Enzyme Catalysis in Organic Synthesis A Comprehensive Handbook

Edited by K. Drauz and H. Waldmann

VCH Weinheim • New York • Basel • Cambridge • Tokyo

Contents

Section A

A.l Introduction 1

A. 1.1 Enzymes as catalysts 1 A. 1.2 Enzyme structure and function 4 A. 1.3 Cofactors and coenzymes 13 A.1.4 Enzyme kinetics 22 A.l.4.1 Reaction rate and Substrate concentration 22 A. 1.4.2 Inhibitors and effectors 25 A. 1.4.3 Influence of pH and buffers 26 A.l.4.4 Temperature 27 A.l.5 Organic solvents as reaction medium 29 A.l.6 Enzyme handling, quality requirements 30 A. 1.7 Enzyme nomenclature 30 A.l.8 Suggested literature for further information 32

A.2 Production and Isolation of Enzymes 35

A.2.1 Introduction 35 A.2.1.1 Sources of enzymes: enzymes from plants and animals 35 A.2.1.2 Microbial enzymes 36 A.2.1.2.1 Strain improvement 36 A.2.1.2.2 Recombinant microorganisms 37 A.2.1.2.3 Engineering proteins for facilitated purification 38 A.2.2 Analytical methods 40 A.2.2.1 Criteria of purity 40 A.2.2.2 Enzyme activity measurement 40 A.2.2.3 Protein determination 41 A.2.2.4 Protein chemical analysis 43 A.2.3 Lab-scale and large-scale enzyme purification 45 A.2.3.1 Introduction 45 A.2.3.1.1 Survey of purification methods 45 A.2.3.1.2 Survey of equipment; from lab scale to large scale 46 A.2.3.2 Harvesting microorganisms 46 A.2.3.3 Cell disintegration and preparation of raw extract 47 A.2.3.3.1 Plant material 47 A.2.3.3.2 Animal tissues 47 A.2.3.3.3 Microorganisms 47 A.2.3.3.4 Liquid phase partitioning 48 A.2.3.4 Concentration and desalting 48 A.2.3.4.1 Concentration by Ultrafiltration 49 A.2.3.4.2 Concentration by precipitation 49 A.2.3.4.3 Desalting of concentrates 49

X Contents

A.2.3.5 Bulk procedures 50 A.2.3.5.1 Precipitation methods 50 A.2.3.5.2 Adsorptive batch methods 51 A.2.3.5.3 Liquid phase partitioning 51 A.2.3.6 Chromatography 52 A.2.3.6.1 Gel filtration 52 A.2.3.6.2 Ion exchange chromatography (IEC) 53 A.2.3.6.3 Hydrophobie chromatography (HIC) 56 A.2.3.6.4 Affinity chromatography 57 A.2.3.6.5 Immobilized metal affinity chromatography (IMAC) 58 A.2.3.6.6 Dye-ligand chromatography 59 A.2.3.7 Electrokinetic methods 60 A.2.3.8 Crystallization 60 A.2.3.9 Protein naturation 61 A.2.4 Enzyme stabilization 62 A.2.4.1 Enzyme stabilization during purification 62 A.2.4.2 Stable storage forms for enzymes 63 A.2.4.2.1 Liquid storage forms 63 A.2.4.2.2 Solid storage forms 63 A.2.4.2.3 Storage conditions 64 A.2.4.3 Chemical modification 64 A.2.4.4 Stabilization by genetic modification 66 A.2.5 Strategie considerations for the design of a purification scheme 66 A.2.6 References 67

A.3 Immobilization of Enzymes 73

A.3.1 Introduction 73 A.3.2 Reasons for using immobilized enzymes 74 A.3.3 Immobilization techniques 75 A.3.3.1 Methods 75 A.3.3.2 Carrier materials and reactive groups 76 A.3.4 Properties of immobilized enzymes 79 A.3.4.1 Binding and mass transfer effects 79 A.3.4.2 Influence of the microenvironment on enzyme activity 80 A.3.4.3 Measurement of enzyme activity 83 A.3.4.4 Stability 85 A.3.5 Applications of immobilized enzymes 85 A.3.5.1 Enzyme reactors 85 A.3.5.2 Examples of technical industrial applications 86 A.3.6 References 86

