
H2 O2 but H2 O. lysis by thin-layer chromotography is recommended. A
silica get plate is soaked in 30 mM borate buffer (pH
9.0) and dried at 110 C for 1 h. Then samples are
VII. QUALITATIVE AND QUANTITATIVE spotted on the plate. The developing system consists
DETERMINATION OF ACTIVITY of: rst dimension with ethyl acetate-pyridine-H2 O
(3:3:2); and second dimension with 1-butanol-metha-
The standard enzyme assay is based on the measure- nol-H2 O (5:3:2). The chromotogram is sprayed with a
ment of hydrogen peroxide generated during the oxi- color reagent, 2% (w/v) diphenyl amine:2% (v/v) ani-
dation of D-sorbitol. The hydrogen peroxide line:15% (v/v) phosphoric acid in acetone. The plate is
oxidatively couples with 4-aminoantipyrine and heated at 100 C for 10 min. Reducing sugars, such as
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline D-glucose, are detected as a dark spot.
(TOOS) in the presence of peroxidase to form a One millimolar Zn2 and Hg2 strongly inhibit
quinoneimine dye, by the method of Allain et al. SOX activity. One millimolar monoiodoacetate (IAA)
(31). The concentration of quinoneimine dye is and N-ethylmaleimide (NEM) also inhibit SOX activ-
measured spectrophotometrically at 555 nm. The ity (Table 2).
reaction mixture in a nal volume of 950 L con-
tains 150 mM KH2 PO4 -KOH (pH 7.0), 47.3 mM D-
sorbitol, 614 M TOOS, 158 M 4-aminoantipyrine, VIII. PURIFICATION
1.9 units/mL horseradish peroxidase, and a suitable
amount of sorbitol oxidase. The incubation is done at The expression level of SOX in Streptomyces sp. H-
37 C for 10 min. The reaction is stopped by the addi- 7775 is very low. In contrast, we succeeded in expres-
tion of 2 mL of 0.5% SDS. Sorbitol oxidase activity has sing about 236-fold higher total enzyme activity in E.
a linear function of both incubation time and protein coli (production of 2360 units/L culture) (5) than that
concentration. One unit of the enzyme activity is of Streptomyces sp. H-7775 (production of 10 units/L
dened as the amount of enzyme that catalyzes the culture) (4). The recombinant SOX was puried as
formation of 1 mol of H2 O2 per min. shown in Table 3; 384 mg of SOX was puried from
SOX is useful for the determination of D-sorbitol 20 L of culture broth of E. coli with a yield of 37.1%.
and D-xylitol concentrations in food. D-sorbitol and The recombinant SOX showed almost the same char-
Table 3 Purication of Recombinant Sorbitol Oxidase from E. coli Harboring pUCSOX
Purication step Total activity Total protein Specic activity Yield

(units) (mg) (units/mg) (%)
Cell-free extract 47,200 42,000 1.1 100

Heat treatment 42,500 8,400 5.1 90.0
Q-Sepharose Big Beads 40,000 3,000 13.3 84.7
Phenyl Sepharose Fast Flow 22,000 1,200 18.3 46.6
Sephadex G-25 21,500 1,100 19.5 45.6
Q-Sepharose HP 18,000 400 45.0 38.1
Sephadex G-25 17,500 384 45.6 37.1

acteristics as those of the authentic SOX from Step 5. Sephadex G-25 Column
Streptomyces sp. H-7775 (5). We describe below a Chromatography
method for the purication of recombinant SOX
SOX fractions obtained at Step 4 are applied onto a
from E. coli. A method for purication of SOX from
Sepharose G-25 column (2 20 cm) equilibrated with
Streptomyces sp. H-7775 is also described in Hiraga et
Buffer A and eluted with the same buffer. SOX frac-
al. (4).
tions are pooled and dialyzed against Buffer A over-
night at 4 C.
A. A Proven Method Leading to Purity
Step 6. Q-Sepharose HP Column
Chromatography
When SOX was expressed in E. coli, SOX activity was
detected in the supernatant of a bacterial sonicate, but The enzyme solution is then loaded on a Q-Sepharose
not in the insoluble fraction or in the culture super- HP column (2:6 30 cm) equilibrated with Buffer A
natant. This suggests that SOX is localized in the cyto- and eluted with a linear 01.0 M NaCl gradient.
sol or periplasmic space. In the case of Streptomyces Active fractions are pooled.
sp. H-7775, SOX is localized in mycelia.
Step 7. Sephadex G-25 column
Chromatography
Step 1. Preparation of Cell-Free Extract
The enzyme solution is then put on a Sepharose G-25
E. coli [pUCSOX] cells from 20 L of culture broth are column (2 20 cm) equilibrated with Buffer A and
washed with distilled water and resuspended in 4 L of eluted with the same buffer. SOX fractions were
10 mM Tris-HCl, pH 7.0 (Buffer A). After the addition pooled and stored at 4 C.
of 0.1% lysozyme and 20 mM EDTA, the cell suspen-
sion is incubated at 37 C for 30 min. The cell debris is B. Stability
removed by centrifugation at 15,000g for 15 min at
4 C. SOX is stable from pH 7.5 to 10.0 after 24 h incubation
at 30 C. SOX is stable below 55 C after 10 min incu-
bation at pH 7.5.
Step 2. Heat Treatment
The cell-free extract from Step 1 is heated at 50 C for 1
h, and centrifuged at 15,000g for 15 min at 4 C. REFERENCES
1. J Jeffery, H Jornvall. Sorbitol dehydrogenase. Adv

Step 3. Q-Sepharose Big Beads Column Enzymol 61:47106, 1988.
Chromatography 2. Y Wen, I Bekhor. Sorbitol dehydrogenase. Full-length
The heated cell-free extract is applied onto a Q- cDNA sequencing reveals a mRNA coding for a pro-
tein containing an additional 42 amino acids at the N-
Sepharose CL-6B Big Beads column (10 20 cm) equi-
terminal end. Eur J Biochem 217:8387, 1993.
librated with Buffer A. The column is washed with the
3. H Wiesinger, B Hamprecht. Purication and charac-
same buffer. The sorbitol oxidase is eluted with a linear terization of sorbitol dehydrogenase from bovine
00.5 M NaCl gradient in buffer A. SOX fractions are brain. J Neurochem 52:342348, 1989.
pooled and dialyzed against Buffer A at 4 C. 4. K Hiraga, M Kitazawa, N Kaneko, K Oda. Isolation
and some properties of sorbitol oxidase from
Streptomyces sp. H-7775. Biosci Biotech Biochem
Step 4. Phenyl Sepharose FF Column 61:16991704, 1997.
Chromatography 5. K Hiraga, T Eto, I Yoshioka, K Oda. Molecular clon-
ing and expression of a gene encoding a novel sorbitol
The SOX fraction obtained from Step 3 was put on a
oxidase from Streptomyces sp. H-7775. Biosci Biotech
Phenyl Sepharose FF column (5 20 cm) equilibrated Biochem 62:347353, 1998.
with Buffer A. The column was washed with the same 6. JFG Vliegenthart, KF Tipton, N Sharon, W Saenger,
buffer. SOX was eluted with a linear 01.0 M NaCl GP Moss, C Liebecq, CR Cantor, AJ Barrett. Enzyme
gradient in Buffer A and eluate was collected in frac- Nomenclature. San Diego: Academic Press, 1992, pp
tions. 24154.

7. RL Bieleski, RL Redgewell. Synthesis of sorbitol in 20. RJ Cook, KS Misono, C Wagner. The amino acid
apricot leaves. Aust J Plant Physiol 4:110, 1977. sequences of the avin-peptides of dimethylglycine
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9. RJ Redgwell, RL Bieleski. Sorbitol-1-phosphate and 21. WC Kenny, WH Walker, TP Singer. Studies on succi-
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17:407409, 1978. the avin site. J Biol Chem 247:45104513, 1972.
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glucose in apple leaf. Plant Cell Physiol 23:891899, sequence encoding the avoprotein and hydrophobic
1982. subunits of the succinate dehydrogenase of
11. A Willie, DE Edmondson, MS Jorns. Sarcosine oxi- Escherichia coli. Biochem J 222:519534, 1984.
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12. M Fukuyama, Y Miyake. Purication and some prop- component. J Biol Chem 254:85908593, 1979.
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Identication and properties of the prosthetic group 26. J Hofsteenge, JM Vereijken, WJ Weijer, JJ Beintema,
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88:197203, 1980. studies of p-hydroxybenzoate hydroxylase from Pseudo-
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DNA encoding rat liver L-gulono-gamma-lactone oxi- 176:44484454, 1994.
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Biol Chem 263:16191621, 1988. covalently-bound avin of hepatic monoamine oxi-
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1200, 1976. Clin Chem 20:470475, 1974.

45
Starch Phosphorylases
Ying Yu
Rutgers University, New Brunswick, New Jersey, U.S.A.
I. INTRODUCTION nomenon observed in stored potatoes. More recently,

there has been increasing interest in the function of
Alpha-1,4-glucan phosphorylases (EC 2.4.1.1) are starch phosphorylase in plant growth and develop-
widely distributed in plants and animals. Starch phos- ment, particularly with respect to starch deposition
phorylase may catalyze its reversible enzymatic reac- and degradation within a range of higher plants.
tion in either a synthetic or degradative mode. The objective of this chapter is to provide an over-
view of the biochemistry, molecular biology, and puta-
Glc-1-P Glcn $ Glcn1 Pi tive functions of starch phosphorylases in plant tissue.
In the synthetic reaction, a glucose unit is transferred The rst part of this chapter focuses on the importance
from Glc-1-P to a growing oligoglucan chain, with of starch phosphorylase to cold storage of food, speci-
release of inorganic phosphate. In the degradative cally potato, and the putative functions of this
mode, addition of inorganic phosphate liberates a enzyme in plant growth and starch deposition. The
molecule of Glc-1-P. With respect to the direction of second part will discuss the biochemical properties of
the starch phosphorylase reaction, the precise physio- starch phosphorylases and the recent information clas-
logical function of starch phosphorylase in higher plants sifying the various isoforms of starch phosphorylase
remains unclear, though the predominating belief is present in higher plants. This information is based lar-
that this enzyme catalyzes the stepwise disassembly of gely on biochemical characterization studies and the
starches and oligoglucans. Evidence for a biosynthetic analysis of the genes and gene families encoding a
role for starch phosphorylases, within the starch bio- range of starch phosphorylase isoforms.
synthetic pathway, has by no means been discounted
and has been the subject of much discussion (1).
The earliest studies on starch phosphorylase focused II. IMPORTANCE OF STARCH
mainly on the interconversion between starch and PHOSPHORYLASE TO COLD
sugars during fruit ripening and cold-sweetening phe- STORAGE OF POTATO AND PLANT
STARCH DEPOSITION
This chapter is dedicated to the memory of my mentor,
Professor Bruce P. Wasserman, who passed away Aug. 26, A. Starch Phosphorylase and the Cold-Induced
1998. Sweetening Phenomenon in Potato

Abbreviations: ADP-Glc, adenosine diphosphoglucose;
AGP, adenosine diphosphoglucose pyrophosphorylase; Glc- Interconversions between starch and sucrose are of
1-P, glucose-1-phosphate; Glc-6-P, glucose-6-phosphate; sh4, great interest, particularly with respect to a phenom-
shunken4 mutant; sh2, shunken2 mutant. enon referred to as cold-induced sweetening in potato.

Cold-induced sweetening generally occurs when sto- phosphorylase genes in potato tubers or maize endo-
rage temperatures are reduced from 10 C to 2 C over sperm have not yet been reported.
the course of several days. Since starch phosphorylase In chloroplasts, starch phosphorylase is regarded as
acts upon oligoglucans to form hexose phosphates, a starch degradative enzyme in the diurnal starch accu-
which are presumed precursors to sucrose, starch phos- mulation cycle (7). However, whether starch phosphor-
phorylase activity has been proposed as a logical cat- ylase plays a similar role in the amyloplasts of storage
alyst for the initial process of cold-induced sweetening tissues has not been determined. Since amyloplasts are
of potato during storage (2). However, despite numer- actively involved in starch biosynthesis, it is conceiva-
ous efforts, a direct role for starch phosphorylase in ble that Pho 1-type starch phosphorylases could play a
cold-induced sweetening has yet to be demonstrated. synthetic role using Glc-1-P as substrate. This is sup-
Temperature shift experiments, either from 10 C to ported by the observation that high levels of the plas-
28C or vice versa, were accompanied by negligible tidic starch phosphorylase transcript levels as well as
changes in starch phosphorylase activity (3). A fol- starch phosphorylase enzyme activity present in sink
low-up study (2) on the dual role of glycolytic inter- tissues of spinach. Source leaves, however, contained
mediates and amyloplast envelope integrity showed low amounts of plastidic starch phosphorylase tran-
that visible deterioration to the double envelope was scripts. In addition, the induction of starch phosphor-
not evident until 38 days of storage at 2 C. While ylase expression was observed when the leaf disks were
these studies cannot exclude a role for starch phosphor- supplied with carbohydrates (8). Some primer-inde-
ylase in the cold-induced sweetening phenomenon, the pendent starch phosphorylase isoforms could also be
precise function of starch phosphorylase in this process involved in the initiation of starch biosynthesis by
remains speculatory. synthesis of glucan primers. Another alternative role
of starch phosphorylases suggested by some scientists
B. Putative Function in the Starch Deposition of is the degradation of starch-derived malto-oligosac-
Higher Plants charides (5).
Starch phosphorylases from higher plants were initially

suggested to be the primary catalysts for starch chain
elongation since in the synthetic direction they catalyze
III. BIOCHEMICAL PROPERTIES OF
the incorporation of Glc-1-P into (1,4)--glucan
STARCH PHOSPHORYLASE
chains. This assumption began to be questioned
when AGP was discovered and the starch-decient
maize mutant, sh2, was characterized (4). Sh2 lacks
the AGP large subunit and is thus unable to convert A. Protein Structure
Glc-1-P into ADP-Glc. The decient starch levels of
this mutant implied that AGP was the enzyme regulat- Phosphorylases from plants, animals, and prokaryotes
ing the ow of carbon into starch. This nding indi- exist as dimers or tetramers containing identical sets of
cates that starch synthesis is predominantly controlled subunits (9). It is generally accepted that glycogen
by transglucosylases rather than phosphorylases. phosphorylase from mammalian tissue participates in
Antisense inhibition of cytosolic starch phosphorylase glycogen degradation and is subject to both allosteric
in potato plants did not cause any signicant changes regulation, and covalent modications such as phos-
of starch levels in leaves or tubers of the transgenic phorylation (10). Three-dimensional crystal structures
plants (5). In addition, no observable alterations of of mammalian glycogen synthases, illustrating the
starch structure or tuber formation were found in the coordinated interaction of subunits, have shed a
transgenic plants. This evidence suggested that cyto- great deal of light on the mechanism of action of gly-
solic starch phosphorylase did not play an essential cogen phosphorylase with respect to glycogen mobili-
role in starch metabolism. Another antisense study zation in mammalian systems (11). X-ray
was conducted on the potato leaf Pho 1-type starch crystallographic studies have revealed the three-dimen-
phosphorylase. Determination of the leaf starch sional crystal structures of mammalian glycogen
content revealed that the antisense inhibition of the synthases. For example, the subunit of rabbit muscle
leaf starch phosphorylase activity had no signicant phosphorylase is composed of two domains. Each
inuence on starch accumulation in leaves (6). The domain consists of a central -sheet core surrounded
effects of antisense suppression of Pho1-type starch by one or more layers of -helices (Fig. 1) (11, 12).

Figure 1 X-ray crystallographic model of a mammalian glycogen synthase. The barrel-and-arrow schematic of rabbit muscle
phosphorylase a was compared with potato phosphorylase. The dark areas represent the regions of strong similarity. (From Refs.
11, 12.)
B. Starch Phosphorylase Isoforms tend to exhibit high afnity for large-highly branched
glucans, such as glycogen. Pho2-type starch phos-
Higher-plant starch phosphorylases are classied into phorylases have an apparent monomer size of 90
two groups based on their subcellular localization kDa (13, 14). The second distinct starch phosphory-
and afnity for various glucans (13). Members of lase group, originally classied as the L-type, but
each group can be readily distinguished by the use now known as the Pho1-type group represents starch
of zymograms (Fig. 2). The cytosolic class of starch phosphorylases localized in plastids such as chloro-
phosphorylases has frequently been referred to as H- plasts and amyloplasts, and are generally character-
type phosphorylases, but more recently the term ized by a monomer size of 100 kDa. Pho1-type
Pho2-type starch phosphorylase, based primarily starch phosphorylases prefer oligoglucans such as
upon the migration behavior on zymograms, has maltodextrins.
replaced the earlier nomenclature. In addition to In spinach leaf or pea seed, one form of each starch
their cytosolic localization, Pho2-type phosphorylases phosphorylase type is present. However, the relative

Figure 2 Classic zymogram illustrating starch phosphorylase activities in various organs of spinach. (From Ref. 8.)
ratio of the two starch phosphorylase types varies dur- C. Starch Phosphorylase Gene Classications:
ing plant development (15, 16). During seed germina- Signicance of the Species Specic Pho 1
tion, the amount of Pho2-type starch phosphorylase is Insertion Sequences
increased while Pho1-type starch phosphorylase
remains at a constant, lower level. The developing cDNA clones representing both Pho1- and Pho2-type
seeds, however, contain much higher amounts of isoforms of starch phosphorylase have been obtained
Pho1-type starch phosphorylase than Pho2-type starch from potato and several other plant species. In potato
phosphorylase. tuber, cDNA clones for one cytosolic (Pho2-type) (22)
In maize, characterization of four starch phosphor- isoform and two plastidial (Pho1-type) isoforms (6, 23,
ylase isoforms has been reported (17, 18). The maize 24) have been characterized. cDNAs for Pho1-type
endosperm is rich in starch phosphorylase activity, starch phosphorylases have also been obtained from
and starch phosphorylase expression patterns corre- sweet potato (25), broad bean (26), and spinach leaf
late closely with starch deposition (19, 20). The four (8). Predicted amino acid sequences are well conserved.
isoforms differ in their pH optima, primer depen- For example, spinach Pho1-type starch phosphorylase
dence, chromatographic properties, development has an overall amino acid sequence identity of 75%
expression patterns, and their localization. Isoforms with potato (6, 8) and sweet potato (27) Pho1-type
I, II, and III occur mainly in the endosperm, while starch phosphorylases. The sequence of spinach
isoform IV is localized in the embryo. The activities Pho1-type starch phosphorylase has 62% identity
of isoforms I, II, and III were each reduced in the with Pho2-type starch phosphorylases from potato
mutant sh4, and their enzymatic properties such as (22) and broad bean (26).
pH optima and sensitivity to activators and inhibitors Starch phosphorylase cDNAs are typied by four
were altered (18). However, the properties of isoform distinct regions (Fig. 3). Region 1, the transit peptide,
IV did not seem to be affected by the sh4 mutation exhibits low homology among species. Regions 2 and
(21). These studies were mainly based upon separa- 4, at the N- and C-termini of the mature protein,
tion of isoforms by ion exchange chromatography. At respectively, are highly conserved. Region 3, distin-
that time, it was not possible to prepare antibodies or guished by a 50- to 80-residue stretch of additional
to clone cDNAs. Therefore, it is not known if these amino acids, is commonly referred to as an insertion
starch phosphorylase isoforms represent distinct gene sequence. Insertion sequences are present only in
products. Pho1-type starch phosphorylases. As with the transit

major starch phosphorylase of maize kernels is a
homodimer with an estimated molecular weight of
223,000 10,000 daltons (29). Pyridoxal phosphate
is a cofactor of the enzyme. The estimated pyridoxal
Figure 3 Schematic diagram of cDNAs encoding the four phosphate content of the puried enzyme was
regions of a typical Pho1-type (plastidial) starch phosphor- 112,000 g protein/mol pyridoxal phosphate (29).
ylase. Pho2-type starch phosphorylases lack the transit pep- The puried enzyme had a Km value of 1.0 mM
tide and insertion sequence. The remaining regions of Pho1- for Glc-1-P when assayed in the synthetic direction
type starch phosphorylases retain strong amino acid at 37 C, pH 5.8. In the phosphorolytic direction, the
sequence similarity with the Pho2-type starch phosphory- puried enzyme had a Km value of 4.2 mM for Pi
lases. (From Refs. 16, 17, 35.) when assayed at 25 C, pH 6.3 (29). Magnesium ion
was shown to be an inhibitor of the enzyme (29).
peptides, the insertion sequences are widely divergent ADP-Glc was found to be an inhibitor with respect
from species to species; however, some common struc- to Glc-1-P in the synthetic reaction (29). Both Pho1-
tural features are noted (28). Each insertion sequence is and Pho2-types of starch phosphorylases have been
highly acidic, with a calculated isoelectric point of 4.0. puried and characterized from spinach leaves (13).
The presence of a transit peptide at the N-terminus The subunit molecular weights of Pho1- and Pho2-
of potato Pho1-type starch phosphorylase was con- types spinach leaf starch phosphorylases were calcu-
rmed by the sequence analysis of the cDNA clone lated to be 108,000 and 92,000 daltons, respectively.
(24). This 50 amino acid transit peptide sequence has Based on their native molecular weights, it was
several structural features common to transit peptides suggested that both starch phosphorylases exist in
of chloroplast proteins. This sequence is rich in hydro- dimeric form. The optimal pH values for Pho1-
philic residues. It contains seven Ser and ve Thr resi- versus Pho2-type enzyme were shown to be 5.7
dues, representing 14% and 10% of the total amino versus 6.1 in the synthetic direction, and 6.2 versus
acid content of the transit peptide. The transit 6.5 in the phosphorolytic direction. At pH 6.6, the
sequence also contains 13 positively charged amino Km values of Pho1-type enzyme were determined to
acids, but no acidic residues or large hydrophobic be 3.1 mM for Glc-1-P in the presence of 10 mg/mL
sequences. The transit peptides are required for trans- soluble starch in the synthetic direction and 2.2 mM
location of plastidic Pho1-type starch phosphorylases for Pi in the presence of 2 mg/mL maltopentose in
from the cytosol into the plastid. the phosphorolytic direction. Under the same con-
With respect to function, the insertion sequences are ditions, the Km values of Pho2-type enzyme were 6.2
hypothesized to inuence the afnity of starch phos- mM for Glc-1-P in the synthetic direction and 6.9
phorylase for substrate. In a benchmark study mM for Pi in the phosphorolytic direction (13).
designed to test the function of an insertion sequence,
a chimeric potato starch phosphorylase, lacking the 78
amino acid insertion of the Pho1-type enzyme was con-
structed, and an extensive kinetic analysis was con- V. QUANTITATIVE DETERMINATION OF
ducted (28). Removal of the insertion sequence STARCH PHOSPHORYLASE ACTIVITY
resulted in a dramatic lowering of the Michaelis con-
stant for glycogen. The authors concluded that the The activity of starch phosphorylase can be measured
insertion sequence served to sterically hinder the inter- either in the synthetic direction or in the phosphoroly-
action of starch phosphorylase with highly branched tic direction. In the synthetic direction, the amount of
substrates such as glycogen. This region is therefore inorganic phosphate released from Glc-1-P can be
thought to reect the afnity of starch phosphorylases determined by a molybdate-based colorimetric assay
for glucan substrate. (30, 31). Alternatively, starch phosphorylase activity
can be determined by measuring the incorporation of
14
C-labeled Glc-1-P into glucan primer (17). In the
IV. PROPERTIES OF STARCH phosphorolytic direction, a continuous assay system
PHOSPHORYLASES is used (13, 14). Glc-1-P is converted to Glc-6-P by
phosphoglucomutase. NADP reduction by Glc-6-P
The enzymatic properties of several starch phosphor- dehydrogenase can then be followed spectrophotome-
ylases have been characterized. For example, the trically at 340 or 265 nm.

VI. PURIFICATION OF STARCH peak of starch phosphorylase activity were pooled and
PHOSPHORYLASES stored (Table 1) (29).
Starch phosphorylases have been puried from a num-

ber of sources, including spinach leaf (13), potato tuber VII. CONCLUSIONS
(32), and maize kernels (29). The purication of the
major starch phosphorylase of maize kernels is Starch phosphorylases from higher plants possess
described below (29). unique features with regard to their structures, cataly-
Protein extracts of maize kernels harvested at 22 tic properties, and subcellular localization patterns.
days after pollination were prepared by homogeniza- Structurally, the phosphorylases have been classied
tion and centrifugation. The extract was brought to into distinct groups based on zymograms that accu-
40% saturation with ammonium sulfate. Following rately pinpoint subcellular localization of the major
centrifugation at 16,000g for 20 min, the superna- isoforms. Deduced amino acid sequences show
tant was brought to 50% saturation with ammo- sequence conservation among a host of species.
nium sulfate and centrifuged again at 16,000g for Genes corresponding to cytosolic and plastidial phos-
10 min. The precipitate was resuspended and dia- phorylase isoforms have been appropriately classied,
lyzed overnight. The sample was then subjected to and expression levels for transcript levels and tissue
a step known as starch absorption. The starch distribution have been carefully quantied. A more
absorption step was adapted from the method of precise role for the insertion sequences located in the
Kamogawa et al. (33). Waxy maize starch was solu- Pho2-type phosphorylases remain to be determined.
bilized in 100 mL water by autoclaving for 1 h at 1 Functionally, however, the various roles of starch
bar (100 kPa). The solution was chilled to 2 C and phosphorylase have yet to be unequivocally sorted out.
15 mL of chilled 95% ethanol was added. The pre- Roles for starch phosphorylases in providing carbon
cipitate was washed once with 100 mL 11% ethanol for seed germination and photosynthetic conversions
at 15 C and resuspended in 100 mL 0.1 M EDTA, seem clear, but other functions for this enzyme remain
pH 7.2. The protein sample was also made 11% vague. For example, the precise role of starch phos-
with ethanol and centrifuged. The chilled superna- phorylase in starch deposition or degradation has
tant was then added to the starch preparation which remained elusive. Transformed potatoes with various
had been chilled to incipient freezing. After mixing forms of starch phosphorylase inserted in the antisense
in an ice bath for 5 min, chilled 95% ethanol was orientation do not appear to exhibit visible effects on
added slowly to the mixture to a nal concentration starch phenotype (5, 6). Such effects could be attribu-
of 12.5%. The resulting precipitate was washed once ted to the presence of multiple starch phosphorylase
with 100 mL of 11% ethanol at 15 C. The precipitate isoforms, which could require co-suppression of
was then dissolved at room temperature with 100 mL 5 several genes.
mM sodium citrate buffer, pH 6.3, and applied to a Moreover, for many years, it was believed that
DEAE-cellulose column. The protein precipitate from starch phosphorylase was the primary catalyst for
the fractions constituting the peak of starch phosphory- chain elongation of amyloses and amylopectins in
lase activity was dissolved in buffer and applied onto a monocots such as corn. This idea quickly fell from
Sephadex G-200 column. The fractions constituting the the forefront when the existence of the AGP pathway
Table 1 Purication of Maize Phosphorylase
Fraction Volume Activity Protein Specic Activity Recovery

(mL) (units/mL) (mg/mL) (units/mg) (%)
Crude homogenate 1427 0.56 5.0 0.047 100

4050% ammonium sulfate 80 8.4 14.0 0.44 85
precipitation
DEAE-cellulose peak 46 8.9 0.17 49 52
Sephadex G-200 peak 7 41 0.72 57 36
Source: Ref. 29.

for starch synthesis was nally characterized (34). 8. E Duwenig, M Steup, J Kossmann. Induction of genes
Nonetheless, maize amyloplasts are rich in starch encoding plastidic phosphorylase from spinach
phosphorylase activity and starch phosphorylase (Spinacia oleracea L.) and potato (Solanum tuberosum
expression patterns correlate closely with starch L.) by exogenously supplied carbohydrates in excised
leaf discs. Planta 203:111120, 1997.
deposition (19, 20). Therefore, more recent thinking
9. T Fukui. Plant phosphorylase. In: T Akazawa, T
retains the possibility that starch phosphorylases may Asahi, H Imaseki, eds. The New Frontiers in Plant
yet prove to be a key component of the amylose and Biochemistry. Tokyo: Japan Scientic Societies
amylopectin biosynthetic system of higher plants (1). Press, 1983, pp 7182.
Similar questions linger with respect to a precise bio- 10. MF Browner, RJ Fletterick. Phosphorylase: a biolo-
chemical function for starch phosphorylase in the cold gical transducer. Trends Biochem Sci 17:6671, 1992.
sweetening process of potato. Continued generation of 11. RJ Fletterick, SR Sprang. Glycogen phosphorylase
mutant cultivars decient in the various isoforms of structures and function. Acc Chem Res 15:361369,
starch phosphorylase should play a major role toward 1982.
further clarifying the potentially interesting and diverse 12. K Nakano, T Fukui. The complete amino acid
functions of this enzyme. sequence of potato -glucan phosphorylase. J Biol
Chem 261:82308236, 1986.
13. S Shimomura, M Nagai, T Fukui. Comparative glu-
can specicities of two types of spinach leaf phosphor-
ACKNOWLEDGMENTS ylase. J Biochem 91:703717, 1982.
14. M Steup. Starch degrading enzymes. In: PJ Lea, ed.
Funding for this research was provided by the U.S. Methods in Plant Biochemistry, Vol 3. London:
Department of Agriculture National Research
15. J van Berkel, J Conrads-Srauch, M Steup. Glucan-
Initiative (95-02531 and 98-01235), and by the New phosphorylase forms in cotyledons of Pisum sativum
Jersey Agricultural Experiment Station. L: localization, developmental change, in-vitro trans-
lation, and processing. Planta 185:432439, 1991.
16. JBW Hammond, J Preiss. Spinach leaf intra- and
REFERENCES extrachloroplast phosphorylase activities during
growth. Plant Physiol 73:709712, 1983.
1. O Nelson, D Pan. Starch synthesis in maize endo- 17. CY Tsai, OE Nelson. Phosphorylases I and II of
sperms. Annu Rev Plant Physiol Mol Biol 46:475 maize endosperm. Plant Physiol 43:103112, 1968.
496, 1995. 18. CY Tsai, OE Nelson. Two additional phosphorylases
2. FA Isherwood. Mechanism of starch-sugar intercon- in developing maize seeds. Plant Physiol 44:159167,
version in Solanum tuberosum. Phytochemistry 15:33 1969.
41, 1976. 19. CY Tsai, F Salamini, OE Nelson. Enzymes of carbo-
3. MGH Kennedy, FA Isherwood. Activity of phos- hydrate metabolism in the developing endosperm of
phorylase in Solanum tuberosum during low tempera- maize. Plant Physiol 46:299306, 1970.
ture storage. Phytochemistry 14:667670, 1975. 20. B Burr, OE Nelson. The phosphorylases of developing
4. CY Tsai, OE Nelson. Starch-decient maize mutant maize seeds. Ann NY Acad Sci 210:129138, 1973.
lacking adenosine diphosphate pyrophosphorylase 21. CY Tsai, OE Nelson. Mutations at the shrunken-4
activity. Science 151:341343, 1966. locus in maize that produce three altered phosphory-
5. E Duwenig, M Steup, L Willmitzer, J Kossmann. lases. Genetics 61:813821, 1969.
Antisense inhibition of cytosolic phosphorylase in 22. H Mori, K Tanizawa, T Fukui. Potato tuber type H
potato plants (Solanum tuberosum L.) affects tuber phosphorylases isozyme: molecular cloning, nucleo-
sprouting and ower formation with only little impact tide sequence, and expression of a full-length cDNA
on carbohydrate metabolism. Plant J 12:323333, in Escherichia coli. J Biol Chem 266:1844618453,
1997. 1991.
6. U Sonnewald, A Basner, B Greve, M Steup. A second 23. K Nakano, H Mori, T Fukui. Molecular cloning of
L-type isozyme of potato glucan phosphorylase: clon- cDNA encoding potato amyloplast -glucan phos-
ing, antisense inhibition and expression analysis. Plant phorylase and the structure of its transit peptide. J
Mol Biol 27:567576, 1995. Biochem 106:691695, 1989.
7. E Beck. The degradation of transitory starch granules 24. N Brisson, H Giroux, A Camirand, C Simard.
in chloroplasts. In: R Heath, J Preiss, eds. Rockville, Maturation and subcellular compartmentation of
MD: American Society of Plant Physiologists, 1985, potato starch phosphorylase. Plant Cell 1:559566,
pp 2744. 1989.

25. CT Lin, MT Lin, HY Chou, PD Lee, JC Su. The gene 31. OH Lowry, JA Lopez. The determination of inorganic
structure of starch phosphorylase from sweet potato. phosphate in the presence of labile phosphate esters. J
Plant Physiol 107:277278, 1995. Biol Chem 162:421428, 1946.
26. P Buchner, L Borisjuk, U Wobus. Glucan phosphor- 32. YP Lee. Potato phosphorylase. I. Purication, physi-
ylases in Vicia faba L.: cloning, structural analysis and cochemical properties and catalytic activity. Biochim
expression patterns of cytosolic and plastidic forms in Biophys Acta 43:1824, 1960.
relation to starch. Planta 199:6473, 1996. 33. A Kamogawa, T Fukui, Z Nikuni. Potato -glucan
27. CT Lin, KW Yeh, PD Lee, JC Su. Primary structure phosphorylase: crystallization, amino acid composi-
of sweet potato starch phosphorylase deduced from its tion and enzymatic reaction in the absence of added
cDNA sequence. Plant Physiol 95:12501253, 1991. primer. J Biochim 63:361369, 1968.
28. H Mori, K Tanizawa, T Fukui. A chimeric -glucan 34. JL Ozbun, JS Hawker, E Greenberg, C Lammel, J
phosphorylase of plant type L and H isozymes: func- Preiss, EYC Lee. Starch synthetase, phosphorylase,
tional role of 78-residue insertion in type L isozyme. J ADP glucose pyrophosphorylase and UDP glucose
Biol Chem 268:55745581, 1993. pyrophosphorylase in developing maize kernels.
29. B Burr, OE Nelson. Maize -glucan phosphorylase. Plant Physiol 51:15, 1973.
Eur J Biochem 56:539546, 1975. 35. E Steup. Starch degradation. In: J Preiss, ed. The
30. CH Fiske, Y Subbarow. The colorimetric determina- Biochemistry of Plants, Vol 14. London: Academic
tion of phosphorus. J Biol Chem 66:375400, 1925. Press, 1988, pp 255296.

46
Amylosucrase
Volker Buttcher
Aventis CropScience GmbH, Frankfurt, Germany
I. INTRODUCTION rhoeae and the N. meningtidis are the only two species
that are pathogenic. All other Neisseria sp. are non-
The enzyme amylosucrase was discovered by Hehre pathogenic and are known to be commensals.
and Hamilton (1). The reaction demonstrated an in
vitro glycogen synthesis without any activated nucleo- B. Molecular Genetics
tide sugars for the rst time. The reaction of amylosu-
crase can be described as follows: The GC content of Neisseria sp. ranges from 49.3 to
Sucrose ! Amylose Fructose 55.6 mol% (3). The size of the chromosome is 1:5
106 bp (4). The gene for amylosucrase was cloned (5, 6)
The amylosucrase splits off the glucosyl part from the and sequenced. Up to now no sequences of other amy-
substrate and links it covalently to an acceptor mole- losucrases have been published, but it is assumed that
cule. During this reaction fructose is released. The the DNA sequences among the Neisseria spp. are very
reaction mechanism is still not proven, but it is similar. The amylosucrase gene of N. polysaccharea
assumed that the glycosyl residue is transferred to the could be strongly expressed in various E. coli strains
nonreducing end of an alpha-1,4-glucan. For the (6). Because of its own secretion-signal-like leader
enzyme reaction no additional energy is needed but a sequence in recombinant E. coli the enzyme is secreted
primer seems to be necessary to trigger the reaction. into the medium, but the bulk of the protein is retained
The best primer is glycogen which shows enormous in the cytosol.
stimulation of the amylosucrase reaction (2).
A. Microbiology
II. PROPERTIES OF THE
AMYLOSUCRASE
Amylosucrase occurs only in prokaryotes and only in
some genera of the genus Neisseria (Table 1). The amy- A. Protein Data
losucrase is located in the cytoplasm of the cells, except
in N. polysaccharea, which secretes the amylosucrase in The puried amylosucrase is a very stable enzyme. It
the medium. remained active for several days in the reaction buffer
The genus Neisseria is the type genus of the family (100 mM Na citrate, pH 6.7) at 37 C. The enzyme
Neisseriaceae which contains 18 species that can be could be stored over 1 year at 20 C without signi-
isolated from humans and animals. The Neisseriaceae cant loss of activity. The MW of the protein is around
are gram-negative bacteria. The morphology in this 72 kDa and the calculated isoelectric point is at pH
family can be diplococci, cocci, or rods. The N. gonor- 5.59.

Table 1 Amylosucrases Are Found in the Following lute requirement for a primer. Up to now it is not
Neisseria Strains absolutely clear if the sucrose itself or an oligoglucan
of a certain length is required obligatorily, because
N. canis ATCC 31005
N. cinerea ATCC 14685 traces of glucans are enough to trigger the reactions
N. cuniculi ATCC 14688 after a very long lag phase. The reaction kinetics
N. denitricans ATCC 14686 depends not only on primer presence or concentration.
N. perava The enzyme and substrate concentrations are very
N. polysaccharea ATCC 43768 important factors in the beginning of the polymeriza-
N. sicca tion reaction.
N. subava ATCC 19243
D. Substrate Specicity
Amylosucrase has a high substrate specicity. Several

substrates like 6-desoxy-6-uorosucrose, 6-desoxysu-
The amylosucrase appears to be tightly bound to its crose, 4,6-dideoxysucrose, 3-desoxysucrose, and -D-
product/substrate, which is amylose (7). allopyranosyl--D-fructofuranoside were used as sub-
strates. None of the tested substrates were incorpo-
B. Detection of the Amylosucrase Activity rated into a polymer, but 6-desoxy-6-uorosucrose,
6-desoxysucrose, and 4,6-dideoxysucrose could inhibit
The simplest way to detect amylosucrase activity is in a the enzymatic reaction (9). Under optimal conditions
small in vitro reaction assay. An aliquot of the enzyme the amylosucrase achieves nearly 100% conversion of
sample is mixed with 100 L reaction buffer (5% (w/v) its substrate sucrose to polymers (or oligomers) and
sucrose, 50 mM Na citrate pH 6.5) and incubated for fructose. Invertase activity (the splitting of sucrose
some minutes up to several days at 37 C. Very high into fructose and glucose without any glycosylation
activity samples show a white precipitate, and samples step) is negligible for the amylosucrase reaction
with a lower activity are mixed with Lugols solutions under all reaction conditions tested. Remaud-Simeon
(0.5 mM I2 KI). This test can be used for relatively et al. (5) showed that under special reaction conditions
rough quantitative determination by comparing the a transglycosylation reaction of oligomaltose may be
color intensity with a calibration row. possible. When the substrate sucrose was lacking, the
A more accurate activity test can be done by mea- amylosucrase could disproportionate oligoglucans.
suring the real-time fructose release in an optically The glycosidic residues were mainly transferred such
coupled enzymatic assay. The reaction cuvette contains that the maltopentaose concentration decreased in
20% sucrose, Na citrate buffer (100 mM, pH 7), glyco- favor of maltohexaose and maltoheptaose.
gen (0.05%), NAD (0.4 mM), ATP (1 mM), and the
helper enzymes hexokinase (1.5 units/mL), phospho-
glucose-isomerase (2 g/mL), and glucose-6-phosphate III. THE PRODUCT OF THE
dehydrogenase (5 g/mL), which are added directly to AMYLOSUCRASE
the reaction mixture (200 L). The assay is started by
addition of the enzyme sample and the OD 340 is The product of the amylosucrase reaction is character-
monitored. The increase of absorption at 340 nm is ized as an -1,4-glycosidic glucan (6). The molecular
equivalent to the production of fructose and can be weight depends on substrate concentration and reac-
transformed mathematically in mol of fructose tion time. The polymer molecular weights are in the
released per minute (units). range from several thousands (103 ) up to several mil-
lions (106 ) g/Mol. The molecular weights reported in
C. Primer Dependence the literature are often overestimated owing to the low
solubility of the polymers and the preference of amy-
The glucan synthesis by amylosucrase is strongly sti- lose to form strong aggregates.
mulated by primers. Glycogen was shown to be one of Older references (10, 11) described the presence of
the best primers (2), but oligoglucans like maltohep- -1,6 linkages, which were generated by very small
taose or branching enzymes can also trigger the glucan impurities of branching activity. The recombinant
synthesis. From studies of MacKenzie et al. (8) it seems enzyme, when produced in a branching enzyme-de-
that the amylosucrase of N. denitricans has an abso- cient strain (glgB), allowed the production of a pure

amylosucrase free from any branching activity. The nant expression of the amylosucrase is an appropriate
product of the puried amylosucrase is an -1,4-glyco- host strain became possible, the very pure enzyme, free
sidic-linked glucose polymer and no -1,6-glycosidic of all disturbing molecules, became available. With this
(or other) linkages could be detected with NMR or recombinant enzyme new products can be synthesized
methylation methods. in a controlled method and with a reliable quality.
The naturally occurring amylose normally must
contain some -1,6-glycosidic branches, because in
vivo the amylose is synthesized in the presence of a
glucan branching enzyme activity. Therefore the in
vitro product of amylosucrase is different from the REFERENCES
naturally found amylose, because of the homogeneity
of its glycosidic linkages. This small but important 1. EJ Hehre, DM Hamilton. Bacterial synthesis of an
amylopectin-like polysaccharide from sucrose. J Biol
difference implies different polymer properties distinct
Chem 166:777778, 1946.
from naturally occurring branched amylose. For
2. G Okada, EJ Hehre. New studies on amylosucrase, a
clear differentiation the product is called Nepo- bacterial alpha-D-glucosylase that directly converts
Amylose (Neisseria polysaccharea-Amylose). sucrose to a glycogen-like alpha-glucan. J Biol Chem
249:126135, 1974.
3. C Hoke, NA Vedros. Taxonomy of the Neisseriae:
IV. APPLICATIONS desoxyribonucleic acid base composition, interspecic
transformation, and deoxyribonucleic acid hybridiza-
The enzyme amylosucrase is not yet used in industry, tion. Int J Syst Bacteriol 32:5766, 1982.
because the product (Nepo-Amylose) from the pure 4. DT Kingsbury. Estimate of the genome size of various
amylosucrase is not well characterized. The formerly microorganisms. J Bacteriol 98:14001401, 1967.
5. M Remaud-Simeon, F Albaret, B Canard, L Varlet, P
used crude enzyme preparations synthesized different
Colonna, RM Willemont, P Monsan. Studies on a
polymers compared to the very pure recombinant amy-
recombinant amylosucrase. In: SB Peterson, B
losucrase. The reason for that is small impurities, e.g., Svenson, S Pederson, eds. Carbohydrate Bioengineer-
glucans and branching enzymes, which get carried over ing. Amsterdam: Elsvier Science, 1995, pp 313320.
from the crude extract. Glucans have a very high bind- 6. V. Buttcher, T Welsh, L Willmitzer, J Kossmann.
ing afnity for the amylosucrase (7). The glucans act in Cloning and characterisation of the gene for amylosu-
an uncontrolled way as primers and serve as starter crase from Neisseria polysaccharea: production of a
molecules for the polymerization. Branching enzymes linear -1,4-glucan. J Bacteriol 179:33243330, 1997.
enhance the polymerization reaction by providing non- 7. CR MacKenzie, KG Johnson, IJ McDonald.
reduced ends, at which the amylosucrase elongates the Glycogen synthesis by amylosurase from Neisseria
growing chain and accelerates the polymerization (12). perava. Can J Microbiol 23:13031307, 1977.
8. CR MacKenzie, JJ McDonald, KG Johnson.
The amylosucrase could, for example, be used in
Glycogen metabolism in the genus Neisseria: synthesis
pharmaceutical formulation processes. The microcrys-
from sucrose by amylosucrase. Can J Microbiol
talline amylose could be easily synthesized around a 24:357362, 1978.
pharmaceutical compound to change the solubility of 9. BY Tao, PJ Reilly, JF Robyt. Neisseria perava amy-
the substance. The wrapped pharmaceutical com- losucrase: characterization of its product polysacchar-
pound could be protected after application and ide and a study of its inhibition by sucrose derivatives.
would release the active substance in a controlled man- Carbohydrate Res 181:163174, 1988.
ner. A second example is the production of indigestible 10. J-Y Riou, M Guibourdenche, MB Perry, LL
amylose, which can be used as a probiotic food ingre- MacLean, DW Grifth. Structure of the exocellular
dient to improve the healthy bacterial ora in the intes- D-glucan produced by Neisseria polysaccharea. Can
tine in order to prevent colon cancer. With a controlled J Microbiol 32:909911, 1986.
11. CR MacKenzie, MB Perry, IJ McDonald, KG
enzymatic process, tailor-made amylose with special
Johnson. Structure of the D-glucans produced by
properties could be produced for paper coating. The Neisseria perava. Can J Microbiol 24:14191422,
coating would improve the printing quality of recycled 1978.
paper used by inkjet printers. 12. V Buttcher, M Quanz, L Willmitzer. Molecular clon-
The investigation of amylosucrase demonstrates ing, functional expression and purication of a glucan
clearly the impact that the recombinant expression of branching enzyme from Neisseria denitricans.
industrial enzymes can have. Only when the recombi- Biochim Biophys Acta 1432:406412, 1999.

47
Dextransucrase
Klaus Buchholz
Braunschweig Technical University, Braunschweig, Germany
Pierre F. Monsan
National Institute for Applied Sciences, Toulouse, France
I. INTRODUCTION by 96 strains of L. mesenteroides, in order to select the

polymers most suitable for medical applications. The
structural characteristics of dextrans from several L.
mesenteroides strains are given in Table 1. Seymour
In 1861, Louis Pasteur discovered the microbial origin
and Knapp (10) suggested classifying dextrans into
of the gelication of cane sugar syrups (1). In 1874, the
three groups, according to the main type of branching:
corresponding product was named dextran because
Group A, -1,2 branching; Group B, -1,3 branching;
of its positive rotatory power. The microorganism
Group C, -1,4 branching.
causing that gelication was isolated in 1878 by Van
The most widely used dextran is produced by the
Tieghem and named Leuconostoc mesenteroides (2).
dextransucrase of the strain L. mesenteroides NRRL
Hehre demonstrated in 1941 that dextran could be
B-512F, which synthesizes a very highly linear polysac-
synthesized from sucrose by a cell-free ltrate (3).
charide containing 95% -1,6 bonds. Besides
The corresponding extracellular enzyme was named
Leuconostoc, dextransucrase is also obtained from
dextransucrase by Hestrin et al. (4).
two other types of lactic bacteriaStreptococcus and
Dextransucrase (DS) catalyzes the following reac-
Lactobacillus (7). Streptococcal glucosyltransferases,
tion:
particularly from Streptococcus mutans, are involved
n sucrose ! dextran n fructose in cariogenesis phenomena. These enzymes synthesize
glucosen from sucrose a glucan polymer which plays a key role
Sucrose is the only natural D-glucosyl unit donor used in the adhesion mechanism of bacteria on the tooth
as substrate by the enzyme. The energy necessary for surface to form the dental plaque (1113).
D-glucose polymerization simply comes from the clea-
vage of the high-energy glycosidic bond. No activated
intermediate or cofactor is needed. B. Substrates and Acceptors
Dextran is a D-glucopyranosyl polymer containing
more than 50% -1,6 glycosidic bonds, with a molecu- The only natural substrate of dextransucrase (DS) is
lar weight ranging from 0.5 to 6:106 kDa (5, 6). Its type sucrose, which serves as a high-energy glucosyl donor
and degree of branching vary according to the origin of for polysaccharide and oligosaccharide synthesis; the
the dextransucrase (Table 1) (7, 8). Jeanes et al. (9) main product is dextran. In addition to sucrose,
have puried and characterized the dextrans produced -D-glucopyranosyl uoride, p-nitrophenyl--D-gluco-

Table 1 Glucosidic Linkages Content in Dextran Polysaccharides from Various Strains of L. mesenteroides and S. mutans
Osidic linkage (%)
-1,6 -1,3 -1,2 -1,4
Leuconostoc mesenteroides NRRL B-512F 95 5

NRRL B-742 87 13
50 50
NRRL B-1299 66 7 27
65 35
NRRL B-1355 95 5
54 46
NRRL B-1191 94 6
NRRL B-1308 95 5
NRRL B-1415 87 1 12
Streptococcus mutans E 49 69 18 13
OMZ176 9 91
66 33
HS6 94 6
Ingbritt 44 56
Source: Refs. 7, 8.
pyranoside, -D-glucopyranosyl--L-sorbofuranoside, C. Classication

and lactulosucrose serve as glucopyranosyl donors (14).
In a secondary reaction, the so called acceptor reac- Dextransucrase is a glucosyltransferase, and more
tion, the glucosyl moiety is transferred from sucrose to precisely a transglucosidase, named sucrose:1,6 -D-
the acceptor molecule instead of the growing dextran glucan 6--D-glucosyltransferase. Its Enzyme Com-
chain to produce the acceptor molecule elongated by mission nomenclature number is EC 2.4.1.5.
one single glucosyl unit as the primary product.
Usually the new bond formed is an -1,6-glucosidic
II. UTILIZATION
bond. In most cases, the product can itself serve as
an acceptor, so that a homologous series of glucosy- A. Synthesis of Nondigestible Oligosaccharides
lated oligosaccharides is formed (15, 16).
Acceptor properties have been reported for a wide The dextransucrase from the soil bacterium
variety of mono-, di-, and oligosaccharides (e.g., glucose Leuconostoc mesenteroides NRRL B-1299 is known
(17), fructose (18), maltose (19), and rafnose [for over- to catalyze the synthesis of dextran polymers contain-
view see 14, 20]), but also for some functionalized ing -1,2-linked branched chains (Table 1). When this
saccharide derivatives, as for example the sugar alcohols specic glucosyltransferase is used in the presence of
D-sorbitol and D-glycerol (17) or the 5-O--D-gluco- maltose as acceptor and of sucrose as D-glucosyl
pyranosyl-arabonic acid (21, 36) or even glucal, an donor, -gluco-oligosaccharides are obtained, which
unsaturated sugar (22, 23). This secondary reaction is contain -1,2 glucosidic bonds (24, 25) at their nonre-
therefore of special technical interest. It offers perspec- ducing end and a maltose residue at the reducing end.
tives for the synthesis of new oligosaccharides, which The presence of these -1,2 linkages results in a very
are not accessible otherwise by feasible technical routes. high resistance of these oligosaccharides to attack by
Maltose and isomaltose are the most efcient accep- the digestive enzymes of humans and animals (26).
tors. In most acceptors, the glucose from sucrose is Such -gluco-oligosaccharides cannot be metabolized,
transferred to the 6-hydroxyl group of the nonreducing as demonstrated by using germ-free rats. That is why
end glucose residue to give a series of isomaltooligo- such -gluco-oligosaccharides were initially developed
saccarides of degree of polymerization of 27 (14). A as low-calorie bulking agents, to be used in food for-
detailed kinetic analysis revealed a nearly equally high mulations in complement of intense sweeteners (24).
acceptor activity for the di- to tetrasaccharides in the But these -gluco-oligosaccharides are specically
reaction mixture (23). metabolized by the positive intestinal bacterial ora.

In contrast to fructo-oligosaccharides and galacto-oli- C. Leucrose
gosaccharides, these -gluco-oligosaccharides are not
bidogenic, but they promote the growth of the cellu- Leucrose (-5-O-[-D-glycopyranosyl]--D-fructopyr-
lolytic intestinal ora. In addition, they induce a anose) is an isomer of sucrose which is obtained via
broader range of glycolytic enzymes than fructo-oligo- glucosyltransfer from sucrose to fructose as an accep-
saccharides and galacto-oligosaccharides, without any tor. Since fructose is a reaction product, the net reac-
important side production of gases and thus any detri- tion is an isomerization of sucrose. Basic data are:

mental effect (27). D 7:5%, melting point 156158 C
spec. rotation [20
The prebiotic effect of -gluco-oligosaccharides has (33).
been demonstrated for piglets, broilers, and calves (28). Pilot production was reported by Schwengers (33)
The addition, for example, of 0.15% (w/w) of -gluco- using dextransucrase (DS) in a 65% aqueous solution
oligosaccharides to young calves feed results in a 20% with 1/3 sucrose and 2/3 fructose (w/w) at 25 C. When
decrease in veterinary costs (28). These oligosacchar- the reaction was complete, the leucrose formed was
ides are currently marketed for human nutritional separated from fructose by chromatography with a
application as food complements, in combination cation exchange resin, followed by an evaporation
with specic microbial ora and vitamins. step and crystallization (30, 33). The formation of leu-
The prebiotic effect of such -gluco-oligosacchar- crose and some reaction conditions, such as the effect
ides has also been demonstrated at the level of skin of fructose concentration on the formation of leucrose
microbial ora (29), in which lactic bacteria also play and dextran, were investigated also by Itoh et al. (34).
a key protective role. This has resulted in the devel-
opment of dermocosmetic applications for the -
III. PROPERTIES AS PROTEIN; DS
gluco-oligosaccharides, under the trade name
CHARACTERISTICS
BioEcolia.
A. Molecular Weight
B. Isomalto-oligosaccharides The size of dextransucrase from L. mesenteroides

NRRL B-512F has been debated for some time.
A product manufactured in Japan as Alo mixture Molecular weights of 65 kDa (35) to 190 kDa (36)
(Anomalously Linked Oligosaccharides) contains a have been reported. Finally, the isolation of the gene
range of isomalto-oligosaccharides to a major extent coding for this enzyme (37) suggested a protein with a
(glucose, isomaltose, and panose as major constitu- total of 1527 amino acids, which corresponds to a 170-
ents) (30). However, it is produced from starch as kDa molecular weight. Lower values are due to pro-
substrate with -amylase, -amylase and a trans- teolytic degradation, while higher values can be attrib-
glucosidase. It is claimed to have favorable properties uted to dextran contamination of the protein
for application in the food industry. preparation. However, one has to be careful in con-
Using glucose as an acceptor, a European patent cluding that the gene size represents the nal MW of
(31) describes the formation of isomalto-oligosacchar- a mature protein, since there often is proteolytic pro-
ides from sucrose with 1020 anhydroglucose units; the cessing (to remove signal peptide and to form subunits)
average molar weight may be in the range of 2000 as well as glycosylation, phosphorylation, and other
5000. The molar ratio of sucrose to glucose is in the posttranslational modications.
range of 25. The sucrose is added to the reaction
solution of 0.20.5 M glucose and 1000 units dextran- B. Gene Structure
sucrase quasi-continuously. The sucrose concentration
should not exceed 25% of the total dry substance; the The structure of the gene coding for dextransucrase
reaction is conducted to high conversion. The product from L. mesenteroides NRRL B-512F (37) is very simi-
mixture contains fructose in a molar range correspond- lar to that of glucosyltransferases from streptococci
ing to that of the sucrose added and may be used as a (3840). Starting from the N-terminal end, they con-
sweetener. Some further data are presented by Pereira secutively contain: a signal peptide with 36 amino acids
et al. (32) with, however, limited yields of oligosacchar- (37) allowing excretion; a variable region with 180200
ides of up to 45%. Systematic investigations concern- amino acids with unknown role (41); an N-terminal
ing reaction engineering were undertaken by K. catalytic domain with 900 amino acids, highly con-
Demuth (23). served, which contains the catalytic groups, and parti-

cularly the Asp551 residue (41); and a C-terminal has been proposed (53, 54). Usually the new bond
domain, with repeated units, involved in the glucan formed is an -1,6-glucosidic bond. In most cases,
binding, but also in the dextran and oligosaccharide the product can itself serve as an acceptor, so that a
synthesis mechanism (42). homologous series of glucosylated oligosaccharides is
The site-directed mutagenesis of amino acid residues formed (15, 16).
Asp511 and Asp513, which were replaced by Asn resi-
dues, completely suppresses dextran and oligosacchar-
ide synthesis activity (43). B. Kinetic Characteristics
Sucrose is the only simple carbohydrate used by dex-

transucrase as a D-glucosyl donor. Initial reaction
IV. PROPERTIES AS ENZYME
rates follow Michaelis-Menten kinetics up to 200
A. Mechanism mM sucrose concentration, but the enzyme is inhibited
by higher substrate concentrations (55). The inhibitor
For dextran formation Ebert and Patat (45) and Ebert constant for sucrose is 730 mM (56). This inhibition
et al. (17, 44) were the rst to propose an insertion can be removed by acceptor or dextran addition (6, 57,
mechanism. This concept was further developed by 58).
Kindler and Ludwig (46), who suggested the existence Table 2 summarizes the Michaelis constant values
of a covalent intermediate glucosyl-enzyme complex. reported in the literature for dextransucrase. Optimal
Several groups provided strong evidence for a covalent pH and optimal temperature with the corresponding
glucosyl enzyme intermediate for S. sanguinis and S. reaction activation energy are also given.
sorbrinus dextransucrase (4750; for overview see 14). Dextransucrase is activated and stabilized by
Robyt et al. (51, 52) presented further evidence for calcium ions (59), but they do not seem to be directly
both covalent glucosyl and glucanyl enzyme intermedi- involved in the catalytic mechanism (60). The kinetic
ates, suggesting two active centers involved in the behavior of dextransucrase has been characterized
growth of the polymer chain via an insertion mechan- and modeled to optimize the production of -gluco-
ism, questioned however by recent gene sequence data oligosaccharides containing -1,2-glucosidic bonds. In
showing one active center with two Asp (511 and 551) fact, three families of oligosaccarides of increasing
involved in glucosyl transfer (41, 43). degree of polymerization are obtained (61), which
In a secondary reaction, the so-called acceptor reac- respectively contain, besides a maltose residue at the
tion, the glucosyl moiety is transferred from sucrose to reducing end (Fig. 1), only -1,6-glucosidic bonds;
the acceptor molecule instead of the growing dextran -1,6-glucosidic bonds and one -1,2-glucosidic
chain to give the acceptor molecule elongated by one bond at the nonreducing end; -1,6-glucosidic bonds
single glucosyl unit as the primary product. A specic and one -1,2-glucosidic bond on the penultimate
acceptor binding site at the active center of the enzyme D-glucosyl unit.
Table 2 Parameter Value of L. mesenteroides NRRL B-512F Dextransucrase
Activation energy
KM (mM) Optimal pH Optimal temperature ( C) (kCal mol1 ) Reference
1216 5.05.5 30 50, 60

46 5.0 30 35, 38
5.2 30 10.3 6
30 5.25.5 30 11.2 51, 62
13 4.55.5 30 10.5 39
28 5.05.6 30 10.0 29, 63
27 55 kJ/mol 20, 54
40 56
(90)a 56
a
Immobilized enzyme, apparent KM .

Figure 1 Types of oligosaccharides formed by dextransucrases.
It must be pointed out that Michaelis kinetics only product formation follow the same scheme with an
apply in the absence of acceptors. Constants like Vmax , even more pronounced tendency; dextran formation
KM , and KI are dependent on acceptor concentrations may be nearly suppressed at high acceptor concentra-
(cf. next paragraph). Thus with good acceptors, appar- tion (Fig. 2) (65).
ent Vmax values increase with increasing acceptor con- The nal product concentrations depend signi-
centration, from 5.8 (U/mL) with substrate only up to cantly on the ratio of substrate and acceptor. The
19.1 (U/mL) at 600 mM maltose concentration (64). rst acceptor product (panose in the presence of mal-
They decrease with increasing concentrations of weak tose) is favored at high maltose excess, whereas tetra-
acceptors like fructose, from 1.3 (U/mL) to 0.38 (U/ and pentasaccharides are formed in comparable
mL) at 2.75 M fructose concentration (54). Apparent amounts, as well as some higher oligosaccharides, at
KM values increase as well with increasing acceptor about equimolar substrate and acceptor concentra-
concentration, from 12 mM at zero to 163 mM at tions.
600 mM maltose (64). A KM value of 27 mM was In the presence of weak acceptors, like fructose,
obtained in the absence of an acceptor whereas this initial overall reaction rates decrease signicantly
value was 40 mM at a fructose concentration of 1.39 with increasing acceptor concentration, whereas
M (54). It is obvious that in the presence of acceptors, they increase with the substrate concentration.
kinetics are complex and Michaelis kinetics do not Initial rates of acceptor product formation increase
apply. with both substrate and acceptor concentration, the
In the presence of strong acceptors, like maltose or dextran formation being suppressed to a major
isomaltose, several general effects may be summar- extent only at very high acceptor concentration
ized: initial overall reaction rates (sucrose consump- (range of 10% at 3 M fructose concentration)
tion or sum of acceptor product and dextran (Fig. 3) (66). The acceptor product of fructose,
formation) signicantly increase both with substrate leucrose, does not act as an acceptor, so high yields
and acceptor concentration. Initial rates of acceptor can be obtained (54).

The acceptors considered, fructose, glucose, and
maltose, represent different types; the rst is a weak
acceptor which slows down the overall reaction rate,
the second is an acceptor of intermediate strength, and
the third represents a strong acceptor which accelerates
the overall reaction rate. These effects are included in
the model. The scheme of reactions catalyzed by dex-
transucrase is shown in Figure 4 (66).
The enzyme is assumed to have three binding sites:
one covalent glucosyl-, one dextran, and one acceptor
(53, 54). Reactions 1, 2, and 3 represent the growth cycle
of dextran formation. A sucrose molecule S is bound at
the enzyme E, fructose F is released, and the glucosyl G
is transferred to the dextran chain Gi , which is always
linked to the enzyme. Fructose can be linked to the
acceptor site, and react with G to form leucrose FG
(reactions 4 and 9). The same reactions are possible
Figure 2 Overall initial reaction rates of acceptor product
with other acceptors A (reactions 6 and 13), and accep-
formation with maltose as an acceptor. (From Ref. 65.) tor products (except leucrose) may again react as accep-
tors (reactions 6 j and 13 2xj). Sucrose is able to
bind at the acceptor site (reaction 5) but does not
C. Mathematical Modeling and Reaction react to an acceptor product, thus leading to inhibition
Engineering Considerations at high concentrations. For approximate modeling,
reactions 1012 and 14 may be neglected.
The synthesis of oligosaccharides by dextransucrase is The decrease of enzyme activity E with time is
a complex series of reactions. The quantitative described by a rst-order reaction (66):
description of it therefore requires a model which
E E0 expkd t
comprises all of the relevant parameters. Such a
model has been developed to predict the optimal reac-
E0 is the initial activity at the time t0 0; E, the
tion conditions and reactor congurations for differ-
remaining activity after time t; and kd , the inactivation
ent acceptors, and notably for the production of
leucrose and isomalto-oligosaccharides (54, 66). The
kinetic parameters of the model identied are based
on a wide range of experimental data (23, 54, 56, 65,
67, 68).
Figure 4 Scheme of reactions catalyzed by dextransucrase

Figure 3 Initial rates of leucrose formation. (From Ref. 66.) (see text for details). (From Ref. 66.)

parameter. For two experiments, the inactivation para- 100200 mM has little effect on leucrose production.
meters have been calculated for the immobilized According to the model, the ratio sucrose/fructose has
enzyme (66, 67). In these experiments, different accept- no effect on the ratio of dextran formation to leucrose
ors were used: fructose in the rst, and maltose in the formation. A tubular ow reactor with immobilized
second. The bead diameters were 0.7 and 1 mm, which enzyme is the best choice for production in a continuous
have been found to be optimal. The inactivation con- mode. So with initial concentrations of 0.6 M sucrose
stants kd were 0.029 (d1 ) in the rst and 0.0135 (d1 ) and 2.2 M fructose the leucrose yield was in the range of
in the second case, respectively. 6472% depending on the sucrose conversion (9555%)
(69). The yield can further be improved to about 85%
by increasing the fructose concentration up to 3 M,
D. Kinetics of Leucrose Formation however, at the expense of enhanced separation efforts.
Byproducts are isomaltulose and trehalulose in the
Boker et al. (54) presented approximate kinetic corre- range of 45% each. The leucrose yield can be further
lations as well as a rational model based on the reac- improved, for example, from 70% to 75%, and the
tion mechanism for different concentration ranges of byproduct concentrations reduced at lower temperature
sucrose and fructose with emphasis on leucrose forma- (5 C instead of 25 C) (54). The productivity of the
tion. If only sucrose and fructose are present in the immobilized enzyme in a continuous tubular reactor
reaction mixture the model may be simplied, so that was found to be 1.5 g leucrose formed per unit dextran-
it comprises only six parameters (Table 3) (54, 66). sucrase (test at 25 C) (69). This type of reactor proved
These equations are similar to an extended to be optimal in model calculations, as is shown in
Michaelis-Menten equation where two substrates Figure 5, where the productivity is shown as a function
(sucrose and fructose as an acceptor) as well as sub- of the initial fructose concentration with an optimum at
strate inhibition are considered. 1:6 M and 1% residual sucrose concentration (or
From the model, it follows that the fructose concen- 2:4 M and 10% residual sucrose concentration) (66).
tration should be as high as possible; for practical rea- Formation of the series of oligosaccharides from
sons, it is limited by solubility and viscosity of the isomaltose to isomaltodecaose is shown in Figure 6
solution considering the sum of substrate, acceptor (23). The rst acceptor products are much stronger
and product. A sucrose concentration higher than acceptors themselves than glucose; therefore isomal-
tose up to isomaltopentaose are formed with nearly
equal concentrations in the range 3545 mM by the
Table 3 Parameters of Simplied Kinetics for Leucrose end of the reaction.
Formationa An overview for the acceptor activity of a range
Sucrose consumption rate: of sugars and sugar derivatives for which kinetic
ds E S p24 p70 F=X

Leucrose formation rate:
dL=dt E p70 S F=X
X 1 p42 S p46 F p47 F S p68 S2
The values of the parameter areb

p24 3:31 108 m3 U min
p42 1:45 102 m3 =mol
p46 7:27 103 m3 =mol
p47 1:11 104 m3 =mol2
p68 3:16 105 m3 =mol2
p70 1:95 108 m6 =U min mol Figure 5 Modeling of the tubular ow reactorleucrose
production rate and formation of byproducts. Kinetics of
a
Parameters pi refer to the model as developed in Ref. 66. the formation of isomaltooligosaccharides and different
b
m3 stands for cubic meters. acceptor products. (From Ref. 66.)

Dextransucrase catalyzes, using sucrose as substrate,

both the synthesis of high-MW dextran polymers and
the synthesis of low-MW oligosaccharides in the pre-
sence of efcient acceptors. Two different sets of reac-
tion conditions are thus necessary to determine
dextransucrase activity.
A. High-MW Dextran Synthesis
Standard reaction conditions are: 20 mM acetate buf-

fer, pH 5.4, containing 100 g/L sucrose, 0.05 g/L
calcium chloride (CaCl2 2H2 O), 1 g/L sodium
azide at 30 C. Dextransucrase activity must be in
the range of 0.31.0 U/mL (1 unit corresponds to
the amount of enzyme which catalyzes the produc-
tion of 1 mol of fructose per min in the above reac-
tion medium).
Dextransucrase activity is measured by determining
the initial rate of production of reducing sugars fol-
lowed by the 3,5-dinitrosalicylic acid (DNS) method
(70). Reaction samples (0.1 mL) are withdrawn at var-
ious time intervals. Dextransucrase reaction is stopped
by addition of 0.1 mL DNS solution (10 g/L DNS, 300
g/L sodium potassium tartarate, 16 g/L sodium hydro-
xide). After heating for 5 min at 95 C followed by ice
cooling, 1 mL water is added and absorbance is read at
540 nm. A standard curve is prepared with standard
fructose solutions ranging from 0 to 2 g/L in fructose
concentration. Any catalytic activity producing redu-
Figure 6 Acceptor production formation with glucose as an cing sugars will interfere with activity determination.
acceptor. (From Ref. 23.) HPLC for analysis of fructose can be applied as an
alternative (see below).
investigations were performed are given in Figure 7 B. Low-MW Oligosaccharide Synthesis

(23). The acceptor activity is based on modeling so
that varying concentrations of substrate, acceptor, Standard reaction conditions are: 20 mM acetate buf-
and acceptor products during the experiments are fer, pH 5.4, containing 100 g/L sucrose, 20100 g/L
taken into account. It is obvious that monosacchar- acceptor (maltose, isomaltose, . . .), 0.05 g/L calcium
ides and their derivatives like mannitol and sorbitol chloride (CaCl2 H2 O), 1 g/L sodium azide at 30 C.
are rather weak acceptors. Disaccharides, including Dextransucrase activity must be in the range of 0.3
acceptor products like isomaltose, are much better 1.0 U/mL. The key factor determining the molecular-
acceptors, except singular molecules like leucrose, weight distribution of the products is the sucrose
which is not an acceptor. Surprisingly, many tri- to donor/acceptor molecular ratio. Reaction samples
pentasaccharides are equally good acceptors as the (0.1 mL) are heated for 5 min at 95 C to stop the
disaccharides, or much better ones in the case of the reaction. They are diluted with ultrapure water to
mannitol and sorbitol acceptor products. It should be reach a total carbohydrate concentration < 5 g/L
noted that the latter ones are nonreducing oligo- and analyzed by HPLC using a C18 column and ultra-
saccharides with presumably increased thermal sta- pure water as eluent. Fructose, maltose, leucrose,
bility. sucrose, and gluco-oligosaccharides with a polymeriza-

Figure 7 Acceptor strength of various mono- and disaccharides and their ensuing acceptor products in relation to the acceptor
efciency of fructose (DP1 signies the monosaccharide, followed by the acceptor products, from di- (DP2) to pentasaccharide
(DP5) (GPM: glucosylmannitol, GPS: glucosylsorbitol, KGPA: potassium glucosylarabonic acid, DFA III: difructoseanhydride
III, 3-KGPM: 3-keto-glucosylmannitol, 3-KP: 3-keto-isomaltulose, n-decyl-malt: n-decyl-maltoside). (From Ref. 23.)
tion degree up to 6 are easily separated within 30 min ity in the range of 510 U/g wet weight) were used.
elution time. Products are detected and quantied by Samples from the reaction digests were taken after
measuring the refractive index of the eluent solution. 15, 30, 45, 60, and 90 min; diluted; and heated at 100
C for 5 min to inactivate the enzyme. After cooling to
ambient temperature, the samples were membrane-l-
C. Immobilized Dextransucrase
tered through a 0.2-m cellulose nitrate lter and ana-
lyzed by HPLC. [Note that prolonged storage of
For immobilized dextransucrase it is obvious that
frozen samples prior to analysis will give wrong results
systematic errors in the activity measurement can be
due to a slowly continuing reaction (72)]. The enzyme
avoided only when dextran formation is suppressed as
activity is calculated from the fructose concentrations
far as possible. This can be achieved by adding an excess
by linear regression. In case of immobilized dextransu-
of good acceptor like maltose (71). A test in which
crase, corrections must be made for the decrease in
maltose was applied in vefold excess was developed
reaction volume by sampling whereas the amount of
for this purpose (56, 68). However, it must be taken
immobilized enzyme remains constant.
into account that in the presence of excess maltose the
maximal activity of dextransucrase is higher by a factor
of 3 at low concentrations (sucrose 28, maltose 50 D. Analytical Methods: HPLC
mmol/L) and a factor of 2 at high concentrations
(sucrose 280, maltose 150 mmol/L) (65). The test solu- Samples were analyzed by HPLC with the
tion was composed of 730 mM maltose and 146 mM LiChrospher 100 NH2 column (Merck, Darmstadt,
sucrose in a 0.025 M calcium acetate buffer at pH 5.4. Germany). Acetonitrile-water eluent mixtures from
To avoid microbial growth the solution furthermore 80:20 to 70:30 (v/v) were used at 0.8 mL/min. A
contained 200 mg/L sodium azide and a droplet of refractometer (Knauer, Berlin, Germany) served as
toluene. detector, while data were acquired with Gynkosoft
With native dextransucrase, 4.8 mL of this solution (Gynkotek, Munich, Germany). The analytical error
was incubated with 0.2 mL dextransucrase solution is 5% at low sugar concentration (< 8 g/L) and 3
(activity in the range of 515 U/mL) at 25 C. When % at higher concentrations. For complex mixtures of
dextransucrasecalcium alginate beads were tested, 20 oligosaccharides high-performance anion chromato-
mL of the maltose test solution and 1.0 g beads (activ- graphy is the optimal method (73).

VI. PRODUCTION AND PURIFICATION sucrase in a continuous packed-bed reactor. The
enzyme is produced by fed-batch cultivation of
A. Dextransucrase Production and Purication
L. mesenteroides NRRL B-1299 (94, 95). Sucrose
Large-scale production of dextransucrase involves plays the simultaneous role of growth substrate for
only L. mesenteroides strains. These gram-positive, the microbial strain, specic inducer for extracellular
nonsporulating bacteria are heterofermentative and dextransucrase production, and substrate for this
produce equimolar amounts of D() lactate and enzyme. The precise characterization of the physiology
acetate or ethanol (74). Their optimal growth of L. mesenteroides NRRL B-1299 has shown that the
temperature is 2530 C, with an optimal pH of 6.5 enzyme production was repressed by D-fructose accu-
(75). Dextransucrase production is specically mulation in the growth medium. But it is possible to
induced by sucrose (76), but the induction mechan- remove this repression effect in the presence of
ism has not been elucidated. Several constitutive D-glucose. For that reason, the simultaneous fed-
mutants have been described for L. mesenteroides batch addition of sucrose and D-glucose to the cultiva-
NRRL B-512 F (7779), B-1355 (80, 81), B-742 tion medium has allowed an increase of 100% in the
(82), and B-1299 (83). amount of dextransucrase activity present in the
Dextransucrase is directly connected with microbial culture medium (96).
growth (84, 85). Lopez (86) obtained a linear relation- More than 90% of the dextransucrase activity is
ship between L. mesenteroides NRRL B-512F growth linked to the microbial cells and/or to the dextran
rate and dextransucrase specic production rate. The polysaccharide associated to the cells. This allows a
optimization of the culture medium of L. mesenteroides very easy recovery of the enzyme from the cultivation
NRRL B-512F has been reported by several groups medium by centrifugation, as well as a very simple
(63, 87, 88). A pH value of 6.7 results in an improved immobilization process by entrapping the dextransu-
dextransucrase activity and stability (87). The higher crase activity in calcium alginate beads. Immobilized
pH value also limits dextran production in the culture dextransucrase is used in a 1 m3 packed-bed reactor.
medium and thus facilitates dextransucrase purica-
tion (89). Culture temperature is generally 26 C or
27 C, although higher enzyme activities have been VII. IMMOBILIZATION
reported at a slower culture growth at 23 C (63, 90),
corresponding to 23 units/mL. Aeration conditions High-MW dextran synthesis can be obtained using
poorly affect cell growth, but carbon dioxide signi- either crude L. mesenteroides culture medium after
cantly increases dextransucrase production (90). Fed- growth on sucrose as carbon source, or the culture
batch sucrose addition results in a sixfold increase in supernatant after microbial cell removal. But in the
enzyme activity (91). This can be coupled together with case of oligosaccharide synthesis through the acceptor
pH regulation by adding an alkaline sucrose solution reaction, the continuous operation of dextransucrase
(63, 85). As sucrose acts simultaneously as an inducer involves its immobilization.
for the enzyme production, a carbon source for the Several attempts have been made for immobilizing
bacterial growth and a substrate for dextransucrase, dextransucrase by covalent grafting onto a variety of
enzyme preparations contain a high concentration of supports, in order to study either the enzyme mechan-
dextran, tightly associated to the protein. ism or the effect of diffusional limitations on enzyme
Dextransucrase from L. mesenteroides NRRL kinetics. The efciency of such methods is always rela-
B-512F can be puried from the culture medium by tively limited. Dextransucrase has been coupled onto
ammonium sulfate or ethanol precipitation (92). It BioGel activated with glutaraldehyde (51, 97).
can also be concentrated by ultraltration and puried Covalent grafting onto DEAE-Sephadex or SP-
by Biogel A-5m chromatography after dextran hydro- Sephadex gives, under the best conditions, a 10%
lysis by dextranase treatment (93). The dextran asso- immobilization yield and a specic activity of 0.5 U/g
ciated with the enzyme allows its very efcient support (98). Dextransucrase adsorption onto phenoxy
concentration/purication by phase partition after acetylcellulose (99) or hydroxyapatite (50) results in
polyethyleneglycol (PEG 1500) addition, resulting in improved immobilization yields, but the enzyme is
a one-step 20-fold concentration from the culture rapidly desorbed from the support. Covalent coupling
supernatant and a 200-fold purication (63). onto porous amino silica carrier activated with glutar-
The production of -gluco-oligosaccharides at the aldehyde gives a specic activity of 40 U/g support, but
industrial scale is achieved using immobilized dextran- with a 2% immobilization yield (100, 101).

The presence of high-MW dextran associated with logical approaches as well as nucleic probes and geno-
the enzyme results in the possibility to very simply mic analysis, and the construction of modied enzymes
and efciently immobilize dextransucrase preparations by site-directed and random mutaganesis, combined
by inclusion into alginate gels. Immobilization yields with directed gene molecular evolution. This will result
>90% are obtained, with a specic activity of 1 up to in the possibility to design improved dextransucrases
10 U/mL gel (69, 102, 103). The apparent volume with controlled selectivity in D-glucosyl residue trans-
of the dextranosylenzyme complex, associated with fer. This will allow the synthesis of a new range of
dextran/alginate interactions, allows enzyme retention poly- and oligosaccharides, and more broadly of glu-
within such gels, while globular enzymes are easily coconjugates of nutritional and/or medical interest.
washed out from the gel (68).
In the case of the production of the dextransucrase
REFERENCES
from L. mesenteroides NRRL B-1299, > 90% of the
activity is associated with the bacterial cells. For that
1. L Pasteur. Sur la fermentation visqueuse. Bull Soc
reason, the continuous industrial production of gluco-
Chim 3031, 1861.
oligosaccharides containing -1,2 linkages involves the 2. P van Tieghem. On sugar-mill gum. Ann Sci Nat Bot
use of the aliginate-entrapped biomass (104). The Biol Veg 7:180203, 1878.
immobilization yield is 93%, with a specic activity 3. EJ Hehre. Production from sucrose of a serologically
of 4.1 U/mL gel. reactive polysaccharide by a sterile bacterial extract.
Science 93:237238, 1941.
4. S Hestrin, S Avireni-Shapiro, M Aschner. The enzy-
VIII. CONCLUSIONS mic production of levan. Biochem J 37:450456, 1943.
5. AJ Groenwall, BGA Ingelman. Manufacture of infu-
Dextransucrase is one of the very few enzymes able to sion and injection uids. U.S. Patent 2,437,518 (1948).
6. KH Ebert, G Schenk. Mechanism of biopolymer
synthesize glucosidic bonds using a substrate as simple
growth: the formation of dextran and levan. Adv
as sucrose. It has the capacity to catalyze both the
Enzymol 30:179210, 1968.
production of high-MW dextran polymers, containing 7. RL Sidebotham. Dextrans. Adv Carbohydr Chem
thousands of D-glucosyl units, and the production of Biochem 30:371444, 1974.
low-MW oligosaccharides with < 10 D-glucosyl units 8. MD Hare, S Svensson, GJ Walker. Characterization
when efcient acceptors, like maltose for example, are of the extracellular, water-insoluble -D-glucans of
added to the reaction medium. oral streptococci by methylation analysis, and by
A variety of dextransucrases have been character- enzymic synthesis and degradation. Carbohydr Res
ized, presenting a wide range of selectivities and thus 66:254264, 1978.
resulting in a large diversity of poly- and oligosacchar- 9. A Jeanes, WC Haynes, CA Williams, JC Rankin, EH
ides. The main microbial sources of dextransucrases Melvin, MJ Austin, JE Cluskey, BE Fischer, HM
Tsuchiya, CE Rist. Characterization and classication
are lactic bacteria from soil (L. mesenteroides) and
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48
Levansucrase
Ki-Bang Song and Sang-Ki Rhee

Korea Research Institute of Bioscience and Biotechnology (KRIBB) and Real Biotech Co., Ltd., KRIBB,
Daejeon, Korea
I. INTRODUCTION levanm levann ! levanm1 levann1
Levansucrase is a kind of transferase which catalyzes a The enzyme catalyzes hydrolysis and polymerization
fructosyl transfer from sucrose to various acceptor reactions concomitantly (Reaction 1), resulting in fruc-
molecules. The product, levan, consists of D-fructofur- tose homopolymer (levan) and free glucose. This reac-
anosyl residues linked predominantly by -(2,6) link- tion occurs when sucrose exists as the sole fructosyl
age as a main chain with some -(2,1) branching donor and acceptor, and involves three steps: initia-
points. Both linkage types are formed by the single tion, propagation, and termination (1). Chains of
enzyme levansucrase. The enzyme catalyzes the follow- levan grow in a stepwise fashion by repeated transfer
ing reactions: of a hexosyl group from the donor to growing acceptor
molecules. The enzyme primarily catalyzes a coupled
1. Polymerization reaction
reaction by a Ping-Pong mechanism, i.e., sucrose
Sucrosen !Glucosen hydrolysis followed by transfructosylation involving a
fructosyl-enzyme intermediate (2).
Levan or Oligosaccharides
When water acts as an acceptor, a free fructose is
2. Hydrolysis reaction generated from both sucrose and levan (Reaction 2).
This reaction occurs in all the levansucrase-catalyzed
Sucrose H2 O ! Fructose Glucose reactions mentioned above, but the rate is much slower
Levann H2 O ! Levann1 Fructose when compared to a sugar acceptor. Reaction 3 occurs
in the presence of an acceptor in the environment. The
3. Acceptor reaction enzyme transfers the fructosyl residue of sucrose spe-
cically to the C-1 OH of aldose in the acceptor.
Sucrose Acceptor Molecules ! Compounds containing hydroxyl groups, such as
Fructosyl-Acceptor Glucose methanol, glycerol, and oligosaccharides, can act as
fructosyl acceptors. The reaction mechanism yields a
4. Exchange reaction nonreducing sugar compound and a series of oligosac-
charides, in which the sugar molecule with one more
Sucrose 14 CGlucose ! fructose moiety remains as a major reaction product.
Fructose-14 CGlucose Glucose The reaction occurs predominantly in the presence of a
high concentration of fructosyl donors, such as sucrose
5. Disproportionation reaction or rafnose. Reaction 4 might be considered analogous

to Reactions 2 and 3, but differs in the regeneration of in vacuoles and plays an important role in temporary
sucrose, which has a high-energy bond. The enzyme storage and partitioning of assimilates and in osmo-
also catalyzes Reaction 5, a disproportionation reac- regulation. It also assists some plants in overcoming
tion, in which the degree of polydispersity of levan or drought and cold stress (6). For example, improved
oligomers is modied. The above ve reactions com- drought resistance is shown upon transformation of
pete with one another, yielding a specic major pro- tobacco, a species normally incapable of forming
duct with some minor products, but they are levan, with a gene encoding a bacterial levansucrase.
predominantly controlled by environmental factors. The enzyme involved in levan biosynthesis in plants,
The enzyme recognizes sugar compounds with a - which may be synthesized by the concerted action of a
1,2 linkage between glucose and fructose residues. The number of distinct fructosyltransferases, is not fully
most abundant substrate for the enzyme in nature is understood.
sucrose. Rafnose also serves as a substrate. Levan There are a number of microorganisms reported as
formed by levansucrase is hydrolyzed by the enzyme levan producers beyond the genus (7). Levansucrases
itself, and the hydrolysis ceases at the branching from some bacteria are inducible and exocellular,
points. The degree of levan hydrolysis depends on whereas some are constitutive and endocellular. The
the enzyme source. For example, Rahnella aquatilis synthesis of levan by levansucrase in a sucrose-contain-
levansucrase has strong activity, but Zymomonas mobi- ing medium gives the microorganisms a mucoid mor-
lis levansucrase shows very little activity even at higher phology. Researchers have demonstrated that this
temperatures (3). mucoid structure of the microorganisms plays a role
Levansucrase (sucrose:2,6--D fructan:6-D-fructo- in the symbiosis (Bacillus polymyxa) or phytopatho-
syltransferase, sucrose 6-fructosyltransferase) is classi- genesis (Erwinia amylovora) of plant interactive bac-
ed EC 2.4.1.10. There are several enzymes that relate teria. In addition, the enzyme also participates in the
to the hydrolysis of sucrose or sucrose-originated sugar defense mechanism against environmental stress
compounds: sucrase (-D-fructofuranosidase, inver- (Bacillus subtilis) (8). The function of levansucrase
tase, EC 3.2.1.26), dextransucrase (EC 2.4.1.5), leva- located intracellularly in some bacteria is not under-
nase (EC 3.2.1.65), inulinase (EC 3.2.1.7), and stood. The diversity of the enzymatic properties of
alternansucrase (EC 2.4.1.140). A new fructosyltrans- levansucrase may be caused by the different biological
ferase (aldose -D-fructosyltransferase, EC 2.4.1.162), function of each enzyme in each microorganism.
which has an exceptionally high propensity to form a Recently, the enzyme was overexpressed in E. coli for
wide range of -(1,2)-linked disaccharides, does not the mass production of levan with high yield and pur-
form appreciable amounts of levan (4). The enzymes ity (9). An approach to produce levan in large quantity
involved in the biosynthesis of fructan (levan and inu- was achieved successfully in transgenic plants (10).
lin) in plants are as follows:
Sucrose:sucrose 1F-fructosyltransferase (1-SST,
III. UTILIZATION OF LEVANSUCRASE
EC 2.4.1.99)
Fructan:fructan 1-fructosyltransferase (EC
Levan, produced by levansucrase from sucrose, con-
2.4.1.100)
sists entirely of fructose and glucose at one terminal
Sucrose:fructan 6-fructosyltransferase (6-SFT)
in each molecule (Fig. 1). Many potential applications
The last one, which has similar enzymatic functions of levan in food and pharmaceutical industries exist,
with microbial levansucrases, is responsible for the but the use of this polymer is not yet practical due to
production of levan in plants (5). the lack of information about its polymeric properties
needed in industrial elds. Novel applications of levan
include such uses as emulsier, formulation aid, stabi-
II. SOURCES AND FUNCTION OF lizer, thickener, surface-nishing agent, encapsulating
LEVANSUCRASE agent, and carrier for colors or avors and fragrances
in the food industry (7). It has been reported that levan
Levansucrase is found in various kinds of microorgan- has certain biological activities, such as the promotion
isms and in higher plants. Levan as a product of levan- of infection and necrosis, tumor inhibition, and an
sucrase-type enzyme reactions is a major non- increase in cell permeability to a cytotoxic agent (11).
structural carbohydrate found in plants such as barley, Calazans et al. (12) postulated that levan showed anti-
wheat, onions, and grass. In plants, levan accumulates tumor activity against sarcoma 180 and Ehrlich carci-

Figure 1 Chemical structure of levan.
noma in Swiss albino mice, and hypothesized that the fructosyl derivatives, alkyl fructosides, which are
effect might be associated with the polymer of a spe- expected to be applicable in similar or improved uses
cic molecular size. of alkyl glucoside and used as a nonionic surfactant
In addition to the above, a novel aqueous two-phase and as raw materials for the production of sugar esters
system has been developed by mixing polyethylene gly- of fatty acids (17).
col (PEG) and levan, which offers a potential applica-
tion of levan in separation and purication of
biological materials, as an alternative to PEG/dextran IV. GENETIC PROPERTIES OF
and PEG/salt two-phase systems (13). The enzymatic LEVANSUCRASE
or chemical hydrolytic products of levan may be used
in the food industry as sweeteners or dietary ber: The molecular weight of bacterial levansucrases is
ultra-high-fructose syrups (UHFS) and -(2,6)-linked reported to be in the range of 4564 kDa. Several ana-
fructofuranosyl oligosaccharides (7). Several microor- logies in their primary structures have conrmed that a
ganisms producing various levan-assimilating enzymes close relationship exists among levansucrases from
such as exo- or endo-type levanase, fructose dimer- or Gram-negative bacteria. The molecular weight of
heptamer-producing levanase, and levan fructotrans- levansucrases from Gram-negative bacteria is nearly
ferase were isolated and characterized (14). Together the same, about 4547 kDa, while those from Gram-
with the difructose anhydride compounds (DFA I and positive bacteria are slightly larger (5464 kDa).
III) from inulin, which are -(2,1)-linked fructans Eight levansucrase genes from microorganisms have
found in plants, DFA IV from levan is an attractive been cloned and characterized to date (18, 19). The
material as low-calorie sugar, non or antitooth- deduced amino acid sequences are aligned in Figure
decaying sweetener, stabilizer, and antianemic agent 2. All levansucrases share several conserved regions,
in the food and pharmaceutical industries (15). which are thought to be important for the enzyme
Another reason for the increasing interest in levan- activity. Although conservation is observed, dissimilar-
sucrase is that the enzyme can catalyze the transfer of ity exists depending on the source of the enzyme.
fructosyl residue to a variety of sugar molecules. Some Levansucrases from Gram-negative origin show rela-
of the resulting hetero-oligosaccharides have potential tively high similarity (> 50%), when compared with
applications as sweeteners and probiotics (16). the Gram-positive bacterial enzymes. However, very
Currently fructo-oligosaccharides and lactosucrose, little similarity (< 30%) exists among enzymes from
which are formed using a fungal fructosyltransferase, the two sources. In the case of protein secretion
are available in the market as a functional food. mechanisms, they are also thought to be different on
Levansucrase enables the formation of a variety of the basis of the presence or absence of a signal peptide.

Figure 2 Multiple alignment of deduced amino acid sequences of bacterial levansucrases. Origins of levansucrases are indicated at
the end of the sequences. Identical and similar residues in all levansucrases are indicated by asterisks. Regions considered to be
important for activity are boxed (IVII). Amino acid residues that are different between Gram-negative and Gram-positive origins
are indicated by dots.

It is generally accepted that levansucrases from Gram- mined in terms of glucose-releasing activity in the
negative bacteria are secreted by a signal peptide inde- hydrolysis reaction is fairly small at lower tempera-
pendent pathway. It is plausible that the Gram-positive tures. The enzyme is thermolabile and loses its activity
and Gram-negative genes diverged at an early stage of at 45 C for 15 min.
evolution and have separately evolved ever since. Interestingly, Z. mobilis enzyme was most active at
Although the amino acid sequences of levansucrases extreme temperatures < 10 C (9). Therefore, levan for-
do not show any considerable homology to those of mation using Z. mobilis enzyme can be conducted in a
sucrose-related enzymes, the third (EWS/AGT/SP/A stable operation and with less contamination pro-
) and the fourth (FRDP) conserved regions are blems.
found in all fructosyl- and glucosyltransferases,
sucrase, sucrose 6-phosphate hydrolase, and even in
fructan-hydrolyzing enzymes. That the regions are pre- C. Cofactors
served in all sucrose-related enzymes implies that they
may be catalytically important regions for the hydro- Sucrose is directly converted to levan by the enzyme
lysis of sucrose. The serine residue in the sixth without the need of any primer, nucleotide sugar inter-
region (YLFTI/DS) has been proposed as the mediate, or sugar phosphate.
putative residue of the catalytic site (20).
D. Inhibitors and Activators

V. BIOCHEMICAL PROPERTIES OF
LEVANSUCRASE There are few reports on inhibitors and activators of
the enzyme. The inhibition of the enzyme by glucose is
The substrate specicity of levansucrase is extremely a function of glucose concentration, and complete inhi-
low. Sucrose and rafnose serve as fructosyl donors. bition occurs at 16% glucose concentration. Tris-HCl
The glucose moiety of sucrose can be replaced by var- buffer is an efcient inhibitor. The activity is inhibited
ious sugars such as D-xylose, L-arabinose, lactose, etc. slightly by EDTA, and no disulde bonds are involved
Substrate specicity for the acceptors is, however, rela- in the activity. Increased thermal stability and
tively broad. Several kinds of alcohol; mono-, di- or enhanced activity of the enzyme in the presence of
oligosaccharides; sugar alcohols; and levan are known Fe2 or Ca2 have been reported, with some excep-
acceptors. The specicity toward acceptor molecules tions (22). Polyethylene glycol 4000 has an activating
differs depending on the catalytic properties of each effect by increasing the maximum velocity. The addi-
enzyme. In B. subtilis levansucrase, a turnover number tion of levan in small quantities to the reaction mixture
of 17,000 moles of sucrose per minute per mole of also stimulates the activity of levansucrase and causes
enzyme at 37 C and pH 6.0 was obtained (21). the synthesis of the high molecular weight levan.
A. pH Optimum
E. Kinetic Constants
The enzyme has a high activity at pH 56, and is stable
at pH 47. No activity is observed below pH 3 and The Km values of levansucrase from B. subtilis are
above pH 9. Thus, the enzyme has a very narrow spec- 20 mM for sucrose and 50 mM for the analog glu-
trum of pH with a trapezoid-shaped curve. cosido-sorboside at pH 6.0 and 37 C. The afnity con-
stant for the short-chain levan (DP 40) acting as an
B. Optimum Temperature acceptor-activator is 5 mM under the same conditions
(21).
The range of optimal temperature for levan formation
is rather uniform (1840 C) with the exception of the
psychrophilic enzyme of Z. mobilis (0 C), B. subtilis F. Storage Property
(> 10 C), and the thermostable enzyme R. aquatilis
(50 C). One striking feature is that the synthesis of The enzyme solution is stable at 4 C for several
levan by levansucrase occurs far more effectively at months without loss of activity. The enzyme was rela-
low temperatures than at room temperature or at tively resistant to freezing, thawing, and protease treat-
higher temperature, though the apparent activity deter- ment.

G. Toxicity First reaction:
Glucose Glucose Oxidase Gluconic acid
Interestingly, the heterologous expression of levansu- ! H2 O2
H2 O O2
crase gene (sacB) from B. subtilis in Gram-negative
bacteria appears to be toxic to the host (23). The leth- Second reaction:
ality of gene expression was observed in a number of H2 O2 Oxidized
Gram-negative bacteria, but not in Gram-positive bac- Peroxidase
o-Dianisidine o-Dianisidine
teria like Streptomyces lividans with the exception of !
Colorless (Brown)
Corynebacterium glutamicum, which contains mycolic
acid in the outer membrane. This makes the bacterium The assay is commercially available as a kit from
behaves as Gram-negative bacterium. Thus, the use of many biological-product manufacturing companies.
the gene as a selection marker has been successful to a Alternatively, the Somogy-Nelson method could be
certain extent, although the molecular and biochemical employed. One unit of enzyme activity is dened as
basis of toxicity is still unclear. the amount or enzyme releasing 1 mol of glucose per
min. The HPLC method is commonly employed for the
quantitative determination of all components in the
VI. DETERMINATION OF reaction mixture, including levan, oligosaccharides,
LEVANSUCRASE ACTIVITY sucrose, fructose, and glucose. The method is described
here in detail (24).
Several analytical methods can be employed for the
measurement of levansucrase activity. Isotopic proce- A. Reagents
dure utilizes [14 C]sucrose as a substrate and measures
the incorporation of isotopes into the products. This 1. Substrate solution: 100 mL solution of 1%
procedure is powerful when a wild-type strain with sucrose and 0.05 M acetate buffer, pH 6.0, is prepared
very little activity is employed as an enzyme source. and stored in refrigerator. The solution is stable for
The measurement of the weight of dried levan that is several months.
formed by the enzyme reaction is conducted after 2. Conditions: The analysis is conducted by HPLC
ethanol precipitation of the reaction mixture. (Gold Systems, Beckman) with a refractive index
Another method for the determination of levan con- detector and Shodex Ionpack KS-820 column (Showa
tent, which gives a linear curve at low concentrations, Denko Co., Japan). Deionized water is used as a
is to measure the optical density at visible range (450 mobile phase at a ow rate of 0.4 mL/min.
550 nm), corresponding to the turbidity of the reac-
tion mixture generated by the formation of levan. By B. Procedure
paper and TLC chromatographic analyses, one can
distinguish the polymeric products (nonmobile), One milliliter reaction mixture containing 1% sucrose
oligomeric products, substrate, and glucose in 0.05 M acetate buffer and enzyme (13 units) is
(byproducts), qualitatively or quantitatively, after incubated for 30 min. The incubation temperature
visualization. Sucrose hydrolysis is commonly related depends on the enzyme source: 10 C for Z. mobilis,
to determination of levansucrase activity. The meth- and 20 C for R. aquatilis. The reaction can be termi-
ods established are based on the fact that glucose is nated by adjusting the pH of the reaction mixture to an
formed stoichiometrically in relation to the amount of alkaline range (above pH 9) with 10 mM NaOH solu-
fructose incorporated into products. The amount of tion. Subsequently, the mixture is ltered using a 0:45
glucose generated by enzymatic reaction can be m pore size membrane lter and analyzed by HPLC.
assayed quantitatively by colorimetric enzymatic ana- Oligosaccharide content is determined by using the cali-
lyses (18). The enzymatic analysis, which is conveni- bration curve of rafnose as a standard. Under these
ent, rapid, and highly accurate, is preferred. In this conditions, a highly polymerized product (levan) and
method, the reaction mixture is incubated at 37 C, at glucose are formed as major reaction products. A small
which the hydrolysis activity of levansucrase is more amount of free fructose, oligosaccharides, and the
active than the polymerization activity (transfructosy- remaining sucrose is also detected (Fig. 3). The oligo-
lation activity). It is followed by the coupled reactions saccharide formed by levansucrase puried from Z.
of glucose oxidase and peroxidase toward the liber- mobilis has been identied mainly as 1-ketose (25).
ated glucose. Sugar components and linkage type of the highly poly-

Figure 3 HPLC analysis of the reaction products formed by levansucrase with sucrose. Chromatographic conditions are
described in the text. (From Ref. 9.)
merized product are determined by acid hydrolysis, VIII. PURIFICATION OF LEVANSUCRASE

methylation, and NMR shift experiments (7). FROM Z. MOBILIS
A. Crude Enzyme
VII. PURIFICATION Z. mobilis ATCC10988 is grown in a rich medium con-
sisting of (per liter); 50 g sucrose, 20 g yeast extract and
Bacterial levansucrases seem to attach on the outer 1 g potassium phosphate (monobasic). Cells are culti-
surface of the membrane as well as secreted extra- vated in a 20-L jar-fermentor (working volume 15 L) at
cellularly (7, 26). In some cases, the enzyme is intra- 30 C. After 24 h, cells are harvested and washed briey
cellular, but its function has not been illustrated. The with distilled water. The pellets from 1 L culture broth
prole of enzyme activity from Z. mobilis culture are resuspended in 50 mL of 100 mM phosphate buffer
shows that the activity is maximal at the late loga- (pH 6.0) and incubated at 30 C for 30 min with mild
rithmic or early stationary phase of cells grown in shaking to release the enzyme from the cell surface and
sucrose medium. The initial recovery of levansucrase the supernatant is saved. Cell pellets are washed once
from the supernatant of cell cultures grown at the late and mixed with the supernatant to yield the crude
logarithmic phase by ammonium precipitation is enzyme.
somewhat difcult. This is likely due to the low
yield of proteins and the presence of polymeric
compound (levan) in the culture medium, in which B. Purication Procedures
levansucrase is inducibly produced in the presence
of sucrose. To circumvent this, the preparation of The crude enzyme is puried according to the follow-
cell-associated levansucrase was employed by Pabst ing procedure: rst ammonium sulfate precipitation
et al. (26). The enzyme bound to levan which is (80% saturation), ion exchange chromatography,
encapsulated outside of the stationary-phase grown hydroxyapatite chromatography, second ammonium
cells can be recovered in signicant amounts. This sulfate precipitation (50% saturation), and gel ltra-
method provides an additional advantage of reducing tion chromatography. Phosphate buffer (20 mM, pH
the number of contaminated proteins. 6.0) is used throughout the purication procedure. A

Table 1 Summary of Levansucrase Purication Steps from Z. mobilis
Volume Units Protein Spec. Act. Yield Purication

Step (mL) (total) (mg/mL) (U/mg) (%) (fold)
Cell washed solutions 1300 a 0.35

1st (NH4 2 SO4 115 1.28
Ion exchange 38 4.35 0.57 0.21 100 1.00
Hydroxyapatite 20 2.58 0.41 0.31 65 1.52
2nd (NH4 2 SO4 2 0.96 0.46 1.04 21 5.07
Superose 12 1.5 0.72 0.13 3.75 16.5 18.3
a
Not determined.
Source: Ref. 24.
summary of the purity and recovery of the enzyme is the puried enzyme is subjected to SDS-PAGE. The
given in Table 1. As a result of chromatography on enzyme can be puried from culture broth 18.3-fold to
DEAE-Toyopearl 650 M column, two distinct peaks a specic activity of 3:75 U mg1 protein, with a yield
of sucrose-hydrolyzing activity can be seen (Fig. 4). of 16.5%. The molecular weight of the enzyme is
The rst peak is responsible for levansucrase; the sec- 91,000 by Superose 12 gel ltration, and 46,000 by
ond one, for sucrase. After the successive purication SDS-PAGE, indicating that levansucrase is a dimer.
steps of the protein by hydroxyapatite chromatogra- The optimum pH for the enzyme activity is around
phy and second ammonium sulfate purication, the pH 4.0 for sucrose hydrolysis, and is around pH 5.0
small amount of contaminated proteins can be elimi- for levan formation (24).
nated from the enzyme solution by gel ltration chro- A method monitoring the presence of sucrose-split-
matography (FPLC). A single band is observed when ting enzymes has been developed using electrophoresis
and zymogram staining with 2,3,5-triphenyltetrazo-
lium chloride (TTC) reagent prior to purication
(27). Levansucrase activity can be easily distinguished
by the formation of a white and opalescent band. The
use of periodic acid Schiff (PAS) reagent to stain levan
formed by levansucrase on polyacrylamide gel has also
been developed (28). Some enzymes form large aggre-
gates in nature or during purication, causing the loss
of enzymes and difculties in purication. A small
amount of unknown carbohydrates may be responsible
for the formation of aggregates. This problem can be
overcome by the treatment with Triton X-100 (29). In
the case of the Z. mobilis enzyme, such aggregation is
not observed during purication. To eliminate the
copurication of other enzymes with sucrose-splitting
activity, the levan formation activity is monitored
using rafnose as a substrate, which does not normally
serve as a substrate for glucosyltransferase or dextran-
Figure 4 DEAE-anion exchange chromatogram of the sucrase.
extracellular saccharolytic enzymes of Z. mobilis ZM1. Cell
washed solution from Z. mobilis ZM1 is put on the column
(2:5 30 cm) pre-equilibrated with 20 mM phosphate buffer
(pH 6.8), and eluted with NaCl gradient (00.5 M). REFERENCES
Levansucrase activity is monitored by sucrose hydrolysis
activity and the type of enzymes contained in fractions is 1. RG Chambert, G Gonzy-Treboul, R Dedonder.
monitored by electrophoresis and zymogram staining with Kinetic studies of levansucrase of Bacillus subtilis.
TTC reagent. (From Ref. 24.) Eur J Biochem 41:285, 1974.

2. RG Chambert, G Gonzy-Treboul. Levansucrase of 17. MG Kim, JW Seo, KB Song, CH Kim, BH Chung,
Bacillus subtilis. Characterization of a stabilized fru- SK Rhee. Levan and fructosyl derivatives formation
tosyl-enzyme complex and identication of an aspar- by a recombinant levansucrase from Rahnella acqua-
tyl residue as the binding site of the fructosyl group. tilis. Biotechnol Lett 20:333, 1998.
Eur J Biochem 71:493, 1976. 18. KB Song, HK Joo, SK Rhee. Nucleotide sequence of
3. KB Song, JW Seo, MK Kim, SK Rhee. Levansucrase levansucrase gene (levU) of Zymomonas mobilis
from Rahnella acquatilis: gene cloning, expression and ZM1(ATCC 10988). Biochim Biophys Acta
levan formation. Ann NY Acad Sci 864:506, 1998. 1173:320, 1993.
4. PJ Cheetham, AJ Hacking, M Vlitos. Synthesis of 19. J Ariesta, L Hernandez, A Coego, V Suarez, E
novel disaccharides by a newly isolated fructosyl Balmori, C Menendez, M Petit-Glatron, R
transferase from Bacillus subtilis. Enzyme Micro Chambert, G Selman-Housein. Molecular characteri-
Technol 11:212, 1989. zation of the levansucrase gene from the endophytic
5. N Sprenger, K Bortlik, A Brand, T Boller, A sugarcane bacterium Acetobacter diazotrophicus
Wiemken. Purication, cloning, and functional SRT4. Microbiology 142:1077, 1996.
expression of sucrose:fructan 6-fructosyltransferase, 20. R Chambert, MF Petit-Glatron. Polymerase and
a key enzyme of fructan synthesis in barley. Proc hydrolase activities of Bacillus subtilis levansucrase
Natl Acad Sci USA 92:11652, 1995. can be separately modulated by site-directed mutagen-
6. GAF Hendry. Evolutionary origins and natural func- esis. Biochem J 279:35, 1991.
tions of fructansa climatological, biogeographic and 21. R Dedonder. Levansucrase from Bacillus subtilis.
mechanistic appraisal. New Phytol 123:3, 1993. Methods Enzymol 8:500, 1966.
7. YW Han. Microbial levan. Adv Appl Microbiol 22. L Hernandez, J Arrieta, C Menendez, R Vazquez, A
35:171, 1990. Coego, V Suarez, G Selman, M Petit-Glatron, R
8. F Kunst, G Rapoport. Salt stress is an environmental Chambert. Isolation and enzymatic properties of
signal affecting degradative enzyme synthesis in levansucrase secreted by Acetobacter diazotrophicus
Bacillus subtilis. J Bacteriol 177:2403, 1995. SRT4, a bacterium associated with sugarcane.
9. KB Song, JW Seo, SK Rhee. Production of levan, a Biochem J 309:113, 1995.
fructose polymer, using an overexpressed recombinant 23. W Jager, A Schafer, A Puhler, G Labes, W
levansucrase. Ann NY Acad Sci 799:601, 1996. Wohlleben. Expression of the Bacillus subtilis sacB
10. MJM Ebskamp, I Van der Meer, BA Spronk, PJ gene leads to sucrose sensitivity in the gram-positive
Weisbeek, SJ Smeekens. Accumulation of fructose bacterium Corynebacterium glutamicum but not in
polymers in transgenic tobacco. Bio/Technology Streptomyces lividans. J Bacteriol 174:5462, 1992.
12:272, 1994. 24. KB Song, JW Seo, HK Joo, SK Rhee. Purication
11. J Leibovici, Y Stark. Increase in cell permeability to a and characterization of an extracellular levansucrase
cytotoxic agent by the polysaccharide levan. Cell Mol from Zymomonas mobilis ZM1(ATCC10988). Kor J
Biol 31:337,1985. Appl Microbiol Biotechnol 26:309, 1998.
12. GMT Calazans, CE Lopes, RMOC Lima, FP de 25. RG Crittenden, HW Doelle. Identication and char-
Franca. Antitumor activities of levans produced by acterization of the extracellular sucrases of
Zymomonas mobilis strains. Biotechnol Lett 19:19, Zymomonas mobilis UQM 2716 (ATCC 39676).
1997. Appl Microbiol Biotechnol 41:302, 1994.
13. BH Chung, WK Kim, KB Song, CH Kim, SK Rhee. 26. MJ Pabst, JO Cisar, CL Trummel. The cell wallasso-
Novel polyethylene glycol/levan aqueous two-phase ciated levansucrase of Actinomyces viscosus. Biochim
system for protein partitioning. Biotechnol Lett Biophys Acta 556:274, 1979.
11:327, 1997. 27. P OMullan, M Szakacs-Dobozi, DE Eveleigh.
14. H Murakami, T Kuramoto, K Mizutani, H Nakano, Identication of saccharolytic enzymes of
S Kitahata. Purication and some properties of a new Zymomonas mobilis CP4. Biotechnol Lett 13:137,
levanase from Bacillus sp. No. 71. Biosci Biotech 1991.
Biochem 56:608, 1992. 28. AW Miller, JF Robyt. Direction of dextransucrase
15. K Saito, A Yokoda, F Tomita. Molecular cloning of and levansucrase on polyacrylamide gels by the
levan fructotransferase gene from Arthrobacter nicoti- periodic acid-Schiff stain: staining artifacts and their
novarans GS-9 and its expression in Escherichia coli. prevention. Anal Biochem 156:357, 1986.
Biosci Biotech Biochem 61:2076, 1997. 29. MF Petit-Glatron, R Chambert, M Steinmetz.
16. JW Yun. Fructooligosaccharidesoccurrence, pre- Levansucrase of Bacillus subtilis. Characterization of
paration, and application. Enzyme Microb Technol a form isolated from phenol-treated cells and acti-
19:107117, 1996. vated by Triton X-100. Eur J Biochem 103:189, 1980.

49
Cyclodextrin Glycosyltransferase
Lubbert Dijkhuizen and Bart A. van der Veen

University of Groningen, Haren, The Netherlands
I. INTRODUCTION and pullulanase (EC 3.2.1.41), both members of family

13. Amylases can be further subdivided into endo- and
Cyclodextrin glycosyltransferase (CGTase) is a mem- exoacting enzymes. A typical endoacting enzyme is -
ber of the -amylase family of glycoside hydrolases amylase (EC 3.2.1.1), cleaving (1-4) bonds randomly
(family 13) (1). Enzymes of this family display a wide in the starch molecule, producing (branched) oligosac-
diversity in reaction specications, catalyzing at least charides of various lengths. Exoacting amylases such
18 different reactions. Whereas amylases generally as -amylase (EC 3.2.1.2, family 14 of glycoside hydro-
hydrolyze (1-4) glucosidic bonds in starch molecules, lases) cleave (1-4) bonds at the nonreducing end of
CGTases mainly catalyze transglycosylation reactions. the starch molecule and hence produce only low-mole-
Structure/function relationships in the -amylase cular-weight products from starch (mostly glucose or
family have been studied extensively, clarifying the maltose). Most of these enzymes are incapable of
mechanistic basis of the unique activities of CGTase bypassing (1-6) linkages; degradation of branched
(25). substrates therefore remains incomplete, leaving high-
Starch and derived linear or cyclic oligosaccharides molecular-weight compounds (limit dextrins). In
are substrates in the CGTase-catalyzed reactions. enzymes also able to cleave (1-6) linkagesfor
Starch consists of two types of glucan polymers: amy- instance, glucoamylase (EC 3.2.1.3, family 15 of glyco-
lose and amylopectin. Potato starch for instance con- side hydrolases) and -glucosidase (EC 3.2.1.20)this
sists of 20% amylose and 80% amylopectin. Amylose reaction is slow compared to hydrolysis of (1-4)
consists mainly of linear chains of (1-4)-linked glu- bonds.
cose residues. Amylopectin is a highly branched (1- CGTase (EC 2.4.1.19) is a unique member of
4) glucan polymer (approximately (1-6) linkage per 20 the -amylase family. Its main products when
glucose residues). acting on starch are cyclodextrins and highly
Figure 1 shows an overview of enzymes of various branched high-molecular-weight dextrins (CGTase
families active on starch (amylopectin in this case). limit dextrins). CGTase has been identied in bacteria
Many starch-degrading enzymes are hydrolytic, cleav- belonging to the genera Bacillus, Clostridium,
ing the linkages in starch; the cleavage product subse- Klebsiella, Micrococcus, Thermoanaerobacter, and
quently reacts with water, resulting in a new reducing Thermoanaerobacterium (6), and in a single archaeon,
end. These enzymes can be roughly divided into amy- Thermococcus (7). CGTase is present as an extracellu-
lases, hydrolyzing (1-4) linkages, and debranching lar enzyme and functions in the initial attack on this
enzymes, hydrolyzing (1-6) linkages. Examples of polymeric substrate. These bacteria subsequently use
debranching enzymes are isoamylase (EC 3.2.1.68) the cyclodextrins produced as carbon and energy

Figure 1 Action of enzymes involved in the degradation of starch. (*) Glucose molecule with a reducing end; (*) glucose
molecule without a reducing end. Arrows indicate preferred cleaving point on the starch molecule. (From Ref. 70.)
sources for growth. This involves a cell-associated CGTase enzymes generally display only a relatively
cyclomaltodextrinase (EC 3.2.1.54), yielding glucose, low starch hydrolytic activity (Fig. 2A). They mainly
maltose, and maltotriose (8). Glucose is metabolized catalyze transglucosylation reactions that can be
intracellularly via glycolysis. described as: Gn Gm ! Gn x Gm x,
in which G(n) is the donor and G(m) the acceptor
oligosaccharide consisting of n and m residues, respec-
tively. Disproportionation (Fig. 2B) can be regarded as
the default reaction, and is also catalyzed by several
other members of the -amylase family (e.g., 4--glu-
canotransferase, EC 2.4.1.25). The specic CGTase
catalyzed reaction is the cyclization reaction (Fig.
2C) in which the part of the donor that has been
cleaved off also acts as the acceptor, resulting in for-
mation of a cyclodextrin. This reaction can be
described as: Gn ! cyclic Gx Gn x. The
reverse reaction, coupling, is also catalyzed by the
enzyme (Fig. 2D).
II. APPLICATIONS OF CGTASE
The cyclodextrins produced by CGTases from starch

via the cyclization reaction provide the basis for most
of the applications of CGTase in the food industry.
The CGTase-catalyzed coupling and disproportiona-
tion reactions, allowing synthesis of modied oligosac-
Figure 2 Schematic representation of the CGTase-catalyzed charides from starch or cyclodextrins and alternative
reactions. Circles represent glucose residues; the white circles acceptor substrates, are drawing increasing attention
indicate the reducing end sugars. (A) hydrolysis; (B) dispro- as well. Applications of CGTase limit dextrins are
portionation; (C) cyclization; (D) coupling. (From Ref. 65.) also being explored.

Cyclodextrins are torus-shaped molecules of cyclic sional form and size. The driving force is the entropic
(1-4)-linked oligosaccharides mainly consisting of six, effect of displacement of water molecules from the
seven, or eight glucose residues (-, -, or -cyclodex- cavity (11). Another possibility is that this water causes
trin, respectively) (Fig. 3). In cyclodextrins the second- strain on the cyclodextrin ring, which is released after
ary hydroxyl groups (C2 and C3) are located on the complexation, producing a more stable, lower energy
wider edge of the ring, and the primary hydroxyl state (9, 11). Other parameters for complexation are
groups (C6) on the other edge; the apolar C3 and C5 charge or polarity of the guest compound and compe-
hydrogens and etherlike oxygens are on the inside of tition with other molecules from the medium. Because
the toruslike molecules. This results in a molecule with the inclusion complexes are quite stable, they can be
a hydrophilic outside, which can dissolve in water, and separated from the medium by crystallization (12).
an apolar cavity, which provides a hydrophobic Entrapment in cyclodextrins often results in changes
matrix, described as a microheterogeneous environ- in the chemical and physical properties of the guest
ment (9). Cyclodextrins form inclusion complexes molecules, suitable for applications in the food indus-
with a wide variety of hydrophobic guest molecules. try. Numerous other cyclodextrin applications, outside
One or two guest molecules can be entrapped by one, the scope of this review, are in analytical chemistry,
two, or three cyclodextrins (10). The most important agriculture, and the pharmaceutical eld (5).
parameter for complex formation with hydrophobic In the food industry, cyclodextrins nd a range of
compounds or functional groups is their three-dimen- applications. Cyclodextrins have a texture-improving
Figure 3 Structure and properties of cyclodextrins. (a) -, -, and -cyclodextrins; (b) 3D form and properties of cyclodextrins;
a: 14.6, 15.4, and 17.5 A for -, -, and -cyclodextrins, respectively; b: 4.75.3, 6.06.5, 7.58.3 A for -, -, and -cyclodextrins,
respectively; (c) formation of inclusion complexes of cyclodextrins and hydrophobic molecules. (From Ref. 71.)

effect on pastry and on meat products. They reduce cyclodextrin yields (6). More recently, CGTases have
bitterness, ill smell, and bad taste, and stabilize avors been characterized from thermophilic anaerobic bac-
when subjected to long-term storage. Emulsions in teria belonging to the genera Thermoanaerobacter
mayonnaise, margarine, or butter creams are stabilized (12, 24) and Thermoanaerobacterium (25) which are
with -cyclodextrin. Furthermore, -cyclodextrin is active and stable at high temperatures and low pH
used to remove cholesterol from milk, yielding dairy values and are able to solubilize starch, thereby elim-
products low in cholesterol. Marketed products inating the need for -amylase pretreatment without
involve reduced cholesterol egg yolks (Simply Eggs) any traces of low-molecular-weight oligosaccharides
by Michael Foods in the United States, cholesterol- produced in the initial stages of the reaction (26). The
reduced butter under Natural and Ballade trademarks use of these thermostable CGTases has the added
by Entremont in France and Corman in Belgium, and advantage that the total cyclodextrin production
a range of avored salts (Compack) in Hungary (13). time can be shortened (6). The Thermoanaerobacter
CGTase is also used in preparation of doughs for CGTase (maximal activities at 90 C and pH 5.8)
baked products; its incorporation into doughs was found commercial application in 1996.
claimed to increase the volume of the baked product
(14).
Conversion of amylose by CGTase not only yields III. CGTASE PROTEIN PROPERTIES
cyclodextrins but initially also larger-size cyclic (1-4)-
glucans with a degree of polymerization ranging from At present, at least 38 CGTases have been identied
nine to > 60 (15). Similar observations have been made and puried, and the corresponding genes character-
with B. stearothermophilus branching enzyme (16) and ized. They are all monomeric enzymes, generally 680
potato disproportionating enzyme (17). These new 685 amino acids in size, yielding molecular weights of
starch structures combine various interesting proper- 75 kDa. CGTases show a clear similarity in amino
tieslow viscosity, high solubility, and a high molecu- acid sequence, ranging from 47% to 99% homology.
lar weightand are expected to nd applications in the Enzymes of the -amylase family share only 30%
food industry (18). overall amino acid sequence similarity. Four highly
The CGTase coupling and disproportionation reac- conserved regions have been identied in members of
tions allow synthesis of a range of novel glycosylated this family (Fig. 4). All four regions contain completely
compounds from starch or cyclodextrins as donor sub- invariant amino acid residues within the -amylase
strates and various acceptor molecules (1921). A com- family, and the functions of most of these have been
mercial example is glycosylation of the intense elucidated by x-ray crystallography, site-directed
sweetener stevioside. This bitter compound is isolated mutagenesis, and chemical modication of various
from leaves of the plant Stevia rebaudiana; it has a low members of this family. These residues are directly
solubility. Glycosylation decreases bitterness and involved in catalysis, either through substrate binding,
increases solubility (6). bond cleavage, transition state stabilization, or as
Like most starch-degrading enzymes, the CGTase ligands of a calcium-binding site present near the active
from B. macerans, which is used for the commercial site. Three carboxylic acid groups, one glutamic acid
production of cyclodextrins (22), is poorly active on and two aspartic acid residues, are essential for cataly-
native starch with its well-organized granular structure tic activity in -amylases and CGTases (Asp229,
held together by internal hydrogen bonds. Heating in Glu257, and Asp328 in CGTase from B. circulans)
water (jet cooking) weakens these hydrogen bonds and (27, 28). Two conserved histidine residues, His140
causes swelling and gelatinization (23), resulting in a and His327 (CGTase numbering), are involved in sub-
very viscous starch solution when performed at starch strate binding and transition state stabilization (29,
concentrations of industrial interest. This initial pro- 30). A third histidine, present only in some -amylases
cessing step is performed at temperatures up to 105- and CGTases (His233), is involved in substrate binding
110 C, and -amylase is added in order to liquefy the and acts as a calcium ligand with its carbonyl oxygen
starch to make it suitable for incubation at the lower (31, 32). Arg227 is important for the orientation of the
temperatures (55 C) required for the CGTase-cata- nucleophile (Asp229; see below) (30). The role of
lyzed production of cyclodextrins. Unfortunately, the Asp135 is not clear, but it is in close proximity to the
-amylase used for liquefaction produces maltodex- catalytic site. Asn139 again is a calcium ligand.
trins, which will act as acceptor molecules in the cou- In contrast to the limited similarity observed in a
pling reaction catalyzed by CGTase, severely reducing primary structure, the three-dimensional structures of

Figure 4 Amino acid sequence alignment of the four conserved regions in members of the -amylase family. TAA: -amylase,
Aspergillus oryzae (Taka-amylase A) (33); CGT: CGTase, Bacillus circulans strain 251 (31); CD: cyclodextrinase, Klebsiella
oxytoca (72); PUL: pullulanase, Klebsiella aerogenes (73); ISO: isoamylase, Pseudomonas amyloderamosa (74). The residues
are numbered according to the CGTase from B. circulans strain 251. An asterisk indicates amino acid identity; a dot indicates
amino acid similarity.
-amylases (33) and CGTases (31, 3437) are quite domain; in glucoamylases (family 15 of glycoside
similar. -Amylases generally consist of three struc- hydrolases) it is attached to the C- or N-terminus of
tural domains, A, B, and C, while CGTases show a the catalytic domain via a glycosylated linker (Fig. 5).
similar domain organization with two additional The E-domain consists of 110 amino acids and is
domains, D and E (Fig. 5). Domain A contains a responsible for the adsorption of the enzyme onto
highly symmetrical fold of eight parallel -strands granular starch (see below).
arranged in a barrel encircled by eight -helices. This All CGTases studied not only catalyze the
so-called (=8 -or TIM-barrel catalytic domain of three transglycosylation reactions but also display
300400 residues is present in all enzymes of the - hydrolytic activity (Fig. 2). Incubation of CGTase
amylase family (38). The =8 barrel was rst with starch thus may not only yield cyclodextrins
found in the structure of chicken muscle triose phos- but also linear products. In some cases this has
phate isomerase (39), but it has been shown to be wide- resulted in misidentication of CGTase enzymes.
spread in functionally diverse enzymes (40). Several The Thermoanaerobacterium thermosulfurigenes EM1
prolines and glycines anking loops connecting the - CGTase was initially identied as an -amylase
strands and -helices have been found to be highly because of its relatively high hydrolytic activity (44).
conserved in these enzymes (41). The catalytic and sub- Further studies showed that it possesses a clear cycli-
strate-binding residues conserved in the -amylase zation activity, as in other CGTases; amino acid align-
family are located in loops at the C-terminal of - ments with other CGTases also show a high overall
strands in domain A. The loop between -strand 3 sequence similarity, with all ve domains present
and -helix 3 of the catalytic domain is rather large (25). The CGTase converts starch not only into cyclo-
and is regarded as a separate structural domain. This dextrins (35%) but also into linear sugars (11%) (45).
B-domain consists of 44133 amino acid residues and In contrast, the CGTase from B. circulans strain 251
contributes to substrate binding. The C-domain is has minor hydrolytic activity; this enzyme exclusively
100 amino acids long and has an antiparallel -sand- forms cyclodextrins (46).
wich fold. Domain C of the CGTase from B. circulans Recently the 3D structure of the Bacillus stearother-
strain 251 contains one of the maltose-binding sites mophilus maltogenic -amylase Novamyl has been
(MBS) observed in the structure derived from mal- determined (47). Unlike the conventional -amylases
tose-dependent crystals (see below) (31). This MBS is of family 13, Novamyl exhibits the ve-domain struc-
involved in raw starch binding (42); the C-domain thus ture associated with CGTase enzymes (Fig. 5, G2A).
contributes in substrate binding. This C-domain may Its overall sequence similarity with CGTase is high,
determine bond specicity, since in enzymes hydrolyz- and the 3D structures are very similar. Most interest-
ing or forming 1,6) bonds (e.g., pullulanase, isoamy- ingly, Novamyl contains a 5 amino acid residue inser-
lase, branching enzyme) the A-domain is followed by a tion in the B-domain, partially enclosing the active site
different domain (Fig. 5) (43). The D-domain is 90 at the 3 subsite. The absence of this insertion in
amino acids in size and is almost exclusively found in CGTases allows for additional subsites, with up to 7
CGTases; it carries an immunoglobulin fold but its characterized crystallographically (32). Novamyl thus
function remains unknown. The E-domain is more is either an -amylase or a CGTase with unusual
widespread in starch degrading enzymes. In enzymes properties. Studies of evolutionary relationships within
of the -amylase family it is present as the C-terminal the -amylase family have provided evidence that

three carboxylic amino acids in CGTase have been
claried by x-ray crystallographic studies with acar-
bose, a potent pseudotetraose inhibitor, bound in the
active site (28). Glu257 (CGTase numbering) is the
general acid catalyst, acting as proton donor; Asp229
serves as the nucleophile, stabilizing the intermediate;
and Asp328 has an important role in substrate binding.
For retaining enzymes the intermediate either could be
an oxocarbonium ion which is electrostatically stabi-
lized by a carboxylate, or involves formation of a cova-
lent bond, in which one of the catalytic aspartates is
presumed to act as a nucleophile. Although initially the
nature of the intermediate was disputed, it is now gen-
erally accepted that the reaction proceeds via a cova-
lent intermediate. Clear evidence for a covalent
Figure 5 Domain level organization of starch degrading glycosylenzyme intermediate in family 13 has been
enzymes. CGT: CGTase, Bacillus circulans; G2A: maltogenic obtained from rapid trapping studies with natural sub-
-amylase, Bacillus stearothermophilus; G4A: maltotetraose strates. Low-temperature 13 C NMR experiments have
forming -amylase, Pseudomonas stutzerl; TAA: -amylase,
provided evidence for the formation of a -carboxyla-
Aspergillus oryzae (Taka-amylase A); CD: cyclodextrinase,
cetal ester covalent adduct between maltotetraose and
Klebsiella oxytoca; ISO: isoamylase, Pseudomonas amyloder-
amosa; PUL: pullulanase, Klebsiella aerogenes: GA: glucoa- porcine pancrease -amylase (53). Conclusive evidence
mylase (family 15 of glycoside hydrolases) from Aspergillus recently came from experiments involving trapping of
niger. (From Ref. 43.) a covalent intermediate with 4-deoxymaltotriosyl -
uoride as a substrate in the virtually inactive
Glu257Gln mutant of B. circulans 251 CGTase (54)
and the x-ray crystallographic structure of the enzyme
CGTase enzymes evolved from -amylases (48). with the covalently linked intermediate (30).
Novamyl may thus be an example of an enzyme at
an intermediary stage in between true -amylases B. Substrate Binding in CGTases
and true CGTases. It appears more likely, however,
that Novamyl is a true CGTase with a few essential The rst important step in enzyme catalysis is binding
mutations modifying product specicity. of the substrate. In several starch-degrading enzymes a
separate domain responsible for adsorption onto raw
starch has been found. Evidence for a starch-binding
IV. PROPERTIES OF CGTASE ENZYMES
site in CGTases separate from the active site was pre-
A. Catalytic Mechanism of the -Amylase sented (55), and fusion of the E-domain of the Bacillus
Family macerans CGTase to -galactosidase demonstrated
that it can indeed function as a starch-binding domain
The reactions catalyzed by CGTase and other enzymes (56). The function of the E-domain has been studied in
of the -amylase family proceed with retention of the more detail with the B. circulans strain 251 CGTase.
substrates anomeric (-)conguration. Since each sub- High concentrations of maltose are required for crys-
stitution at a chiral center results in inversion of con- tallization of this protein (57). Three maltose-binding
guration, catalysis must proceed through a double sites (MBS) were observed at the protein surface, two
displacement reaction (49). The rst step involves a of which (MBS1 and MBS3) contribute to intermole-
protonation of the glycosidic oxygen by a general cular crystal contacts. MBS1 and MBS2 are both
acid catalyst, creating an oxocarbonium transition located on the E-domain, suggesting a role in the raw
state which subsequently collapses into an intermediate starch-binding function of this domain (31). Indeed,
(5052). This intermediate is attacked by a water mutational studies revealed that MBS1 is important
nucleophile (or the C4-OH at the nonreducing end of for (raw) starch binding, while MBS2 assists in guiding
another oligosaccharide in case of transglycosylases, the linear starch chains into the active site via a groove
e.g., CGTase) in the second step, assisted by the base at the surface of the CGTase protein (see below) (31,
form of the acid catalyst. The mechanistic roles of the 42). These CGTase E-domain MBS sites interact

strongly with cyclodextrins and oligosaccharides (58). more detail the mode of substrate binding in the
Recent studies have revealed that the raw starch-bind- groove (32). The substrate binding sites are numbered
ing domain also functions in the disruption of the 2 to 7 [numbering according to Davies et al. (60)],
structure of granular starch (59). with the catalytic site between sites 1 and 1. The
The A-domain contains the catalytic residues of nonreducing end of the oligosaccharide is bound at
CGTases, while domain B is involved in substrate subsite 7, which agrees with the formation of mainly
binding. X-ray crystallographic studies have revealed -cyclodextrin from starch by the enzyme. Factors
a groove on the surface of these enzymes formed on determining cyclodextrin product specicity in
one side by loops of the A-domain and on the other CGTases are discussed in more detail by Van der
side by the B-domain. In crystal structures of CGTase Veen et al. (4). The glucose residue at subsite 7 is
from B. circulans strain 251 (31), where maltose mole- located at the end of the substrate binding groove,
cules serve as contact points between the enzyme mole- interacting with amino acid residues of the B-domain.
cules in the crystals, the functionality of this groove in The glucose residues bound at subsites 1 and 1 only
substrate binding has been shown nicely. From soak- interact with amino acids conserved in the whole -
ing experiments with the CGTase from B. circulans amylase family.
strain 251 the structure of this enzyme with a malto-
nonaose inhibitor was obtained (Fig. 6), revealing in C. Effects of pH and Temperature on CGTase
Activity
CGTase enzymes have been found in both neutrophilic

and alkaliphilic bacteria (6, 61, 62). The optimum pH
for activity is generally around pH 56. CGTase of the
alkaliphile Bacillus sp. 1011 also shows maximum
activity at pH 5.5, but its pH activity prole is very
broad; the activity at pH 8.0 is 90% of that at pH 5.5.
The mutation Phe283Leu shifted the pH curve over
both acidic and alkaline pH ranges (63). Mutant
His327Asn has strongly reduced activity in the alkaline
pH range, down to 30% of that at pH 5.5 (29). The low
and the high slopes of the pH optimum curve of T.
thermosulfurigenes EM1 CGTase clearly changed by
mutations close to the catalytic residue Glu257, the
proton donor in the rst step of the cyclization reac-
tion (45). The pH optimum curve of CGTase thus may
be determined by the protonation state of Glu257.
Changes in the environment of Glu257 before and
after substrate binding can account for the broad pH
optima of CGTase.
CGTase enzymes have been characterized from
both mesophilic and thermophilic sources. The
Thermoanaerobacter sp. (12) and Thermoanaero-
bacerium thermosulfurigenes (25) CGTases display
maximal activities at 8090 C and pH 5.56.0. The
Thermococcus sp. strain B1001 CGTase has a tem-
perature optimum at 90100 C and a pH optimum
at 5.05.5 (7). Substrate (starch) and calcium ions
generally stabilize these enzymes. Fortunately, ve
Figure 6 Schematic representation of the hydrogen bonds
between the Bacillus circulans 251 CGTase and a maltono- different CGTase 3D structures have been solved
naose inhibitor bound at the active site. The subsites are (31, 3437), with amino acid sequence similarities
numbered according to the general subsite labeling scheme > 60%, of which two are thermostable enzymes.
recently proposed for all glycoside hydrolases (60). (From These structures were extensively compared by
Ref. 32.) Knegtel et al. (36), in combination with biochemical

data, identifying many sites contributing to thermo- Table 2 Kinetic Properties of the Coupling Reaction of
stability. Uitdehaag and Dijkstra (64) extended these CGTase from Bacillus circulans Strain 251
studies, focusing on the atomic B factors in any of the
-CD -CD -CD
ve CGTase x-ray structures. They concluded that coupling coupling coupling
Nature has chosen a strategy of stabilizing exible
loops near the active site to develop more thermo- KmCD (mM) 2:2 0:3 0:32 0:02 0:13 0:02
stable CGTase enzymes. KmMDG (mM) 0:45 0:05 18:1 1:4 16:6 3:0
0
KmCD (mM) 5:3 1:2 0:15 0:04 0:12 0:03
0
D. Kinetic Analysis of CGTase KmMDG (mM) 1:09 0:26 8:25 2:2 15:7 6:1
Transglycosylation Reactions Vmax (U/mg) 192 5:7 294 7:6 150 9:0
kcat (sec1 ) 240 7:1 368 9:5 188 11:3
CGTase catalyzes three transglycosylation reactions
(cyclization, coupling, disproportionation; Fig. 2) Source: Ref. 65.
with starch and derived products. In the industrial pro-
Table 3 Kinetic Properties of the Disproportionation
duction process for cyclodextrins, all three reactions
Reaction of CGTase from Bacillus circulans Strain 251
participate in starch degradation and affect the overall
efciency and outcome of the process. Rational engi- KmEPS (mM) 0:223 0:015
neering of specic CGTases thus requires detailed Kmmaltose (mM) 0:827 0:050
knowledge of the kinetic mechanisms of the different Vmax (U/mg) 970 17:6
reactions. The three transglycosylation reactions
kcat (sec1 ) 1213 22
catalyzed by CGTase of B. circulans 251 have been
analyzed in detail (65). Cyclization, coupling, and dis- Source: Ref. 65.
proportionation all involve the same catalytic residues
(Asp328, Glu257, and Asp229). The chemical mechan-
charide to the acceptor substrate methyl -D-glucopyr-
ism of catalysis by CGTase is a double displacement
anoside [MaDG]) proceeds according to a random
reaction involving a covalent enzyme intermediate
ternary complex mechanism. Thus, both substrates
complex (30). Nevertheless, the transglycosylation
can bind simultaneously and in random order to the
reactions were found to proceed via different kinetic
active site cleft of CGTase. This leads to two apparent
mechanisms. The kinetic data are summarized in
afnity constants for cyclodextrins and acceptor, one
Tables 13. Cyclization (cleavage of an -glycosidic
in the absence (KM ) and one in the presence (KM0 ) of
bond in amylose or starch and subsequent formation
the other substrate (see Table 2). Disproportionation
of a cyclodextrin) is a single-substrate reaction with an
(cleavage of an -glycosidic bond of a linear malto-
afnity for the high-molecular-weight substrate used
oligosaccharide and transfer of one part to an acceptor
(Paselli SA2, a starch derivative with an average degree
substrate), using 4-nitrophenyl--D-maltoheptaoside-
of polymerization of 50) which was too high to allow
4-6-)-O-ethylidene (EPS, a maltoheptasaccharide
elucidation of the kinetic mechanism. Michaelis-
blocked at the nonreducing end and with a p-nitro-
Menten kinetics, however, has been observed when
phenyl group at its reducing end) as the donor
using shorter amylose chains. The B. circulans 251
molecule and maltose as the acceptor molecule,
CGTase mainly produces -cyclodextrin. Highest turn-
proceeds according to a Ping-Pong mechanism.
over rates were observed for this product. Coupling
Despite the two-substrate character of the dispro-
(cleavage of an -glycosidic bond in a cyclodextrin
portionation reaction, its kcat value is threefold higher
ring and transfer of the resulting linear malto-oligosac-
than that for the cyclization reaction (Table 3). This
may be explained by the involvement of merely linear
Table 1 Cyclization Activities of CGTase from Bacillus substrates and products in the disproportionation reac-
circulans Strain 251 tion. Consequently, the reaction rates of cyclization
and coupling should be determined by the rate at
-Cyclization -Cyclization -Cyclization which the conformation changes from linear substrate
to circular product and from circular substrate to lin-
Vmax (U/mg) 20 2:0 276 4:4 53 3:1
ear product, respectively. This rate of conformational
kcat (s1 ) 25 2:5 345 5:5 66 3:6
change presumably differs for the various cyclodex-
Source: Ref. 65. trins, explaining the variation in cyclization and cou-

pling activities among the cyclodextrins. Also the rates cyclodextrins are produced simultaneously from the
of linearization and circularization for the individual starch substrate, the -cyclodextrin formed (measured
cyclodextrins are different; especially, for -cyclodex- using the phenolphthalein method) can be used as an
trin the rate of formation through cyclization is much internal standard. One unit of activity is dened as the
lower than that of degradation through coupling amount of enzyme able to produce 1 mol of cyclodex-
(Tables 13). In view of the different kinetic mechan- trin per min.
isms observed for the various reactions, which can be Coupling activity, in which cyclodextrins (donor) are
related to differences in substrate binding in the active cleaved and the resulting linear oligosaccharide is
site, it appears possible to mutagenize CGTase in such linked to another (oligo)saccharide (acceptor), can be
a manner that a single reaction is affected most determined by measuring the amount of glucose pre-
strongly. This opens possibilities for the rational design sent in the linear products formed. In this assay -, -,
of enzymes displaying specic activities. Recent work, and -cyclodextrins are used as donors in the reaction;
based on the detailed knowledge of the active-site methyl -D-glucopyranoside (MDG) is used as
architecture, has led to the construction of CGTase acceptor substrate. This modied glucose does not
mutants with enhanced -cyclodextrin production react in the glucose detection reaction involving glu-
(66). Current work involves rational design of mutants cose oxidase, and therefore acceptor concentrations do
with decreased coupling activities and mutants with not interfere with the activity measurements.
decreased transferase activities in favor of hydrolysis. Cyclodextrin and MDG are incubated in 10 mM
citrate (pH 6.0) for 10 min at 50 C before the reaction
is started with appropriately dilute CGTase. At regular
V. DETERMINATION OF CGTASE time intervals (0.5 min) 100-L samples are added to
ACTIVITY 20 L 1.2 N HCl (4 C) followed by incubation at 60 C
for 10 min to inactivate the CGTase. Subsequently, the
Since CGTase catalyzes four different reactions (see samples are neutralized with 20 L 1.2 N NaOH and
above), specic assays for the determination of the subjected to a 30-min incubation on ice with 60 L
various activities have been developed. The assays (0.25 U) amyloglucosidase (EC 3.2.1.3; Sigma A-
described are used for B. circulans strain 251 3514) in 167 mM NaAc (pH 4.5) to convert the pro-
CGTase, which requires incubation of appropriately ducts (linear oligosaccharides) to single glucose resi-
diluted enzyme (0.10.2 units of specic activity for dues. The glucose concentration is determined with
the specic reactions) at 50 C with substrate solutions the glucose/GOD-Perid method (Boehringer
in 10 mM sodium citrate (pH 6.0) (65). Mannheim 124036). One unit of activity is dened as
Cyclization activity, resulting in formation of cyclo- the amount of enzyme coupling of 1 mol of cyclodex-
dextrins, can be determined based on the ability of the trin to MDG per min.
different (-, -, -) cyclodextrins to form inclusion Disproportionation activity can be measured using 4-
complexes with specic guest molecules. Paselli SA2, nitrophenyl--D-maltoheptaoside-4-6-O-ethylidene
partially hydrolyzed potato starch with an average (EPS) as a donor substrate and glucose or malto-oli-
degree of polymerization of 50 (Avebe, Foxhol, gosaccharides as acceptor substrate. EPS is a malto-
Netherlands), is used as a substrate (up to 5% w/v). heptasaccharide blocked at the nonreducing end and
The Paselli SA2 solution is incubated in 10 mM citrate with a p-nitrophenyl group at its reducing end, which is
buffer (pH 6.0) for 10 min at 50 C before the reaction generally used for the detection of amylase activity.
is started with appropriately diluted CGTase. At reg- The reaction mixtures are incubated for 10 min at 50
ular time intervals (0.250.5 min) 100-L samples are C before the reaction is started with appropriately
taken and added to 900 L specic detection reagent: diluted CGTase. At regular time intervals (0.5 min)
phenolphthalein (67) or bromocresol green (68) for the 100-L samples are added to 20 L 1.2 N HCl (4 C)
detection of - or -cyclodextrin, respectively. - followed by incubation at 60 C for 10 min to inactivate
Cyclodextrin formation can be analyzed by complexa- the CGTase. Subsequently, the samples are neutralized
tion with Methyl Orange which is, however, not very with 20 L of 1.2 N NaOH and incubated for 60 min
specic and whose sensitivity is low. Therefore the use at 37 C with 60 L (1 unit) -glucosidase
of high-performance liquid chromatography is pre- (E.C.3.2.1.20; Boehringer Mannheim Biochemica
ferred, using a 25-cm Econosphere NH2 5-m column 1630385) in 833 mM potassium phosphate (pH 7.0)
(Alltech Associates) eluted with acetonitrile/water (60/ to liberate p-nitrophenol from the product of the dis-
40, v/v) at a ow rate of 1 mL/min. Since -, -, and - proportionation reaction, nonblocked linear oligosac-

charide. The pH of the samples is raised above 8 by ferase reaction and product specicity. Biochim
adding 1 mL of 1 M sodium carbonate, and the absor- Biophys Acta Protein Structure and Molecular
bance at 401 nm is measured (
401 18:4 mM1 ). One Enzymology 1543:336360, 2000.
unit of activity is dened as the amount of enzyme 5. RD Wind. Starch-converting enzymes from thermo-
philic microorganisms. Ph.D. Thesis. University of
converting 1 mol of EPS per min.
Groningen, 1997.
6. S Pederson, L Dijkhuizen, BW Dijkstra, BF Jensen,
ST Jorgensen. A better enzyme for cyclodextrins.
VI. PURIFICATION OF CGTASE Chemtech 25:1925, 1995.
7. Y Tachibana, A Kuramura, N Shirasaka, Y Suzuki, T
CGTase enzymes occur extracellularly, making puri- Yamamoto, S Fujiwara, M Takagi, T Imanaka.
cation relatively easy. The method used has been devel- Purication and characterization of an extremely ther-
oped for B. circulans strain 251 wild-type CGTase mostable cyclomaltodextrin glucanotransferase from a
enzyme and involves expression of the cgt gene in B. newly isolated hyperthermophilic archaeon, a
subtilis, which also excretes the enzyme (42). Thermococcus sp. Appl Environ Microbiol 65:1991
Production strains are grown in a batch fermentor to 1997, 1999.
an optical density at 600 nm of 1113 (for 24 h). 8. H Bender. Purication and characterization of a
cyclodextrin degrading enzyme from Flavobacterium
Under these conditions high extracellular CGTase
sp. Appl Microbiol Biotechnol 39:714719, 1993.
levels generally are produced. Cultures are centrifuged 9. W Saenger. Structural aspects of cyclodextrins and
at 4 C for 30 min 16,000g and (mutant) CGTases their inclusion complexes. In: JL Atwood, JED
present in the supernatants are further puried to Davies, DD MacNicol, eds. Inclusion Compounds.
homogeneity by afnity chromatography, using a London: Academic Press, 1984, pp 231259.
30-mL -cyclodextrin Sepharose-6FF column 10. G Wenz. Cyclodextrins as building blocks for supra-
(Pharmacia, Sweden) (69) with a maximal capacity of molecular structures and functional units. Angew
3.5 mg protein/mL. After washing with 10 mM sodium Chem Int Ed 33:803822, 1994.
acetate buffer (pH 5.5), bound CGTase is eluted with 11. W Saenger. Cyclodextrin inclusion compounds in
the same buffer containing 10 mg/mL -cyclodextrin. research and industry. Angew Chem 19:344362,
This allows purication to homogeneity of up to 100 1980.
12. RL Starnes. Industrial potential of cyclodextrin glyco-
mg of stable (mutant) CGTase proteins, with a vefold
syl transferases. Cereal Foods World 35:10941099,
purication and a yield close to 90%, from 1-1 culture 1990.
supernatants. 13. M Allegre, A Deratani. Cyclodextrin uses: from con-
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14. JH van Eijk, JHGM Mutsaers. Bread improving com-
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50
Limonoid Glucosyltransferase
Shin Hasegawa
Masayuki Kita
National Institute of Fruit Tree Science, Tsukuba, Ibaraki, Japan
Mitsuo Omura
National Institute of Fruit Tree Science, Shimizu, Shizuoka, Japan
I. INTRODUCTION fruit ripens, the concentration of LARL decreases. The

mechanism of this natural debittering process was not
determined until the discovery of limonoid glucosides
Limonoids are a group of chemically related tetracyclic
in Citrus (11). As maturation progresses, limonoids are
triterpenoids present in the plants of the Rutaceae and
glucosylated to form nonbitter 17-d-glucopyranoside
Meliaceae. As the bitter constituents in citrus juices,
derivatives (12). This reaction is catalyzed by limonoid
excessive limonoid levels lower the quality and value
glucosyltransferase (LGTase) (EC 2.4.1.); UDP glu-
of citrus juices and have a signicant negative eco-
cose:limonoid -d-glucosyltransferase) (13).
nomic impact on the citrus industry worldwide (1).
However, limonoids have been shown to have antic-
arcinogenic activity in laboratory animals (25) and in A. Chemical Reaction Catalyzed
cultured human breast cancer cells (6). Limonoids also
have been shown to possess antifeedant activity against LGTase catalyzes the glucosylation of limonoid agly-
insects (7) and are excellent chemotaxonomic markers cones to form their respective 17-d-glucopyranoside
in the Rutaceae family plants (8). Hence, limonoids are derivatives (1214). Seventeen limonoid glucosides
important functional chemicals in the agriculture and have been isolated from Citrus and its closely related
food industries. genera (15). Each has a single glucose molecule attached
Intact citrus fruit tissues do not contain bitter limo- to the 17-position of the limonoid molecule via a -glu-
nin but contain the nonbitter precursor, limonoate A- cosidic linkage, such as limonin 17-d-glucopyranoside
ring lactone (LARL). In juice this precursor is gradu- (Fig. 1). The isolated enzyme from the albedo tissues of
ally converted to limonin (9) (Fig. 1). This process navel oranges takes both limonoate A-ring lactone and
proceeds under acidic conditions and is accelerated nomilinoate A-ring lactone as its substrates (13). This
by the action of limonin D-ring lactone hydrolase indicates that the enzyme is able to convert all limonoid
(10). This phenomenon is generally referred to as aglycones present in Citrus sp. to their respective gluco-
delayed bitterness. sides. Southern blot analysis of genomic DNA supports
Delayed bitterness is a problem in the early- to mid- this observationthe gene for this enzyme is present
season fruits, but not in the late-season fruit. As the only as a single copy in the citrus genome (16).

