Professional Documents
Culture Documents
Tyler Johannes
Michael R. Simurdiak
Huimin Zhao
Department of Chemical and Biomolecular Engineering, University of Illinois,
Urbana, Illinois, U.S.A.
Biocatalysis may be broadly defined as the use of Advantages and Disadvantages of Biocatalysis
enzymes or whole cells as biocatalysts for industrial vs. Chemical Catalysis
synthetic chemistry. They have been used for hundreds
of years in the production of alcohol via fermentation, Similar to other catalysts, biocatalysts increase the
and cheese via enzymatic breakdown of milk proteins. speed in which a reaction takes place but do not affect
Over the past few decades, major advances in our the thermodynamics of the reaction. However, they
understanding of the protein structure–function relation- offer some unique characteristics over conventional
ship have increased the range of available biocatalytic catalysts (Table 1). The most important advantage of
applications. In particular, new developments in protein a biocatalyst is its high selectivity. This selectivity
design tools such as rational design and directed is often chiral (i.e., stereo-selectivity), positional
evolution have enabled scientists to rapidly tailor (i.e., regio-selectivity), and functional group specific
the properties of biocatalysts for particular chemical (i.e., chemo-selectivity). Such high selectivity is very
processes. Rational design involves rational alterations desirable in chemical synthesis as it may offer several
of selected residues in a protein to cause predicted benefits such as reduced or no use of protecting
changes in function, whereas directed evolution, some- groups, minimized side reactions, easier separation,
times called irrational design, mimics the natural and fewer environmental problems. Other advantages,
evolution process in the laboratory and involves like high catalytic efficiency and mild operational
repeated cycles of generating a library of different pro- conditions, are also very attractive in commercial
tein variants and selecting the variants with the desired applications.
functions (see the entry ‘‘Protein Design’’). Enzyme The characteristics of limited operating regions,
properties such as stability, activity, selectivity, and substrate or product inhibition, and reactions in
substrate specificity can now be routinely engineered in aqueous solutions have often been considered as the
the laboratory. Presently, approximately 100 different most serious drawbacks of biocatalysts. Many of these
biocatalytic processes are implemented in pharma- drawbacks, however, turn out to be misconceptions
ceutical, chemical, agricultural, and food industries.[1] and prejudices.[2,3] For example, many commercially
The products range from research chemicals to com- used enzymes show excellent stability with half-lives
modity chemicals and the number of applications con- of months or even years under process conditions. In
tinue to grow very rapidly. In spite of these successes, addition, there is an enzyme-catalyzed reaction equiva-
however, the vast potential of biocatalysis has yet to lent to almost every type of known organic reaction.
be fully realized. Many enzymes can accept non-natural substrates and
In this entry, we briefly outline the scope of biocata- convert them into desired products. More importantly,
lysis and discuss its advantages and disadvantages as almost all of the biocatalyst characteristics can be
compared to chemical catalysis. We then review such tailored with protein engineering and metabolic
topics as enzyme and whole-cell based biocatalysis, engineering methods (refer to the section Biocatalyst
biocatalysts used in nonaqueous media, biocatalyst Engineering and see also the entry ‘‘Protein Design’’)
immobilization, discovery and engineering of novel to meet the desired process conditions.
enzymes, and hybrid approaches combining chemical Biocatalytic processes are similar to conventional
and biological synthesis. An overview of the six general chemical processes in many ways. However, when
classifications of enzymes along with their relative use considering a biocatalytic process one must account
in industry is discussed. Selected industrial applications for enzyme reaction kinetics and enzyme stability
of whole-cell based biocatalysis including the produc- for single-step reactions, or metabolic pathways for
tion of lactic acid and 1,3-propanediol are also studied. multiple-step reactions.[4] Fig. 1 shows the key steps
Encyclopedia of Chemical Processing DOI: 10.1081/E-ECHP-120017565
Copyright # 2006 by Taylor & Francis. All rights reserved. 101
102 Biocatalysis
Escherichia coli or Saccharomyces cerevisiae for gene in organic solvents.[6] One such method improves
expression. The overexpressed enzymes are purified enzyme activity in organic solvents by lyophilizing
from the cell extracts based on their chemical and (freeze-drying) an aqueous biocatalyst solution in the
physical properties. The most commonly used enzyme presence of organic and inorganic molecules called excip-
purification techniques include electrophoresis, centri- ients. These excipients include nonbuffer salts, crown
fugation, and chromatography. Centrifugation sepa- ethers, cyclodextrins, and solid-state buffers.[7] Some
rates enzymes based on their differences in mass or remarkable results have also been achieved by using
shape, whereas electrophoresis separates enzymes ionic liquids as solvents in biocatalytic reactions.[8]
based on their differences in charge. Liquid chromatog-
raphy separates enzymes based on their differences
Biocatalyst Immobilization
in charge (ion-exchange chromatography), in mass
(gel filtration chromatography), or in ligand-binding
Immobilization is the process of adhering biocatalysts
property (affinity chromatography).
