Glycoside Hydrolases

Prof. Arun Goyal, M.Sc., M.Tech., Ph.D., is currently a Professor in the Department of Biosciences and Bioengineering, at the Indian Institute of Technology Guwahati, in Assam, India. Prof. Goyal has over 18 years of teaching and 30 years of research experience. His research mainly focuses on structure and functional analysis of plant cell wall degrading enzymes and their applications. Prof. Goyal obtained his M.Sc. and M.Tech. degrees from the Indian Institute of Technology Delhi and Ph.D. from the Indian Institute of Technology Kanpur, India. Prof. Goyal has handled many national and international collaborative research projects, sponsored by various funding agencies of the Government of India such as DBT, DST and CSIR. So far 30 students have obtained Ph.D. under his supervision and 12 students are continuing their Ph.D. Prof. Goyal has published more than 265 research papers in various National and International reputed journals and over 30 book chapters. Dr. Kedar Sharma, M.Sc., Ph.D. currently working as Visiting Fellow at National Institute of Environmental Health Sciences, North Carolina, USA. He worked as Postdoctoral Research Associate, at Department of Biological Engineering, at Indian Institute of Technology Gandhinagar, Gujarat, India. Dr. Sharma has over 7 years of research experience. He completed his Bachelors of Science in Biotechnology from University of Rajasthan, Jaipur and his Masters in Biotechnology from Guru Ghasidas Vishwavidyalaya (Central University) Bilaspur, Chhattisgarh, India. He completed Ph.D. from Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati. His Ph.D. work as focused on structural and functional characterization of xylanolytic enzymes and other glycoside hydrolases and their application in production of xylooligosaccharides. Currently, he is working on elucidating the structure and fusion mechanism of viral entry into the host cell. He received a IUCr Young Scientist award from International Union for Crystallography, and several best poster and oral presentation awards from different organizations. Dr. Sharma has published 33 research papers in various international reputed journals and 5 book chapters.

Contributors About the Editors Preface

CHAPTER 1 Carbohydrates and Carbohydrate-Active enZymes (CAZyme): An overview Parmeshwar Vitthal Gavande, Arun Goyal, and Carlos M.G.A. Fontes

1.1 Introduction 1.1.1 Various carbohydrate polymers present in nature 1.1.2 Natural source of polysaccharides 1.1.3 Requirement for deconstruction of carbohydrates 1.1.4 Carbohydrate-active enzymes 1.1.5 Carbohydrate-active enzyme database (CAZy) 1.1.6 Multienzyme complexes of CAZyme: The cellulosome 1.1.7 Commercially available CAZyme libraries 1.2 Conclusion References

CHAPTER 2 Glycoside hydrolases: Mechanisms, specificities, and engineering Antoni Planas

2.1 Structures, functions, and classifications 2.2 Glycosidase mechanisms for hydrolysis of glycans and glycoconjugates 2.2.1 General mechanisms: Inverting vs. retaining 2.2.2 Retaining glycosidases with enzyme nucleophile: Ring distortion and covalent intermediate 2.2.3 Retaining glycosidases by substrate-assisted catalysis: Oxazoline/oxazolonium intermediate 2.2.4 Retaining glycosidases by neighboring-group participation through a 1,2-epoxide intermediate 2.2.5 Retaining glycosidases by an unusual NAD+-dependent mechanism 2.2.6 Inverting glycosidases 2.3 Protein engineering of glycosidases for improved and novel properties 2.3.1 Thermostability 2.3.2 Substrate specificity 2.4 Glycosidases acting in reverse for glycosynthesis: Transglycosidases and glycosynthases 2.4.1 Transglycosidases 2.4.2 Glycosynthases 2.5 Concluding remarks References

CHAPTER 3 Endo-β-1,4-glucanase Parmeshwar Vitthal Gavande and Arun Goyal

3.1 Introduction 3.1.1 Cellulase 3.1.2 Cellulase evolution and conservation in nature 3.1.3 Endo-β-1,4-glucanase 3.1.4 Exoglucanase 3.1.5 β-glucosidase 3.1.6 Cellulosome 3.2 Endoglucanases belong to various GH families 3.2.1 GH5 family 3.2.2 GH6 family 3.2.3 GH7 family 3.2.4 GH8 family 3.2.5 GH9 family 3.2.6 GH12 family 3.2.7 GH44 family 3.2.8 GH45 family 3.2.9 GH48 family 3.3 Synergism of endo-β-1,4-glucanase with exoglucanase and β-glucosidase 3.4 Endo-β-1,4-glucanase-producing microorganisms 3.4.1 Biochemical properties, kinetics, and catalytic efficiency of endoglucanases 3.5 Structure of endo-β-1,4-glucanases 3.5.1 Mechanism of cellulose hydrolysis in endoglucanases 3.6 Multifunctionality of endoglucanases 3.6.1 Broad substrate specificity of various endoglucanases 3.6.2 Significance of multifunctional endoglucanases 3.7 Processivity of endoglucanases 3.8 Applications of endoglucanases 3.9 Conclusion Authors’ contribution References

