Biocatalysis in Non-Conventional Media (eBook)

Front Cover 1
Biocatalysis in Non–Conventional Media 4
Copyright Page 5
Table of Contents 10
Preface 6
Acknowledgements 9
OPENING OF THE SYMPOSIUM 18
Chapter 1.Medium and biocatalyst engineering 20
1. INTRODUCTION 20
2. THEORETICAL CONSIDERATIONS 21
3. THE ROLE OF WATER 21
4. CHOICE OF ORGANIC SOLVENT 22
5. IMPORTANCE OF THE SUPPORT MATERIAL FOR ENZYMATIC ACTIVITY IN ORGANIC MEDIA 23
6. MOLECULAR IMPRINTING 23
7. EFFECTS OF ORGANIC SOLVENTS ON THE ENANTIOSELECTIVITY OF ENZYMES 24
8. ONE AND TWO LIQUID-PHASE SYSTEMS 24
9. ENZYME DESIGN FOR NON-AQUEOUS MEDIA 25
10. CONCLUDING REMARKS 25
REFERENCES 26
SESSION I: PHYSICAL-CHEMICAL ASPECTS 28
Chapter 1.Physical-chemical nature of low water systems for biocatalysis: especially 
30 
1. SOME GENERAL THERMODYNAMIC PRINCIPLES 30
2. EFFECTS OF WATER: GENERAL 31
3. WATER EFFECTS ON ACTIVITY 31
4. WATER ACTIVITY CONTROL 34
5. MEASUREMENT AND EFFECTS OF pH 36
6. REFERENCES 38
Chapter 2.Usefulness of NMR methods for assaying cutinase catalysed synthesis of ester in organic media 40
INTRODUCTION 40
MATERIALS AND METHODS 41
RESULTS AND DISCUSSION 41
CONCLUSION 46
AKNOWLEDGMENTS 46
REFERENCES 46
Chapter 3.Membrane concentrations of primary alcohols which inhibit progesterone 11a-hydroxylase in Rhizopus nigricans 48
1. IITTRODOCTION 48
2. RELATIONSHIP BETWEEN Poct and P....e.... 49
3. CORRELATION WITH ANAESTHESIA AND OTHER MEMBRANE CHEMISTRY 52
REFERENCES 53
Chapter 4.A comparison of enzymatic reactions in aqueous, organic and multiphase systems 54
Multiphase systems 54
Comparison of the enantioselective production of ...ß...-hydroxycarbonic acids in organic and aqueous reaction phases 56
A biosensor for the determiantion of enantiomeric excesses 56
Conclusions 58
Acknowledgement 61
References 61
Chapter 5.Biocatalysis in non-conventional media: Effect of enzyme microenvironment 62
1. INTRODUCTION 62
2. EFFECT OF SPECIFIC LIGANDS 63
3. EFFECT OF NON SPECIFIC LIGANDS 64
4. CONCLUSION 69
5· REFERENCES 69
SESSION II: BIOCATALYST ENGINEERING 70
Chapter 1.On the importance of the support material for enzymatic synthesis in 
72 
1. INTRODUCTION 72
2. INDIRECT SUPPORT EFFECTS 73
3. DIRECT SUPPORT EFFECTS 76
4. CONCLUSIONS 78
5. ACKNOWLEDGEMENTS 78
6. REFERENCES 78
Chapter 2.Enzyme design for nonaqueous media 80
1. INTRODUCTION 80
2. EXPERIMENTAL 80
3. RESULTS AND DISCUSSION 81
4. REFERENCES 83
Chapter 3. Application of Q.S.A.R. methodology to the biocatalysis. I. Hydrolysis of esters 84
1.INTRODUCTION 84
2.EXPEREMENTAL 85
3.RESULTS AND DISCUSSION 85
4.PREFERENCES 90
Chapter 4. Application of Q.S.A.R. methodology to the biocatalysis. II. Synthesis of peptides 92
1.INTRODUCTION 92
2.EXPERIMENTAL 93
3.RESULTS AND DISCUSSION 93
ACKNOWLEDGEMENT 98
REFERENCES 98
SESSION III: GASEOUS AND (NEAR)-SUPERCRITICAL MEDIA 100