A.4 Reaction Engineering for Enzyme-Catalyzed Biotransformations 89

A.4.1 Introduction 89 A.4.2 Steps of process optimization 89 A.4.3 Investigation of the reaction System 94 A.4.3.1 Properties of the enzyme 94 A.4.3.2 Properties of the reaction System 96 A.4.3.2.1 Thermodynamic equilibrium of the reaction 96

Contents XI

A.4.3.2.2 Complex reaction Systems: The existence of parallel- and consecutive reactions 98

A.4.3.2.3 Other properties of the reaction System 106 A.4.3.2.4 Application of organic solvents 107 A.4.4 Investigation of enzyme kinetics 110 A.4.4.1 Methods of parameter identification 111 A.4.4.2 The kinetics of one-enzyme Systems 113 A.4.4.2.1 Michaelis-Menten kinetics 113 A.4.4.2.2 Competitive inhibition 119 A.4.4.2.3 Non-competitive inhibition 120 A.4.4.2.4 Uncompetitive inhibition 121 A.4.4.2.5 Reversibility of one-substrate reactions 122 A.4.4.2.6 Two-substrate reactions 123 A.4.4.2.7 Kinetics of aminoacylase as an example of a random uni-bi mechanism 125 A.4.4.3 The kinetics of multiple enzyme Systems 129 A.4.5 Enzyme reactors 132 A.4.5.1 Basic reaction engineering aspects 132 A.4.5.2 Reactors for soluble enzymes 138 A.4.5.2.1 Reactor optimization exemplified by the enzyme membrane reactor . . . 140 A.4.5.2.2 Control of conversion of a continuously operated EMR 148 A.4.5.2.3 Scale-up of the EMR 149 A.4.5.3 Reactor Systems for immobilized enzymes 149 A.4.5.4 Reaction techniques for enzymes in organic solvent 150 A.4.6 Outlook 152 A.4.7 References 152

A.5 Microbial Transformations Using Growing or Resting Cells 157

A.5.1 The advantages of using cells instead of isolated enzymes 157 A.5.2 Obtaining cultures and growth conditions 158 A.5.3 Use of resting or growing cells 159 A.5.4 References 160

Section B

B.l Hydrolysis and Formation of C - O-Bonds 165

B.l.l Hydrolysis and formation of carboxylic acid esters 178 B.l. 1.1 Hydrolysis of carboxylic acid esters 178 B. 1.1.1.1 Pig liver esterase 178 B.l.1.1.2 a-Chymotrypsin 196 B. 1.1.1.3 Acetylcholine acetylhydrolases 200 B.l.1.1.4 Lipases 201 B.l. 1.2 Formation of carboxylic esters 226 B.l.1.2.1 Lipases 226 B.l. 1.3 Inter- and intramolecular alcoholysis 250 B.l. 1.4 Technical applications 261 B.l.1.4.1 Introduction 261

XII Contents

B.1.1.4.2 Examples 265 B.l.1.4.3 Conclusions and future expectations 270 B. 1.2 Hydrolysis of epoxides 271 B.l.2.1 Epoxide hydrolases from liver 271 B.l.2.1.1 Hydrolysis of epoxides to give 1,2-diols 272 B.l.2.1.2 Opening of epoxides with amines using liver microsomes or lipase . . . . 276 B. 1.2.2 Microbial epoxide hydrolases 277 B.l.3 Hydrolysis and formation of glycosidic bonds 279 B.l.3.1 Introduction 279 B.l.3.2 Glycosyltransferases of the Leloir pathway 281 B.l.3.2.1 Synthesis of sugar nucleoside phosphates 284 B.l.3.2.2 Substrate specificity and synthetic applications of glycosyltransferases . 289 B.l.3.2.3 In Situ cofactor regeneration 297 B.l.3.2.4 Cloning and expression of glycosyltransferases 298 B. 1.3.3 Non-Leloir glycosyltransferases: transfer of glycosyl donors from glycosyl

