Industrial Enzymes

Industrial Enzymes

Structure, Function and Applications

Edited by

Julio Polaina

and

Andrew P. MacCabe

Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Valencia, Spain

ISBN 978-1-4020-5376-4 (HB) ISBN 978-1-4020-5377-1 (e-book)

Published by Springer,

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Cover illustration: Crystal structure of xylanase B fromBacillussp. BP-23.

Courtesy of Julia Sanz-Aparicio.

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Preface. Industrial Enzymes in the 21st Century ix Julio Polaina and Andrew P. MacCabe

Contributors xi

SECTION A. CARBOHYDRATE ACTIVE ENZYMES

Chapter 1. Amylolytic Enzymes: Types, Structures and Specificities 3 Martin Machoviˇc and Štefan Janeˇcek

Chapter 2. The Use of Starch Processing Enzymes in the Food Industry 19 Józef Synowiecki

Chapter 3. Cellulases for Biomass Conversion 35

Qi Xu, William S. Adney, Shi-You Ding and Michael E. Himmel

Chapter 4. Cellulases in the Textile Industry 51

Arja Miettinen-Oinonen

Chapter 5. Xylanases: Molecular Properties and Applications 65 F. I. Javier Pastor, Óscar Gallardo, Julia Sanz-Aparicio

and Pilar Díaz

Chapter 6. Microbial Xylanolytic Carbohydrate Esterases 83 Evangelos Topakas and Paul Christakopoulos

Chapter 7. Structural and Biochemical Properties of Pectinases 99 Sathyanarayana N. Gummadi, N. Manoj and D. Sunil Kumar Chapter 8. -L-rhamnosidases: Old and New Insights 117

Paloma Manzanares, Salvador Vallés, Daniel Ramón and Margarita Orejas

v

Chapter 9. Application of Glycosidases and Transglycosidases

in the Synthesis of Oligosaccharides 141

Francisco J. Plou, Aránzazu Gómez de Segura and Antonio Ballesteros

SECTION B. PEPTIDASES

Chapter 10. An Introduction to Peptidases and theMEROPSDatabase 161 Neil D. Rawlings, Fraser R. Morton and Alan J. Barrett

Chapter 11. Cysteine Proteases 181

Zbigniew Grzonka, Franciszek Kasprzykowski and Wiesław Wiczk

Chapter 12. Subtilisin 197

John Donlon

Chapter 13. Aspartic Proteases Used in Cheese Making 207 Félix Claverie-Martín and María C. Vega-Hernández

Chapter 14. Metalloproteases 221

Johanna Mansfeld

Chapter 15. Aminopeptidases 243

Yolanda Sanz

SECTION C. LIPASES

Chapter 16. Lipases: Molecular Structure and Function 263 Marina Lotti and Lilia Alberghina

Chapter 17. Use of Lipases in the Industrial Production

of Esters 283

Soundar Divakar and Balaraman Manohar

Chapter 18. Use of Lipases in Organic Synthesis 301

Vicente Gotor-Fernández and Vicente Gotor

Chapter 19. Use of Lipases for the Production of Biodiesel 317 Andrea Salis, Maura Monduzzi and Vincenzo Solinas

Chapter 20. Use of Lipases in the Synthesis of Structured

Lipids in Supercritical Carbon Dioxide 341

José da Cruz Francisco, Simon P. Gough and Estera S. Dey

SECTION D. NUCLEIC ACIDS ENZYMES

Chapter 21. Restriction and Homing Endonucleases 357 Krzysztof J. Skowronek and Janusz M. Bujnicki

Chapter 22. DNA Polymerases for PCR Applications 379 Régen Drouin, Walid Dridi and Oumar Samassekou

Chapter 23. Prokaryotic Reverse Transcriptases 403

Bert C. Lampson

Chapter 24. Dicer: Structure, Function and Role in RNA-Dependent

Gene-Silencing Pathways 421

Justin M. Pare and Tom C. Hobman

SECTION E. OXIDOREDUCTASES AND OTHER ENZYMES OF DIVERSE FUNCTION

Chapter 25. Hydrogen Peroxide Producing and Decomposing Enzymes:

Their Use in Biosensors and Other Applications 441 Nóra Adányi, Teréz Barna, Tamás Emri, Márton Miskei

and István Pócsi

Chapter 26. Laccases: Biological Functions, Molecular Structure and

Industrial Applications 461

Miguel Alcalde

Chapter 27. High Redox Potential Peroxidases 477

Ángel T. Martínez

Chapter 28. Amino Acid Dehydrogenases 489

Stephen Y.K. Seah

Chapter 29. Phytase: Source, Structure and Application 505 Xin Gen Lei, Jesus M. Porres, Edward J. Mullaney

and Henrik Brinch-Pedersen

Chapter 30. Nitrile Hydrolases 531 Praveen Kaul, Anirban Banerjee and Uttam Chand Banerjee Chapter 31. Aspartases: Molecular Structure, Biochemical Function

and Biotechnological Applications 549

Tomohiro Mizobata and Yasushi Kawata

Chapter 32. Transglutaminases 567

María Jesús Arrizubieta

Chapter 33. Penicillin Acylases 583

David W. Spence and Martin Ramsden

Chapter 34. Hydantoinases 599

Yun-Peng Chao, Chung-Jen Chiang, Jong-Tzer Chern and Jason T.C. Tzen

Subject Index 607

Organism Index 637

INDUSTRIAL ENZYMES IN THE 21st CENTURY

Man’s use of enzymes dates back to the earliest times of civilization. Important human activities in primitive communities such as the production of certain types of foods and beverages, and the tanning of hides and skins to produce leather for garments, involved the application of enzyme activities, albeit unknowingly.

