Biocatalysis for Practitioners

Gonzalo de Gonzalo is Associate Professor at the Departamento de Química at the University of Seville, Spain. He obtained his Degree and his Ph. D. in chemistry at the University of Oviedo (Spain). He spent his postdoctoral stage at Consiglio Nazionale delle Ricerche (Milan, Italy), moving back to University of Oviedo with a Juan de la Cierva Fellowship. In 2010, he spent one year at the University of Groningen working tin the research of novel oxidative enzymes. He worked two years for the pharmaceutical company Antibióticos S.A.U. (León, Spain), moving to Seville in 2014. His research is focused on asymmetric synthesis by using different approaches, including biocatalytic and organocatalytic procedures, as well as the development of concurrent chemo- and biocatalytic reactions. He has published over 70 scientific publications and has recently been coeditor of the book "Biocatalysis: an Industrial Perspective".Iván Lavandera completed his Ph. D. studies in Chemistry in 2003 with Prof. Vicente Gotor and Prof. Miguel Ferrero. He continued in Prof. Gotor's group as a researcher until 2005, and then he moved to the University of Graz as a postdoctoral researcher under the supervision of Prof. Wolfgang Kroutil. He returned to Oviedo in 2008, where he became first as a Clarín and then as a Ramón y Cajal post-doctoral researcher. Since 2015, he is Associate Professor at the Organic and Inorganic Department at the University of Oviedo, where he got the habilitation in 2017. He has been co-author of two patents and more than 90 publications. His research interests are focused on Biocatalysis, especially the use of oxidoreductases and transferases to achieve green processes and to develop new synthetic routes combining bio- and chemocatalysis in a concurrent manner.

• Foreword xviiPart I Enzyme Techniques 11 Techniques for Enzyme Purification 3Adrie H. Westphal and Willem J. H. van Berkel1.1 Introduction 31.2 Traditional Enzyme Purification 41.2.1 Ion Exchange Chromatography 71.2.2 Gel Filtration 91.2.3 Bio-affinity Chromatography 111.2.4 Hydrophobic Interaction Chromatography 141.2.5 Hydroxyapatite Chromatography 151.3 Example of a Traditional Enzyme Purification Protocol 171.4 Purification of Recombinant Enzymes 181.4.1 Immobilized Metal Affinity Chromatography 181.4.2 Affinity Chromatography with Protein Tags 201.5 Column Materials 221.6 Conclusions 24References 252 Enzyme Modification 33Antonino Biundo, Patricia Saénz-Méndez, and Tamas Görbe2.1 Introduction 332.2 Practical Approach: Experimental Information, Analytical Methods, Tips and Tricks, and Examples 342.2.1 Directed Evolution 342.2.1.1 (Ultra)High-Throughput Screening and Selection 352.2.1.2 Applications of Directed Evolution Methodology 362.2.2 Semi-rational Design 372.2.2.1 Applications of Semi-rational Design Methodology 382.2.3 De Novo Enzyme Design 392.2.3.1 Applications of De Novo Enzyme Design Methodology 402.2.4 Rational Enzyme Design 402.2.4.1 Applications of Rational Design Methodology 412.3 Expectations and Perspectives 492.4 Concluding Remarks 50References 513 Immobilization Techniques for the Preparation of Supported Biocatalysts: Making Better Biocatalysts Through Protein Immobilization 63Javier Rocha-Martín, Lorena