phosphates and glycosides 301 B.l.3.4 Glycosidases 303 B.l.3.4.1 Equilibrium controlled synthesis 304 B.l.3.4.2 Kinetically controlled synthesis 305 B.l.3.4.3 Selectivity 305 B.l.3.5 Synthesis of N-glycosides 306 B.l.3.5.1 Nucleoside Phosphorylase 306 B.l.3.5.2 NAD hydrolase 314 B.l.3.6 Biological applications of synthetic glycoconjugates 315 B.l.3.6.1 Glycosidase and glycosyl transferase inhibitors 315 B.l.3.6.2 Glycoprotein remodeling 315 B.l.3.7 Future opportunities 315 B.l.3.8 Natural Polysaccharide degrading enzymes 316 B.l.3.8.1 Introduction 316 B.l.3.8.2 Starch 316 B.l.3.8.3 Cellulose 323 B.l.3.8.4 Xylan 327 B.l.3.8.5 Pectin 332 B.1.4 Addition of water to C = C-bonds 340 B.l.4.1 Formation of malic acid 340 B.1.4.1.1 L-Malic acid 340 B.l.4.1.2 D-Malic acid 340 B.l.4.2 Formation of 3-substituted malic acids 341 B.l.4.3 Formation of L-( — )-carnitine 341 B.l.4.4 Miscellaneous 342 B.1.4.4.1 (R)-[10-2H] Hydroxystearic acid 342 B.l.4.4.2 2-Keto-4-hydroxycarboxylic acids 342 B.1.5 References 343

B.2 Hydrolysis and Formation of C - N-Bonds 365

B.2.1 Hydrolysis of nitriles 365 B.2.1.1 Introduction 365

Contents XIII

B.2.1.2 Types of nitrile hydrolyzing enzymes 365 B.2.1.2.1 Enzymatic hydrolysis of organic nitriles 365 B.2.1.2.2 Enzymatic hydrolysis of Cyanide 366 B.2.1.3 Examples of enzymatic nitrile hydrolysis 367 B.2.1.3.1 Asymmetrie hydrolysis of nitriles 367 B.2.1.3.2 Monohydrolysis of dinitriles 368 B.2.1.3.3 Substrate and produet inhibition of nitrile hydrolysis 370 B.2.1.3.4 Nitrile hydrolysis in solvents 372 B.2.1.3.5 Large scale production of acrylamide 373 B.2.1.4 Availability of nitrile hydrolyzing biocatalysts 375 B.2.1.5 Industrial future of nitrile hydrolyzing enzymes 375 B.2.2 Formation and hydrolysis of amides 376 B.2.2.1 Introduction 376 B.2.2.2 Enzymatic formation of amides 376 B.2.2.3 Enzymatic enantioselective hydrolysis of amides 377 B.2.2.3.1 Hydrolysis of carboxylic amides 377 B.2.2.3.2 Hydrolysis of amino aeid amides 379 B.2.2.3.3 Hydrolysis of cyclic amides 384 B.2.2.4 Selective cleavage of the C-terminal amide bond 386 B.2.2.5 Hydrolysis of antibiotics and formation of semisynthetic antibiotics by

amidases 386 B.2.2.5.1 Enzymatic production of 6-APA, 7-ADCA and 7-ACA using amidases 387 B.2.2.5.2 Enzymatic formation of semisynthetic antibiotics 389 B.2.2.6 Conclusions and future prospects 392 B.2.3 Hydrolysis of A?-acyl amino aeids 393 B.2.3.1 Introduction 393 B.2.3.2 Acylase I (A'-acylamino aeid amidohydrolase, E.C. 3.5.1.14) 394 B.2.3.3 Acylase II (W-acyl-L-aspartate amidohydrolase, aspartoaeylase

