Preface ......................................................... v
1 Introduction ................................................. 1
1.1 Advantages and Disadvantages of Biocatalysts ............ 3
1.2 Biocatalysis - An Interdisciplinary Science ............. 6
1.3 The Impact of Biocatalysis on Teaching Natural
Science ................................................. 8
2 History of Biocatalysis ..................................... 11
3 Classification of Enzymes ................................... 15
4 Non-protein Groups in Biocatalysis .......................... 22
4.1 Thiamine ............................................... 24
4.2 Niacin and Riboflavin .................................. 28
4.3 Vitamin B6 ............................................. 31
4.4 Folic Acid, Tetrahydrofolate and Dihydrobiopterin ...... 38
4.5 Cofactors in Methanogenesis ............................ 45
4.6 Ascorbic Acid Exerts a Protective Function ............. 50
5 Introduction into Kinetics .................................. 54
5.1 Rate of Reactions and Rate Equations ................... 54
5.2 Reaction Mechanisms .................................... 56
5.3 Steady-state ........................................... 58
5.4 Pre-equilibria ......................................... 59
5.5 Temperature Dependence ................................. 60
5.5.1 Chemical equilibria and temperature ............. 61
5.5.2 Arrhenius parameters ............................ 64
5.5.3 Transition state theory ......................... 65
5.6 Reactions in Solutions ................................. 68
5.6.1 Diffusion controlled reactions .................. 70
6 Enzyme Kinetics ............................................. 74
6.1 The Michaelis-Menten Equation .......................... 75
6.1.1 Determination of KM and rmax ..................... 79
6.1.2 Integrated Michaelis-Menten equation ............ 82
6.1.3 Reversible equilibria ........................... 83
6.1.4 Inhibition ...................................... 87
6.1.4.1 Non-competitive inhibition ............. 91
6.1.4.2 Competitive inhibition ................. 94
6.1.4.3 Uncompetitive inhibition ............... 95
6.1.4.4 Substrate inhibition ................... 97
6.1.5 Formation of intermediates ...................... 98
6.1.6 Вisubstrate reactions .......................... 100
6.1.7 Multi-substrate reactions ...................... 105
6.1.8 King and Altman method ......................... 110
6.1.9 Importance of KM and kcat ....................... 113
6.2 Parameters Affecting Enzymatic Activity ............... 120
6.2.1 pH-dependence .................................. 121
6.2.2 Temperatute-dependence ......................... 126
6.3 The Kinetic Isotope Effect and Hydrogen Tunneling ..... 129
6.3.1 The kinetic isotope effect ..................... 129
6.3.1.1 Kinetic isotope effect and reaction
mechanisms ............................ 131
6.3.2 Hydrogen tunnelling ............................ 134
7 Mechanisms in Enzymatic Catalysis and Enzyme Models ........ 142
7.1 Enzyme-substrate Interactions ......................... 142
7.2 Enzymes and Other Catalysts - The Entropy Effect ...... 150
7.3 Single Mechanisms with Examples ....................... 151
7.3.1 Acid-base catalysis ............................ 152
7.3.2 Covalent catalysis ............................. 153
7.3.3 Metal ion catalysis ............................ 156
7.3.3.1 Methylmalonyl-CoA mutase -
a cobalamin enzyme .................... 165
7.4 Metalloenzyme of Вiotechnological Relevance ........... 170
7.4.1 Hydrogenases ................................... 170
7.4.2 Enzymes of the nitrogen cycle .................. 178
7.4.3 Dinitrogenoxide reductase ...................... 185
7.4.4 Vanadium and vanadium-containing enzymes ....... 186
7.4.4.1 Vanadium-nitrogenases ................. 189
7.4.4.2 Fe-nitrogenases ....................... 192
7.4.4.3 VNase model compounds ................. 193
7.4.4.4 Vanadium haloperoxidases .............. 196
8 Synthesis of Peptide Antibiotics ........................... 201
8.1 Mechanism of Nonribosomal Peptide Synthesis ........... 207