However, not until the 19th century with the development of biochemistry and the pioneering work of a number of eminent scientists did the nature of enzymes and how they work begin to be clarified. In France Anselme Payen and Jean-François Persoz described the isolation of an amylolytic substance from germinating barley (1833). Shortly afterwards the Swedish chemist Jöns Jacob Berzelius coined the term catalysis (1835) to describe the property of certain substances to accelerate chemical reactions. In Germany the physiologist Theodor Schwann discovered the digestive enzyme pepsin (1836), Wilhelm Kühne proposed the term ‘enzyme’ (1877), and the brothers Hans and Eduard Buchner demonstrated that the transformation of glucose into ethanol could be carried out by chemical substances (enzymes) present in cell-free extracts of yeast (1897). In the 1870’s the Danish chemist Christian Hansen succeeded in obtaining pure rennet from calves’ stomachs, the use of which in cheese-making resulted in considerable improvements in both product quantity and quality. Shortly thereafter he industrialised the production of rennet thus setting in motion the first enzyme production industry.

During the 20th century the recognition that enzymes are proteins along with the design of techniques for their purification and analysis, principally the work of James B. Sumner and Kaj Linderstrøm-Lang, paved the way for the development of procedures for their industrial production and use. The nineteen-sixties witnessed two major breakthroughs that had a major impact on the enzyme industry: the commercialisation of glucoamylase which catalyses the production of glucose from starch with much greater efficiency than that of the chemical procedure of acid hydrolysis, and the launch of the first enzyme-containing detergents. The devel- opment of genetic engineering in the eighties provided the tools necessary for the production and commercialisation of new enzymes thus seeding a second explosive expansion to the current billion dollar enzyme industry. Recent advances in X-ray crystallography and other analytical methods in the field of protein chemistry along with the ever increasing amounts of biological information available from genomics

ix

programs and molecular techniques such as directed evolution and gene and genome shuffling, are bringing powerful means to bear on the study and manipulation of enzyme structure and function. The search for improvements in existing enzyme- catalysed procedures, the need to develop new technologies and the increasing concern for responsible use and reuse of raw materials can be expected to stimulate not only the rational modification of enzymes to match specific requirements but also the design of new enzymes with totally novel properties.

The aim of this book is to provide in a single volume an updated revision of the most important types of industrial enzymes based on consideration of their physicochemical and catalytic properties, three-dimensional structure, and the range of current and foreseeable applications. The first section of this volume is dedicated to the carbohydrate active enzymes which are extensively used not only in many food industry applications (baking, beverage production, starch processing, etc.) but also in the industrial production of textiles, detergents, paper, ethanol, etc.

The second section, on peptidases, begins with an introductory chapter about the MEROPS database which constitutes the current classification of reference for this important group of enzymes, and subsequent chapters review the most industrially relevant types of peptidases. The section on lipases places special emphasis on the increasing application of these enzymes in synthetic processes. Nucleic acid modifying activities are considered in the fourth section. Whilst the nature of the applications and scale of use of the latter are not yet comparable to those of the enzymes considered in the preceding sections, they are of growing in importance given the indispensability of some in highly specialised fields including basic and applied research, medicine, pharmaceuticals, agronomy and forensics. The final section considers a number of important enzymes that cannot be classified into any of the other sections.

We wish to thank everyone involved in making this book possible and hope that it will become a tool equally useful to researchers, industrialists and students.

Julio Polaina Andrew P. MacCabe

Adányi, Nóra, sect. E, ch. 25, p. 439 Adney, William S., sect. A, ch. 3, p. 35 Alberghina, Lilia, sect. C, ch. 16, p. 263 Alcalde, Miguel, sect. E, ch. 26, p. 459 Arrizubieta, María J., sect. E, ch. 32, p. 565 Ballesteros, Antonio, sect. A, ch. 9, p. 141 Banerjee, Anirban, sect. E, ch. 30, p. 529 Banerjee, Uttam Chand, sect. E, ch. 30, p. 529 Barna, Teréz, sect. E, ch. 25, p. 439