E.C. 3.5.1.15) 399 B.2.3.4 Proline acylase (N-acyl-L-proline amidohydrolase) 400 B.2.3.5 Dehydroamino aeid aeylases 403 B.2.3.6 D-specific aminoaeylases 403 B.2.3.7 Acylase process on a large scale 406 B.2.4 Hydrolysis and formation of hydantoins 409 B.2.4.1 Classification and natural occurrence of hydantoin cleaving and related

enzymes 409 B.2.4.2 D-Hydantoinases — Substrate speeificity and properties 417 B.2.4.3 D-A^-Carbamoylases — Substrate speeificity and properties 422 B.2.4.4 L-Hydantoinases — Substrate speeificity and properties 425 B.2.4.5 L-JV-Carbamoylases — Substrate speeificity and properties 426 B.2.4.6 Hydantoin racemases 429 B.2.4.7 Conclusions 431 B.2.5 Hydrolysis and formation of peptides 431 B.2.5.1 Introduction 431 B.2.5.2 Hydrolysis of peptides 432 B.2.5.2.1 Peptide-cleaving enzymes 432 B.2.5.2.2 Importance of proteolysis 439 B.2.5.3 Formation of peptides 441 B.2.5.3.1 Tools for peptide synthesis 441

XIV Contents

B.2.5.3.2 Choice of enzyme 442 B.2.5.3.3 Principles of enzymatic peptide synthesis 443 B.2.5.3.4 Planning and process developments 451 B.2.5.3.5 Evaluation of the enzymochemical approach 455 B.2.5.4 Conclusion and outlook 458 B.2.6 Chemistry of yS-lactams 458 B.2.6.1 Introduction 458 B.2.6.2 Biosynthesis of penicillins and Cephalosporins 459 B.2.6.3 Isopenicillin N synthase (IPNS) 460 B.2.6.3.1 Mechanism 460 B.2.6.3.2 Modified Substrates for IPNS 461 B.2.6.4 Deacetoxycephalosporin synthase (DAOCS) and deacetylcephalosporin

synthase (DACS) 466 B.2.6.4.1 Mechanism and cofactors 466 B.2.6.4.2 Isopenicillin analogs (a-aminoadipic acid) 466 B.2.6.4.3 Transformation of Cephalosporin analogs B.2.6.5 Isopenicillin N epimerase 468 B.2.6.6 ACV synthetase 468 B.2.6.7 Clavaminic acid synthase 470 B.2.7 Transaminations and miscellaneous reactions 470 B. 2.7.1 Introduction 470 B.2.7.2 Mechanism of transamination 471 B.2.7.3 Transaminases from E. coli 472 B.2.7.4 Transaminases in the synthesis of amino acids 473 B.2.7.4.1 Equilibrium in transamination 473 B.2.7.4.2 Transamination coupled reactions 473 B.2.7.5 Tyrosine phenol-lyase (yS-tyrosinase) 477 B.2.7.6 L-Tryptophan indole-lyase (tryptophanase) 477 B.2.7.7 Chiral amines by transamination 478 B.2.8 Addition of amines to C = C-Bonds 480 B.2.8.1 Formation of aspartic acid 481 B.2.8.1.1 L-Aspartic acid 481 B.2.8.1.2 Labeled L-aspartic acids 482 B.2.8.1.3 D-Aspartic acid 482 B.2.8.2 Formation of 3-substituted L-aspartic acids 483 B.2.8.3 Formation of L-histidine 484 B.2.8.4 Formation of L-phenylalanine 484 B.2.8.5 Miscellaneous 485 B.2.8.5.1 L-Arginosuccinic acid 485 B.2.8.5.2 Adenylosuccinic acid 486 B.2.8.5.3 2-(Trifluoromethyl)-3-aminopropanoic acid derivatives 486 B.2.8.5.4 (-)-Ethyl 3-diethylamino-4,4,4-trifluorobutanate 487 B.2.9 References 487

B.3 Formation and Cleavage of P - O Bonds 505

B.3.1 Introduction 505 B.3.1.1 Enzymes forming or cleaving phosphorous-oxygen bonds 505 B.3.1.2 Biological phosphorylating agents 508

