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Industrial enzyme applications Ole Kirk*, Torben Vedel Borchert and Claus Crone Fuglsang
The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used.
Addresses Research and Development, Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark *e-mail: oki@novozymes.com Current Opinion in Biotechnology 2002, 13:345351 0958-1669/02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0958-1669(02)00328-2

century, aimed specifically at the production of enzymes by use of selected production strains, made it possible to manufacture enzymes as purified, well-characterized preparations even on a large scale. This development allowed the introduction of enzymes into true industrial products and processes, for example, within the detergent, textile and starch industries. The use of recombinant gene technology has further improved manufacturing processes and enabled the commercialization of enzymes that could previously not be produced. Furthermore, the latest developments within modern biotechnology, introducing protein engineering and directed evolution, have further revolutionized the development of industrial enzymes (Figure 1). These advances have made it possible to provide tailor-made enzymes displaying new activities and adapted to new process conditions, enabling a further expansion of their industrial use. As illustrated in Table 1, the result is a highly diversified industry that is still growing both in terms of size and complexity. The majority of currently used industrial enzymes are hydrolytic in action, being used for the degradation of various natural substances. Proteases remain the dominant enzyme type, because of their extensive use in the detergent and dairy industries. Various carbohydrases, primarily amylases and cellulases, used in industries such as the starch, textile, detergent and baking industries, represent the second largest group [1]. As illustrated in Figure 2, the technical industries, dominated by the detergent, starch, textile and fuel alcohol industries, account for the major consumption of industrial enzymes. Overall, the estimated value of the worldwide use of industrial enzymes has grown from $1 billion [1] in 1995 to $1.5 billion in 2000 [2]. This growth, however, has stagnated in some of the major

Introduction
The enzyme industry as we know it today is the result of a rapid development seen primarily over the past four decades thanks to the evolution of modern biotechnology. Enzymes found in nature have been used since ancient times in the production of food products, such as cheese, sourdough, beer, wine and vinegar, and in the manufacture of commodities such as leather, indigo and linen. All of these processes relied on either enzymes produced by spontaneously growing microorganisms or enzymes present in added preparations such as calves rumen or papaya fruit. The enzymes were, accordingly, not used in any pure or well-characterized form. The development of fermentation processes during the later part of the last
Figure 1 The steps involved in classical versus state-ofthe-art development of enzymes.

Present enzyme development Creating biological diversity Primary screening Secondary screening Creating expression system Fermentation Up-scaling process Production Purification Formulation

Classical enzyme development Nature's diversity

Molecular evolution

Classical mutagenesis

Up-scaling process Production


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Protein technologies and commercial enzymes

Table 1 Enzymes used in various industrial segments and their applications. Industry Detergent (laundry and dish wash) Enzyme class Protease Amylase Lipase Cellulase Mannanase Amylase Amyloglucosidase Pullulanase Glucose isomerase Cyclodextrin-glycosyltransferase Xylanase Protease Protease Lipase Lactase Pectin methyl esterase Pectinase Transglutaminase Amylase Xylanase Lipase Phospholipase Glucose oxidase Lipoxygenase Protease Transglutaminase Phytase Xylanase -Glucanase Pectinase Amylase -Glucanase Acetolactate decarboxylase Laccase Cellulase Amylase Pectate lyase Catalase Laccase Peroxidase Lipase Protease Amylase Xylanase Cellulase Lipase Phospholipase Lipase Acylase Nitrilase Protease Lipase Amyloglucosidase Glucose oxidase Peroxidase Application Protein stain removal Starch stain removal Lipid stain removal Cleaning, color clarification, anti-redeposition (cotton) Mannanan stain removal (reappearing stains) Starch liquefaction and saccharification Saccharification Saccharification Glucose to fructose conversion Cyclodextrin production Viscosity reduction (fuel and starch) Protease (yeast nutrition fuel) Milk clotting, infant formulas (low allergenic), flavor Cheese flavor Lactose removal (milk) Firming fruit-based products Fruit-based products Modify visco-elastic properties Bread softness and volume, flour adjustment Dough conditioning Dough stability and conditioning (in situ emulsifier) Dough stability and conditioning (in situ emulsifier) Dough strengthening Dough strengthening, bread whitening Biscuits, cookies Laminated dough strengths Phytate digestibility phosphorus release Digestibility Digestibility De-pectinization, mashing Juice treatment, low calorie beer Mashing Maturation (beer) Clarification (juice), flavor (beer), cork stopper treatment Denim finishing, cotton softening De-sizing Scouring Bleach termination Bleaching Excess dye removal Pitch control, contaminant control Biofilm removal Starch-coating, de-inking, drainage improvement Bleach boosting De-inking, drainage improvement, fiber modification Transesterification De-gumming, lyso-lecithin production Resolution of chiral alcohols and amides Synthesis of semisynthetic penicillin Synthesis of enantiopure carboxylic acids Unhearing, bating De-pickling Antimicrobial (combined with glucose oxidase) Bleaching, antimicrobial Antimicrobial

