Enzyme Catalysis in Organic Synthesis

A Comprehensive Handbook

Volume III

Edited by Karlheinz Drauz and Herbert Waldmann

Second, Completely Revised and Enlarged Edition

^WILEY-VCH IIX

Contents

Foreword V Preface VII

Volume I

1 Introduction 1 Maria-Regina Kula 1.1 Enzymes as Catalysts 1 1.2 Enzyme Structure and Function 4 (X^ 1.3 Cofactors and Coenzymes 12 1.4 Enzyme Nomenclature 21 1.5 Enzyme Kinetics 23 1.5.1 Reaction Rate and Substrate Concentration 23 1.5.2 Inhibitors and Effectors 26 1.5.3 Influence of pH and Buffers 27 1.5.4 Temperature 28 1.6 Organic Solvents as Reaction Media 31 1.7 Enzyme Handling: Quality Requirements 32 1.8 Biotransformation Using Whole Cells 33 1.8.1 General Aspects 33^ 1.8.2 Biotransformation with Growing Cells 36 1.8.3 Biotransformation with Resting Cells 37 1.8.4 Biotransformations with Permeabilized or Dried Cells 37 Bibliography 38

2 Production and Isolation of Enzymes 41 Yoshihiko Hirose 2.1 Introduction 41 2.2 Enzyme Suppliers for Biotransformation 44 2.3 Origins of Enzymes 45 2.3.1 Microbial Enzymes 45 2.3.2 Plant Enzymes 46 2.3.3 Animal enzymes 46 X| Contents

2.4 Fermentation of Enzymes 46 2.4.1 Liquid Fermentation 46 2.4.2 Solid Fermentation 47 2.4.3 Extraction of Enzymes 47 2.5 ExtractionofEnzym.es 47 2.5.1 Microbial Enzymes 47 2.5.2 Plant Enzymes 48 2.5.3 Animal Enzymes 48 2.6 Concentration 48 2.7 Purification of Enzymes 49 2.7.1 Chromatography 49 2.7.1.1 Ion Exchange Chromatography (IEX) 49 2.7.1.2 Hydrophobic Interaction Chromatography (HIC) 54 2.7.1.3 Gel Filtration (GF) 56 2.7.1.4 Reversed Phase Chromatography 58 , 2.7.1.5 Hydrogen Bond Chromatography 59 2.7.1.6 Affinity Chromatography 59 2.7.1.7 Salting-out Chromatography 62 2.7.2 Precipitation 62 2.7.2.1 Precipitation by Salting out 62 2.7.2.2 Precipitation by Organic Solvents 63 2.7.2.3 Precipitation by Changing pH 63 2.7.2.4 Precipitation by Water-Soluble Polymer 63 2.7.3 Crystallization 64 2.7.4 Stabilization During Purification 64 2.7.5 Storage of Enzymes 64 2.7.5.1 Storage in Liquids 64 2.7.5.2 Storage in Solids 65 2.8 Commercial Biocatalysts 65 References 66

3 Rational Design of Functional Proteins 67 Tadayuki Imanaka and Haruyuki Atomi

3.1 Protein Engineering 67 3.2 Gene Manipulation Techniques in Enzyme Modification 68 3.3 Protein Crystallization 70 3.4 Comparative Modeling of a Protein Structure 73 3.5 What is Needed to Take a Rational Approach? 75 3.6 Examples of Protein Engineering 76 3.6.1 Protein Engineering Studies: Providing a Rational Explanation for Enzyme Specificity 76 3.6.2 Enhancing the Thermostability of Proteases 78 3.6.3 Contribution of Ion Pairs to the Thermostability of Proteins from Hyperthermophiles 79 Contents XI

3.6.4 Thermostability Engineering Based on the Consensus Concept 80 3.6.5 Changing the Optimal pH of an Enzyme 81 3.6.6 Changing the Cofactor Specificity of an Enzyme 82 3.6.7 Changing the Substrate Specificity of an Enzyme 84 3.6.8 Changing the Product Specificity of an Enzyme 85 3.6.9 Combining Site-directed Mutagenesis with Chemical Modification 86 3.6.10 Changing the Catalytic Activity of a Protein 087 3.7 Conclusions 89 References 90

4 Enzyme Engineering by Directed Evolution 95 Oliver May, Christopher A. Voigt and Frances H. Arnold 4.1 Introduction 95 4.2 Evolution as an Optimizing Process 96 4.2.1 The Search Space of Chemical Solutions 97 4.2.2 The Directed Evolution Algorithm 98 4.3 Creating a Library of Diverse Solutions 99 4.3.1 Mutagenesis 99 4.3.1.1 Random Point Mutagenesis of Whole Genes 99 4.3.1.2 Focused Mutagenesis 104 4.3.1.3 Calculation of Mutagenesis Hot-Spots 105 4.3.2 Recombination 107 4.3.2.1 In Vitro Recombination 107 4.3.2.2 In vivo Recombination 110 4.3.2.3 Family Shuffling 111 4.4 Finding Improved Enzymes: Screening and Selection 112 4.4.1 You Get What You Screen For 113 4.4.2 Screening Strategies 113 4.4.2.1 Low-Throughput Screening 114 4.4.2.2 High-Throughput Screening 115 4.4.2.3 Choosing Low versus High Throughput 116 4.4.2.4 Analyzing the Mutant Fitness Distribution 117 4.4.3 Selection and Methods to link Genotype with Phenotype 119 4.5 Applications of Directed Evolution 121 4.5.1 Improving Functional Enzyme Expression and Secretion 122 4.5.2 Engineering Enzymes for Non-natural Environments 127 4.5.3 Engineering Enzyme Specificity 129 4.5.3.1 Substrate Specificity 129 4.5.3.2 Enantioselectivity 131 4.6 Conclusions 132 References 133 XIII Contents

5 Enzyme Bioinformatics 139 Kay Hofmann • - 5.1 Introduction 139 5.2 Protein Comparison 140 5.2.1 Sequence Comparison versus Structure Comparison 140 5.2.2 Substitution Matrices in Sequence Comparisons 141 5.2.3 Profile Methods 142 5.2.4 Database Searches 144 5.3 Enzyme-specific Conservation Patterns 145 5.3.1 General Conservation Patterns 145 5.3.2 Active Site Conservation Patterns 146 5.3.3 Metal Binding Conservation Patterns 146 5.3.4 Making Use of Conservation Patterns 148 5.4 Modular Enzymes 149 5.4.1 The Domain Concept in Structure and Sequence 149 5.4.2 A Classification of Modular Enzymes 150 5.4.3 Inhibitory Domains 151 5.5 Enzyme Databases and Other Information Sources 151 5.5.1 E. C. Nomenclature and ENZYME Database 152 5.5.2 BRENDA 152 5.5.3 KEGG and LIGAND database 153 5.5.4 UM-BBD 153 5.5.5 Structural Databases 153 5.5.6 Metalloprotein Databases 154 5.5.7 Databases for Selected Enzyme Classes 154 5.6 Protein Domain and Motif Databases 154 5.6.1 PROSITEea55 5.6.2 PFAM 156 5.6.3 Other Related Databases 156 5.7 Enzyme Genomics 156 5.7.1 Ortholog Search 157 5.7.2 Paralog Search 157 5.7.3 Non-homology Based methods 159 5.8 Outlook 159 References 161