8.2 Other Multi-functional Enzymes ........................ 211
8.2.1 Fatty acid synthetases ......................... 211
8.2.2 Polyketide synthetases ......................... 213
8.3 Naturally Occurring Peptide/Polyketide Hybrids ........ 219
8.4 Application of Nonribosomal Peptide Synthetases ....... 220
8.5 The Case Mycosubtilin ................................. 224
8.6 Ribosomal Synthesis of Antibiotics .................... 228
9 Immobilized Biocatalysts ................................... 231
9.1 Definition and Characterization ....................... 232
9.2 Reasons for Immobilizing Biocatalyst .................. 234
9.3 Properties of Carriers for Immobilization ............. 235
9.4 Different Types of Carrier Materials .................. 237
9.5 Immobilization Methods ................................ 238
9.5.1 Immobilization by adsorption ................... 239
9.5.2 Covalent immobilization ........................ 241
9.5.2.1 Optimizing experimental parameters .... 245
9.5.3 Immobilization by crosslinking ................. 246
9.5.4 Entrapment of biocatalysts ..................... 249
9.5.4.1 Sol-gel processes - inorganic
polymers .............................. 250
9.5.4.1.1 Silicon-based matrixes ..... 251
9.5.5 Encapsulation .................................. 252
9.6 Kinetics of Immobilized Biocatalysts .................. 253
9.6.1 Conformational changes ......................... 253
9.6.2 Partitioning effects ........................... 254
9.6.3 Diffusion limitation ........................... 255
9.7 Improving the Properties of Enzymes by
Immobilization ........................................ 257
9.8 Carrier-free Immobilizates ............................ 263
9.8.1 Crosslinked enzyme crystals .................... 264
9.8.1.1 Application of CLECs .................. 265
9.8.2 Crosslinked enzyme aggregates .................. 277
10 Structure, Function, and Application of Enzymes ............ 289
10.1 Hydrolases ............................................ 292
10.1.1 Lipases - general aspects ...................... 293
10.1.1.1 Reaction behavior of lipases .......... 293
10.1.1.2 Stereoselectivity of lipases .......... 294
10.1.1.3 Different types of lipases and
nomenclature .......................... 295
10.1.1.4 Kinetic resolution .................... 297
10.1.1.5 The Kazlauskaz rules .................. 301
10.1.1.6 Dynamic kinetic resolution ............ 304
10.1.1.7 Enantioselectivity .................... 307
10.1.2 Esterases ...................................... 312
10.1.2.1 Structural aspects and mechanism ...... 313
10.1.2.2 Application of estereases ............. 314
10.1.2.3 Screening assays ...................... 318
10.1.2.4 Pig liver esterase .................... 320
10.1.2.4.1 Pig liver esterase
model ..................... 325
10.1.2.5 Acetylcholinesterase .................. 327
10.1.2.5.1 AChE/CdS-nanoparticle
hybrids ................... 332
10.1.2.6 Cholesterol esterase and
carbohydrate esterases ................ 334
10.1.2.7 The salicylic acid-binding protein .... 336
10.1.3 Epoxide hydrolases and epoxides ................ 337
10.1.3.1 Chemical synthesis of epoxides ........ 338
10.1.3.2 Biological synthesis of epoxides ...... 341
10.1.3.2.1 Bacterial
monooxygenases ............ 342
10.1.3.3 Structural aspects of epoxide
hydrolases ............................ 345
10.1.3.4 Application of epoxide hydrolases in
synthesis ............................. 354
10.1.3.5 Epoxid hydrolases from yeasts ......... 367
10.1.4 Nitrile-converting enzymes ..................... 371
10.1.4.1 Nitrile hydratases; Fe- and
Co-NHases ............................. 373
10.1.4.1.1 Mechanism of NHases ........ 378
10.1.4.1.2 Co-nitrile hydratase ....... 382
10.1.4.1.3 Application of NHases and
amidases in organic
synthesis .................. 384
10.1.4.1.4 Desymmetrization ........... 391
10.1.4.1.5 Nitrile hydratase active
site models ................ 393
10.1.4.2 Nitrilases ............................ 395
10.1.4.2.1 Reaction mechanism of
nitrilases ................. 397
10.1.4.2.2 Application of
nitrilases ................. 399
