Directed Evolution Of Selective Enzymes

Authored by one of the world's leading organic chemists, this authoritative reference provides an overview of basic strategies in directed evolution and introduces common gene mutagenesis, screening and selection methods. Throughout the text, emphasis is placed on methodology development to maximize efficiency, reliability and speed of the experiments and to provide guidelines for efficient protein engineering. Professor Reetz highlights the application of directed evolution experiments to address limitations in the field of enzyme selectivity, substrate scope, activity and robustness. He critically reviews recent developments and case studies, takes a look at future applications in the field of organic synthesis, and concludes with lessons learned from previous experiments.

This book focuses on some of the most significant advances in enzyme engineering that have been achieved through directed evolution and hybrid approaches. On the 25th anniversary of the discovery of directed evolution, this volume is a tribute to the pioneers of this thrilling research field, and at the same time provides a comprehensive overview of current research and the state of the art. Directed molecular evolution has become the most reliable and robust method to tailor enzymes, metabolic pathways or even whole microorganisms with improved traits. By mirroring the Darwinian algorithm of natural selection on a laboratory scale, new biomolecules of invaluable biotechnological interest can now be engineered in a manner that surpasses the boundaries of nature. The volume is divided into two sections, the first of which provides an update on recent successful cases of enzyme ensembles from different areas of the biotechnological spectrum, including tryptophan synthases, unspecific peroxygenases, phytases, therapeutic enzymes, stereoselective enzymes and CO2-fixing enzymes. This section also provides information on the directed evolution of whole cells. The second section of the book summarizes a variety of the most applicable methods for library creation, together with the future trends aimed at bringing together directed evolution and in silico/computational enzyme design and ancestral resurrection.

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

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.

A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.

Biocatalysis has become an essential tool in the chemical industry and is the core of industrial biotechnology, also known as white biotechnology, making use of biocatalysts in terms of enzymes or whole cells in chemical processes as an alternative to chemical catalysts. This shift can be seen in the many areas of daily life where biocatalysts—with their environmentally friendly properties—are currently employed. Drivers are the big societal challenges resulting from concerns about the global climate change and the need for an assured energy supply. Modern biocatalysis relies to a large extent on the tremendous advances in the so-called omics techniques and the structural elucidation of biomolecules, which have led to synthetic biology and metabolic engineering as new research fields with high application potential for the rational design of enzymes and microbial production strains. In this book, renowned scientists discuss the actual developments in these research fields together with a variety of application-oriented topics.

Miniturization and high throughput assay technology have brought the power of molecular evolution to the bioscience laboratory. Applied wisely, the evolutionary approach can quickly yield the desired result even where other methods have failed. From library generation by random or directed mutagenesis to screening and selection techniques -- the crucial steps for successful evolutionary biotechnology are described in detail in this practical guide that also includes valuable troubleshooting hints on frequently encountered problems. Modern methods for the surface display of peptides and proteins, selective enrichment of nucleic acid aptamers and high-throughput screening of industrial biocatalysts are explained, and computer-based methods for in silico protein and RNA engineering are described as an alternative to in vitro approaches. A special section covers the patenting regulations with regard to biotechnological innovations derived from directed evolution. As an added bonus, a CD-ROM is included that contains software tools for library design, selection of mutagenesis positions, and various predictive algorithms. In short, this practice oriented handbook is an indispensable tool for every scientist working in this interdisciplinary research area.

The Arthur M. Sackler Colloquia of the National Academy of Sciences address scientific topics of broad and current interest, cutting across the boundaries of traditional disciplines. Each year, four or five such colloquia are scheduled, typically two days in length and international in scope. Colloquia are organized by a member of the Academy, often with the assistance of an organizing committee, and feature presentations by leading scientists in the field and discussions with a hundred or more researchers with an interest in the topic. Colloquia presentations are recorded and posted on the National Academy of Sciences Sackler colloquia website and published on CD-ROM. These Colloquia are made possible by a generous gift from Mrs. Jill Sackler, in memory of her husband, Arthur M. Sackler.