Part I: Enzyme Libraries Preparation and Screening
1. Preparation of Soil Metagenome Libraries and Screening for Gene Specific Amplicons
Luke J. Stevenson, David F. Ackerley, and Jeremy G. Owen
2. Ultrahigh-Throughput Screening of Metagenomic Libraries Using Droplet Microfluidics
Davide Agostino Cecchini, Mercedes Sánchez-Costa, Alejandro H. Orrego, Jesús Fernández-Lucas, and Aurelio Hidalgo
3. Synthetic DNA Libraries for Protein Engineering toward Process Improvement in Drug Synthesis
Michele Tavanti
Part II: Directed Evolution
4. In Silico Prediction Methods for Site-Saturation Mutagenesis
Ge Qu and Zhoutong Sun
5. Recombination of Compatible Substitutions by 2GenReP and InSiReP
Haiyang Cui, Mehdi D. Davari, and Ulrich Schwaneberg
Part III: Semi-Rational and De Novo Design
6. Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP)
Connie M. Ross, Gabriel Foley, Mikael Boden, and Elizabeth M.J. Gillam
7. Resurrecting Enzymes by Ancestral Sequence Reconstruction
Maria Laura Mascotti
8. Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors
Paul Curnow and J.L. Ross Anderson
Part IV: Rational Design
9. Rational-Design Engineering to Improve Enzyme Thermostability
Vinutsada Pongsupasa, Piyanuch Anuwan, Somchart Maenpuen, and Thanyaporn Wongnate
10. Using Molecular Simulation to Guide Protein Engineering for Biocatalysis in Organic Solvents
Haiyang Cui, Markus Vedder, Ulrich Schwaneberg, and Mehdi D. Davari
11. In Silico Engineering of Enzyme Access Tunnels
Alfonso Gautieri, Federica Rigoldi, Archimede Torretta, Alberto Redaelli, and Emilio Parisini
12. Quantum-Mechanical/Molecular-Mechanical (QM/MM) Simulations for Understanding Enzyme Dynamics
Rimsha Mehmood and Heather J. Kulik
13. Computational Enzyme Design at Zymvol
Emanuele Monza, Victor Gil, and Maria Fatima Lucas
Part V: Biocatalytic Process Development
14. Advanced Enzyme Immobilization Technologies: An Eco-Friendly Support, a Polymer-Stabilizing Immobilization Strategy, and an Improved Cofactor Co-Immobilization Technique
Ana I. Benítez-Mateos and Francesca Paradisi
15. Chemical Reaction Engineering to Understand Applied Kinetics in Free Enzyme Homogeneous Reactors
This book provides comprehensive methods and protocols about enzyme design. The chapters are grouped by main topic, starting with methodologies describing library preparation and screening, state of the art techniques in directed evolution and rational design, followed by examples of immobilization of enzymes on sustainable polymers, as well as biocatalytic conversions mediated by homogenous enzymatic preparations or whole cells. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and timely, Enzyme Engineering: Methods and Protocols is an ideal guide for both the novice and the veteran researcher interested in biocatalysis.
Chapter 13 is available open access under a CC BY 4.0 license.