(isolated enzymes or whole cells) to a solid support.
The whole-cell biocatalysis approach is typically
The solid support can be an organic or inorganic mate-
used when a specific biotransformation requires multi-
rial, such as derivatized cellulose or glass, ceramics,
ple enzymes or when it is difficult to isolate the enzyme.
metallic oxides, and a membrane. Immobilized bioca-
A whole-cell system has an advantage over isolated
talysts offer several potential advantages over soluble
enzymes in that it is not necessary to recycle the cofac-
biocatalysts, such as easier separation of the biocata-
tors (nonprotein components involved in enzyme catal-
lysts from the products, higher stability of the biocata-
ysis). In addition, it can carry out selective synthesis
lyst, and more flexible reactor configurations. In
using cheap and abundant raw materials such as
addition, there is no need for continuous replacement
cornstarches. However, whole-cell systems require
of the biocatalysts. As a result, immobilized biocata-
expensive equipment and tedious work-up because
lysts are now employed in many biocatalytic processes.
of large volumes, and have low productivity. More
More than one hundred techniques for immobiliz-
importantly, uncontrolled metabolic processes may
ing enzymes have been developed which can be divided
result in undesirable side reactions during cell growth.
into five major groups summarized in Table 2.[9]
The accumulation of these undesirable products as well
Adsorption of the enzyme onto a surface is the easiest
as desirable products may be toxic to the cell, and these
and the oldest method of immobilization. Entrapment
products can be difficult to separate from the rest of
and cross-linking tend to be more laborious enzyme
the cell culture. Another drawback to whole-cell
fixation methods, but they do not require altering the
systems is that the cell membrane may act as a mass
enzyme as much as other techniques. The formation
transport barrier between the substrates and the
of the covalent linkage often requires harsh conditions,
enzymes.
which can result in a loss of activity because of confor-
mational changes of the enzyme. It is important to note
that most of these techniques can also be used to
Nonaqueous Biocatalysis
immobilize whole cells. In addition, although these
types of immobilization are considered to be relatively
Historically, enzymes have been used extensively in
old and well established, the emerging field of nano-
aqueous media. Enzymes are well suited to their
tube biotechnology has created another possible means
natural aqueous environment; however, biotransfor-
of immobilizing biocatalysts.[10]
mations in industrial synthesis often involve organic
molecules insoluble in water. More importantly,
because of its high boiling point and high heat of Biocatalyst Discovery: Sources
vaporization, water is usually the least desired solvent and Techniques
of choice for most organic reactions. Thus, shifting
enzymatic reactions from an aqueous to an organic Traditionally, potentially commercial enzymes are
medium is highly desired. identified by screening micro-organisms, which are
Over the past 15 yr, studies have shown that frequently isolated from extreme environments, for
enzymes can work in organic solvents.[5] However, biocatalytic activity. Commercial enzymes are selected
the enzymatic activity is quite low in an organic solvent by probing libraries of related enzymes for a range of
compared to that in water. Recent advances in protein properties, including activity, substrate specificity, sta-
engineering and directed evolution have aided in the bility over a temperature range, enantio-selectivity, or
development of enzymes that show improved activity compatibility under various physical and chemical
in organic solvents. Progress has also been made in conditions. Unfortunately, most of the commercially
developing simple, scalable, and low-cost techniques to viable enzymes have been isolated in only a few micro-
produce highly active biocatalyst preparations for use bial species such as Bacillus and Pseudomonas because
104 Biocatalysis
of the limitations in micro-organism cultivation tech- such as low stability and activity. Moreover, naturally
niques.[11] It has been widely acknowledged that the occurring biocatalysts may not catalyze the reaction
majority of microbial species (up to 99%) have never with the desired non-natural substrates or produce
been cultivated and thus have never been investigated. the desired products. To address these limitations,
To access this vast untapped microbial diversity, molecular techniques have been developed to create
several companies such as Diversa Corporation improved or novel biocatalysts with altered industrial
(San Diego, California, U.S.A.) and TerraGen Dis- operating parameters. It should be noted that, for
covery (Vancouver, British Columbia, Canada) have enzyme based biocatalysts, many molecular techniques
successfully developed modern bioprospecting tech- have been developed for engineering enzymes with
niques such as multiple metagenome cloning to isolate novel or improved characteristics. Readers are referred
novel industrial enzymes.[12] to the entry ‘‘Protein Design.’’ In this section, we
New methods for exploring natural biodiversity mainly discuss the molecular techniques used for
have been greatly facilitated by high-throughput whole-cell based biocatalyst engineering, or metabolic
screening technologies and robust expression in recom- engineering.
