Foreword ....................................................... ix
1.Introduction .................................................. 1
Andrés Illanes
1.1.Catalysis and Biocatalysis ................................ 1
1.2.Enzymes as Catalysts. Structure-Functionality
Relationships ............................................. 4
1.3.The Concept and Determination of Enzyme Activity .......... 8
1.4.Enzyme Classes. Properties and Technological
Significance ............................................. 16
1.5.Applications of Enzymes. Enzyme as Process Catalysts ..... 19
1.6.Enzyme Processes: the Evolution from Degradation to
Synthesis. Biocatalysis in Aqueous and
Non-conventional Media ................................... 31
References ................................................... 39
2.Enzyme Production ............................................ 57
Andrés Illanes
2.1.Enzyme Sources ........................................... 57
2.2.Production of Enzymes .................................... 60
2.2.1.Enzyme Synthesis ................................... 61
2.2.2.Enzyme Recovery .................................... 65
2.2.3.Enzyme Purification ................................ 74
2.2.4.Enzyme Formulation ................................. 84
References ..................................................... 89
3.Homogeneous Enzyme Kinetics ................................. 107
Andrés Illanes, Claudia Altamirano, and Lorena Wilson
3.1.General Aspects ......................................... 107
3.2.Hypothesis of Enzyme Kinetics. Determination of
Kinetic Parameters ...................................... 108
3.2.1.Rapid Equilibrium and Steady-State Hypothesis ..... 108
3.2.2.Determination of Kinetic Parameters for
Irreversible and Reversible One-Substrate
Reactions ......................................... 112
3.3.Kinetics of Enzyme Inhibition ........................... 116
3.3.1.Types of Inhibition ............................... 116
3.3.2.Development of a Generalized Kinetic Model
for One-Substrate Reactions Under Inhibition ...... 117
3.3.3 Determination of Kinetic Parameters for
One-Substrate Reactions Under Inhibition .......... 120
3.4.Reactions with More than One Substrate .................. 124
3.4.1.Mechanisms of Reaction ............................ 124
3.4.2.Development of Kinetic Models ..................... 125
3.4.3.Determination of Kinetic Parameters ............... 131
3.5 Environmental Variables in Enzyme Kinetics .............. 133
3.5.1.Effect of pH: Hypothesis of Michaelis and
Davidsohn. Effect on Enzyme Affinity and
Reactivity ........................................ 134
3.5.2.Effect of Temperature: Effect on Enzyme
Affinity, Reactivity and Stability ................ 140
3.5.3 Effect of Ionic Strength .......................... 148
References .................................................. 151
4.Heterogeneous Enzyme Kinetics ............................... 155
Andrés Illanes, Roberto Fernández-Lafuente,
Jose M.Guisán, and Lorena Wilson
4.1.Enzyme Immobilization ................................... 155
4.1.1.Methods of Immobilization ......................... 156
4.1.2.Evaluation of Immobilization ...................... 166
4.2.Heterogeneous Kinetics: Apparent, Inherent and
Intrinsic Kinetics; Mass Transfer Effects in
Heterogeneous Biocatalysis .............................. 169
4.3.Partition Effects ....................................... 171
4.4.Diffusional Restrictions ................................ 172
4.4.1.External Diffusional Restrictions ................. 173
4.4.2.Internal Diffusional Restrictions ................. 181
4.4.3.Combined Effect of External and Internal
Diffusional Restrictions .......................... 192
References .................................................. 197
5.Enzyme Reactors ............................................. 205
Andrés Illanes and Claudia Altamirano
5.1.Types of Reactors, Modes of Operation ................... 205
5.2.Basic Design of Enzyme Reactors ......................... 207
5.2.1.Design Fundamentals ............................... 207
5.2.2.Basic Design of Enzyme Reactors Under Ideal
Conditions. Batch Reactor; Continuous Stirred
Tank Reactor Under Complete Mixing; Continuous
Packed-Bed Reactor Under Plug Flow Regime ......... 209
5.3.Effect of Diffusional Restrictions on Enzyme
Reactor Design and Performance in Heterogeneous
Systems. Determination of Effectiveness Factors.