Chapter 1.The role of water in gaseous biocatalysis 102
REFERENCES 108
Chapter 2.Pressure control of reactions in supercritical fluids: thermodynamics and kinetics. 110
1 Introduction 110
2 Examples of Enzymatic Catalysis in Supercritical Fluids 111
3 Pressure Effects on Reactions in Supercritical Fluids 112
4 Conclusions 116
References 116
SESSION IV: ONE-LIQUID-PHASE SYSTEMS 120
Chapter 1. Enzyme mechanisms in homogeneous hydro-organic solutions. Solvents,temperature and pressure effects 120
1. INTRODUCTION 120
2. EXPERIMENTAL APPROACHES 121
3. SPECIFIC TECHNOLOGIES 121
4. INTERPRETATION OF DATA 122
5. COSOLVENTS AND TEMPERATURE AS TOOLS TO KINETIC STUDIES 123
6. IMPROVEMENT OF DATA USING THE PRESSURE PARAMETER 125
7. CONCLUSIONS 126
8. ACKNOWLEDGEMENTS 128
9. REFERENCES 128
Chapter 2. Effect of reaction conditions on the activity and enantioselectivity of lipases in organic solvents 129
INTRODUCTION 129
RESULTS AND DISCUSSION 130
REFERENCES 137
Chapter 3.Correlations between enzyme activity, water activity, and Log in one-liquid-phase systems 139
1. INTRODUCTION 139
2. MATERIALS AND METHODS 140
3. RESULTS AND DISCUSSION 141
4. CONCLUSION 145
5. RFERENCES 145
Chapter 4.Microenvironmental effects on steroid ....1-dehydrogenation in organic media using immobilized whole cells 147
1. INTRODUCTION 147
2. MATERIALS AND METHODS 148
3. RESULTS AND DISCUSSION 149
4. CONCLUSIONS 153
5. REFERENCES 154
Chapter 5.Enzyme kinetics in monophasic and biphasic aqueous-organic systems 155
1. INTRODUCTION 155
2. CHOICE OF THE THERMODYNAMIC STANDARD STATE 155
3. ACTIVITY COEFFICIENTS 156
4. CALCULATION OF EQUILIBRIA IN AQUEOUS-ORGANIC MONOPHASIC AND BIPHASIC SYSTEMS 157
5. RATE EQUATIONS IN MONOPHASIC ORGANIC SOLVENTS 158
6. RATES IN AQUEOUS-ORGANIC BIPHASIC SYSTEMS 160
7. KINETIC RESOLUTION 161
8. REFERENCES 162
SESSION V: TWO-LIQUID-PHASE SYSTEMS I 163
Chapter 1.Process engineering of two-liquid phase biocatalysis 165
1. INTRODUCTION 165
2. ESTABLISHED PRINCIPLES 166
3. RULES 166
4. EXPERIMENTAL TOOLS 167
5. PROCESS APPLICATION 171
6. FORWARD LOOK 171
7. REFERENCES 171
Chapter 2.The effect of organic solvents on enzymatic esterification of polyols 173
INTRODUCTION 173
THEORY 174
MATERIALS AND METHODS 174
RESULTS AND DISCUSSION 175
CONCLUSIONS 179
ACKNOWLEDGEMENTS 179
REFERENCES 179
Chapter 3.Process development for the optical resolution of phenylalanine by means of 
181 
1. INTRODUCTION 181
2. PROPERTIES OF THE REACTION SYSTEM 182
3. MATERIALS AND METHODS 186
4. MASS-TRANSFER STUDIES IN THE TWO-PHASE SYSTEM 186
5. MASS-TRANSFER STUDIES IN THE THREE-PHASE SYSTEM 187
6. ACKNOWLEDGEMENT 188
7. NOMENCLATURE 188
8. REFERENCES 188
SESSION VI: TWO-LIQUID-PHASE SYSTEMS II 189
Chapter 1.Understanding protein performance in reversed micelles: the contribution of transport rate, local concentration and water content to enzyme kinetics 191
