Industrial Enzyme Applications

Andreas Vogel is Head of R&D Enzyme Development at c-LEcta in Leipzig, Germany. After his Ph.D. degree he worked at the EMBL outstation in Hamburg, where he focused on structure-function relationships of enzymes. He moved to the Max-Planck-Institute for Coal Research with Prof. M. T. Reetz. In 2006 he started to work in the c-LEcta company where he successfully developed a series of enzymes for various industrial applications. Oliver May is currently Senior Science Fellow and responsible for DSM´s global Biochemistry and Microbiology competence field. He has more than 15 years experience in industrial enzymes from his work at Evonik Industries and DSM and published more than 30 enzyme related scientific publications.

• Preface xiiiPart I Overview of Industrial Enzyme Applications and Key Technologies 11.1 Industrial Enzyme Applications – Overview and Historic Perspective 3Oliver May1.1.1 Prehistoric Applications 31.1.2 Growing the Scientific Basis 51.1.3 The Beginning of Industrial Applications and the Emerging Enzyme Industry 12References 211.2 Enzyme Development Technologies 25Andreas Vogel1.2.1 Introduction 251.2.2 Identification of Wild-Type Enzymes 261.2.2.1 Selection Parameters for Starting Enzymes 281.2.3 Enzyme Engineering 301.2.3.1 Types of Enzyme Modifications 301.2.3.2 General Engineering Strategies. Library Design and Generation 301.2.3.3 Screening for Better Enzymes 371.2.4 Impact of Enzyme Development Technologies Today and Tomorrow 38Acknowledgments 41References 411.3 Eukaryotic Expression Systems for Industrial Enzymes 47Lukas Rieder, Nico Teuschler, Katharina Ebner, and Anton Glieder1.3.1 Eukaryotic Enzyme Production Systems 471.3.2 Special Considerations for Working with Eukaryotic Expression Systems 471.3.2.1 Choice of Expression Host 471.3.2.2 Comparison of Cell Structure and Their Influence on Molecular Biology 491.3.3 Differences in Vector Design for Eukaryotic and Prokaryotic Hosts 511.3.4 Differences in Regulation of Gene Expression in Eukaryotes and Prokaryotes 561.3.4.1 Different Types of Promoters 581.3.5 Industrial Enzyme Production 581.3.6 Enzyme Production on Industrial Scale 611.3.6.1 Homologous Protein Production 611.3.6.2 Heterologous Protein Production 62References 631.4 Process Considerations for the Application of Enzymes 71Selin Kara and Andreas Liese1.4.1 Biocatalyst Types Used in Industrial Processes 711.4.2 Enzyme Immobilization for Biocatalytic Processes 741.4.3 Reaction Medium Applied in Enzymatic Catalysis 761.4.3.1 Monophasic Systems – Organic Media 771.4.3.2 Multiphasic Systems – Liquid/Liquid Mixtures 801.4.3.3 Multiphasic Systems – Gas/Liquid Mixtures 831.4.3.4 Multiphasic Systems – Solid/Liquid Mixtures 841.4.4 Appropriate Reactor Types in Enzyme Catalysis 871.4.5 Assessment Criteria for Enzymatic Applications 90References 92Part II Enzyme Applications for the Food Industry 952.1 Enzymes Used in Baking 97Joke A. Putseys and Margot E.F. Schooneveld-Bergmans2.1.1 Introduction 972.1.2 The Baking Process –The Baker’s Needs 982.1.2.1 Flour Quality and Standardization 982.1.2.2 Mixing and Dough Handling 1002.1.2.3 Fermentation and Dough Stability 1052.1.2.4 Baking and Oven Spring 1092.1.3 The Bread Quality – The Consumers’ Needs 1112.1.3.1 Color and Flavor 1112.1.3.2 Shelf Life 1122.1.4 Trends and Opportunities for Baking Enzymes 1162.1.4.1 Fine Baking and Confectionary 1162.1.4.2 Consumer Preference: Health, Individual Values, and Convenience 1172.1.5 Conclusion 118References 1192.2 Protein Modification to Meet the Demands of the Food Industry 125Andrew Ellis2.2.1 Food Proteins 1252.2.2 Processing of Food Protein 1272.2.3 Enzymes in the Processing of Food Proteins 1272.2.4 Food Protein Value Chain 1302.2.5 Recent Enzyme Developments 1312.2.5.1 Simple Protein Modification (Value Level 3) 1312.2.5.1.1 Developing Microbial Alternatives to Plant and