Barrett, Alan J., sect. B, ch. 10, p. 161

Brinch-Pedersen, Henrik, sect. E, ch. 29, p. 503 Bujnicki, Janusz M., sect. D, ch. 21, p. 355 Chao, Yun-Peng, sect. E, ch. 34, p. 597 Chern, Jong-Tzer, sect. E, ch. 34, p. 597 Chiang, Chung-Jen, sect. E, ch. 34, p. 597 Christakopoulos, Paul, sect. A, ch. 6, p. 83 Claveríe-Martín, Félix, sect. B, ch. 13, p. 207 Dey, Estera S., sect. C, ch. 20, p. 339 Díaz, Pilar, sect. A, ch. 5. p. 65 Ding, Shi-You, sect. A, ch, 3, p. 35 Divakar, Soundar, sect. C, ch. 17, p. 283 Donlon, John, sect. B, ch. 12, p. 197 Dridi, Walid, sect. D, ch. 22, p. 377 Drouin, Régen, sect. D, ch. 22, p. 377 Emri, Tamás, sect. E, ch. 25, p. 439

Francisco, José da Cruz, sect. C, ch. 20, p. 339 Gallardo, Óscar, sect. A, ch. 5, p. 65

Gómez de Segura, Aránzazu, sect. A, ch. 9, p. 141 Gotor, Vicente, sect. C, ch. 18, p. 301

Gotor-Fernández, Vicente, sect. C, ch. 18, p. 301 Gough, Simon P., sect. C, ch. 20, p. 339

Grzonka, Zbigniew, sect. B, ch. 11, p. 181

Gummadi, Sathyanarayana N., sect. A, ch. 7, p. 99 Himmel, Michael E., sect. A, ch. 3, p. 35

xi

Hobman, Tom C., sect. D, ch. 24, p. 419 Janecek,ˇ Stefan, sect. A, ch. 1, p. 3ˇ

Kasprzykowski, Franciszek, sect. B, ch. 11, p. 181 Kaul, Praveen, sect. E, ch. 30, p. 529

Kawata, Yasushi, sect. E, ch. 31, p. 547 Kumar, D. Sunil, sect. A, ch. 7, p. 99 Lampson, Bert, sect. D, ch. 23, p. 401 Lei, Xin Gen, sect. E, ch. 29, p. 503 Lotti, Marina, sect. C, ch. 16, p. 263 Machovic, Martin, sect. A, ch. 1, p. 3ˇ Manohar, Balaraman, sect. C, ch. 17, p. 283 Manoj, N., sect. A, ch. 7, p. 99

Mansfeld, Johanna, sect. B, ch. 14, p. 221 Manzanares, Paloma, sect. A, ch. 8, p. 117 Martínez, Ángel T., sect. E, ch. 27, p. 475 Miettinen-Oinonen, Arja, sect. A, ch. 4, p. 51 Miskei, Márton, sect. E, ch. 25, p. 439 Mizobata, Tomohiro, sect. E, ch. 31, p. 547 Monduzzi, Maura, sect. C, ch. 19, p. 315 Morton, Fraser R., sect. B, ch. 10, p. 161 Mullaney, Edward J., sect. E, ch. 29, p. 503 Orejas, Margarita, sect. A, ch. 8, p. 117 Pare, Justin M., sect. D, ch. 24, p. 419 Pastor, F. I. Javier, sect. A, ch. 5, p. 65 Plou, Francisco J., sect. A, ch. 9, p. 141 Pócsi, István, sect. E, ch. 25, p. 439 Porres, Jesus M., sect. E, ch. 29, p. 503 Ramón, Daniel, sect. A, ch. 8, p. 117 Ramsden, Martin, sect. E, ch. 33, p. 581 Rawlings, Neil D., sect. B, ch. 10, p. 161 Salis, Andrea, sect. C, ch. 19, p. 315 Samasekou, Oumar, sect. D, ch. 22, p. 377 Sanz, Yolanda, sect. B, ch. 15, p. 243 Sanz-Aparicio, Julia, sect. A, ch. 5, p. 65 Seah, Stephen Y. K., sect. E, ch. 28, p 487 Skowronek, Krzysztof J., sect. D, ch. 21, p. 355 Solinas, Vincenzo, sect. C, ch. 19, p. 315 Spence, David W., sect. E, ch. 33, p. 581 Synowiecki, Jósef, sect. A, ch. 2, p. 19 Topakas, Evangelos, sect. A, ch. 6, p. 83 Tzen, Jason T. C., sect. E, ch. 34, p. 597 Vallés, Salvador, sect. A, ch. 8, p. 117

Vega-Hernández, María C., sect. B, ch. 13, p. 207 Wiczk, Wiesław, sect. B, ch. 11, p. 181

Xu, Qi, sect. A, ch. 3, p. 35

CARBOHYDRATE ACTIVE ENZYMES

AMYLOLYTIC ENZYMES: TYPES, STRUCTURES AND SPECIFICITIES

MARTIN MACHOVI ˇC¹AND ŠTEFAN JANE ˇCEK^12∗

1Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia and

2Department of Biotechnologies, Faculty of Natural Sciences, University of St. Cyril and Methodius, Trnava, Slovakia

∗stefan.janecek@savba.sk

1. INTRODUCTION

Cellulose and starch are the most abundant polymers on Earth. They both consist of glucose monomer units which are, however, differently bound to form polymer chains: starch contains the glucose linked up by the-glucosidic bonds, while the glucose in cellulose is bound by the -glucosidic linkages. Therefore these two important sources of energy for animals, plants and micro-organisms are biochemi- cally hydrolysed by two different groups of enzymes: starch by-glycoside hydro- lases, and cellulose by-glycoside hydrolases. Starch (amylon in Greek) consists of two distinct fractions: amylose – linear-1,4-linked glucans, and amylopectin – linear-1,4-linked glucans branched with-1,6 linkages (Ballet al., 1996; Mouille et al., 1996), therefore the enzymes responsible for its hydrolysis are called amylolytic enzymes or – simply – amylases. Amylolytic enzymes form a large group of enzymes among which the most common and best known are-amylases, -amylases and glucoamylases.