10.1.5 Hydantoinases .................................. 402
10.1.5.1 Application of hydantoinases .......... 404
10.1.5.2 Structural and functional aspects of
hydantoinases ......................... 407
10.1.5.3 Engineering of hydantoinases and
related enzymes ....................... 412
10.2 Carbohydrate-modifying Enzymes ....................... 424
10.2.1 The glycosidic linkage ........................ 427
10.2.2 Glycosidases .................................. 428
10.2.2.1 Application of glycosidases ........... 434
10.2.2.2 Synthesis of glycoconjugates .......... 437
10.2.2.3 Stable glycosidic bonds ............... 441
10.2.2.4 Examples of unusual glycosidases ...... 449
10.2.3 Glycosyltransferases ........................... 455
10.2.3.1 Structural and mechanistic aspects .... 467
10.2.3.2 Synthesis of glycoconjugates
catalyzed by GTs ...................... 474
10.2.3.3 High-throughput screening for GTs ..... 480
10.2.3.4 Bidirectional glycosyltransferases .... 482
10.2.4 Applications of glycoconjugates ................ 492
10.3 Catalysts for Redox Reactions ......................... 509
10.3.1 Oxidoreductases - general aspects .............. 509
10.3.1.1 Stereochemical aspects ................ 511
10.3.1.2 Co-factor regeneration ................ 512
10.3.1.2.1 Electrochemical methods
of cofactor regeneration
or bypassing ............... 514
10.3.1.3 Examples of oxidoreductases ........... 522
10.3.2 Dehydrogenases ................................. 525
10.3.2.1 NAD(P)+-dependent dehydrogenases ...... 525
10.3.2.2 Flavin-dependent dehydrogenases ....... 527
10.3.2.3 Application of dehydrogenases ......... 528
10.3.3 Oxygenases ..................................... 532
10.3.3.1 Monooxygenases ........................ 532
10.3.3.1.1 Cytochrom P-450
oxygenases ................. 533
10.3.3.1.2 Aromatic hydroxylases ...... 536
10.3.3.1.3 Baeyer-Villiger
monooxygenases ............. 538
10.3.3.2 Dioxygenases .......................... 559
10.3.3.2.1 Application of
dioxygenases ............... 562
10.3.3.2.2 Dioxygenases in
environmental
biotechnology .............. 567
10.3.3.2.3 Catechol dioxygenases ...... 572
10.3.3.2.4 Lipoxygenases .............. 577
10.4 Lyases ................................................ 610
10.4.1 Aldolases ...................................... 610
10.4.1.1 The aldol reaction .................... 610
10.4.1.2 Aldolases - mechanisms and mimics ..... 612
10.4.1.3 Aldolases in organic synthesis ........ 622
10.4.1.3.1 DHAP dependent aldolases ... 622
10.4.1.3.2 DHAP - problems and
solutions .................. 641
10.4.1.3.3 Aldolases accepting DHA .... 645
10.4.1.3.4 Pyruvate and
phosphoenolpyruvate
dependent aldolases ........ 647
10.4.1.3.5 DER aldolases .............. 659
10.4.1.3.6 Glycine-dependent
aldolases .................. 666
10.4.1.4 Aldolases - environmental aspects ..... 669
10.4.2 Hydroxynitril lyases ........................... 675
10.4.2.1 Mechanism of hydroxynitrile lyases
and structural aspects ................ 677
10.4.2.2 Application of hydroxynitrile
lyases ................................ 680
10.4.2.3 Engineering of HNLs ................... 688
10.4.2.4 Further modifications of
cyanohydrins .......................... 695
10.4.2.5 Immobilized (R)-oxynitrilases ....... 700
10.5 Epimerases and Racemases .............................. 706
10.5.1 Racemases ...................................... 708
10.5.2 Conversion of a racemase into an aldolase ...... 713
10.5.3 Industrial application of racemases ............ 715
10.5.4 Isomerases ..................................... 718
10.6 Transaminases - Catalysts for Amino Acid Synthesis .... 722
11 Enzymes in Non-conventional Media .......................... 731
11.1 Enzymes in Organic Solvents ........................... 731
11.1.1 Thermal stability of enzymes in organic
solvents ....................................... 735
11.1.2 The role of water .............................. 738
11.1.3 The role of solvent properties ................. 741
11.1.4 pH-memory in organic solvents .................. 746
11.1.5 (Low) enzymatic activity in organic solvents ... 746
11.1.6 Structure of enzymes in organic solvents ....... 750