6 Immobilization of Enzymes 163 James Lalonde 6.1 Introduction 163 6.2 Methods of Immobilization 164 6.2.1 Non-Covalent Adsorption 165 6.2.2 Covalent Attachment 168 6.2.2.1 Carriers for Enzyme Immobilization 170 6.2.3 Entrapment and Encapsulation 171 Contents XIII

6.2.4 Cross-Linking 175 6.3 Properties of Immobilized Biocatalysts 175 6.3.1 Mass Transfer Effects 176 6.3.2 Partition 176 6.3.3 Stability 177 6.3.4 Activity of Immobilized Enzymes 177 6.4 New Developments and Outlook 178 6.4.1 Cross-linked Enzyme Crystals (CLEC®) 179 6.4.2 Sol-Gel 181 6.4.3 Controlled Solubility "Smart Polymers" 181 References 182

7 Reaction Engineering for Enzyme-Catalyzed Biotransformations 185 Manfred Biselli, Udo Kragl and Christian Wandrey

7.1 Introduction 185 7.2 Steps of Process Optimization 186 7.3 Investigation of the Reaction System 190 7.3.1 Properties of the Enzyme 190 7.3.2 Properties of the Reaction System 193 7.3.2.1 Thermodynamic Equilibrium of the Reaction 193 7.3.2.2 Complex Reaction Systems: The Existence of Parallel and Consecutive Reactions 195 7.3.2.3 Other Properties of the Reaction System 204 7.3.2.4 Application of Organic Solvents 204 7.4 Investigation of Enzyme Kinetics 208 7.4.1 Methods of Parameter Identification 209 7.4.2 The Kinetics of One-Enzyme Systems 210 7.4.2.1 THE Michaelis-Menten Kinetics 210 7.4.2.2 Competitive Inhibition 214 7.4.2.3 Non-Competitive Inhibition 215 7.4.2.4 Uncompetitive Inhibition 216 7.4.2.5 Reversibility of One-Substrate Reactions 217 7.4.2.6 Two-Substrate Reactions 218 7.4.2.7 Kinetics of Aminoacylase as Example of a Random Uni-Bi Mechanism 223 7.4.3 Kinetics of Multiple Enzyme Systems 230 7.5 Enzyme Reactors 232 7.5.1 Basic Reaction Engineering Aspects 232 7.5.2 Reactors for Soluble Enzymes 238 7.5.2.1 Reactor Optimization Exemplified by the Enzyme Membrane Reactor 241 7.5.2.2 Control of Conversion in a Continuously Operated EMR 249 7.5.3 Reactor Systems for Immobilized Enzymes 250 7.5.4 Reaction Techniques for Enzymes in Organic Solvent 251 XIV I Contents

7.6 Conclusions and Outlook 253 References 254

8 Enzymic Conversions in Organic and Other Low-Water Media 259 Peter Hailing 8.1 Introduction 259 8.2 Enzyme Form 260 8.2.1 Lyophilized Powders 260 8.2.2 Immobilized Enzymes 261 8.2.3 Cross-Linked Crystals 261 8.2.4 Direct Precipitation in Organic Solvents 262 8.2.5 Additives in Catalyst Powders 262 8.2.6 Solubilized Enzymes 263 8.3 Residual Water Level 264 8.3.1 Fixing Initial Water Activity of Reaction Components 266 • 8.3.2 Control of Water Activity During Reaction 269 8.3.3 "Water Mimics" 273 8.4 Temperature 274 8.5 Substrate (Starting Material) Concentrations 274 8.6 Solvent Choice 276 8.6.1 Effects on Equilibrium Position 276 8.6.2 "Solvent Effects" that Really are Not 276 8.6.3 Solvent Polarity Trend and Recommended Choices 277 8.6.4 Solvent Parameters 279 8.6.5 Solvent Effects on Selectivity 280 8.6.6 No Solvent or Little Solvent Systems 280 8.7 Acid-Base Conditions 281 8.7.1 pH Memory 281 8.7.2 Processes Erasing pH Memory 282 8.7.3 Systems for Acid-Base Buffering 283 References 285

9 Enzymatic Kinetic Resolution 287 Jonathan M.J. Williams, RebeccaJ. Parker, and Claudia Neri 9.1 Introduction 287 9.2 Alcohols and their Derivatives 288 9.2.1 Cyanohydrins 289 9.2.2 Other Readily Racemized Substrates 290 9.2.3 Enzyme and Metal Combinations 293 9.3 Carboxylic Acids and their Derivatives 297 9.3.1 Readily Enolized Carboxylic Acid Derivatives 297 9.3.2 Amino-Esters and Related Compounds 301 9.3.3 Reactions of cyclic amino acid derivatives 302 9.4 Reduction of P-Ketoesters 307 Contents |XV

9.5 Conclusion 309 References 310

10 Enzymes from Extreme Thermophilic and Hyperthermophilic Archaea and Bacteria 313 Costanzo Bertoldo and Carabed Antranikian 10.1 Introduction 313 10.2 Starch-Processing Enzymes 315 10.2.1 Thermoactive Amylolytic Enzymes 316 10.2.1.1 Heat-Stable Amylases and Glucoamylases 316 10.2.1.2 a-Glucosidases 317 10.2.1.3 Thermoactive Pullulanases and CGTases 317 10.3 Cellulose-Hydrolyzing Enzymes 0321 10.3.1 Thermostable Cellulases 321 10.4 Xylan-Degrading Enzymes 324 10.4.1 Thermostable Xylanases 324 10.5 Chitin Degradation 325 10.6 Proteolytic Enzymes 326 10.6.1 Stable Proteases 327 10.7 Intracellular Enzymes 329 References 331

Volume II

11 Hydrolysis and Formation of C-O Bonds 335 11.1 Hydrolysis and Formation of Carboxylid Acid E sters 335 Hans-Joachim Cais and Fritz Theil 11.1.1 Hydrolysis and Formation of Carboxylic Acid Esters 351 11.1.1.1 Hydrolysis of Carboxylic Acid Esters 351 11.1.1.2 Formation of Carboxylic Esters 472 11.1.1.3 Inter-and Intramolecular Alcoholysis 545 References 574 11.2 Hydrolysis of Epoxides 579 Kurt Faber and Romano V. A. Orru 11.2.1 Epoxide Hydrolases in Nature 581 11.2.1.1 Isolation and Characterization of Epoxide Hydrolases 582 11.2.1.2 Structure and Mechanism of Epoxide Hydrolases 584 11.2.1.3 Screening for Microbial Epoxide Hydrolases 587 11.2.2 Microbial Hydrolysis of Epoxides 588 11.2.2.1 Fungal Enzymes 588 11.2.2.2 Bacterial Enzymes 590 11.2.2.3 Yeast Enzymes 591 11.2.3 Substrate Specificity and Selectivity 592 11.2.3.1 Asymmetrization of meso-Epoxides 592 XVI Contents