II. IMPORTANCE TO QUALITY OF
CITRUS FRUIT AND PROCESSED
PRODUCTS
Delayed bitterness lowers the quality and value of

citrus juices. Also, under unusual conditions such as
freezing or mechanical damage, the formation of the
bitter forms of limonoids occurs even in the fruit tis-
sues. Once the intact fruit tissues are disrupted, the
mixture of acidic pH and limonin D-ring lactone
hydrolase activity in the fruit tissues makes the conver-
sion of limonoate A-ring (LARL) to limonin inevita-
ble. Limonin bitterness is a major problem in the citrus
industry world-wide and decreases the market value of
citrus juices.
Limonin aglycones are glucosylated in the fruit tis-
sues during late stages of fruit growth and maturation
(12, 14). Figure 2 shows the changes in LARL and
limonin glucoside (LG) content of California-grown
navel orange during fruit growth and maturation
(12). The glucosylation of LARL begins in
September and continues until the fruit is harvested
which begins in November. Juice from fruit harvested
in November and December has a serious delayed bit-
terness problem due to the incomplete conversion of
LARL to the glucosides. In Valencia orange, glucosy-
lation of aglycones also begins in September and is
able to continue until fruit is harvested beginning in
March (14). The Valencia orange has an additional 4
months of maturation time over that of the navel
orange. During this extra time, the aglycones are
almost completely converted to the tasteless gluco-
sides. As a result, the juice from Valencia orange
Figure 1 Mechanism of delayed bitterness in extracted
citrus juice and the mechanism of glucosylation (a naturally
occurring limonin debittering process) in Citrus.
B. Chemical Structures of Substrates
Limonoids are tetracyclic triterpenoid dilactones.

Thirty-six limonoids have been isolated from Citrus
and its closely related genera (15). An open D-ring is
necessary for the glucosylation of limonoid aglycones
to proceed via the LGTase reaction. In intact fruit
tissue, limonoid aglycones are present in the open D-
ring form, whereas in seeds they are present in both
open and closed D-ring forms (1, 15). Alkaline condi-
tions open the D-ring, whereas acidic conditions close
it. The enzyme, limonoid D-ring lactone hydrolase cat- Figure 2 Changes in limonoate A-ring lactone (LARL) and
alyzes both the lactonization of the open D-rings and limonoid glucoside (LG) contents of California-grown navel
the hydrolysis of the D-ring lactones (10). oranges during fruit growth and maturation. (From Ref 12.)

does not have the bitterness problem. Many winter Limonoid aglycones are present in high concentra-
citrus fruits including certain varieties of Murcott tions in citrus seeds (1, 21). Isolated aglycones gener-
orange, Shamouti orange, Iyokan, grapefruit, ally are water insoluble, and among 36 aglycones
Natsudaidai, pummelo, Melogold, and Oroblanco isolated from Citrus and its closely related genera,
have a limonoid bitterness problem (1, 17, 18). six are intensely bitter in taste (1, 22). Mass conver-
The rate of glucosylation of limonoid aglycones is sion of these aglycones to limonoid glucosides is
inuenced by a number of factors, but the presence of pharmacologically an important future project. It
LGTase is vitally important. Any stimulation of this would enhance the utilization of these byproducts
enzyme activity by bioregulation or genetic engineering of citrus juice processing. Limonoid glucosides are
should result in the earlier reduction of LARL concen- excellent candidates for new food additives as they
trations via conversion to the glucoside form in the provide anticarcinogenic activity. They are soluble
fruit tissue, and consequently reduce or eliminate limo- in water and are practically tasteless. An approach
nin bitterness. would be to use transgenic E. coli cultures containing
the LGTase gene to convert limonoid aglycones iso-
lated from seeds into limonoid glucosides in fermen-
III. LGTase ACTIVITY IN COMMERCIAL
tation tanks. The LGTase gene has been successfully
CITRUS
expressed in E. coli (16).
Mandarin orange, pummelo, and citron are believed to
be the three original species of Citrus (19). The other
species and varieties are the result of centuries of nat-
ural and articial breeding. Mandarins and citrons tend V. ISOLATION OF THE LGTase GENE
to have relatively high levels of LGTase activity, while
pummels have relatively low levels. Consequently, Using a pair of degenerated oligonucleotide primers
pummelo juices, for instance, extracted from fruits har- which were synthesized based on N-terminal and inter-
vested very late in the season have a severe limonoid nal amino acid sequences of the puried LGTase iso-
bitterness problem (17). Sweet oranges, hybrids of lated from albedo tissue of navel oranges, a cDNA
mandarin oranges, in general have relatively high levels clone encoding citrus LGTase has been isolated from
of LGTase activity and consequently contain high Satsuma mandarin (Citrus unshiu Marc) (16) (Fig. 3).
concentrations of limonoid glucosides (20). In contrast, The isolated cDNA is 1732 bp in length encoding 511
hybrids such as grapefruit and the new varieties amino acids with a predicted molecular mass of 57.5
Oroblanco grapefruit and Melogold grapefruit more kDa.
closely resemble the pummelo and have low levels of Figure 4 compares the amino acid sequences of the
this enzyme activity (17, 18). putative UDP-glucose binding domain of the LGTase
gene with those of other glucosyltransferases (16).
IV. UTILIZATION OF LGTase This region shows 4368% identity in amino acid
sequences to those of previously reported glucosyl-
An attractive approach to solving the bitterness pro- transferases, indicating that the isolated clone con-
blem at the present is through the use of genetic engi- serves the features of the glucosyltransferase family.
neering techniques. LGTase was chosen for genetic The coding region of the LGTase cDNA was ligated
manipulation to create transgenic citrus trees that pro- with an expression vector and transferred into E. coli.
duce fruits free from limonin bitterness. The insertion The transformed E. coli was grown and assayed for
of multiple copies of a constitutively controlled gene LGTase activity. The active protein fraction prepared
encoding for LGTase into commercial cultivars may from these cultures, when incubated with UDP-glu-
result in enhancing the conversion of LARL to LG cose and limonoate A-ring lactone, produced limonin
(Fig. 2) and may prevent the accumulation of signi- glucoside.
cant levels of any LARL at time of early season har- Work is under way to transform and produce trans-
vest. This enhancement of the LGTase activity in genic plants which express a constitutively controlled
transgenic plants may also increase the concentration LGTase activity. It is hoped that the resulting trans-
of limonoid glucosides in the fruit tissues, which are genic citrus will produce fruit that is substantially free
important constituents for human health and nutri- of LARL and delayed bitterness, but retain high levels
tion. of the limonoid glucosides.

Figure 3 The nucleotide sequence of the cDNA encoding LGTase. (From Ref. 16.)

Figure 4 Comparison of the amino acid sequences of the UDP binding domain of LGTase. STSGT: solanidine UDP-glucosyl-
transferase from potato; CASGT: UDP-glucosyltransferase from cassava; IAAGT: IAA glucosyltransferase from Arabidopsis
thaliana; IPFGT: avonoid 3-O-glucosyltransferase from Ipomoea purpurea; PLZGT: zeatin O-glucosyltransferase from
Phaseolus lunatus.
VI. PROPERTIES HCOOH (5:3:1:1). In this solvent system the Rf for

nomilin is 0.88 and that for nomilin glucoside is 0.42.
A. Properties as Protein
The developed plates are scanned for radioactivity with
a Berthold Automatic TLC-Linear Analyser, model
SDS-PAGE reveals a single protein band having an
LB 2832 (13).
apparent Mr of 5658 kDa (13), in agreement with
Radioactive nomilin can be produced by feeding
the estimated Mr from DNA sequencing data dis-
labeled acetate to stems of lemon seedlings (25).
cussed above. LGTase is a single subunit enzyme.
Fresh stems are cut to 1 cm length and fed with ran-
Glucosyltransferases from citrus sources that utilize
domly labeled [14 C]Na-acetate. After 2 days of incuba-
avonoids and other small molecules as substrates
tion with a continuous supply of water, up to 5% of
are generally single-subunit enzymes. Flavanone
the original activity is incorporated into nomilin.
glucosyltransferase from pummelo leaves has a Mr of
Recently, a liquid chromatographymass spectro-
52 kDa (23) and avanone glycosyltransferase from
metry (LC-MS) technique has been developed to ana-
grapefruit seeds has Mr of 54.9 kDa (24).
lyze limonoids in levels as low as 40 pg (26). Thus, the
method could be amended to include the use of unla-
B. Properties as Enzyme
beled limonoid aglycones.
The enzyme displays broad activity between pH 6.5
and 9.0 with an optimal activity at pH 8.0. Activity
VIII. PURIFICATION
below pH 6.5 is completely inhibited by a closed D-
ring. The enzyme requires Mn2 ion for its activity,
The albedo tissue of navel oranges is homogenized in
and appears to be very sensitive to Cl . Partially pur-
buffer consisting of 50 mM Tris-HCl, pH 8.0, 5 mM
ied enzyme placed into 50 mM Tris-HCl, pH 8, and
DTT, 15 mM KCl, 0.5% PVP (w/v) using a Polytron
stored at 80 C showed a complete loss of activity.
at low-medium speed for 4 min. The resulting slurry is
Replacing Tris buffer with 10 mM Mes-KOH, pH 7,
centrifuged at 8000g for 20 min. The supernatant is
allowed frozen storage of the enzyme with full recovery
brought to 40% saturation (NH4 2 SO4 and stirred in
of activity (13).
a 0 C bath for 1 h. The precipitate is removed by
centrifugation at 20,000g for 30 min. The supernatant
VII. QUALITATIVE AND QUANTITATIVE is taken to 80% saturation with NH4 2 SO4 and stirred
DETERMINATION OF ACTIVITY in a 0 C bath for 1 h and the precipitate is collected by
centrifugation as before.
LGTase activity can be assayed at pH 7.5 in a volume The protein precipitate is redissolved in 10 mM
of 50100 L. The assay solution contains 100 M Mes/KOH buffer, pH 7.0, containing 15 mM 2-mer-
UDP-glucose, 50 M Mn2 , and 39 M captoethanol and 50 M MnCl2 (Buffer A). The dis-
14
[ C]nomilinoate A-ring lactone. Assays are incubated solved protein is desalted on PD-10 gel ltration
at 34 C for 1530 min. The reaction is terminated by column (Pharmacia), equilibrated with Buffer A. The
adding 3 L of 1 M HCl, which also closes the D-ring desalted, buffer-exchanged sample is loaded onto a
of the unreacted nomilinoate A-ring lactone. Aliquots uridine 5 0 -diphosphoglucuronic acid afnity column
of the enzyme reaction mixture are spotted on TLC (Sigma), having a 75-mL bed volume that is pre-equi-
plates and developed with EtOAcMeEtCOH2 O librated with Buffer A. The column is washed with

buffer and eluted with 10 mM UDP-glucose dissolved Washington: ACS Symposium Series 758, 2000, pp
in Buffer A. Fractionation on a UDP-glucuronic acid 164174.
afnity column results in a 55-fold increase in enzyme 8. MA Berhow, S Hasegawa, K Kwan, RD Bennett.
purication over the NH4 2 SO4 fraction. Limonoids and the chemotaxonomy of Citrus and
the Rutaceae family. In: MA Berhow, S Hasegawa,
The active fractions are pooled and loaded onto an
GD Manners, eds. Citrus Limonoids: Functional
HPLC Bio-Gel TSK-IEX DEAE 5PW (75 7:5 mm) Chemicals in Agriculture and Foods. Washington:
column (BioRad) which has been equilibrated with 50 ACS Symposium Series 758, 2000, pp 212229.
mM Tris-HCl, pH 7, with 2 mM DTT (Buffer B). A 9. VP Maier, GD Beverly. Limonin monolactone, the
linear salt gradient, 0400 mM NaCl, is used to elute nobitter precursor responsible for delayed bitterness
the column over a 20-min period at a ow rate of 1.0 in certain citrus juice. J Food Sci 33:488492, 1968.
mL/min using Buffer B. The active fractions are col- 10. VP Maier, S Hasegawa, E Hera. Limonin D-ring lac-
lected, diluted 10-fold into Buffer C (50 mM/Tris-HCl, tone hydrolase. A new enzyme from citrus seeds.
pH 8, with 2 mM DTT), and loaded onto the above Phytochemistry 8:405407, 1969.
DEAE column and eluted with Buffer C with a linear 11. S Hasegawa, RD Bennett, Z Herman, CH Fong, P
NaCl gradient, 0400 mM, over a 20-min period at a Ou. Limonoid glucosides in Citrus. Phytochemistry
28:17171720, 1989.
ow rate of 1 mL/min. This procedure results in a 452-
12. S Hasegawa, P Ou, CH Fong, Z Herman, CW
fold increase in enzyme purication (13). Coggins Jr, DR Atkin. Changes in the limonoate A-
ring lactone and limonin 17-d-glucopyranoside con-
tent of navel oranges during fruit growth and matura-
tion. J Agric Food Chem 39:262265, 1991.
REFERENCES 13. S Hasegawa, CG Suhayda, WJ Hsu, GH Robertson.
Purication of limonoid glucosyltransferase from
1. VP Maier, RD Bennett, S Hasegawa. Limonin and navel orange albedo tissues. Phytochemistry 46:33
other limonoids. In: S Nagy, P Shaw, MK Velduis, 37, 1997.
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Agric Food Chem 37:878880, 1989. 40:11781181, 1992.
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51
Transglutaminase
Chang-Rak Ha and Ichiro Iuchi

Sophia University, Tokyo, Japan
I. INTRODUCTION A. Classication and Nomenclature of TGase
Transglutaminase (TGase, EC 2.3.2.13, -glutamyl- Classication and nomenclature of TGases have been
peptide, amine--glutamyl-transferase) is a class of traditionally based on the function of the TGase (blood
enzymes that catalyze the acyl transfer between the coagulation Factor XIII), on cell, tissue, or organ con-
-carboxyamine group of a peptide-bound glutamine taining the TGase (keratinocyte TGase, liver TGase, or
residue and the primary amino group of various sub- prostate gland TGase), or subcellular distribution of
strates in a calcium-dependent reaction. the TGase (extracellular matrix TGase, cytosolic
TGase was rst discovered in guinea pig liver and TGase, or membrane-bound TGase). Recently, some
characterized by Waelsch and coworkers (1, 2), and TGases have been frequently called by abbreviations
identied as an enzyme catalyzing incorporation of such as TGase K (18) or C (19), and TGase 1 (3), 2
polyamines into glutamine residues of proteins or (34) or 3 (19, 20). However, these recent names are
peptides. Since then, the reaction of blood-clotting somewhat inadequate to classify the many TGases
Factor XIIIcatalyzing polymerization of brin has from various sources including bacteria and plants.
been found to be identical to that of the liver Therefore, the traditional names will be used here.
TGase. The reaction is also involved in keratiniza-
tion of epidermis (3), hair formation (4), or copula-
B. Reaction Catalyzed by TGase
tory plug formation (5). More recently, TGase has
been implicated as a widely distributed enzyme that
TGase catalyzes the following reactions.
participates in various biological phenomena such as
apoptosis (6), cell growth and differentiation (7), 1. Formation of
-(-glutamyl) lysine isopeptide
embryonic cell differentiation (8), retinoic acid crosslinks between proteins or peptides, resulting in
induced abnormalities of sh embryo during the polymerization (Fig. 1).
early development (9), and fertilization envelope for- 2. Incorporation of amines into proteins or pep-
mation (1015, 81). tides. This reaction plays a role in deamination or
There are some studies on TGase from an aspect of amine xation. (Fig. 2).
food science (16, 17). The TGase reaction must be 3. Formation of N,N-bis-(-glutamyl) derivative
closely related to the visco-elasticity of food materials crosslinks (Glu-CONH-R-NHCO-Glu) between pro-
such as meat and sh eggs. teins or peptides. (Fig. 3) (21).

II. PROPERTIES AS PROTEIN
A. Physicochemical Properties of TGase
Properties of TGase as a protein are very variable from

TGase to TGase. For example, liver TGase of guinea
pig is monomeric and its molecular size is 75 kDa.
On the other hand, human blood coagulation Factor
XIII (300350 kDa) consists of two A (or a) subunits
where the catalytic site is located and two B (or b)
subunits. Although more investigations are necessary,
it seems reasonable to conclude that TGase belongs to
a highly heterogeneous family or superfamily. Some
characteristics of various TGases are shown in Table 1.
Figure 1 Formation of
-(-glutamyl) lysine isopeptide Isoelectric points of various TGases are somewhat
crosslinks between proteins or peptides, resulting in polymer- variable. Using agarose gel electrophoresis at pH 7.4,
ization.
human blood clotting Factor XIII and rat tissue
TGase migrate toward the anode (22), while two iso-
forms of secretory TGase in rat prostate gland (coagu-
lating gland) move toward the cathode (5, 23). Using
isoelectric focusing, TGases in the crude extracts were
focused at pH 7.8. On the contrary, the analysis of
pure enzymes revealed an additional isoelectric point
at pH 8.7 (5, 23). Rat testicular tissue TGase was
focused at pH 5.25 (5). Isoelectric points of many
TGases have not been accurately determined.
However, they can be calculated from data of their
amino acid compositions (24).
B. Primary Structure of TGase
The primary structures of various TGases have been

predicted by nucleotide sequence analysis of cDNAs or
genomic DNAs: blood coagulation Factor XIII
[human, A subunit (25, 26); B subunit (27)], keratino-
Figure 2 Incorporation of amines into proteins or peptides. cyte TGase [rat (28); mouse (20)], rat prostate gland
Figure 3 Formation of N,N-bis-(-glutamyl) derivative crosslinks between proteins or peptides.

Table 1 Some Characteristics of Various Transglutaminases (TGases)
Molecular
Source (cell, tissue, weight
organ, etc.) Organisms (daltons) Natural substrate Remarks References
Blood plasma Human Fibrin, brinogen Secretory TGase

Factor XIII. Tetramer (A2 B2 ;
300350 kDa).
The A subunit has catalytic 25
activity 26
(platelet) Human 83231 Unknown Zymogen form of Factor XIIIA.
Dimer (A2 ; 150 kDa).
75490 B subunit 27
Prostate gland Rat 75471 Dorsal protein 1 29
Functions as a secretory TGase
Secretion II protein. *108
Egg envelope Rainbow trout Egg envelope Extracellular matrix TGase
P1 (Oncorhynchus 82 and subunit proteins 14
mykiss) 76 kDa (45, 56 and 65 kDa)
P2 76 kDa 13, 14
Keratinocytes Human 89338 This group of TGases has been 18
frequently called epidermal
Ivolucrin TGase or TGase K. *109
SKALP/Elan *110
(hair follicle) Mouse 77276 TGase E, TGase 3 20
Involucrin *111
Small, proline-rich proteins
(Cornin B, SPR1B
Cornin A, SPR1A)
Keratinocytes Rat 90768 Involucrin 28
Cornin
Tracheal Rabbit 92 kDa 112
epithelial cells (subunit)
Cronin , *113
Liver Guinea pig 77140 Unknown This group of TGases has been 33
Liver Rabbit 80 kDa Unknown frequently called tissue TGase 114
Liver Rat 76889 Unknown or TGase C, and inhibited by 62
Liver Red sea bream 78214 Unknown nucleotides such as ATP and 39
(Pagrus major) GTP.
Liver Salmon 75859 Unknown 40
(Oncorhychus
keta)
Brain Rat 75 kDa Unknown Tissue TGase, TGase C 52
inhibited by GTP.
Brain
(N1) Rat 45 kDa Unknown 115
(N2) 29 kDa Unknown
Macrophage Mouse 77237 Unknown Tissue TGase, TGase C 34
Lens Rabbit 78 kDa Crystallins Not inhibited by GTP 116
Endothelial cells Human 77256 Unknown Tissue TGase, TGase C 34
Aortic and Bovine 77111 Unknown 35
endothelial cells
Erythrocyte Human 76972 Unknown Similar to band 4.2 proteins 30
(pallidin)
(Continued)

Table 1 Continued
Molecular
Source (cell, tissue, weight
organ, etc.) Organisms (daltons) Natural substrate Remarks References
Erythrocyte Mouse 76738 Unknown Pallidin 31

Erythrocyte Chicken 78739 Unknown Tissue TGase, TGase C 32
Muscle Some shes, 80100 Probably muscle proteins 86
shrimp, or kDa
molluscans
Mesenchyme cells Embryo of an 87193 Unknown Tunica formation (?) 37
ascidian (Ciona
intestinalis)
Hemocyte Horseshoe crab 87021 8.6-kDa protein Membrane-bound TGase 38, 117
(Tachypleus
tridentatus)
Boundary cells of American grass- 85940 Unknown Annulin 8
limb segments hopper
of embryos (Schistocerca
americana)
Nematode 61 kDa Unknown Involved in programmed cell 66
(Caenorhapditis death
elegans)
Leaves Soybean 80 kDa Unknown Inhibited by GTP 53
(Glycine max)
Slime mold 77 kDa Unknown A probable role in the 54
(Physarum construction of spherules
polycephalum)
101 kDa LAV1-2 65
Bacteria 42445 Unknown 118
(Streptoverti-
cillium
mobaraense)
The molecular weights of TGases were calculated from the amino acid sequences predicted from cDNAs or genomic DNAs. The molecular sizes
(kDa) were those of the TGases isolated by ordinary biochemical methods. The complete sequences of them have not been determined. The
asterisk in the column of References indicates the literature where only natural substrates for TGase are described.
TGase (29), erythrocyte band 4.2 [pallidin; human 60 amino acids longer than those of other vertebrate
(30); mouse (31)], chicken erythrocyte tissue TGase TGases, respectively. It may be predicted that they
(32), guinea pig liver TGase (33), mouse macrophage have a large insertion at the N-terminal regions, or at
TGase (34), bovine aorticendothelial cell TGase (35), the C-terminal regions. In addition, the multiple align-
chicken limb TGase (36), mesenchyme TGase of Ciona ment reveals that the sequence of a novel human
intestinalis embryo (37), Limulus hemocyte TGase (38), TGase X or 5 is especially characteristic in a large
and TGase of grasshopper embryo [annulin (8)]. The deletion of about 90 amino acids at the N-terminal
amino acid sequences of liver TGases of shes [red sea region and an insertion of about 50 amino acids near
bream (39); a salmon, Oncorhynchus keta (40)] are the central region.
available. We tentatively analyzed molecular phylogenetic
As shown in Figure 4, amino acid sequences of var- relationships of various TGases of vertebrates. The
ious TGases were aligned by the program CLUSTAL human TGase X or 5 was removed from the analysis.
W ver 1.7 (41) in DNA Data Base of Japan (DDJB). In addition, the amino acids corresponding to gaps
The overall sequences of keratinocyte TGases, human were removed from the analysis. A tree was con-
blood clotting Factor XIIIA, and mesenchyme TGase structed by the neighbor-joining method (42) using
of Ciona intestinalis embryo were 130150, 50, and the program MEGA (43). The tree was rooted with

the sequence of the Ciona TGase (Fig. 5) or Limulus well-characterized protein disulde isomerases, not to
hemocyte TGase (data not shown). At least, ve para- any of known TGases (47).
logous groups are denitely distinguishable in verte- Thus, the enzyme having TGase activity is not
brates; the groups of blood clotting Factor XIIIA, necessarily restricted to a distinct single type of
keratinocyte TGases, prostate gland TGases, erythro- enzyme. TGase is highly polymorphic in catalytic
cyte membrane band 4.2 recently named pallidin, property, active site motif, and primary structure.
and so-called tissue TGases.
A conserved amino acid sequence GQCWVF is C. Three-Dimensional Structure of TGase
found at the central region (e) panel of any sequence
in Figure 4, which is deduced as a putative active site The three-dimensional structures of human blood coa-
region for the TGase reaction. The cysteine residue in gulation Factor XIIIA and its zymogen have been
the motif is considered to be a catalytic site. However, determined by x-ray crystallography. The overall struc-
the comparable site of the pallidin is GQAWVL for ture of the dimer A2 is shown in Figure 6. The mono-
human and TQAWVS for mouse. Thus, pallidin is mer molecule is characterized by the activation peptide
unique among them. The cysteine residue as a cata- from residues 1 to 37, domain 1 from 38 to 185,
lytic site is replaced by alanine residue. The pallidin domain 2 from 197 to 503, domain 3 from 517 to
has no TGase activity, and is considered to play a 628, and domain 4 from 632 to 731. The connecting
role in the regulation of erythrocyte shape and loops run between the domains (44, 48). Domain 1 (-
mechanical properties (30, 31). Elucidation of three sandwich) contains almost exclusively -structure con-
dimensional structure of human blood clotting sisting of two four-stranded -sheets and forms an
Factor XIIIA suggests that the catalytic site for its open barrel. Domain 3 (barrel 1) and 4 (barrel 2) con-
TGase activity is constructed from three residues, tain seven -strands and are topologically identical.
Cys314, His373, and Asp396 (44). Among the cataly- Both domains interact closely with domain 2.
tic triad, the Cys314 corresponds to a cysteine residue Domain 2 (catalytic core) is constructed from a central
in the motif GQCWVF. In addition, the His373 and six-stranded -sheet, a second -sheet of three strands,
the Asp396 in the Factor XIIIA are conserved in all a -hairpin, and a total of nine -helical segments. The
TGase activity enzymes. However, the former is longest -helix of 17 amino acid residues in the center
replaced by glutamine (Q) in human and mouse pal- of the molecule contains the active-site cysteine Cys314
lidins, the latter is by histidine (H) in mouse pallidin constituting the above-mentioned motif GQCWVF.
(Fig. 4). The other members of catalytic triad are His373 and
The complete amino acid sequence of Asp396. The catalytic triad is not accessible to solvent.
Streptoverticillium TGase has been determined (45). The calcium is located in the core domain, near the
The TGase is a Ca2 -dependent SH enzyme and con- surface of the protein.
tains a sole cysteine residue. The active site sequence of The stereostructure of its catalytic site region is
the TGase was predicted to be YGCVGV, which is highly similar to that of cysteine proteinase, containing
highly homologous to that of thiol proteases such as a cysteine at its catalytic site similarly to the TGase. A
papain and cathepsin H. The overall sequence is also similar active site in the two different enzyme families
signicantly different from those of vertebrate TGases catalzying a reaction in opposite directions suggests
having the sequence GQCWVF as an active site. that the two enzymes are in a common evolutionary
Recently, three novel protein disulde isomerases lineage (49). In addition, there is a recent report that
were cloned from the protozoan parasite Giardia lam- cytosolic TGase of guinea pig liver and a coagulation
blia (46). The enzymes are well known to be essential factor XIIIA can hydrolyze the
- linkage of
-(-
for forming disulde bonds during nascent protein glutamyl) lysine isopeptides (129). TGase may play a
folding in the endoplasmic reticulum. Unlike other dynamic role not only in promoting the formation of
protein disulde isomerases, the giardial enzymes isopeptide bonds but also in the breaking of the bonds.
have only one active site. The active-site sequence The main calcium-binding site in each monomer
motif is CGHC characteristic of eukaryotic protein involves the main chain oxygen atom of Ala457 and
disulde isomerases. Interestingly, all three enzymes also the side chains from residues Asn436, Asp438,
have a Ca2 -dependent TGase activity. There is Glu485, and Glu490 (44).
another example like this. It has been claried that In the processing of a zymogen to the mature form,
TGase of heartworm Dirolaria immitis (Filarial para- an amino terminal activation peptide consisting of 37
site of dog heart) has signicant sequence similarity to residues is cleaved by thrombin and is released from

Figure 4 Alignment of amino acid sequences of various transglutaminases. A multiple alignment of the sequences was per-
formed by the program CLUSTAL W ver 1.7 (41) in DNA Data Base of Japan (DDBJ). The identical amino acid residues in all
sequences are indicated by asterisk, and conservative changes are shown by dots. In addition, the alignment gaps are expressed by
hyphens. The N-terminal 37 amino acid sequence of human blood clotting factor XIIIA (underlined in the sequence 21) is the
activation peptide. The C-terminal side of arginine residue (R-37) is hydrolyzed by thormbin, and the TGase activity for the
Factor XIIIA becomes apparent. The active site for TGase activity in the factor consists of the amino acid residues, Cys314 in the
consensus sequence GQCWVF, His373, and Asp396 (44, 48). These triad residues are conserved in TGases other than erythro-
cyte band 4.2 (pallidins, 1 and 2). Such active-site motif, His and Asp are boxed.

Figure 4 Continued.

the rest of the molecule. However, the 3D structure of polymerization of proteins (Fig. 2). Thus, amines are
thrombin-cleaved Factor XIIIA is remarkably similar competitive inhibitors for the TGase reaction, and can
to that of the zymogen. The activation peptide remains be utilized as one of substrates as described above. For
associated with the rest of the molecule (50, 51). example, there is a report that Km values of rat brain
TGase for putrescine and N,N-dimethylcasein were 0.26
and 0.065 mM, respectively (52). The Km values of mus-
III. PROPERTIES AS ENZYME cle TGases of some animals will be described later. In a
case of the TGase from soybean (Glycine max) leaves,
The main function of TGase is formation of
-(-gluta- the Km values for putrescine, spermidine, and spermine
myl) lysine isopeptide crosslinks between naturally were determined to be 0.109, 0.042, and 0.069 mM,
occurring proteins or peptides, resulting in polymeriza- respectively (53). Although this plant enzyme does not
tion (Figs. 1, 3). Amines such as hydroxylamine, mono- essentially require Ca2 , the Km values are similar to
dansylcadaverine, cadaverine, spermine, spermidine, those of tissue TGases known in animals. Slime
and putrescine can be incorporated into the protein sub- mold Physarum polycephalum TGase has Km values
strate by the TGase reaction to bind covalently to glu- of 0.049, 0.021, and 0.032 mM for [14 C]putrescine,
tamine residues resulting in the inhibition of the [14 C]spermidine, and [14 C]spermine, respectively (54).

Figure 5 Molecular phylogenetic relationship among vertebrate transglutaminases. A tree was constructed by the neigbor-
joining method (42) using the program MEGA (43). Bootstrap values > 50% are shown above or below branches. Filled
diamond, open circle, open diamond, open square, and lled circles indicate blood-clotting Factor XIIIA, keratinocyte
TGases, prostate gland TGases, erythrocyte band 4.2 (pallidin), and tissue TGases, respectively. The groups of them are
considered to be paralogous to each other. The evolutionary distance is expressed in terms of amino acid substitutions per
site. The tree is rooted with mesenchyme TGase of Ciona intestinalis embryo.
Inhibitors or cofactors were searched for mainly by ATP or GTP has been well elucidated for various tis-
an assay method utilizing amine incorporation. Metal sue TGases [guinea pig liver TGase (5); erythrocyte
ion chelating reagents such as EDTA and EGTA inhi- TGase (59); Factor XIII (60); rat brain TGase (52)].
bit the activity of various TGases, which can be Moreover, it has been demonstrated that a human
restored by the addition of Ca2 . In addition, iodoa- tissue TGase has nucleotide hydrolysis activity similar
cetic acid, iodoacetamide, and p-chloromercuribenzo- to ATPase or GTPase (61). This raises the possibility
ate inhibit TGase activity, while 2-mercaptoethanol that the enzyme regulates cell receptor signaling (62).
and dithiothreitol (DTT) stabilize the activity. Studies show that the GTP and ATP hydrolysis
Therefore, TGase is well known as a Ca2 -dependent sites are localized within the core domain of the
SH enzyme. tissue TGase (63). Recently, a 77-kDa GTP-binding
On the other hand, some synthetic compounds such protein, Gh5, was isolated from pig heart membranes
as 2-[3-(diallylamino)-propionyl]benzothiophene (55), with a binding afnity of nucleotides in order:
and 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazo- GTP > GDP > ITP ATP, which is similar to that
nium chloride or L-68277 (56) are well-known non- observed for other G-proteins involved in receptor sig-
competitive inhibitors of TGase. naling. The Gh5 also exhibits TGase activity (64).
Tissue TGase is inhibited by nucleotides. ATP inhi- The optimum pH of the TGase activity was highly
bits the activity of rat liver TGase in a concentration- variable as follows: 7.5 for 101-kDa Physarum polyce-
dependent way. Complete inhibition was obtained with phalum TGase (65); 8.0 for Caenorhabditis elegans
3 mM ATP. ADP inhibited the TGase activity simi- TGase (66); 9.09.5 for red sea bream TGase (67),
larly to ATP, but AMP had much less inhibition. and 56 for egg envelope TGases of rainbow trout
There was no signicant inhibition by adenosine and (1315).
adenine. CTP possessed the same inhibitory activity as For guinea pig liver TGase, the optimal incorpora-
that of ATP, while GTP and UTP had 50% of the tion of amines into proteins occurs at pH 7.28.5 (2).
ATP-induced inhibition (57). Such inhibitory effect of However, the pH of optimal ammonia liberation (see

Figure 6 Three-dimensional structure of human blood-clotting Factor XIIIA. The coordinate le for the human blood clotting
factor XIIIA dimer [PDB Id., 1GGU (44)], which is supported by Brookhaven Protein Databank, was obtained from National
Library of Medicine (NCBI), and the 3D structure was displayed by Molecular Visualization Program, RasMol ver 2.5 (128).
The overall structure is characterized by the activation peptide (AP, arrow heads) and four domains, the -sandwich, the catalytic
core, barrels 1 and 2 (44, 48). Due to unresolved problems, it appears that each monomer is missing the eight amino acid residues
from the N-terminus (18), the linker between the activation peptides and -sandwich (3043), the sequence between the core and
barrel 1 (508516), and the C-termini (728731). (A) Overall structure of the Factor XIIIA dimer. Two monomer molecules are
visible in parallel, symmetrically with respect to the center of the gure . The cysteine residue in the catalytic site (Cys314) is
represented by a solid sphere (SH). (B) Left side view of A. The left and right chains in A are displayed in light and dark,
respectively. Two spheres (Ca) are Ca2 ions in both molecules.
Fig. 1) varies with the substrate: 7.07.5 for unmodi- A. TGase, Reinoic Acid, and Induction of
ed insulin; 6.57.0 for acetylated insulin (68). On the Apoptosis
other hand, there is a report for the same enzyme that
the optimal incorporation of hydroxylamine into N- Clarication of the compounds involved in natural cell
carbobenzoxy (CBZ)-L-glutaminylglycine occurs near death including apoptosis (programmed cell death) has
pH 6.0 (69). become one of the most interesting subjects in biochem-
istry and molecular biology. One of the effective com-
pounds in the death pathway is considered to be tissue
TGase, because of a parallel induction of apoptosis and
IV. BIOLOGICAL FUNCTION OF TGase activity [see the review of Fesus and coworkers
TRANSGLUTAMINASE (6)]. Tissue TGase-mediated crosslinking of some intra-
cellular proteins occurs in cells undergoing apoptotic or
As described in the Introduction, TGase participates in programmed cell death (70, 71). Such protein crosslink-
various biological phenomena such as blood clotting, ing may stabilize the apoptotic bodies and prevent the
keratinization, hair formation, and copulatory plug leakage of cytosolic proteins into the extracellular space
formation. In the present article, we focus our atten- during fragmentation of cell (72). The induction and
tion on the following subjects to briey review the activation of TGase-mediated apoptosis are also sup-
recent development of TGase studies; apoptotic cell ported by a recent study that TGase is involved in pro-
death, and egg envelope hardening in sh. grammed cell death of C. elegans (66).

Retinoic acids, vitamin A derivatives, are known to proteins and the hardening of chorion (11). These stu-
participate in various biological phenomena by media- dies have raised a possibility that TGase is responsible
tion of their receptors, retinoic acid receptors (RARs), for chorion hardening (79, 81).
and retinoid X receptors (RXRs). Especially, the nd- Further studies showed that the activity of TGase
ings are interesting that retinoids induce apoptotic was not localized in unfertilized egg cytoplasm but was
death in various cells [human nueroblastoma cells exclusively in the egg chorion (11, 12); that is, the
(73): human myeloma cell line RPMI 8226 (74); TGase was an extracellular matrix TGase. Thus, the
embryonic stem cells (75): mouse thymocyte (76)]. For chorion is a functional structure in coexistence with the
example, there is a report that apoptosis is in part inhib- enzyme and the substrate.
ited by the antisense oligonucleotides for RAR message Two TGases, P1 and P2, were extracted and puried
during the differentiation of mouse embryonic stem from the chorion and were partially characterized.
cells into neuronal cells; that is, the decreased expression They had an ability to catalyze the formation of
-(-
of RARs may inhibit apoptosis (75). glutamyl) lysine isopeptide crosslinks among 49-, 56-,
In the pathway of apoptotic cell death, retinoids have and 65-kDa subunit proteins of the chorion, to poly-
been suggested to induce the expression of TGase in the merize all the subunits, and to harden the chorion. The
cells; that is, the TGase gene, especially tissue TGase activity of P1 enzyme was apparent exclusively in the
gene, is one of the retinoid-responsive genes. The induc- medium of low ionic strength such as 10 mM Tris-HCl
tion is not related to binding of retinoid receptor mono- (pH 7.2), while that of P2 was apparent both in 10 mM
mers to the DNA. Homo- and heterodimeric retinoid Tris-HCl (pH 7.2) and in isotonic saline (0.143 M
receptor complexes bind to distinct retinoids response NaCl-10 mM Tris-HCl, pH 7.2). P1 consisted of 82-
elements (RREs) embedded in the regulatory regions of and 76-kDa proteins, but the amino acid compositions
retinoid-responsive genes (77). Recently, such RREs were highly identical. P2 consisted of a homogenous 76-
have been identied in the promoter regions of mouse kDa protein (13, 14). After egg activation, P1 enzyme
tissue TGase gene and characterized (78). The element was processed into a 48-kDa enzyme and its activity
has been suggested to be capable of binding to and was enhanced. As the 48-kDa TGase remains localized
being activated by both RAR/RXR heterodimer and in the chorion, it must crosslink the subunit proteins at
RXR homodimer receptor complexes. the restricted sites of the chorion. On the contrary, P2
enzyme was released into perivitelline space after acti-
B. Egg Envelope TGases vation of the egg. It may uniformly polymerize the
chorion subunit proteins (15). The released P2 enzyme
Recently, TGases in eggs of the rainbow trout corresponds to the chorion-hardening enzyme initially
Oncorhynchus mykiss have been well characterized suggested by Zotin (80, 81).
with special reference to egg envelope (chorion) hard- It has been suggested that the processing of chorion
ening. Some properties of the TGases will be described TGases after egg activation is caused by the action of
in detail because it is considered to be important in an EDTA- or leupeptin-sensitive proteinase(s). In
food science. addition, the enzymes become active by Ca2 released
Unfertilized eggs are very fragile, while fertilized/ from the activated eggs (15, 81). Thus, a main biologi-
activated eggs are very tough. An unfertilized egg of cal function of chorion TGases is the fertilization/egg
rainbow trout was broken by pressing it with the activation-associated hardening of the chorion to pre-
weight of less than 20 g. A fertilized/activated egg vent the eggs from environment-derived mechanical
incubated at 10 C overnight was resistant to the weight and chemical hazard (81).
of 23 kg. Such toughness was due to the hardening of
the egg envelope (chorion). During the hardening pro-
cess,
-(-glutamyl) lysine isopeptide crosslinks in the V. TRANSGLUTAMINASE STUDY IN
chorion increased 6 (unfertilized egg chorion) to 43 FOOD SCIENCE
nmol/mg dry chorion (fertilized egg chorion).
Moreover, 49-, 56-, and 65-kDa proteins constituting Unfertilized eggs of salmonid shes including rainbow
the unfertilized egg chorion were gradually polymer- trout are frequently treated with a high concentration of
ized, and the chorion became insoluble in protein salt ( 7% NaCl) and are utilized as food. During food
denaturants such as 3% SDS, 8 M guanidium hydro- processing, the activity of the chorion TGases survives,
chloride, and 12 M urea. In addition, monodansylca- resulting in a gradual formation of isopeptide crosslinks
daverine inhibited both the polymerization of chorion between egg proteins (82). It is conceivable that the

TGase reaction contributes to the viscoelastic character dansylcadaverine into a protein such as N,N-dimethyl-
of salted eggs. In fact, unfertilized eggs are very fragile, casein (Fig. 2). The standard reaction mixture, slightly
while salted eggs have hardness as a desirable food. modied by Ha and Iuchi (11, 12) is as follows:
Although more investigations are necessary, TGases
have been found in unfertilized eggs of other shes: Volume Final
Oncohynchus masou, Salvelinus leucomaenis (82), carp Stock solution (L) concentration
Cyprinus carpio (83), medaka Oryzias latipes (79, 84), Tris-HCl (pH 7.2), 200 mM 250 50 mM
cod Gadus morhua (85), and sea urchin 2-mercaptoethanol, 200 mM 25 5 mM
Stronglyocentrotus purpuratus (10). TGase in sh eggs CaCl2 , 1.0 M 5 5 mM
must play a role in forming a desirable texture as food Monodansylcadaverine, 2.0 mM 250 0.5 mM
as well as in the hardening of egg envelope after ferti- N,N-dimethylcasein, 1.0% 200 0.2%
lization. However, changes of egg proteins induced by Enzyme sample and some reagent 270
food processing remain unclear.
TGases were prepared from muscles of scallop The mixture was incubated at appropriate tempera-
(Patinopecten yessoensis), botan shrimp (Pandalus nip- ture for various times, and the reaction was terminated
ponensis), squid (Todarodes pacicus), carp, rainbow by adding 1.0 mL of 10% trichloroacetic acid. After
trout, and atka mackerel (Pleurogrammus azonus), and centrifugation, the precipitate was washed several
their properties were characterized (86). The Km value times with ethanol-ether (1:1), dried by vacuum
of carp TGase for monodansylcadaverine was 0.33 mM, pump, and dissolved into 3 mL of 8 M urea-5% sodium
while the other enzymes had values of 0.010.03 mM. dodecylsulfate (SDS)-50 mM Tris-HCl (pH 8.6). The
The Km values for succinylated casein of scallop, botan intensity of uorescence of the solution was measured
shrimp, squid, carp, rainbow trout, and atka mackerel by excitation at 355 nm and with emission at 525 nm,
enzymes were 1.2, 0.3, 1.8, 0.3, 0.2, and 0.1 mg/mL, using 1 nmol/mL monodansylcadaverine as a standard.
respectively. In the presence of 0.5 M NaCl or 0.5 M For human blood coagulation Factor XIII (brinoli-
KCl (isotonic to sea water), the activities of TGases gase), the unit of TGase activity has been dened as
from marine invertebrates, scallop, botan shrimp, and amine incorporation unit (AIU) (87). For egg envelope
squid were enhanced about 11-, two-, and sixfold, TGase of rainbow trout, 1 unit of the activity was
respectively. On the contrary, there was no effect of dened as 1 nmol of monodansylcadaverine incorpo-
such concentrations of NaCl on the sh enzymes. rated into dimethylcasein per hour at 10 C (MIU:
Muscle TGases may play a role in food processing. monodansylcadaverine incorporation unit) (13).
High pressure at near 300 Mpa is considered to Monodansylcadaverine in the above reaction mix-
induce gelation and denaturation of salted paste sur- ture can be replaced by biotinylated cadaverine (88, 89)
imi, a myobrillar concentrate of sh muscle. and other amine derivatives. A novel, sensitive chemi-
Endogenous TGase that survives at the high pressure luminescent assay was recently developed to quantitate
promotes gelation of Alaska pollock surimi (17). In the acyl transfer activity of blood coagulation Factor
addition, there is a review article with regard to food XIIIA or liver TGase, utilizing 6-[N-(4-aminobutyl)-
processing that TGase is applicable for food protein N-ethylaminol]-2,3-dihydrophthalazine-1,4-dione as a
deamination to improve solubility, emulsication, substrate (90). As a more sensitive method, a 14 C- or
3
foaming, and other functional properties of the pro- H-labeled putrescine or spermidine substrate has been
teins (16). The use of the enzyme must be more desir- frequently used (91, 92).
able than other chemical treatments in speed, mild A uorescent dipeptide compound, 1-N-(carboben-
reaction conditions, and their high specicity. zoxy-L-glutaminylglycyl)-5-N-(5 0 -N 0 , N 0 -dimethylamino-
Especially, it may prevent foods from contamination 1 0 -naphthalenesulfonyl)-diamidopentane (CBZ-Gln-
of pollutants such as endocrine disturbers. Gly-C-DNS 1), was synthesized and was used as a
substrate for assaying the activity of bacterial TGase
or guinea pig liver TGase (93). The glutamine peptides
VI. DETERMINATION OF were incorporated into lysine residues of s1 -casein.
TRANSGLUTAMINASE ACTIVITY Thus, the method is based on measuring incorporation
of a glutamine residue into a protein or peptide.
A convenient assay method for measuring the TGase A uorometric, high-performance liquid chromato-
activity was developed by Lorand and Gotoh (87). graphic (HPLC) assay for TGase activity has been
This method is based on the incorporation of mono- described (94). This method used the small synthetic

peptide benzyloxycarbonyl-L-glutaminylglycine and nium sulfate precipitation; 2550% saturation for
monodansylcadaverine as substrates. The reaction pro- TGase of secretions of coagulating glands and dorsal
duct was separated by reverse phase HPLC, and was prostate (5), or 5060% saturation for testicular tissue
quantitated by uorometry. An apparent Km of puri- TGase of rat (5) and bovine (95). Such enrichment has
ed guinea pig liver TGase was 35 mM for the pep- been performed by protamine precipitation (liver
tide substrate. TGases of rodents) (97) or by ethanol precipitation
A microtiter plate assay using human brinogen (Factor XII of human placenta) (98). In addition, ion
as an immobilized substrate has been developed for exchange column chromatography may be available
coagulation Factor XIII (88). Factor XIII was acti- for concentration of TGase.
vated by Ca2 and thrombin, added to brinogen Conventional chromatographic methods such as gel
immobilized on a plate, and then assayed by adding ltration, ion exchange, and hydrophobic column
biotinylated cadaverine. After the reaction was termi- chromatography are applicable for isolation or puri-
nated by adding EDTA, the quantity of incorporated cation of TGases, as summarized in Table 2.
biotin was determined with streptavidin--galacto- Purication methods based on isoelectric points of
sidase. Microtiter plates coated with N,N-dimethyl- TGases also have been useful. Preparative isoelectric
casein are also available as an immobilized substrate. focusing in a granulated Sephadex G-75 gel bed was
performed for isolation of TGase from secretions of
coagulating glands and dorsal prostate of rat (5, 23).
VII. ISOLATION OF In addition, epidermal TGase from cow muzzle was
TRANSGLUTAMINASE nally puried using zone electrophoresis in combina-
tion with gel ltration chromatography on a Sephadex
Methods used for isolation of different types of G-200 column (99).
TGases have been recently reviewed by Bergamini Afnity chromatography is particularly useful for
and Signorini (95) and by Wilhelm and coworkers purifying various TGases. The Ca2 -dependent afnity
(92). Some data for the purication procedures are chromatography using casein-Sepharose was employed
summarized in Table 2. for the isolation of rat liver cytosolic TGase (100). In
In addition to secretory TGases such as blood-clot- the presence of 5 mM Ca2 , the enzyme was bound to
ting Factor XIII and copulatory plug-forming TGase the column, and elution was achieved using 5 mM
in secretions from prostate gland, tissue TGases fre- EGTA. Recently, TSKgel Heparin-5PW (Tosoh,
quently occur in cytosol and are therefore extractable Tokyo) or GTP-agarose (Sigma) was used to purify
by homogenization of materials such as tissue and rat brain TGases (52). GTP-agarose afnity chromato-
organ with conventional buffer solutions. Some graphy was desirable because GTP has been known to
TGases bind to cell membrane, cellular particles, or bind to tissue TGases to inhibit their activities.
extracellular matrix. As described above, egg envelope Immunoafnity column chromatography has been
TGases of rainbow trout, extracellular matrix TGases, employed for one-step purication of guinea pig
were nearly completely extracted by repeated homoge- TGase, where the supernatants of guinea pig liver
nization with isotonic saline (0.143 M NaCl, 10 mM homogenates were applied to an afnity column con-
Tris-HCl, pH 7.2) (1114). Rat liver particulate TGase jugating a monoclonal antibody against guinea pig
has been partially extracted by three times homogeni- liver TGase (101).
zation of pellet fractions with a sucrose buffer.
However, the extraction was not complete. The
TGase was further extracted by homogenization of
the remaining particulate fractions with 1% Lubrol- VIII. COMMENTS
WX (96). In contrast to these TGases, the TGase activ-
ity in egg envelope of the sh medaka (Oryzias latipes) 1. As shown in Table 1, natural substrates for
was never extracted by homogenization with 5 mM 2- many TGases remain unidentied. Recently, a novel
mercaptoethanol, 0.5 M NaCl, 1% Tween 20 or 0.6 M histone modication was found in the testis of the star-
CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1- sh (Asterina pectinifera) (102, 103), involving forma-
propane-sulfonate) (Ha and Iuchi, unpublished data). tion of an
-(-glutamyl) lysine isopeptide crosslink
At the next step of purication, TGases in the between a glutamine residue of histone H2B and a
extracts are frequently concentrated by various meth- lysine residue of histone H4. Although a biological
ods. A usual method for the concentration is ammo- role of the TGase has not been well claried, there is

Table 2 Practical Methods for the Purication of Transglutaminases (TGases)
Purication Steps Transglutaminases Remarks References
Extraction Conventional buffer Secretory TGases, tissue TGases,

and some particulate or extra-
cellular matrix TGases
Detergent Rat keratinocyte TGase Lubrol-WX (1%) 96
(particle-associated)
Rat keratinocyte TGase NP-40 (0.3%) 119
(particle-associated)
Concentration Ammonium sulfate Rat prostate gland TGases 2550% saturation 5, 23
precipitation (puried from the secretions)
Rat testicular tissue TGase 5060% saturation 5, 23
Coagulation factor XIII of 40% saturation 98
human placenta
Protamine precipitation Tissue TGase of rodent liver 97
Ethanol precipitation Coagulation factor XIII of 98
human placenta
Gel ltration column Sephdex G-200 Rat prostate gland TGases 5,23
chromatography (puried from the secretions)
Sephacryl S-300 Tissue TGase of human 120
erythrocyte
Toyopearl SW55S Egg envelope TGases of rainbow 13, 14
trout
TSK 125 Tissue TGase of human erythrocyte HPLC 120
TSK-gel Egg envelope TGases of rainbow HPLC 13, 14
trout
Ion exchange column DEAE-cellulose Human blood-clotting Factor XIII Anion exchange column 98
chromatography Rat keratinocyte TGase chromatography 96
Guinea pig liver TGase [Most tissue TGases are 97
Human epidermal TGase eluted with 0.250.45 121
Limulus hemocyte TGase M NaCl using a Tris 117
buffer containing
EDTA and DTT at
pH 7.5.)
DEAE-Sephadex A50 Bovine epidermal TGase 99
DEAE-Biogel A Tissue TGase of 122
human erythrocyte
DEAE-EMD-Factogel Rat prostate gland TGases 92
650 (puried from the secretions)
DEAE-Sepharose Human blood-clotting Factor XIII 123
QAE-Sephadex Guinea pig liver TGase 124
Q-Sepharose Egg envelope TGases of rainbow 13, 14
trout
Mono Q Rat liver TGase 125
CM-cellulose Human epidermal TGase Cation exchange column 121
chromatography
Phosphocellulose Rat prostate gland TGases 126
(puried from the secretions)
SP-Sepharose Egg envelope TGases of rainbow Not retained 13, 14
trout
Hydrophobic column Phenylalanine- Liver TGase 124
chromatography Sepharose 4B
Phenyl-Sepharose Rat testicular tissue TGase 5, 23, 95
(Continued)

Table 2 Continued.
Purication Steps Transglutaminases Remarks References
Afnity column Casein-Sepharose Guinea pig liver TGase 100

chromatography Heparin-Sepharose Tissue TGase of human 120
erythrocyte
TSK heparin 5PW Rat brain TGase 52
GTP-agarose Rat brain TGase 52
Blue-Sepharose CL-6B Human erythrocyte 122
Immunoafnity Guinea pig liver TGase Antibody to cytosolic 125
TGase
Guinea pig liver TGase Monoclonal antibody to 101
guinea pig liver TGase
Other column Hydroxyapatite on Guinea pig liver TGase 124
chromatography Biogel-HTP
Rat liver TGase 127
Zinc-chelating Limulus hemocyte TGase 117
Sepharose 6B
Preparative Rat prostate gland TGases pH 4.09.0 5, 23
isoelectric focusing (puried from the secretions)
a possibility that the TGase-mediated crosslink forma- Sports, and Culture of Japan to I.I. Thanks are
tion of nuclear proteins is closely related to some also due Irikawa Trout Hatchery, Tokyo, and Fuji
important cellular events. Trout Farm, Shizuoka, for supplying rainbow trout
2. Antibodies specic to several TGases are com- eggs.
mercially available: antitransglutaminase II (Mono)
(Quartett GmbH, Berlin, Germany); and antitransglu-
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52
Feruloyl Esterases
Craig B. Faulds
Institute of Food Research, Colney, Norwich, England
Gary Williamson
Nestle Research Centre, Lausanne, Switzerland
I. INTRODUCTION Arabinoxylans and certain pectins contain ester-linked

ferulic acid as shown in Figures 1 and 2 (1). The ferulic
Feruloyl esterases (ferulic acid esterase, cinnamoyl acid can be either monomeric or in one of several
esterase, cinnamoyl ester hydrolase, FAE, FE, etc.) dimeric forms (2) (Fig. 3). Free ferulic acid and two
belong to the class of carboxylesterases (EC 3.1.1.1). of the ferulate dimers (5-5 0 mainly, but also the 8-0-4 0 -
These esterases catalyze the hydrolysis of ester-linked linked dimer) are released from arabinoxylans in wheat
ferulic acid groups from a wide range of substrates. The bran or from pectins in sugar beet pulp by some fer-
product is a phenolic carboxylic acid and an alcohol. uloyl esterases. This activity is greatly enhanced by
xylanases (3).
II. IMPORTANCE TO FOOD QUALITY AND

UTILIZATION IN FOOD PROCESSING
The action, amount, and importance of endogenous

feruloyl esterase in foods are unknown. Plants, espe-
cially cereals, produce feruloyl esterases (4). The role of
these enzymes in plant tissue has yet to be elucidated.
Endogenous activity could inuence structural and tex-
tural properties during processing. Most microbial fer-

uloyl esterases are secreted into the culture medium.
Abbreviations: AEBSF, 4-(2-amino-ethyl)-benzenesulfonyl
Generally, monosaccharides and oligosaccharides do
uoride; AraF, ferulic acid-arabinose ester; BLAST, basic
not induce production of feruloyl esterases but may
local alignment search tool; CinnAE, type-B feruloyl esterase
from Aspergillus niger; DFP, di-isouorophosphate; EDTA, be involved in other aspects of their regulation.
ethylenediaminetetra-acetic acid; FAE, feruloyl esterase; Polymeric substrates such as oat spelt xylan, wheat
FAEA, type-A feruloyl esterase from Aspergillus niger; FE, bran, pectins, and maize bran induce production of a
feruloyl esterase; HPLC, high-pressure liquid chromatogra- mixture of different forms of feruloyl esterase.
phy; MFA, methyl ferulate; SDS-PAGE, sodium dodecylsul- Addition of free ferulic acid to the culture medium
fate polyacrylamide gel electrophoresis. further stimulates production of feruloyl esterases (5).

Figure 1 Simplied structure of arabinoxylan, emphasizing the attachment of ferulic acid.
The commercial production of vanillin from agro- Other enzymes that have been puried are listed in
industrial wastes such as cereal brans and sugar beet Table 1. FAEA from A. niger and from A. tubingesis
pulp requires treatment of the starting materials with show 92% homology in amino acid sequence (12), but
enzyme mixtures containing xylanase and feruloyl there is very little similarity between the feruloyl
esterase. The released ferulic acid is bioconverted by esterases sequenced to date. The primary protein
microbes (Aspergillus niger, Pycnoporus cinnabarinus, sequence of FAEA shows some homology to lipases.
Pseudomonas spp.) into vanillin (6, 7). Other possible Based on this homology, FAEA possesses conserved
uses are in baking or any food process involving plant residues at the active site: the nucleophilic serine (resi-
cell walls. due 133), the catalytic acid (Asp174), and the catalytic
base (His247). Based on modeling studies using the
lipase template and on chemical modication using
III. PROPERTIES OF THE PROTEIN dithiobisnitrobenzoic acid treatment of denatured
enzyme, FAEA has three intramolecular disulde
In recent years, the number of microbial cinnamoyl bonds and one free cysteine. CinnAE has not yet
esterases identied has reached > 30, with 10 genes been cloned, but BLAST searches of short protein
sequenced. Some esterases are part of a modular com- sequences show no homology with any other protein.
plex (89), contain cellulose-binding modules (10, 11), There is limited glycosylation of FAEA, while
or exist as only a catalytic domain (12). No structural CinnAE is extensively glycosylated, possibly up to
data have been published, although recently a crystal 40% (ww). A. tubingensis FAEA is more susceptible
of the cinnamoyl esterase domain of XynZ from the to proteolysis than A. niger FAEA, and this can give
anaerobic bacteria Clostridium thermocellum was rise to multiple bands on Western blots in culture
obtained (13). Two feruloyl esterases will be considered supernatants. This susceptibility may be due to residue
here: feruloyl esterase A (FAE-III as puried from differences at locations on the protein surface (16).
culture supernatants of A. niger CBS120.49 or recom- Although no more than one gene encoding feruloyl
binant FAEA; Mr 29,700) (14), and CinnAE esterases have been sequenced from a single source,
(Mr 150,000 [dimer]), also from A. niger (15). there is some evidence of enzymatic isoforms existing.

Figure 2 Simplied structure of sugarbeet pectin, emphasizing the attachment of ferulic acid.
Figure 3 The most abundant diferulic acids found in plant cell walls: 8-0-4 0 , 8-5 0 benzofuran and 5-5 0 diferulic acids linked to
sugars.

Table 1 Ferulic Acid Esterases Puried from Microbial Sources
Organism Enzyme Native Mr (kDa) Reference
Neocallimastix MC-2 FAE-I 69 28

Neocallimastix MC-2 FAE-II 24 28
Neocallimastix MC-2 pCAE 11 34
P. pinophilum p-CAE/FAE 57 29
P. funiculosum FaeB 53 11
P. expansum p-CAE/FAE 57.5 33
A. awamori FAE 112 30
A. awamori p-CAE 75 30
A. oryzae FAE 30 31
A. niger FAE-I/CinnAE 145 15, 17
A. niger FAE-II/FAE-III/FAEA 29.7 12, 14, 17
B. brisolvens CEH (cinA) 27 25
B. brisolvens CEH (cinB) 31.4 32
Piromyces equis EstA 37 10
S. olivochromogenes FAE 29 35
Ps. uorescens XYLD 58.5 36
FAE-I was puried from a commercial source of pec-

tinase originally obtained from a. niger (17). On the
basis of physical properties and substrate specicity,
it may be a posttranslationally modied form of
CinnAE. Likewise, FAE-II was also puried from
the pectinase preparation and may be a posttransla-
tionally modied form of FAEA.
IV. ENZYMATIC PROPERTIES
Although there have yet to be any reports on active site

inhibitor kinetics or active site mutagenesis of feruloyl
esterases, the mechanism of action of these esterases is Figure 4 Summary of the specicity and preferences of
probably analogous to other serine esterases and FAEA for substrates.
lipases, and involves a nucleophilic serine (Ser133 in
FAEA), which can be modied by the reagent, di-iso-
uorophosphate (DFP). By analogy with lipases, a cat-
alytic acid and base are also essential. CinnAE activity
is lost on modication with AEBSF, which also indi-
cates the involvement of an active site nucelophilic ser-
ine. Feruloyl esterases isolated to date do not require
the presence of a cofactor.
Tables 28 show the activity of FAEA and CinnAE
on a range of different substrates. The substrate can be
considered to be two moieties, a phenolic and an ester-
ied group, either a sugar or alkyl substituent. A sum-
mary of the specicity of each enzyme is shown in
Figures 4 and 5. Methyl caffeate is not a substrate
for FAEA, but competitively inhibits it with a Ki of
1:5 mM (18). Based on the selective hydrolytic activity Figure 5 Summary of the specicity and preferences of
shown in these tables, a putative classication of fer- CinnAE for substrates.

Table 2 Inuence of Length and Saturation in the Aliphatic Chain on Activity of FAEA and CinnAE
on Methyl Esters
FAEA Km FAEA Vmax OH k2 103 CinnAE Km CinnAE

Substrate (mM) (sec1 ) (dm3 mol1 sec1 ) (mM) Vmax sec1 )
2.1 87 2.5 1.3 12
3.2 289 120 2.0 610
11
07
a
Comparison, the rate of deesteriation with NaOH is also shown.
Source: Ref. 18.
Table 3 Inuence of Length of the Aliphatic Chain on Activity of FAEA and CinnAE on Methyl Esters

8 0.79 56
4.4
a
For comparison, the rate of deesteriation with NaOH is also shown.
Source: Ref. 18.
uloyl esterases has been proposed: type A (e.g., A. niger Like many esterases, feruloyl esterases catalyze the
FAEA) and type B (e.g., A. niger CinnAE, P. funicu- reverse reactioni.e., synthesis of an ester bond (21).
losum FAEB), although there are exceptions (e.g., P. Although this reaction has only been reported with
uorescens XYLD), and not all enzymes displaying pentanol, the potential is there to make feruloylated
feruloyl esterase activity have been tested for activity esters with novel biological activities and applications.
using all the substrates used for FAEA and CinnAE.
There is clearly a dependence of the rate of reaction on
the number and nature of sugars in the substrate
V. MEASUREMENT OF FERULOYL
(Tables 6, 7) (19). However, it is not known if this is
ESTERASE ACTIVITY
due to the presence of subsites on the esterases. The pH
optimum of FAEA is 5.0, and that of CinnAE is 6.0.
To measure feruloyl esterase activity, a range of low-
The stability of FAEA to thermal and chemical
molecular-weight synthetic methyl esters can be used
denaturation has been studied. Thermodynamic para-
as substrates, as described above (Fig. 6). Suitable nat-
meters are shown in Table 8. FAEA is most stable at
ural substrates, which are feruloylated, are wheat ara-
pH 56, where it retains 50% of its catalytic activity
binoxylan or sugar beet pulp (Figs. 1, 2). The rate of
after 1 h incubation at 60 C (20).

Table 4 Inuence of Number of Methoxy Groups on Activity of FAEA and CinnAE on Methyl Esters
Substrate FAEA Km FAEA Vmax OH k2 103 CinnAE Km CinnAE

(mM) (sec1 ) (dm3 mol1 sec1 ) (mM) Vmax sec1 )
8 0.8 55
2 9.9 61
1.4 61 33
1.6 880 45
a
For comparison, the rate of deesteriation with NaOH is also shown
Source: Ref. 18.
Table 5 Inuence of Position of a Single Methoxy Group on Activity of FAEA and CinnAE on Methyl
Esters

2 9.9 61
22 0.31 99
41 0.85 65
a
Source: Ref. 18.
reaction is increased in the presence of a xylanase or total assay volume is 0.6 mL in disposable semimicro
arabinofuranosidase/arabinanase respectively. cuvettes with a 1-cm path length. For higher substrate
Table 9 shows the activity of native FAEA and concentrations, 0.3 mL in 0.1-cm path length cuvettes
recombinant FAEA on methyl ferulate at pH 6.0, 37 can be used. Dissolve methyl ferulate in methanol and
8C (12). The assay for feruloyl esterases can be per- add a small amount to 100 mM MOPS, pH 6.0.
formed in several ways, but the most convenient is to Determine the A335 of a solution of 100 M
use methyl ferulate as a substrate and monitor the (A335 1:4; E335 for MFA is 1400 M1 cm1 ). Add
reaction in a spectrophotometer (19). The limitation 0100 L of the enzyme solution to appropriate
of this method is that the absorbance of methyl fer- volumes of assay mixture so that nal volume is 0.6
ulate makes it difcult to use the substrate at concen- mL (1-cm path length) or 0.3 mL (0.1 cm) and incu-
trations about the Km . As a standard method, the bate at 378C. Calculate the rate using E of

Table 6 Inuence of Number of Hydroxyl Groups on Activity of FAEA and CinnAE on Methyl Esters

8 0.79 55
6 0.014 54
2 0.22 85
a
Source: Ref. 18.
Table 7 Inuence of Sugars on Activity of FAEA and Table 8 Inuence of Sugars on Activity of FAEA and
CinnAE on Feruloylated Oligosaccharides; All Ferulic CinnAE on Feruloylated Oligosaccharides; All Ferulic
AcidSugar Linkages are 1 ! 5 (substrates derived from AcidSugar Linkages are 1 ! 2 (substrates derived from
limited hydrolysis of wheat or maize bran) limited hydrolysis of sugarbeet pulp)
FAEA CinnAE FAEA CinnAE

Substrate (all 1 ! 5 linked) (Vmax =Km (Vmax =Km ) Substrate (all 1 ! 2 linked) (Vmax =Km (Vmax =Km )
1728 3.5 0 14
2448 30 0 204
7600 34
0 109
1086 17
3270 nd
The most accurate and time-consuming assay for
the measurement of the release of ferulic acid is by
HPLC. This is the only assay that can be used when
measuring the release of phenolic acids (ferulic acid
and dimers) from insoluble plant cell wall-derived
material (26). Samples of plant material (e.g., 10
mg) and enzyme preparation diluted in buffer (in a
9300 M1 cm1 at 335 nm (19). Other assays reported total volume of 0.5 mL) are incubated at a specic
include, using a microtiter plate containing AraF (22), temperature for selected times. It is important to
ethyl cinnamate in agar with pH sensitive dye (23), mix the reaction tube during this step to ensure
separation of substrate and product by capillary complete interaction between the enzyme and the
zone electrophoresis (24) or MUTMAC (4-methylum- substrate. Reactions are stopped by the addition of
belliferoyl [p-trimethyl ammonium cinnamate chlor- glacial acetic acid (0.2 mL) and centrifuged, and the
ide]) with detection by uorescence (25). supernatant is ltered through a 0.22-m lter. The

Table 9 Thermodynamic Parameters for FAEA
G (H2 O) (kJ/mol) Slope (m) GuHCl1=2 (M) kunfolding (sec1 ) kunfolding sec1 )
with GuHCl as (kJ/mol.M) with (30 C, pH 6.0) (60 C, pH 6.0) (60 C, pH 6.0)
denaturant GuHCl as with 0.1 mM
(30 C, pH 6.0) denaturant sinapic acid
30 C, pH 6.0)
16.5 11:9 1.38 0.0076 0.0066
The rate of irreversible thermal denaturation is given by the rst-order rate constant, kunfolding , and the stability is
slightly increased in the presence of sinapic acid, which is a product of the hydrolysis of methyl sinapate.
Source: Ref. 20.
Table 10 Kinetic Parameters for FAE-III and VI. PURIFICATION PROCEDURES

Recombinant FAEA from A. niger Show That the Two
Enzymes Are Identical Most microbial feruloyl esterases are extracellular. In
this section, we describe the purication of FAEA
FAE-III FAEA from A. niger
from Aspergillus niger (14). Slight variations of these
Mr (SDS-PAGE) 36000 36000 procedures have been used for a number of microbial
Mr (mass-spectrometry) 29740 16 29738 50 feruloyl esterases.
pI 3.3 3.3 Prepare 20 L of liquid media (2 L media in each 5-L
pH optimum 5.0 5.0 conical ask) containing 1.0% (w/v) oat spelt xylan,
temperature optimum 5560 C 60 C incubate with Aspergillus niger (CBS, 120.49) at 25 C
Vmax (MFA) 147 143 and 120 rpm for 4 days. At the end of the incubation,
Km (MFA) 0.72 0.76 pass the culture supernatant though a double layer of
Source: Ref. 12. muslin, and clarify by centrifugation (4000 rpm/15 min
at 4 C). The culture supernatant is concentrated to
800 mL (10,000 Mr cutoff). Concentrated culture super-
natant is fractionated by 5080% saturated ammonium
sulfate, and the pellet from 80% saturation is resus-
pended in 80100 mL of 50 mM sodium phosphate,
pH 7.0, 1 mM EDTA, the solution made up to 1 M
(NH4 2 SO4 and then ltered through a 0.45-m cellu-
lose acetate lter. Hydrophobic interaction chromato-
graphy (HIC) is carried out using two buffers: Buffer
A50 mM phosphate, pH 7.0, 1 mM EDTA, 1.7 M
NH4 2 SO4 ; Buffer B50 mM phosphate, pH 7.0, 1
mM EDTA. The prepared sample is loaded into a
Figure 6 Using methyl esters of ferulic (MFA), caffeic HiLoad column (200 mL). FAEA elutes at 100%
(MCA), sinapic (MSA), and p-coumaric (MpCA) acids to Buffer B. The fractions containing activity are then
ngerprint esterases from Streptomyces olivochromogenes, again subjected to a repeat hydrophobic interaction
Pseudomonas uorescens, and Aspergillus niger. The y axis
chromatography step. The pooled FAEA sample
shows specic activity (U/mg protein) for the A. niger
enzymes, and the specic activity (U/mg) multiplied by
from the previous step is made 1 M NH4 2 SO4 , ltered
100-fold for the S. olivochromogenes and P. uorescens through a 0.45-m cellulose acetate lter, and then
enzymes. applied again to a smaller HiLoad (50 mL) column
with the same gradient. The active fractions are then
applied to an anion exchange column using MonoQ
supernatant is then injected onto an HPLC column (10/10) with a gradient of Buffer A: 20 mM Tris-HCl,
(e.g., Spherisorb ODS 3), and the amount of free pH 8.5, 1 mM EDTA to Buffer B: 20 mM Tris-HCl, pH
phenolic acid released is quantied from a standard 8.5, 1 mM EDTA, 1 M KCl. FAEA elutes at 35%
curve (27). Buffer B. At this stage, the enzyme is essentially pure

by SDS-PAGE. Feruloyl esterases are relatively stable and binds crystalline cellulose contains a carbohydrate
when stored frozen between pH 5 and 6. binding module. Eur J Biochem 267:67406752, 2000.
12. RP deVries, B Michelsen, CH Poulsen, PA Kroon,
RHH van den Heuvel, CB Faulds, G Williamson,
JPTW van den Hombergh, J Visser. The faeA genes
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Microb Technol 14:875884, 1992. 355, 1993.

53
Lipase
Dominic W. S. Wong
I. INTRODUCTION bonds. Lipases hydrolyze carboxylic ester bonds of

triacylglycerols to diacylglycerols and carboxylates.
Lipases (EC 3.1.1.3) are key enzymes involved in fat According to IUBMB nomenclature (3), triacylgly-
digestion in vertebrates by converting insoluble triacyl- cerol lipase or lipase (trivial name) has the systematic
glycerols into more soluble products, fatty acids and name of triacylglycerol acylhydrolase, and a code num-
monoacylglycerols, that can easily be assimilated by ber of EC 3.1.1.3. The pancreatic enzyme acts only on
the organism. The lipolysis process is controlled by an ester-water interface, with the outer ester links pre-
gastric lipase in the stomach that hydrolyzes dietary ferentially hydrolyzed. Other important lipolytic
fat (triacylglycerols) to fatty acids and diacylglycerols, enzymes include phospholipases A1 (EC 3.1.1.32)
which are further converted by pancreatic lipase in the and A2 (EC 3.1.1.4), which are phosphoglyceride acyl-
intestine to fatty acids and monoacylglycerols for hydrolases, and phospholipases C (EC 3.1.4.3) and D
intestinal absorption. Most lipases have a basic pH (EC 3.1.4.4), which are phosphoric diester hydrolases.
optimum. Only lipase isolated from lingual or gastric Triacylglycerol lipases are distributed widely in ani-
tissue have an acidic pH optimum. Lipases for food mals, plants, and microorganisms. Animal lipases
processing are obtained from edible forestomach tissue including pancreatic, gastric, intestinal, and milk
of calves, kids, or lambs, and animal pancreatic tissues have been investigated. Microbial lipases are found
as puried edible tissue preparations or as aqueous in Rhizomucor miehei, Rhizopus delemar, R. niveus,
extracts (1). Industrial lipases are also produced by Geotrichum candidum, Candida rugosa (C. cylindracea),
the controlled fermentation of Aspergillus niger var., C. antarctica, Humicola lanuginosa, Pseudomonas glu-
Aspergillus oryzae var., Candida rugosa, and mae, Ps. aeruginosa, Ps. cepacia, Ps. fragi, and
Rhizomucor miehei as a powder or liquid. Typical Staphylococcus hyicus. The present discussion will
applications in the food industry include lipid hydro- focus on pancreatic lipases and fungal lipases which
lysis, manufacture of cheese and cheese avors, mod- have been extensively investigated in terms of their
ication of lipids, manufacture of dairy products and reaction mechanisms and known crystal structures.
confectionery goods, and development of avors in
processed foods (2).
III. INTERFACIAL ACTIVATION
II. CLASSIFICATION Lipases constitute a special class of esterases that acts

specically on water-insoluble esters at oil-water inter-
Esterases are classied under hydrolases which are sub- faces. Sarda and Desnuelle (4) rst demonstrated in a
divided according to the action on the type of ester quantitative way that pancreatic lipase exhibits very

pancreatic; Sus scrofa (pig) pancreatic; Candida rugosa
(yeast) (formerly Candida cylindracea); C. albicans
(yeast); Moraxella sp; Geotrichum candidum (Oospora
lactis); Myocastor coypus (Coypu) (Nutria) pancreatic,
pancreatic lipase-related protein; Chromobacterium vis-
cosum; Rhizomucor miehei; Rhizopus oryzae (R. dele-
mar); R. niveus; Staphylococcus epidermidis; S. hyicus;
S. aureus; Saccharomyces cerevisiae (bakers yeast);
Photohabdus luminescens (Xenorhabdus luminescens);
Yarrowia lipolytica (Candida lipolytica); Aeromonas
hydrophila extracellular lipase; Burkholderia cepacia
(Pseudomonas cepacia); Ps. aeruginosa lactonizing
lipase; Pseudomonas sp. (strain KWI-56); Ps. uores-
cens; Ps. glumae; Ps. fragi; Pseudomonas sp. (strain
109) lactonizing lipase; Psychrobacter immobilis;
Canadida antarctica (yeast) (Trichosporon oryzae);
Bacillus subtilis; Vibrio cholerae lactonizing lipase.
The crystal structures of a number of lipases have
Figure 1 Hydrolysis of triacetin by pancreatic lipase and
been solved, including those of Bos taurus (bovine)
esterase. (Adapted from Ref. 4.)
pancreatic lipase; Sus scrofa (pig) pancreatic lipase;
Rattus norvegicus (rat) pancreatic lipase-related pro-
tein; Homo sapiens (human) gastric, pancreatic lipase,
little activity when triacetin, a short fatty acid chain
lipase complexed with colipase and phospholipid,
triacylglycerol, is in a monomeric (soluble) solution
lipase complexed with colipase and inhibited by unde-
(Fig. 1). However, when the triacetin concentration
cane phosphonate methyl ester; Cavia porcellus (guinea
exceeds the solubility limit and the insoluble substrate
pig) pancreatic lipase related protein; Equus caballus
forms emulsied particles separated from water by an
(horse) pancreatic lipase; Canis familiaris (dog) pan-
interface, the lipase activity increases dramatically.
creatic lipase related protein; Chromobacterium viso-
Adsorption of lipase at the interface is followed by a
sum lipase; Candida antarctica lipase with
103 -fold activation. This unique phenomenon of
phosphonate inhibitor; Rhizopus niveus lipase; R. dele-
lipases in catalyzing a heterogeneous reaction that
mar lipase; Candida rugosa (formerly C. cylindracea)
involves interfacial activationa sharp increase in
lipase, lipase complexed with (IR)-methyl hexyl phos-
the enzyme activity once the substrate solubility is
phonate, lipase complexed with doecanesulfonate,
exceeded and a lipid-water interface appearsis in
lipase complexed with hexadecanesulfonate, lipase
great contrast to esterases that act on water-soluble
complexed with hexadecanesulfonate, lipase com-
substrates and show a normal Michaelis-Menten-type
plexed with (IS)-menthyl hexyl phosphonate;
dependence on substrate concentration. (See Sec. V for
Pseudomonas cepacia lipase; Ps. glumae lipase;
more details.)
Candida antarctica lipase; Rhizomucor miehei lipase,
complex with diethylphosophate, complexed with n-
IV. MOLECULAR STRUCTURE hexylphosphonate ethyl ester; Geotrichum candidum
strain atcc 34614 lipase; and Humicola lanuginosa
Numerous amino acid sequences of lipases are known, lipase.
primarily deduced from DNA sequencing. A total of
58 entries are listed in the Swiss-Prot: Bos tauru A. Pancreatic Lipases
(bovine) pregastric, pancreatic lipase; Rattus norvegi-
cus (rat) hepatic, pancreatic, lingual, pancreatic lipase The porcine pancreatic enzyme is a glycoprotein com-
related protein; Mus musculus (mouse) hepatic, pancr- posed of 450 amino acids with a calculated MW of
reatic lipase; canis familiaris (dog) gastric, pancreatic 50,084 daltons. The enzyme consists of six disulde
lipase-related protein; Homo sapiens (human) hepatic, bridges, Cys4-Cys10, Cys91-Cys102, Cys238-262,
pancreatic, gastric, pancreatic lipase-related protein; Cys286-297, Cys300-Cys305, and Cys434-450, with
Cavia porcellus (guinea pig) pancreatic; Oryctolagus two free cysteines, Cys 104 and Cys 182 (58) (see
cuniculus (rabbit) pancreatic; Equus caballus (horse) also Swiss-Prot). The porcine pancreatic enzyme is

N-glycosylated at Asn167. The glycosylation site ing the signal peptide of 13 residues), with a calculated
Asn167-Gly168-Thr169 in the porcine enzyme is con- MW of 59,085, containing two and three N-glycosyla-
served in the human and canine lipases, and the glycan tion sites, respectively (15). However, lipases I and II,
structure of porcine pancreatic lipase has been eluci- with an overall 84% identity in the two sequences, are
dated (9). In contrast to the human, porcine, and encoded by separate genes, lip1 and lip2 (16, 17). The
canine pancreatic lipases, the horse, ovine, and bovine asporogenic yeast Candida rugosa (formerly C. cylin-
enzymes are not glycosylated. Heterogeneity of the gly- dracea) produces extracellular lipase in multiple forms,
can moiety at Asn167 gives rise to 4 isoforms, LA1 , ve of which have been cloned and sequenced: lipase I
LA2 , LB , and LC , with the less anionic LB the major (pI 4.5), II (pI 4.9), III (pI 5.1), IV (pI 5.7), and V (pI
form (70%) in the porcine enzyme (10). Unlike most of 5.5) (18, 19). All ve genes code for 57-kDa proteins of
the pancreatic enzymes which are secreted as proen- 534 amino acid residues.
zymes and further activated by proteolytic cleavage The Rhizomucor miehei lipase, similar to pancreatic
in the small intestine, pancreatic lipases are directly lipases, is an =-type protein. The enzyme molecule
secreted as a 50-kDa active enzyme. consists of a central parallel eight-stranded -sheet
The porcine lipase comprises two domains (7). The folded onto a highly amphipathic N-terminal helix
larger, N-terminal domain (residues 1336) has an = (Fig. 2) (2022). All the connecting loops are right-
structure, containing the catalytic triad: Ser153, handed, and hence located on one side of the -sheet.
Asp177, and His264. The C-terminal domain (residues Three disulde bonds, Cys29Cys268, Cys40Cys43,
337450) assumes a -sandwich, and contains the bind- and Cys235Cys244, provide a global stabilization to
ing site for colipase. In the human pancreatic lipase, the protein. The active center containing the catalytic
the central parallel -sheet of the /-domain has nine triad (Ser144 His257 Asp203) is buried under a
strands cross-connected by -helices or loops, whereas helical lid that is amphipathic in nature. When the
the C-terminal -sandwich is formed by two layers of enzyme is adsorbed at the lipid-water interface, the lid
-sheet, each of four antiparallel strands (11). The undergoes conformational changes to roll back from
crystal structure of the horse enzyme shows close the active site. The hydrophobic side of the lid is
resemblance (8). A signicant discovery in the x-ray exposed to the interface, thereby expanding the non-
crystallographic investigation is the existence of a sur- polar surface area surrounding the active site. This
face loop or ap covering the active site. This lid stabilization of the open form of the lipase at the inter-
must be repositioned via a conformational change dur- face in effect creates a catalytically active enzyme to
ing interfacial activation to allow the substrate to enter attack the triacylglycerol molecule with the lipid phase.
the active site. In a lipase-colipase system, the lid, The Geotrichum candidum lipase has an = struc-
together with the colipase, forms a continuous hydro- ture with a central mixed -sheet formed by 11 strands,
phobic surface which serves as the lipid binding site of seven of which are parallel (23, 24). The helices and
the complex at the interface. loops connecting the strands are found on both sides of
the -sheet. Two disulde bonds are present: Cys61
B. Microbial Lipases Cys105, and Cys276Cys288. The active site, consist-
ing of the catalytic triad Ser217His463Glu354 is con-
Microbial lipases have been extensively studied owing cealed by two nearly parallel helices.
to their potential industrial applications. The phyco- The general molecular architecture has been
mycete fungus Rhizomucor miehei (formally Mucor observed in lipases from other microbial sources,
miehei) produces an extracellular lipase that hydrolyzes including Candida rugosa (25), C. antarctica (26),
a broad spectrum of lipid substrates found in animal Rhizopus delmar (27), Pseudomonas glumae (28), and
plants. The lipase is synthesized as a precursor contain- Ps. aeruginosa (29). The major difference between the
ing a signal peptide and a propeptide, in addition to molecular structures of the pancreatic and the micro-
269 amino acid residues of the mature enzyme which bial enzymes is that the former contains a C-terminal
has a calculated MW of 29,472 daltons (12). The domain serving as the binding site for colipase which is
secreted lipases were puried in two isoforms, A required for facilitating the interfacial catalysis in the
(pI 3:9) and B (pI 4:3), because of partial degly- presence of bile salts, whereas the latter is devoid of
cosylation in posttranslational processing (13). The this domain. It should also be noted that lipases do not
Geotrichum candidum lipase also exists in two isoforms, all subscribe to the phenomenon of interfacial activa-
I (pI 4:56) and II (pI 4:46) (14). Both have the tion. For example, the Psedomonas aeruginosa lipase
same chain length of 544 amino acid residues (exclud- lacks a lid-like helical loop structure covering the

Figure 2 A stereo view of Candida rugosa lipase (PDBid: 1trh).
active site, and does not show interfacial activation water interface. The inhibitory action of bile salts is
(30). A lipase isolated from guinea pig was found to observed on both microbial and pancreatic lipases,
carry a deletion in the lid sequence, and shows no but the activity of the pancreatic enzyme can only be
interfacial activation (31). In addition to providing restored by the addition of pancreatic colipase, indicat-
accessibility of the active site for the substrate, the ing that specic interactions occur between the pan-
opening of the lid causes a concomitant conforma- creatic lipase and colipase (35).
tional change for proper positioning of the residues Porcine pancreatic colipase is a single polypeptide
forming the oxygen hole during catalysis (22, 25). chain containing 96 amino acid residues with ve dis-
ulde bridges, and has an isoelectric point of 5.0 (36).
Its primary structure has been determined by protein
V. COLIPASE sequencing (37, 38). The protein is synthesized as a
procolipase of 101 amino acid residues, and the active
Adsorption of protein molecules at the lipid-water form, colipase96 , is produced by proteolytic (tryptic)
interface often leads to unfolding and denaturation. removal of the N-terminal pentapeptide (Val-Pro-
For pancreatic lipases, adsorption at the interface Asp-Pro-Arg) (39, 40).
results in decreased activity and consequently inactiva- Colipase consists of a small N-terminal region and a
tion. In the physiological environment, the activity of three-ngers region held together by disulde bridges
pancreatic lipases is regulated by the presence of bile (41, 42). Colipase exhibits an overall amphipathic char-
salts (cholic and chenodeoxycholic acids conjugated acter with most hydrophilic residues found in the N-
with glycine or taurine, such as taurodeoxycholate, terminal region, whereas most of the hydrophobic resi-
glycodeoxycholate, or taurocholate) and other amphi- dues are located at the tip of the ngers in the opposite
philic compounds, such as phospholipids and proteins. side that constitute the lipid-binding site (4346).
The presence of bile salts creates a negatively charged Binding of colipase to lipase occurs between two
lm on the triacylglycerol micelle that prevents lipase hairpin loops (residues 4446, 6567) in the N-terminal
adsorption (32, 33). The cooperative formation of an region of colipase, and the -sheet of the C-terminal
enzymebile salt complex with a dissociation constant domain of the lipase molecule (41, 46). The interac-
of 1:4 1015 M prevents the adsorption to the inter- tions are mostly hydrophilic with the formation of
face (34). For a pancreatic lipase to act at the interface, two salt bridges and six hydrogen bonds. Porcine pan-
the enzyme requires the binding of colipase, a 10-kDa creatic lipase binds to colipase to form a 1:1 complex
protein also secreted by the exocrine pancreas, to facil- with a Kd of 5 107 M in the absence of an interface
itate adsorption of the enzyme at bile salt-coated lipid- (34, 47, 48). In the presence of 2 mM sodium tauro-

deoxycholate and 150 mM NaCl, binding of lipase to phosphatidylcholine and glycerol showed a Kd of 1:2
colipase at pH 7.0 has a Kd of 2 106 M (49). 106 M in the presence of 4 mM sodium taurodeoxy-
The binding of colipase to the bile saltliquid sub- cholate and 150 mM NaCl at pH 7.0.
strate at the interface involves the three-nger region Colipase also binds to the surface helix (the lid)
that forms a hydrophobic surface interacting with the covering the catalytic triad of lipase in its open con-
lipid (Fig. 3) (41). The central nger consists of three formation (54, 55). The interaction is mediated by
highly conserved Tyr residues (Tyr55, Tyr58, Tyr59 in three hydrogen bonds between Arg38, Leu16, and
porcine prolipase) in a strongly hydrophobic loop (also Glu15 of colipase, and Val246, Ser343, and Asn240
designated as the tyrosine loop region) that has long of the lid. The binding serves to stabilize the lid during
been implicated as the lipid-binding site (5052). The activation, and provides an extended hydrophobic sur-
binding of colipase to the substrate, tributyrin, at pH face that constitutes the lipid-water interface binding
7.0 in the presence of 4 mM sodium taurodeoxycholate site, which is more than 50 A in length and has a sur-
and 150 mM NaCl has a dissociation constant Kd face area of approximately 965A2 (42, 56).
3:3 107 M (53). The binding of colipase to an
emulsion of long-chain triacylglycerols stabilized with
VI. REACTION MECHANISM
All lipases and esterases contain an active Ser in a

consensus sequence G-X-S-X-G which is reminiscent
of the pentapeptides in serine proteases (Gly-Asp-
Ser-Gly-Gly in the trypsin family, and Gly-X-Ser-X-
Ala in subtilisin) (Fig. 4) (57). That most lipases are
susceptible to inactivation with classic serine protease
inhibitors, such as diisopropylphosphouoridate and
diethyl-p-nitrophenyl phosphate, has long been impli-
cated as evidence that lipases belong to the mechanistic
class of serine proteases. Recent structural analyses
have been shown conclusively that lipases contain the
same constellation of the catalytic triad, Ser His
Asp, present in all serine proteases, although the topo-
logical position of the individual residues side chain
varies (11, 20). It has also been demonstrated that the
Figure 3 The structure of human pancreatic lipase-colipase

complex and its comformational change in the presence of
mixed phospholipid/bile salt micelles. (Reprinted from
Riddihough 1993, Nature 362, 793, with permission. Copy- Figure 4 Active serine sequences of lipases and esterases
right 1993 Macmillan Magazine, Ltd.) compared with serine proteases.

hydrolysis of dissolved p-nitrophenyl acetate by pan- charge builds up on the carbonyl oxygen from the for-
creatic lipase proceeds via an acyl enzyme intermediate mation of the new carbon-oxygen bond via the nucleo-
(Fig. 5) with k3 kcat deacylation 0:11 philic attack of the Ser-O at the carbonyl carbon of
0:01 min1 , k2 (acylation 7 0:7 min1 , Kmapp the substrate.
0:22 0:02 mM, and KS 15 mM, and that the rela- Taking into account that the lipase-catalyzed hydro-
tion kcat =Km equals k2 =Ks is consistent with an acyl- lysis of monomeric substrates involves the Ser His
enzyme mechanism (58). Using p-nitrophenyl butyrate Asp triad and the formation of an acylenzyme, the
as the substrate, the kcat =Km values of Candida cylin- following mechanism can be envisioned to occur based
dracea lipases A and B have been reported to be on the catalytic pathway of serine proteases (Fig. 6).
2:51 107 , and 0:42 107 sec1 M1 , respectively
1. The enzyme rst binds the substrate to form a
(59). In the hydrolysis of p-nitrophenyl laurate in
Michaelis-Menten adsorption complex.
mixed micelles with Triton X-100, the apparent kcat =
2. Nucleophilic attack by the essential Ser-OH on
Km values are 3:0 105 and 5:6 105 sec1 M1 ,
the acyl carbon of the substrate yields a covalent tetra-
respectively for lipase A and B (60).
hedral intermediate. This step is facilitated by general
Since the pioneering work by Matthews et al. (61)
base catalysis by the His in the triad.
on the 3D structure of chymotrypsin, the mechanistic
3. A collapse of the intermediate to an acylenzyme
nature of the catalytic triad during catalysis by serine
involves the His-catalyzed protonation of the ester
proteases has been the focus of extensive investiga-
oxygen of the leaving group.
tions. In the original model of double proton transfer,
4. In the following step, deacylation of the acylen-
the catalytic triad is described as a charge relay system
zyme occurs with H2 O assisted by general base cataly-
involving a concerted double transfer of protons from
sis involving the His, resulting in the formation of a
Ser-OH to His-N
2 and from His-N1 to Asp-O2 with
tetrahedral intermediate.
a schematic representation (Asp-COOH N-Im-
5. Finally, protonation of the Ser causes the break-
NH O-Ser). The negative charge on the carboxylate
down of the intermediate, resulting in liberation of the
anion is transferred fully to the carbonyl oxygen of the
product as a carboxylic acid.
substrate (6264). An alternative mechanism proposes
that the proton of Ser-OH is transferred to His-N
2 It has been well established that oxyanion-binding
without the concomitant transfer of the His-N1 pro- sites in the proteases, for example, Ser221 and Asn155
ton to the adjacent carboxylate anion (65, 66). NMR in subtilisin, form hydrogen bonds to the carbonyl
and neutron diffraction studies, as well as theoretical oxygen of the scissile peptide bond, contributing to
calculations of the energetics, suggest that the Asp resi- the stabilization of the charge distribution and the
due remains ionized and the imidazole is protonated at reduction of the ground-state energy of the tetrahedral
the transition state to form a charged structure [Asp intermediate (71). In human lipase, the repositioning of
His Ser ] (6770). In this model, positive char- the lid induces a second conformational change in the
acter will develop on the His imidazole, as negative vicinity of the active site. This brings the Phe78 sided
Figure 5 Lipase-catalyzed hydrolysis of p-nitrophenyl acetate.

Figure 6 Proposed reaction mechanism showing acylation and deacylation via the formation of an acylenzyme intermediate.
(From Ref. 122.)
chain into a position for its main chain nitrogen to and the desorption of the bound enzyme at the
stabilize the negatively charged oxyanion (Ser153-O) interface occur after each catalytic turnover cycle.
developed in the tetrahedral intermediate (8). In the The Pseudomonas glumae lipase exhibits a kinetic
Rhizomucor miehei lipase, the movement of the lid in behavior which is explained by the hopping model
the active conformation results in the positioning of (74). Lipoprotein lipase shows a similar mode of action
the hydroxyl and amide bond nitrogen of Ser82 for (75).
its interactions with and stabilization of the ester car-
bonyl oxygen of the substrate complex (22). In the
Candida rugosa lipase, the open state involves a change VII. ACYL TRANSFER REACTIONS
in the conformation of the lid, leading to a movement
of the NH of Ala132 into a proper position for the In the reaction mechanism described above for lipase-
oxyanion hole (25). catalyzed hydrolysis of lipids, deacylation involves
The kinetic behavior of lipases at the lipid-water nucleophilic attack of the acylenzyme by H2 O. This
interface is far more complicated than what is step can also be accomplished by other nucleophiles,
described above for the hydrolysis of dissolved sub- such as alcohols and amines, resulting in an acyl trans-
strates. The lipase in the aqueous phase must rst fer instead of hydrolysis. In an aqueous environment,
bind to the interface (E ! E ) before it can bind to hydrolysis is the favorable route, whereas in organic
the substrate. Physically, it involves movement of the media, the acylenzyme is exposed to nucleophiles with-
lid, and the enzyme assumes an open conformation. In out competition from H2 O, and acyl transfer prevails.
addition, the lipase regenerated in the deacylation step It has been shown that Rhizomucor miehei lipase
can either diffuse from the interface or stay bound retains high rates in esterication and transesterica-
(Fig. 7). The pathway through which E is recycled tion at water activity < 0:0001 (76).
can be described by two different models (72, 73). In The acyl transfer reaction provides a novel use of
the scooting model, E stays in the interface between lipases as catalysts in organic synthesis. Using amines
catalytic turnovers. In the extreme situation, it remains as nucleophiles, esters can be converted to the corre-
bound to the interface until all substrate has been sponding amides in organic media at ambient tempera-
depleted. In the hopping model, only a small ture. For example, Candida antarctica lipase-catalyzed
amount of the substrate will be hydrolyzed in each aminolysis of ethyl octanoate gives a 95% yield of
binding event. In the extreme situation, the binding octanamide (77). Peptide bonds can be formed

Figure 7 Scooting and hopping models of interfacial catalysis. (Adapted from Ref. 72, 73.)
between N-acetyl-L-(amino acid)-2-chloroethyl esters among the triacylglycerol molecules is nonspecic

and amides of L or D amino acids (78). This reaction with random distribution of the acyl fatty acids. By
can be utilized in the production of optically active exploiting lipases as catalysts, it is possible to generate
amides by taking advantage of the regio- and stereo- products enriched with the new fatty acids at specic
specicity of the enzyme (79). Using hydroxyacid positions, thereby changing the physicochemical prop-
esters, bifunctional molecules containing both a methyl erties of triacylglycerols (87, 88). For example, in the
ester and an OH group as substrates, lipases can cata- interesterication of a triacylglycerol/fatty acid mix-
lyze intermolecular or intramolecular condensation to ture catalyzed by 1,3-specic lipase, the product con-
give corresponding oligomers or lactones as the sists of novel triacylglycerols with the fatty acid
product (80, 81). The preference for oligomerization selectively incorporated to the 1- and 3-positions, and
or lactonization depends on the chain length and no enrichment at the 2-position. Using Geotrichum
degree of substitution of the substrate. This reaction candidum lipase which has specicity for cis-9 unsatu-
has been exploited for the synthesis of optically active rated fatty acids, linolenic acid, for example, can be
-substituted lactones from symmetrical hydroxy- selectively exchanged and enriched in an interestied
carboxylic acid esters (82). Lipases have been used in triacylglycerol mixture. Miller et al. (89) studied the
the regioselective acylation of sugars, the process of kinetics of interesterication of trilaurin and lauric
which can rarely be effectively performed in organic acid catalyzed by Candida cylindracea lipase in cyclo-
synthesis and often requires multisteps of protection hexane. The reaction follows a two-step mechanism:
and deprotection of the various hydroxyl groups. initial hydrolysis of the trilaurin to dilaurin and lauric
Porcine pancreatic lipase catalyzes transesterication acid with Km 7:8 103 M, and kcat 0:9 sec1 , and
between monosaccharides (glucose, galactose, man- subsequent reesterication of dilaurin and lauric acid
nose) and trichloroethyl carboxylates in pyridine with Km values of 1:7 102 M (lauric acid) and 0.16
resulting in the selective acylation at the C6-OH (83). M (dilaurin), and kcat 3 sec1 . The use of microbial
Sucrose esters, used as emulsiers in food, cosmetics, lipases in nonconventional solvents, such as supercri-
and medicines, can be synthesized by the lipase- tical uids, has also been investigated as a means of
catalyzed transesterication of sucrose with esters of improving the activity and stability of the enzyme for
fatty acids (84). In similar reactions, nucleosides and promoting acyl transfer reactions in an anhydrous
furanose and pyranose derivatives can be selectively environment (90).
acylated (85, 86).
One of the lipase-catalyzed reactions important to
the food industry is the interesterication of triacylgly- VIII. LIPASE SPECIFICITY
cerols. In these reactions, a triacyglycerol reacts with a
fatty acid, alcohol, or fatty acid alkyl ester, to produce The types of specicity that can be ascribed to lipases
a mixture of new triacylglycerols resulting from a rear- include:
rangement of the fatty acid moieties. Chemical cata- 1. Substrate specicity: The enzyme shows a dif-
lysts such as sodium metal or sodium alkoxide can ferent rate of lipolysis of various acylglycerols, tri-, di-,
promote interesterication; however, acyl migration and monoacylglycerols, or various types of fatty acids.

2. Positional specicity: The enzyme catalyzes the change in the stereoselectivity. The preference of
release of fatty acids preferentially at the primary ester, Chromobacterium viscosum lipase is shifted from sn-3
secondary ester, or nonspecic at all esters. to sn-1 when the sn-2 acylester is replaced by a non-
3. Stereospecicity: The enzyme hydrolyzes the ester alkyl chain (106).
two primary esters (sn-1 or sn-3) at different rates
(91).
Porcine pancreatic lipase has specicity for IX. MEASUREMENT OF LIPASE
acylglycerols in the order: 1,3-triacylglycerol > 1(3)- ACTIVITY
monoacylglycerol > 2-monoacylglyercol (91). Eico-
sapentaenoic and docosahexaenoic acids at the 1(3)- Numerous methods have been used for measuring the
position are resistant to the action of lipase (92). The activity of lipases, and can be classied into the follow-
enzyme shows the same reactivity on palmitoyl, lino- ing groups: (a) titrimetry based on the neutralization of
leoyl, and linolenoyl groups at the 1(2)-position. the free fatty acids released with time in the hydrolysis
However, triacylglycerols containing hydroperoxy of triacylglycerols by titration with NaOH; (b) use of
linoleic or linolenic acids at the 1(3)-position are chromogenic reagents to form color complexes with
hydrolyzed at rates about two times higher than free fatty acids, or substrates conjugated with a
those of the unoxidized fatty acids in the correspond- chromophore or uorophore to form fatty acid
ing positions (93). Lipolysis rates of the porcine pan- products, that can be monitored spectrophoto-
creatic lipase on esters of saturated fatty acids increase metrically or uorimetrically (107, 108); (c) use of
with chain length until C9, which approaches the rates radioactive triacylglycerols and measurement of the
of the oleates. Substitutions or unsaturation, in parti- release of radiolabeled fatty acids (109); (d) chroma-
cular at C2-C5, lead to increased resistance to hydro- tographic analysis of the fatty acid released (110); and
lysis (94). It has been demonstrated that human and (e) enzymatic conversion of the release fatty acid to
rabbit gastric lipases have a stereospecicity for the sn- products that can be measured by spectrophotometry
3 ester bond of trioctanoin or triolein (95). Pancreatic (111). For a comprehensive review of this subject, refer
lipase cleaves the ester bonds in the sn-1 and sn-3-posi- to Beisson et al. (112).
tions without selectivity (96). The following is a procedure for measuring the rate
The Geotrichum candidum lipase is known to show of hydrolysis of emulsied trioleoylglycerol titri-
a rather unique specicity for the hydrolysis of fatty metrically based on Vorderwulbecke et al. (113). A
acids with a cis-9 or cis,cis-9,12 unsaturations in mixture of trioleoylglycerol (1 g) and Triton X-100
preference to the trans-isomers or corresponding (30 mL) is heated to 55 C and added to a NaCl solution
saturated fatty acids (97, 98). The isoform lipase B (0.9%, 200 mL H2 O, preheated also to 55 C) slowly
exhibits a very high degree of specicity for mono- under stirring. The nal mixture is cooled to room
unsaturated fatty acid esters of cis-9 double bonds, temperature and the pH adjusted to 8.0 with 1 M
whereas lipase A hydrolyzes a wide variety of fatty NaOH. For enzyme activity assay, 25 mL of this
acid esters (99, 100). Similar to pancreatic lipases, a solution is prewarmed to 37 C, adjusted to pH 8.0,
majority of the microbial lipases attack preferentially before the addition of lipase. Fatty acids released are
the 1(3)-position of triacylglycerols, with exceptions, continuously titrated with 10 mM NaOH at pH 8.0 and
such as Corynebacterium acnes, Staphylococcal aureus, 37 C using a recording pH stat. The lipase activity is
and Candida cylindracea lipases which hydrolyze all expressed as International Enzyme Units where 1 unit
three positions (101103). The isoforms III and IV corresponds to the release of 1 mol of fatty acid per
from Geotrichum candidum have been shown to cleave min related to the volume of the reaction mixture or to
the 2-position ester bond of triolein at nearly twice the amount of substrate transformed. The specic
the rate of the cleavage of the 1(3)-position (104). activity is expressed as U/mg of pure lipase.
Candida natarctica lipase A shows a preference for Trioleoylglycerol (18:1/18:1/18:1) exists in a liquid
sn-2 when trioctanoin and triolein are used as sub- form at the assay temperature and can be readily emul-
strates, whereas lipase B is selective for sn-3 (105). sied. It satises the denition of a true substrate for
The Rhizopus arrhizus lipase shows stereospecicity lipases, in particular if it is hydrolyzed in an emulsion.
with sn-1 preference toward trioleoylglycerols, while Emulsiers other than Triton include Tweens, SDS,
the Chromobacterium viscosum enzyme has sn-3 bile salts, and stabilizers such as gum arabic which
selectivity (105). It has been demonstrated that can be used. The high salt in the reaction mixture is
structural variations at the sn-2 position cause a used to suppress interfacial charge effects, otherwise

causing inhibition of the enzyme. Tributyrylglycerol essentially includes an aqueous extraction of a
(4:0/4:0/4:0) is another substrate used in many investi- defatted pancreas powder, followed by DEAE-cellu-
gations because of the convenience that it can be dis- lose and Sephadex G-100 chromatographic steps.
persed without the use of emulsiers. However, it does Various modications of the purication have been
not meet the denition of a substrate for lipase, and it described (10, 118, 119). The procedure, according to
is also hydrolyzed by some esterases. The choice of pH, Brockman (119), consists of (a) delipidation of fresh
temperature, substrate types, inclusion/exclusion of porcine pancreas tissue by chloroform-n-butanol; (b)
emulsiers, and other parameters, depends on the extraction of lipase with potassium phosphate buffer
aim of the investigation. Refer to Jensen (114) for a containing NaCl and diisopropyluorophosphate; (c)
critical assessment of this subject. afnity chromatography using hydrophobic glass
Lipase activity can be screened using a zymogram beads; and (d) DEAE-cellulose chromatography
procedure where the PAGE gel is overlaid with a chro- (Table 1). Colipase contamination can be removed
mogenic substrate, such as resorun ester (1,2-O- by additional chromatography on concanavalin A
dilauryl-rac-glycero-3-glutaric acid resorun ester) dis- Sepaharose or hydroxyapatite (120). The use of
solved in dioxane with deoxycholic acid. Bands con- fresh pancreas is critical, as most commercial pan-
taining lipase activity show up as a pink band over a creatic powders may contain degraded lipase, and so
white background (115). A plate assay has been used to is the addition of di-isopropyluorophosphate to the
detect lipase-producing bacteria or to quantify lipase buffer for inhibition of proteolytic enzymes present in
activity in the culture (116). Lipase activity is detected the extract.
by the formation of orange uorescent halos around Purication of extracellular lipases from microbial
lipase-producing colonies when incubated in agar sources is less cumbersome than from animal tissues.
plates containing trioleoylglycerol and rhodamine B. Rhizomucor miehei lipase is puried by passing the
The logarithm of lipase activity is linearly related to culture supernatant onto an anion exchange column,
the zone diameter. following by afnity chromatography on Con A
Sepharose, hydrophobic interaction chromatography
(Phenyl-Sepharose), and gel ltration (12, 13). The
X. PURIFICATION OF LIPASE Geotrichum candidum lipases I and II have been
obtained by ethanol precipitation, gel ltration
Triacylglycerol lipase has been puried to homogene- (Sephacryl-200 HR), anion exchange (Mono Q), and
ity from pancreas of a number of species, with the chemofocusing (Polybuffer exchanger 94) (14). A simi-
porcine enzyme the most thoroughly investigated. The lar scheme has been used to purify multiple isoforms of
procedure originally developed by Verger et al. (117) Candida rugosa lipase (25).
Table 1 Purication of Triacylglycerol Lipase from Porcine Pancreas
Total Total Specic

Volume activity protein activity Yield Colipase
(mL) 104 U) (mg) (U/mg) (%) (mol%)
1. Extraction of delipidated pancreas 190 7 96 13 1:1 0:1 104 87 100

2. Concentration by 3.5 mM 107 16 35 14 220 90 1590 36
taurodeoxycholate eluate from
hydrophobic beads
3. DEAE chromatography
Fraction I (lipase B) 189 21.1 33.0 6360 7.3 1:2
Fraction II (lipase B A) 170 9.7 17.1 5610 3.3 12.3
Fraction III (lipase B A) 224 12.4 24.0 5120 4.3 62.0
Enzyme U 1 mmol of butyric acid released per min. Volume, activity, and protein are expressed as average SD from three preparations
subsequently combined for DEAE chromatography.
Source: Ref. 119.

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Biophys Acta 242:381394, 1971. 119. HL Brockman. Triglyceride lipase from porcine pan-
103. J Rollof, SA Hedstrom, P Nilsson-Ehle. Positional creas. Methods Enzymol 71:619627, 1981.
specicity and substrate preference of puried 120. J Rietsch, F Pattus, P Desnuelle, R Verger. Further
Staphylococcus aureus lipase. Biochim Biophys Acta studies of mode of action of lipolytic enzymes. J Biol
921:370377, 1987. Chem 252:4313, 1977.
104. A Sugihara, Y Shimada, M Nakamura, T Nagao, Y 121. G Riddihough. Picture an enzyme at work. Nature
Tominaga. Positional and fatty acid specicities of 362:793, 1993.
Geotrichum candidum lipases. Protein Eng 7:585588, 122. DWS Wong. Food Enzymes. NY: Chapman & Hall,
1994. 1995, pp 180181.

54
Chlorophyllase
Roger F. McFeeters
U.S. Department of Agriculture and North Carolina State University, Raleigh, North Carolina, U.S.A.
I. INTRODUCTION II. MEASUREMENT OF

CHLOROPHYLLASE ACTIVITY
Chlorophyllase (EC 3.1.1.14) was rst described in
1913 (1). When attempts have been made to detect it, Chlorophylls or pheophytins are the usual substrates
chlorophyllase has been found in all green plants and for chlorophyllase activity assays. These compounds
algae. Chlorophyllase can hydrolyze alcohols esteried are insoluble in water. Therefore, either reactions
to the propionic acid side chain on C7 of the porphyrin must be done with the substrate solubilized into an
ring of chlorophylls, pheophytins, and various deriva- aqueous buffer system using a surfactant, or there
tives of these compounds. In addition, it can catalyze must be sufcient organic solvent, usually acetone,
transesterication reactions (2) that result in the addi- added to the reaction mixture to dissolve the substrate.
tion of a variety of side chains to the porphyrin ring. McFeeters et al. (4) provided an example of the aqu-
See Table 1 and Figure 1 for the nomenclature of eous buffer system adapted from the earlier procedure
chlorophyllase substrates. A previous review of this of Klein and Vishniac (5). Examples of the use of acet-
enzyme has been published by Drazkiewicz (3). one to solubilize substrates are Weast and Mackinney
Table 1 Structures for Substrates and Inhibitors of Chlorophyllasea
Mg2 7, 8 Position
Compound () reduced () R R1 R2 R3 R4
Chlorophyll a CH3 CH2 CH3 O

CO2 CH3 phytyl
Chlorophyll b CHO CH2 CH3 O
CO2 CH3 phytyl
Pheophytin a CH3 CH2 CH3 O
CO2 CH3 phytyl
Methyl chlorophyllide a CH3 CH2 CH3 O
CO2 CH3 CH3
Ethyl chlorophyllide a CH3 CH2 CH3 O
CO2 CH3 CH2 CH3
Methyl pheophorbide a CH3 CH2 CH3 O
CO2 CH3 CH3
9-Hydroxyl methyl pheophorbide a CH3 CH2 CH3 OH CO2 CH3 CH3
Pyropheophytin a CH3 CH2 CH3 O
H phytyl
I
Protochlorophyll a CH3 CH2 CH3 O
CO2 CH3 phytyl
I
4-Vinyl protochlorophyll a CH3 CH3
CH O
CO2 CH3 phytyl
a
See Figure 1 for the ring structure common to these compounds
I
Inhibitor

chlorophyllase isozymes from mature Chenopodium
album leaves using hydrophobic chromatography,
ConA Sepharose, heparin, and ion exchange HPLC
on two different columns. The presence of isozymes
was conrmed by N-terminal sequencing of the sepa-
rated chlorophyllases and by showing that the 10th
amino acid from the N-terminus was different (Fig.
2). However, the N-terminal sequences of the
Chenopodium isozymes had no homology with the
sequence from orange chlorophyllase (11) or with
any other published protein sequence (Fig. 2).
Figure 1 Ring structure for compounds that are substrates IV. PROPERTIES
or inhibitors of chlorophyllase
The molecular weight for chlorophyllase has been
reported to range from 27 to 65 kDa. Chlorophyllase
(6) and Shioi et al. (7). The reaction is stopped, and the from citrus fruit peel was found to be 27 kDa in one
product is separated from the remaining substrate by study (13) and 35 kDa in another (11). Phaeodactylum
partitioning between an organic phase and an aqueous was reported to have two enzymes that were 43 and 46
acetone phase after KOH solution is added to raise the kDa in size (14). Isozymes from Chenopodium album
pH. For measurement of pheophytin or chlorophyll were 41.3 and 40.2 kDa (12). The largest size chloro-
hydrolysis, McFeeters et al. (4) added 60% hex- phyllase reported to date was 65 kDa in Chlorella reg-
ane:40% acetone and KOH solution to raise the reac- ularis (15). Most frequently, however, molecular size
tion mixture pH to 8.5. The nal proportion of has been found to be near 38 kDa. This includes the
solvents was 50% hexane, 33.3% acetone, and 16.7% chlorophyllase from Chlorella protothecoides (7), tea
water. After separation of the two phases, substrate leaf sprouts (10), sugar beet leaves (16), and rye seed-
depletion or, preferably, product formation is mea- lings (17).
sured spectrophotometrically. To measure hydrolysis Most often, chlorophyllases have been found to
of methyl and ethyl chlorophyllides or pheophorbides, have pH optima near pH 7. However, McFeeters et
McFeeters (8) used a mixture of hexane, acetone, and al. (4) and Ogura (18) found chlorophyllases from
2-butanone to separate reaction products from the Ailanthus altissima and tea, respectively, had acidic
substrates. optima. McFeeters et al. (4) showed a pH optimum
near 7 could be obtained erroneously if the pH of the
aqueous phase in the usual organic solvent/aqueous
III. PURIFICATION partitioning mixtures was not raised by the addition
of KOH. Therefore, earlier reports of pH optima for
Chlorophyllase was rst puried to homogeneity by chlorophyllases may be in error. This work indicated
Moll and Stegwee (9). Subsequently, Shioi et al. (7) the absence of ionizable groups in the enzyme that
developed a somewhat simplied method. A homoge- affected substrate binding, but there did appear to be
neous preparation of the enzyme was prepared from a pKa 3.4 group in the active site of the enzyme
Chlorella protothecoides with over a 50% yield using a involved in substrate hydrolysis. Use of acetone in
butanol extraction, ammonium sulfate precipitation, the reaction mixture when determining the optimum
and chromatography on size exclusion columns pH of chlorophyllase could raise the apparent pKa of
(Table 2). The same procedure, except that the butanol this group and give a higher pH optimum in the pre-
solubilization step was unnecessary, resulted in puri- sence of acetone than in the absence of this solvent.
cation of the enzyme from tea leaf sprouts but with The effect of different substituents of the chloro-
only a 9.6% yield (10). Trebitsch et al. (11) puried phyll a molecule on the ability of chlorophyllase to
chlorophyllase from the avedo of mature green bind and hydrolyze potential substrates has been inves-
oranges, determined the N-terminal sequence of the tigated. The picture that emerges is that of a rather
puried enzyme, and used it for generation of antibo- high specicity esterase. First, chlorophyllase does
dies to chlorophyllase. Tauchiya et al. (12) puried two not require the presence of a metal ion in the center

Table 2 Purication of Chlorophyllase from Chlorella protothecoidesa
Purication step Protein Total activity Specic activity Purication Yield

(mg) (units) (U/mg protein) (-fold) (%)
Butanol extract 1105 1436 1.3 1 100

Ammonium sulfate 12.4 1561 126 97 109
(030% saturation)
First Sephadex G-200 5.50 1359 247 190 95
Sepharose CL-6B 1.31 902 689 530 63
Second Sephadex G-200 0.79 758 960 738 53
a
Chlorophyllase was extracted from 50 g (wet weight) of cells.
Source: Ref. 7.
of the tetrapyrrole ring. This is demonstrated by the (19) found that chlorophylls a 0 and b 0 were not hydro-
fact that chlorophyllase has been found to hydrolyze lyzed by chlorophyllase from two different plants and
chlorophylls and pheophytins with similar Km and that the presence of these isomers did not affect the
Vmax value (4). Kinetic data suggested that the Km is hydrolysis rates of the corresponding chlorophylls.
a binding constant for those substrates. Data have not These results suggested that a carbomethoxy group
been obtained to determine the effect of different metal on the opposite side of ring V, as occurs in chlorophyll,
ions in the porphyrin ring on chlorophyllase hydroly- prevents both binding and hydrolysis of chlorophylls
sis. Transesterication experiments have shown that a 0 and b 0 . Comparison of the kinetics of hydrolysis of
chlorophyllase can use a variety of primary and sec- methyl pheophorbide a with 9-hydroxy methyl pheo-
ondary alcohols in place of phytol alcohols as sub- phorbide a showed that introduction of a hydroxyl
strates (2). This implies that the enzyme can also group at C9 in ring V reduced the binding afnity of
hydrolyze chlorophyllide esters of the same alcohols. substrate dramatically while, at the same time, having
Protochlorophyll and 1-vinyl protochlorophyll are not little effect on substrate hydrolysis rate (8).
hydrolyzed by chlorophyllases, but they bind with af- There is little information available on the stability
nities similar to pheophytin a (4). This showed that a of chlorophyllase from different plants. In general,
double bond between C7 and C8 does not affect bind- assays for activity have been performed at 30 C at a
ing to chlorophyllase but that it prevents hydrolysis of pH near 7. Recently, a 30 C optimal temperature was
phytol. Similarly, removal of the carbomethoxy group reported for chlorophyll hydrolysis by crude chloro-
from C10 of pheophytin greatly reduces hydrolysis phyllase extracts from artichoke (20). Chlorophyllase
rates with little effect upon binding (4). Fiedor et al. activity was nearly inactivated during a 1-h incubation
Figure 2 N-terminal sequences of chlorophyllases from orange (Citrus sinensis L.) (A) and Chenopodium album (B and C).
(From Refs. 11, 12.)

at 45 C. Partially puried chlorophyllase from They suggest that the initiation of chlorophyll degra-
Ailanthus altissima retained > 90% of its activity dur- dation must involve a mechanism to transport chlor-
ing a 1-h incubation between pH 3.7 and 9.2 (4). ophyll from the thylakoid pigmentprotein complexes
to the chloroplast envelope where chlorophyllase is
located.
V. FUNCTION IN CHLOROPHYLL
Trebitsh et al. (11), using chlorophyllase antibodies
METABOLISM
to detect chlorophyllase molecules, found that chloro-
phyllase was synthesized de novo when green oranges
There has been uncertainty for many years whether
were exposed to ethylene to speed ripening. The senes-
chlorophyllase is active in chlorophyll biosynthesis or
cence-delaying plant regulators gibberellin A3 and N6 -
degradation in plants. However, Rudiger et al. (21)
benzyladenine inhibited the synthesis of chlorophyl-
have demonstrated the existence of a chlorophyll
lase, which could be induced by ethylene.
synthase which catalyzes esterication of chlorophyll
using geranyl geranyl pyrophosphate as the substrate.
The geranyl geranyl ester is then reduced to phytol, the
VII. SIGNIFICANCE IN FOOD
usual side chain in the chlorophylls of higher plants.
PROCESSING
There is the possibility that chlorophyll synthase
and chlorophyllase are two names given to the
Chlorophyllase appears to be of limited signicance in
same enzyme investigated in different ways, as was
food processing and storage. Dephytylated chlorophyll
assumed by Dogbo et al. (22).
derivatives, particularly pyropheophorbide a, have
However, the information available on the sub-
been implicated in photosensitization reactions in
strate specicity of chlorophyllase described above
humans. It has been observed that higher levels of
compared to the substrate specicity of chlorophyll
these derivatives in food supplements prepared from
synthase activity strongly suggests that these are dif-
leaf protein concentrate (29) or dried Chlorella cells
ferent enzymes. For example, pheophorbide a is not a
(30) is correlated with chlorophyllase activity in the
substrate for chlorophyll synthase (23), but the pheo-
plant or alga.
phytins are readily hydrolyzed to pheophorbides by
Pheophorbides derived from chlorophylls have been
chlorophyllase (4). In addition, pyrochlorophyllide a
observed in pickles (31), fermented olives (32), and
is a good substrate for chlorophyll synthase (24), but
coleslaw (33). Modeling of these data suggested that,
the Vmax for pyropheophytin hydrolysis by chloro-
in coleslaw, chlorophyllase converted pheophytins to
phyllase is only 2.5% as large as the Vmax for pheo-
pheophorbides after the chlorophylls were initially
phytin hydrolysis (8). Langmeier et al. (25) concluded
converted to pheophytins by the low pH (34). In the
there is little double that chlorophyllase is indispen-
fermented vegetables, chlorophylls were rst converted
sable for chlorophyll degradation. This was based
to chlorophyllides by chlorophyllase, and, as acid
upon evidence that pheophorbide a is a required
formed during fermentation, the chlorophylides con-
intermediate in the degradation of chlorophyll to
verted to pheophorbides (34). While pheophytins and
uorescent linear tetrapyrrole compounds. However,
the corresponding pheophorbides have the same visible
it may not be the rst enzyme in the degradative
spectra in organic solvents, Heaton et al. (33) observed
pathway because Mg2 removal may occur prior to
that the color of coleslaw changed as chlorophyllase
phytol hydrolysis (25).
hydrolyzed pheophytins to the corresponding pheo-
phorbides.
VI. LOCATION AND PHYSIOLOGICAL Efforts have been made to promote retention of
CHANGES green color in processed vegetables by treating them
to increase formation of chlorophyllides from chloro-
Brandis et al. (26) found chlorophyllase to be asso- phylls (35). This was done on the assumption that
ciated with the chloroplast envelope and not with magnesium ions would be removed more slowly from
chlorophyllprotein complexes, as has been reported chlorophyllides than chlorophylls. However, it has
earlier (27). This has been conrmed by Matile et al. been shown that chlorophyllides lose the magnesium
(28). They also found that pheophorbide a oxygenase, ion more rapidly than do chlorophylls (36). Ihl et al.
the enzyme responsible for degrading the cyclic tetra- (20) found that inactivation of chlorophyllase in green
pyrrole chlorophyll degradation product to a linear artichokes correlated with optimum color retention for
tetrapyrrole, is located in the chloroplast envelope. microwave and boiling-water blanching procedures.