Starch and fuel

Food (including dairy)

Baking

Animal feed

Beverage

Textile

Pulp and paper

Fats and oils Organic synthesis

Leather Personal care

technical industries, first of all the detergent industry [2]. The fastest growth over the past decade has been seen in the baking and animal feed industries, but growth is also being generated from applications established in a wealth of other industries spanning from organic synthesis to paper and pulp and personal care. This review will, segment by segment, discuss the most important recent developments in the technical use of enzymes and will consider the

 

most recent technological advances that have facilitated these developments.

New technologies for enzyme discovery


Natural microorganisms have over the years been a great source of enzyme diversity. The developments in bioinformatics and the availability of sequence data have increased immensely the efficiency of isolating an interesting gene

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from nature. Rational protein engineering and the possibility of introducing small changes to proteins, on the basis of their structure and the related biochemical and biophysical properties, introduced a new valuable tool to enzyme optimization in the 1980s. Directed evolution is the latest addition to the toolbox (for a recent review see [3]). The more or less random introduction of mutations generates variant libraries that are subsequently exposed to a screening or selection procedure. The isolated, improved variants from one round of screening are then used as starting material in the following rounds of recombination and/or new diversity generation (Figure 1). Recently, various attempts at understanding the important parameters in directed evolution have emerged and successful examples of combining rational engineering with directed evolution have been reported [4,5]. Usually, the new, exciting technology is predicted to out-compete the existing technologies, but we expect that time will demonstrate how the combined use of rational design, directed evolution and natures diversity will be far superior to any lone-standing technology.

Figure 2

Food

Technical

Animal feed

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Segmentation of the industrial enzyme market. In the year 2000, the enzyme market totalled $1.5 billion. The technical industries segment comprises the detergent, starch, textile, fuel alcohol, leather, and pulp and paper industries.

The detergent industry


Their use as detergent additives still represents the largest application of industrial enzymes, both in terms of volume and value. The major component is proteases, but other and very different hydrolases are introduced to provide various benefits, such as the efficient removal of specific stains (Table 1). Constantly, new and improved engineered versions of the traditional detergent enzymes, proteases and amylases, are developed. These new second- and third-generation enzymes are optimized to meet the requirements for performance in detergents, the composition of which is also constantly developed. In particular, the compatibility of enzymes with detergent components (i.e. typically stability properties) is addressed, but their ability to function at lower temperatures has also been amongst the recently reported improvements. To save energy, the temperature used in household laundering and automated dishwashers has been reduced in recent years. This often results in problems with efficient cleaning and stain removal that enzyme technology can help overcome. Recent examples of second-generation detergent enzymes include the development of novel amylases that have enhanced activity at lower temperatures and alkaline pH, while maintaining the necessary stability under detergent conditions. These enzymes were developed by the combined use of microbial screening and rational protein engineering [6]. Proteases displaying activity at low temperatures have been isolated from nature, but have also been evolved in the laboratory by a directed evolution approach [7]. Furthermore, from a starting material of 26 subtilisin proteases Ness and coworkers [8] utilized one round of DNA shuffling to isolate new proteases with various improved properties. The improvements included characteristics very relevant for detergent proteases (i.e. improved activity and stability at alkaline pH). The most recent introduction of a new enzyme class into a detergent has been the addition of a mannanase the result of a joint development between Procter and Gamble and Novozymes [9]. This enzyme helps remove various food stains containing guar gum, a commonly used stabilizer and thickening agent in food products.