11.2.3.2 Resolution of Racemic Epoxides 592 11.2.3.3 Deracemization Methods 596 11.2.4 Use of Non-Natural Nudeophiles 599 11.2.5 Applications to Asymmetric Synthesis 600 11.2.6 Summary and Outlook 604 References 605 11.3 Hydrolysis and Formation of Glycosidic Bonds 609 Chi-Huey Wong 1.3.1 Introduction 609 11.3.2 Glycosyltransferases of the Leloir Pathway 611 11.3.2.1 Synthesis of Sugar Nucleoside Phosphates 613 11.3.2.2 Substrate Specificity and Synthetic Applications of Glycosyltransferases 619 11.3.2.3 In Situ Cofactor Regeneration 626 11.3.2.4 Cloning and Expression of Glycosyltransferases 628 11.3.3 Non-Leloir Glycosyltransferases: Transfer of Glycosyl donors from Glycosyl Phosphates and Glycosides 631 11.3.4 Glycosidases 633 11.3.4.1 Equilibrium-controlled Synthesis 633 11.3.4.2 Kinetically Controlled Synthesis 634 11.3.4.3 Selectivity 634 11.3.5 Synthesis of N-glycosides 637 11.3.5.1 Nucleoside Phosphorylase 638 11.3.5.2 NADHydrolase 639 11.3.6 Biological Applications of Synthetic Glycoconjugates 639 11.3.6.1 Glycosidase and Glycosyl Transferase Inhibitors 639 11.3.6.2 Glycoprotein Remodeling 641 11.3.7 Future Opportunities 642 References 643 11.4 Natural Polysaccharide-degrading Enzymes 653 Constanzo Bertoldo and Carabed Antranikian 11.4.1 Introduction 653 11.4.2 Starch 653 11.4.2.1 Classification of Starch-degrading Enzymes 654 11.4.2.2 a-Amylase (l,4-a-D-Glucan,4-Glucanhydrolase, E.C. 3.2.1.1) 655 11.4.2.3 P-Amylase (1,4-a-D-Glucan Maltohydrolase, E.C. 3.2.1.2) 656 11.4.2.4 Glucoamylases (1,4-a-D-glucanglucohydrolase, E.C. 3.2.1.3) 656 11.4.2.5 a-Glucosidase (a-8-Glucoside Glucohydrolase, E.C. 3.2.1.20) 657 11.4.2.6 Isoamylase (Glycogen 6-Glucanohydrolase, E.C. 3.2.1.68) 657 11.4.2.7 Pullulanase Type I (a-Dextrin 6-Glucanohydrolase, E. C. 3.2.1.41) 657 11.4.2.8 Pullulanase Type II or Amylopullulanase 658 11.4.2.9 Pullulan Hydrolases (Type I, Neopullulanase; Type II, Isopullulanase, E. C. 3.2.1.57, Pullulan Hydrolase Type III) 659 11.4.2.10 Cyclodextrin Glycolsyltransferase (1,4-a-D-Glucan 4-a-D-(l,4-a-D- Glucano)-Transferase, E.C. 2.4.1.19) 659 Contents IXVII

11.4.2.11 Biotechnological Applications of Starch-degrading Enzymes 659 11.4.3 Cellulose 661 11.4.3.1 Cellulose-degrading Enzyme Systems 663 11.4.3.2 Endoglucanase (1,4-P-D-Glucan-Glucanohydrolase, E.C. 3.2.1.4) 663 11.4.3.3 Cellobiohydrolase (1,4-P-D-Glucan Cellobiohydrolase, E.C. 3.2.1.91) 663 11.4.3.4 p-Glucosidase (P-D-Glucoside Glucohydrolase, E.C. 3.2.1.21) 664 11.4.3.5 Fungal and Bacterial Cellulases 664 11.4.3.6 Structure and Synergistic Effect of Cellulases 665 11.4.4 Xylan 667 11.4.4.1 The Xylanolytic Enzyme System 668 11.4.4.2 Endoxylanase (1,4-P-D-XylanXylanohydrolase, E.C. 3.2.1.8) 670 11.4.4.3 P-Xylosidase (P-D-Xyloside Xylohydrolase, E.C. 3.2.1.37) 670 11.4.4.4 a-L-Arabinofuranosidase (E.C. 3.2.1.55) 671 11.4.4.5 a-Glucuronidase (E.C. 3.2.1.136) 671 11.4.4.6 Acetyl Xylan Esterase (E. C. 3.1.1.6) 672 11.4.4.7 Mechanism of Action of Endoxylanase 672 11.4.4.8 Biotechnological Applications of Xylanases 672 11.4.5 Pectin 673 11.4.5.1 Classification of Pectic Substances 675 11.4.5.2 Pectolytic Enzymes 675 11.4.5.3 Classification of Pectolytic Enzymes 676 11.4.5.4 Protopectinase 676 11.4.5.5 Pectin Methylesterase 677 11.4.5.6 Pectin and Polygalacturonate Depolymerizing Enzymes 677 11.4.5.7 Pectin and Polygalacturonate Hydrolase 678 11.4.5.8 Pectin and Polygalacturonate Lyase 679 11.4.5.9 Biotechnological Applications of Pectolytic Enzymes 680 References 681 11.5 Addition of Water to C=C Bonds 686 Marcel Wubbolts 11.5.1 Addition of Water to Alkenoic Acids 686 11.5.2 Addition of Water to Alkene-Dioic Acids 687 11.5.2.1 L-and D-Malic Acid 687 11.5.1.2 Substituted Malic Acids 688 11.5.3 Addition of Water to Alkene-Tricarboxylic Acids 688 11.5.3.1 Citric Acid and Derivatives 688 11.5.4 Addition of Water to Alkynoic Acids 690 11.5.5 Addition of Water to Enols 690 11.5.5.1 Carbohydrates: Addition of Water to 2-Keto-3-Deoxysugars 690 11.5.5.2 Addition/Elimination of Water with Other Enols 691 11.5.6 Addition of Water to Unsarurated Fatty Acids 693 11.5.6.1 CoA and ACP Coupled Fatty Acid Hydratases 693 11.5.6.2 Hydratases Acting on Free Fatty Acids 695 11.5.7 Addition of Water to Steroids 695 References 696 XVIII Contents