1. INTRODUCTION 191
2 . THEORY 192
3. ADVANTAGES AND DRAWBACKS OF THE MODEL 194
4. w0 DEPENDENCE 194
5. STRUCTURAL STUDIES 195
6. CONCLUSION 196
ACKNOWLEDGEMENTS 197
REFERENCES 197
Chapter 2.Protein-interface interactions in reverse micelles 199
1. INTRODUCTION 199
2. REVERSE MICELLES 199
3. CONCLUSIONS 205
4. ACKNOWLEDGEMENTS 205
5. REFERENCES 206
Chapter 3.Kinetics of enzyme-catalysed reactions in water-in-oil microemulsions 207
1. INTRODUCTION 207
2. THEORY 208
3. INTERPRETATION OF THE RATE EQUATION 212
4. EXAMPLES O F SYSTEMS OBEYING TYPE (ii) KINETICS 213
5. CONCLUDING REMARKS 214
Acknowledgements 214
6. REFERENCES 216
Chapter 4.CONCLUDING REMARKS 219
1.Phase and reaction equilibrium "engineering" 219
2. Biocatalyst stability and activity in non-conventional media 221
3. Biocatalyst engineering 223
4. Experimental tools 224
5 . Fundamentals vs. Applications 224
Chapter 5.POSTER PAPERS 225
A: PHYSICAL-CHEMICAL ASPECTS 227
Chapter 1.Enzyme kinetics in a self evolving microstructured medium 229
INTRODUCTION 229
RESULTS AND DISCUSSION 229
CONCLUSION 230
REFERENCES 230
Chapter 2.Enzyme deactivation phenomena in solid-state and organic solvents 231
1. INTRODUCTION 231
2. METHODS 231
3. EXPERIMENTAL RESULTS AND DISCUSSION 232
4. CONCLUSIONS 237
5. REFERENCES 238
Chapter 3.Insolubilized enzyme derivatives in organic solvents: Mechanisms of 
239 
1.- INTRODUCTION. 239
EXPERIMENTAL 240
2.- CYCLES OF INACTIVATION-REACTIVATION. 243
3 . - UREA-INDUCED REACTIVATION. 243
DISCUSSION 244
REFERENCES. 246
Chapter 4.Relation of enzymatic reaction rate and hydrophobicity of the solvent 247
1. INTRODUCTION 247
2. THEORY 248
3 . RESULTS 249
ACKNOWLEDGEMENTS 253
LITERATURE 253
Chapter 5.Kinetic resolution of racemic glycidyl esters with porcine pancreatic lipase: A major effect of ping-pong kinetics 255
1. INTRODUCTION 255
2. PPL-CATALYZED RESOLUTION OF RACEMIC GLYCIDYL BUTYRATE 256
3. CHEMICAL EQUILIBRIUM 257
4. HETEROGENEITY OF PPL 257
5. ESTEROLYTIC AND LIPOLYTIC ACTIVITY OF PPL 257
6. PARTITIONING OF REACTION COMPONENTS 258
7. PING-PONG KINETICS OF PPL-CATALYZED HYDROLYSIS 259
8. CONCLUDING REMARKS 261
ACKNOWLEDGEMENTS 261
REFERENCES 261
Chapter 6.Biocatalysts operating at high substrate concentrations 262
1. INTRODUCTION 262
2. PHENOMENOLOGY OF SI AND SA 262
3. AETIOLOGY OF SI AND SA 263
4. CONCLUDING REMARKS 268
5. REFERENCES 268
Chapter 7.Regulation of allosteric enzymes in water-restricted media 270
1.INTRODUCTION 270
2. EXPERIMENTAL 271
3. RESULTS AND DISCUSSION 271
4. CONCLUSION 276
5. REFERENCES: 277
Chapter 8.Quantitative deuterium NMR of protein hydration in air and organic solvents 278
1. INTRODUCTION 278
2. EXPERIMENTAL PROCEDURE 279
3. RESULTS AND DISCUSSION 279
Acknowledgements 283
4. REFERENCES 283
Chapter 9.Effects of temperature on stereochemistry of alcohol dehydrogenases 