Since starch (like the structurally related glycogen) is an essential source of energy, amylolytic enzymes are produced by a great variety of living organisms (Vihinen and Mäntsäla, 1989). Although the different amylases mediate the same reaction – they all catalyse the cleavage of the -glucosidic bonds in the same substrate – structurally and mechanistically they are quite different (MacGregor et al., 2001). Both-amylase and -amylase adopt the structure of a TIM-barrel fold (for a review see Pujadas and Palau, 1999),i.e.their catalytic domain consists of a (/₈-barrel formed by 8 parallel -strands surrounded by 8 -helices

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J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 3–18.

© 2007Springer.

(Matsuuraet al., 1984; Mikamiet al., 1993). The barrels are, however, not similar in their details (Jespersenet al., 1991). Glucoamylase on the other hand possesses the structure of an (/₆-barrel, consisting of an inner barrel composed of 6-helices which is surrounded by 6 more (Aleshinet al., 1992). Strands and helices of the (/₈-barrel domain as well as the helices of the (/₆-barrel are connected by loop regions of various lengths.

Based on the similarities and differences in their primary structures, amylolytic enzymes have been classified into families of glycoside hydrolases (GH) (Henrissat, 1991): (i) -amylases – family GH13; (ii) -amylases – family GH14; and (iii) glucoamylases – family GH15. This classification, available on- line at the CAZy (Carbohydrate-Active enZymes) internet site (Coutinho and Henrissat, 1999), reflects the differences in the reaction mechanisms and catalytic machinery employed by the three types of amylase (Davies and Henrissat, 1995).

Due to the enormous accumulation of new sequence data in recent years,-amylase family GH13 has expanded so that it now contains almost 30 different enzymes and proteins (e.g.pullulanase, isoamylase, neopullulanaseetc.) exhibiting sequence relatedness to-amylases (MacGregoret al., 2001). At present all these enzymes are classified into families GH13, GH70 and GH77 which together constitute glycoside hydrolase clan GH-H (Coutinho and Henrissat, 1999). Moreover, families GH31 and GH57 contain a few amylolytic specificities with no sequence similarity to family GH13 (Henrissat and Bairoch, 1996).

The present review focuses on structural characteristics of the GH families of amylases. Its main goal is to provide a brief overview of the best-known glycoside hydrolases families GH13, GH14, GH15, GH31, GH57 GH70 and GH77. Emphasis is placed on the description of their: (i) specificities with regard to the EC numbers;

(ii) three-dimensional structures; and (iii) catalytic domain architecture.

2. CLAN GH-H: FAMILIES GH13, GH70 AND GH77

A recent list of members of clan GH-H is shown in Table 1. There are not only hydrolases (EC 3) but also transferases and isomerases from enzyme classes 2 and 5, respectively (Fig. 1). The GH13, GH70 and GH77 families constitute the members of the GH-H clan – the so-called the-amylase family (MacGregoret al., 2001).

This clan now covers about 30 different enzyme specificities (MacGregor, 2005). All GH-H clan members share several characteristics: (i) the catalytic domain is formed by the (/₈-barrel fold (i.e.TIM-barrel) with a longer loop connecting strand3 to helix3 known as domain B; (ii) a common catalytic mechanism in which the 4-strand aspartate acts as a base (nucleophile) and the5-strand glutamate acts as a proton donor (acid/base catalyst) with the help of the third residue, the7-strand aspartate, essential for substrate binding (transition state stabiliser); (iii) they employ the retaining mechanism for the cleavage of the -glycosidic bonds (Matsuura et al., 1984; Buisson et al., 1987; Machius et al., 1995; Aghajari et al., 1998;

Matsuura, 2002).

Besides the requirements for classification, it is practically impossible to study the-amylase family without taking into account the conserved sequence regions

Table 1.-Amylase family (clan GH-H)