Enzymes for starch conversion


The enzymatic conversion of starch to high fructose corn syrup is a well-established process and provides a beautiful example of a bioprocess in which the consecutive use of several enzymes is necessary. The enzymes utilized in the starch industry are also subjected to constant improvements. The first step in the process is the conversion of starch to oligomaltodextrins by the action of -amylase. The concomitant injection of steam puts extreme demands on the thermostability of the enzyme. Using traditional -amylases, the pH has to be adjusted to an undesirable high level and calcium must be added to stabilize the enzyme. New -amylases with optimized properties, such as enhanced thermal stability, acid tolerance, and ability to function without the addition of calcium, have recently been developed [6,10,11] offering obvious benefits to the industry. Engineering efforts have also been undertaken to develop improved versions of the enzymes used later in the process (i.e. glucoamylase and glucose isomerase [12,13]).

Fuel alcohol production


In the alcohol industry, the use of enzymes for the production of fermentable sugars from starch is also well established. Over the past decade, there has been an increasing interest in fuel alcohol as a result of increased environmental concern, higher crude oil prices and, more acutely, by the ban in certain regions of the gasoline additive methyl

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Figure 3 Peroxidase (removal of excess dye) Acid cellulase (biopolishing) Enzymes used in various unit operations in textile wet processing and the manufacturing of Denim.

-Amylase

Pectinase

De-sizing

Scouring

Bleaching

Dyeing

Finishing

Raw fabric

Catalase (bleach clean-up)

Finished fabric

De-sizing

Stone-wash

Bleaching

Finished blue jeans

Neutral cellulase

Laccase/mediator
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tert-butyl ether (MTBE), which can be interchanged directly with ethanol [1416]. Therefore, intense efforts are currently being undertaken to develop improved enzymes that can enable the utilization of cheaper and partially utilized substrates such as lignocellulose, to make bio-ethanol more competitive with fossil fuels [17,18]. The cost of enzymes needed to turn lignocellulose into a suitable fermentation feed-stock is a major issue, and current work focuses both on the development of enzymes with increased activity and stability as well as on their efficient production. Huge governmental programs have been launched in the United States by the Department of Energy to support these developments, spurred by the general emphasis on reducing pollution and the need to work towards fulfilling the Kyoto protocol.

Enzymes for the feed industry


The use of enzymes as feed additives is also well established. For example, xylanases and -glucanases have been used throughout the past decade in cereal-based feed for monogastric animals which, contrary to ruminants, are unable to fully degrade and utilize plant-based feeds containing high amounts of cellulose and hemicellulose. During recent years focus has been on the utilization of natural phosphorus bound in phytic acid in cereal-based feed for monogastrics. Better utilization of total plant phosphorus, of which 8590% is bound in phytic acid, is only obtained by adding the enzyme phytase to the feed. The attention on this enzyme has increased dramatically during recent years owing to the bovine spongiform encephalopathy (BSE)-related bone-meal ban in many countries, which has effectively removed the main traditional source for inorganic phosphorus. Also, several western countries with intensive animal production have adopted standards for the release of phosphorus into the environment. As the addition of phytase to feeds results in a significant reduction of the phosphorus outlet from monogastrics, the phytases have grown to become the largest enzyme segment in the feed industry. Besides the direct effects on phosphorus uptake and secretion, indirect effects on the uptake of other nutrients are also being recognized [20,21]. The most recent advances in feed enzymes have been aimed at improvements in the applicability and performance of phytases [22]. New fungal phytases have been identified with 450-fold higher specific activities than previously reported [23]. Alternative approaches for the development of more effective enzymes have been to increase the catalytic activity of fungal phytases by site-directed mutagenesis; for example, on the basis of three-dimensional structural studies, the specific activity of the Aspergillus fumigatus phytase was increased fourfold [24]. In order for the enzymes to be applicable for use in

Textile applications
In the textile industry a completely new enzymatic activity has recently been introduced. This industry is under considerable environmental pressure owing to its large energy and water consumption and subsequent environmental pollution. One of the most energy- and water-consuming steps in the processing of cotton is the scouring step, the removal of various remaining cell-wall components on the cellulose fibers performed at high temperature and under strong alkaline conditions. An alternative, enzyme-based process performed at much lower temperatures and using less water has now been developed based on a pectate lyase [19]. The positive environmental impact of the new process was recognized by a grant of the United States Presidential Green Chemistry Challenge Award in 2001. Following the introduction of this step, enzymes have now been introduced into most of the major steps in the manufacturing of cotton textiles (see Figure 3). The use of these enzymes has benefited both the textile industry and the environment.