REFERENCES 16. MF Bacon, M Holden. Chlorophyllase of sugar-beet
leaves. Phytochemistry 9:115125, 1970.
1. R Willstatter, A Stoll. Untersuchungen uber 17. K Tanaka, T Kakuno, J Yamshita, T Horio.
Chlorophyll. Berlin: Springer-Verlag, 1913, pp. 172 Purication and properties of chlorophyllase from
193. greened rye seedlings. J Biochem 92:17631773,
2. TJ Michaelski, JR Hunt, C Bradshaw, AM Wagner, 1982.
JR Norris, JJ Katz. Enzyme-catalyzed organic synth- 18. N Ogura. Studies on chlorophyllase in tea leaves. III.
eses: transesterication reactions of chlorophyll a, Properties of soluble and insoluble chlorophyllases.
bacteriochlorophyll a, and derivatives with chloro- Plant Cell Physiol 13:971979, 1972.
phyllase. J Am Chem Soc 110:58885891, 1988. 19. L Fiedor, V Rosenbach-Belkin, A Scherz. The stereo-
3. M Drazkiewicz. Chlorophyllase: occurrence, func- specic interaction between chlorophylls and chloro-
tions, mechanism of action, effects of external and phyllase. J Biol Chem 267:2204322047, 1992.
internal factors. Photosynthetica 30:321331, 1994. 20. M Ihl, M Monsalves, V Bifani. Chlorophyllase inacti-
4. RF McFeeters, CO Chichester, JR Whitaker. vation as a measure of blanching efcacy and colour
Purication and properties of chlorophyllase from retention of artichokes (Cynara scolymus L.).
Ailanthus altissima (Tree of Heaven). Plant Physiol Lebensm-Wiss U-Technol 31:5056, 1998.
47:609618, 1971. 21. W Rudiger, J Benz, C Guthoff. Detection and partial
5. AO Klein, W Vishniac. Activity and partial purica- characterization of activity of chlorophyll synthetase
tion of chlorophyllase in aqueous systems. J Biol in etioplast membranes. Eur J Biochem 109:193200,
Chem 236:25442547, 1961. 1980.
6. CA Weast, G Mackinney. Chlorophyllase. J Biol 22. O Dogbo, F Bardat, B Camara. Terpenoid metabo-
Chem 133:551558, 1940. lism in plastids: activity, localization and substrate
7. Y Shioi, H Tami, T Sasa. A simple purication specicity of chlorophyll synthetase in Capsicum
method for the preparation of solubilized chlorophyl- annum plastids. Physiol Veg 22:7582, 1984.
lase from Chlorella protothecoides. Anal Biochem 23. M Helfrich, W Rudiger. Various metallopheophor-
105:7479, 1980. bides as substrates for chlorophyll synthetase. Z
8. RF McFeeters. Substrate specicity of chlorophyllase. Naturforsch 47C:231238, 1992.
Plant Physiol 55:377381, 1975. 24. J Benz, W Rudiger. Chlorophyll biosynthesis: various
9. WA Moll, D Stegwee. The activity of Triton X-100 chlorophyllides as exogenous substrates for chloro-
soluble chlorophyllase in liposomes. Planta 140:7580, phyll synthetase. Z Naturorsch 36C:5157, 1981.
1978. 25. M Langmeier, S Ginsburg, P Matile. Chlorophyll
10. M Kuroki, Y Shioi, T Sasa. Purication and proper- breakdown in senescent leaves: demonstration of
ties of soluble chlorophyllase from tea leaf sprouts. Mg-dechelatase activity. Physiol Plant 89:347353,
Plant Cell Physiol 22:717725, 1981. 1993.
11. T Trebitsh, EE Goldschmidt, J Riov. Ethylene induces 26. A Brandis, A Vainstein, EE Goldschmidt.
de novo synthesis of chlorophyllase, a chlorophyll Distribution of chlorophyllase among components of
degrading enzyme, in citrus fruit peel. Proc Natl chloroplast membranes in Citrus sinensis organs. Plant
Acad Sci USA 90:94419445, 1993. Physiol Biochem 34:4954, 1996.
12. T Tsuchiya, H Ohta, T Masuda, B Mikami, N Kita, Y 27. LG Rarasenko, EV Khodasevich, KI Orlovskaya.
Shioi, K Takamiya. Purication and characterization Location of chlorophyllase in chloroplast membranes.
of two isozymes of chlorophyllase from mature leaves Photobiochem Photobiophys 12:119121, 1986.
of Chenopodium album. Plant Cell Physiol 38:1026 28. P Matile, M Schellenberg, F Vicentini. Localization of
1031, 1997. chlorophyllase in the chloroplast envelope. Planta
13. K Shimokawa. Hydrophobic chromatographic 210:9699, 1997.
purication of ethylene-enhanced chlorophyllase 29. M Holden. Chlorophyll degradation products in leaf
from Citrus unshiu fruits. Phytochemistry 21:543 protein preparations. J Sci Food Agric 25:14271432,
545, 1982. 1974.
14. A Khalyfa, S Kermasha, P Marsot, J Goetghebeur. 30. Y Tamura, T Maki, Y Shimamura, S Nishigaki, Y
Purication and characterization of chlorophyllase Naoi. Causal substances of photosensitivity dermatitis
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native electrophoresis. Appl Biochem Biotech 53:11 20:173180, 1979.
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32. MI Minguez-Mosqera, J Garrido-Fernandez, G 34. JW Heaton, RW Lencki, AG Maragoni. Kinetic
Gandul-Rojas. Pigment changes in olives during fer- model for chlorophyll degradation in green tissue. J
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33. JW Heaton, RY Yada, AG Maragoni. Discoloration Maintenance of colour in processed green vegetables.
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Rev Food Sci Nutr 29:117, 1990.

55
Phytase
Onno Misset
DSM Patents & Trademarks, Delft, The Netherlands
I. INTRODUCTION mercial phytase products have been developed and

used as a feed additive since 1991.
Phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen For reviews of phytate and phytases, the reader is
phosphate) is the primary source of inositol and sto- referred to (1) and (2, 3), respectively.
rage form of phosphorus in plant seeds.
Approximately 75% of the total phosphorus in plants
is in the form of phytic acid. Animal feed is primarily A. Chemical Reaction Catalyzed
of vegetal origin (i.e., oilseed meals, cereal grains, and
legumes) and will therefore contain a lot of phytate. Phytase catalyzes the hydrolysis of the 6-monopho-
In contrast to polygastric animals such as rumi- sphate esters in phytate (myo-inositol 1,2,3,4,5,6-hexa-
nants, monogastric animals like humans, pigs, and kis dihydrogen phosphate) via intermediary penta-,
poultry have only a limited microbial population in tetra-, tri-, di-, and monophosphates ultimately to
the upper part of the digestive tract and are not able inorganic phosphate and myo-inositol.
to use phytate as a phosphorus source. Consequently,
to supply these animals with phosphate, inorganic B. Chemical Structure of Substrate
phosphate has to be added separately to the feed.
Phosphorus from phytate ends up in the manure, Phytic acid was described for the rst time in 1903, and
which gives rise to serious environmental pollution. its chemical structure (Fig. 1) was elucidated in 1914
Degradation of phytate by exogenous enzymes would (3). The chemical formula is C6 H18 O24 P6 and it has a
therefore avoid or lower the necessity to add inorganic molecular weight of 659.86 Da. Because phytate carries
phosphate and reduce the amount of phosphorus from six phosphate groups, it can be regarded as a multi-
phytate in the manure. substrate molecule. Upon successive hydrolysis, the
Phytate is also considered an antinutritional factor resulting penta-, tetra-, tri-, di-, and monophosphate
because of its strong chelating properties. The phos- forms of myo-inositol are again substrates for phytase.
phate moieties of phytic acid are able to bind di- and
trivalent metal ions such as calcium, magnesium, zinc,
and iron. The chelating properties of inorganic phos- C. Classication According to Enzyme
phate are much less than those of phytate. Degradation Commission Nomenclature
of phytate in this case would considerably reduce the
metal chelation properties of plant-derived animal feed. Two different types of phytases are known and classi-
Phytases are enzymes that are capable of hydrolyzed accordingly: EC 3.1.3.8 and EC 3.1.3.26. They
ing the phosphate groups from phytate. Several com- belong to the class of hydrolases (group 3), acting on

Table 1 Production of Intra- and Extracellular Phytases
by Microorganisms
Intra- or
Organism extracellular Reference
Fungi
Agrocybe pediades Extra 6
Aspergillus spp. Extra 7
Aspergillus amstelodami Extra 8
Aspergillus awamori Extra 9
Aspergillus chevalieri Extra 8
Aspergillus candidus Extra 8
Aspergillus cuum Extra 8
Aspergillus fumigatis Extra 10, 11
Aspergillus avus Extra 7, 8
Figure 1 Structure of phytic acid. Carbon 1 of the myo- Aspergillus niger Extra 7, 8, 14
inositol ring has been indicated. Aspergillus repens Extra 8
Aspergillus sydowi Extra 8
Aspergillus terreus Extra 7, 12, 15
Aspergillus vesicolor Extra 7, 8
ester bonds (subgroup 1), in particular phosphomo- Aspergillus wentii Extra 8
noesters (subsubgroup 3). Botrytis cinerea Extra 8
EC 3.1.3.8 is a 3-phytase (recommended name) and Emericella nidulans Extra 13
hydrolyzes rst the ester bond at the 3-position of the Geotrichum candidum Extra 8
substrate. The systematic name is myo-inositol hexaki- Mucor species Extra 7
sphosphate 3-phosphohydrolase. EC 3.1.3.26 is 6-phy- Mucor proformis Extra 8
tase (recommended name) and hydrolyzes rst the Mucor racemosus Extra 8
Myceliophtora thermophila Extra 10, 12
ester bond at the 6-position of the substrate. The sys-
Neurospora crassa Extra 16
tematic name is myo-inositol hexakisphosphate 6- Paxillus involtus Extra 6
phosphohydrolase. Penicillium species Extra 7
Penicillium caseicolum Extra 17
Peniophora lycii Extra 6, 18
II. NATURAL OCCURRENCE OF
Rhizopus oryzae Extra 8
PHYTASE Rhizopus oligosporus Extra 8
Rhizopus stolonifer Extra 8
Phytases are found in both the plant and microbial Talaromyces fumigatus Extra 10, 13
kingdoms. While most plant phytases belong to the Thermomyces lanugosis Extra 19, 20
class of 6-phytases (EC 3.1.3.26), most microbial phy- Trametes pubescens Extra 6
tases are considered to be 3-phytases (EC 3.1.3.8). Ruminant microorganisms Extra 21
However, not all publications mention whether the Selenomonas
phytase involved is a 3- or 6-phytase; the reason for Prevotella
this is that it is rather complicated to determine the Treponema
Megasphaera
nature of the myo-inositol pentaphosphate (1,2,4,5,6
Yeast
or 1,2,3,4,5) generated by the enzymes.
Saccharomyces cerevisiae Extra 8
The occurrence of phytase in germinating plants has Schwanniomyces occidentalis Extra 2224
been reviewed recently (2, 5). The rst phytase pre- Bacteria
parations were of plant origin; Suzuki et al. in 1907 Aerobacter aerogenes Intra 25
isolated them from rice and wheat bran (3). All pre- Bacillus subtilis Extra 26, 27
sent-day commercial phytase products are derived Bacillus natto N-77 Extra 28, 29
from lamentous fungi, in particular from Aspergillus Bacillus DS11 Extra 30, 31
spp. For this reason this chapter will further focus on Escherichia coli Intra 32
the microbial, in particular fungal, phytases. Escherichia coli B Intra 33
Extensive screening programs have been carried out Klebsiella aerogenes Intra 34
Pseudomona species Intra 35
in the past and are still carried out today. These pro-

grams have resulted in a long list of bacteria, yeasts, In addition to its application as a feed additive, it
and fungi that produce 3-phytase or 6-phytase either has been suggested that phytase can also be used
intra- or extracellular phytase (Table 1). Many fungal advantageously in the starch-processing industry dur-
phytases were identied a few decades ago (7, 8), and ing the liquefaction step of cornstarch (42). The func-
resulted in most of the Aspergillus phytases known tion of phytase is mainly to degrade the phytate that
today. There is a tendency to screen more for phytases has been shown to be an EIC (enzyme-inhibiting com-
from thermophilic strains or from microorganisms that position) inhibiting especially the alpha-amylase that is
are present in the gastrointestinal tract of ruminants. used in the liquefaction step.
As can be seen in Table 1, fungal phytases are more
abundant than phytases derived from yeasts or bac-
teria. IV. PROPERTIES AS PROTEIN
In addition to screening, protein engineering via
A. Molecular Weight
site-directed mutagenesis of existing phytases has
resulted in enzymes with increased specic activity
Most of the phytases from fungi and yeast are glyco-
(36) and thermostability (37).
sylated. This means that their molecular weight is com-
posed of the total of the amino acids of the (mature)
protein and the molecular weight of the attached sugar
III. INDUSTRIAL APPLICATION OF chains. Furthermore, since most phytases are extracel-
PHYTASE lular enzymes, their genes are encoding for a precursor
protein that is slightly larger than the nal mature
The most important industrial application of phytase is secreted enzyme, the difference being the signal peptide
in animal nutrition for reasons set out in Section I. that is the responsible signal for the excretion process.
When added to pig and poultry feed, the normal addi- Therefore, we can discriminate among the following
tion of inorganic phosphate can be lowered consider- molecular weights for phytases:
ably, and the amount of undigested phytate in the
1. Precursor phytase, not glycosylatedcalculated
manure of the animals is reduced substantially (30
from the amino acid sequence derived from the gene
60% depending on the type of diet composition and
sequence.
animal). These and other related subjects have been
2. Mature phytase, not glycosylatedcalculated
documented in great detail in the BASF Reference
from the amino acids (gene) sequence and the experi-
Manual entitled Phytase in Animal Nutrition and
mentally determined maturation site (e.g., via N-term-
Waste Management (38) and will not be further dealt
inal amino acid sequencing of the puried enzyme).
with here.
3. Mature, glycosylated phytaseexperimentally
At present only a limited number of phytase pro-
determined by various methods (see below).
ducts are commercially available (Table 2). The rst
phytase product, which entered the market in 1991, is Table 3 summarizes the availability of amino acid
manufactured by DSM (Gist-brocades) and sold by sequences derived from either the corresponding gene
BASF under the trade name Natuphos. This product sequences or from Edman degradation. The fungal
contains the phytase PhyA from Aspergillus niger PhyA and PhyB genes encode precursor phytases
(cuum) NRRL3135 and is available as powder, gran- with 439487 amino acids, corresponding to molecular
ulate, or liquid (e.g., see www.natuphos.com). weights ranging from 47 to 53 kDa (Table 4). In the
Table 2 Commercially Available Phytase Products
Produced by Production
Company Trademark GMOa organism Phytase from Reference
DSM (Gist-brocades)/BASF Natuphos Yes Aspergillus niger Aspergillus niger cuum 39

NRRL 3135, van Tieghem
Novozymes A/S Phytase Novo Yes Aspergillus oryzae Aspergillus niger 40
Rohm (formerly from Alko) Finase P Yes Trichoderma Aspergillus awamori 41
a
Genetically modied organism.

Table 3 Phytase Amino Acid Sequence Entries in the Protein Information Resource (PIR) and SWISS-PROT/TrEMBL
Databases Available on the Web (http://www-nbrf.georgetown.edu/pirwww/pirhome.shtml and http://www.expacy.ch
respectively)
Gene Organism Abbr. SWISS PROT TrEMBL PIR Ref
Phy Bacillus strains DS11 B.DS11 PHYT_BACSP 30, 31

Phy Bacillus subtilis B.sub PHYT_BACSU
PhyA Agrocybe pediades A.ped 6
PhyA Aspergillus awamori A.awa PHYA_ASPAW JN0889 9
PhyA Aspergillus cuum A.nig02 46
PhyA Aspergillus fumigatus A.fum O00092 10, 11
PhyA Aspergillus nidulans A.nid PHYB_EMENI 14
PhyA Aspergillus niger (cuum) NRRL 3135 A.nig01 PHYA_ASPNG JN0656 43, 44
JN0482 45
PhyA Aspergillusterreus 9A1 A.ter02 O00085 12
PhyA Aspergillus terreus CBS 116.46 A.ter01 O00100 48
PhyA Myceliophtora thermophila M.the O00107 12
PhyA Peniophora lycii P.lyc 6, 18
PhyA Talaromyces thermophilus T.the O00096 13
PhyA Thermomyces lanuginosis T.lan 19, 20
PhyA Trametes pubescens T.pub 6
PhyA1 Paxillus involtus 6
PhyB Aspergillus awamori A.awa PHYB_ASPAW JN0890 9
PhyB Aspergillus niger (cuum) NRRL 3135 A.nig PHYB_ASPNG JN0715 47
case of the PhyA phytase of Aspergillus cum (strain B. Primary, Secondary, Tertiary, and
NRRL 3135), the molecular weight of the precursor Quaternary Structures
phytase (51,086 daltons; Table 4) could be calculated
from the gene-derived sequence (43, 44). In addition, The primary sequences of some 2025 phytases are
the mature protein was also sequenced completely by known. Table 2 shows that many of these sequences
Edman degradation (45). A comparison of the two are available at the Web from sequence databases such
sequences allowed identifying the position of the as SWISS-PROT, TrEMBL, and PIR. Yet, many
maturation site; it was found that the signal peptide (new) sequences are available from the patent literature
consists of 23 amino acids, resulting in a mature pro- only.
tein of 444 amino acids with a calculated molecular Pairwise alignment of the amino acid sequences and
weight of 48,846 daltons. The chemically and gene- calculation of the homology revealed that the phytase
derived sequences of the mature protein were found sequences can be derived into three groups: fungal
to be identical. phyA sequences, fungal phyB sequences, and others
In a recent study, Wyss et al. (49) determined the such as the sequences from Bacillus. The homology
molecular weights of several fungal phytases by SDS- within a group can be as high as 4597% as is illu-
PAGE, analytical ultracentrifugation, and gel ltra- strated in Table 5 for the fungal PhyA phytases.
tion analysis (see Table 4 for a summary). The experi- Between the groups the homology is much lower; i.e.,
mentally determined molecular weights are much between phyA and phyB the homology is 2327%
larger than the values calculated on the basis of the whereas no signicant homology exists between the
amino acid sequences. The difference in molecular Bacillus phytases and the fungal phyA and/or phyB
weight must be attributed to the glycosyl chains phytases (not shown). Figure 2 shows a multiple align-
that are attached to the enzymes. The authors con- ment of fungal PhyA sequences. The N-terminal resi-
cluded that all phytases are monomers and that the dues with a gray background have been shown or are
contribution of the sugar chains to the molecular suggested to belong to the signal peptide. Residues in
weight of the glycosylated mature enzyme could white with a black background are conserved in at
amount to 2065%. least ve sequences.

Table 4 Molecular Weights of Phytases
Precursor Mature
Calculated Calculated Analytical References for

MW (Da); no MW (Da); no ultra- #AA and
Gene Organism Abbr. #AA glycosylation #AA glycosylation SDS-PAGE centrifuge Gel ltration calculated MW
PhyA Aspergillus awamori A.awa 467 51,075 444 48,851 9

PhyA Aspergillus cuum A.nig02 467 51,164 444 48,864 46
PhyA Aspergillus fumigatus A.fum 465 50,836 439 48,270 72,360 520 (49) 70,740 (49) 90,500 1; 630 (49) 10, 11
60,770 2; 150 (49)
PhyA Aspergillus nidulans A.nid 463 51,786 441 49,355 66,430 2; 300 (49) 67,400 (49) 77,850 600 (49) 13
86,930 1,330 (49)
PhyA Aspergillus niger A.nig01 467 51,086 444 48,846 66,360 2,440 (49) 64,890 (49) 82,360 1,710 (49) 43
(cuum) NRRL 3135 441 48,524 (45) 85,000 (44) 45
PhyA Aspergillus terreus 9A1 A.ter02 446 51,093 447 49,128 12
PhyA Aspergillus terreus A.ter01 466 51,055 447 49,174
CBS116.46
PhyA Agrocybe pediades A.ped 454 50,030 427 47,256 6
PhyA Myceliophtora M.the 487 52,537 467 50,518 62,890 1,210 (49) 66,150 (49) 73,800 200 (49) 12
thermophila
PhyA1 Paxillus involtus P.inv 442 47,552 423 45,468 6
PhyA Peniophora lycil P.lyc 439 47,563 413 44,845 6, 18
PhyA Talaromyces T.the 466 51,450 452 50,082 128,400 1,700 (49) 137,140 (49) 13
thermophilus
PhyA Thermomyces T.lan 475 53,279 444 50,009 19, 20
lanuginosis
PhyA Trametes pubescens T.pub 443 47,773 426 45,908 6
PhyB Aspergillus awamori A.awa 479 52,678 460 50,848 9
PhyB Aspergillus niger A.nig 479 52,611 460 50,781 45
(cuum) NRRL 3135 476 52,595
Phy Bacillus strain DS11 B.DS11 383 41,802 357 39,230 30, 31
Phy Bacillus subtilis B.sub 382 41,946 356 39,359

Table 5 Amino Acid Sequence Homology Between Various Fungal PhyA Phytases
A.nig01 A.nig02 A.awa A.fum A.nid A.ter01 A.ter02 T.the T.lan M.the P.lyc P.inv 1 P.inv 2 T.pub A.ped
A.nig01
A.nig02 96
A.awa 97 95
A.fum 66 65 66
A.nid 62 62 62 67
A.ter01 62 62 63 60 59
A.ter02 60 60 61 59 57 87
T.the 61 61 61 60 59 59 87
T.lan 54 53 54 52 54 49 48 59
M.the 46 46 46 49 48 45 47 45 46
P.lyc 40 41 41 40 40 43 43 43 38 43
P.inv 1 41 41 40 40 40 41 41 37 40 41 55
P.inv 2 38 39 38 39 39 39 39 36 39 40 55 85
T.pub 41 39 41 40 39 39 39 39 40 41 51 62 63
A.ped 38 38 38 37 42 38 38 37 37 39 51 58 58 61
The secondary and tertiary structures of phytase are 3). The homology between these two types of phytases
known in great detail because the rst 3-D structure of is relatively low, and the enzymological properties are
phytase from A. niger (cuum) was recently determined very different (51).
by x-ray crystallography at a resolution of 2.5 A by In relation to phytases, several publications also
researchers of HoffmanLa Roche (50). To be success- refer to acid phosphatases (EC 3.1.3.2). However,
ful in crystallizing the phytase, the enzymes had to be these enzymes show no amino acid sequence homol-
deglycosylated completely. ogy with phytases whatsoever, and also their sub-
The structure has an =-domain similar to that of strate spectrum differs from phytase since they
rat acid phosphatase and an -domain with a new fold cannot use phytate as a substrate (51, 53).
(Fig. 3). The high homology that exists between fungal Therefore, fungal PhyA phytases can be regarded as
PhyA phytases suggests that all these phytases (Table 5 very specialized acid phosphatases because of their
and Fig. 2) will have the same secondary structural unique property to efciently dephosphorylate phytic
elements and tertiary fold and topology. acid.
Not all the 444 amino acids could be detected in the
electron density map: residues 16 and 249252 (num-
2. From Posttranslational Modication
bering of the mature protein; add 23 to obtain the
precursor protein numbering) were not visible, pre- Extracellular phytases undergo two posttranslational
sumably because of too high a mobility. All 10 cysteine modications: glycosylation and maturation.
residues are involved in a disulde bridges: Cys817, Glycosylation takes place in the Golgi apparatus of
Cys48391, Cys192442, Cys241259, and Cys413 eukaryotic cells like fungi and yeast (and not in pro-
421. The amino acid alignment in Figure 2 shows karyotic cells such as bacteria), and it involves only
that, with the exception of the cysteines involved in those proteins that are secreted by the cell.
bridge Cys817, the eight other cysteines and therefore Glycosylation is known to generate a heterogeneous
the four disulde bridges are completely conserved in population of glycoproteins. Since the glycosyl chains
the fungal PhyA phytases. Amino acids involved in contribute substantially to the molecular weight of
catalysis and substrate binding will be discussed later. phytase, the molecular weight will show a distribution
around an average value instead of having one single
C. Isoforms value.
Glycosylation of different phytases depends on the
1. From Different Genes
microbial expression system (49). In Aspergillus, glyco-
In Aspergillus cuum and A. awamori, two phytase sylation was found to be moderate, whereas it was
genes have been identied: PhyA and PhyB (Table excessive and highly variable in Humicola polymorpha

Figure 2 Amino acid sequence alignment of fungal PhyA phytases. For abbreviations and references see Table 3. The sequences
were aligned using the Multiple Alignment Tool from PIR at http://www.nbrf.georgetown.edu/pirwww/search/multaln.html
which makes use of Clustal W. White text and a black background mark conserved amino acid residues in at least ve sequences.
A gray background marks signal peptides. The -helices and -strands are denoted by and , respectively.

Figure 2 Continued

and Saccharomyces cerevisae. On the basis of the con- ferent glycosylation patterns will certainly affect prop-
sensus Asn-Xxx-Ser and Asn-Xxx-Thr sequences, erties such as molecular weight, isoelectric point,
potential N-linked glycosylation sites in A. niger phy- solubility, and possibly also thermostability but much
tase were identied at positions 27, 59, 105, 120, 207, less likely the enzyme activity.
230, 339, 352, 376, and 388. However, the multiple Translocation of the protein over the cell membrane
alignment of the amino acid sequences shows that is mediated by the signal peptide. During this process,
only the consensus sequences at two positions: the signal peptide is cleaved off by signal peptidases.
Asn207 and Asn339 are conserved. Apparently, differ- This process can also give rise to heterogeneity at the
ent phytases can become glycosylated at different posi- N-terminus of the mature protein (49). In Figure 2, a
tions. Although the effect of glycosylation on enzyme gray background marks the (putative) signal peptides
structure and function is always under debate, the dif- of the various phytases.

to hydrolyze many other phosphate esters. Table 6
summarizes the kcat , KM , and kcat =KM values of the
phytase from A. niger strain NRRL3135 for various
phosphate ester substrates. Phytase has the highest
afnity for phytate with six phosphate groups (IP6)
as can be deduced from the low KM values compared
with the values for the other substrates. The highest
kcat values are found with the phytate substrates, in
particular IP4 and IP3, for which kcat is even much
higher than for the IP6 substrate. The highest speci-
city constants (kcat =KM ) are observed for the phytate
substrates that are 10 to 1000-fold higher than for the
other substrates. In conclusion, phytate is the best
substrate for phytase.
Figure 3 Schematic representation of the phytase structure. In a comparative study, Wyss et al. (53) measured
The cylinders represent the -helices and the arrows the - the specic activities of nine fungal and bacterial phy-
strands. The left-hand side of the molecule is the =-domain tases and acid phosphatases with 14 different sub-
and the right-hand side the -domain. (Picture courtesy of strates at a xed concentration of 5 mM and at pH
Dr. Jan-Metske van der Laan.) 5.0 and 37 C (Fig. 4). The results clearly show that
the phytases from A. niger, A. terreus, and E. coli
have a narrow specicity compared to the other
enzymes. Their highest specic activities are obtained
3. Proteolysis with phytate while 30% or lower values of the phy-
When fungal phytases were expressed in an Aspergillus tate specic activity were found with the other 13
niger strain and stored as concentrated culture super- substrates. On the contrary, the phytases from A.
natants, Wyss et al. (49) found that the enzymes were fumigatis, E. nidulans, and M. thermophilus, and the
susceptible to limited proteolytic cleavage. In some acid phosphatases from A. niger (measured at pH 5.0
cases, this proteolysis was accompanied with activity and pH 2.5) display higher activity toward a broader
loss (A. fumigatis) but not in others (E. nidulans). In the range of substrates. However, their activity toward
latter case, the fragments remained associated with phytate is much lower than the activity of A. niger,
each other as was shown by gel ltration experiments, A. terreus, and E. coli phytase, or not even detectable
and the complex was as active as the intact enzyme. (acid phosphatase).
Determination of the cleavage sites was done by N-
terminal sequencing of the proteolytic fragments.
Comparison with the 3-D structure of the A. niger B. Effect of Environmental Factors
enzyme showed that proteolysis occurred at exposed
loops on the surface of the molecule. By site-directed Environmental factors such as pH and temperature
mutagenesis of the cleavage site amino acid residues it exert their effect on two enzyme properties: activity
was possible to reduce the proteolytic susceptibility and stability. Both properties are important since
without affecting the specic activity of the mutant they determine the nal application properties of the
enzyme (49). enzyme.
When expressed in Hansenula polymorpha, no pro- The pH dependence of the phytase activity has been
teolysis of the fungal phytases was observed. studied extensively for various phytases from fungal
and bacterial origin (Table 7). In general, the fungal
phytases are active in the acid-neutral region, while the
bacterial phytases behave the same (E. coli) or display
their activity in the alkaline region (e.g., Bacillus).
A. Substrate Specicity Various research groups found that the PhyA phytase
from Aspergillus niger NRRL3135 has a broad pH
Phytases are known as acid phosphatases capable of optimum ranging from pH 2.5 to 6. The prole is
cleaving phosphate monoester bonds. In addition to remarkable in the sense that two optima are found
phytic acid, phytases have been reported to be able one at pH 4 and another at pH 5.5.

To be active at the pH of the digestive tract of ani- was higher than of the other phytases (up to 63:3 C
mals (i.e., neutral in the chickens crop and acidic in the for the A. niger phytase).
stomachs), a phytase is required to possess the pH
activity prole as displayed by PhyA phytase from A.
niger (cuum) NRRL3135. C. Specic Mechanism of Action
The activity of phytase increases with temperature
until a maximum is observed. This maximum is Phytases hydrolyze phosphomonoester bonds in a vari-
usually caused by inactivation of the enzyme due to ety of substrates and as such belong to the class of
instability. Table 7 shows that the temperature opti- phosphatases. This class can be divided into three
mum of the various phytases ranges from 45 C to groups: alkaline, acid, and protein phosphatases (56).
65 C. The acid phosphatases can be further subdivided into
The stability of phytase, in particular its thermo- low- and high-molecular-weight ( 18 kDa and 5060
stability, is an important property in view of its appli- kDa, respectively). Examples of the last group are the
cation as a feed additive. Namely, during the feed extracellular fungal enzymes and the purple acid phos-
pelleting step, steam is injected which results in an phatases with binuclear Fe-Fe and Fe-Zn centers in
increase of the temperature up to 6595 C and some- their active site.
times even higher. Consequently, phytase, which has Phytases belong to the group of histidine high-
already been added to the feed at this stage, must be molecular-weight acid phosphatases. They hydrolyze
able to withstand these temperatures; otherwise, too phytate and other phosphoesters in a two-step
much activity will be lost. This demand for thermo- mechanism (Ping-Pong) involving a covalent phos-
stability explains the continuous screening efforts for phorylated histidine adduct enzyme intermediate
more thermostable phytases and/or the protein engi- (57, 58). In the rst partial reaction, the phosphory-
neering activities to improve the thermostability of lated substrate molecule binds to the enzyme to give
existing phytases. Also, improved formulations of phy- the noncovalent Michaelis complex which is then fol-
tase comprising stabilizing additives are continuously lowed by transfer of the phosphate group to the
developed in order to deliver stability to the enzyme active-site histidine. In the second partial reaction, a
during the feed pelleting step. water molecule dephosphorylates the enzyme, result-
Wyss et al. (55) compared the thermostability of A. ing in free enzyme and inorganic phosphate. The
niger phytase (the currently used commercial phy- consequence of this mechanism is that in case of
taseTable 2), the A. fumigatis phytase, and A. chiral substrates, net retention of the conguration
niger acid phosphatase. Both in solution studies and at the phosphorus atom will occur, consistent with
in feed pelleting trials, the A. fumigatis enzyme two successive associative in-line phosphoryl trans-
proved to be more thermostable than the A. niger fers, each of which occurs with inversion of the con-
enzyme. This better thermostability, however, is coun- guration.
teracted by a vefold lower specic activity of the A. Sequence alignment of phytases shows that there is
fumigatis enzyme compared with the A. niger enzyme a conserved R81 H82 G83 A84 R85 sequence (residues 81
(Table 8). 85 in the A. niger precursor sequence numbering). On
The same research group also used protein engi- the basis of chemical modication studies of arginines
neering to improve the thermostability of phytase and lysines, Ullah et al. suggested that this sequence
(37). A so-called consensus phytase was designed contains an essential arginine and histidine residue
that has an amino acid sequence consisting of the (59, 60), which was conrmed by site-directed muta-
most conserved residues found at each position in genesis experiments in the E. coli acid phosphatase
the phytases from A. niger (two sequences), A. awa- (57). The 3D structure of A. niger phytase indeed
mori, A. terreus (two sequences), A. fumigatis (ve conrmed that this peptide was involved in substrate
sequences), A. awamori, A. terreus (two sequences), binding (50). Therefore, it is very likely that the his-
A. fumigatis (ve sequences), A. nidulans, T. thermo- tidine in the peptide mentioned is involved in the
philus, and M. thermophila. This consensus phytase phosphorylation/dephosphorylation cycle. The con-
proved to have the highest thermostability among served peptide, at least residues R81 H82 G83 , has
all the other phytases mentioned. The temperature been found to be conserved in other members of
optimum of the activity was 70 C (compare with the group of acid phosphatases, some bisphospha-
Table 7) and also the melting temperature as mea- tases, and some mutases which all make use of a
sured with differential scanning calorimetry (78 C) phosphohistidine intermediate (59, 61).

Table 6 Substrate Specicity of PhyA Phytase from Aspergillus niger (cuum) NRRL 3135
Spec. act.
kcat KM kcat =KM at Vmax
Substrate (sec1 ) (mM) (mM1 :sec1 ) (U/mg) Conditions Reference
Phytate:
Dodeca-sodium phytate (IP6) 216 0.040 5400 126 58 C, pH 5:0 52
Dodeca-sodium phytate (IP6) 348 0.027 12888 58 C, pH 5:0 51
Dodeca-sodium phytate (IP6) 171 0.250 684 100 37 C, pH 5:5 44
Dodeca-sodium phytate (IP6) < 0:005 103 37 C, pH 5:0 53
Mono-potassium phytate (IP6) 138 0.025 5520 81 58 C, pH 5:0 52
Dipotassium-tetramagnesium 222 0.070 3171 130 58 C, pH 5:0 52
phytate (IP6)
Myo-inositol-1,2,4,5,6- 477 0.16 2980 58 C, pH 5:0 51
pentakisphopate (IP5)
Myo-inositol-1,2,5,6- 1554 1.00 1554 58 C, pH 5:0 51
tetrakisphophate (IP4)
Myo-inositol-triphophate (IP3) 164 0.20 820 58 C, pH 5:0 51
Myo-inositol 2-monophosphate 12 4.50 3 7 58 C, pH 5:0 52
(IP1)
Para-nitrophenylphosphate 104 0.27 385 60 58 C, pH 5:0 52
Beta-glycerophosphate 17 1.66 10 10 58 C, pH 5:0 52
Na2 H2 pyrophosphate 42 0.29 145 24 58 C, pH 5:0 52
ATP 104 0.35 300 60 58 C, pH 5:0 52
Beta-NADP 17 0.50 34 10 58 C, pH 5:0 52
Glucose-6-phosphate 28 8.30 3 2 58 C, pH 5:0 52
Phenylphosphate 103 0.53 194 6 58 C, pH 5:0 52
4-nitrophenylphosphate bis 200 0.72 278 117 58 C, pH 5:0 52
D-fructose-1,6-bisphosphate 59 1.60 37 34 58 C, pH 5:0 52
Adenosine 5 0 -diphosphate 22 0.39 56 13 58 C, pH 5:0 52
Beta-naphthylphosphate 22 0.53 42 13 58 C, pH 5:0 52
The structure of phytase was compared with the active-site residues (Arg81, His82, Arg85, Arg165,
structure of rat acid phosphatase (50). The substrate His361, and Asp362) are involved in electrostatic
prole of phytase differs from the rat acid phosphatase interactions with the scissile 3-phosphate group of
in that phytase is able to hydrolyze the large phytate the substrate. Furthermore, His82 is in a favorable
substrate compared to the smaller substrates of the rat position to make the nucleophilic attack at the
enzyme. Inspection of the active site of the two phosphorus and Asp362 is in a position to proto-
enzymes reveals on one side of the substrate-binding nate the leaving alcohol [for a further discussion see
pocket the following conserved residues (numbering of (50)].
the A. niger precursor protein): Arg81 (Arg11 in the rat
acid phosphatase), His82 (His12), Gly83 (Gly13),
Arg85 (Arg15), Pro87 (Pro17) of the RHGxRxP pep- VI. PHYTASE ASSAYS
tide as well as Arg165 (Arg79), His361 (His257), and
Asp362 (Asp258) which all belong to the =-domain The (specic) activity of phytase is usually determined
of the enzyme. On the other side of the substrate-bind- with its natural substrate phytate or the chromogenic
ing pocket (the -domain) no similarities were found. substitute para-nitrophenylphosphate. The rst assay
Here, the active site of the phytase is much larger than involves the detection of the liberated inorganic phos-
that of the rat acid phosphatase, thus allowing the phate while the second one detects the yellow para-
accommodation of the larger phytate substrate. nitrophenol spectrophotometrically.
A molecular modeling study of the phytasephy- Many conditions have been reported in the litera-
tate complex showed that all conserved charged ture for the phytase assay using phytate as a sub-

Figure 4 Substrate specicities of wild-type phytases and A. niger pH 2.5 acid phosphatase. All substrates were used at a
concentration of 5 mM. 1, Phytic acid; 2, para-nitrophenyl phosphate; 3, phenyl phosphate; 4, fructose 1,6-bisphosphate; 5,
fructose 6-phosphate; 6, glucose 6-phosphate; 7, ribose 5-phosphate; 8, -glycerophosphate; 9, -glycerophosphate; 10, 3-phos-
phoglycerate; 11, phosphoenolpyruvate; 12, AMP; 13, ADP; 14, ATP. (A) A. fumigatis phytase; (B) A. nidulans phytase; (C) M.
thermophila phytase; (D) A. niger pH 2.5 acid phosphatase; (E) A. niger pH 2.5 acid phosphatase (measurements obtained at pH
2.5 instead of pH 5.0); (F) A. niger phytase; (G) A. terreus CBS phytase; (H) A. terreus 9A1 phytase; (I) E. coli phytase. (Courtesy
of the American Society for Microbiology.)
Table 7 pH Proles of Phytasesa
pH optimum Temperature
Phytase from at 37 C Ref. optimum (pH) Ref.
A. niger (cuum) NRRL 3135 2.56 54 58 (5.0) 54
2.56 44 50 (5.5) 44
2.56 53 55 (5.0) 37
26 19 55 (5.5) 19
A. fumigatis 38 53 55 (5.0) 37
37.5 11, 36
A. terreus 9A1 47 53 50 (5.0) 37
A. terreus CBS 47 53 45 (5.0) 37
E. nidulans 5.57 53 45 (5.0) 37
M. thermophila 3.56.5 53
T. lanuginosis 38 19 65 (5.5) 19
A. pediades 36 6 50 (5.5) 6
P. lycii 36 18 50 (5.5) 18
B. subtilis 69 27
E. coli 2.56 53
a
In all cases, phytate was used as substrate.

Table 8 Specic Activities of Various Phytases with Phytate as Substrate
Phytase from Spec. act. (U/mg) Conditions Ref.
Aspergillus niger (cuum)

PhyA 103 20 37 C, pH 5:0 53
PhyA 180 37 C, pH 5:5 19
PhyA 100 37 C, pH 5:5 44
PhyA 126 58 C, pH 5:0 62
Aspergillus fumigatis 27 5 37 C, pH 5:0 36, 53
Aspergillus nidulans 29 4 37 C, pH 5:0 53
Aspergillus terreus 9A1 142 6 37 C, pH 5:0 53
Aspergillus terreus CBS 196 18 37 C, pH 5:0 53
Myceliophtora thermophila 42 4 53
Thermomyces lanuginosis 37 C, pH 6:0 19
Peniophora lycii 987 37 C, pH 5:5 6, 18
Paxillus involtus PhyA1 810 37 C, pH 5:5 6
Paxillus involtus PhyA2 1370 37 C, pH 5:5 6
Trametes pubescens 1450 37 C, pH 5:5 6
Escherichia coli 811 216 37 C, pH 5:0 53
Bacillus subtilis B13 88 37 C, pH 7:5 27
strate. The basic principle, however, is the same. phate, pH 4.0, and 100 L 10 mM PNPP. The absor-
Variations exist with respect to the incubation tem- bance of the liberated para-nitrophenol is measured
perature, pH, and choice of buffer, substrate concen- spectrophotometrically at 30 nm as a function of
tration, and the like. A suitable assay is carried out as time. The absorbance increase (A/min) can be con-
follows (44): 100 L enzyme solution or distilled verted to a rate expressed in M/min using an extinc-
water as a blank is added to 900 L of a solution tion coefcient of 3690 M1 cm1 . One unit of phytase
composed of 0.25 M sodium phosphate buffer, pH is dened as the formation of 1 mole of para-nitro-
5.5, and 1 mM phytic acid (sodium salt). The result- phenol per minute.
ing mixture is incubated for 30 min at 37 C. The
reaction is stopped by the addition of 1 mL of 10%
trichloroacetic acid. After the reaction has termi- VII. PURIFICATION
nated, 2 mL of a reagent composed of 3.66 g of
FeSO4 7 H2 O in 50 mL of ammonium molybdate Numerous purication methods for the various phy-
solution (2.5 g (NH4 )6 Mo7 O24 4 H2O and 8 mL tases discussed here have been published in the litera-
H2 SO4 , diluted to 250 mL with demineralized ture; for a summary see Table 9. In general, the
water), is added. The intensity of the blue color is complexity of the method and number of steps
measured spectrophotometrically at 750 nm. The involved depend on the expression level of the enzyme.
measurements are indicative of the quantity of phos- This means that for the purication of phytase from
phate released in relation to a calibration curve of nonrecombinant strains, more steps are required to
inorganic phosphate in the range of 01 mM. One obtain a homogeneous phytase preparation than pure
unit of phytase is dened as the amount of enzyme phytase from overexpressing strains.
that is capable of forming 1 mole of phosphate per
minute. Specic activities of various phytases are
summarized in Table 8. ACKNOWLEDGMENTS
The activity of phytase can also be determined con-
veniently by using para-nitrophenylphosphate (PNPP) I would like to acknowledge my colleagues Dr. Rob
as a substrate. The conditions for this determination Beudeker and Dr. Jan-Metske van der Laan for
are as follows: 10 L of an enzyme solution is added to helpful discussions and critically reading the manu-
a solution containing 890 L of 0.25 M sodium phos- script.

Table 9 Overview of Phytase Purication Methodsa
Original strain or
Phytase from overexpressed in Method Remarks Reference
A. fumigatis A. niger NW205 1. Desalting Fast-Desaltin HR 10/10 11

ATCC13073 2. Cation-ion exchange chromatography Poros HS/M
A. niger NRRL 3135 Original strain 1. Methanol treatment of the culture ltrate Mixture of phytase and 54
2. Cation-ion exchange chromatography over SP-Trisacryl M acid phosphatase that
3. Anion exchange chromatography over DEAE-Trisacryl M copuried along with
4. Chromatofocusing the phytase (42)
1. Filtration of the culture broth 44
2. Cation-ion exchange chromatography S-Sepharose
Fast-Flow
3. Anion exchange chromatography Q-Sepharose Fast-Flow
4. Isoelectric focusing (42)
1. Immunoafnity column chromatography using monoclonal Simple one-step 44
antibodies against phytase that were covalently attached to procedure
to CNBr-activated Sepharose 4B. Elution of active phytase
with a pH 2.5 buffer.
A. niger CB S. cerevisaea 1. Hydrophobic interaction chromatography Butyl Sepharose 49
Fast Flow
2. Gel permeation chromatography Sephacryl S-300
A. niger NW205 or 1. Desalting East-Desaltin HR 10/10 or Sephadex G-25 superne 49
H polymorpha 2. Anion exchange chromatography Poros HQ/M
A. pediades S. cerevisiae W3124 1. Anion exchange chromatography Q-Sepharose Fast ow 6
2. Hydrophobic interaction chromatography Phenyl
Toyopearl 650s
3. Anion exchange chromatography Source 30Q
4. Anion exchange chromatography Hightrap Q
A. terreus S. cerevisaea 1. Hydrophobic interaction chromatography Butyl Sepharose 49
Fast Flow
A. niger NW205 or 1. Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 superne 49
H. polymorpha 2. Anion-ion exchange chromatography Poros HQ/M
E. nidulans S. cerevisaea 1. Hydrophobic interaction chromatography Butyl Sepharose 49
Fast Flow
A. niger NW205 or 1. Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 superne 49
H. polymorpha 2. Anion-ion exchange chromatography Poros HQ/M
E. coli E. coli BL21 1. Sonication of cell suspension + centrifugation Enzyme expressed 49
2. Afnity chromatography Ni2 chromatography intracellulary
continued

702
Table 9 Continued
Original strain or
Phytase from overexpressed in Method Remarks Reference
M. thermophila S. cerevisaea 1. Hydrophobic interaction chromatography Butyl Sepharose 49

Fast Flow
A. niger NW205 or 1. Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 49
H. polymorpha superne
2. Anion exchange chromatography Poros HQ/M
P. involtus Phy 1 A. cryzae IFO4177 1. Anion exchange chromatography Q-Sepharose Fast Flow 6
2. Hydrophobic interaction chromatography Butyl Toyopearl
650S
3. Desalting Sephadex G25
4. Anion exchange chromatography Q- Sepharose Fast Flow
Toyopearl 650S
P. involtus Phy 2 A. oryzae IFO4177 1. Hydrophobic interaction chromatography Phenyl Sepharose 6
Fast Flow
2. Hydrophobic interaction chromatography Butyl
Toyopearl 650S
Misset
4. Anion exchange chromatography Q-Sepharose Fast Flow
Phytase
P. lycii A. oryzae IFO4177 1. Anion exchange chromatography Q-Sepharose Fast Flow 18
Toyopearl 650S
T. pubescens A. oryzae IFO4177 1. Anion exchange chromatography Q-Sepharose Fast Flow 6
2. Hydrophobic interaction chromatography Butyl
Toyopearl 650S
T. lanuginosis Fusarium venenatum 1. Anion exchange chromatography Q-Sepharose Big Beads 19
2. Ultraltration
3. Anion exchange chromatography MonoQ HR10/16 column
4. Ultraltration
5. Cation exchange chromatography MonoS HR 5/5 column
T. thermophilus S. cerevisaea 1. Hydrophobic interaction chromatography Butyl Sepharose 49
Fast Flow
A. niger NW205 or 1. Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 49
H. polymorpha superne
2. Anion exchange chromatography Poros HQ/M
a
Most methods comprise as a rst step the removal of the cells by centrifugation or ltration. The clear, occasionally ultraltrated, culture supernatant is then subjected to the further
stages as indicated.
703
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56
-Amylases

I. INTRODUCTION and -amylase is used for desizing to nish the fabric.

-Amylase is one of the enzymes incorporated into
-Amylases (EC 3.2.1.1) are endoglucanases that cata-
laundry detergents to remove starchy stains from fab-
lyze the cleavage of internal glucosidic bonds in starch
rics, and to increase the efciency and reduce chemical
and related polysaccharides to yield dextrins and oli-
ingredients in detergents.
gosaccharides with the anomeric C1-OH in the -con-
guration. The -1,4 bonds near -1,6 branches are
resistant to hydrolysis. Extensive hydrolysis of amylo- II. CLASSIFICATION
pectins with -amylases produce -limited dextrins,
as cleavage terminates at the -1,6 bonds. -Amylase Glycosidases are classied under hydrolases which are
plays a central role in carbohydrate metabolism of subdivided into those hydrolyzing O-glycosyl, N-gly-
microorganisms, plants, and animals. Microbial - cosyl, and S-glycosyl compounds (EC 3.2.1, 3.2.2,
amylases derived from Bacillus licheniformis or related and 3.2.3). -Amylase hydrolyzes internal 1,4--D-
organisms for commercial applications are stable at glucosidic linkages in oligosaccharide or polysacchar-
temperatures > 90 C, relatively less dependent on ide in a random manner with the reducing groups
Ca2 ions, and active over a wide pH range. liberated in the -conguration. According to the
-Amylase for food processing is obtained by con- IUBMB recommendation (1), -amylase (trivial
trolled fermentation of Bacillus stearothermophilus, name) therefore has the systematic name of 1,4--D-
Bacillus subtilis containing a Bacillus megaterium or glucan glucanohydrolase, and a code number of EC
Bacillus stearothermphilus -amylase gene, as an off- 3.2.1.1. This classication scheme is based on the type
white to brown, amorphous powder or a liquid. of reaction catalyzed and the substrate specicity of
Typical applications include preparation of starch the individual enzyme.
syrup, dextrose, alcohol, beer, and bakery products. More recently, an alternative classication of gly-
-Amylase is used industrially in the liquefaction of cosyl hydrolases based on amino acid sequence simi-
starch to produce dextrins which are further sacchar- larities has been proposed (24). There are 70
ied by glucoamylase to yield glucose feed stock for families according to this classication, and most
corn syrup, fuel ethanol, or beverage alcohol produc- -amylases belong to family 13, together with pull-
tion. In the baking industry, -amylase is added to the ulanases (EC 3.2.1.41), cyclomaltodextrin glucano-
dough to ensure a continuous supply of fermentable transferase (EC 2.4.1.19), cyclomaltodextrinase (EC
sugar for yeast growth and gas production. In the tex- 3.2.1.54), and trehalose-6-phosphate hydrolase (EC
tile industry, mechanical weaving of fabrics often 3.2.1.93). However, -amylases from Dictyoglomus
requires the coating of threads with a starch size, thermophilum, Methanococcus jannaschii, Pyrococcus

furiosus, and Pyrococcus horikoshii belong to family aromycopsis buligera (yeast), Debaryomyces occiden-
57. This classication scheme is based on the direct talis (yeast) (Schwanniomyces occidentalis), Oryza
relationship between sequence and folding similari- sativa (rice), Triticum aestivum (wheat), Hordeum vul-
ties, and it provides a convenient tool to predicting gare (barley), Vigna mungo (rice bean) (black gram),
mechanistic information. However, classication Drosophila melanogaster (fruit y), D. mauritiana, D.
based on sequence and structural similarities may yakuba, Aedes aegypti (yellow fever mosquito),
cause unnecessary confusion because in this scheme, Dermatophagoides pteronyssinus (house-dust mite),
an enzyme is dened by its primary sequence as a Tribolium castaneum (red our beetle), Pecten maxi-
protein, but not directly by its intrinsic catalytic mum (king scallop) (pilgrims clam), Tenebrio molitor
functions. (yellow mealworm), Sus scrofa (pig), Homo sapiens
(human), Rattus norvegicus (rat), and Mus musculus
(mouse).
III. MOLECULAR STRUCTURES The sequences of -amylases from different origins
have very few discernible similarities, with the excep-
Numerous amino acid sequences of -amylases are tion of four short, highly conserved segments (515)
known, primarily deduced from DNA sequencing. A (Fig. 1). The three catalytic residues (two Asp and
total of 70 entries are listed in Swiss-Prot: Aeromonas one Glu), as well as residues involved in substrate
hydrophila, Alteromonas haloplanktis, Dictyoglomus binding, are located in these four highly conserved
thermophilum, Escherichia coli, Bacillus amyloliquefa- regions which are also found in GGTase (Bacillus
ciens, B. megaterium, Bacillus sp. (strain B1018), B. macerans), pullulanase (Klebsiella aerogenes), isoamy-
circulans, B. stearothermophilus, B. licheniformis, B. lase (Psudomonas amyloderamosa), -glucosidase
subtilis, Paenibacillus polymyxa (Bacillus polymyxa), (Saccharomyces carlsbergenesis), and cyclodextrinase
Butryrivibrio brisolvens, Methanococcus jannaschii, (Thermoanaerobacter ethanolicus), suggesting that
Streptomyces lividans, S. violaceus (Streptomyces vene- these enzymes may participate in the same type of
zuelae), S. griseus, S. limosus (Streptomyces albidoa- reaction mechanism (16). Several microbial -amy-
vus), S. hygroscopicus, S. thermoviolaceus, Clostridium lasesBacillus circulans (17), Bacillus subtilis (18),
acetobutylicum, Thermoanaerobacter thermosulfuro- Bacillus stearothermophilus (19), Streptomyces limosus
genes (Clostridium thermosulfurogenes), T. ethanolicus (20), Chalara paradoxa (21), Lactobacillus amylovorus
(Clostridium thermohydrosulfuricum), T. thermohydro- (22), and Cryptococcus sp. S-2 (23)are known to
sulfuricus (Clostrium thermohydrosulfuricum), T. sac- digest not only soluble starch, but raw starch gran-
charolyticum, Thermomonospora curvata, Pyrococcus ules. In addition, porcine pancreatic -amylase (24)
furiosus, P. horikoshii, Salmonella typhimurium, and barley -amylase (25) contain C-terminal seg-
Aspergillus niger, A awamori, A. oryzae, A. shirousami, ments that have been identied to be a raw starch
Schizosaccharomyces pombe (ssion yeast), Sacch- binding site. The raw starchbinding sequences found
Figure 1 Comparison of regions of sequence similarities among: Bacillus stearothermophilus (5), Bacillus amyloliquefaciens (6),
Bacillus licheniformis (7), Aspergillus oryzae (8), Micrococcus sp. 207 (9), Saccharomycopsis buligera (10), barley isozyme 1 (11),
Drosophila melanogaster (fruit y) (12), yellow mealworm (13), porcine pancreatic (14), human pancreatic (15). The three residues
involved in catalysis are indicated by asterisks. Numbering of the sequences starts with N-terminus of the mature protein.

in -amylases from microbial sources exhibit a high wide variety of reactions. The rst protein discovered
degree of similarity to the starch binding segments in a to contain such folding was triose phosphate isomer-
number of bacterial -amylase and GGTases and ase, hence the name TIM barrel (31). The evolution-
fungal glucoamylases. The alignment suggests an ary history of this = barrel family of enzymes has
evolution path according to species rather than been the subject of continuous interest. Arguments in
individual enzyme specicity (26). Some microbial support of convergence, as well as divergent evolution
enzymes, for example, Streptomyces -amylase, have been presented (3235). For most = barrel
contain a glycine rich segment segregating the proteins, including the -amylase family, the active
C-terminus from the rest of the molecule. This site is located at the C-terminus of the parallel -
arrangement is reminiscent of glucoamylases where strands, with the catalytic and substrate-binding resi-
the catalytic domain and the starch-binding domain dues in the loop regions. -Amylases also contain a
are attached via a highly glycosylated linker (27, 28). small structural domain (domain B) inserted between
However, the domain-linker-domain architecture is -strand 3 and helix 3. This protruding domain B
not a universal requirement for raw starch digestion, varies both in length and sequence, and is implicated
because the majority of the -amylases that are in functional diversity and stability. In the Aspergillus
known to degrade starch granules do not contain a oryzae enzyme, the main feature of domain B is a
linker sequence. short, three-stranded antiparallel -sheet (36). In the
Despite the low sequence similarities, the crystal Bacillus licheniformis -amylase, domain B possesses a
structures of -amylases from various organisms exhi- -sheet of six loosely connected twisted -strands
bit similar tertiary folding. The central core is an forming a twisted barrel with a large interior hole
(=8 barrel (domain A), consisting of eight parallel (37). For several -amylases with known three-dimen-
strands alternating with eight helices joined by sional structures, including Bacillus licheniformis,
loops (29, 30) (Fig. 2). About 10% of all known Aspergillus oryzae, and porcine pancreatic, domain
enzymes have an = barrel domain, catalyzing a B contains an invariant Asp residue (equivalent to
Figure 2 A stereo view of the barley -amylase (isoform 2). The structure consists of three domainsthe central (=8 -barrel
(domain A), a ve-stranded antiparallel -sheet domain C at the bottom, and a protruding loop at the top (domain B). (From
Ref. 29, PDBid: 1 amy.)

Asp175 of the A. oryzae enzyme) involved in calcium IV. REACTION MECHANISMS
binding. The =8 barrel is followed by domain C, a
distinct globular unit of 100 amino acid residues -Amylases hydrolyze the glucosidic bond with reten-
with a folding of an antiparallel -sandwich of the tion of the anomeric conguration. The retaining and
Greek key topology. inverting actions of glycosidases have been the subject
The three-dimensional structures of several -amy- of numerous investigations, and several mechanisms
lases have been solved: Aspergillus oryzae, A. niger, have been proposed to explain the stereochemistry of
Bacillus licheniformis, B. subtilis, B. stearothermophilus, the catalytic process.
yellow mealworm, porcine pancreatic, human pancrea-
tic, Alteromonas haloplanctis, and barley isozyme 2. A. Nucleophilic Displacement Mechanism
The following is a functional description of the enzyme
structure of barley -amylase (isoform AMY2) (29, 30, For retaining glycosidases, the reaction proceeds via a
38). AMY2 assumes the same basic features in the double displacement (4345). The protonation of the
overall structure (Fig. 2). Domain A forms an =8 glucoside oxygen is concerted with the nucleophilic
barrel of 286 residues, with a loop of 64 residues attack of a carboxylate anion at C1, resulting in the
(domain B) protruding between -strand 3 and helix formation of a covalent glucosyl-enzyme intermediate
3. The C-terminal 53 residues form domain C with a (Fig. 3). A second displacement by a general base
ve-stranded antiparallel -sheet. The active site is hydrolyzes the carboxyl-acetal bond of the glucosyl-
located on the C-terminal side of the =8 barrel, enzyme intermediate to yield a product with -cong-
with the catalytic residues Asp179, Glu204, and uration.
Asp289 positioned at A-4 , A-5 , and A-7a , respec- For inverting glycosidases, the reaction involves a
tively. Several invariant residues, including Tyr51, single displacement. The glucosidic oxygen is proto-
His92, Arg177, and His288, form hydrogen bonds nated by a general acid, resulting in a weakened mal-
with the substrate (39). tosyl CO bond. The attack of a water molecule occurs
The -amylase contains a calcium-binding site at the -side of substrate assisted by a general base. It
structurally similar to that of other amylolytic has been suggested that both reactions involve transi-
enzymes (40). The calcium atom is coordinated by tion states with oxocarbonium ion character. It is inter-
amino acid side chains located in both domains esting to note that although both reactions can be
A and B, providing structural stability to the called displacement, they are obviously very different.
enzyme. Removal of calcium from the barley In the single displacement, one of the carboxylic cata-
enzyme results in irreversible inactivation. In addi- lytic residues acts as a general acid and the other as a
tion to this high-afnity binding site, two other general base, whereas in the double displacement, one
calcium-binding sites have been located with ligands residue acts as a general acid/base, and the other acts
entirely in the domain B. The calcium-binding inter- as a nucleophile. Furthermore, the covalent enzyme-
actions seem to contribute to the folding stability of substrate intermediate invoked for the double displace-
domain B. ment is not formed in the single displacement.
A surface-located starch granulebinding site
characteristic of cereal -amylase has been identied B. Oxocarbonium Ion Mechanism
that interacts with starch granules. This site also
binds -cyclodextrin, an analog of amylose, in com- In this model, the reaction is described by SN 1 substi-
petition with starch granules. Chemical modication tution (Fig. 4). Protonation of the glucosidic oxygen
and site-directed mutagenesis suggest that two con- results in bond cleavage, leading to the formation of an
secutive Trp residues, Trp276 and Trp277, are oxocarbonium ion intermediate that is stabilized by a
involved (41, 42). In a three-dimensional structure carboxylate anion residue (4648). It has also been
of AMY2 in complex with the inhibitor acarbose, suggested that binding of the sugar substrate to the
the two Trp residues stack onto the substrate sugar enzymes active site induces a distortion of the sugar
rings, a typical feature of proteincarbohydrate ring into a half-chair conformation, thereby weakening
interactions (30). However, this structural arrange- the C1O4 bond as shown in the case of lysozyme (49).
ment involving consecutive Trp residues protruding The stereoselectivity of the reaction then depends on
from the helix A-6b is not found in the microbial the direction of the nucleophilic attack of the water
or animal -amylases that also hydrolyze raw starch molecule assisted by a general base. In -amylases,
granules. the water molecule attacks from the front (original

Figure 3 (A) A single-displacement mechanism. (B) A double-displacement mechanism with the formation of a glycosyl enzyme
intermediate via oxocarbonium-like transition rates.
glucoside oxygen side), with the product maintaining lent intermediate if nucleophilic attack occurs prior to
the -conguration. In inverting glycosidases, such as the complete formation of the cation.
-amylase and glucoamylase, a back-side attack results
in an inversion of the conguration. The appealing C. Ring-Opening Reaction
aspect of this model is that the catalytic reactions of
both inverting and retaining glycosidases can be An alternative mechanism that is not as well received
explained by a single unied mechanism (50). as the above two models involves endocyclic carbon
The main difference between this model and the oxygen cleavage as the rate-determining step, leading
displacement mechanism lies in whether a transient to the formation of an acyclic oxocarbonium ion inter-
or stable oxocarbonium ion intermediate exists (51, mediate (44, 53). In this model, there is no distortion of
52). The formation of a stable oxocarbonium ion inter- the substrate into a twist-boat conformation as shown
mediate is preferred if bond breaking precedes the by molecular dynamics simulation. In the chair con-
attack of water facilitated by general base catalysis. formation of both and -anomers, the antiperiplanar
The oxocarbonium ion is readily collapsed into a cova- orientation of the exocyclic O4 lone pair fullls the

Figure 4 Reaction mechanism of carbohydrases involving a stabilized oxocarbonium intermediate.
stereoelectronic requirement in the fragmentation of proteinaceous inhibitors (61, 62). Analysis of the crys-
the ring CO bond (54). It has been also postulated tal structure indicates that the head of Tendamistat
that protonation occurs at the cyclic oxygen instead of containing the highly conserved triplet residues binds
the glucosidic oxygen, and the water nucleophilic directly to the active site of the porcine pancreatic -
attack is followed by a rotation of the hemiacetal cen- amylase, blocking the ve binding subsites 3 to 2,
ter to position for cleavage (55). with Arg19 forming a salt bridge with the catalytic
residue, Glu233 (for subsite nomenclature, refer to
Ref. 62). In the formation of the enzymeinhibitor
V. -AMYLASE INHIBITORS complex, the conformation of the enzyme active site
is induced to accommodate tight binding of
Two families of -amylases inhibitors (-AI), including Tendamistat and related inhibitors.
carbohydrate- and protein-based inhibitors, have been The seeds of common beans contain a family of
studied in detail. The knowledge of the inhibitory proteins involved in a defense mechanism, composed
action of these compounds is of great interest in med- of phytohemaglutinin, and -amylase inhibitor. The
icine in the attempt to develop therapeutics for dia- -AIs inhibit the activity of some mammalian and
betes, obesity, and hyperlipemia. Proteinaceous -AIs insect -amylases, but not of the endogenous plant
are widespread in microorganisms and higher plants. enzyme. Red kidney bean -AI has a KD of 3:5
Tendamistat, one of the most potent inhibitors of 1011 M at pH 6.9 and 30 C (63), and black bean
mammalian -amylases, is an acidic protein of 74 -AI 4:4 109 M at pH 6.9 at 37 C (64). There
amino acids isolated from the culture medium of are at least ve known sequences derived from -
Streptomyces tendae (56, 57). This class of inhibitors AIs of common domesticated beans, wild common
also includes HAIM (S. grieseosporeus) (58), AI-3688 bean, white kidney bean, and black bean (65). The
(S. aureofaciens) (59), and PAIM (S. griseosporeus) inhibitors are glycoproteins synthesized as prepropro-
(60). Tendamistat binds tightly with porcine pancreatic teins which require proteolytic cleavage for activation
-amylase with a very high inhibition constant of 9 (66). Translational processes produce the active inhi-
1012 M (56). The molecular structure assumes a - bitor formed by the association of two polypeptide
barrel of two -sheets, each consisting of three antipar- chains, and -subunits (67). In the crystal structure
allel strands. The -strands are connected by loops and of the enzymeinhibitor complex, -AI-I exists as a
turns which contain the active-site triplet Trp18- dimer that binds two porcine pancreatic enzyme
Arg19-Tyr20 characteristics of all Tendamistat-type molecules (68). Each monomer consists of a concave

12-stranded antiparallel -sandwich fold. Two hairpin potent inhibitor via transglycosylation or condensation
loops protruding from the fold interact directly with of the cleaved products (36, 75). The additional sugar
the active-site region of the enzyme. A large confor- rings of the product may be stabilized by secondary
mational change occurs in the enzyme active site binding sites on the surface of the enzyme molecule
upon -AI-1 binding, resulting in extensive interac- (76). Another carbohydrate inhibitor, cyclohepta-amy-
tions with the inhibitory site of -AI. Site-directed lose (CHA), has been known to inhibit -amylase
mutagenesis studies suggest the involvement of digestion of raw starch granule by interfering with -
Trp188, Arg74, and Tyr190 in AI, analogous to amylase adsorption to raw starch granules. Hydrolysis
the Trp-Arg-Tyr motif of Tendamistat (69). X-ray of starch granules by wheat -amylase is inhibited 40%
structural analysis conrms that Tyr190 forms strong by 0.88 mM CHA (41). The granular starch-binding
contacts with the catalytic residue Asp300. domain of Aspergillus niger glucoamylase 1 binds -
A variety of -AIs, both endogenous and exogen- cyclodextrin with a dissociation constant Kd of
ous, occur in barley and wheat kernels. The -amy- 1:68 M, and a competitive inhibition constant (Ki )
lase/subtilisin inhibitor (BASI) from barley has been of 11:0 M for raw starch adsorption showing that
extensively investigated and its three-dimensional -cyclodextrin and granular starch bind to the same
structure is known. BASI is active against the plant site of the enzyme molecule (28). It has been proposed
endogenous -amylase 2 (one of the two major barley that the cyclodextrin seven-member ring structure
amylase isozymes). It consists of a single polypeptide mimics the helical amylose with binding in the cavity
of 181 residues with a calculated MW of 19,685 dal- (77).
tons. Its amino acid sequence shows 26% identical
residues compared to that of the soybean trypsin inhi-
bitor (Kunitz) (70). Inhibitors with a dual function of
protease and -amylase have also been identied in VI. MEASUREMENT OF -AMYLASE
wheat and ragi (71, 72). BASI forms an inactive 1:1 ACTIVITY
complex with -amylase 2, with a dissociation con-
stant of 2:2 1010 M at pH 8 and 37 C (73) but The kinetic parameters of barley -enzyme were mea-
does not inhibit -amylase 1, the other major barley sured using various concentrations of the substrate
isozyme. The crystal structure of BASI is composed ET-G7 PNP. A Km of 0.45 mM and a kcat of 1:2 102
of a 12-stranded -barrel, with three sets of four sec1 were obtained for the -amylase, with kcat =Km of
sequential -strands arranged in a threefold symmetry 2:7 102 mM1 sec1 . Barley malt -amylase 1 has a
(38). The hydrophobic core is composed of six - kcat =Km value of 1:75 102 mM1 sec1 using p-nitro-
strandsS1, S4, S5, S8, S9, and S12and one end phenyloligosaccharide (p-NPGlc7 ) as the substrate
of the barrel is closed by triangular array consisting (78). The catalytic efciency is generally consistent
of the remaining -strands. BASI binds tightly to with those obtained for -amylases from other organ-
domains A and B of the enzyme but does not directly isms. For example, a kcat =Km of 2:5 102 mM1 sec1
interact with the catalytic residues at the enzyme has been reported for Saccharomycopsis buligera -
active site. A calcium ion is trapped at the interface amylase in the hydrolysis of malto-oligosaccharides
of the enzymeinhibitor complex. (79), and a higher value of 1:3 103 mM1 sec1
The second type of inhibitors are carbohydrate- has been observed for the same enzyme with amylose
based derived from the trestatin family and are found A as the substrate (80). Porcine pancreatic -amylase
also in Streptomyces. The most common inhibitor in has a kcat =Km of 5:8 103 mM1 sec1 in the hydro-
this class is acarbose, a pseudo-tetrasaccharide with a lysis of p-NPGlc7 (81). Increasing chain length of the
cyclitol unit and an amino sugar unit at the nonredu- substrate decreases the Km value and increases the kcat ,
cing end. Cyclitol ring exists as a half-chair resembling as demonstrated in the Aspergillus oryzae enzyme: Km
the conformation of the oxocarbonium ion. Binding of 9:0 104 and 9:5 106 mM for maltotetraose
the inhibitor to the enzyme induces structural changes and maltodextrin VI (n 117), respectively, and kcat
at the active site, involving extensive hydrogen bonds, 1:2 104 and 9:9 103 min1 (82).
hydrophobic stacking, and water-bridged contacts The method of -amylase activity measurement is
(74). It has been shown that pancreatic -amylase characterized by the type of substrate employed in a
can hydrolyze the reducing end glucose units, and sev- particular procedure. All three methods described
eral x-ray crystal structural analyses revealed that the below have been used routinely by the authors for
original substrate acarbose is transformed into a more the assay of plant and recombinant -amylases.

A. Reducing Sugar Method covalent bonds (89). The substrate forms an insoluble
network of polymers that swells in water or buffer,
Reducing sugar assays measure the increase of new resulting in a suspension. The substrate is known to
reducing ends on fragments produced by the hydrolytic be very resistant to -amylase, and is particularly sui-
action of -amylase on soluble starch. In the 3,5-dini- table for the analysis of -amylase in cereal grains
trosalicyclic acid method, the concentration of the which commonly contain considerable amounts of -
nitroaminosalicyclic acid formed at the reducing hemi- amylase as a common contaminant in enzyme extracts.
acetal is measured colorimetrically (83). The measure-
ment of -amylase activity by this method is accurate 2. Dye-Labeled Non-crosslinked Starch
only if the sample is not contaminated with other amy-
Dye-labeled non-crosslinked starch is made by labeling
lolytic enzymes. A modied procedure for small-scale
non-crosslinked starch with Remazol Brilliant Blue by
analysis of cereal -amylases is described: 250 L of
covalent bonds. This is a soluble substrate (90). The
enzyme in buffer (200 mM sodium acetate, pH 5.5;
substrate is also susceptible to -amylase action, and
10 mM CaCl2 ) was combined with 250 L of 1% solu-
therefore this assay is nonspecic for -amylase.
ble starch and incubated at 37 C for 10 min. To the
A microassay has been developed using dye-labeled
reaction mixture 500 L of dinitrosalicyclic acid
crosslinked starch (Phadebas, Pharmacia) for rapid
reagent (1% nitrosalicyclic acid, 30% potassium
screening of -amylase activity (91). Linear correlation
sodium tartrate, 40% I N NaOH) was added, heated
between the absorbance and enzyme concentrations in
for 5 min in a 100 C water bath, cooled to room tem-
the 50-ng range was observed. The assay has been
perature in cold water, and then claried by centrifu-
adapted for semiautomation and routinely used in
gation. The absorbance at 547 nm was measured, and
monitoring the production of recombinant -amylase
the activity was calculated using a maltose standard
produced in transformed yeast cells. One unit of -
curve. One enzyme unit is dened as the amount of
amylase activity is dened as the amount of enzyme
enzyme catalyzing the release of 1 mole of reducing
that gives an absorbance value of 0.100 (92). This
groups from soluble starch, measured as maltose, per
denition is the most useful in the measurement of
min at 37 C. In some cases, it may be desirable to treat
relative activities, such as monitoring chromatographic
the starch with sodium borohydride to remove term-
column fractions. The absorbance values obtained in
inal reducing glucosyl residues that may interfere with
the dyed-substrate-based assay can be converted to
the reducing sugar measurement (84). The reducing
International Units using a standard curve.
groups can also be quantitated by the alkaline ferricya-
nide method (85, 86), or the Nelson-Somogyi method
(87, 88). Benedicts solution can detect 0.01% glucose C. Dened Substrate Method
in water.
The use of low-MW synthetic oligosaccharides with a
dened structure has the advantage of elimination of
B. Dyed Starch Substrate Method
substrate variability. However, these are not natural
substrates, and activity measurement may fail to reveal
This procedure involves the hydrolysis of dyed starch
some unique enzyme properties, such as raw starch
substrate by -amylase and the measurement of absor-
binding and hydrolysis. Dened substrates have been
bance at 610 nm of the soluble colored fragments
initially developed with a chromophore such as p-
released into solution from degradation of the sub-
nitrophenol group attached to the reducing-end glu-
strate. This method is unaffected by reducing sub-
cose unit. These substrates, however, are nonspecic
stances and is therefore particularly applicable to the
for -amylases, since other amylases, such as -amy-
measurement of -amylase activity in cell culture
lase or glucoamylase, also can readily cleave the sub-
media containing glucose or other reducing sugars.
strate. More recently, a specic substrate has been
Two types of dyed starch substrates are available com-
developed with the additional attachment of a chemi-
mercially.
cal blocking group at the non-reducing-end glucose
residue (93, 94). We use 4,6-ethylidene-(G7 )-p-nitro-
1. Dye-Labeled Crosslinked Starch
phenyl-(G1 )-,D-maltoheptaside (Sigma) as the sub-
Dye-labeled crosslinked starch is made by crosslinking strate. The ethylidene group in the 4,6-position of G7
partially hydrolyzed potato starch using 1,4-butandiol- blocks the action of exoamylases. -Amylase hydro-
diglycidether, and labeling with Cibachron Blue by lyzes the substrate to G2-, G3-, and G4-p-nitrophenol

fragments, which are further hydrolyzed by -glucosi- umn of cyclohepta-amylose-substituted epoxy-
dase to p-nitrophenol and glucose. Enzyme activity Sepharose 6B according to Silvanovich and Hill (96).
corresponds to the increase in absorbance at 405 nm The concentrated enzyme preparation (100 mL) was
owing to the liberation of p-nitrophenol, and the che- loaded onto CHA-Sepharose afnity column
mically blocked nonreducing end renders the substrate (1 8 cm), followed by successive washings with 50
unsusceptible to the action of -amylase and glucoa- mL buffer, 50 mL buffer containing 4 mM NaCl,
mylase. and 100 mL buffer. The adsorbed enzyme was eluted
with buffer containing 8 mg/mL CHA which was sub-
sequently removed by dialysis in 20 mM sodium acet-
VII. PURIFICATION OF -AMYLASE ate, pH 5.0, buffer containing 2 mM CaCl2 .
The following method, based on MacGregor and

Morgan (95), has been used routinely in our lab to
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Saccharomycopsis -amylase secreted from

57
b-Amylases

I. INTRODUCTION end. According to the IUBMB classication (1), -

amylase has the systematic name of 1,4--D-glucan
The distribution of -amylase is limited to higher maltohydrolase, and a code number of EC 3.2.1.2.
plants and bacteria. It is an exoenzyme that catalyzes A classication of glycosyl hydrolases based on
the hydrolysis of 1,4--D-glucosidic linkages in poly- similarities in amino acid sequence has been proposed
saccharides to remove successively -anomeric maltose and yields useful information on the structure and
units from the nonreducing end of -1,4-glucans such function of these enzymes (24). -Amylase belongs
as starch and glycogen. Sweet potato contains an to family 14 in this scheme. This classication scheme
abundant -amylase which accounts for 5% of the should only be used as a supplement to the IUBMB
total soluble proteins in the tuber; by contrast, only a classication.
trace amount of -amylase is present. The sweetness of
sweet potato is the result of the hydrolysis of starch to
maltose by the endogenous -amylase during cooking. III. MOLECULAR STRUCTURE
-Amylase is employed by the starch industry in the
production of high maltose syrups. Compared with Several amino acid sequences of -amylases are
glucose syrups, maltose syrups have a higher viscosity known, primarily deduced from the gene sequences.
and lower hygroscopicity, less tendency to crystallize, These include (as entries are listed in Swiss-Prot):
and more resistance to browning. These characteristics Ipomoea batatas (sweet potato), Glycine max (soy-
make them particularly suitable for use in the confec- bean), Hordeum vulgare (barley), Triticum aestivum
tionery and baking industry. -Amylase is highly sig- (wheat), Zea mays (maize), Secale cereale (rye),
nicant in determining the fermentability of worts for Vigna unquiculata (cowpea), Medicago sativa (alfalfa),
brewing. Trifolium repens (creeping white clover), Arabidopsis
thaliana (mouse ear cress), Bacillus cereus, B. rmus,
B. circulans, B. polymyxa, and Thermoanaerobacter
II. CLASSIFICATION thermosulfurogenes (Clostridium thermosulfurogenes).
Several conserved regions exist in all -amylases,
Glycosidases are classied under hydrolases and are although the sequences between plant and bacterial
subdivided into those hydrolyzing O-glycosyl, N-glyco- -amylases show only 30% similarities (Fig. 1).
syl, and S-glycosyl compounds (EC 3.2.1., 3.2.2, 3.2.3). The three-dimensional structures of -amylases
-Amylase is an exoglucanase that catalyzes the hydro- from soybean (57), sweet potato (8), barley (9),
lysis of 1,4--D-glucosidic bonds in polysaccharides to and Bacillus cereus (10) have been reported.
cleave successively maltose units from the nonreducing Soybean -amylase consists of 495 amino acid resi-

Figure 1 Comparison of regions of sequence similarities among nine -amylases, including sweet potato (P10537), soybean
(P10538), barley (P16098), corn (P55005), wheat (P93594), Arabidopsis thaliana (P25853), Bacillus cereus (P36924), Bacillus
circulans (P06547), and Bacillus polymyxa (P21543). The catalytic Glu are marked as general acid () and general base (#).
Numbering of sequences starts with the N-terminus of the mature protein.
dues with a molecular weight of 56 kDa. The enzyme Waals contacts between Val99 and the substrate. The
consists of a large =8 -barrel core; a smaller lobe L3 lid serves to shield the active site from being
formed by three long loops (L3, L4, and L5) extend- exposed to the solvent, and also positions the sub-
ing from 3, 4, and 5 strands; and a C-terminal tail strate in proximity to the two catalytic residues
loop of 50 residues extending from 8 and covering Glu186 (acting as a general acid) and Glu380 (acting
the entrance of the barrel. Between the surface of the as a general base). Glu186 has been identied as the
lobe region and the core is a cleft that opens into a catalytic residue by afnity labeling with 2,3-epoxy-
pocket 18 A deep that contains the catalytic resi- propyl--D-glucopyranoside (13). The binding energy
dues Glu186 and Glu380, and an essential residue upon closing the lid for -maltose estimated in an
Asp101. This architecture contrasts to that of -amy- automated docking experiment is 22 kcal/mol, indi-
lases which have the reactive center located in a long cating considerable enhancement of interactions
cleft open at both ends (11). The deep pocket con- between the substrate and the subsites (14, 15). The
formation found in -amylase is also present in glu- active site of soybean -amylase can accommodate
coamylase, although the latter has an =8 -core two maltose molecules, corresponding to a maltote-
domain (12). That the catalytic site is situated in a traose. The two maltose molecules bind in tandem in
cavity allows endwise hydrolysis of the starch chain the active site cleft occupying subsites 1, 2 and 1,
with the length of glucose units cleaved in a con- 2, respectively, with the nonreducing end at the 1
trolled manner. Substrate binding causes a movement subsite and cleavage occurring between subsite 1
of some segments in L3 (residues 96103) to close and 1 (for subsite nomenclature, refer to 16).
over the bound substrate like a hinged lid. In the Substrates with longer chain length may have the
crystal structure of soybean -amylase complexed outer glucose units anchored by hydrophobic interac-
with maltose, L3 in the closed conformation interacts tion with the methyl groups of Leu383 at the cleft
with maltose forming hydrogen bonding between entrance, as revealed in the crystal structure of soy-
Asp101 and O-2 of the glucose unit 1, and Van der bean -amylase complex with -cyclodextrin (6).

All plant -amylases with known crystal structures plant -amylases do not contain a raw starchbinding
have the same molecular architecture, except for the site, and are incapable of digesting raw starch.
sweet potato enzyme which is a tetramer having a sub-
unit MW of 55,800 daltons (598 amino acid residues)
(8, 17). Each subunit consists of a central =8 -barrel,
a small domain formed by three long loopsL3 (resi- IV. REACTION MECHANISMS
dues 91150), L4 (residues 183258), and L5 (residues
300327)and a long C-terminal loop (residues 445 In a single nucleophilic displacement mechanism, -
493). The active site is located at a deep cleft between amylase catalyzes hydrolysis of the glucosidic bond
the =8 -barrel and the small domain -amylase (Fig. with inversion of the anomeric conguration (21, 22).
2) shows an architecture similar to that of the soybean The initial step involves protonation of the glucosidic
enzyme, except that the C-terminal sequence contains oxygen by a general acid which weakens the maltosyl
Gly-rich repeats (9). Deletion of the C-terminal 45 resi- C-O bond. This is followed by a general acid catalysis
dues led to a decrease in the thermostability of the with a water molecule attacking from the -side of the
enzyme (18). A similar extended sequence is found in substrate to release a maltose unit with -conguration.
endosperm-specic rye -amylase (19). In the soybean enzyme, the carboxylic group of Glu186
Bacterial -amylases share similar tertiary structure acts as a general acid, and the carboxylate anion of
with plant -amylases, in spite of the low sequence Glu380 serves as a general base, according to afnity
identity ( 30%) with those of plant -amylases. The labeling studies and x-ray structural analysis (7, 13).
crystal structure of -amylase from Bacillus cereus In an equally viable mechanism, protonation of the
var. mycoides reveals a C-terminal domain with two glucosidic oxygen results in a glucosyl oxocarbonium
antiparallel -sheets, similar to the raw starchbind- ion intermediate with an enzyme-induced distortion of
ing domains of glucoamylase and cyclodextrin glyco- the substrate in a half-chair conformation (2325). A
syltransferase (10, 20). The possession of this unique backside attack by a water molecule assisted by a gen-
C-terminal domain provides bacterial -amylases with eral base produces an inversion of the conguration.
the ability to bind and digest raw starch. In contrast, (See Chapter 56, Sec. IV.)
Figure 2 A stereo view of barley -amylase (isoform 2). The structure consists of a large =8 -barrel core, a smaller lobe
formed by three long loops (L3, L4, and L5), and a C-terminal tail loop residues extending from 8 and covering the entrance of
the barrel (Ref. 9, PDBid: 1bly).

V. INHIBITORS soybean and pea enzymes. In the Triticeae (barley and
wheat), -amylase is synthesized in the endosperm in a
-Cyclodextrin has long been known to be a competi- bound form which is posttranslationally modied by
tive inhibitor of -amylase, and the enzyme-inhibitor proteolytic cleavage to yield a free and more active
binding has been postulated as evidence for the form, during germination (37, 38). Common to all cer-
induced t theory (26, 27). The observed Kd of soybean eals is the ubiquitous -amylase that exhibits a tran-
-amylase for -cyclodextrin is 0.5 mM. The three- sitory presence in various tissues of the developing
dimensional structure of soybean -amylase com- kernels and in other seedling organs (39). All known
plexed with -cyclodextrin indicates that two glucose plant -amylases exist in several isoforms. The barley
units of the -cyclodextrin molecule interact with the enzyme has four isozymes with pI values of 4.65, 4.75,
enzyme at loops L5 and L6 which form the entrance of 4.90, and 5.20 (40). All four isoforms have been shown
the active-site cleft (5, 6). The inhibitor forms an inclu- to be products of proteolysis of the C-terminus of the
sion complex with the methyl groups of Leu383 (28). full-length enzyme (MW 57 kDa), and exhibit optimal
In contrast to the binding of the maltose which is posi- activity between pH 4.5 and 7.5, with a Km of 2.5
tioned deep in the cleft, -cyclodextrin inhibits the cat- mg mL1 (soluble starch), and Vmax of 17 mol mal-
alytic function by physically blocking the entrance to tose min1 (41). Soybean contains several isozymes,
the active site. with isozymes 2 (pI 5.25) and 4 (pI 5.5) the most pre-
-Amylases contain a conserved cysteine, and its dominant. The differences between the two isozymes
modication by various sulfhydryl reagents, such as are due to the substitution of Glu152 and Lys399 of
iodoacetamide, N-ethylmaleimide, p-chloromercuric- isozyme 2 with Lys and Arg in isozyme 4 (42). The
benzoate (PCMB), and 5,5 0 dithiobis-(2-nitrobenzoic corn enzyme consists of two isoforms with pI values
acid) (DTNB), causes a decrease of the enzyme activity of 4.25 and 4.40, with MW of 65 kDa (43). Soybean -
and a slight change in the Michaelis constant (29). amylase has a Km of 4:9 102 mM using reduced
Studies of the interaction of native and SH-modied amylodextrin substrate of average degree of polymer-
-amylases of soybean with -cyclodextrin and mal- ization of 21.7 (29). Using reduced soluble starch as the
tose suggest that the cysteine is located near the bind- substrate, Totsukan and Fukazawa (28) reported a Km
ing site of the two substrates, but is not involved in of 0.17 mM and a kcat of 410 sec1 , with a kcat =Km
catalysis (3032). Selective modications of all three value of 2:4 103 sec1 mM1 . Shorter-chain maltoo-
cysteine residues in Bacillus polymyxa -amylase yield ligosaccharides, such as G7, showed an increasing Km
the same conclusion (33, 34). It is believed that the value of 1:9 101 M, and a kcat of 610 sec1 (44). A
attachment of bulky alkylating agents used in chemical kcat =Km of 1:39 103 sec1 mM1 has also been
modication introduces steric stresses that cause a reported using a similar G7 substrate.
structural distortion that in turn prevents the sub-
strates from productive binding (35). This suggestion
has been conrmed by the crystal structure of soybean
-amylase complexed with -cyclodextrin where Cys95 VII. MEASUREMENT OF ACTIVITY
and Cys343 are located in the L3 and L5 loops near the
maltose and -cyclodextrin binding site, respectively Several methodologies are available for the determina-
(5). tion of -amylase activity. Some are based on determi-
nation of the increase in reducing groups produced by
the hydrolytic action of -amylase on suitable 1,4--D-
VI. PHYSIOCHEMICAL PROPERTIES glucans, such as soluble starch. In the 3,5-dinitrosali-
cyclic acid method, the concentration of the nitroami-
Plant -amylases are 55 kDa, with the exception of nosalicyclic acid formed at the reducing hemiacetal is
sweet potato -amylase, which is a tetramer of 215 measured colorimetrically (46). The reducing groups
kDa. Microbial -amylases have MW of 30160 kDa can also be quantitated by the alkaline ferricyanide
with the high value representing possible association of method (47) or the Nelson-Somogyi method (48, 49).
subunits. the tetrameric structure of the sweet potato The reducing sugar assay is nonspecic, and the mea-
enzyme is not essential for the catalytic function, surement of -amylase activity by these methods is
because the monomer retains full activity (36). The accurate only if the sample is devoid of -amylase
pH optimum for activity ranges from 5.0 for wheat, and other amylolytic enzymes. For more details, refer
barley malt, and sweet potato -amylases, to 6.0 for to Chapter 56, Sec. VI.

A specic assay for -amylase in the presence of strating the -amylase activity (using dened sub-
cereal -amylases has been developed using a colori- strates) from the total amylolytic activity.
metric substrate consisting of p-nitrophenyl oligosac-
charides (50, 51). The substrate used is p-nitrophenyl
-maltopentaoside (PNPG5), which is too short to be VIII. PURIFICATION
effectively hydrolyzed by cereal -amylases but is
readily cleaved by -amylase to maltose and p-nitro- -Amylase has been prepared in a crystalline form
phenyl maltotriose (PNPG3 ). A subsequent reaction from a variety of plant sources including germinated
of the product, PNPG3 , with added -glucosidase barley (40), sweet potato (36), and soybean (52).
produces glucose and p-nitrophenol. The latter pro- Purication of -amylase from various sources
duct, with a molar extinction coefcient of reported in the literature mostly involves multisteps
1:8 103 M1 cm1 , can be measured at 410 nm. In of fractionation by acetone, ethanol, or NH4 2 SO4 ,
a standard assay (51), the enzyme preparation or followed by cation/anion exchange chromatography
extract (0.2 mL) in a buffer containing 100 mM and gel ltration. The following describes a three-
sodium maleate, 1 mM diNaEDTA, 1 mg/mL BSA, step procedure of cereal -amylase purication consist-
and 3 mM NaN3 , pH 6.2, is incubated with 0.2 mL of ing of NH4 2 SO4 fractionation, chromatofocusing,
pre-equilibrated substrate mixture containing 5 mM and hydroxyapatite adsorption chromatography
PNPG5 , 20 units of -glucosidase, at 40 C for 10 (Table 1) (43).
min. The reaction is terminated and color developed Cereal grains were sterilized for 30 min with 1%
by the addition of 1% w/v Tris base (3.0 mL, NaOCl, germinated on water-saturated cotton wool
pH > 10). The absorbance of p-nitrophenol is mea- in the dark at 2030 C for 6 days, and homogenized
sured at 410 nm. One enzyme unit is dened as the in 100 mM Tris buffer, pH 8.1. The extract was cen-
amount of enzyme that catalyzes the release of 1 mol trifuged at 65,000g for 20 min, to yield 200 mL
of p-nitrophenol per min under the assay condition. It supernatant from 40 g of germinated grains. The
should be noted that although PNPG5 is not an effec- majority of the -amylase as precipitated by
tive substrate for cereal -amylases, it has been found NH4 2 SO4 at 4060% saturation was redissolved in
to be readily attacked by some other -amylases, such 25 mM histidine-HCl buffer, pH 6.2, and dialyzed in
as Aspergillus niger -amylase. The procedure the same buffer. The dialyzed sample was applied to a
described above can therefore only be applied for 35-mL Polybuffer Exchanger Gel column, and eluted
the selective measurement of -amylase in cereal with polybuffer PB 74 diluted 10 with water, pH 4.
our and products. For a precise quantitation of - Active fractions were pooled and dialyzed in 1 mM
amylase, it may be necessary to determine the degree sodium phosphate buffer, pH 6.8, and applied onto a
of interference of -amylase in the sample. If neces- 4.5-mL hydroxyapatite column pre-equilibrated with
sary, the -amylase in the sample should be measured the same buffer. Elution was performed using a linear
using dened substrates, such as end-blocked p-nitro- gradient of sodium phosphate (150 mM) with a ow
phenol maltosaccharide, that are not susceptible to rate of 4 mL/h. Active fractions were pooled and the
the action of -amylase and glucoamylase (see purity was analyzed by SDS-PAGE and isoelectric
Chapter 56). The -amylase is then obtained by sub- focusing.
Table 1 Purication of -Amylase from 40-g Germinating Maize Grains
Total protein Total activity Specic activity Yield

Purication steps (mg) (EUa ) (EU mg1 ) (%)
Crude extract 604 397 0.66 100

NH4 2 SO4 precipitate 110 320 2.91 81
Chromatofocusing NDb 192 48
Hydroxyapatite 0.38 105 276 26
Source: Ref. 43.

a
EU one enzyme unit corresponds to the release of 1 mol min1 of p-nitrophenol at pH 6 at an
incubation temperature of 30 C.
b
ND Not determined because of polybuffer interference.

Although not as frequently used as in the purica- apparent roles in catalysis. Biochemistry 33:7779
tion of -amylases, the use of afnity chromatography 7787, 1994.
for -amylases has been reported. Subbaramaiah and 8. CG Cheong, SH Eom, C Chang, DH Shin, HK Song,
Sharma (53) described the afnity binding of plant - K Min, JH Moon, KK Kim, KY Hwang, W Suh.
Crystallization, molecular replacement solution, and
amylases (mustard, barley, and sweet potato) to soluble
renement of tetrameric -amylase from sweet potato.
starch (amylose) or native starch (potato starch grains) Proteins Struct Funct Genet 21:105117, 1995.
and a rapid elution of the enzyme by 1% (w/v) white 9. B Mikami, H-J Yoon, N Yoshigi. The crystal struc-
dextrin. Hoshino et al. (54) used raw rice starch for the ture of the sevenfold mutant of barley -amylase with
adsorption, hence the purication of Bacillus -amy- increased thermostability at 2.5 A resolution. J Mol
lases. This procedure is made possible because micro- Biol 285:12351243, 1999.
bial -amylases contain a raw starchbinding site as 10. T Oyama, M Kusunoki, Y Kishimoto, Y Takasaki, Y
revealed by their sequences and crystal structures. Nitta. Crystal structure of -amylase from Bacillus
Saha et al. (55) puried the Clostridium thermosulfuro- cereus var. mycoides at 2.2 A resolution. J Biochem
genes -amylase taking advantage of the binding of the 125:11201130, 1999.
enzyme onto raw starch granules and its desorption by 11. G Buisson, E Duee, R Haser, F Payen. Three-dimen-
sional structure of porcine pancreatic -amylase at 2.9
soluble starch. Raw starch adsorption/desorption also
A resolution. Role of calcium in structure and activity.
provides a useful technique for separating a mixture of EMBO J 6:39093916, 1987.
- and -amylases of plant origins. A number of 12. A Aleshin, A Golubev, LM Firsov, RB Hozaiko.
investigations employed the competitive inhibitor Crystal structure of glucoamylase from Aspergillus
-cyclodextrin as a possible ligand for the afnity awamori var. X100 to 2.2-A resolution. J Biol Chem
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58
Glucoamylase
Peter J. Reilly
Iowa State University, Ames, Iowa, U.S.A.
I. INTRODUCTION -Glc-1; 4; 1; 6-Glcn H2 O ! Glc Glcn

Glc Glcn -Glc-1; 4; 1; 6; -1; 1; 1; 3;
Glucoamylase [-(1,4)-d-glucan glucohydrolase, EC
3.2.1.3; GA], discovered in 1951 (1) and known 1; 2-Glcn H2 O
industrially as amyloglucosidase, attacks from the
GA also can produce disaccharides of glucosyl residues
nonreducing end of starch-derived substrates to pro-
linked to reducing-end arabinosyl, fructosyl, galacto-
duce glucose (2). Hydrolysis is by multichain attack,
syl, myo-inositol, lyxosyl, mannosyl, ribosyl, or xylosyl
different substrate molecules being more or less
residues, as well as galactosyl-galactose, galactosyl-
equally susceptible to attack after each bond cleavage
glucose, and mannosyl-glucose disaccharides (8).
(3). Although GAs natural substrates are chains of
Although it was thought for many years that these
glucosyl residues linked through -(1,4) glycosidic
so-called reversion reactions were catalyzed by gluco-
bonds, with branch points initiated by -(1,6) glyco-
syltransferase impurities in GA preparations, it is now
sidic bonds, its action is more general, as it catalyzes
quite clear that GA itself is responsible for them (7, 8).
the hydrolysis of -(1,4), ; -(1,1), -(1,6), -(1,3),
and -(1,2) glycosidic bonds between adjacent gluco-
syl residues, in order of decreasing rate (4, 5). At
higher glucose concentrations, GA reforms all the II. GLUCOAMYLASE IN FOODS
glycosidic bonds that it hydrolyzes, and therefore it
condenses glucose to isomaltose (6-O--d-glucopyra- GA is produced by some eubacteria, a few archaea, a
nosyl-d-glucose), isomaltotriose (6-O--d-isomaltosyl- number of yeasts, and many lamentous fungi.
d-glucose), kojibiose (2-O--d-glucopyranosyl-d-glu- Although there are reports of animal and plant GAs,
cose), nigerose (3-O--d-glucopyranosyl-d-glucose), these appear to be fundamentally different enzymes
maltose (4-O--d-glucopyranosyl-d-glucose), ; -tre- with kinetic properties that overlap those of true
halose (-d-glucopyranosyl -d-glucopyranoside), GAs. GA is therefore not a signicant factor in most
panose (4-O--d-isomaltosyl-d-glucose), and isomal- unprocessed foods.
totetraose (6-O--d-isomaltotriosyl-d-glucose) in GA plays a major role in food processing. In Asia,
order of decreasing equilibrium concentration (6, 7). lamentous fungi from Aspergillus and Rhizopus and
GA catalyzes the following reactions starting with lamentous yeast such as Saccharomycopsis buligera
higher concentrations of starch and related sub- that secrete GA are used to saccharify steamed rice and
stances: potato, uncooked ground grains, and amylopectin-rich

rice. In addition, black koji molds from Aspergillus Glcn1 eq
H2 Oeq
species are employed in Japan to make many tradi- Keq
Glceq Glcn eq
tional foods and beverages, such as soy sauce, sake,
and sochu (9). is pseudorst order in product concentration, the
The greatest use of puried GA has been to make water concentration being high and relatively constant
glucose syrups from maltodextrins produced by - during the reaction or even with substantial changes in
amylase from puried starch. Originally Rhizopus dele- initial reactant concentration, while the reactant con-
mar (now R. oryzae) and R. niveus GAs did this in centrations are second order overall, being second
Japan, but since the identical GAs from Aspergillus order in glucose concentration if disaccharides are
awamori and A. niger are more thermostable and being produced, or rst order each in glucose and oli-
have a wider optimal pH range, they are now used gosaccharide concentrations if higher-molecular-
almost universally for starch saccharcation. Because weight products are being formed. The reaction is
glucose not only is used in syrup and crystalline form stopped at peak glucose yield, when in 30% solids
in foods but also serves as a raw material for high- about 9596% of the sugars are glucose. Glucose
fructose syrups, light beer, and pure fructose, as well yield decreases thereafter because of continuing bypro-
as for fuel ethanol, GA is one of the largest-tonnage duct formation. It is possible to increase glucose yield
enzymes in present use. slightly by adding pullulanase or isoamylase, de-
The standard industrial saccharication process branching hydrolases active on -(1,6) glycosidic
using GA starts with dextrin of DE 10-15, with DE bonds between adjacent glucosyl residues, to the reac-
standing for dextrose equivalent, where DE 0 is a dex- tion mixture. These enzymes break the branch points
trin of zero reducing power and therefore theoretically remaining from the -amylase-catalyzed hydrolysis of
composed of innitely long chains, and DE 100 is glu- amylopectin more rapidly than does GA. This allows
cose (dextrose in industrial usage). A DE 10 dextrin the peak glucose yield to be reached earlier, before the
has 10% the reducing power of glucose and therefore byproducts have as much opportunity to be formed as
has an average chain length of 10 glucosyl residues. A when GA alone is present.
lower initial DE risks retrogradation, a pseudo-crystal-
lization of long malto-oligosaccharide chains. On the
III. PROTEIN PROPERTIES
other hand, a high initial DE leads to a lower nal DE
and glucose yield after GA hydrolysis, because the A. General Properties and Isoforms
extended -amylase hydrolysis at pH 66.5 and
90105 C required to produce high-DE starting mate- The most thoroughly studied GA, that from A.
rial allows more of the glucosyl residues at the reducing awamori and A. niger, is composed of three separate
ends of the malto-oligosaccharide molecules to be con- structures (11): catalytic and starch-binding domains
verted to fructosyl residues by alkaline catalysis (10). and a rigid, highly glycosylated linker between them
The GA hydrolysis process is conducted batchwise (Fig. 1). When produced industrially, it has two
in large tanks for 2 days or so at 5560 C, a high common forms, the complete 616-residue mature
enough temperature for microbial contamination to protein (GA I) and a proteolytically cleaved form
be insignicant but not so high that the enzyme is with 512 residues (GA II), missing all but a few
inactivated rapidly. The pH is 4.34.5, the optimal residues of the starch-binding domain but containing
pH for GA activity and stability and, fortuitously, the whole catalytic domain and linker. GA II has little
the pH where glucose and malto-oligosaccharides are activity on raw starch (14) but has kinetics on smaller
most stable. substrates identical to those of GA I (5). A third form
To reduce enzyme inactivation, microbial contami- of 470 residues, also formed by proteolysis (15), was
nation, and the cost of boiling off large amounts of the one whose crystals were used to obtain the rst
water so that the product can be economically shipped, three-dimensional structure of the GA catalytic
all operations in the conversion of starch to sugars are domain (12). Crystals of GA I and GA II have never
carried out at high solids concentrations, characteristi- been obtained.
cally 3040% (w/v). This has the unwanted effect of When expressed by yeast, the A. awamori/A. niger
allowing GA-catalyzed condensation reactions to pro- GA catalytic domain has a molecular weight by
ceed more rapidly and to a greater extent than at low matrix-assisted laser desorption/ionization time-of-
concentrations. This occurs because the equilibrium ight mass spectroscopy of 56.2 kDa, while those of
constant of the condensation reaction GA II and GA I are 68.7 and 81.7 kDa, respectively

Figure 1 Space-lling model of the A. awamori var. X100 GA catalytic domain (12), the linker, and the A. niger GA starch-
binding domain (13). A postulated structure of the A. niger GA linker based on scanning tunneling electro micrograph (11) is
shown.
(16). These values can vary slightly because of different occur. To overcome this handicap, many lamentous
degrees of O-glycosylation produced under different fungi have evolved the ability to produce very large
culture conditions. Isoelectric focusing also indicates amounts of extracellular GA. The placement of the
multiple forms of GA I and GA II (17), presumably active site in a well explains both the exo-acting nat-
also caused by glycosylation differences. GAs from ure of GA and its multichain attack pattern.
other organisms have widely varying molecular A. niger and A. awamori GAs were the rst GAs
weights, depending on their primary structures and sequenced, in 1983 and 1984, respectively (25, 26). Of
degrees of glycosylation, and more specically on the 21 archaeal, eubacterial, yeast, and lamentous
whether they possess linkers and starch-binding fungal GAs whose full catalytic domain primary struc-
domains. tures are known at present, computational analysis
shows that all except those from Schwanniomyces occi-
B. Catalytic Domain dentalis and Schizosaccharomyces pombe are substan-
tially homologous, and have essentially the same
The crystal structure of the catalytic domain of GA secondary and tertiary structures as the A. awamori
from a strain of A. awamori var. X100, a slightly var. X100 GA catalytic domain (18, 27, 28). -
different strain from the A. awamori/A. niger form Helices 2 and 3 are shortened and fused in the GA
used industrially, was the rst GA structure to be from the lamentous fungus Humicola grisea var. ther-
elucidated (12). The domain is an (; 6 barrel, moidea, and the peripheral -helix 11 is missing from
with six interior -helices surrounded by six exterior the GAs from the eubacterium Clostridium var. G0005
-helices (Fig. 2), a structure found elsewhere among and the archaeon Methanococcus jannaschii (18). In
glycohydrolases only in Clostridium thermocellum addition, a number of loops vary in length in different
endo-1,4--glucanases A (22) and D (23) (CelA and GA catalytic domains.
CelD, respectively) and in Thermomonospora endo/ GA is a member of family 15 of glycosyl hydrolases
exo-cellulase E4 (24). There are 13 helices in all, (2932), the families being classied by primary
with helix 11, counting from the N-terminus of the sequence homology. Although nearly all the members
domain, peripheral to the rest. The helices form a bed of this family are GAs, a glucodextranase (EC 3.2.1.70)
supporting a network of loops, in the center of which from Arthrobacter globiformis is also present (32).
is found the active site. This is a well 10 A deep Of the other three glycosidases with ; 6 barrel
and 15 A wide at its mouth, a very primitive struc- structures, C. thermocellum endoglucanases CelA
ture ensuring that the enzyme will have a low turn- and CelD are in families 8 and 9, respectively, and
over number, since the substrate chain must penetrate T. fusca endo/exocellulase E4 is in family 9, so
the well deeply and undergo cleavage, followed by the their primary structures are not closely related to
remaining chain and then the liberated glucose mole- that of GA. However, they may well share a distant
cule leaving the well before the next reaction can ancestor.

strongly decreased (33, 34). In addition to 10 O-gly-
cosylated Ser and Thr residues in the belt, many GAs
have two N-glycosylated residues, Asn171 and Asn395
in A. awamori/A. niger GA numbering (12, 25), with
N-acetylglucosamine residues attached in ve- and
eight-residue chains, respectively (35). Elimination of
the latter chain by mutation of Asn395 greatly reduces
enzyme secretion and thermostability (36). GAs from
Neurospora crassa, Hormiconis resinae P, R. oryzae,
and two forms of S. buligera appear to have another
N-glycosylated chain attached to Asn72 (18).
GAs from most lamentous fungi except R. oryzae
have catalytic domains with three disulde bridges
Cys210213, Cys262270, and Cys222449 (28). The
last serves to anchor the N-terminal end of the belt
to the rest of the catalytic domain. The GAs from C.
rolfsii and N. crassa have an additional Cys2027
bridge (18).
C. O-Glycosylated Linker
In GAs from lamentious fungi except R. oryzae, the

O-glycosylated belt continues into an O-glycosylated
linker between the catalytic domain and a starch-bind-
ing domain, longer in most Aspergillus GAs than in
other fungi (18). In its longest form, 40 residues to
about residue 508 (A. awamori/A. niger GA number-
ing), it extends 95 A, as measured by scanning tun-
neling electron microscopy (11). Deletions of residues
in this linker also decrease A. awamori GA secretion
from yeast (34). O-Glycosylation of both the belt and
Figure 2 Stereoscopic views (18) from the front (a) and side the linker occurs on Ser and Thr residues, and in GAs
(b) of the A. awamori var. X100 GA catalytic domain and of secreted by A. awamori or A. niger this is composed of
the A. niger starch-binding domain (c) visualized by mannosyl residues either singly or in very short chains
Molscript (19). Panels (a) and (b) show d-gluco-dihydroacar- (37). It appears that glycosylation stiffens the linker
bose (20) in the active site (center and upper center, respec- (38, 39) and that longer linkers allow the catalytic
tively). Increasing hydrophobicities of different folding units domain in nature to attack a larger area of a starch
are denoted by increasingly dark shading. Panel (c) shows granule while tethered to its surface by the starch-bind-
maltose in each of the two surface binding sites of the ing domain.
starch-binding domain (13), as found in a highly homologous
cyclodextrin glucosyltransferase (21). (From Ref. 18.)
D. Starch-Binding Domain
Filamentous fungi except R. oryzae have GAs with

GAs from lamentous fungi other than R. oryzae starch-binding domains of 108 residues, extending
have a highly O-glycoslated belt of 30 amino acid to the end of the enzyme at residue 616. O-
residues. It extends from about residue 436 to about Glycosylation extends into the rst few residues of
residue 467 (A. awamori/A. niger GA numbering), this domain, and a disulde bridge is found between
although it is shorter in Corticium rolfsii GA, and it Cys509 and Cys604 in all starch-binding domains
appears to protect one side of the catalytic domain except that from C. rolfsii (18). A three-dimensional
from proteolytic attack (18). When portions of this structure of the A. niger starch-binding domain has
belt are deleted by site-directed mutagenesis, A. awa- been elucidated (13) (Fig. 2); both its sequence and
mori GA thermostability and secretion from yeast are structure, with its eight -strands, are highly homolo-

gous with Bacillus circulans cyclodextrin glucosyltrans- lies each have a single member. They appear somewhat
ferase (21, 40). closely related, even though Rhizopus is a basidiomy-
The A. awamori/A. niger GA starch-binding domain cete and Arxula is an ascomycete. The
has two binding sites for starch and related substances Saccharomycopsis and Saccharomyces subfamilies,
such as maltose (Fig. 2). Its binding characteristics each with two representatives, are fairly homologous
have been most completely determined by Dalmia with each other. Finally, the Clostridium and
and Nikolov (41). Methanococcus subfamilies, the only ones with nonfun-
In R. oryzae GA, a glycosylated linker and starch- gal members, are more closely related to each other
binding domain are found to the N-terminal side of the than to any other subfamily. It does appear that GA
catalytic domain (28). The yeast Arxula adeninivorans has an ancient eubacterial origin, not only because it is
has a GA with an N-terminal region somewhat homo- found among eubacteria, archaea, and fungi, but also
logous to that of R. oryzae, but the enzyme cannot because it possesses a rare tertiary structure, an ; 6
degrade raw starch (42). The GAs from the yeasts S. barrel otherwise found at present among glycosyl
buligera, Saccharomyces cerevisiae, and S. diastaticus, hydrolases only in C. thermocellum and T. fusca,
the archaeon M. jannaschii, and the eubacterium both eubacteria.
Clostridium var. G0005 also continue beyond the N-
termini of GAs from most lamentous fungi (18).
The functions of these regions are unknown except in IV. ENZYME PROPERTIES
the Clostrium GA, where it links the catalytic domain A. Enzyme Mechanism
to the cell wall (43, 44).
As mentioned above, the GA active site is a well,
E. Evolution and Phylogenetic Tree ensuring both multichain attack and exo-action. Two
residues, Glu179 (45) and Glu400 (46), are the catalytic
Seven subfamilies of GAs can be delineated through general acid and general base, respectively, and are
parsimony analysis (Fig. 3) (18). All members of each 10 A apart (Fig. 4). Hydrolysis is by single-displace-
subfamily are closely related to each other by sequence ment mechanism, with a proton being donated to the
homology. The Aspergillus subfamily has the most oxygen atom of the glycosidic bond by the acid, while
members, containing all the Aspergillus GAs along the hydrolytic water is coordinated by the base, which
with single representatives from N. crassa, H. grisea accepts the proton (48) (Fig. 5). Inversion of cong-
var. thermoida, H. resinae P, and C. rolfsii. The sec- uration occurs, a non-reducing-end -glycosidic bond
ondary and tertiary structures of these GAs are very being cleaved to yield -glucose (6). The limiting step
similar, with the linker becoming progressively shorter appears to be the release of the remaining substrate
in the order listed. The Rhizopus and Arxula subfami- chain from the active site (49).
Figure 3 Phylogenetic tree (18) showing the seven subfamilies of GAs from eubacteria (Clostridium), archaea (Methanococcus),
yeast (Saccharomyces, Saccharomycopsis, and Arxula), and lamentous fungi (Rhizopus and Aspergillus). Dark circles: catalytic
domains; medium-light ovals: starch-binding domains; light rods: O-glycosylated regions or linkers; black ovals: other domains;
vertical bar: cell wall. (From Ref. 18.)

Figure 4 Stereoscopic view (47) of the crystal structure of A. awamori var. X100 GA with d-gluco-dihydroacarbose (20) (dark
gray: more abundant conformer at pH 4.0; light gray: less abundant conformer at pH 4.0) and the automated docking structure
of methyl -maltotrioside (black). Also shown is the catalytic water (lled circle). The scissile glycosidic bond is immediately to
the left of the rightmost glucosidic residue. (From Ref. 47.)
All but one of the many GAs that have been sub-
jected to subsite mapping with various substrates
appear to have six or seven subsites, with the rst,
binding the nonreducing glucosyl residue of the sub-
strate, having a small negative or positive binding free
energy, and the ve or six subsites to the other side of
the cleavage point having progressively smaller but
always negative binding free energies (17, 5064).
This causes substrates with more glucosyl residues to
be bound more rmly and cleaved at higher rates than
those with fewer residues. With all substrates and all
but one GA, values of KM decrease essentially mono-
tonically with increasing substrate chain length, while
values of kcat increase until reaching a maximum at
substrates with four or ve glucosyl residues (Figs. 6,
7).
The reason for the uniformity of subsite structure
with different GAs has now become clear, in that all
have essentially the same tertiary structure, with speci-
cally the same active-site structure and general acidic
and basic catalytic residues (18). The one exception to
this uniformity is the Swanniomyces castelli GA, which
has different kinetics (65) and which is not homolo-
gous with the other GAs.
The number of substrate-binding subsites in GA,
obtained by mathematical analysis of the kinetics of
the hydrolysis of oligosaccharides of different lengths,
does not agree with either the crystal structures of GA
Figure 5 Schematic of GA mechanism. with various ligands or with simulated docking studies,

cleaved substrate chain from the active site (49), as
mentioned above.
B. Effect of Environmental Factors
Many studies have shown GAs from A. awamori, A.

niger, and other lamentous fungi to have optimal
pHs for activity and thermostability near 4.5. The opti-
mal pH for activity is halfway between the pKa of the
catalytic acid Glu179, 6.0 in the presence of maltose
and 5.8 when it is absent, and that of the catalytic base
Glu400, 2.4 in the presence of maltose and 2.7 when it
is absent (67). Substrates of higher chain length have
slightly higher optimal pHs (67).
Activation energies for GA-catalyzed hydrolysis of
many di- and trisaccharides range from 60 to 80 kJ/
mol (5). Those for GA thermoinactivation in buffer are
310 kJ/mol (36), so increasing the temperature leads
to much higher rates of enzyme inactivation but only
moderate increases in enzyme activities.
Increasing substrate and product concentrations
strongly protect GA against inactivation, so that at
30% solids the optimal operating temperature of the
A. awamori/A. niger form over 48 h is 60 C. With
buffer rather than substrate or product, serious inacti-
vation occurs within 30 min at 60 C and pH 4.5, since
the rst order thermoinactivation rate coefcient is
104 sec1 under these conditions (36).
Figure 6 Kinetic parameters of different GAs as a function GA has no cofactors, nor is it stabilized by either
of chain length of maltooligosaccharide substrates. a: values cations or anions. It has a number of inhibitors, the
of kcat (sec1 ); b: values of KM (mM). Symbols: &
most potent being the pseudo-tetrasaccharide acarbose
, A. niger G1, pH 4.5, 35 C (17); ^ , A. niger
G2, pH 4.5, 35 C (17); - e -, Clostridium G0005, pH 4.5,
and its various derivatives, that presumably mimic the
25 C (44); , R. delemar (R. oryzae), pH 4.5, 25 C (50); transition-state form of the glucosyl residues about the
L
---- ~ ----, A. awamori, pH 4.5, 37 C (51); , A. glycosyl bond being cleaved.

saitoi, pH 5.0, 25 C (52); ---- ----, R. delemar (R. oryzae),
pH 4.5, 40 C (53); ! , A. awamori wt, pH 4.4, 50 C (54); C. Kinetic Properties
---- & ----, A. niger, pH 4.5, 55 C (56); * , A. niger G2,
pH 4.5, 25 C (57); --- ~ --- , A. niger G1 wt, pH 4.5, 4 An exhaustive listing of the kinetic properties of many
5 C (58); ---- ) ----, Clostridium G0005 wt, pH 4.5, 25 C (59); different GAs with different substrates and inhibitors
---- ^ ----, A. awamori wt, pH 4.5, 45 C (60); --- ---, A. is found in the doctoral dissertation of P. M. Coutinho
awamori wt, pH 4.5, 45 C (61); - * - , A. awamori (68). The sheer bulk of the data set (48 pages of tables)
wt, pH 4.4, 45 C (62).
suggests that noting many specic values here would
be futile. Most data have been gathered at pHs
between 4 and 5 and temperatures between 25 C and
which show that only two subsites are within the 50 C (Figs. 6, 7). Substrates range from maltose
active-site well, with a third subsite being near the through maltodecaose to various types of starch and
enzyme surface and being composed of different glycogen, but also include oligosaccharides with differ-
amino acid residues depending on the pH (20, 4) ent glycosidic bonds. Most data are on GAs from var-
(Fig. 4). Furthermore, standard subsite theory is ious Aspergillus and Rhizopus spp. but there are some
based on the assumption that the limiting reaction is data from virtually every GA that has been sequenced.
the cleavage of the glycosidic bond (66), while instead To concentrate on the kinetics of A. awamori/A.
the limiting reaction appears to be the release of niger GA at 45 C and pH 4.5 (68), average values of

fungi is about 100 mM at pH 4.5 and a range of
temperatures. Data are also rather sparse for GA
kinetics on soluble starch, amylose, and amylopectin,
with kcat at pH 4.5 and extrapolated to 45 C being
60 sec1 , the same value as for maltoheptaose.
Values of KM are too widely scattered for an average
value to be reported, with most values being in weight
percent, since substrate molecular weights were often
not determined.
Among inhibitors, acarbose has a Ki of 1012 M
on A. awamori/A. niger GA at pH 4.5 and 27 C (68),
while equivalent values for d-gluco-dihydroacarbose,
methyl -arcarviosinide, 1-deoxynojirimycin, and d-
glucono-1,5-lactone are 3 108 M (64), 5 108 M
(64), 3 105 M (64), and 1 103 M (68), respec-
tively.
A general observation based on fewer kinetic and
inhibition data is that values for GAs from other
sources are not greatly different from those of A. awa-
mori/A. niger GA, although there are a number of
single values under different conditions, often at
lower temperatures, that vary from the average values
above.
Kinetic data for the GA-catalyzed condensation of
glucose to di- and trisaccharides are available for A.
niger GA at pHs 3.5, 4.5, and 5.5, at 25, 35, and 45 C,
and at 20%, 30%, and 40% (w/v) initial glucose con-
centrations (7). Based on maltose formation as 100, the
Figure 7 Kinetic parameters of different GAs as a function average initial rates of formation of ; -trehalose,
of chain length of isomalto-oligosaccharide substrates kojibiose, nigerose, and isomaltose across the different
(except where noted). a: Values of kcat (sec1 ); b: Values of pHs, temperatures, and initial concentrations are
KM (mM). Symbols: & , A. niger G1, pH 4.5, 35 C 0.027, 0.010, 0.087, and 1.45, respectively, with relative
(17); ^ , A. niger G2, pH 4.5, 35 C (17); ~ rates for isomaltotrioside increasing from 0.05 to 0.085
, H. resinae P, pH 4.3, 25 C (44); ! , H. resinae with increasing initial glucose concentrations because
S, pH 4.3, 25 C (55); *, p-nitrophenyl--d-malto-oligo- of their second-order dependence on solids concentra-
saccharides, A. niger G2, pH 4.5, 25 C (57); , A. tion, and that of panose being too small to measure.
awamori wt, pH 4.5, 45 C (61); * , R. niveus, pH 4.5, Equilibrium constants, dened as above (Sec. I), aver-
25 C (63). H. resinae S, pH 4.3, 25 C (55); *, p-nitro-
age 0.082 for ; -trehalose, 0.20 for kojibiose, 0.18 for
phenyl--d-malto-oligosaccharides, A. niger G2, pH 4.5, 25
nigerose, 0.091 for maltose, 2.2 for isomaltose, 3.3 for
C (57); , A. awamori wt, pH 4.5, 45 C (61);
* , R. niveus, pH 4.5, 25 C (63). panose, and 2.3 for isomaltotriose; little change occurs
with changes of initial glucose concentration, pH, or
temperature. The last three values should be similar
since -1,6 bonds are formed in each case.
kcat and KM for maltoheptaose hydrolysis are 60 sec1
and 0.13 mM, respectively, while for maltose hydroly-
sis they are 10 sec1 and 1.2 mM, respectively. For the V. DETERMINATION OF
major byproduct isomaltose the equivalent values are GLUCOAMYLASE KINETIC
0:37 sec1 and 23 mM, showing the large effect that PROPERTIES AND THERMOSTABILITY
decreasing substrate chain length and a change of gly-
cosidic bond have in decreasing kcat and increasing KM . GA activity is easy to determine, since the enzyme
Fewer data are available for the primary product glu- exclusively produces glucose, which can be detected
cose, but it appears that Ki for GAs from lamentous with minimal interference using glucose oxidase (69,

70). Assays are conducted at pH 4.44.5 in 0.05 M in high amounts, so in those cases no cell breakage
sodium acetate buffer and at 50 C or below, where is necessary unless the small amounts remaining
enzyme inactivation over short periods is minimal. inside the cell are to be studied. GA I and GA II
Substrates are either 4% maltose or 2% soluble starch, can be separated from each other and from an
both concentrations being well above their correspond- acidic -amylase by a linear gradient from pH 8
ing KM values, which with A. awamori/A. niger GA are to pH 3 by column chromatography with DEAE
1.2 mM (0.4%) for maltose, and 0.1% or less for solu- or another weak base on a suitable support (71, 72),
ble starch near pH 4.5 and 2550 C (68). However, the elution order being GA II, -amylase, and GA
both values can vary when GAs from other sources I. Meagher et al. (17) have completely described the
or different conditions are used, so assay substrate purication of a sample containing both GA I and
concentrations should be modied with this in mind GA II by (a) precipitation of extraneous protein
so that measured activities approach their maximal with NH4 2 SO4 at 80% of saturation; (b) desalting
values. Six or more samples are taken at short time by passage through a Sephadex G-25 column; (c)
intervals to make substrate conversions sufciently separation of the two GA forms from each other
low that increase in glucose concentration is linear and from other proteins by DEAE-cellulose chroma-
with time. Samples are added to pH 7 Tris at 1 M tography using a citrate-phosphate buffer eluant
(nal concentration) buffer to stop the hydrolysis reac- whose pH decreases linearly from 7.9 to 3.6; (d)
tion (14), and the quenched sample is treated with glu- concentration by ultraltration; (e) desalting of
cose oxidase, peroxidase, and one of several each fraction with a Sephadex G-25 column; (f)
chromophores, the most common being o-dianisidine, rechromatography for each fraction with DEAE-cel-
found in different commercial kits. lulose; and (g) further desalting and concentration
If values of kcat and KM are to be determined, by ultraltration.
substrate concentrations covering at least the range Protein in a 20-g sample of a commercial prepara-
from 0.2 KM to 5 KM should be used. Since GA follows tion decreased from 1.57 g to 1.19 g after step (a), to
Michaelis-Menten kinetics with all common pure 1.02 g after step (b), to 313 mg and 217 mg in the GA I
substrates, at least six activity points should be plotted and GA II fractions, respectively, after step (c), and to
against their corresponding substrate concentrations 173 mg and 94 mg for the two fractions after step (f).
and a hyperbolic function should be tted through GA specic activities increased from 7.9 U/mg for the
them with a nonlinear regression algorithm. This is starting material to 9.8 U/mg after the second step, to
far superior to use of various linearizing routines 9.8 U/mg and 12.3 U/mg for the GA I and GA II
such as Lineweaver-Burk, Eadie-Hofstee, and Hanes fractions, respectively, after the third step, and to
plots, as the data are not distorted by algebraic manip- 10.8 U/mg and 13.2 U/mg for the two fractions after
ulations. the sixth step. One unit was the GA activity that lib-
GA thermostability in buffer without substrate can erated 1 mol/min glucose from 4% maltose at pH 4.5
be measured by incubating samples at different tem- and 35 C, while protein concentration was determined
peratures or different pHs. At least six samples taken by UV spectroscopy using a previously measured
at different times are treated as above to obtain resi- molar extinction coefcient for GA (14).
dual activities. Since GA loses activity by a rst-order Alternatively, complete purication of each GA
mechanism, the slope of a ln (activity) vs. time curve form can be achieved after dialysis by acarbose-
gives the negative inactivation rate coefcient. Sepharose chromatography eluted with 1.7 M Tris
Obtaining GA thermostability in substrate or glucose at pH 7.6 (73). This is especially efcient if wild-
solutions is much more difcult, since glucose, the pro- type GA containing only GA I is produced from
duct being measured in the residual activity assay, is yeast (54), but may also be used after the third step
initially present. This problem can be ameliorated but above if production is from A. niger or another la-
not eliminated by using assay samples of very low resi- mentous fungus.
dual activity and incubating for long periods. In some cases it is important to separately establish
that seemingly homogeneous GA preparations are free
of glucosyltransferase activity. This may be accom-
VI. PURIFICATION OF GLUCOAMYLASE plished by measuring the conversion of panose in the
presence of 0.1 g/L or more of acarbose, which com-
GA can be brought to homogeneity in several steps. pletely inhibits GA but only slightly inhibits glucosyl-
Many lamentous fungi produce GA extracellularly transferase (17, 74).