Contents XV

B.3.2 Phosphorylation 510 B.3.2.1 Regeneration of nucleoside triphosphates 510 B.3.2.1.1 Regeneration of ATP from ADP and AMP 511 B.3.2.1.2 Regeneration of other nucleoside triphosphates 515 B.3.2.2 Applications 516 B.3.2.2.1 Phosphorylations with ATP as a cofactor 516 B.3.2.2.2 P —O bond formation with other nucleoside triphosphates than ATP. . 518 B.3.2.2.3 Other phosphorylating agents 518 B.3.2.3 Tables containing typical examples ordered according to the classes of

Compounds 519 B.3.3 Cleavage of P - O bonds 527 B.3.3.1 Hydrolysis of phosphate and pyrophosphate monoesters 527 B.3.3.2 Hydrolysis of S- and N-substituted phosphate monoester analogs 529 B.3.3.3 Hydrolysis of phosphate and phosphonate diesters 531 B.3.3.3.1 Nucleic acids and their analogs 531 B.3.3.3.2 Other phosphate and phosphonate diesters 532 B.3.3.4 Other P - O bond cleavages 532 B.3.4 Potential technical applications 535 B.3.4.1 Introduction 535 B.3.4.2 Applications of biocatalytic P —O-bond hydrolysis for waste degradation

or detoxification 535 B.3.4.2.1 Reactions utilizing free or immobilized enzymes 535 B.3.4.2.2 Biodegradation by intact microorganisms 536 B.3.4.3 Synthetic applications of biocatalytic P —O-bond hydrolysis 537 B.3.4.4 Synthetic applications of biocatalytic P — O-bond formation 538 B.3.4.4.1 Reactions utilizing free enzymes 538 B.3.4.4.2 Reaction sequences catalyzed by intact microorganisms 539 B.3.5 References 540

B.4 Formation of Carbon-Carbon Bonds 547

B.4.1 Aldol reactions 547 B.4.1.1 DHAP-utilizing aldolases 547 B.4.1.1.1 Fructose 1,6-diphosphate (FDP) aldolase (E.C. 4.1.2.13) 547 B.4.1.1.2 Synthesis of dihydroxyacetone phosphate (DHAP) 555 B.4.1.1.3 The other DHAP-utilizing aldolases: 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 tagatose 1,6-diphosphate (TDP) aldolase 559

B.4.1.2 Pyruvate/phosphoenolpyruvate-utilizing aldolases 562 B.4.1.2.1 N-Acetylneuraminate (NeuAc) aldolase (E.C. 4.1.3.3) and NeuAc syn­

thetase (E.C. 4.1.3.19) 562 B.4.1.2.2 3-Deoxy-D-/wa««o-2-octulosonate aldolase (E.C. 4.1.2.23) and 3-deoxy-

D-wa,««o-2-octulosonate 8-phosphate synthetase (E.C. 4.1.2.16) 564 B.4.1.2.3 3-Deoxy-D-arabino-2-heptulosonic acid 7-phosphate (DAHP) synthetase

(E.C. 4.1.2.15) 567 B.4.1.2.4 2-Keto-4-hydroxyglutarate (KHG) aldolase (E.C. 4.1.2.31) 567 B.4.1.2.5 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (E.C. 4.1.2.14) . . . 568 B.4.1.2.6 2-Keto-3-deoxy-D-glutarate (KDG) aldolase (E.C. 4.1.2.20) 569 B.4.1.3 2-Deoxyribose 5-phosphate aldolase (DERA) (E.C. 4.1.2.4) 569