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pelleted feed products, they have to survive high temperatures (above 80C) during pelleting for short periods of time. A novel approach used to achieve thermal stabilization has been the construction of a consensus phytase based on homologies among various phytases. This enzyme exhibits an increase in thermal stability to around 80C [25]. Still, phosphorus utilization is not the only issue of concern to the animal feed industry; continuous effort is put into obtaining increased nutritional value from various feed sources, for example, by increasing the digestibility of the protein in soybean meal. It is likely that in the future we will see new and different hydrolytic enzymes applied in the feed industry to increase the value of feed stock, thus lowering the energy consumption and pollution per live stock to the benefit of the environment.

amylopectin to a degree that prevents re-crystallization after gelatinization, without completely degrading the amylopectin network which provides the bread with elasticity. Besides the above-mentioned advances, a few entirely new applications within the food industry should be mentioned, although little, if any, literature is publicly available. The use of laccase for clarification of juice (laccases catalyze the cross-linking of polyphenols, resulting in an easy removal of polyphenols by filtration) and for flavor enhancement in beer are recently established applications within the beverage industry. It is likely that the functional understanding of different enzyme classes will provide new applications within the food industry in the future.

Processing of fats and oils


In the fat and oil industries, several new enzyme-based processes have recently been introduced. Even though the use of immobilized lipases in the interesterification of triglycerides was first described in the 1980s, the process has not been sufficiently cost-effective to be introduced in true large-scale applications, for example, in the production of margarine. Although enzyme production has become much more efficient, the cost of immobilization has remained an obstacle. Recent developments have, however, changed this picture. A new process for immobilizing lipases based on the granulation of silica has dramatically lowered process costs, and procedures based on this new material are now being implemented for the production of commodity fats and oils with no content of trans-fatty acids [32]. Another recently introduced process is the removal of phospholipids in vegetable oils (de-gumming), using a highly selective microbial phospholipase [33]. This is yet another example where the introduction of an enzymebased step has enabled both energy and water savings for the benefit of both the industry and the environment.

Enzymes for the food industry


As indicated in Table 1, applications of enzymes in the food industry are many and diverse, ranging from texturizing to flavoring. Common to more or less all food applications, the enzymes are applied to processed food products as processing agents upstream from the final product. Several advances have been made in the optimization of enzymes for existing applications and in the use of recombinant protein production to provide efficient mono-component enzymes that do not have potential detrimental side-effects. Recently, much work has been carried out on the application of transglutaminase as a texturing agent in the processing of, for example, sausages, noodles and yoghurt, where cross-linking of proteins provides improved viscoelastic properties of the products [26]. An obstacle, which may prevent even wider usage, is the currently limited availability of the enzyme in industrial scale. At present only the transglutaminase from Streptoverticillium sp. is commercially available at a reasonable scale, and work is ongoing to increase the availability of the enzyme by recombinant production in Escherichia coli [27]. Within the baking industry there is an increasing focus on lipolytic enzymes [28,29]. Recent findings suggest that (phospho)lipases can be used to substitute or supplement traditional emulsifiers, as the enzymes degrade polar wheat lipids to produce emulsifying lipids in situ. Also, efforts are currently devoted towards the further understanding of bread staling and the mechanisms behind the enzymatic prevention of staling when using -amylases and xylanases [30]. Studies have confirmed previous findings showing that water-binding capacity and retention in the starch and hemicellulose fractions of the bread, being the substrates of -amylases and xylanases, respectively, to be critical for maintaining softness and elasticity. The recently determined three-dimensional structure of the widely applied amylase for antistaling (Novamyl) provided further insight into the mechanism of enzyme action [31]. This amylase is probably capable of degrading