11.1.7 Improving enzymatic activity in organic
solvents ....................................... 751
11.2 Other Non-conventional Media .......................... 752
11.2.1 Supercritical fluids ........................... 752
11.2.2 Ionic liquids .................................. 759
11.2.2.1 Ionic liquids in enzyme catalysis .... 763
11.2.3 Combination of ILs and scCO2 ................... 771
12 Methods to Improve Biocatalysts ............................ 777
12.1 Protein Design and Related Aspects .................... 777
12.2 Fundamentals of Genetic Engineering ................... 780
12.2.1 Transcription .................................. 780
12.2.2 Translation .................................... 783
12.2.3 Transformation and expression .................. 788
12.2.4 Recombinant techniques ......................... 789
12.2.4.1 The PCR reaction ...................... 791
12.2.4.2 Restriction enzymes and DNA ligases ... 793
12.3 Engineering of New Proteins ........................... 799
12.4 Evolutionary Methods in Biocatalysis .................. 802
12.4.1 Generating diversity ........................... 805
12.4.1.1 Saturation mutagenesis ................ 805
12.4.1.2 Error-prone PCR ....................... 808
12.4.1.3 Gen-shuffling, recombining methods .... 811
12.4.2 Recent developments ............................ 817
12.4.2.1 Substrate acceptance .................. 817
12.4.2.2 Improvement of enantioselectivity ..... 819
12.4.2.3 Thermal stability ..................... 824
13 Metabolic Pathway Engineering .............................. 830
13.1 Metabolic Engineering in Oligosaccharide Synthesis .... 838
13.2 Further Applications of Pathway Engineering ........... 843
13.3 Novel Carotenoids ..................................... 850
13.4 Combining Transketolase and Transaminase Activity ..... 854
14 Catalytic Antibodies ....................................... 857
14.1 Antibodies ............................................ 860
14.2 Some Historical Aspects ............................... 863
14.3 Cleavage of C-O and C-N Bonds ......................... 866
14.4 Further Examples ...................................... 870
14.5 Catalysis of Unfavored Reactions ...................... 887
14.6 Catalytic Antibodies in Medicine ...................... 889
14.6.1 Catalytic antibodies and detoxification ........ 889
14.6.2 Activation of prodrugs by antibodies ........... 893
14.7 Evolution of Binding Energy and Catalysis ............. 897
15 Nucleic Acids as Catalysts ................................. 908
15.1 Nucleic Acid Catalysts and Protein Enzymes ............ 914
15.2 Examples of the Mechanism of RNA Catalysis ............ 920
15.3 Characterization of a Ribozyme ........................ 930
15.4 Selection of Catalytic Nucleic Acids .................. 940
15.5 Expanding the Scope of Catalysis by Nucleic Acids ..... 945
15.6 Examples of Catalytic RNA and DNA ..................... 948
15.6.1 RNA-based catalysts - ribozymes ................ 948
15.6.2 DNA-based catalysts ............................ 960
16 Use of Enzymes in Industry ................................. 968
16.1 Industrial Biotechnology in Numbers ................... 968
16.2 Enzymes for Starch Conversion ......................... 972
16.3 Milk Processing ....................................... 977
16.4 Cheesemaking .......................................... 978
16.5 Enzymes and Beer ...................................... 980
16.6 Bread Processing ...................................... 982
16.7 Application in Fruit Juice Production ................. 986
16.8 The Detergent Industry ................................ 987
16.9 Textile Applications .................................. 988
16.10 Pulp and Paper Processing ............................ 990
17 White Biotechnology ........................................ 993
Outlook ...................................................... 1008
List of Abbreviations ........................................ 1011
Index ........................................................ 1015
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