Enzyma class Enzyme EC GH family

Hydrolases -Amylase 3.2.1.1 13

Oligo-1,6-glucosidase 3.2.1.10 13

-Glucosidase 3.2.1.20 13

Pullulanase 3.2.1.41 13

Amylopullulanase 3.2.1.1/41 13

Cyclomaltodextrinase 3.2.1.54 13

Maltotetraohydrolase 3.2.1.60 13

Isoamylase 3.2.1.68 13

Dextranglucosidase 3.2.1.70 13

Trehalose-6-phosphate hydrolase 3.2.1.93 13

Maltohexaohydrolase 3.2.1.98 13

Maltotriohydrolase 3.2.1.116 13

Maltogenic-amylase 3.2.1.133 13

Maltogenic amylase 3.2.1.133 13

Neopullulanase 3.2.1.135 13

Maltooligosyltrehalose hydrolase 3.2.1.141 13

Maltopentaohydrolase 3.2.1.- 13

Transferases Amylosucrase 2.4.1.4 13

Glucosyltransferase 2.4.1.5 70

Sucrosea phosphorylase 2.4.1.7 13

Glucan branching enzyme 2.4.1.18 13

Cyclodextrin glucanotransferase 2.4.1.19 13

4--Glucanotransferase 2.4.1.25 13, 77

Glucan debranching enzyme 2.4.1.25/3.2.1.33 13

Alternansucrasee 2.4.1.140 70

Maltosyltransferase 2.4.1.- 13

Isomerases Isomaltulose synthase 5.4.99.11 13

Trehalose synthase 5.4.99.15 13

Maltooligosyltrehalose synthase 5.4.99.16 13

(Janeˇcek, 2002). It has been known for some time that the sequence similarity is extremely low (about 10%) even for the-amylases alone (i.e.for EC 3.2.1.1). This was described for-amylases from different micro-organisms, plants, and animals (Nakajimaet al., 1986). With subsequent expansion of the family,i.e.when many sequences from various sources and with different enzyme specificities became available, the number of identical residues among the-amylase family enzymes had decreased to 8-10 amino acids by 1994 (Janeˇcek, 1994; Svensson, 1994).

The conserved sequence regions of those-amylase family members whose three- dimensional structures have already been solved are shown in Fig. 2. The regions of the GH70 glucan-synthesising glucosyltransferase are based on the prediction study by MacGregoret al., (1996) and site-directed mutagenesis (Devulapalleet al., 1997) since no three-dimensional structure is currently available for a GH70 member. It is clear that the GH-H clan contains the invariant catalytic triad consisting of two aspar- tates (in strands4 and7) and one glutamate (in strand5). The two functionally important histidines (in strands 3 and 7) – although strongly conserved and

Figure 1.Evolutionary tree of the-amylase family,i.e.clan GH-H. For the sake of simplicity, the tree is based on the alignment of conserved sequence regions (see Fig. 2),i.e.it does not reflect the complete amino acid sequences

apparently essential for several specificities (MacGregor et al., 2001) – are not present in GH13 maltosyltransferase (both His are missing) nor in the members of both GH70 and GH77 families (the3 His is missing) (Fig. 2). The histidines have nevertheless been demonstrated to be critical in transition-state stabilisation (Søgaardet al., 1993). The fourth invariant residue of the-amylase family seemed to be the arginine in the position i−2 with respect to the catalytic 4-strand aspartate (Janeˇcek, 2002). However, this is no longer sustainable (Machoviˇc and Janeˇcek, 2003) because the sequences of the GH77 4--glucanotransferase from Borrelia burgdorferiandBorrelia gariniihave the arginine substituted by a lysine (Fig. 3). This substitution is not a general feature characteristic of GH77 since it was not possible to detect more examples with such Arg/Lys substitution in the sequence databases. Moreover, the two putative Borrelia4--glucanotransferases exhibit several additional remarkable sequence features that distinguish them from the rest of the GH77 enzymes. These are (Fig. 3): Pro/Ala in region VI (2), Asp/Asn in region I (3), Ile(Leu)/Trp and Leu-Gly/Phe-Gln(Glu) in region III (5), and His/Gly in region IV (7). With regard to protein function, catalytic activity and enzyme specificity of the two Borrelia 4--glucanotransferases, it

Figure 2.Conserved sequence regions in the-amylase family. One representative for each enzyme specificity is presented. For those with three-dimensional structures already determined, the year when the structure was solved is shown in the first column. The important residues are highlighted in black;

the catalytic triad is identified by asterisks. The other residues are coloured grey if conserved in at least 50% of the sequences. Figure adapted from Janeˇcek (2002)

Figure 3.Selected conserved sequence regions in representative GH77 4--glucanotransferases. The regions I, II, III, IV and VI correspond to the strands3,4,5,7 and2, respectively, of the catalytic /₈-barrel domain. The members shown above the two Borrelia representatives are confirmed 4--glucanotransferases, whereas the members shown below are putative proteins only with GH77-like sequences. The invariant catalytic triad of the GH-H clan is identified by asterisks and bold characters.

The important substituted residues in the twoBorrelia4--glucanotransferases are highlighted in black, the most interesting mutation (Arg/Lys) being emphasized by an arrow

is worth mentioning that these amino acid sequences were deduced from the nucleotide sequence of the Lyme disease spirochete and related genomes (Fraser et al., 1997; Glöckneret al., 2004), i.e. they are only translated ORFs. The 4-- glucanotransferase specificities in both cases were thus assigned only by virtue of sequence similarities with other GH77 4--glucanotransferases/amylomaltases. The conserved catalytic triad, however, supports the possibility that the functions have been maintained. For example, the Arg/Lys mutant ofBacillus stearothermophilus -amylase had 12% of the specific activity of the parental enzyme (Vihinen et al., 1990) and the same mutant of the maize branching enzyme retained also some residual activity (Libessart and Preiss, 1998). The possibility of a sequencing error (Arg/Lys exchange) can be disregarded because the Borrelia burgdorferi 4--glucanotransferase was recently cloned, expressed in Escherichia coli and sequenced (Godany et al., 2005). All the substitutions highlighted in Fig. 3 have been experimentally confirmed.