Contents

4.2 Ketol and aldol transfer reactions 573 4.2.1 Transketolase (TK) (E.C. 2.2.1.1) 573 4.2.2 Transaldolase (TA) (E.C. 2.2.1.2) 574 4.3 Acyloin condensation 575 4.4 C —C bond formation reactions involving acetylCoA 576 4.5 Isoprenoid and Steroid synthesis 578 4.6 yß-Replacement of chloroalanine 580 4.7 Synthesis of cyanohydrins 580 4.7.1 (/?)-Oxynitrilase catalyzed addition of hydrogen Cyanide to aldehydes . . 581 4.7.2 (5)-Oxynitrilase catalyzed addition of hydrogen Cyanide to aldehydes . . 582 4.7.3 (Ä)-Oxynitrilase catalyzed addition of hydrogen Cyanide to ketones . . . . 583 4.7.4 Enantioselective preparation of (R)- and (S)-cyanohydrins by catalysis

with esterases and lipases, respectively 584 4.7.5 Stereoselective reactions of optically active cyanohydrins 585 4.8 Conclusions 586 4.9 References 587

5 Reduction Reactions 595

5.1 Reductions of aldehydes and ketones 595 5.1.1 Introduction 595 5.1.2 Reductions with isolated enzymes 596 5.1.2.1 Regeneration of reduced nicotinamide cofactors: NAD(P)H from

NAD(P) 596 5.1.2.2 Reductions catalyzed by alcohol dehydrogenases 600 5.1.2.3 Reductions catalyzed by dehydrogenases other than ADH 616 5.1.3 Yeast mediated reductions 620 5.1.3.1 Introduction to yeast Systems 620 5.1.3.2 Reductions of a,ß- and J-oxoesters and oxoacids 621 5.1.3.3 Reduction of diketones 625 5.1.3.4 Reductions of a-hydroxy ketones, aromatic and aliphatic ketones and

others 628 5.2 Reduction of carboxylic acids to the alcohols 628 5.3 Reduction of C = N bonds 633 5.3.1 Introduction 633 5.3.2 Structural features of amino acid dehydrogenases 634 5.3.3 Thermodynamics and mechanism of reductive amination 634 5.3.4 Individual amino acid dehydrogenases 636 5.3.4.1 Leucine dehydrogenase (LeuDH, E.C. 1.4.1.9) 636 5.3.4.2 Alanine dehydrogenase (AlaDH, E.C. 1.4.1.1) 636 5.3.4.3 Phenylalanine dehydrogenase (PheDH, E.C. 1.4.1.-) 637 5.3.5 Processing: cofactor regeneration and enzyme membrane reactor (EMR) 638 5.4 Reductions of carbon-carbon double bonds 641 5.4.1 Reductions of a,/?-unsaturated aldehydes and ketones and other activated

C - C double bonds 641 5.4.2 Reductions of a,/?-unsaturated enoates 647 5.4.3 Reductions of substituted fumaric acids 653 5.4.4 Reductions of C —C double bonds in Steroids 653 5.5 References 656