binant organisms. Recombinant DNA technology Metabolic engineering is a rapidly growing area
makes it possible to produce enzymes at levels 100-fold with great potential to impact biocatalysis.[13] It has
greater than native expression and allows expression been broadly defined as ‘‘the directed improvement
of genes from organisms that cannot be cultured. of product formation or cellular properties through
Although some problems may be resolved by screening modifications of specific biochemical reaction(s) or
larger libraries of DNA, this may not be the most the introduction of new one(s) with the use of recombi-
efficient or expedient method of obtaining a viable nant DNA technology.’’[14] In an industrial context,
biocatalyst. A more efficient means of obtaining a the ultimate goal of metabolic engineering is the devel-
good biocatalyst may involve engineering the catalyst opment of optimal biocatalysts. In the past two
itself using various protein engineering and metabolic decades, metabolic engineering has been successfully
engineering techniques (refer to the section Biocatalyst used to engineer micro-organisms to produce a
Engineering and see also the entry ‘‘Protein Design’’). wide variety of products, including polymers, aro-
matics, carbohydrates, organic solvents, proteins, anti-
biotics, amino acids, and organic acids. According to
Biocatalyst Engineering the approach taken or the aim, these applications
can be classified into seven groups: 1) expression of
Nature has supplied us with a vast array of biocatalysts heterologous genes for protein production; 2) exten-
capable of catalyzing numerous biological reactions. sion of the range of substrate for cell growth and
Unfortunately, naturally occurring biocatalysts are often product formation; 3) design and construction of path-
not optimal for many specific industrial applications, ways for the production of new products; 4) design and
Biocatalysis 105
Transferases
Fig. 2 The relative use of enzyme classes in industry. (From
Ref.[2].) Transferases catalyze the transfer of functional groups
such as methyl, hydroxymethyl, formal, glycosyl, acyl,
alkyl, phosphate, and sulfate groups by means of a
nucleophilic substitution reaction. They are not widely
regeneration systems have been developed, the most
used in industrial processes; however, there are a few
widely used being the formate=formate dehydrogenase
examples of industrial processes that utilize transferases.
(FDH) system.[17]
A classical example of industrial application of
An example of a pharmaceutical synthesis reaction
transferases is the use of various glycosyltransferases
involving an oxidoreductase is the synthesis of
for the synthesis of oligosaccharides. Oligosaccharides
3,4-dihydroxylphenyl alanine (DOPA).[2] 3,4-Dihy-
and polysaccharides are important classes of naturally
droxylphenyl alanine is a chemical used in the treat-
occurring compounds, which play vital roles in cellular
ment of Parkinson’s disease. The industrial process
recognition and communication processes.[19] Because
that synthesizes DOPA utilizes the oxidoreductase
of the required use of many protection and deprotec-
polyphenol oxidase. As shown in Fig. 3, the monohy-
tion groups, chemical synthesis of complex oligosac-
droxy compound is oxidized by the regio-specific
charides represents a daunting challenge in synthetic
addition of a hydroxyl group. It is worth mentioning
organic chemistry. By contrast, enzymatic synthesis
that epinephrine (adrenaline) can also be synthesized
of oligosaccharides by glycosyltransferases requires
by a similar reaction path using the same enzyme.[2]
very few protection and deprotection steps because of
Another example is the use of leucine dehydrogen-
the high regio- and stereoselectivity of glycosyltrans-
ase coupled with FDH for the reductive amination of
ferases, thus offering an attractive alternative.[2]
trimethylpyruvate to L-tert-leucine (Fig. 4). The whole
Another example is the use of a glucokinase (a trans-
ferase) in combination with an acetate kinase for
the production of glucose-6-phosphate (Fig. 5). This
process is carried out in multikilogram scale by the
Japanese company Unitika.[1]
Hydrolases
Fig. 6 Regio-selective ester-hydrolysis catalyzed by subtilisin. Fig. 8 Enzymatic synthesis of high-fructose corn syrup.