284 
1. INTRODUCTION 284
2. EXPERIMENTAL 285
3. RESULTS AND DISCUSSION 285
4. CONCLUSIONS 289
5. REFERENCES 290
Chapter 10.Comparative influence of microenvironment on the activity of two enzymes: lipoxygenase and thermolysin 292
1. INTRODUCTION 292
2. MATERIALS AND METHODS 292
3. RESULTS 293
4. DISCUSSION 298
5.REFERENCES 299
Chapter 11.On the crucial role of water in the lipase catalysed isomerisation of l,2-(2,3)-diglyceride into 1,3-diglyceride 300
Summary 300
Introduction 300
Results and discussion 301
Experimental 306
Conclusion 306
Aknowledgements 307
References 307
Chapter 12.Rapid determination, using dielectric spectroscopy, of the toxicity of organic solvents to intact cells 308
1. INTRODUCTION 308
2. MATERIALS AND iMETHODS 309
3. RESULTS AND DISCUSSION 310
Acknowledgments 313
References 314
Chapter 13.Factors affecting protein transfer from an aqueous phase into a reversed micellar phase 316
1. INTRODUCTION 316
2. MATERIALS AND METHODS 318
3. RESULTS AND DISCUSSION 319
ACKNOWLEDGEMENT 322
4. REFERENCES 322
Chapter 14.Cryo-bioorganic synthesis - Enzyme catalysis at low and in low water content environmments 324
INTRODUCTION 324
MATERIALS AND METHODS 325
RESULTS AND DISCUSSION 326
CONCLUDING REMARKS 328
ACKNOWLEDGEMENTS 329
REFERENCES 329
Chapter 15.Photoinduced charge separation in microemulsions 330
1. INTRODUCTION 330
2. MATERIALS AND METHODS 331
3 .RESULTS AND DISCUSSION 333
4. CONCLUSIONS 336
5. ACKNOWLEDGEMENTS 337
6. REFERENCES 337
Chapter 16.Induced stereo- and substrate selectivity of bio-imprinted a-chymotrypsin in anhydrous organic media 338
1. INTRODUCTION 338
2. MATERIALS AND METHODS 338
3. RESULTS AND DISCUSSION 339
Acknowledgement 344
4. REFERENCES 344
B: BIOCATALYST ENGINEERING 346
Chapter 1.The effect of attachment of hydrophobic modifiers on the catalytic activities of lipase and trypsin 348
INTRODUCTION 348
MATERIAL AND METHODS 349
RESULTS AND DISCUSSION 349
ACKNOWLEDGEMENT 354
REFERENCES 354
Chapter 2.Influence of the solvent and the solid support on the microenvironment of immobilized ...a-chymotrypsin 356
SUMMARY 356
INTRODUCTION 356
MATERIALS AND METHODS 358
RESULTS AND DISCUSSION 358
SYNTHETIC ACTIVITY 361
ACKNOWLEDGMENT 363
REFERENCES 363
Chapter 3.Hydrophilic gels as immobilization materials and stabilizers for 
364 
1. INTRODUCTION 364
2. MATERIALS AND METHODS 365
3 . RESULTS AND DISCUSSIONS 367
4. REFERENCES 371
Chapter 4.Effect of polyhydroxy compounds on the activity of lipase from Rhizopus arrhizus in organic solvent 372
INTRODUCTION 372
MATERIALS AND METHODS 373
RESULTS 373
DISCUSSION 376
REFERENCES 378
Chapter 5.Complex formation between chymotrypsin and polymers as a means 
380 
1. INTRODUCTION 380
2. MATERIALS AND METHODS 380
3. RESULTS AND DISCUSSION 382
4. CONCLUSIONS 386
5. ACKNOWLEDGEMENTS 386
6. REFERENCES 386