ACKNOWLEDGMENT 12. A Aleshin, A Golubev, LM Firsov, RB Honzatko.
Crystal structure of glucoamylase from Aspergillus
The author thanks Pedro Coutinho for his advice awamori var. X100 to 2.2-A resolution. J Biol Chem
during the preparation of this manuscript and for pro- 267:1929119298, 1992.
13. K Sorimachi, AJ Jacks, M-F Le GalCoeffet, G
ducing Figures 14.
Williamson, DB Archer, MP Williamson. Solution
structure of the granular starch binding domain of
glucoamylase from Aspergillus niger by nuclear mag-
netic resonance spectroscopy. J Mol Biol 259:970987,
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59
Limit Dextrinase/Pullulanase
E. Ann MacGregor
University of Manitoba, Winnipeg, Manitoba, Canada
I. INTRODUCTION proteins, particularly since there are some specicity

differences, with plant enzymes in general being less
The enzymes known as pullulanase (EC 3.2.1.41) or active on glycogen than the microbial pullulanases (1).
limit dextrinase (EC 3.2.1.142) catalyze hydrolysis of The minimum structural requirement for a branched
-1,6-D-glucosidic linkages in pullulan, amylopectin, oligosaccharide to be a substrate for pullulanase/limit
and the - and -amylase limit dextrins of amylopectin dextrinase is a maltose residue on either side of the -1,6
and glycogen (Fig. 1), with limited or no action on linkage (Fig. 2); i.e., the enzymes cannot remove a 1,6-
glycogen. They are usually more active on oligosac- linked glucosyl stub from a chain of -1,4-linked glucose
charide dextrins than on polysaccharide substrates residues, or remove a 1,4-linked glucose chain attached
hence the systematic name -dextrin 6-glucanohydro- to carbon 6 of a single glucose unit. The enzymes have
lase. The names limit dextrinase and R-enzyme have high activity on 63 --maltosyl maltotetraose and 63 --
both been applied to enzymes of plant origin; the for- maltotriosyl maltotetraose (2, 3) (Fig. 2).
mer name is preferred since it is related to the natural The limited activity of pullulanase/limit dextrinase
substrate. In plants, the function of the enzyme is to on glycogen distinguishes these enzymes from another
help bring about complete degradation of starch to important starch-debranching enzyme, isoamylase (EC
glucose and maltose by hydrolyzing the -1,6-glucosi- 3.2.1.68), which brings about appreciable hydrolysis of
dic bonds of small branched dextrins resulting from the -1,6-glucosidic bonds in glycogen but has little effect
combined action of - and -amylase on starch. The on pullulan. Furthermore pullulanase, hydrolyzing -
name pullulanase, in contrast, has been used for many 1,6 links in pullulan to yield maltotriose, should not be
years for microbial enzymes important for the break- confused with neopullulanase (EC 3.2.1.135) or isopul-
down of branched polysaccharides such as the amylo- lulanase (EC 3.2.1.57), which hydrolyze the -1,4-glu-
pectin of starch. The EC recommended name is - cosidic bonds in pullulan to give panose or isopanose,
dextrin endo-1-6--glucosidase, but it is little used. respectively (Fig. 3). Enzymes known as amylopullula-
Studies of the primary structures of limit dextrinases nases or -amylase-pullulanases can bring about
and pullulanases have shown appreciable sequence hydrolysis of both -1,4 and -1,6 linkages in glucans,
similarity between the plant and microbial enzymes, and therefore can be distinguished from true pullula-
and it is now common to refer to plant pullulanases nases, which hydrolyze -1,6-glucosidic bonds only. In
where the name limit dextrinase has been used in the some cases, however, an enzyme acting on pullulan is
past. There has been, however, a recommendation that not always tested for activity on -1,4- and -1,6-glu-
limit dextrinase continue to be used for the plant cosidic linkages, and confusion exists in the literature
enzymes to distinguish between these and the microbial where several amylopullulanases are described as pull-

Figure 2 (a) Oligosaccharides resistant to pullulanase/limit
dextrinase action. Symbols are as in Figure 1, with an arrow
representing an -1,6-glucosidic linkage. 1: Isomaltose, 2:
panose, 3: 61 --glucosyl maltose, 4: isopanose, 5: 62 --gluco-
syl maltotriose, 6: 63 --glucosyl maltotriose. (b) Substrates
for pullulanase/limit dextrinase. 7: 62 --maltosyl maltose, 8:
63 --maltosyl maltotriose, 9: 62 --maltosyl maltotriose, 10:
63 --maltosyl maltotetraose, 11: 63 --maltotriosyl maltote-
traose.
importance is the limit dextrinase of barley, which plays

Figure 1 (a) Representation of glycogen or amylopectin a role in the brewing and distilling industries (see Sec.
molecule. *=glucose residue; B reducing glucose resi-
IV). The enzyme is known to occur in mature barley
due; =-1,4-glucosidic bond; & -1,6-glucosidic bond. In
glycogen there is approximately one -1,6-linkage for every kernels, and additional limit dextrinase is synthesized
12 -1,4-linkages; in amylopectin the ratio is 1:25. (b) -Limit when barley seeds germinate. Much of the limit dextri-
dextrin of amylopectin or glycogen. Outer chains are nase activity of germinated barley is also found in
trimmed to one to three glucose residues in length. (c) brewers malt, i.e., barley germinated under carefully
Pullulan. controlled conditions and dried by kilning. During
brewing, the enzyme, in theory, acts in conjunction
with malt - and -amylases to break down the starch
ulanases (e.g., 4). In other cases, enzymes are described from the malt itself and any added starch to glucose and
as type I or type II pullulanases. The type I enzymes maltose, prior to fermentation with yeast to produce
are true pullulanases, specic for - 1,6-glucosidic alcohol. In practice, however, the dextrinase has very
bonds, while type II pullulanases are, in fact, amylo- limited action, since a potent inhibitor of the enzyme
pullulanases (5). (see Secs. VI and VII) also exists in malt (6).
II. IMPORTANCE IN FOOD III. SOURCES OF PULLULANASE/LIMIT

DEXTRINASE
Microbial pullulanases are used, as puried enzymes, in
the production of glucose, fructose, and maltose syrups True pullulanases have been found in, and prepared
(see Sec. IV). The plant enzyme of greatest commercial from, a variety of bacteria. The enzyme is synthesized

(9). The enzymes from Bacillus acidopullulyticus and B.
deramicans (10, 11) are efcient under the optimum
conditions for glucoamylase, but are not heat stable at
temperatures much above 60 C. Enzymes from ther-
mophilic bacteria, with potentially greater heat stabi-
lity, have been investigated, but most have been found
to have high activity only at pH 6.0 or higher. A pull-
ulanase from Fervidobacterium pennavorans is, how-
ever, very active in the pH range 4.55.0 and is stable
at 70 C (12).
Amylopullulanases could be used in place of pull-
ulanases if they are active under the conditions
required for dextrin hydrolysis by glucoamylase;
enzymes from Clostridium thermohydrosulfuricum
and Thermoanaerobium Tok6-B1 appear to have
these properties (13, 14).
Limit dextrinases are known to occur in several
plants, but only the enzyme of barley is of commercial
Figure 3 (a) Hydrolysis of pullulan at 1 by neopullulanase;
at 2 by pullulanase; at 3 by isopullulanase. (b) 1. Panose, importance. The enzyme is found in mature barley
product of neopullulanase action; 2. maltotriose, product kernels, mainly in the endosperm (15), and additional
of pullulanase action; 3. isopanose, product of isopullulanase limit dextrinase is synthesized in the aleurone cells dur-
action. Symbols are as in Figure 1 with an arrow representing ing germination (16). The location of the enzyme
an -1,6-glucosidic bond. within the seeds has been determined by dissection of
the seeds and evaluation of enzyme levels within each
tissue (15, 16).
intracellularly, then is usually secreted to give an extra-
cellular pullulanase that can be puried from the
supernatant of the culture. IV. UTILIZATION OF ENZYMES IN
Bacterial pullulanases that are to be used as puried FOODS
enzymes in the production of glucose syrups must act
in conjunction with a fungal glucoamylase, and so are Before microbial pullulanases can be used in the pre-
used at 60 C and pH 4.55.0, conditions where the paration of glucose and maltose syrups, starch in a
glucoamylase is most active (see Sec. IV). The most slurry must be gelatinized by heat treatment and lique-
widely studied pullulanases, those from Klebsiella ed using a heat-stable -amylase. In the liquefaction
pneumoniae or related bacteria, lose activity at 60 C stage, the amylose and amylopectin are hydrolyzed to
oligosaccharides of average chain length of 812 glu-
cose units, if a glucose syrup is required, or slightly

There is confusion about the nomenclature of bacteria in higher chain length for production of a maltose
which important pullulanases can be found. The rst pull- syrup (17, 18). These oligosaccharides are a mixture
ulanase, studied by Bender and Wallenfels (7), was said of linear dextrins containing -1,4-glucosidic linkages
to have been prepared from Aerobacter aerogenes. This is only, and branched dextrins containing all the -1,6-
now reclassied as Klebsiella pneumoniae (8). The bacterium glucosidic bonds of the amylopectin, since such bonds
Klebsiella aerogenes, frequently cited as a source of pullula- are not hydrolyzed by -amylase. The second stage in
nase, should also, more properly, be called Klebsiella pneu- glucose syrup production is saccharication, where sin-
moniae (8). In 1982, however, a new species of Klebsiella, K. gle glucose residues are removed from the nonreducing
planticola, was distinguished from K. pneumoniae (8). Thus
ends of the dextrins by an exo-acting enzyme, a glu-
some pullulanases, prepared before 1982 and said to have
coamylase. This enzyme, however, acts slowly on -1,6
originated from K. pneumoniae, may actually derive from
K. planticola. Indeed the strain of Aerobacter aerogenes linkages and, when glucose concentrations become
used by Bender and Wallenfels (7) for the original pullula- high, can catalyze a reverse reaction to give isomaltose.
nase preparation is now believed to be K. planticola (Culture Addition of a bacterial pullulanase to hydrolyze -1,6-
No. 15050, American Type Culture Collection; Web site glucosidic bonds allows reaction times to be reduced,
www.atcc.org/). while higher substrate and lower glucoamylase concen-

trations can be used. This lowers isomaltose and glucose, maltose, and linear and small branched
increases glucose yields. Because the glucoamylase dextrins are usually present in wort (aqueous extract
and pullulanase act simultaneously, the two enzymes of malt) before yeast is added, and since the yeast is
must be compatible with respect to optimum pH and able to utilize only glucose, maltose, and maltotriose
temperature. The saccharication reaction is usually for alcohol production, the larger dextrins appear in
carried out at 60 C and pH 4:5 for maximum activ- a traditional beer (26, 27). Lowering the inhibitor
ity of the glucoamylase. Pullulanases active under these level, or raising limit dextrinase activity, would
conditions can be obtained from B. acidopullulyticus allow for greater breakdown of branched dextrins
(10, 17) and B. deramicans (11), and both are consid- and greater fermentability of a wort, essential for
ered safe for food use (17, 19). If required, fructose the production of low-carbohydrate beers, but also
syrup can subsequently be prepared from glucose of economic importance to the distilling industry
syrup by isomerization with glucose isomerase. (6, 28). Alternatively, an extraneous pullulanase or
For maltose syrups, the dextrins obtained by lique- limit dextrinase could be added to increase hydro-
faction of starch are treated simultaneously with - lysis of branched oligosaccharides (29).
amylase and pullulanase. The -amylase produces mal-
tose from the nonreducing ends of -1,4-linked glucose
chains, but can neither hydrolyze nor bypass -1,6- V. PROTEIN PROPERTIES
glucosidic bonds. The pullulanase is therefore essential
to convert branched dextrins in the liqueed starch The molecular weight of pullulanase/limit dextrinase
mixture to linear dextrins to allow the -amylase to varies with the source of the enzyme, and discrepancies
have maximum effect (17, 20). exist in the value quoted for one pullulanase, particu-
Although puried enzymes are usually added to the larly the Klebsiella pullulanases, depending on the
liqueed starch in a batch process, various studies have method used for molecular-weight determination.
been carried out to investigate the feasibility of using Calculations from amino acid sequence for the pull-
immobilized pullulanase, or plant limit dextrinase, or ulanases of B. acidopullulyticus and B. deramicans
pullulanase plus glucoamylase for hydrolysis (2123). suggest the molecular weights should be 100,000 dal-
Cyclodextrins can be used for avor delivery in tons, while experimental determinations gave values of
food, but limited solubility of the cyclodextrins can 116,000 and 102,000, respectively (10, 11). A molecular
pose problems. Means of increasing the water solubi- weight of 105,000 was found experimentally for barley
lity of cyclodextrins have been studied, and for this limit dextrinase (30), while predictions from the
purpose glucosyl or maltosyl cyclodextrins have been sequence would lead to a value 97,000 (31, 32).
prepared, where the glucose or maltose is attached by Polysaccharide-hydrolyzing enzymes have been clas-
an -1,6 linkage to a glucose residue of the cyclodex- sied into families, based on amino acid sequence simi-
trin ring. Here the reversibility of the hydrolysis reac- larities (33). It is now thought that the enzymes of one
tion catalyzed by pullulanase has been employed, family share a common tertiary structure. The primary
where the enzyme synthesizes rather than ruptures - structures of several pullulanases/limit dextrinases are
1,6 bonds (24, 25). Such products may, in the future, now known, including those of B. acidopullulyticus, B.
acquire some commercial importance. deramicans, and barley (11, 31, 32, 34), and the
In the process of brewing beer, barley malt starch is enzymes are believed to belong to family 13 of the car-
gelatinized in warm water, and precooked adjunct bohydrases, which includes many other starch-degrad-
starch, often from corn, may be added. The starches ing enzymes such as -amylase and isoamylase (35).
are broken down by the hydrolytic enzymes of the While the three-dimensional structure of a pullula-
malt, principally - and -amylase, to glucose and nase/limit dextrinase molecule has not been determined,
maltose plus smaller quantities of longer -1,4-linked it is expected to be similar to those of other family 13
linear maltodextrins and branched dextrins containing enzymes for which x-ray crystallographic studies have
the -1,6 bonds of the amylopectin of the original been carried out (see, e.g., 36). The family 13 enzymes
starches, since neither - nor -amylase attacks these are, in general, multidomain proteins, and in pullula-
latter bonds. In principle, the limit dextrinase of the nase/limit dextrinase, as in isoamylase, an N-terminal
malt is able to hydrolyze the -1,6-glucosidic linkages domain precedes the catalytic domain. The catalytic
of the branched dextrins, but the presence of a domain itself is likely to take the form of a =8 -barrel,
proteinaceous inhibitor (see Secs. VI and VII) in the i.e., a barrel of eight parallel -strands, usually with at
malt limits the efcacy of the enzyme (6). Thus least one helix located in the primary sequence between

> 140,000 daltons. These enzymes, however, also
belonging to family 13, are predicted to have the char-
acteristic =8 -barrel catalytic domain, and contain
the four conserved sequence segments typical of the
family (Fig. 5) (35, 37, 38).
Phylogenetic trees have been constructed for family
13 enzymes in general and starch-debranching enzymes
in particular (31, 35, 38). These show that the plant
limit dextrinases are closely related to one another,
with the bacterial pullulanases being nearest neighbors,
and isoamylases next closest in a tree of the whole
family. Amylopullulanases, in contrast, are much
more distantly related, and are closer to neopullula-
nases than pullulanases.
In neither bacteria nor plants is there evidence for
more than a single gene for pullulanase/limit dextri-
nase in each organism. Multiple forms of an enzyme
Figure 4 Secondary structure of catalytic domain of a
family 13 enzyme. B1B8 represent parallel -strands; H1
can exist, however, but these have not been well char-
H8 represent helices of the =8 -barrel. Arrows joining a - acterized. Klebsiella pullulanases are believed to be
strand to the following helix represent loops in the enzyme modied by attachment of lipid at the N-terminal
structure on which are situated amino acid residues of the end (40), while the differences between two apparent
active site. forms of B. acidopullulyticus pullulanase have not been
explained (10). Several forms of barley limit dextrinase,
separated by electrophoresis and detected using anti-
each pair of adjacent -strands (Fig. 4) (37). In general, bodies (15), possibly arise because of the presence of
the active site of family 13 enzymes is made up of amino free limit dextrinase and limit dextrinase bound to a
acid residues situated on loops linking the C-terminal proteinaceous inhibitor (41). In another study, three
end of a -strand to the N-terminal end of the adjacent forms of puried barley limit dextrinase have been
helix (36) (Fig. 4). Amino acids on -strands 3, 4, 5, and found, but the differences among them have not been
7, or on loops immediately following these strands, are elucidated (42), although they are believed to be pro-
found at the active site in all family 13 enzymes. The ducts of a single gene (31, 32).
presence and location of active-site acids on other loops
vary from one enzyme to another. An obvious charac-
teristic of this family is a long loop between the third -
strand of the barrel and the third helix, amino acid resi- VI. ENZYME PROPERTIES
dues on this loop making up much of one side of the
active site. In the known three-dimensional structures of While pullulanase/limit dextrinase can degrade pullu-
family 13 enzymes, at least one domain containing a - lan, the importance of these enzymes for food and
sheet structure follows the catalytic domain, and is also beverages lies in their ability to hydrolyze the -1,6-
likely to be found in pullulanases/limit dextrinases. glucosidic bonds of amylopectin and branched dextrins
There is limited sequence homology among family resulting from amylopectin breakdown. It is difcult to
13 enzymes, but all have four well-conserved short measure quantitatively the increase in reducing power
sequence segments around -strands 3, 4, 5, and 7 of produced by limited pullulanase action on amylopec-
the barrel that contain amino acid residues thought to tin. It is also difcult to obtain a standard sample of
be critically important for the catalytic action of the amylopectin, or a pure, small, branched dextrin to be
enzymes (Fig. 5) (38). used as substrate. Thus, absolute values of pullulanase
The amylopullulanases tend to have larger mole- activity are rarely determined, and it is difcult to com-
cules than pullulanases, with polypeptide chains of pare results from different laboratories, since different
up to 1500 amino acids (39), while true pullulanases samples of pullulan or amylopectin may have been
usually have 7001100 amino acid residues. Hence the used. It is generally agreed, however, that pullula-
molecular weights of amylopullulanases are expected nase/limit dextrinase is more active on small branched
to be higher than those of pullulanases, in general dextrins than on pullulan or amylopectin (Table 1).

Figure 5 Four short sequence segments conserved in all members of family 13 carbohydrases. Taka: -amylase from Aspergillus
oryzae, Genbank sequence No. D00434; Kleb: pullulanase from Klebsiella pneumoniae, Genbank sequence No. M16187; Bacid:
pullulanase from Bacillus acidopullulyticus, sequence from (34); Bderam: pullulanase from B. deramicans, sequence from (11);
BLD: barley limit dextrinase, sequence from (31); Amypull: amylopullulanase from Clostridium thermohydrosulfuricum, sequence
from (39); Isoamy: isoamylase from Pseudomonas amyloderamosa, Genbank sequence No. A10909. Amino acid residues are
numbered from the N-terminal end of the protein. Indicates residues conserved in all enzymes shown. The three residues
involved in catalysis are shown in bold lettering; D in sequence segment II, E in segment III, and D in segment IV.
Values of Km for pullulan for Klebsiella pullulanase active between pH 5.5 and 7, and is stable in the
are given as 2.5 mM (10), 78 M, or 21 M (43) in pH range 512 (9); the B. acidopullulyticus and B.
glucose equivalents, and 70 M (44) in terms of con- deramicans enzymes have maximum activity at pH
centration of hydrolyzable -1,6-glucosidic bonds. For 46 and pH 3.754.9, respectively, and are stable at
the B. acidopullulyticus enzyme, Km for pullulan is said pH 49 (10) and 3.56 (11), respectively. The limit
to be 2.5 mM glucose equivalents (10), while barley dextrinase of barley, on the other hand, has optimum
limit dextrinase has a Km for amylopectin of 26 mM activity at pH 5.56.5 in the presence of bovine serum
glucose equivalents (3). albumin, which seems to stabilize the active enzyme
The turnover number (kcat ) for Klebsiella pullula- (30).
nase has been measured as 99 sec1 or 220 sec1 at 30 Temperature optima for Klebsiella and B. acidopul-
C (43), 53 sec1 at 25 C, 132 sec1 at 40 C (44), and lulyticus pullulanases are 50 C and 65 C, respectively,
290 sec1 at 60 C, while measured values for the B. and both enzymes are stable at temperatures up to 55
acidopullulyticus enzyme are 2:9 103 or 3:6 103 C at pH 5 (9, 10). The enzyme from B. deramicans is
sec1 at 60 C, all on pullulan (10). For barley limit said to retain 75% of its activity when held at 60 C for
dextrinase acting on a well-dened dextrin, 62 -0-- 24 h at pH 4.5, and has optimum activity at 60 C (11).
maltotriosyl maltotriose, at 40 C, the number is The barley limit dextrinase shows maximum activity at
90 sec1 (42). 4050 C (45, 46) and is stable under the mashing con-
The response to pH and temperature varies with ditions in a brewery for at least 1 h at 60 C, but loses
the enzyme source. Klebsiella pullulanase is most activity rapidly at 65 C (47).
Table 1 Relative Activity of Pullulanase/Limit Dextrinase on Different Substrates
Source of enzyme K. pneumoniae (2) B. acidopullulyticus (10) Barley (3)
Activitya on 63 --maltotriosyl 146 250

maltotetraose
Activitya on 15 1516 13
amylopectin
a
Activity on pullulan taken as 100.

Activation energies, Ea , can be calculated from pub- trinase, since germination for malt production is
lished data on the variation of activity with tempera- stopped before inhibitor disappears (6).
ture, and are shown in Table 2.
Several enzymes of family 13 are known to require
Ca2 for activity, but although a Klebsiella pullulanase VII. DETERMINATION OF ACTIVITY
was found to be activated by Ca2 (48), little effect was
found for barley limit dextrinase (42). EDTA gave Well-dened substrates for pullulanase/limit dextri-
some inhibition of both Klebsiella and barley enzymes nase, particularly small branched dextrins, are not
(42, 48). Cyclodextrins are known to be strong inhibi- readily available, and so activity is usually determined
tors of pullulanase/limit dextrinase, with -cyclodex- on pullulan or a commercially available modied pull-
trin being the most effective. Ki for this dextrin is ulan.
around 1 M at 25 C for a Klebsiella pullulanase There seems to be no generally accepted standard
(44) and 40 M at 40 C for barley limit dextrinase (42). procedure for measurement of activity on pullulan.
No investigations of subsite structure of pullula- Temperature and pH conditions are usually chosen
nase/limit dextrinase have been carried out because to suit the particular enzyme under study. Pullulan
of the difculties of obtaining well-dened substrates. concentration is usually in the range 2.55 mg/mL in
In common with other family 13 enzymes, however, buffer, often acetate of concentration 20100 mM and
these enzymes would be expected to have several sub- a pH in the range 5.05.6 (10, 44, 50). Enzyme is added
sites for glucose binding at the active site. In general, to the pullulan and after a set time a sample is with-
family 13 enzymes are believed to operate by a drawn and may be added to 0.1 M NaOH to stop the
mechanism involving retention of anomeric congura- reaction, and reducing power in the sample is deter-
tion. This has been demonstrated experimentally for a mined by the Nelson-Somogyi method (51).
bacterial pullulanase (49) and barley limit dextrinase Calculation of activity depends on the assumption
(42). Two acid groups at the active site, one to act as that pullulanase/limit dextrinase converts pullulan
proton donor and the other as catalytic nucleophile, mainly to maltotriose (Fig. 3) and that measurement
are expected to be found in family 13 enzymes. In fact, of reducing power gives an indication of the amount of
three acid groups (Fig. 5) are found to be essential for maltotriose present. If the pullulanase is not pure,
enzymic activity; the conserved glutamic acid is however, and contains a glucosidase, then reducing
thought to be the proton donor, while the conserved power determination may seriously overestimate mal-
aspartic acid in sequence segment II acts as the nucleo- totriose concentration and activity of the pullulanase.
phile. The role of the second aspartic acid residue in In plants, -amylase may be present in impure sam-
sequence segment IV is not clear. ples of limit dextrinase, and in high enough concentra-
In barley, germinated barley, and malt, a proteinac- tion, -amylase also can hydrolyze maltotriose.
eous inhibitor of barley limit dextrinase has been Methods of determining limit dextrinase activity in
found. The inhibitor levels decrease at later stages of plant extracts have been developed based on the use
germination (6), and it therefore appears likely that the of modied pullulans (50). In one method, a soluble
uninhibited limit dextrinase can then continue the red-dyed pullulan (Red Pullulan, Megazyme, Bray,
degradation of barley starch begun by the - and - Ireland) is dissolved in 0.5 M KCl solution to a con-
amylases of the grain. The resulting small linear mal- centration of 20 mg/mL. Enzyme solution in acetate
todextrins can be further hydrolyzed by -glucosidases buffer (0.2 M, pH 5.0) containing 20 mM cysteine and
to provide glucose for the growth of a new barley 0.02% sodium azide is added to substrate solution and
plant. Under the conditions used for brewing beer, incubated at 40 C for a set time. High-molecular-
this inhibitor severely restricts the action of limit dex- weight substrate is then precipitated by addition of
Table 2 Activation Energies
Enzyme source K. pneumoniae (9) B. acidopullulyticus (10) B. deramicans (11) Barley (46)
Activation energy (kJ) 33 45 23 37

Calculated over temperature 3050 4055 5560 3050
range ( C)

ethanol (95% v/v), the mixture is left at room tempera- cult, unless polyacrylamide gel electrophoresis is used
ture for 10 min, and centrifuged at 1000g for 10 min. (53). Red Pullulan (Megazyme, Bray, Ireland) can be
Small fragments of dyed pullulan, hydrolyzed by the incorporated into a gel, and after electrophoresis in e.g.,
enzyme from the original high-molecular-weight pull- Tris-HCl buffer (pH 7.4), the gel should be immersed in
ulan, remain in the supernatant giving a colored solu- acetate buffer (0.2 M, pH 5.5) at 40 C to allow the
tion. Absorbance of this solution is measured at 510 enzyme to hydrolyze the pullulan and leave a yellow
nm against distilled water. A blank without enzyme band on the red background. Amylopullulanase may
must also be prepared. Absorbance can then be related also give a yellow band, but enzymes such as -amylase,
to pullulanase activity in terms of moles of glucose -amylase, and -glucosidase should have no effect on
reducing sugar equivalents released per min, using a the red pullulan substrate.
standard curve. The standard curve is prepared by A major problem in determining the activity of limit
measuring reducing sugar released from pullulan by dextrinase in a plant extract is the possible presence of
puried enzyme in acetate buffer (0.2 M, pH 5.0) at strong inhibitors. This has been demonstrated to be the
40 C over a set time period. The Nelson-Somogyi (51) case for germinated barley, for example, and it has
method is used for reducing sugar determination. been found that prolonged incubation (16 h) at 40 C
In a second method involving a modied pullulan in the presence of 25 mM dithiothreitol (30) or treat-
(50), enzyme solution in sodium maleate buffer (0.1 ment overnight at 4 C with 5 mM ascorbic acid (42) is
M, pH 5.5) containing 25 mM dithiothreitol and necessary to disrupt completely the inhibitorlimit dex-
0.02% sodium azide is incubated at 40 C for 5 min. A trinase complex. If the complex is not destroyed, then
tablet of insoluble blue-dyed pullulan (Limit Dextri- any measure of limit dextrinase activity gives an under-
Zyme tablet, Megazyme, Bray, Ireland) is added and estimate of the amount of enzyme actually present.
the mixture is incubated for a set time. The reaction is
terminated by stirring with Tris base solution (1% w/v),
and the mixture is left at room temperature for 5 min, VIII. PURIFICATION
then stirred and ltered. In this case, the enzyme hydro-
lyzes some of the insoluble substrate to give soluble Details of the method used for purication of a pull-
blue-dyed fragments that remain in the ltrate, and ulanase/limit dextrinase depend on the source of the
measurement of absorbance of the ltrate at 590 nm enzyme and the response of the enzyme to pH and
gives a measure of pullulanase activity. Again, a suitable temperature.
blank must be prepared by adding Tris base solution to B. acidopullulyticus pullulanase, for example, can be
enzyme solution before addition of substrate. Limit dex- puried from Promozyme (Novozymes North
trinase activity in terms of absorbance at 590 nm must America, Inc., Franklinton, NC, U.S.A.), a commer-
be converted to glucose reducing power equivalents cially available source of the enzyme (10). After dialy-
using a standard graph. Preparation of the standard sis of Promozyme overnight against acetate buffer (50
graph differs from the method given for the red pullulan mM, pH 5.0), the pullulanase can be precipitated by
graph only in that buffer at pH 5.5 must be used. addition of cold acetone (20 C) to a nal concentra-
Once standard graphs have been prepared, both tion of 50% (v/v). The precipitate is collected by cen-
methods using modied pullulan have the advantage trifugation at 10,000g for 30 min and dried in vacuo.
that the assays are not affected by other enzymes such The powder is dissolved in acetate buffer (20 mM, pH
as -amylase, -amylase, or -glucosidase that could 5.0) and applied to a CM-Toyopearl 650 S column
be present in impure preparations of plant limit dex- equilibrated with the same buffer. The enzyme is eluted
trinase. (Both modied pullulans are available com- with the acetate buffer containing a linear gradient of
mercially, and detailed instructions for their use are 00.5 M NaCl. Active fractions are pooled and con-
provided by the supplier.) It has been found, however, centrated, then applied to a Toyopearl HW-55S col-
that the presence of maltodextrins in a limit dextrinase umn equilibrated with acetate buffer (20 mM, pH 6.0)
assay system (such as would occur in a malt extract) containing 0.8 M NaCl. Enzyme is eluted from the
interferes with activity determinations using the dyed column using this buffer, and active fractions are
pullulans as substrate (52). In such cases, pretreatment pooled and concentrated, then dialyzed against phos-
of extracts with amyloglucosidase gives a more accu- phate buffer (20 mM, pH 7.0). Solid ammonium sul-
rate estimate of limit dextrinase activity. fate is added to 25% saturation, the precipitate is
Screening for pullulanase/limit dextrinase activity in removed by centrifugation at 10,000g for 20 min, and
the presence of other starch-degrading enzymes is dif- the supernatant is applied to a Butyl Toyopearl 650 S

column equilibrated with phosphate buffer (20 mM, nity column [Sepharose CL6B covalently linked to -
pH 7.0) containing ammonium sulfate at 25% satura- cyclodextrin (54)] is prepared and equilibrated with
tion. The column is eluted with the same buffer using a acetate buffer (0.02 M, pH 4.0) containing 1 mM
linear gradient of 25% to 0% saturation of ammonium monothioglycerol. The dialyzed enzyme solution is
sulfate. Two active fractions are obtained from the adjusted to pH 4.0 with acetic acid, centrifuged at
column, but the properties of the two fractions are 10,000g for 10 min, and loaded onto the afnity col-
very similar, and apparently there is no gain in purity umn. The column is washed with acetate buffer (0.02
by use of the Butyl Toyopearl column (10). Yield of M, pH 5.5) containing 1 mM monothioglycerol, and
active enzyme decreases appreciably, however, when enzyme is eluted with the buffer using a linear gradient
the latter column is used (10). of 00.5 mg/mL -cyclodextrin. Fractions are collected
Barley limit dextrinase is most easily prepared from into tubes containing acetate buffer (0.02 M, pH 5.5)
green malt, and two slightly different methods have containing 1 mM monothioglycerol to minimize time
been published (30, 42). In the procedure of spent by the enzyme at pH 4.0. Active fractions are
MacGregor et al. (30), ground green malt is extracted pooled, concentrated, and dialyzed overnight at 4 C
with acetate buffer (0.1 M, pH 5.5) containing 25 mM against the 0.02 M acetate buffer. The enzyme solution
dithiothreitol at 40 C for 16 h. The slurry is centri- is then further concentrated and applied to a Sephacryl
fuged at 9000g for 15 min and the supernatant col- S-200 gel permeation column equilibrated with the
lected. The pellet is re-extracted for 1 h at 40 C with same acetate buffer. The column is eluted with the
buffer and recentrifuged, and the supernatants are buffer, and active enzyme fractions are pooled and
combined. Limit dextrinase is precipitated overnight concentrated. Typical recovery of protein and activity
at 4 C by addition of ammonium sulfate to 80% during the purication are given in Table 3.
saturation. The precipitate is obtained by centrifuga- In a second method (42), green malt our is
tion at 9000g for 15 min and dissolved in phosphate- extracted with acetate buffer (0.2 M, pH 5.0) contain-
citrate buffer (0.02 M, pH 6.0) containing 1 mM ing 5 mM ascorbic acid for 24 h at room tempera-
monothioglycerol. The solution is dialyzed at 4 C ture and allowed to settle overnight at 4 C. The
against the same buffer to remove ammonium sulfate supernatant is concentrated and ammonium sulfate
and centrifuged at 8000g for 15 min, and the super- is added to 20% saturation. The precipitate is dis-
natant is ltered through glass wool. The dialyzed carded after centrifugation, and ammonium sulfate
extract is then loaded onto a diethylaminoethyl cellu- is added to the supernatant to 70% saturation. The
lose ion exchange column (Whatman DE52) equili- precipitate is collected and dissolved in acetate buffer
brated with the phosphate-citrate buffer containing 1 (50 mM, pH 5.0) containing 0.5 M CaCl2 . The solu-
mM monothioglycerol. The column is washed with this tion is desalted by dialtration and applied to a
buffer until the absorbance of the eluate at 280 nm DEAE-Fractogel 650S column equilibrated in acetate
returns to baseline. The column is then eluted with buffer (20 mM, pH 5.0). The column is washed with
the same buffer using a linear gradient of 00.5 M this buffer and eluted using a linear gradient of 01
NaCl. Active fractions are pooled, concentrated, and M NaCl. Active fractions are pooled and ammonium
dialyzed against acetate buffer (0.02 M, pH 5.5) con- sulfate is added to 1.52 M. The solution is applied to
taining 1 mM monothioglycerol. A -cyclodextrin af- a -cyclodextrin Sepharose column equilibrated in
Table 3 Purication of Limit Dextrinase
Total activity Total protein Specic activity Purication

Stage of purication (mU)a (mg) (mU/mg) (fold)
Extract 412,000 50,200 8.21 1

Ammonium sulfate precipitate 230,000 7,740 29.7 3.6
After ion exchange chromatography 161,000 144 1,120 136
After afnity chromatography 116,000 13.1 8,860 1080
After gel permeation chromatography 70,400 8.32 8,460 1030
a
Activity was measured using Limit DextriZyme tablets and converted to glucose reducing power equivalents using a standard graph supplied
with the substrate (see Sec. VII).

acetate buffer (50 mM, pH 5.0) containing 2 M 10. S. Kusano, N Nagahata, S Takahashi, D Fujimoto, Y
ammonium sulfate. The column is washed with this Sakano. Purication and properties of Bacillus acido-
buffer and then with acetate buffer without the pullulyticus pullulanase. Agric Biol Chem 52:2293
ammonium sulfate until the absorbance of the eluate 2298, 1988.
11. P Deweer, A Amory. Pullulanase producing microor-
at 280 nm is < 0:05. Limit dextrinase is then eluted
ganisms. U.S. patent 5817498 (1998).
with the acetate buffer containing 7 mM -cyclodex-
12. R Koch, F Canganella, H Hippe, KD Jahnke,
trin. Active enzyme fractions are pooled and applied G Antranikian. Purication and properties of a
to a DEAE-Fractogel 650S column in acetate buffer thermostable pullulanase from a newly isolated ther-
(20 mM, pH 5.5). The column is washed with the 20 mophilic anaerobic bacterium, Fervidobacterium
mM acetate buffer, and enzyme is eluted using this pennavorans Ven 5. Appl Environ Microbiol
buffer with a linear gradient of 00.5 M NaCl. Active 63:10881094, 1997.
fractions are pooled and may be stored at 18 C. 13. H Melasniemi. Characterization of -amylase and
In each preparation, the initial presence of dithio- pullulanase activities of Clostridium thermohydrosul-
threitol or ascorbic acid is necessary to disrupt limit furicum. Biochem J 246:193197, 1987.
dextrinaseinhibitor complexes and release free, active 14. AR Plant, RM Clemens, RM Daniel, HW Morgan.
Purication and preliminary characterization of an
limit dextrinase.
extracellular pullulanase from Thermoanaerobium
Tok6-B1. Appl Microbiol Biotechnol 26:427433,
1987.
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Pseudomonas isoamylase on various branched oligo- 17. BF Jensen, BE Norman. Bacillus acidopullulyticus
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4. Y Suzuki, K Hatagaki, H Oda. A hyperthermostable 18. WD Crabb, C. Mitchinson. Enzymes involved in the
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Appl Microbiol Biotechnol 34:707714, 1991. ulanase enzyme preparation derived from Bacillus
5. A Spreinat, G Antranikian. Purication and proper- licheniformis containing the pullulanase gene from
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Biotechnol 33:511518, 1990. lar structure of dextrins in enzymatic high maltose
6. AW MacGregor, LJ Macri, SL Bazin, GW Sadler. syrups. Starch 42:437444, 1990.
Limit dextrinase inhibitor in barley and malt and its 21. AC Chakrabarti, KB Storey. Co-immobilization of
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7. H Bender, K Wallenfels. Pullulan. II. Specic decom- 22. L Furegon, ADB Peruffo, A Curioni. Immobilization
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26. BS Enevoldsen. Degradation of starch by amylases in J Bacteriol 166:10831088, 1986.
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39. H Melasniemi, M Paloheimo, L Hemio. Nucleotide
sequence of the -amylase-pullulanase gene from

60
Limit Dextrinase
James Hutchison Bryce

Heriot-Watt University, Edinburgh, Scotland
I. INTRODUCTION lopectin, and -1,6-glucosidic bonds in branched dex-

trins derived from amylopectin (2). The structures of
My aim is to provide an outline of the reactions of pullulan and amylopectin are shown in Figure 1.
limit dextrinase and consider how our present knowl-
edge about limit dextrinase will affect its use during the
processing of starch in the food industry. My focus will II. IMPORTANCE TO QUALITY AND
be upon the effect of limit dextrinase in the production EFFICIENCY OF ALCOHOLIC
of alcoholic beverages from malted barley. However, BEVERAGE PRODUCTION
my comments are relevant to anybody involved in pro-
cessing starch from any cereal to produce alcoholic Limit dextrinase is necessary for complete starch
beverages or syrups. Furthermore, an understanding breakdown because starch consists not only of amy-
of the properties and regulation of limit dextrinase is lose, linear chains of glucose, but also of amylopectin,
essential to any project where the aim is to produce a which contains 45% of -1,6-glucosidic linkages
genetically modied organism with altered limit dextri- (3). The precise proportion of amylose to amylopectin
nase activity. varies with the source of the starch, but generally is in
A plant with genetically modied limit dextrinase the range of 2030% for normal cereal starches (4).
activity may have altered starch degradation and/or In processing cereals to produce alcoholic bev-
modied starch synthesis. Such genetically modied erages, a wort is normally produced by mixing the
plants would be an important source of material for ground cereal with hot water ( 65 C) for at least 1
studying starch metabolism. Also, such plants have the h. The temperature is selected to achieve gelatinization
potential to be the breeding material for new varieties of starch granules while maintaining, at least briey,
of crops which could produce products with improved some activity of the hydrolytic enzymes that will
yields or processing properties. degrade carbohydrate and protein (5). In a wort pro-
Limit dextrinase (-1,6-glucanohydrolase, EC duced entirely from malted barley, a typical carbohy-
3.2.1.142) is a debranching enzyme which selectively drate prole following starch degradation is 75%
catalyzes the hydrolysis of -1,6-glucosidic linkages glucose, maltose, and maltotriose, and 25% dextrins,
and has no action on -1,4-glucosidic linkages (1). about half of which have multiple branches (6, 7).
Limit dextrinase is the name commonly used for the Limit dextrinase activity, or indeed lack of activity,
debranching enzyme derived from plants, whereas the is of fundamental importance in the production of
bacterial enzyme is known as pullulanase (EC wort from cereals in the brewing and distilling indus-
3.2.1.41). Both these enzymes degrade pullulan, amy- tries. Dextrins with -1,6-glucosidic bonds are not fer-

Figure 1 Representations of the structures of amylopectin and pullulan. Circular symbols denote glucose residues. A line ()
denotes an -1,4-glucosidic bond, and line with an arrow (!) denotes an -1,6-glucosidic bond. All glucose residues are linked
via their hemiacetyl hydroxyl groups at carbon 1 except for the residues shown with a diagonal line through them (\) (the
reducing end of each molecule). The residues shown in black contain hydroxyl groups at carbon 4 which are not linked to other
residues.
mented by yeast, except in the rare cases of yeast cular weight of 103105 kDa with a pH optimum of
strains that secrete enzymes capable of hydrolyzing 5.05.5 (5.56.5 in the presence of bovine serum albu-
-1,6-glucosidic bonds. Normally, therefore, if min [BSA]). The pI values reported in the literature
branched dextrins are present in brewers wort, they vary from 4.2 to 4.6.
will be found in the nal beer. In beer, dextrins con- The gene-encoding limit dextrinase has been iso-
tribute to caloric value and may also impart fullness lated (11). In an independent study, the cDNA isolated
or body (8). In contrast, during fermentation of distil- by Burton et al. (11) has been used to show that there is
lers wort, dextrin degradation may continue because only one limit dextrinase gene in barley and that the
the wort is not boiled and therefore malt enzymes that gene is located on the long arm of chromosome 4H,
survive mashing temperatures can remain active. where it has now been mapped [C-D Li, X-Q Zhang,
However, dextrins remaining after fermentation will RCM Lance, LC MacLeod, GB Fincher, P Langridge,
contribute nothing to the new make distilled spirit. unpublished data; cited by Burton et al. (11)].
Therefore, the requirements for limit dextrinase activ- Molecular techniques have been used to establish a
ity differ between brewers and distillers. A brewer may clear distinction between strictly -1,6-specic de-
desire only limited activity of limit dextrinase to ensure branching enzymes, such as limit dextrinase, and
that adequate fermentable sugars are produced during dual-bond type enzymes that attack -1,4-glucosidic
mashing (but sufcient branched dextrins remain), and -1,6-glucosidic bonds (12).
whereas a distiller would wish for complete hydrolysis When amino acid sequences of debranching
of -1,6-glucosidic bonds so that the greatest possible enzymes were examined for phylogenic relatedness
amount of fermentable sugar is available to yeast (9). using the PileUp program (13), two distinct groups
were distinguished (11). Debranching enzymes that
attacked pullulan were grouped together, whereas the
III. PROPERTIES OF LIMIT DEXTRINASE isoamylase type fell into a second group.
PROTEIN Partial amino acid sequence analysis provides evi-
dence that barley limit dextrinase belongs to the -
The properties of limit dextrinase have been thor- amylase family of multidomain amylolytic enzymes
oughly reviewed by Stenholm (10). In summary, the with a characteristic =8 -barrel catalytic domain
enzyme from malted barley is reported to have a mole- (12, 14, 15).

IV. SUBSTRATES, SPECIFICITY, AND side of the -1,6-glucosidic bond if it is to hydrolyze
ROLE OF DEBRANCHING ENZYME IN the -1,6-bond.
BARLEY The question arises as to why enzymes formerly clas-
sied as EC 3.2.1.41 should have had two names, limit
The specicity and nomenclature of debranching dextrinase (EC 3.2.1.142) (from plants) and pullulanase
enzymes have been claried by Manners (16). (EC 3.2.1.41) (from microbes). Manners (16) suggested
Enzymes formerly classied as EC 3.2.1.41 included that the two different names were appropriate because
limit dextrinase (from plants) which does not act on pullulanase is capable of attacking both amylopectin
glycogen, and pullulanase of microbial origin and glycogen whereas limit dextrinase has very limited
which attacks both amylopectin and glycogen. activity, if any, with glycogen. Furthermore, the
Isoamylase enzymes (EC 3.2.1.68) hydrolyze inter- nomen- clature maintained the subgroups of enzymes
chain linkages in glycogen, amylopectin, and certain that attack pullulan in different ways, namely pullula-
derived dextrins, but have no action on pullulan. nase that produces maltotriose, and isopullulanase and
Barley limit dextrinase has 50% and 85% neopullulanase that act on -1,4-linkages to produce
sequence identity to bacterial pullulanases and rice isopanose (22) and panose (23), respectively. However,
starch debranching enzyme, respectively (17). This a distinction has now been drawn between these
sequence similarity between barley limit dextrinase enzymes with their allocation of separate EC numbers.
and bacterial pullulanases and the similarity of their Although the debranching enzyme from germinating
action (1821) added weight to the appropriateness barley grain falls into the pullulanase group of de-
of the former classication of barley limit dextrinase branching enzymes; it should be emphasized that the
with pullulanases (EC 3.2.1.41). natural substrates of the enzyme are amylopectin or
It was believed in the early 1970s that there were oligomeric limit dextrins rather than pullulan. In vivo,
two plant debranching enzymes, limit dextrinase and the debranched oligosaccharides are susceptible to
R-enzyme. R-enzyme has been found to act on amy- further hydrolysis by amylases and -glucosidases (24).
lopectin and its -limit dextrin, and to act on -dex- In addition to a role in hydrolysis of starch degra-
trins from both amylopectin and glycogen, but it did dation products during germination, limit dextrinase
not act on undegraded glycogen. Thus, the substrates has been implicated in starch synthesis where it is pro-
of limit dextrinase and R-enzyme are identical. posed that an appropriate balance between branching
Indeed, it is now well established that limit dextrinase and debranching enzymes is required to achieve the
and R-enzyme are the same enzymes, and the name nal degree of branching in amylopectin (2530).
limit dextrinase is preferred since this name relates to Furthermore, a debranching enzyme would also be
its natural substrate (16). In the context of starch capable of producing primers for the starch synthase
breakdown to fermentable sugars, it is important to reaction (31), a function that could explain the pre-
note that the smallest substrate for limit dextrinase is sence of limit dextrinase in developing rice grain (32).
62 --maltosyl-maltose (Fig. 2) (20). Limit dextrinase Burton et al. (11) have obtained mRNA encoding
requires at least one -1,4-glucosidic bond on either barley limit dextrinase from overlapping cDNA clones
from PCR amplication. The cDNA sequence has an
open reading frame that encodes a putative transit
peptide of 78 amino acid residues and a mature poly-
peptide of 884 amino acid residues. The calculated
molecular weight of the mature polypeptide is 97.4
kDa and the calculated pI is 5.0 (assuming all the
acidic and basic groups are on the surface in the ter-
tiary structure). These are values comparable to the
molecular weight of 105 kDa and pI values of 4.2
4.6 reported for puried limit dextrinase (17, 33, 34).
The discrepancy between the deduced and measured
values of molecular weight could be due to glycosyla-
tion of the enzyme; seven potential N-glycosylation
sites are present in the deduced amino acid sequence.
Figure 2 Structure of 62 --maltosyl-maltose. (Symbols are A number of features relating to the function of the
as for Figure 1.) gene stand out. First, limit dextrinase mRNA is abun-

dant in gibberellic acid (GA)-treated aleurone layers V. MEASUREMENT OF LIMIT
and in germinated grain. Furthermore, abscisic acid DEXTRINASE ACTIVITY
(ABA) abolishes the GA induction of limit dextrinase
A. Assay
mRNA in isolated aleurone cells. This would be
expected since ABA often acts as an antagonist to
The extraction and measurement of limit dextrinase
GA in plant tissues. These observations would tend
have advanced recently with analytical procedures
to conrm that limit dextrinase should participate in
employing pullulan reacted with dye molecules (36,
starch degradation during endosperm mobilization in
37). The sensitivity of these methods is better than
germinating grains. However, if this is so, then it would
that of methods based on measurement of reducing
be expected that limit dextrinase would carry a signal
sugars (10). Two commercial products, Red-Pullulan
peptide to target the nascent polypeptide to the endo-
(soluble Reactive Red 120-labeled pullulan, Megazyme
plasmic reticulum for eventual secretion from the
Ltd) and Limit DextriZyme tablets (insoluble cross-
aleurone to the endosperm. No such signal peptide
linked azurine-pullulan, Megazyme Ltd), are now
has been identied (11). Therefore, since there is no
widely used for assay of limit dextrinase activity in
evidence that aleurone cells become leaky to metabo-
barley and malt extracts. The Limit-DextriZyme
lites as they senesce, the mechanism of limit dextrinase
tablets have the advantage of greater sensitivity (10-
release into the endosperm, if it does occur, remains to
to 15-fold), lower blank values, greater ease of use,
be established.
and greater stability than Red-Pullulan (37).
The question of the potential role of limit dextrinase
There are other potential methods for the measure-
in starch synthesis also arises. Low levels of limit dex-
ment of limit dextrinase. Sissons et al. (38) developed a
trinase mRNA were detected in the developing endo-
rapid enzyme-linked immunosorbant assay (ELISA)
sperm of barley until 20 days post anthesis (DPA),
for measuring limit dextrinase. The assay was sup-
after which time the levels rapidly declined. Limit
posed to be specic and sensitive and to recognize
dextrinase activity was detected in the developing
the active enzyme, but values do not correlate with
grain. This expression pattern corresponds closely to
the Red Pullulan assay (10).
the timing of starch synthesis in developing barley
Earlier assays were based on the measurement of
grains, although the precise timing in any give grain
maltotriose released from pullulan by limit dextrinase
is dependent to some extent on growth temperatures
(24, 39). The drawback of this procedure was the pro-
(35).
duction of additional reducing groups if the sample to
Starch synthesis occurs in an organelle known as an
be analyzed contained -glucosidase. Thus, for crude
amyloplast and limit dextrinase has a presequence typi-
extracts containing endogenous -glucosidase, Lee and
cal of transit peptides that target nascent polypeptides
Pyler (40) developed a two-step assay method in which
to amyloplasts (11). In the developing endosperm of
the limit dextrinase reaction products from pullulan
barley, limit dextrinase is thus targeted to the organelle
were hydrolyzed to glucose by added yeast -glucosi-
in cells whose primary role is to synthesize starch and is
dase, and the glucose was then assayed. Problems with
expressed at a time coinciding with starch synthesis.
this approach included (a) the fact that -glucosidase
This suggests a role for limit dextrinase in starch synth-
does not hydrolyze the higher oligosaccharides, which
esis in the developing endosperm.
are initial hydrolysis products of pullulan (37), and (b)
While the above molecular evidence points to a
the requirement to remove endogenous reducing
role for limit dextrinase in starch synthesis, the
sugars by dialysis (41).
biochemical evidence is much less convincing. In the
model of amylopectin biosynthesis involving deb-
ranching enzyme activity (28), it is proposed that B. Extraction
there is intensive branching generating phyto-
glycogenlike structures. These structures are then From the foregoing discussion, it should be evident
trimmed by debranching enzymes. This requires very that while the assay of limit dextrinase can be difcult,
precise regulation of the activities of the enzymes the use of dyed pullulan has been a signicant advance.
involved. The lack of evidence for such precise regu- However, just as important as assaying the enzyme
lation of these enzymes, plus the lack of limit dextri- correctly is the correct extraction of the enzyme. It is
nase activity with phytoglycogen (16), means that a thought that in vivo in germinating barley, the enzyme
role of limit dextrinase in starch synthesis is as yet exists in three formsbound and inactive, soluble and
unresolved. active (known as free), and soluble and inactive (42,

43). Furthermore, limit dextrinase inhibitors have been thermostability properties during kilning that are inter-
detected in barley grain extracts (44). While barley mediate between -amylase (heat stable) and -gluca-
grains at maturity have a high level of limit dextrinase nase (relatively heat labile and inactivated by kilning)
inhibitor, the level is lower in wheat, and the inhibitor (37). However, whereas in the past it was thought that
is absent in rice and maize (45). All these factors com- limit dextrinase was denatured during mashing, there is
plicate the interpretation of limit dextrinase activity now good evidence that limit dextrinase survives mash-
measurements. However, it is currently believed that ing temperatures of 6365 C.
the total activity of limit dextrinase in germinating
barley grain can be determined by extraction for 5 h
3. Measurement in Crude Extracts
with a reducing agent, whereas extraction for 5 h with-
out a reducing agent provides a measure of the free The measurement of limit dextrinase is most simply
activity (soluble and active). done by determining the total activity of the enzyme
(extraction with a reducing agent) and activity of the
free enzyme (extraction without a reducing agent).
C. Properties
In the recommended procedures, limit dextrinase is
1. Inhibitors and Activators extracted from ground grain for 5 h at 40 C in 0.1
0.2 M NaOAc (pH 5.05.5). This extracts the free
The effect of inhibitors and activators of limit dextri-
enzyme (soluble-active) (37), whereas total activity is
nase is reviewed by Stenholm (10). One potentially
obtained after similar extraction with a reducing agent,
important feature to emerge is the inhibition of rice
e.g., 25 mM dithiothreitol (DTT). Soluble-inactive
limit dextrinase by maltose, maltotriose, and a number
enzyme is the total activity minus the free activity.
of small maltosaccharides. This suggests that rice limit
Excessive extraction of limit dextrinase in the presence
dextrinase contains binding sites for at least two, and
of DTT can start to lead to a decline in the apparent
probably more, adjacent -1,4-linked glucose residues
total activity of limit dextrinase, a decline that is prob-
(19). If barley limit dextrinase were inhibited by these
ably due to the activation of nonspecic proteases that
low-molecular-weight oligosaccharides during mashing
start to degrade limit dextrinase.
in the brewing process, then it could reduce the con-
Sjoholm et al. (48) measured limit dextrinase activ-
version of nonfermentable branched dextrins to fer-
ity during mashing. In a 1 h mash, limit dextrinase
mentable sugars.
activity remained stable at 60 C, declined by 55% at
Cyclodextrins inhibit limit dextrinase, with -cyclo-
62:5 C, and declined by 65% at 65 C. Limit dextrinase
dextrin being very signicantly more inhibitory than -
was only fully inactivated in 1 h by temperatures
cyclodextrin or -cyclodextrin (21, 34, 46, 47). Puried
> 65 C. Similar evidence for the stability of limit dex-
limit dextrinase (200 mU/mL) was inhibited 50% by
trinase at mashing temperatures was obtained by
75 g/mL -cyclodextrin (34). All three cyclodextrins
MacGregor et al. (45) and Bryce et al. (49).
are competitive inhibitors of limit dextrinase.
Furthermore, when MacGregor et al. (45) collected
The properties of limit dextrinase differ signicantly
samples mashed at 63 C and incubated the sample
from those of -amylase. There is no effect of Ca2 (1
supernatants at 40 C with 25 mM DTT (dithiolthrei-
100 mM) on activity and EDTA (10 mM) causes only a
tol) the measured activity of limit dextrinase increased
15% decrease in activity. Also, the pseudo-tetrasach-
dramatically. This shows that a large portion of the
haride acarbose, a potent inhibitor of -1,4-specic
limit dextrinase in the malt was solubilized during
amylolytic enzymes such as -amylase and glucoamy-
mashing but had remained in the wort as inactive
lase, reduces the activity of limit dextrinase by only
enzyme.
30% at a high concentration of 10 mM (34).
In some of the experiments just described (45, 48,
49), samples were taken directly from wort for mea-
2. Temperature Stability
surement of limit dextrinase activity using dyed pull-
In the past, there have been major differences in the ulan as substrate. The aim of this was to provide a
reported heat stability of limit dextrinase (10). If limit measure of limit dextrinase activity in the wort rather
dextrinase is to be active in mashing, then it must sur- than its potential activity. MacGregor et al. (45) were
vive kilning of the grain where temperatures will nor- able to obtain much higher activities when the sample
mally exceed 60 C and may even exceed 80 Cfor was extracted with DTT prior to assay. This makes an
example, if an ale malt is being produced. At present, important point; if limit dextrinase is to be assayed, the
it is generally agreed that barley limit dextrinase has researcher must decide whether to determine the activ-

ity present or the potential free or total activity. trinase activity is regulated. However, present knowl-
Potential free activity would be that obtained by edge should make it possible to measure limit
further extraction in the absence of a reducing agent. dextrinase activity and how it changes either during
Furthermore, the presence of limit dextrinase inhi- the processing of food, for example, cereal grains or
bitor in certain cereals means that in any process to following the production of genetically modied plants
which limit dextrinase or pullulanase is being added, it with altered limit dextrinase expression.
is necessary to determine whether or not the enzyme
will be inhibited upon addition to the process. Bryce et
al. (49) produced a lactic malt with a free limit dex- 6. Extraction and Purication of Limit
trinase activity of 800 mU/g. A recombination experi- Dextrinase
ment was carried out in which an extract from this
malt with high free activity was mixed with an equal The key to extraction of limit dextrinase from germi-
proportion of a more typical malt where a high per- nating barley is to do it in the presence of a reducing
centage of limit dextrinase is in the soluble but inactive agent such as DTT; also, if a good yield of enzyme is
form. The result was an inhibition of the free limit desirable, it should be done after at least 810 days of
dextrinase from the lactic malt > 50%. This shows germination. There is evidence that sulfhydryl pro-
that the typical malt contains inhibitor capable of inhi- teases, activated by a reducing agent, are necessary
biting free limit dextrinase. It also suggests that suppo- during extraction to activate limit dextrinase solubi-
sedly free activity in malt could be activity of the lized from germinating barley grains (50), and such
soluble and inactive enzyme. However, pullulanase proteases are also necessary to solubilize bound limit
from Bacillus acidopullulyticus (Promozyme 200 L, dextrinase associated with the insoluble fraction of ger-
Novo Nordisk, Denmark) was not inhibited by malt minating barley grains (43).
extracts (49). Therefore, it appears that pullulanase is The extraction process should also aim to minimize
not inhibited by limit dextrinase inhibitor. the effect of heat-stable endogenous inhibitor(s) of
McCafferty and Bryce (unpublished) have found 15 kDa (44) that are reported to form soluble but
activities of limit dextrinase from malted barley sub- inactive complexes with limit dextrinase (51). Two fac-
stantially higher than those of free limit dextrinase tors could reduce the effect of these inhibitors. First,
by rapid extraction (a few minutes) and assay at pH activity of sulfhydryl proteases could upset the
4.4. Therefore, any analysis of limit dextrinase activ- enzymeinhibitor complex, and second, an extended
ities of crude extracts should be accompanied by care- period of germination could lead to breakdown of
ful checking of the optimum pH and time for the inhibitor. MacGregor et al. (45) showed a very
extraction and assay. low level of inhibitor to be present after 6 days of
germination. The problem of the inhibitor will only
4. Measurement of Puried Enzyme Activity occur in extracts from cereal grains of barley, triticale,
durum, wheat, and oats, since the inhibitor has been
A further factor that should be taken into account in
detected in these grains. The inhibitor has not been
assaying limit dextrinase is the presence of protein. BSA
detected in rice, sorghum, millet, or maize.
(0.05 mg/mL) increased the activity of puried limit
Homogeneous barley limit dextrinase has been iso-
dextrinase threefold (34). This stabilization or activa-
lated on a large scale with a yield of 9 mg/kg from 10-
tion effect was not specic to BSA; proteins from crude
day germinated green (unkilned) malt (17). This repre-
barley extracts had the same effect of boosting the
sented a 9400-fold purication with a 29% recovery of
activity of puried limit dextrinase (provided endogen-
activity compared to that of a our extracted in 0.2 M
ous inhibitor was rst removed from the extracts). To
NaOAc (pH 5.0) containing 5 mM ascorbic acid. The
avoid any apparent loss of activity of limit dextrinase
purication protocol consisted of precipitation of limit
following purication 0.5 mg/mL BSA was added rou-
dextrinase from an extract at 2070% saturated
tinely to assay buffers to overcome any effects that
ammonium sulfate (AMS), followed by diethylami-
could arise owing to variable protein levels (34).
noethyl (DEAE) 650S Fractogel anion exchange chro-
matography, and afnity chromatography on -
5. Measurement in Food and Food
cyclodextrin-Sepharose in the presence of 2 M AMS.
ProcessingSummary
Limit dextrinase was eluted by 7 mM -cyclodextrin
From the above discussion, it should be apparent that and contained a single polypeptide chain of 105 kDa
there is an incomplete understanding of how limit dex- (SDS-PAGE) and pI of 4.3.

MacGregor et al. (34) puried limit dextrinase by 3. DJ Manners. Starch degradation during malting and
extraction of grist in 0.1 M NaOAc, pH 5.5 at 40 C for mashing. Brew Dig 49:5662, 1974.
16 h with 25 mM DTT, followed by ammonium sulfate 4. DJ Manners. Some aspects on the structure of starch.
precipitation, and chromatography on ion exchange Cereal Foods World 30:461467, 1985.
5. MJ Lewis, TW Young. Brewing. New York:
(DEAE, pH 6.0), afnity (-cyclodextrin Sepharose
Chapman and Hall, 1995.
with pH 4.0 for loading and pH 5.5 for elution), and
6. BS Enevoldsen, F Schmidt. Dextrins in brewing. II.
gel ltration (Sephacryl S-200, pH 5.5) columns. The Distribution of oligo- and megalosaccharides during
enzyme had high specic activity, was homogeneous, mashing, in wort and beer. Proceedings of 14th
and gave single bands of protein when examined by Congress, European Brewery Convention, Salzburg.
SDS-PAGE ( 105 kDa) and IEF (pI 4.6). Amsterdam: Elsevier Scientic, 1974, pp 135148.
Binding of limit dextrinase to the -cyclodextrin 7. BS Enevoldsen, F Schmidt. Dextrins in brewing.
column was improved in two ways. First, the afnity Studies on singly-branched and multiply-branched
column was equilibrated to pH 4.0, and the pH of the dextrins in brewing. J Inst Brew 80:520533, 1974.
DEAE puried pool was adjusted to pH 4.0 just before 8. CW Bamforth. Beer. Tap into the Art and Science of
loading to the column. Seventy percent of the limit Brewing. London: Plenum Press, 1998.
9. GN Bathgate. Cereals in Scotch whisky production.
dextrinase applied to the column was recovered in
In: GH Palmer, ed. Cereal Science and Technology.
the postafnity pool. This degree of binding was simi-
Aberdeen: Aberdeen University Press, 1989, pp 243
lar to that reported by Maeda et al. (52) but higher 278.
than that reported by researchers who used a higher 10. K Stenholm. Malt Limit Dextrinase and Its
pH (5.5 or 6.0) for -cyclodextrin columns (33, 43, 53). Importance in Brewing. Espoo, Julkaisija-Utgivare,
Limit dextrinase activity is lost quickly at pH 4.0; Finland: VTT Publication 323, Technical Research
therefore it was necessary to bring the pH of the eluted Centre of Finland, 1997.
enzyme back to pH 5.5 as quickly as possible by col- 11. R Burton, X-Q Zhang, M Hrmova, GB Fincher. A
lecting column efuent into tubes containing a small single limit dextrinase gene is expressed both in the
volume of strong pH 5.5 buffer. developing endosperm and in germinated grains of
Binding of limit dextrins to the afnity column was barley. Plant Physiol 119:859-871, 1999.
12. HM Jespersen, EA MacGregor, B Henrissat, MR
also improved by rst removing from the preparation,
Sierks, B Svensson. Starch- and glycogen-debranching
by DEAE chromatography, other -cyclodextrin-bind- and branching enzymes. Prediction and structural fea-
ing enzymes, such as -amylase. -Amylase binds to - tures of the catalytic (=8 -barrel domain and evolu-
cyclodextrin columns much more tightly than limit tionary relationships to other amylolytic enzymes. J
dextrinase (53), and would be eluted after limit dextri- Prot Chem 12:791805, 1993.
nase in the -cyclodextrin gradient. 13. J Devereux, P Haeberli, O Smithies. A comprehensive
Limit dextrinase was eluted from the column by a set of sequence analysis programs for the VAX.
low concentration of -cyclodextrin. This step Nucleic Acids Res 12:387395, 1984.
achieved a large increase in the specic activity of 14. HM Jespersen, EA MacGregor, MR Sierks, B
the enzyme, conrming the effectiveness of an appro- Svensson. Comparison of the domain level organiza-
priate afnity chromatography method of protein tion of starch hydrolases and related enzymes.
Biochem J 280:5155, 1991.
purication. Gel permeation chromatography on
15. B Svensson. Protein engineering in the -amylase
Sephacryl S-200 was used as the nal step in the
family: catalytic mechanism, substrate specicity,
purication of the enzyme to remove -cyclodextrin, and stability. Plant Mol Biol 25:141157, 1994.
as well as a small amount of a low-molecular-weight 16. DJ Manners. Observations on the specicity and
contaminant. nomenclature of starch debranching enzymes. J Appl
Glycosci 44:8385, 1997.
17. M Kristensen, V Planchot, J-I Abe, B Svensson.
Large-scale purication and characterization of barley
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26. MG James, DS Robertson, AM Meyers. 40. WJ Lee, RE Pyler. Improved assay procedure for limit
Characterization of the sugary-1, a determinant of dextrinase in malt extracts. Brew Dig 57:2426, 1982.
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117:425435, 1998. possible role in malting and brewing. Proceedings of
31. CM Duffus, MP Cochrane. Development, structure 25th Congress, European Brewery Convention,
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61
Methodologies for Assaying the Hydrolysis of Cellulose by

Cellulases
David Johnston
U.S. Department of Agriculture, Wyndmoor, Pennsylvania, U.S.A.
I. INTRODUCTION classes, based on their positions of hydrolysis and the

products they produce.
This chapter does not intend to be a complete descrip- The rst class comprises the endoglucanases, which
tion of all cellulase assays available. The methods are enzymes that hydrolyze the internal bonds of the
described here are generally regarded as the most use- cellulose chain (endo-1,4[1,3;1,4]--D-glucan 4-gluca-
ful or commonly employed assays. Many of the assays nohydrolases, EC 3.2.1.4). The second class is the
are useful from an empirical standpoint, but they can- exocellobiohydrolases (1,4--D-glucan cellobiohydro-
not be used to compare results among laboratories. An lases, EC 3.2.1.91). This class of enzymes releases pre-
attempt has been made to include methods applicable dominately cellobiose from cellulose molecules.
to a variety of research interests. Previously this group consisted of enzymes that
The analysis of cellulase activity presents a number released cellobiose from the nonreducing end; how-
of unique problems that are not typically observed in ever, the class is now being subdivided into two sepa-
the study of other enzymes. Cellulose, the native sub- rate groups of enzymes that release cellobiose from
strate for cellulases, is insoluble and varies signicantly either the reducing or the non-reducing end of the
in its structure. This complexity makes the kinetic ana- cellulose molecule. The third class is the exoglucohy-
lyses difcult since concentration and form of substrate drolases (1,4--D-glucan glucobiohydrolase, EC
are variable. Additionally, the presence of trace 3.2.1.74). This group is composed of enzymes that
amounts of other cellulases may enhance hydrolysis release predominantly glucose from the nonreducing
in a synergistic fashion, altering experimental results. end of the cellulose molecule. There are some
Enzymes such as -glucosidase or cellobiose phosphor- researchers who dispute the existence of these
ylase, which are not strictly cellulases, can also enhance enzymes, with the contention that they are actually
cellulose hydrolysis by the removal of products that cellobiohydrolases contaminated with -glucosidase
cause inhibition. or cellobiohydrolases that have some ability to hydro-
The unifying characteristic of all cellulases is their lyze cellobiose. The fourth class is the -glucosidases
ability to hydrolyze the -1,4-glucosidic linkage in cel- (-glucoside glucohydrolase, EC 3.2.1.21). This group
lulose. Cellulases are classied into four separate hydrolyzes cellobiose, and to a much lesser extent
cellotriose, to produce glucose. Technically these
Mention of brand or rm name does not constitute an endor- enzymes may not be considered cellulases (since
sement by the U.S. Department of Agriculture above others they do not hydrolyze cellulose); they are included
of a similar nature not mentioned. with the cellulases because they hydrolyze a -1,4-

glucosidic linkage and their action is important for weighed into a vacuum ask and degased with stirring
complete hydrolysis of cellulose to glucose. for 1 h.
The classication above is currently under revision
as increased information about cellulase specicity, B. Phosphoric Acid Swollen Cellulose (4)
hydrolytic activity, products formation, hydrolytic
stereochemistry, and structural similarities is gathered. Phosphoric acid swollen cellulose (PSC) is commonly
The current trend in the literature is to classify cellu- used as an example of an amorphous cellulose sub-
lases based on the comparison of their deduced amino strate in cellulase hydrolysis experiments. This proce-
acid sequences. In 1993 the comparison of 120 primary dure is a slight modication of Stallberg et al. (3)
sequences of cellulases and xylanases dened a total of producing a more uniform substrate than by other
11 families (designated AK) (1). methods, without requiring blending or ltering (3, 4).
Cellulases have a variety of commercial applications A suspension of cellulose is prepared by addition of
and many potential applications. In the food industry, 3.00 g Sigmacell 20 to 5 mL deionized water. The cel-
cellulases are used in the processing of some fruits to lulose suspension is then added slowly to 300 mL of
aid in peeling or to increase juice yield. In the textile cold 85% (w/v) phosphoric acid with stirring in an ice
industry, cellulases are used to treat dyed fabrics to bath. The container of the cellulose suspension is
give a look similar to stone washing and to alter the rinsed with an additional 5 mL water to remove all
properties of some regenerated bers. One of the more of the cellulose. The cellulose solution is stirred on
exciting potential uses of cellulases is in the conversion ice for 2 h. No undissolved cellulose should be visible
of cellulosic waste material to glucose for fermentation to the naked eye. The solution is then slowly added to 4
into fuel ethanol. While cellulases are currently used in L cold water with constant stirring on a magnetic stir
the detergent and textile industries, their application plate. The precipitated cellulose is allowed to settle and
for waste conversion is still not considered economic- the supernatant is removed by vacuum aspiration. An
ally viable because of the large quantities of enzymes additional 4 L water is added with mixing, the suspen-
necessary for the complete conversion of cellulosic sion is allowed to settle, and the supernatant is
material to glucose. removed by vacuum aspiration. This procedure is
repeated a total of 10 times. After the nal settling of
the suspension, the cellulose is transferred to 1-L grad-
uated cylinder. The supernatant is removed, and 500
II. SUBSTRATE PREPARATION
mL of 0.10 M acetate buffer, pH 5.0, is added and the
A. Carboxymethyl Cellulose (CMC) and volume adjusted to 1 L. The nal substrate concentra-
Hydroxyethyl Cellulose (HEC) (2) tion is determined using the Dubois method (5) for
total carbohydrate.
Many different procedures are cited for preparation of
substrates for viscosity determinations; however, most C. Dyed Avicel (6)
do not yield a substrate with a stable viscosity. The
procedure described here is a slight modication Dyed Avicel is a convenient substrate for many appli-
from Garcia et al. (2) and does yield a solution with cations. It is particularly useful for assays where redu-
stable viscosity for several weeks. CMC or HEC can be cing group measurements give excessively high blank
used interchangeably without modication. Higher- values (e.g., inhibition by glucose or cellobiose).
molecular-weight substrates permit greater sensitivity A suspension of 100 g of Avicel is prepared in 1 L
in viscosity methods. CMC is the preferred choice water. The suspension is heated to 50 C with vigorous
when using capillary viscometry since HEC tends to stirring. One liter of 1% (w/v) Remazol Brilliant Blue
form bubbles. dye is prepared in water and added to the Avicel sus-
The substrate solution is prepared by slow addition pension. Vigorous stirring is continued for 45 min,
of CMC 7H3SF from Aqualon to water heated to 80 followed by addition of 200 g Na2 SO4 in several por-
C and stirred for 1 h to completely dissolve the sub- tions. Following the Na2 SO4 addition, 100 mL of a
strate. After completing the addition, the solution is 100% solution of Na3 PO4 is added (pH should now
ltered through a sintered glass funnel (coarse) into a be 12) and the mixing continued for an additional 75
vacuum ltering ask and then allowed to cool to min. The suspension is now ltered through a sintered
room temperature. Equal weights of the substrate glass funnel (porosity 1) and washed with 60 C dis-
and a 0.1 M sodium acetate buffer (pH 5.0) are tilled water until the ltrate is colorless. While still in

the funnel, the dyed Avicel is washed with acetone and group methods described here for measurement of cel-
then ether. The prepared substrate is then vacuum lulase activity, the disodium 2,2 0 -bicinchoninate (BCA)
dried. method gives the greatest sensitivity and little or no
difference in response among different DPs.
III. METHOD TYPES

1. BCA Method
A. Viscosity (2)
Reagent A. The reagent is prepared by dissolving
1. Capillary 54.28 g/L (512 mM) of Na2 CO3 , 24.2 g/L (288 mM)
of NaHCO3 and 1.942 g/L (5 mM) of disodium 2,20 -
A 22-mL (22.00 g) aliquot (delivered by weight) of
bicinchoninate. The solution is stored in a foil-covered
0.5% CMC solution (prepared as described in Sec.
bottle at room temperature. It is stable for several
II.A) is placed in a screw-cap tube in a 40 C water
months.
bath and preequilibrated for 15 min. After equilibra-
Reagent B. The reagent is prepared by dissol-
tion, 220 L enzyme solution is added and the reaction
ving 1.248 g/L (5 mM) of CuSO4 5H2 O and 1.262
timer activated. The cap is secured and the tube
g/L (12 mM) of L-serine. Solution is stored in a foil-
inverted gently 10 times. The solution is removed
covered bottle at 4 C. The solution is stable for up to 2
from the tube using a syringe, and 20 mL is injected
months. A new solution should be made when reagent
into the capillary tube (Ubelode type). The reaction
blank value increases.
mixture is drawn up into the capillary using suction,
Sodium carbonate/bicarbonate solution. The solu-
and the drain time measured. This is repeated consecu-
tion is prepared by dissolving 54.28 g/L (512 mM) of
tively over the desired time frame. The viscosity is then
Na2 CO3 , and 24.2 g/L (288 mM) of NaHCO3 in the
calculated using the capillary tube calibration con-
same solution. Final pH of the solution is 10.
stant. The reaction time used for each measurement
BCA Working Reagent. Equal volumes of reagents
is the time at one-half of the drain time (reaction
A and B are mixed. The BCA Working Reagent solu-
time at beginning of measurement plus 0.5 the drain
tion should be prepared daily.
time).
a. Sampling Using CMC (2). Twenty milliliters
2. Rotational (20.00 g) of CMC solution is transferred to a 50-mL
screw-cap tube gravimetrically and incubated at 40 C.
A rotating spindle viscometer (Brookeld DV-III rhe-
Two 1-mL aliquots are taken as controls after 15 min
ometer) is used to measure viscosity. Eight milliliters
and transferred to 13 100 mm test tubes containing 2
(8.00 g) of 0.5% CMC solution (prepared as described
50 L Na2 CO3 =NaHCO3 solution. The enzyme reac-
in Sec. II.A) is weighed into a chamber for small sam-
tion is initiated by addition of 180 L enzyme solution
ple volumes (Brookeld spindle model SC4-18) and
to the 50-mL tube and mixed by gently inverting several
preequilibrated with the spindle at 40 C for 15 min.
times. One-milliliter aliquots are removed at timed inter-
After equilibration, an 80 L aliquot of CMC solution
vals and transferred to tubes containing 250 L of the
is removed and 80 L enzyme solution is added. The
Na2 CO3 =NaHCO3 solution to inactivate the enzyme.
sample is mixed for 1 min by moving the chamber up
and down in the thermostated jacket. The spindle is b. Sampling Using PSC (4). One milliliter of
then started at 60 rpm; after 2 min the rst reading is PSC suspension is transferred into a microcentrifuge
taken, and subsequently each minute thereafter, in tube from a ask mixing on a magnetic stir plate. The
order to follow the rate of CMC hydrolysis. tubes are preequilibrated to 40 C for 10 min. The
enzyme reaction is initiated by addition of 540 L
B. Reducing-Group Methods enzyme plus buffer followed by thorough mixing.
The mixture is incubated at 40 C for a specied time
Reducing-group measurements are the best methods and then inactivated by addition of 250 L
for general cellulase activity determinations. One of Na2 CO3 =NaHCO3 solution followed by mixing.
the greatest difculties with this type of method is To determine the total reducing groups formed dur-
the nonuniform response between oligosaccharides ing the enzymatic reaction, the inactivated sample is
with different degrees of polymerization (DP). vortexed (< 5 min before sampling) and hand-mixed
Another difculty is the incompatibility of some meth- immediately before removing a 100 L sample. The
ods with certain substrates. Among the three reducing- sample is added to 900 L of water in a 13 100

mm test tube. Soluble reducing groups can be deter- c. Procedure. Sampling using CMC and PSC is
mined after centrifuging for 5 min prior to removing done as described under the BCA-reducing group
the 100-L sample to pellet the insoluble cellulose. assay with the modication of enzyme inactivation
Total and soluble reducing groups can be determined by heating in a boiling water bath for 5 min prior to
on the same sample by rst sampling for total reducing reducing group determination.
groups followed by sampling of soluble reducing Somogyi reagent (0.5 mL) is added to 0.5 mL of
groups. sample or standard in a 16 100 mm test tube. The
tubes are mixed by vortexing, covered with marbles to
c. Determination of Reducing Group (4). Reduc-
prevent evaporation, and placed in a boiling water
ing groups are determined by addition of 1 mL of BCA
bath for 10 min. The tubes are removed and cooled
Working Reagent to each tube containing 1 mL of
to room temperature. After cooling, 0.5 mL of the
sample or standard and incubated in a water bath at
Nelson reagent is added to each tube followed by mix-
80 C for 30 min. Tubes are mixed before incubation
ing using a vortex mixer until no bubbles remain. A
and covered with marbles to minimize evaporation. A
standard curve using glucose from 0 to 180 g/mL is
standard curve of glucose (055 nmoles/mL) is pre-
prepared and heated at the same time as the samples.
pared and incubated at the same time as the samples.
The absorbance values are measured at 560 nm in a
Following heating, the tubes are cooled to room
spectrophotometer, and the number of glucose equiva-
temperature in a water bath, sealed with Paralm,
lents (moles of reducing groups/mL) is calculated by
and mixed by inverting several times. The absorbance
comparison to the standard curve.
values are measured in a spectrophotometer at 560 nm
against a buffer blank. The number of glucose equiva-
lents (nmoles of reducing groups/mL) is calculated by 3. DNS Method (9)
comparison to the standard curve. To prevent varia-
The 3,5-dinitrosalicyclic acid reagent is prepared by
tion due to turbidity, samples using PSC as the sub-
dissolving 1.0 g of 3,5-dinitrosalicylic acid (DNS) in
strate should be allowed to settle and the supernate
50 mL water followed by addition of 20 mL of 2 N
removed for absorbance measurements using a
NaOH. After all the material is dissolved, 30 g of
Pasteur pipette.
sodium potassium tartrate (KNaC4 H4 O6 ) is added
and dissolved. The solution is diluted to 100 mL with
water. The solution is stable for several weeks when
2. Nelson-Somogyi Method (7, 8)
stored in an amber bottle protected from CO2 .
Sampling using CMC and PSC is done as described
a. Somogyi Reagent. The reagent is prepared by
under the BCA reducing group assay (Sec. III.B.1)
dissolving 12.0 g KNaC4 H4 O6 (potassium sodium tar-
with the modication of enzyme inactivation by heat-
trate) and 24.0 g anhydrous Na2 CO3 in 250 mL deio-
ing in a boiling water bath for 5 min prior to reducing
nized water. In a separate container, 4.0 g of
group determination.
CuSO4 5H2 O is then dissolved in minimal water
DNS reagent (1.00 mL) is added to 1.00 mL of
and added to the mixture. Sixteen grams of
sample or standard in a 18 150 mm test tube. The
NaHCO3 is then added and dissolved. Deionized
tubes are heated for 5 min in a boiling water bath and
water (500 mL) is boiled for 10 min with 180 g
then cooled in a water bath to room temperature. The
Na2 SO4 to expel air and then added to the mixture.
solutions are diluted to 3 mL with water and read in a
The nal volume is then diluted to 1.0 L and the
spectrophotometer at 540 nm against a blank in which
solution aged 1 week. The aged solution is ltered
1.00 mL water is used in place of the sample. The
before using.
concentration of reducing groups is determined from
b. Nelson Reagent. The reagent is prepared by a standard curve of glucose (01 mg).
dissolving 50.0 g ammonium heptamolybdate
[(NH4 6 Mo7 O24 4H2 O in 900 mL deionized water.
4. Filter Paper Method
Concentrated H2 SO4 (42.0 mL) is then added slowly
with mixing. In a separate container, 6.0 g While this method is considered standard, many
Na2 HAsO4 H2 O is dissolved in 50 mL deionized researchers have made extensive modications to
water and added to the mixture. The nal volume is meet various needs. The method described here is
adjusted to 1.0 L and aged 24 h at 37 C in the dark based on the standard method of Mandelis et al. (10)
before use. as described by Ghose (11).

To a 25-mL or larger test tube is added 1.0 mL of solution 2 mL is pipetted into a test tube and equili-
0.05 M sodium citrate buffer, pH 4.8. A 0.5-mL ali- brated to 50 C. Three milliliters of the substrate sus-
quot of enzyme diluted in sodium citrate buffer is pension (equilibrated to 50 C) is added. The enzyme-
added and the tube equilibrated at 50 C. (A minimum substrate mixture is incubated for 12 h with or with-
of two different dilutions is necessary for each enzyme out stirring using a magnetic stirrer at 400 rpm.
to be tested. One dilution should produce slightly > 2 Following incubation, the suspension is heated in a
mg of glucose and the other slightly less.) A 1:0 6:0 boiling water bath for 5 min and then ltered through
cm ( 50 mg) Whatman No. 1 lter paper strip is Whatman No. 1 lter paper. The ltrate is allowed to
rolled around a glass rod and added to the test tube cool to room temperature, and the absorbance at 595
and incubated at 50 C for 60 min. The reaction is nm is determined. Activity is expressed in arbitrary
stopped by the addition of 3 mL DNS mixture. All units of absorbance.
tubes are then boiled together (including standards
and blanks) for 5 min and cooled in a cold-water 3. MeUmb Method (13, 14)
bath. The reactions are then diluted with 20 mL
The use of 4-methylumbelliferyl -D-glycosides as sub-
water, mixed by inverting several times, read in a spec-
strates can be used to monitor cellulase activities. The
trophotometer at 540 nm, and compared with a stan-
cellobiose and cellotriose derivatives are commercially
dard curve of glucose (13.35 mg).
available or can be prepared according to Tilbeurgh et
The absolute amount of glucose released is deter-
al. (14).
mined after subtraction of the appropriate enzyme
blank. A semilog plot (to improve linearity) of the a. Continuous Method, OD. At pH 5.0, the
glucose released against the fold enzyme dilution is release of 4-methylumbelliferone (MeUmb) can be
made and the amount of enzyme necessary to release followed continuously by difference absorption
exactly 2.0 mg of glucose is determined. The lter spectrophotometry at 347 nm. Enzyme assays are
paper units (FPU) in U/mL are calculated by dividing done at 25 C in 50 mM sodium acetate buffer, pH
0.37 by the determined enzyme concentration neces- 5.0, using substrate concentration of 102000 M.
sary to release 2.0 mg glucose. [Note: The FPU is Concentrations of MeUmb glycosides can be deter-
derived from the International Unit (IU). When the mined using the molar absorption coefcient at 316
product is glucose, 1 IU is equal to 0.18 mg min1 . nm;
m 13,600 M 1 cm1 .
At the dened dilution (4% hydrolysis), 2 mg of glu-
b. Fixed-Point Method, Fluorescent. Using a dis-
cose is produced from 0.5 mL of enzyme in 60 min.
continuous method, taking samples, and adjusting the
Therefore, the enzyme necessary to release 2.0 mg of
pH to 10.0, the release of MeUmb can be monitored
glucose in the reaction contains 0.37 units (IU mL1 ).]
uorometrically. The sensitivity of the discontinuous
uorometric method is signicantly greater (< 1 M
C. Chromophore and Fluorescent Group Release of MeUmb) than the continuous method at pH 5.0.
Methods The reaction conditions used are the same as in the
continuous assay, but the reaction is stopped by the
1. Dye ReleaseCellulose Azure Method (12)
addition of an equal volume of 50 mM Na glycine, pH
Cellulose azure (commercially available) is weighed 10, in 50% ethanol. The relative uorescence is then
into a test tube (100 mg), and 3 mL of 0.05 M sodium measured (excitation 366 nm, emission > 400 nm). A
citrate buffer, pH 4.8, is added. The tube is equili- calibration curve is made using standard solutions of
brated to 50 C and a stirring bar is added. Two milli- MeUmb (recrystallized from ethanol).
liters of enzyme (equilibrated to 50 C) is added and
incubated with stirring for 10 min. After incubation,
D. Chromatographic Methods
the suspension is ltered through Whatman No. 1 lter
paper and the OD at 595 nm is measured in a spectro- 1. HPLC Method
photometer.
Separation of substrates and products by HPLC can be
used to analyze product distribution of individual cel-
2. Dye ReleaseAvicel Method (12)
lulases for classication and to determine specicity of
A substrate suspension is prepared in 50 mM sodium cellulases when using cello-oligosaccharides. Several
citrate buffer, pH 4.8, using 50100 mg/mL dyed different procedures have been used successfully with
Avicel (prepared as described in Sec.IIC). Enzyme a variety of different separation procedures. Labeled

and unlabeled substrates and products have also been oxidized with nitroblue monotetrazolium (NBMT)
used. producing an insoluble blue precipitate.
Standard gel electrophoresis is carried out using
a. Unlabeled Sugar Substrates (15). Separation
nondenaturing PAGE or isoelectric focusing systems.
of unlabeled sugars using Aminex columns with detec-
The incubation solution is prepared by rst dissolving
tion by refractive index is one of the more common
5 mg of 5-bromoindoxyl--D-cellobioside and 20 mg
procedures used. These columns have the distinct
NBMT in 0.5 mL dimethylformamide followed by
advantage that they use water, or in some applications,
mixing with 20 mL of 0.1 M sodium acetate buffer,
dilute sulfuric acid as the eluent. In application to cel-
pH 5.0. The gel is added to the solution and
lulases analysis, the HPX-87P (Pb form) and the HPX-
incubated in the dark at 40 C until dark blue bands
42A (Ag form) for monosaccharides and oligosacchar-
appear. The development time is 215 h and is depen-
ides analyses, respectively, are used most frequently.
dent on the concentration of active enzyme in the gel.
An HPLC system using an Aminex column (ideally
Gels can be scanned or photographed for preserva-
with a deashing guard column), a column heater, and a
tion. The staining solution can be used many times
refractive index detector is required. Samples are pre-
before discarding.
pared using an appropriate hydrolysis system followed
by inactivation of the enzyme by heating (5 min in
3. Gel Electrophoresis of Products (1719)
boiling water). If the nal pH is not between 5 and 9
for the HPX-87P or between 6 and 8 for the HPX-42A,
a. ANTS Reagents. A 0.2-M solution is prepared
the pH should be adjusted to prevent loss of column
by dissolving 0.086 g of 8-aminonaphthyl-1,3-6-trisul-
performance. Samples are then ltered through 0.45-
fonate (ANTS) in 5 mL of a solution of glacial acetic
m lters and injected (1020 L) into the HPLC sys-
acid/water (3:17; v/v).
tem. Flow rates of 0.40.8 mL/min are used with col-
umn temperatures of 6085 C. Quantication of b. Reducing Agent. A 1.0-M solution of sodium
results is accomplished by running external standards cyanoborohydride (NaCNBH4 is prepared in HPLC-
and constructing response factors for each sugar. grade dimethylsulfoxide (DMSO) by the addition of
2.5 mL DMSO to 0.25 g NaCNBH4 .
b. MeUmb-Labeled Substrates (13). MeUmb-
labeled cello-oligosaccharides are prepared according c. Procedure. Enzymatic hydrolysis reactions are
to Tilbeurgh et al. (14), or they are available commer- carried out using 0.05 M ammonium acetate buffer,
cially. Enzymes are mixed with substrates in 0.05 M pH 5.0, and inactivated by a change in pH using
acetate buffer, pH 5.0, with substrate concentration ammonium hydroxide. The use of this volatile buffer
from 300500 M and incubated at 25 C. Samples system prevents high salt concentrations and allows
are taken from the reaction mixture (at 5 to 10-min samples to be dried before labeling with the ANTS
intervals for 3060 min) and diluted 1:3 in acetonitrile reagent. Following hydrolysis, the inactivated reaction
to stop the reaction. Inactivated samples (20 L) are is centrifuged and the necessary volume (1050 nmoles
injected into an HPLC system consisting of a 25 0:46 reducing sugar) is transferred to a microcentrifuge tube
cm Rsil-Polyol 5-m particle-size column from Alltech and dried using a vacuum centrifuge.
and a UV detector monitoring at 313 nm. The eluent is The dried sample containing 1050 nmoles of redu-
a mixture of acetonitrile and water (39:11, v/v) at a cing sugar is labeled by adding 510 L of the ANTS
ow rate of 1.5 mL/min. Quantication of results is reagent and 510 L of the reducing agent. This mix-
accomplished by running external standards and con- ture is vortexed for 10 sec and centrifuged briey to
structing response factors for each sugar using inte- bring reagents together. The reaction is incubated at
grated peak areas. Using the same column and 40 C for 15 h.
eluent, the unmodied cello-oligosaccharides can be Electrophoresis is done using a 536% gradient
analyzed using a refractive index detector. acrylamide gel with a Tris-glycine buffer system, pH
8.3, and a 5% stacking gel, pH 6.8 (no SDS is incor-
porated). [The acrylamide stock solution used to pre-
2. Enzyme Detection in Polyacrylamide Gels (16)
pare gels is acrylamide/bis-acrylamide (39:2 w/w).] The
The detection of cellulases in polyacrylamide gels is labeled sample is diluted with four times loading buffer
based on the hydrolysis of 5-bromoindoxyl--D-cello- (40% glycerol in 0.2 M Tris-HCL buffer, pH 6.8) and
bioside by endo-1,4--D-glucanases to produce 5-bro- applied to sample wells. Current is applied and the
moindoxyl. The product 5-bromoindoxyl is then migration monitored by use of a hand-held UV moni-

tor. The gel is removed when the uorescent dye front a. Glucose Reagent. Dissolve 1.5 U/mL peroxi-
reaches the bottom of the gel. Gels are documented by dase, 9 U/mL glucose oxidase, and 0.5 mg/mL
photography on a UV gel box. A Polaroid camera is of 2,2 0 -azino-di-(3-ethylbenzthiazoline)-6-sulfonate
used with a 4 5 lm holder model 545, type 55 lm, (ABTS) in 0.12 M phosphate buffer, pH 7.0. A few
which gives a negative and a positive image, and a No. drops of chloroform can be added to avoid bacterial
8 lter. growth. When stored at 4 C the solution is stable for
several weeks.
4. Capillary Electrophoresis of Products (17, 19)
b. Procedure. Glucose reagent (3 mL) is added to
1.0 mL of sample or standard in a 16 100 mm test
a. Saturated Trisodium Phosphate (TSP). An
tube. The tubes are incubated for 30 min at room tem-
excess of TSP is added to water and mixed. Solution
perature and the absorbance measured at 450 nm. A
is kept at room temperature.
standard curve of glucose (075 g) is used to deter-
b. Phosphoric Acid. Commercial 85% phospho- mine the concentration in unknown samples.
ric acid (8.67 M) is diluted to 1 M with deionized
water.
2. Cellobiose Dehydrogenase (Discussion Only)
c. Running Buffer. Phosphoric acid 100 mM is
titrated to pH 2.5 using triethylamine (TEA). The Coupled enzyme methods using cellobiose dehydro-
nal solution is 100 mM phosphate, 36 mM TEA. genase for determining cellulase activity have been
published (21, 22). The methods require the use of
d. Procedure. Hydrolysis samples are labeled as
enzymes from Phanerochaeta chrysosporium or
above for gel electrophoresis but diluted with water
Humicola insolens that are not commercially available
(1:10) rather than loading buffer. A capillary electro-
and therefore their purications will be required to use
phoresis system with an inactivated capillary column
this method. These are among the few methods permit-
(60 cm 50 m) and phosphate-TEA buffer, pH 2.5, is
ting a continuous measurement of cellulase activity
used. Injection is done using pressure at 20 psi sec.
and could prove extremely valuable in kinetic compar-
Separation is monitored at 235 nm and samples are run
ison between puried cellulases.
at 20 kV with the polarity () to (). Capillary and
sample chamber temperatures are set at 35 C. Run
times are 15 min. The column is ushed with fresh
buffer between runs. F. Special Methods
The capillary is conditioned and cleaned before
separation by sequentially ushing the column with In a detailed study of a puried cellulase, additional
saturated TSP, deionized water, 1 M phosphoric details about the enzyme activity can contribute to the
acid, deionized water, and running buffer for 5 min characterization and classication of the enzyme.
each. When the column performance decreases, as Several techniques will be discussed that have been
indicated by a change in elution times, the column is developed for determination of some important fac-
again cleaned. tors.
E. Coupled Enzyme Methods 1. Inverting or RetainingNMR Method (22, 23)

1. Glucose Oxidase/Peroxidase Method (20) During the course of hydrolysis, the stereochemistry of
The enzymatic determination of glucose concentra- the anomeric carbon formed is dependent on the struc-
tions for estimating cellulase activity is a useful method ture and topology of the enzyme active site. Utilizing
when reducing group determinations are not possible proton NMR, the hydrolysis of reduced cellodextrins
or when total cellulose conversion determinations are (i.e., cellohexaitol) can be monitored for the formation
required. Several variations of the glucose oxidase/per- of - or -anomers. The enzymes can then be classied
oxidase assay for the determination of glucose concen- as inverting or retaining based on the product stereo-
tration have been published. The primary difference is chemistry. Dividing cellulases based on the primary
the particular compound used as the indicator. Kits are sequences into families shows that there is very strong
available from some chemical supply companies relationship between the family and the stereochemical
(Sigma). course of hydrolysis.

2. Cellulose Chain End Determination Method (24) 6. T Wood. Preparation of crystalline, amorphous, and
dyed cellulase substrates. In: Methods in
Until recently it was believed that the mechanism of Enzymology, Vol 160. San Diego: Academic Press,
hydrolysis for exocellulases (cellobiohydrolases) was 1988, pp 1925.
solely from the nonreducing end of the cellulose mole- 7. N Nelson. A photometric adaptation of the Somogyi
cule. In 1996, Barr et al. (24) used 18 O and 14 C labeling method for the determination of glucose. J Biol Chem
of cellooligosaccharides in conjunction with ion-spray 153:375380, 1944.
mass spectrometry to determine the precise position 8. M Somogyi. Notes on sugar determination. J Biol
and direction of hydrolysis of ve different exocellu- Chem 195:1923, 1951.
lases from Thermonospora fusca and Trichoderma ree- 9. GL Miller. Use of dinitrosalicylic acid reagent for
determination of reducing sugar. Anal Chem 31:426
sei. Their results support the conclusion that there are
428, 1959.
two different classes of exocellulases each working
10. M Mandelis, R Andreotti. C Roche. Biotechnol
from different ends of the cellulose molecules. Bioeng Symp 6:17, 1976.
11. TK Ghose. Measurement of cellulase activities. Pure
3. Dialysis Reactor Cell Method (25) Appl Chem 59(2):257268, 1987.
12. M Leisola, M Linko. Determination of the solubiliz-
Synergism is an interesting observation shown with ing activity of a cellulase complex with dyed sub-
cellulases and with other enzymes, such as amylase, strates. Anal Biochem 70:592599, 1976.
that have insoluble substrates and multiple types of 13. H van Tilbeurgh, M Claeyssens, CK De Bruyrne. The
enzymes working on the same substrate. use of 4-methylumbelliferyl and other chromophoric
Determination of synergism is difcult, especially glycosides in the study of cellulolytic enzymes. FEBS
using a native substrate. Baker et al. (25) developed a Lett 149:152156, 1982.
14. H van Tilbeurgh, FG Loontiens, CK De Bruyne, M
microdialysis device for studying synergism using
Claeyssens. Fluorogenic and chromogenic glycosides
native or pretreated substrates, under conditions in as substrates and ligands of carbohydrases. In:
which product inhibition is minimized. The reactor Methods in Enzymology, Vol 160. San Diego:
cell is loaded with substrate and enzymes and held Academic Press, 1988, pp 4559.
under constant temperature. The cell is continually 15. B Nidetzky, W Zachariae, G Gercken, M Hayn, W
ushed with fresh buffer, and the eluent is collected Steiner. Hydrolysis of cellooligosaccharides by
and assayed using HPLC sugar analysis. Trichoderma reesei cellobiohydrolases: experimental
data and kinetic modeling. Enzyme Microbiol
Technol 16:4352, 1994.
16. VM Chernoglazov, OV Ermolova, YV Vozny, AA
Klyosov. A method for detection of cellulases in poly-
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cellulase action on microcrystalline cellulose. Appl Identication of two functionally different classes of
Microb Biotech 37:750755, 1992. exocellulases. Biochemistry 35:586592, 1996.
22. C Schou, G Rasmussen, M Kaltoft, B Henrissat, M 25. JO Baker, TB Vinzant, CI Ehrman, WS Adney, ME
Schulein. Stereochemistry, specicity and kinetics of Himmel. Use of a new membrane-reactor saccharica-
the hydrolysis of reduced cellodextrins by nine cellu- tion assay to evaluate the performance of cellulases
lases. Eur J Biochem 217:947953, 1993. under simulated SSF conditions. Effect on enzyme
23. M Claeyssens, P Tomme, CF Brewer, JE Hehre. quality of growing Trichoderma reesei in the presence
Stereochemical course of hydrolysis and hydration of targeted lignocellulosic substrate. Appl Biochem
reactions catalyzed by cellobiohydrolases I and II Biotechnol 63/65:585595, 1997.
from Trichoderma reesei. FEBS Lett 263:8992, 1990.

62
Cellulases in Food Processing
Maija Tenkanen, Marja-Leena Niku-Paavola, Markus Linder, and Liisa Viikari

VTT Biotechnology, Espoo, Finland
I. INTRODUCTION line cellulose. Some bacteria, especially Acetobacter

xylinum, are also able to synthesize cellulose.
Plant materials contain different -glucans, mainly Cellulose is chemically a very simple polymer. It is a
celluloses and mixed 1,3-1,4--glucans. Cellulases homopolymer consisting of up to 1000 -1,4-linked
belong to the group of -glucan hydrolases that have anhydroglucopyranoside units. However, its physical
the ability to degrade cellulose. Enzymes often called state makes it a very challenging substrate for enzymes.
-glucanases that are able to hydrolyze -1,4-linkages Single glucose polymers are packed onto each other to
in other materials, such as in water-soluble -glucans, form a highly crystalline brillar material in which the
but not in cellulose, are not covered in this chapter. individual cellulose chains are held together by hydro-
Cellulases are not among the main enzymes used in the gen bonds. Cellulose microbrils also contain some
food industry. They are, however, applied in pro- amorphous regions, the amount of which depends on
cessing of cereal-based beverages and foods such as the source. The most crystalline cellulose is produced
beer and bread. Cellulases are used in rather large by algae, and the least crystalline by plants.
amounts in animal feed. As a consequence of modern Animals do not possess digestive enzymes able to
enzyme production technologies, which enable the pro- degrade crystalline cellulose. However, several micro-
duction of more selective, tailor-made enzyme pro- organisms, including lamentous fungi, yeast, and bac-
ducts, and owing to the increased knowledge of the teria, existing in the intestine and colon of monogastric
structure-function relationship of food components, animals are able to hydrolyze cellulose to oligosacchar-
the use of cellulases may increase in the future in ides and eventually to glucose, which they can then use
food processing. as a carbon source. Ruminants, particularly, are able
to fully degrade cellulose in their rumen by the wide
spectrum of microorganisms present. To meet the chal-
II. CELLULOSE: A CHEMICALLY SIMPLE lenge of degradation of crystalline and water-insoluble
BUT STRUCTURALLY COMPLEX cellulose, microorganisms produce a complex mixture
SUBSTRATE of several enzymes, which act synergistically. There are
also several reports on plant cellulases, which are
Cellulose is the most abundant biopolymer on earth. It involved in plant cell expansion for loosening the cell
is mainly produced in higher plants, e.g., woods as well walls during the growth of cell (1). Cellulase from dif-
as annual plants in which it forms the rigid skeleton of ferent microorganisms have been thoroughly reviewed
the plant. In addition, all algae produce highly crystal- during the last decade (27).

III. CLASSIFICATION OF CELLULASES reaction mechanisms. So far, some families contain
only enzymes with the same catalytic activity, but
All cellulases act on the chemically identical bond, i.e., most contain both endo- and exoglycanases being
the -1,4-linkage between two anhydroglucose units. able to act on different polymers. For example, family
However, they differ in terms of their site of attack 5 contains endoglucanases, exoglucanases, endoman-
on the cellulose chain. Enzymes acting on cellulose nanases, and endoxylanases. Currently, there are cellu-
have traditionally been classied by the International lases in 11 glycosyl hydrolase families (families 510,
Union of Biochemistry (IUB) (www.expacy.ch.cg1- 12, 26, 44, 45, and 48).
bin/enzyme-search-cl) into two distinct groups, endo- The new suggestion for the naming of different cel-
glucanases and exoglucanases, which act in the middle lulases is based purely on their assignment to these
of the cellulose chain or at either end of the cellulose glycosyl hydrolase families (11). If there is more than
chain, respectively (Fig. 1; Table 1). However, this clas- one enzyme belonging to the same family from one
sication is not very satisfactory, as some exogluca- organism, the enzymes are further named with running
nases have also been claimed to possess endoactivity alphabets in the order that they have been classied.
(8, 9). The classication of cellulases becomes even For example Trichoderma reesei Cel7A (CBH I) and
more complicated as some also have activity on other Cel7B (EG I) are two cellulose degrading enzymes in
polysaccharides, such as xylan (10). Therefore, a new the glycosyl hydrolase family 7. In this chapter, the old
classication of these enzymes, as well as of other gly- naming system using endoglucanases and cellobiohy-
cosyl hydrolases, have been suggested based on their drolases is used since most publications still use this
structural similarities (11). This, however, requires nomenclature.
information on their amino acid sequence which is The endoglucanases (EG) (1,4--D-glucan-4-gluca-
not always available. nohydrolase, EC 3.2.1.4) catalyse random cuts in cel-
At present, the different glycosyl hydrolases are lulose chains, thereby producing shorter and shorter
grouped according to the structures of their catalytic cello-oligomers, which can be further degraded by exo-
domains into more than 70 families (afmb.cnrs-mrs.fr/ glucanases. The trivial name given by the IUB for
cazy/CAZY/index.html). In addition, some of the endoglucanases is cellulases, which is very misleading
families have further subfamilies. The classication is as cellobiohydrolases (CBH) (1,4--D-glucan cellobio-
based on sequence alignments and hydrophobic cluster hydrolase, EC 3.2.1.91) also act on cellulose and are
analysis (HCA) (12), but it has been proven to corre- actually the main enzymes responsible for the degrada-
late well with the three-dimensional structures and tion of crystalline cellulose. Therefore, in this chapter
the name cellulase covers all enzymes that are able to
degrade polymeric celluloses, i.e., both EGs and CBHs.
The nal degradation of cello-oligomers produced by
EGs and CBHs to glucose is accomplished by -gluco-
sidases (BGL) (EC 3.2.1.21). In addition to -1,4-lin-
kages, the latter enzymes are often also able to
hydrolyze other -glucosidic linkages, and although
they are not able to hydrolyze polymeric cellulose
they are, owing to their action on cello-oligosacchar-
ides, usually considered as part of cellulolytic enzyme
system.
CBHs cleave predominantly cellobiose units from
either the reducing or nonreducing the ends of cellulose
chains and cello-oligosaccharides (Table 1). CBHs nor-
mally produce a small amount of cellotriose when act-
ing on cello-oligosaccharides. Substitution of cellulose
chains blocks the action of CBHs, but most of them
are able to hydrolyze 1,4--linkages in mixed 1,3-1,4--
glucans, which exist in high quantities in the cell walls
of grains (Table 2).
EGs are mainly active on amorphous cellulose, thus
Figure 1 Hypothetical picture of cellulose degradation. breaking down the less ordered amorphous parts of

Table 1 Classication of Enzymes Acting on 1,4--Glucans
Action Systematic name Trivial name(s) EC No. Substrate specicity Linkage hydrolyzed Main products formed
Endo 1,4--D-glucan-4- Cellulase, endoglucanase 3.2.1.4 Cellulose, 1,3-1,4-- 1,4- 1,4--dextrins, mixed 1,3-1,4--
glucanohydrolase (EG) glucans dextrins
1,3-1,4--D-glucan- Lichenase, -glucanase 3.2.1.73 Lichenin, 1,3-1,4-- Next 1,4- after 1,3- Mixed 1,3-1,4--dextrins
4-glucanohydrolase glucans -linkage
1,3--D-glucan-3/4- Laminarinase 3.2.1.6 Laminarin, 1,3-1,4- Next 1,4-- or 1,3-- Mixed 1,3-1,4--dextrins
glucanohydrolase -glucans after 1,3--linkage
1,3--D-glucan-3- Laminarinase 3.2.1.39 Laminarin, pachyman, 1,3- 1,3--dextrins
glucanohydrolase (1,3-1,4--glucans) (1,4--dextrins)
Exo 1,4--D-glucan- Cellobiohydrolase 3.2.1.91 Cellulose, 1,3-1,4-- 1,4- Cellobiose
cellobiohydrolase (CBH) glucans
1,4--D-glucan- Exoglucanase 3.2.1.74 1,4--glucans 1,4- Glucose
glucohydrolase
-Glucosidase Cellobiase 3.2.1.21 Wide variety of -D- 1,4- Glucose
glucosides 1,3-
1,6-

Table 2 Specic Activities of the Major T. reesei high quantities in some plants, such as in konjac
Cellulases Against Different Substrates tubers. Some EGs act also on other -1,4-glycosidic
linkages. One example of this lack of specicity is
Specic activitya (nkat/mg)
the EG I from T. reesei which is also able to hydro-
Enzyme HEC -GLC XYL MUC lyze -1,4-xylosidic linkages in xylans (Table 2) (see
Chapter 71).
EG I 540 3920 550 60
EG II 1170 6850 3 3.4
CBH I 2 12 3 3.2 IV. METHODS TO ASSAY CELLULASES
CBH II 2 72 2 0
a
Substrates used in the measurement: HEC hydroxyethylcellulose; The determination of cellulase activities is not a very
-GLC -glucan from barley; XYL birch xylan; MUC easy task, as the degradation of the water-insoluble,
methylumbelliferyl cellobiose. highly crystalline cellulose is not linear with time and
different enzyme dosages. Therefore, several nonna-
tural substrates are normally used. As mentioned ear-
lier, soluble cellulose derivatives are often used to assay
cellulose bers (13). Some bacterial EGs are also EG activity. These are rather specic to EGs, as CBHs
claimed to be able to degrade crystalline parts of cel- are not generally able to degrade substituted celluloses
lulose microbrils (14). EGs are highly active on var- (Table 2). The international standard assay for mea-
ious water-soluble cellulose derivatives such as suring EGs is based on the hydrolysis of HEC (15).
carboxymethyl cellulose (CMC) and hydroxyethyl cel- Soluble cereal, 1,3-1,4--glucans can also be used as
lulose (HEC), which are often used as substrates for substrates. They are degraded by EGs and by many
determination of the endoglucanase activity (see CBHs. However, 1,3-1,4--glucans are not a specic
Chapter 61). In addition, EGs hydrolyze -1,4 linkages substrate for cellulases as other -glucanases are also
in mixed 1,3-1,4- glucans. There are also other able to degrade it. Amorphous cellulose has been used
enzymes, which are able to hydrolyze -1,4-glucosidic as a substrate mainly for EGs, but in many cases the
linkages in soluble -glucans (Table 1) (Fig. 2). enzymatic reaction is not linear or is linear only at the
However, as they are not able to hydrolyze cellulose, short initial reaction stage. Recently, a novel method
these enzymes are not discussed here (see Chapter 74). for the preparation of totally amorphous cellulose
EGs are basically able to hydrolyze all polysacchar- beads has been developed (Pettersson, unpublished).
ides containing -1,4-linked glucopyranosyl units, such These beads can also be made from dyed cellulose
as xyloglucans (1,4--glucan backbone with side chains and were shown to be excellent substrates for EGs.
made up of xylose, galactose, and occasionally fucose) There are several ways to analyze the degree of
(see Chapter 73). These are present in small quantities degradation of the substrate. It can be based on the
in plants but are the predominant polysaccharides in reduction in viscosity, on the formation of new redu-
tamarind seeds. Many EGs are also able to hydrolyze cing groups, or on the liberation of dyed oligosacchar-
polymers in which glucose is -1,4-linked to other gly- ides from chromophoric polymers. The viscosity
cosyl units. Thus, for example, the -1,4-linkages method is the most sensitive one and can be used to
between glucose and mannose units in glucomannans measure very low levels of EGs, for example, from
are cleaved by EGs (see Chapter 75). Glucomannans plant extracts. It is also highly specic to EGs, as exo-
are interesting food hydrocolloids and they exist in glycanases do not affect the viscosity of the polymer.
Figure 2 Endoglucanases acting on 1,3-1,4--glucans.

The action of EGs and CBHs is difcult to differenti-
ate in the methods based on the formation of reducing
groups.
The activity of CBHs is often measured by using
small uorogenic substrates such as methylumbelliferyl
cellobioside or methylumbelliferyl lactoside. However,
these substrates are specic not only to CBHs as some
EGs also degrade them (Table 2). The activity of -
glucosidases is in most cases determined using p-nitro-
phenyl--D-glucopyranoside as a substrate and mea-
suring the color formed by the liberated p-nitrophenol.
EGs and CBHs do not hydrolyze this substrate.
Naturally soluble oligosaccharides up to DP 6 can
be used as substrates for all cellulases, and the formed
degradation products are normally analyzed by HPLC.
In some cases it is important to know the overall cel-
lulolytic capacity of the sample, i.e., how well real cel-
lulose is degraded by a mixture of different cellulases.
This is normally measured by the international stan- Figure 3 Model showing the two-domain structure of CBH
dard method using lter paper as a substrate and the from T. reesei. The larger catalytic domain is connected by
the linker to the smaller cellulose-binding domain (CBD).
activity is expressed as lter paper units (FPU) (15).
Model coordinates provided by Christina Divne, Uppsala
The different methods used for assaying the hydrolysis
University.
of cellulose by cellulases are covered in more detail in a
separate chapter (see Chapter 61).
also proteins involved in binding and targeting the cel-
lulosome to the substrate. The individual enzymes
V. STRUCTURE-FUNCTION alone in the complex normally have no signicant
RELATIONSHIPS IN CELLULASES activity against crystalline cellulose, but the cellulo-
some hydrolyzes it actively. Each enzyme component
A two-domain structure consisting of a catalytic is attached to a nonenzymatic protein called scaffoldin,
domain and a separate substrate-binding domain is via a conserved docker sequence. The scaffoldin poly-
typical for cellulose-degrading enzymes from both bac- peptide contains a CBD and internal repeats (cohesins)
teria and fungi. The existence of substrate-binding which interact with enzymes. The molecular weight of
domains is also common in other enzymes which cellulosomes can be as high as 2000 kDa.
degrade solid substrates. In fungal enzymes the cellu- Although 13 families of CBDs have been identied
lose-binding domains (CBDs) are typically linked to based on sequence homology, only one of them (family
the catalytic domain (catalytic core) by linkers which 1) occurs in fungi (20). The fungal CBDs are distin-
clearly separate the two domains (Fig. 3). The CBDs guished by their small size (< 40 amino acid residues).
can be found in either the C or N terminus of the Like most other carbohydrate-binding proteins, the
enzyme. In some cases, such as in the bacterium binding to cellulose has been shown to be largely domi-
Thermomonospora fusca enzyme E4, the CBD forms nated by interactions from aromatic amino acid resi-
a more integral part of the enzyme (16). Some bacteria dues. The CBDs have been shown to be important for
have been reported to produce multidomain proteins the function of cellulases on solid substrates (Fig. 4). If
which contain different, covalently linked catalytic they are removed either by proteolysis or protein engi-
domains hydrolyzing cellulose and hemicelluloses as neering, both activity and binding to solid substrates
well as several binding domains (17). are greatly decreased (21, 22). In many experiments the
Many aerobic organisms produce large complexes binding of cellulases to cellulose has been seen to be
called cellulosomes, which are clusters of different non- irreversible, which has sometimes been ascribed to the
covalently linked proteins (18, 19). The assembly and CBD. The interaction is complex, however, and is seen
action of cellulosomes have been studied by cloning the as interplay between the catalytic and binding domains
genes of the different subunits. Cellulosomes may con- which results in a synergistic binding to cellulose (23).
sist of several EGs, CBHs, and other glycanases, but At least in the case of fungal enzymes, no evidence has

Figure 4 Hydrolysis of bacterial crystalline cellulose and amophous cellulose by T. reesei cellulases and the corresponding core
proteins (300 nmol/g, pH 5, 50 C, 20 h).
been presented which would show that CBDs have one this enzyme a much more open active site (Fig. 5). The
active role in hydrolysis of the crystalline cellulose, e.g., tunnel shape of the active site in the CBH I, as well as
breaking down the hydrogen bonds. CBDs have been that found in other CBHs, gives a structural explana-
reported to lower the apparent Km , but also to reduce tion for the action of the enzyme. It is thought that the
the apparent kcat probably by slowing down the mobi- long tunnel keeps the enzyme bound to the same cel-
lity of the enzyme (24). lulose chain by multiple interactions as it progressively
Owing to several three-dimensional protein struc- moves along the chain hydrolyzing cellobiose units.
tures of cellulases that are available together with The open active site groove found in EGs allows
detailed biochemical characterization, many special them to hydrolyze in the middle of cellulose chains
structural features of cellulases have been revealed. (2527). It has been speculated that cellulases can dis-
For example the EG I and the CBH I of T. reesei play more or less a continuum of structures with active
have a sequence identity of 45%. Comparison of sites covered to different degrees, giving more endo-
their structures shows that the active site of CBH I is like or exo-like enzymes, making the division
located in a tunnel formed by surface loops, while unclear as mentioned earlier. The degree of endo- or
many of these loops are missing in EG I, which gives exo-character can be determined by either the pre-
Figure 5 Comparison of the structure of endo- and exoglucanases. (A) Core domain from CBH I; (B) core domain of EG I from
T. reesei.

sence/absence of the loops, or their exibility. An issue, VI. FUNGAL CELLULASES
which is still not understood for CBHs is how the cel-
lulose chain is picked up from the crystalline cellulose Cellulolytic fungi occur in all major taxa (30, 31), but
and how it is targeted to the tunnel. In the case for the current knowledge of fungal cellulases has been
CBH II from T. reesei, a region on the outer side of the obtained mainly by studying deuteromycetes
tunnel has been shown to be important for the hydro- Trichoderma (21), Penicillium (13), and Humicola (24).
lysis of crystalline cellulose, but not for soluble sub- Other signicant fungi-producing cellulase are the plant
strates or amorphous cellulose (28). pathogen Fusarium solani (32), Aspergillus aculeatus
Cellulases, like all glycosidases, catalyze the hydro- (33), and the asomycete Neurospora crassa (34).
lysis of the glycosidic bonds by general acid catalysis Cellulase producers have also been found among anae-
(29). The reaction involves two essential catalytic car- robic rumen fungi, the rst example of which was
boxylates, the proton donor and the nucleophile, Neocallimastix frontalis (35). Studies on the white rot
which are generally located on opposite sides of the fungi Sporotrichum pulverulentum (anamorph of
scissile bond. Depending on the structure of the active Phanerochaete chrysosporium) (36) and Phanerochaete
site, the cleavage proceeds by either retention or inver- chrysosporium (37) have revealed that lignin-degrading
sion of the conguration of the () anomeric carbon fungi are also able to produce a complete set of cellu-
(29). Based on the structure of the active-site tunnel, lolytic enzymes depending on the culture conditions.
some cellulases are proposed to twist the cellulose The studies on brown rot fungi suggest that these
chain at the active site in order to expose the scissile organisms produce only EGs and -glucosidases (38).
bond better (25). The active sites have been shown to Most cellulolytic fungi produce a mixture of several
bind up to 10 glucose units. Since the active sites con- EGs, CBHs, and -glucosidases to be able to degrade
tain several sugar binding sites, it is convenient to have crystalline cellulose into glucose (Table 3). Fungal
a common nomenclature for them. The bond cleavage enzymes are normally glycosylated, and the isoforms,
occurs at the bond between the 1 and 1 sites so that having different isoelectric points, make the purica-
the direction is toward the reducing end of the sugar tion and characterization of individual enzymes dif-
chain and the direction is toward the nonreducing cult. In addition, the culture ltrates often contain the
end. catalytic core domains of cellulases as the native
Table 3 T. reesei and H. insolens Cellulases
Fungi Enzyme New name Size (kDa) pI Remarks Optimal pH Reference
T. reesei EG I Cel7B 5055 4.6 4.55.5 101

EG II Cel5A 48 5.5 4.55.5 102
EG III Cel12A 25 7.4 No CBDa 4.55.5 103
EG IV Cel61A 37 nd 4.55.5 104
EG V Cel45A 23 2.83.0 4.55.5 105
CBH I Cel7A 5968 3.54.2 4.55.5 106
CBH II Cel6A 5058 5.16.3 4.55.5 107
BGL I 71 8.7 Family 3 4.6 108
BGL II 52 4.8 Family 1 4.0 108, 109
(114) Dimer
H. insolens EG I Cel7B 50 5.5 No CBD 7.08.5 110
EG II Cel5A 50 7.0 7.08.5 110
EG III Cel12 26 5.2 No CBD 7.08.5 110
EG V Cel45 43 5.2 7.08.5 110, 111
EG VI Cel6B 43 5.0 7.08.5 110
CBH I Cel7A 72 4.5 5.5 110
CBH II Cel6A 65 4.65.2 9.0 110
BGL IV 54 Family 1 5.0 109, 112
(250295) Multimer
a
CBD cellulose binding domain.
Source: From Refs. 101112.

enzymes are sometimes cleaved at the linker region by gene encoding CBH I has resulted in > 70% reduction
secreted proteases. Therefore, confusing literature in the lter paper degrading activity, whereas deletion
exists on the number of individual enzymes produced of other cellulases had only a minor effect (45).
by different fungi. After cloning the genes encoding the Electron microscopy studies have shown that CBH I
corresponding cellulases, the picture will become causes lateral thinning of cellulose microbrils,
clearer. whereas CBH II has been reported to sharpen the crys-
The commercial cellulases are mainly produced with tal ends (46, 47). CBH I seems to be a more processive
T. reesei and H. insolens. In addition, individual H. enzyme than CBH II. This might be due to a longer
insolens genes have been cloned into a noncellulolytic tunnel in CBH I having 10 glucose binding sites (25).
Aspergillus strain and produced as monocomponent However, the action of CBHs on crystalline cellulose is
enzymes. However, these enzymes are not yet available at least 10 times slower than their action on soluble
for food use. The current T. reesei strains, which have cellulose derivatives (28). Thus, it seems that stripping
been developed by classical mutagenesis from the the cellulose chain from the crystal and targeting the
native strain, are among the most powerful producers chain end to the active site is the rate-limiting step. It
of extracellular proteins (39, 40). When grown on cel- might also be that the transfer of the cellulose chain
lulose, 90% of the extracellular protein consists of along the active site for the next cut is a slow process
four major cellulases ( 50% CBH I, 20% CBH II, compared to the actual hydrolytic action. T. reesei
10% EG I, and 10% EG II). Several tailor-made T. CBH I is also very sensitive to end-product inhibition
reesei strains have been recently developed by deleting (48). The Trichoderma enzymes usually exhibit the
the genes encoding nonwanted enzymes, thus leaving highest activity at acid pH ( 5) and at temperatures
for example only one of the four major cellulases (40). < 55 C.
T. reesei is also nowadays used as a host organism for
the production of other glycosyl hydrolases (41, 42). B. Cellulases from H. insolens
A. Cellulases from T. reesei The other commercially interesting fungus, H. insolens,

produces an almost identical pattern of cellulases to T.
T. reesei is the most thoroughly studied cellulolytic reesei, having at least ve EGs, two CBHs, and several
organism. It is reported to produce at least ve differ- -glucosidases. These enzymes are also structurally
ent endoglucanases (EG I through EG V), two cello- similar, thus belonging to same glycosyl hydrolase
biohydrolases (CBH I and CBH II) and two - families (Table 3). In addition, there exists remarkable
glucanases (BGL I and BGL II) (Table 3). All EGs, homology (> 50%) in the amino acid sequences
except EG III, and both CBHs have a modular struc- between the corresponding cellulase component of T.
ture with a separate catalytic core domain and a CBD. reesei and H. insolens. However, several H. isolens cel-
EG I differs from the other EGs in being able to also lulases work at higher pH and higher temperatures
hydrolyze xylan (10). Its specic activity on the water- than T. reesei enzymes and have therefore found com-
soluble xylan is about the same as the activity on the mercial applications, especially in textile treatments
soluble cellulose derivative HEC (Table 2). All EGs act (49).
mainly on amorphous cellulose (Fig. 4). However, EG Two H. insolens cellulases, EG I and EG III, are
II has been shown to reduce the degree of polymeriza- lacking the CBD. EGs IIII show similar activity
tion much faster than EG I (43). toward CMC and amorphous cellulose, while EGs
CBH I and CBH II from T. reesei both act on the VVI have the highest catalytic activity on amorphous
cellulose chain ends, but they clearly have different cellulose. None of them has been reported to be active
substrate specicities. CBH I acts from the reducing in hydrolyzing xylan. H. insolens CBH I degrades crys-
end of the cellulose chain whereas CBH II starts the talline cellulose, but it is clearly less sensitive to end
action from the nonreducing end (7, 44). Thus, all product inhibition than CBH I from T. reesei.
these cellulases act on different parts of the cellulose
chain and complement each other in the total hydro- C. Other Fungal Cellulases
lysis of cellulose. CBH I has a rather unique action as
most exoglycanases act from the nonreducing end. Many Aspergillus spp. produce cellulases. A. aculeatus
CBH I, which is clearly the major enzyme produced produces several cellulases (33) of which EG V, EG I,
by T. reesei, has been found to be the key enzyme in and CBH I have clear similarities to EG II, EG III, and
the degradation of crystalline cellulose. Deletion of the CBH I from T. reesei, respectively (afmb.cnrs-mrs.fr/

cazy/CAZY/index.html). Noncellulolytic Aspergillus have also been studied in detail (57, 58). Several
spp., mainly A. oryzae, are often used as hosts for Streptomyces spp. have been reported to produce cellu-
transformed cellulase genes from other fungi (50). lases. These lamentous bacteria are able to secrete
Penicillium strains are also good cellulase producers. both EGs and CBHs.
P. pinophilum produces an extensive set of endogluca- Some bacteria do not produce cellulolytic enzymes
nases (13). EG II and CBH I of P. janthinellum are separately but rather as large cellulosomes (18). The
homologous to EG II and CBH I of T. reesei (51). most thoroughly studied cellulosome is from
Among anaerobic cellulolytic rumen fungi, Clostrium thermocellum. The individual cellulase com-
Neocallimastix frontalis has been studied most exten- ponents cloned from C. thermocellum are three CBHs,
sively (35). It is reported to produce a cellulosome-like 21 EGs, six xylanases, two -glucosidases, and two
protein complex containing EG, CBH, and xylanase lichenases (59). The main cellulase component is the
(52, 53). Other rumen fungi, e.g., Piromyces sp., pro- exoglucanase CelS, which alone is able to slowly hydro-
duce similar cellulase complexes. It has been speculated lyze microcrystalline cellulose, but when linked to the
that cellulases from anaerobic rumen fungi would anchoring protein containing the CBD it hydrolyzes
hydrolyze crystalline cellulose more actively than this substrate extensively (60). However, none of the
enzymes from aerobic fungi (35). bacterial CBHs characterized until now belong to the
Studies on the white rot fungi Sporotrichum pulver- family 7, but several are in the family 6 (afmb.cnrs-
ulentum (anamorph of Phanerochaete chrysosporium) mrs.fr/cazy/CAZY/index.html). Cellulosomes are
and Phanerochaete chrysosporium have revealed that also formed by the following bacteria: Bacteroides,
lignin-degrading fungi are able to produce a complete Acetivibrio, Bacillus, Butyrivibrio, Cellulomonas,
set of cellulolytic enzymes (36, 37). Four P. chrysospor- Fibrobacter, and Thermomonospora (18). Thermo-
ium CBH Is are classied into family 7 and CBH II into philic bacterium Caldocellulosiruptor saccharolyticus
family 6 (afmb.cnrs-mrs.fr/cazy/CAZY/index.html). produces an interesting multidomain enzyme that con-
Interestingly one of the CBH Is is lacking the CBD. tains two catalytic domains, EG and mannanase, and
The studies on the brown rot fungi suggest that two substrate-binding domains (17).
these fungi possess both an oxidative and hydrolytic Bacterial cellulases have in many cases been
pathway in cellulose degradation. The initial step in the reported to have much higher specic activities than
hydrolysis of crystalline cellulose is speculated to be fungal cellulases. However, bacteria generally produce
nonenzymatic. Brown rot fungi have been reported extracellular cellulases in much lower quantities than
to produce only EGs and -glucosidases (38). The fungi. In addition, several bacterial cellulases exist as
degradation of cellulose by white rot and brown rot part of a cellulosome. Therefore, their production,
fungi is different. The action of brown rot fungi is especially by using native strains, is difcult and une-
directed toward the whole cellulose microbrils, conomic at large scale compared to the production of
whereas white rot fungi attack the surfaces of the fungal cellulases. Individual bacterial cellulases have,
microbril, producing progressive erosion (54). however, been cloned and produced at an industrial
scale in more suitable host organisms. These types of
products are entering the markets, but they are still
VII. BACTERIAL CELLULASES used only in nonfood applications.
Bacteria harbor several systems for cellulose degrada-

tion. They can produce extracellular EGs and CBHs, VIII. ENDOGENOUS CELLULASES IN
cell-bound enzymes, or powerful enzyme complexes. PLANTS
For example, the following bacterial species secrete
low amounts of EGs and CBHs: Clostridium, Several plants, for example cereals, produce endogen-
Acetovibrio, Ruminococcus, Streptomyces, Microspora, ous enzymes acting on -1,4-glucosyl linkages (see
and Cellulomonas (55). Cellulomonas mi cellulases are Chapter 74). However, these enzymes are reported to
among the most extensively studied bacterial cellulases. be mainly active on mixed-linked 1,3-1,4--glucans and
Four secreted EGs (CenA, -B, -C, -D), one CBH do not possess activity on cellulose. There are also
(CbhA), and one cellulase-xylanase (Cex) have been several reports on true cellulases in plants (1, 61). So
characterized both at the biochemical and genetic levels far only EGs have been found in plants; there are no
(56). The cellulases of Actinomycetes, Corynebacteria, reports on CBHs. In contrast to fungal and bacterial
Thermomonospora fusca, and Microbispora bispora EGs, plant enzymes lack the CBD. Plant EGs have

been found to degrade only amorphous cellulose in key enzymes are CBHs possessing activity on crystal-
vitro. The exact substrate in vivo is not known, but it line cellulose. These enzymes are sometimes even able
is thought to be xyloglucan (1). Endogenous plant EGs to degrade crystalline cellulose totally on their own,
have been suggested to play a role in plant develop- but the hydrolysis is very slow (60, 68). CBHs are
ment, e.g., in fruit ripening and leaf abscission, or in thought to be processive enzymes, remaining bound
the rearrangement of polysaccharides in growing cells. to the substrate until it is totally degraded (69). They
The mode of action of EGs in cell expansion could be start their action from the ends of the cellulose chain,
either to promote wall loosening or to contribute to the liberate cellobiose, and decrease the degree of poly-
incorporation of new microbrils in the growing wall merization (DP) of the substrate very slowly. EGs
(61). There are several projects ongoing around the cleave bonds along the length of the cellulose chains,
world screening different plant genomes. Owing to resulting in a faster decrease in DP of the substrate.
fast, modern screening techniques, several new Most EGs attack only the amorphous regions of cel-
enzymes, which are able to hydrolyze cellulose, will lulose and have different specicities toward the oli-
almost surely be found in the near future. gosaccharides liberated during hydrolysis. They are,
One interesting group of proteins is the expansins, however, highly active on polymeric water-soluble -
which have been found in several plants (62). The glucans such as carboxymethyl cellulose and cereal -
action of these proteins is not well understood, but glucans, and even very low dosages of EGs reduce the
they are believed to break down the hydrogen bonds DP rapidly.
between individual cellulose chains during growth of Conventional kinetic parameters for cellulase reac-
the plant cells. Expansins are currently not classied as tions are not obtained while the substrate is changing
enzymes as they seem not to act on any covalent lin- during hydrolysis, and thus, only apparent values can
kages. One study has been carried out on the produc- be measured. Furthermore, the degradation of cellu-
tion of an expansins type protein by the fungus T. lose by individual cellulases is seldom linear, making
reesei (Markku Saloheimo, unpublished). kinetic measurements difcult. Therefore, water-solu-
ble cellulose derivatives and cello-oligosaccharides up
to DP 6 are normally used in kinetic studies for EGs
IX. ROLE OF DIFFERENT CELLULASES and CBHs, respectively.
IN THE HYDROLYSIS OF CELLULOSE Cellulases have not been reported to have any spe-
cic inhibitors. Hg compounds, which inactive many
To study the biochemical properties of cellulases, abso- glycosyl hydrolases, have been reported to affect the
lutely pure enzymes are needed. The purication of activity of CBH I more than EGI from T. reesei (70).
individual cellulases from the mixture is often very dif- End-product inhibition has often been considered as
cult even using specic afnity chromatography tech- one reason for inefcient hydrolysis. The Ki values of
niques (63). It has been shown that minor (0.4%) cellobiose for different CBHs cannot be strictly com-
contamination of an EG makes a signicant difference pared because they have been estimated using different
in the action of a CBH (64). Cross-contamination can substrates. A very low Ki value (0.02 M) has been
be avoided by gene cloning and expressing the indivi- reported for T. reesei CBH I in the hydrolysis of
dual cellulases in noncellulolytic hosts (65). Another methylumbelliferyl -glucosides (48), whereas the Ki
possibility is to delete most of the cellulase genes was 0.4 mM in the hydrolysis of cellodextrins (71).
from the cellulolytic organism and leave only the one CBH I from H. insolens is less sensitive to the end-
encoding the acceptable activity (66). The enzyme pre- product inhibition, having a Ki value of 0.65 mM in
paration used in the studies must also carefully be the hydrolysis of cellotriose (24).
characterized and conrmed to be a whole, intact pro- When the cellulase components act together in the
tein since core parts are produced by proteolysis during hydrolysis of cellulose, they enhance the efciency of
cultivation. It has clearly been demonstrated that each other signicantly (72, 73). EGs assist the action
CBDs are necessary in the hydrolysis of both crystal- of CBHs by liberating new chain ends from which
line and amorphous cellulose (Fig. 4) (67). However, CBHs can continue the hydrolysis towards more crys-
the hydrolysis of soluble substrates, which are often talline parts of the cellulose (Fig. 1). In general, only a
used in the activity assays, is not dependent on the small amount of EG is needed to improve the hydro-
presence of CBDs. lysis rate signicantly. For example, the optimal mix-
For complete hydrolysis of crystalline cellulose an ture of T. reesei EG I and CBH II in the hydrolysis of
effective mixture of different cellulases is needed. The crystalline cellulose is 2 : 8 (74). The synergism is com-

pleted by -glucosidase, which is needed for the nal X. APPLICATION OF CELLULASES IN
step in the hydrolysis of cello-oligosacchares to glu- FOOD PROCESSING
cose. The presence of -glucosidase also enhances the
action of other enzymes by diminishing the end-pro- Cellulases are not among the major enzymes used in
duct inhibition through the hydrolysis of cellobiose, food production (Table 4). In many food applications
which is a strong inhibitor especially for CBHs. cellulases are not used alone but are added to boost the
Cooperative action is mainly found between EGs and action of other enzymes, mainly pectinases and/or
CBHs, but also exo-exo synergism has been reported xylanases. As all EGs are active on 1,3-1,4--glucans,
(75). The degree of synergism is dependent on the rela- they can be used in the same applications as the more
tive amounts of the enzymes and on the substrate used. specic 1,3-1,4--glucanases e.g., lichenases and lami-
The synergy is greatest on crystalline cellulose (76). narinases (Table 1; Fig. 2). 1,3-1,4--Glucans and ara-
The synergy in general can be explained when differ- binoxylans are the main components in cereal cell walls
ently acting enzymes provide good substrates and (see Chapter 74) and 1,3-1,4--glucans exist in high
binding sites for each other by degrading different quantities especially in barley and oat (Table 5).
parts of cellulose crystals. EGs and CBHs also assist Thus, cellulases are useful enzymes in the processing
each other in the hydrolysis of soluble 1,3-1,4--glu- of cereals. Most mixed-linked -glucans are water solu-
cans. However, the synergism is much smaller than in ble and form viscous solutions. In some cases this is
the case of cellulose as EGs are alone very effective in desiredfor example, as soluble nondigestible ber
the degradation of water-soluble -glucans (see within the foodbut in other cases they create pro-
Chapter 73). blems during processing of cereal materials.
Cross-synergism between cellulases from different
organisms has also been demonstrated (76). This A. Brewing
makes it possible to optimize the mixture of supercel-
lulase components originating from different sources. Brewing is largely based on the action of endogenous
In fact, the degradation of cellulose in nature obviously barley and yeast enzymes which are activated during
proceeds by mixed microbial populations. malting, mashing, and fermentation. During malting,
Table 4 Possible Uses of Cellulases in Food and Feed
Process Action Benets
Food Baking Degradation of cell walls Softer dough

Shorter fermentation time
Increased loaf volume
Brewing Degradation of soluble -glucan Better lterability
Higher production efciency
Wine and juice production Degradation of cell walls Better extraction
Higher yield
Enhanced color and/or aroma
Extraction of oils Degradation of cell walls Better extraction
Higher yield
Extraction of starch and other Degradation of cell walls Better extraction
polysaccharides Higher yield
Upgrading agricultural residues Degradation of cellulose to Better economics
glucose Less waste production
Utilization of renewable materials
Feed Animal farming Degradation of cell walls Better release of nutrients
Enhanced feed conversion rate
Less waste
Pet animals Degradation of cell walls Better release of nutrients
Healthier animals

Table 5 Chemical Composition of Cereal Grains EG I was cloned > 15 years ago into an industrial
Starch -Glucan Xylan Protein Fat brewers yeast strain (83). The EG I secreted during
Grain (%) (%) (%) (%) (%) fermentation decreased the viscosity of beer, which
resulted in a markedly improved lterability with unal-
Wheat 6073 0.53.8 5.57.8 917 2.13.8 tered quality (84).
Barley 5367 3.010.6 4.011.0 813 0.94.6
Oat 3955 2.25.4 3.212.6 1115 5.09.0 B. Wine and Juice Production
Rye 5063 1.03.5 8.010.0 812 2.03.5
Even though the amount of -glucans in grapes and
fruits is signicantly lower than in cereals, cellulases
have also found applications in the production of
the grain polymers are mainly degraded to smaller wines and fruit juices where they are used in combina-
fragments by amylases, proteases, and -glucanases. tion with pectinases and hemicellulases. In wine mak-
The main separation problems during brewing pro- ing, the enzymes are generally used for better skin
cesses are connected to the water-soluble cell wall poly- maceration, improved color extraction, easier must
saccharides, i.e., -glucan and xylan. They both may clarication and ltration, and increased wine quality
increase viscosity, form gels, and cause serious pro- and stability (85). The use of -glucosidases for aroma
blems including poor ltration of the wort, slow run- development from naturally present glucosylated pre-
off times, low extract yields, or the development of cursors has also been described (86). Macerating
haze in the nal product. Mashing performance is enzyme cocktails with cellulases are also used in fruit
determined largely by the raw materials used. In addi- juice production. In addition, the use of these enzymes
tion to malt, alternative sources of fermentable carbo- offers advantages in the treatment of by products of
hydrates, such as corn, rice, and wheat starch from the fruit industry by improving the extraction yield and
various origins and syrups, are used in many breweries the overall process efciency. Cellulases and hemicel-
depending on the beer type, availability of starchy raw lulases can be used for the isolation of pectin from
material, and the price of the extract. There is natural citrus fruits to break down the cell walls, increasing
seasonal variation in the quality of barley and malt, the pectin yield. Cellulases can be used alone or in
due to variations in weather conditions during growth combination with other cell wall degrading enzymes
and storage. Barley cultivars also differ from each in all processes in which valuable compounds such as
other in the potential to produce endogenous enzymes juice, oil, polysaccharides, protein, etc., are extracted
during germination. from the plant material.
Plant enzymes are not very heat stable and are
mostly inactivated in kilning after germination, C. Baking
whereas most microbial enzymes can tolerate much
higher temperatures. Thus, exogenous microbial Added enzymes are used increasingly in baking for
enzymes are often used in brewing. -Glucanases (cel- improved processing and product quality.
lulases) are mainly added in the mashing (77), but they Traditionally, the main enzymes used in wheat baking
can also be used during malting (78) or after mashing are amylases and xylanases. -Glucanases (cellulases)
in primary fermentation (77). T. reesei cellulases have have gained less attention. Enzymes acting on cell walls
been found to be better than other commercial -glu- are used especially in the manufacture of high-ber
canases in both pilot and industrial-scale trials in terms bread to overcome processing problems caused by
of higher ltration rate and overall brewhouse ef- the high content of cell wall material in the dough.
ciency (79). Addition of cell wall components decreases the bread
The other option to increase the level of -gluca- volume, and the breadcrumb loses its elasticity.
nases is to enhance the level of endogenous plant Soluble, nondigestable polysaccharides, which are
enzymes by genetic engineering (see also Chapter 75) often called dietary ber, are believed to be benecial
or by cloning a microbial enzyme to barley. The rst for human health by lowering cholesterol levels (espe-
enzyme to be successfully cloned into barley was the cially soluble -glucan), preventing colon cancer, and
EG I from T. reesei (80, 81). An even more thermo- acting as specic growth substrates (prebiotics) for
stable bacterial -glucanase has later been expressed in benecial intestinal bacteria (probiotics, mainly lactic
barley (82). Another sophisticated option is to con- acid bacteria and bidobacteria) (87). Thus, breads
struct a glucanolytic brewers yeast strain. T. reesei with a high ber content are gaining growing interest.