Enzymes for organic synthesis


Chemical synthesis is an area where the use of enzyme catalysis has long been seen as having great promise. Even so, the chemical industry has been slow to implement enzyme-based processes and the use of enzymes in the chemical industry is still low compared with other industries. At present, however, we are seeing very significant growth in this area and enzymebased processes are now, finally, being widely introduced for the production of a diversity of different chemicals; one key example is in the production of single-enantiomer intermediates used in the manufacture of drugs and agrochemicals [34]. This market is characterized by a very high degree of fragmentation, as very few enzymes have applicability in a broad range of different processes. Recently introduced enzymebased processes include the use of lipases for the production of enantiopure alcohols and amides, nitrilases for the production of enantiopure carboxylic acids, and acylases for the production of new semisynthetic penicillins [34]. As many companies are currently at an early stage in the exploitation of enzyme-based catalysis, many new developments are expected in this area over the next few years.

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Conclusions and perspectives


As outlined above, enzymes are currently used in several different industrial products and processes and new areas of application are constantly being added. Thanks to advances in modern biotechnology, enzymes can be developed today for processes where no one would have expected an enzyme to be applicable just a decade ago. Common to most applications, the introduction of enzymes as effective catalysts working under mild conditions results in significant savings in resources such as energy and water for the benefit of both the industry in question and the environment. In a world with a rapidly increasing population and approaching exhaustion of many natural resources, enzyme technology offers a great potential for many industries to help meet the challenges they will face in years to come.

14. Jolly L: The commercial viability of fuel ethanol from sugar cane. Int Sugar J 2001, 103:117-143. 15. Taylor F, Mcaloon AJ, Craig JC, Yang P, Wahjudi J, Eckhoff SR: Fermentation and costs of fuel ethanol from corn with quick-germ process. Appl Biochem Biotechnol 2001, 94:41-49. 16. Taylor F, Kurantz MJ, Goldberg N, Mcaloon AJ, Craig JC: Dry-grind process for fuel ethanol by continuous fermentation and stripping. Biotechnol Prog 2000, 16:541-547. 17. Zaldivar J, Nielsen J, Olsson L: Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 2001, 56:17-34.

18. Wheals AE, Basso LC, Alves DMG, Amorim AV: Fuel ethanol after 25 years. Trends Biotechnol 1999, 17:482-487. 19. Tzanov T, Calafell M, Guebitz GM, Cavaco-Paulo A: Biolpreparation of cotton fabrics. Enzyme Microb Technol 2001, 29:357-362. This work describes the successful substitution of traditional chemical processes by the introduction of pectinases for biopreparation of cotton fabrics. 20. Lei XG, Stahl CH: Nutritional benefits of phytase and dietary determinants of its efficacy. J Appl Anim Res 2000, 17:97-112. The paper discusses the beneficial gains of utilizing phytase for animal feed, in a fair and critical manner, and provides a nice overview on this particular usage of phytase. 21. Kies AK, van Hemert KHF, Sauer WC: Effect of phytase on protein and amino acid digestibility and energy utilization. Worlds Poult Sci J 2001, 57:109-126. 22. Lei XG, Stahl CH: Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 2001, 57:474-481. 23. Lassen SF, Breinholt J, stergaard PR, Brugger R, Bischoff A, Wyss M, Fuglsang CC: Expression, gene cloning and characterization of five novel phytases from four Basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp. and Trametes pubescens. Appl Environ Microbiol 2001, 67:4701-4707. Describes an entirely new group of fungal phytases and their properties. One of these phytases has recently been commercialized for application in animal feed. 24. Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L, van Loon APGM, Pasamontes L: Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure. Protein Sci 2000, 9:1304-1311. 25. Lehmann M, Kostrewa D, Wyss M, Brugger R, DArcy A, Pasamontes L, van Loon APGM: From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng 2000, 13:49-57. An interesting new approach for designing enzymes with improved properties. A significant thermal stabilization is obtained compared with the parent phytase backbones. 26. Kuraishi C, Yamazaki K, Susa Y: Transglutaminase: its utilization in the food industry. Foods Rev Int 2001, 17:221-246. Industrial application of transglutaminase is still in its infancy. This paper provides a nice overview of some of the first applications of this enzyme in the food industry. 27. Yokoyama K, Nakamura N, Seguro K, Kubota K: Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form. Biosci Biotechnol Biochem 2000, 64:1263-1270.