Batch Reactor; Continuous Stirred Tank Reactor
Under Complete Mixing; Continuous Packed-Bed
Reactor Under Plug Flow Regime .......................... 223
5.4.Effect of Thermal Inactivation on Enzyme Reactor
Design and Performance .................................. 224
5.4.1.Complex Mechanisms of Enzyme Inactivation ......... 225
5.4.2.Effects of Modulation on Thermal Inactivation ..... 231
5.4.3.Enzyme Reactor Design and Performance Under
Non-Modulated and Modulated Enzyme Thermal
Inactivation ...................................... 234
5.4.4.Operation of Enzyme Reactors Under
Inactivation and Thermal Optimization ............. 240
5.4.5.Enzyme Reactor Design and Performance
Under Thermal Inactivation and Mass Transfer
Limitations ....................................... 245
References .............................................. 248
6.Study Cases of Enzymatic Processes .......................... 253
6.1.Proteases as Catalysts for Peptide Synthesis ............ 253
Sonia Barberis, Fanny Guzmán, Andrés Illanes, and
Joseph López-Santín
6.1.1.Chemical Synthesis of Peptides .................... 254
6.1.2.Proteases as Catalysts for Peptide Synthesis ...... 257
6.1.3.Enzymatic Synthesis of Peptides ................... 258
6.1.4.Process Considerations for the Synthesis of
Peptides .......................................... 263
6.1.5.Concluding Remarks ................................ 267
References .............................................. 268
6.2 Synthesis of β-Lactam Antibiotics with Penicillin
Acylases ................................................ 273
Andrés Illanes and Lorena Wilson
6.2.1.Introduction ...................................... 274
6.2.2.Chemical Versus Enzymatic Synthesis of
Semi-Synthetic β-Lactam Antibiotics ............... 274
6.2.3.Strategies of Enzymatic Synthesis ................. 276
6.2.4.Penicillin Acylase Biocatalysts ................... 277
6.2.5.Synthesis of β-Lactam Antibiotics in
Homogeneous and Heterogeneous Aqueous
and Organic Media ................................. 279
6.2.6.Model of Reactor Performance for the
Production of Semi-Synthetic β-Lactam
Antibiotics ....................................... 282
References .............................................. 285
6.3 Chimioselective Esterification of Wood Sterols with
Lipases ................................................. 292
Gregorio Álvaro and Andrés Illanes
6.3.1.Sources and Production of Lipases ................. 293
6.3.2.Structure and Functionality of Lipases ............ 296
6.3.3 Improvement of Lipases by Medium and
Biocatalyst Engineering ........................... 299
6.3.4.Applications of Lipases ........................... 304
6.3.5.Development of a Process for the Selective
Transesteritication of the Stanol Fraction
of Wood Sterols with Immobilized Lipases .......... 308
References .............................................. 315
6.4.Oxidoreductases as Powerful Biocatalysts for
Green Chemistry ......................................... 323
Jose M. Guisán, Roberto Fernández-Lafuente,
Lorena Wilson, and César Mateo
6.4.1.Mild and Selective Oxidations Catalyzed by
Oxidases .......................................... 324
6.4.2.Redox Biotransformations Catalyzed by
Dehydrogenases .................................... 326
6.4.3.Immobilization-Stabilization of Dehydrogenases .... 329
6.4.4.Reactor Engineering ............................... 330
6.4.5.Production of Long-Chain Fatty Acids with
Dehydrogenases .................................... 331
References .............................................. 332
6.5.Use of Aldolases for Asymmetric Synthesis ............... 333
Josep López-Santfn, Gregorio Álvaro, and Pere Gapés
6.5.1.Aldolases: Definitions and Classification ......... 334
6.5.2.Preparation of Aldolase Biocatalysts .............. 335
6.5.3.Reaction Performance: Medium Engineering
and Kinetics ...................................... 339
6.5.4.Synthetic Applications ............................ 346
6.5.5.Conclusions ....................................... 352
References .............................................. 352
6.6.Application of Enzymatic Reactors for the
Degradation of Highly and Poorly Soluble
Recalcitrant Compounds .................................. 355
Juan M.Lema, Gemma Eibes, Carmen Lopez,
M.Teresa Moreira, and Gumersindo Feijoo
6.6.1.Potential Application of Oxidative Enzymes
for Environmental Purposes ........................ 355
6.6.2.Requirements for an Efficient Catalytic Cycle ..... 357
6.6.3.Enzymatic Reactor Configurations .................. 358
6.6.4.Modeling of Enzymatic Reactors .................... 364
6.6.5.Case Studies ...................................... 365
6.6.6.Conclusions and Perspectives ...................... 374
References ................................................. 375
Index ...................................................... 379
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