In wheat baking, the addition of water-soluble - target polysaccharide can be further isolated.
glucans has shown to improve breadcrumb grain Cellulases can also be used for controlled degradation
(88). Treatment of the -glucans with -glucanase of some water-adsorbing polysaccharides used in food
before their addition to the bread dough resulted in processing, e.g., CMC, konjac glucomannan, and some
bread with poor crumb grain. In wheat dough supple- bacterial exopolysaccharides, such as xanthan, which
mented with rye bran, xylanases and cellulases have contain -1,4-glucosidic linkages.
been shown to make the cell wall polysaccharides
more soluble and functional (89). The added enzymes E. Animal Feed Production
made the dough softer, specic volume of the bread
and crumb structure were improved, and the staling A large quantity of cereals or various side fractions of
rate was reduced. Enzyme mixtures were found to be grains from food processing are used as animal feed. In
more efcient than individual enzymes. many cereals, part of their energy content is locked up
Wholemeal rye baking differs signicantly from in the form of nonstarch polysaccharides, undigestible
wheat baking as rye proteins do not form a gluten for several animals. Therefore, selected enzymes, such
matrix. Instead of gluten, the amounts of soluble as cellulases and hemicellulases, can be added to break
xylans and endogenous enzymes are very important down the cell walls leading to increased metabolizable
for the baking quality of rye since the gums in the energy (93). The viscous arabinoxylan and 1,3-1,4--
rye dough have an important role in stabilizing the glucan also creates difculties in the adsorption and
gas cells. The water-extractable xylans improve the digestion of nutrients, especially by monogastric ani-
baking quality by increasing dough viscosity. In rye mals such as chicken and pig. The viscosity is consid-
meal slurries -glucan and xylan extractability was ered to be an important constraint to animal digestion
increased by 90% and 40%, respectively, by EG II of by interfering with the diffusion of pancreatic enzymes,
T. reesei (90). Both xylanase and EG II from T. reesei substrates, and reaction products (94). As a conse-
are reported to make the rye dough softer, increase quence, the presence of high levels of these components
dough volume, and shorten fermentation time (91). in feed causes poor feed conversion rates, slow weight
The most marked effect of the enzymes was the reduc- gain, and wet droppings, particularly with poultry. The
tion of proong time. Cell walldegrading enzymes addition of enzymes, mainly xylanases and -gluca-
softened the bread crumb and reduced the staling nases (cellulases), in feed enhances the breakdown of
rate, but had a negative effect on oven rise. From the the corresponding polysaccharides and helps the
published reports on -glucanases in baking it appears adsorption of all feed components including proteins
that the enzymes, including EGs, are useful especially and fats, thereby improving feed conversion and
when used in combination with other baking enzymes. weight gain. It is assumed that the benecial effects
of enzyme addition are due to both reduced intestinal
D. Isolation of Starch and Other viscosity and release of nutrients from grain cells.
Polysaccharides Other benets of the cell walldegrading enzymes are
greater exibility of diet formulations, the possibility
The major application of food enzymes is in starch of using cheaper raw materials, and production of less
processing, i.e., hydrolysis of starch to glucose by amy- waste. In addition, with chickens the color of egg yolk
lolytic enzymes (see Chapters 56 and 57) and in the is better and cleaner eggs are produced.
further isomerization of glucose to high-fructose The liquid enzyme preparations are normally mixed
syrup by glucose isomerase. The starch is mainly iso- in feed before pelleting. However, the current trend is
lated from wheat or corn by milling and extraction. to use higher pelleting temperatures to eliminate
The isolation procedure can be further improved by Salmonella contamination. Thus, normal fungal cellu-
cell wall degrading enzymes, i.e., by -glucanases (cel- lases, such as those from T. reesei, may not survive the
lulases), xylanases, or their mixture (92). high temperature (95). The other option is to use
Several food hydrocolloids, mainly carrageenan, granulated enzymes or to spray liquid enzyme prepara-
alginate, and agar, are isolated from algae. After initial tions on the pellets at the cooling phase. The most
isolation steps, the products often contain some con- advanced option is to clone new enzymes directly
taminating cellulose from algae cell walls that impairs into animals. A transgenic mouse expressing a micro-
the functional properties of these polysaccharides. bial cellulase in the exocrine pancreas has been con-
Cellulases are in some cases used in further purication structed (96). The enzyme was secreted into the small
steps to degrade the residual cellulose, after which the intestine, demonstrating the feasibility of generating

nonruminant animals with a capacity to utilize cell wall raw materials in the chemical industry will increase in
polysaccharides. the future. Agricultural residues from farming and
Fungal enzymes are very suitable as feed addi- food processing, such as husks, straws, stems, brans,
tives owing to their acidic pH optimum which facil- peels, etc., are normally fed to animals. These side
itates their action in the upper intestine. Some fractions are rich in plant cell walls and could be
enzyme action already takes place during the feed upgraded with the aid of enzymes to several products
production, especially at and after the pelleting including glucose. The hydrolyzed glucose can be
stage, but mostly it occurs in the animals digestive further transformed by microorganisms to several
track. valuable products, such as ethanol for fuel or lactic
The optimal enzyme mixture varies depending on acid for bioplastics.
the animals the feed is made for. Different enzyme
cocktails are used for pigs and chickens. In addition, G. Commercial Cellulase Products
the xylanase : cellulase ratio is carefully designed for
each diet. Broilers and laying and breeding hens fed Several enzyme companies produce commercially
on diets based on barley, wheat, or wheat and barley available cellulases. The main producers are Nozymes
mixtures each have their own optimal enzyme formu- (Denmark) and Genencor International (USA). Other
lation. Postweaning young piglets can obviously bene- companies manufacturing industrial enzymes are AB
t most from enzyme supplementation to their diet. Enzymes (Germany/Finland/UK), DSM Bakery
Cellulases and xylanases are often used in combination Ingredients (Netherlands), ICI-Quest (Netherlands/
with proteases and amylases, and the cereal diet com- UK), Danisco (Denmark), Le Saffre (France), Beldem
position will determine the optimal enzyme mixture to (Belgium), Frimond (Belgium), Biocatalysts (UK),
be used (85). Adult pigs are less susceptible to viscose Stern-Enzyme (Germany), Muhlenchemie (Germany),
polysaccharides because of the longer retention time in Enzyme Bio-Systems (USA), Valley Research (USA),
their digestive system and more effective dilution of the Amano (Japan), Meiji (Japan), and Sankyo (Japan).
viscose polymer (97). However, several commercial cellulase products are
not accepted for food use. Most cellulases that are
F. Other Uses of Cellulases now marketed for food applications are mixtures
containing several EGs, CBHs, and -glucosidases, as
Clearly the largest industrial use of cellulases is outside well as many other enzymes such as xylanases, pro-
the food and feed sector in textile processing of natural teases, amylases, etc., as minor components. This
textile bers composed of cellulose (85, 98). Cellulases must be kept in mind when use of these enzymes in
have also found potential applications in the pulp and selective degradation of cellulose and/or 1,3-1,4--glu-
paper industry (99). The textile and pulp bers must can is desidered. Some puried enzymes are also avail-
retain their strength after cellulase treatment. Thus, able in small quantities mainly for research purposes
novel commercial products with altered cellulase pro- from Megazyme (Ireland) and Sigma (USA).
les have been developed. The detergent industry is the
other major user of enzymes. The main enzymes used
are proteases, but cellulases are also added in some
detergents. Enzymes are now entering dishwashing XI. CONCLUSIONS
detergents, and cellulases could also nd applications
there. Considering the signicant variability of cellulolytic
The total enzymatic hydrolysis of agricultural and enzymes and the complexity of their substrates, it can
forest residues to monosaccharides is an actively stu- be concluded that these enzymes still offer scientic
died eld. For this purpose a complete mixture of all challenges. Because of the better production techni-
individual cellulolytic enzymes is desired. The cellu- ques, complex enzyme mixtures will be in the future
lase mixtures are capable of hydrolyzing cellulose to replaced by well-dened cellulases or tailor-made
glucose with good yields, but the hydrolysis requires a enzyme cocktails, which will enable better control the
large enzyme dosage or a very long hydrolysis time process and the quality of the end product. Modern
and is therefore economically not yet competitive with genetic engineering techniques offer tools for improv-
the enzymatic production of glucose from starch ing cellulase properties to better suit different applica-
(100). However, better enzymes are being intensively tions. As the structure-function relationships in food
developed, as the use of renewable biomaterials for materials will be further understood, enzyme products

can be specically designed to achieve targeted 10. P Biely, M Vrs anska, M Claeyssens. The endo- 1,4--
changes. Cellulases are very versatile enzymes and D-glucanase from Trichoderma reesei: action on -1,4-
thus can offer practical solutions for several food oligomers derived from D-glucose and D-xylose. Eur J
applications. Biochem 200:157163, 1991.
11. B Henrissat, TT Teeri, RAJ Warren. A scheme for
designating enzymes that hydrolyse the polysacchar-
ides in the cell walls of plants. FEBS Lett 425:352354,
ACKNOWLEDGMENTS 1998.
12. B Henrissat. A classication of glycosyl hydrolases
based on amino acid sequence similarities. Biochem
We are thankful to Martin Schulein from Novozymes
J 280:309316, 1991.
for providing information on Humicola cellulases and 13. TM Woods, S McCrae, M Bhat. The mechanism of
to our colleagues Silja Home, Marjatta Salmenkallio- fungal cellulase action. Biochem J 260:3743, 1989.
Marttila, and Anu Koivula for valuable information 14. K Bronnenmeier, WL Staudenbauer. Cellulose hydro-
and comments. lysis by a highly thermostable endo-1,4--glucanase
(avicelase 1) from Clostridium stercorarum. Enzyme
Microb Technol 12:431436, 1990.
15. IUPAC. Measurement of cellulase activities. Pure
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63
b-Glucosidase
Asim Esen
Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U.S.A.
I. INTRODUCTION wall degradation in the endosperm during germination

(9); and (e) quality enhancement in wine, fruit juices
-Glucosidase (-D-glucoside glucohydrolase, EC (10; Chapter 21), and tea (12). -Glucosidases hydro-
3.2.1.21) catalyzes the hydrolysis of the -glycosidic lyze either O-linked -glycosidic bonds, as is done by
bond between two glycone residues (e.g., cellobiose -glucosidases (-D-glucoside glucohydrolase, EC
and other -linked oligosaccharides) or that between 3.2.1.21), or S-linked -glycosidic bonds, as is done
glucose and an aryl or alkyl aglycone (e.g., many natu- by -thioglucosidases (myrosinase, or -D-thiogluco-
rally occurring substrates in plants). The enzyme con- side glucohydrolase, EC 3.2.3.1). Thus, specic physio-
stitutes a major group among glycoside hydrolases and logical functions and multiplicity of -glycosidases in
occurs universally in all three domains (Eukarya, an organism depend on the nature and diversity of the
Archaea, and Eubacteria) of living organisms. A aglycone (aryl or alkyl) or glycone (Glc, Gal, Fuc, Xyl)
nomenclature system classifying glycosyl hydrolases moiety of their substrates.
into 82 families based on amino acid sequence simila-
rities has been proposed (13) and is now widely used.
A continuously updated server giving access to these II. USE OF b-GLUCOSIDASES IN FOOD
families is available at URL://http://afmb.cnrs-mrs.fr/ PROCESSING AND QUALITY
pedro/CAZY/db.html. Under this classication ENHANCEMENT
scheme, -glucosidases belong to families 1 and 3 of
the clan GH-A. Unless otherwise stated, this chapters The importance of -glycosidases to food quality and
emphasis will be family 1 enzymes. processing is ably reviewed and discussed by Gunata
-Glucosidases have been the focus of much (see Chapter 21) with emphasis on avor enhancement
research recently because of their key roles in a variety in fruit juices and various beverages derived from
of fundamental biological (e.g., growth and develop- them. Therefore, the reader is referred to Chapter 21
ment, chemical defense, hostparasite interactions, cel- for more information on this aspect. There are several
lulolysis, lignication, glycosylceramide and vitamin hundred different -glucosidic avor precursors iden-
B6 metabolism, signaling, etc.) and biotechnological tied from plants whose aglycones are products of
(biomass conversion, food detoxication, and beverage mevalonate or shikimate pathways. Obviously, there
quality enhancement) processes. For example, plant - are -glucosidases in source plant tissues that hydro-
glycosidases have been either implicated or shown to lyze these avor precursors. Thus, in each case there is
be involved in (a) defense against pests (46); (b) phy- need for isolating and characterizing the specic
tohormone activation (7); (c) lignication (8); (d) cell enzyme that hydrolyzes a -glucoside whose aglycone

moiety is of interest to food quality and processing. origin associated with glucosinolates, direct ingestion
Such biochemical data are crucial to making practical of large amounts of cruciferous vegetables is thought
decisions as to whether or not enzymes from host to cause endemic goiter in man, as well as toxicity in
plants or other sources should be added to drinks laboratory animals. Similarly, claims have been made
and beverages before, during, or after processing to on anticarcinogenic effects of glucosinolates and their
enhance avor, aroma, and other quality factors. breakdown products in humans. Although the precise
Likewise, such data are essential for targeting enzymes mechanism of action is not clear, studies on rodents
with desirable properties for overproduction in trans- showed that raw or cooked cruciferous vegetables (e.g.,
genic microbial or plant hosts and improvement of cabbage, broccoli, turnip, and cauliower) increased
their catalytic properties and stability for specic uses aryl hydrocarbon hydroxylase activity (14).
by genetic engineering.
Another aspect of -glucosidases that pertains to
food processing and quality is that edible portions of
III. BIOCHEMISTRY
some plants contain compartmentalized -glucosidase-
-glucoside systems that produce toxic aglycones and/ A. Structure
or HCN when tissue is macerated during preparation
or by chewing. This is exemplied by cassava roots and Family 1 -glucosidase monomers have molecular
leaves, lima beans, and ax seeds. Of these, cassava is a weights ranging from 55 to 65 kDa as estimated by
food staple in tropical regions of Africa, Asia, and SDS-PAGE, which is consistent with the determined
South America; consumption reaches about 1 kg/per polypeptide lengths ranging from 447 (e.g., Bacillus
capita per day in some parts of Africa (e.g., Congo). polymyxia) to 527 (e.g., white mustard myrosinase)
It contains the cyanogenic -glucoside linamarin and amino acids long, depending on source organism of
the corresponding -glucosidase linamarase. When the enzyme. Not surprisingly, the longest polypeptide
consumed raw, cyanide poisoning can occur depending chains are found in eukaryotic enzymes while the
on the amount ingested, where symptoms are difculty shortest ones are in eubacteria and archaeabacteria
in breathing, paralysis, convulsion, coma, and even (Fig. 1). All -glucosidases isolated from animals and
death. Similarly, certain processing methods (e.g., dicotyledonous plants (except indigo plant) so far are
maceration of roots followed by washing) remove the glycosylated and thus have estimated monomeric
components and products of cyanogenesis as well as molecular weights 35 kDa larger than those calcu-
many nutritional factors, thus reducing quality. lated from sequences of mature polypeptide chains
Cooking inactivates the enzyme and eliminates the deduced from cDNA or genomic DNA sequences.
possibility of cyanogenesis. Similar symptoms can The catalytically active form of -glucosidases is a
arise when bitter almonds are eaten and ingested with- homodimer of 120 kDa or its multimers. The
out roasting. amino acids and sites that contribute to the dimer
The myrosinaseglucosinolate (or -thioglucosi- interface are not well conserved based on crystallo-
dase-thioglucoside) system, which occurs in crucifer- graphic data from eight enzymes (see below). High-
ous vegetables (e.g., mustard, cabbage, kale, broccoli, molecular-weight quaternary complexes of -glucosi-
rapeseed, horseradish, etc.), also has importance for dases are either homocomplexes differing from each
food quality and processing because the aglycone moi- other by a dimer or its multiples as is the case for
ety and its breakdown products from enzymatic hydro- example with avenacosidase from oats, or heterocom-
lysis of glucosinolates are responsible for the bitter, plexes formed between -glucosidase dimers and cer-
pungent taste and aroma associated with these vegeta- tain specic proteins associated with them. For
bles, as well as the processed foods and relishes that example, in maize -glucosidase forms large, insoluble
include them (13). The distinct avor associated with or poorly soluble aggregates resulting from a specic
glucosinolates comes primarily from isothiocyanates interaction between the enzyme and a 32-kDa small
and is believed to have evolved to serve as a repellent heat shock protein, which is expressed at elevated
against microorganisms and herbivores. Gluco- levels in certain maize inbred lines (15, 16). Similarly,
sinolates and their breakdown products may impart high-molecular-weight aggregates of -glucosidases
undesirable avors to milk, meat, and eggs when have been described from oat (17), ax (18), and cab-
farm animals graze on cruciferous plants or when bage (19) where in the case of cabbage the aggregates
their feed includes seed meals from such plants. were shown to be due to interaction with specic myr-
Besides off-odors and off-avors in foods of animal osinase-binding proteins.

Figure 1 Sequence alignment of family 1 -glycosidases from representative species of three organismic domains (Eukarya,
Archaea, and Eubacteria). TR-1CBG, white clover (Trifolium repens L.) linamarase; SA-1MYR, white mustard (Sinapis alba)
myrosinase; ZMGlu1, maize (Zea mays L.) Glu1; CP-Glu, guinea pig (Cavia porcellus) cytosolic -glucosidase; BP-1BGA,
Bacillus polymyxa -glucosidase; BC-1QOX, Bacillus circulans sp. alkalophilus -glucosidase; SS-1GOW, Sulfolobus solfataricus
-glucosidase; and TA-1QVB, Thermosphaera aggregans -glucosidase. Regions with black background show the universally
conserved sites, whereas regions with dark gray and light gray show highly conserved and moderately conserved sites, respec-
tively. Regions with white background show variable sites, while dashes indicate the gaps (deletions) that the alignment software
(ClustalW) introduced to optimize the alignment. The alignment was produced with ClustalW and formated with Boxshade, both
of which are free downloads from the Web.

The primary structure of each family 1 -glucosi- while blotting techniques are limited to those genes
dase monomer contains the highly conserved peptide or mRNAs that have sufcient sequence similarity to
motifs SAYQI, YRFSI, TFNEP, LGLNYY, the probe sequence. Therefore, the number of isoforms
YITENG, and DNFEW, which serve as ngerprints detected is very likely to be lower than the number of
to identify an unknown protein as a family 1 -gluco- those that actually exist. Indeed, the data from genome
sidase (Fig. 1). Of these, TFNEP and YITENG are the sequencing projects appear to support this conclusion.
most conserved; they make up a part of the active site For example, Arabidopsis thaliana genome sequence
and contain the two catalytic glutamates (2023). data so far indicate that there are at least 40 different
Furthermore, the 3D structures of eight family I - genes encoding family 1 -glucosidase in this plant,
glycosidases [1CBG, white clover (Trifolium repens) which has the smallest and most streamlined genome
linamarase; 1MYR, white mustard (Sinapis alba) myr- among plants. Whether or not all of these putative
osinase; ZMGlu1, maize (Zea mays L., see Fig. 2); genes are actually expressed and code for a functional
1BGA, Bacillus polymyxa -glucosidase; 1QOX, enzyme remains to be shown by functional genomics
Bacillus circulans sp. alkalophilus -glucosidase; studies. However, it would not be too surprising if
1PBG, Lactococcus lactis phospho-galactosidase; there are more -glucosidase isoforms than expected
1GOW, Sulfolobus solfataricus -glucosidase; and in view of the possibility that there may be tens of
1QVB, Thermosphaera aggregans -glucosidase] have different physiological substrates (-glucosides or glu-
recently been solved (2027). All eight enzymes have coconjugates) produced during the life of, especially, a
essentially the same basic (=8 -barrel fold structure multicellular organism in specic tissues, organs, devel-
(Fig. 2) although they share only 1763% sequence opmental stages, and environmental stresses. Some if
identity (Fig. 1). It is remarkable that the basic cataly- not all of these substrates may have a corresponding
tic machinery, reaction mechanism, and 3D structure specic -glucosidase isoform hydrolyzing them under
have been rigidly retained in the three domains of liv- specic conditions and with specic kinetics.
ing organisms in spite of large amounts of sequence In addition to genetically determined -glucosidase
divergence through eons of evolution. multiplicity that can be demonstrated by DNA sequen-
cing and blotting, there are many examples of -glu-
cosidase multiplicity generated by posttranslational
B. Multiple Molecular Forms (Isoforms) modications (e.g., glycosylation and proteolytic mod-
ication in vitro) during isolation and subsequent
-Glucosidase is ubiquitous, and thus a given organism manipulations of enzyme preparations. For example,
is expected to have at least one molecular form of the as many as six different forms of the maize -glucosi-
enzyme. When the enzyme occurs in multiple molecu- dase isozyme Glu1 can be detected on zymograms in a
lar forms (isoforms) as detected by electrophoretic time- and pH-dependent manner. Five of these bands
mobility or ion exchange chromatography, typically are artifactual, and they result from the activity of an
two or three forms are observed whose existence is endogenous cysteine protease in buffers of pH 56 con-
also conrmed by molecular biological techniques taining a reducing agent such as 2-ME (28). These data
(e.g., Southern blotting and Northern blotting) and suggest that multiple forms of -glucosidase detected
DNA sequencing. However, electrophoretic and chro- on zymograms or after chromatography should not be
matographic detections are limited to those enzymes interpreted as being distinct isozymes or allozymes
that hydrolyze the test substrate used for detection without ruling out articial heterogeneity due to pro-
teolysis and differential posttranslational modication
on the same polypeptide chain.

Abbreviations: AH, amygdalin hydrolase; AS, ammonium
sulfate; 6BNGlc, 6-bromo-2-naphthyl--D-glucoside; C. Catalytic Site and Machinery
DIMBOA, 2,4-dihydroxy-7methoxy-1,4-benzoxazin-3-one;
DIMBOAGlc, 2-O--D-glucopyranosyl-4-hydroxy-7-meth-
All family 1 -glycosidases are retaining enzymes in
oxy-1,4-benzoxazin-3-one; HIC, hydrophobic interaction
that the anomeric conguration of the glycone (e.g.,
chromatography; IEF, isoelectric focusing; 2-ME, 2-mercap-
toethanol; 4MUGlc, 4-methylumbelliferyl--D-glucoside; glucose) is the same in the product (-D-glucose) as
NP, nitrophenol; NPGlc, nitrophenyl glucoside; PAGE, it was in the substrate (a -D-glucoside). Substrate
polyacrylamide gel electrophoresis; PH, prunasin hydrolase; hydrolysis involves two steps (enzyme glycosylation
SB, Sorghum bicolor; SDS, sodium dodecyl sulfate; Xglc, 5- and deglycosylation) and requires participation of
bromo-4-chloro-3-indolyl--D-glucoside; ZM, Zea mays. two specic glutamic acid residues in the active site.

Figure 2 Ribbon diagram representation of the tertiary structure of the maize -glucosidase Glu1 monomer, showing the
catalytic glutamates E191 and E406, four residues (F198, F205, W378, and F466) forming the aglycone-binding pocket, and
two other residues (A467 and Y473) that are probably important for aglycone recognition. Note the (=8 fold barrel structure
with alternating -strands and -helices and the four loops surrounding and contributing to the structure of the active site.
Different colors and the color transitions in -helices and -strands in the original diagram trace the polypeptide backbone in the
barrel-shaped 3-D structure from the N-terminal to C-terminal direction. The gure was produced with the softwares
MOLSCRIPT and Raster3D.
In the glycosylation step, the nucleophilic glutamate the nucleophilic glutamate residue. In all structures
residue in the motif YI/VTENG attacks at the anome- except myrosinase, a -S-glucosidase, the residues of
ric carbon (C1 ) of the substrate and forms a covalent the TFNEP and I/VTENG motifs are involved in gly-
glycosyl-enzyme intermediate with concomitant release cone binding and catalysis within a crater-shaped
of the aglycone after protonation of the glycosidic oxy- active site (31). The two catalytic glutamic acid resi-
gen by the acid catalyst glutamic acid residue in the dues (i.e., the nucleophile and the acid/base catalyst)
motif (T(F/L/M)NEP (29, 30). In the deglycosylation are positioned within the active site at expected dis-
step, the second catalytic glutamate residue, now an tances ( 5:5 A) (Fig. 2). In myrosinases, the motif
anion and a base catalyst, removes a proton from that is homologous to TFNEP of -O-glucosidases is
water, and the resulting OH group performs a nucleo- TINQL, in which the acid/base catalyst glutamic acid
philic attack on the covalent bond between the glycone residue is replaced by a glutamine residue. There is no
and the enzyme, releasing the glycone and regenerating need for protonic assistance for aglycone departure in

myrosinase, as the aglycone of -glucosinolates is, by As for the so-called broad-specicity plant -gluco-
its chemical nature, an excellent leaving group. sidase, such as almond emulsin, Poulton and associates
(37) showed that the black cherry homolog of the
emulsin actually includes two distinct -glucosidases,
D. Substrate Specicity each with multiple forms and catalyzing different steps
in amygdalin hydrolysis. One of these enzymes is
Substrate specicity in -glycosidases literally means amygdalin hydrolase (AH), and it has four isozymes
specicity for the chemical group attached to glycone and hydrolyzes amygdalin (mandelonitrile -1,6-gen-
through -glycosidic linkage since the glycone moiety tiobioside) to prunasin (mandelonitrile -Glc) and glu-
in a -glycosidic substrate is an invariant monosac- cose. The second enzyme is prunasin hydrolase (PH),
charide (e.g., glucose in O- and S-linked -glucosides). and it has three isozymes, each hydrolyzes prunasin to
The group attached to glucose is either another gly- glucose and mandelonitrile. Both AH and PH hydro-
cone as in -linked disaccharides (e.g., cellobiose) lyze only their own natural substrates in vivo, and each
and oligosaccharides or an aglycone as in glucoconju- shows varying levels of activity on articial substrates.
gates. The aglycone moiety of the substrate is either an The above-cited examples support Conns conclu-
alkyl group (e.g., linamarin) or an aryl group (e.g., sion (33) that plant -glycosidases exhibit narrow spe-
prunasin, dhurrin, and DIMBOAGlc). The subtle sub- cicity when one uses puried enzyme in assays. Conn
strate specicity differences exhibited by -glucosi- also points out that almost every research paper deal-
dases in general and plant -glucosidases in ing with plant -glucosidases used an NPGlc as sub-
particular, and the importance of the aglycone moiety strate. In some studies, both articial and natural
in determining the specicity of -glycosidases for their substrates were used during enzyme purication
physiological substrates, have been well documented in while in others either a physiological substrate was
the literature and reviewed (32, 33). The authors cited not known or it was unavailable. In such cases two
many cases of subtle substrate specicities and urged unfortunate outcomes are possible: (a) purifying and
the use of physiological substrates during enzyme pur- describing an enzyme that has no activity on an abun-
ication and characterization. dant natural substrate, and (b) failing completely to
Conn (33) explained the historical reasons for the detect a physiologically relevant -glucosidase that
inaccurate perception that plant -glycosidases have does not hydrolyze the articial substrate used in the
broad substrate specicity and cited as examples the assay. Thus, the broad substrate specicity often
popular plant -glucosidase almond emulsin and crude attributed to -glucosidases does not mean that the
plant extracts. Both of these sources contain mixtures enzymes catalyze the hydrolysis of a large number of
of enzymes with -glucosidase activity and exhibit physiological substrates in vivo. Rather, it means that
broad specicity on articial chromogenic (e.g., nitro- a large number of test substrates whose aglycone struc-
phenyl--D-glucosides) or uorogenic (e.g., 4MUG1c) ture is similar to that of the physiological substrate can
substrates. However, examples of narrow substrate be hydrolyzed by an enzyme.
specicity can be demonstrated if one uses puried In fact, even some nonphysiological substrates may
enzyme in assays. Two such examples are -glucosi- be hydrolyzed with a much higher catalytic efciency
dases (also called dhurrinase) of sorghum and the than the physiological substrate. This is because the
Glu2 isozyme of maize whose primary structures nonphysiological substrate may have better shape
have been determined in the authors laboratory (34, and functional group complementarity for binding
35). In the rst case, one isozyme (Dhr1) represents an with high afnity and its aglycone moiety may be a
extreme; it hydrolyzes only the natural substrate dhur- better leaving group in the glycosylation step of the
rin and shows no activity toward other aryl or alkyl - reaction than that of the physiological substrate. For
glucosides, including the popular articial substrate example, the maize -glucosidase isozyme Glu1 hydro-
pNPGlc. The second dhurrinase isozyme (Dhr2) also lyzes such natural and articial substrates with aryl
shows narrow specicity for dhurrin but it also hydro- aglycone moieties as p- and o-NP, 4-methylumbelli-
lyzes pNPGl1 and 4MUGlc, albeit at a very low rate feryl, 6-bromo-2-naphthyl, indoxyl, 5-bromo-4-
(35, 36). Similarly, the maize Glu2 isozyme hydrolyzes chloro-3-indolyl, and cytokinin in addition to its abun-
the natural substrate DIMBOAGlc with the same ef- dant natural substrate DIMBOAGlc. Moreover, the
ciency as does the other maize isozyme (Glu1) but maize enzyme hydrolyzes pNP-fucosides about 10
hydrolyzes poorly or not at all other substrates that times more efciently than pNP-glucosides. However,
are readily hydrolyzed by the Glu1 isozyme. none of these substrates hydrolyzed by the enzyme

except DIMBOAGlc occur in maize. The only thing nor within the extreme 47-amino-acid-long C-terminal
they have in common with the natural substrate is an domain of ZMGlu1.
aryl aglycone moiety that mimics DIMBOA. In fact, a Questions about the mechanism and the site of sub-
research group (38) isolated Glu1 as an auxin-binding strate (i.e., aglycone) specicity and afnity could be
protein using an auxin analog as an afnity matrix directly addressed if it were possible to crystallize the
because the enzyme was bound to the matrix owing enzyme-aglycone, inactive enzyme-substrate, or
to the similarity of the auxin analog to the aglycone enzyme-unhydrolyzed competitive inhibitor complexes
(DIMBOA) of the natural substrate. and identify the residues that are interacting with the
A simple and effective assay to search for natural aglycone in the 3D structure. Recently, the authors
substrates of -glucosidases is to prepare an alcohol laboratory in collaboration with that of Bernard
extract of the tissue of interest, concentrate, and frac- Henrissat in Marseille (AFMB-CNRS) was able to
tionate it over a column (e.g., Sephadex LH-20) in directly investigate the mechanism and the site of sub-
70% methanol. The resulting fractions are then dried strate (i.e., aglycone) recognition and specicity in
and tested with a crude protein extract of the same maize -glucosidase by x-ray crystallography using
tissue for glucose production, by a coupled PGO co-crystals of a catalytically inactive mutant
assay (see below) using appropriate controls. Once glu- (Glu1E191D) in complex with the natural substrate
cose production has been detected and conrmed in a DIMBOAGlc, the free aglycone DIMBOA, and unhy-
fraction or crude organic extract, one can purify and drolyzed competitive inhibitor para-hydroxy-S-mande-
identify the source glucoside by further analytical stu- lonitril -glucoside (dhurrin). The structures of these
dies. Similarly, one can purify and identify the -glu- complexes and uncomplexed mutant were solved at
cosidase that is hydrolyzing an isolated and identied 2.0, 2.1, and 2.2 A resolution, respectively. The struc-
physiological substrate by subjecting crude protein tural data from the complexes for the rst time allowed
extracts of the same tissue or organ to purication by us to visualize an intact substrate, free aglycone, or an
conventional biochemical procedures. unhydrolyzed competitive inhibitor in the slotlike
active site of a -glucosidase (42). These data show
that the aglycone moiety of the substrate is sandwiched
E. Mechanism of Substrate Specicity between W378 on one side and F198, F205, and F466
on the other. Thus, specic conformations of these
Much progress has been made in understanding the four hydrophobic amino acids and the shape of the
mechanism of catalysis and dening the roles of the aglycone binding site they form determine aglycone
two catalytic glutamates within the active site that recognition and substrate specicity in maize
are involved in catalysis (29, 30, 39, 40). However, ZMGlu1. In addition to these four residues, A467
until recently, there was little or no information as to interacts with the 7-methoxy group of DIMBOA. All
how -glucosidases recognize and interact with their but W378 of these sites are variable among -glucosi-
substrates, specically the aglycone moiety, which is dases that differ in substrate specicity, supporting the
the basis of tremendous diversity in natural substrates conclusion that these sites are the basis of aglycone
and is responsible for the subtle substrate specicity recognition and binding (i.e., aglycone substrate speci-
differences among -glucosidases. None of the seven city) in -glucosidases. The data also provide a plau-
structures published on family 1 enzymes contained sible explanation for the competitive binding of
either an intact substrate or an aglycone product to dhurrin to maize -glucosidases with high afnity
address the issue of substrate specicity. without being hydrolyzed.
Studies with reciprocal ZMGlu1/SBDhr1 chimeric
enzymes indicated that the aglycone (i.e., substrate) F. Kinetic Constants
specicity determining sites are different in maize -
glucosidase isozyme ZMGlu1 and sorghum -glucosi- -Glucosidases show variable afnity toward -gluco-
dase isozyme SBDhr1 (41). These data show that spe- sides. The Km values for good substrates, including
cicity for dhurrin hydrolysis residues in a C-terminal natural substrates, are typically 1 mM or less.
region octapeptide (462 SSGYTERF469 ) of SBDhr1 Similarly, -glucosidases have, relatively speaking,
where SBDhr1 and ZMGlu1 sequences differ from low kcat values ( 300 s1 or lower). It is believed
each other by four amino acid substitutions, while spe- that the physiological function of these enzymes for
cicity for DIMBOAGlc hydrolysis is not within the the cell dictates slow hydrolysis of the substrates rather
ZMGlu1 homolog of the aforementioned octapeptide than bursts and natural selection favored such slow

rate of hydrolysis. The best way to compare substrate (45). Similarly, the presence of Mn2 in the crystal struc-
specicities and afnities is to compare the kcat =Km ture of myrosinase suggests that this divalent cation
(efciency coefcient) values, as two substrates with might be required for activity by myrosinases.
similar Km values may yield as much as an order of
magnitude differences in catalytic efciency owing to H. Heat and pH Stability and Resistance to
the aglycone moiety (better or worse as a leaving Proteases
group) or the glycone moiety. It is obvious that high
kcat and low Km values would increase catalytic ef- -Glucosidases are stable in the pH range 410 at 0
ciency. When substrates differ with respect to leaving- 4 C, with highest stability at pH 7. The sources of
group ability of their aglycones, the rate-limiting or instability during storage are pH extremes, co-purifying
enhancing step will be the glycosylation reaction. If proteases, and microbial contamination. Although
they differ with respect to the glycone moiety, either most -glucosidases are extremely resistant to pro-
glycosylation or deglycosylation step, or both, would teases because of their tightly folded core structure,
be rate limiting. For example, maize -glucosidases which exposes only the extreme N- and C-terminal
(along with most other -glucosidases) hydrolyze p- regions to proteases, they are slowly degraded by con-
nitrophenyl -fucoside 10 times more efciently than taminating or co-purifying proteases during prolonged
pNPGlc although their Km values are similar. The dif- storage without protease inhibitors in the medium. For
ference in this case is clearly apparent in Vmax values, example, maize Glu1 and almond emulsin lose only
although how the fucopyranosyl moiety enhances the 1015% of their activity after 18 h of exposure to
reaction rate is not known. one of the most potent proteases (proteinase K;
Esen, unpublished) at room temperature. Under simi-
G. Inhibitors and Cofactors lar conditions, almost all of the other proteins in a
crude extract are digested. -Glucosidases are resistant
-Glucosidases are inhibited by transition state sugar to denaturation by ionic detergents such as SDS, which
analogs (e.g., -gluconolactone), substrate analogue allows extraction with buffers containing up to 3%
glucosides, free aglycones of their substrates, and sub- SDS and zymogram development after SDS-PAGE
strate analogs. Since the active site has distinct agly- when samples are applied without heating.
cone- and glycone-binding pockets, sugars and sugar As for thermostability, mesophilic -glucosidases are
analogs with half-chair conformation can bind to the irreversibly inactivated at and above 5560 C, depend-
glycone-binding site and inhibit the enzyme, as can free ing on the source, although they may show highest cata-
aglycones, and the aglycone moiety of substrate ana- lytic activity at 5055 C (43). Increased catalytic activity
logs bind to the aglycone-binding site. at such high temperatures as 5055 C is not physiologi-
Free glucose is a poor inhibitor Ki 100200 mM) cally and practically meaningful if the half-life of the
because glucose must be in a half-chair conformation activity is only 10 min or so due to thermal denaturation
for binding to the glycone binding site, which it and inactivation, as is the case with maize -glucosidase
acquires after the aglycone moiety binds to the agly- (43). However, -glucosidases of thermophilic bacteria
cone-binding site and distorts the conformation of the (e.g., Thermosphaera spp. and Sulpholubus spp.) have
attached glucose to half-chair. In contrast, free agly- temperature optima 85 C (46). The basis of this ther-
cones are potent competitive inhibitors because their mostability is attributed to the increase in the number of
ground-state conformation is sufcient to recognize proline residues, internal water molecules, internal and
and bind to the aglycone binding site. surface electrostatic bridges, and the decrease in the sol-
Metal ions, primarily Ag and Hg2 , are also potent vent-accessible surface area by a tetrameric quaternary
-glucosidase inhibitors. The inhibition can be comple- structure as opposed to the dimeric structure of meso-
tely reversed by reducing agents such as 2-ME (43). The philic enzymes (25).
target of these cations on the enzyme is not known
although the catalytic nucleophile glutamate is a poten-
tial target. There are also reports of inhibition by Cu2 IV. ASSAYS FOR b-GLUCOSIDASE
and Fe3 (44). A complete listing of -glucosidase inhi- ACTIVITY
bitors is given in Zollner (44). There is no well-documen-
ted case of a cofactor requirement for a -O- There are well-established procedures for assay of -
glucosidase, whereas the requirement of ascorbate for glucosidase activity, and they can be used to determine
activity of -S-glucosidases (myrosinases) is well known not only whether or not a crude tissue extract or sam-

ple has enzyme activity, but also to quantify the time, and add it to the wells that had received 70 L
amount of enzyme activity present in a preparation. of enzyme solution (Solution 4) or Buffer 4 before.
To this end, solution assays utilizing a substrate 4. Place in the incubator and shake at 50 rpm until
whose aglycone moiety is either chromogenic or exactly 5 min have elapsed from the time of substrate
uorogenic are desirable. Almost all published solution addition.
assays use the commercially available nitrophenyl glu- 5. Remove the plate, add to each well 70 L of
cosides as substrate and commercially available Solution 3, and mix gently.
almond emulsin as enzyme source. The assay can be 6. Read the absorbance at 410 nm in the micro-
performed in cuvettes or in 96-well microtiter plates plate reader.
and the absorbance of the nitrophenolate ion 7. Compute the mean and standard deviation of
(pKa 7:16) can be read in a spectrophotometer or the four values for each sample (see step 8).
microtiter plate reader for quantication. A sample 8. Express (calculate) the enzyme activity as below.
solution assay protocol for the microtiter plate format One unit of enzyme activity is dened as the amount
is give below. This procedure can be scaled up for test of enzyme that hydrolyzes 1 mole of pNPG/h at
tube or cuvette format. 25 C. It has been determined that 1:69 g (or
0.0121 mole) pNP in 210 L of volume in a 96-well
A. Spectrophotometric Assays microplate reader well gives an absorbance of 1000 at
410 nm. Example: A tissue extract, after diluting 100,
The following is a solution assay procedure that is yielded 0:711 A410 units of -glucosidase activity in 5
applicable to any family 1 -glucosidase after changing min at 25 C.
the pH of the buffer to one that is optimum for the Enzyme activity a b c d 0:0121 0:711
enzyme of interest. All reagent chemicals and sub- 100 12 10:32 U/h/70 L of original enzyme solu-
strates mentioned are available from Sigma Chemical tion, or 10:32 1000=70 147:5 U/mL.
Company. The solutions needed are: Specic activity 147:5=1:5 98:3 U/mg protein if
the protein content of the original extract is 1.5 mg/mL.
1. 50 mM citrate 100 mM phosphate buffer, pH
To calculate the amount of pNP produced from
5.8 (also known as McIlvaine Buffer). Titrate
pNPG by -glucosidase activity, it is best to prepare
100 mM citric acid solution with 200 mM
serial dilutions of pNP, read their absorbance in the
Na2 HPO4 , until pH is 5.8 (Buffer 1).
volume to be used in enzyme assays, and convert the
2. 5 mM p-nitrophenyl--D-glucopyranoside
reading to the amount of the pNP (moles) from the
(pNPGlc) prepared in Buffer 1.
extinction coefcient (
410 18,300 M1 cm1 ) (47) of
3. 0.4 M sodium carbonate (Na2 CO3 ) prepared in
the nitrophenolate ion.
water (stored at room temperature).
The above spectrophotometric assay may be chan-
4. Tissue extracts or puried -glucosidase pre-
ged to a uorometric assay, for even higher sensitivity,
parations, diluted with Buffer 1 to yield 0.1
using the uorogenic substrate 4MUGlc and a uo-
1.0 A410 units of activity in 5 min at 25 C.
rometer. Although most -glucosidases hydrolyze
(Initially, tissue extracts may be diluted 10- to
pNPGlc and 4MUGlc, albeit at varying rates, some
100-fold in Buffer 1 and assayed to decide the
such as dhurrinase-1 of sorghum may not have any
appropriate dilution for quantitative assay.)
detectable activity on these popular articial sub-
The procedure is as follows. strates. Therefore, an assay measuring the amount of
1. Add 70 L of enzyme solution (Solution 4) to glucose rather than the aglycone is desirable to detect
the wells of a 96-well microtiter plate in quadruplicate. the activity of a known or unknown enzyme on known
Add 70 L Buffer 1 to four blank wells (this is a minus or unknown natural substrates that are not chromo-
enzyme control). Place the plate on rotary shaker-incu- genic or uorogenic. Coupling -glucosidase assay to
bator set at 25 C for temperature equilibration ( 5 glucose oxidase/peroxidase assay meets the require-
min). ment of measuring glucose produced by -glucosidase
2. Pour sufcient volume of Solution 2 (substrate) activity by oxidizing glucose to -gluconolactone by
in a 25-mL pipetting reservoir and leave it at 25 C for
5 min to equilibrate to room temperature.
a, Amount of nitrophenolate (0.0121 mole) yielding
3. Draw 70 L of Solution 2 (substrate) with a 1:0 A410 ; b, measured A410 (0.711); c, enzyme dilution factor
multichannel pipette (if available, otherwise use a sin- (100); d, time multiplication factor (12) to convert assay time
gle-channel micropipette) from the trough, mark the (5 min) to 1 h.

glucose oxidase. The latter reaction produces hydrogen fuge tube. Usually 1 mL of 1 mg/mL solution
peroxide, which is used by peroxidase as a hydrogen will be sufcient for two gels.
acceptor to oxidize a chromogenic peroxidase sub- 4. Fast Blue BB salt. Dissolve 150 mg in 200 mL of
strate 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic 50 mM citrate100 mM phosphate buffer, pH
acid) (ABTS). The amount of oxidized peroxidase pro- 5.8, in a 250-mL ask by stirring.
duct is determined by measuring the absorbance and 5. Fixative. Acetic acid methanol water mixed in
relating it to equivalent amount of glucose using a 1 : 1 : 5 ratio. Store at room temperature.
glucose calibration curve.
Alternative substrate: 1 mM 4-methylumbelliferyl-
B. Solid-Phase Assays (Dot Blots and -D-glucoside (4 MUGlc) in 50 mM citrate100 mM
Zymograms) phosphate buffer, pH 5.8. This substrate is more sen-
sitive and detects more -glucosidases than 6BNGlc,
Solid-phase assays are performed in the dot-blot for- but gels cannot be stored and dried. One needs to
mat using nitrocellulose, polyvinyldene diuoride photograph the gel within 520 min under UV light.
(PVDF), or ordinary chromatography paper. The test The procedure is as follows.
protein solutions are directly spotted on the matrix 1. Place the gel in a glass or clear plastic tray after
(sheet or strips) and incubated with a substrate electrophoresis or isoelectric focusing. Equilibrate
whose aglycone forms an insoluble colored or uoro- in two changes of 50 mM citrate100 mM phosphate
genic spot at the site of enzyme activity. The most buffer, pH 5.8 15 min each, at room temperature.
suitable chromogenic substrates for spot assay are 2. Pour off the last (second) change of the equili-
6BNGlc and XGlc. The aglycone of 4MUGlc may be bration buffer and then add freshly mixed substrate-
detected by viewing under a UV lamp or on a UV box coupling dye solution onto the gel in the dish. For this,
within the rst 5 min or so, but it is soluble and cannot add the substrate solution to the Fast Blue RR salt
be xed and detected later. solution, mix in ask, and then pour onto the gel.
Make sure that there is enough solution to cover the
gel and permit free movement of the gel in solution
C. Detection of -Glucosidase Activity on during incubation and shaking.
Polyacrylamide Gels 3. Cover the tray with Saran wrap and aluminium
foil; incubate in a shaker-incubator (set at 50 rpm) in
Zymogram assays are also routinely used for detec- the dark at 37 C for 216 h.
tion of the presence and multiplicity of -glucosidase 4. Stop the shaker-incubator at intervals, remove
activity after native polyacrylamide gel electrophor- foil, and check for the appearance and intensity of
esis (PAGE), isoelectric focusing gel (IEF), or sodium enzyme bands. If no bands develop or they are too
dodecyl sulfatepolyacrylamide gel electrophoresis light after 2 h, it is best to continue incubation over-
(SDS-PAGE) under nondenaturing conditions. A night.
sample protocol for native PAGE is given. The 5. When enzyme bands develop to a desired inten-
same protocol can be used for IEF and SDS gels if sity and resolution, pour off the substrate-dye coupling
samples are applied to SDS gels without boiling. The solution, and rinse the gel several times with water in
following is a gel assay procedure that is applicable to order to wash away the precipitated dye-substrate
all family 1 -glucosidases after changing the pH of complex.
the buffer to the one that is optimum for the enzyme 6. Add the xative solution (#5) and store at room
of interest (modied from 48). The solutions and temperature for several hours before photographing,
reagents needed are: scanning, or imaging. Note that enzyme bands are
1. 100 mM citrate200 mM phosphate buffer, pH completely stable in xative solution up to several
5.8. Titrate 200 mM citric acid solution with 400 months, and the gel may be dried for long-term storage
mM sodium phosphate (dibasic) until pH is 5.8. and record keeping.
2. 50 mM citrate100 mM phosphate buffer. When ones interest is surveying a food source for -
Dilute the above buffer with water at a ratio glucosidase activity, a simple, rapid, and inexpensive
of 1 : 1. assay procedure is needed. Clearly, chromogenic solu-
3. 6-Bromo-2 naphthyl -D-glucopyranoside tion assay in 96-well microtiter plates and dot-blot
(6BNGlc). Prepare just before using in dimethyl assay on nitrocellulose or ordinary chromatography
formamide at 100 mg/mL in a 1.5-mL micro- paper are the best choices as long as the enzyme of

interest hydrolyzes the test substrate at detectable laboratory, a number of maize and sorghum -gluco-
levels. In certain applications, one might be interested sidases and their chimeras and mutants have been suc-
in detecting a -glucoside present in a food sample cessfully puried to homogeneity using just a
using a specic enzyme that hydrolyzes and detects combination of differential solubility and hydrophobic
it. In this case, the PGO assay will be the choice interaction chromatography (41, 49). Needless to say,
since it measures glucose production. the rst step in purication protocol should start with
differential solubility fractionation. To that end, a sur-
D. Factors Interfering with Assays vey of buffers with different pH and ionic strengths to
solubilize the -glucosidase of interest while keeping
The presence of -glucosidase inhibitors in buffers or most of the contaminating proteins insoluble or partly
tissue extracts can interfere with solution assays, espe- soluble would be worthwhile. For example, Mkpong et
cially when extracts are not diluted much. In this case, al. (50) were able to isolate cassava -glucosidase by
endogenous substrates, substrate analog (aryl or alkyl extraction with a buffer (0.1 M Na phosphate) of pH
glycosides), and glucose and its analog (e.g., -gluco- 3.5 with very high specic activity because the enzyme
nolactone) can competitively inhibit the hydrolysis of was stable and soluble at such a low pH, whereas con-
test substrate such that the enzyme activity is neither taminating proteins were not. Once conditions to
detected nor underestimated. This problem can be alle- extract the enzyme with high specic activity have
viated by diluting crude extracts 50- to 100-fold if they been dened, such conditions can be used in large-
have high activity levels or removing interfering sub- scale purication projects. The next step would be to
stances by precipitating the enzyme with ammonium make a 4060% ammonium sulfate (AS) cut on the
sulfate (AS) and resolubilizing to the original volume crude extract (prepared with a buffer of pH 57) at
and/or dialysis. 04 C as all family -glucosidases precipitate between
40% and 60% AS concentration and can be fully
recovered from the pellet by suspension in a suitable
V. PURIFICATION buffered solution. This step can increase specic activ-
ity signicantly and serves as a stepping stone to a
When it comes to purication, every protein becomes a chromatographic separation method such as ion
specic project and challenge to the skills and ingenu- exchange, chromatofocusing, and hydrophobic inter-
ity of protein biochemists, although one can prescribe action chromatography (HIC). Of these, HIC is the
a general scheme for purication of related proteins. most effective as the pellet can be solubilized with 1.5
Having said that, it should be noted that -glucosi- M AS in a buffer of pH 68, which itself removes a
dases are relatively easier to purify than most proteins. substantial amount of contaminants and then is
The reader is referred to the primary literature for directly loaded onto the column. In the authors
detailed procedures to design a protocol to purify a laboratory HIC is performed on a Toyo-Pearl butyl
specic -glucosidase of interest. In the authors column, and the eluate from this column, if necessary,
Table 1 Puricationa of the Sorghum -Glucosidase Isozyme Dhr2 after Expression in E. coli
Enzyme activity Protein Specic activity Purication

Purication steps (mole o-NP h1 ) (mg/mL) (mole o-NP h1 mg1 ) (fold)
1. Crude extract (before sonication) 17.54 0.98 17.90 1.0

2. Crude extract (after sonication) 82.05 4.90 16.74 0.94
3. 30% AS supernatant 82.01 4.50 18.22 1.02
4. 65% AS, pellet suspended 417.36 10.50 39.75 2.22
5. Butyl-HIC 119.70 0.74 161.76 9.04
6. Ether-HIC 68.02 0.24 283.42 15.83
a
E. coli cell pellet was lysed in the extraction buffer (100 mM Tris-HCl, pH 8, and 50 mM NaCl), sonicated, and centrifuged. Ammonium sulfate
(AS) was added to the supernatant (30% nal concentration) and centrifuged. The supernatant was transferred to a fresh tube and sufcient AS
was added to obtain 65% nal concentration. The slurry was centrifuged and the resulting pellet was suspended in buffer, cleared by centrifuga-
tion, and subjected to hydrophobic interaction chromatography (HIC) on Toyopearl Butyl 650M and Toyopearl Ether 650M columns, respec-
tively. Enzyme activity and protein content were determined after each step during purication.

may be rechromatographed on Toyo-Pearl Ether- 13. GR Fenwick, RK Heaney, WJ Mullin. Glucosinolates
M650. This two-step procedure after optimization of and their breakdown products in food and food
elution conditions for HIC yields homogeneous or plants. CRC Crit Rev Food Sci Nutr 18:123201,
near homogeneous preparations for characterization 1983.
14. LW Wattenberg. Studies on polycyclic hydrocarbon
and crystallization studies. A summary of purication
hydroxylases of the intestine possibly related to can-
steps and increase in specic activity using the cer. Effect of diet on benzopyrene hydroxylase activ-
described procedure for the sorghum -glucosidase iso- ity. Cancer 28:99102, 1971.
zyme Dhr2 expressed in E. coli is given as an example 15. A Esen, DJ Blanchard. A specic -glucosidase-aggre-
in Table 1. gating factor is responsible for the -glucosidase null
phenotype in maize. Plant Physiol 122:110, 2000.
16. DJ Blanchard, M Cicek, J Chen, A Esen.
Identication of -glucosidase aggregating factor
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64
b-D-Fructofuranoside Fructohydrolase
Laura Cantarella
University of Cassino, Cassino, Italy
Francesco Alfaniy and Maria Cantarella
University of LAquila, LAquila, Italy
I. INTRODUCTION Acid Fs are able to catalyze transfructosylation reac-

tions. At high sucrose concentration (1 M), the -D-
-D-Fructofuranoside fructohydrolases (-Fs) are fructofuranosyl residue is transferred to a primary
sucrose-hydrolyzing enzymes found in microbial, alcohol (methanol, ethanol, n-propanol) and to only
plant, and animal sources. Trivial names are -fructo- isopropanol as a secondary alcohol (3).
furanosidase, formerly called invertase or saccharase,
and sucrase. The enzyme was rst isolated by Berthelot B. Chemical Structure of Substrates
in 1860 (1). Fs occur in Saccharomyces cerevisiae and
in strains used in brewing, distilling, wine making, and 1. Sucrose, -D-glucopyranosyl [1 ! 2--D-
the baking of bread, as an extracellular glycoprotein, fructofuranoside, also indicated as [Glc 1,2
the predominant form, and an intracellular protein (2, Fru]
3). Several molecular forms exist differing in molecular 2. Rafnose, O--D-galactopyranosyl [1 ! 6]--
weight and cell location even within the same yeast D-glucopyranosyl [1 ! 2--D-fructofurano-
culture. Most Fs in plants and tissues (59) are multi- side, also indicated as [Gal 1,6 Glc 1,2 Fru]
meric proteins discernible on their pH optima (acid, 3. Stachyose, -D-galactopyranosyl [1 ! 6]--D-
4.55.0, and neutral (alkaline), 7.07.8) (1013), glyco- galactopyranosyl [1 ! 6]--D-glucopyranosyl
sylation state, subcellular location, and pI values. Fs [1 ! 2]--D-fructofuranoside, also indicated
from S. cerevisiae or S. carsbergensis are commercially as [Gal 1,6 Gal 1,6 Glc 1,2 Fru]
available. 4. Substituted -D-fructofuranosides, such as
methyl--D-fructofuranoside, and p-nitrophe-
A. Chemical Reaction Catalyzed nyl--D-fructofuranoside
Sucrose hydrolysis is the main reaction catalyzed by

F. Rafnose and stachyose are hydrolyzed to a lesser C. Classication According to Enzyme
extent. Commission Nomenclature
-fructofuranosidase
Sucrose H2 O ! D-Glucose All Fs are classied as EC 3.2.1.26. True Fs have to
show no activity with nonsubstrates such as cellobiose
D-Fructose [Glc 1,4 Glc], gentobiose [Glc 1,6 Glc], lactose [Gal
y
Deceased.

1,4 Glc], maltose [Glc 1,4 Glc], melezitose [Glc 1,3 stored roots awaiting processing (19). During the pre-
Fru 2,1 Glc], -methyl-D-glucopyranoside, -methyl- storage phase of Vicia faba, high levels of hexoses in
D-glucopyranoside, ,-trehalose [Glc 1,1 Glc] or tur- the cotyledons are correlated with the activity of seed
anose [Glc 1,3 Fru], and p-nitrophenyl--glucoside (- coat-associated invertases. The degradation of the
fructofuranosides, fructopyranosides, -L-sorbofura- thin-walled parenchyma expressing the invertase
nosides, -D-xyloketofuranosides, and sugars with sub- (developmentally regulated) apparently initiates the
stituent on the -fructofuranosyl residue). storage phase and is characterized by a switch to a
low sucrose/hexose ratio (8). During the storage of
raw juice extracts from healthy tubers of Cyperus escu-
II. IMPORTANCE TO QUALITY OF FOOD lentus Linn. (earth almond), yeasts possibly play a
DURING GROWTH, MATURATION, greater role than bacteria in the hydrolysis of sucrose.
STORAGE, AND PROCESSING Saccharomyces rouxii, an osmophilic yeast strain, was
isolated from dried prunes where the natural environ-
In plants, the different F isoforms independently con- ment typically has a high concentration of sucrose. The
trol import sucrose metabolism, sugar composition in species survives by initially metabolizing other compo-
storage organs, translocation from source tissues to nents in the medium. After death of a fraction of the
sink tissues, osmoregulation, and gravitropism (6, population, cryptic F hydrolyzes sucrose, and the
1416). Compositional changes observed in several osmotic pressure is gradually lowered. Competition
developing fruits are associated with the changes in with other microorganisms in the ecological niche is
enzyme activities related to sucrose metabolism. Acid thus delayed.
and neutral F activities are very high in immature In plant intact tissue nearly all the F activity can be
fruits of Cucumelis melo L. and decline rapidly and attributed to alkaline F, while in the case of injury,
concomitantly with the accumulation of sucrose, as acid Fs are rapidly formed in response to wounding
the fruit matures (7). The activation of preexisting and bacterial infection, suggesting their possible role in
invertase in dormant Helianthus tuberosus (Jerusalem pathogen defense mechanisms (20). The active defense
artichoke) tubers takes place at the beginning of the response can be induced by chemical stimuli (elicitors)
germination process. derived from fungi (21). -Chymotrypsin cleavage of
The absence of F contributes to the accumulation yeast F generates small glycopeptides, acting as elici-
of sucrose rather than hexoses as a source of carbon tors in tomato cells, inducing the biosynthesis of ethy-
and energy. Vacuolar F controls the sugar composi- lene (plant stress hormone) and of phenylalanine
tion in tomato species (15). Lycopersicon esculentum ammonia-lyase. By contrast, the isolated glycan side
has high F activity, accumulates high levels of glucose chains act as suppressors.
and fructose, and stores little sucrose. In contrast, L. Freezing-tolerant wheat cultivars may respond to
peruvianum and L. chmielewskii (15) have low F activ- subzero temperatures and increase hardiness by the
ity, accumulate high levels of sucrose and store little conversion of fructan to cryoprotective sugars, such
glucose and fructose. Vacuolar Fs are also involved in as fructose and sucrose, whereas snow mold-resistant
glucose and fructose accumulation in grape berries that cultivars continue to accumulate fructan. This differ-
is one of the main features of the ripening process and ence in response to subzero temperature may depend
is a major commercial consideration for grape growers, on differences in gene expression of enzymes in fructan
wine makers, and dried-fruit producers (17). Alkaline metabolism, which involves several enzymes (22).
F activities increase during the development and
maturation of tissues in sugar cane stalks, carrot, and
sugar beet roots and young versus mature leaves of III. RAW-FOODS ENZYMES FOUND
Citrus sinensis.
During cold storage (13 C), potato tuber F iso- In Saccharomyces cerevisiae, internal F is found in the
forms cause the accumulation of reducing sugars that cytoplasm whereas external F resides on the cell wall
are responsible for the development of unpalatable a- (cw-F), and functions to cleave extracellular sucrose to
vor and texture in cooked potatoes (18). Darkening of fructose and glucose, sugars that yeast can import (2, 3).
the potatoes due to a nonenzymatic Maillard reaction In plants, the subcellular location of soluble F is
also occurs. Sugarbeet roots contain an endophytic controversial: the enzyme is described either as cyto-
population of sucrose-hydrolyzing bacteria (Pseudo- plasmic, despite its glycoprotein nature, or as vacuolar.
monas uorescens) that possibly utilize the sucrose in Spatially, Fs occur as intra- and extracellular forms.

Both soluble and cw-Fs are found (2327). Two types of invert sugars from sucrose at concentrations up to 3
of acid F (N-linked glycoproteins) are located either M (30).
as soluble proteins in the vacuole or ionically bound to Brewing, distilling, wine making, and the baking of
the cell wall. Acid and neutral Fs may coexist even in bread all require freshly grown, specially developed
the same tissue (11). However, acid F is mainly found yeast strains (S. cerevisiae) to inoculate each fermenta-
in immature tissues while neutral F is conned to tion. The activity of F and of -glucosidase deter-
mature storage tissues of sucrose-accumulating plants. mines the physical and organoleptic properties of
In contrast, neutral F in the mesocarp of developing bread. The capacity of a given strain for sugar uptake
muskmelon declined with the accumulation of sucrose and the further conversion of the sugars present in
(7). Soluble acid F also occurs in the apoplast of plant bread dough are of primary importance for its bread-
tissues (papaya, sugar cane, oat, radish, wheat, maize, making suitability (31).
artichoke, barley, potato, carrot, cherry, tomato, Fructo-oligosaccharides (1-kestose, nystose, and 1F -
peach, strawberry). Acid F is extremely low in the -fructofuranosyl nystose), produced by transferring
reserve cotyledon tissue throughout development, one, two, or three molecules of fructose to the fructose
and activity appears to be correlated with the growth residue in sucrose by the action of fungal F (32), have
and differentiation of certain plant tissues, particularly been shown to decrease the levels of cholesterol and
involved in organ elongation and cell enlargement (6). lipid in the serum and to stimulate the growth of intest-
It declines rapidly when roots begin to enlarge, and is inal bidobacteria.
hardly detectable in mature roots and organs. Acid cw- Transgenic tomato plants that expressed a yeast F
F plays a key role in the generation of sink strength of show a decrease in the growth rate. Accumulation of
storage sinks (photosynthetically less active or inactive hexoses and amino acids (proline) is up to 40-fold
tissues such as stems, owers, and roots) during devel- higher than in the wild type. The fruits were 30%
opment of Vicia faba L. seeds (28). Less information is smaller than controls. Soluble acid F controls this
available on neutral or alkaline Fs (10) present in the differential growth which correlates with high rates
cytoplasm of most living cells of higher plants. of sugar accumulation during the last stage of devel-
opment (33). Potato tubers expressing yeast F in the
cytosol result in a reduction in tuber size and an
IV. UTILITY/UTILIZATION OF ENZYMES increase in tuber number per plant, whereas apoplastic
IN FOODS targeting leads to an increase in tuber size and a
decrease in tuber number per plant (18).
In the food industry, the main use of F is for produ-
cing chocolate-coated candies with a soft and creamy
V. PROPERTIES AS PROTEIN
center. A hard and rm center is prepared with sucrose
and molded into the desired shape. After coating with A. Molecular Weight
chocolate the F is inoculated into the center to change
its consistency to a permanent and noncrystallizable The molecular mass of Fs varies considerably: in
cream. Puried F is preferred to yeast since variations plants from 42 kDa to 504 kDa, in microorganisms
in both the added amount and in the strains of yeast from 37560 kDa. The glycosylated nature may lead
utilized does not assure uniform quality of softer cen- to a lack of precision in estimating the molecular
ters. Other F uses are related to the production of weight. Values are reported in Tables 1 and 2.
articial honey and to the enzymatic determination
of glucose and sucrose in dietetic foods (29). The pro- B. Primary, Secondary, Tertiary, Quaternary,
ductivity value, the operational stability and mechan- and Macromolecular Structures
ical stability of F, the low cost of the immobilization
on a variety of carrier, and the absence of undesirable Internal and external yeast Fs appear with identical
reaction byproducts make the scale production of protein moieties. The same (SUC2) gene is translated
inverted syrups from sucrose and nonrened sugars from different start codons resulting in one or two
economically very attractive. In the confectionery additional amino acids (a.a.) at the N-terminus of
industry fructose is preferred over sucrose because it external Fs (44). The subunit has an MW of 58
is sweeter and does not crystallize as easily. Health and kDa. Internal F exists in its native state as a nongly-
taste reasons require highly puried F. Invertase from cosylated active monomer. A signal sequence directs
S. cerevisiae is utilized for the commercial production external F into the endoplasmic reticulum, where it

Table 1 Major Structural Characteristics of Some Puried F from Different Plants
Native protein SDS-PAGE
F puried MW MW Protein
Source [pl] Carbohydrate Form (kDa) (kDa) band Other isoforms [pI]
Arabidopsis thaliana L., mature green acid [4.75] positive monomer 58 52 4 acid [4.65; 4.70; 4.85;
leaves (9) 4.95] and 1 cw [9.0]
Avena sativa, oat seedling (5) acid [8.6] 59 single
acid [4.4] 108 single
Cichorium intybus L., roots (10) neutral tetramer 260 65 single multiple peaks
Daucus carota L., cell culture (12) neutral octamer 456 57 single multiple peaks
alkaline tetramer 504 126 single
Seed seedling (23, 24) acid [5.7] positivea monomer 68 68, 43, 25 3 bands acid [3.8] cw bound
Glycine max, sprouting hypocotyl (6) alkaline negativeb tetramer 240 58 single acid
Hordeum vulgare L., leaves (34) acid positiveb monomer 64 64 single
multimer 116 multi
multimer 155 multi
Lycopersicon esculentum, pericarp acid monomer 52 52 single 2
tissue (17)
Prunus avium L., mesocarp (35) acid positiveb multimeric 400 63 single 1
Solanum tuberum, tuber (36) acid 10.9%b dimer 60 30 single
Sugar cane juice (37) neutral 22 monomer 15
dimer 35
tetramer 66
decamer 160
Vicia faba L., cotyledon (8) alkaline [5.2] homotetramer 238 53.4 acid
a
N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans.
b
Is a glycoprotein as indicated by positive reaction with periodic acidSchiff reagent and its ability to bind ConA.

Table 2 Major Structural Characteristics of Some Puried F from Different Microorganisms
Native protein SDS-PAGE
Protein Other
Source Location Carbohydrate Form MW (kDa) MW (kDa) band isoforms
Arthrobacter sp. K-1 (38) 2.4% Glc monomer 51 52 single 1

monomer 58 58 single
Aspergillus nidulans (39) secreted S-F 14% Man 5% Gal dimer 185 78 single 3
secreted F-F 29% Gal 12% Man monomer 94 110 single 3
Aspergillus niger (40) secreted Man, Glc, Gal, N- dimer 250 125 single
acetylglucosamine
secreted N-linked glycans, Man, Glc, Gal, N- dimer 120 90
O-linked glycans acetylglucosamine
Candida utilis (41) secreted positivea dimer 150 single
Pichia anomala (42) 30% Man multimer 254 86.5 single
Saccharomyces cerevisiae wild internal/protoplast < 3% carbohydrate dimer 135 60 single 6
type X2180 (4, 43) external cw bound/periplasm 50% Man 3% dimer 270 140 single
glucosamineb tetramer 360
hexamer 560
a
Positive Lecitin afnity recognition on blotting using ConA-HRP conjugate.
b
Nine high mannose oligosaccharide chains each linked to an asparagine residue by a di-N-acetylchitobiosyl core.

is glycosylated (45). The enzyme consists of 140-kDa Signicant sequence homologies around the cataly-
subunits, which are in excess of 50% by weight man- tic residues (Asp23 and Glu204) has been shown with
nose glycan and 3% glucosamine. The carbohydrate is other fructosylhydrolases form different sources.
attached as short oligosaccharides (Man8-14 GlcNAc2 ) Inulinase from Kluyveromyces marxianus and F
to hydroxyamino acids or as polysaccharide chains from Pichia anomala share 53% and 44% homology,
(Man>50 GlcNAc2 ) to asparagine through N-acetylglu- respectively, and the inulinase contains a single
cosamine. The protein consists of 13 or 14 potential cysteine residue that corresponds to Cys205 in the
glycosylation sites with an Asn-X-Ser (or Thr) F. Yeast F is closer to levanases and fructosyl-
although only 910 appear to be glycosylated (45). transferase from bacteria (3435% homology) than
Yeast F presents a continuous spectrum of molecular bacterial sucrases (3031% homology) and plant
forms that might represent different degrees of glyco- Fs (2729% homology (47).
sylation during the secretion process, which culminates In plants, alkaline Fs are thought to be nonglyco-
in the formation of the heavy extracellular enzyme (4). sylated proteins (6). The glycosylation of cytoplasmi-
The oligosaccharide moiety maintains external Fs in cally synthesized acid F is necessary for its transport
an oligomeric equilibrium of dimer, tetramer, hexamer, across either the tonoplast or the plasma membrane.
and octamer, equally active, and facilitates, without Nucleotide and predicted amino acid sequences of acid
affecting the active sites of F, subunit interactions invertase cDNA are known for tomato fruit, carrot,
to form active oligomers. The very stable dimers potato tuber, and grape berries (17, 4850) with the
present a spherical nature presumably with the two following homologies in the amino acid sequences:
monomers tightly bound (46), occluding some oligo- tomato acid F and carrot extracellular acid F are
saccharides at the interacting subunit surfaces. 40.5% identical; isoenzyme I of carrot soluble acid
Dissociation to a monomer results in the generation F and acid F from tomato are closely related; iso-
of a form devoid of enzyme activity (43). enzyme II and acid F from bean show the highest
In contrast to Saccharomyces, SUC2 F secreted by homology; carrot extracellular cw-F and acid F
Pichia pastoris is a homogeneous product of 85-kDa potato share high homology. A phylogenetic tree was
subunits associated with 810 oligosaccharides of the generated (49). Table 3 reports some identied genes
size Man8-14 GlcNAc2 . However, the majority of com- producing F in plants and microorganisms together
ponent of Man8-10 GlcNAc family has structures iden- with molecular characteristics.
tical to those found associated with Saccharomyces F.
The protein produced by A. niger F gene (SUC1)
C. Isoforms
contains 14 potential glycosylation sites with equal
numbers of the two sequences Asn-X-Thr and Asn-
Some of the identied F isoforms in plants and
X-Ser, for N-linked glycosylation. The F a.a.
microorganisms are reported in Tables 1 and 2.
sequences from S. cerevisiae and Schwanniomyces occi-
Growth conditions and glucose concentration deter-
dentalis contain large conserved domains but do not
mine the cellular content of internal and external
exhibit extensive homology in any region of the protein
forms and their distribution in yeasts. In repressed S.
with the predicted sequence of the A. niger enzyme. Six
cerevisiae cells, most of the enzyme is intracellular; in
domains of S. cerevisiae F and Schw. occidentalis are
derepressed cells, 95% of the total enzyme is mainly
conserved in Fs from prokaryotes, Zymomonas mobi-
located outside the cytoplasmic membrane. In
lis, Bacillus subtilis, Streptococcus mutans, and Vibrio
Saccharomyces wild type six different genes encode
alginolyticus. By contrast, only one could be partially
six isoforms of F. The existence of multiple forms
identied in the A. niger F since only six a.a. were
of F is common also in fungi (40). F isoforms are
identical of the nine a.a. which comprised the domain.
not uncommon in plants with their location in tissues
Homologous sequences in 14 S. cerevisiae strains
well distinct, and concentration levels within cells
and in 10 closely related Saccharomyces spp. are
appeared to be regulated during development.
detected by using cloned SUC2 DNA probes. The
SUC DNA sequence is highly conserved within the
1. From Different Genes
genus Saccharomyces; only the DNA from
Saccharomyces kluyveri UCD51-242 fails to hybridize Six F structural genes in unlinked loci, similar in
with the SUC2 DNA probe. During evolution its SUC structure and expression but not identical, are known
gene has diverged sufciently to leave no detectable in the yeast S. cerevisiaeSUC1SUC5 and SUC7.
homology (44). Each SUC gene encodes both the constitutive

Table 3 Some Identied Genes Producing -D-Fructofuranosidase in Microorganisms and Plants
F encoding Signal peptide

Source (location) gene MW Polypeptide size Active sitea a.a. Glycosylation
Bacillus subtilis SACA or 54,888 480 D-43

IPA-50D
Escherichia coli (cytoplasm) CSCA 54,363 477 D-39
Hansenula anomala INV1 63,188 550 22 10
Klebsiella pneumoniae (cytoplasm) SCRB 52,708 466 D-41
Lactococcus lactis (cytoplasm) SACA 54,785 473 D-47
Saccharomyces cerevisiae SUC1b 60,570 20532 D-42 19 12
(external) SUC2 or 60,639 20532 D-42 19 13
YIL 162Wb
(internal) SUC2b 21532 D-42 19
SUC3b 20 > 74 (fragment) D-42 19
SUC4b 60,575 20532 D-42 19 14
Schwanniomyces occidentalis INV 60,839 23533c D-50 22 8
Staphylococcus xylosus SCRB 57,371 494 D-48
Streptococcus mutans (cytoplasm) SCRB 51,754 455 D-47
Vibrio alginoliticus (cytoplasm) SCRB 55,657 484 D-51
Zymomonas mobilis (cytoplasm) INVA 58,725 512 D-43
(cytosplasm) SACA 58,397 511d D-43
Daucus carota L. INV1 66,813 49592 D-74 131 of 39 (propeptide 5
(cw-F of leaves roots of young plants) 3248 or 40)
e
(cw-F II) INV2 67,397 592 D-75 potential 5
e
(cw-F) INV3 66,381 583; insoluble (propeptide potential) 5
isoenzyme 3
Lycopersicon esculentum (tissue-fruit vacuole) TIV1 or 70,097 93636 D-118 potential and propeptide 4
AIV-1 from e92
Phaseolus aurens (vacuole) INVA 72,167 heterodimer: D-130 potential from 1e 3f
102328 (30 kDa)
329649 (38 kDa)
monomer: (70 kDa)
Pisum sativum BFRUCT1 62,655 23555 D-140 22 3
e
Zea mays (vacuole) IVR1 71,932 670 D-139 potential 5
(cw-F) 65,198 29590 D-68 28 2
a
Conservative domain, NDPNG.
b
SUC1, SUC3, SUC4, SUC5 compared with SUC2, SUC7, all are highly homologous within the coding region.
c
45% identity with S. cerevisiae.
d
The a.a. sequence showed strong homologies with sucrases from Bacillus subtilis, Salmonella thyphimurium, and Vibrio alginolyticus.
e
Not identied.
f
(2 complex-type glycan and 1 high-mannose type glycan).
Source: SwissProt database.

nonglycosylated F and the glycosylated one (4, 44). F oligosaccharides shield the subunit from large inter-
The different yeast strains contain zero, one, or several actions with NaCl. The formation of higher oligomers
of these genes, and the possession of one active SUC is also facilitated by KCl. Concentrations up to 0.4 M
gene enables a yeast strain to ferment sucrose and raf- of CH3 COONa, NH4 2 SO4 , K3 PO4 , and NaF have
nose. Strains expressing different SUC genes reveal no effect, indicating that chloride ions are primarily
different specic F activities under both repressing responsible for promoting self-association. External
and derepressing conditions. SUC4 determines the F exists mostly as tetramers in 0.8 M GuHCl, reect-
highest F activity on rafnose as carbon source. ing the opposing effects of dissociation by guanidium
Strains expressing SUC7 are unable to grow on raf- and association by chloride ions (46).
nose as the sole carbon source. SUC4 and SUC5 differ Soluble F of carrot isoenzyme I (68 kDa) and
in their F activity about vefold. SUC2, the most tomato are cleaved into an N-terminal and a shorter
extensively studied, encodes the enzyme F, required C-terminal fragment, neither of which separates during
for growth of yeast cells on sucrose. SUC2 is transcrip- protein purication (16, 24, 49). Whether this proteo-
tionally repressed in high glucose whereas it is dere- lytic cleavage occurs in vivo or during protein purica-
pressed in low glucose, and transcription is increased tion is not known. The monomeric nature of the carrot
> 100-fold (44). enzyme suggests a labile bond such as a disulde
In many plants F is encoded by a multigene family bridge between the two polypeptide fragments.
(9). The different isoforms of tomato fruit F may Maturation of tomato acid F may involve additional
originate from a single gene or from closely related proteolytic processing at the C-terminus (48) since the
genes (16). In Daucus carota L. different genes encode MW of predicted mature polypeptide (60 kDa) exceeds
acid F while the neutral F is encoded either by dif- that determined by SDS (52 kDa).
ferent genes or by the same gene and is generated by
differential splicing or proteolytic cleavage of alkaline
VI. PROPERTIES AS ENZYME
F (12, 24). Only one gene appears to encode F in
Helianthus tuberosus, although it is possible that sev- A. K m , V max , Substrates, Nonsubstrates, and
eral genes produce transcripts of a similar size. The Activation Energy
transcript size (2.5 kb) is close to those shown in
tomato fruit (2.4 kb), carrot (2.0 kb), and yeast (1.8 Tables 4 and 5 report the Michaelis constant for
and 1.9 kb). Multiple F cDNAs were isolated from sucrose, KmSuc , and rafnose, KmRaf , of Fs from several
carrot tissue and exhibited between 44% and 99.1% plants and microorganisms, other identied substrates
identity of amino acid sequence. Two cDNAs (GIN1 and nonsubstrates, the optimal pH, and maximal velo-
and GIN2) were cloned from berries; the translation city. In plants, KmSuc for alkaline F is higher than that
products are 62% identical to each other, and both of the acid form. The activation energy, Ea of plant F
appear to be vacuolar forms of F (17). has been reported to be 57.3 kJ/mol for Ricinus com-
munis (25) and 147 kJ/mol for nodules of Cicer arieti-
2. From Posttranslational Modication num L. (51). The Ea value is 37.63 kJ/mol in whole
yeast cell for acid F (56) and 29.30 kJ/mol for a solu-
The most posttranslational modication of Fs is the
ble preparation of acid F from yeast (57).
extensive glycosylation that could affect the efciency
of biosynthesis and secretion of both the heterologous
B. Effect of Environmental Factors
and homologous proteins. During secretion and trans-
port to the periplasm the signal peptide is cleaved and
The optimal pH depends on the form and the source of
the external yeast F becomes glycosylated. The poly-
F as presented in Tables 4 and 5. Saccharomyces cer-
mannose chains aid the self-association of homodimer
evisiae F exhibits relatively high activity over a broad
into tetramer, hexamer, and octamer (46, 47).
range of pH (3.55.5), with the optimum near pH 4.5.
The enzyme activity reaches a maximum at 55 C.
3. Artifacts
Certain ions affect the pH optimum of F. The symbol
Freezing or addition of PEG promotes the self-associa- () in Table 6 indicates a weak inhibition, with at the
tion of only external yeast F subunits. Low NaCl most 20% loss of F activity. Inhibition constants and
concentration favors the presence of internal F inhibition mechanism, when identied, are reported in
dimers while concentration as high as 0.8 M yields Table 7. Plant Fs are characteristically inhibited by
the maximum octamer-hexamer mixture. In external heavy metals and reagents that react with sulfhydryl

Table 4 Some Values of Km for Puried F from Different Plants
KmSuc KmRaf Other Optimal

Source F puried (mM) pH (mM) substrates Nonsubstratesd Vmax pH
Avena sativa cv. (5) soluble acid 2.4 4.5 2.9 Sta Inu 4.5
soluble acid 6.7 5.0 17 Inu 5.0
Beta vulgaris L. (11) soluble acid 3.8 5.0
alkaline 33.3
acid NaClreleased 0.56
acid EDTAreleased 4.2
extracellular acid I 12.3
extracellular acid II 4.4 4.8a 7.0
Cicer arietinum L. (51) neutral 14.2
Cichorium intybus (10) neutral 1020 Raf Cel, Inu, Kes, Mal, Nys, 7.07.5
Tre
Cucumis melo L. (7) soluble acid 2.2 Raf, Sta
cw acid EDTAreleased 3.2
cw acid NaClreleased 2.2
soluble neutral 10 6.57.0
Daucus carota L. (12) neutral 18.7 7.5 Raf
14.3 6.8 Sta
alkaline 20.7 7.5 Raf, Sta
15.1 8
Daucus carota L. (24) soluble acid 5 Raf Inu 5
Fragaria ananassa (14) soluble acid 3.5 Raf, Sta 4.6
cw NaClreleased 3.7 5.0
cw EDTAreleased 4.4 4.2
Glycine max L. (52) alkaline 10 Sta, Raf Cel, Gen, Mal, Tur, Lac,
Mele, Tre, -CH3 -D-Glc,
-CH3 -D-Glc
(6) alkaline 10 Cel, Lac, Mal, Raf 7.07.6
Lycopersicon soluble acid 7.9 9.23b 4.5
Esculentum (16) soluble acid 7.7 9.08b
soluble acid 7.4 8.42b
Oryza sativa (26) alkaline 70.1 Raf, Mal 7.0
soluble acid 0.9 4.7 Raf Mal 3.54.0
soluble acid 12.1 30.2 Raf Mal 5.0
acid cw- bound 4.3 Mal 4.5
Phaseolus vulgaris (53) alkaline 8.9 7.7
soluble acid 2.4 5 14 [5.0] Sta, Kes Mele
Prunus avium L. (35) soluble acid 4 Sta Tre, Mal, Lac, Mel, Mele
Ricinus communis (25) soluble acid 8.7 3.5 Raf Mele, -Gal, - and -
Glc
Solanum tuberum (36) soluble acid 16 4.7 5.0
Sugar cane (37) soluble acid 2.8 2.7c
neutral 0.32 2.8c
Vicia faba L. (8) alkaline 10.1 Lac, Mal, Raf, Sta, Tre 0.58b 7.4
Wheat (27) soluble acid 3.5 Raf Mel, Mele
cw acid 1.7
a
mole/h.
b
mole/min.
c
mole sucrose hydrolyzed/h/mg protein.
d
Cellobiose (cel), -galactosides (-Gal), gentiobiose (Gen), - and -glucosides (--Glc), -methyl-D-glucopyranoside (-CH3 -D-Glc), -
methyl-D-glucopyranoside (-CH3 -D-Glc), inulin (Inu), 1-kestose (Kes), lactose (Lac), maltose (Mal), melibiose (Mel), melezitose (Mele), 1, 1-nystose
(Nys), rafnose (Raf); stachyose (Sta), turanose (Tur), , -trehalose (Tre).

Table 5 Some Km Values of Puried F from Different Microorganisms
Optimal Optimum pH
KmSuc KmRaf d
Vmax temperature
Source Form (mM) (mM) Nonsubstratesc (mM min1 ) ( C) Activity Stability
Arthrobacter sp. K-1a (38) 9.1 15.1 Pal, Inul, Cel, Mal 100% (Suc) 55 6.56.8 5.510
49% (Raf)
Aspergillus niger (40) Specic F 30160b Mal, Inu, Mele 6.0
Nonspecic F 40 Mal, Mele 5.0
Candida utilis (41) 11 150 333 (Suc) 5.5 3.06.0
215 (Raf)
Kluyveromyces fragilis (54) -Mercaptoethanolreleased 13.6 46.1 5.0
Pichia anomala (42) 16 82 Inu, Mele 38 4.5 4.56.5
Pseudomonas uorescens (19) Internal 90 6.5
Saccharomyces rouxii (55) Internal 83 5.5 4.57.5
Saccharomyces cerevisiae (2) Internal 25 150 3.55.5 6.09.0
External 26 15 3.55.5 3.07.5
a
Other substrates: erlose, fructosylxyloside, neokestose, lactosylfructoside, stachyose.
b
Non-Michaelis-Menten kinetics.
c
Cellobiose (Cel), inulin (Inu), inulobiose (Inul), maltose (Mal), melezitose (Mele), palatinose (Pal).
d
Rafnose (Raf), sucrose (Suc).

Table 6 Inhibitory Effect on the Activity of Puried F from Some Microorganisms and Plants
Source F puried AgNO3 CaCl2 CoCl2 CuSO4 HgCl2 I2 KCl MgCl2 MnCl2 NaCl NH4 Cl ZnCl2 MgSO4 EDTA Aniline Fructose Glucose Tris
Arthrobacter sp.K-1
(38)
Aspergillus oryzae (58)
Kluyveromyces fragilis
(54)
Avena sativa cv., oat soluble acid
seedlings (5) soluble acid
Cichorium intybus, neutral
chicory roots (10)
Cucumis melo L., soluble acid
mesocarp (7) cw EDTAreleased
cw NaClreleased
soluble neutral
Daucus carota L., neutral
cell culture (12) alkaline
Fragaria ananassa, soluble acid
fruit (14) cw NaClreleased
cw EDTAreleased
Glycine max L., alkaline
nodule (52)
Prunus avium L., soluble acid
mesocarp (35)
Wheat, coleoptiles soluble acid
(27)

Table 7 Some Ki Values (mM) for F from Different Sources
Cichorium Daucus carota Daucus carota

intybus L. neutral L. alkaline Glycine max L. Potato Sugar cane Sugar cane Vicia faba L. Neurospora Yeast
Inhibitor (5) (12) (12) (6) (59) (37) (37) (8) (59) (59)
Lauryl sulfate 0.54a 12

Metasilicate 0.96b 0.28
Pirydoxal 4 21 29
Pyridoxine 18 24 27
Deoxypyridoxine 17 30 15
Pyridoxal phosphate 0.5 0.005 6 > 150 56
Pyridoxamine 60 > 150 > 150
Aniline 110 1.5 1.7
Fructose 16c 13.4b 16.3b 8:6 0:6
Glucose 16 28b 33b
Tris 1.2 4
HgCl2 0.025
a b
Inhibition mechanism: uncompetitive; noncompetitive; c competitive.

groups. Hg2 , p-chloromercuribenzoate (100 M), and and tryptophan residues, is essential for F activity.
p-chloromercuriphenyl sulfonic acid (100 M) comple- An ester linkage of the carboxylate group with condur-
tely inhibit the F. Co2 and Zn2 are inhibitors to a itol B epoxide causes an irreversible inactivation of F.
weaker extent. Ag ions attach to the histidine side The participation of carboxyl imidazole groups in cat-
chains of the F molecule and render it inactive. alysis is suggested for Neurospora Fs and in some
Dithiobis 2-nitrobenzoic acid and iodoacetamide, higher plants (47).
reagents known to inactive enzymes that require free
sulfhydryl groups for activity, have no effect because D. Unique Properties
such groups are perhaps inaccessible under the assay
conditions (10). Inhibitors of plant Fs are pyridoxal, Yeast Fs have a transferring activity for -fructofur-
pyridoxine, and pyridoxamine (57, 26, 27). Yeast anosyl residues that is negligible under the usual assay
external F is weakly inhibited by p-hydroxymercuri- conditions (2). Fs from barley leaves exhibit a fruc-
benzoate, N-ethylmaleimide, iodoacetic acid, and tosyltransferase activity, at high concentrations of
iodoacetamide. Alkaline metal ions and related cations sucrose, forming mainly 1-kestose (34). The action
affect alkaline F activity from Ipomoea batatas in the of transglycosylation or transfructosylation of differ-
order of Li > Na > K > Rb > NH
4 > Tris , ent microorganisms F (61, 62) leads to fructosyl

while anions affect the activity in order of F > and oligofructosyl trehaloses. Methyl fructosides are
NO3 > Br > I > Cl . The interrelated effect of prepared from sucrose and methanol by Fs from
cations and anions on F activity prevents distinction S. cerevisiae (63). Mold Fs also (A. niger, A. syduwi,
between activator (suggested Na and K ) and inhibi- A. japonicus, A. oryzae, Penicillium oxalicum,
tor (suggested Cl ) (20). Some alkaloids found in Aureobasidium sp.) show high transfructosylating
vacuoles (caffeine, berberine, strychnine, morphine, activity (38).
ethyl-narceine, and nornicotine) produce an inhibiting The hydrolysis of inulin is also catalyzed, albeit very
effect on F activity of Ricinus communis, Solanum slowly, by Fs. Unlike that in higher plants, the major-
tuberosum, Oryza sativa, and Carica papaya (60). ity of microbial inulinases also possess sucrose hydro-
Acid Fs activity is curtailed by endogenous proteinac- lytic activity and are thus classied as nonspecic Fs.
eous inhibitors (36) as it occurs in red beet, sugar beet, The reverse situation, that all microbial Fs possess
and sweet potato and in maize endosperm. Plant Fs inulinase activity, has not been veried (40). The ques-
are activated by exogenous proteins (R. communis F tion in debate is whether inulinase should be regarded
activity is increased 30%), PVP, and dextran sulfate as a special type of F or as a different enzyme with an
(25), which suppress the inhibitory action of glucose, analogous mode of action. Inulinases are characterized
fructose, and organic acids. Since the vacuoles contain by the ratio of the activities with sucrose versus inulin
19% of the total protein of the protoplast, it appears as substrates < 50. The physiological role of F mainly
unlikely that fructose or the other vacuolar metabolites concerns the hydrolysis of sucrose within the cell wall.
could exert control on F activity. The main physiological role of inulinase is the break-
down of fructans outside the cell wall. Difructose dia-
C. Specic Mechanism of Action nhydride is hydrolyzed to D-fructose in two reaction
steps by the enzyme from Arthrobacter aureafaciens
The mechanism of yeast F action involves a nucleo- presumed to be a -fructofuranosidase, which is mark-
phile and a proton donor in a two-step reaction. The edly different from yeast enzyme. It is not clear
Glu204 residue acts as a proton donor to the glycosidic whether Arthrobacter enzyme is some other fructan
oxygen of sucrose. The C-2 of the fructose moiety is hydrolase.
attacked by the nucleophile Asp23. A covalent fructo-
sylenzyme complex is formed and glucose is released.
In the second step a proton from the water molecule is
VII. QUALITATIVE AND QUANTITATIVE
abstracted by Glu204. The hydroxyl group displaces
fructose, and the original active center residues are
restored. Glu204 is essential for sucrose hydrolysis A. Assay Conditions
while Cys205 possibly plays a supportive role in cata-
lysis by maintaining a suitable microenvironment in Approximately 0.030.05 units of acid F are incu-
the active site, or perhaps in aiding substrate binding. bated in 0.05 M acetate buffer, pH 4.6, with 100200
A carboxylate group, along with histidine, methionine, mM sucrose for 1060 min at 30 or 37 C. Phosphate

buffer, pH 7.5, is used for alkaline F. Crude extracts inhibit the nonspecic reoxidation of NADPH are
require 0.1 M buffer. recommended.
The amount of glucose or fructose released can be 3. Dinitrosalicylic reagent (DNS): Dissolve 40 g
estimated by enzymatic quantitative assay or by any of dinitrosalicylic acid, 8 g phenol, 2 g Na2 SO3 , 800 g
the procedures commonly used for determining redu- Rochelle salt (NaKC4 H4 O6 4H2 O in 2 L of 2% (w/
cing sugars such as the dinitrosalicylic acid (DNS) v) NaOH solution and then dilute it to 4 L with dis-
method (64) or the Nelson-Somogyi method (65). tilled water. Procedure: Add 1 mL DNS reagent to 0.5
Glucose always serves as standard in the calibration mL assay mixture, and it stops the action of F by
curve. shifting the pH of the medium into the alkaline
1. -D-Glucose is determined by coupled reactions range. Heat 5 min in a boiling water bath, then dilute
involving glucose oxidase (GOD), peroxidase (POD), with 210 mL water, and measure the absorbance at
and o-dianisidine dihydrochloride (DH2 ) as chromo- 540 nm. The molar extinctions of glucose and fructose
gen. are identical.
GOD
4. Nelson-Somogyi reagents. Reagent A: Dissolve 25
-D-GlucoseH2 O O2 ! g Na2 CO3 , 25 g NaKC4 H4 O6 4H2 O (Rochelle salt),
20 g NaHCO3 , and 200 g anhydrous Na2 SO4 into dis-
D-Gluconic acid H2 O2
tilled water (1 L nal volume). Store at room tempera-
POD
H2 O2 DH2 ! 2H2 O D ture. Reagent B: Dissolve 15% (w/v) of CuSO4 5H2 O
in water with few drops of concentrated H2 SO4 .
The F activity is stopped by changing the pH with the Reagent C: Dissolve 50 g (NH4 2 MoO4 in 900 mL
0.5 M addition of Na2 HPO4 , pH 8.83 (which also water. Add slowly under stirring 42 mL concentrated
accelerates the mutarotation of -D-glucose), and H2 SO4 , then add 6 g Na2 HAsO4 7H2 O in 50 mL
heating in a boiling bath for 10 min. A 0.4-mL volume water. Mix, incubate at 37 C for 2448 h, and store
of GOD-POD mixture (0.4 unit of each enzyme and in a dark bottle.
400 g of DH2 ) is mixed with 0.8 mL incubation mix- Procedure: The enzyme reaction (1.0 mL) is stopped
ture and kept for 30 min at 37 C. Then 0.3 mL of 6N with 1 mL of a freshly prepared solution (25 mL
HCl is added. The color developed is measured at 460 reagent A 1 mL reagent B) and heated for 20 min
nm against blanks. in a boiling bath. Cool on ice bath, add 1 mL reagent C
2. The D-glucose and D-fructose released are with immediate mixing to develop a blue color and
determined using a commercial kit (Boehringer CO2 . Dilute with water to 25 mL and mix. Read absor-
Mannheim) based on hexokinase (HK), phosphoglu- bance at 520 nm against reagent blank.
cose isomerase (PGI), and glucose-6-phosphate dehy-
drogenase (G6P-DH). B. Specic Activity
HK
D-Glucose D-Fructose 2ATP ! One unit of activity is dened as the amount of enzyme
that hydrolyzes 1 mol substrate (expressed as glucose
Glucose-6-P Fructose-6-P P 2ADP
equivalent) per min under the assay conditions.
Alternately, units can be expressed in microkatals
PGI
Fructose-6-P ! Glucose-6-P i.e., the amount of enzyme catalyzing the hydrolysis
of 1 mol substrate per sec under the assay conditions.
G6P-DH The specic activity of F is expressed as units of
2Glucose-6-P 2NADP ! katals/mg protein as measured by the method of
2Glucoonate-6-P 2NADPH 2H Lowry et al. (66) with bovine serum albumin as a stan-
dard protein.
A 0.01-mL volume of the sample for F activity assay
is added with 2.5 mL triethanolamine buffer (0.1 M,
pH 7.6), 0.1 mL MgCl2 (0.2 M), 0.1 mL ATP Na salt, VIII. PURIFICATION
0.1 mL NADP Na salt, 0.01 mL HK, 0.01 mL PGI (all
10 mg/mL), and 0.01 mL G6P-DH (5 mg/mL). The Most purication schemes of F from different
reaction is kept at 25 C for 15 min and the absorbance sources are adapted from the procedure of Goldstein
is read at 340 nm. Deproteinization of biological sam- and Lampen (3) with extensive modications.
ples (e.g., food) and addition of 0.5 mM EDTA to Purication-fold, P, and percentage of activity recov-

Table 8 Purication Yield and Recovery of F from Some Microorganisms and Plants
Assay
conditons
Crude extract Puried F Purication Recovery

Source U mg1
protein U mg1protein (fold) % T ( C) pH
Aspergillus niger (66) secreted 143 3,290 23 9.9 30 5.0

Bidobacterium infantis (68) internal 25.8 437 17 30.9 37 6.2
Candida utilis (41) periplasmic 32 3,100 97 34 60 5.1
Pichia anomala (42) internal 29 1,482 51 46 30 4.5
Saccharomyces cerevisiae (3) internal 3 4,700 1,567 32 30 4.9
strain FH4C (3) external 19 4,800 253 37 30 4.9
(43) internal 0.18 3,600 19,390 19 37 5.0
Thermomyces lanuginosus (69) internal 0.5 132 275 3.4 6.5
Arabidopsis thaliana (9) soluble acid inv1 0.16 431 2,694 1.7 37 5.5
Daucus carota (24) soluble acid Iso1 0.93 600 650 4.5 37 4.6
(13) acid 0.001 0.62 400 6.91 4.5
(12) alkaline 0.2 15 75 6.2 37 8.0
(12) neutral 0.2 23.4 117 4.4 37 6.8
Hordeum vulgare L. (34) soluble acid 0.14 192 1,370 1.2 25 5.6
Oryza sativa (26) soluble acid 0.08 4.21 50 7 37 5.0
cw 0.30 5.6 6.8 37 7.0
Phaseolus vulgaris (53) alkaline 0.13 14.5 110 0.1 25
Ricinus communis (25) 0.038 166 4,368 23.3 37 5.5
Solanum tuberosum (36) 0.042 65.5 1,560 29 37 4.7
Glycine max (52) alkaline 0.27 10.4 38 40 30 7.5
(6) alkaline 0.49 48 98 11 25 7.0
Wheat (27) 7.5 1,305 174 26.4 37 5.5
ery, R, are listed in Table 8 for F from some micro- formed and is discarded. The supernatant, containing
organisms and plants. F, is adjusted to pH 7.3 with a Trizma base solution.
The procedure of Chu et al. (43) for internal F 5. The supernatant is loaded onto a Whatman
from S. cerevisiae, leading to a 19,390-fold purication microgranular DE 52 column (2 18 cm). This is
and 19% activity recovery, is hereafter outlined. All developed with 1 L linear gradient of 50350 mM
operations are carried out at 05 C. NaCl in buffer 1. External F is eluted at 0:1 M
1. Yeast paste is suspended in equal weight of 50 NaCl, while the internal form is eluted at 0.2 M
mM Tris-HCl pH 7.5, with 5 mM MgCl2 , 40 mM NaCl (R 54; P 1:5).
MSH, 1 mM phenylmethylsulfonyluoride (PMSF, 6. Fractions exhibiting internal F activity are
a protease inhibitor), and broken in a Bead-Beater. pooled and concentrated by precipitation with solid
Cell lysate is centrifuged 20 min at 30,000g (R 100; (NH4 2 SO4 up to 80% saturation (R 35;
P 1). P 5024).
2. 1% streptomycin sulfate is added to the super- 7. The precipitate is dissolved in 12 mL buffer 1
natant and after 1 h in an ice bath; nucleic acids and with 0.1 M NaCl and loaded onto a Ultrogel AcA 44
ribosomes are removed by centrifugation (R 98; column (1:5 98 cm) equilibrated with the same buf-
P 1:04). fer. The F fractions are pooled and added with
3. A precipitate is obtained from streptomycin NH4 2 SO4 to a nal concentration of 20%, then chro-
supernatant at 4085% (NH4 2 SO4 saturation and is matographed on a 1 4 cm column of phenyl-
then dissolved in 10 mM Tris-HCl buffer, pH 7.3 (buf- Sepharose CL-4B equilibrated with buffer 1, plus
fer 1) containing 1 mM MSH and 0.5 mM PMSF, 20% (NH4 2 SO4 . A linear gradient, 200%
and dialyzed thoroughly against this buffer. (NH4 2 SO4 in buffer 1, is applied for eluting internal
4. 2 M acetic acid solution is added to the dialy- F in a pure form at 5% (NH4 2 SO4 (R 22;
sate to bring the pH to 4.9. A proteic precipitate is P 19,610).

8. Concentration by ultraltration and dialysis 3. A Goldstein, JO Lampen. -D-Fructofuranosidase
against 10 mM NaH2 PO4 =Na2 HPO4 , pH 7.2 fructohydrolase from yeast. Methods Enzymol
(R 19; P 19,390). 42:504511, 1975.
The procedure suggested by Bracho and Whitaker 4. F Moreno, AG Ochoa, S Gascon, JR Villanueva.
Molecular forms of yeast invertase. Eur J Biochem
(36) for a plant glyco-F and outlined hereafter allows
50:571579, 1975.
good purication-fold (1560) and enzyme recovery
5. R Pressey, JK Avants. Invertases in oat seedlings.
(29%). All steps are performed at 05 C. Plant Physiol 65:136140, 1980.
1. Potato slices (1 kg) are homogeneized with 100 6. JQ Chen, CC Black. Biochemical and immunological
mL NaHSO3 (0.1 M) in a Waring blender. The ltrate properties of alkaline invertase isolated from sprout-
through eight-layer cheesecloth is centrifuged and the ing soybean hypocotyls. Arch Biochem Biophys
supernatant is dialyzed against water. The formed pre- 95:6169, 1992.
cipitate is removed by centrifugation. The supernatant 7. AP Ranwala, S Iwanami, H Masuda. Acid and neu-
is lyophilized and suspended in buffer A (20 mM Tris- tral invertases in the mesocarp of developing muskme-
HCl/500 mM NaCl, pH 7.4) containing CaCl2 , MgCl2 , lon (Cucumis melo L. cv. Prince) fruit. Plant Physiol
and MnCl2 (1 mM of each) (R 100; P 1). 96:881886, 1991.
8. HA Ross, D McRae, HV Davies. Sucrolytic enzyme
2. The sample is loaded onto an afnity column
activities in cotyledons of faba bean. Plant Physiol
(2:5 10 cm) Con ASepharose 4B equilibrated and
111:329338, 1996.
eluted with buffer A. 30 mM of methyl -D-manno- 9. X Tang, HP Ruffner, JD Scholes, SA Rolfe.
pyranoside in buffer A allows enzyme elution. F is Purication and characterisation of soluble invertases
concentrated by ultraltration and then dialyzed from leaves of Arabidopsis thaliana. Planta 198:1723,
against buffer B (20 mM NaH2 PO4 =Na2 HPO4 , pH 1996.
6.0) (R 110; P 11). 10. W Van den Ende, A Van Laere. Purication and
3. The solution is chromatographed on a DEAE- properties of a neutral invertase from the roots of
Sephadex A-50-120 column (2:5 18 cm) equilibrated Cichorium intybus. Physiol Plant 93:241248, 1995.
in buffer B and eluted with a linear gradient of 0250 11. H Masuda, T Takahashi, S Sugawara. Acid and alka-
mM NaCl. The active fractions are pooled and con- line invertases in suspension cultures of sugar beet
cells. Plant Physiol 86:312317, 1988.
centrated by ultraltration. The concentrate is dialyzed
12. HS Lee, A Sturm. Purication and characterization of
three times against buffer B (R 69; P 193). neutral and alkaline invertase from carrot. Plant
4. The sample is loaded on a Sephadex G-150 col- Physiol 112:15131522, 1996.
umn (1:5 90 cm), equilibrated, and eluted with 50 13. JR Stommel, PW Simon. Multiple forms of invertase
mM NaH2 PO4 =Na2 HPO4 =100 mM NaCl buffer (pH from Daucus carota cell cultures. Phytochemistry
7.2). F fractions are pooled and concentrated 29:20872089, 1990.
(R 51; P 210). 14. AP Ranwala, C Suematsu, H Masuda. Soluble and
5. The puried enzyme is obtained upon a second wall-bound invertases in strawberry fruit. Plant Sci
run on a DEAE-Sephadex A-50-120 column (1:5 18 84:5964, 1992.
cm), equilibrated in buffer B, and eluted with buffer B/ 15. EM Klann, RT Chetelat, AB Bennett. Expression of
100 mM NaCl (R 29; P 1560). acid invertase gene controls sugar composition in
tomato (Lycopersicon) fruit. Plant Physiol 103:863
870, 1993.
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Physiol 95:10261035, 1991.
17. C Davies, SP Robinson. Sugar accumulation in grape
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1968. Biochem Biophys 327:98106, 1996.

65
b-Galactosidase
Raymond R. Mahoney
University of Massachusetts, Amherst, Massachusetts, U.S.A.
I. INTRODUCTION the enzyme is used in clinical settings to aid lactose

digestion and in commercial operations to produce
-Galactosidase (-D-galactoside galactohydrolase, low-lactose diary products.
EC 3.2.1.23) is often known as lactase because it
catalyzes the hydrolysis of lactose into its constituent
sugarsviz., lactose H2 O ! galactose glucose. II. IMPORTANCE TO FOOD QUALITY
All lactases are -galactosidases but some -galactosi-
dases, including those from plant cells and mammalian Enzymatic hydrolysis of lactose is of interest in both
organs other than the intestine, have little or no activ- food science and nutrition, as well as in waste manage-
ity on lactose because their function is to hydrolyze ment and byproduct utilization. The product sugars
other galactosyl moieties, including glycolipids, glyco- are sweeter, more soluble, more easily fermented, and
proteins, and mucopolysaccharides (1). more readily absorbed from the intestine.
-Galactosidase hydrolyzes O-glycosyl bonds and Consequently, hydrolysis of lactose in milk and whey
exhibits strict specicity for the glycone part of the provides new functional properties for these ingredi-
substrate (Fig. 1). The only changes that allow activity ents which can be exploited in a variety of products
(albeit reduced) are replacement of the hydroxylmethyl (see Sec. IV below).
group on carbon 6 with a methyl group or a hydrogen The principal reason for hydrolyzing lactose in uid
atom. Consequently, the enzyme shows some activity milk, however, is to overcome the problem of lactose
on -L-arabinosides and -D-fucosides. Other changes
such as methylation of the hydroxyl on carbons 2, 3, 4,
or 6; loss of the pyranose ring structure; or conversion
to the -anomeric form, lead to loss of activity (1).
Replacement of the glycosidic oxygen with sulfur
causes loss of activity but not loss of binding afnity
(1). The enzyme shows wide tolerance for the structure
of the aglycone which may be another sugar, an alkyl
group or an aryl group. The nature of the aglycone,
however, strongly inuences the kinetic parameters (1).
Activity of intestinal lactase in mammals decreases
after weaning, and this leads to some degree of lactose
intolerance in many population groups. Consequently, Figure 1 Hydrolysis of lactose by -galactosidase.

intolerance, which is widespread among non-Caucasian IV. USE IN FOODS
adults and leads to reduced milk consumption. This is
of concern since milk is a good source of high-quality Reduction of the lactose content of milk or whey by
protein and an especially good source of calcium. hydrolysis with -galactosidase leads to several pro-
Whey is the principal byproduct of cheese manufac- ducts/applications, as shown in Table 2. These applica-
ture and contains > 70% lactose on a dry-weight basis. tions have been reviewed extensively elsewhere (2).
Hydrolysis of the lactose improves whey utilization in
a variety of products and processes (see Sec. IV).
V. PROPERTIES AS A PROTEIN
III. SOURCES The best-understood -galactosidase in terms of pro-

tein structure is the enzyme from Escherichia coli
-Galactosidases are widely distributed in nature encoded by the lac Z gene. This enzyme has a molecu-
according to their various functions, which include lar weight of 464 kDa and is composed of four iden-
digestion, lysosomal degradation, and catabolism. tical subunits (each 116 kDa), each containing 1023
However, lactases are found essentially only in the residues (3). X-ray crystallography conrms that the
mammalian intestinal tract and in various microorgan- protein is a tetramer with 222 point symmetry (4).
isms, including many fungi, yeasts, and bacteria (2). Each subunit contains a binding site for Mg2 and
In the intestine, the enzyme is located in the micro- has ve essentially independent domains. The key
villi of the mucosal cells of the brush border mem- active site residues are located in a depression formed
brane. In microorganisms, the enzyme is usually from loops connecting the C-terminal ends of -
intracellular in bacteria and yeasts but may be intra- strands to surrounding -helices in an = barrel.
or extracellular in fungi. Subunits and dimers of subunits are inactive whereas
For commercial use lactase is extracted from a few activity in the tetramer state is due to completion of
microbial sources, primarily yeasts and fungi, which each active site by a loop donated from a neighboring
are considered safe as hosts for food grade enzymes monomer (4).
(see Table 1). It may be possible to use -galactosidases At the primary sequence level, each subunit con-
from other sources by cloning them into one of these tains 16 half-cystine residues per 116-kDa monomer;
hosts. The commercial enzymes are usually classied as virtually none of these residues appear to be involved
acid pH (pH optimum < 5) for use in acid whey, or in disulde linkages (5).
neutral pH (pH optimum 5.57.0) for use in milk or
sweet whey. The enzyme is also produced from
Escherichia coli for biochemical and analytical uses.
Table 2 Applications of Lactose Hydrolysis
Table 1 Commercial Sources of -Galactosidase for Product/process Advantages

Hydrolysis of Lactose in Foods
Low-lactose milk Overcomes the problem of
Molecular lactose intolerance
Organism pH Optimuma weight (kDa) Sweetened condensed milk, Reduce lactose crystallization
ice cream, dulce de leche in concentrated or frozen
Bacteria milk products
Bacillus spp. related to 5.56.5 116 Yogurt and cheese Accelerates ripening by
B. stearothermophilus production of more easily
Yeasts fermented sugars
Kluyveromyces fragilis 6.57.5 201 Sugars syrups from Increased sweetness of
K. lactis 6.57.0 117 deproteinized whey glucose and galactose
Candida psuedotropicalis 6.2 b allows multiple uses in ice
Fungi cream, baked goods,
Aspergillus niger 2.54.0 109112 confectionery, and soft
A. oryzae 4.55.0 90 drinks
a
Fermentation to ethanol Fermentation yeasts grow
Dependent on strain. more easily on the glucose
b
Not known.
produced

In contrast to the above, very little is known enzyme , enzyme-lactose
about the protein structure of -galactosidase
enzyme-lactose!enzyme-galactose
from eukaryotes. However, amino acid sequences
have been established for -galactosidase from sev- +glucose enzyme-galactose!
eral other bacteria and from the yeast
enzyme-galactose enzyme-galactose
Kluyveromyces lactis (6). Comparison of the yeast
-galactosidase with the subunits of -galactosidase H2 O ! enzyme galactose
from prokaryotes show similarity in molecular 1
weight (117 kDa) and extended sequence homology,
suggesting close structural and evolutionary rela- Reaction of the enzyme-galactose complex with
tionships (6). another sugar (such as lactose, galactose, or glucose)
Extracts of E. coli subjected to electrophoresis instead of water allows for the synthesis of oligosac-
and stained histochemically for -galactosidase charides by the transferase reaction, which can become
show multiple bands (7), and a similar phenom- signicant at high substrate concentrations and/or
enon has been seen with both crude and puried when the degree of substrate conversion is high (13).
enzyme from Kluyveromyces fragilis (8). Purication There are several substrate/product analogs which
and characterization of the isoforms from E. coli inhibit -galactosidase. Galactose is a competitive inhi-
have shown them to be polymers and have not bitor, but glucose is often ineffective except at very
revealed compositional differences (9). In the case high concentrations, where it is noncompetitive (14).
of fungal enzymes, however, isoforms have been Thiogalactosides such as p-aminophenyl -D-thio-
observed which do differ in the extent of glycosy- galactoside are weak competitive inhibitors (15, 16).
lation (10). The most powerful inhibitors are the 1-4 and 1-5 galac-
tonolactones, especially the latter, which, it has been
suggested, may resemble the transition state form of
VI. PROPERTIES AS AN ENZYME the substrate (17).
The enzymatic properties of -galactosidase vary with

the source. These properties are summarized in Table 3 VII. DETERMINATION OF ACTIVITY
for three different -galactosidases (one each from bac-
teria, yeasts, and molds). -Galactosidase activity can be followed quite readily
Comparison of kcat =Km shows that the synthetic by following the disappearance of substrate or the
substrate ONPG (o-nitrophenyl -D-galactopyrano- appearance of product, but it is usually easier to follow
side) is a better substrate than lactose in all cases. product formation.
The fungal enzyme from Aspergillus niger is the most Determination of lactose hydrolysis is complicated
thermostable and is unaffected by cations or sulfhydryl by transferase activity, although the latter is minimal
modiers such as p-chloromercuribenzoate. However, (and can often be ignored) when the lactose concentra-
it is the most susceptible to product inhibition by tion is 5% or less, or only initial rates are being mea-
galactose. sured. Under these conditions, it is easy to assay
The mechanism of action is not completely activity on lactose by measuring the production of
understood, but the E. coli enzyme appears to glucose or galactose, preferably enzymatically. The
work in a fashion analogous to that of lysozyme. most commonly used method is determination of glu-
Thus, one group acts as a general acid, donating a cose with glucose oxidase coupled to peroxidase (see
proton to the glycosidic oxygen, and another, nega- Sec. VII.A below).
tively charged group stabilizes a positively charged Where a large percentage of the original lactose is
carbonium galactosyl-enzyme intermediate for reac- hydrolyzed, determination of total monosaccharides is
tion with water (11). Site-directed substitutions in a good practical indicator of the extent of hydrolysis,
the E. coli enzyme indicate that Tyr503 functions but it may underestimate the number of glycosyl bonds
as the general acid and that Glu461 functions as broken in lactose, owing to the formation of oligosac-
the stabilizer for the intermediate which alternates charides by the transferase reaction.
between a carbocation and a covalently bound Hydrolysis of lactose can also be followed by polari-
form (12). The hydrolytic reaction [Eq. (1)] can metry due to a small change in specic rotation (from
then be described as: 52:5 to 67:0 ). However, this method is not

Table 3 Enzymatic Properties of -Galactosidase
Escherichia coli Kluyveromyces fragilis Aspergillus niger

(14) (8, 15) (10, 27)
Molecular weight (kDa) 464 201 109112

pI 4.61 5.1 4.64
pH optimum 7.27.4 6.26.4 2.54.0
pH stability 68 6.57.5 28
Temperature optimum ( C) 40 37 5560
Activation energy (kJ/mole) 12.6 38.1 35.1
kcat ONPGa sec1 1:38 106 3:41 103 2:19 105
kcat lactose sec1 5:10 103 1:551 103 1:91 105
Km ONPGa (mM) 0.161 2.72 2.22
Km lactose (mM) 1.9 13.9 85125
kcat =Km PNPGa (sec1 M1 ) 8:57 109 1:25 106 98:6 106
kcat =Km lactose (sec1 M1 ) 2:68 106 1:08 105 1:91 106
Ki galactose (mM) 21 27.7 4
Ki PAPTGb (mM) 5 6.05 c
Activators Na , Mg2 K , Mg2 , Mn2 None
Inhibitors
Galactono-1-4-lactone Yes Yes c
p-Chloromercuribenzoate Yes Yes No
a
ONPG, o-nitrophenyl--D-galactoside.
b
PAPTG, p-aminophenylthio--D-galactoside.
c
Not known.
used much compared to chemical estimation of the analyzed for glucose, preferably by the use of glucose
products. oxidase and peroxidase in conjunction with a dye such
Synthetic substrates such as o-nitrophenyl -D- as o-dianisidine [Eqs. (2) and (3)].
galactopyranoside (ONPG) and p-nitrophenyl -D-
galactopyranoside (PNPG) are usually hydrolyzed glucose
D-glucose+O2 ! H2 O2 D-gluconolactone
faster than lactose and provide an easy, convenient, oxidase
colorimetric assay since the nitrophenol released
absorbs in the range 400420 nm when in alkaline 2
solution.
peroxidase
For detection of very low levels of activity, uori- H2 O2 o-dianisidine ! oxidized o-dianisdine
metric methods of assay can be used with substrates
such as 4-methylumbelliferone -D-galactoside (8). max 436 nm 3
The conditions for assay vary with the source of the
enzyme and the substrate; representative examples are Alternatively, the galactose produced can be esti-
given below. mated indirectly by use of galactose dehydrogenase
[Eq. (4)].
A. Assay of b-Galactosidase Activity on Lactose
with Neutral-pH Enzyme galactose
D-galactose NAD !
dehydrogenase
4
Enzyme 100 L ( 30 U/mL) is added to 4 mL of 5%
lactose in 0.1 M potassium phosphate buffer, pH 6.6, D-galactonolactone NADH H
containing 3.2 mM MgCl2 , at 30 C, or to 4 mL milk at
the same temperature. The reaction is stopped after 10 The increase in absorbance at 340 nm is a measure
min by adding 0.1 mL of 4 N HCl and then neutralized of the NADH produced and of the lactose hydro-
by adding 0.1 mL of 6.4 N NaOH. The solution is then lyzed.

B. Assay of b-Galactosidase Activity on o- cipal stabilizer, but its effect is markedly lactose
Nitrophenylgalactoside (ONPG) Using dependent.
Neutral-pH Enzyme The -galactosidase from Streptoccocus thermophi-
lus can be stabilized by a number of proteins, such as
Enzyme (100 L ( 1 U/mL) is added to 4 mL of 2 bovine serum albumin and casein, via hydrophobic
mM ONPG in 0.1 M potassium phosphate buffer, pH interactions (24). The -galactosidase from K. lactis
6.6, containing 0.1 mM MnCl2 and 0.5 mM dithioer- is stabilized by several amino acids, notably histidine,
ythritol. The reaction is stopped after 5 min by adding which decreases the rate of unfolding of the enzyme
1 mL of 0.5 M Na2 CO3 containing 15 mM EDTA, and during heat denaturation (25).
the absorbance is determined at 420 nm. Activity is
calculated using a molar extinction coefcient of 4500
M1 cm1 for o-nitrophenol at pH 10 (15). For acid-
VIII. PURIFICATION
pH enzymes, the substrate buffer is changed to 100
mM sodium acetate buffer, pH 4.0, and the reaction
-Galactosidase can be puried by the conventional
is stopped as described above.
methods of protein separation such as gel ltration
and ion exchange chromatography and also, very
C. Other Aspects effectively, by afnity chromatography. Examples are
shown below.
-Galactosidase activity in tissues can be detected by
histochemical staining with 6-bromo-2-naphthyl--
galactosidase. The product 6-bromo-2-naphthol, reacts A. Purication of b-Galactosidase from
with tetrazotized o-dianisidine (Diazo Fast Blue B) in Kluyveromyces lactis (26)
alkaline solution to give a blue-purple color at the site
of enzyme activity (19). The same method can be used The yeast cells were disrupted in a steel press, and the
to detect activity of -galactosidase in electrophoresis ruptured cells were extracted with 50 mM phosphate
gels (7). buffer, pH 7.0, containing 10 mM MgCl2 (Buffer 1).
Neutral-pH enzymes require Mg2 or Mn2 for After centrifugation to remove cell debris, the super-
maximum activity, but acid-pH enzymes (from fungi) natant was brought to 55% saturation with
do not require metal ions. Incorporation of a sulfhy- (NH4 2 SO4 to precipitate the enzyme. The precipitate
dryl reagent (2-mercaptoethanol or dithiothreitol) was dialyzed against Buffer 1 containing 1 mM 2-mer-
often increases activity with neutral-pH enzymes. captoethanol to stabilize the enzyme against activity
Activity and stability of -galactosidase in milk loss. The enzyme was then puried by gel ltration
and whey can be very different from that in lactose chromatography on Sephadex G-100 using Buffer 1
solutions. Activity is often lower in milk or whey followed by ion exchange chromatography on
(20, 21) owing to the high level of calcium, which DEAE-Sephadex A50. The enzyme was applied in
exerts a strong inhibitory effect (21, 22). Stability Buffer 1 and was eluted with a gradient of 00.5 M
against thermal denaturation is markedly NaCl in Buffer 1. A summary of the purication is
increasedup to 100-fold in milk (23). This appears shown in Table 4. Overall, this enzyme was puried
to be due to several milk constituents: salts, lactose, 79-fold with a yield of 19% and was judged homoge-
and proteins, acting in concert. Casein is the prin- neous by polyacrylamide gel electrophoresis.
Table 4 Purication of -Galactosidase from Kluyveromyces lactis
Volume Protein Activity Specic activity

Fraction (mL) (mg/mL) (U 103 ) (U/mg)
Centrifuged cell extract 46 77 14.9 4.22

Ammonium sulfate fractionation 30 48 12.7 8.25
Sephadex G-100 123 2.7 14.5 43.7
DEAE Sephadex-A50 26 0.55 2.04 143
Source: Ref. 26

B. Purication of -Galactosidase from 10. F Widmer, JL Leuba. -Galactosidase from
Escherichia coli by Afnity Chromatography Aspergillus niger. Separation and characterization of
(16) three multiple forms. Eur J Biochem 100:559567,
1979.
11. ML Sinnott. Ions, ion pairs and catalysis by the lacZ
Cells of E. coli were suspended in 50 mM Tris-HCl, pH
-galactosidase of Escherichia coli. FEBS Lett 94:19.
7.5, containing 100 mM NaCl and 10 mM MgCl2 12. CG Cupples, JH Miller, RE Huber. Determination of
(Buffer 2) and disrupted by sonication. After centri- the roles of Glu-461 in -galactosidase (Escherichia
gation to remove cell debris, the supernatant was coli) using site-specic mutagenesis. J Biol Chem
brought to 50% saturation with NH4 2 SO4 and cen- 265:55125518, 1990.
trifuged, and the pellet was dissolved in Buffer 2 and 13. RR Mahoney. Galactosyl-oligosaccharide formation
dialyzed against the same buffer. The enzyme solution during lactose hydrolysisa review. Food Chem
was then applied to a column of an afnity matrix 63:147154, 1998.
consisting of the inhibitor p-aminophenyl--D-galacto- 14. K Wallenfels, OP Malhotra. -Galactosidase. In: PD
pyranoside (Ki 5 mM) attached to Sepharose 4B via Boyer, H Lardy, K Myrback, eds. The Enzymes, Vol 4
a long spacer arm (3-aminosuccinyl-3-aminodipropyla- (2nd ed). New York: Academic Press, 1960, p 409.
15. RR Mahoney, JR Whitaker. Stability and enzymatic
mine). The column was washed with Buffer 2, and the
properties of -galactosidase from Kluveromyces fra-
enzyme was eluted by 0.1 M borate buffer, pH 10; it gilis. J Food Biochem 1:327350, 1977.
was homogeneous as judged by gel electrophoresis. 16. E Steers Jr, P Cuatrecasas, HB Pollard. The purica-
The yield was 95% compared with a yield of 50% tion of -galactosidase from Escherichia coli by af-
using conventional procedures (5). nity chromatography. J Biol Chem 246:196200, 1971.
17. J Conchi, AJ Hay, I Strachan, GA Levvy. Inhibition
of glycosidases by aldonolactones of corresponding
REFERENCES conguration. Biochem J 102:929941, 1967.
18. JW Woolen, PG Walker. Fluorimetric estimation of
1. K Wallenfels, R Weil. -Galactosidase. In: PD Boyer N-acetyl beta-glucosaminidase and beta-galactosi-
ed. The Enzymes, Vol 7 (3rd ed). New York: dase in blood plasma. Clin Chem Acta 12:647
Academic Press, 1972, pp 617663. 665, 1965.
2. RR Mahoney. Lactose: enzymatic modication. In: 19. MS Burstone. Enzyme Histochemistry. New York:
PF Fox, ed. Advanced Dairy Chemistry3. Academic Press, 1962, p 375.
London: Chapman and Hall, 1977, pp 77118. 20. NA Greenberg, RR Mahoney. The activity of lactose
3. AV Fowler, I Zabin. Amino acid sequence of -galac- (Streptococcus thermophilus) in milk and sweet whey.
tosidase. XI. Peptide ordering procedures and the com- Food Chem 15:307313, 1984.
plete sequence. J Biol Chem 258:1020410207, 1978. 21. RR Mahoney, C Adamchuk. Effect of milk constitu-
4. RH Jacobson. The dimensional structure of -galac- ents on the hydrolysis of lactose by lactase from
tosidase. PhD thesis, University of Oregon, Corvallis, Kluyveromyces fragilis. J Food Sci 45:962969, 1980.
OR, 1993. 22. EJ Guy, EW Bingham. Properties of -galactosidase
5. GR Craven, E Steers Jr, CB Annsen. Purication, of Saccharomyces lactis in milk and milk products. J
composition and molecular weight of the -galactosi- Dairy Sci 61:147151, 1978.
dase of Escherichia coli K12. J Biol Chem 240:2468 23. RR Mahoney, T Wilder. Stabilization of lactase
2477, 1965. (Escherichia coli) by milk components and related
6. O Poch, HL Hote, V Dallery, F Debeaux, R Fleer, R compounds. J Food Sci 45:899901, 1989.
Sodoyer. Sequence of the Kluyveromyces lactis - 24. BS Chang, RR Mahoney. Enzyme thermostabiliza-
galactosidase: comparison with prokaryotic enzymes tion by bovine serum albumin and other proteins: evi-
and secondary structure analysis. Gene 118:5563, dence for hydrophobic interactions. Biotechnol Appl
1992. Biochem 22:203214, 1995.
7. SH Appel, DH Alpers, GM Tomkins. Multiple mole- 25. SS Surve, RR Mahoney. Thermostabilization of
cular forms of -galactosidase. J Mol Biol 11:1221, Kluveromyces marxianus -galactosidase by histidine:
1965. physical studies. Biotechnol Appl Biochem 23:155
8. RR Mahoney, JR Whitaker. Purication and physio- 162, 1996.
cochemical properties of -galactosidase from 26. J Burstone, MD Glantz. Isolation and characteriza-
Kluyveromyces fragilis. J Food Sci 43:584591, 1978. tion of -galactosidase from Saccharomyces lactis.
9. SL Marchesi, E Steers Jr, S Shifrin. Purication and Biochim Biophys Acta 167:373377, 1968.
characterization of the multiple forms of -galactosi- 27. YC Lee, V Wacek. Galactosidases from Aspergillus
dase of Escherichia coli. Biochim Biophys Acta niger. Arch Biochem Biophys 138:264271, 1970.
181:2034, 1969.

66
Pectic Polysaccharides
H. A. Schols and Alphons G. J. Voragen

Wageningen University, Wageningen, The Netherlands
I. INTRODUCTION wounding. As constituents of plant cell walls and due

to their anionic nature, pectic polysaccharides are con-
In the vast amount of literature on pectic enzymes an sidered to be involved in the regulation of ion trans-
unambiguous interpretation of presented results is port; they determine the porosity of the walls, and they
often hampered by the fact that ill-dened substrates may thus control the permeability of the walls for
and inadequate methodology have been used. The enzymes. They also determine the water-holding capa-
importance of the use of well-dened substrates and city of the wall (1, 2).
adequate methods is demonstrated by the facts that The following groups of polysaccharides are com-
in the last decade more than ve new enzymes active prised in the term pectic substances:
on specic structural elements of pectin have been
identied and that within classes of specic enzymes
a further differentiation became possible based on dif- 1. Linear homogalacturonan consisting of -
ferences in substrate specicity. So can polygalacturo- (1 ! 4-linked D-galacturonic acid units in
nases be differentiated with respect to their tolerance of which varying proportions of the carboxyl
methyl esteried galacturonic acid residues in their groups are esteried with methanol, and sec-
active sites (Chapter 69). Also, to understand the tech- ondary hydroxyl groups at O-2 and O-3 with
nological role of a specic enzyme in an application, its acetyl groups.
action at a molecular level should be known. We there- 2. Linearly branched xylogalacturonan carrying
fore felt the need to devote a chapter to the description single-unit xylopyranosyl residues and the
of the structural elements present in pectins, which rarely occurring apiogalacturonan carrying
variations are found in nature in these structural ele- mono- and diapiosyl side chains (2).
ments, and the methodology to measure the activity 3. Highly branched galacturonan, in the literature
and mode of action of the enzyme families active on in retrospect wrongly termed rhamnogalacturo-
pectins. nan II (RG-II).
Pectins or pectic substances are collective names for 4. Rhamnogalacturonan having a backbone of
a group of closely associated polysaccharides present alternating -(1 ! 2-linked L-rhamnosyl resi-
in plant cell walls, where they contribute to complex dues and -(1 ! 4)-linked D-galacturonosy-
physiological processes like cell growth and cell differ- luronic acid residues ramied at O-4 of the
entiation and so determine the integrity and rigidity of rhamnosyl residue with neutral sugar side
plant tissue. They also play an important role in the chains of varying composition and size (1 to
defence mechanisms against plant pathogens and 50 sugar moieties).