108 Biocatalysis
are important in certain cellular processes, such as con- fermentation (whole-cell biocatalysis) several years
necting nucleotides in DNA replication. However, ago. A production plant was built in Blair, NE, in
similar to isomerases, ligases have very few industrial 2001 and it now produces 140,000 metric tons of poly-
applications.[2] It is important to note that DNA lactic acid per year. It is predicted that the eventual
ligases are essential tools in recombinant DNA tech- cost of polylactic acid will be between $0.50 and
nology and are used almost in every biology-related $0.75 per pound (http:==www.cargilldow.com).
laboratory. One of the drawbacks in the current commercial
fermentation process is that the predominant form of
the product is the deprotonated lactate rather than
INDUSTRIAL APPLICATIONS OF WHOLE-CELL lactic acid, requiring more expensive and wasteful
BASED BIOCATALYSIS product purification steps. This is because the Lacto-
bacillus fermentation operates at a minimum pH of
Whole-cell based biocatalysis utilizes an entire micro- 5.0–5.5 which is above the pKa of lactic acid (3.87).
organism for the production of the desired product. To overcome this limitation, a powerful strain
One of the oldest examples for industrial applications improvement method, genome shuffling, was used to
of whole-cell biocatalysis is the production of acetic improve the acid tolerance of a poorly characterized
acid from ethanol with an immobilized Acetobacter industrial strain of Lactobacillus.[24] A population of
strain, which was developed nearly 200 yr ago.[1] The strains with subtle improvement in pH tolerance was
key advantage of whole-cell biocatalysis is the ability isolated using classical strain improvement methods
to use cheap and abundant raw materials and catalyze such as chemostats, and were then shuffled by recursive
multistep reactions. Recent advances in metabolic pool-wise protoplast fusion to create mutant strains that
engineering have brought a renaissance to whole-cell grow at substantially lower pH than does the wild-type
biocatalysis. In the following sections, two novel indus- strain.
trial processes that utilize whole-cell biocatalysis are
discussed with emphasis on the important role played
by metabolic engineering. 1,3-Propanediol
pathway for production of 1,3-propanediol from 5. Klibanov, A.M. Improving enzymes by using
glycerol was found in Klebsiella pneumoniae, which them in organic solvents. Nature 2001, 409
consists of glycerol dehydratase and 1,3-propanediol (6817), 241–246.
dehydrogenase. The genes encoding these two natural 6. Schoemaker, H.E.; Mink, D.; Wubbolts, M.G.
pathways were cloned and expressed in E. coli. E. coli Dispelling the myths—biocatalysis in industrial
was chosen as the production strain because it has been synthesis. Science 2003, 299 (5613), 1694–1697.
used in large-scale production on an industrial level, it 7. Lee, M.Y.; Dordick, J.S. Enzyme activation for
has many genetic tools, and its metabolism and physiol- nonaqueous media. Curr. Opin. Biotechnol.
ogy are well characterized. This engineered E. coli was 2002, 13 (4), 376–384.
found to produce over 120 g=L of 1,3-propanediol in 8. Kragl, U.; Eckstein, M.; Kaftzik, N. Enzyme cata-
40 hr fed-batch fermentation.[13] lysis in ionic liquids. Curr. Opin. Biotechnol.
2002, 13 (6), 565–571.
9. Klibanov, A.M. Immobilized enzymes and cells
CONCLUSIONS as practical catalysts. Science 1983, 219 (4585),
722–727.
Biocatalysis has become an important tool for indus- 10. Martin, C.R.; Kohli, P. The emerging field of nano-
trial chemical synthesis and is on the verge of signifi- tube biotechnology. Nat. Rev. Drug Discovery
cant growth. In the past several decades, many 2003, 2 (1), 29–37.
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duce a wide variety of products in various industries. ques to identify new microbial biocatalysts.
Most of them use naturally occurring enzymes or Trends Biotechnol. 1998, 16 (6), 265–272.
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