Enzyme Engineering : Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science

Enzyme Engineering An authoritative and up-to-date discussion of enzyme engineering and its applications In Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science , a team of distinguished researchers deliver a robust treatment of enzyme engineering and its applications in various fields such as biotechnology, life science, and synthesis. The book begins with an introduction to different protein engineering techniques, covers topics like gene mutagenesis methods for directed evolution and rational enzyme design. It includes industrial case studies of enzyme engineering with a focus on selectivity and activity. The authors also discuss new and innovative areas in the field, involving machine learning and artificial intelligence. It offers several insightful perspectives on the future of this work. Readers will also find: A thorough introduction to directed evolution and rational design as protein engineering techniques Comprehensive explorations of screening and selection techniques, gene mutagenesis methods in directed evolution, and guidelines for applying gene mutagenesis in organic chemistry, pharmaceutical applications, and biotechnology Practical discussions of protein engineering of enzyme robustness relevant to organic and pharmaceutical chemistry Treatments of artificial enzymes as promiscuous catalysts Various lessons learned from semi-rational and rational directed evolution A transdisciplinary treatise, Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science is perfect for protein engineers, theoreticians, organic, and pharmaceutical chemists as well as transition metal researchers in catalysis and biotechnologists. Cover 1 Title Page 5 Copyright 6 Contents 7 Preface 11 About the Authors 13 Chapter 1 Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques 15 1.1 Methods and Aims of Directed Enzyme Evolution 15 1.2 History of Directed Enzyme Evolution 18 1.3 Methods and Aims of Rational Design of Enzymes 33 References 35 Chapter 2 Screening and Selection Techniques 43 2.1 Introductory Remarks 43 2.2 Screening Methods 43 2.3 Selection Methods 52 2.4 Conclusions and Perspectives 66 References 67 Chapter 3 Gene Mutagenesis Methods in Directed Evolution and Rational Enzyme Design 73 3.1 Introductory Remarks 73 3.2 Directed Evolution Approaches 73 3.2.1 Mutator Strains 73 3.2.2 Error‐Prone Polymerase Chain Reaction (epPCR) 74 3.2.3 Whole Gene Insertion/Deletion Mutagenesis 80 3.2.4 Saturation Mutagenesis as a Privileged Method: Away from Blind Directed Evolution 87 3.2.5 DNA Shuffling and Related Recombinant Gene Mutagenesis Methods 103 3.2.6 Circular Mutation and Other Domain Swapping Techniques 108 3.2.7 Solid‐Phase Combinatorial Gene Synthesis as a PCR‐Independent Mutagenesis Method for Mutant Library Creation 110 3.2.7.1 Introductory Remarks 110 3.2.7.2 The Sloning Approach to Solid‐Phase Gene Synthesis of a Mutant Library: Comparison with the Respective Molecular Biological Saturation Mutagenesis Library 111 3.2.7.3 The Twist Approach to Solid‐Phase Gene Synthesis of a Mutant Library: Comparison with Molecular Biological Saturation Mutagenesis Library 113 3.2.8 Computational Tools and the Role of Machine Learning (ML) in Directed Evolution and Rational Enzyme Design 116 3.2.8.1 Introductory Remarks 116 3.2.8.2 Designing Mutant Libraries and Estimating Library Completeness 117 3.3 Diverse Approaches to Rational Enzyme Design 126 3.3.1 Introductory Remarks 126 3.4 Merging Semi‐rational Directed Evolution and Rational Enzyme Design by Focused Rational Iterative Site‐Specific Mutagenesis (FRISM) 128 3.5 Conclusions and Perspectives 134 References 134 Chapter 4 Guidelines for Applying Gene Mutagenesis