12 Hydrolysis and Formation of C-N Bonds 699

12.1 Hydrolysis of Nitriles 699 Birgit Schulze 12.1.1 Introduction 699 12.1.2 Types of Nitrile Hydrolyzing Enzymes 700 12.1.2.1 Enzymatic Hydrolysis of Organic Nitriles 700 12.1.2.2 Enzymatic Hydrolysis of Cyanide 702 12.1.3 Examples of Enzymatic Nitrile Hydrolysis 703 12.1.3.1 Enantioselective Hydrolysis of Nitriles 703 12.1.3.2 Monohydrolysis of Dinitriles 705 12.1.3.3 Substrate and Product Inhibition of Nitrile Hydrolysis 708 12.1.3.4 Activation and Stabilization of Nitrile Hydratases 710 12.1.3.5 Nitrile Hydrolysis in Organic Solvents 710 12.1.3.6 Large Scale Production of Acrylamide 711 12.1.4 Availability and Industrial Future of Nitrile Hydrolyzing Biocatalysts 713 References 713 12.2 Formation and Hydrolysis of Amides 716 Birgit Schulze and Erik de Vroom 12.2.1 Introduction 716 12.2.2 Enzymatic Formation of Amides 716 12.2.3 Enzymatic Enantioselective Hydrolysis of Amides 719 12.2.3.1 Hydrolysis of Carboxylic Amides 719 12.2.3.2 Hydrolysis of Amino Acid Amides 720 12.2.3.3 Hydrolysis of Cyclic Amides 727 12.2.4 Selective Cleavage of the C-Terminal Amide Bond 728 12.2.5 Amidase Catalyzed Hydrolytic and Synthetic Processes in the Production of Semi-synthetic Antibiotics 729 12.2.5.1 Enzymatic Production of 6-APA, 7-ADCA and 7-ACA Using Amidases: Hydrolytic Processes 730 12.2.5.2 A New Fermentation-based Biocatalytic Process for 7-ADCA 735 12.2.5.3 Enzymatic Formation of Semi-synthetic Antibiotics: Synthetic Processes 735 12.2.6 Conclusions and Future Prospects 737 References 738 12.3 Hydrolysis of N-Acylamino Acids 741 Andreas S. Bommarius 12.3.1 Introduction 741 12.3.2 Acylase I (N-Acylamino Acid Amidohydrolase, E. C. 3.5.1.4.) 742 12.3.2.1 Genes, Sequences, Structures 743 12.3.2.2 Substrate Specificity 744 12.3.2.3 Stability of Acylases 746 12.3.2.4 Thermodynamics and Mechanism of the Acylase-catalyzed Reaction 748 12.3.3 Acylase II (AT-Acyl-L-Aspartate Amidohydrolase, Aspartoacylase, E.C. 3.5.1.15.) 749 Contents XIX

12.3.4 Proline Acylase (N-Acyl-L-Proline Amidohydrolase) 752 12.3.5 Dehydroamino Acid Acylases 753 12.3.6 D-Specific Aminoacylases 754 12.3.7 Acylase Process on a Large Scale 757 References 758 12.4 Hydrolysis and Formation of Hydantoins 761 Markus Pietzsch and Christoph Syldatk 12.4.1 Classification and Natural Occurrence of Hydantoin Cleaving and Related Enzymes 761 12.4.2 D-Hydantoinases - Substrate Specificity and Properties 773 12.4.3 DN-Carbamoylases - Substrate Specificity and Properties 777 12.4.4 L-Hydantoinases - Substrate Specificity and Properties 784 12.4.5 L-N-Carbamoylases - Substrate Specificity and Properties 786 12.4.6 Hydantoin Racemases 792 12.4.7 Conclusions 794 References 796 12.5 Hydrolysis and Formation of Peptides 800 Hans-Dieter Jakubke 12.5.1 Introduction 800 12.5.2 Hydrolysis of Peptides 801 12.5.2.1 Peptide-Cleaving Enzymes 801 12.5.2.2 Importance of Proteolysis 813 12.5.3 Formation of Peptides 818 12.5.3.1 Tools for Peptide Synthesis 818 12.5.3.2 Choice of the Ideal Enzyme 822 12.5.3.3 Principles of Enzymatic Synthesis 823 12.5.3.4 Manipulations to Suppress Competitive Reactions 831 12.5.3.5 Approaches to Irreversible Formation of Peptide Bond 840 12.5.3.6 Irreversible C-N Ligations by Mimicking Enzyme Specificity 842 12.5.3.7 Planning and Process Development of Enzymatic Peptide Synthesis 851 12.5.4 Conclusion and Outlook 858 References 859 12.6 Addition of Amines to C=C Bonds 866 Marcel Wubbolts ' 12.6.1 Addition of Ammonia to Produce Amino Acids 866 12.6.1.1 AsparticAcid 866 12.6.1.2 Aspartic Acid Derivatives 868 12.6.1.3 Histidine Ammonia Lyase 869 12.6.1.4 Phenylalanine, Tyrosin and L-DOPA 870 12.6.1.5 Serine and Threonine Deaminases 871 12.6.1.6 Ornithine Cyclodeaminase 871 12.6.2 Ammonia Lyases that Act on Other Amines 871 12.6.2.1 Elimination of Ammonia from Ethanolamine 871 References 872 XX Contents

12.7 Transaminarions 873 J. David Rozzell and Andreas S. Bommarius 12.7.1 Introduction 873 12.7.2 Description of Transaminases 875 12.7.2.1 Homology and Evolutionary Subgroups of Aminotransferases 875 12.7.2.2 Mechanism of Transamination 875 12.7.2.3 Protein Engineering and Directed Evolution with Aminotransferases 876 12.7.3 Use of Aminotransferases in Biocatalytic Reactions 878 12.7.3.1 Synthesis of a-L-Amino Acids 878 12.7.3.2 Synthesis of Enantiomerically Pure Amines 880 12.7.3.3 Other Preparative Applications of Aminotransferases 881 12.7.4 Driving the Reaction to Completion 884 12.7.5 Production of L-Amino Acids Using Immobilized Transaminases 885 12.7.6 D-Amino Acid Transferases 889 12.7.7 Synthesis of Labeled Amino Acids 891 12.7.8 Availability of Enzyme 892 References 892

13 Formation and Cleavage of P-O Bonds 895 George M. Whitesides

13.1 Introduction 895 13.1.1 Enzymes Forming or Cleaving Phosphorous-Oxygen Bonds 896 13.1.2 Biological Phosphorylating Agents 899 13.2 Phosphorylation 901 13.2.1 Regeneration of Nucleoside Triphosphates 901 13.2.1.1 Regeneration of ATP from ADP and AMP 902 13.2.1.2 Regeneration of other Nucleoside Triphosphates 906 13.2.2 Applications 907 13.2.2.1 Phosphorylations with ATP as a Cofactor 907 13.2.2.2 P-0 Bond Formation with other Nucleoside Triphosphates than ATP 909 13.2.2.3 Other Phosphorylating Agents 910 13.2.3 Tables Containing Typical Examples Ordered According to the Classes of Compounds 918 13.3 Cleavage of P-0 bonds 918 13.3.1 Hydrolysis of Phosphate and Pyrophosphate Monoesters 919 13.3.2 Hydrolysis of S- and N-substituted Phosphate Monoester Analogs 920 13.3.3 Hydrolysis of Phosphate and Phosphonate Diesters 922 13.3.3.1 Nucleic Acids and their Analogs 922 13.3.3.2 Other Phosphate and Phosphonate Diesters 922 13.3.4 Other P-0 Bond Cleavages 923 References 926 Contents XXI