Betancor, and Fernando López-Gallego3.1 Introduction 633.2 General Aspects to Optimize Enzyme Immobilization Protocols 643.2.1 Carrier Nature 643.2.2 Immobilization Chemistry 643.2.3 Protein Orientation 643.2.4 Multivalence of the Protein Attachment 653.2.5 Chemical and Geometrical Congruence 653.2.6 Enzyme Spatial Organization 653.3 Type of Carriers for Immobilized Proteins 663.3.1 Types of Materials 663.3.1.1 Organic Materials 663.3.1.2 Inorganic Materials 663.3.2 Geometry 673.3.2.1 Beads 673.3.2.2 Monoliths 673.3.2.3 Membranes 673.3.3 Dimensions 673.3.4 Commercially Available Porous Carriers for Enzyme Immobilization 683.4 Immobilization Methods and Manners 683.5 Evaluation of the Enzyme Immobilization Process 703.5.1 Considerations Before Immobilization 713.5.1.1 Preparation of the Enzymatic Solution to Be Immobilized 713.5.1.2 Stability of the Soluble Enzyme Under Immobilization Conditions 713.5.2 Parameters Required to Define an Immobilization Process 713.5.2.1 Immobilization Yield 723.5.2.2 Expressed Activity or Apparent Activity 723.5.2.3 Specific Activity of the Immobilized Biocatalyst 733.6 Applied Examples of Immobilized Enzymes 733.6.1 Characterization of the Immobilized Biocatalyst 743.6.1.1 Determination of the Catalytic Activity of the Final Immobilized Biocatalyst and Maximum Protein Loading Capacity 743.6.1.2 Apparent Kinetic Parameters of the Immobilized Enzyme 763.6.1.3 Biocatalyst Stability 773.6.1.3.1 The Half-life Time of Biocatalysts 783.7 Challenges and Opportunities in Enzyme Immobilization 793.8 Conclusions 81List of Abbreviations 82References 824 Compartmentalization in Biocatalysis 89Robert Kourist and Javier González-Sabín4.1 Introduction 894.2 Cell as a Compartment 934.3 Compartmentalization Using Protein Assemblies 954.4 Compartmentalization Using Emulsion and Micellar Systems 964.5 Compartmentalization Using Encapsulation 1004.6 Compartmentalization Using Tea Bags and Thimbles 1034.7 Separation of Reaction Steps Using Continuous Flow 1054.8 Conclusions and Prospects 107References 108Part II Enzymes Handling and Applications 1135 Promiscuous Activity of Hydrolases 115Erika V. M. Orozco and André L. M. Porto5.1 Introduction 1155.2 Catalytic Promiscuity 1165.3 Hydrolases 1175.3.1 Applications of Hydrolases to Organic Synthesis 1185.3.2 Lipases and Their Hydrolysis Mechanism 1225.3.3 Catalytic Promiscuity of Hydrolases 1225.3.4 Promiscuous Aldol Reaction Catalyzed by Hydrolases 1305.3.5 Aldol Reaction Between 4-Cyanobenzaldehyde and Cyclohexanone Catalyzed by Porcine Pancreatic Lipase (PPL-II) and Rhizopus niveus Lipase (RNL) 1355.4 Conclusions 136References 1376 Enzymes Applied to the Synthesis of Amines 143Francesco G. Mutti and Tanja Knaus6.1 Introduction 1436.2 Hydrolases 1456.2.1 Practical Approaches with Hydrolases 1456.2.1.1 Kinetic Resolution 1456.2.1.2 Dynamic Kinetic Resolution 1466.2.2 Practical Examples with Hydrolases 1486.2.2.1 Kinetic Resolution of Racemic α-Methylbenzylamine Through the Methoxyacetylation Catalyzed by a Lipase 1486.2.2.2 Dynamic Kinetic Resolution for the Synthesis of Norsertraline 1496.3 Amine Oxidases 1496.3.1 Practical Approaches with Amine Oxidases 1506.3.1.1 Kinetic Resolution and Deracemization 1506.3.2 Practical Examples with Amine Oxidases 