3. FAMILY GH13

GH13 ranks among the largest GH families with almost 30 enzyme specificities and more than 2,000 sequences (Coutinho and Henrissat, 1999; MacGregoret al., 2001;

Pujadas and Palau, 2001; Svensson et al., 2002). It is the principal and most important family of the entire GH-H clan. In addition to-amylase (EC 3.2.1.1), it contains (Table 1) cyclodextrin glucanotransferase (CGTase), -glucosidase, amylopullulanase, neopullulanase, amylosucrase,etc. (MacGregoret al., 2001). It seems reasonable to group the very closely related GH13 members into subfamilies, e.g.the oligo-1,6-glucosidase-like and neopullulanase-like members (Oslancova and Janeˇcek, 2002; Oh, 2003).

Not all GH13 enzymes attack the glucosidic bonds in starch. However they do have a number of features in common (Svensson, 1994; Janeˇcek, 1997; Kuriki and Imanaka, 1999; MacGregor, 2005): (i) sequence similarities (the so-called conserved sequence regions) covering the equivalent elements of their secondary structure (especially the -strands); (ii) catalytic machinery (Asp, Glu and Asp residues in-strands 4, 5 and7, respectively); (iii) retaining reaction mechanism (the resulting hydroxyl group retains the -configuration); (iv) the three-dimensional fold (TIM-barrel). The first three-dimensional structure of an-amylase to be solved was that of Taka-amylase A,i.e.the-amylase fromAspergillus oryzae(Matsuura et al., 1984) (Fig. 4a). The enzyme adopts the so-called TIM-barrel fold which was first identified in the structure of triosephosphate isomerase (Banneret al., 1975) and now found in about 50 different enzymes and proteins (Reardon and Farber, 1995;

Janeˇcek and Bateman, 1996; Pujadas and Palau, 1999). The (/₈-barrel motif consists of eight parallel-strands forming the inner-barrel which is surrounded by the outer cylinder composed of eight-helices so that the individual-strands and-helices alternate and are connected by loops. Although all the members of the-amylase family (Table 1) should share the characteristics given above, some have been classified into the new GH families (Coutinho and Henrissat, 1999). Thus

Figure 4.Three-dimensional structures of (a) GH13-amylase fromAspergillus oryzae(PDB code:

2TAA; Matsuuraet al., 1984) and (b) GH77 amylomaltase fromThermus aquaticus(1CWY; Przylas et al., 2000)

the sucrose-utilising glucosyltransferases (EC 2.4.1.5) have been placed in family GH70 because their catalytic domain was predicted to contain a circularly permuted version of the-amylase type (/₈-barrel (MacGregoret al., 1996). This is also the case for one of the very recent members of the-amylase family, alternansucrase (Argüello-Moraleset al., 2000). Furthermore, some amylomaltases (EC 2.4.1.25), whose sequences exhibit low similarities with the most representative members of the-amylase family, have been grouped into the new GH77 family (Coutinho and Henrissat, 1999). However, the three-dimensional structure of amylomaltase from Thermus aquaticus(Przylaset al., 2000) confirmed that this enzyme also possesses the regular (/₈-barrel structure (Fig. 4b) with the arrangement of the catalytic side-chains (two Asp residues and one Glu residue) being similar to that found in the-amylase family.

With regard to quaternary structure, many members are able to form oligomers (Robyt, 2005). The most remarkable examples are cyclomaltodextrinases (for details, see Leeet al., 2005a; Turneret al., 2005).

4. FAMILIES GH14 AND GH15

There are two other amylolytic GH families in CAZy (Coutinho and Henrissat, 1999), GH14 and GH15, covering-amylases and glucoamylases, respectively. They both employ the inverting mechanism for cleaving the-glucosidic bonds,i.e.the products of their reactions are-anomers (Sinnot, 1990; Kuriki, 2000; MacGregoret al., 2001).

From an evolutionary point of view,-amylases seem to be a ’solitary’ GH family since they do not exhibit an obvious structural similarity to other glycoside hydrolases (Pujadas et al., 1996; Coutinho and Henrissat, 1999). By contrast, glucoamy- lases from GH15 form clan GH-L together with family GH65 (Egloffet al., 2001).

As regards sequence, these two types of amylase do not contain any of the conserved regions characteristic of the-amylase family (Fig. 2). Although they are both exo-amylases their amino acid sequences and three-dimensional structures are different (Aleshinet al., 1992; Mikamiet al., 1993). Structurally,-amylase (Fig. 5a) ranks along with -amylase among the large family of parallel (/₈-barrel proteins (Pujadas and Palau, 1999), while glucoamylase (Fig. 5b) belongs to a smaller family of proteins adopting the (/₆-barrel fold (Aleshinet al., 1992).