Contents XVII

B.6 Oxidation Reactions 667

B.6.1 Oxygenation of C —H and C=C bonds 667 B.6.1.1 Introduction 667 B.6.1.2 Hydroxylating enzymes 668 B.6.1.3 Mechanistic aspects 670 B.6.1.4 Hydroxylation of non-activated carbon atoms 672 B.6.1.4.1 Hydroxylation of monoterpenes 672 B.6.1.4.2 Hydroxylation of higher terpenes 676 B.6.1.4.3 Hydroxylation of Steroids 679 B.6.1.4.4 Miscellaneous Compounds 680 B.6.1.5 Epoxidation of olefins 686 B.6.1.5.1 Epoxidation of straight-chain terminal olefins 686 B.6.1.5.2 Mechanistic studies 690 B.6.1.5.3 Short-chain alkenes 690 B.6.1.5.4 Terpenes 694 B.6.1.5.5 Cyclic sesquiterpenes 699 B.6.1.6 Cis hydroxylation of aromatic double bonds 701 B.6.1.6.1 Introduction 701 B.6.1.6.2 Preparation of cis dihydrodiols 702 B.6.2 Oxidations of alcohols 705 B.6.2.1 Introduction 705 B.6.2.2 Oxidations catalyzed by oxidases 709 B.6.2.3 Oxidations catalyzed by NAD(P) + independent dehydrogenases 715 B.6.2.4 Oxidations catalyzed by NAD(P)+ dependent dehydrogenases 717 B.6.2.4.1 Enzymatic regeneration Systems 717 B.6.2.4.2 Regeneration with quinoid Systems 723 B.6.2.4.3 Direct electrochemical regeneration 725 B.6.2.4.4 Indirect electrochemical NAD(P)+ regeneration 727 B.6.2.4.5 Dehydrogenase catalyzed synthesis 731 B.6.3 Oxidation of phenols 739 B.6.3.1 Oxidases and hydroxylases as catalysts 739 B.6.3.2 NAD(P)+ dependent dehydrogenases as catalysts 744 B.6.4 Oxidations of aldehydes 744 B.6.5 Baeyer-Villiger oxidations 745 B.6.5.1 Introduction 745 B.6.5.1.1 Steroidal Substrates 745 B.6.5.1.2 Aliphatic Substrates 748 B.6.5.1.3 Alicyclic Substrates 749 B.6.5.2 Baeyer-Villiger monooxygenases 755 B.6.5.3 Synthetic applications 762 B.6.6 Oxidations of carboxylic acids 772 B.6.6.1 Oxidases as catalysts 772 B.6.6.2 NAD(P)+ dependent dehydrogenases as catalysts 774 B.6.7 Oxidations of C - N bonds 774 B.6.7.1 Oxidases as catalysts 774 B.6.8 Oxidation at sulfur 780 B.6.8.1 Enzymes oxidizing at sulfur and their sources 780 B.6.8.2 Oxidation of Sulfides 781

XVIII Contents

B.6.9 Halogenation 783 B.6.9.1 Classification of halogenating enzymes 783 B.6.9.2 Sources and production of enzymes 784 B.6.9.3 Substrates for halogenating enzymes and reaction products 784 B.6.9.3.1 Halogenation of aromatic Compounds 784 B.6.9.3.2 Halogenation of aliphatic Compounds 787 B.6.9.4 Regio- and stereospecificity of enzymatic halogenation reactions 790 B.6.9.5 Comparison of chemical with enzymatic halogenation 790 B.6.10 References 791

B.7 Isomerizations 809

B.7.1 Introduction 809 B.7.2 Racemizations and epimerizations 810 B.7.2.1 Pyridoxal 5'-phosphate dependent amino acid racemases and

epimerases 811 B.7.2.1.1 Alanine racemase (E.C. 5.1.1.1) 811 B.7.2.1.2 Amino acid racemase with low Substrate specificity (E.C. 5.1.1.10) 817 B.7.2.1.3 a-Amino-e-caprolactam racemase 818 B.7.2.2 Cofactor-independent racemases and epimerases acting on

amino acids 820 B.7.2.2.1 Glutamate racemase (E.C. 5.1.1.3) 820 B.7.2.2.2 Aspartate racemase (E.C. 5.1.1.13) 824 B.7.2.2.3 Diaminopimelate epimerase (E.C. 5.1.1.7) 826 B.7.2.2.4 Proline racemase (E.C. 5.1.1.4) 826 B.7.2.3 Other racemases and epimerases acting on amino acid derivatives 827 B.7.2.3.1 2-Amino-A2-thiazoline-4-carboxylate racemase 827 B.7.2.3.2 Hydantoin racemase 828 B.7.2.3.3 Af-Acylamino acid racemase 830 B.7.2.3.4 Isopenicillin N epimerase 831 B.7.2.4 Racemization and epimerization at hydroxy group bearing carbons . . . . 833 B.7.2.4.1 Mandelate racemase (E.C. 5.1.2.2) 833 B.7.3 Isomerizations 836 B.7.3.1 D-Xylose (glucose) isomerase (E.C. 5.3.1.5) 836 B.7.3.2 Phosphoglucose isomerase (E.C. 5.3.1.9) 841 B.7.3.3 Triosephosphate isomerase (E.C. 5.3.1.1) 842 B.7.3.4 Maleate cis-trans-isomemse (E.C. 5.2.1.1) 842 B.7.3.5 Reactions with catalytic antibodies 843 B.7.4 Conclusion 844 B.7.5 References 845