CHAPTER 4 Cellobiohydrolases Tulika Sinha, Kanika Sharma, and Syed Shams Yazdani

4.1 Introduction 4.2 Structure and mode of action of cellobiohydrolases 4.2.1 The catalytic domain (CD) 4.2.2 The carbohydrate-binding module (CBM) 4.2.3 The linker 4.2.4 The dissociation mechanism of processive CBH1 4.3 Biochemical and biophysical properties of cellobiohydrolases 4.3.1 pH and temperature 4.3.2 Metal ions 4.3.3 Surfactants 4.4 Protein engineering and strain improvement for higher enzyme activity and productivity 4.4.1 Enhanced activity 4.4.2 Enhanced thermostability 4.4.3 Enhanced performance in nonconventional media 4.4.4 Engineering cellulase for pH stability 4.5 Industrial applications of CBH 4.5.1 Bioconversion 4.5.2 Pulp and paper industry 4.5.3 Food processing industry 4.5.4 Textile industry 4.5.5 Agriculture 4.5.6 Animal feed 4.5.7 Detergent industry 4.6 Conclusion and future perspective References

CHAPTER 5 β-Glucosidase: Structure, function and industrial applications Sauratej Sengupta, Maithili Datta, and Supratim Datta

5.1 Introduction 5.2 Classification 5.3 Structure 5.4 Reaction mechanism 5.4.1 Substrate recognition and specificity 5.4.2 Glycone and aglycone specificity 5.5 Function and distribution 5.6 Characteristics 5.6.1 Biophysical characteristics 5.6.2 Biochemical characteristics 5.6.3 Product inhibition and enhancement of activity in the presence of glucose 5.6.4 Substrate inhibition 5.7 Industrial applications 5.7.1 Biofuels 5.7.2 Food industry 5.7.3 Pharmaceutical industries Acknowledgments References

CHAPTER 6 Endo-β-1,3-glucanase Parmeshwar Vitthal Gavande and Arun Goyal

6.1 Introduction 6.2 The role of endo-β-1,3-glucanase in nature 6.2.1 β-1,3-Glucan 6.2.2 Exo-β-1,3-glucanase 6.2.3 Endo-β-1,3-glucanase 6.2.4 Classification of endo-β-1,3-glucanases 6.3 Sources of endo-β-1,3-glucanase 6.4 Endo-β-1,3-glucanases of different families, their structure, and mechanism 6.4.1 The family GH5 6.4.2 The family GH16 6.4.3 The family GH17 6.4.4 The family GH55 6.4.5 The family GH64 6.4.6 The family GH81 6.4.7 The family GH128, GH152, GH157, GH158 6.5 Applications of endo-β-1,3-glucanases 6.6 Conclusion References Further reading

CHAPTER 7 Diversity of microbial endo-β-1,4-xylanases Peter Biely, Katarı´na Sˇuchova´, and Vladimı´r Puchart

7.1 Introduction 7.2 Chemical structure of plant xylans 7.3 Enzymes of xylan hydrolysis 7.4 Endoxylanases—Xylan depolymerizing enzymes 7.4.1 Molecular architecture of xylanases 7.4.2 Classification into glycoside hydrolase families 7.4.3 Mode of action and structure-function relationship 7.5 Synergism of endoxylanases with debranching xylanolytic enzymes 7.6 Application of xylanases 7.7 Conclusions and future prospects References

CHAPTER 8 β-D-Xylosidases: Structure-based substrate specificities and their applications Satoshi Kaneko and Zui Fujimoto