1516.3.2.1 One-pot, One-enzyme Oxidative Pictet–Spengler Approach Combined with Deracemization 1516.3.2.2 Desymmetrization of meso-compounds 1526.4 Transaminases (or Aminotransferases) 1526.4.1 Practical Approaches with Transaminases 1536.4.2 Practical Examples with Transaminases 1536.4.2.1 Kinetic Resolution and Deracemization 1536.4.2.2 Asymmetric Synthesis from Prochiral Ketone 1556.5 Amine Dehydrogenases, Imine Reductases, and Reductive Aminases 1556.5.1 Practical Approaches with Amine Dehydrogenases, Imine Reductases, and Reductive Aminases 1566.5.2 Practical Examples with Amine Dehydrogenases, Imine Reductases, and Reductive Aminases 1606.5.2.1 IRed-Catalyzed Reductive Amination of an Aldehyde Combined with KR of a Racemic Amine 1606.5.2.2 Asymmetric Reductive Amination Catalyzed by AmDH 1626.6 Ammonia Lyases 1626.6.1 Practical Approaches with Ammonia Lyases 1636.6.1.1 Aspartase, 3-Methylaspartate Ammonia Lyase, and Related Enzymes 1636.6.1.2 Aromatic Amino Acid Ammonia Lyases and Mutases 1656.6.2 Practical Examples with Ammonia Lyases 1666.6.2.1 Chemoenzymatic Synthesis of (S)-2-Indolinecarboxylic Acid 1666.6.2.2 Synthesis of L-Aspartate from Fumarate 1666.6.2.3 Enzymatic and Chemoenzymatic Synthesis of Toxin A and Aspergillomarasmine A and B 1666.7 Pictet–Spenglerases 1676.7.1 Practical Approaches with Pictet–Spenglerases 1676.7.2 Practical Examples with Pictet–Spenglerases 1696.7.2.1 Biocatalytic Synthesis of (R)-Harmicine 1696.7.2.2 Biocatalytic Synthesis of (S)-Trolline and Analogs 1696.8 Engineered Cytochrome P450s (Cytochrome “P411”) 1696.8.1 Practical Approaches with Engineered Cytochrome P450s 1706.9 Protocols for Selected Reactions 1716.9.1 Hydrolases 1716.9.1.1 Kinetic Resolution rac-Methylbenzylamine (rac-1) 1716.9.1.2 Dynamic Kinetic Resolution of Norsertraline Intermediate (rac-3) 1716.9.2 Monoamine Oxidases 1726.9.2.1 Chemoenzymatic Deracemization of Harmicine (rac-8) 1726.9.3 ω-Transaminases 1726.9.3.1 Deracemization of Mexiletine (rac-9, Kinetic Resolution, Followed by Formal Reductive Amination) 1726.9.4 Imine Reductases and Amine Dehydrogenases 1726.9.4.1 Reductive Amination of Aldehyde (11) with Kinetic Resolution of Amine Nucleophile (rac-trans-12) 1726.9.4.2 Asymmetric Reductive Amination of Acetophenone (14) Using Amine Dehydrogenase 1736.9.5 Ammonia Lyases 1736.9.5.1 Asymmetric Ammonia Addition to 2′-Chlorocinnamic Acid (17) 1736.9.6 Pictet–Spenglerases 1736.9.6.1 Asymmetric Pictet–Spengler Reaction with Strictosidine Synthase 1736.9.7 Engineered Cytochrome P450s 1746.9.7.1 Intermolecular Alkane C–H Amination Using Cytochrome P411 1746.10 Conclusions 174Acknowledgments 175References 1757 Applications of Oxidoreductases in Synthesis: A Roadmap to Access ValueAdded Products 181Mélanie Hall7.1 Introduction 1817.2 Reductive Processes 1847.2.1 Reduction of C¨TO Bonds 1847.2.1.1 Selection of Alcohol Dehydrogenase (ADH) for Stereoselective Reduction Reactions 1857.2.1.1.1 Absolute Configuration of the Product 1857.2.1.1.2 Substrate Type 1867.2.1.1.3 Thermostability 1877.2.1.1.4 Cofactor Preference 1877.2.1.1.5 Kits 1877.2.1.2 Practical Approach 1877.2.1.2.1 Montelukast 1887.2.1.2.2 Atorvastatin 1897.2.1.2.3 Dynamic Kinetic Resolutions 1897.2.1.2.4 Disproportionation 1907.2.1.2.5 Redox