5. Polymeric side chains of pectin usually dis- II. HOMOGALACTURONAN
tinctly referred to as arabinans, galactans, and
arabinogalactans. As described above, the linear homogalacturonan
dominates in commercial pectins and plays a promi-
Next to the xyloglucan-cellulose network, these pectic nent role in plant physiology (e.g., ripening).
polysaccharides are suggested to form an independent Therefore, both the chemical ne structure of homo-
network determining, e.g., the porosity of the cell wall galacturonans (Fig. 2) and enzymes specic to (methyl
(3). esteried) galacturonans are studied in great detail. In
The texture of fruits and vegetables during growth, addition to the type and amount of neutral sugars pre-
ripening, and storage is strongly inuenced by the sent, the major structural differences among various
amount and composition of the pectic molecules pre- commercial pectins (in essence homogalacturonans)
sent. Consequently, the structure of pectins present in are explained by the level of methyl esters present at
fruit and vegetables depends on enzymatic and chemi- C6 of the galacturonic acid residues and the distribu-
cal modications occurring during these processes. tion of these esters and neutral sugar side chains over
Endogenous as well as exogenous enzymes (processing the galacturonan backbone. Usually, the degree of
aids) play an important role in determining the pectic methyl esterication (DM) is expressed as moles of
structures present in plant tissues or food products at a methanol present per 100 moles of galacturonic acid.
given time. When the DM is 50 or higher, pectins are called high-
Pectin used in the food industry as natural ingredi- methoxyl (HM) pectins, whereas the term low-meth-
ents for their gelling, thickening, and stabilizing prop- oxyl (LM) pectins indicates a DM < 50.
erties are extracted from suitable agrobyproducts like Methyl esters may be distributed over the homoga-
citrus peel and apple pomace (2, 4). Commercially pro- lacturonan quite differently where the two extremes are
duced pectins usually contain > 65% of galacturonic formed by a random distribution where all esters are
acid by weight, since the extraction conditions used spread out over the polymer in a rather statistical
remove the greatest part of the neutral side chains pre- way or by a blockwise distribution where blocks of
sent in native pectin rather specically (2, 4, 5). nonesteried galacturonic acid residues are inter-
The chemistry and technology of pectin are discussed spersed with segments having a rather high degree of
extensively by Walter (6). A general representation of methyl esterication (2, 10). Such distributions are
native pectins is given in Figure 1. This gure shows that strongly inuenced by the presence of endogenous
the major part of the galacturonosyl residues is present enzymes like plant pectin methylesterase, whose activ-
in homoglacturonan segments (smooth regions of pec- ity may cause a blockwise distribution or by the con-
tin), whereas only a relatively small part of the uronic ditions under which the raw material is processed or
acids is present in so-called hairy regions (7, 8). the pectin extracted (2). Pectin lyase can therefore be
The proportion of the various structural elements of active on an LM pectin with a blockwise distribution
pectin may differ strongly from one pectin to another and polygalacturonase on an HM pectin with block-
(Table 1). The proportion of homogalacturonan can be wise distribution. In addition to methyl esterication,
> 65% for commercially extracted pectins but can also depending on the origin of the pectin, galacturonic acid
be zero in the case of soybean pectin. Large variations moieties may be substituted with acetyl groups at O-2
are also found for xylogalacturonan, arabinan, or or O-3 (e.g., sugar beet pectin, potato pectin) (2).
galactan segments (Table 1) (8, 26).
The various different structural elements present in
pectic substances will be discussed in more detail III. XYLOGALACTURONANS
below. Enzyme systems which are able to (specically)
degrade or modify homogalacturonans, xylogalactur- In xylogalacturonans, the amount of -xylose substitu-
onans, rhamnogalacturonans, arabinans, and (ara- tion to the O-3 position of galacturonic acid residues in
bino) galactans are covered in Chapters 6770, 73, the backbone may vary signicantly (Fig. 3), depend-
and 80 of this book. ing on their origin (e.g., apple, pea hulls, watermelon,

Figure 1 Structural elements of pectin. Occurrence, amount, and chemical ne structure of the individual segments may vary
signicantly depending on the origin of the pectin. Gal, galactose; GalA, galacturonic acid; Rha, rhamnose; Fuc, fucose, Xyl,
xylose; DHA, 3-deoxy-D-lyxo-2-heptulosaric acid; KDO, 2-keto-3-deoxy-D-manno-octulosonic acid; Api, apiose; AceA, aceric
acid, GlcA, glucuronic acid, Ac, acetyl group; Me, methyl ester; 4-O-Me, 4-O-methyl ether. (From Refs. 7, 8.)
soybeans). This is also the case for the amount and apiose, aceric acid, KDO, and DHA) in a dened and
distribution of methyl esters present, since for apples rather conserved structure (Fig. 1). This structural ele-
various populations of xylogalacturonans with a rather ment is involved in the crosslinking of two pectin mole-
distinctive methyl ester level have been reported (7). cules within the cell wall through a borate diester (11,
12). No enzymes able to degrade the complex HBG
structure have been described in detail.
IV. HIGHLY BRANCHED
GALACTURONAN (HBG) OR
RHAMNOGALACTURONAN II V. RHAMNOGALACTURONAN
HBG or RG-II consists of a backbone of about nine - Rhamnogalacturonan has a backbone of up to 300
(1 ! 4)-linked galacturonosyl residues carrying four repeating units of alternating -(1 ! 2)-linked rham-
side chains containing a number of rare sugars (e.g., nosyl and -(1 ! 4)-linked galacturonosyluronic acid

Table 1 Occurrence and Proportion of the Various Structural Elements of
Native Pectin in Apple, Sugar Beet, and Soybean
Soybean Sugar beat Apple

meal pulp
Total polysaccharide (w/w% dry matter) 16 67 20

Pectic substances (% of total 59 40 42
polysaccharides)
Structural element (% of pectic substances)
Homogalacturonan 0 29 36
Xylogalacturonan 21 <1 4
Rhamnogalacturonan II 4 4 10
Rhamnogalacturonan backbone 15 8 4
arabinan 60 46 27
arabinogalactan I 12 20
arabinogalactan II 0 0
Source: Ref. 8.
residues, which may be methyl esteried and/or acety- RG segments. A common feature of these RG struc-
lated (Fig. 4) (7, 13, 14). Depending on the origin of the tures is that they are also (highly) substituted with
cell walls examined (type of cell wall, tissue, plant seg- acetyl groups, either attached to O-2 or to O-3 or to
ment, etc.), the length of the side chains attached to both O-2 and O-3 of the galacturonic acid moieties (7,
2080% of the rhamnose residues can vary from one 14) which inhibits RG-hydrolase and -lyase (see Chap.
single galactose residue up to chains of 50 residues or 73).
more being composed of arabinose (e.g., sugar beet),
galactose (e.g., ax, garlic, onion) or both (e.g., soy,
potato) (7, 13). Taking all possible variations into VI. ARABINANS
account, it can be stated that rhamnogalacturonans
are a family of quite complex structures with only Arabinans are branched homoglycans mainly com-
the rhamnogalacturonan backbone in common. posed of a backbone of -L-(1 ! 5)-linked arabinosyl
Recently, a whole family of rhamnogalacturonan- residues and in native form substituted with -arabi-
specic carbohydrases has been recognized which is nofuranosyl residues at O-2 and/or O-3 position. Such
discussed in Chapter 73. Using a specic rhamnogalac- a substitution can be a single arabinosyl residue, but
turonan hydrolase, it was shown that rhamnogalactur- longer (branched) chains can also be present mostly
onan contains blocks highly ramied with long neutral one to three arabinosyl residues (see Fig. 5), resulting
sugar side chains, and in addition segments of alternat- in complex structures (see the reviews 1517 and refer-
ing rhamnose and galacturonic acid sequences with or ences herein).
without single-unit galactose substitution on O-4 of the Arabinans have been described to be present in cell
rhamnosyl residues up to 30 residues in length. Some walls from apples, sugar beet, rapeseed, carrot, cow-
rhamnogalacturonan preparations (e.g., from syca- pea, azuki- and soybean, rape, mustard, lemon, grape,
more cells) were resistant to degradation by RG hydro- cabbage, onion, potato, and others (17). Arabinans
lases (7), demonstrating the absence of low-substituted can have molecular weights up to 10 kDa and are
Figure 2 Homogalacturonan fragment of pectin. Acetyl groups may be present at O-2, O-3 or both at O-2 and O-3 for some
pectins from some sources. (From Ref. 2.)

Figure 3 Schematic structure of a xylogalacturonan. The position of methyl esters is chosen arbitrarily.
often covalently linked to galacturonic acidrich poly- arabinofuranosyl residues attached through the O-3
mers. Although arabinans have been isolated in a pure position (20). O-6 substitution of the galactan back-
form, the extraction conditions used may have resulted bone with -galactose is also found. Figure 6 shows
in chemical (-elimination, chain peeling) or enzymatic the schematic structure of type I arabinogalactans.
degradation of any attached pectin structures (17 and Depending on the way of extraction, remnants of
references herein). the pectic backbone may still be present.
VII. GALACTANS B. Arabinogalactan Type II
Galactans and arabinogalactans in which the galactan Arabinogalactans of type II are highly branched poly-
backbone is substituted with various amounts of ara- saccharides with ramied chains of -D-galactopyra-
binose or galactose have been found in a broad range nose residues joined by 1,3 and 1,6 linkages. They are
of higher plants (1820). Aspinall (21) classied the more common in plants than type I and have been
arabinogalactans as type I and type II arabinogalac- reported in leaves, stems, roots, oral parts, seeds,
tans, where both types may be present as side chains of and media of suspension cultured cells (20).
pectin or as a single polymer. Type II arabinogalactans In general, the interior backbone of a type II galac-
may also be present as side chains of a protein segment tan consists mainly of -(1 ! 3)-linked galactose moi-
and as such be referred to as arabinogalactan proteins eties; -(1 ! 6)-linked galactopyranosyl residues occur
(AGPs). mainly in the exterior chains which may be terminated
with an L-arabinopyranosyl residue. The (1 ! 3)-
A. Arabinogalactan Type I galactan may be branched through O-6 with arabino-
furanose residues or (to a lesser extent) with arabino-
Arabinogalactans of type I are quite common for var- pyranose units, while also longer -(1 ! 3)-linked
ious types of plant tissues (20) and consist of a arabinose chains may be present. In general, galactose
(1 ! 4)-linked linear chain of -D-glactopyranosyl is more abundantly present than arabinose, although
residues. Pure galactans have been isolated from the ratio of arabinose to galactose may differ signi-
lupine, potato, and tobacco, although type I galactans cantly. Arabinogalactans of type II may be present
usually are substituted with short chains of (1 ! 5)- within the ramied regions of pectins but are also
Figure 4 The alternating structure of rhamnogalacturonan segments of pectin. The number, length, and structure of the side
chains may vary signicantly depending on the origin of the pectin. The acetyl groups may be substituted to O-2, O-3 or both O-2
and O-3 of the galacturonic acid residues. (From Refs. 7, 13, 14.)

Figure 5 Schematic structure of a highly branched -L-(1 ! 5)-arabinan.
often reported to be linked to a hydroxyproline-rich followed by precipitation with alcohol and possibly
protein (AGP), of which gum arabic from Acacia sene- treatment with acidic alcohol to remove salts like
gal is a well-known example. The hypothetical struc- potassium, sodium, and calcium. The precise condi-
ture of a type II arabinogalactan is shown in Figure 7. tions (pH, time, temperature) will determine the mole-
It should be emphasized that many variations with cular weight, the amount of neutral sugars still present,
respect to the model shown have been reported includ- and the degree of methyl esterication (2, 5).
ing substitution with glucuronic acid (22, 23). Arabinans and (arabino)galactans are not yet being
used routinely by the food industry. However, for
research purposes, these polysaccharides are commer-
VIII. ISOLATION OF POLYSACCHARIDE
cially available and are usually obtained by alkaline
STRUCTURES
treatment at increased temperatures to enhance degra-
dation of the pectic backbone and lowering the
It should be realized that all commercially available
strength of hydrogen bridges between the neutral poly-
polymeric substrates to be used in enzyme screening
saccharides and the cellulose matrix. Further purica-
and characterization have been isolated from the com-
tion of neutral polysaccharides includes preparative
plex plant material. As mentioned above, pectins with
anion exchange chromatography to remove charged
a high galacturonic acid content (> 70%) are extracted
impurities.
under acidic conditions from suitable raw materials,
Figure 6 Schematic structure of a type I -(1 ! 4)-linked arabinogalactan.

Figure 7 Hypothetical structure of type II -(1,3,6)-arabinogalactan. (From Refs. 22, 23.)
Dened polysaccharides with various degrees of given rather than extensive descriptions of the method
methyl esterication or branching are usually not com- mentioned.
mercially available and should be prepared in the
laboratory. Purication of polysaccharides is often a
A. Substrates
compromise: extensive handling in order to obtain
pure polysaccharides often changes the chemical com-
Pectic substrates can be purchased from several (spe-
position (as compared to the polysaccharide in its
cialized) suppliers (e.g., Megazyme, Ireland; Sigma-
native form), whereas mild procedures may result in
Aldrich Fine Chemicals, USA), but the origin, purity,
the presence of contaminating polysaccharides, ori-
and specications are not always clear and straightfor-
ginally linked to the polysaccharide of interest. This
ward. When monitoring crude enzyme mixtures, rela-
latter phenomenon may result in the presence of galac-
tively small amounts of other polysaccharides
turonic acids in an arabinan or galactan preparation.
accompanying the polysaccharide indicated on the
More universal assays to monitor enzyme action as
label may give erroneous results.
based on the release of reducing end groups may so
A broad range of synthetic substrates like p-nitro-
produce unreliable results because they do not given
phenyl- and p-methylumbelliferylglycosides can be
information as to which glycosidic bond in the sub-
obtained (e.g., Sigma-Aldrich) to assay glycosidase
strate is split. Analysis of the degradation product by
activity. Specic oligosaccharides may also be pur-
high-performance liquid chromatography (HPLC) or
chased from the same companies, although the choice
mass spectrometry (MS) will avoid such a confusion
and purity are limited. Alternatively, oligosaccharides
in most cases.
can be produced and puried in the laboratory (24).
Branched polysaccharides like arabinan or arabino-
IX. METHODS TO MONITOR PECTIC galactans can be rather easily de-branched by mild acid
ENZYMES treatment (17, 20, 25, 26) to obtain a more suitable
substrate for endoacting enzymes. A wide variety of
It should be realized that the summary of frequently both soluble azopolysaccharides and insoluble azur-
used methods given below is only a short list selected ine-crosslinked polysaccharides which can be used to
from the literature and from our own laboratory and is screen for specic classes of carbohydrases are com-
not complete. In most cases, adequate references are mercially available from Megazyme.

When a series of pectins is needed with various approach and method. Some of the methods and
degrees of methyl esterication, it is better to prepare approaches used in the characterization of enzymes
such a series in-house than to buy pectins with a dif- are described below.
ferent DM. In the former situation, relatively homo-
geneous substrates are obtained; in the latter situation
these preparations may be very heterogeneous owing A. Reducing End Groups
to their different history (origin, MW, neutral sugars),
and these different characteristics may inuence the Carbohydrases increase the number of reducing ends
outcome of the experiments. Lowering the DM in a when degrading their substrates. Many methods have
random way is possible by controlled alkali treatment been described to determine the amount of reducing
(27) or by using fungal pectin methylesterase (see sugars released including the Somogyi-Nelson proce-
Chapter 68). A more blockwise-distributed methyl dure, neocuproine assay, ferricyanide, and the 2,4-dini-
esterication can be obtained using pectin methylester- trosalicylic acid test (29; Chapter 71). One has to be
ase from plant origin (Chapter 68). Degrees of ester- aware that the outcome of the different assays may
ication up to 8590% can be obtained by methyl depend on the type of sugar moieties present in the
esterication using a MeOHH2 SO4 treatment as oligomers formed and the length of the oligomers
described previously (28). Polygalacturonic acid can (29, Chap. 71). Recently, the use of the very sensitive
be obtained from chemical supply stores and is a rather bicinchoninic acid (BCA) reducing value assay has
well-dened substrate for endopolygalacturonase been described to detect any carbohydrase activity
owing to the absence of methyl esters and neutral degrading any polysaccharide by which both endo-
sugars (as a result of a bleaching step). and exoenzymes can be measured (30). The reaction
can be carried out in wells of microtiter plates so
B. Substrate Solubility and Stability that large numbers of samples can be analyzed simul-
taneously. Because of the sensitivity, several precau-
Not all polysaccharides, when added to cold water, will tions should be taken to prevent too high
readily dissolve but they may cause lump formation. background levels as a result of the enzyme protein
This phenomenon can be avoided by premoistening the itself. Furthermore, depending on the polysaccharide
sample with a drop of ethanol prior to adding the used, the background caused by the reducing end
water/buffer. Heat treatment (80100 C) may also be group of the polymer can also be too high. This can
helpful. However, for esteried pectins, care should be be avoided by choosing a rather low substrate concen-
taken to avoid circumstances which allow -elimina- tration or, alternatively, by reducing the reducing end
tive degradation (T; pH > 5). Especially when the of the polymer by treatment with NaBH4 prior to use
solubilization of insoluble cell wall polysaccharides as (31).
consequence of enzyme action is monitored, it is
important not to have inaccurate results caused by
cleaving a few pectin bonds chemically. Depending B. Assays Using Synthetic or Dyed Substrates
on the structure of the substrate, the history of the
sample (cold storage, freezing, etc.) may inuence The most commonly used assays to determine glycosi-
aggregation/association behavior of the polymeric dases and exocarbohydrases make use of synthetic
molecules, which may result either in a rather poor substrates like p-nitrophenyl- and p-methylumbellifer-
solubility or in a high apparent molecular weight. ylglycosides where the release of the glycosyl group
When only part of the substrate is soluble and only results in a colored solution, which can be quantied
this part is used further for enzyme assays, it is relevant using a spectrophotometer set at the appropriate wave-
to examine whether this part is representative for the length.
desired substrate. Endoacting enzymes can be measured using poly-
saccharides to which usually an azocompound has
been attached. Degradation with endoenzymes results
X. ANALYSIS OF DIGESTS OF PECTIC in the release of dyed fragments which remain soluble
POLYSACCHARIDES after the addition of ethanol to precipitate the remain-
ing polymer. The color of the supernatant (and thus
Enzymic digests of pectic substances can be analyzed the enzyme activity) is measured spectrophotometri-
from different points of view, each requiring its own cally (Megazyme).

C. Solubilizing Activity of Carbohydrases products from their specic substrate (
235 4600 M1
cm1 for pectate and 5200 M1 cm1 for pectin (39
When enzymes are tested on insoluble substrate (e.g., 41).
cell wall polysaccharides), a relatively simple assay to
monitor the solubilization of pectic material is by using 2. Pectin Methyl Esterase
(automated) colorimetric assays like the m-hydroxybi-
phenyl assay (32, 33) for uronic acids and the phenol- The action of pectin methyl esterase results in the
sulfuric acid or orcinol-sulfuric acid assay for neutral release of methanol and consequently in the formation
sugars (34, 35). Obviously, the latter two methods are of carboxylic acid groups, and allows two ways of
not very discriminative for the various neutral sugars, monitoring the action of the enzyme. The release of
but can be used quite satisfactorily to monitor enzyme the H can simply be monitored in a nonbuffered sys-
activity. More information on the enzyme is obtained tem using a pH indicator which corresponds to the
when the determination of concentration of the solu- optimal pH range of the pectin methyl esterase assayed
bilized carbohydrates is combined with information on (42, 43). Furthermore, a more quantitative assay to
their size (e.g., reducing sugar assay or size exclusion measure the action of the enzyme includes an auto-
chromatography). mated titration at the optimal pH (nonbuffered)
where the amount of NaOH necessary to compensate
the pH changes caused by the enzyme is measured (43,
D. Molecular-Weight Measurement
44).
The release of methanol can be measured using gas-
To establish whether the (puried) carbohydrase is an
liquid chromatography (GLC) directly (45) or after
exo- or an endoacting enzyme, a relatively simple assay
conversion of the methanol to methyl nitrite (46).
is based on the measurement of viscosity reduction
Although this latter method is quite sensitive and reli-
during the enzymatic degradation, since endoacting
able, it is also quite laborious (42). Alternatively,
enzyme activity results almost immediately in a loss
methanol can also be determined by HPLC on an
of viscosity. However, using this method, quantica-
Aminex column 87H column (47) although this
tion of the enzyme activity is rather difcult and the
method may not be sensitive enough for all purposes.
method is not very specic in case of mixtures of pectic
Colorimetric methods have been developed in which
enzymes (36). Using high-performance size exclusion
methanol is oxidized to formaldehyde (43, 4850),but
chromatography, a more accurate impression of the
they are not easy to perform (51).
mode of action of the enzyme is obtained. Although
many different types of columns can be used for such
HPSEC measurements (37), fairly good results to 3. Acetyl Esterases
determine the enzymic degradation of pectic polysac- The release of acetyl groups by enzymatic action is
charides includes elution on three Bio-Gel TSK col- usually followed by HPLC using the so-called organic
umns in series (40 XL, 30 XL, 20 XL; identical to acid column (Aminex HPX 87H) and refractive index
TSK-Gel G4000, 3000, 2000 PWXL, respectively), detection (47; Chap. 71). An alternative procedure is
eluted with 0.4 M acetic acid, pH 3, or 0.2 M NaNO3 an enzymic assay for acetic acid including a NADH/
and monitored using a refractive index detector (38). NAD conversion step using a commercial test kit
In a time course experiment, HPSEC allows quanti- (Boehringer-Mannheim, Germany). Obviously, care
cation of the enzyme activity fairly well, whereas also should be taken when using the frequently applied
endo- and exoactivity can be distinguished rather easily acetate buffers when using these methods. This is less
(even in enzyme mixtures having both activities). important (although product inhibition may occur)
when using colorimetric assays based on p-nitrophenyl
E. Assays for Specic Pectic Enzymes acetate, p-naphtyl acetate, or p-methylumbelliferylace-
tate (Chap. 71). Another synthetic substrate used for
1. Lyases
the determination of acetylesterases is triacetine, where
Enzymes acting by -eliminative cleavage like pectin the acetic acid released has to be measured by one of
and pectate lyase or rhamnogalacturonan lyase may the methods mentioned. However, these latter sub-
be monitored quite easily by measuring the absorbance strates are usually also deesteried by nonpectin
at 235 nm due to the formation of 4,5-unsaturated esterases.

XI. CHROMATOGRAPHY AND MASS disadvantage of the method is that the separation is
SPECTROMETRY OF COMPLEX inuenced by the size, sugar (linkage) composition,
ENZYME DIGESTS and degree of branching of the oligomer (55).
Figure 8 shows the dependency of the elution beha-
Chromatography in its wide variety is a powerful tech- viour of various holologous series of oligosaccharides
nique to detect activities of specic enzymes and to on the elution power of the gradient expressed as
obtain important information on their substrate speci- NaOAc. This can result in a rather unpredictable elu-
city and mode of action. Especially for heterogeneous tion behavior when not much is known about the com-
carbohydrate substrates, chromatography (with or plexity of the oligosaccharide mixtures under
without MS detection) is almost indispensable to deter- investigation (55), and other methods of identication
mine enzymes substrate requirements. High-perfor- like mass spectrometry might be necessary. However,
mance anion exchange chromatography (HPAEC) as when the polymeric substrate is well dened and the
introduced by Dionex in the late 1980s is able to sepa- (class of) enzyme is known, even complex elution pat-
rate oligosaccharides having the same size and sugar terns might be interpreted reasonably. This is illu-
composition but differing in branching and/or linkage strated in Figure 9, where a complex HPAEC elution
composition. A similar breakthrough in oligosacchar- pattern of a pectic arabinogalactan type I from soy-
ide characterization and identication as achieved by bean after digestion with arabinan- and galactan-
the introduction of HPAEC was obtained by the intro- degrading enzymes is shown (26). The more intensive
duction of matrix-assisted laser desorption/ionization peaks eluting between 10 and 25 min represent linear
time of ight mass spectrometry (Maldi Tof MS) dur- galactose oligomers, whereas the smaller peaks eluting
ing the last 5 years enabling molecular-mass measure- in this time frame represent mixed oligomers con-
ments of oligosaccharide mixtures rather accurately sisting of both arabinose and galactose. Oligomers of
and easily. Nowadays, HPAEC, Maldi Tof MS, and arabinose are eluted after 25 min.
LC-MS techniques have been developed into quick, The broad applicability of HPAEC/PAD is demon-
reliable, and easy-to-operate techniques allowing a strated by the fact that also the release of the various
more detailed examination of the mode of action of monomeric sugar residues can easily be monitored
enzymes on complex substrates by characterizing the using weaker elution conditions (58). In case of UV-
enzymic degradation products. absorbing oligosaccharides (e.g., after lyase action),
degradation products can be monitored using an UV
A. High-Performance Anion Exchange detector set to 235 nm (59). HPAEC-PAD may give
Chromatography of Oligosaccharides some problems when quantifying unknown com-
pounds where response factors are not available,
Although many different types of chromatography since such response factors may also depend on size
have been used to separate oligosaccharides (52, 53), and structure of the analyte (55).
HPAEC using alkaline elution conditions in combina- When the oligomeric degradation products released
tion with pulsed amperometric detection (PAD) is pre- by the enzyme under investigation are expected to be
ferred for specically monitoring oligosaccharides (55 negatively charged, HPAEC can be performed under
57). The principles for both separation and detection neutral elution conditions (56, 60). Such conditions are
are nicely described by Hensall (56). In principle, relevant when the oligomers are substituted with
separation is based on interaction with a strong alkali-labile groups (e.g., methyl-esteried galacturonic
anion exchange resin, and usually salt (sodium acetate) acid oligomers). Identication of these partially ester-
gradients in 100 mM NaOH are used to elute the oli- ied oligomers may give information about the precise
gosaccharides. Since elution is usually performed in mode of action of the enzyme (10, 6163).
alkali, even neutral carbohydrates bind to the column
and can be eluted later on. HPAEC allows separation B. Matrix-Assisted Laser Desorption/Ionization
of oligosaccharides with a DP up to 50. PAD detection Time of Flight MS
is direct (no derivatization required), highly sensitive,
and compatible with the gradients commonly used in Maldi Tof MS is a rather new mass-spectrometric tech-
HPAEC. When neutral eluents are used to separate nique, enabling collection of valuable data concerning
charged oligosaccharides, alkali should be added post the molecular masses of compounds present in a com-
column prior to detection since PAD detection of (part plex mixture of biomolecules in the range of 400
of the) sugar residues requires a high pH (56, 57). A 300,000 daltons (64, 65). Maldi Tof MS gives a good

Figure 8 Total sodium concentration at which series of linear oligosaccharides elute in HPAEC as a function of the degree of
polymerization (DP). Gradient: 0600 mM NaAc in 100 mM NaOH in 40 min. CarboPac PA100 column. (From Ref. 54.)
mass accuracy, is easy to operate, and requires rela- of galacturonic acid oligomers having methyl or ethyl
tively little sample preparation. For oligosaccharides, esterication, glycosidation, or amidation, and oligo-
mass determinations in the range of 4004000 are mers as present in an enzyme digest have been studied
rather easy to perform. by Maldi Tof MS and nanoelectrospray MS in detail
Figure 10 shows a Maldi Tof mass spectrum for the (61, 63, 6770).
pentamer/hexamer fraction of enzymatically degraded
soybean arabinogalactan, obtained after size exclusion
chromatography of the whole digest, as shown in C. Screening Using Plate Assays and
Figure 9. It can be seen that the different oligomers Chromogenic Substrates
consisting of arabinose and galactose can be easily
recognized. Obviously, isomers having the same mole- When huge amounts of enzyme samples have to be
cular mass cannot be distinguished from each other. If screened for specic activities (e.g., cDNA libraries),
necessary, electrospray tandem MS analysis following commonly used assays are based on the property of
HPAEC analysis may be used to verify the precise the substrate to specically bind to a coloring agent
structure of different isomers present (26). Kabel et like Congo Red (7173) or Ruthenium Red (74), or
al. (66) described the off-line coupling of HPAEC by using polysaccharides where a chromogen is cova-
and Maldi Tof MS where on-line desalting by mem- lently attached (Megazyme catalog). In specic cases,
brane suppressors, microtiterplate fraction collection, enzyme action may result in a modied substrate which
and automated sample handling followed by Maldi can specically be precipitated by metal complexation
Tof MS analysis allow determination of the molecular in a plate assay (75). Also the use of synthetic sub-
mass of individual peaks within a complex HPAEC strates in plates like p-methylumbelliferylglycosides
elution pattern rather routinely. The precise structure and p-nitrophenylglycosides to screen for glycosidases

Figure 9 HPAEC elution pattern for the oligosaccharide-containing pool from digest obtained after incubation of soybean-
chelating agent extractable pectin with endogalactanase, exogalactanase, endoarabinanase, and arabinofuranosidase B. Gn , linear
-1,4-linked galactose oligomers; Am , linear -1,5-linked arabinose oligomers. (From Ref. 26.)
Figure 10 MALDI-Tof MS of a size exclusion chromatography fraction containing mainly pentamers and hexamers released
from soybean pectic arabinogalactan type I by arabinogalactan-degrading enzymes. (From Ref. 26.)

is quite common. The same holds for the inclusion of a 14. P Komalavilas, AJ Mort. The acetylation at O-3 of
pH indicator to the medium to detect esterase activity. galacturonic acid in the rhamnose-rich portion of pec-
tins. Carbohydr Res 189:261272, 1989.
15. JR Whitaker. Pectin substances, pectic enzymes and
haze formation in fruit juices. Enzyme Microb
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66. MA Kabel, HA Schols, AGJ Voragen. Mass determi- University, 1997, pp 6373.
nation of oligosaccharides by matrix-assisted laser

67
Pectic Enzymes
Jacques A. E. Benen and Alphons G. J. Voragen

Jaap Visser
Fungal Genetics and Technology Consultancy, Wageningen, The Netherlands
I. INTRODUCTION that cannot be extracted from the plant tissue by che-

mical or enzymatic treatment without degradation.
The pectic enzymes comprise a diverse group of The action of a protopectinase results in the release
enzymes. In view of the complexity of the substrate, of soluble pectin or pectin constituents. Several proto-
the multitude of different enzymes active toward pectin pectinases have been studied in detail, and it turned
can easily be envisaged. Formally, the enzymes acting out that most of them belong to either the polygalac-
on the side chains, galactanases and arabinases, do not turonases, rhamnogalacturonases, pectate lyases, or
belong to the pectic enzymes although these substrates the pectin lyases (3). One of the protopectinases was
are in general strongly associated with pectin as side in fact an arabinase whereas another was a galacta-
chains. nase. The nondiscriminative term protopectinase will
The pectic enzymes consist of main-chain depoly- therefore not be used.
merases and esterases active on methyl- and acetylesters In recent years knowledge about pectic enzymes has
of galacturonosyl uronic acid residues in the galactur- increased signicantly. Numerous genes encoding pec-
onan and rhamnogalacturonan structures. The depoly- tic enzymes of all classes have been cloned from bac-
merizing enzymes comprise hydrolases as well as lyases. terial, fungal, and plant origin. Several of these genes
Both classes of enzymes contain enzymes that act on the have been overexpressed, and the corresponding
homogalacturonan backbone or smooth part, and enzymes have been puried and extensively character-
enzymes that degrade the rhamnogalacturonan part, ized. Three dimensional structures have now been
also known as hairy regions. The methylesterases solved for representatives from many classes of pectic
hydrolyze the methylester at O6 of a galacturonic enzymesviz., pectate and pectin lyases (47), polyga-
acid residue whereas acetylesterases hydrolyze the lacturonases (8, 9), a rhamnogalacturonase (10), a pec-
acetylester at O2 and/or O3 of a galacturonic acid. tin methylesterase (11), and a rhamnogalacturonan
Acetylesterases specic for the smooth regions as well acetyl esterase (12). Surprisingly, except for the rham-
as for the hairy regions have been identied (1). It is not nogalacturonan acetyl esterase, all the enzymes show
known whether a hairy regionspecic methylesterase the same basic -helical architecture. This new archi-
exists. A list of pectic enzymes is provided in Table 1. tecture was rst described for a pectate lyase by Jurnak
In the literature a class of enzymes named protopec- and coworkers (4). Despite the rather low degree of
tinases is described (2, 3). Protopectinases are those sequence similarity between different classes of pectic
enzymes active on protopectin, the insoluble pectin enzymes, ranging from 10% to 25%, the common -

Table 1 Enzymes Involved in Pectin Degradation
Hydrolases Lyases Esterases Auxiliary enzymes
endopolygalacturonase endopectate lyase pectin methyl esterase galactanase

exopolygalacturonase exopectate lyase pectin acetyl esterase arabinanase
exo-poly--D-galacturonosidase pectin lyase (endo) rhamnogalacturonan acetyl -galactosidase
rhamnogalacturonan hydrolase rhamnogalacturonan esterase -L-arabino-
rhamnogalacturonan rhamnohydrolase lyase furanosidase
xylogalacturonanhydrolase feruloyl esterase
coumaryl esterase
helical structure suggests that these pectinases have grinding of intact tissues into a semiuid system in
evolved from one ancestral gene (13). The unique which cells and cell wall fragments are suspended in
structural features of each class will be described in cell liquid. From this pulp a crude juice can be
the corresponding chapters. obtained by mechanical separation methodse.g.,
Coutinho and Henrissat (14) have classied the pressing, sieving, or centrifugation. Clear juices can
pectic enzymes based on amino acid sequence be obtained from the crude juice by additional clari-
similarities with the intention that such similarities cation treatments: cloudy or pulpy juices by adjust-
reect structural features of these enzymes. The ment of the amount of neness of the pulp particles
classication can be found at the following URL: through mechanical treatments in which large and
http://afmb.cnrs-mrs.fr/CAZY/CAZY/db.html insoluble particles are removed. Clarication is
obtained through reduction of the viscosity of the
cloudy and pectin-rich crude juice by treatment with
II. ENDOGENOUS AND EXOGENOUS enzymes, which causes coagulation and precipitation
PECTIC ENZYMES IN FRUIT AND of the cloud particles. The viscosity reduction also
VEGETABLE PROCESSING allows the production of fruit juice concentrates
which, for economic and technological reasons, can
Pectic enzymes occur naturally in many fruits and be stored and transported.
vegetables (endogenous enzymes), but they are also From the pulp of apples, grapes, and pears, juice
added as processing aids (exogenous enzymes). The can be obtained quite easily by pressing. In the manu-
latter are mostly derived from food-grade fungi, e.g., facture of juices from berries or bananas, grinding
Aspergillus niger, A. aculeatus. In higher plants, parti- results in a highly viscous juice which sticks as a gel
cularly pectin methyl esterase and polygalacturonase, to the pulp particles. A mechanical separation of juice
both endo- and exoacting, are present. Pectin acetyl and pulp particles is here impossible. To facilitate this
esterase has been found in citrus fruit and more separation the pulps are treated with enzymes, which
recently also pectate lyase and rhamnogalacturonan are able to degrade the polysaccharides forming the
hydrolase have been found in plants. The endogenous gel. This enzyme treatment results in higher juice yields
enzymes are assumed to play important roles in plant and improves the color of the juice. This treatment is
development and during ripening. The modern geno- also successfully used for grapes and for stored over-
mic approach will enable us to better understand their ripe apples.
presence in plants in numerous isoforms and their roles Industrial enzymes used in these applications con-
in plant developments. Pectic enzymes are also pro- tain pectin methyl esterases, polygalacturonases, pectin
duced by many microorganisms, and here also numer- lyases, rhamnogalacturonan hydrolases, rhamno-
ous isoforms have been described. The impact of galacturonan lyases, and rhamnogalacturonan acetyl
endogenous enzymes on fruit and vegetable processing esterases in varying amounts, as well as arabinanases,
will be discussed in the chapters devoted to the various galactanases, xylanases, -1,4-glucanases, amylases,
pectic enzymes (1517). many glycosidases, and proteases.
As processing aids, pectic enzymes have predomi- The enzymes of technological relevance in the var-
nantly found applications in the fruit juice industry. ious steps of fruit juice manufacture have been identi-
Traditional processes to manufacture fruit juices in ed and we are now more and more able to formulate
essence consist of mechanical destruction of cells by enzyme cocktails tailored to specic applications. It

was established that enzyme mixtures able to readily formulations have to be established depending on the
degrade high esteried pectins (pectin methyl esterase, starting raw material. In the following chapters the dif-
endopolygalacturonase, and/or pectin lyase) are also ferent classes of enzymes active toward pectin are cov-
very suitable for improving the pressability of fruit ered. In these chapters also specic application aspects
pulps and for clarication of fruit juices. The presence as processing aids are mentioned. A separate chapter is
of rhamnogalacturonan hydrolase or rhamnogalactur- devoted to the description of the characteristics of the
onan lyase and rhamnogalacturonan acetyl esterase in structural units making up pectins and of methods to
these formulations further enhances juice extraction monitor activity of pectic enzymes.
but also the solubilization of cell wall polysaccharides
(18, 19). Pectic enzymes (This chapter)
Food-grade fungal enzyme preparations which pre- Pectic polysaccharides (Schols and Voragen, Chap. 66)
dominantly contain endopolygalacturonase activity Polygalacturonases (Benen and Visser, Chap. 69)
and no pectin methyl esterase and pectin lyase are suc- Pectate and pectin (Benen and Visser, Chap. 80)
cessfully used as macerating enzymes for the produc- lyases
tion of pulpy nectars. These nectars are characterized Pectic esterases (Benen, van Alebeek, Visser,
by a creamy consistency and have higher contents of and Voragen, Chap. 68)
fruit solids, more pigments, and more nutrients than Enzymes degrading (Vincken, Voragen, and
nectars prepared by thermomechanical treatments. rhamnogalacturonan Beldman, Chap. 73)
Cloudy or pulpy products can be obtained by further and xylogalacturonan
grinding, nishing, and homogenization. Maceration Enzymes releasing L- (de Vries and Visser, Chap. 70)
arabinose and D-
can also be achieved by endopectin lyase and endopec-
galactose from the
tate lyase. side chains of pectin
By combining pectin-degrading enzymes like endo-
polygalacturonase, pectin methyl esterase, and/or REFERENCES
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osmotic pressure, and they collapse. The phenomenon pectins. In: J Visser, AGJ Voragen, eds. Progress in
of complete liquefaction of the fruit tissues can be Biotechnology 14: Pectins and Pectinases.
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Pectin pectinase, and protopectinase: production,
onan lyase and rhamnogalacturonan acetyl esterase in
properties, and applications. Adv Appl Microbiol
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nan-, and arabinan-, and galactan-degrading enzymes. 4. MD Yoder, F Jurnak. The rened three dimensional
The clarication of the juices by ultraltration is structure of pectate lyase C from Erwinia chrysanthemi
substantially improved by avoiding fouling of the mem- at 2.2 A resolutionimplications for an enzymatic
branes (19). The liquefaction technology is particularly mechanism. Plant Physiol 107:349364, 1995.
suitable for fruits from which no juice can be obtained 5. R Pickersgill, J Jenkins, G Harris, W Nasser, J
by pressing or for which no presses have yet been devel- Robert-Baudouy. The structure of Bacillus subtilis
oped, e.g., tropical fruits such as mango, guava, and pectate lyase in complex with calcium. Nature Struc
banana (1921). The liquefaction process limits losses Biol 1:717723, 1994.
6. O Mayans, M Scott, I Connerton, T Gravesen, J
of nutrients and makes them even more bioavailable
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(22). The liquefaction process can also be used for
structures of pectin lyase A from Aspergillus reveal a
extraction of oils from oleaginous fruits and seeds or pH driven conformational change and striking diver-
for the extraction of other valuable cell constituents. gence in the substrate-binding clefts of pectin and pec-
In the conversion of agricultural biomass to sugars or tate lyases. Structure 5:677:6891997.
alcohol, enzymatic liquefaction is the rst step to further 7. J Vitali, B Schick, HCM Kester, J Visser, F Jurnak.
saccharication. For each of these applications optimal The three-dimensional structure of Aspergillus niger

pectin lyase B at 1.7-A resolution. Plant Physiol 15. AGJ Voragen, W Pilnik. Pectin-degrading enzymes in
116:6980, 1998. fruit and vegetable processing. In: JR Whitaker, PE
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9. Y van Santen, JAE Benen, K-H Schroter, KH Kalk, S ous and exogenous pectic enzymes in fruit and vege-
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directed mutangenesis. J Biol Chem 274:3047430480, vegetable juice manufacture. In T Nagodawithana, G
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Aspergillus aculeatus: a right-handed parallel beta Dupuy, ed. Use of Enzymes in Food Technology.
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Rhamnogalacturonan acetylesterase elucidates the Pectinases. Amsterdam: Elsevier, 1996, pp 453463.
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12.

68
Pectic Esterases
Jacques A. E. Benen, Gert-Jan W. M. van Alebeek, and Alphons G. J. Voragen

Jaap Visser
I. INTRODUCTION plants and represent pectin methylesterases and pectin

acetylesterases. As for the endopolygalacturonases, by
Pectic esterases comprise enzymes that hydrolyze the far the largest portion is taken by open reading frames
esters present in the pectin backbone (EC 3.1.1.). (putative genes) from Arabidopsis thaliana.
Currently three classes of esterases have been identi-
ed: (a) the pectin methylesterases (EC 3.1.1.11) that A. Pectin Methylesterase
hydrolyze methylesters from O6 of galacturonic acid in
the homogalacturonan part; (b) the pectin acetyles- Apart from the presence of many genes encoding puta-
terases (EC 3.1.1.6); and (c) the rhamnogalacturonan tive pectin methylesterases (PMEs), these enzymes
acetylesterases (3.1.1.). The acetylesterases remove have also been detected in various plants by their activ-
acetyl groups from O2 and/or O3 of galacturonic ity, which conrms their wide distribution. They have
acid residues either in the homogalacturonan part or been identied in higher plants, particularly in apple,
in the rhamnogalacturonan part. Until now, no pectin banana, berries, citrus (lime, orange, grapefruit, and
methylesterase active toward methylesters in the rham- mandarin), cherry, grape, mango, papaya, passion
nogalacturonan part has been identied. The pectin fruit, pear, plum, beans, carrot, cauliower, cucumber,
methylesterases belong to family 8 of the carbohydrate leek, onion, pea, potato, radish, and tomato. Within
esterase classication (1). The microbial rhamnogalac- each species multiple forms of PME can be present as
turonan and homogalacturonan acetylesterases have was shown for example for citrus fruits (2, 3), mung
been grouped into family 12 whereas the plant pectin bean hypocotyl (46), and ax seedlings (7). In general,
acetylesterases make up family 13 (1). Other esterases, basic, neutral, and acidic isoforms are found, which
like feruloylesterase, which are not active on the back- differ in various biochemical properties, such as rela-
bone but rather on side chains, will be covered else- tive molecular mass and pH optimum. PMEs are
where in this volume. found in various tissues and are mainly associated by
ionic interactions with cell wall proteins, although
some soluble forms have been detected as well. Their
II. ENDOGENOUS PECTIC ESTERASES IN exact role is not known, but since they are able to de-
PLANTS esterify pectin and are often present in the cell wall, it is
obvious that they are involved in pectin modication.
A major part of the pectic esterase nucleotide The degree of methylesterication is very important for
sequences present in the databases originate from the gelling properties of pectin. Highly methylated pec-

tins form gels via hydrogen bonds and are generally the other isoforms. It was found to occur in all the
associated with young, differentiating and elongating component parts of the orange fruit. In the traditional
cells, whereas Ca2 -pectate gels are formed by low- French cider industry, the endogenous PME of apple
methoxyl pectins in older, elongated, and differen- was used for the self-clarication of apple juice. Today
tiated cells (8). Thus, it can be inferred that pectin fungal PME preparations free of depolymerizing activ-
methylesterases serve important developmental pro- ity are available for industrial application in cider and
cesses in plants by modulating the degree of methyles- lemon juice clarication (13).
terication. The calcium pectate coagulation phenomenon as a
During growth, ripening, and storage, the texture of result of PME action improves the pressing character-
the fruit changes, and fruit softening occurs. These istics of citrus peel and lowers costs when the peel has
alterations are believed to be due to changes in the to be dried for use as cattle feed. Before drying the peel
turgor of the cell and cell wall metabolism. In tomato is ground up together with calcium hydroxide. This
these are caused by the degradation of both pectins limiting operation activates the enzyme, pectin coagu-
and hemicelluloses (9, 10). Pectin degradation is caused lates as calcium pectate, and peel juice is released easily
by the combined action of PME and PG in the fruit. before and during pressing (13). Endogenous PME can
But since PG activity appears to be limited by some also be used for protecting and improving the texture
chemical or physical restrictions despite being present and rmness of several processed fruits and vegetables
in high abundance, pectin degradation is limited. As a such as apple slices, cauliower, carrots, potatoes,
consequence, antisense suppression of PG activity or beans, and peas. Through adjustment of the blanching
PME activity in tomato results in only a small decrease temperature and time, PME is activated. By the
in pectin degradation and a very small increase in fruit demethylation of cell wall pectin, the cation uptake is
rmness later, during ripening (11). Despite the low increased. Firming effects by the addition of Ca2 ions
effect of the antisense downregulation of PG or are well established and have long been used to prevent
PME, in normal tomato fruits, during processing, excess vegetable softness. Fungal PME preparations
these enzymes are liberated and, without precautions, have also been developed for these applications, for
will degrade the pectin. This may result in either desir- improving the consistency of tomato pastes, and also
able or undesirable changes (11). for the manufacture of low-ester pectins with reduced
In the processing of tomatoes a heat shock treat- Ca2 sensitivity (13).
ment called hot break is used to inactivate PME
and PG and keep a high consistency and viscosity of B. Pectin Homogalacturonan Acetylesterase
the juice. When this high consistency is not desired, a
cold break is used during which PME and PG Pectin homogalacturonan acetylesterases (PAE) have
degrade pectin, thereby lowering the viscosity (11). so far been puried from orange (3) and mung bean
During fruit fermentation PME action is responsi- hypocotyl (14, 15). In orange the enzyme was distrib-
ble for methanol release (12). To keep methanol levels uted over the whole fruit, with higher levels in the
below legal limits, fruit pulps have to be pasteurized outer parts of the peel (albedo and avedo) and in
before fermentation. PMEs play important roles in the segments. In mung bean the PAE expression was
fruit and vegetable processing. In citrus processing shown to be tissue specic. The highest expression was
they cause cloud loss due to precipitation of pectin found in the hypocotyl, whereas expression was signif-
demethylated by endogenous PME with calcium ions icantly lower in the leaves and roots and almost absent
and negatively charged proteins. This is desirable in the in the cotyledons (15). PAE activity has been detected
production of lemon juice but causes a serious quality in carrots as well (16), and it is likely that this activity is
defect of orange juice. In orange juice concentrates more widespread among plants.
strong calcium pectate gels may form which prevent
reconstitution of single-strength juice. The problem C. Rhamnogalacturonan Acetylesterase
can be overcome either by heat inactivation of PME,
which will harm the avor, or by keeping the concen- Thus far, rhamnogalacturonan acetylesterase (RGAE)
trate at 20 C. Another possibility is the addition of activity has been demonstrated only for the fungus
endopolygalacturonase, which breaks down the low- Aspergillus (17, 18). Despite the absence of any reports
ester pectin formed before it can coagulate with cal- describing this activity in plants, it is reasonable to
cium (13). Versteeg et al. (2) identied a high-MW iso- assume plants have this activity, as it has recently
form of PME, which was much more heat stable than been shown that rhamnogalacturonan degrading

enzymes, which require RGAE activity for optimal B. Pectin Homogalacturonan Acetylesterase
activity, are present in carrot (16).
Sugar beet pectin is rich in methylated and acetylated
galacturonic acid residues and hence has poor gelling
III. APPLICATION OF ESTERASES
properties with either acid/sugar or Ca2 ions (22).
A. Pectin Methylesterases PME treatment of sugar beet pectin results in partial
hydrolysis of the methylesters, resulting in better gel-
Pectin enzymes nd a wide application in fruit and ling properties using Ca2 ions. Upon addition of PAE
vegetable juice manufacturing, like fruit juice clarica- isolated from A. niger (23), part of the acetyl groups
tion, enzymic pulp treatment for juice extraction, are removed as well. This action in turn allows PME to
liquefaction, and maceration (19). In most of these further remove methylesters. This synergistic effect
applications pectic enzymes are used for the complex results in an increase of the stiffness of the Ca2 -
degradation of pectin. Since these juices are generally formed gel (24).
acidic, the depolymerization of the pectin is accom-
plished by polygalacturonases which are generally spe-
cic for low-methylated polygalacturonic acid. To
C. Rhamnogalacturonan Acetylesterase
convert the pectin into a PG-accessible substrate, the
action of PME is required. For maceration of fruits
Rhamnogalacturonan acetylesterase is essential for the
and vegetables, complete pectin degradation is not
breakdown of rhamnogalacturonan. After deacetyla-
desired. Only pectin in the middle lamella should be
tion of the galacturonic acid residues in rhamnogalac-
degraded, which can be accomplished by either PG or
turonan, the hairy regions can be further degraded by
PL action alone. Therefore, endogenous pectin methy-
rhamnogalacturonan hydrolase (25) and rhamnogalac-
lesterase has to be inactivated.
turonan lyase (26), which is of technological impor-
Pectin is widely used in industry because of its
tance for the extraction of fruit and fruit juice
gelling properties. High-methoxyl (HM) pectins are
ultraltration (18).
able to form sugar acid gels (jams) in which cosolutes
like sugar and acid are present to lower water activity
or diminish electrostatic repulsion, respectively. LM
pectins (degree of methylation [DM < 50%) can gel
in the presence of divalent ions. In food gels this ion is IV. BIOPHYSICAL PROPERTIES OF
usually Ca2 . The Ca2 gel-forming ability increases PECTIC ESTERASES
with decreasing DM, although the distribution of the
methylesters along the homogalacturonan backbone Pectin methylesterases have been known and charac-
strongly inuences the gelling properties of the pectin. terized for several decades, which has yielded a wealth
LM pectins with a blockwise distribution of the of information compared to the recently discovered
carboxyl groups (long stretches of de-esteried homo- pectin and rhamnogalacturonan acetylesterases. As
galacturonan) are extremely sensitive to low calcium with many enzyme families, the more enzymes one
levels, whereas randomly de-esteried pectins are far studies, the more the variety in biochemical and bio-
less sensitive. This distribution of methylesters can be physical properties one observes within the family.
controlled by the manner of de-esterication of HM The pectic esterases are medium-size enzymes as
pectin. De-esterication of HM pectin by plant PME their MWs are 2554 kDa. These enzymes are active
results in blocks of unmethylesteried galacturonic as monomers. Many PMEs from eukaryotic origin
acid residues, whereas NaOH or fungal PME yields are glycoproteins, and there is even a report of a
a random distribution of free carboxyl groups. Thus, bacterial PME from E. chrysanthemi that is in fact
pectin methyl esterases are of major importance for a lipoprotein (27). The isoelectric point of the pectic
the preparation of pectins for specic applications. esterases varies from as low as 3.1 for fungal PME to
Recently, techniques like MALDI-TOF MS and 11 for a tomato PME. The pH stability depends on
HPAEC (pH 5) have been developed to discriminate the source of the enzyme, and the range for all PMEs
among the various types of pectins and thus allow stretches from pH 1.1 to pH 10.0. The temperature
correlation of functional properties of the pectin stability is moderate (4070 C) with a slightly higher
to the degree and distribution of methylesterication stability for the plant enzymes compared to the
(20, 21). microbial enzymes.

A. Three-Dimensional Structures of Pectic V. BIOCHEMICAL PROPERTIES OF
Esterases PECTIC ESTERASES
Currently, 3D structures are only available for the In the premolecular biology era, numerous pectin
Erwinia chrysanthemi PME (PDB code: 1QJV) (28) methylesterases, primarily of plant origin, were char-
and the Aspergillus aculatus rhamnogalacturonan acet- acterized. In the past few years the pectin acetylesterase
ylesterase (RGAE) (PDB codes: 1DEO and 1DEX) and rhamnogalacturonan acetylesterase activities were
(29). The structure of the PME reveals a similar topol- discovered and studied, but the number of reports on
ogy as found for the pectate and pectin lyases, and these enzymes is limited (3, 1418).
polygalacturonases/rhamnogalacturonases (3033). Pectin methylesterases hydrolyze the ester bond
This topology is characterized by a right-handed par- between methanol and the carboxylic function of
allel beta-helix structure with loops protruding from galacturonic acid. The action of the enzyme thus
the helix core that form the substrate-binding cleft. results in the formation of a free carboxylic function,
The conservation of the unique topology among methanol, and H3 O . In general, the plant and bacter-
these enzymes strongly indicates that a complete set ial PMEs have pH optima between pH 6 and 8 whereas
of different pectinases have evolved from the same some fungal PMEs have pH optima between pH 4 and
ancestral gene. 6. The plant enzymes often require the addition of 0.1
The structure adopted by RGAE is completely dif- 0.2 mM NaCl for the optimal catalysis. Action of any
ferent. This enzyme folds into an == structure (29), PME on (highly) methylated pectins does not result in
which is different from the normally observed = full demethylation. Generally, the residual DM is
structure common to esterases and lipases. As for the between 20% and 30%. In part this is due to the pre-
= enzymes in the RGAE, the active-site residues are sence of acetyl groups in certain pectins (24).
Ser, Asp, and His, in the same sequential order, but the Pretreatment or simultaneous treatment with PAE
Asp and His residues are only three residues apart increases the amount of methyl groups removed, but
instead of being located on different loops (29). still not all groups will be hydrolyzed (2).
Based on the strict sequence conservation in the ester- Plant and microbial PMEs differ in their mode of
ase family 12 of Ser and His and two additional resi- action. Whereas plant enzymes generally remove
dues, Gly and Asn, which both act as hydrogen bond blocks of methyl groups on a single chain, fungal
donors to the oxyanion hole, it has been proposed that PMEs attack the methyl groups on the pectin ran-
this group of esterases be named the SGNH hydrolase domly, resulting in a random distribution of the
family (29). unmethylated galacturonic acid groups. Recently,
three different isoforms from mung bean hypoco-
tylsPME, PME, and PMEwere subjected to
B. Isoforms of Pectic Esterases a detailed analysis of their mode of action (35). This
elaborate study in which PME activities were measured
The Arabidopsis genome sequencing project has at pH 5.6 and 7.6 revealed that the three isoforms have
resulted in > 50 putative pectin methylesterases and different substrate specicities and rates. Strikingly, for
numerous pectin acetylesterases. The databases also PME at pH 5.6, a sharp drop in activity was observed
contain multiple (putative) PME sequences from when the DM decreased below 70% whereas at pH 7.6
other plants. The presence of isoforms at the genetic the reaction rate remained constant down to 40% DM.
level therefore seems a common theme among plants. For PME and PME this effect was less pronounced.
Such a large variety of pectic esterases is not present in However, PME showed at both pH values the stron-
microorganisms. At the genetic level, two PME iso- gest preference for highly methylesteried pectin. Not
forms have been identied in E. chrysanthemi (27, 34) only in this respect did the enzymes differ; in their
whereas all other microorganisms studied so far have mode of action a clear difference was also observed.
only one pme, pae, or rgae gene. The mode of action of the enzymes was simulated and
Eukaryotic enzymes and proteins are prone to N- tted to the actual distribution of methylesters after
and O-linked glycosylation. Heterogeneous glycosyla- partial hydrolysis of various DM pectins as recorded
tion can often lead to multiple isoforms of the same by NMR (35).
enzyme. There are no reports describing the analysis of For PME at both pH values the reaction was best
(heterogeneous) glycosylation patterns of pectic ester- described by a single-chain mechanism. The enzyme
ase isoforms. remains attached to one substrate molecule and con-

tinues to demethylate the pectin molecule toward the was established by peptide sequencing (38), which now
reducing end until the end of the molecule is reached. allows in vitro gene synthesis. Primary sequence
Similar behavior was concluded for PME and PME homology searches have revealed signicant sequence
at pH 5.6, but at pH 7.6 the single-chain mechanism homology with some plant invertase inhibitors (38).
changed into a multiple-attack mechanism. According Thus, the presence of a family of inhibitor proteins
to this mechanism, only a limited number of methyl may be common in plants.
groups is removed during each encounter.
Furthermore, the studies revealed that for catalysis B. Specic Mechanism of Action
the presence of an unesteried galacturonic acid resi-
due is required for all three enzymes. For PME and Based on the 3D structure of PME and primary
PME, this unesteried residue should be present two sequence alignments, Pickersgill and Jenkins
residues toward the nonreducing end from the methy- hypothized a possible mechanism (28). Two aspartates
lated residue to be attacked. For PME, this distance and one arginine are strictly conserved among PMEs.
was three residues in the same direction (35). One of the Asp residues makes a hydrogen bond to the
From a practical point of view, pectins treated with Arg and is therefore most likely unprotonated. The
either plant or microbial PME result in pectins with other Asp is likely to be protonated. A water molecule
different gelling properties and thus are suitable for adjacent to the unprotonated Asp may be activated by
different applications. Recently, a PAE encoding transferring its proton to the Asp. The hydroxyl gen-
gene (paeY) was cloned from the phytopathogenic bac- erated can then attack the carbonyl carbon.
terium Erwinia chrysanthemi, sequenced, and overex- Simultaneous protonation of one of the oxygens
pressed in Escherichia coli (36). An E. chrysanthemi results in the formation of a tetrahedral intermediate,
strain with a disrupted pae gene showed a reduced which collapses with the release of methanol and thus
invasion of the host plant tested, demonstrating the results in demethylation (28).
important role of PAEY in the soft-rot disease (36). For RGAE it can be postulated that catalysis will
proceed via the general mechanism observed for lipases
A. Effect of Environmental Factors where the catalytic triad Asp-His-Ser, which forms a
charge-relay system, will attack the ester bond via the
To date no pectic esterasespecic low MW inhibitory activated serine (29).
compounds have been reported. However, there are For PAE no mechanism has been proposed yet.
numerous compounds (Hg2 , Al3 , NH 4 , sugar, gallic
acid) that inhibit some PMEs, whereas other PMEs are C. Subsites in Binding to the Substrate
not inhibited (reviewed in Ref. 2). Many PMEs are
also inhibited by polygalacturonic acid, the product For PAE and RGAE no studies have been reported
of its action. NaCl and KCl have been reported to with respect to the number of subsites involved in sub-
stimulate activity of PME and it has been argued strate binding. For PME such studies have not been
that this stimulation is a result of reduced inhibition carried out either, but some reports allow inferring the
by polygalacturonic acid in the presence of NaCl or number of subsites. Kester and coworkers (39)
KCl (2). reported that the rate of de-esterication by fungal
Recently, a glycoprotein inhibitor of PME has been A. niger PME of individual fully esteried oligomers
identied in kiwi fruit (37, 38). This 16.7-kDa protein increased by a factor of 100 when the chain length
was puried by afnity chromatography using par- increased from n 3 to n 6. The increase in reaction
tially puried tomato PME coupled to Sepharose. rate for n 5 to n 6 still amounted to a factor of 2.
The interaction between the PME and the inhibitor These results indicate that the number of subsites for
appeared to be a 1-to-1 complex formation. This inter- A. niger PME is at least ve and may even be six.
action is very strong and the complex was only dissoci- Catoire et al. (35) simulated the mode of action of
able at high pH and high ionic strength (pH 9.5, 1.5 M PME isoforms from mung bean hypocotyls using
NaCl). The physiological function of the protein may NMR data of (partially) de-esteried pectins. For
be in the defense mechanism of plants against phyto- one of the PME forms, PME, successful simulation
pathogens, similar to the polygalacturonase-inhibiting of the data was only possible by allowing an unester-
proteins (PGIPs) or, alternatively, in the regulation of ied residue to interact with the enzyme at a subsite
endogenous PME activity in relation to cell wall devel- three subsites in the direction of the nonreducing end
opment. The primary sequence of the PME inhibitor away from the active site (35). Thus, for this particular

enzyme, at least four subsites are involved in binding of orange fruit acetylesterase. In: J Visser, AGJ
the substrate. Voragen, eds. Progress in Biotechnology 14: Pectins
and Pectinases. Amsterdam: Elsevier Science, 1996,
D. Synthetic Substrates pp 723730.
4. M Bordenave, R Goldberg. Purication and charac-
terization of pectin methylesterases from mung bean
Using monomethylesteried trigalacturonides carrying
hypocotyl cell walls. Phytochemistry 33:9991003,
the monomethyl group at a dened position (40) and 1993.
fully methylesteried oligogalacturonides of chain 5. M Bordenave, R Goldberg. Immobilized and free
length n 36, Kester et al. (39) studied the mode of apoplastic pectinmethylesterases in mung bean hypo-
action of Aspergillus niger PME. These studies revealed cotyl. Plant Physiol 106:11511156, 1994.
that this fungal enzyme cannot remove a methyl group 6. M Bordenave, C Breton, R Goldberg, JC Huet, S
from a galacturonic acid residue at the nonreducing Perez, JC Pernollet. Pectinmethylesterase isoforms
end. It was further shown that an unesteried residue from Vigna radiata hypocotyl cell walls: kinetic prop-
somewhere along the chain is not necessary for this erties and molecular cloning of a cDNA encoding the
enzyme. A. niger PME preferentially attacked a methyl most alkaline isoform. Plant Mol Biol 31:10391049,
1996.
group in the middle of the methylesteried oligomers.
7. F Alexandre, O Morvan, J Gaffe, A Mareck, A
The distribution of the products observed after partial Jauneau, H Dauchel, AP Balange, C Morvan. Pectin
saponication demonstrates that this enzyme acts methylesterase pattern in ax seedlings during their
according to a multiple-chain mechanism: each development. Plant Physiol Biochem 35:427436,
encounter results in the removal of only one methyl 1997.
group (39). 8. P Albersheim, AG Darvill, MA ONeill, HA Schols,
Triacetin is often used as a substrate for esterases. AGJ Voragen. An hypothesis: the same six polysac-
For A. niger esterase, the substrate was specically charides are components of the primary cell walls of
used by pectin acetylesterase, whereas it was not all higher plants. In J Viser, AGJ Voragen, eds.
hydrolyzed by RGAE (23). Alternative substrates for Progress in Biotechnology 14: Pectins and
acetylesterases are paranitrophenyl acetate and 4- Pectinases. Amsterdam: Elsevier Science, 1996, pp
4755.
methylumbelliferyl acetate.
9. DJ Huber, EM ODonoghue. Polyuronides in avo-
cado (Persea Americana) and tomato (Lycopersicon
VI. QUALITATIVE AND QUANTITATIVE esculentum) fruits exhibit markedly different patterns
of molecular weight downshifts during ripening. Plant
Physiol 102:473480, 1993.
10. G Maclachlan, C Brady. Endo-1,4--glucanase and
In order to determine the different enzymes qualita- xyloglucanase and xyloglucan endo-transglycosylase
tively and/or quantitatively, specic substrates and activities versus potential substrates in ripening toma-
assays are needed. This preparation of substrates, toes. Plant Physiol 105:965974, 1994.
together with the assays, is described by Schols and 11. GA Tucker, H Simons, N Errington. Transgenic
Voragen in Section IX. in Chapter 66. tomato technology: enzymic modication of pectin
pastes. Biotechnol Genet Eng Rev 16:293307, 1999.
12. C Frenkel, JS Peters. Pectin methylesterase regulates
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69
Polygalacturonases
Jacques A. E. Benen
Jaap Visser
I. INTRODUCTION dance of genes indicates a profound developmental

role of the corresponding enzymes. Contrary to plant
Polygalacturonases cleave the -1,4-D-galacturo- polygalacturonases, microbial polygalacturonases
nosidic linkage by hydrolysis. Whereas endopolygalac- serve to mobilize nutrients from pectin to support
turonases (EC 3.2.1.15) hydrolyze the polymer growth. For several yeasts such as Kluyveromyces
substrate randomly, the exopolygalacturonases are marxianus (7) and Saccharomyces cerevisiae (8), poly-
conned to cleave off galacturonic acid monomers galacturonase production has been documented and
(EC 3.2.1.67) or digalacturonides (EC 3.2.1.82) from respective genes have been cloned, whereas no obvious
the nonreducing end. The names of these enzymes physiological function is served since those yeasts can
suggest that polygalacturonic acid is their sole sub- not utilize galacturonic acid.
strate. However, as found for the pectate lyases, endo-
polygalacturonases are also active on pectins with a
lower or moderate degree of esterication. Recently it II. ENDOGENOUS
was shown that endopolygalacturonases A and B from POLYGALACTURONASES IN PLANTS
Aspergillus niger even prefer a low degree of esterica-
tion (1). In view of the important role of pectin in the primary
The polygalacturonases have all been grouped into cell wall of plants, it is obvious that endogenous
family 28 of the general glycosyl hydrolase classica- polygalacturonases play important roles in the
tion (2). In addition to the endopolygalacturonases, development of plants. Polygalacturonase activity has
this family also comprises the rhamnogalacturonases been demonstrated in almost any plant tissue. Based
and the recently cloned xylogalacturonase (3). Three- on mRNA expression studies in Arabidopsis thaliana
dimensional structures of two endopolygalacturonases (9), tomato [Lycopersicon esculentum (10)], and lemon
(4, 5) and a rhamnogalacturonase have been solved [Cucumus melon (11)] among others, it was shown that
(6), and their close resemblance indeed justies the there are at least three types of polygalacturonases: the
grouping in one family. By far the largest group of pollen-specic polygalacturonases, the fruit-specic
potential polygalacturonase genes (52 open reading enzymes, and the abscission-specic polygalacturo-
frames) is formed by those from Arabidopsis thaliana. nases. For tomato it was established that the specic
This large amount originates from the recent expression, i.e., auxin repression and ethylene and
Arabidopsis genome-sequencing project. Such an abun- abscisic acid induction, resides in the regulatory

elements within the individual promoters of the poly- A. Three-Dimensional Structures of
galacturonase-encoding genes (10). Polygalacturonases
A phylogenetic analysis by Hadeld and coworkers
(11) revealed that the plant polygalacturonases belong Currently, 3-D structures are only available for
to three clades. Clade A comprises fruit- and abscision- the Erwinia carotovora endopolygalacturonase [PDB
specic polygalacturonases without pro-sequence, code: 1 BHE (4)] and the Aspergillus niger endo-
clade B consists of fruit- and abscision-specic polyga- polygalacturonase II [PDB code: 1CZF (5)]. Despite
lacturonases with a pro-sequence, and clade C contains limited sequence identity between the two enzymes
all the pollen-specic polygalacturonases. It has been (19%), a striking similarity of the two -helical
hypothesized that all clade C enzymes are exopolyga- structures is observed at the C-alpha trace level
lacturonases, as those enzymes characterized were of although certain differences can be observed as well,
the exolytic type. In an excellent review Hadeld and notably in the loop regions. Details are discussed by
Bennet (12) have compiled the major ndings on endo- Van Santen et al. (5). Unfortunately, no structure is
genous plant polygalacturonases of the past two dec- available for any enzyme-substrate complex. However,
ades. In a more recent review Lang and Dornenburg Van Santen et al. (5) and Armand et al. (15) present
(13) further elaborate on this and also pay attention to evidence that the substrate binds to the enzyme at the
the role of polygalacturonases in wounding of plants, observed cleft with the reducing end of the substrate
the interaction of phytopathogenic fungi and plants, facing toward the C-terminal part of the enzyme. This
and the plant response to this. orientation was also observed for the topologically
To the food enzymologist the most interesting endo- highly similar pectate lyase C in complex with the
genous polygalacturonases are those present in the substrate (16).
part of the plant that is used as or in food. One of
the best-characterized subjects in this respect is the B. Isoforms of Polygalacturonases
tomato. As a major food, either as the complete fruit
or as puree, catsup, or juice, the inuence of endogen- The rapidly expanding family 28 of glycosyl hydrolases
ous polygalacturonases on fruit ripening and proces- clearly demonstrates that many species analyzed have
sing has been studied extensively. To this end gene- at least two genes encoding polygalacturonases. Thus,
silencing techniques have been employed that have as for the pectic lyases, isoforms at the genetic level are
revealed that endopolygalacturonases are not neces- plentiful. Wubben et al. (17) constructed a phyloge-
sary for fruit ripening (reviewed in 1214). However, netic tree of 35 endopolygalacturonases of yeast and
owing to the gene silencing it turned out that the shelf fungi which appeared to group into ve monophylo-
life of the tomato increased profoundly as a result of genetic groups. Hadeld et al. (11) analyzed the plant
reduced cracking of the tomato. Upon homogenization polygalacturonases in this respect which appeared to
of nongenetically modied tomatoes, the deleterious group in three clades.
effect of endogenous polygalacturonases becomes evi- For eukaryotic polygalacturonases, many reports
denti.e., a decrease in viscosity of the homogenate describe heterogeneity of polygalacturonase prepara-
upon aging. To prevent the action of these enzymes, tions as a result of glycosylation. To date, the N-gly-
tomatoes are homogenized after heat pretreatment to cosylation patterns of only two polygalacturonases
inactive the enzymes and yield the so-called hot break have been analyzed in detail, viz., endopolygalacturo-
pastes. nases I and II from Aspergillus niger (18, 19). The
single N-glycosylation site in endopolygalacturonase
II appeared to be heterogeneously glycosylated with
III. BIOPHYSICAL PROPERTIES OF glycans of the high mannose type (18), whereas of
POLYGALACTURONASES the two N-glycosylation sites of endopolygalacturo-
nase I only one was similarly decorated as the one of
Although all polygalacturonases belong to family 28 of polygalacturonase II and the other site was not glyco-
glycosyl hydrolases, considerable variation in molecu- sylated (19). For neither enzyme was O-glycosylation
lar mass (3075 kDa) and isoelectric point (pI 3.88.8) found.
can still be obtained from the databases going from In contrast to A. niger pectic lyases, the A. niger
enzyme to enzyme. Currently no relation has been polygalacturonases all contain a prosequence in addi-
established between molecular mass and/or isoelectric tion to the presequence normally found in secreted
point and/or specicity. enzymes. The role of the prosequence is not clear but

is removed by a KEX-like protease in most cases (20, pressed in view of their potential industrial
21). Also, in Botrytis cinerea the prosequence was application and to facilitate production, purication,
reported in four of the six endopolygalacturonase and characterization (1, 20, 27, 28). Endopolygalac
genes cloned (17). turonase D appeared quite different from the other
Sheehy et al. 922) and DellaPenna et al. (23) were A. niger endopolygalacturonases as it carries an 136
among the rst to analyze processing of a tomato amino acid N-terminal extension whose function is
polygalacturonase. In addition to glycosylation, not clear but which is not processed as evidenced by
evidence for proteolytic processing was obtained. In sequencing the N-terminus by Edman degradation
addition to a presequence, a prosequence was reported (27).
as well. Moreover, Sheehy et al. (22) also observed Several basic biochemical properties of the seven
C-terminal processing. It has been proposed that the enzymes are listed in Table 1. As can be seen, there is
prosequence is necessary for the targeting of the only little variation in pH optima. However, there is an
particular enzyme to a certain part of the cell wall enormous difference in maximal turnover, ranging
(23), but it has also been hypothesized that the from 25 U/mg for endopolygalacturonase C to 4000
prosequence keeps the enzyme in an inactive state U/mg for endopolygalacturonase II. For the three
until the prosequence is processed at the proper enzymes with the lowest turnover rates, endopolyga-
location (12). As mentioned in Section II, the enzymes lacturonase C, D, and E, it was proposed that polyga-
of clade B all have a prosequence in addition to the lacturonic acid was not the natural substrate. All three
presequence. enzymes still retain some activity on pectins with a low
degree of esterication (DE), making it unlikely that
these are the preferred substrates (Table 2). This in
IV. BIOCHEMICAL PROPERTIES OF contrast to endopolygalacturonases A and B, which
POLYGALACTURONASES both prefer low-DE pectin.
For these latter two enzymes that is quite under-
In the past, several polygalacturonases have been standable since these enzymes are constitutively
characterized biochemically. However, the most expressed, thus making their role as scouting
comprehensive and in-depth analyses have been enzymes which encounter primarily medium DE pec-
carried out for polygalacturonases from the genus tin quite likely (1). They are supposed to be involved
Aspergillus and notably for the seven-membered in producing the inducers required to obtain the
endopolygalacturonase family from A. niger N400 complete pectinase spectrum (1). The natural sub-
and for exopolygalacturonase from A. tubingensis strates for endopolygalacturonases C and E have
NW752. The variety of biochemical properties of yet to be identied. For endopolygalacturonase D,
these enzymes is such that it covers essentially most
other endo- and exopolygalacturonases. The discus-
sion of biochemical properties will therefore be con-
ned to this group of enzymes and will be
Table 1 General Properties and Kinetics of Aspergillus
complemented with additional data for other poly- niger Endopolygalacturonases
galacturonases where appropriate.
Mr S.A.b Km b Vmax b
A. A. niger endopolygalacturonases Enzyme (kDa) pH a
(U/mg) (mg/mL) (U/mg)
PG I 34.9 4.2 800 < 0:15 800

An early investigation of a commercial A. niger pecti-
PG II 34.9 4.2 4000 < 0:15 4000
nase preparation by Kester and Visser (24) revealed the PG A 35.5 4.0 1030 < 0:15 1200
presence of six different endopolygalacturonases with PG B 34.9 5.0 520 0.9 900
different specic activities and different modes of PG C 36.2 4.1 25 < 0:15 25
action (cleaving patterns) on oligomeric substrates. PG D 50.8 4.2 90 0.2 96
Using a reversegenetics approach, genes encoding PG E 35.6 3.8 38 2.5 80
endopolygalacturonases I and II were cloned (21, 25), a
Determined in McIlvaine buffers using 0.25% polygalacturonic acid
and, using the endopolygalacturonase II gene as a
at 30 C.
probe, genes encoding endopolygalacturonases A to b
Conditions: 50 mM sodium acetate, pH 4.2, 30 C. PG B: 50 mM
E (1, 20, 26, 27) were cloned. All the endopolygalac- sodium acetate, pH 5.0. S.A.: specic activity using 0.25% polyga-
turonase-encoding genes were individually overex- lacturonic acid.

Table 2 Performance of Aspergillus niger proposed to form part of the plants immune sys-
Endopolygalacturonases on Pectins with Various Degrees tem (31). It had been known for quite some time
of Esterication that oligogalacturonides with degree of polymeriza-
Degree of esterication (%) tion > 10 could effectively induce phytoalexin accu-
mulation in plants (32). Cervone and coworkers
Endopolygalacturonase 0 7 22 45 60 75 proposed that inhibition of endopolygalacturonase
activity by PGIP would increase the amount of larger
Relative specic activity oligogalacturonides and hence stimulate the phytoa-
I 100 97 87 43 18 3 lexin accumulation which in turn would trigger the
II 100 68 50 24 9 2 PGIP production (33). The effect of PGIP on endo-
A 100 125 135 52 7 6 polygalacturonase activity was demonstrated in vitro
B 100 150 168 131 62 27 (34). Bergmann et al. (35) showed that the oligoga-
C 100 102 86 36 16 5
lacturonides indeed exerted such an elicitor function
D 100 97 93 71 44 25
by analyzing PGIP mRNA accumulation. Not only
E 100 103 71 38 16 4
were the oligogalacturonides active in this respect, but
Enzymes were incubated with 0.25% (w/v) substrate in 50 mM wounding as well as infection resulted in a similar
sodium acetate buffer, pH 4.2, at 30 C. observation. Devoto et al. (36) demonstrated by
using a PGIP1 promoter glucuronidase (GUS) repor-
ter construct that of the minimally ve PGIP genes
present in Phaseolus vulgaris L. at least the PGIP1
it has been proposed that the enzyme is actually an gene is only induced by wounding and not by elicitor
oligogalacturonan hydrolase (27) based on the esti- molecules or infection.
mated number of subsites and other features (see Since phytopathogenic fungi generally have a family
below). of endopolygalacturonases at their disposal, different
specicities of PGIPs are expected. Indeed, Desiderio
et al. (37), Cook et al. (38) and Stotz et al. (39) demon-
1. Effect of Environmental Factors
strated that the inhibition depends on the type of PGIP
Endopolygalacturonases are generally active between and the type of endopolygalacturonase. The most com-
pH 3.5 and pH 6.0. Some enzymes have a quite nar- prehensive study in this respect so far was carried out
row pH optimum whereas others have a relatively by Leckie and coworkers (40). They studied P. vulgaris
broad pH optimum. As the majority of endopolyga- PGIP1 and PGIP2 of which only the former cannot
lacturonases have been isolated from mesophilic interact with the Fusarium moniliforme endopolygalac-
organisms, it is not surprising that the enzymes are turonase. In a loss-of-function approach they deter-
also mesophilic. Many polygalacturonases tested in mined that Q253 of PGIP2 was important for
this respect have their highest activity between 40 C binding to the F. moniliforme endopolygalacturonase.
and 55 C. However, it should be noted that at these By mutagenizing K253 of PGIP1 into Q253, PGIP1
temperatures denaturation sets in. Generally, assays acquired inhibitory properties toward F. moniliforme
are performed between 25 C and 37 C. endopolygalacturonase. By modeling studies it was
Low-molecular-mass inhibitors for endopolygalac- shown that K253 is exposed to the surface of the pro-
turonases have not been reported, although some inhi- tein (40).
bitory effect can be expected from divalent ions that Stotz et al. (39) not only demonstrated the differ-
interact with the substrate. The known inhibitors of ences in specicity of PGIPs and endopolygalacturo-
polygalacturonases are the polygalacturonase inhibitor nases, they also analyzed the evolution of 22 PGIP
proteins (PGIPs) produced by plants. genes and 19 fungal endopolygalacturonase genes of
Polygalacturonases have long been implicated in the different origin. It was demonstrated that advanta-
infection process of plants by phytopathogenic micro- geous amino acid substitutions dominate the molecular
organisms and not until recently has direct evidence of evolution of both PGIP and endopolygalacturonases
this been obtained via gene knock-out studies in A. and that the proteins are likely to evolve adaptively
avus (29) and B. cinerea (30). in response to natural selection. Unlike the demonstra-
PGIPs belong to the leucine-rich repeat proteins tion of Q253 to be critical for F. moniliforme recogni-
and are produced by nongraminaceous monocotyle- tion (40), this residue was not signicant in the
donous and dicotyledonous plants. They have been evolutionary analysis (39).

2. Specic Mechanism of Action contribution to the binding of the substrate by subsites
2, 1, and 1 was only small (1.1 kJ/mole). The
Endo- and exopolygalacturonases hydrolyze the gly-
major contribution of the substrate-binding energy,
cosidic bond between two adjacent 1,4-linked D-
14.4 kJ/mole, originated from subsite 3. The low
galacturonic acid residues. Biely et al. (41) demon-
afnity at subsites 2, 1, and 1 can be due to the
strated that for both exoPG and endopolygalacturo-
fact that the substrates used, oligogalacturonates, are
nase, hydrolysis proceeds with inversion of the
not the natural substrates for endopolygalacturonase
anomeric conguration. According to the general
E. For endopolygalacturonases I, II, A, B, and C,
concept of glycosyl hydrolytic action inverting hydro-
the number of subsites was estimated to be at least
lases should have two acidic residues, of which one
seven, from 5 to 2 (1, 28) and for endopoly-
activates the water that acts as a nucleophile and the
galacturonase D the number of subsites is four, from
other serves as the base, spaced 99.5 A apart. In
3 to 1 (27).
neither the E. carotovora endopolygalacturonase (4)
For endopolygalacturonases I and II subsite energy
and A. niger endopolygalacturonase II 3D structures
maps were calculated as well (28). A major difference
(5) nor in the A. aculeatus rhamnogalacturonase 3D
was observed for subsite 5. At this particular sub-
structure (6) are acidic residues spaced at such a dis-
site, quite remote from the active site, the contribu-
tance that they could play the proposed role. By site-
tion to binding of the substrate was 6 kJ/mole higher
directed mutagenesis studies of A. niger endopoly-
for endopolygalacturonase I than for endopoly-
galacturonase II and by comparison with the phage
galacturonase II. It was proposed that this higher
22 tailspike rhamnosidase, Van Santen et al. (5) and
afnity at subsite 5 is the underlying principle for
Armand et al. (15) were able to propose the catalytic
the processive (or multiple attack on a single chain)
mechanism. A triad of three strictly conserved Asp
behavior of endopolygalacturonase I (28). Owing to
residues at the bottom of the substrate-binding cleft
the high afnity after hydrolysis, the product remains
of endopolygalacturonases forms the catalytic
bound from subsites 5 to 1 instead of diffusing
machinery. Two Asp residues, Asp180 and Asp202
away, and subsequently shifts to subsites 4 to 1
(A. niger endopolygalacturonase II numbering),
for another hydrolytic event. Thus, the processivity
together activate the water that acts as the nucleo-
appears as an exolytic action from the reducing end
phile whereas Asp201 serves as the base that proto-
and continues until the degree of polymerization of
nates the leaving group (15). Asp201 is assisted in its
the substrate has reduced so far (n 5 for endopoly-
role by an adjacent His residue (His223). A His resi-
galacturonase I) that it does not cover the remote
due has long been thought to be critical for catalysis
high-afnity subsite anymore. For endopolygalac-
(4244). By chemical modication Stratilova et al.
turonases A, C, and D, processive behavior was
(45) demonstrated the involvement of a tyrosine in
observed as well (1, 27, 28). For endopolygalacturo-
catalysis of an endopolygalacturonase. Recently,
nases A and C, the minimum chain length is 6 and for
Page`s et al. (46) indeed demonstrated the indirect
endopolygalacturonase D this is 4. Upon hydrolysis
involvement in catalysis of a strictly conserved Tyr
of polymeric substrates the product progression of
residue (Tyr291, A. niger endopolygalacturonase II
typical endoacting enzymes such as endopolygalactur-
numbering). Replacing Tyr291 by Phe reduced the
onases II, B, and E is characterized by a transient
activity to 7% of the wild-type enzyme (46).
increase of oligomers with n > 6, which are gradually
converting into smaller products whereas for the pro-
cessive enzymes the product progression typically
3. Subsites in Binding to Substrate
shows a strong increase of monomers from the begin-
The A. niger endopolygalacturonase II was the rst ning of the reaction with hardly any transient accu-
enzyme to be characterized with respect to number of mulation of longer oligomers.
subsites, which was estimated at four (47). More Of the processive A. niger endopolygalacturonases,
recently the entire A. niger endopolygalacturonase endopolygalacturonase D is the most extreme in this
family was characterized in this respect (1, 20, 27, respect. Furthermore, endopolygalacturonase D is the
28). For endopolygalacturonase E the number of only A. niger endopolygalacturonase capable of hydro-
subsites was estimated to be at least ve, stretching lyzing dimers. This, in conjunction with only four sub-
from 4 to 1 (20) and subsite afnities were calcu- sites, prompted Par enicova` et al. (27) to propose
lated according to the method outlined by Suganuma endopolygalacturonase D as an oligogalacturonan
et al. (48). The calculation revealed that the total hydrolase.

B. A. tubingensis Exopolygalacturonase Erwinia chrysanthemi (54, 55), Clostridium thermosac-
charolyticum (56), and Ralstonia solancearum (57).
The A. tubingensis exopolygalacturonase (EC 3.2.1.67) By using a mixture of 4,5-unsaturated oligomers
hydrolyzes monomers from the nonreducing end of the (generated by pectate lyase action) and saturated oli-
substrate (49). Like the majority of A. niger endopoly- gomers, Collmer et al. (54) were able to deduce that the
galacturonases, exopolygalacturonase is optimally Erwinia enzyme preferred hydrolysis of 4,5-unsatu-
active between pH 4 and 5. The calculated subsite rated dimers, attacked the nonreducing end, and was a
map reveals four subsites, stretching from 1 to 3. single-attack enzyme. The preferred action of this
Subsite 1 has a very high afnity for a galacturonic enzyme on unsaturated substrates is understandable
acid unit (24.5 kJ/mole) whereas subsite 1 even has in view of the enormous amount of different pectate
some repelling force (1:6 kJ/mole). The high afnity lyases produced by this organism (see Chapter 80).
at subsite 1 and negative afnity at subsite 1 are Shevchik et al. (55) demonstrated that hydrolysis of
probably why nonproductive complex formation saturated oligomers indeed occurs at the nonreducing
occurs readily and why therefore the actual turnover, end by using reduced hexagalacturonate. In addition,
220 sec1 , is much lower than the potential intrinsic Kester et al. (53) showed that on puried 4,5-unsa-
rate (716 sec1 ) (49). Also, galacturonic acid appeared turated trigalacturonate the enzyme is slightly more
to be quite a strong inhibitor (Ki 0.3 mM). active than on the saturated counterpart (0.32 U/mL
Both the A. tubingensis and A. aculeatus exopolyga- vs. 0.2 U/mL) and that the enzyme can not accommo-
lacturonase are able to release xylogalacturonic acid date a methylated galacturonic acid residue at subsites
dimers (-xylose-1,3-galacturonic acid) from apple 2, 1, or 1.
pectin (50) or soy bean pectin (51). This ability may For C. thermosaccharolyticum, Van Rijssel et al.
be a reection of the low afnity at subsite 1. It is not (56) were able to show that the exopolygalacturonase
known whether xylose-substituted galacturonic acid activity is associated with a pectin methylesterase
may be present at subsite 1. In view of the high af- activity in a very large complex (1200 kDa).
nity for galacturonic acid it is likely that this may not Although this enzyme primarily hydrolyzed dimers
be the case. Korner and coworkers (52) have shown by from the nonreducing end, the formation of trimers
analysis of reaction products of partially methylated was observed as well. It is not known whether the
galacturonic acid oligomers by mass spectrometry formation of trimers is a result of a specic binding
that A. tubingensis exopolygalacturonase does not mode or is due to condensation or tranglycosylation.
hydrolyze a nonmethylated galacturonic acid from The latter is highly unlikely to invert. It is therefore
the reducing end when the following residue is methy- interesting that yet another D-galacturonan, digalac-
lated. However, using dened monomethylated dimers turonohydrolase, has been studied that does transgly-
and trimers, Kester et al. (53) showed that exopolyga- cosylate (58). This enzyme, puried from Selenomonas
lacturonase can accommodate such residues at subsite ruminatium, released dimers from tri-, tetra-, and
1 and 1 albeit with reduced efciency (< 20% of pentagalacturonates, but at high trimer concentrations
nonmethylated trimer and < 36% of nonmethylated bimolecular reactions were observed. This demon-
dimer). The discrepancy between these two studies ori- strates that the particular exopolygalacturonase
ginates from the presence of galacturonic acid mono- acts via retention of the anomeric conguration and
mers, potent inhibitors of exopolygalacturonase, in the thus should not be included in family 28 of glycosyl
oligomer mixture used by Korner et al. (52). In prac- hydrolases.
tice, during pectin hydrolysis, exopolygalacturonase
will cease action when a methylated galacturonic acid D. Synthetic Substrates
has to bind at subsite 1 thus leaving a nonmethylated
residue at the nonreducing end. Using monomethylesteried di- and trigalacturonides
carrying the monomethyl group at a dened position
(59), Kester et al. (53) studied the specicity of
C. Exo-Poly--D-Galacturonosidase Aspergillus polygalacturonases. The data obtained for
the A. tubingensis exopolygalacturonase were discussed
Exo-poly--D-galacturonosidase (EC 3.2.1.82) cata- above, as were the data for endopolygalacturonase D
lyzes the hydrolysis of digalacturonate from the non- (27). For all six remaining endopolygalacturonases (I,
reducing end of polygalacturonic acid (pectate). So far, II, A, B, C, and E) it was shown that at subsite 1 a
the activity has only been detected in bacteria like nonmethylated galacturonate residue is mandatory

(53). At subsite 1 only endopolygalacturonase I, II, pectin methylesterase activity are used successfully for
and B were able to accommodate a methylated galac- the production of pulpy nectars. These comminuted
turonate albeit very poorly (activities < 0:6% com- fruit juices are viscous, pulpy drinks and are usually
pared to nonmethylated trimer). At subsite 2 all six prepared by a mechanical-thermal dispersion process.
enzymes tolerated fairly well a methylated galacturo- However, products prepared with enzymes are super-
nate (activities between 6% and 38%). This was also ior in cloud stability and smooth consistency and have
shown by Korner et al. (52), who used partially methy- higher contents of soluble solids and pigments. These
lated oligomers mixtures as substrate and analyzed the suspensions of loose cells from fruit and vegetable tis-
products of endopolygalacturonase II action by mass sue are obtained by weakening the cell cohesion by a
spectrometry. limited pectin breakdown, particularly in the middle
Strictly speaking, almost all commercially available lamella. Nutrients like vitamin A or proteins are
pectin compounds are synthetic substrates, as they all protected inside the cells. Enzyme preparations,
have undergone some chemical treatment. This holds which have only one depolymerase system (commonly
even more for pectin series of various degree of methy- Endopolygalactronase(s)), are chosen. When endo-
lation that have been either methylated by chemical genous pectin methylesterase is present in the fruit,
treatment (acidic methanol) or demethylated by blanching is indicated. Maceration can also be
sodium hydroxide treatment or pectin methylesterase achieved with endopectin lyase or exopectate lyase.
action. Especially the pectin methylesterase-treated These enzymes might have potential application
pectins can give valuable information about substrate for processing of vegetables with a higher pH (62).
preference of pectic depolymerases as a blockwise dis- Endopolygalacturonase(s) added to citrus concentrates
tribution of methyl groups can be obtained by plant will reduce the viscosity of these concentrates and con-
pectin methylesterase action and a random distribution tribute(s) to extend the cloud stability of citrus juices.
by fungal pectin methylesterase. By using a series of Endopolygalacturonases can also be used for the man-
demethylated pectins which were differently prepared, ufacture of less calcium-sensitive pectin preparations
Page`s et al. (46) demonstrated that endopolygalactur- that can be used in systems where the viscosity aspect
onase II prefers blockwise demethylated substrate but is not important. Endopolygalacturonases will hydro-
that a single mutation (Glu252Ala) can change the lyze the galacturonan backbone preferentially in the
preference profoundly toward less block specicity or nonesteried, calcium-sensitive regions.
to even more block specicity (Gln186Glu). Although Polygalacturonases are widely distributed in higher
these studies are based on kinetic studies (initial rates), plants. They are believed to contribute to fruit soften-
valuable information on substrate specicity is also ing of pears, peaches, and avocado (62, 64).
obtained by analyzing the products after various Polygalacturonases and also pectin methylesterase
times of hydrolysis of pectins with various degree of activities are abundant in tomatoes. Ingenious systems
methylation (52, 60, 61). are used in the tomato processing industry to heat-
inactivate instantaneously (hot break process) to
obtain the highly viscous juice preferred by the consu-
V. APPLICATION OF mer and the high-consistency concentrated juice
POLYGALACTURONASES (tomato paste) used for sauces, soups, catsup, and
similar products. When tomatoes are used for color
Polygalacturonases have found widespread application and avor only, and consistency is provided by other
in many industrial processes. The enzymes applied are ingredients such as starch, a more easily handled, thin,
mostly obtained from the lamentous fungi of the cold break juice is the starting material for paste
genus Aspergillus. These preparations generally con- production. A holding time is introduced between
tain a whole range of cell walldegrading and mod- crushing and heat treatment to ensure breakdown of
ifying enzymes and are predominantly applied in the the pectins by combined pectin methylesterase/polyga-
fruit juice industry. Together with pectin methylester- lacturonase action (64).
ase, endopolygalacturonases are essential in most of
these applications e.g., clarication of fruit juices,
enzymatic juice extraction, liquefaction of plant tissues VI. QUALITATIVE AND QUANTITATIVE
(Chap. 67). DETERMINATION OF ACTIVITY
Fungal pectinase preparations containing predomi-
nantly endopolygalacturonases and which are free of See Chapter 66 on pectic polysaccharides.

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36. A Devoto, F Leckie, E Lupotto, F Cervone, G De site of an Aspergillus niger extracellular endopolyga-
Lorenzo. The promoter of a gene encoding a polygalacturonase. Eur J Biochem 39:109115, 1973.
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is activated by wounding but not by elicitors or patho- study of the mechanism of action of Taka-amylase
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7378, 1997.

70
Enzymes Releasing L-Arabinose and D-Galactose from the Side

Chains of Pectin
Ronald P. de Vries
Utrecht University, Utrecht, The Netherlands
Jaap Visser
I. INTRODUCTION cleave the internal linkages in the galactans; exo-

galactanases; and -D-galactosidases which remove
Owing to the abundance of arabinose containing terminal galactose residues and short galacto-oligo-
structures in pectin and other plant cell wall polysaccharides (in the case of the exogalactanases) from
saccharides, arabinose-releasing enzymes (arabinan- galactose containing oligo- and polysaccharides.
ases) play an important role in the degradation of These enzymes have been identied in both pro-
plant material. These enzymes and the polysaccharides karyotes and lower eukaryotes, as well as in some
on which they are active have recently been reviewed plants, and have been studied in detail. The enzymes
(1). Arabinanases can be divided into four classes: from microbial origin are those most commonly used
in food technology, particularly enzymes from species
1. Endoarabinanases (often referred to as endoar-
of the lamentous fungus Aspergillus. Therefore the
abinases) cleave the internal bonds of the arabi-
emphasis in this chapter will be on these enzymes,
nan chains.
although enzymes from other origins will also be
2. Exoarabinanases release terminal arabinose
mentioned. An overview of arabinose- and galac-
residues or short arabino-oligosaccharides
tose-releasing enzymes from Aspergillus and their
from arabinan.
encoding genes can be found in recent review on
3. -L-Arabinofuranosidases release only mono-
plant cell wall polysaccharide degrading enzymes
meric terminal arabinose residues from arabi-
from Aspergillus (2).
nose containing oligo- and polysaccharides.
The more specic experimental procedures involved
4. Arabinoxylan arabinofuranohydrolase, another
in the purication and characterization of galactanases
exoacting type of enzyme specic for xylan and
and arabinanases will be given. The different classes of
xylan-derived oligosaccharides. The latter
enzymes will be discussed separately, followed by
enzyme will be discussed in Chapter 71.
information about the induction of the production of
A second group of enzymes that is important for these enzymes, the cooperative or synergy of these
the hydrolysis of pectin side chains are the galactose enzymes in the degradation of the polymeric sub-
releasing enzymes (galactanases), which can be strates, and the applications of the enzymes in food
divided in three classes: endogalactanases which technology.

II. EXPERIMENTAL PROCEDURES FOR juice) (7). More advanced methods to determine arabi-
THE PURIFICATION AND nanase activity (including activity on polymeric sub-
CHARACTERIZATION OF THE strates) use FPLC and HPLC. Methods for the
ARABINANASES AND GALACTANASES analysis of the delignication activity of arabinofura-
nosidases (8) and action on monoterpenyl arabinofur-
A. Purication
anosylglucosides (9) have also been described but will
not be discussed here.
Since most microbial arabinanases and galactanases
Several methods have been described for the
are extracellular enzymes, they can be isolated from
determination of galactanase activity. The 1 6-
culture ltrates using classical purication techniques,
galactanase activity was derived from the amount
such as precipitation, ultraltration, gel ltration, ion
of 1 ! 6-galacto-oligosaccharides liberated from
exchange chromatography, isoelectric focussing, and
grape galactan (5). The amount of 1 ! 6-galacto-
adsorption. However, some materials for afnity
oligosaccharides was measured by oxidizing non-
chromatography have been developed specically for
reducing terminal galactose residues using the
these enzymes. Arabinanases and galactanases are
D-galactose dehydrogenase method of Finch et al.
commonly produced by microorganisms during
(10). Using the same method, 1 ! 3-exogalactanase
growth on crude plant cell wall material. The crude
activity was measured using a galactan prepared from
enzyme preparations obtained this way also contain
acacia gum (11, 12). Recently, a wide spectrum of sub-
many other cell walldegrading enzymes. During
strates has become commercially available. These
growth on sugar beet pulp, which is rich in pectin,
include different arabanans, galactans and arabinoga-
polygalacturonase and arabinanase are produced
lactans, as well as arabino- and galacto-oligosacchar-
abundantly. Using chromatography over a cross linked
ides. Additionally, several chromogenic substrates
alginate column (3), these two classes of enzymes have
have been developed for the rapid analysis of arabina-
been efciently separated. Divinylsulfone-substituted
nase and galactanase activity. McCleary (13) devel-
Sepharose and chelating Sepharose have been applied
oped a specic assay for endoarabinanase activity
to crude enzyme preparations high in both galacta-
using a dye-crosslinked substrate (Azurine-crosslinked
nases and arabinanases. Using this material an efcient
linear arabinan) which is commercially available
separation of arabinanases and galactanases from A.
(Megazyme, Ireland). The same company also devel-
niger was obtained (4). This column has also been used
oped a similar assay for 1 ! 4-galactanase activity
to remove contaminating arabinanase activities during
using Azurine-crosslinked potato galactan. In both
the purication of a 1 ! 6-endogalactanase (5).
assays the enzyme activity is measured by the amount
Crosslinked arabinan or arabinan linked to activated
of color released from the insoluble substrate. The
Sepharose 6B (6) was demonstrated to be able to bind
same company also markets an endoarabinanase, an
one of the two arabinofuranosidases commonly pre-
arabinofuranosidase, a 1 ! 4-D-endogalactanase,
sent in crude enzyme preparations from Aspergillus.
and a -galactosidase, all puried from A. niger.
B. Qualitative and Quantitative Determination of

III. a-L-ARABINOFURANOSIDASES
Activity
A. Substrate Specicity and Action Pattern
Arabinofuranosidase and -galactosidase activity is
generally assayed by using p-nitrophenyl--L-arabino- -L-Arabinofuranosidases are exoacting enzymes that
furanoside and p-nitrophenyl--L-galactopyranoside remove terminal arabinofuranose residues from the
as their respective substrates. The activity of the nonreducing end of arabinan and arabinose containing
enzymes is determined by measuring the release of p- oligosaccharides. In pectin these residues are linked via
nitrophenol spectrophotometrically at 400420 nm an 1 ! 2-, 1 ! 3- or an 1 ! 5-linkage to
after adjustment of the pH to 9.0. arabinan and arabinogalactan side chains. Arabino-
An assay in which both arabinofuranosidase activ- furanosidases differ with respect to their ability to
ity and endoarabinanase activity can be determined is release 1 ! 3- or 1 ! 5-linked arabinose. Two
measuring the appearance of reducing sugars from ara- distinctly different arabinofuranosidases have been
binans with different degrees of branching (e.g., linear described (1). Type A is not active on polymeric sub-
arabinan, heavily branched sugar beet arabinan, and strates, only on oligomeric substrates. Type B, how-
moderately branched arabinans isolated from apple ever, is able to release arabinose from both

oligomeric and polymeric substrates. Some -L-arabi- whereas the variation among bacterial arabinofurano-
nofuranosidases are able to release arabinose not only sidases is much wider (from 3.8 to 9.0). No apparent
from arabinan but also from arabinoxylan (14, 15). All correlation exists between the pI and A or B type ara-
arabinofuranosidases described so far are able to binofuranosidases. Differences observed in the molecu-
hydrolyze the synthetic substrate p-nitrophenyl--L- lar mass and pI of arabinofuranosidases from the same
arabinofuranoside. This substrate can be used to dis- (eukaryotic) organism are most likely due to posttran-
tinguish between arabinofuranosidases and arabinoxy- slational modications of the enzymes, especially gly-
lan arabinofuranohydrolases, since the latter enzyme is cosylation. Differences in glycosylation of extracellular
not able to hydrolyze p-nitrophenyl--L-arabinofura- fungal enzymes have been observed depending on the
noside. culture conditions of the strain. This can explain, to
some extent, the different molecular mass reported for
the arabinofuranosidases from two Aspergillus niger
B. Properties strains (Table 1) but not the large difference in pI,
indicating signicant differences in the enzymes pro-
In general, type A arabinofuranosidases in general duced by the different strains.
have a molecular weight in the range of 6080 kDa. A comparison of the kinetic properties of the two
Some gel ltration studies assessed the molecular mass types of arabinofuranosidases is made for the well-stu-
to be > 120 kDa, suggesting that these enzymes may be died enzymes from A. niger (1). Using p-nitrophenyl--
present as homodimers (Table 1). The native form of a L-arabinofuranoside as a substrate the Km for AbfA
type A arabinofuranosidase was demonstrated to be an and AbfB are 0.6 and 0.48 mM, respectively (Table 1).
octamer composed of eight subunits of 62 kDa (16). The kcat for AbfA and AbfB are 262 and 243 (sec1 ),
The variation in molecular weight of type B arabino- respectively (1). These data indicate that despite the
furanosidases is wider (between 34 and 94 kDa; Table strong differences in their ability to hydrolyze natural
1). For these enzymes, higher molecular masses were substrates, their activity on p-nitrophenyl--L-arabino-
also observed suggesting that they are also present as furanoside is nearly identical. The Km and kcat of ara-
multimeric proteins. Nearly all fungal arabinofurano- binofuranosidases of other organisms show differences
sidases have an acidic pI (between 3.2 and 6.0), up to a factor of 30 compared to the A. niger enzymes.
Table 1 Comparison of the Properties of Some -L-Arabinofuranosidases
Organism Type MW (kDa)a pI pHopt b Km (mM) Ref.
Bacterial
Bacillus subtilus A 61 7.0 66
Bacillus subtilus B 65 5.3 6.5 67
Streptomyces massasporeus B 54 (gf) 5.0 1.67 68
Streptomyces purpurascens A 62 3.9 6.5 16
495 (gf)
Thermomonospora fusca A 46 6.0 0.1 69
92 (gf)
Fungal
Aspergillus niger A 128 66.5 4.1 0.6 3
Aspergillus niger B 60 5.56 3.7 0.48 3
Aspergillus niger A 83 3.3 3.4 0.68 70
Aspergillus niger B 67 3.3 3.4 0.52 70
Aurobasidium pullulans B 105 4.04.5 71
210 (gf)
Trichodema reesei B 53 7.5 4.0 1.2 72
Plant
Daucus carota (carrot) B 94 4.7 4.2 1.33 73
a
MW is determined by SDS-PAGE, unless otherwise indicated (gf gelfiltration chromatography:
b
Determined using p-nitrophenyl--L-arabinofuranoside as a substrate.

Despite the signicant differences in pI between the Inhibition of -L-arabinofuranosidase III from
enzymes puried from the two A. niger strains (Table Monilinia fructigena was demonstrated using 1,4-
1), nearly identical Km values were observed. dideoxy-1,4-imino-L-threitol and 1,4-dideoxy-1,4-
imino-L-arabinitol (25). However, the data presented
C. Classication and Mechanism there indicated that efcient inhibition will only occur
when the pKa of the iminoalditol is below that of the
Based on the classication and nomenclature of the acid-catalytic group of the target enzyme.
Commission of Enzymes of the International Union Consequently these compounds will not inhibit most
of Biochemistry, -L-arabinofuranosidases (EC fungal arabinofuranosidases.
3.2.1.55) belong to the class of hydrolases (group 3),
more specically the O-glycosylases (subgroup .2.1.).
The distinction between the two types of arabino-
IV. ENDOARABINANASES
furanosidases from Aspergillus niger in the ability to
hydrolyze polymeric substrates reects the assignment A. Substrate Specicity and Mode of Action
of these enzymes to the different glycoside hydrolase
families as described by Henrissat and Bairoch (17) at Endoarabinanases act on the arabinan side chains of
URL http://afmb.cnrs-mrs.fr/cazy/cazy/index.html. pectins releasing arabinose oligosaccharides. They
This assignment of enzymes to the different families hydrolyze the internal 1 ! 5-linkages of the main
is made based on homology of the deduced amino acid chain of arabinan resulting in arabino-oligosacchar-
sequence derived from the genes encoding these ides. The activity of this enzyme signicantly enhances
enzymes. AbfA is assigned to family 51, whereas the activity of the -L-arabinofuranosidases and is
AbfB is assigned to family 54. The genes encoding therefore essential for an efcient degradation of ara-
the other puried type A or type B arabinofuranosi- binan. Endoarabinanases prefer unsubstituted linear
dases have not all been isolated, so a conclusion substrates and therefore also benet form the action
whether this is a general correlation cannot yet be of arabinofuranosidase (AbfB), removing the side
made. Families 51 and 54 contain only arabinofurano- chains from polymeric branched arabinan. Bacterial
sidases, indicating that these enzymes are not very endoarabinanases produce predominantly arabinose
similar to other proteins. An arabinofuranosidase and arabinobiose as end products (26, 27), while action
from family 54 has also been shown to possess -xylo- of an endoarabinanase from A. niger results in the
sidase activity (18). The same is observed for some accumulation of arabinobiose and arabinotriose in
bacterial arabinofuranosidases (1921), which have the reaction mixture (3).
been assigned to family 43. This family also contains Two endoarabinanases from closely related
endoarabinanases and -xylosidases. Aspergillus spp. have been studied with respect to
The reaction mechanism of some members of these their action pattern and substrate specicity (28). The
families has been determined Arabinofuranosidases A. aculeatus enzyme contains six subsites, while the A.
from families 51 and 54 work via a retaining mechan- niger endoarabinanase contains only ve subsites.
ism, whereas enzymes from family 43 have an inverting Different action patterns of the two enzymes resulted
reaction mechanism (17). The reaction mechanism of in different initial hydrolysis products, but for both
the arabinofuranosidase from Pseudomaonas uores- enzymes the predominant nal hydrolysis products
cens subsp. cellulosa (22), the only member of family were arabinobiose and arabinotriose.
62, has not been determined. This enzyme contains a
putative cellulose-binding domain. Additionally, an
arabinofuranosidase has been identied which con- B. Properties
tains a xylan-binding domain (23), but this enzyme
has not yet been assigned to a glycosylase family. Endoarabinanases are relatively small monomeric pro-
teins with a molecular mass between 30 and 45 kDa
D. Inhibitors (Table 2). The fungal endoarabinanases have an acidic
pI (3.03.3), whereas bacterial endoarabinanases have
L-Arabinose has been demonstrated to be a compati- an alkaline pI (7.99.3). Although many organisms
ble inhibitor (KI 16:4 mM) of arabinofuranosidase produce several arabinofuranosidases, no evidence
II from Penicillium casulatum, but did not affect arabi- for multiple forms of endoarabinanase has been
nofuranosidase I from this fungus (24). reported for any organism.

Table 2 Comparison of the Properties of Some can also not be assigned to one of the glycosyl hydro-
Endoarabinanases lase families.
Organism MW (kDa) pI pHopt Ref.
VI. ENDO- AND EXOGALACTANASES
Bacillus subtilis 32 9.3 6.0 67
Aspergillus nidulans 40 3.25 5.5 61 A. Substrate Specicity and Mode of Action
Aspergillus niger 43 3.0 4.6 70
Endogalactanases cleave within the galactan chains
reacting with galacto-oligosaccharides, whereas exoga-
lactanases attack the substrate from the terminal non-
C. Classication and Mechanism reducing endreleasing galactose or short galacto-
oligosaccharides. While most of these enzymes work
Like the -L-arabinofuranosidases, endo--L-arabina- specically by an endo- or exomechanism, some bac-
nases (EC 3.2.1.99) belong to the O-glycosylases. The terial enzymes are able to work via both mechanisms
systematic name of endoarabinanase is arabinan endo- (32, 33). Different types of endo- and exogalactanases
1,5--L-arabinosidase, but this name is rarely used. In have been identied with respect to their specicity for
this chapter this enzyme will therefore also be referred the various -D-galactan linkages. These enzymes
to as endoarabinanase. therefore have different specicities with respect to
Based on the sequence of the encoding genes, all naturally occurring substrates. Type I arabinogalac-
these enzymes belong to family 43 of the glycoside tans only contain 1 ! 4-galactan linkages and
hydrolases (17). This is a very heterogeneous family can therefore only be hydrolyzed by 1 ! 4-endo-
containing also (bacterial) arabinofuranosidases and and exogalactanases. Type II arabinogalactans contain
-xylosidases. The enzymes from this family work via mainly 1 ! 3- and 1 ! 6-galactan linkages. The
an inverting mechanism (17). No three-dimensional hydrolysis of pectins containing both types of arabino-
structure has been reported for an endoarabinanase, galactans requires the joint action of all three types of
but crystals have been obtained for the endoarabina- galactanases.
nase produced by Pseudomonas uorescens (29). In general, bacterial 1 ! 4-galactanases release
mainly galactotriose or galactotetrose (3236) from
galactan, while some also release galactobiose (32,
V. EXO-a-L-ARABINANASES 36, 37). Eukaryotic 1 ! 4-galactanases release pre-
dominantly galactobiose and galactose from galactan
Exoarabinanases are enzymes acting on polymeric and (4, 38, 39). Fewer studies have been reported on galac-
oligomeric substrates and have no, or very low, activity tanases working against type II arabinogalactan. They
against the synthetic substrate p-nitrophenyl--L-ara- have been shown to release mainly galactose and galac-
binofuranoside. So far, two exoarabinanases have been tobiose from the polymeric substrate (5, 12, 40).
identied. An exoarabinanase from Erwinia carotovora
produced arabinotriose from sugar beet arabinan (30). B. Properties
The enzyme was specic for 1 ! 5-linked arabino-
furanosyl residues but did not hydrolyze linear arabi- Properties of some endo- and exogalactanases are
nan. Therefore, this enzyme most likely releases side given in Table 3. The molecular mass of the enzymes
chains of sugar beet arabinan. differs strongly among different organisms and
A second exoarabinanase was characterized from A. depends on the linkage they are active on. The
niger (var. aculeatus) (31). This enzyme produced 1 ! 4-endogalactanases from Aspergilli all have
mainly arabinobiose from sugar beet arabinan, molecular masses 40 kDa and an acidic pI. The
although a small amount of arabinotriose was also enzymes are monomers and prefer unsubstituted galac-
detected. tan chains as a substrate (41). The A. niger 1 ! 6-
Exoarabinanases have not yet been assigned a num- endogalactanase has a higher molecular mass (60
ber according to the Commission on Enzymes of the kDa), and its activity is inhibited by the presence of
International Union of Biochemistry. However, they arabinose residues on the 1 ! 6-, 1 ! 3-galac-
will surely be added to the group of O-glycosylases tan chain. Both types of endogalactanases release pre-
(EC 3.2.1.). So far no genes encoding exoarabina- dominantly galactobiose, but also release some
nases have been described, and therefore the enzyme galactose, demonstrating a different action pattern

Table 3 Comparison of the Properties of Some Endo- and Exogalactanases
MW
Organism Active against Action (kDa) pI pHopt Topt Km (mM) Ref.
Aspergillus aculeatus 1 ! 4 endo 44 2.85 4.04.5 4565 41

Aspergillus niger 1 ! 4 endo 43 46 4.0 5055 0.77 52
Aspergillus sojae 1 ! 4 endo 39.7 3.6 4.5 50 0.82 58
Aspergillus niger 1 ! 4 exo 120 3.8 3.5 60 43
90 4.1
Bacillus subtilis 1 ! 4 exo 36 7.9 6.57 60 37
Lupinus angustifolius L. 1 ! 4 exo 60 7.0 74
Tomato 1 ! 4 exo 75 9.8 4.5 75
Aspergillus niger 1 ! 3 exo 62 5.0 40 1.3 76
Irpex lacteus 1 ! 3 exo 4251 8.2 4.6 2.13 12
Aspergillus niger 1 ! 6 endo 60 3.5 60 4.5 5
compared to the Aspergillus endoarabinases. The and EC 3.2.1.90, respectively. The exogalactanases and
1 ! 3-exogalactanase from A. niger (11) and Irpex the 1 ! 6)-endogalactanases should also be included
lacteus (12) differ signicantly in their molecular mass in this class (EC 3.2.1.).
(66 and 51 kDa, respectively). They are both inhibited For only one type of galactanases, the 1 ! 4-
in their activity by branching of the 1 ! 3-galactan endogalactanases, have genes been characterized. On
chain. the basis of the sequence the encoded enzymes are all
For some galactanases transglycosylation activity assigned to family 53 of the glycosyl hydrolases (17).
has been observed. The 1 ! 4-endogalactanase These enzymes have a retaining mechanism, and for
from Penicillium citrinum produced several oligosac- the Pseudomonas uorescens subsp. cellulosa
charides using soybean arabinogalactan as donor, 1 ! 4-endogalactanase the catalytic residues have
and mono- or disaccharide derivatives containing - been determined (45). For the 1 ! 4-endogalacta-
galactosyl residues as acceptors (42). The 1 ! 4- nase for A. aculeatus crystals have been obtained (46),
exogalactanase from A. niger synthesized oligosacchar- but a three-dimensional structure has not yet been
ides with a higher degree of polymerization when incu- reported.
bated with short galacto-oligosaccharides (43). A
comparison was made of the transfer products of gly-
cerol formed by Bacillus subtilis 1 ! 4-exogalacta- VII. b-D-GALACTOSIDASES
nase, E. coli -galactosidase, and P. citrinum 1 ! 4-
endogalactanase (44). The -galactosidase transferred Endogalactanases enhance the release of monomeric
90% of the galactose residues to the primary hydroxyl galactose by -D-galactosidases, which remove sin-
group of glycerol, whereas the 1 ! 4-endogalacta- gle-terminal galactose residues. -Galactosidases have
nase transferred 80% of the galactose residues to the been detected in prokaryotes and lower and higher
secondary hydroxyl group. The 1 ! 4-exogalacta- eukaryotes and are active on a wide range of substrates
nase was less specic. Both products were formed, as discussed in other chapters of this book. This chap-
although the amount of transfer to the secondary ter will therefore only briey mention the function of
hydroxyl group was approximately twice as high. -galactosidases in pectin hydrolysis.
Plant -galactosidases are believed to be involved in
fruit ripening. -Galactosidase was demonstrated to
C. Classication and Mechanism increase in parallel with an increase in tissue softness
(4749). This is most likely caused by removal of the
So far, only 1 ! 4- and 1 ! 3)-endogalactanases galactose residues from pectin, thus altering the cell
have been included in the classication and nomencla- wall structure. The -galactosidase from A. niger was
ture of the Commission on Enzymes of the shown to be highly active on the side chains of sugar
International Union of Biochemistry. Like all other beet pectin (15). It enhanced the activity of the A. niger
enzymes discussed in this chapter, they belong to the 1 ! 4-endogalactanase, resulting in an efcient
group of O-glycosylases and are numbered EC 3.2.1.89 hydrolysis of the galactan chains.

-D-Galactosidases (EC 3.2.1.23) are also members
of the O-glycosylases in the classication and nomen-
clature of the Commission on Enzymes of the
International Union of Biochemistry. All -galactosi-
dases for which the encoding gene has been cloned have
so far been assigned to family 35 of the glycosyl hydro-
lases (17). They work via a retaining mechanism, and
contain a highly conserved region (G-G-P[LIVM]2 -X2 -
Q-X-E-N-E-[FY]). Based on the similarity with other
glycosyl hydrolases, the second glutamic acid residue
most likely acts as the proton donor in the catalytic
mechanism (50).
VIII. COOPERATIVELY (SYNERGY) OF

THE ENZYMES IN THE
DEGRADATION OF POLYMERIC Figure 1 Synergy between A. niger enzymes in the release of
SUBSTRATES arabinose from sugar beet pectin. AbfB, arabinofuranosidase
B; AbnA, endoarabinanase; GalA, 1 ! 4-endogalacta-
Positive interaction among enzymes, most commonly nase; LacA, -galactosidase; FaeA, feruloyl esterase A.
(From Ref. 15.)
referred to as synergy, is a general phenomenon in
polysaccharide biodegradation. Synergy is important
for the efcient action of both arabinofuranosidases
and endoarabinanases. These two classes of enzymes from the side chains of pectin positively affected the
positively affect each other in the hydrolysis of arabi- activity of all four enzymes.
nan. Additionally, the presence of -galactosidase and Less specic interactions have also been reported.
endogalactanase also enhances the activity of arabina- The addition of BSA (100 g/mL) had a stimulating
nases on sugar beet pectin (15) and arabinogalactan effect on the hydrolysis of p-nitrophenyl--L-arabino-
protein (51). furanosidoside by arabinofuranosidase (51), most
Synergy has also been reported for 1 ! 4-endo- likely by stabilizing the enzyme in the reaction mixture.
galactanase and -galactosidase from A. niger, and for
1 ! 4)-endogalactanase and endoarabinanase from
A. niger (52). The rst effect is mostly likely due to the
removal of single-unit galactose side chains from the
galactan backbone, whereas the second effect indicates
that linear arabinan chains limit the activity of
1 ! 4-endogalactanase on galactan. A recent
study (15) reported the synergy of endoarabinanase,
arabinofuranosidase B, -galactosidase, 1 ! 4-
endogalactanase, and feruloyl esterase A from A.
niger in the hydrolysis of the side chains of sugar
beet pectin. Synergy was observed between most
enzymes, although not always to the same extent.
Endoarabinanase and arabinofuranosidase B posi-
tively inuenced each other (Fig. 1), as did -galacto-
sidase and 1 ! 4-endogalactanase (Fig. 2).
Additionally, endoarabinanase and arabinofuranosi-
dase B strongly inuenced the release of galactose by Figure 2 Synergy between A. niger enzymes in the release of
-galactasidase and 1 ! 4-endogalactanase, galactose from sugar beet pectin. AbfB, arabinofuranosidase
whereas the latter two enzymes did not signicantly B; AbnA, endoarabinanase; GalA, 1 ! 4-endogalacta-
inuence arabinose release. Feruloyl esterase A, an nase; LacA, -galactosidase; FaeA, feruloyl esterase A.
enzyme capable of removing ferulic acid residues (From Ref. 15.)

IX. PRODUCTION OF ARABINANASES brane fouling. Adequate formulations of enzyme mix-
AND GALACTANASES tures are required (77).
Arabinanases and galactanases used in food processing

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71
Xylanolytic Enzymes
Peter Biely
Slovak Academy of Sciences, Bratislava, Slovakia
I. INTRODUCTION insects (8). Most, if not all, fungal plant pathogens

produce and secrete enzymes that degrade cell wall
A. Xylanthe Second Most Abundant Plant
hemicellulose (9). Xylanolytic enzymes also occur in
Cell Wall Component and Natural Polymer
plants. The enzymes participate in the process of cell
wall extension, cell division, seed germination, and
Plant cell walls can be considered to be the main
other morphological and physiological events in
renewable resource formed in the process of photosyn-
plants. During germination endogeneous enzymes cat-
thetic xation of carbon dioxide. They are composed
alyze hydrolysis of these polysaccharides to remove the
of three major polymeric constituents: cellulose, an
physical barrier imposed by the walls on the free diffu-
insoluble skeletal polysaccharide composed of -D-
sion of starch and storage protein-degrading enzymes
glucopyranosyl residues linked -1,4-glycosidically;
through the germinated grains (1012). Enzymes
hemicellulose, a series of matrix and crosslinking het-
degrading hemicellulose polysaccharides were also
eropolysaccharides that include a variety of glucans,
reported to be present in wheat grain (13) and wheat
mannans, arabinans, galactans, and xylans; and lignin,
our (14).
a complex polyphenol. As a part of the carbon cycle,
all three constituents are degraded by specialized
enzyme systems produced by microorganisms. This
C. Application of Xylanolytic Enzymes
chapter is devoted to the enzymes that degrade the
major plant hemicellulose, xylan. After cellulose,
The past two decades have seen a growing interest in
xylan is the most abundant polysaccharide in nature.
microbial enzyme systems that degrade plant xylan, a
Xylanolytic enzymes have been extensively reviewed in
polymer of the ve-carbon sugar D-xylose. Xylose can
the past (15). Books covering xylan and xylanases
be converted to a variety of useful products, including
exclusively have also been published (6, 7).
ethanol, the engine fuel of the future (15, 16). Enzymic
saccharication of agricultural, industrial, and munici-
B. Occurrence of Xylanolytic Enzymes pal wastes may provide sugar syrups for human and
animal consumption, and carbon sources for industrial
Many bacteria, fungi, yeasts, and protozoa are able to fermentations. Xylo-oligosaccharides have a variety of
degrade xylan (1, 8). Some of these are saprophytic soil uses as food additives (17). Plant structural polysac-
or aqueous microorganisms, some grow aerobically, charides provide the major source of nutrients for rum-
some grow at room temperature, and others show ther- inal livestock and also play an important role in animal
mophilicity. Some occur in the digestive track of rumi- fodder. Pretreatment of forage crops with polysacchar-
nant animals and in the intestines of wood-eating ide-degrading enzymes improves the nutritional qual-

ity and digestibility of ruminant fodder and facilitates D. Chemical Structure of Xylan
composting (18, 19). Enzymic hydrolysis of highly vis-
cous arabinoxylans originating in cereal endosperms Xylan is structurally similar to cellulose, but instead of
improves nutrient uptake and digestion in broiler D-glucose, its main chain is built from -1,4-linked
chickens (2023). Other applications of xylanolytic xylopyranosyl residues. In contrast to cellulose, how-
enzymes include improvement of the baking process ever, the xylan structure is extremely variable and
and modication of baked products (2430). depends on its source. The structure ranges from an
Xylanases are also important components of enzyme almost linear unsubstituted chain, e.g., in some grasses,
systems used for liquefaction of vegetables and fruit, to highly branched heteropolysaccharides in cereal
and for clarication of juices (3, 18). In all of the seeds. The prex hetero indicates the presence of sugars
above-listed processes, microbial xylanolytic systems other than D-xylose. The main chain is usually substi-
can be applied together with other enzymes hydrolyz- tuted to various degrees by residues of 4-O-methyl-D-
ing plant polysaccharides such as amylases, cellulases, glucuronic acid, D-glucuronic acid, or L-arabinofura-
and pectinases. There are, however, applications in nose, and in some cases is also esteried by acetyl
which xylanases should not be contaminated by cellu- groups. The substituents may be also represented by
lases to preserve the polymerization degree of cellulose. oligosaccharides and esteried by cinnamic (phenolic)
Xylanases free of cellulases have applications with acids. The principal types of xylan found in plants are
important ecological implications in pulp and paper well known and have been extensively reviewed (39
industry. They facilitate lignin extraction, reducing 41). They differ in the character of their side chains as
the consumption of toxic chemicals required for pulp indicated in Table 1.
bleaching (3134). The mechanism of this process, A hypothetical fragment of a plant xylan which
called enzymic prebleaching or bleachbusting, is not shows the major structural features found in this
completely understood. However, it is believed that group of hemicelluloses is depicted in Figure 1.
xylanase attacks the xylan moiety of lignin-carbohy- Xylans backbone is built of -1,4-linked D-xylopyra-
drate complexes (31). Xylanases also have potential nosyl residues. In hardwood xylans the side chains are
in the purication of dissolving pulp from hemicellu- -1,2-linked 4-O-methyl-D-glucuronic acid residues
lose (35, 36), in the recovery of cellulose textile bers and acetyl groups partially esterifying the two avail-
(37), and in paper recycling (38). able hydroxyl groups of xylosyl residues. Owing to
Table 1 Examples of Major Types of Plant Xylans
Approximate ratio
Structural type Source Nature of side chains Xyl:side chain subst.
Linear -1,4-D-xylan Esparto grass Substituents are rare

Tobacco stalk
O-Acetyl-4-O-methyl-D- Hardwoods Acetyl, 2-O--MeGlcA Xyl:Ac:MeGlcA 10:7:1
glucuronoxylan
O-Acetyl-4-O-methyl-D- Gramineae Acetyl, 2-O--MeGlcA Not specied
glucuronoxylan Legumes Acetyl, 2-O--GlcA
L-Arabino-4-O-methyl-D- Softwoods 3-O--L-Araf , 2-O--MeGlcA Xyl:Ara:MeGlcA 10:1:3:2
glucuronoxylan
O-Acetyl-L-arabinoxylan Gramineae 2-O--L-Arfa, 3-O--L-Araf Xyl:Ara varies from 0.9 to 2.2
(monocots Substitution of Xyl in the main depending on source.
primary walls, chain: single and double Double substitution of Xyl with
ours) Ara is more frequent than single
substitution.
Pericarp L-Arabinan oligomers Not specied
Source: Refs. 4244.

Figure 1 Hypothetical plant xylan and the enzymes required for its complete hydrolysis.
the ease of migration of acetyl groups among the E. Xylanolytic System and Its Components
hydroxyl groups, it is difcult to determine their actual
distribution between positions 2 and 3. Softwood Depending on its complexity, the complete breakdown
xylans are similar to hardwood xylans, but contain of xylan requires the action of several enzyme compo-
-1,3-linked L-arabinofuranosyl residues instead of nents which can be considered as a xylanolytic system
acetyl groups. Xylans from grasses contain a smaller (3). As depicted in Figure 1, the more complex the
proportion of uronic acids, but are more highly xylan structure, the more components of the xylanoly-
branched with L-arabinofuranosyl residues. As indi- tic system are required. The crucial enzyme for xylan
cated in Table 1, the arabinofuranosyl residues also depolymerization is endo--1,4-xylanase (-1,4-xylan
can be linked to the main chain by -1,2-linkages, xylanohydrolase; EC 3.2.1.8, abbreviated EX). EX
often to D-xylopyranosyl residues already substituted attacks the main chain most easily and therefore
by -1,3-linked L-arabinofuranosyl residues. In cer- most rapidly at non-substituted regions, generating
eals, the ratio of Xyl : Ara is low, ranging from 1 to nonsubstituted and branched or esteried oligosac-
2.2 (44). Xylans from graminaceous plants also contain charides. The main chain substituents are liberated
25% by weight of acetyl groups and 12% of pheno- by corresponding glycosidases or esterases: -L-arabi-
lic (cinnamic) acids esterifying the C-5 position of ara- nosyl residues by -L-arabinofuranosidases (-L-ara-
binose side chains (45). In sugar beet pectins, ferulic binofuranoside arabinofuranosidase; EC 3.2.1.55), D-
acid esteries the OH-2 of L-arabinofuranose or the glucuronosyl and 4-O-methylglucuronosyl residues by
OH-6 of D-galactose (46). Ferulic acid [3(3-methoxy- -glucuronidase (EC 3.2.1.139), acetic acid, and ferulic
4-hydroxy phenyl)-2-propanoic] is the major cinnamic acid and p-coumaric acid residues by corresponding
acid found in cereals and in the Gramineae (47). In esterases (EC numbers not assigned). -Xylosidase
wheat bran, one of 150 of xylose residues is substituted (xylobiase or exo--1,4-xylanase) (-1,4-xylan xylohy-
with L-arabinose esteried with ferulic acid (45). drolase; EC 3.2.1.37) attacks xylo-oligosaccharides
Ferulic acid is synthesized in plants via the shikimic from the nonreducing end, liberating D-xylose.
pathway and is an intermediate in lignin biosynthesis The hydrolases removing xylan side chains have
(48). In some plants it has been shown to be involved in been referred to as accessory or auxiliary xylano-
the chemical linkages between xylan and lignin, con- lytic enzymes. Two categories of accessory enzymes
tributing to integrity of the plant cell walls. can be recognized: those that operate on both poly-
Intermolecular crosslinking may result from the dimer- meric and oligomeric substrates, and those that are
ization of feruoyl and p-coumaroyl residues or from active only on branched or substituted oligosacchar-
the formation of ester linkages between polysaccharides generated by EXs. There is usually no sharp
ides and lignin (49, 50). boundary between these groups. The enzymes operat-

ing on polymeric substrates can be distinguished from acetateacetic acidwater (18:7:8, always v/v). A simi-
those of the second group by their ability to catalyze lar efciency of separation can be achieved on thin
precipitation of the originally highly soluble branched layers of microcrystalline cellulose (Merck,
polymeric xylan. This precipitation is due to removal Darmstadt, Germany) in ethyl acetateacetic acid
of substituents that hinder the association of the xylan water (3:2:2) or ethyl acetateacetic acidformic acid
main chains by hydrogen bonding (analogous to that water (18:3:1:5). These systems separate well neutral
found in cellulose). This precipitation can be used for oligosaccharides, aldouronic acids, and partially acety-
specic procedures to detect debranching enzymes in lated xylo-oligosaccharides. Acetylated xylo-oligosac-
the absence of depolymerizing endoxylanases. charides show higher mobility than the
Accessory enzymes further differ in their requirements corresponding deacetylated products. These systems
for the ne structure of the xylan main chain and the may also be used to follow the liberation of 4-O-
position of the group to be removed. methyl-D-glucuronic acid, which migrates in all the
above solvent systems ahead of D-xylose. The use of
F. Synergism of Xylanolytic Enzymes aniline-hydrogenphthalate reagent provides a color
differentiation of the acid from xylose and xylo-oligo-
The synergistic action of two or more enzymes during saccharides (orange-brown versus dark-brown colors).
hydrolysis of xylan is evident when the rate of hydro- The reagent, which can be poured onto dry chromato-
lysis of the polysaccharide with two or more enzymes is grams, is prepared by dissolving 1 g of phthalic acid in
faster than the sum of the rates of hydrolysis by indi- 50 mL acetone followed by addition of 0.9 mL aniline.
vidual enzymes. Several types of synergism have been Visualization of reducing sugars is obtained on heating
recognized among the xylanolytic enzymes (5153). at 110 C for a few min.
The most important type concerns the cooperativity Good separation of neutral products of xylan
between the enzymes attacking the main chain (EXs) hydrolysis is obtained by thin-layer chromatography
and enzymes that liberate side chain substituents, such on silica gel (Merck) in such solvent systems as 1-buta-
as acetyl (54) and arabinofuranosyl residues (51). The nolethanolwater (10:8:7), 1-propanolethyl acetate
action of EX releases substituted xylo-oligosacchar- ethanolpyridinewater (7:3:3:2:1) or nitromethane
ides, which are more readily diffusible and more favor- acetonitrileethanolwater (1:4:3:2), the last being the
able substrates for accessory enzymes. On the other fastest system. Reducing sugars can be visualized with
hand, the removal of side chain substituents by acces- an aniline/diphenylamine reagent, consisting of a solu-
sory enzymes creates new sites on the main chain for tion of aniline (2 mL) and diphenylamine (2 g) in acet-
productive binding with EX. From some accessory one (100 mL) containing 15 mL of 85% H3 PO4 .
enzymes the cleavage of the main chain is indispensa- Visualization of all sugars, both reducing and nonre-
ble. Individual components of xylanolytic systems are ducing, is done with 1% orcinol solution in ethanol/
discussed below. conc. H2 SO4 (9:1). Both reagents can be poured onto
well-dried chromatograms, which is more convenient
G. Analysis of Xylan Hydrolysates than spraying. The solvent system 1-butanolethanol
water (10:5:1) is suitable to follow the reaction of xyla-
It is always important to conrm that the apparent nases with 4-nitrophenyl -xylopyranoside. 4-
xylanolytic activity of any preparation is really con- Nitrophenol and 4-nitrophenyl glycosides also can be
nected with xylan depolymerization and not with detected directly under UV light (360 nm).
degradation of some other polysaccharides present as Other, more up-to-date methods of monitoring the
impurities. This aspect is especially important for plant enzymic hydrolysis of xylan include a variety of liquid
enzyme preparations which contain high levels of amy- chromatographies, such as gel permeation chromato-
lolytic enzymes. Almost all commercial xylan prepara- graphy, ion exchange chromatography, adsorption
tions contain a small amount of starch, which must be chromatography, reversed-phase chromatography,
removed by -amylase treatment before testing for the and partition chromatography, usually in HPLC set-
presence of xylanases. ups. All of these methods require the proper choice of
The traditional method of analyzing enzymic hydro- sorbents and eluent, and sophisticated equipment.
lysates of xylan is by paper and thin-layer chromato- Paper and thin-layer chromatography are the most
graphy. A suitable solvent system for separation of D- simple and least expensive separation techniques for
xylose and -1,4-xylooligosaccharides (linear or substi- the simultaneous analysis of a large number of sam-
tuted) on Whatman No. 1 or 3 MM paper is ethyl ples.