14 Formation of C-C Bonds 931 Chi-Huey Wong 14.1 Aldol Reactions 931 14.1.1 DHAP-Utilizing Aldolases 931 14.1.1.1 Fructose 1,6-Diphosphate (FDP) Aldolase (E. C. 4.1.2.13) 931 14.1.1.2 Fuculose 1-Phosphate (Fuc 1-P) Aldolase (E.C. 4.1.2.17), Rhamnulose 1-Phosphate (Rha 1-P) Aldolase (E.C. 4.1.2.19) and Ragatose 1,6-Diphosphate (TDP) Aldolase 939 14.1.1.3 Synthesis of Dihydroxyacetone Phosphate (DHAP) 943 14.1.2 Pyruvate/Phosphoenolpyruvate-Utilizing Aldolases 944 14.1.2.1 N-Acetylneuraminate (NeuAc) Aldolase (E. C. 4.1.3.3) and NeuAc Synthetase (E. C. 4.1.3.19) 944 14.1.2.2 3-Deoxy-D-monno-2-octulosonate Aldolase (E. C. 4.1.2.23) and 3-Deoxy- D-manno-2-octulosonate 8-Phosphate Synthetase (E. C. 4.1.2.16) 946 14.1.2.3 3-Deoxy-D-arabino-2-heptulosonic Acid 7-Phosphate (DAHP) Synthetase (E. C. 4.1.2.15) 947 14.1.2.4 2-Keto-4-hydroxyglutarate (KHG) Aldolase (E. C. 4.1.2.31) 948 14.1.2.5 2-Keto-3-deoxy-6-phosphogluconate (KDPG) Aldolase (E. C. 4.1.2.14) 949 14.1.2.6 2-Keto-3-deoxy-D-glucarate (KDG) Aldolase (E. C. 4.1.2.20) 950 14.1.3 2-Deoxyribose 5-phosphate Aldolase (DERA) (E. C. 4.1.2.4) 950 14.2 Ketol and Aldol Transfer Reactions 960 14.2.1 Transketolase (TK) (E.C. 2.2.1.1) 960 14.2.2 Transaldolase (TA) (E.C. 2.2.1.2) 962 14.3 Acyloin Condensation 962 14.4 C-C Bond Forming Reactions Involving AcetylCoA 963 14.5 Isoprenoid and Steroid Synthesis 965 14.6 P-Replacement of Chloroalanine 966 References 966 14.7 Enzymatic Synthesis of Cyanohydrins 974 Martin H. Fechter and Herfried Cringl 14.7.1 The Oxynitrilases Commonly Used for Preparative Application 975 14.7.2 Oxynitrilase Catalysed Addition of HCN to Aldehydes 976 14.7.3 HNL-Catalyzed Addition of Hydrogen Cyanide to Ketones 978 14.7.4 Transhydrocyanation 978 14.7.5 Experimental Techniques for HNL-Catalysed Biotransformations 981 14.7.6 Resolution of Racemates 982 14.7.7 Follow-up Chemistry of Enantiopure Cyanohydrins 985 14.7.8 Safe Handling of Cyanides 985 14.7.9 Conclusions and Outlook 986 References 986 XXII Contents

Volume III

15 Reduction Reactions 991

15.1 Reduction of Ketones 991 Kaoru Nakamura and Tomoko Matsuda 15.1.1 Introduction 991 15.1.1.1 Enzyme Classfication and Reaction Mechanism 991 15.1.1.2 Coenzyme Regeneration 992 15.1.1.3 Form of the Biocatalysts: Isolated Enzyme vs. Whole Cell 995 15.1.1.4 Origin of Enzymes 996 15.1.2 Stereochemical Control 997 15.1.2.1 Enantioselectivity of Reduction Reactions 997 15.1.2.2 Modification of the Substrate: Use of an "Enantiocontrolling" Group 998 15.1.2.3 Screening of Microorganisms 1000 15.1.2.4 Treatment of the Cell: Heat Treatment 1001 15.1.2.5 Treatment of the Cell: Aging 1001 15.1.2.6 Treatment of the Cell: High Pressure Homogenization 1002 15.1.2.7 Treatment of the Cell: Acetone Dehydration 1002 15.1.2.8 Cultivation Conditions of the Cell 1003 15.1.2.9 Modification of Reaction Conditions: Incorporation of an Inhibitor 1004 15.1.2.10 Modification of Reaction Conditions: Organic-Solvent 1005 15.1.2.11 Modification of Reaction Conditions: Use of a Supercritical Solvent 1006 15.1.2.12 Modification of Reaction Conditions: Cyclodextrin 1007 15.1.2.13 Modification of Reaction Conditions: Hydrophobic Polymer XAD 1007 15.1.2.14 Modification of Reaction Conditions: Reaction Temperature 1008 15.1.2.15 Modification of Reaction Conditions: Reaction Pressure 1009 15.1.3 Improvement of Dehydrogenases for use in Reduction Reactions by Genetic Methods 1010 15.1.3.1 Overexpression of the Alcohol Dehydrogenase 1010 15.1.3.2 Access to a Single Enzyme Within a Whole Cell: Use of Recombinant Cells 1011 15.1.3.3. Use of a Cell Deficient in an Undesired Enzyme 1012 15.1.3.4 Point Mutation for the Improvement of Enantioselectivity 1012 15.1.3.5 Broadening the Substrate Specificity of Dehydrogenase by Mutations 1012 15.1.3.6 Production of an Activated Form of an Enzyme by Directed Evolution 1014 15.1.3.7 Change in the Coenzyme Specificity by Genetic Methods: NADP(H) Specific Formate 1014 15.1.3.8 Use of a Mutant Dehydrogenase for the Synthesis of 4-Amino-2-Hydroxy Acids 1014 15.1.3.9 Catalytic Antibody 1015 15.1.4 Reduction Systems with Wide Substrate Specificity 1016 Contents XXIII

15.1.4.1 Bakers'Yeast 1016 15.1.4.2 Rodococcus Erythropolis 1016 15.1.4.3 Pseudomonas sp. Strain PED and Lactobacillus Kefir 1017 15.1.4.4 Thermoanaerobium Brockii 1018 15.1.4.5 Geotrichum Candidum 1019 15.1.5 Reduction of Various Ketones 1021 15.1.5.1 Reduction of Fluoroketones 1021 15.1.5.2 Reduction of Fluoroketones Containing Sulfur Functionalities 1024 15.1.5.3 Reduction of Chloroketones 1025 15.1.5.4 Reduction of Ketones Containing Nitrogen, Oxygen, Phosphorus and Sulfur 1028 15.1.5.5 Reduction of Diketones 1028 15.1.5.6 Reduction of Diaryl Ketones 1029 15.1.5.7 Diastereoslective Reductions (Dynamic Resolution) 1030 15.1.5.8 Chemo-enzymatic Synthesis of Bioaktive Compounds 1031 15.2 Reduction of Various Functionalities 1033 15.2.1 Reduction of Aldehydes 1033 15.2.2 Reduction of Peroxides to Alcohols 1034 15.2.3 Reduction of Sulfoxides to Sulfides 1034 15.2.4 Reduction of Azide and Nitro Compounds to Amines 1035 15.2.5 Reduction of Carbon-Carbon Double Bonds 1036 15.2.6 Transformation of a-Keto Acid to Amine 1037 15.2.7 Reduction of Carbon Dioxide 1038