Chapter 6.Synthesis of enkephalins using modified proteases in organic media 388
1. INTRODUCTION 388
2.MATERIALS AND METHODS 388
3. RESULTS AND DISCUSSION 390
4.REFERENCES 393
Chapter 7.Stabilization of adsorbed enzymes used as biocatalysts in organic solvents 394
1. INTRODUCTION 394
2. MATERIALS AND METHODS 394
3. RESULTS AND DISCUSSION 395
4. CONCLUSION 399
5. ACKNOWLEDGEMENTS 399
6. REFERENCES 399
C: GASEOUS AND (NEAR-)SUPERCRITICAL MEDIA 400
Chapter 1.The use of amylolytic and proteolytic enzymes in art restoration 402
A new tool in art restoration 402
Pastes 402
Papers 403
Paste hydrolysis in low water environment 403
Enzyme activity on pasted paper 404
Diffusion of enzymes through papers 404
RESULTS 404
Alcohols and á-amylase activity 407
The effect of Brij 35 on removal 407
Removal with alcoholic solutions and surfactant 408
Conclusion 409
References 409
Chapter 2.Methyl isobutyl and methyl ethyl ketone biodégradation in biofilters 410
1. INTRODUCTION 410
2. MATERIALS AND METHODS 412
3. RESULTS 413
4. CONCLUDING REMARKS 416
5. REFERENCES 416
Chapter 3.Lipase catalysed esterification in supercritical carbon dioxide 418
1. INTRODUCTION 418
2. MATERIALS AND METHODS 419
3. RESULTS AND DISCUSION 420
4. PROCES OUTLINE 422
5. CONCLUSION 423
6. REFERENCES 423
Chapter 4.Effect of a near- critical and supercritical fluid on the viability ratio of microbial cells 424
1. INTRODUCTION 424
2. MATERIALS AND METHODS 426
3. RESULTS AND DISCUSSION 427
4. CONCLUSIONS 432
5. REFERENCES 432
Chapter 5.Enzymatic reaction in organic solvents and supercritical gases 434
1. Introduction. 434
2. Experimental 435
3. Results of preliminary experiments and calculations 435
4. Conclusions. 439
Acknowledgments 439
Nomenclature 439
Greek Letters 440
Superscripts and Subscripts 440
References 440
Chapter 6.Fatty acid esterification in supercritical carbon dioxide 442
1. INTRODUCTION 442
2. MATERIALS AND METHODS 444
3. RESULTS OF THE BATCH STUDY IN AGITATED VESSEL 444
4. STUDY OF THE CONTINUOUS REACTION 445
5. POST REACTIONAL SEPARATION 448
References 449
D: ONE-LIQUID-PHASE SYSTEMS 450
Chapter 1.Influence of organic solvents on the specificity of ...a-chymotrypsin and subtilisin 
452 
1· INTRODUCTION 452
2. RESULTS AND DISCUSSION 453
3· CONCLUSIONS 457
4. ACKNOWLEDGEMENTS 459
5· REFERENCES 459
Chapter 2.Peptide synthesis in organic-aqueous media catalysed by ...a-chymotrypsin immobilised over different supports 460
l.INTRODUCTION 460
2. EXPERIMENTAL 461
3· RESULTS AND DISCUSSION 463
REFERENCES 467
Chapter 3.Control of water activity by using salt hydrates in enzyme catalysed esterifications in organic media 468
INTRODUCTION 468
RESULTS AND DISCUSSION 469
CONCLUSION 473
ACKNOWLEDGEMENTS 473
EXPERIMENTAL 473
REFERENCES 474
Chapter 4.Enzymatic peptide synthesis using new water-soluble amino acid derivatives 476
1. INTRODUCTION 476
2. MATERIALS AND METHODS 477