Methods in Organic Chemistry, Pharmaceutical Applications, and Biotechnology 155 4.1 Some General Tips 155 4.1.1 Rational Design 155 4.1.2 Directed Evolution 163 4.2 Rare Cases of Comparative Directed Evolution Studies 166 4.2.1 Converting a Galactosidase into a Fucosidase 166 4.2.2 Enhancing and Inverting the Enantioselectivity of the Lipase from Pseudomonas aeruginosa (PAL) 170 4.3 Choosing the Best Strategy When Applying Saturation Mutagenesis 177 4.3.1 General Guidelines 177 4.3.2 Choosing Optimal Pathways in Iterative Saturation Mutagenesis (ISM) and Escaping from Local Minima in Fitness Landscapes 182 4.3.3 Systematization of Saturation Mutagenesis with Further Practical Tips 188 4.3.4 Single Code Saturation Mutagenesis (SCSM): Use of a Single Amino Acid as Building Block 197 4.3.5 Triple Code Saturation Mutagenesis (TCSM): A Viable Compromise When Choosing Optimal Reduced Amino Acid Alphabets in CAST/ISM 199 4.4 Techno‐economical Analysis of Saturation Mutagenesis Strategies 201 4.5 Generating Mutant Libraries by Combinatorial Solid‐Phase Gene Synthesis: The Future of Directed Evolution? 204 4.6 Fusing Directed Evolution and Rational Design: New Examples of Focused Rational Iterative Site‐Specific Mutagenesis (FRISM) 206 References 208 Chapter 5 Tables of Selected Examples of Directed Evolution and Rational Design of Enzymes with Emphasis on Stereo‐ and Regio‐selectivity, Substrate Scope and/or Activity 217 5.1 Introductory Explanations 217 References 234 Chapter 6 Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology 247 6.1 Introductory Remarks 247 6.2 Rational Design of Enzyme Thermostability and Resistance to Hostile Organic Solvents 248 6.3 Ancestral and Consensus Approaches and Their Structure‐Guided Extensions 255 6.4 Further Computationally Guided Methods for Protein Thermostabilization 256 6.4.1 SCHEMA Approach 257 6.4.2 FRESCO Approach 259 6.4.3 FireProt Approach 261 6.4.4 Constrained Network Analysis (CNA) Approach 263 6.4.5 Alternative Approaches 265 6.5 Directed Evolution of Enzyme Thermostability and Resistance to Hostile Organic Solvents 267 6.6 Application of epPCR and DNA Shuffling 269 6.7 Saturation Mutagenesis in the B‐FIT Approach 272 6.8 Iterative Saturation Mutagenesis (ISM) at Protein–Protein Interfacial Sites for Multimeric Enzymes 277 6.9 Conclusions and Perspectives 279 References 279 Chapter 7 Artificial Enzymes as Promiscuous Catalysts in Organic and Pharmaceutical Chemistry 293 7.1 Introductory Background Information 293 7.2 Applying Protein Engineering for Tuning the Catalytic Profile of Promiscuous Enzymes 299 7.3 Applying Protein Engineering to P450 Monooxygenases for Manipulating Activity and Stereoselectivity of Promiscuous Transformations 313 7.4 Conclusions and Perspectives 321 References 322 Chapter 8 Learning Lessons from Protein Engineering 331 8.1 Introductory Remarks 331 8.2 Additive Versus Nonadditive Mutational Effects in Fitness Landscapes Revealed by Partial or Complete Deconvolution 332 8.3 Unexplored Chiral Fleeting Intermediates and Their Role in Protein Engineering 341 8.4 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo‐ and/or Regioselectivity 343 8.4.1 Esterase 343 8.4.2 Epoxide Hydrolase 345 8.4.3 Ene‐reductase of the Old Yellow Enzyme (OYE) 349 8.4.4 Cytochrome P450 Monooxygenase 357 8.4.5 Analysis of Baeyer–Villiger Monooxygenase with Consideration of Fleeting Chiral Intermediates 364 8.5 Conclusions and Suggestions for Further Theoretical Work 372 References 374 Chapter 9 Perspectives for Future Work 381 9.1 Introductory Remarks 381 9.2 Extending Applications in Organic and Pharmaceutical Chemistry 381 9.3 Extending Applications in Biotechnology 386 9.4 Patent Issues 390 9.5 Final Comments 390 References 391 INDEX 395 EULA 402