II. ENDO-b-1,4-XYLANASE (EC 3.2.1.8) designed to predict protein folding based on hydropho-
bic/hydrophilic patterns and is used to compare mem-
A. Chemical Reaction Catalyzed
bers of a protein group of similar functions (60). Acidic
high-molecular-mass EXs (> 30 kDa) were found to be
H2 O assigned to glycanase family 10 (59) (formerly family
R1 -Xyl1-4Xyl1-4Xy11-4Xyl-R2 ! R1 -Xyl1- F), and basic, low-molecular-mass EXs (< 30 kDa), to
4Xyl Xyl-4Xyl-R2 glycanase family 11 (formerly family G). While family
10 also contains other glycosyl hydrolases, family 11 is
R1 and R2 , substituted or unsubstituted portions of the exclusively made up of EXs.
main chain of xylan; reducing end. EXs belonging to the two glycosyl hydrolase families
show remarkable differences in their tertiary structures
B. Classication as established by crystallography (Fig. 2). Members of
individual families have a similar protein folding pat-
-1,4-Xylan xylanohydrolase, EC 3.2.1.8. terni.e., a similar three-dimensional structure. These
tertiary structures are more conserved than amino acid
C. Properties as Proteins sequences. The most conserved region within a family
appears to be the catalytic domain. EXs of family 11
1. EX Families
appear to be smaller, well-packed molecules, formed
A careful comparison of microbial EXs on the basis of mainly of -sheets (6164). These enzymes appear
their different physicochemical properties, such as very small in their native states (65, 66). The catalytic
molecular mass and isoelectric point, was carried out groups are present in a cleft that accommodates a chain
by Wong et al. (55). This analysis indicated that these of ve to seven xylopyranosyl residues.
enzymes can be divided into two groups, one consisting The tertiary structure of EXs of family 10 (Fig. 2) is
of high-molecular-mass enzymes with low pI values, typically an eightfold = barrel =8 , resulting in a
and the other consisting of low-molecular-mass salad bowl shape (6770). The substrate binds to a
enzymes with high pI values. As usual, there are excep- shallow groove on the bottom of the bowl, which is
tions, particularly concerning pI values. Interestingly, shallower than the substrate binding cleft of family 11
this grouping of EXs (55) was found to be in agree- EXs (9). This feature, together with the generally
ment with the general classication of glycanases on greater conformational exibility of larger enzymes,
the basis of hydrophobic cluster analysis and sequence may account for the lower substrate specicity and
similarities (5659). Hydrophobic cluster analysis is greater catalytic versatility of family 10 EXs.
Figure 2 Tertiary structures of endoxylanases of families 10 and 11.

A comparison of the tertiary structure of EXs with domains (modules) of EXs act independently of the
other classied glycosyl hydrolases reveals that the two catalytic domain as polysaccharide-binding domains
EX families evolved from different ancestors. EXs of (carbohydrate-binding modules; CBMs). Catalytic
family 10 are closely related to the endo--1,4-gluca- domains are connected to CBMs to which they are
nases of family 5, with which they share some common linked by linker sequences rich in proline and hydroxy
catalytic properties (see below). Both enzyme families 5 amino acids. The linkers function as exible, extended
and 10 belong to a larger clan of enzymes possessing hinge regions between functional domains (57).
the eightfold = barrel architecture (69, 71), known as Originally, it was believed that CBMs increased the
clan GH-A (59). EXs of family 11 show a certain catalytic rate against insoluble substrates. however,
degree of similarity to the low-molecular-mass endo- later observations suggested that binding domains
-1,4-glucanases of family 12 (formerly H) (72). may also facilitate the hydrolysis of soluble polysac-
charides (75, 77).
2. Multiplicity of Forms Surprisingly, there are several EXs that contain
binding modules that bind exclusively to cellulose (cel-
Genetic differences are not the only source of hetero-
lulose-binding domain; CBD) and not to xylan (57,
geneity among EXs. A multiplicity of forms is fre-
74). This is likely related to the biological role of
quently observed among the products of one gene.
these enzymes in degrading plant cell walls which are
Several molecular forms of enzymes puried to homeo-
predominantly built from cellulose closely associated
geneity often can be revealed by isoelectric focusing.
with hemicellulose. Targeting of EX to cellulose may
The observed multiplicity of forms may be a conse-
bring the enzyme in proximity of its substrate. There is
quence of several factors. The most common reasons
a family of CBMs (CBD9) that are found only in EXs
include differential mRNA processing, partial proteo-
(73). CBMs of EX B (XlnB) from Streptomyces lividans
lysis, and variable degrees of amidation and glycosyla-
and EX D from Cellulomonas mi appear to be specic
tion. Additional causes may include autoaggregation
for soluble and insoluble xylan (75, 77). The 3D struc-
and aggregation with other proteins (51).
ture of this xylan-binding domain (XBD) is very simi-
lar to that of CBD. A single amino acid change
3. Multiple Domains
converts the protein from a XBD to a CBD (75). An
The molecular architectures of EXs range from a single example of an EX containing two CBMs has been
catalytic domain to a complex arrangement of catalytic found in Pseudomonas uorescens subsp. cellulose (78).
and noncatalytic domains (7376), recently named Nature also has designed multifunctional enzymes
modules (73, 74). The catalytic domain (module) with two catalytic domains connected by linker
includes the part of enzyme (Fig. 3) that contains the sequences. Some bacteria produce bifunctional EXs
active site, i.e., the substrate-binding site in the classi- containing two identical (79) or two different EX
cal sense, and the catalytic groups. Noncatalytic domains (80, 81). Other enzymes possess catalytic
Figure 3 Molecular architecture of some endo--1,4-xylanases. (From Ref. 76.)

domains of two different xylanolytic enzymesone some exceptions, also from acetylxylan, than do EXs
catalyzing the xylan depolymerization, and the other of family 11. EXs of family 10 can further hydrolyze
deacetylation of the xylan main chain (8284). Such the shortest branched or isomeric xylooligosaccharides
bifunctional enzymes may have an advantage over which are released by EXs of family 11 (87).
enzymes containing a single catalytic domain in the Characterization of the shortest acidic fragments
hydrolysis of native acetylated xylan. Multidomain released from 4-O-methyl-D-glucurono-D-xylan after
xylanases also occur as subunits of cellulosomes and a long-term hydrolysis (88, 89) suggests which lin-
xylanosomes. These supramolecular extracellular cell- kages are accessible to hydrolysis by the two types
associated protein complexes of thermophilic clostridia of EXs. EXs of family 10, unlike EXs of family 11,
can efciently degrade cellulose, xylan, and related are capable of attacking the glycosidic linkage next
plant cell wall polysaccharides (85, 86). to the branch and toward the nonreducing end.
While EXs of family 10 require two unsubstituted
D. Properties as Enzymes xylopyranosyl residues between the branches, EXs
of family 11 require three unsubstituted consecutive
1. Catalytic Properties of EXs Belonging to
xylopyranosyl residues (Scheme 1). Kinetic data for
Two Families
cleavage of individual glycosidic linkages of the
EXs belonging to glycanase family 10 hydrolyze het- xylan main chain are not available, but time course
eroxylans and rhodymenan (a linear algal -1,3--1,4- of product analyses suggest that linkages closer to
xylan) to a greater extent than EXs of family 11 (87, substituents are hydrolyzed more slowly than more
88). The EXs of family 10 are better able to cleave distant linkages.
EXs 10 # # # # #
-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-
EXs 11 " 2 " 2 2
j j j
1 1 1
MeGlcA MeGlcA MeGlcA
Scheme 1
glycosidic linkages in the xylan main chain that are The degradation of a cereal L-arabino-D-xylan with
near substituents, such as MeGlcA and acetic acid. two EXs of Aspergillus awamori (EXI, 39 kDa, pI 5.7
The alteration of the xylan main chain by replacing 6.7, most probably a member of family 10, and EXIII,
-1,4-linkages by -1,3-linkages, as it is in rhodyme- 26 kDa, pI 3.33.5, most probably a member of family
nan, represents a more serious steric barrier for EXs of 11) (90) is shown in Scheme 2. The cleavage site speci-
family 11 than for EXs of family 10. Consequently, cities L-arabinosyl-D-xylan resemble those on 4-O-
EXs of family 10 liberate smaller products from 4-O- methyl-D-glucurono-D-xylan. The L-arabinosyl sub-
methyl-D-glucurono-D-xylan, rhodymenan, and, with stituents represent a more serious steric hindrance for
Araf
1
j
EXI # # # # 2 # #
-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-
EXIII " 3 " 3 3
j j j
1 1 1
Araf Araf Araf
Scheme 2

the formation of productive complexes of the polysac- of subsites and their binding afnities can be determined
charide with EXIII. Only the larger type of the enzyme by a procedure that is called subsite mapping (9698)
is capable of attacking the linkages between substituted which is complementary to the image of the substrate
and unsubstituted xylopyranosyl residues. binding site provided by x-ray crystallography (99).
Studies of the action of EXs on rhodymenan are EXs of both families catalyze the hydrolysis of gly-
complicated by the fact that EXs of family 10 are cosidic linkages with the retention of anomeric cong-
also capable of cleaving -1,3-linkages (91, 92). uration (100, 101). The reaction involves a double-
Generally, EXs of family 10 do not attack the poly- displacement mechanism leading to a new reducing
saccharide at -1,4-linkages which follow -1,3-lin- end with -anomeric conguration (102) (Fig. 4). The
kages toward the reducing end (91), liberating catalytic amino acids in the substrate binding site
xylotriose, Xyl1-3Xyl-4Xyl, as the shortest isomeric divide the subsites to the left (numbered with minus
product EXs of family 11 liberate from rhodymenan Roman numbers) and to the right of the catalytic
isomeric products larger than xylotriose. groups (numbered with positive Roman numbers)
Consistent with the fact that EXs of family 10 are (Fig. 5). The catalytic residues have been identied as
related to endo--1,4-glucanases and some glycosi- two conserved glutamate residues (in a few cases one
dases belonging to the clan of glycosyl hydrolases asparate replaces a glutamate), which are located
GH-A (59), EXs of family 10 catalyze hydrolysis of opposite each other in the active site. One residue
aryl -D-xylopyranosides and low molecular mass functions as a general acid catalyst protonating the
cellulase substrates. These include aryl -D-cellobio- glycosidic oxygen, while the other functions as a
sides, aryl -D-lactosides (93, 88), and cellooligosac- nucleophile, attacking the anomeric center to form a
charides (94). The degradation of aryl xylosides, e.g. covalent enzyme-glycosyl intermediate (Fig. 4). In the
of 4-nitrophenyl -D-xylopyranoside, also involves a second step a water molecule attacks the intermediate
complex reaction pathway including glycosyl transfer in a general base-catalyzed process to yield the product
reactions leading to xylooligosaccharides and their gly- of retained anomeric conguration (99, 103). The
cosides (94, 95). The EXs of family 11 do not possess catalytic residues have been identied by chemical
these catalytic abilities. modication, site-directed mutagenesis, and use of
stabilized enzyme-glycosyl intermediate (76, 103).
Glycosyl transfer reactions catalyzed by both types of
2. Mechanism of Substrate Degradation
EXs at high substrate concentrations are also evidence
The hydrolytic reaction takes place in the active site of for the enzyme-glycosyl intermediate. Using linear -
the enzyme consisting of the substrate-binding site and 1,4-xylo-oligosaccharides as substrates, it has been
catalytic groups. Consistent with their tertiary struc- established that EXs utilize multiple pathways of sub-
tures, EXs of family 10 appear to have a smaller substrate degradation (104, 105). Unimolecular hydrolysis
strate-binding site than do EXs of family 11. EXs of takes place at low substrate concentrations. Different
family 11 contain a larger number of subsites (ve to cleavage of the substrate occurs at so-called shifted
seven) than EXs of family 10 (four to ve subsites). A binding, which is observed at higher substrate concen-
subsite is a part of the binding site that interacts with trations and which is, generally, accompanied by for-
one xylopyranosyl residue of the substrate. The interac- mation of products larger than the substrate. An
tion may not be only attractive, it can also be repulsive, example of such reactions of xylotriose catalyzed by
particularly on the site of the leaving group. The number EX from Cryptococcus albidus is shown in Figure 5.
Figure 4 Mechanism of hydrolysis of glycosidic linkage with endo--1,4-xylanases.

Figure 5 Possible binding and degradation of xylotriose by EX from Cryptococcus albidus. (A) Productive complexes at low
substrate concentration and probability of their formation; (B) shifted two-one binding of xylotriose at high substrate concen-
tration and subsequent xylosyl transfer to second xylotriose molecule to give xylotetraose, which is nally cleaved to two
molecules of xylobiose. (From Ref. 104.)
At sufciently high concentrations of xylotriose, xylo- cellulose and xylan (106, 107). The best-known enzyme
biose initially is generated as the only product of xylo- in this regard is endoglucanase I from Trichoderma
triose degradation. Analogous reaction pathways are reesei, a member of family 5 glycosyl hydrolases. The
also observed with longer oligosaccharides. The main enzyme catalyzes the hydrolysis of both polysacchar-
molecular and catalytic properties of EXs of families ides in the same active site (108). Although the enzyme
10 and 11 are summarized in Table 2. belongs to the same clan as EXs of family 10, the
products the enzyme generates from glucuronoxylan
and rhodymenan are more similar to those liberated
3. Xylan-Degrading Endo--1,4-Glucanases
by EXs of family 11 (87). In T. reesei the enzyme is
It has been unambiguously established that at least one coinduced as a component of the cellulolytic and not
type of endo--1,4-glucanase exhibits activity on both of the xylanolytic system (3).
Table 2 Properties of EXs of Families 10 and 11
Properties EXs of family 10 EXs of family 11
Molecular mass usually > 30 kDa usually < 30 kDa

Isoelectric points usually < 7 usually > 7
Protein fold (catalytic module) 8 two -sheets and one -helix
Substrate binding site shallow groove deep cleft
Catalytic amino acids two glutamic acid residues (occasionally two glutamic acid residues (occasionally
one glutamate and one asparate) one glutamate and one aspartate)
Number of subsites 45 57
Substrate specicity less specic more specic
heteroxylans active active
aryl--xylosides active inactive
aryl--cellobiosides active inactive
aryl--lactosides active inactive
Stereochemistry of hydrolysis retaining retaining
Multiple reaction pathway at yes yes
high substrate concentrations

E. Qualitative and Quantitative Determination of 2. D-Xylose, 10 mM calibration solution in 0.05
Activity M citrate buffer, pH 5.3 (or other buffer).
3. DNS reagent prepared according to Sumner
1. Reductometric Assays
(112) or Miller (113).
The most common way to follow EX activity is to
Procedure. Preheated xylan solution (1.8 mL; 50 C)
determine the reducing sugars formed from selected
is mixed with 0.2 mL preheated enzyme solution
type of xylan. Two basic procedures for determination
(50 C) and incubated for 5 min at 50 C. The reaction
of reducing sugars have been widely used: the
is stopped by addition of 3 mL of the DNS reagent.
Somogyi-Nelson procedure (109111), and the 2,4-
The mixture is heated for 5 min at 100 C (boiling water
dinitrosalicylic acid (DNS) test (112114). The meth-
bath) and then cooled in cold water. Absorbance of
ods are sufcient to prove the presence of the enzyme,
samples is measured at 540 nm against the substrate
to assay its activity, and to evaluate the extent of poly-
blank and the values are corrected for absorbance of
saccharide hydrolysis, which may be the rst criteria
the enzyme blank. Calibration samples with known
for xylanase differentiation. The two methods for the
amounts of xylose are prepared in the presence of the
determination of reducing sugars have been compared
substrate.
in an interlaboratory evaluation organized by Finnish
One unit of xylanase activity is dened as the
scientists (115). Although the Somogyi-Nelson pro-
amount of enzyme liberating 1 mol of xylose equiva-
cedure is almost 10 times more sensitive than the
lent under the experimental conditions in 1 min. (Note:
DNS method, the latter always gives higher values of
The volume of the assay can be reduced.)
xylanase activity. When standardized with respect to
substrate and procedure, the assay employing the DNS
method afforded reproducible results in various b. Xylanase Assay Using the Somogyi-Nelson
laboratories with a standard deviation of 17% (115). Method (120)
Recently Jeffries et al. (116) reported that the differ- Reagents
ences between the Somogyi-Nelson and DNS methods 1. Birchwood or beechwood 4-O-methyl-D-glu-
are due to the fact that the rst method is less reactive curonoxylan (Roth 7500, Germany; or Sigma, USA),
and the second method more reactive with xylooligo- 0.4% solution in 0.1 M acetate buffer, pH 5.4, or 0.2%
saccharides than with xylose. The prevalence of oligo- solution in 0.05 M acetate buffer (or other buffer). The
saccharides among the products of xylan hydrolysis is solution is prepared under heating up to boiling point
thus why the Somogyi-Nelson method underestimates, with stirring until the polysaccharide dissolves. The
and the DNS method overestimates enzyme activity. solution can be stored in the presence of 0.010.02%
The most sensitive method for photometric determi- sodium azide.
nation of reducing sugars is that using 2,2 0 -bicinchoni- 2. D-Xylose, 1 mM calibration solution.
nate (117, 118). However, it has not been widely
adopted for determination of xylanase activity. Procedure. Xylan solution (0.25 mL, 0.4%) is mixed
Nevertheless, the method should be listed here because with 0.25 mL enzyme solution, or 0.5 mL of 0.2%
of its potential for miniaturization to the scale of a xylan solution is mixed with 1050 L enzyme solution
microplate reader (119). and incubated at 30 C for an appropriate time. The
reaction is terminated by addition of 0.5 mL of the
Somogyi reagent, mixed well, and heated for 10 min
a. DNS Xylanase Assay (115) on a boiling water bath. After cooling in cold water the
samples are vigorously mixed with 0.5 mL of the
Reagents Nelson reagent. After 15 min standing with occasional
1. Birchwood 4-O-methyl-D-glucuronoxylan (Roth stirring, cloudy samples are centrifuged to remove the
7500, Germany; or Sigma, USA), 1% solution in ne precipitate. Absorbance at 560 nm of the super-
0.05 M citrate buffer, pH 5.3 (in the case of fungal natants is measured. Blanks to correct the values for
enzymes or other buffers with bacterial enzymes). reducing power of the substrate and the enzyme are
The solution is prepared under heating up to boiling run in parallel. Calibration is done in the range 0.05
point with stirring until the polysaccharide dissolves. 0.5 mol of xylose. One unit of xylanase is dened
Clarication by centrifugation is not necessary but similarly as above. (Note: Exact duration of heating
may be useful. Cooled solution can be stored in the at 100 C with the Somogyi reagent is critical for repro-
presence of 0.010.02% sodium azide. ducibility of results.)

2. Other Xylanase Assays and Specic EX Assays respond to 5 mg/mL of undyed xylan) or solution in
0.1 M acetate buffer (1011 mg/mL).
It should be noted that reductometric assays are not
2. Ethanol, 96% or absolute.
specic for EXs and, when used with crude xylanolytic
systems or EX preparations containing other xylano- Procedure. The enzyme solution or suspension (10
lytic enzymes, they cover the overall saccharication 50 L) is mixed in 1.5 or 2.0 mL plastic microtest tubes
ability of the tested enzyme preparations. Frequently, with 0.5 mL of preheated (30 C) solution of RBB-
reductometric xylanase assays may not be sufciently xylan solution (56 mg/mL), or 0.25 mL of enzyme
sensitive or may be hampered by a high background of solution or suspension is mixed with 0.25 mL RBB-
reducing sugars in the enzyme preparations. In such xylan solution in 0.1 M acetate buffer and incubated
cases, a viscosimetric method of xylanase assay can at 30 C for an appropriate time. The reaction is termi-
be recommended. This method is based on measure- nated by the addition of 1 mL ethanol to precipitate
ment of the decrease of viscosity of a soluble xylan, unhydrolyzed RBB-xylan and its high-molecular-
e.g., arabinoxylan (121) or soluble xylan derivative, weight fragments. After standing at room temperature
like carboxymethylxylan (122, 123). Viscosity can be for 2030 min in closed test tubes, the mixtures are
measured using various viscosimeters or by modern mixed again and centrifuged at 2500g for 1.53 min.
electronic devices employing vibrating metal rods. The absorbance of supernatants is then measured at
Changes in the resistance of the medium to the vibrat- 595 nm against the respective substrate blank. (Note:
ing or rotating rods are recorded and transformed into The assay does not give direct data suitable for calcu-
activity units. Viscosimetric measurements are impor- lation of the enzyme activity. However, the assay can
tant to establish the endocharacter of glycanases. Plots be calibrated by a reductometric assay carried out
of specic uidity (1=) versus the amount of liberated under the same experimental conditions with undyed
reducing sugars give linear relationships, the slopes of xylan.
which correspond to the randomness of the xylan
attack by the enzymes (89).
Another specic EX assay employs a soluble cova- 3. Synthetic Substrates
lently dyed xylan, Remazol Brilliant Bluexylan (RBB-
Synthetic substrates of glycosyl hydrolases usually
xylan), a substrate that is precipitable from aqueous
include chromogenic and uorogenic glycosides
solution by ethanol (124). The assay is based on the
which are widely used to quantify particularly glycosi-
photometric determination of enzyme-released dyed
dases. The most common substrates are 4-nitrophenyl
fragments that remain soluble in the presence of etha-
and 4-methylumbelliferyl glycosides. Liberation of 4-
nol. This method allows the determination of xylanase
nitrophenol is followed photometrically at 405410 nm
activity in the presence of larger amounts of reducing
after termination of the enzymic reaction with alkaline
sugars, in membrane fractions of cells, and even in the
reagents. Fluorimetry is used to follow the liberation of
presence of viable cells utilizing liberated xylan frag-
4-methylumbelliferone (excitation at 365 nm and emis-
ments. Limitations of the method are its sensitivity to
sion at 448 nm at pH 1011). To achieve maximal
temperature and ionic strength. Testing in 15 labora-
sensitivity, permitting work in the nM concentration
tories showed the method reproducible to 30% rela-
range of substrates and products, basidication of the
tive standard deviation, suggesting that RBB-xylan
reaction mixtures is required (126, 127).
assay could be a useful alternative assay for xylanases
Similar aryl glycosides of oligosaccharides derived
(115). A number of covalently dyed soluble and inso-
from polysaccharides can be used as chromogenic and
luble xylans are available from Megazyme (Wicklow,
uorogenic substrates of endoacting glycosyl hydro-
Ireland). The company offers also a specic tablet test
lases. Signicant progress in differentiation of cellulo-
for EX activity based on wheat arabinoxylan. The
lytic glycanases was achieved by the introduction of 4-
nephelometric assay of xylanase (125) could be suitable
nitrophenyl- and 4-methylumbelliferyl--glycosides or
for differentiation of xylanases with respect to their
cellobiose, cellotriose, and lactose (93, 128). Of these
performance on insoluble xylan.
substrates, only 4-nitrophenyl--cellobioside has been
occasionally used for determination of activity of cel-
a. RBB-Xylan Assay (115, 124)
lobiohydrolase. This assay is not specic, because aryl
Reagents -cellobiosides are attacked at the agluconic linkage
1. RBB-xylan, solution in 0.05 M acetate buffer, also by endo--1,4-glucanases (128) and EXs of family
pH 5.4, 56 mg/mL (the concentration should cor- 10 (88). Liberation of the aryl aglycone from cellobio-

sides occurs also after a two-step hydrolysis of the A soluble undyed glucuronoxylan (0.10.2%) can
substrate by -glucosidase. be used instead of RBB-xylan. The production of EX
Aryl -glycosides of xylo-oligosaccharides have also is revealed after an appropriate time by ooding the
been described as potential substrates of EXs plate rst with 1 M NaCl and then with 0.1% aqueous
(129135). However, because they have not been com- solution of Congo Red (C.I. 22120; Sigma, Aldrich,
mercialized, their use remains very limited in spite of Calbiochem). After 15 min the dye solution is poured
their great analytical potential and unusually high off and areas with hydrolyzed polysaccharides are
sensitivity. 4-Nitrophenyl -xylobioside was used for destained for 10 min with two changes of 1 M NaCl
characterization of substrate requirements of a non- with a 20-min treatment with 5 M NaCl.
specic -xylanase from Cellulomonas mi (129). Umb-Xyl2 or Umb-Xyl3 gives the best results when
4-Methylumbelliferyl --xylobioside and -xylotrioside used in the form of solutions (0.10.5 mM) applied to
were successfully used for detection of EXs in electro- the surface of solid growth medium after colonies are
phoretic gels (130) and for differentiation of EXs (133). clearly visible. Incorporation of the uorogenic sub-
strates into the solid growth media may not give
straightforward results due to the rapid diffusion of
4. Assays for Detection and Screening Purposes
the uorogenic aglycone in comparison with the rate
A number of different procedures have been developed of colony growth.
over the years for detection of EXs in gels. The same
principles can be used both for the detection of the Semiquantitative gel diffusion assay (141, 142). A
enzyme in electrophoretic gels and for screening of solution of 0.150.2% RBB-xylan in an appropriate
microorganisms and genetically transformed cells on buffer is solidied with agar or agarose in petri dishes.
solid agar media. The detection and screening techni- Wells 4 mm in diameter are cut using a cork borer or
ques employ soluble xylan, insoluble xylan, soluble suitably shortened disposable plastic micropipette tip
covalently dyed xylan, and uorogenic -glycosides and the gel is removed by suction or by a needle.
of xylo-oligosaccharides. Enzyme solutions (10 L) are pipetted into the wells,
In agar gels incorporating insoluble xylan, EX activ- and closed dishes are left to incubate at appropriate
ity is visualized as the appearance of clearing zones temperature for several hours. Diffusion-zone dia-
(136). On solid growth media containing a soluble meters are proportional to the logarithm of enzyme
xylan-dye conjugate, such as RRB-xylan (124), xyla- concentrations.
nase-positive colonies are identied by pale-blue or A soluble glucuronoxylan in combination with the
colorless zones resulting from diffusion of dyed poly- above described Congo Red complexing can be used
saccharide fragments generated by EXs (137, 138). The for the same purpose. More sensitive and faster are
advantage of these two screening substrates is that the similar plate assays with gels containing Umb-Xyl2
degradation of the substrate can be followed progres- or Umb-Xyl3 (0.10.5 mM). Liberation of the uoro-
sively without any further treatment which might pre- genic 4-methylumbelliferone around the wells is
vent the isolation of the positive colonies directly from observed under UV light (365 nm). Since the liberation
the screening plates. A replica plating is required in the of the aglycone can be a result of -xylosidase action,
case of screens employing the soluble xylan complexed enzyme samples should be tested in a parallel plate
with Congo Red (139, 138) or precipitation of soluble containing a specic -xylosidase substrate, 4-methy-
xylan with ethanol (140). Replica plating is avoided in lumbelliferyl -xyloside.
plate assays with the uorogenic glycosides 4-methy-
lumbelliferyl -xylobioside (Umb-Xyl2 ) or -xylotrio- Detection of EX in electrophoretic gels (142). A
side (Umb-Xyl3 ) (130). warm 1.5% aqueous solution (10 mL) of RBB-xylan
is mixed well with 3% molten agar in appropriate buf-
a. Procedures
fer (20 mL) and poured between two glass plates sepa-
Detection of microbial producers of xylanase (137, rated by 0.75 to 1.0-mm spacers and left to solidify.
138). Solid media containing 0.150.2% RBB-xylan The glass plates can be covered by transparent plastic
(can be autoclaved) are suitable for the detection of sheets, so the gel can be easily stored after glass
microbial producers of extracellular EX. Surrounding removal. If not used immediately, the agar gel should
colonies of EX producers, pale-blue zones are formed be supplied with 0.010.02% sodium azide. Native
as a result of diffusion of secreted enzymes and PAGE or isoelectrofocusing gels (agarose or polyacry-
enzyme-released dyed fragments of RBB-xylan. lamide) are washed twice for 510 min (depending on

their thickness) with a buffer corresponding to pH longer oligosaccharides are referred to as exo--xyla-
optimum of the tested EXs. They are then brought nases. A careful literature search suggests, however,
into contact with the RBB-xylan containing detection that in spite of the existence of several glycosyl hydro-
gel. The presence of EX leads to a change in the shade lase families into which -xylosidases have been
of the blue background, due to a diffusion of enzyme- assigned, and in spite of the fact that the enzymes
released dyed fragments. The agar detection gel is then behave as both retaining and inverting hydrolases,
separated from the electrophoretic gel and, if needed, there is no sharp boundary between -xylosidase and
further incubated in a wet chamber. It is then dipped exo--xylanase with respect to their substrate prefer-
into the solvent ethanol0.05 M acetate buffer (pH 5.4) ence. Therefore, this section considers all enzymes cat-
2:1 (v/v). Zones with enzyme-degraded substrate are alyzing successive removal of -xylosyl residues from
destained as a result of the solubilization of dyed frag- non-reducing termini.
ments. The background with unhydrolyzed substrate
remains unchanged. The dyed fragments liberated B. Classication
from the blue detection gel diffuse into the separation
gel, where they mark the position of the enzyme. -1,4-Xylan xylohydrolase EC 3.2.1.37.
Individual xylanase forms can be subsequently isolated
from the separation gel, e.g., by electroelution. C. Properties as Proteins
The use of RBB-xylan enables visual monitoring of
substrate hydrolysis, which is important for appropri- -Xylosidases are, with few exceptions, larger enzymes
ate timing of contact between the substrate and than EXs. Their molecular mass ranges between 26
enzymes. The detection of EXs by means of Congo and 360 kDa (144), and they are monomeric or dimeric
Red (143) requires preliminary experimentation or proteins as shown by SDS-PAGE. Molecular masses
knowledge of enzyme activity to terminate the reaction exceeding 120 kDa usually correspond to dimeric/tet-
at the appropriate time. rameric aggregates. All thus far isolated enzyme spe-
The fastest procedure for the detection of EXs in cies showed acidic pI values (3.256.1). The properties
electrophoretic gels can be achieved using Umb-Xyl2 of these enzymes vary considerably, which complicates
or Umb-Xyl3 (130). Either uorogenic substrate at a their classication. Moreover, -xylosidase belongs to
concentration of 0.10.5 mM can be incorporated into those xylanolytic enzymes which exhibit variable cellu-
a 0.75 to 1-mm thick agar or agarose detection gel lar localization. In several species of bacteria and
which is laid upon the rebuffered separation gel. A yeasts, the enzyme is localized strictly intracellularly
simpler approach is to bring the washed separation and is not glycosylated. In these organisms, xylo-oli-
gel into contact with a solution of substrates on a gosaccharides generated outside the cell by EXs must
at glass plate. Fluorescence appears rapidly under enter the cell by transport systems localized in plasma-
UV light ( 360 nm) in the regions of EXs. The uor- membrane (3). In other organisms, like Aureobasidium
escence intensity can be enhanced by ammonium pullulans, -xylosidase is found outside and inside the
hydroxide vapors. cells. It remains to be established whether some organ-
isms produce two different -xylosidases. In laboratory
cultures of mycelial fungi, -xylosidase remains asso-
III. b-XYLOSIDASE (EC 3.2.1.37) ciated with mycelia during early stages of growth and
is released into the media later either by true secretion
or as a result of cell lysis.
Only a small fraction of known -xylosidases have
Xyl1-4Xyl ! 2 Xyl Xylnn ! Xyln1 Xyl been classied on the basis of hydrophobic cluster
analysis and comparison of amino acid sequences.
NPh--Xyl ! Xyl NPh-OH
They have been assigned to four glycosyl hydrolase
-Xylosidase hydrolyzes xylobiose and higher linear - families: 3, 39, 43, and 52 (58, 59). Family 3, which
1,4-xylo-oligoaccharides to monomer and also releases contains mainly -glucosidases (EC 3.2.1.21), includes
xylose from branched or substituted xylo-oligosacchar- a nonspecic -glucosidase/-xylosidase from Erwinia
ides produced by the action of EXs. -Xylosidases vary chrysanthemi (145) and the best-known fungal -xylo-
in their afnity toward short and long oligosacchar- sidases from Aspergillus and Trichoderma species
ides. Those which prefer xylobiose as a substrate are (146, 147). A common feature of the members of
referred to as -xylosidase, and those which prefer this family is their retaining character. -Xylosidases

of thermophilic bacteria have been assigned to family information, it is not possible to correlate the catalytic
39 (148150). All -xylosidases of this family are properties of enzymes with their glycosyl hydrolase
localized intracellularly and belong to retaining glyco- families. Therefore, only the properties of best
syl hydrolases. Their molecular mass is between 55 known -xylosidases will be described.
and 60 kDa. One of the best-characterized -xylosidases is an
Family 43 contains 50 to 60-kDa -xylosidases of intracellular enzyme from Bacillus pumilus (154, 155).
several Bacillus spp. and from Butyrivibrio brosolvens. It shows absolute specicity for -xylopyranosyl resi-
The latter enzyme has been shown to operate by a dues but does not catalyze glycosyl transfer reactions.
mechanism which results in inversion of the anomeric This suggests that the enzyme belongs to family 43 of
conguration (151). If the unequivocal relationship glycosyl hydrolases (Table 3). The best-studied fungal
between family classication and molecular mechan- -xylosidases are extracellular enzymes produced by
ism is valid, other members of family 43 should also Aspergillus and Trichoderma spp., i.e., the enzymes
operate with retention of the anomeric conguration. from family 3. Their retaining character is revealed in
The amino acid sequences of two Bacillus stear- glycosyl transfer reactions in high concentrations of
othermophilus (7080 kDa of unprocessed precursor) xylo-oligosaccharides and 4NPh--Xyl (156159).
-xylosidases (152, 153) are so unique that they were These enzymes do not show high specicity for the
grouped into a special family, assigned as family 52. glycan moiety. -Xylosidases from several Tricho-
The stereochemistry of hydrolysis by these enzymes derma strains exhibit -L-arabinofuranosidase activity
has not been established yet. Three-dimensional struc- (160162). This has been conrmed by cloning the T.
tures are not available for any members of these four reesei -xylosidase gene in yeast (147). The -L-
families. Some characteristic properties of individual arabinofuranosidase from T. reesei hydrolyzes only
-xylosidase families are listed in Table 3. -L-arabinofuranosides and is not able to liberate
L-arabinose from arabinoxylan or arabinose-
D. Properties as Enzymes substituted xylo-oligosaccharides. Aryl -xylosidase
activity of Trichoderma strains is inhibited by
There is limited knowledge about structure-function D-xylose. Fungal -xylosidases were shown to have a
relationships in the glycosyl hydrolase families con- specic requirement for structural arrangement of
taining -xylosidases. All -xylosidases thus far branched oligosaccharides. The enzyme from A. awa-
described hydrolyze not only xylobiose and xylooligo- mori was tested on different arabinose-substituted
saccharides but also aryl -D-xylopyranosides. xylooligosaccharides. The enzyme was able to remove
Consequently, 4-nitrophenyl -D-xylopyranoside has terminal xylopyranosyl residues from the nonreducing
become the most common substrate for -xylosidase end of branched oligosaccharides only when two con-
assay. There are, however, examples of aryl -xylosi- tiguous unsubstituted xylopyranosyl residues were
dases which do not attack xylo-oligosaccharides. linked to single- or double-substituted xylopyranosyl
Therefore, it is always important to test the enzymes residues (163). This is similar to the behavior of -
directly on xylobiose and xylo-oligosaccharides. xylosidase from T. reesei (159, 164). The enzyme
Several -xylosidases are reported also to attack poly- does not liberate -1,4-xylopyranosyl residues linked
meric xylan in an exofashion. Because of the limited to an -1,3- or -1,3-substituted xylopyranosyl residue
Table 3 Families of Glycosyl Hydrolases Containing -Xylosidases
Common microbial Molecular mass range Stereochemistry Other enzymes

Enzyme family producers of listed enzymes of hydrolysis in the family
3 Erwinia, Aspergillus, 60122 kDa retaining -glucosidase

Trichoderma
39 Thermophilic bacteria 5560 retaining -L-iduronidase
43 Butylvibrio, Baccillus 5060 inverting -L-arabinanase
EX
52 Bacillus stearothermophilus 7080 (precursor) unknown none
Source: http://expasy.hcuge.ch/cgi-bin/list/glycosid.txt.

(compounds of the type Xyl-4[Ara-3]Xyl- or Xyl- region of the spectrum. In a similar assay, phe-
4[Xyl-3]Xyl-). In such compounds the substituent is nyl--D-xylopyranoside is used as a substrate
apparently so close to the site of the enzymic attack (167). Liberated phenol is determined using the
(i.e., glycosidic oxygen) that the formation of the phenol reagent from the well-known Lowry proce-
enzyme-substrate complex is sterically impaired. The dure for protein determination.
-1,4-glycosidic linkage becomes susceptible to the
enzyme attack only if the substituent is linked to a 1. -Xylosidase Assay Using 4-Nitrophenyl--
more distant C-2 position (Scheme 3). D-Xylopyranoside/Na2 CO3
Araf Xyl
a. Reagents
1 non-hydrolyzed 1
j substrates j 1. 4-Nitrophenyl--D-xylopyranoside, 4 mM solu-
3 3 tion in 0.05 M acetate buffer, pH 4.5 (or other buffer).
Xyl1-4Xyl1-4Xyl- Xyl1-4Xyl1-4Xyl- 2. 2 M solution of Na2 CO3 .
3. 4-Nitrophenol, 2 mM stock solution in the acet-
Xyl1-4Xyl1-4Xyl- Xyl1-4Xyl1-4Xyl-
ate buffer; 0.2 mM calibration solution in the acetate
" 2 " 2 buffer.
j hydrolyzed j
b. Procedure. Preheated substrate solution (0.9
1 substrates 1
mL, e.g., at 50 C) is mixed with 0.1 mL appro-
MeGlcA Xyl
priately diluted enzyme solution and incubated for
Scheme 3 suitable time at 50 C. The reaction is terminated by
addition of 1 mL of 2 M Na2 CO3 . The absorbance
E. Determination of b-Xylosidase Activity is measured at 410 nm against the substrate blank.
The values have to be corrected for enzyme blanks
The activity of -xylosidases, which hydrolyze xylo- if they show absorbance at 410 nm. Calibration
biose, xylo-oligosaccharides, eventually linear portions with 4-nitrophenol is done in the range of 0.02
of xylan, can be determined reductometrically using 0:2 mol per assay mixture. One unit of -xylosi-
xylobiose and xylotriose, which are poor substrates dase activity is dened as the amount of enzyme
for EXs. The sensitivity of assays can be increased by liberating 1 mol of 4-nitrophenol in 1 min. (Note:
eliminating the background of oligosaccharides by The assay can be miniaturized to the scale of a
their reduction to sugar alcohols with NaBH4 . The microplate reader.)
reductometric procedures described in the section
devoted to EXs can be adapted for the use of xylobiitol 2. -Xylosidase Assay Using 4-Nitrophenyl--
or xylotriitol as substrates. Xylobiase activity can also D-Xylopyranoside/Na2 B4 O7
be determined using xylobiose as a substrate in an
a. Reagents
enzyme-coupled assay which employs NAD -depen-
1. 4-Nitrophenyl--D-xylopyranoside, 10 mM
dent glucose dehydrogenase. The dehydrogenase is
solution in 0.05 M acetate buffer, pH 5.0 (or other
not specic for glucose and catalyzes conversion of
buffer).
generated xylose to xylonolactone (165, 166). The for-
2. Saturated solution of Na2 B4 O7 .
mation of NADH is followed at 340 nm to estimate -
3. 4-Nitrophenol, 1 mM stock solution in the acet-
xylosidase activity. Two side reactions, the oxidation
ate buffer; 0.1 mM calibration solution in the acetate
of xylobiose to lactone by the dehydrogenase and
buffer.
hydrolysis of the lactone by -xylosidase, have to be
taken into consideration. b. Procedure. Preincubated substrate solution
However, the most common assay of -xylosi- (0.1 mL 50 C) is mixed with 0.1 mL tested enzyme
dase employs 4-nitrophenyl--D-xylopyranoside as sample (50 C) in an Eppendorf test tube and incubated
a substrate. There are a great variety of condi- for appropriate time at 50 C. After addition of 1.0 mL
tions under which the assay can be done involving saturated solution of Na2 B4 O7 , the absorbance is mea-
the substrate concentration, pH and type of the sured at 410 nm against a substrate blank. Calibration
assay buffer, and various alkaline reagents to is done with 4-nitrophenol in the range of 0.01
terminate the reaction and to shift the absorption 0:1 mol. One unit of -xylosidase activity is dened
maximum of liberated 4-nitrophenol to a visible as the amount of enzyme liberating 1 mol of 4-nitro-

phenol in 1 min. (Note: This assay is carried out in B. Classication
microcentrifuge test tubes and is suitable for determi-
nation of cell-bound -xylosidase activity. The cells -D-Glucuronidase (EC 3.2.1.139).
used as the enzyme source must be separated by cen-
trifugation before measurement.) C. Occurrence of a-Glucuronidase
-Glucuronidase activity has been detected in cellulo-

F. Assays for Screening Purposes lytic and xylanolytic systems of a variety of fungi (168
176), actinomyces, and bacteria (177184). Two
Detection of -xylosidase activity in solid growth reports mention -glucuronidase in snail digestive
media or in electrophoretic gels usually involves juice (184, 185). There is no report on the occurrence
incorporation of chromogenic and uorogenic sub- of the enzyme in plants. In most microorganisms -
strates, 4-nitrophenyl--D-xylopyranoside (15 mM) glucuronidase was found to be secreted into culture
or 4-methylumbelliferyl--D-xylopyranoside (0.10.5 medium. However, there are examples in which the
mM). Activity is revealed by ooding the growth enzymes has been found conned to the cells (172,
media or gels with alkaline solutions (1 M Na2 CO3 , 180, 181).
alkaline buffers) or exposing them to ammonium
hydroxide vapors. Replica plating is required before D. Properties as Proteins
alkalication when working in acidic pH regions.
The liberation of 4-methylumbelliferone is revealed -Glucuronidase has been puried from a relatively
under UV light (360 nm). Alkalication of the solid small number of microorganisms. All eukaryotic -glu-
medium or gel, e.g., exposure to ammonium hydroxide curonidases, with the exception of the enzyme from
vapors, increases the uorescence. As mentioned with Agaricus bisporus (196), are monomeric enzymes with
uorogenic substrates of EXs, uorogenic glycosides molecular masses between 91 and 150 kDa. Their iso-
for screening purposes give better results when applied electric points are acidic, between values 4.6 and 5.3
as overlay at the later stages of colony development (146). The -glucuronidase puried from the snail
than when incorporated into growth media before Helix pomatia (185) also shares these properties.
inoculation. Bacterial -glucuronidases are usually found as dimers
with subunits of a molecular mass 70 kDa. pI values
of bacterial enzymes are similar to those of fungal
enzymes. -Glucuronidase from the thermophilic bac-
IV. a-GLUCURONIDASE, 4-O-METHYL-a-
terium Thermotoga maritima shows a variable oligo-
GLUCURONIDASE (EC 3.2.1.139)
meric structure depending on the salt concentration
A. Chemical Reactions Catalyzed (183).
So far only ve amino acid sequences of
Xyl-R Xyl-R -glucuronidases are known, all based on gene
2 sequencing (146, 176). The sequences show a high
j ! degree of similarity (4060% identity) and have been
1 assigned to a separate glycosyl hydrolase family 67,
MeGlcA MeGlcA which does not contain any other hydrolases. A
three-dimensional structure of -glucuronidase has
-R 1-4Xyl; 1-4Xyl1-4Xyl; 1-4Xyln not been reported; however, the enzyme from
Bacillus stearophilus has been successfully crystallized
One of the best substrates and subjected to x-ray analysis (186).
Xyl1-4Xyl1-4Xyl Xyl1-4Xyl1-4Xyl E. Properties as Enzymes

2
j ! -Glucuronidases seem to belong to those accessory
1 xylanolytic enzymes that do not operate on polymeric
MeGlcA MeGlcA substrates. With exception of -glucuronidase from

Reducing end Thermoascus aurantiacus (170), -glucuronidases failed
to liberate 4-O-methyl-D-glucuronic (MeGlcA) acid or

D-glucuronic acid at an appreciable rate from poly- 5.0 for fungal enzymes. Km values for aldouronic
meric substrates. In a few cases, an extremely low acids as substrates were reported in the range of
rate was found. However, it should be stressed that 0.140.95 mM.
even a slight contamination of -glucuronidase by The stereochemistry of hydrolysis of glycosidic link-
EX and -xylosidase would generate polysaccharide age by -glucuronidase was recently investigated with
fragments that would serve as -glucuronidase sub- puried Aspergillus tubingensis enzyme, which belongs
strates. -Glucuronidases, regardless of their eukaryo- to family 67 of glycosyl hydrolases. By NMR spectro-
tic or prokaryotic origin, are very selective toward the scopy it has been established that the enzyme uses a
structure of aldouronic acids. They are capable of single displacement reaction mechanism, leading to
removing MeGlcA or GlcA from aldouronic acids in inversion of the anomeric conguration (189). Based
which MeGlcA or GlcA residues are linked to a single on this nding, inverting character can be expected
xylopyranosyl residue or to a nonreducing terminal with other -glucuronidases.
xylopyranosyl residue of xylo-oligosaccharides. Such
fragments can be generated from glucuronoxylan by F. Determination of a-Glucuronidase Activity
the action of EXs of family 10 or by the action of a
combination of EXs of family 11 and -xylosidase As mentioned above, the activity of -glucuronidase
(Scheme 4) (88, 187). cannot be determined directly on glucuronoxylan or
Xyl1-4Xyl1-4Xyl Xyl Xyl1-4Xyl1-4Xyl1-4Xyl Xyl1-4Xyl1-4Xyl1-4Xyl

2 2 2 2
j j j j
1 1 1 1
MeGlcA MeGlcA MeGlcA MeGlcA
Hydrolyzed compound Nonhydrolyzed compounds which become substrates after removal

of xylopyranosyl residues left from the branch by -xylosidase
Scheme 4
An interesting question related to the substrate spe- a related polymeric substrate. Glucuronoxylan can
cicity of -glucuronidase is whether the enzyme is be used for the -glucuronidase assay only in the
specic for GlcA or MeGlcA. The enzyme from presence of xylan-depolymerizing enzymes which
Aspergillus niger hydrolyzed GlcA-Xyl3 about two generate aldouronic acids of the required structures
times faster than MeGlcXyl3 (172). This example sug- (190). The most common substrate for -glucuroni-
gests that the 4-O-methyl group in the acid is not deci- dase assays are linear aldouronic acids obtained by
sive for the action of the enzymes. enzymic or acid hydrolysis of glucuronoxylan. From
A surprising property of microbial -glucuronidases such aldouronic acids, liberation of MeGlcA is
is their inability to hydrolyze 4-nitrophenyl--D-glu- usually followed by determination of the reducing
curonide, a compound that could otherwise serve as power of the free uronic acid by a modication of
a convenient chromogenic substrate. The only -glu- the Somogyi copper reagent and the Nelson phos-
curonidase capable of hydrolyzing this compound was phomolybdenic acid reagent according to Milner
the snail enzyme (185). Microbial -glucuronidases and Avigad (191). The method is quite specic for
require uronic acid to be linked to at least one xylo- uronic acids and suffers only slightly from the pre-
pyranosyl residue, which can be in the form of an aryl sence of neutral reducing sugars. The assay could be
glycoside as in 4-nitrophenyl-2-O-(methyl-4-O-methyl- affected by some salts (citrate, phosphate), however.
-D-glucuronosyl)--D-xylopyranoside (188). Alternative methods include HPLC determination of
Optimal pH values for bacterial -glucuronidases free MeGlcA (173, 174) or the equivalent amount of
are in the range of 5.46.5 and in the range of 3.5 xylo-oligosaccharides (169), or GLC of trimethylsilyl

oxim derivatives of uronic acids (178, 179, 182, 184). of the developed reaction mixture with water. Some
Unfortunately, there is no direct -glucuronidase authors stop the reaction by boiling and clarify the
assay available that would utilize a chromogenic sub- mixture by centrifugation before addition of the cop-
strate. As we have already mentioned, 4-nitrophenyl- per reagent (146).)
-glucuronide does not serve as substrate of micro-
bial -glucuronidases. Recently, a new -xylosidase-
coupled -glucuronidase assay has been introduced
that uses 4-nitrophenyl-2-O-(4-O-methyl--D-glu- 2. -Xylosidase-Coupled Assay of -
curonosyl)--D-xylopyranoside (NPh-Xyl-MeGlcA) Glucuronidase (188)
(188). Liberation of MeGlcA from the compound a. Reagents
yields an equimolar amount of 4-nitrophenyl--xylo- 1. 4-Nitrophenyl-2-O-(4-O-methyl--D-glucuro-
pyranoside. In the presence of excess of -xylosidase nosyl)--D-xylopyranoside (NPh-Xyl-MeGlcA), pre-
to hydrolyze the xyloside, the amount of liberated pared by alkaline deesterication of its methyl ester
4-nitrophenol becomes a measure of -glucuronidase (191, 192), 4 mM solution in 0.05 M acetate buffer,
activity (Scheme 5). pH 5.0.
NPh-Xyl-MeGlcA ! MeGlcA NPh-Xyl ! NPh-OH Xyl

-glucuronidase -xylosidase
Scheme 5
1. Determination of -Glucuronidase Activity 2. Recombinant -xylosidase of Aspergillus niger

Using the Milner-Avigad Copper Reagent produced in Saccharomyces cerevisiae (191), a solution
2 U/mL of 0.05 M acetate buffer, pH 5.0 (one unit of
a. Reagents -xylosidase is dened as the amount of enzyme liber-
1. A mixture of aldotriuronic and aldotetrauronic ating 1 mol of 4-nitrophenol from 4-nitrophenyl--D-
acid (ratio 8 : 2) (Megazyme, Ireland), a solution 2 xylopyranoside).
mg/mL in 0.05 M acetate buffer pH 5.0. 3. Saturated solution of Na2 B4 O7 .
2. Copper reagent prepared according to Milner 4. 4-Nitrophenol, 1 mM solution in the acetate
and Avigad (191). buffer, calibration in the range 0.010:1 mol.
3. Arsenomolybdate reagent of Nelson reagent (110).
b. Procedure. 0.1 mL of 4 mM solution of NPh-
4. D-Glucuronic acid, 1 mM calibration solution
Xyl-MeGlcA is mixed with 0.1 mL of the -xylosidase
in 0.05 M acetate buffer (calibration in the range 0.02
solution (200 mU per reaction mixture), preheated to
0:2 mol).
37 C, and the reaction is started with addition of 52
0 L of the tested enzyme solution. After 10 min or
b. Procedure. Enzyme (0.04 mL) is added to a longer incubation at 37 C 1 mL saturated solution of
preheated solution of aldouronic acids (0.16 mL, Na2 B4 O7 is added and the absorbance is measured at
40 C) and incubated at 40 C for an appropriate time. 410 nm. An alternative procedure is to add 0.1 mL
The reaction is stopped by adding 0.6 mL of the copper enzyme solution to 0.05 mL double-concentrated sub-
reagent. The mixture is heated in a boiling water bath strate and -xylosidase solution in 0.1 M acetate buf-
for 10 min and then cooled in an ice-water bath. fer. One unit of -glucuronidase is the amount of
Subsequently 0.4 mL of the Nelson reagent is then enzyme required to release 1 mol of 4-nitrophenol.
added and after mixing the absorbance at 600 nm is (Note: The assay was applied to three different -glu-
measured against the substrate blank. One unit of - curonidases. Their Km values for NPh-Xyl-MeGlcA
glucuronidase is dened as the amount of enzyme lib- were in the range 0.210.58 mM, which is in the
erating 1 mol of glucuronic or 4-O-methylglucuronic range of Km values of -glucuronidases for aldouronic
acid per 1 min under the selected assay conditions. acids. The new substrate is still not commercially avail-
(Note: The original procedure (191) involves dilution able.)

G. Assays for Screening Purposes of hardwood holocellulose, e.g., birchwood or beech-
wood holocellulose (198, 199). An analogous polysac-
No direct in situ detection of -glucuronidases has charide of a lower polymerization degree (average DP
been reported. 25) and acetyl content of 13% can be obtained as a
nondialyzable fraction of a water-soluble, noncellulo-
sic polymer by steaming birch at 200 C for 10 min
(200). An aqueous thermochemical treatment of hard-
V. ACETYLXYLAN ESTERASE (3.1.1.72)
wood leads to a similar material with a higher acetyl
A. Chemical Reactions Catalyzed content (201). Since none of the above-mentioned
AcXE substrates are commercially available and their
isolation is difcult or dependent on availability of
H2 O
special equipments, some authors have replaced nat-
R1 -Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl-R2 !
ural acetylxylan by a chemically acetylated polysac-
2 3
charide. A widely used method of chemical
j j
acetylation of xylan was published by Johnson et al.
Ac Ac
(202). There are also examples of the use of fully che-
mically acetylated hardwood xylan as a substrate for
R1 -Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl-R2 2AcH
AcXE (203, 204). As will be shown below, a majority
of AcXEs can be assayed on a variety of aryl acetates
H2 O
which serve as substrates of nonhemicellulolytic acet-
Xyl1-4Xyl ! Xyl1-4Xyl AcH
ylesterases and lipases.
2
j
C. Classication
Ac
H2 O
Acetylxylan esterase (EC 3.1.1.72).
Xyl1-4Xyl ! Xyl1-4Xyl AcH
3
D. Properties as Proteins
j
Ac Reducing end
AcXEs show the greatest diversity in molecular orga-
nization among all components of microbial xylanoly-
Acetylxylan esterases (AcXEs) are components of tic systems. On the basis of amino acid sequence
microbial xylanolytic and cellulolytic systems which similarity AcXEs have been assigned to seven families
are able to remove acetyl groups esterifying D-xylopyr- of 13 so far recognized families of carbohydrate
anosyl residues of xylan main chain at positions 2 or 3 esterases (73, 205) (Table 4).
(193195). AcXEs also deacetylate partially acetylated The best-known fungal AcXEs from Aspergillus,
xylo-oligosaccharides. The enzyme action on polysac- Penicillium, Schizophyllum and Trichoderma spp. can
charide substrates creates new sites on the xylan main be found in carbohydrate esterase families 1 and 5
chain, suitable for productive binding with depolymer- together with bacterial acetylxylan esterases. The
izing EXs. This effect is reected in a clear coopera- majority of these represents one catalytic domain of
tivity of the two enzymes during acetylxylan bifunctional acetylxylan-degrading enzymes. Diverse
degradation, a typical example of enzyme synergy AcXEs of the anaerobic fungus Neocallimastix patri-
(54, 196, 197). Acetylxylan degradation with EXs pro- ciarum (214) can be found in families 2, 3, and 6.
ceeds faster and to a higher degree in the presence of AcXEs produced by Streptomyces are grouped in
AcXEs (54). Deacetylation of xylo-oligosaccharides family 4 together with similar AcXE modules of bacter-
makes the oligosaccharides fully susceptible to the ial bifunctional enzymes. Their sequences are similar to
action of -xylosidase. those of nodulating proteins (NodB) from Rhizobium
spp. and with some yeast enzymes, identied as chito-
B. AcXE Substrates oligosaccharide and chitin deacetylases (205).
Fungal AcXEs, and some bacterial enzymes, are
The substrate similar to the native O-acetyl-4-O- usually monomeric enzymes, while some AcXEs from
methyl-D-glucurono-D-xylan present in plant cell thermophilic anaerobic bacteria occur as oligomers of
walls can be prepared by dimethylsulfoxide extraction smaller subunits (224). As already mentioned, numer-

Table 4 Families of Carbohydrate Esterases Containing Acetylxylan Esterases
Microbial producer of Mw Other enzymes

Family acetylxylan esterase (kDa) pI in the family References
1 Aspergillus niger 30.5 3.1 None 206

Schizophyllum communea 30 3.4 203, 207
Penicillium purpurogenum AcXE I 48 7.8 208
Pseudomonas uorescens 209
Catalytic domains of bifunctional 210212
enzymes of anaerobic bacteria
2 Ruminal anaerobic bacteria and None 213, 214
fungi
3 Ruminal anaerobic bacteria and None 213215
fungi
4b Streptomyces lividans 34 9.0 Chitin and chito- 216, 217
Streptomyces thermoviolaceus 34.3 oligosaccharide 218
deacetylase
Catalytic domains of bifunctional 82, 212, 219
enzymes of anaerobic bacteria
5 Trichoderma reesei 34 6, 8, 7.0 Cutinase 220, 221
Penicillium purpurogenum AcXE II 23 7.8 208, 222
6 Anaerobic ruminal fungi 214, 223
7 Thermoanaerobacterium 32, 26 4.2, 4.3 224, 225
Thermotoga neapolitana
a
Assignment based on a sequence of fty NH2 -terminal amino acids (226).
b
This may be the only family, members of which do not attack synthetic low-molecular-mass substrates, such as aryl acetates (226).
Source: http://afmb.cnrs-mrs.fr/pedro/CAZY/db.html.
ous enzymes have modular architecture. Some of AcXEs existing as single-domain enzymes have a
AcXEs, such as those from Pseudomonas uorescens molecular mass in the range of 2348 kDa and acidic
(209) or Trichoderma reesei (221), contain cellulose- or neutral pI values. There does not seem to be a spe-
binding domains (CBDs) separated from the catalytic cial relationship between these two parameters and
domain by linker regions. Others, like AcXE from their assignment into carbohydrate esterase families
Streptomyces lividans (217), contain a xylan-binding (Table 4).
domain (77). AcXE of Cellulomonas mi is a part of Three-dimensional structures are known only for
a bifunctional enzyme which contains both CBD and AcXEs belonging to family 5. Crystallization has
XBD (82). The two-catalytic module architecture, been reported for AcXEII from P. purpurogenum
represented by various combinations of EXs linked (227) and AcXE from T. reesei (228). The protein
to AcXEs, is very common particularly among acetyl- from P. purpurogenum shows an = hydrolase fold,
xylan-degrading bifunctional enzymes of ruminal similar to that of cutinase (227). Cysteine residues form
anaerobic bacteria (212). AcXE domains of such ve S-S bridges which makes the structure very rigid
bifunctional enzymes can be found in carbohydrate and tightly packed. The substrate binding site appears
esterase families 1, 2, 3, and 4 (Table 4). With the to be small and able to accommodate only an acetyl
bifunctional EX-AcXE enzyme from Clostridium group, thus giving a relatively high substrate specicity
thermocellum (83) it has been demonstrated that the to the enzyme.
two catalytic domains act synergistically in the degra-
dation of acetylxylan. This observation again suggests E. Properties as Enzymes
that the production of an enzyme having both deb-
1. Substrate Specicity
ranching and depolymerizing activity can be a great
advantage for a microorganism competing for a Substrate specicity of AcXEs is one of the least-clar-
carbon source with microorganisms using separate ied aspects of this xylanolytic component. There is
enzyme components. very limited knowledge on the structure-function rela-

tionship of these enzymes, particularly considering that that at least some AcXEs are quite specic for acetyl-
they have been assigned to seven different families xylan (197, 230).
(Table 4). The situation is complicated because there
are esterases and lipases which cannot be strictly con-
2. Specicity for the Position of the Acetyl
sidered as components of xylanolytic systems, but
Group in the Main Chain
which could participate in the later stages of acetylxy-
lan degradation, by deacetylation of xylooligosacchar- Xylose residues in xylan are acetylated at positions 2
ides. It is known that nonhemicellulolytic esterases and and/or 3, and the acetyl groups are more or less evenly
lipases operate quite efciently on low-molecular-mass distributed between these positions. This raises ques-
acetylated carbohydrases (226, 229). As a consequence, tions of whether the AcXEs are capable of releasing
we shall limit the discussion here to the catalytic prop- acetyl groups bound only at one position or at both
erties of enzymes that deacetylate polymeric substrates positions, and to what extent is deacetylation inu-
as such or coinduced with other plant cell walls hydro- enced by the migration of acetyl groups between posi-
lyzing enzymes. tions 2 and 3. Studies of three AcXEs on acetylated
Several lines of evidence suggest that there are two methyl glycosides showed the regioselective removal of
major types of acetylesterases that participate in acet- acetyl group from positions 2 and 3 (229, 231, 232).
ylxylan degradation (197). They differ in their afnity AcXE from S. commune AcXE exhibited a preference
toward polymeric and oligomeric substrates. The for position 3 (229), while the T. reesei AcXE deacety-
enzymes that perform well on polymeric substrates lated the model compounds sequentially, rst at posi-
are capable of causing precipitation of xylan from tion 2 and then rapidly at position 3 (231). S. lividans
solution due to deacetylation (207, 226). The removal AcXE worked almost simultaneously on positions 2
of acetyl groups results in the association of xylan and 3, essentially catalyzing a double deacetylation of
molecules into insoluble aggregates. This property is compounds at both positions (232). Two methyl xylo-
very typical of AcXEs of family 4 (Table 4), which pyranoside diacetates, which had a free hydroxyl
appear to be most specic for acetyl xylan and do group at position 2 or 3, i.e., the derivatives that
not hydrolyze low-molecular-mass substrates such as most closely mimicked monoacetylated xylopyranosyl
4-nitrophenyl- or 4-methylumbelliferyl acetate (226). residues in acetylxylan, were deacetylated by 1 or 2
The enzymes of other families share properties compa- orders of magnitude faster than 2,3,4-tri-O-acetyl- or
tible with general acetylesterases and acylesterases 2,3-di-O-acetyl--D-xylopyranoside. These observa-
(226). They are active on a variety of synthetic aryl tions explained the double deacetylation. The second
acetates and acylates (226). This second group of acetyl group was removed immediately after the rst
enzymes do not perform well on polymeric acetylxylan one. An implication of these results is that the deace-
but are more active on acetylated oligosaccharides gen- tylation mechanism of positions 2 and 3, when the
erated from acetylxylan by EXs (197, 226, 230). Thus neighboring positions 3 and 2 are nonacetylated,
EXs would synergistically facilitate the release of acetic might involve an enzyme-catalyzed formation of a
acid in concert with this type of acetylesterases. ve-member transition state intermediate (Scheme 6).
Additional studies are also required to dene the spe- Such intermediates are believed to be involved in the
cicity of AcXEs with respect to the type of polysac- spontaneous migration of acetyl group along the gly-
charide that they deacetylate. There are indications copyranoid ring in aqueous media. If this hypothetical
Scheme 6

mechanism is experimentally conrmed, the question 2. 1 N H2 SO4 .
of regiospecicity of AcXE at positions 2 and 3 will 3. Acetic acid, 0.1.0% (w/v).
become irrelevant. 4. Glycerol, 1% (w/v).
b. Procedure. One volume of acetylxylan solu-
3. Reaction Mechanism
tion or suspension is mixed with one volume of enzyme
The inactivation of AcXEs with phenyl methyl sulfonyl solution and incubated at 50 C. At time intervals, 0.2-
uoride (221, 227) and the occurrence of the serine mL aliquots are taken, mixed with 10 L of 1 N
sequence GXSXG in active site of AcXEs (195) suggest H2 SO4 to stop the reaction, claried by a short centri-
that AcXEs utilize a catalytic mechanism similar to fugation, and supernatants analyzed for acetic acid by
lipases and serine proteases. This mechanism involves HPLC. Standards of acetic acid are analyzed under the
two major elements, a nucleophilic serine and a general same conditions. Glycerol can be used as an internal
acid-base catalyst, usually a histidine. It remains to be standard at 0.1% or 0.2% concentration. One unit of
established whether all types of AcXEs contain the AcXE is dened as the amount of enzyme liberating
serine motif (GXSXG) in their catalytic site. 1 mol of acetic acid per minute. (Note: Termination
of the reaction is done by heating of the sample at
G. Determination of AcXE Activity 100 C in case the acetic acid is determined enzymically
(200).)
The most specic AcXE assay employs as substrates
hardwood acetylxylans extracted either by DMSO or
2. Acetylesterase Assay on 4-Nitrophenyl
obtained by steaming wood. The naturally occurring
Acetate (233)
acetylxylan can be replaced by chemically acetylated
xylan. In the presence of EXs, both types of polysac- a. Reagents
charides are also suitable for enzymes that prefer acety- 1. 4-Nitrophenyl acetate, saturated solution in 0.2
lated xylo-oligosaccharides as substrates. The amount M potassium phosphate buffer, pH 6.5 (freshly pre-
of released acetic acid can be determined by HPLC pared solution).
chromatography, or enzymically using the Boeringer 2. 4-Nitrophenol, 1 mM standard solution in 0.2
Test Combination No. 148,261. Practically all HPLC M potassium phosphate buffer, pH 6.5.
procedures employ for analysis of acetic acid a Bio-
b. Procedure. A volume of 1050 L appropri-
Rad (Richmont, CA) Aminex HPX-87H column
ately diluted enzyme solution is mixed with 1 mL clar-
eluted with 0.01 N H2 SO4 (193, 202), connected to a
ied saturated solution of 4-nitrophenyl acetate in a 1
refractometric detector. Glycerol may be recom-
cm light-path microcuvette and incubated at 22 C.
mended as internal standard. The conditions of
Absorbance at 410 nm is measured at time intervals
AcXE assay on acetylxylan vary considerably among
against a substrate blank. Alternatively, the absor-
different laboratories. Typical conditions include a
bance can be followed continuously. The amount of
substrate concentration in the range of 0.210%, pH
substrate hydrolyzed is calculated from a calibration
in the range of 5.07.0, and temperature in the range of
curve prepared using 4-nitrophenol in the 0.05
3075 C.
0:3 mol/mL range in 0.2 M phosphate buffer. The
Since most AcXEs exhibit general acetylesterase
reaction cannot be terminated chemically because of
activity [the exception is family 4 (226)], their activity
the labile nature of the substrate. One unit of acetyles-
also can be determined using chromogenic or uoro-
terase activity is dened as the amount of enzyme lib-
genic acetylesterase substrates such as 4-nitrophenyl
erating 1 mol of 4-nitrophenol in 1 min.
acetate, - or -naphthyl acetate, and 4-methylumbel-
liferyl acetate. Examples of selected AcXE assays are
given below. 3. Acetylesterase Assay on 4-Nitrophenyl
Acetate (202)
1. AcXE Assay on Acetylxylan (202, 233)
a. Reagents
a. Reagents 1. 4-Nitrophenyl acetate, 50 mM solution in
1. Acetylxylan obtained from hardwood by DMSO (stable at 4 C), 2 mM solution in water pre-
DMSO extraction or steaming, or chemically acety- pared freshly by diluting 1 volume of the DMSO solu-
lated xylan, 10% solution or suspension in 0.4 M tion with 24 volumes of water.
potassium phosphate buffer, pH 6.0 or 6.5. 2. 0.1 M potassium phosphate buffer, pH 6.5.

3. 4-Nitrophenol calibration solution in 0.05 M H. Assays for Screening and Detection Purposes
phosphate buffer.
All substrates used for AcXE assays can also be
b. Procedure. Enzyme present in 1.5 mL of 0.1 M
employed for detection of the enzyme in gels.
phosphate buffer is mixed with 1.5 mL of 2 mM solu-
Detection of AcXE using polymeric acetylxylan is
tion of 4-nitrophenyl acetate and incubated at 25 C.
based on the precipitation of the polysaccharide due
The increase in absorbance at 410 nm is measured
to deacetylation (207). This approach is applicable
against a substrate blank. Enzyme activity is calculated
only in those cases when the AcXEs located in the
from the slopes of absorbance versus time and calibra-
gel are free of EXs, which would prevent the precipita-
tion values of 4-nitrophenol obtained under the same
tion by hydrolyzing the deacetylated polymer. Such a
conditions. One unit of acetylesterase activity is
situation occurs after separation of EXs from AcXEs
dened as above.
by electrophoretic methods. This means that precipita-
tion of acetylxylan can be used for screening purposes
4. Acetylesterase Assay on -Naphthyl Acetate (234) only in the case of naturally occurring and recombi-
a. Reagents nant strains that do not produce EXs.
1. -Naphthyl acetate, 1 mM solution in 0.05 M For initial screens of microorganisms and transfor-
sodium citrate buffer, pH 5.3. mants producing AcXEs belonging to other families
2. Fast Corinth V Salt (Sigma F-6389), 0.1% solu- than family 4 (Table 4), it is possible to use chromo-
tion in 1 M acetate buffer, pH 4.3, containing 10% genic and uorogenic aryl acetates, such as - or -
Tween. naphthyl acetate and 4-methylumbelliferyl acetate
3. -Naphthol, calibration solution in the citrate (214). An alternative is to screen for the production
buffer. of enzymes clearing of milky solid media containing
insoluble per-O-acetylated carbohydrates, e.g., penta-
b. Procedure. Enzyme solution (0.2 mL) is incu- O-acetyl-D-glucose (236). Subsequent screening of
bated with 1.8 mL -naphthyl acetate solution at 50 C positive colonies for the ability to deesterify acetylxy-
for 10 min, after which 1 mL dye solution is added. lan itself is necessary. The principles outlined above
The absorbance at 535 nm is read exactly 10 min after can be used for detection of AcXEs in electrophoretic
addition of the dye. The calibration curve is con- gels.
structed under the same conditions.
1. Detection of AcXE in Gels Using
E. Fluorimetric Acetylesterase Assay on 4- Acetylxylan as a Substrate (233)
Methylumbelliferyl Acetate (235)
An approximately 1-mm-thick at 1.52.0% agarose
a. Reagents. 4-Methylumbelliferyl acetate, 10 gel containing 0.52.0% of acetylxylan (DMSO
mM solution in methanol. 0.5 M potassium phosphate extracted or obtained by steaming wood) and 0.01%
buffer, pH 6.5. sodium azide in 0.1 M phosphate buffer, pH 6.5, is
prepared by casting between glass plates. Visualiza-
b. Procedure. 4-Methylumbelliferyl acetate solu-
tion of AcXE is carried out by superimposing the
tion (10 L) is mixed with 10 L phosphate buffer
detection and rebuffered separation gel in a plastic
and made up to 100 L with the enzyme sample
bag and incubating at 3050 C until cloudy areas
and water. After an appropriate time of incubation
appear. The precipitate forms in both the separation
at 40 C, the mixture is diluted with 2.9 mL distilled
and detection gels because of cross-diffusion of
water. The uorescence is measured at an excitation
enzyme and substrate. The method fails when esterase
wavelength of 330 nm and an emission wavelength at
is located close to EXs. Gels with precipitates are
445 nm using 4-methylumbelliferone as the calibra-
photographed in diffuse light similarly as immunopre-
tion standard. One unit of esterase is dened as
cipitation bands.
1 mol of the product formed per minute. (Note: A
much less sensitive acetylesterase assay using 4-methy-
2. Detection of Acetylesterase in Gels by -
lumbelliferyl acetate is a photometric assay which
Naphthyl Acetate (237)
exploits the fact that 4-methylumbelliferone shows
considerable absorbance at 354 nm (not an absorp- A solution of 20 mg -naphthyl acetate in 4 ml of
tion maximum) while its acetate does not absorb at N,N 0 -dimethylformamide is mixed with 36 mL of a
this wavelength (224).) solution of 20 mg of Diazo Blue R (Sigma) in 0.05

M phosphate buffer, pH 7.4. An electrophoretic or bino-D-galactans. Other natural substrates of -L-ara-
isoelectrofocusing gel is washed with 0.1 M phosphate binofuranosidase are glycosylated terpenols of grape,
buffer, pH 7.4, for 15 min. The gel is then placed in a important precursors of grape and wine aromas. Many
freshly prepared substrate-dye solution and incubated of these terpenols are linked to disaccharide moieties in
for 1 h at 40 C. The reaction is stopped by decanting which the nonreducing terminal sugar is frequently L-
the staining solution and covering the gel with a solu- arabinofuranose. Thus, -L-arabinofuranosidase can
tion of 2% acetic acid. be used for enhancement of wine avor by the release
of free terpenols (240242).
Since the enzymes degrading arabinans and arabi-
3. Detection of Acetylesterase Using 4-
nogalactaus were specically covered in Chapter 70,
Methylumbelliferyl Acetate (233)
here we shall focus on -L-arabinofuranosidases
A at detection agarose gel containing 0.1% 4-methy- involved in the degradation of polysaccharides con-
lumbelliferyl acetate in 0.1 M phosphate buffer is taining main chains built from -1,4-linked xylopyra-
freshly prepared. The gel is placed in contact with a nosyl residues, specically, softwood arabinoglu-
washed electrophoretic gel and incubated at room tem- curonoxylans and cereal arabinoxylans. Some of
perature until a bright uorescent zone appears under these enzymes, particularly those which attack low-
UV light at 360 nm on both separation and detection molecular-mass fragments of these polysaccharides,
gels. An alternative procedure is to bring the separa- participate in the degradation of all types of L-arabi-
tion gel into contact with a saturated solution of 4- nose-containing polysaccharides. However, there are
methylumbelliferyl acetate in 0.1 M phosphate buffer -L-arabinofuranosidases specic for arabinoxylans.
on a glass plate. In the softwood polysaccharide the L-arabinofurano-
syl residues are linked to the main chain -1,3- or -
1,2 glycosidically as single substituents. Cereal arabi-
noxylans contain, in addition to -1,3- and -1,2-
VI. a-L-ARABINOFURANOSIDASE (EC
linked L-arabinofuranosyl residues as single substitu-
3.2.1.55)
ents, doubly arabinosylated xylopyranosyl residues of
A. Chemical Reactions Catalyzed the main chain.
B. Properties as Proteins
Xyl1-4Xyl1-4Xyl-R
3 2 Fungal -L-arabinofuranosidases occur mostly as
j ! Xyl1-4Xyl1-4Xyl-R Ara monomeric enzymes with molecular masses between
32 and 84 kDa and pI values between 3.2 and 7.5
1 (44). The majority of described fungal enzymes are
Araf products of Aspergillus spp. (146). and have pI values
3:5. Bacterial and Streptomyces -L-arabinofurano-
R 0 -Xyl1-4Xyl1-4Xyl-R sidases frequently occur as oligomers, form dimers up
32 to octamers, with subunits of molecular mass between
j ! R 0 -Xyl1-4Xyl1-4Xyl-R Ara 33 and 75 kDa. pI values were reported in the range of
3.89.0.
1 Some -L-arabinofuranosidases show modular
Araf architecture similar to other xylanolytic enzymes.
Interesting in this regard is -L-arabinofuranosidase
from T. reesei (244, 245). Two different forms of the
-L-Arabinofuranosidase is a component of hemi- enzyme have been isolated from a culture uid of the
cellulolytic systems that participates in hydrolysis of fungus. The 25-kDa form corresponds to the catalytic
plant polysaccharides built from or containing nonre- domain only (245). The 53-kDa form, the rst form of
ducing terminal -L-arabinofuranosyl residues (44, the enzyme described (244), also contains a noncataly-
238). Such polysaccharides have been recognized as tic xylan-binding domain (245). The large 53-kDa form
part of pectic substances, gum exudates, and wood can be converted to the shorter one by proteolytic
and cereal hemicelluloses (239). The pectic substances digestion (245). -L-Arabinofuranosidase from P.
mainly include L-arabinan and various types of L-ara- uorescens was reported to contain a cellulose-binding

domain in the N-terminal portion of the polypeptide attack not only NPh-Araf and arabinose-containing
chain (246). A xylan-binding domain was detected in oligosaccharides, but also polymeric substrates such
C-terminal region of -L-arabinofuranosidase B from as arabinoxylans and arabinoglucuronoxylans (239).
S. lividans (247). The enzymes containing the noncata- These enzymes catalyze hydrolysis of -1,5-, -1,3-
lytic polysaccharide-binding domains are specic for and also -1,2-linked terminal L-arabinofuranosyl resi-
release of L-arabinofuranose from polymeric sub- dues with retention of the anomeric conguration
strates. (248).
The family 62 of -L-arabinofuranosidases (Table
C. Properties as Enzymes 5) includes enzymes which preferentially attack poly-
meric substrates, i.e., arabinoxylans. The best-known
Microbial -L-arabinofuranosidases can be divided members of this family are enzymes from Aspergillus
into three different groups according to substrate spe- spp., assigned as AXHs (249), -L-arabinofuranosi-
cicities (239). The groups seem to coincide with three dase B from Streptomyces lividans (247), and an
different glycosyl hydrolase families into which -L- enzyme from Ps. uorescens subsp. cellulosa having a
arabinofuranosidases belong (Table 5). modular architecture (246). The enzymes show a clear
The enzymes that perform best on short -1,5- preference for -1,2-linked and -1,3-linked -L-arabi-
linked arabinooligosaccharides and are active on 4- nofuranosyl residues of arabinoxylan main chain.
nitrophenyl--L-arabinofuranoside (NPh-Araf ) and However, the enzymes of this family do not remove
inactive on polymeric substrates are grouped in family -L-arabinofuranosyl residues from doubly substituted
51. They include both fungal and bacterial -L-arabi- xylopyranosyl residues.
nofuranosidases of molecular mass 5772 kDa, which An arabinofuranose-releasing enzyme of novel sub-
catalyze the hydrolysis of the glycosidic linkage with strate specicity was isolated from Bidobacterium
retention of the anomeric conguration (248). These adolescentis DSM 20083 (250). This enzyme specically
are apparently the enzymes designed to degrade more releases terminal arabinofuranosyl residues linked -
arabinans rather than arabinoxylans. 1,3 to xylopyranosyl residues of main chains that
Family 54 includes mainly fungal -L-arabinofura- also carry -1,2-linked arabinofuranose. Wood and
nosidases (M.m. 4953 kDa) which exhibit somewhat McCrea (251) reported the occurrence of a hydrolase
broader substrate specicities than the enzymes of releasing feruloylated L-arabinofuranosyl residues
family 51. The 54 family -L-arabinofuranosidases from wheat straw arabinoxylan. Additional evidence
Table 5 Families of -L-Arabinofuranosidases and Differences in Their Properties
Producing Molecular Mechanism of

Family microorganism mass (kDa) Hydrolyzed substrates hydrolysis Ref.
51 A. niger (enz. A) 5772 NPh--Araf , arabino- Retaining 205

Bacillus oligosaccharides (enzymes are
Streptomyces inactive on polymers)
Thermotoga
Clostridium
54 A. niger (enz. B) 4953 NPh--Araf , arabino- Retaining 205, 253
T. reesei oligosaccharides, arabinoxylan
Ewericella nidulans (attack mainly on -1,3-linked
Synchocystis sp. Araf , no attack on double
arabinosylated xylopyranosyl
residues.
62 Aspergillus (AXH) 4759 arabinoxylan (the enzymes remove Unknown 246, 247,
Streptomyces (enz. B), -1,3- and -1,2-linked L-Araf 249
Ps. uorescens residues, no attack on double
arabinosylated xylopyranosyl
residues)

for hydrolysis of substituted arabinofuranosyl residues (pH 4.0) or in other buffer depending on the pH opti-
has been reported for -L-arabinofuranosidase from mum of the assayed enzyme.
A. terreus (252). The mode of action of enzymes 2. 1.0 M sodium carbonate.
operating on high-molecular-mass arabinoxylans and
their relationship to glycosyl hydrolase families is b. Procedure. 4-Nitrophenyl -L-arabinofurano-
illustrated in Scheme 7. Based on the complexity of side solution (0.9 mL) is mixed with 0.1 mL of the
structures of plant arabinoxylan, one can anticipate enzyme sample, incubated at 50 C or an appropriate
the discovery of arabinofuranosidases with other time, and the reaction is stopped by addition of 0.5 mL
substrate specicities. of 1 M solution of Na2 CO3 . Liberated 4-nitrophenol is
Scheme 7
D. Determination of -L-Arabinofuranosidase measured spectrophotometrically at 400 nm. One unit

Activity of -L-arabinofuranosidase activity is dened as the
amount of enzyme liberating 1 mol of 4-nitrophenol
As indicated in Table 5, two families of -L-arabino- in 1 min. (Note: The literature offers a variety of mod-
furanosidases can be assayed easily using the chromo- ication of the above procedure with respect to buffer,
genic substrate 4-nitrophenyl -L-arabinofuranoside substrate concentration, and the reaction terminating
or the uorogenic substrate 4-methylumbelliferyl -L- agent. Termination of the reaction using a saturated
arabinofuranoside. One should be aware of the fact solution of sodium tetraborate is recommended.)
that these substrates can also be attacked by -xylosi-
dases of family 43, which exhibit aryl -L-arabinofur-
anosidase activity (Table 3). Therefore, it is imperative
2. Fluorimetric Assay of -L-
to conrm the identity of the enzyme by determining
Arabinofuranosidase (254)
its ability to release arabinose from arabino-sub-
stituted xylans or xylooligosaccharides (5). -L- a. Reagents
Arabinofuranosidases of family 62 must be assayed 1. 4-Methylumbelliferyl -L-arabinofuranoside,
on natural substrates, arabinoxylans, and oligosac- 10 mM solution in dimethyl formamide.
charides derived from arabinoxylans. Released arabi- 2. 0.02 M acetate buffer, pH 4.5.
nose is determined using HPLC or as reducing sugar. 3. 0.2 M sodium carbonate buffer, pH 10.0.
Examples of determination of -L-arabinofuranosi- 4. 4-Methylumbelliferone.
dase activity are given below.
b. Procedure. Enzyme solution, 520 L, is made
up to 200 L with 0.02 M acetate buffer (pH 5.5) con-
1. -L-Arabinofuranosidase Assay Using 4-
taining 4 L of 10 mM solution of the substrate in
Nitrophenyl -L-Arabinofuranoside (244)
dimethyl formamide. To monitor the enzymic activity
a. Reagents 20-L aliquots of the reaction mixture are diluted with
1. 4-Nitrophenyl -L-arabinofuranoside, 2 mM 2 mL sodium carbonate buffer, and the liberated 4-
solution in 0.05 M sodium citrate phosphate buffer methylumbelliferone is determined uorimetrically.

One unit is dened as the amount of enzyme that tion of an intensely colored indigo precipitate. The
releases 1 mol of 4-methylumbelliferone per minute. glycoside was suggested for use in cloning experi-
ments in which -L-arabinofuranosidase will be used
3. Assay of 1,4--Arabinoxylan as a reporter gene (254).
Arabinofuranohydrolase (249)
a. Reagent. Arabinoxylan (wheat, rye, or oats;
Megazyme), 0.1% solution in 0.05 M acetate buffer, ACKNOWLEDGMENT
pH 5.0.
The author thanks Dr. Timothy D. Leathers from the
b. Procedure. The solution of oats spelt arabi- Biopolymer Unit of the USDA, ARS, in Peoria, IL, for
noxylan is mixed with a small volume of enzyme solu- generous help with English in this chapter.
tion, incubated at 30 C for an appropriate time, and
analyzed for L-arabinose by HPLC.
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72
Enzymes with Activity Toward Xyloglucan
Jean-Paul Vincken
I. INTRODUCTION these seeds, a battery of enzymes is induced to mobi-

lize the xyloglucan food reserves of the hypocotyl.
Xyloglucans are plant cell wall polysaccharides, The storage xyloglucans, in particular, are used as
which belong to the hemicellulose class; i.e., they food ingredients or in pharmaceutical applications.
can bind to cellulose. They are the most abundant The decoration (or side-chain conguration) of these
hemicellulose in the walls of many nongraminaceous xyloglucans determines to a large extent their func-
species. Xyloglucans can function both as a struc- tional properties. Depending on the application, the
tural and as a reserve polysaccharide. In the pri- decoration may be adjusted by using enzymes of
mary cell wall, xyloglucans can crosslink cellulose either plant or fungal origin. With the discovery of
bers, yielding a network that determines to a the rst enzymes in xyloglucan biosynthesis, it may be
large extent the strength of the wall. This complex possible in the future to realize these structural mod-
usually constitutes approximately half of the amount ications in the plant itself. Biosynthetic enzymes
of wall polysaccharides, the cellulose (30%) being a using nucleoside-diphosphate sugars will not be dis-
bit more abundant than the xyloglucan (20%). cussed in this chapter.
Plants possess many enzymes to remodel this net- In the paragraphs below, the structural variations
work. Certain degradation products of xyloglucan of xyloglucans will be reviewed rst. As we will see
may serve as signaling molecules, the activity of later, substitution determines the rate at which xylo-
which can be regulated by various glycosidases. glucan molecules are degraded. Subsequently, the
Structural xyloglucans are also important from a anchoring of xyloglucan in the plant cell wall will
food industry point of view. They are an important be discussed. Some background information on this
target for fungal enzymes in applications, which aim is provided, because certain enzyme purication pro-
at a complete degradation of the plant cell wall, cedures and enzyme assays make use of these princi-
such as the liquefaction process for fruit juice ples. The following sections deal with (plant and
manufacturing. Further, xyloglucans might play a microbial) enzymes involved in degradation or mod-
role in determining the shelf life of fruits and vege- ication of xyloglucan. After this, a number of meth-
tables. ods for activity measurements are summarized. These
Xyloglucans can also be deposited in the secondary methods deal predominantly with the determination
cell walls of seeds of several species, such as of transferase activity, because these assays follow
Tamarindus indica, Tropaeolum majus, Hymenaea different procedures from those used for degradative
courbaril, and Copaifera langsdorfi. The seeds can enzymes. Finally, a few applications in the food
contain > 50% xyloglucan. Upon germination of industry will be described.

II. STRUCTURE AND PROPERTIES OF two general branching patterns can be distinguished,
XYLOGLUCANS XXXG and XXGG (see Fig. 1B) (1). Thus, xyloglu-
cans seem to have a subunit length of four glucosyl
Xyloglucans are polysaccharides with a highly residues in common. In the XXXG-type xyloglucans
branched -(1 ! 4-Glcp or cellulosic backbone. (most nongraminaceous species), three contiguous Glc
Most side chains contain -Xylp residues, which are residues are branched, followed by an unbranched one.
attached to the 6-position of the Glc residues (Fig. The XXGG-type xyloglucans (solanaceous plants)
1A). The -Xylp residues are distributed in the glucan contain clusters of two branched Glc residues, which
backbone according to regular patterns. In principle, alternate with a sequence of two bare Glc residues.
Figure 1 (A) Structure of the cellobiosyl building unit of cellulose, the xylobiosyl building unit of xylan, and a trisaccharide XG
subunit of xyloglucan. Note that glucose and xylose are very similar except for C-6. (B) Two general branching patterns
of xyloglucans, XXXG and XXGG. Small shaded circles represent acetyl groups. (C) Hypothetical (nonexisting) molecule
showing the different structural elements encountered in xyloglucans. G -D-Glcp; X -D-Xylp-1 ! 6--D-Glcp;
L -D-Galp-1 ! 2--D-Xylp-1 ! 6--D-Glcp; F -L-Fucp-1 ! 2--D-Galp-1 ! 2--D-Xylp-1 ! 6--D-Glcp; S
-L-Araf -1 ! 2--D-Xylp-1 ! 6--D-Glcp; A -D-Xylp-1 ! 6--D-Glcp-2 1--L-Araf ; B -D-Xylp-1 ! 6-
-D-G l cp-2 1--D-X ylp; C -D-X y lp-1 ! 6--D-G l cp-2 1--D-Xylp-3 1--L-Araf ; J -L-Galp-1 ! 2-
-D-Galp-1 ! 2--D-Xylp-1 ! 6--D-Glcp; T -L-Ar af -1 ! 3--L-Ar af -1 ! 2--D-X y lp-1 ! 6--D-G l c p. ( D )
Flat backbone conformation of the xyloglucan heptadecamer GXXFGXXXG [adapted from (4)]. NR, nonreducing end of
the polymer; R, reducing end. (E) Schematic representation of a part of the cellulose-xyloglucan network in the primary cell of
plants [adapted from Roberts and McCann (9)].

An exception to this rule is the xyloglucan from groups can be attached to the C-6 position of Glc
Hymenaea courbaril, which contains similar amounts residues, mainly as in XX(G-ac)G, and to a minor
of XXXG- and XXXXG-type subunits (2). Another extent as in (G-ac)XXG. Further, acetyl groups can
exception might be the xyloglucan from certain grami- be present on the C-5 of terminal arabinosyl units.
naceous species (rice, barley), which are thought to Despite their heavily branched nature, xyloglucans
have a degree of branching of 40% or lower (1). can interact intimately with cellulose. Molecular mod-
The variation in xyloglucan structure is much larger eling studies with the heptadecamer GXXFGXXXG
than the two general branching patterns suggest. The have shown that xyloglucan molecules can adopt a
xyloglucan skeleton can be decorated with many dif- so-called at-backbone conformation, which facilitates
ferent residues. However, the sequence in which these their binding to cellulose (4, 5). In this conformation,
residues occur in the side chain is very consistent. In the xylosyl residues point outwards in an alternate
1993, a concise nomenclature was developed to unam- fashion (Fig. 1D). The fucosylated side chain folds
biguously designate xyloglucan structures (3). A xylo- onto the backbone toward the nonreducing side,
glucan molecule is named by partitioning the backbone whereas the opposite face of the molecule is relatively
into segments consisting of a single glucosyl residue at. The structure of the side chains plays an important
and its pendant side chains. Each segment is given a role in the interaction with cellulose. The fucosylated
specic code letter, depending on the side chain con- sidechain in particular seems to induce the at-back-
guration. For example, the letters G and X refer to bone conformation. Indeed, binding studies have
the unbranched -D-Glcp residue and the -D-Xylp- shown that fucosylated xyloglucans display a higher
(1 ! 6)--D-Glcp segment, respectively. The letter F afnity binding than nonfucosylated xyloglucans (5,
refers to the -L-Fucp-(1 ! 2)--D-Galp-(1 ! 2)-- 6). This may be consistent with the function of these
D-Xylp-(1 ! 6)--D-Glcp segment of the fucosylated xyloglucans in the plant cell wall. The former is
nonasaccharide (XXFG) commonly found in xyloglu- encountered as a crosslinker of cellulose microbrils
cans. The different structural elements which are cur- in the primary wall. The latter serves as a reserve poly-
rently known are summarized in the hypothetical saccharide in the secondary wall of seeds, which needs
(nonexisting) xyloglucan molecule in Figure 1C. to be easily accessible to glycanases during germina-
The variation in xyloglucan structure is not only tion. At the moment, the binding characteristics of
species dependent, but also relates to the physiological XXGG-type xyloglucan are unknown, as is the contri-
state of the cell wall of that particular species (1). bution of arabinosyl substitution to binding. Next to
Xyloglucans that are present as storage polysacchar- the side chain conguration, the length of the xyloglu-
ides in seeds are of the XXXG type. Unlike the xylo- can molecule is also an important parameter for inter-
glucan derived of the primary cell wall, these are not action with cellulose. It has been shown that molecules
fucosylated (Fig. 1B). Depending on their origin, they composed of (at least) four oligosaccharide binding
contain different proportions of XXXG, XLXG, units (equivalent to a backbone length of 16 Glc resi-
XXLG, and XLLG. In addition to these, the primary dues) bind quantitatively to cellulose (7, 8).
cell wall xyloglucan can contain the fucosylated oligo- The cellulose-xyloglucan network comprises 50%
saccharides XXFG, XLFG, and XFFG, as well as lar- of the primary cell wall (width 75 nm). The wall is
ger oligosaccharides with the more rare segments A, B, anked by a plasma membrane and a middle lamella,
and C. Smaller oligosaccharides, like FG, GFG, XFG, and can accommodate three to four lamellae of cellu-
and XXG, also occur, but these are most likely a result lose microbrils (diameter 512 nm; indeterminate
of postdepositional modication by endogenous length). The microbrils in one layer run parallel,
enzymes. Finally, the galactosyl residue of the F ele- and no weaving between layers is observed (9).
ments can be acetylated. Xyloglucans coat the cellulose surface; they are also
The XXGG-type xyloglucans have so far only been incorporated in the microbrils during the synthesis
found in solanaceous plants (potato, tomato, tobacco) of the wall. The length of individual xyloglucan mole-
(1). Fucosylated trisaccharide side chains are not pre- cules (30400 nm) generally exceeds the distance
sent in these xyloglucans. Instead, they possess a fair between lamellae many times, which suggests that
amount of arabinosyl-containing disaccharide side these polysaccharides can interlink several microbrils
chains. It seems that all combinations of the elements (Fig. 1E). Typically, xyloglucans seem to be built of
X, L, and S can occur (XX, LX, XL, LL, SX, XS, SS, blocks of 30 nm repeats (10); one such repeat corre-
SL, and LS). The structural elements A, B, and C have sponds to 60 Glc residues (or 15 xyloglucan oligo-
not been demonstrated in the XXGG type. Acetyl saccharides), which is similar to the length of a

crosslink or the distance between two lamellae. The sources. Arabidopsis thaliana alone already contains >
attachment of xyloglucan to cellulose provides impor- 12 endoglucanases (15). Virtually nothing is known
tant mechanical properties such as extensibility, which about the substrate specicity of these enzymes.
can not be realized with cellulose alone (11). Microbial endoglucanases are often modular pro-
teins; i.e., they are composed of separate domains,
which are connected by linker peptides (16). The sub-
III. ENZYMIC DEGRADATION OF strate specicity of the endoglucanase resides in the
XYLOGLUCANS catalytic core. One or more cellulose-binding domains
(CBDs) determine whether the enzyme can attach itself
Over the years, many xyloglucan hydrolases of both to, and degrade insoluble substrates like cellulose. The
fungal and plant origin have been described. In this fact that EGI from Trichoderma reesei has a CBD,
section, most attention will be devoted to endogluca- whereas EGIII has not, shows that xyloglucanases
nases, which can rapidly depolymerize xyloglucan. with and without CBD can occur naturally. So far,
Next to these, a number of exoacting enzymes will be no plant endoglucanases with a CBD have been
reviewed, the majority of which only act on oligosac- described (15). However, these enzymes can contain
charides (such as xyloglucosidase, -xylosidase, and - (putative) signal sequences for cell attachment or mem-
fucosidase). -Galactosidase is an exception to this, brane anchoring. This implies that the action of the
and can be used to adjust the physical properties of endoglucanases with a signal sequence is restricted to
polymeric xyloglucan. Hydrolases with the ability to specic locations within the wall.
split off -L-Araf from XXGG-type xyloglucan have As we have seen above, the answer to the question
not been described so far. whether an endoglucanase has xyloglucanase activity
lies in the topology of the catalytic core. These cataly-
A. Endoglucanase tic cores can be grouped in families, based on simila-
rities in apparent secondary structures of proteins
In the older literature endoglucanases are sometimes (http://afmb.cnrs-mrs.fr/ pedro/CAZY/). In principle,
referred to as cellulases or Cx factor. In principle, cores with very low amino acid sequence identity can
both cellulase and Cx are a collective noun for a be attributed to the same glycosyl hydrolase family;
group of enzymes, including endoglucanases, which i.e., the fold of the core proteins seems to be much
have in common that they hydrolyze -D-Glcp- better conserved than their sequence. Within one
(1 ! 4) linkages. Endoglucanases have long been family, the catalytic mechanism (retaining or inverting)
characterized by their activity on carboxymethyl cellu- of the hydrolysis reaction is the same. So far, endoglu-
lose (CMC). Unfortunately, determination of xylo- canases with xyloglucanase activity have been attribu-
glucanase activity is not a common practice, and for ted to two families-7 and 12 (both retaining).
many well-characterized cellulases it is not known if Important characteristics of enzymes belonging to
they are able to degrade xyloglucan. However, it is these families are summarized in Table 1.
important to realize that not every endoglucanase is Typically, the Trichoderma reesei EGI and EGIII
a xyloglucanase (12). Recently, an endoglucanase can cleave both xyloglucan and xylan (12), suggesting
with xyloglucanase, but no CMCase, activity has that the presence of CH2 OH groups or a xylosyl resi-
been described (13). This suggests that there are at due protruding from the backbone do not matter for
least two types of xyloglucanasesa highly specic these enzymes (see also Fig. 1A). However, EGII from
one, and one with a more promiscuous substrate Aspergillus aculeatus is highly specic for xyloglucan
specicity. and is unable to degrade xylan (13). This shows that
Bacteria and fungi usually possess several endoglu- activity toward xylans is not a common feature of
canases. For instance, the fungus Trichoderma reesei xyloglucanases.
has four endoglucanasesEGI, EGII, EGIII, and Another important conclusion from Table 1 is that
EGVof which at least two have xyloglucanase activ- the substrate specicity of endoglucanases is not con-
ity (EGI and EGIII; for EGV this has not been deter- served within each family. For instance, family 12 con-
mined) (12). With this set of different endoglucanases, tains many different kinds of endoglucanases. Either
Trichoderma seems very well equipped to efciently they are mainly active on CMC, or they are active only
degrade plant cell wall cellulose, i.e., cellulose that is on xyloglucan. Also, there are endoglucanases, which
coated with hemicellulose (14). The variety of endoglu- can degrade both substrates. This suggests that only
canases in plants is even larger than that in microbial subtle changes in the structure of endoglucanases can

Table 1 Overview of Selected Endoacting Enzymes with Activity Toward -(1 ! 4)-D-Glucans
Enzyme Organism EC No. XG CMC Xylan MWcore Family Cat. mech. Cat. res. CBD? Anchors?
EG I T. reesei 3.2.1.4 yes yes yes 40 7 retaining E,E yes no

EG plant 3.2.1.4 ?? ?? ?? 5060 9 inverting E,D no possible
EG III T. reesei 3.2.1.4 yes yes yes 25 12 retaining E,E no no
EG II A. aculeatus 3.2.1.4 yes no no 25 12 retaining E,E no no
EG S. lividans 3.2.1.4 ?? yes ?? 24 12 retaining E,E yes no
XET plant 2.4.1.207 yes no no 32 16 retaining E,E no no
determine whether these enzymes have xyloglucanase means that xyloglucanase activity may not be reserved
activity. Probably, it is important that the substrate- to retaining enzymes.
binding groove is lined with the appropriate amino In the previous paragraphs it was shown that the
acid residues at certain positions; this may determine structure of xyloglucan determines the properties of
whether the xylosyl side chains on the glucan backbone these molecules, such as binding to cellulose surfaces.
are tolerated in the groove. Also, xylosyl groups may Next to this, the structure also greatly inuences the
be involved in the molecular recognition of the sub- enzymic digestibility; i.e., the nature of the sidechains
strate by xyloglucanases. From the examples above it determines how fast xyloglucan is degraded by endo-
is clear that the family classication does not have a glucanases. It is difcult to relate the tolerance to par-
predictive value with respect to the substrate specicity ticular side chains to individual endoglucanases,
of endoglucanases. The xyloglucanase activity of each because most studies have been carried out with rather
individual endoglucanase should be veried experi- crude mixtures of enzymes from Trichoderma.
mentally. There are a number of reports showing In Figure 2 a number of rules that seem to have a
that there are enzymes in plant extracts that have xylo- general validity are summarized. The presence of fuco-
glucanase activity. Possibly, some of these xylogluca- sylated side chains can hinder cleavage by the endoglu-
nases will be classied in family 9. At this moment, canase (17). Two adjacent fucosylated side chains as in
there is no experimental evidence supporting this XFFGXXXG can give partial resistance to endogluca-
remark. It is interesting to note that the enzymes of nase, meaning that prolonged incubations are needed
family 9 act with an inverting mechanism. This to generate the constituent oligosaccharides XFFG
Figure 2 Overview of xyloglucan structures, which (a) are easily cleaved by endoglucanases (solid arrow), (b) are partially
resistant to endoglucanase (thin arrow), and (c) are completely resistant to endoglucanases (thin arrow with cross). For details on
xyloglucan structure, see Figure 1C. Small shaded circle on open box (bottom right structure) represents an acetyl group attached
to a glucosyl residue.

and XXXG (18). Molecular modeling studies suggest EGI and the higher specicity EGIII are common fea-
that, in solution, the fucosylated side chain folds tures of, respectively, family 7 and 12 endoglucanases.
toward the reducing end of the molecule, i.e., over From the above it is clear that the activity of endo-
the linkage to be cleaved (4). This probably decreases glucanases toward xyloglucan is greatly affected by the
the accessibility of the glucan backbone to endogluca- ne structure of the polysaccharide. Much more work
nases. remains to be done to establish the structure-function
The presence of A elements adjacent to unsubsti- relationships of endoglucanases, and to answer the
tuted glucosyl residues (as in XXFGAXXG or important question of what determines whether an
XXAGXXFG) blocks cleavage by endoglucanases endoglucanase has xyloglucanase activity.
completely (19). In Solanaceous spp., two vicinal
unbranched glucosyl residues can occur as in B. Glucosidase
XSGGXXGG (20). In this case the endoglucanase
has a choice in cleavable linkages, either Many of the glycosidases described to date play a role
XSG # GXXGG or XSGG # XXGG. The presence in mobilizing seed reserves upon germination. The -
of other structural elements (L or S) at the position glucosidase from Tropaeolum majus is probably the
of the X elements probably inuences the preference best-characterized one described in the literature (22).
of the endoglucanase for a particular linkage. There is Therefore, the discussion of glucosidases will be
some evidence indicating that the presence of acetyl restricted to this enzyme. The enzyme belongs to gly-
groups (as in XS(G-ac)GXXGG) inhibits cleavage of cosyl hydrolase family 3 (retaining), and shows strong
the adjacent glycosidic bond (21). sequence homology to -glucan exohydrolase from
Because most xyloglucans are of the XXXG type, barley. The glucosidase contains two highly conserved
not many people have realized that endoglucanases motives, SDW(24 aa)GIDM, in which the aspar-
may actually display differences in their mode of action tates have been implicated as the catalytic residues.
towards xyloglucan. When it appeared that solanac- Several -linked glucose disaccharides were tested as
eous xyloglucans were less heavily substituted than a substrate. From this it was concluded that the enzyme
others, and that they had a regular branching pattern hydrolyzes the various linkages at different rates:
(XXGG type), it became possible to investigate this (1 ! 3 > 1 ! 4 > 1 ! 2 > 1 ! 6. Xyloglucan
(20). It was demonstrated that two endoglucanases oligosaccharides were also tested as a substrate. The
from Trichoderma reesei, EGI and EGIII, respond dif- glucosidase was active against GXXG, GXLG, and
ferently to substitution of the glucan backbone (Fig. GXG, but not against XXXG, XXLG, XLXG,
3). EGIII predominantly released an XXGG type of XLLG, GLXG, or GLLG. Thus, the enzyme requires
oligosaccharide, whereas EGI also released GXXG an unsubstituted glucosyl residue at the nonreducing
and XXG types of oligosaccharides (20). At this end of the oligosaccharide. Next to this, the sidechain
moment it is unknown whether the promiscuity of conguration of the penultimate glucosyl residue at the
Figure 3 Schematic illustration of mode of action of endoglucanases (EGI and EGIII) from Trichoderma reesei on two different
types of xyloglucan (XXXG and XXGG). For details on xyloglucan structure, see Figure 1C.

nonreducing end is important. Enzyme action is inhib- bial -xylosidases (2832), PNP--Xyl and isoprime-
ited when this side chain contains a galactosyl residue. verose were not substrates for the plant -xylosidases.
Another important observation is that the glucosidase
shows transglycosylation activity with cello-oligosac-
E. Galactosidase
charides, but not with xyloglucan oligosaccharides.
The cello-oligosaccharide transglycosylation products
Both fungal and plant -galactosidases have received
can be extended with Glc-(1 ! 4)--, and possibly
attention. The fungal enzymes are sometimes used for
with Glc-(1 ! 6-.
structural characterization of xyloglucan oligosacchar-
ides. The plant enzymes play a role in mobilization of
C. Xyloglucosidase seed reserves, together with the XET (see further), glu-
cosidase, and xylosidase. York and coworkers (33)
This enzyme is discussed in a separate paragraph, demonstrated that the -galactosidase from
because contrary to glucosidase it seems to have a Aspergillus niger catalyzed the following reactions:
requirement for xylosyl side chains. Xylogluco- XLXG ! XXXG and XLLG ! XXLG. The enzyme
sidase was puried from a preparation of Asper- was not capable of degalactosylating sidechains at the
gillus oryzae (23) and is often used for structural char- penultimate glucosyl residue at the reducing end, under
acterization (sequencing) of xyloglucan oligosac- the conditions used. Similar results have been reported
charides. It releases isoprimeverose (-D-Xylp- for a plant -galactosidase from Copaifera langsdorfi
(1 ! 2)--D-Glcp) from the nonreducing terminus (34). The activity of this enzyme toward polymeric
of XXXG (! XXG X, XXG (! XG X, or xyloglucan and other galactosyl-containing substrates
XG (! G X) oligosaccharides. Glucose release was not further investigated. The substrate specicity
from cellotetraose is considerably slower ( 50 of -galactosidase from Aspergillus oryzae seems to be
times), which demonstrates that the enzyme is rather different. This preparation was successfully used to
specic for xyloglucan. Also PNP--Glc was a very remove all galactosyl residues from three-oligo frag-
poor substrate. Xyloglucosidase is unable to by-pass ments of tamarind xyloglucan (8). The -galactosidase
galactosyl-containing side chains. It is unclear if this from Tropaeolum majus was active toward PNP--Gal
enzyme is active toward polysaccharides, because the as well as polymeric xyloglucan (35). Unfortunately,
polymeric substrates tested contain galactosyl resi- the exact specicity was not further investigated. It is
dues. A similar activity is present in a preparation unknown if this enzyme is capable of removing both
from Irpex lacteus (known under the commercial galactosyl residues from, for instance, XLLG-building
name Driselase), which is used by many plant blocks.
researchers (24, 25). As of now, no such enzymes
from plant origin are known.
F. Fucosidase
D. Xylosidase Fucosidases have received some interest from plant

researchers because it has been suggested that particu-
-Xylosidases have been puried from Pisum sativum lar fucosylated xyloglucan oligosaccharides have bio-
(26) as well as from Tropaeolum majus (27), and the logical activity (so-called oligosaccharins). The -
two enzymes were shown to have very similar proper- fucosidase from Pisum sativum has rather narrow sub-
ties. The enzyme releases the xylosyl residue attached strate specicity. The enzyme recognizes -L-Fucp-
to the non-reducing glucosyl residue of a xyloglucan (1 ! 2)--D-Galp-(1 ! structures in xyloglucan oli-
oligosaccharide (for instance XXXG ! GXXG gosaccharides. No activity toward PNP--Fuc or poly-
Xyl). No measurable amounts of xylose were split off meric xyloglucan was observed (36). It appeared that
from polymeric xyloglucan, which shows that no inter- 6-O-acetylation of subterminal galactose of a synthetic
nal xylosyl residues are removed. However, the release substrate enhanced fucosidase activity. Interestingly, 6-
of one xylosyl residue from the nonreducing terminus O-acetylated XXFG naturally occurs as a building unit
can not be excluded. The -xylosidase may play a role of sycamore cell wall xyloglucan (37). This enzyme
in deblocking the nonreducing termini to facilitate probably plays a role in controlling the half-life of
-glucosidase action. Contrary to a number of micro- certain oligosaccharins (XXFG), which antagonize

auxin-induced growth. Fucosidases from other sources tion, the molecular-weight distribution of xyloglucan
have also been described, but these will not be dis- molecules broadens, but no net change in molecular
cussed here. weight is observed (38, 41). Polysaccharide-to-
oligosaccharide transglycosylation results in a large
decrease in molecular weight. This reaction has practi-
IV. XYLOGLUCAN cally the same effect as endohydrolysis catalyzed by
ENDOTRANSGLYCOSYLASE AND endoglucanases.
RELATED ENZYMES Experiments with plant extracts have shown that
XET is highly specic for xyloglucan. Only xyloglucan
A separate section has been devoted to xyloglucan serves as a donor substrate, whereas carboxymethyl
endotransglycosylase (XET), mainly because it is a cellulose and cellulose are not recognized (38).
very interesting enzyme from an enzymological point Several oligosaccharide have been tested as acceptor
of view. The enzyme occurs throughout the plant king- substrates. No transglycosylation was observed with
dom, although some plants (for instance tomato) seem cellobiose, cellotetraose to cellohexaose, laminarihex-
a richer source than others (38). XET or XET-related aose, FG, XGG, and GXG (38, 4143). Some xyloglu-
(referred to as XTR) proteins probably play a role in a can oligosaccharides appeared to be more efcient
number of important plant processes: (a) anchoring than others (efciency in decreasing order XLLG,
newly (synthesized and) deposited xyloglucan into the XXXG and XXFG) (38). In a separate study it was
plant cell wall; (b) ripening; and (c) mobilization of shown that XXXG was a better acceptor than XXG
seed reserves (39, 40). In the older literature, XET is (42). From this it was concluded that XET requires
sometimes referred to as xyloglucan-specic endoglu- two adjacent xylosyl residues as in XXG, which is
canase, oligosaccharide-activatable endoglucanase, or the smallest acceptor molecule.
endo-xyloglucan transferase (EXT or EXGT). XET proteins can be puried relatively easily from a
XET catalyzes the cleavage of a xyloglucan mole- crude mixture of proteins, such as a plant extract. Two
cule (donor substrate), and attaches the nonreducing procedures have been described; both are based on the
moiety of this molecule to an acceptor, either water fact that XET can form a relatively stable enzyme-sub-
(hydrolysis reaction) or another xyloglucan molecule strate complex with polymeric xyloglucan. In the rst
(transferase reaction of transglycosylation). Only procedure, the molecular weight of XET is increased
XXXG-type donor molecules have been tested sofar, articially by adding polymeric xyloglucan, which
and these are cleaved by XET at a similar position as nests in the active site. Subsequently, the XET-xyloglu-
by endoglucanases. The acceptor can be an oligosac- can complex is puried by size exclusion chromatogra-
charide or a polysaccharide. The three possible reac- phy. Finally, XET is released from the polymer by
tions of XET are shown in Figure 4. During addition of xyloglucan oligosaccharides, followed by
polysaccharide-to-polysaccharide endotransglycosyla- a second chromatography step (44). The second pro-
Figure 4 Schematic illustration of three reactions that can be catalyzed by XET. NR, nonreducing end of the polymer; R,
reducing end. Vertical dotted line represents site of cleavage by XET.

cedure utilizes the afnity of xyloglucan for cellulose makes it difcult to make an extensive comparison.
(45). The enzyme-substrate complex is adsorbed to cel- The enzymes have a high transferase activity in com-
lulose. Subsequently, XET is released by addition of mon, and they do not seem to catalyze the hydrolysis
xyloglucan oligosaccharides. reaction (see Fig. 4). Both fucosylated and nonfucosy-
Sequence comparisons have shown that the differ- lated xyloglucan can be used as donor. The activity of
ent XETs can be assigned to the same family as bac- the Vigna angularis XET decreases dramatically when
terial -(1 ! 3)(1 ! 4)-glucanases, namely glycosyl the donor substrate becomes shorter (41). Only very
hydrolase family 16 (39, 40). Reactions catalyzed by little activity was found on donor substrates of 10
members of this family proceed with retainment of kDa (approximately eight-oligo fragments). Possibly,
conguration, which is in line with the fact that this this preference for high-molecular-weight xyloglucan
family contains many transferases. The three-dimen- is related to the conformation of xyloglucan in solu-
sional structure of two Bacillus -glucanases has been tion, but this requires further investigation. Similar
solved (46, 47). The enzymes are compact proteins observations have been made by Tabuchi et al. (49),
with a sandwichlike jellyroll architecture. A groove, but in this case the activity cannot be related to a
where the actual catalysis takes place, spans the consequence. The EXGT from Arabidopsis thaliana prefers
cave side of the molecule. The other side of the the acceptor substrate XLLG over XXXG. XXXG
enzyme contains a Ca2 -binding site, which probably seems to be preferred over XXFG (50).
plays a role in stabilizing the protein together with Three group II XETs from Arabidopsis thaliana
one disulde bridge. Because bacterial -glucanases have been characterized biochemically: TCH4, Meri-
and XET have been assigned to the same family, it 5, and XTR9 (50, 51). These enzymes have a pH
is expected that certain structural characteristics (e.g., optimum between 6.0 and 6.5, and have low (if any)
fold) will also apply for XET. XET has been sug- hydrolytic activity. TCH4 and XTR9 have a relatively
gested to contain at least one disulde bond, which low temperature optimum of 18 C, whereas Meri-5 is
appears to be important for maintaining enzyme most active at 28 C. TCH4 and Meri-5 are not inu-
activity. Most XETs contain a consensus sequence enced by fucosylation of the donor substrate. In con-
of DEIDFEFLG. Bacterial -glucanases contain a trast to this, XTR9 has a clear preference for
sequence very similar to this, DEIDIEFLG. The nonfucosylated donor substrate. The three XETs pre-
DEIDIE residues are located along the bottom of fer XLLG over XXXG and XXFG as an acceptor
the active site of these enzymes. The second glutamate substrate. However, only XTR9 seems to prefer
(E) in this motif has been implicated as one of the XXFG over XXXG. Further, XTR9 has a lower
catalytic residues of the enzyme. For XET it has been Km value for the XLLG acceptor and is signicantly
shown that the rst glutamate is important for inhibited by high levels of XLLG. The activity of
enzyme activity (48). TCH4 and Meri-5 is abolished upon removal of N-
The sequence of many XETs or XET-related pro- linked glycosylation, whereas the activity of XTR9 is
teins have been described. For Arabidopsis it has been unaffected by such treatment.
shown that it contains > 20 different XET (or XET- The best-characterized member of group III is nas-
related) genes. The various XET sequences can be clas- turtium (Tropaeolum majus) XET, often referred to as
sied into four subgroups (I through IV) (40). Most NXG1 (52). This enzyme is characterized by its rela-
sequence divergence is found in the carboxy terminal tively higher preference for water as acceptor substrate;
part of the proteins. Arabidopsis sequences are present i.e., it can catalyze hydrolysis reactions next to trans-
in groups IIII, suggesting that plants have acquired glycosylation reactions. It is unlikely that NXG1
specialized XETs to catalyze particular processes. requires high-molecular-weight donor substrates. This
Group III XETs differ from the enzymes in the other enzyme plays a role in the mobilization of xyloglucan
groups in that they have a C-terminal extension and a reserves in seeds during germination, and has long
putative (N-linked) glycosylation site at a different been regarded as a xyloglucan-specic endoglucanase.
position in the protein. Enzymes from these groups Typically, the highly conserved active site motif is
will be discussed in more detail below. Group IV slightly different in NXG1 (and also in other members
only contains two barley XET sequences, and will of group III), DELDIEFLG instead of DEIDFEFLG
not be discussed further. (40). In principle, one could say that the bacterial -
The biochemical properties of two XETs in group I glucanases hold an intermediate position between
have been mapped. Unfortunately, different experi- NXG1 and most other XETs with respect to this
mental procedures/conditions were used, which motif (compare with motif above). In addition, the

group III XETs have a small loop of three extra Important questions to be answered are what deter-
amino acids upstream of this motif. At the moment mines the hydrolytic activity of some XETs? and
it is still unclear whether these special NXG1 features what underlies the requirement for high molecular
are responsible for its relatively high hydrolytic activ- weight donor substrates for some XETs? For family
ity. The substrate specicity of NXG1 was investigated 12 endoglucanases, we have discussed already (see pre-
by Fanutti et al. (52). A xylosidase (see previously) was vious section) that subtle differences between these
used to tailor the specic substrates GXXGXXXG enzymes can determine whether or not they have xylo-
and GLLGXLLG, which were subsequently treated glucanase activity. In analogy to this, it may be
with NXG1. It appeared that GXXGXXXG was expected that particular amino acid substitutions
converted to GXXGGXXGXXXG and XXXG. underlie donor and acceptor substrate specicity of
GXXG(#)G#XXGXXXG could be cleaved as indi- XETs. In addition, it remains to be established whether
cated (cleavage site between brackets is likely but XETs can display a different mode of action toward
does not follow unambiguously from the experiments). XXXG-type or XXGG-type xyloglucan.
GLLGXLLG was converted to GLLGGLLGXLLG
and XLLG. GLLG(#)GLLGXLLG was degraded in
a different manner as GXXGGXXGXXXG (cleavage
site between brackets is likely but does not follow V. DETERMINATION OF ENZYME
unambiguously from the experiments). From these ACTIVITY ON XYLOGLUCANS
and other experiments a number of important conclu-
sions with respect to donor and acceptor substrate spe- Many different assays can be used for studying conver-
cicity could be drawn: sions of xyloglucans. Simple reducing end-group ana-
1. NXG1 can use acceptor substrates, which do lysis can point out whether enzymes or enzyme extracts
not carry a xylosyl residue on the nonreducing glucosyl contain xyloglucanase activity. It should be realized
residue. that xyloglucans contain relatively few cleavable gly-
2. NXG1 does not require xylosyl substitution at cosidic linkages (less than one out of seven!). As a result
glucose residues 4, 2, 1, and 3 (for nomenclature of this, it may be necessary to use higher substrate
see Fig. 5). concentrations than one would use for unbranched
3. Xylosyl substitution at 2 (and possibly 3) is substrates (to avoid substrate limitation). High-
a requirement for NXG1. performance size exclusion chromatography (HPSEC)
4. Galactosyl substitution of a xylosyl residue at with refractive index (RI) detection may be used
1 prevents, and at 2 modies, chain cleavage by to determine differences in molecular-weight distribu-
NXG1. tions. High-performance anion exchange chromatogra-
The overview above shows that there can be impor- phy (HPAEC) with an electrochemical detection unit is
tant differences in the properties of XETs, even within suitable for monitoring the release of oligosaccharides
one subgroup. Much more research is needed to under- from a polymer. Appropriate reference oligosacchar-
stand the structure-function relationships of XETs. ides cannot be obtained commercially, and should be
puried by oneself. Mass spectrometry (MS) is a very
useful tool in structural elucidation of reaction pro-
ducts. In particular, liquid chromatography/tandem
mass spectrometry (LC-MSMS) in combination with
18
O-labeling of reaction products may detect differ-
ences in mode of action between two endoglucanases.
In this case, incubations are done in the presence of
18
O-labeled water; tandem-MS is used to sequence
the individual oligosaccharides (53).
Reducing end-group assays are not applicable for
determining XET activity (except for NXG1-like
Figure 5 Xyloglucan fragment illustrating the pattern of enzymes catalyzing hydrolysis reactions), because the
xylose substitution of the glucan backbone around the site number of reducing ends remains constant during
of chain cleavage (arrow). For details on xyloglucan struc- transglycosylation. Several methods to quantify the
ture, see Figure 1C. For signicance of numbering, see text. transglycosylation activity of XET have been
(From Ref. 52.) described. These protocols are all based on a polysac-

charide-to-oligosaccharide transglycosylation. Three Many processes in the food industry aim at a com-
methods are discussed below. plete degradation of the plant cell wall (the so-called
1. Xyloglucan oligosaccharides are radiolabeled liquefaction process). For instance, in fruit juice man-
by exchanging the hydrogen at the reducing terminus ufacturing enzyme preparations are applied to increase
of the molecule with 3 H (39, 40). Radiolabeled juice yield. Also the recovery of important metabolites
oligosaccharides are subsequently incorporated into from plant material can be facilitated by using mix-
polymeric xyloglucan by transglycosylation. The tures of cell wall-degrading enzymes. In many cases,
radiolabeled products are sufciently long to be xyloglucanases are an important component of these
adsorbed on a piece of lter paper (cellulose). After a cocktails. In apple cell wall degradation, a Trichoderma
washing step to remove the unreacted 3 H-oligosacchar- reesei endoglucanase with xyloglucanase activity works
ides, the transglycosylation activity can be simply mea- particularly well with pectinases (55). The ratio of var-
sured by counting the radioactivity of the lter paper ious endoglucanases in commercial enzyme prepara-
with a scintillation counter. tions also deserves attention. It is known for quite
2. In the second method, the xyloglucan (acceptor) some time that cellulose is most efciently degraded
oligosaccharides are tagged at the reducing end with a by a mixture of glucanases: endoglucanase and cello-
UV or uorescent label (e.g., 2-aminopyridine) (39, biohydrolase (CBH). The endoglucanase produces new
40). The transglycosylation reaction produces uores- chain termini for the CBH to work on. It is less known
cent xyloglucans with an increased molecular weight. that efcient degradation of cell wallembedded cellu-
These are separated by HPSEC, and quantied by a lose (i.e., cellulose which is coated with xyloglucan)
uorescence or UV detector. It may be advantageous requires (at least) one more glucanase, namely xyloglu-
to couple a RI detector to the HPSEC/UV system in canase. The xyloglucanase increases the accessibility of
order to visualize a shift in molecular-weight distribu- cellulose by removing its xyloglucan coating (14).
tion of the polymeric xyloglucan. In the previous sections, we have seen that many
3. The third method is probably the easiest, endoglucanases and XETs can be present in plant tis-
because it does not involve radiolabeling or compli- sues. It has been suggested that these enzymes may act
cated derivation procedures of the acceptor oligosac- in concert during fruit ripening. In this process, the EG
charides. The method is based on the fact that generates xyloglucan oligosaccharides, which are sub-
xyloglucans with a molecular weight > 10 kDa form sequently used as acceptor substrates by XET in a
a blue-green complex with iodine, whereas fragments polysaccharide-to-oligosaccharide transglycosylation
< 10 kDa remain unstained (54). XET activity can be (56). As a result of this, xyloglucan will be more
determined colorimetrically by measuring the disap- rapidly depolymerized. This rapid breakdown of xylo-
pearance of the blue-green-colored iodine:xyloglucan glucan may be related with fruit softening, although
complex in the presence of xyloglucan acceptor oligo- also other enzymes like pectinases are known to play
saccharides at 620 nm. an important role in this process. It is expected that the
For a more qualitative analysis of XET action LC- shelf life of fruits can be extended when these enzyme
MSMS is a valuable analytical tool (see previous para- activities can be kept at a low level. It has also been
graph) (53). This method could, for instance, be used shown that the level of XET in apple fruit can be an
to detect differences in mode of action of various important factor in the liquefaction process. The endo-
XETs. genous XET may act in concert with exogenous (fun-
gal) endoglucanases (57, 58). The endoglucanases
generate xyloglucan oligosaccharides on the outside
of the apple tissue. These oligosaccharides diffuse
VI. APPLICATIONS IN THE FOOD into the tissue and serve as acceptor substrates for
INDUSTRY XET. High-XET apple fruit could be processed
much faster than low-XET apple fruit, because it
Enzymes with activity toward xyloglucan can be used is degraded from both the inside and the outside dur-
in a number of processes which are relevant for the ing liquefaction. This may explain observations by the
food industry. Most applications are related to proces- industry that the processing time can vary from one
sing of vegetables and fruits. Other applications are fruit batch to another.
more at the ingredient level. Some examples in which Xyloglucans, which are stored in the secondary cell
enzymic modication of xyloglucans can play a role wall of seeds (for instance, tamarind seed), are often
will be discussed below. used as a viscosifyer in the food industry. The gelling

properties of these polysaccharides can be modulated 7. J-P Vincken, A de Keizer, G Beldman, AGJ Voragen.
by treatment with -galactosidase from plant (59) or Fractionation of xyloglucan fragments and their inter-
fungal origin (60). Removal of 40% of the galactosyl action with cellulose. Plant Physiol 108:15791585,
residues from xyloglucan resulted in self-association of 1995.
8. T Hayashi, T Takeda, K Ogawa, Y Mitsuishi. Effects
the polymers, and consequently the formation of a gel.
of the degree of polymerization on the binding of
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( 100 C); i.e., there are two sol-gel transition points 9. MC McCann, B Wells, K Roberts. Direct visualiza-
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73
Enzymes Degrading Rhamnogalacturonan and Xylogalacturonan
Jean-Paul Vincken, Alphons G. J. Voragen, and Gerrit Beldman

I. INTRODUCTION identication. It is expected that the interest in these

enzymes will continue to grow, because they may also
In a previous section of this book (Chap. 66), the struc- be important in tailoring bioactive oligosaccharides.
ture of pectin was discussed in detail. Besides homo- With the completion of the Arabidopsis genome-
galacturonans (often referred to as the smooth regions sequencing project, plant biologists have obtained
of pectin), also branched or hairy regions occur (1). clues that homologs of the enzymes discussed in this
The hairy regions are composed of a collection of poly- chapter are also present in plants; these enzymes may
saccharides: rhamnogalacturonan (RG), xylogalactur- be involved in remodeling plant cell wall structure dur-
onan (XGA), arabinan, and (arabino)galactan. This ing development.
chapter deals with carbohydrases having activity As a consequence of their fairly recent discovery,
toward the former two. Both polysaccharides can be the picture of RG- and XGA-degrading enzymes is
referred to as heterogalacturonans. A third heteroga- rather incomplete when compared to that of the
lacturonan present in plant cell walls, rhamnogalactur- more classical pectinases such as endopolygalacturo-
onan II, has a very complex structure. At this moment, nase (endoPG), endopectin lyase (endoPL), and endo-
no enzymes with activity toward this polysaccharide pectate lyase (endoPAL). Much less sequence
have been characterized. Homogalacturonan-degrad- information is available for these enzymes, and sys-
ing enzymes, as well as arabinan- and galactan-degrad- tematic studies to delineate the principles underlying
ing enzymes, are described elsewhere in this book their structure-function relationships still have to get
(Chap. 70). started. Pectinases can be assigned to various enzyme
In comparison with homogalacturonan-degrading families. This family classication is based on simila-
enzymes, enzymes with activity toward RG and rities in apparent secondary structure characteristics of
XGA are newcomers in the eld of carbohydrates. proteins (http://afmb.cnrs-mrs.fr/ pedro/CAZY/) and
Reasons for this are the following: (a) industrial inter- is updated regularly. In principle, pectinases with very
est in these enzymes has only recently been roused by low amino acid sequence identity can be attributed to
new fruit juice manufacturing techniques (see further); the same family; i.e., the fold of the proteins seems to
(b) the enzymes are usually minor components of com- be much better conserved than their sequence. Within
mercial enzyme preparations used by the food indus- one family, the catalytic mechanism (retaining or
try; (c) substrates for these enzymes are often difcult inverting) and the three-dimensional structure of the
to obtain from commercial resources; and (d) the protein are thought to be similar.
enzymes produce complex mixtures of degradation A selection of classes of pectin-degrading enzymes is
products, requiring sophisticated analytical tools for summarized in Table 1. Enzymes with activity toward

Table 1 Overview of Galacturonan-Degrading Enzymesa
Enzyme Organism Fam. No. entries Comments
endoPG Aspergillus niger GH28 7b 3D structure available

exoPG Aspergillus tubingensis GH28 1 Active toward xylogalacturonan
endoXGH Aspergillus tubingensis GH28 1 Homologous to endoPG, presumably
requires xylosyl sidechains for
activity
put. PG Arabidopsis thaliana GH28 55 Very limited biochemical data
available, sequences cluster in
various groups
endoRGH Aspergillus niger GH28 2 RGHs differ in the presence of a C-
terminal extension, 3D structure of
Aspergillus aculeatus RGH is
available
RG Rha-hydrolase Aspergillus aculeatus ??? n.a. No sequence information available
RG GalA-hydrolase Aspergillus aculeatus ??? n.a. No sequence information available,
inverting enzyme
endoPAL Erwinia chrysanthemi L1 10 3D structure available, various strains
put. endoPAL Arabidopsis thaliana L1 23 Very limited biochemical data
available
endoPL Aspergillus niger L1 3 3D structure available
exoPAL Erwinia chrysanthemi L2 1
endoPAL Erwinia chrysanthemi L3 1
endoRGL Aspergillus aculeatus L4 1
put. endoRGL Arabidopsis thaliana L4 7
endoPAL Erwinia chrysanthemi L9 1
exoPAL Erwinia chrysanthemi L9 1
endoPAL Pseudomonas cellulosa L10 1 Modular protein
endoRGL Pseudomonas cellulosa L11 1 Modular protein
a
Enzymes with activity against rhamnogalacturonan are in boldface. For more details, see text.
b
Number indicates that family GH28 contains seven different endoPGs from Aspergillus niger. EndoPGs from many other species are also
members of this family, but these are not shown.
branched galacturonans are shown in boldface. lyases (L1 and L4). Again, at present the plant
Glycosyl hydrolase family 28 (GH28) is a particularly enzymes have not been characterized. Despite the
large family, comprising enzymes with a very diver- large differences in substrate specicity (homogalac-
gent substrate specicity. Hydrolases with activity turonan, XGA, and RG) and catalytic mechanism
toward homogalacturonan, XGA, and RG are pre- (hydrolase versus lyase), it turns out that the three-
sent within this family. Further, GH28 contains dimensional structures of pectin-degrading enzymes
many putative proteins from plant origin. An inter- are very similar (2). All the enzymes investigated so
esting observation is that Arabidopsis members of far possess the characteristic right-handed parallel -
GH28 outnumber those derived from one fungus helical structure, which has been discussed for homo-
(e.g., Aspergillus niger). It remains to be established galacturonan-degrading enzymes elsewhere in this
whether these Arabidopsis proteins represent an array book. In this chapter, we will focus on microbial
of different pectinases with activity toward the var- enzymes, because there are currently no biochemical
ious galacturonan-containing polymers in plants. data available for the plant enzymes. XGA-degrading
Surprisingly, galacturonan lyases are distributed enzymes will be discussed rst, followed by RG-
over as many as seven different families (L1L4 and degrading enzymes. Subsequently, a few words will
L9L11). Also in this case the Arabidopsis genome- be said on the detection of enzyme activity toward
sequencing effort has provided clues on the possible heterogalacturonans. Finally, applications with rele-
existence of an abundance of plant galacturonan vance to the food industry will be discussed.

II. ENZYMIC DEGRADATION OF product. The exoPG can release Xyl-GalA disacchar-
XYLOGALACTURONANS ides and monogalacturonic acid from the nonreducing
terminus of xylogalacturonan (see Fig. 1). Thus, it
In the literature, there are a number of reports describ- seems that the A. aculeatus exoPG is tolerant to xylosyl
ing a homogalacturonan with -D-Xylp-(1 ! 3) side substitution of the homogalacturonan backbone, a fea-
chains (3, 4). This polysaccharide is referred to as xylo- ture also encountered in other Aspergillus exoPGs (10).
galacturonan (XGA). Besides the xylosyl branches,
methoxyl groups can be attached to the O-6 of GalA B. Endopolygalacturonase
residues (3). So far, the presence of XGA in plants
seems to be conned to reproductive organs (fruits, EndoPGs can also show activity toward saponied
seeds) (5), such as apple (3), pea (4), and watermelon XGA; however, this seems to depend greatly on its
(6), and to exudates from Astragalus trees (7, 8). It degree of xylosyl branching. Apple XGA (saponied)
seems that the degree of xylosylation of the homo- was resistant to all endoPGs tested, including endoPGI
galacturonan backbone can differ with the origin of and edoPGII from Aspergillus niger. The less branched
the XGA. For instance, in apple XGA three out of watermelon XGA can be degraded to GalA, GalA2 ,
four GalA residues carry a xylosyl sidechain, whereas GalA3 , and a series of Xyl-containing oligogalacturo-
in watermelon XGA this is only one out of four. In nides by a commercial endoPG preparation (6).
apple, XGA seems to be covalently linked to RG (3); Oligogalacturonides with a Xyl:GalA ratio of 1:4 and
for other species, this has not been investigated in so 1:5 seem to be abundant in the XGA digest. These
much detail. results suggest that endoPGs are not as tolerant to
xylosyl substitution as exoPG. They require a number
A. Exopolygalacturonase of contiguous, unbranched GalA residues (presumably
more than three) for activity.
Exopolygalacturonase (exoPG) from Aspergillus acu-
leatus was the rst enzyme of which activity toward C. Endoxylogalacturonan Hydrolase
unesteried XGA was demonstrated (9). This enzyme
is not specic for XGA, as (unesteried) homogalac- Very recently, an endoacting enzyme with activity
turonans are also degraded, with GalA as the only toward XGA was discovered by screening an
Figure 1 Schematic illustration of xylogalacturonan degradation by exopolygalacturonase (closed arrowheads) and endoxylo-
galacturonan hydrolase (open arrowheads). Both enzymes are hindered by the presence of methoxyl groups. Numbers indicate
1st, 2nd, 3rd, etc., cleavage of the substrate by the enzyme. Open circles represent -D-GalpA residues, shaded circles -D-Xylp
residues, and small closed triangles methoxyl groups. NR indicates the nonreducing end; R, the reducing end.