Animal Enzymes 1312.2.5.2 Specialized Enzyme Modification (Value Level 4) 1342.2.5.2.1 Whey Protein Hydrolysates 1342.2.5.2.2 Plant Protein Hydrolysates 1342.2.5.3 Highly Specific Protein Modification (Value Level 5) 1352.2.5.3.1 Gluten Modification 1352.2.5.3.2 Acrylamide Reduction 1352.2.5.3.3 Bioactive Peptides 1362.2.6 Enzymes to Meet Future Needs 137Acknowledgments 139References 1392.3 Dairy Enzymes 143Peter Dekker2.3.1 Introduction 1432.3.2 Coagulants 1452.3.2.1 Traditional Rennets 1472.3.2.2 Microbial Rennets 1482.3.2.3 Fermentation Produced Chymosin 1512.3.3 Ripening Enzymes 1522.3.3.1 Proteases/Peptidases 1532.3.3.2 Lipases/Esterases 1542.3.4 Lactases 1542.3.4.1 Neutral Lactase 1562.3.4.2 Acid Lactase 1582.3.4.3 GOS Production 1582.3.5 Miscellaneous Enzymes 1612.3.5.1 Oxidases/Peroxidases 1612.3.5.2 Phopholipases 1622.3.5.3 Cross-linking Enzymes 1622.3.5.4 Preservation 1632.3.6 New Developments 163References 1632.4 Enzymatic Process for the Synthesis of Cellobiose 167Birgit Brucher and Thomas Häßler2.4.1 Enzymatic Synthesis of Cellobiose 1672.4.2 Cellobiose – Properties and Applications 1682.4.3 Existing Routes for Cellobiose Synthesis 1702.4.4 Enzyme Development 1712.4.5 Process Development 1732.4.5.1 Synthesis of Cellobiose 1742.4.5.2 Purification of Cellobiose 1742.4.6 Summary and Future Perspective 176References 1762.5 Emerging Field – Synthesis of Complex Carbohydrates. Case Study on HMOs 179Dora Molnar-Gabor, Markus J. Hederos, Sebastian Bartsch, and Andreas Vogel2.5.1 Introduction to Human Milk Oligosaccharides (HMOs) 1792.5.1.1 Discovery and Function of HMOs 1792.5.1.2 Structure of HMOs 1802.5.1.3 HMO Production, Regulatory Authorizations, and Commercial Launch – Historical Overview 1812.5.2 Glycom A/S Technologies Toward Commercial HMO Production 1842.5.2.1 Whole Cell Microbial Fermentation to HMOs (In Vivo Process) 1852.5.2.2 The Glycom In Vitro Concept to Diversify HMO Blends 1872.5.2.3 Validation of the HMO Diversification Concept with Non-optimized Enzymes 1872.5.3 Enzyme Development 1892.5.3.1 Optimization of the α1-3/4 Transfucosidase 1892.5.3.2 Optimization of the α2-6 Transsialidase 1922.5.4 Applications of the Optimized Enzymes for the HMO Profiles 1952.5.4.1 Scale-Up of the Lacto-N-fucopentaose III (LNFP-III), Sialyl Lacto-N-neotetraose (LST-c), and Sialyl Lacto-N-tetraose (LST-a) HMO Profiles 1952.5.5 Conclusion and Perspective 197References 198Part III Enzyme Applications for Human and Animal Nutrition 2033.1 Enzymes for Human Nutrition and Health 205Yoshihiko Hirose3.1.1 Introduction 2053.1.2 Current Problems of Enzymes in Healthcare Business 2053.1.3 Enzymes in Existing Healthcare Products 2063.1.3.1 Digestive Enzymes 2063.1.3.1.1 Digestive Enzymes in United States 2063.1.3.1.2 Therapeutic Digestive Enzymes 2073.1.3.2 Acid Lactase 2073.1.3.3 α-Galactosidase (ADG) 2083.1.3.4 Dextranase 2083.1.3.5 Glucose Oxidase 2083.1.3.6 Acetobacter Enzymes 2103.1.3.7 Laccase (Polyphenol Oxidase) 2103.1.4 New Enzyme Developments in Healthcare Products 2113.1.4.1 Transglucosidase 2113.1.4.2 Laccase 211References 2153.2 Enzyme Technology for Detoxification of Mycotoxins in Animal Feed 219Dieter Moll3.2.1 Introduction to Mycotoxins 2193.2.2 Mycotoxin Mitigation Strategies 2203.2.3 Enzyme Applications 2243.2.4 FUMzyme® 2253.2.4.1 The Substrate: Fumonisins 2253.2.4.2 Enzyme Discovery 2273.2.4.3 Enzyme Selection 2303.2.4.4 Enzyme Activity Assays 2323.2.4.5 Enzyme Characterization and Evaluation 2333.2.4.6 Enzyme Feeding Trials and Biomarker Analysis 2343.2.4.7 Enzyme Engineering 2373.2.4.8 Enzyme Production 2383.2.4.9 Enzyme Registration 2393.2.5 Future Mycotoxinases 2403.2.6 Conclusions 242References 2433.3 Phytases for Feed Applications 255Nikolay Outchkourov and Spas Petkov3.3.1 Phytase As a Feed Enzyme: Introduction and Significance 2553.3.2 Historical Overview of the Phytase Market Development 2563.3.3 