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

Cover
Title Page
Copyright
Contents
Preface
About the Authors
Chapter 1 Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques
	1.1 Methods and Aims of Directed Enzyme Evolution
	1.2 History of Directed Enzyme Evolution
	1.3 Methods and Aims of Rational Design of Enzymes
	References
Chapter 2 Screening and Selection Techniques
	2.1 Introductory Remarks
	2.2 Screening Methods
	2.3 Selection Methods
	2.4 Conclusions and Perspectives
	References
Chapter 3 Gene Mutagenesis Methods in Directed Evolution and Rational Enzyme Design
	3.1 Introductory Remarks
	3.2 Directed Evolution Approaches
		3.2.1 Mutator Strains
		3.2.2 Error‐Prone Polymerase Chain Reaction (epPCR)
		3.2.3 Whole Gene Insertion/Deletion Mutagenesis
		3.2.4 Saturation Mutagenesis as a Privileged Method: Away from Blind Directed Evolution
		3.2.5 DNA Shuffling and Related Recombinant Gene Mutagenesis Methods
		3.2.6 Circular Mutation and Other Domain Swapping Techniques
		3.2.7 Solid‐Phase Combinatorial Gene Synthesis as a PCR‐Independent Mutagenesis Method for Mutant Library Creation
			3.2.7.1 Introductory Remarks
			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
			3.2.7.3 The Twist Approach to Solid‐Phase Gene Synthesis of a Mutant Library: Comparison with Molecular Biological Saturation Mutagenesis Library
		3.2.8 Computational Tools and the Role of Machine Learning (ML) in Directed Evolution and Rational Enzyme Design
			3.2.8.1 Introductory Remarks
			3.2.8.2 Designing Mutant Libraries and Estimating Library Completeness
	3.3 Diverse Approaches to Rational Enzyme Design
		3.3.1 Introductory Remarks
	3.4 Merging Semi‐rational Directed Evolution and Rational Enzyme Design by Focused Rational Iterative Site‐Specific Mutagenesis (FRISM)
	3.5 Conclusions and Perspectives
	References
Chapter 4 Guidelines for Applying Gene Mutagenesis Methods in Organic Chemistry, Pharmaceutical Applications, and Biotechnology
	4.1 Some General Tips
		4.1.1 Rational Design
		4.1.2 Directed Evolution
	4.2 Rare Cases of Comparative Directed Evolution Studies
		4.2.1 Converting a Galactosidase into a Fucosidase
		4.2.2 Enhancing and Inverting the Enantioselectivity of the Lipase from Pseudomonas aeruginosa (PAL)
	4.3 Choosing the Best Strategy When Applying Saturation Mutagenesis
		4.3.1 General Guidelines
		4.3.2 Choosing Optimal Pathways in Iterative Saturation Mutagenesis (ISM) and Escaping from Local Minima in Fitness Landscapes
		4.3.3 Systematization of Saturation Mutagenesis with Further Practical Tips
		4.3.4 Single Code Saturation Mutagenesis (SCSM): Use of a Single Amino Acid as Building Block
		4.3.5 Triple Code Saturation Mutagenesis (TCSM): A Viable Compromise When Choosing Optimal Reduced Amino Acid Alphabets in CAST/ISM
	4.4 Techno‐economical Analysis of Saturation Mutagenesis Strategies
	4.5 Generating Mutant Libraries by Combinatorial Solid‐Phase Gene Synthesis: The Future of Directed Evolution?
	4.6 Fusing Directed Evolution and Rational Design: New Examples of Focused Rational Iterative Site‐Specific Mutagenesis (FRISM)
	References
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
	5.1 Introductory Explanations
	References
Chapter 6 Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology
	6.1 Introductory Remarks
	6.2 Rational Design of Enzyme Thermostability and Resistance to Hostile Organic Solvents
	6.3 Ancestral and Consensus Approaches and Their Structure‐Guided Extensions
	6.4 Further Computationally Guided Methods for Protein Thermostabilization
		6.4.1 SCHEMA Approach
		6.4.2 FRESCO Approach
		6.4.3 FireProt Approach
		6.4.4 Constrained Network Analysis (CNA) Approach
		6.4.5 Alternative Approaches
	6.5 Directed Evolution of Enzyme Thermostability and Resistance to Hostile Organic Solvents
	6.6 Application of epPCR and DNA Shuffling
	6.7 Saturation Mutagenesis in the B‐FIT Approach
	6.8 Iterative Saturation Mutagenesis (ISM) at Protein–Protein Interfacial Sites for Multimeric Enzymes
	6.9 Conclusions and Perspectives
	References
Chapter 7 Artificial Enzymes as Promiscuous Catalysts in Organic and Pharmaceutical Chemistry
	7.1 Introductory Background Information
	7.2 Applying Protein Engineering for Tuning the Catalytic Profile of Promiscuous Enzymes
	7.3 Applying Protein Engineering to P450 Monooxygenases for Manipulating Activity and Stereoselectivity of Promiscuous Transformations
	7.4 Conclusions and Perspectives
	References
Chapter 8 Learning Lessons from Protein Engineering
	8.1 Introductory Remarks
	8.2 Additive Versus Nonadditive Mutational Effects in Fitness Landscapes Revealed by Partial or Complete Deconvolution
	8.3 Unexplored Chiral Fleeting Intermediates and Their Role in Protein Engineering
	8.4 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo‐ and/or Regioselectivity
		8.4.1 Esterase
		8.4.2 Epoxide Hydrolase
		8.4.3 Ene‐reductase of the Old Yellow Enzyme (OYE)
		8.4.4 Cytochrome P450 Monooxygenase
		8.4.5 Analysis of Baeyer–Villiger Monooxygenase with Consideration of Fleeting Chiral Intermediates
	8.5 Conclusions and Suggestions for Further Theoretical Work
	References
Chapter 9 Perspectives for Future Work
	9.1 Introductory Remarks
	9.2 Extending Applications in Organic and Pharmaceutical Chemistry
	9.3 Extending Applications in Biotechnology
	9.4 Patent Issues
	9.5 Final Comments
	References
INDEX
EULA