3. RESULTS AND DISCUSSION 477
4. CONCLUSION 483
REFERENCES 483
Chapter 5.Lipase-catalyzed resolution of 1,2-diols 484
INTRODUCTION 484
MATERIALS AND METHODS 485
RESULTS AND DISCUSSION 486
CONCLUSIONS 490
ACKNOWLEDGMENT 491
REFERENCES 491
Chapter 6.Partitioning of water during the production of terpene esters using immobilized lipase 492
INTRODUCTION 492
MATERIALS AND METHODS 493
RESULTS AND DISCUSSION 494
CONCLUSIONS 499
ACKNOWLEDGEMENTS 499
REFERENCES 499
Chapter 07.Thermoinactivation of polyphenol oxidase in organic solvents with low water content 500
1. INTRODUCTION 500
2. MATERIALS AND METHODS 501
3 . RESULTS AND DISCUSSION 502
4. REFERENCES 506
Chapter 8.Continuous enzymatic transesterification of rapeseed oil and lauric acid in a solvent-free system 508
1. INTRODUCTION 508
2. MATERIALS AND METHODS 509
3. RESULTS AND DISCUSSION 510
4. REFERENCES 513
Chapter 9.Effect of the solvent on enzyme enantioselectivity 514
1. INTRODUCTION 514
2. EXPERIMENTAL 515
3. RESULTS A N D DISCUSSION 516
4. CONCLUSIONS 519
5. REFERENCES 520
Chapter 10.Modification of waste fats by lipase-catalyzed reaction in solvent-free substrate 522
1· INTRODUCTION 522
1· INTRODUCTION 522
2. MATERIALS AND METHODS 523
3. RESULTS AND DISCUSION 525
4. CONCLUSIONS 529
5. ACKNOWLEDGEMENTS 529
6. REFERENCES 529
Chapter 11.Thermolysin- and chymotrypsin-catalyzed peptide synthesis in the presence of salt hydrates 530
Introduction 530
Results and Discussions 531
References 535
Chapter 12.Lipase catalyzed triglyceride synthesis. The role of isomerisation 536
1. INTRODUCTION 536
2. MATERIAL AND METHODS 536
3. MODELLING 537
4. RESULTS 539
5. CONCLUSIONS 540
6. REFERENCES 541
Chapter 13.Resolution of l-benzamido-4-carboxymethyl-cyclopent-2-ene using pig-liver esterase 542
1. INTRODUCTION 543
2. MATERIALS AND METHODS 543
3. RESULTS AND DISCUSSION 544
4. CONCLUSIONS 548
Acknowledgements 548
References 548
Chapter 14....a-Substituted primary alcohols as substrates for enantioselective 
550 
Introduction 550
Materials and Methods 551
PFL-Catalyzed Transesterification: General Procedure. 551
Results and Discussion 551
Conclusions 556
Acknowledgements 556
REFERENCES 556
Chapter 15.Soluble and immobilized saccharidases in water-miscible organic solvents 558
SUMMARY 558
INTRODUCTION 558
RESULTS AND DISCUSSION 559
REFERENCES 564
Chapter 16.Effect of water activity on rate of lipase-catalysed esterification 566
1. INTRODUCTION 566
2. METHODS 567
3. RESULTS AND DISCUSSION 568
4. ACKNOWLEDGEMENTS 571
5. REFERENCES 572
Chapter 17.Synthesis of triacylglycerols. The crucial role of water activity control 574
INTRODUCTION 574
EXPERIMENTAL 576
RESULTS AND DISCUSSION 577
CONCLUSIONS 578
LITERATURE 579
Chapter 18.Chemo-enzymatic synthesis of monosaccharide fatty acid esters and 
580 
Introduction 580
Materials and Methods 581
Results 581