Aspergillus tubingensis expression library in the yeast (1 ! 4] or longer neutral sidechains. The latter are
Kluyveromyces lactis (11). A modied gum tragacanth often referred to as the hairs, and are mainly com-
(saponied and treated with dilute acid; Xyl:GalA ratio posed of galactosyl and/or arabinosyl residues. The
of 1:2) was used as a substrate, and activity was detected GalA residues can be acetylated at the O-2 and/or
using a reducing end-group assay (12). Amino acid O-3 position. This section will focus on the enzymes
sequence comparisons have shown that endoxylogalac- which are required to convert RG to its constituent
turonan hydrolase (endoXGH) is a GH28 member, just monosaccharides.
like endoPG, exoPG, and endorhamnogalacturonan
hydrolase (endoRGH). Typically, it shares the highest A. Endoacting Rhamnogalacturonan-Degrading
homology with exoPG, which is in accordance with its Enzymes
substrate specicity (11). EndoXGH can degrade sapo-
nied apple hairy regions (containing XGA); the di- The rst rhamnogalacturonan-degrading enzyme was
saccharide Xyl-GalA and an unknown oligosaccharide discovered during fractionation of a commercial
are the main end products of this treatment. Digestion enzyme preparation derived from Aspergillus aculeatus
of the modied gum tragacanth results in a complex (13). The enzyme acted exclusively on RG, and showed
mixture of many xylosylated oligogalacturonides (11). no activity on homogalacturonan. A few years later,
The low activity of endoXGH toward unesteried this enzyme, as well as another rhamnogalacturonase
homogalacturonan and its much higher activity toward from A. aculeatus, was cloned. The enzymes were
saponied XGA suggest that the enzyme has a require- termed RGaseA and RGaseB, respectively (14, 15). It
ment for xylosyl side chains (contrary to exoPG, which appeared that the two enzymes differed in their cata-
has a tolerance to xylosyl branches). Therefore, it is lytic mechanism. RGaseA is a hydrolase, meaning that
believed that endoXGH cleaves XGA between two it cleaves the RG backbone with the concomitant
xylosylated GalA residues (see Fig. 1), but this needs uptake of a water molecule. Degradation of RG by
to be further substantiated. RGaseB follows a -eliminative reaction; a double
bond is formed between C-4 and C-5 of the nonredu-
D. Other Enzymes cing GalA residues of the reaction products (16, 17).
This reaction can be monitored spectrophotometrically
It seems likely that more enzymes exist with activity on at 235 nm. Therefore, RGaseA and RGaseB were
XGA. In apple hairy regions, XGA can be highly ester- renamed to endorhamnogalacturonan hydrolase
ied with methoxyl groups. This polymer can not be (endoRGH) and endorhamnogalacturonan lyase
deesteried by pectin methylesterase (PME) (3), sug- (endoRGL), respectively. Thus, it seems that fungi
gesting that this enzyme is also hindered by xylosyl side have acquired a similar set of enzymes for RG degra-
chains, just like endoPG (10). At this moment it is dation as for that of homogalacturonan (endoPG,
unknown whether nature offers PMEs which are tol- endoPAL, endoPL).
erant to xylosyl branching. The Xyl-GalA disaccharide Over the years, a number of new sequences have
can be cleaved by -xylosidase (10). It is unknown been added to the sequence databases. The GH28
whether this enzyme is also active toward polymeric family contains four entries of endoRGHs: the one
XGA. Possibly, XGA may become digestible by from A. aculeatus (14, 15); rhgA and rhgB from A.
endoPG after treatment with an appropriate -xylosi- niger (18); and one from Botryotinia fuckeliana (19).
dase. Note that A. niger rhgB is not related to A. aculeatus
RGaseB. Comparison of the primary structure of the
enzymes reveals a number of regions, some of which
III. ENZYMIC DEGRADATION OF are pectinase specic, and some of which are
RHAMNOGALACTURONANS endoRGH specic (19). Typically, rhgB from A.
niger and endoRGH from B. fuckeliana contain C-
Rhamnogalacturonan (RG, also referred to as RG-I) is terminal extensions, which are not present in the two
the second heterogalacturonan of which the enzymic other RGHs. It is currently unknown whether this
degradation is described in this chapter. This polysac- extension affects the properties of the enzymes. The
charide can have a backbone composed of as many as endoRGLs have so far been assigned to two polysac-
100 repeats of the disaccharide [! 2--L-Rhap- charide lyase families. The A. aculeatus endoRGL
(1 ! 4)--D-GalpA-(1 !] (5). The rhamnosyl residues belongs to family L4, whereas the recently discovered
can be substituted at O-4 with single unit [-D-Galp- Pseudomonas cellulosa endRGL forms a new family

(L11), together with the uncharacterized proteins are presumably the catalytic ones. The distance
YesW and YesX from Bacillus subtilis, and PSP from between Asp156 and Asp180 is 10 A, which is con-
Streptomyces coelicolor (20). It is interesting to note sistent with the spacing between catalytic residues gen-
that the P. cellulosa endoRGL is equipped with a cel- erally found for inverting enzymes. Another striking
lulose-binding module, which is very uncommon for feature of endoRGH is its high degree of glycosylation.
pectinases. The signicance of this is unknown. The The enzyme contains almost 6 kDa of carbohydrates
P. cellulosa endoRGL differs from the one from A. on a total molecular mass of 52 kDa. As a result of
aculeatus in having a higher pH optimum (9.5 vs. 6) this, endoRGH is highly water soluble. N-glycan trees
and an absolute requirement for Ca2 . [Man1-6(Man1-3)Man1-4GlcNAc1-4GlcNAc]
The three-dimensional structure of the A. aculeatus are linked to Asn32 and Asn299. The C-terminus of
endoRGH has been solved (21), and appears to be very the molecule (amino acid residues 367422) contains
similar to that of other pectinases (2). EndoRGH folds all O-linked glycosylation sites (Thr, Ser), which are
into a large right-handed parallel helix (four parallel monosubstituted with mannosyl residues. Figure 2
sheets), with a core composed of 13 turns of clearly shows that the glycosylation sites are not uni-
strands (see Fig. 2) (21). The potential active site is a formly distributed over the enzyme. Despite the fact
groove, oriented almost perpendicular to the helical that the structure of endoRGH has been solved, the
axis. Four amino acids with acidic side chains are important question of which structure-function rela-
located in the groove. Of these, Asp156 and Asp180 tionships determine whether an GH28 member has
Figure 2 The three-dimensional structure of A. aculeatus endoRGH. (A1) Cartoon indicating the four parallel sheets; the
putative catalytic amino acids, Asp156 and Asp180, are indicated as black sticks. (A2) View into the superhelix of the molecule
at a 90 rotation to A1. (B1) Space-lling model of endoRGH (similar view as with cartoon A1) clearly showing the substrate-
binding groove of the enzyme in which the catalytic residues (in black, only one visible) are located. The illustration also shows
that endoRGH is heavily glycosylated. Amino acids with O-linked glycosylation (Thr, Ser) and those with N-linked glycosylation
(Asn) are shown in black. (B2) View of endoRGH at a 180 rotation to B1 to show that glycosylation is conned to one side of
the molecule.

endoPG or endoRGH activity remains to be answered. at the nonreducing end, and a Rhap at the reducing
At this moment, no structural information is available terminus (17, 24). Similar products are released from
for endoRGL. saponied hairy regions by the P. cellulosa rgl11A (20).
Detailed structural characterization of reaction pro- In addition, this enzyme seems to produce smaller
ducts has shown that endoRGH and endoRGL cleave fragments, presumably with a 4,5GalpA-Rhap di-
the RG backbone at different positions (17, 2224). saccharide backbone. The difference in specicity of
The oligomeric products of A. aculeatus endoRGH endoRGH and endoRGL are illustrated in Figure 3.
consist of two or three Rhap-GalpA disaccharide From the formation of oligomeric products dis-
units, with a rhamnosyl at the nonreducing end and cussed above, it might be concluded that endoRGH
a galacturonosyl residue at the reducing end (22, 23). and endoRGL are active toward a similar part of the
The rhamnosyl residues can be substituted with single RG backbone. However, it is possible that the various
units of galactose. The two A. niger endoRGHs release RG-degrading enzymes recognize parts of the back-
a similar set of oligosaccharides from saponied hairy bone. When the same saponied hairy regions are
regions (this is a RG preparation containing arabinan degraded with various RG-degrading enzymes, differ-
and galactan side chains, after treatment with alkali to ent shifts in the molecular-weight distribution of the
remove acetyl groups) as the A. aculeatus enzyme (18), polymeric products are evidenced (14, 20). Removal
but the ratio between the various oligosaccharide proof the arabinan hairs from saponied hairy regions
ducts is different for the three enzymes. It seems as if increased the catalytic efciency of A. aculeatus
A. niger rhgB accumulates the largest products of the endoRGL, whereas removal of galactan hairs
three endoRGHs, including a number of larger oligo- decreased the efciency (24). The P. cellulosa rgl11A
saccharides that have not yet been characterized. The can degrade a galactosylated oligosaccharide with a
oligosaccharides produced by A. aculeatus endoRGL [Rhap-GalpA]3 backbone, whereas this oligosacchar-
differ from those formed by endoRGH in that they ide is not degraded anymore after treatment with a
contain an unsaturated GalpA residue (4,5GalpA) -galactosidase (20). These examples show that side
Figure 3 Schematic illustration of enzymes involved in the degradation of rhamnogalacturonan. Both endoRGH and endoRGL
are hindered by the presence of acetyl groups. Numbers indicate 1st, 2nd, 3rd, etc., cleavage of the substrate by the enzyme. Open
circles represent -D-GalpA residues, closed boxes -L-Rhap residues, shaded triangles -D-Galp, and small shaded circles acetyl
groups. NR indicates the nonreducing end; R, the reducing end.

chains may play an important role in molecular recog- Figure 4 summarizes the mode of action of three dif-
nition. The various RG-degrading enzymes may differ ferent enzymes toward linear RG oligosaccharides of
considerably in their tolerance to side chains, or they different length. Both endoRGHs require a backbone
may even have a requirement for side chains. of at least nine or ten glycosyl residues for activity,
From a quantitative analysis of the total number of whereas endoRGL requires longer ones (12 glycosyl
polymeric and oligomeric reaction products the num- residues). The A. aculeatus endoRGH prefers to cleave
ber of splits an enzyme makes per encounter with its four or ve glycosyl residues away from the nonredu-
substrate can be estimated. It appears that endoRGH cing terminus of the oligosaccharide, and shows multi-
and endoRGL from A. aculeatus differ in their degree ple attack toward the substrates offered (26). The B.
of multiple attack: 4.0 and 2.5, respectively (24). This fuckeliana enzyme cleaves ve or seven glycosyl resi-
implies that endoRGL is a more randomly acting dues away from the reducing terminus of the substrates
(or less processive) enzyme than endoRGH. Linear (19). The activity of this enzyme is distributed over a
RG oligosaccharides have been used to further sub- number of linkages, instead of consistently attacking
stantiate the mode of action of RG-degrading enzymes. the same linkage of a particular substrate (see Fig. 4).
Such substrates can be prepared by treating pectin with In this case, it may be more appropriate to speak of
dilute acid, and subsequent fractionation of the forth- bond cleavage frequencies. The B. fuckeliana
coming fragments by chromatography (25). endoRGH does not show processivity. The A. aculea-
The linkage between Rha and GalA is more easily tus endoRGL prefers to cleave the smaller substrates
hydrolyzed by acid than the one between GalA and four residues from the reducing rhamnose. The longest
Rha. As a result of this, the linear RG oligosaccharides oligosaccharide (DP 16) is cleaved at 6 units from the
carry a rhamnosyl residue at their reducing terminus. reducing terminus. These examples demonstrate that
Figure 4 Mode of action of Aspergillus aculeatus and Botryotinia fuckeliana endoRGH and Aspergillus aculeatus endoRGL on
degalactosylated rhamnogalacturonan oligosaccharides of various length. Thick arrows indicate preferred cleavage sites; less
preferred sites are indicated with thinner arrows. Closed arrowheads marked with 2 indicate the second cleavage site of the
enzyme. Open circles represent -D-GalpA residues; and closed boxes -L-Rhap residues.

RG-degrading enzymes can differ considerably in their cated in the paragraphs above. These enzymes are
mode of action. dealt with in more detail elsewhere in this book.
Another important enzyme for the degradation of
B. Rhamnogalacturonan Rhamnohydrolase RG in its natural setting is RG acetyl esterase
(RGAE). This enzyme specically can remove 70%
RG-rhamnohydrolase was puried from a commercial of the acetyl groups present in hairy regions; acetyl
pectinase preparation (27). Characteristic of this esters present in homogalacturonans are not hydro-
enzyme is its absolute preference for terminal rhamno- lyzed (30). Acetyl groups hinder the action of both
syl residues (1,4)-linked to -galacturonosyl residues A. aculeatus endoRGH and endoRGL. When hairy
(see Fig. 3). The enzyme was unable to split p-nitro- regions are incubated with a combination of
phenyl--rhamnoside. The RG-rhamnohydrolase can endoRGH and RGAE, a more extensive decrease of
degrade the products formed by endoRGH, together the molecular weight is found than with endoRGH
with the enzymes mentioned in the following para- alone (31). Similar results were obtained with
graphs. Prior to the action of the rhamnohydrolase, endoRGL. RGAE is discussed in more detail in the
removal of galactosyl residues from the RG oligosac- esterase section of this book (Chap. 67).
charides is required. At this moment, no sequence
information on this enzyme is available, and conse-
quently the enzyme has not been attributed to a glyco- IV. DETERMINATION OF ENZYME ACTIVITY
syl hydrolase family. The enzyme acts with inversion of ON HETEROGALACTURONANS
the anomeric conguration (28).
Many different assays can be used for studying con-
version of rhamnogalacturonans. Simple reducing end-
C. Rhamnogalacturonan Galacturonohydrolase group analysis can point out whether enzymes or
enzyme extracts contain RG-degrading activity. In
The RG-galacturonohydrolase can act in concert with the case of lyase activity, spectrophotometric measure-
RG-rhamnohydrolase in the degradation of products ments at 235 nm is a convenient option (17, 24). High-
released from hairy regions by endoRGH (see Fig. 3). performance size exclusion chromatography (HPSEC)
This enzyme has a striking specicity for the release of with refractive index (RI) detection may be used to
galacturonosyl residues from the nonreducing end of determine differences in molecular-weight distributions
RG oligosaccharides (29). Oligogalacturonides were (13, 14). High-performance anion exchange chromato-
not degraded by the enzyme. Also, no activity was graphy (HPAEC) with an electrochemical detection
detected toward the products released from hairy unit is suitable for monitoring the release of oligosac-
regions by endoRGL, showing that the enzyme is charides from a polymer (14, 23, 24). Appropriate
unable to release 4,5GalpA residues. From kinetic reference oligosaccharides cannot be obtained com-
experiments with linear RG oligosaccharides it could mercially, and should be puried by oneself. Mass
be concluded that low-molecular-weight substrates spectrometry (MS) is a very useful tool in structural
were hydrolyzed with the highest catalytic efciency. elucidation of reaction products. In particular, liquid
At this moment, no sequence information for RG- chromatography/tandem mass spectrometry (LC-
galacturonohydrolase is available, and it is therefore MSMS) in combination with 18 O-labeling of reaction
unknown whether this enzyme has homology with products may detect differences in mode of action
for instance exoPG. The initial product of hydrolysis between two rhamnogalacturonases. In this case, incu-
was -galacturonic acid, showing that RG-galacturo- bations are done in the presence of 18 O-labeled water;
nohydrolase is an inverting enzyme (28). tandem-MS is used to sequence the individual oligo-
saccharides (32).
D. Synergism in Rhamnogalacturonan
Degradation
V. APPLICATIONS IN THE FOOD
Many enzymes are involved in the degradation of hairy INDUSTRY
regions. Endogalactanase, endoarabinanase, arabino-
furanosidase, and -galactosidase may all facilitate Degradation of branched pectins, hairy regions, or
the action of RG-degrading enzymes, as has been indi- heterogalacturonans is at this moment probably

mainly applied in fruit juice manufacturing, in parti- structure of this plant material. Novel auxiliary
cular when the juice is made by the so-called liquefac- enzymes remain to be discovered for improving the
tion process (33). In this process, fruit cell walls are recovery of valuable nutrients from soy.
extensively degraded by a mixture of pectinases,
hemicellulases, and cellulases. Eventually, the cells
cannot withstand the osmotic pressure anymore, and REFERENCES
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up to 100%), this process has also introduced some Pectinases. Amsterdam: Elsevier, 1996, pp 319.
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the nal clarication step. In apple juice manufac- xylogalacturonan subunit present in the modied
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8. DMW Anderson, DAD Grant. The chemical charac-
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Undoubtedly, the heterogalacturonan-specic exoen- 712, 1996.
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to note that soy pectin is different from that of other xylogalacturonan. Biotechnol Appl Biochem 30:53
57, 1999.
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11. CJB van der Vlugt-Bergmans, PJA Meeuwsen, AGJ
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of RG and XGA. Soy is an extremely important hydrolase, a novel pectinolytic enzyme. Appl
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two rhamnogalacturonan hydrolase genes from syluronohydrolase. An enzyme that specically
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74
Enzymic Hydrolysis of Cereal (1 ! 3,1 ! 4)-b-Glucans
Maria Hrmova and Geoffrey Fincher

University of Adelaide, Glen Osmond, South Australia, Australia
I. INTRODUCTION ents of the dietary ber component of human foods,

which is considered to be of salutary importance in
The 1 ! 3; 1 ! 4)--glucans are important compo- several areas of human digestion and health (3).
nents of cell walls in members of the Poaceae family Against this background, we will describe in this
of higher plants (1). The 1 ! 3; 1 ! 4)--glucans chapter the enzymes that are responsible for the com-
consist predominantly of long, linear chains of glucosyl plete depolymerization of cereal 1 ! 3; 1 ! 4)--glu-
residues that are linked via 1 ! 3)- and 1 ! 4-- cans to glucose. This description will be focused on
glucosidic linkages; small amounts of protein may be well-characterized enzymes from germinated grain
associated with the polysaccharide (2). Although their and young seedlings of barley and maize and is likely
distribution in higher plants is restricted to cell walls of to be equally relevant to the hydrolysis of
the Proaceae, the 1 ! 3; 1 ! 4)--glucans are consti- 1 ! 3; 1 ! 4)--glucans from wheat, rice, and other
tuents of most human diets and many animal feed cereals. Finally, we will present selected examples that
formulations, because the Poaceae include key cereal illustrate how a detailed understanding of enzyme
species, such as wheat, rice, maize, barley, rye, sor- structures and substrate specicities can provide
ghum, and millet. As cell wall components, the opportunities for the manipulation of commercially
1 ! 3; 1 ! 4)--glucans usually make a relatively important enzymes via newly emerging technologies,
minor contribution to the total weight of cereal grains, which can be used to enhance performance of these
but they can have a disproportionately large impact on enzymes in the food industry.
grain technology, utilization, and nutrition. This
impact is largely attributable to the propensity of
these polysaccharides to be extracted from walls with II. STRUCTURE AND PROPERTIES OF
aqueous solvents and thereafter to form solutions of CEREAL (1 ! 3; 1 ! 4)-b-GLUCANS
high viscosity. Thus, in baking with cereal ours, the
1 ! 3; 1 ! 4)--glucans and other wall polysacchar- Before embarking on a description of 1 ! 3; 1 ! 4)-
ides will inuence dough rheology, and in malting and -glucan endo- and exohydrolases, it is necessary to
brewing they can adversely affect the efciency of malt provide a little more information on the structure
extraction, ltration processes, and the quality of the and properties of the polysaccharide itself. This infor-
nal beer. Similarly, 1 ! 3; 1 ! 4)--glucans can mation will be important in subsequent discussions on
have undesirable effects on the digestibility of cereal- substrate specicity and substrate binding of individual
based stockfeeds by monogastric animals such as pigs enzymes that are involved in 1 ! 3; 1 ! 4)--glucan
and poultry. In contrast, they are important constitu- depolymerization.

A. Cell Walls of the Poaceae cosyl residues are separated by variable numbers of
adjacent (1 ! 4)--glucosyl residues.
In the primary cell walls of higher plants, a network of In 90% of cases, the single (1 ! 3)--glucosyl resi-
cellulosic microbrils is embedded in a matrix which dues are separated by two or three adjacent (1 ! 4)--
consists mainly of polysaccharides, but may also con- glucosyl residues, as shown in Figure 1, but up to 10%
tain a secondary network of structural proteins (1, 4, of the water-soluble 1 ! 3; 1 ! 4)--glucan from
5). Matrix-phase polysaccharides include xyloglucans, barley consists of blocks containing four to 14 contig-
heteroxylans, and pectic polysaccharides. Cell walls of uous (1 ! 4)-glucosyl residues (9). Similar cellulosic
the Poaceae are characterized by a matrix phase which blocks of adjacent (1 ! 4)--glucosyl residues are
consists predominantly of 1 ! 3; 1 ! 4)--glucans detected in 1 ! 3; 1 ! 4)--glucans from oats (11).
and arabinoxylans; xyloglucans and pectic polysac- The linkage sequence in barley 1 ! 3; 1 ! 4)--glu-
charides are relatively less abundant than they are in cans has been studied at two levels. At the rst level,
walls of many dicotyledonous plants (1, 4). The major the absence of adjacent (1 ! 3)--glucosyl residues
wall polysaccharides found in the starchy endosperm indicates that (1 ! 3)- and (1 ! 4)-linkages are
of various cereal grains are compared in Table 1, where arranged nonrandomly, because if 30% of -glucosyl
it can be seen that 1 ! 3; 1 ! 4)--glucan content residues are (1 ! 3)-linked, one would expect to nd
generally ranges from trace amounts to 75% of the frequent blocks of two or more advance (1 ! 3)--
wall, and from 0.1% to 8% of total grain weight. glucosyl residues. At the second level, however, math-
ematical analyses of the arrangement of the blocks of
two or three adjacent (1 ! 4)--glucosyl residues,
B. Chemical Properties of Cereal which together account for 90% by weight of the
1 ! 3; 1 ! 4)-b-Glucans polysaccharide, shows that these cellotriosyl and
cellotetraosyl blocks (Fig. 1) are randomly distribu-
In considering the chemical structures of cereal ted (10).
1 ! 3; 1 ! 4)--glucans it must be remembered that
these polysaccharides occur as a heterogeneous family
with varying molecular sizes and ne structural fea- C. Physical Properties of Cereal 1 ! 3; 1 ! 4)-
tures. However, several 1 ! 3; 1 ! 4)--glucan frac- b-Glucans
tions have been characterized in detail, and their
overall structures are similar, if not identical. The Estimates of molecular masses of cereal
1 ! 3; 1 ! 4)--glucans from oats, wheat, and barley 1 ! 3; 1 ! 4)--glucans vary widely, mainly because
generally contain 70% (1 ! 4)--glucosyl residues of the different polysaccharide fractions that have been
and 30% (1 ! 3)--glucosyl residues (7, 8). Adjacent examined and because of the different methods used to
1 ! 3--glucosyl residues are seldom if ever detected determine molecular mass values. Sedimentation equi-
in these polysaccharides, but the single 1 ! 3)--glu- librium ultracentrifugation indicates that water-soluble
Table 1 Relative Abundance of 1 ! 3; 1 ! 4)--Glucans in Cereal Grains and in Cell Walls from the Starchy Endosperm
Approximate cell wall composition
Overall (1 ! 3,1 ! 4)-

-glucan contenta (1 ! 3; 1 ! 4-
Cereal species (% grain weight) -Glucan Arabinoxylan Pectin Glucomannan Xyloglucan Cellulose
Wheat 0.50.75 20% 70% 7% 4%

Barley 4.57.7 75% 20% 2% 2%
Rice 0.13 trace 40% 10% NDb trace 48%
Oats 2.56.6 ND 60% ND ND ND ND
Rye 1.92.9 ND 65% ND ND ND 20%
a
Range of mean values from many varieties.
b
Not determined.
Source: Ref. 6.

Figure 1 Distribution of linkages in barley (1 ! 3; 1 ! 4)--glucans. G -glucosyl residues; 3 1 ! 3-linkages;
4 1 ! 4-linkages; red reducing end; arrows sites of hydrolysis by (1 ! 3; 1 ! 4)--glucan endohydrolases (EC
3.2.1.73). (From Ref. 10.)
barley 1 ! 3; 1 ! 4)--glucans have weight average grain, where a signicant proportion of total endo-
molecular weights in the range 200,000300,000 (12, sperm glucosyl residues are embodied in cell wall
13). These values correspond to polysaccharides con- 1 ! 3; 1 ! 4)--glucan (15), it might be expected
taining 12001800 glucosyl residues. that a battery of enzymes would catalyze the complete
The high viscosities of cereal 1 ! 3; 1 ! 4)--glu- conversion of the polysaccharide to glucose, which
can solutions can be attributed not only to the high could be translocated as an energy source to the devel-
molecular masses of the polysaccharides, but also to oping seedling. In elongating coleoptiles, only partial
their high degree of molecular asymmetry (Fig. 2). hydrolysis of the 1 ! 3; 1 ! 4)--glucan might be
Woodward et al. (9) showed that the water-soluble required to loosen crosslinking polysaccharides in
barley 1 ! 3; 1 ! 4)--glucan has an axial ratio the wall during turgor-driven cell expansion.
(average length:average width) of 100. It might be
anticipated that such long, extended molecules would A. Enzymes That Release Large Fragments of
aggregate and precipitate from aqueous solution. 1 ! 3; 1 ! 4)-b-Glucans
However, the irregular spacing of the (1 ! 3)-linkages
along the polysaccharide chain introduces irregularly The rst step involves a hypothetical endoacting
spaced links that preclude extensive intermolecular enzyme (designated Endo-X in Fig. 3) that releases
associations and prevent precipitation from solution. polymeric wall 1 ! 3; 1 ! 4)--glucan into solution,
Thus, the arrangement of the (1 ! 3)- and (1 ! 4)-- without hydrolyzing it to low-molecular-mass oligo-
glucosyl residues along the polysaccharide chain saccharides. An enzyme with this action pattern in
accounts for the solubility of large 1 ! 3; 1 ! 4)-- germinated barley grain has been the subject of persis-
glucan molecules in water, their extended exible chain tent reports in the malting and brewing literature, and
conformations, and their tendency to form aqueous has been given the name -glucan solubilase (16). The
solutions of high viscosity (6). It is precisely these enzyme has not yet been puried for detailed charac-
properties that enable relatively low abundance terization, and in one report it was suggested that the
1 ! 3; 1 ! 4)--glucans to exert such a large inu- enzyme originated from microorganisms that reside on
ence on cereal grain utilization in food and related the surface of the barley grain (17). However, another
industries. 1 ! 3; 1 ! 4)--glucan endohydrolase extracted
from maize coleoptiles also releases relatively high-
III. OVERVIEW OF ENZYMIC molecular-mass 1 ! 3; 1 ! 4)--glucan from cell
HYDROLYSIS OF 1 ! 3; 1 ! 4)-b- walls (18). It is unlikely that the maize coleoptiles
GLUCANS would be heavily contaminated with microorganisms.
Furthermore, the NH2 -terminal sequence of the maize
Enzymes that mediate the depolymerization of wall- endohydrolase (19) matches the sequences of numer-
bound 1 ! 3; 1 ! 4)--glucans and their degradation ous cDNAs in the rice and maize EST databases, and
products have been mostly extracted from germinated is clearly of plant origin.
barley grain, young barley seedlings, or maize or barley
coleoptiles. Several different types of enzymes would be B. 1 ! 3; 1 ! 4)-b-Glucan Endohydrolases
required to completely depolymerize 1 ! 3; 1 ! 4)--
glucans to glucose. Candidate enzymes and their action Much more precise information is available for the
patterns are summarized in Figure 3. In germinated 1 ! 3; 1 ! 4)--glucan endohydrolases of the EC

Figure 2 Computer-generated instantaneous conformation of barley (1 ! 3; 1 ! 4)--glucan. (From Ref. 14.)
3.2.1.73 group. These enzymes catalyze the hydrolysis C. Hydrolysis of Oligosaccharides

of (1 ! 4)--glucosyl linkages but only where these
linkages are immediately adjacent, on the reducing The 1 ! 3; 1 ! 4)--oligoglucosides released by
terminal side, to (1 ! 3)--glucosyl residues (Fig. 1). 1 ! 3; 1 ! 4)--glucan endohydrolases can be
Thus, the 1 ! 3; 1 ! 4)--glucan endohydrolases of further hydrolyzed to glucose (Fig. 3). This process
this group have a strict requirement for adjacent will be particularly important in the germinated
(1 ! 3- and 1 ! 4)--glucosyl residues (Fig. 1), grain. The enzymes responsible for the hydrolysis of
and the enzymes release a series of oligosaccharides the 1 ! 3; 1 ! 4)--oligoglucosides have not been
that contain (1 ! 4)--glucosyl residues and a single identied unequivocally, although -glucosidases (EC
(1 ! 3)--glucosyl residue at the reducing terminus 3.2.1.21) and a group of broad-specicity -glucan
(Figs. 1, 3). Tri- and tetrasaccharides are the most exohydrolases are likely to be involved (Fig. 3). The
abundant products of hydrolysis, but higher oligosac- latter enzymes can hydrolyze not only the
charides of up to 10 or more (1 ! 4)--glucosyl resi- 1 ! 3; 1 ! 4)--oligoglucosides but also polymeric
dues, again with a single reducing terminal (1 ! 3)-- 1 ! 3; 1 ! 4)--glucans and other polysaccharides
glucosyl residue, are released from the cellulosic (18, 24, 25).
regions of cereal 1 ! 3; 1 ! 4)--glucans (20, 21). Because the substrate specicities, action patterns,
The 1 ! 3; 1 ! 4)--glucan endohydrolases are and three-dimensional structures of the
abundant in germinated cereal grains (20, 22), where 1 ! 3; 1 ! 4)--glucan endohydrolases, the -gluco-
they are clearly key enzymes in the degradation of cell sidases, and the -glucan exohydrolases are now well
walls of the starchy endosperm. They are also detected dened, these will be discussed individually and in
in young leaves and roots, but are not detected in detail in the sections below. However, the denition
elongating barley coleoptiles (23). of these enzymic properties depends on the availability

lases hydrolyze polymeric (1 ! 3; 1 ! 4--glucans,
(1 ! 3; 1 ! 4--oligoglucosides, 40 -nitrophenyl -
glucoside (4NPG), and a range of other oligo- and
polysaccharides (25). The (1 ! 3; 1 ! 4--glucan
endohydrolases are absolutely specic for (1 ! 3;
1 ! 4--glucans. Putative -glucosidases hydrolyze
not only 4NPG, but also (1 ! 4--oligoglucosides
(27). During the early stages of purication, it may
therefore be necessary to assay column fractions with
a number of substrates in order to monitor the activity
of a particular glucan endo- and/or exohydrolase.
Figure 3 Enzymic hydrolysis of cell wall (1 ! 3; 1 ! 4)-- Huber and Nevins (28) specically measured the
glucans. For explanation see text in Section III. Symbols are maize coleoptile (1 ! 3; 1 ! 4--glucan endohydro-
as described in legend to Figure 1. lase by adding HgCl2 to the cereal (1 ! 3; 1 ! 4--
glucan substrate; HgCl2 inhibits the -glucan exohy-
drolases, but not the endohydrolases.
of highly puried enzyme preparations and a range of
additional analytical techniques. Some methodological
strategies for the purication and characterization of B. Problems with Enzyme Classication
1 ! 3; 1 ! 4)--glucan hydrolases are outlined in the
next section. The broad specicity of certain cereal (1 ! 3; 1 ! 4-
-glucan hydrolases not only presents problems for
the selection of a diagnostic substrate during purica-
IV. EXPERIMENTAL APPROACHES TO
tion and characterization, as described above, but
ENZYME CHARACTERIZATION
also creates problems for the classication of the
A. Enzyme Purication and Assay Procedures enzyme into existing Enzyme Commission categories
(29). Thus, the cereal -glucan exohydrolases have
The development of a protocol for the preparation of such a broad specicity that they do not t any EC
highly puried enzyme is considered of paramount classication.
importance for studies on the enzymic hydrolysis of Similarly, cereal -glucosidases are difcult to
cereal 1 ! 3; 1 ! 4)--glucans. Enzymes extracted place in existing EC categories (27), because they
from germinated barley grain and elongating coleop- hydrolyze cello-oligosaccharides at signicantly higher
tiles are generally amenable to purication by frac- rates than synthetic aryl -glucosidic substrates such
tional precipitation with ammonium sulfate, cation as 4NPG. Partly because of these difculties, a new
and/or anion exchange chromatography, and size method for the classication of glycoside hydrolases
exclusion chromatography (18, 24, 26, 27), but has gained considerable support in recent years. This
additional steps involving hydrophobic interaction method is based on sequence alignments and hydro-
chromatography, chromatofocusing, or afnity phobic cluster analysis (HCA) (30), and has been used
chromatography are usually required to achieve high to group the glycoside hydrolases into 90 families
levels of purity. (http://afmb.cnrs-mrs.fr/CAZY) (31). It is becoming
Enzyme purity can be rigorously demonstrated clear that HCA is particularly useful for identifying
through gel electrophoresis, provided sufcient similarities in enzymes for which amino acid sequence
enzyme is loaded onto the gel. A single protein similarities are relatively low, and that members of
band on an overloaded gel, and NH2 -terminal each family have similar three-dimensional conforma-
amino acid sequence that shows a single sequence at tion (32). Thus, the structural classication pro-
the expected yields, are sound complementary proce- vided by HCA usefully complements the EC
dures through which enzyme purity can be con- substrate specicity classication. For example,
dently assessed. the HCA method shows that -glucosidases can be
Another potential complication during the purica- classied into families 1 and 3, and this further
tion of cereal (1 ! 3; 1 ! 4--glucan hydrolases is emphasizes the inadequacies of designating an enzyme
that assay systems may not be specic for a particular as a -glucosidase simply because it hydrolyzes the
enzyme type. Thus, the cereal -glucan exohydro- synthetic substrate of convenience, 4NPG (27).

C. Primary Structures of Enzymes To solve the 3D structures of enzymes that partici-
pate in cereal (1 ! 3; 1 ! 4--glucan hydrolysis, x-
Preliminary amino acid sequence information can be ray crystallography remains the method of choice;
obtained by direct NH2 -terminal sequencing of the indeed, the structures of a barley (1 ! 3; 1 ! 4--glu-
puried enzyme or by sequencing peptides generated can endohydrolase and a barley -glucan exohydrolase
from the puried enzyme by peptidase digestion. To have now been solved by x-ray crystallography (34,
dene the complete primary structure of the enzyme 35). The major bottleneck in the technology is prob-
it is usually necessary to isolate and sequence a corre- ably associated with difculties in obtaining high-qual-
sponding cDNA. If the amino acid sequence deduced ity crystals. Crystals can take up to several months to
from the nucleotide sequence of the cDNA exactly grow to the 0.21 mm size required by most x-ray
matches the NH2 -terminal and peptide amino acid crystallographers (36). If a good-quality native data
sequences determined directly from the puried set can be collected from the x-ray diffraction patterns,
enzyme, then the primary structure of the enzyme together with data sets for heavy-metal derivatives of
can be considered solved. However, it is often at this the enzymes, crystallographers can generally solve the
stage that we discover that multiple isoforms of an structure of the enzyme.
enzyme exist. Indeed, most enzymes involved in cereal A nal point to be made here is that protein model-
(1 ! 3; 1 ! 4--glucan hydrolysis are encoded by ing is nding an increasing important place in dening
multigene families, and care must be taken to dene likely 3D structures of enzymes for which no crystal-
exactly which isoenzyme is under examination. lographical data are available. If the complete primary
structure of an enzyme can be deduced from a cDNA,
and if the 3D structure of a closely related enzyme
D. Specicity and Action Pattern
from the same family of glycoside hydrolases has
been solved, it is possible to use modeling software
Once the purity of an enzyme is clearly established, its
programs to generate a likely 3D structure for the
activity on a broad range of alkyl and aryl -glucosidic
enzyme. The limitations and constraints of modeling
substrates, oligosaccharides, and polysaccharides can
must be acknowledged, but there are several techni-
be checked quickly and easily. Analysis of the products
ques that can be used to evaluate the reliability of
released during hydrolysis by thin-layer chromatogra-
the model (37).
phy or other simple procedures will reveal whether the
enzyme exhibits an endo- or an exohydrolytic pattern.
Proton NMR can be used to measure the anomeric V. CEREAL (1 ! 3; 1 ! 4-b-GLUCAN
conguration of released reducing-end monosacchar- ENDOHYDROLASES
ide residues, and hence to classify the particular
A. Substrate Specicity and Action Pattern
enzyme into retaining or inverting groups (27,
33).
Two (1 ! 3; 1 ! 4--glucan 4-glucanohydrolases
(EC 3.2.1.73) from barley have been puried from
E. Three-Dimensional Structures extracts of germinated grain and characterized (20,
21). Both are members of the family 17 group of glyco-
Perhaps the ultimate data that allow catalytic side hydrolases (31). Similar enzymes are found in ger-
mechanisms, substrate specicity, and substrate bind- minated wheat (22, 38), rye (39), sorghum (40), and
ing to be dened in precise molecular terms are pro- other cereals (41). The properties of the two barley
vided by detailed knowledge of the 3D structure of isoforms are compared in Table 2. Unlike other gluco-
the enzyme of interest. Furthermore, 3D structural side hydrolases that hydrolyze cereal (1 ! 3; 1 ! 4-
information can quickly reveal rational design oppor- -glucans, the substrate specicity of the EC 3.2.1.73
tunities that can be used to engineer enhanced perfor- enzymes from barley is absolute; the enzymes will only
mance into a commercially important enzyme. hydrolyze (1 ! 4--glucosyl linkages where these are
Increased temperature or pH stability, and altered immediately adjacent, toward the reducing end of the
substrate specicity are but a few examples of how polysaccharide, to a (1 ! 3)--glucosyl residue, as
3D structural information can be applied for the shown in Figure 1. Reaction products are
manipulation of enzyme utilization in food industries. (1 ! 3; 1 ! 4--oligoglucosides which have a single
These possibilities are explored further in later sec- (1 ! 3)--linkage at their reducing termini (Figs. 1,
tions of this chapter. 3). This indicates that the enzymes are endohydrolases,

Table 2 Properties of Barley (1 ! 3; 1 ! 4)--Glucan Endohydrolases
Property Isoenzyme EI Isoenzyme EII
Apparent molecular mass 30,000 32,000

Amino acids 306 306
Isoelectric point 8.5 10.6
Carbohydrate 0 4% by weight
Substrate specicity Absolute for (1 ! 3; 1 ! 4)--glucans Absolute for (1 ! 3; 1 ! 4)--glucans
Anomeric conguration Retained during hydrolysis Retained during hydrolysis
Glycosyl hydrolase classication Family 17 Family 17
Protein fold (=8 Barrel (=8 Barrel
Catalytic acid/base Glu93 Glu93
Catalytic nucleophile Glu232 Glu232
Substrate binding subsites 58 58
Expression sites Scutellum, young vegetative tissue, Aleurone
aleurone
Source: Refs. 20, 21. 23, 33, 34, 41, 42.
as does their ability to very rapidly reduce the viscosity enzyme-substrate interactions by diffusing nonhydro-
of (1 ! 3; 1 ! 4--glucan solutions (20). Chen et al. lyzable S-glycoside substrate analogs (44) into barley
(33) used proton NMR to show that the anomeric (1 ! 3; 1 ! 4--glucan endohydrolase crystals, in
conguration of released products is retained during the expectation that the substrate analogs will be
hydrolysis. bound but that their S-glycosidic linkages will not
be cleaved by the enzyme. Thus, the substrate analogs
B. Three-Dimensional Structure should remain associated with the enzyme during the
generation of x-ray diffraction data.
The 3D structure of barley (1 ! 3; 1 ! 4--glucan Measurements of the substrate binding cleft that
endohydrolase isoenzyme EII has been determined at runs across the surface of the barley (1 ! 3; 1 ! 4-
2:22.3 A resolution by x-ray crystallography (34). -glucan endohydrolase indicate that it is long enough
The enzyme adopts a (=8 TIM barrel fold. The most to accommodate up to eight glucosyl residues of the
striking feature of the enzyme is a deep substrate-bind- (1 ! 3; 1 ! 4--glucan substrate (34).
ing cleft that extends across its surface (Fig. 4). The
open cleft is consistent with the enzymes endohydro-
VI. CEREAL b-GLUCOSIDASES
lytic pattern, because it would allow the enzyme to
bind its substrate at essentially any position along the A. Substrate Specicity and Action Pattern
polysaccharide backbone for the hydrolysis of internal
glycosidic linkages. Two -glucosidases puried from extracts of germi-
nated barley grain have been designated isoenzymes
C. Substrate Binding I and II (26, 27, 45), and their properties are
shown in Table 3. The enzymes are members of the
The precise details of substrate binding and the iden- family 1 group of glycoside hydrolases (31), although
tity of amino acid residues involved have not been it is difcult to classify them into existing Enzyme
dened. When relatively long (1 ! 3; 1 ! 4--oligo- Commission groups. The enzymes hydrolyze 4NPG
glucoside substrates are allowed to diffuse into and are therefore referred to as -glucosidases of the
crystals, they clearly bind to the enzyme. However, EC 3.2.1.21 group (26, 27, 37). However, more detailed
they are subsequently hydrolyzed and products studies on their substrate specicities show that the
diffuse away, even at low temperatures and sub- enzymes hydrolyse a range of di- and oligosacharides.
optimal pHs, and it has therefore not been possible Highest activity is observed for cellodextrin substrates,
to generate diffraction data for the enzyme-substrate for which the rate of hydrolysis increases as the length
complex (M Hrmova, JN Varghese, GB Fincher, of the substrate increases from cellotriose to cellohex-
unpublished). We are now attempting to dene aose (Fig. 5) (26, 27). In all cases, the barley enzymes

Figure 4 Three-dimensional structure of the barley (1 ! 3; 1 ! 4)--glucan endohydrolase. Stereoview space-lling represen-
tation of an (=8 TIM barrel fold. The drawing was generated using RasMol (43). (From Ref. 34.)
remove single glucose units from the nonreducing ends glucosidases are more typical of polysaccharide exo-
of the substrates, and anomeric conguration is hydrolases of the (1 ! 4)--glucan glucohydrolase
retained during hydrolysis (27). The barley -glucosi- group (EC 3.2.1.74). The uncertainties associated
dases also catalyze glycosyl transfer reactions, through with the classication of the barley enzymes are
which higher oligosaccharides containing (1 ! 3)-, acknowledged by Hrmova et al. (37), who propose
1 ! 4)-, and (1 ! 6)--linkages are generated during that the enzyme be referred to as a -glucosidase/-glu-
hydrolysis of 4NPG (37). The increased activity of the can exohydrolase until its native substrate in germi-
enzymes as chain length of cello-oligosaccharides nated barley grains has been identied unequivocally.
increase (Fig. 5) is suggestive of a polysaccharide exo- The preference of barley grain -glucosidases for
hydrolase rather than an enzyme that is specic for (1 ! 4)--oligoglucosides of increasing chain length
low-molecular-mass -oligoglucosides (27). Indeed, suggests that it might have an extended substrate-bind-
the action patterns and specicities of the barley - ing region.
Table 3 Properties of Barley -Glucosidases
Property Isoenzyme BI Isoenzyme BII

Amino acids 471 471
Carbohydrate Not known Not known
Substrate specicity
4NPG Active Active
laminarin Not active Not active
(1 ! 3; 1 ! 4--glucans Not active Not active
(1 ! 4)--oligosaccharides Active Active
Glucosyl hydrolase classication Family 1 Family 1
Protein fold (=8 Barrel =8 Barrel
Catalytic nucleophile Glu391 Glu391
Subsite binding subsites 6 6
Expression sites Developing endosperm Developing endosperm
Source: Refs. 26, 27, 37, 45.

is revealed in these modeling experiments, and this can
be reconciled with the substrate specicity. When
(1 ! 4)--oligoglucosides were used as molecular
rules in the active-site pocket, it became clear that at
least six glucosyl residues would be closely associated
with the enzyme surface (37). This suggests that the
enzyme has ve or six glucosyl-binding subsites.
Furthermore, catalytic amino acid residues are located
near the bottom of the pocket, and the inside surface of
the pocket is lined with aromatic amino acid residues
(Fig. 6) that could be involved with substrate binding,
through stacking interactions with nonpolar surfaces
of glucosyl residues (47). In contrast to the cleft
observed on endoacting polysaccharide hydrolases
(Fig. 4), the dead-end tunnel geometry (Figure 6) is
consistent with an exo-action pattern, through which
Figure 5 Relative rates of hydrolysis of -oligoglucosides by single glucosyl residues are released from one end of
the barley -glucosidase. (From Ref. 27.) the polysaccharide.
VII. THE BROAD-SPECIFICITY b-GLUCAN

B. Three-Dimensional Structure
EXOHYDROLASES
There are no reports of 3D structures for cereal -glu- A. Substrate Specicity and Action Pattern
cosidases. When the primary structure of the barley -
glucosidase isoenzyme II deduced from a cDNA Two barley -glucan exohydrolases, designated isoen-
sequence (26) was analyzed using molecular modeling zymes ExoI and ExoII, have been characterized in
software programs (37), 3D models with very high detail (24, 25, 27). Similar enzymes are found in elon-
reliability scores were built using as a template the gating maize coleoptiles (18) and in dicotyledonous
crystal structure of a cyanogenic -glucosidase from plants (4850). Although two -glucan exohydrolase
white clover (46) (Fig. 6). A deep, funnel-shaped isoenzymes have been puried from barley seedlings,
pocket, or dead-end tunnel, in the barley -glucosidase Southern hybridization analyses suggest that the
Figure 6 Three-dimensional modeled structure of the barley -glucosidase. Stereoview space-lling representation of (=8
TIM barrel fold. The aromatic amino acid residues lining the surface of the active site pocket are colored in black. The drawing
was generated using RasMol (43). (From Ref. 37.)

enzymes are encoded by a family of ve to six genes enzymes have no difculty hydrolyzing mixed linkage
(AJ Harvey, M Hrmova, GB Fincher, unpublished). (1 ! 3; 1 ! 4--oligoglucosides (25, 27). This is in
The properties of barley -glucan exohydrolase isoen- marked contrast to the barley -glucosidase, for
zymes ExoI and ExoII are shown in Table 4. which rates of hydrolysis increase with the chain length
The barley -glucan exohydrolases are exoacting of cello-oligosaccharides (Fig. 5), but (1 ! 3)--oligo-
enzymes that hydrolyze the nonreducing terminal saccharides, (1 ! 3)--glucans, and (1 ! 3; 1 ! 4--
glycosidic linkage in a broad range of aryl -glyco- glucans are hydrolyzed very slowly, if at all. The small
sides, -oligoglucosides, and polymeric -glucans. effect of substrate chain length on hydrolytic rate
The anomeric conguration of the released glucose would suggest that the enzyme has a relatively short
unit is retained, and, at high substrate concentra- substrate-binding region, and indeed, subsite mapping
tions, the enzymes catalyze a range of glycosyl trans- experiments indicate that only two or three glucosyl-
fer reactions (25, 27). The enzymes can be binding subsites are present (M Hrmova, GB Fincher,
condently classed as polysaccharide exohydrolases unpublished). Once these subsites are fully occupied,
rather than -glucosidases because of their ability reaction rates would be maximal and would be
to rapidly release glucose from a number of polysac- expected to be unaffected by any increases in length
charide substrates, including laminarin and cereal of substrates.
(1 ! 3; 1 ! 4--glucans (25). The barley -glucan
exohydrolases can be classied into the family 3
group of glycoside hydrolases (31), but again their B. Three-Dimensional Structure
broad substrate specicities make it difcult to assign
them to existing EC groups. The barley -glucan exohydrolase isoenzyme ExoI was
The barley -glucan exohydrolases also catalyze the recently crystallized by vapor diffusion in the presence
hydrolysis of (1 ! 3)--glucosidic linkages at higher of ammonium sulfate and polyethylene glycol (36).
rates than other linkage types (Fig. 7) (25, 27). Platinum and mercury derivatives of the crystals were
However, hydrolytic rates are relatively independent subsequently obtained, and this allowed the 3D struc-
of the length of substrates, especially at degrees of ture of the enzyme to be solved by x-ray crystallogra-
polymerization of 3 and above (Fig. 7), and the phy at 1.82.2 A resolution (35).
Table 4 Properties of Barley -Glucan Exohydrolases
Property Isoenzyme ExoI Isoenzyme ExoII

Amino acids 605 602
Carbohydrate 4.7% by weight at 3 N-glycosylation Not known
sites
Substrate specicity
4NPG Active Active
(1 ! 3-, (1 ! 3, 1 ! 4-,
(1 ! 3,1 ! 6)--glucans Active Active
(1 ! 2)-, (1 ! 3)-,
(1 ! 4- (1 ! 6)--oligosaccharides Active Active
Glucosyl hydrolase classication Family 3 Family 3
Protein fold (=8 Barrel and (=6 sandwich (=8 Barrel and (=6 sandwich
Catalytic nucleophile Asp285 Asp284
Subsite binding subsites 23 23
Expression sites Mainly in scutellum and coleoptiles, Mainly in scutellum and coleoptiles,
also in young leaves and roots also in young roots and leaves
Source: Refs. 24, 25, 27, 35.

which consists of residues 374599 arranged in an =
6 sandwich. The (=6 sandwich has a six-stranded
-sheet, with three -helices on either side of the sheet
(Fig. 8) (35). At the COOH-terminal region of the
enzyme (residues 500605) is a long, antiparallel
loop. Carbohydrate can be detected on each of three
potential N-glycosylation sites at Asn221, Asn498,
Asn600 (35). Of particular interest in the crystal struc-
ture is the presence of a glucose molecule at the bot-
tom of a surface pocket on the enzyme. The entrance
to the pocket is shaped like a coin slot in a vending
machine and is located near the interface of domains 1
and 2. The glucose is tightly bound to the enzyme and
is likely to be the product of the enzyme-catalyzed
reaction that has not been released after hydrolysis is
complete (Fig. 8). It follows, therefore that the pocket
occupied by the glucose represents the substrate-bind-
Figure 7 Relative rates of hydrolysis of -oligoglucosides by ing region of the enzyme.
the barley -glucan exohydrolase. (From Ref. 27.)
As already noted for the barley -glucosidase (Sec.
VI.B), the 3D structure of the barley -glucan exohy-
The enzyme is globular in shape but has two distinct drolase (Fig. 8) and the positioning of catalytic resi-
domains. The rst consists of 357 amino acid residues dues in its substrate-biding pocket can be reconciled
that fold into a =8 TIM barrel. A 16 amino acid with its substrate specicity (Table 4). At 13 A in
linker joins the rst domain to the second domain, depth, the catalytic pocket could accommodate two
Figure 8 Three-dimensional structure of the barley -glucan exohydrolase. Stereoview space-lling representation of the overall
structure. The (=8 TIM barrel (colored in white) and the (=6 sandwich (colored in light gray) represent domains 1 and 2,
respectively. The linker region connecting the two domains is on the left and is marked in black. Active-site region of the enzyme,
which is bounded by Trp286 and Trp434 (colored in dark gray), contains a trapped glucose moiety (in a dark gray wireframe
representation). The drawing was generated using RasMol (43). (From Ref. 35.)

to three glucosyl residues, given that the distance tohormone gibberellic acid (GA). Expression occurs in
between glycosidic oxygen atoms of two adjacent resi- the scutellar epithelium and in the aluerone layers,
dues in (1 ! 3- or (1 ! 4)--glucans is 56 A (51). where it is under tight spatial and temporal control
Furthermore, the length of the substrate-binding (52, 53). Isoenzyme EI is the predominant isoform
region estimated by subsite mapping is also two to found in the scutellum, while isoenzyme EII is secreted
three glucosyl residues (M Hrmova and GB Fincher, only from the aleurone layer. The genes encoding both
unpublished). In addition, if the pocket is deep enough barley isoenzymes have been isolated, and various
for only two glycosyl residues, the remainder of the sequence motifs that might be related to GA induction
oligomeric or polymeric substrate would project out and tissue specicity of expression have been identied
of the pocket and away from the enzyme surface. (5456).
Thus, substrate binding would be relatively indepen- While the (1 ! 3; 1 ! 4--glucan endohydrolases
dent of the overall substrate shape for the -glucan are clearly involved in depolymerization of wall
exohydrolases, in contrast to the -glucosidases, (1 ! 3; 1 ! 4--glucans in germinated grain, it is
where the substrate has to have the correct shape to not so clear whether or not another endohydrolase
t into the much longer substrate-binding tunnel (Fig. might be responsible for the initial release of relatively
6). Because substrate shape is determined in large part high-molecular-mass (1 ! 3; 1 ! 4--glucan from
by the type of glycosidic linkage, the lack of strict the walls (Fig. 3). As noted in Figure 3, the nal pro-
shape requirements for substrates of the -glucan exo- ducts of hydrolysis of the endohydrolases will be a
hydrolases could explain why substrates with many family of (1 ! 3; 1 ! 4--oligoglucosides. These
different linkage types can be hydrolyzed by this still contain a signicant proportion of total grain glu-
broad-specicity -glucan exohydrolase. cose (15), but their complete hydrolysis to glucose
requires the activity of other enzymes.
In barley, (1 ! 3; 1 ! 4--glucan endohydrolase
VIII. BIOLOGICAL ROLES OF THE isoenzyme EI is also expressed in young leaves and
DIFFERENT ENZYMES IN CEREAL roots, where transcription of the gene is mediated by
(1 ! 3; 1 ! 4-b-GLUCAN auxins (23, 53). The enzyme is also found in young
HYDROLYSIS roots of rice (57). Auxins participate in several physio-
logical processes that are related to wall metabolism in
The availability of precise information on the plants, including cell elongation, vascular differentia-
specicity, action pattern, and kinetics of key tion, phototropic responses, and geotropism. Because
-glucan exo- and endohydrolases from higher plants (1 ! 3; 1 ! 4--glucan endohydrolases of the EC
provides important clues on the biological roles of 3.2.1.73 group appear to be completely absent from
individual enzymes during the hydrolysis of cereal elongating barley coleoptiles (23), a role in cell elonga-
(1 ! 3; 1 ! 4--glucans. The potential roles of the tion seems unlikely.
enzymes in germinated grain, where cell walls of the
starchy endosperm are completely degraded, are com- B. b-Glucosidases
pared in the sections below with their potential roles in
elongating vegetative tissues, where partial hydrolysis The functional role of -glucosidases in cereals is more
of cell wall polysaccharides is believed to loosen the difcult to dene. In barley, the genes encoding the
wall structure sufciently to allow turgor-driven cell enzyme are expressed only in the maturing endosperm
expansion during normal growth and development. of the grain (26). The enzyme itself is also detected in
the late stages of grain development and in ungermi-
A. (1 ! 3; 1 ! 4-b-Glucan Endohydrolases nated grain; activity does not increase after germina-
tion (26, 45). Possible functions of the -glucosidases
It can be condently concluded that the primary func- in developing and germinated grain have been
tion of the EC 3.2.1.73 group of (1 ! 3; 1 ! 4--glu- reviewed by Leah et al. (26), and some of these are
can endohydrolases in germinated grain is in the related to cell wall metabolism. The -glucosidases
hydrolysis of cell wall (1 ! 3; 1 ! 4--glucans. are expressed at a time when (1 ! 3; 1 ! 4--glucan
Both the (1 ! 3; 1 ! 4--glucan substrate and the is being deposited in the walls of starchy endosperm
(1 ! 3; 1 ! 4--glucan endohydrolases are restricted cells (26), and might be involved in trimming or turn-
in their distribution to the Poaceae. The enzymes are over of wall (1 ! 3; 1 ! 4--glucans during their
expressed in germinated grain in response to the phy- synthesis.

Finally, the -glucosidases could be responsible for microbrils to slip past each other if the strength of the
salvaging glucose units from the (1 ! 3; 1 ! 4--oli- crosslinking or matrix phase between the microbrils
goglucosides released from wall (1 ! 3; 1 ! 4--glu- were weakening (5). It is not easy to visualize how an
cans by endohydrolases. The -glucosidases are able to exoacting hydrolase of the -glucan exohydrolase type
hydrolyze not only the more abundant tri- and tetra- would be able to cleave crosslinking polysaccharides
saccharides released by the endohydrolases, but also between cellulosic microbrils. It might also be argued
the longer-chain cellulosic -oligoglucosides that that this fundamental concept as to how cell wall com-
have single (1 ! 3)--glucosyl residues at their redu- ponents might behave during cell expansion is simplis-
cing termini (Figs. 1, 3) (27). The detection of the - tic, and that our inability to describe the enzymology
glucosidases in germinated grain is consistent with of the process reects our generally inadequate under-
such a role in the complete depolymerization of wall standing of the role of walls in cell expansion as a
(1 ! 3; 1 ! 4--glucans to glucose, and, based on whole.
current evidence, we believe that this might be a likely Another possible function of the -glucan exohy-
function of the barley -glucosidases. drolases could be linked to defense strategies devel-
oped by plants to counter pathogen attack. The
C. b-Glucan Exohydrolases enzymes can hydrolze (1 ! 3)-- and (1 ! 3; 1 ! 6-
-glucans of the type commonly found in fungal cell
The -glucan exohydrolases from barley hydrolyze walls (61), and could act in synergy with the better-
polysaccharides such as laminarin and cereal known pathogenesis-related (PR) proteins, (1 ! 3)--
(1 ! 3; 1 ! 4--glucans, as well as a range of -oli- glucan endohydrolases (EC 3.2.1.39), to degrade walls
goglucosides, including (1 ! 3; 1 ! 4--oligogluco- of invading fungi and thus to arrest their growth (25).
sides (Table 4). This broad specicity, together with a
distribution that includes not only the Poaceae but also
many dicotyledons, makes it difcult to assign a single, IX. APPLICATIONS IN THE FOOD
unequivocal function to the enzyme. INDUSTRY
Because the -glucan exohydrolases hydrolyze
(1 ! 4)--glucosidic linkages more slowly than Cell wall (1 ! 3; 1 ! 4--glucans are widely recog-
(1 ! 3)--glucosidic linkages (Fig. 7), it might be nized for their effects on extract viscosity during the
argued that these enzymes are less likely to play a commercial utilization of cereals. Although the
role in hydrolysis of (1 ! 3; 1 ! 4--oligoglucosides (1 ! 3; 1 ! 4--glucans and their highly viscous
in germinated grain than are the -glucosidases. The solutions are considered benecial in human diets (3),
genes encoding the -glucan exohydrolases are tran- in most commercial applications the high viscosity of
scribed in the scutellum of germinated grain, but (1 ! 3; 1 ! 4--glucan solutions is undesirable.
their mRNAs are most abundant in the elongating
coleoptile (AJ Harvey, M Hrmova, GB Fincher, A. Malting and Brewing Industries
unpublished). This latter observation has led to the
suggestion that -glucan exohydrolases function in Attempts by the food and beverage industries to
auxin-mediated cell elongation in growing coleoptiles address these problems through the application of
(24, 58: AJ Harvey, M Hrmova, GB Fincher, unpub- modern technologies have been concentrated so far
lished), where the amount of (1 ! 3; 1 ! 4--glucan in the malting and brewing industries. Specications
in walls decreases markedly during coleoptile growth for barley quality are tailored predominantly for
(59) in a process that has been linked to the wall loos- these industries, and emphasis has been placed on
ening believed to be necessary for cell elongation (60). parameters such as grain size, dormancy, malt
In concluding that the broad-specicity -glucan extract, grain protein content, development of
exohydrolases are likely to play a role in cell elongation starch- degrading enzymes (diastatic power), and
during normal growth and development of plants, it removal of (1 ! 3; 1 ! 4--glucans. High levels of
must be acknowledged that the actual enzymic (1 ! 3; 1 ! 4--glucans in barley malt are considered
mechanism of wall loosening cannot be precisely undesirable because they are often indicative of incom-
explained. Once can visualize wall loosening as a par- plete cell wall breakdown in the starchy endosperm
tial endohydrolysis of polysaccharides that noncova- and are therefore correlated with low values for malt
lently crosslink cellulosic microbrils in the wall; extract (41). Further, undegraded (1 ! 3; 1 ! 4--
turgor pressure-driven forces would allow the cellulose glucans can cause ltration difculties in the brewery,

again because of their propensity to form solutions of powerful promoters on the barley (1 ! 3; 1 ! 4--
high viscosity, and can contribute to the formation of glucan endohydrolase genes, even if these promoters
undesirable hazes in the nal product (62). direct appropriate tissue-specic and temporal expres-
The quality specication relating to the removal of sion patterns, is the potential to upset the metabolic
(1 ! 3; 1 ! 4--glucans can clearly be tackled on two balance in the germinated grain. Given that the grain
fronts. Firstly, breeders can select for barley with has a nite energy potential and well-evolved regula-
inherently low (1 ! 3; 1 ! 4--glucan contents (3), tory mechanisms to ensure that the correct balance of
or for barleys with the potential to produce high levels individual hydrolytic enzymes is obtained, higher levels
of (1 ! 3; 1 ! 4--glucan hydrolases after germina- of (1 ! 3; 1 ! 4--glucan endohydrolases might be
tion. The importance of these two parameters in bar- achieved at the expense of other enzymes, which
ley-breeding programs is underscored by the efforts to might then become rate limiting and cause unpredicted
place on detailed genetic maps the quantitative trait metabolic imbalances and associated difculties in the
loci (QTLs) that control(1 ! 3; 1 ! 4--glucan con- malting and brewing processes.
tent and (1 ! 3; 1 ! 4--glucan endohydrolase activ-
ity (64, 65).
The second approach to the (1 ! 3; 1 ! 4--glu- C. Increased Stability of (1 ! 3; 1 ! 4-b-
can problem in the malting and brewing industries Glucan Endohydrolases
is to produce an improved barley through genetic
engineering technologies. The genes encoding A second approach to increasing (1 ! 3; 1 ! 4--
(1 ! 3; 1 ! 4--glucan synthases have not yet been glucan endohydrolase activity for the malting and
isolated, and manipulation of initial (1 ! 3; 1 ! 4- brewing industries could be to increase the stability
-glucan levels by downregulating this gene is thereof the existing enzyme, without trying to increase
fore not possible at this stage. However, the two expression levels at the gene level. In this approach
genes encoding barley (1 ! 3; 1 ! 4--glucan endo- the enzyme itself would be engineered for increased
hydrolases have been cloned (5456), as have stability, but the native (1 ! 3; 1 ! 4--glucanase
(1 ! 3; 1 ! 4--glucan endohydrolase genes from gene promoter would be retained so that the meta-
various Bacillus spp. (66, 67). Thus, genetic manipu- bolic balance of the germinated grain would not be
lation of genes encoding these endohydrolases could perturbed. The amount of (1 ! 3; 1 ! 4--glucan
result in elevated levels of the enzyme in malt, and endohydrolase in germinated barley grains will clearly
this is in turn could result in the rapid removal of meet the normal physiological requirements for star-
residual, high-molecular-mass (1 ! 3; 1 ! 4--glu- chy endosperm mobilization and successful germina-
can molecules in malt extracts. tion of the grain. However, these enzymes are
irreversibly denatured and almost completely inacti-
B. Increased Levels of (1 ! 3; 1 ! 4-b-Glucan vated at the temperatures used during kilning (up to
Endohydrolases 85 C) or during mashing (usually 65 C) (21, 69).
Thus, if the stability of the enzymes could be
There are several approaches to increasing increased at these temperatures so that more of the
(1 ! 3; 1 ! 4--glucanase activity via genetic engi- enzyme survived the kilning and mashing steps, the
neering. One would be to attach a more powerful pro- higher levels of surviving enzyme could hydrolyze
moter to the native barley (1 ! 3; 1 ! 4--glucan residual high-molecular-mass (1 ! 3; 1 ! 4--glu-
endohydrolase genes. The promoter would need to be cans after mashing.
activated in the appropriate tissue at the appropriate How then, could the loss of (1 ! 3; 1 ! 4--glu-
time, so powerful constitutive promoters that were can endohydrolase activity at elevated temperatures be
active in many tissues throughout growth and devel- addressed? Firstly, heat-stable (1 ! 3; 1 ! 4--gluca-
opment would be of little value in this approach. nases occur naturally in various microbial species. An
However, if a barley -amylase promoter were extremely thermostable (1 ! 3; 1 ! 4--glucanase
attached to a barley (1 ! 3; 1 ! 4--glucan endohy- has been engineered by intragenic recombination of
drolase gene, it might be anticipated that high levels of gene segments from Bacillus macerans and B. amyloli-
the enzyme would be expressed in aleurone cells after quefaciens (67). The gene encoding this hybrid, thermo-
germination, because the expression patterns of -amy- stable (1 ! 3; 1 ! 4--glucan endohydrolase, has
lases and (1 ! 3; 1 ! 4--glucanases in barley are been expressed in barley protoplasts (70 and in trans-
similar (68). The major disadvantage of using more genic barley (71).

Another approach to preventing the loss of Mr. Andrew Harvey and Mr. Richard Stewart for
(1 ! 3; 1 ! 4--glucan endohydrolase activity at the their contributions to various aspects of the work,
temperatures encountered during malting and brewing and Dr. Jose Varghese for his patience in teaching us
would be to engineer enhanced thermostability into the the principles of x-ray crystallography.
barley enzyme itself. Random mutagenesis of the
cDNA or gene encoding the barley enzyme could be
used to generate thermostable mutants. Alternatively,
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75
Enzymology of Endo-1,4-b-Mannanases
Henrik Stalbrand
Lund University, Lund, Sweden
I. INTRODUCTION different monomeric sugar units linked with O-glyco-

sidic linkages (1, 2). The two major groups of hemi-
Mannan-based polysaccharides are abundant in nat- cellulose, heteroxylans and heteromannans, have,
ure; galactoglucomannan is the major softwood hemi- respectively, predominantly xylose units or mannose
cellulose, and galactomannans are common storage units in the main chain. The major softwood hemicel-
polysaccharides in certain plant seeds. The major lulose is acetylated galactoglucomannan which com-
depolymerazing enzyme of these and similar polysac- prises up to 20% of the softwood content (2). It
charides is endo-1,4--mannanase (-mannanase, EC has -1,4-linked mannose and glucose residues in the
3.2.1.78), which hydrolyzes randomly internal 1,4-- main chain and is substituted with -1,6-linked galac-
D-mannosidic linkages within the backbone of the tosyl sidegroups. Two major types of acetyl galactoglu-
polysaccharide (see Secs. VI and VII). Further hydro- comannans occur in softwoods, one which can be
lysis into monomeric sugars is accomplished by the solubilized in water and one which is alkali soluble
exohydrolases -galactosidase (EC 3.2.1.22) and - (mannosyl:glucosyl:galactosyl ratio of 3:1:1 and
mannosidase (EC 3.2.1.25). Heteromannan degrada- 3:1:0.1, respectively) (1). Hardwoods contain 35%
tion is important in exogenous and endogenous con- unsubstituted glucomannan (2). The degree of poly-
version of plant polysaccharides. merization (DP) of galactoglucomannan and gluco-
Several -mannanase-encoding genes from bacteria, mannan hemicellulose is 100 200 (1, 2).
fungi, and plants have been isolated. Generally, - Certain plants contain other types of mannan and
mannanase catalytic modules have molecular masses heteromannans present especially in seeds as storage
of 3045 kDa. Like many glycoside hydrolases, -man- polysaccharides (3). Leguminous seeds from several
nanases may be modular proteins with molecular species contain galactomannan, which has a backbone
masses up to or even above 100 kDa. -Mannanases of -1,4-linked mannose residues with -1,6-galactosyl
(i.e., their catalytic modules) are retaining enzymes side groups. Carob (Ceratonia siliqua) seed galacto-
classied in family 5 and family 26 of glycoside hydro- mannan (contained in locust bean gum) and guar
lases (see Sec. IX). galactomannan have a DP of 1500 and 900, respec-
tively (4). The mannosyl to galactosyl ratio is 5=1 for
locust bean gum and 2/1 for guar gum (4). The galac-
II. THE SUBSTRATES tomannans can be extracted from the milled seeds (5).
These polysaccharides can form highly viscous solu-
Hemicellulose is a collective term for a group of diverse tions and are important owing to their use as thick-
plant cell wall polysaccharides, consisting of several eners in the food industry and other industrial sectors.

They are also used in combination with xanthan gum Shibata (9) screened for and detected -mannanase
to form strong gels and in combination with kappa activity in cultures of several fungi using ivory nut
carrageenan to form stronger and more elastic gels mannan or carob galactomannan as carbon source.
(68). Ivory nut (Phytelephas macrocarpa) mannan is In this study, two fungi produced moderate -man-
a linear -1,4-linked insoluble mannan (9). nanase activity when grown on cellulose. Media con-
taining cellulose have also been used for -mannanase
production by Trichoderma reesei (41). Higher levels
III. OCCURRENCE OF b-MANNANASES
of -mannanase were produced on cellulose when
compared to locust bean gum (42). Trichoderma
Both aerobic and anaerobic microorganisms produce
reesei -mannanase production appears to increase
plant cell wall polysaccharide-degrading enzymes,
when glucose is depleted (36). The plant-pathogenic
reviewed in Warren (10). Some of these microorgan-
fungi Sclerotsium rolfsii produced -mannanase
isms are present in decaying plant material, in soil, or
when grown on several carbon sources, including
in the rumen; others are plant pathogens, reviewed in
sorbose, cellulose, mannan, and heteromannans
Warren (10) and Klein and Eveleigh (11).
(43). -Mannanase was produced by the yeast
-Mannanases are produced by species of bacteria,
Aureobasidium pullulans in media containing either
fungi, and plants, and several -mannanase genes from
galactomannan, galactoglucomannan, -1,4-manno-
these organisms have been cloned (1221).
oligosaccharides, or a synthetic nonmetablized glyco-
Furthermore, -mannanase activity has been puried
side: methyl -D-mannopyranoside (44).
from a gut preparation of the snail Helix pomatia (22).
Also, some extremophiles produce -mannanase; the
genes have been cloned from thermophilic bacteria
V. b-MANNANASE MULTIPLICITY
(2325).
-Mannanase activity has also been detected in sev-
Frequently, polysaccharidases are produced in multi-
eral plant seeds, which is correlated to their germina-
ple forms, which at least in some cases, is a reection
tion (26, 27). For example, -mannanase activity has
of the composition and complexity of naturally occur-
been detected in seeds of tomato (2830), Coffea ara-
ring substrates. As an example, usually several geneti-
bica (31) carob, and fenugreek (32). Low levels of -
cally distinct endoglucanases and cellobiohydrolases
mannanase activity were detected in banana fruits dur-
are secreted by cellulolytic organisms (10, 45). -
ing ripening (33). The -mannanase encoding gene
Mannanases are commonly extracellularly produced
from tomato (Lycopersicon esculentum) has been iso-
in multiple forms as observed for some bacteria (20,
lated (17). Several tomato -mannanase isoforms have
46), several plant seeds (26, 29), and several fungi,
been observed during germination (29).
including Scelortium rofsii (47) and Trichoderma
spp. (39, 41). -Mannanase isoforms may be post-
IV. PRODUCTION OF MICROBIAL b- translationally modied products of the same gene
MANNANASES (16) or products of separate genes (21).
Expression of hemicellulases and cellulases among

fungi like Trichoderma and Aspergillus spp. is com- VI. CATALYTIC MECHANISM AND
monly glucose repressed (3437). However, variations MANNAN HYDROLYSIS
even within a species have been observed, partly due to
the development of mutant strains, reviewed by -Mannanases hydrolyze randomly internal 1,4--D-
Kubicek and Penttila (37). -Mannanases are extracel- mannosidic linkages in mannan and heteromannan
lular proteins, but may be cell associated as indicated polysaccharides. The A. niger and the T. reesei -man-
for anaerobes. nanases can efciently hydrolyze manno-oligosacchar-
The most commonly used carbon sources for ides with a DP of 4 or higher; the major hydrolysis
microbial -mannanase production are galactoman- products of ivory nut mannan after extensive incuba-
nan and other mannans. Fungal -mannanases have tion are mannobiose and mannotriose (38, 41).
been produced in submerged cultures on locust bean Mannotriose can only be slowly hydrolyzed and,
gum by, for example, Aspergillus niger (38), owing to secondary hydrolysis of mannotriose (38,
Trichoderma harzianum (39), and Aspergillus awamori 48, 49) mannose may also be produced from ivory
for which also wheat bran was used (40). Reese and nut mannan hydrolysis (50).

Glycoside hydrolases acting on polymeric substrates The pattern of galactomannan hydrolysis and the
often have several substrate binding subsites. Each apparent subsite-binding requirements have been inter-
subsite binds one backbone monosaccharide unit. preted from structural analysis by NMR of isolated
The kinetics of oligosaccharide hydrolysis suggest at hydrolysis products from the galactomannan and glu-
least four subsites for the T. reesei (48) and the A. comannan incubated with A. niger -mannanase (49).
niger (49) -mannanases It is suggested that the enzyme has ve substrate bind-
Each glycoside hydrolase family comprises enzymes ing subsites, , , , and
which is equivalent to
that hydrolyze the glycosidic bond by either of two 3, 2, 1, 1, and 2, according to the nomencla-
catalytic mechanismsone that yields a net retention ture given by Davies et al. (52). Binding to at least four
of the anomeric conguration, and one that yields a subsites is required for efcient hydrolysis.
net inversion (51, 52). Two catalytic residues partici- Substitution of the substrate monomers at two of the
pate in the mechanism: a nucleophile and an acid/base subsite positions (but not at the other) restricted
catalyst. The retaining mechanism is a two-step pro- hydrolysis, most likely by preventing binding (49).
cess. A covalent glycosyl enzyme intermediate is The hydrolysis of pine kraft pulp galactoglucomannan
formed, which is hydrolyzed in the second step (51, by the T. reesei -mannanase was also studied by
52). Retaining glycoside hydrolases may perform NMR analysis of the produced mixed oligosaccharides
transglycosylation (53). Hitherto -mannanases have (55). Judging from this study, the T. reesei enzyme also
been classied in glycoside hydrolase families 5 and appears to be restricted by galactosyl sidegroups in a
26 (see Sec. IX), which have the retaining mechanism. similar way to the A. niger enzyme. Also, the restric-
Accordingly, transglycosylation has been observed for tion by the substrate main-chain glucose residues has
several -mannanases including those from T. reesei been studied for some -mannanases (49, 55).
and A. niger (38, 48, 49).
VIII. MODULAR STRUCTURE OF b-

VII. HETEROMANNAN HYDROLYSIS MANNANASES
-Mannanases also hydrolyze heteromannans. Glycoside hydrolases like cellulases and hemicellulases
However, their activity is generally restricted by the commonly have a modular structure. The catalytic
galactosyl sidegroups and the main-chain glucose resi- modules may be attached to other module(s), such as
dues of the substrate (22). The subsite-binding proper- carbohydrate-binding modules (10). The modules are
ties may vary among enzymes of different origin. The often connected by linker peptides. Furthermore, as
major hydrolysis products from incubation of fungal reviewed (10, 45), anaerobic cellulolytic organisms
as well as bacterial -mannanase with heteromannans like Clostridium spp. frequently form large cell-asso-
are mixed oligosaccharides, mannotriose, and manno- ciated multienzyme complexes. Although several -
biose, sometimes with the production also of mannose mannanases are sole catalytic modules of 3045
(49, 54, 55), as reviewed by Viikari et al. (56). These kDa (13, 15, 18), some -mannanases are modular
products are hydrolyzed further by exohydrolases, also proteins with molecular masses from 50 kDa up to
secreted by the microorganisms. Thus, the cooperation 100 kDa (12, 14, 16, 19, 20, 23, 25, 65). Examples of
of several enzymatic activities is needed for the degra- modular -mannanases from anaerobes are enzymes
dation of heteromannans. In addition to -mannanase which carry an additional endoglucanase catalytic
(EC 3.2.1.78), -galactosidase (EC 3.2.1.22) and - module and cellulose binding modules (12) or putative
mannosidase (EC 3.2.1.25) are also needed for the protein binding modules which may be involved in the
complete hydrolysis of galactoglucomannan into formation of multienzyme complexes (19, 65).
monomeric sugars (57, 58). -Mannosidases hydrolyze Also, some aerobic organisms produce modular -
terminal nonreducing mannose residues, and -galac- mannanases (14, 16, 20). The T. reesei family 5 -man-
tosidases hydrolyze -1,6-linked galactosyl side groups nanase has a C-terminal cellulose-binding module (16,
from oligomeric and sometimes polymeric substrate 38, 66). Biely and Tenkanen (58) discussed the possi-
(5962). -Glucosidase (EC 3.2.1.21) may be needed bility that the presence of a cellulose-binding module
for the hydrolysis of non-reducing-end glucose resi- on the T. reesei -mannanase is part of the reason for
dues. For the complete hydrolysis of acetylated galac- the superior performance in bleaching experiments for
toglucomannan, acetyl esterase is also needed (63, 64). this enzyme (67). The Cellulomonas mi enzyme is the

rst -mannanase reported to contain a mannan-bind- X. STRUCTURES OF b-MANNANASE
ing module (68). It interacts with galactomannan. CATALYTIC MODULES
Generally, endoacting cellulases and hemicellulases are

expected to have an active site which is present in a
IX. CATALYTIC MODULES OF b- quite open cleft, as is the case for structurally deter-
MANNANASES mined family 5 endoglucanases. Judging from the three
-mannanase structures available (71, 72, 76), these
On the basis of sequence and, where available, struc- enzymes also have roughly this general architecture.
tural similarities, the catalytic modules of glycoside These catalytic modules form the Thermomonospora
hydrolases has been classied into different families fusca and Trichoderma reesei -mannanases belong to
by Henrissat et al. (69) (see also http://afmb.cnrs- family 5 of glycoside hydrolases, and thus both struc-
mrs.fr/pedro/CAZY/ghf.html). Enzymes in a parti- tures are =8 -barrels and share the same overall
cular family have a conserved overall fold, conserved fold. In both -mannanase structures the two catalytic
catalytic mechanism and conserved catalytic and glutamates are situated in a groove across the surface
other residues. Known -mannanases belong to of the enzymes. As expected, the conserved tryptophan
family 5 or family 26 of glycoside hydrolases; both in family 5, probably forming the 1 subsite, is present
contain enzymes which have the retaining mechanism. in both structures. It is unclear if the other subsites also
Family 5 and family 26 enzymes belong to the clan are conserved or similar for the two -mannanases.
GH-A (4/7 /-barrel superfamily) of glycoside For the T. fusca enzyme complex structures with man-
hydrolases (70). notriose enabled the identication of subsites 3 and
2 (71). The 1 and 2 subsites were identied for the
T. reesei -mannanase (72).
A. Glycoside Hydrolase Family 5
XI. b-MANNANASE ASSAYS
Family 5 includes several enzymatic activities for
which the corresponding genes have been cloned. A. Conditions for Catalysis
These are, for example, several endo-1,4--glucanases
and cellobiohydrolases in addition to -mannanases. As previously reviewed (22, 56), generally, compared
The genes of family 5 -mannanase have been isolated to bacterial mannanases, fungal -mannanases have
both from prokaryotes (1214, 24) and eukaryotes, i.e., lower pH optima, between 3:0 and 5.5. Among the
from fungi (15, 16) and from a planttomato (17). highest yet reported pH optima for a -mannanase is
The three-dimensional structure of two -mannanases pH 9, for the enzyme from an alkalophilic Bacillus sp.
of family 5 has been determined (Sec. X) (71, 72). (46). In many cases, -mannanases are stable for hours
Structures of family 5 enzymes are (=8 -barrels. at their optimal pH up to a temperature of at least
Eight residues are conserved, including two catalytic 50 C. The Km values reported for Trichoderma and
glutamates (73, 74). Aspergillus -mannanases on locust bean gum varies
between 0.0015 to 0.7 g/L (15, 22, 42).
B. Glycoside Hydrolase Family 26 B. Assays Based on Detection of Reducing

Sugars Produced by Hydrolysis
Family 26 contains several -mannanases from bac-
teria (1820) and from one species of anaerobic fungi The following assay procedure is used for the
(21) for which the genes have been isolated. Two glu- Trichoderma reesei -mannanase (41), and can be
tamates have been identied as the putative catalytic used generally (adjustments of buffer and pH may be
residues (75). At least two additional residues are con- needed depending on the enzyme). -Mannanase activ-
served. Crystallization and preliminary x-ray diffrac- ity is assayed using 0.5% (w/v) locust bean gum
tion studies have been reported for a -mannanase of (SIGMA G-0753). The substrate is suspended in 50
Pseudomonas uorescence subsp. cellulosa (76). Family mM citrate or acetate buffer, pH 5.3, by homogenizing
26 enzymes have been predicted to belong to the clan at 80 C and heating to the boiling point, cooled, and
GH-A and thus to have the (=8 -barrel structure stored overnight with continuous stirring, then centri-
(75). fuged and the pellet discarded. The enzyme sample (0.2

mL) is incubated with 1.8 mL substrate solution at 5 fully washed off and the plate is incubated with 0.1%
0 C for 530 min. The reducing sugars liberated in the Congo Red solution, then destained with 1 M NaCl. -
enzymatic reaction are assayed by the addition of 3 mL Mannanase activity is detected as clear halos. Locust
DNS-reagent, boiling for 5 min, cooling, and measur- bean gum dyed with Remazol Brilliant Blue R can also
ing the absorbance at 540 nm. Recipe for the DNS be used for detection of clones with -mannanase (24).
reagent can be found in Meth Enzymol, Vol 160 -Mannanase activity in single plant seeds can be
(1988), pp 87112. A standard curve is prepared with detected by the plate assay using locust bean gum
D-mannose. The enzymatic activity is given in nano- and Congo Red staining (77).
katals (nkat). An assay based on the quantication of
the released reducing sugars using the Somogyi-Nelson
F. Assays of Other Enzymes Active on
copper agent has also been described (22).
Heteromannans
C. Assay Based on Detection of Dyed
The exoglucosidases -galactosidase, -mannsosi-
Oligosaccharides Produced by Hydrolysis
dase, and -glucosidase can be assayed using colori-
metric detection using nitrophenylglycosides as the
Carob galactomannan dyed with Remazol Brilliant
substrate, as described for example by Ratto and
Blue R (AZO-carob galactomannan, Megazyme,
Poutanen (40).
Wicklow, Ireland) is used as the substrate. After
enzyme substrate incubation for 10 min, ethanol is
added to stop the reaction and to precipitate high-
molecular-weight galactomannan. Low-molecular- XII. APPLICATIONS OF b-MANNANASES
weight fragments stay soluble and can be spectropho-
tometrically quantied at 590 nm, due to the attached -Mannanases have several potential and existing
dye (22). This assay is relative. To relate to enzyme applications in particular in the food and feed, pulp,
activity units, a standard curve (supplied by and paper industries. The industrial applications have
Megazyme for each batch of substrate) with known been reviewed by several authors (56, 78, 79). -
enzyme amounts should be prepared. Mannanase can solubilize softwood pulp mannan
(80, 81) and can be used as an aid in pulp bleaching
D. Zymogram (67, 80, 82). Improvement of animal feed with -man-
nanase has been reported (83). Coffee beans contain
After running a protein sample with isoelectric focus- heteromannan which is extractable with hot water
ing (IEF), bands with -mannanase activity can be from the roasted bean (84). Extracted green coffee
identied by the use of 2% agar overlay gel (2 mm bean mannan has been hydrolyzed with -mannanase
thick) containing 0.5% locust bean gum (41). When (85). A potential application for -mannanase is to
the IEF is done the overlay gel is incubated with the reduce the viscosity of the extract during production
separation gel at 50 C for 45 min; then the overlay gel of instant coffee (78). -Mannanase and other hemi-
is stained with 0.1% Congo Red solution and cellulases may also be used to reduce the viscosity of
destained with 1 M NaCl. -Mannanase activity pineapple juice during processing (86). Over the past
appears as clearing zones. The overlay gel should be few years, attention has been directed toward endogen-
made in a 50-mM buffer with a pH suitable for enzyme ous -mannanase in plant seed germination (27).
activity and stability (50 mM citrate, pH 5.3, for the T. Furthermore, -mannanases may be used in the con-
reesei and A. niger -mannanases). The method also version of biomass and for the hydrolysis and modi-
works with native polyacrylamide gel electrophoresis. cation of polysaccharides. The potential application of
Zymograms can also conveniently be made with AZO- -mannanase in the hydrolysis of galactomannan-
carob galactomannan (20). based uids used in oil and gas well stimulation has
been shown (87).
E. Plate Assays Other hemicellulases, endo-1,4--xylanases, have
after a relatively short time of research and develop-
-Mannanase activity secreted from bacterial or yeast ment, reached major applications especially in pulp
colonies can be detected by growing the microorgan- bleaching (88) and in cereal processing (89).
isms on agar plates containing 0.5% locust bean gum Increased attention for -mannanases will likely open
(16). After making replicas, the microorganism is care- up new applications for these enzymes.

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76
Lysozyme
Akio Kato
Yamaguchi University, Yamaguchi, Japan
I. INTRODUCTION perature, as shown in Figure 1. The activity is retained

> 80% during storage at low temperature for 1 week,
Fleming found the lytic action of human nasal mucus while it is decreased < 60% during storage at room
and tear on Micrococcus cells in 1922 and he reported temperature for 1 week (4). The loss of lysozyme activ-
the similar lytic action of egg white constituent on ity results from the increases in pH during storage of
Gram-positive bacteria. He proposed the name lyso- egg. The pH increases up to 9.3 within several days
zyme for the enzyme and Micrococcus lysodeikticus for storage at room temperature. On the other hand, lyso-
the organism serving as the substrate (1). Lysozyme zyme is stable in acidic pH in the presence of CO2 gas,
cleaves the -(1,4)-glycoside linkages between N-acet- as shown in Figure 1. Lysozyme interacts with ovo-
ylglucosamine and muramic acid in bacterial cell wall mucin in thick egg white. It is well known that the
and chitin or other oligomers. In addition to glycosi- interaction is responsible for the gel structure of
dase activity, this enzyme has transglycosylation activ- thick egg white (5). The ovomucin-lysozyme interac-
ity (2) and esterase activity (3). Thus, numerous names tion decreases with egg white thinning.
have been proposed for this enzyme. The Commission
on Enzyme Nomenclature in 1964 recommended E.C.
3.2.1.17. Systematic name: mucopeptide N-acetylmur- III. UTILIZATION OF LYSOZYME IN
amyl hydrolase; trivial name: mucopeptide glucohy- FOODS
drolase, lysozyme. Lysozyme is easily crystallized
from chicken egg white, and a large amount of the Hen egg white lysozyme is utilized as a food preserva-
enzyme can be obtained. Therefore, lysozyme has tive with antimicrobial effects without any food toxi-
been one of the most intensively investigated and char- cities. The bactericidal action of lysozyme is intensive
acterized enzymes. on Gram-positive bacteria but much weaker on Gram-
negative bacteria, because the former expose the cell
wall consisting of peptidoglycan, while the latter has a
II. LYSOZYME IN FOODS unique cell envelope, which consists of the outer mem-
brane, inner membrane, and peptidoglycan layer.
Lysozyme exists abundantly in chicken egg white and Therefore, the perturbation of the outer membrane
constitutes 3.5% of the total egg white proteins when structure is needed to enhance the susceptibility to
calculated on the basis of lytic activity. Since lysozyme lysozyme of the peptidoglycan layer in Gram-negative
interacts with ovomucin gel in egg white, the content bacteria. Despite this disadvantage, lysozyme is gener-
may be > 3:5%. The lysozyme activity in hen egg white ally used as a food antiseptic. The antimicrobial action
decreases during storage of egg dependent on the tem- may be efcient on Gram-positive bacteria but inef-

greatly enhanced the bactericidal action on E. coli (6).
Nakamura et al. in 1991 reported that the Maillard-
type lysozyme-polysaccharide conjugate also enhanced
the bactericidal action on various Gram-negative bac-
teria (7). The antimicrobial action of lysozyme-dextran
conjugate is shown in Figure 2. The bactericidal action
is enhanced by heating at 50 C. The polysaccharide
attachment seems to stabilize lysozyme and to have
stronger afnity to the outer membrane of Gram-nega-
tive bacteria. These modied lysozymes can be used as
food antiseptics effective on various bacteria.
Figure 1 Effects of the temperature and pH on the relative Lysozyme consists of 129 amino acids and the molecu-
activity of lysozyme in egg white during storage. **, 5 C lar weight is 14,307 daltons. The protein contains many
(nal pH 9.0); **, 20 C (nal pH 9.3); *- - -*, 30 C more basic amino acids (11 arginines and six lysines)
(nal pH 9.4); *- - -*, 40 C (nal pH 9.5); * - *, than acidic amino acids (six aspartic acids and two glu-
stored in an atmosphere of CO2 at 30 C (nal pH 6.5). (From tamic acids), and the isoelectric point is 11.2. The
Ref. 4.) amino acid and nucleotide sequences of prelysozyme
are shown in Figure 3. The sequence 1 to 18 is the
signal peptide, and the N-terminus of mature lysozyme
cient on Gram-negative bacteria. Some attempts to is the position 1 (lysine). Lysozyme has four disulde
enhance the antimicrobial action for Gram-negative bonds: Cys6127, Cys30115, Cys6480, and Cys76
bacteria have been done by our colleagues. Ibrahim 94. Human lysozyme, which consists of 130 amino
et al. in 1991 found that the covalent attachment of acids, is very similar to hen egg white lysozyme in its
palmitic acid residues to the lysyl residues of lysozyme primary and tertiary structures and the positions of
Figure 2 Antimicrobial activity of lysozyme-dextran conjugate for ve Gram-negative bacteria. (a) V. parahaemolyticus IFO
12713; (b) E. coli IFO 12713; (c) A. hydrophila IFO 13286; (d) P. mirabilis IFO 12668; (e) K. pneumoniae IFO 14438. *, Control
(medium without lysozyme or conjugate); *, addition of 0.05% lysozyme; | addition of 0.05% lysozyme-dextran conjugate.
The abscissa is the heating time at 50 C. (From Ref. 7.)

Figure 3 Amino acid and nucleotide sequences of hen egg white pre-lysozyme. The cDNA is inserted between Kpn1 and BamH1
in pUC18. Numbering of the nucleotide sequence starts at the most likely position for the 5 0 end of mRNA derived from the
sequence of the genomic clone. The Met (18) to Gly (1) is the signal peptide and correctly processed in eukaryote cell,
resulting in the N-terminal lysine.
disulde bonds. -Lactalbumin, which consists of 123 mined by x-ray crystallography (8). The ribbon draw-
amino acids, is also homologous to lysozyme in its pri- ing model is shown in Figure 4. As shown in Figure 4, it
mary and tertiary structure and in the position of di- divides the molecule into two partsa predominantly
sulde bonds. The x-ray analysis of hen egg white helical core, and an irregular -strand core. The helical
lysozyme was carried out by Blake et al. in 1965 as core consists of four -helices (515, 2535, 8899, and
the rst enzyme to have its tertiary structure deter- 108115), and the -strand core consists of three -

strand regions (3846, 5054, and 5760). The two
structural domains ( and ) of lysozyme appears to
be formed prior to docking of the domains to generate
the native closed-packed structure (9). The domain
forms persistent structure during folding more rapidly
than the domain. In addition to a -helical domain,
lysozyme contains two 310 helices (7984 and 119124).
The most striking feature of the molecule is the cre-

vice running across the waist of the egg-shaped mole-
cule. The crevice is the active site of lysozyme which
has the binding area for the substrate of hexasacchar-
ide (N-acetylglucosamine/N-acetylmuramic acid)3 .
The space-lling CPK model of lysozyme (left) and
its interaction with substrate are shown in Figure 5
(10). The substrate is compactly bound to the crevice
of the lysozyme molecule. A schematic view of the
substrate hexamer, NAG-NAM-NAG-NAM-NAG-
NAM, and its interactions with the enzyme are
shown in Figure 6 (10). The view is nearly straight
into the crevice, with the heavy edges of the sugar
Figure 4 Ribbon drawing model of hen egg white lysozyme. rings on the exterior. The site of catalysis was identi-
The two catalytic residues, glutamic acid 35 and aspartic acid ed to be between D and E rings. When a search was
52, are represented as small spheres. made in the vicinity of the D/E ring for possible cat-
Figure 5 Space-lling CPK model of hen egg white lysozyme. Left: Enzyme without substrate, showing active site crevice.
Right: Enzyme-substrate complex, with hexamer NAG substrate. (From Ref. 10.)

alytic groups, two candidates came to light: Asp52, in
a polar environment where it will be ionized, and
Glu35, in largely hydrophobic surroundings where it
might easily remain protonated. The mechanism of
cleavage of the polysaccharide bond in lysozyme is
suggested as shown in Figure 7 (10). The proton of
Glu35 rst attacks the linkage oxygen and weakens
the C1 -O bond (a). If the bond is cleaved, ring D
forms a carbonium ion. The nearby charged Asp52
helps to stabilize the carbonium ion (b). The Glu35
proton is replaced by another from an ionizing water
molecule, and the resultant hydroxyl ion then attacks
the carbonium ion and completes the reaction (c).
This proposal of Phillips and coworkers (1113) has
been supported by a number of chemical and kinetic
experiments.
The enzyme activity of lysozyme can be easily mea-
sured by the lytic action on M. lysodeikticus (M.
luteus). However, the change in the turbidity of M.
luteus does not necessarily reect the real enzyme
activity, because the lysis is a rupture of the bacterial
cell wall and is not equal to the cleavage of glycosidic
bonds. Nevertheless, the lytic action is generally used
for the measurement of lysozyme activity because of
the ease of the assay. The decrease in the turbidity of
the suspension of M. luteus cell wall is followed by
the absorbance at OD450 and the enzyme activity can
be represented as the initial rate. In order to get a
linear curve for the decrease, a small amount of lyso-
zyme (4 g) should be added into the cell suspension
(OD450 0:7). When the enzyme reaction is done at
room temperature for 1 min, a linear decrease curve is
obtained and used to determine the initial rate. The
optimal pH is 7:0 using lytic activity. On the other
hand, when the activity is measured using synthetic
substrates, p-nitrophenyl N-acetylglucosamine oligo-
mer (14) and glycol chitin (15), the optimal pH is 5
and 5.3, respectively. Therefore, the optimal pH of
lytic activity is different from that of the glycosidic
activity. The glycosidic activity is determined by mea-
suring the reducing power produced by the glycolysis
of ethylene glycol chitin (15). One milliliter of 0.5%
ethylene glycol chitin is added to 0.5 mL lysozyme
solution in sodium acetate buffer (pH 4.5). The mix-
ture is incubated at 40 C for 30 min. After the reac-
tion, 2 mL of the color reagent (prepared by
dissolving 0.5 g potassium ferricyanide in 1 L of 0.5
Figure 6 Substrate interactions with the active crevice in M sodium carbonate) is added and the mixture is
lysozyme. The dark edges of the carbohydrate rings are immediately boiled for 15 min to estimate the redu-
exposed to the outside, and the lighter ones are buried at cing power resulting from the hydrolysis of ethylene
the crevice bottom. (From Ref. 10.) glycol chitin.

egg white is stirred for 48 h at 4 C. The resulting crys-
tals are collected and solubilized in a half-volume of
acetate buffer (pH 4.5). The solution is recrystallized at
least ve times. Thus, the puried lysozyme is obtained
and the purity is 100%. On the other hand, lysozyme
can be puried by cation exchange chromatography,
because the enzyme is a basic protein. For example,
when the recombinant lysozyme is expressed in S. cer-
evisiae, the yeast medium is applied to CM-toyopearl
equilibrated with a buffer of low ionic strength, and
the adsorbed basic proteins are eluted in a gradient
manner.
VII. RECENT BREAKTHROUGH STUDIES

ON LYSOZYME
Since lysozyme is easily obtained in a puried form,

it has been studied comprehensively as a model pro-
tein for structure, dynamics, and folding. The recent
development of recombinant techniques has enabled
elucidation of the molecular mechanism of structural
and functional properties of lysozyme. In early stu-
dies, recombinant lysozyme was investigated in the
E. coli expression system. However, since the prokar-
yotic cells have a different secretion system from
eukaryotic for correct foldings cells, the correctly
folded lysozyme could not be expressed. It seems
likely that the folding of disulde-rich protein such
as lysozyme is difcult in E. coli because of the
absence of endoplasmic reticulum (ER) in which pro-
teins are posttranslationally folded. Although the
enzyme is obtained as an inclusion body, the yield
of refolding is at a very low level. The N-terminus of
recombinant lysozyme is methionine, while that of
native lysozyme is lysine. Since the N-terminus lysine
is essential for correct folding, the substitution of N-
terminus lysine with another amino acid results in a
signicant decrease in the stability of lysozyme.
Kumagai et al. in 1987 found that hen egg white
lysozyme was correctly processed and folded in S.
cerevisiae (16). The N-terminus of recombinant lyso-
Figure 7 Mechanism of cleavage of the polysaccharide bond zyme is lysine and the stability is the same as the
in lysozyme. (From Ref. 10.) native lysozyme. Since S. cerevisiae is a typical
eukaryotic cell with an ER system, lysozyme is cor-
rectly processed and folded in the yeast. Taniyama et
al. in 1992 reported the folding mechanism of disul-
VI. PURIFICATION OF LYSOZYME de bonddecient human lysozyme C77/95A
mutant secreted in S. cerevisiae (17). Although the
Lysozyme is easily crystallized from fresh chicken egg decient mutants of other disulde bonds could not
white. A 5% sodium chloride (w/w) is added to the be secreted, only C77/95A mutant secreted by eight-
homogenized egg white adjusted to pH 9.59.8. The fold greater than wild-type in yeast. The stability of

C77/95A mutant greatly decreased despite the correct 4. A Kato, T Wakinaga, N Matsudomi, K Kobayashi.
folding. These observations show that the disulde Changes in lysozyme during egg white thinning. Agric
bond Cys7795 contributes to the stabilization of Biol Chem 42:175176, 1978.
the folded form of human lysozyme. 5. OJ Cotterill, AR Winter. Egg white lysozyme. 3. The
effect of pH on the lysozyme-ovomucin interaction.
Recently, the identication of human lysozyme as
Poultry Sci 37:679686, 1955.
an amyloidogenic protein which causes serious dis-
6. HR Ibrahim, A Kato, K Kobayashi. Antimicrobial
eases is of particular interest. The two unknown nat- effects of lysozyme against Gram-negative bacteria
ural mutations (Ile56Thr and Asp67His) in the due to covalent binding of palmitic acid. J Agric
human lysozyme gene both cause autosomal-domi- Food Chem 39:20772088, 1991.
nant hereditary amyloidosis (18). It has been sug- 7. S Nakamura, A Kao, K Kobayashi. New antimicro-
gested that the lysozyme amyloid bril may be bial characteristics of lysozyme-dextran conjugate. J
formed by the intermolecular -sheet association Agric Food Chem 39:647650, 1991.
due to -strand exposed to the molecular surface by 8. CCF Blake, DF Koenig, GA Mair, ACT North, DC
a single amino acid substitution. Phillips, VR Sarma. Structure of hen egg-white lyso-
We have developed new approaches for industrial zyme. A three-dimensional Fouier synthesis at 2 A
resolution. Nature 206:757761, 1965.
application using genetic modications of lysozyme.
9. SE Radford, CM Dobson, PA Evans. The folding of
Nakamura et al. (19) reported that the large molecu- hen lysozyme involves partially structured intermedi-
lar size of N-glycosylated lysozyme with a poly- ates and multiple pathways. Nature 358:302307,
mannose chain was predominantly expressed in 1992.
yeast carrying the lysozyme gene modied to have 10. RE Dickerson, I Geis. The Structure and Action of
N-linked signal sequence Asn-X-Thr/Ser at the Proteins. New York: Harper & Row, 1969, pp
position of the molecular surface. The polymannosyl 6978.
lysozyme showed remarkable heat stability in that no 11. RU Rupley, V Gates. Studies on the enzymic activity
coagulation was observed by heating at 100 C. In of lysozyme. II. The hydrolysis and transfer reactions
addition to heat stability, the lysozyme revealed excel- of N-acetylglucosamine oligosaccharides. Proc Natl
lent emulsifying properties, superior to the commer- Acad Sci USA 57:496510, 1967.
12. TY Lin, DE Koshland Jr. Carboxyl group modica-
cial emulsiers (20).
tion and the activity of lysozyme. J Biol Chem
On the basis of the idea that the covalent attach- 244:505508, 1969.
ment of palmitic acid residues to the lysysl residues of 13. SM Parsons, MA Raftery. The nature of amino acid
lysozyme stabilizes it and makes it more active (6), side chains which are critical for the activity of lyso-
Ibrahim et al. (21) attempted the genetic fusion of a zyme. Biochemistry 8:700712, 1969.
hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro), 14. MA Raftery, FW Dahlquist, SM Parsons, RG
which forms a -strand conformation with the same Wilcott. The use of nuclear magnetic resonance to
length as a palmitic acid residue, to its C-terminus. describe relative modes of binding to lysozyme of
As expected, the hydrophobic peptide fusion greatly homologous inhibitors and related substrates. Proc.
enhanced the bactericidal action to E. coli. These mod- Nat. Acad. Sci. USA 62:4451, 1969.
ied lysozymes having antimicrobial action against 15. T Imoto, K Yagishita. A simple activity measure-
ment of lysozyme. Agric Biol Chem 35:11541156,
both Gram-positive and Gram-negative bacteria and
1971.
can be used for industrial applications in future. 16. I Kumagai, S Kojima, E Tamaki, K Miura.
Conversion of Try 62 of hen egg-white lysozyme to
Tyr by site-directed mutagenesis. J Biochem 102:733
17. Y Taniyama, K Ogasawara, K Yutani, M Kikuchi.
Folding mechanism of mutant human lysozyme secre-
1. A Fleming. On a remarkable bacteriolytic element
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found in tissues and secretions. Proc R Soc Lond
1992.
Ser B 93:306317, 1922.
18. DR Booth, M Sunde, V Bellotti, CV Robinson, WL
2. DM Chipman, N Sharon. Mechanism of lysozyme
Huchinson, PE Fraser, PN Hawkins, CM Dobson, SE
action. Science 165:454465, 1969.
Radford, CCF Blake, MB Pepys. Instability, unfold-
3. D Pirzkiewiez, TC Bruice. Interaction of cellodextrins
ing and aggregation of human lysozyme variants
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19. S Nakamura, H Takasaki, K Kobayashi, A Kato. 21. HR Ibrahim, M Yamada, K Matsushita, K
Hyperglycosylation of hen egg white lysozyme in Kobayashi, A Kato. Enhanced bactericidal action of
yeast. J Biol Chem 268:1270612712, 1993. lysozyme to Escherichia coli by inserting a hydropho-
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262, 1993.

77
Ribonucleases
Jaap J. Beintema and Wei Zhao

University of Groningen, Groningen, The Netherlands
I. INTRODUCTION C. Classication According to Enzyme

Commission Nomenclature
Section II of this handbook is organized according to
Ribonucleases depolymerize ribonucleic acids by clea-
the classication of enzymes as presented in Enzyme
vage of phosphoric diester bonds between nucleotide
Nomenclature (2). In this classication hydrolases
residues. This means that they are specialized phospho-
(EC group 3) are listed separately from transferases
transferases or phosphoric diester hydrolases. A
(EC group 2). However, for several enzymes this dis-
further specication is that they are endonucleases, as
tinction is not correct as they are transferases reacting
exonucleases degrade nucleic acids from one of the two
with compounds with general formula R-OH in which
termini, which results in little depolymerization.
R may also be H, (H-OH) as will be discussed below.
Cleavage may occur between phosphate and the 3 0 -
The best-characterized ribonucleases belong to the
oxygen of the ribose, producing 5 0 -phosphomonoesters
endoribonucleases producing 3 0 -phosphomonoesters,
(EC 3.1.26) or between phosphate and the 5 0 -oxygen of
with 2 0 , 3 0 -cyclic phosphate as intermediates. This
the ribose, producing 3 0 -phosphomonoesters (EC
feature indicates that they will not be active on
3.1.27). Enzymes with the latter cleavage specicity
deoxyribonucleic acids. These ribonucleases have
often are transferases producing 2 0 ,3 0 -cyclic phosphate
been classied earlier also as nucleotidyl transferases
as intermediates, which generally are hydrolyzed in a
(EC 2.7.7) or phosphoric diester hydrolases (EC 3.1.4).
second reaction catalyzed by the same enzyme. Figure
Three superfamilies with this cleavage pattern are
1 shows as an example the two successive reactions
those of ribonucleases A (311), T1 (12, 13), and T2
catalyzed by bovine pancreatic ribonuclease.
(12), respectively. Mammalian pancreatic ribonu-
cleases belong to the ribonuclease A superfamily and
the two successive catalyzed reactions are shown in
B. Chemical Structure of Substrates Figure 1. Enzymes belonging to this family have a
strict specicity for a pyrimidine base at the 3 0 -side
Generally, nucleases are specic for either desoxyribo- of the cleaved phosphoric diester bond and a general
nucleic acids (deoxyribonucleases) or ribonucleic acids preference for a purine base at the 5 0 -side. Taking into
(ribonucleases), although nucleases also occur which account the reversibility of chemical reactions, the sec-
cleave both substrates producing either 5 0 -phosphomo- ond reaction with H-OH as reacting molecule can be
noesters (EC 3.1.30) or 3 0 -phosphomonoesters [micro- considered as the reverse of the rst reaction with R-
coccal nuclease (1); EC 3.1.31.1]. OH as leaving group. Generally the second (hydroly-

Figure 1 The two successive reactions catalyzed by bovine pancreatic ribonuclease. This ribonuclease belongs to the ribonu-
clease A superfamily, which has a strict specicity for a pyrimidine base at the 3 0 -side of the cleaved phosphoric diester bond.
Within the superfamily this ribonuclease belongs to the family of pancreatic-type, secretory ribonucleases 1, which have a higher
preference for cytosine than uracil as pyrimidine. These enzymes have a general preference for a purine base at the 5 0 -side of the
diester bond, with highest activities with an adenine at that side. The pyrimidine-binding site includes a disulde-linked loop
structure in the molecule, which is absent in mammalian angiogenins and in non-mammalian members of the superfamily. These
enzymes do not have a strong adenine preference and in several cases even prefer guanine over adenine.
sis) reaction is much slower than the rst (transferase), could be isolated in relatively large amounts from
and several members of the ribonuclease A superfamily bovine pancreas, a major product of the beef industry
do not even hydrolyze the intermediate cyclic phos- (14). This enzyme has played a key role in the devel-
phates (6). This example shows that the classication opment of the study of proteins, with four Nobel
of these ribonucleases as hydrolases (EC 3.1.27) is less Prizes in chemistry (in 1972 and 1984) awarded for
correct and that the previous classication as trans- research on it. However, it was later found that ribo-
ferases (EC 2.7.7) is more appropriate. nuclease is only present in high amount in the pancreas
of ruminants and species that have a ruminantlike
digestion, and in a number of species with cecal diges-
II. IMPORTANCE OF RIBONUCLEASES tion (15, 16). For instance, the ribonuclease content in
TO QUALITY OF FOOD AND IN FOOD human pancreas is < 1% of that in bovine pancreas,
DIGESTION and in the 1970s ribonuclease disappeared again as an
important digestive pancreatic enzyme in textbooks for
Enzymes to be included as topics in a Handbook of medical students.
Food Enzymology should have either a role (or poten- Ribonuclease was considered around 1980 as a dull
tial role) in food production or processing, or be enzyme, without a relevant biological function and not
important in digestion of food components in the very interesting for chemical investigations. However,
human species, in its domesticated animals, or in bio- since then the enzyme underwent a renaissance by the
technology. In this respect the history of ribonuclease discovery of proteins with novel biological actions,
research is a disappointing one. One of the rst which after structural studies proved to be homologs
enzymes to be isolated and puried in crystalline of ribonucleases with their ribonuclease activities being
form has been ribonuclease, a digestive enzyme which a condition of these special actions (17, 18).

III. RIBONUCLEASES ARE PROTEINS OR also called ribonuclease 3). EDN and ECP are the
CATALYTICALLY ACTIVE NUCLEIC products of a gene duplication which occurred in an
ACIDS ancestor of Old World monkeys and apes after their
divergence from the ancestor of New World monkeys.
Not all ribonucleases are proteins. Catalytically active Recently, another human enzyme (ribonuclease 6)
nucleic acids include predominantly ribonucleases, belonging to this family with low enzymatic activity
among which ribonucleases P are very well character- has been discovered and characterized.
ized ones. Ribonucleases P (EC 3.1.26.5) consist of a 3. Mammalian ribonucleases 4 (8). These are basic
protein and a ribonucleic acid component, of which the uridyl-preferential enzymes, isolated from the liver of
latter is the catalytically active part, requiring a metal several mammalian species.
ion for its activity, and producing 5 0 -phosphomonoe- 4. Mammalian angiogenins (or ribonucleases 5) (9,
sters (19). 23). These proteins stimulate blood vessel formation.
They are virtually inactive toward RNA. However,
their biological activity requires intact enzymic active
IV. OCCURRENCE OF RIBONUCLEASES
sites and involves very specic cleavage of RNA mole-
A. Digestion of Extracellular RNA cules.
5 and 6. Ribonucleases from chicken liver (24) and
The three best-characterized superfamilies of ribonu- chicken transformed bone marrow cells (25, 26).
cleases are those of the already-mentioned ribonu- 7 and 8. Pancreatic ribonucleases from the pan-
clease A, T1 , and T2 . All members of these creas of reptiles [turtle (27) and iguana (28)].
superfamilies are extracellular ones, have sequences 9. Several amphibian ribonucleases (5, 29).
coding for signal peptides at the DNA level, and are Ribonucleases T1 and T2 were discovered originally
primarily digestive enzymes for foreign RNA, originat- in the commercial digestive Taka-Diastase from
ing from dead cell material. Aspergillus oryzae (30). Enzymes homologous with
The ribonuclease A superfamily has a strong speci- RNase T1 with a preference for guanine have been
city for endonucleolytic cleavage with a pyrimidine at isolated from several fungi (EC 3.1.27.3; EC 3.1.27.4)
the 3 0 -side of the cleaved phosphodiester bond (EC (12), but this superfamily also includes homologous
3.1.27.5) and includes homologous enzymes isolated prokaryotic representatives such as enzymes from sev-
from many mammalian, avian, reptilian, and amphi- eral Bacillus spp.[EC 3.1.27.2; barnase and binase (13)],
bian sources. No representatives have yet been found which have a less strict preference for guanine. A spe-
in shes or nonvertebrate taxa. Other enzymes men- cialized fungal member of this superfamily isolated
tioned under EC 3.1.27.5 in Enzyme Nomenclature (2) from Aspergillus spp. is -sarcin (EC 3.1.27.10),
are not well characterized and probably are not homo- which has cytotoxic properties resulting from a very
logous. Several representatives have specic functions specic cleavage pattern of ribosomal RNA (31).
and have a very low or no apparent activity with more The ribonuclease T2 superfamily is the most ubiqui-
conventional ribonuclease substrates (10). tous one in living nature. Members of this superfamily
Nine separate families with sequences that are > 50% (EC 3.1.27.1) with a preference for purines (A or G)
identical in each family can be distinguished in the ribo- have been isolated, sequenced, and characterized from
nuclease A superfamily: a large number of fungi, plants, and animals (including
1. Mammalian (pancreatic-type or secretory) ribo- mammals) (12). The active site of members of this
nucleases 1. This family, with a rather high preference superfamily consists of two very diagnostic cassettes
for cytidine relative to uridine, includes digestive pan- with primary structures: I/L-H-G-L-W-P and W-X-
creatic enzymes (4), but also paralogous ribonucleases H-E-W/Y-X-K/T-H-G, respectively. Similar cassettes
isolated from bovine semen (20) and brain (21). can be recognized even in the sequences of prokaryote
2. Mammalian (nonsecretory or neurotoxin-type) and virus ribonucleases, which show no recognizable
ribonucleases 2 (7, 22). This family includes both homology to the eukaryote enzymes in the remainder
enzymes isolated from tissues such as liver and kidney, of the sequences. These prokaryote and virus ribonu-
and more specialized ones, isolated from eosinophil cleases, but also several animal members of the RNase
cells, such as the enzymatically active human eosino- T2 superfamily, are not purine preferential anymore
phil derived neurotoxin (EDN), which has also been (12).
isolated from other human sources, and the enzymati- Pancreatic enzymes in the ribonuclease A superfam-
cally inactive human eosinophil cationic protein (ECP; ily and microbial (especially fungal) representatives in

the other two may be classied as digestive enzymes, B. Role of Pancreatic Ribonucleases as Digestive
depolymerizing foreign RNA. Also an extracellular T2 Enzymes in Mammals
ribonuclease from tomato (RNase Le) has a digestive
role, as its expression and excretion from cells are sti- In Section II of this chapter it has already been men-
mulated by shortage of inorganic phosphate in the tioned that pancreatic ribonuclease is only present in
extracellular environment, necessitating digestion of high amounts in the pancreas of a limited number of
organic phosphate sources (32). mammalian taxa. Large quantities of this enzyme are
However, even pancreatic ribonucleases do not have found in ruminants and species that have a ruminant-
only a digestive function as they have been identied in like digestion, and in a number of species with cecal
other tissues and body uids as well, often in compar- digestion (Fig. 2). However, there are also herbivores
able quantities as observed in the pancreas (see Sec. with cecal digestion with extremely low ribonuclease
IV.B). Other ribonucleases are synthesized exclusively contents in the pancreas, such as lagomorphs (rabbit),
in tissues which have no digestive function. Highly hyraxes, manatee, and elephant (16). Barnard (15) pro-
interesting are proteins which were isolated and char- posed that an elevated level of pancreatic ribonuclease
acterized at rst because of specic biological proper- is the response to the necessity of digesting large
ties (18). Surprisingly, sequence analyses showed them amounts of ribonucleic acid derived from the micro-
to be members of one of the three extracellular ribo- ora of the stomach of ruminants. This explanation
nuclease superfamilies, which was conrmed by agrees with the elevated level of stomach lysozyme in
demonstrating ribonuclease activity, often at a low several ruminants and species that have a ruminantlike
level or only with very specic cleavage patterns. digestion (37). As far as we know, however, these roles
Generally these ribonuclease activities were found to of ribonuclease and lysozyme have never been investi-
be a condition of the specic biological actions. gated by comparative physiologists studying digestive
Ribonucleases endowed with these special bioactions systems of mammals.
[RISBases (18)] are angiogenins (9, 23), the cytotoxic The most striking differences between pancreatic
and aspermatogenic bovine seminal ribonuclease (20), ribonucleases concern the presence or absence of cova-
amphibian members of the ribonuclease A superfamily lently attached carbohydrate. These differences may be
such as onconase (33) and lectins from frog oocytes (5), related to the digestive system and the diet of the
self-incompatibility proteins in plants, which belong to species involved. A number of pancreatic ribonucleases
the ribonuclease T2 superfamily (12), and the already- have carbohydrate attached to asparagine residues at
mentioned -sarcin, which is a ribonuclease T1 super- positions 21, 22, 34, 62, 76, and/or 88 (Fig. 3). These
family member (31). positions are part of highly variant sequences at the
A general feature of these ribonucleases endowed surface of the molecule, far from the active site.
with special bioactions is that available evidence indi- Only asparagine residues in Asn-X-Ser/Thr
cates that they exert their cytotoxic actions by cleavage sequences have been found to act as attachment sites,
of intracellular RNA in their target cells. This means where X may be any residue except proline (Fig. 3).
that in some way these extracellular enzymes are inter- However, not all Asn-X-Ser/Thr sequences possess
nalized again, for which several specic receptor- carbohydrate chains. Other features of the ribonu-
mediated processes have been proposed and also clease molecule and/or the effectiveness of the carbo-
experimentally demonstrated (23, 33). Members of hydrate-attaching system apparently inuence the
the ribonuclease A superfamily bind cytoplasmic pro- glycosylation process.
tein ribonuclease inhibitors (RI) with very high af- Figure 4 summarizes the occurrences of an Asn-X-
nities (34). In a previous review we pointed to the Ser/Thr sequence in several investigated mammalian
paradox that these cytoplasmic inhibitors bind to non- ribonucleases and their glycosylation states. The
cytoplasmic ribonucleases (35), but recent studies sugar composition of the carbohydrate chains is also
about uptake in foreign cells point to a biological func- indicated schematically in this gure. Both simple-type
tion of these inhibitors. Within mammals there is little chains containing only glucosamine and mannose, and
specicity in ribonuclease inhibitor interactions, also complex-type chains also containing fucose, galactose,
not with respect to the different ribonuclease families and sialic acid occur.
occurring in this vertebrate class. But ribonucleases All six carbohydrate sites in ribonucleases are
from one vertebrate class (mammals, birds, amphi- located at external bends of the folded chain. This
bians) and inhibitors from another one do not bind localization ensures maximal exposure of the carbohy-
to each other (5, 36). drate moieties to the external solvent. In Figure 5 a

Figure 2 Pancreatic ribonuclease content in mammalian taxa. XXX, ruminants; XX, ruminantlike digestion; X, (mainly)
herbivorous. Pancreas badly autolyzed.
space-lling model of carbohydrate-free bovine ribo- (40). This would be analogous to the function of ribo-
nuclease is compared with the same model with three nucleases in the digestion of ribonucleic acid from the
biantennary complex-type oligosaccharide chains stomach microora in ruminants and herbivores with
attached at positions 22, 34, and 62, representing the ruminantlike digestion postulated by Barnard (15).
minor glycosylated component of horse ribonuclease. Ruminants and species with ruminantlike digestion
Probably the carbohydrate moieties do not occupy (camel, hippopotamus, sloth, kangaroo) have less car-
xed positions because of the presence of a number bohydrate attached to their ribonucleases. Therefore, it
of exible bonds, but the latter model shows the seems that glycosylation of pancreatic ribonuclease
increase in volume as a result of the addition of carbo- may not be advantageous for species with stomach
hydrate. fermentation. These species escaped glycosylation of
The glycosylation states of the ribonucleases inves- their ribonucleases not only by amino acid replace-
tigated were correlated with the type of fermentation ments in the protein (camel, reindeer), but probably
(stomach or cecal) used by the species involved (Fig. also by developing a much less effective carbohy-
4). It was found that species with cecal digestion, such drate-attaching enzyme system in the pancreas than
as pig, horse, and the hystricognath rodents (guinea encountered in other species. Hippopotamus ribonu-
pig and relatives), produce ribonucleases with large clease is an example of the second solution. This
carbohydrate chains attached to several positions at enzyme has four carbohydrate attachment sites, with
the surface. Therefore, it was suggested that the pre- carbohydrate attached to only one of them in only a
sence of carbohydrate protects ribonuclease from fraction of the molecules.
absorption in the gut, enabling it to be transported If glycosylation of a ribonuclease is avoided by
to the large intestine where it should hydrolyze the amino acid replacements, the enzyme will also not be
ribonucleic acid derived from the cecal microora glycosylated if its gene is expressed at other sites of the

Figure 3 Amino acid sequence of the carbohydrate attach-
ment site 7678 in ribonucleases. CHO, carbohydrate.

Partially glycosylated.
body, but other pancreatic enzymes may be glycosy-

lated extensively in the species involved. However, a
less effective carbohydrate-attaching enzyme system
in the pancreas will result in a general scarcity of gly-
cosylated secretory pancreatic proteins, but the same
gene product may be extensively glycosylated at other
sites of the body. An example is human ribonuclease, Figure 4 Occurrence of Asn-X-Ser/Thr sequences at the six
which has three carbohydrate attachment sites, one carbohydrate sites (residues 2123 or 2224, 3436, 6264,
being completely and one partially glycosylated in 7678, and 8890) in ribonuclease and compositions of car-
the pancreatic enzyme (41). The urinary enzyme, bohydrate moieties. &, completely glycosylated Asn-X-Ser/
Thr sequence; , part of the molecules are glycosylated; &,
k
which is not synthesized in the pancreas, is glycosy-

Asn-X-Ser/Thr sequence without carbohydrate attached; ,
lated completely at all three sites (42). no Asn-X-Ser/Thr sequence present. Carbohydrate composi-
These examples show dramatic secondary effects of tions: &, present in all molecules; &, present in all glycosy-
apparently neutral replacements. Strong positive or lated molecules (only part of the molecules are glycosylated);
negative selection may inuence the presence of Asn- & , only present in a minor glycosylated component, not
X-Ser/Thr sequences in proteins. However, the pre- present in the major glycosylated component; , carbohy-
sence of a short oligosaccharide chain in bovine ribo- drate present, but composition not determined.

ogy and stomach structure (47), moose and giraffe are
grouped together as large concentrate selectors with
a diet containing relatively little cellulose ber and a
simple (primitive) stomach.
Strongly positively charged ribonucleases are con-
siderably more active on double-stranded RNA than
ribonucleases with a smaller excess of positive charges
on the surface (48). We do not know whether the activ-
ity of the ribonucleases on double-stranded RNA has
any function. The ribonucleases with the larger excess
of positive charges are the ribonucleases from human
and whale pancreas, bovine semen, and ruminant
brain. These enzymes have no function in the digestion
of microoral RNA from the stomach or cecum of
Figure 5 Space-lling models of bovine ribonuclease (left)
herbivores. This suggests that there is a relationship
and of a glycosylated ribonuclease with three biantennary
complex-type oligosaccharide chains attached to the surface between the excess of positive charges of ribonucleases
(right). This gure is a collage of the gure on p. 81 in Ref. 38 and nondigestive functions.
(ribonuclease) and Fig. 11B in Ref. 39 (spatial conformation Man and langur monkey are two species in the same
of a biantennary glycan of the N-acetyllactosamine type in mammalian order, of which the latter possesses all
the T-conformation). features of having ruminantlike digestion. The amino
acid sequence of their ribonucleases differ at 14 (11%)
of the positions (49). This leaves no doubt that they are
located on the same branch in a most parsimonious
nuclease B, the minor glycosylated component, may be tree. The hypothesis that species with ruminantlike
a neutral property. The three-dimensional structure of digestion not only have a higher ribonuclease content
this protein was found to be identical to that of bovine in their pancreas, but also have less excess of positive
ribonuclease A, with the carbohydrate chain extending charge and fewer attached carbohydrates, is in agree-
into the solvent. This chain is disordered for most part ment with data presented in Table 1.
and exerts no inuence on the structure of the protein
(43, 44). C. Intracellular RNA Processing and Turnover
Evolutionary selection against the occurrence of
Asn-X-Ser/Thr sequences should occur if glycosylation Many RNA species in living cells are continuously
is disadvantageous for the stability or function of the synthesized, processed and degraded, in which ribonu-
protein. Evidence of this has been presented by Hunt cleases should play a major role. However, disappoint-
and Dayhoff (45) and Sinohara and Maruyama (46), ingly much less is known about these ribonucleases
who observed that Asn-X-Ser/Thr sequences occur less than about those discussed in the previous sections.
frequently than expected by chance in extracellular Deutscher (50) and Nicholson (51) list endoribonu-
proteins from eukaryotes. cleases identied and characterized in the prokaryote
Similar glycosylation requirements for ribonu- Escherichia coli. These lists include the excreted peri-
cleases from different taxa may explain convergences plasmic enzyme ribonuclease I (EC 3.1.27.6), which is a
in functional and structural characteristics. The pan- member of the ubiquitous ribonuclease T2 superfamily
creatic ribonuclease of giraffe is positioned with that of (12) discussed in the previous section. Interestingly,
pronghorn and not with those of deer in most parsi- there occurs a modied form of this ribonuclease, ribo-
monious trees of the sequences (21) as a consequence nuclease I , which has no signal peptide sequence on its
of a number of rather rarely observed amino acid coding DNA, is not secreted through the cytoplasmic
replacements. However, since these shared replace- membrane, and may exert its function intracellularly.
ments occur at internal positions, the relationship Less well characterized enzymes are ribonucleases M
does not come to clear expression in many of the prop- and R, which are similar to ribonuclease I.
erties of these enzymes. Thus, giraffe ribonuclease is The major participant in mRNA decay in E. coli is
very similar to that of moose (a deer species) in glyco- ribonuclease E. This enzyme also plays a role in rRNA
sylation and other properties. It may be signicant that maturation. This is a divalent metal requiring enzyme,
in a classication of ruminants based on feeding ecol- producing 5 0 -phosphomonesters (EC 3.1.26). It has a

Table 1 Several Characteristics of Human and Langur Pancreatic Ribonucleases
Man Langur
g/g Tissue 5 280

(Lys Arg)(Asp Glu 6 1
Number of possible glycosylation
Sites (Asn-X-Ser/Thr sequences) 3 2
Extent of glycosylation All molecules; Part of the molecules;
extensive glycosylation of one simplea chain
one site in all molecules and
of another site in about half
of the molecules
a
Simple-type carbohydrate chains only contain glucosamine and mannose.
polypeptide sequence of 1061 amino acids, much Moiseyev et al. (55, 56) have isolated, characterized
longer than the extracellular enzymes discussed in the and sequenced two plant ribonucleases isolated from
previous section. A similar activity is exhibited by the ginseng calluses. These enzymes, with polypeptide
already mentioned ribonuclease P (EC 3.1.26.5), of lengths of 150 residues, are homologous with cyto-
which the ribonucleic acid component is the catalyti- toxic intracellular pathogenesis-related proteins (IPR
cally active one. or PR-10) from several plants such as parsley (57)
More specialized E. coli ribonucleases are ribonu- and bean (58), and with pollen allergens from tree spe-
cleases III (EC 3.1.26.3) and H1 and H3 (EC 3.1.26.4), cies such as birch (59) and alder (60). This suggests that
which cleave double-stranded (ds) RNA molecules and the cytotoxic properties of these latter proteins are
the RNA strand of RNA-DNA hybrids, respectively. caused by their ribonuclease activities. However, this
Other, less well characterized ribonucleases in E. coli hypothesis is still being met with skepticism. In addi-
are ribonucleases IV and F (producing 3 0 -phosphate tion, this protein family does not contain any con-
termini; EC 3.1.27.7), P2, O, PIV, PC and N. served histidine residues, a universal active-site
Few intracellularly synthesized eukaryotic ribonu- residue in all other investigated ribonuclease families
cleases have been identied and characterized. Two so far investigated.
of the few examples are the 2-5A (=oligoadenylate So, although much knowledge has already been col-
with 2 0 ! 5 0 internucleotide linkages)dependent lected about intracellular RNA processing and degrad-
human and murine ribonucleases L (52) which cleave ing activities in eukaryotes (61, 62), little is known
RNA with the production of 3 0 -phosphoryl and 5 0 - about the responsible enzymes. Part of these enzymes
hydroxyl groups. The polypeptide chains of these will be proteins, but many others may be enzymatically
enzymes have chain lengths of 750 residues, and active RNA molecules (ribozymes). Penny and cowor-
each includes a ribonuclease domain of 200 residues, kers recently published a model for the evolution of life
with some sequence similarity with ribonuclease E. from an RNA world to the emergence of eukaryotes
This suggested homology, however, is not in agreement and prokaryotes (63), and summarized the presence of
with the different cleavage specicities of these mam- RNAs in modern organisms that are relics from the
malian and E. coli enzymes. Another ribonuclease E ancient RNA world. Proteins have gradually replaced
related activity in mammalian cells is the product of RNAs in catalysis by virtue of their superior catalytic
the human ard-1 gene, which encodes a basic, proline- properties. However, in the case of large substrates
rich 13.3-kDa molecular-weight protein (53). such as RNA the rate of diffusion is the slowest reac-
Expression of this gene in E. coli can complement a tion step, and catalytic perfection will not help much in
mutant of the ribonuclease E gene, but there are no speeding up the reaction rate. Therefore, it is not sur-

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University of CaliforniaDavis

Davis, California, U.S.A.

U.S. Department of Agriculture

Marcel Dekker, Inc. New York Basel

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Library of Congress Cataloging-in-Publication Data

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PART I. GENERAL ASPECTS

Protein Structure and Kinetics of Enzyme Reactions: A Historical Perspective

History of Enzymology with Emphasis on Food Production

Enzyme-Catalyzed Reactions: Experimental Factors that Affect Rates

Inactivation of Enzymes: From Experimental Design to Kinetic Modeling

Regulatory Issues of Food Enzymes Used in the United States

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

THE FOOD CHAIN AND ENZYMES

Nitrogen Fixation and the Enzyme Nitrogenase

Enzymes in Amylose and Amylopectin Biosynthesis

Fatty Acid Biosynthesis and Triacylglycerol Assembly

Phospholipid Biosynthetic Enzymes in Plants

Applications of Oxidoreductases in Foods

Transgenic Plants for Production of Enzymes

Enzymes in Protein Biosynthesis

Nucleic Acid Biosynthesis

ENZYMES AS TOOLS TO MODIFY FOOD COMPONENTS

Protein Modication to Optimize Functionality: Protein Hydrolysates

Production and Modication of Acylglycerides

ENZYMES IN FOODS POSTHARVEST

Signicance of Indigenous Enzymes in Milk and Dairy Products

Exogenous Enzymes in Dairy Technology

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Entrapment of Biocatalysts by Prepolymer Methods

Genetic Immobilization of Enzymes on Yeast Cell Surface

Use of Immobilized Enzymes in the Food Industry

ENZYME UTILIZATION AND DEVELOPMENT

Enzymes and Food Analysis

Recent Advances in Enzyme Development

PART II. SPECIFIC ENZYMES

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Mammalian Sulfhydryl Oxidase

Lipoxygenase and Associated Enzymes

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Methodologies for Assaying the Hydrolysis of Cellulose by Cellulases

Cellulases in Food Processing

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Enzymes with Activity Toward Xyloglucan

Enzymes Degrading Rhamnogalacturonan and Xylogalacturonan

Enzymic Hydrolysis of Cereal (1!3, 1!4)--Glucans

Pectate and Pectin Lyases

Cystine Lyases in Plants

Xylose (Glucose) Isomerase

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Jaap J. Beintema Department of Biochemistry, University of Groningen, Groningen, The Netherlands

Rob F. Beudeker DSM Food Specialties, Delft, The Netherlands

Peter Biely Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia

Yvonne Bruggeman Functional Biomolecules, Unilever Research, Vlaardingen, The Netherlands