15.2.7.1 Reduction of CO2 to Methanol 1038

15.2.7.2 Reductive fixation of CO2 1039 References 1040 15.3 Reduction of C=N bonds 1047 Andreas S. Bommarius 15.3.1 Introduction 1047 15.3.2 Structural Features of Amino Acid Dehydrogenases (AADHs) 1049 15.3.2.1 Sequences and Structures 1050 15.3.3 Thermodynamics and Mechanism of Enzymatic Reductive Animation 1050 i 15.3.3.1 Thermodynamics 1050 15.3.3.2 Mechanism, Kinetics 1051 15.3.4 Individual Amino Acid Dehydrogenases 1052 15.3.4.1 Leucine Dehydrogenase (LeuDH, E.C. 1.4.1.9.) 1052 15.3.4.2 Alanine Dehydrogenase (AlaDH, E.C. 1.4.1.1.) 1053 15.3.4.3 Glutamate Dehydrogenase (GluDH, E.C. 1.4.1.2-4) 1054 15.3.4.4 Phenylalanine Dehydrogenase (PheDH, E.C. 1.4.1.20) 1054 15.3.5 Summary of Substrate Specificities 1056 15.3.6 Process Technology: Cofactor Regeneration and Enzyme Membrane Reactor (EMR) 1058 15.3.6.1 Regeneration of NAD(P)(H) Cofactors 1058 15.3.6.2 Summary of Processing to Amino Acids 1060 References 1061 XXIV Contents

16 Oxidation Reactions 1065

16.1 Oxygenationof C-H and C=C Bonds 1065 Sabine Flitsch 16.1.1 Introduction 1065 16.1.2 Hydroxylating Enzymes 1066 16,1.2 Hydroxylating Enzymes 1068 16.1.4 Hydroxylationof Non-Activated Carbon Atoms 1069 16.1.4.1 Hydroxylationof Monoterpenes 1069 16.1.4.1 Hydroxylationof Monoterpenes 1075 16.1.4.3 Hydroxylationof Steroids 1078 16.1.4.4 Miscellaneous Compounds 1079 16.1.5 EpoxidationofOlefins 1084 16.1.5.1 Epoxidation of Straight-Chain Terminal Olefins 1084 16.1.5.2 Short-Chain Alkenes 1088 16.1.5.3 Terpenes 1090 16.1.5.4 Cyclic Sesquiterpenes 1096 16.1.6 Conclusions, Current and Future Trends 1097 16.1.7 Cis Hydroxylation of Aromatic Double Bonds 1099 16.1.7.1 Introduction 1099 16.1.7.2 Preparation of cis Dihydrodiols 1100 References 1103 16.2 Oxidation of Alcohols 1108 Andreas Schmid, Frank Hollmann, and Bruno Buhler 16.2.1 Introduction 1108 16.2.2 Dehydrogenases as Catalysts 1108 16.2.2.1 Regeneration of Oxidized Nicotinamide Coenzymes 1108 16.2.2.2 Dehydrogenases as Regeneration Enzymes 1109 16.2.2.3 Molecular Oxygen as Terminal Acceptor 1111 16.2.2.4 Electrochemical Regeneration 1112 16.2.2.5 Photochemical Regeneration 1114 16.2.2.6 Oxidations Catalyzed by Alcohol Dehydrogenase from Horse Liver (HLADH) 1115 16.2.2.7 Alcohol Dehydrogenase from Yeast (YADH) 1120 16.2.2.8 Alcohol Dehydrogenase from Thermoanaerobium brokii (TBADH) 1120 16.2.2.9 Glycerol Dehydrogenase (GDH, E.C. 1.1.1.6) 1122 16.2.2.10 Glycerol-3-phosphate Dehydrogenase (GPDH, E. C. 1.1.1.8) 1124 16.2.2.11 Lactate Dehydrogenase (LDH, E. C. 1.1.1.27) 1125 16.2.2.12 Carbohydrate Dehydrogenases 1126 16.2.2.13 Hydroxysteroid Dehydrogenases (HSDH) 1127 16.2.2.14 Other Dehydrogenases 1127 16.2.3 Oxidases as Catalysts 1129 16.2.3.1 General Remarks 1129 16.2.3.2 Methods to Diminish/Avoid H2O2 1129 Contents XXV

16.2.3.3 Pyranose Oxidase (P2O, E.C. 1.1.3.10) 1132 16.2.3.4 Glycolate Oxidase (E.C. 1.1.3.15) 1135 16.2.3.5 Nucleoside Oxidase (E.C. 1.1.3.28) 1138 16.2.3.6 Glucose Oxidase (E.C. 1.1.3.4) 1138 16.2.3.7 Alcohol oxidase (E.C. 1.1.3.13) 1139 16.2.3.8 Galactose Oxidase (E.C. 1.1.3.9) 1141 16.2.3.9 Cholesterol Oxidase (ChOX, E. C. 1.1.3.6) 1142 16.2.4 Peroxidases as Catalysts 1142 16.2.4.1 Introduction 1142

16.2.4.2 Methods to Generate H2O2 1143 16.2.4.3 Chloroperoxidase (CPO, E.C. 1.11.1.10) 1145 16.2.4.4 Catalase (E.C. 1.11.1.6) 1145 16.2.5 Quinoprotein Dehydrogenases (QDH) 1146 16.2.5.1 General Remarks 1146 16.2.5.2 Methanol Dehydrogenase (E.C. 1.1.99.8) 1147 16.2.5.3 Glucose Dehydrogenase (E.C. 1.1.99.17) 1148 16.2.6 Whole-Cell Oxidations 1148 16.2.6.1 Stereoselective Oxidation of (-)-Carveol to (-)-Carvone 1148 16.2.6.2 Sugar Dehydrogenases Applied in Whole Cells 1149 16.2.6.3 Oxidation of Aromatic and Aliphatic Alcohols to Corresponding Aldehydes and Acids 1150 16.2.6.4 Enantiospecific Reactions 1154 16.2.6.5 Stereoinversions using Microbial Redox Reactions 1157 16.2.7 Miscellaneous 1162 16.2.7.1 Biofuel Cells 1162 16.2.7.2 Biomimetic Analogs to Nicotinamide Co-nzymes 1163 References 1164 16.3 Oxidation of Phenols 1170 Andreas Schmid, Frank Hollmann, and Bruno Buhler 16.3.1 Introduction 1170 16.3.2 Oxidases 1170 16.3.2.1 Vanillyl oxidase (E.C. 1.1.3.38) 1170 16.3.2.2 Laccase (E.C. 1.10.3.2) 1174 16.3.3 Monooxygenases 1176 16.3.3.1 Tyrosinase (E.C. 1.10.3.1) 1176 16.3.3.2 2-Hydroxybiphenyl-3-monooxygenase (HbpA, E.C. 1.14.13.44) 1179 16.3.4 Peroxidases 1185 16.3.4.1 Oxidative Coupling Reactions 1185 16.3.4.2 Hydroxylationof Phenols 1186 16.3.4.3 Nitration of Phenols 1187 16.3.5 Other Oxidoreductases 1188 16.3.5.1 4-Cresol-oxidoreductase (PCMH, E.C. 1.17.99.1) 1188 16.3.5.2 4-Ethylphenol Oxidoreductas 1189 16.3.6 In vivo Oxidations 1190 16.3.6.1 Phenoloxidase of Mucuna pruriens 1190 XXVI Contents