Discussion 583
Acknowledgements 585
References 585
Chapter 19.Reversing an ...a-chymotrypsin catalyzed reaction, by substituting a water / 
586 
1. INTRODUCTION 586
2. MATERIALS AND METHODS 587
3. RESULTS AND DISCUSSION 588
4. CONCLUSION 593
REFERENCES 593
Chapter 20.Engineering aspects of the lipase-catalyzed production of 
594 
1. Introduction 594
2. Influence of the water content on enzyme activity 595
3. Kinetics 597
4. Enzyme Immobilization 597
5. Continuous reaction system 599
6. Conclusions 601
7. References 601
Chapter 21.Variation of tyrosinase activity with solvent at a constant water activity 602
INTRODUCTION 602
EXPERIMENTAL 603
RESULTS AND DISCUSSION 604
REFERENCES 608
Chapter 22.Hydrolase activity of Pseudomonas fluorescens lipase in organic media 610
1. INTRODUCTION 610
2. MATERIALS AND METHODS 611
3. RESULTS AND DISCUSSION 613
4. CONCLUSION 617
5. REFRENCES 617
Chapter 23.Factors affecting lipase catalyzed n-butyl oleate synthesis 618
1. INTRODUCTION 618
2. MATERIALS AND METHODS 619
3. RESULTS AND DISCUSSION 620
4. REFERENCES 625
Chapter 24.Behaviour of soluble aminoacylase in water-organic solvent mixtures 626
INTRODUCTION 626
MATERIALS AND METHODS 627
RESULTS 627
DISCUSSION 628
REFERENCES 629
E: TWO-LIQUID-PHASE SYSTEMS I 632
Chapter 1.Effect of organic solvents on growth and anthraquinone production in Morinda citrifolia cell cultures 634
Introduction 634
Materials and methods 634
Results and discussion 636
Conclusions 639
References 639
Chapter 2.Functional stability of cytoplasmic enzymes in aqueous and mixed-phase solvents 640
INTRODUCTION 640
METHODS 640
RESULTS 641
DISCUSSION and CONCLUSIONS 644
REFERENCES 644
Chapter 3.Studies on papain catalyzed synthesis of Gly-Phe in a two-liquid-phase system 646
1. INTRODUCTION 646
2. MATERIALS & METHODS
3. RESULTS AND DISCUSSION 648
4. CONCLUSIONS 652
5. ACKNOWLEDGEMENTS 653
6. BIBLIOGRAPHY 653
Chapter 4.The effect of alkanes on viability, enzyme induction and 
654 
1. INTRODUCTION 654
2, MATERIALS AND METHODS 655
3. RESULTS 656
4. DISCUSSION 659
5. REFERENCES 660
Chapter 5.Kinetics and engineering studies of lipase-catalyzed transesterification in organic solvent 662
1. INTRODUCTION 662
2. THEORY 664
3. RESULTS AND DISCUSSION 666
4. REFERENCES 669
Chapter 6.Kinetic study of enzymatic reaction in aqueous-organic two-phase systems - 
670 
1. INTRODUCTION 670
2. MATERIALS AND METHODS 670
3. RESULTS AND DISCUSSION 671
4. CONCLUSIONS 675
REFERENCES 675
Chapter 7.The influence of organic cosolvents on the lipase catalyzed hydrolysis of decylchloroacetate 676
INTRODUCTION 676
MATERIALS AND METHODS 677
RESULTS AND DISCUSSION 678
CONCLUDING REMARKS 682
ACKNOWLEDGEMENT 682
REFERENCES 682
Chapter 8.Biotransformation of benzaldehyde to benzyl alcohol by whole cells and 
684 
INTRODUCTION 684
MATERIALS AND METHODS 685
RESULTS 686
DISCUSSION 689