Isomerization 1907.2.2 Reduction of C¨TC Bonds 1917.2.2.1 Mechanism 1917.2.2.2 Enzymes and Substrates 1937.2.2.2.1 Enzymes 1937.2.2.2.2 Substrates 1937.2.2.3 Practical Approach 1967.2.2.3.1 Stereocontrol 1967.2.2.3.2 (Dynamic) Kinetic Resolution 1977.3 Oxidative Processes 1987.3.1 Oxygenations 1987.3.1.1 Baeyer–Villiger Oxidations 1987.3.1.1.1 Regiopreference 2007.3.1.1.2 Stereoselectivity 2017.3.1.1.3 Practical Approach 2037.3.1.2 Epoxidation of Alkenes 2047.3.2 Heteroatom Oxidation 2067.3.2.1 Reaction 2067.3.2.2 Substrates 2077.3.3 Peroxygenases: One Catalyst – Many Reactions 2077.4 Protocols for Selected Reactions Employing Oxidoreductases 2097.4.1 Alcohol Dehydrogenase (ADH): Disproportionation of rac-2-Phenylpropanal 2097.4.1.1 Biotransformation 2097.4.1.2 Product Recovery and Purification 2107.4.2 Ene-reductase/Old Yellow Enzyme (OYE): Dynamic Kinetic Resolution of a γ-substituted Lactone 2107.4.2.1 Biotransformation 2107.4.2.2 Product Recovery and Purification 2107.4.3 Baeyer–Villiger Monooxygenase (BVMO): Kinetic Resolution of a Racemic Ketone 2107.4.3.1 Biotransformation 2117.4.3.2 Product Recovery and Purification 2117.4.4 Baeyer–Villiger Monooxygenase (BVMO): Asymmetric Sulfoxidation 2117.4.4.1 Biotransformation 2117.4.4.2 Product Recovery and Purification 2117.5 Conclusions 211Acknowledgments 212References 2128 Glycosyltransferase Cascades Made Fit For the Biocatalytic Production of Natural Product Glycosides 225Bernd Nidetzky8.1 Introduction: Glycosylated Natural Products and Leloir Glycosyltransferases 2258.2 Glycosylated Flavonoids and Nothofagin 2278.3 Glycosyltransferase Cascades for Biocatalytic Synthesis of Nothofagin 2298.4 Enzyme Expression 2308.5 Solvent Engineering for Substrate Solubilization 2328.6 Nothofagin Production at 100 g Scale 2338.7 Concluding Remarks 237References 237Part III Ways to Improve Enzymatic Transformations 2459 Application of Nonaqueous Media in Biocatalysis 247Afifa A. Koesoema and Tomoko Matsuda9.1 Introduction 2479.2 Advantages and Disadvantages of Reactions in Nonaqueous Media 2489.3 Nonaqueous Media Used for Biocatalysis 2489.4 Enzymatic Activity and Inactivation in Nonaqueous Media 2519.4.1 Enzymatic Activity in Nonaqueous Media 2519.4.2 Factors Causing Inactivation of Enzymes in Nonaqueous Media 2529.5 Practical Approaches to Stabilize Enzymes in Nonaqueous Media 2529.5.1 Utilization of Nonaqueous Media-Tolerant Enzymes or Host Cells 2529.5.2 Enzyme Immobilization 2539.5.3 Modification of the Enzyme Preparation 2549.5.4 Protein Engineering 2559.6 Examples of Biocatalyzed Reactions in Solvent-Free Systems 2569.7 Examples of Reactions in Micro-aqueous Systems 2589.8 Examples of Reactions in Bio-Based Liquids 2609.8.1 2-Methyltetrahydrofuran (MeTHF) 2609.8.2 Cyclopentyl Methyl Ether (CPME) 2619.8.3 Potential Application of other Bio-based Liquids 2629.9 Examples of Reactions in Liquid CO+ 2629.10 Examples of Reactions in CO2-Expanded Bio-based Liquids 2649.11 Examples of Reactions in Natural Deep Eutectic Solvents 2659.12 Conclusions and Future Perspectives 267References 26710 Nonconventional Cofactor Regeneration Systems 275Jiafu Shi, Yizhou Wu, Zhongyi Jiang, Yiying Sun, Qian Huo, Weiran Li, Yang Zhao, and Yuqing Cheng10.1 Introduction 27510.2 Basics of