B.8 Introduction and Removal of Protecting Groups 851

B.8.1 Introduction 851 B.8.2 Protection of amino groups 851 B.8.2.1 ATerminal protection of peptides 851 B.8.2.2 Protection of the side chain amino group of lysine 855 B.8.2.3 Protection of amino groups in /9-lactam chemistry 856 B.8.2.4 Protection of amino groups of nucleobases 857

Contents XIX

B.8.3 Protection of thiol groups 857 B.8.3.1 Protection of the side chain thiol group of cysteine 857 B.8.4 Protection of carboxy groups 858 B.8.4.1 C-Terminal protection of peptides 858 B.8.4.2 Protection of the side chain groups of glutamic and aspartic acid 864 B.8.5 Protection of hydroxy groups 865 B.8.5.1 Protection of carbohydrates 866 B.8.5.1.1 Monosaccharides 866 B.8.5.1.2 Di- and Oligosaccharides 875 B.8.5.1.3 Nucleosides 877 B.8.5.1.4 Further aglycon glycosides 879 B.8.5.2 Protection of polyhydroxylated alkaloids 881 B.8.5.3 Protection of Steroids 883 B.8.5.4 Protection of phenolic hydroxy groups 883 B.8.6 Outlook 885 B.8.7 References 886

B.9 Catalytic Antibodies 891

B.9.1 Introduction 891 B.9.2 Antibodies 893 B.9.3 Hydrolysis and formation of ester and amide bonds 894 B.9.4 Claisen rearrangements 900 B.9.5 Redox reactions 901 B.9.6 Decarboxylations 904 B.9.7 Eliminations and rearrangements 904 B.9.8 Conclusions 906 B.9.9 References 907

B.10 Enzymatic Analysis and Biosensors 911

B.10.1 Introduction 911 B.10.2 Determination of analyte concentrations using enzymes 912 B.10.2.1 Introduction 912 B. 10.2.2 End-point assays 913 B. 10.2.3 Kinetic assays 916 B.10.2.4 Catalytic assays 917 B. 10.2.5 Enzyme inhibitors/activators 917 B.10.2.6 Enzyme immunoassay 918 B.10.3 Determination of the catalytic activity of enzymes 919 B. 10.4 Measurement techniques 921 B. 10.4.1 Photometrie and fluorimetric assays 921 B.10.4.2 "Dry-Chemistry" assays 922 B.10.4.3 Biosensors 922 B.10.4.3.1 Amperometric enzyme electrodes 923 B. 10.4.3.2 Surface waveguides based on antibodies 924 B.10.4.3.3 Outlook 925 B.10.5 References 926

XX Contents

B.l l Protein Engineering 929

B. l l . l Introduction 929 B.11.2 Preparation and analysis of modified enzymes 930 B.ll.2.1 Tyrosyl tRNA-synthetase 933 B.ll.2.2 Serine proteases 935 B.ll.2.3 Enzyme engineering for nonaqueous solvents 939 B.ll.2.4 Ligation of peptide bonds 939 B.11.2.5 Cofactor design 940 B.ll.2.6 Purification tag 940 B. 11.2.7 De novo design 941 B.ll .3 Conclusion 941 B.ll .4 References 942

B.12 Enzymes from Extreme Thermophilic and Hyperthermophilic Archaea and Bacteria 945

B.12.1 Introduction 945 B. 12.2 Starch hydrolyzing enzymes 946 B.12.3 Cellulolytic and hemicellulolytic enzymes 951 B. 12.4 Proteolytic enzymes 956 B. 12.5 Intracellular enzymes 958 B.12.6 Conclusion 959 B.12.7 References 960

Section C

C.l Tabular Survey of Commercially Available Enzymes 963

C. 1.1 Introduction 963 C.l.2 Tabular survey of commercially available enzymes by type and reaction

catalyzed 964 C. 1.3 List of suppliers 973

Indices 977