Acknowledgement 690
REFERENCES 690
Chapter 9.Production of phenylacetyl carbinol by biotransformation using baker's yeast two-phase systems 692
INTRODUCTION 692
MATERIALS AND METHODS 693
RESULTS 693
DISCUSSION 697
Acknowledgement 697
REFERENCES 697
F: TWO-LIQUID-PHASE SYSTEMS II 698
Chapter 1.Stability and activity of cholesterol oxidase in supramolecular systems 700
1. INTRODUCTION 700
2. EXPERIMENTAL 701
3. RESULTS AND DISCUSSION 702
4. ACKNOWLEDGEMENTS 706
5. REFERENCES 706
Chapter 2.Crown ethers can enhance enzyme activity in organic solvents 708
1. INTRODUCTION 708
2. RESULTS AND DISCUSSION 709
3 . ACKNOWLEDGEMENTS 712
4. REFERENCES 712
Chapter 3.Dynamics, structure and stability of ...a-chymotrypsin in aqueous solution and in reverse micelles as studied by fluorescence spectroscopy 714
1.INTRODUCTION 715
2. MATERIALS AND METHODS 716
3. RESULTS 716
4. DISCUSSION 720
5. ACKNOWLEDGEMENT 721
6. REFERENCES 721
Chapter 4.Comparison of activity and stability of enzymes suspended in 
722 
1. INTRODUCTION 722
2. ACTIVITY STUDIES ON ENZYME SUSPENSIONS IN ORGANIC SOLVENTS 723
3. INACTIVATION IN W/O MICROEMULSIONS 725
4. INACTIVATION IN LIQUID-SOLID TWO PHASE SUSPENSIONS 728
Acknowledgements 729
Abbreviations 729
REFERENCES 729
Chapter 5.Batch and continuous lipolysis/product separation in a reversed micellar membrane bioreactor 730
1. INTRODUCTION 730
2. MATERIALS AND METHODS 731
3. RESULTS 733
4. CONCLUSIONS 735
5. REFERENCES 735
ACKNOWLEDGEMENTS 735
Chapter 6.Synthesis of fatty acid esters by a recombinant cutinase in reversed micelles 736
1. INTRODUCTION 736
2.MATERIALS AND METHODS 737
3. RESULTS AND DISCUSSION 737
Acknowledgements 741
4. REFERENCES 741
Chapter 7.Application of fractional factorial design to the study of enzymatic dipeptide synthesis in reverse micelles 742
INTRODUCTION 742
2. MATERIALS AND METHODS 743
3.RESULTS AND DISCUSSION 744
4. CONCLUSIONS 749
5. REFERENCES 749
Chapter 8.Studies on the specificity of Pénicillium simplicissimum lipase catalyzed 
750 
1. INTRODUCTION 750
2. MATERIALS AND METHODS 751
3. RESULTS AND DISCUSSION 752
ACKNOWLEDGMENTS 754
REFERENCES 755
Chapter 9.Enzymes entrapped in liquid crystals - a novel approach for biocatalysis in non-aqueous media 756
1. INTRODUCTION 756
2. T H E MODEL REACTIONS 757
3. MATERIALS AND METHODS 759
4. RESULTS AND DISCUSSION 759
5. REFERENCES 762
Chapter 10.Synthesis of phosphatidylcholine with polyunsaturated fatty acids by phospholipase A2 in an organic solvent 764
INTRODUCTION 764
MATERIAL AND METHODS 765
RESULTS AND DISCUSSION 767
CONCLUSIONS 769
REFERENCES 770
ACKNOWLEDGEMENTS 770
Chapter 11.Catalysis of polyphenol oxidase in a ternary system of reverse vesicles in organic solvents 772
1. INTRODUCTION 772
2. MATERIALS AND METHODS 773
3 . RESULTS AND DISCUSSION 774
4. ACKNOWLEDGEMENTS 777
5. REFERENCES 777
AUTHOR INDEX 778