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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.
List of contents
Preparation of Soil Metagenome Libraries and Screening for Gene Specific Amplicons.- Ultrahigh-Throughput Screening of Metagenomic Libraries Using Droplet Microfluidics.- Synthetic DNA Libraries for Protein Engineering toward Process Improvement in Drug Synthesis.- In Silico Prediction Methods for Site-Saturation Mutagenesis.- Recombination of Compatible Substitutions by 2GenReP and InSiReP.- Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP).- Resurrecting Enzymes by Ancestral Sequence Reconstruction.- Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors.- Rational-Design Engineering to Improve Enzyme Thermostability.- Using Molecular Simulation to Guide Protein Engineering for Biocatalysis in Organic Solvents.- In Silico Engineering of Enzyme Access Tunnels.- Quantum-Mechanical/Molecular-Mechanical (QM/MM) Simulations for Understanding Enzyme Dynamics.- Computational Enzyme Design at Zymvol.- Advanced Enzyme Immobilization Technologies: An Eco-Friendly Support, a Polymer-Stabilizing Immobilization Strategy, and an Improved Cofactor Co-Immobilization Technique.- Chemical Reaction Engineering to Understand Applied Kinetics in Free Enzyme Homogeneous Reactors.- Enzymatic Oxidative Cascade for Oxofunctionalization of Fatty Acids in One-Pot.- CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440.