16.3.6.2 Monohydroxylation of (i?)-2-Phenoxypropionic Acid and Similar Substrates 1191 16.3.6.3 Biotransformation of Eugenol to Vanillin 1191 References 1192 16.4 Oxidation of Aldehydes 1194 Andreas Schmid, Frank Hollmann, and Bruno Buhler 16.4.1 Introduction 1194 16.4.2 Alcohol Dehydrogenases 1194 16.4.3 Aldehyde Dehydrogenases 1196 16.4.4 Monooxygenases 1198 16.4.4.1 Luciferase (E.C. 1.14.14.3) 1198

16.4.4.2 P450BM-3 1199 16.4.5 Oxidases 1201 16.4.5.1 Xanthine Oxidase (E.C. 1.1.3.22) 1201 16.4.6 Oxidations with Intact Microbial Cells 1201 References 1201 16.5 Baeyer-Villiger Oxidations 1202 Sabine Flitsch and Gideon Grogan 16.5.1 Introduction 1202 16.5.1.1 Steroidal Substrates 1202 16.5.1.2 Aliphatic Substrates 1205 16.5.1.3 Alicydic Substrates 1207 16.5.1.4 Polycyclic Molecules 1212 16.5.2 Baeyer-Villiger Monooxygenases 1213 16.5.2.1 TypelBVMOs 1214 16.5.2.2 Type2BVMOs 1216 16.5.2.3 Mechanism of the Enzymatic Baeyer-Villiger Reaction 1216 16.5.3 Synthetic Applications 1222 16.5.4 Models for the Action of Baeyer-Villiger Monooxygenases 1234 16.5.5 Conclusion and Outlook 1238 References 1241 16.6 Oxidation of Acids 1245 Andreas Schmid, Frank Hollmann, and Bruno Buhler 16.6.1 Introduction 1245 16.6.2 Pyruvate Oxidase (PYOx, E.C. 1.2.3.3) 1246 16.6.3 Formate Dehydrogenase (FDH, E.C. 1.2.1.2) 1247 16.6.4 Oxidations with Intact Microbial Cells 1247 16.6.4.1 Production of Benzaldehyde from Benzoyl Formate or Mandelic Acid 1247 16.6.4.2 Microbial Production of cis,cis-Muconic Arid from Benzoic Acid 1248 16.6.4.3 Biotransformation of Substituted Benzoates to the Corresponding cis-Diols 1249 References 1249 Contents XXVII

16.7 Oxidation of C-N Bonds 1250 Andreas Schmid, Frank Hollmann, and Bruno Buhler 16.7.1 Introduction 1250 16.7.2 Oxidations Catalyzed by Dehydrogenases 1251 16.7.2.1 L-Alanine Dehydrogenase (L-Ala-DH, E.C. 1.4.1.1) 1251 16.7.2.2 Nicotinic Acid Dehydrogenase (Hydroxylase) (E. C. 1.5.1.13) 1252 16.7.3 Oxidations Catalyzed by Oxidases 1254 16.7.3.1 Amino Acid Oxidases 1254 16.7.3.2 Amine Oxidases 1256 16.7.4 Oxidations Catalyzed by Transaminases 1260 References 1261 16.8 Oxidation at Sulfur 1262 Karl-Heinz van Pee 16.8.1 Enzymes Oxidizing at Sulfur and their Sources 1262 16.8.2 Oxidation of Sulfides 1263 16.8.2.1 Oxidation of Sulfides by Monooxygenases and by Whole Organsims 1263 16.8.2.2 Oxidation of Sulfides by Peroxidases and Haloperoxidases 1264 References 1266 16.9 Halogenation 1267 Karl-Heinz van Pee 16.9.1 Classification of Halogenating Enzymes and their Reaction Mechanisms 1267 16.9.1.1 Haloperoxidases and Perhydrolases 1267 16.9.1.2 FADH2-dependent Halogenases 1268 16.9.2 Sources and Production of Enzymes 1268 16.9.2.1 FADH2-dependent Halogenases 1268 16.9.2.2 Haloperoxidases and Perhydrolases 1269 16.9.3 Substrates for Halogenating Enzymes and Reaction Products 1271 16.9.3.1 Halogenation of Aromatic Compounds 1271 16.9.3.2 Halogenation of Aliphatic Compounds 1273 16.9.4 Regioselectivity and Stereospecificity of Enzymatic Halogenation Reactions 1275 16.9.4.1 FADH2-dependent Halogenases 1275 16.9.5 Comparison of Chemical with Enzymatic Halogenation 1277 References 1278

17 Isomerizations 1281 Nobuyoshi Esaki, Tatsuo Kurihara, and Kenji Soda

17.1 Introduction 1281 17.2 Racemizations and Epimerizations 1282 17.2.1 Pyridoxal 5'-phosphate-dependent Amino Acid Racemases and Epimerases 1283 17.2.1.1 Alanine Racemase (E.C. 5.1.1.1) 1283 XXVIII Contents

17.2.1.2 Amino Acid Racemase with Low Substrate Specificity (E.C. 5.1.1.10) 1289 17.2.1.3 a-Amino-e-caprolactam Racemase 1292 17.2.2 Cofactor-independent Racemases and Epimerases Acting on Amino Acids 1293 17.2.2.1 Glutamate Racemase (E.C. 5.1.1.3) 1293 17.2.2.2 Aspartate Racemase (E.C. 5.1.1.13) 1297 17.2.2.3 Diaminopimelate Epimerase (E.C. 5.1.1.7) 1299 17.2.2.4 Proline Racemase (E.C. 5.1.1.4) 1301 17.2.3 Other Racemases and Epimerases Acting on Amino Acid Derivatives 1301 17.2.3.1 2-Amino-A2-thiazoline-4-carboxylate Racemase 1301 17.2.3.2 Hydantoin Racemase 1303 17.2.3.3 N-Acylamino Acid Racemase 1306 17.2.3.4 Isopenicillin N Epimerase 1308 17.2.4 Racemization and Epimerization at Hydroxyl Carbons 1310 17.2.4.1 Mandelate Racemase (E.C. 5.1.2.2) 1310 17.3 Isomerizations 1312 17.3.1 D-Xylose (Glucose) Isomerase (E.C. 5.3.1.5) 1313 17.3.1.1 Properties 1313 17.3.1.2 Reaction Mechanism 1314 17.3.1.3 Production of Fructose 1316 17.3.1.4 Production of Unusual Sugar Derivatives 1316 17.3.2 Phosphoglucose Isomerase (E.C. 5.3.1.9) 1318 17.3.3 Triosephosphate Isomerase (E.C. 5.3.1.1) 1320 17.3.4 L-Rhamnose Isomerase (E.C. 5.3.1.14) 1321 17.3.5 L-Fucose Isomerase (E.C. 5.3.1.3) 1323 17.3.6 N-Acetyl-D-glucosamine 2-Epimerase 1324 17.3.7 Maleate cis-trans Isomerase (E. C. 5.2.1.1) 1324 17.3.8 Unsaturated Fatty Acid cis-trans Isomerase 1325 17.4 Conclusion 1326 References 1326

18 Introduction and Removal of Protecting Croups 1333 Dieter Kadereit, Reinhard Reents, Duraiswamy A. Feyaraj, and Herbert Waldmann