References and recommended reading


Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest of outstanding interest


1. Godfrey T, West SI: Introduction to industrial enzymology. In Industrial Enzymology, edn 2. Edited by Godfrey T, West S. London: Macmillan Press; 1996:1-8. McCoy M: Novozymes emerges. Chem Eng News 2000, 19:23-25. Tobin MB, Gustafsson C, Huisman GW: Evolution: the rational basis for irrational design. Curr Opin Struct Biol 2000, 10:421-427. Voigt CA, Kauffman S, Wang ZG: Rational evolutionary design: the theory of in vitro protein evolution. Adv Protein Chem 2000, 55:79-160.

2. 3. 4.

5.

Altamirano MM, Blackburn JM, Aguayo C, Fersht AR: Directed evolution of a new catalytic activity using the /-barrel scaffold. Nature 2000, 403:617-622. The elegant combination of rational engineering and directed molecular evolution are used for the introduction of a new catalytic activity in an enzyme. 6. Bisgaard-Frantzen H, Svendsen A, Norman B, Pedersen S, Kjrulff S, Outtrup H, Borchert TV: Development of industrially important -amylases. J Appl Glycosci 1999, 46:199-206. Wintrode PL, Miyazaki K, Arnold FH: Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution. J Biol Chem 2000, 275:31635-31640.

7.

Ness JE, Welch M, Giver L, Bueno M, Cherry JR, Borchert TV, Stemmer WPC, Minshull J: DNA shuffling of subgenomic sequences of subtilisin. Nat Biotechnol 1999, 17:893-896. This work describes the shuffling of a large family of homologous genes and analysis of the resulting functional diversity. Screening of a rather small library resulted in improvements for five different properties. 9. McCoy M: Soaps & detergents. Chem Eng News 2001, 20:19-32. An update on the latest developments within the detergent industry also introducing the latest new detergent enzyme, a mannanase. 10. Shaw A, Bott R, Day AG: Protein engineering of -amylases for low pH performance. Curr Opin Biotechnol 1999, 10:349-352. 11. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C: Probing structural determinants specifying high thermostability in Bacillus licheniformis -amylase. J Mol Biol 2000, 301:1041-1057. The elegant use of suppressors aided the construction and analysis of thermostability of 175 amylase variants. Several stabilizing mutations were identified. 12. Sauer J, Sigurdskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B: Glucoamylase: structure/function relationships and protein engineering. Biochem Biophys Acta 2000, 1543:275-293. 13. Hartley BS, Hanlon N, Jackson RJ, Rangrajan M: Glucose isomerase: insight into protein engineering for increased thermostability. Biochem Biophys Acta 2000, 1543:294-335.

8.

28. Collar C, Martinez JC, Andreu P, Armero E: Effect of enzyme associations on bread dough performance. A response surface study. Food Sci Technol Int 2000, 6:217-226. 29. Monfort A, Blasco A, Sanz P, Prieto JA: Expression of LIP1 and LIP2 genes from Geotricum species in bakers yeast strains and their application to the bread-making process. J Agric Food Chem 1999, 47:803-808. 30. Andreu P, Collar C, Martnez-Anaya MA: Thermal properties of doughs formulated with enzymes and starters. Eur Food Res Technol 1999, 209:286-293. 31. Dauter Z, Dauter M, Brzozowski AM, Christensen S, Borchert TV, Beier L, Wilson KS, Davies GJ: X-ray structure of Novamyl, the five-domain maltogenic -amylase from Bacillus

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stearothermophilus: maltose and acarbose complexes at 1.7 resolution. Biochemistry 1999, 38:8385-8392. Describes the determination of the three-dimensional structure of a maltogenic -amylase widely applied for providing antistaling effects in white bread. Structural insight into the unique specificity and performance of this enzyme is also provided. 32. Christensen MW, Andersen L, Kirk O, Holm HC: Enzymatic interesterification of commodity oils and fats: approaching the tonnes scale. Lipid Technol News 2001,7:33-37.

33. Clausen K: Enzymatic oil-degumming by a novel microbial phospholipase. Eur J Lipid Sci Technol 2001, 103:333-340. The use of phospholipases for oil-degumming is described with focus on the introduction of the first enzyme of microbial origin for this application. 34. Schmidt A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B: Industrial biocatalysis today and tomorrow. Nature 2001, 409:258-268.