From Phytate to Phosphorus: Step by Step Action of the Phytase 2593.3.3.1 Properties of Phytate 2593.3.3.2 Phytases Structural and Functional Classification 2603.3.3.2.1 Phytases from the Histidine Acid Phosphatases (HAP) Superfamily 2613.3.3.2.2 β-Propeller Phytase (BPP) 2613.3.3.2.3 Cysteine Phytase (CPhy) 2633.3.3.2.4 Purple Acid Phytases (PAPhy) 2633.3.3.2.5 Classification of the Phytases Based on Phytate Dephosphorylation Steps 2633.3.4 Nutritional Values of Phytase in Animal Feed 2653.3.5 Phytase Application As Feed Additive 2653.3.6 Effective Phytate Hydrolysis in the Upper Digestive Tract of the Animal 2663.3.7 Kinetic Description of Ideal Phytases 2693.3.8 Resistance to Low pH and Proteases 2713.3.9 Temperature Stability 2713.3.10 In lieu of Conclusion: Lessons from Phytase Super Dosing Trials 274References 275Part IV Enzymes for Biorefinery Applications 2874.1 Enzymes for Pulp and Paper Applications 289Debayan Ghosh, Bikas Saha, and Baljeet Singh4.1.1 Refining and Fiber Development Enzyme 2904.1.1.1 Microscopic Evaluation 2914.1.1.2 Evaluation of Enzyme-Treated Handsheets 2934.1.1.2.1 Case Study 1 2934.1.1.2.2 Case Study 2 2954.1.2 Drainage Improvement Enzyme 2964.1.2.1 Case Study 3 2994.1.2.2 Case Study 4 3004.1.3 Stickies Control Enzyme 3014.1.3.1 Case Study 5 3034.1.4 Deinking Enzymes 3064.1.4.1 Case Study 6 3074.1.5 Hardwood Vessel Breaking Enzyme 3084.1.5.1 Fiber Tester Image Analysis 3084.1.6 Native Starch Conversion Enzyme 3104.1.7 Bleach Boosting Enzyme 3124.1.7.1 Common Bleaching Agents 3124.1.7.1.1 Case Study 7 3134.1.7.2 Overcoming Challenges Faced by Bleaching Enzymes in Pulp and Paper industry 3154.1.8 Paper Mill Effluent Treatment Enzymes 3154.1.8.1 Case Study 8 3164.1.9 Slushing Enzyme 3174.1.9.1 Case Study 9 3174.1.9.2 Role of Enzymes in Pulp and Paper Industry – End Note! 318References 3194.2 Enzymes in Vegetable Oil Degumming Processes 323Arjen Sein, Tim Hitchman, and Chris L.G. Dayton4.2.1 Introduction 3234.2.2 General Seed Oil Processes 3244.2.2.1 Phospholipids 3254.2.2.2 A Molecular View of the Degumming Process 3274.2.3 Enzymatic Degumming 3304.2.3.1 Phospholipase C 3314.2.3.2 Ways to Cope with Poor Conversion/Poor Quality Oils in PLC-Based Processes 3334.2.3.3 Phospholipase A 3364.2.4 Enzymatic Degumming in Industrial Practice 3374.2.4.1 Introduction Hurdles 3414.2.5 Other Applications of Enzymes in Oil – Outlook 3434.2.5.1 Enzymatic Interesterification of Triglyceride Oils 3434.2.5.2 Biodiesel 3444.2.5.3 Enzyme-Assisted Decoloring 3444.2.5.4 Enzyme-Assisted Oil Extraction 3444.2.6 Conclusion 345Acknowledgments 345References 345Part V Enzymes used in Fine Chemical Production 3515.1 KREDs: Toward Green, Cost-Effective, and Efficient Chiral Alcohol Generation 353Chris Micklitsch, Da Duan, and Margie Borra-Garske5.1.1 Introduction 3535.1.2 Ketoreductases 3555.1.3 Cofactor Recycling 3565.1.4 CodeEvolver® Protein Engineering Technology 3585.1.5 Reduction of a Wide Range of Ketones/Aldehydes 3585.1.6 Critical Selectivity Tools for Enantiopure Asymmetric Carbonyl Reduction 3645.1.7 Examples of Improved KREDs for Improved Manufacturing 3695.1.8 KREDs: Going Green and Saving Green 373References 3775.2 An Aldolase for the Synthesis of the Statin Side Chain 385Martin Schürmann5.2.1 Introduction – Biocatalysis 3855.2.1.1 Enzymes as Biocatalysts in Chemical Process 3855.2.1.2 Biocatalytic Routes to the Statin Side Chain 3875.2.2 The Aldolase DERA in Application 3875.2.2.1 DERA-Catalyzed Aldol Reactions 3875.2.2.2 Feasibility Phase of DERA-Enabled Statin Side Chain Process 3905.2.3 Directed Evolution and Protein Engineering to Improve DERA 3925.2.3.1 Rational Design 3925.2.3.2 Directed Evolution of DERA 3945.2.3.3 Other Approaches to Suitable or Improved DERAs 3965.2.3.4 Other Applications of Process Intermediates and the DERA Technology 3975.2.4 Conclusions 398Acknowledgments 400References 401Index 405