8.1 Introduction 8.2 Structures of β-xylosidases 8.2.1 GH3 8.2.2 GH39 8.2.3 GH43 8.2.4 GH52 8.2.5 GH120 8.2.6 Other families 8.3 Substrate specificities of the β-xylosidases 8.3.1 GH1 8.3.2 GH2 8.3.3 GH3 8.3.4 GH5 8.3.5 GH10 8.3.6 GH11 8.3.7 GH30 8.3.8 GH39 8.3.9 GH43 8.3.10 GH51 8.3.11 GH52 8.3.12 GH54 8.3.13 GH116 8.3.14 GH120 8.4 Applications of β-xylosidases References

CHAPTER 9 Arabinofuranosidases Priyanka Pisalwar, Austin Fernandes, Devashish Tribhuvan, Saurav Gite, and Shadab Ahmed

9.1 Introduction 9.2 Classification 9.2.1 Classification on the basis of substrate specificity and mechanism of action 9.2.2 Classification on the basis of amino acid sequencing and structural similarity 9.3 Structural and functional characteristics of arabinofuranosidases 9.3.1 Effect of metal ions 9.3.2 Carbohydrate-binding modules (CBM) associated with arabinofuranosidases 9.4 Substrate specificity and biochemical properties of arabinofuranosidases 9.4.1 Substrate specificity 9.4.2 Physical and chemical properties 9.5 Industrial applications of arabinofuranosidase 9.5.1 Biofuel and biochemical industry 9.5.2 Food and animal feed industry 9.5.3 Beverage industry 9.5.4 Paper and pulp industry 9.5.5 Probiotic and pharmaceutical industry 9.6 Future trends and scope of arabinofuranosidases 9.6.1 Protein engineering 9.6.2 Development of new modular enzymes with enhanced substrate degradation potential 9.7 Conclusions References

CHAPTER 10 Glycoside hydrolase family 16—Xyloglucan:xyloglucosyl transferases and their roles in plant cell wall structure and mechanics Barbora Stratilova´, Stanislav Kozmon, Eva Stratilova´, and Maria Hrmova

10.1 Plant cell walls are protective multicomposite hydrogels 10.1.1 Plant cell wall composition and function 10.1.2 Plant cell wall structure and organization 10.2 Plant xyloglucan:xyloglucosyl transferases 10.2.1 Nomenclature and classification 10.2.2 Catalytic mechanism 10.2.3 Structural properties 10.2.4 Enzyme activity methods 10.2.5 Reactions with xyloglucan-derived and other substrates 10.2.6 Genetics approaches to the XTH gene function 10.3 The function of XTH enzymes in plant cell walls 10.3.1 Plant cell wall dynamics 10.3.2 Roles of XTH enzymes in cell wall restructuring 10.4 Conclusions and future directions Author contributions Funding Conflict of interest References

CHAPTER 11 Endo-arabinase: Source and application Dixita Chettri and Anil Kumar Verma

11.1 Introduction 11.2 Hemicellulose structure and hydrolysis of arabinans 11.3 Source and biochemical characteristics 11.4 Structure and mechanism of action 11.5 Application of arabinase 11.6 Safety assessment 11.7 Conclusion and future prospects Acknowledgment Conflict of interest References

CHAPTER 12 Overview of structure-function relationships of glucuronidases Samar Ballabha Mohapatra and Narayanan Manoj

12.1 Introduction 12.2 Xylanolytic α-glucuronidases 12.2.1 GH67 α-glucuronidases 12.2.2 GH115 α-glucuronidases 12.3 Non-xylanolytic GH4 α-glucuronidase 12.3.1 Active site architecture and the substrate specificity of GH4 TmAgu4B 12.3.2 Mechanism of hydrolysis by GH4 AguA 12.4 β-Glucuronidases 12.4.1 GH1 β-glucuronidase 12.4.2 GH2 β-glucuronidases 12.4.3 GH30 β-glucuronidase 12.4.4 GH79 β-glucuronidases 12.4.5 GH154 β-glucuronidase 12.4.6 GH169 β-glucuronidase 12.5 Perspectives on the development of applications of glucuronidases 12.5.1 Xylanolytic α-glucuronidases 12.5.2 Inhibitors of β-glucuronidases Credit References

CHAPTER 13 Mannanases and other mannan-degrading enzymes Caio Cesar de Mello Capetti, Andrei Nicoli Gebieluca Dabul, Vanessa de Oliveira Arnoldi Pellegrini, and Igor Polikarpov