Photocatalytic NADH Regeneration 27910.2.1 Processes and Mechanism Associated with Photocatalytic NADH Regeneration 27910.2.2 Aspects of Measuring Photocatalytic NADH Regeneration 28110.3 Advancements in Photocatalytic NADH Regeneration 28210.3.1 Nature Photosensitizers 28210.3.2 Organic Molecular Photosensitizers 28210.3.3 Inorganic Semiconductors 28510.3.4 Organic Semiconductors 28810.4 Expectations 29010.5 Conclusions and Prospects 29210.5.1 Conclusions 29210.5.2 Prospects 292List of Abbreviations 292References 29311 Biocatalysis Under Continuous Flow Conditions 297Bruna Goes Palma, Marcelo A. do Nascimento, Raquel A. C. Leão, Omar G. Pandoli, and Rodrigo O. M. A. de Souza11.1 Introduction 29711.2 Practical Approach for Biocatalysis Under Continuous Flow Conditions 29911.2.1 Esterification 29911.2.1.1 Experimental Procedure 30111.2.2 Transesterification 30211.2.2.1 Experimental Procedure 30311.2.3 Kinetic Resolutions 30311.2.3.1 Kinetic Resolution of Amines Employing Lipases 30411.2.3.1.1 Experimental Procedure 30411.2.3.2 Kinetic Resolutions Employing ω-Transaminases 30511.2.3.2.1 Experimental Procedure 30511.2.3.3 Kinetic Resolution of Alcohols Using Lipases 30711.2.3.3.1 Experimental Procedure 30711.2.4 Dynamic Kinetic Resolutions 30811.2.4.1 Experimental Procedure 30911.2.5 Asymmetric Synthesis 30911.2.5.1 Experimental Procedure 31111.2.5.1.1 Protein Immobilization 31111.2.5.1.2 Ion Exchange of NADPH on Ag-DEAE 31111.2.5.1.3 General Procedure for the Continuous Asymmetric Reduction 31111.3 Conclusions and Perspective 311References 312Part IV Recent Trends in Enzyme-Catalyzed Reactions 31712 Photobiocatalysis 319Martín G. López-Vidal, Guillermo Gamboa, Gabriela Oksdath-Mansilla, and Fabricio R. Bisogno12.1 Introduction 31912.2 Oxidative Processes 32112.2.1 Baeyer–Villiger Oxidation 32112.2.2 Alkane Hydroxylation 32212.2.3 O-Dealkylation 32612.2.4 Decarboxylation 32712.2.4.1 Alkene Production 32712.2.4.2 Alkane Production 32812.2.5 Epoxidation 33012.3 Reductive Processes 33212.3.1 Carbonyl Reduction 33212.3.2 Olefin Reduction 33612.3.3 Imine Reduction 34212.3.4 Reductive Amination 34412.3.5 Dehalogenation 34512.3.6 Deacetoxylation 34712.4 Combination of Photooxidation and Enzymatic Transformation 34812.5 Summary and Outlook 352Abbreviations 352References 35413 Practical Multienzymatic Transformations: Combining Enzymes for the Onepot Synthesis of Organic Molecules in a Straightforward Manner 361Jesús Albarrán-Velo, Sergio González-Granda, Marina López-Agudo, and Vicente Gotor-Fernández13.1 Introduction 36113.2 Non-stereoselective Bienzymatic Transformations 36313.2.1 Amine Synthesis 36313.2.2 Bienzymatic Linear Cascades Toward the Production of Other Organic Compounds 36513.3 Stereoselective Bienzymatic Transformations 36713.3.1 Stereoselective Amine Synthesis Through Concurrent Processes 36813.3.1.1 Amination of Alcohols 36813.3.1.2 Deracemization of Amines 37113.3.1.3 Amino Alcohol Synthesis 37213.3.1.4 Other Bienzymatic Stereoselective Synthesis of Amines 37413.3.2 Stereoselective Bienzymatic Cascades Toward the Production of Other Organic Compounds 37713.3.2.1 Synthesis of Organic Compounds Other Than Amino Acids 37713.3.2.2 Amino Acid Synthesis 38313.4 Multienzymatic Transformations: Increasing Synthetic