Family GH14 includes-amylases (EC 3.2.1.2) and hypothetical proteins with sequence similarity to-amylases. Half of the family members are experimentally verified enzymes having-amylase activity. -Amylases are especially produced by plants: Arabidopsis thaliana, Oryza sativa, Triticum aestivum and Solanum tuberosum. Family GH15 includes glucoamylases (EC 3.2.1.3), two glucodex- tranases (EC 3.2.1.70) and hypothetical proteins with sequence similarity to GH15.

Again, about 50% of the family members are experimentally verified enzymes having glucoamylase or glucodextranase activities.

The first determined three-dimensional structure of a -amylase was that of soybean (Mikami et al., 1993). At present, the structures of -amylases from sweet potato (Cheong et al., 1995), barley (Mikami et al., 1999b) and Bacillus cereus (Mikami et al., 1999a; Oyama et al., 1999) are also known. The core of the-amylase structure is formed by the catalytic (/₈-barrel domain (Fig. 5a) followed by the C-terminal loop region. Although this loop surrounds the N-terminal side of the (/₈-barrel and may stabilise the whole -amylase molecule, it is not involved in catalysis (Mikami, 2000). As has been pointed out above, the -amylase (/₈-barrel differs from that of -amylase and all other enzymes of clan GH-H, resembling more the single-domain structure of triosephosphate isomerase (Mikami, 2000). The two amino acid residues responsible for catalysis are the two glutamates, Glu186 and Glu380 (soybean-amylase numbering), positioned

Figure 5.Three-dimensional structures of (a) GH14 -amylase from soybean (1BYA; Mikami et al., 1993) and (b) GH15 glucoamylase fromAspergillus awamori(1AGM; Aleshinet al., 1992)

near the C-terminus of strands 4 and 7 of the (/₈-barrel domain, respec- tively (Mikamiet al., 1994). Totsuka and Fukazawa (1996) described further the indispensable roles for Asp101 and Leu383 in addition to the two catalytic gluta- mates. Analyses of the (/₈-barrel fold of-amylases from both the evolutionary and structural points of view are available (Pujadas et al., 1996; Pujadas and Palau, 1997).

Glucoamylase structures have been solved for two fungal enzymes:Aspergillus awamori(Aleshinet al., 1992) and the yeastSaccharomycopsis fibuligera(Sevcik et al., 1998), and one bacterial enzyme fromThermoanaerobacterium thermosac- charolyticum(Aleshinet al., 2003). The glucoamylase catalytic domain is composed of 12-helices that form the so-called (/₆-barrel fold (Fig. 5b). It consists of an inner core of six mutually parallel -helices that are connected to each other through a peripheral set of six-helices which are parallel to each other but approx- imately antiparallel to the inner core of the-helices (Aleshin et al., 1992). This fold is not as frequent as the TIM-barrel fold (Farber and Petsko, 1990; Janeˇcek and Bateman, 1996; Pujadas and Palau, 1999), however, the (/₆-barrel has also been found in different proteins and enzymes, for example in the enzymes from families GH8 and GH9 (Juyet al., 1992; Alzariet al., 1996). Some glucoamylases, like some-amylases (and related enzymes from the clan GH-H) and-amylases, contain starch-binding domains (Svenssonet al., 1989; Janeˇcek and Sevcik, 1999) which can be of various types (for a review, see Rodriguez-Sanojaet al., 2005).

The starch-binding domain may be evolutionarily independent from the catalytic domain (Janeˇceket al., 2003). It should also be possible to add a starch-binding domain artificially to an amylase (or eventually to any other protein) to improve its amylolytic and raw starch-binding and degradation abilities (Ohdanet al., 2000; Ji et al., 2003; Huaet al., 2004; Levyet al., 2004; Kramhøftet al., 2005; Latorre- Garciaet al., 2005). Recently, it seems evident that some amylases may contain starch-binding activity without a specific structural module (Hostinovaet al., 2003;

Tranieret al., 2005).

Based on the analysis of glucoamylase amino acid sequences, Coutinho and Reilly (1997) described seven subfamilies taxonomically corresponding to bacterial (1), archaeal (1), yeast (3) and fungal (2) origins. As evidenced by the crystal structures of the glucoamylases from Aspergillus awamori (Harris et al., 1993;

Aleshin et al., 1994, 1996; Stoffer et al., 1995) andSaccharomycopsis fibuligera (Sevcik et al., 1998), the two glutamates, Glu179 and Glu400 (Aspergillus enzyme numbering), act as the key catalytic residues. The next most well-studied glucoamylase is that from Aspergillus niger (Christensen et al., 1996; Frandsen et al., 1996) which is highly similar to theAspergillus awamoricounterpart.

5. FAMILY GH31

There are some glucoamylases that have been classified into family GH31 together with -glucosidases, -xylosidases and glucan lyases (Yu et al., 1999; Lee et al., 2003; 2005b). These enzymes act through a retaining mechanism like the

Figure 6.Three-dimensional structure of GH31 -xylosidase from Escherichia coli (1XSI;

Loveringet al., 2005)

members of clan GH-H (Chiba , 1997; Nakaiet al., 2005). GH31 was considered to be a member of clan GH-H because of remote sequence homologies between GH31 and GH13 enzymes (Rigden, 2002). This assumption has recently been supported by the resolution of the three-dimensional structure of a GH31-xylosidase from Escherichia coli(Loveringet al., 2005) and-glucosidase from Sulfolobus solfa- taricus (Ernst et al., 2006) showing the expected (/₈-barrel catalytic domain (Fig. 6). Interestingly, the domain arrangement of the GH31 members strongly resembles that of GH13 enzymes (Fig. 3), especially regarding domain B protruding out of the (/₈-barrel in the place of loop 3 (Loveringet al., 2005).