13.1 Mannan structure 13.2 Enzymes involved in the mannan degradation 13.2.1 β-mannanases 13.2.2 Other enzymes important for mannan degradation 13.3 Production of β-mannanases 13.4 Industrial applications of β-mannanases 13.4.1 Oil drilling 13.4.2 Biofuel production 13.4.3 Production of manno-oligosaccharides 13.4.4 Paper and pulp production 13.4.5 Textile industry 13.4.6 Detergents 13.4.7 Pharmaceutical and food industry 13.5 Concluding remarks References

CHAPTER 14 Structure, function, and protein engineering of GH53 β-1,4-galactanases Sebastian J. Muderspach, Kenneth Jensen, Kristian B.R.M. Krogh, and Leila Lo Leggio

14.1 Introduction, classification, and structure overview of β-1,4-galactanases 14.2 Biological functions and diversity 14.2.1 Galactans in the plant cell walls 14.2.2 Degradation of plant cell wall galactans in plant pathogens via GH53 enzymes 14.2.3 Characterized GH53 galactanases from human gut microbiome 14.2.4 Plant cell wall remodeling for mobilization of energy resources or fruit ripening 14.2.5 GH53 galactanases from extremophiles 14.3 Related enzyme activities 14.3.1 Other microbial endo-galactanases 14.3.2 β-galactosidases and exo-β-1,4-galactanases 14.3.3 α-L-arabinofuranosidase and endo-1,5-α-L-arabinanase 14.4 GH53-associated modules and domains 14.4.1 Association of GH53 with carbohydrate-binding modules 14.4.2 Association of GH53 with other domains 14.5 Biotechnological applications 14.5.1 GH53 galactanases in enzymatic degradation of biomass 14.5.2 Prebiotic galactooligosaccharide production 14.5.3 Other industrial uses 14.6 Structure-function studies 14.6.1 Conformation of substrate in a computationally derived BlGal-galactononaose complex 14.6.2 Substrate-binding sites in GH53 galactanase crystal structures and their implication on product profile 14.6.3 Structural features inducing thermostability in GH53 galactanases 14.6.4 Prediction of structural features from sequence alignments and AlphaFold models 14.7 Protein engineering 14.7.1 Modulating thermostability and pH optimum 14.7.2 Changing the product profile 14.8 Conclusions and future directions References

CHAPTER 15 Structural and functional insights and applications of β galactosidase Azra Shafi and Qayyum Husain

15.1 β Galactosidase 15.2 Glycoside hydrolase families 15.3 Sources of β-galactosidases 15.3.1 Bacterial β-Gals 15.3.2 β-Gals from filamentous fungi 15.3.3 β-Gals from yeasts 15.3.4 β-Gals from plants 15.3.5 β-Gals from animals 15.3.6 Recombinant β-Gals 15.4 Lactose intolerance 15.5 Structural characterization of β-Gal 15.5.1 The active site 15.5.2 Metal binding sites 15.6 Functional characterization of β-Gal 15.6.1 Mode of action and reaction mechanism 15.6.2 Hydrolysis and transgalactosylation activities of β-Gal 15.7 Applications of β-Gal 15.7.1 Lactose-hydrolyzed milks 15.7.2 β-Gal supplements 15.7.3 Treatment of industry effluents 15.7.4 Synthesis of GOS 15.7.5 Reactors and biosensors 15.8 Conclusion References

CHAPTER 16 α-L-Rhamnosidases: Structures, substrate specificities, and their applications Satoshi Kaneko and Zui Fujimoto

16.1 Introduction 16.2 Structure of α-L-rhamnosidases 16.2.1 GH78 16.2.2 GH106 16.3 Substrate specificities of α-L-rhamnosidases 16.3.1 GH78 16.3.2 GH106 16.3.3 Unknown family 16.4 Applications of α-L-rhamnosidases References

CHAPTER 17 Diversity and biotechnological applications of microbial glucoamylases Sanjeev Kumar, Priyakshi Nath, Arindam Bhattacharyya, Suman Mazumdar, Rudrarup Bhattacharjee, and T. Satyanarayana

17.1 Introduction 17.2 Production of glucoamylase: Microbes, substrate, nutrients, and fermentation system 17.3 Thermophilic and mesophilic fungal glucoamylases 17.4 Production of native glucoamylases 17.5 Recombinant glucoamylases 17.6 Multiple molecular forms of glucoamylases 17.7 Structural characteristics of glucoamylases 17.8 Biotechnological applications of glucoamylase 17.9 Role of glucoamylase in starch conversion to sugar syrup 17.10 Role of glucoamylase in HFCS 17.11 Role of glucoamylase in the brewing and baking industry 17.12 Conclusion References

Index