18.1 Introduction 1333 18.2 Protection of Amino Groups 1334 18.2.1 N-Terminal Protection of Peptides 1334 18.2.2 Enzyme-labile Urethane Protecting Groups 1338 18.2.3 Protection of the Side Chain Amino Group of Lysine 1341 18.2.4 Protection of Amino Groups in P-Lactam Chemistry 1341 18.2.5 Protection of Amino Groups of Nucleobases 1343 18.3 Protection of Thiol Groups 1343 Contents IXXIX

18.3.1 Protection of the Side Chain Thiol Group of Cysteine 1343 18.4 Protection of Carboxy Groups 1344 18.4.1 C-Terminal Protection of Peptides 1344 18.4.2 Protection of the Side Chain Groups of Glutamic and Aspartic Acid 1352 18.5 Protection of Hydroxy Groups 1353 18.5.1 Protection of Monosaccharides 1354 18.5.2 Deprotection of Monosaccharides 1369 18.5.3 Di- and Oligosaccharides 1378 18.5.4 Nudeosides 1380 18.5.5 Further Aglycon Glycosides 1383 18.5.6 Polyhydroxylated Alkaloids 1386 18.5.7 Steroids 1388 18.5.8 Phenolic Hydroxy Groups 1390 18.6 Biocatalysis in Polymer Supported Synthesis: Enzyme-labile Linker Groups 1392 18.6.1 Endo-linkers 1393 18.6.2 Exo-linkers 1402 18.7 Outlook 1408 References 1409

19 Replacing Chemical Steps by Biotransformations: Industrial Application and Processes Using Biocatalysis 1419 Andreas Liese

19.1 Introduction 1419 19.2 Types and Handling of Biocatalysts 1420 19.3 Examples 1421 19.3.1 Reduction Reactions Catalyzed by Oxidoreductases (E. C. 1) 1422 19.3.1.1 Ketone Reduction Using Whole Cells of Neurospora crassa (E.C. l.l.l.l)p 1422 19.3.1.2 Ketoester Reduction Using Cell Extract of Acinetobacter calcoaceticus (E.C. 1.1.1.1) 1423 19.3.1.3 Enantioselective Reduction with Whole Cells of Candida sorbophila (E.C. 1.1.X.X) 1424 19.3.2 Oxidation Reactions Catalyzed by Oxidoreductases (E. C. 1) 1425 19.3.2.1 Alcohol Oxidation Using Whole Cells of Gluconobacter suboxydans (E.C. 1.1.99.21) 1425 19.3.2.2 Oxidative Deamination Catalyzed by Immobilized D-Amino Acid Oxidase from Trigonopsis variabilis (E.C. 1.4.3.3) 1426 19.3.2.3 Kinetic Resolution by Oxidation of Primary Alcohols Catalyzed by Whole Cells from Rhodococcus erythropolis (E. C. l.X.X.X) 1427 19.3.2.4 Hydroxylation of Nicotinic Acid (Niacin) Catalyzed by Whole Cells of Achromobacter xylosoxidans (E.C. 1.5.1.13) 1428 XXX Contents

19.3.2.5 Reduction of Hydrogen Peroxide Concentration by Catalase (E.C. 1.11.1.6) 1428 19.3.3 Hydrolytic Cleavage and Formation of C-O Bonds by Hydrolases (E.C. 3) 1430 19.3.3.1 Kinetic Resolution of Glycidic Acid Methyl Ester by Lipase from Serratia marcescens (E. C. 3.1.1.3) 1430 19.3.3.2 Kinetic Resolution of Diester by Protease Subtilisin Carlsberg from Bacillus sp. (E. C. 3.4.21.62) 1431 19.3.3.3 Kinetic Resolution of Pantolactones and Derivatives thereof by a Lactonase from Fusarium oxysporum (E. C. 3.1.1.25) 1433 19.3.3.4 Hydrolysis of Starch to Glucose by Action of Two Wnzymes: a-Amylase (E. C. 3.2.1.1) and Amyloglucosidase (E. C. 3.2.1.3) 1433 19.3.4 Formation or Hydrolytic Cleavage of C-N Bonds by Hydrolases (E.C. 3) 1435 19.3.4.1 Enantioselective Acylation of Racemic Amines Catalyzed by Lipase from Burkholderiaplantarii (E.C. 3.1.1.3) 1435 19.3.4.2 7-Aminocephalosporanic Acid Formation by Amide Hydrolysis Catalyzed by Glutaryl Amidase (E. C. 3.1.1.41) 1436 19.3.4.3 Penicillin G Hydrolysis by Penicillin Amidase from Escherichia coli (E.C. 3.5.1.11) 1438 19.3.4.4 Kinetic Resolution of a-Amino Acid Amides Catalyzed by Aminopeptidase from Pseudomonas putida (E. C. 3.4.1.11) 1439 19.3.4.5 Production of L-Methionine by Kinetic Resolution with Aminoacylase of Aspergillus oryzae (E. C. 3.5.1.14) 1441 19.3.4.6 Production of D-p-Hydroxyphenyl Glydne by Dynamic Resolution with Hydantoinase from Bacillus brevis (E.C. 3.5.2.2) 1441 19.3.4.7 Dynamic Resolution of a-Amino-e-caprolactam by the Action of Lactamase (E. C. 3.5.2.11) and Racemase (E. C. 5.1.1.15) 1442 19.3.4.8 Synthesis of P-Lactam Antibiotics Catalyzed by Penicillin Acylase (E.C. 3.5.1.11) 1444 19.3.4.9 Synthesis of Azetidinone p-Lactam Derivatives Catalyzed by Penicillin Acylase (E.C. 3.5.1.11) 1444 19.3.4.10 Enantioselective Synthesis of an Aspartame Precursor with Thermolysin from Bacillusproteolicus (E.C. 3.4.24.27) 1446 19.3.4.11 Hydrolysis of Heterocydic Nitrile by Nitrilase from Agrobacterium sp. (E.C. 3.5.5.1) 1447 19.3.5 Formation of C-0 Bonds by Lyases 1447 19.3.5.1 Synthesis of Carnitine Catalyzed by Carnitine Dehydratase in Whole Cells (E.C. 4.2.1.89) 1447 19.3.6 Formation of C-N Bonds by Lyases (E. C. 4) 1448 19.3.6.1 Synthesis of L-Dopa Catalyzed by Tyrosine Phenol Lyase from Erwinia herbicola (E. C. 4.1.99.2) 1448 19.3.6.2 Synthesis of 5-Cyano Valeramide by Nitrile Hydratase from Pseudomonas chlororaphis B23 (E. C. 4.2.1.84) 1449 Contents XXXI

19.3.6.3 Synthesis of the Commodity Chemical Acrylamide Catalyzed by Nitrile Hydratase from Rhodococcus rodochrous (E. C. 4.2.1.84) 1450 19.3.6.4 Synthesis of Nicotinamide Catalyzed by Nitrile Hydratase from Rhodococcus rodochrous (E. C. 4.2.1.84) 1451 19.3.7 Epimerase 1452 19.3.7.1 Epimerization of Glucosamine Catalyzed by Epimerase from E. coli (E.C. 5.1.3.8) 1452 19.4 Some Misconceptions about Industrial Biotransformations 1453 19.5 Outlook 1454 References 1454

20 Tabular Survey of Commercially Available Enzymes 1461 Peter Rasor

Index 1519