6. FAMILY GH57

For a long time GH57 has been one of the most popular GH families, attracting much scientific interest. More than 15 years ago the sequence of a heat-stable -amylase from the thermophilic bacterium Dictyoglomus thermophilum was published (Fukusumiet al., 1988). Despite the fact that this sequence encoded an- amylase, its analysis did not reveal any detectable similarity with GH13-amylases.

Later, a similar sequence encoding the -amylase from the hyperthermophilic archaeon,Pyrococcus furiosus, was determined (Ladermanet al., 1993). These two sequences became the basis for the new amylolytic family, GH57, established in 1996 (Henrissat and Bairoch 1996). In the last few years, when entire genomes

of many micro-organisms have been sequenced, family GH57 has expanded. Its members are all prokaryotic enzymes, most of them from hyperthermophilic archaea (Zonaet al., 2004). At present the GH57 family consists of about 100 members (Coutinho and Henrissat, 1999) and five enzyme (Janeˇcek, 2005; Murakamiet al., 2006): -amylase (EC 3.2.1.1), -galactosidase (EC 3.2.1.22), amylopullulanase (EC 3.2.1.1/41), branching enzyme (EC 2.4.1.18) and 4--glucanotransferase (EC 2.4.1.25). Only about 10% of the family sequence entries are enzymes; all others are hypothetical proteins without known activity (Zonaet al., 2004). GH57 sequences are highly heterogeneous: some of them have less than 400 residues whereas others have more than 1,500 residues (Zonaet al., 2004).

Structural information for GH57 members is scarce. To date, only the structures of the 4--glucanotransferase fromThermococcus litoralis(Imamuraet al., 2003) and AmyC enzyme fromThermotoga maritima(Dickmannset al., 2006) have been determined. They both revealed a (/₇-barrel fold (Fig. 7), i.e. an incomplete TIM-barrel. Glu123 and Asp214 (T. litoralisenzyme numbering) which define the

Figure 7.Three-dimensional structure of GH57 4--glucanotransferance fromThermococcus litoralis (1K1W; Imamuraet al., 2003)

catalytic centre of the enzyme, are arranged at a distance of less than 7 Å (Imamura et al., 2003), thus confirming that GH57 also employs a retaining mechanism for -glycosidic bond cleavage.

New information about GH57 has arisen from a bioinformatic study focused on the conserved sequences containing the pair of catalytic residues (Zonaet al., 2004).

In addition to T. litoralis 4--glucanotransferase, both catalytic residues were experimentally identified in two amylopullulanases fromThermococcus hydrother- malis(Zonaet al., 2004) andPyrococcus furiosus(Kanget al., 2005). The catalytic nucleophile was found also in the-galactosidase fromPyrococcus furiosus(Van Lieshout et al., 2003). Biochemical analysis indicates that family GH57 enzymes may lack a genuine-amylase specificity (Janeˇcek, 2005).

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THE USE OF STARCH PROCESSING ENZYMES IN THE FOOD INDUSTRY

JÓZEF SYNOWIECKI^∗

Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, Gdansk University of Technology, Gdansk, Poland

∗synowiec@chem.pg.gda.pl

1. INTRODUCTION

Starch, the main component of many agricultural products, e.g. corn (maize), potatoes, rice and wheat, is deposited in plant cells as reserve material for the organism in the form of granules which are insoluble in cold water. This carbo- hydrate is the main constituent of food products such as bread and other bakery goods or is added to many foods for its functionality as a thickener, water binder, emulsion stabilizer, gelling agent and fat substitute. Starch granules consist of two types of molecules composed of-D-glucose units called amylose and amylopectin.

In amylose almost all the glucose residues are linked by-1,4-glycosidic bonds, whereas in amylopectin about 5 % of the carbohydrate units are also joined by-1,6- linkages forming branch points. The relative contents of amylose and amylopectin depend on the plant species. For example, wheat starch contains about 25% amylose while waxy corn starch is more than 97–99% amylopectin. Starch origin also makes differences to the size, shape and structure of the polysaccharide granules, their swelling power, gelatinisation temperature, extent of esterification with phosphoric acid, and the amounts of lipids and other compounds which are retained inside the hydrophobic inner surface of the amylose helices.

Expanding starch functionality can be achieved through chemical or enzymatic modifications. The most important methods of enzymatic starch processing (Fig. 1) are the production of cyclodextrins and the hydrolysis of starch into a mixture of simpler carbohydrates for the production of syrups having different compositions and properties. These products are used in a wide variety of foodstuffs: soft drinks, confectionery, meats, packed products, ice cream, sauces, baby food, canned fruit,

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