Complexity 38613.5 Summary and Outlook 395References 39514 Chemoenzymatic Sequential One-Pot Protocols 403Harald Gröger14.1 Introduction: Theoretical Information and Conceptual Overview 40314.2 State of the Art in Sequential Chemoenzymatic One-Pot Synthesis: Selected Examples and Historical Overview About Selected Contributions 40614.2.1 Sequential Chemoenzymatic One-Pot Synthesis Combining a Metal-Catalyzed Reaction with a Biotransformation 40614.2.2 Sequential Chemoenzymatic One-Pot Synthesis Combining an Organocatalytic Reaction with a Biotransformation 41114.2.3 Sequential Chemoenzymatic One-Pot Synthesis Combining a Reaction Catalyzed by a Heterogeneous Chemocatalyst with a Biotransformation 41614.2.4 Sequential Chemoenzymatic One-Pot Synthesis Combining a Reaction Catalyzed by a Heterogeneous Biocatalyst with a Chemocatalytic Transformation 41714.2.5 Sequential Chemoenzymatic One-Pot Synthesis Combining More than Two Reactions 41814.3 Practical Aspects of the Development of Sequential Chemoenzymatic One-Pot Syntheses 42014.4 Conclusions and Outlook 423References 424Part V Industrial Biocatalysis 42715 Industrial Processes Using Biocatalysts 429Florian Kleinbeck, Marek Mahut, and Thierry Schlama15.1 Introduction 42915.2 Biocatalysis in the Pharmaceutical Industry 43015.2.1 Pregabalin 43115.2.2 Vernakalant 43215.2.3 Sitagliptin 43315.2.4 Esomeprazole 43515.2.5 Montelukast 43615.2.6 Boceprevir 43915.3 Aspects to Consider for Development of a Biocatalytic Process on Commercial Scale – A Case Study 44215.3.1 Identification of a Suitable Enzyme 44315.3.2 Process Development 44315.3.3 Control Strategy and Regulatory Considerations 44515.3.3.1 Impurities 44615.3.3.2 Types of Biocatalysts 45015.3.3.3 Type of Expression System 45115.3.3.4 Route of Administration 45115.3.3.5 Position of the Biocatalytic Step in the Synthesis and Downstream Transformations 45115.3.3.6 Summary of the Case Study 45215.3.4 Health, Process Safety and Environmental Aspects 45315.3.4.1 Health 45315.3.4.2 Process Safety 45315.3.4.3 Environmental Aspects 45415.3.5 Equipment Utilization and Throughput Time 45515.3.6 Equipment Cleaning 45515.3.7 Enzyme Release Testing 45615.3.8 Transport and Storage 45715.4 Conclusions, Expectations, and Prospects 458Acknowledgments 460List of Abbreviations 460References 46116 Enzymatic Commercial Sources 467Gonzalo de Gonzalo and Iván Lavandera16.1 Introduction 46716.2 European Companies 46816.2.1 AB Enzymes 46816.2.2 Almac 46816.2.3 Biocatalysts 46916.2.4 c-Lecta GmbH 46916.2.5 Enzymicals 47016.2.6 Evoxx Technologies GmbH 47016.2.7 GECCO 47116.2.8 Inofea AG 47216.2.9 Johnson-Matthey 47216.2.10 Metgen Oy 47316.2.11 Novozymes 47416.2.12 Prozomix 47416.2.13 Royal DSM 47516.3 American Companies 47516.3.1 Codexis Inc. 47516.3.2 Dupont Nutrition and Biosciences 47616.3.3 IBEX Technologies 47616.3.4 MP Biomedical 47716.3.5 Sigma-Aldrich 47716.3.6 Strem Chemicals, Inc. 47816.3.7 Worthington Biochemical Corp 47916.4 Asian Enzyme Suppliers 48016.4.1 Advanced Enzymes Technologies, Ltd. 48016.4.2 Amano Enzyme Co., Ltd. 48016.4.3 Aumgene Biosciences 48116.4.4 EnzymeWorks 48116.4.5 Meito Sangyo Co., Ltd. 48116.4.6 Oriental Yeast Co., Ltd. 48216.4.7 Takabio 48216.4.8 Toyobo Co., Ltd. 48216.5 Outlook 483References 484Index 487