Biocatalysts and Enzyme Technology

Klaus Buchholz, Volker Kasche, Uwe Theo Bornscheuer

• Publisher: Wiley-Blackwell
• Year: 2012
• Print ISBN: 9783527329892
• eBook ISBN: 9783527672004
• Edition: 2
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

This second edition of a bestselling textbook offers an instructive and comprehensive overview of our current knowledge of biocatalysis and enzyme technology.
The book now contains about 40% more printed content. Three chapters are completely new, while the others have been thoroughly updated, and a section with problems and solutions as well as new case studies have been added.
Following an introduction to the history of enzyme applications, the text goes on to cover in depth enzyme mechanisms and kinetics, production, recovery,
characterization and design by protein engineering. The authors treat a broad range of applications of soluble and immobilized biocatalysts, including wholecell
systems, the use of non-aqueous reaction systems, applications in organic synthesis, bioreactor design and reaction engineering. Methods to estimate the
sustainability, important internet resources and their evaluation, and legislation concerning the use of biocatalysts are also covered.

1

Introduction to Enzyme Technology

1.1 Introduction

Biotechnology offers an increasing potential for the production of goods to meet various human needs. In enzyme technology – a subfield of biotechnology – new processes have been and are being developed to manufacture both bulk and high added-value products utilizing enzymes as biocatalysts, in order to meet needs such as food (e.g., bread, cheese, beer, vinegar), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals. Enzymes are also used to provide services, as in washing and environmental processes, or for analytical and diagnostic purposes. The driving force in the development of enzyme technology, both in academia and in industry, has been and will continue to be

• the development of new and better products, processes, and services to meet these needs, and/or
• the improvement of processes to produce existing products from new raw materials such as biomass.

The goal of these approaches is to design innovative products and processes that not only are competitive but also meet criteria of sustainability. The concept of sustainability was introduced by the World Commission on Environment and Development (WCED, 1987) with the aim to promote a necessary “...development that meets the needs of the present without compromising the ability of future generations to meet their own needs.” This definition is now part of the Cartagena Protocol on Biosafety to the Convention on Biological Diversity, an international treaty governing the movements of living modified organisms (LMOs) resulting from modern biotechnology from one country to another. It was adopted on January 29, 2000 as a supplementary agreement to the Convention on Biological Diversity and entered into force on September 11, 2003 (http://bch.cbd.int/protocol/text/). It has now been ratified by 160 states. To determine the sustainability of a process, criteria that evaluate its economic, environmental, and social impact must be used (Gram et al., 2001; Raven, 2002; Clark and Dickson, 2003). A positive effect in all these three fields is required for a sustainable process. Criteria for the quantitative evaluation of the economic and environmental impact are in contrast with the criteria for the social impact, easy to formulate. In order to be economically and environmentally more sustainable than an existing process, a new process must be designed not only to reduce the consumption of resources (e.g., raw materials, energy, air, water), waste production, and environmental impact, but also to increase the recycling of waste per kilogram of product (Heinzle, Biwer, and Cooney, 2006).

1.1.1 What are Biocatalysts?

Biocatalysts either are proteins (enzymes) or, in a few cases, may be nucleic acids (ribozymes; some RNA molecules can catalyze the hydrolysis of RNA). These ribozymes were detected in the 1980s and will not be dealt with here (Cech, 1993). Today, we know that enzymes are necessary in all living systems, to catalyze all chemical reactions required for their survival and reproduction – rapidly, selectively, and efficiently. Isolated enzymes can also catalyze these reactions. In the case of enzymes, however, the question whether they can also act as catalysts outside living systems had been a point of controversy among biochemists in the beginning of the twentieth century. It was shown at an early stage, however, that enzymes could indeed be used as catalysts outside living cells, and several processes in which they were applied as biocatalysts have been patented (see Section 1.3).

These excellent properties of enzymes are utilized in enzyme technology. For example, they can be used as biocatalysts, either as isolated enzymes or as enzyme systems in living cells, to catalyze chemical reactions on an industrial scale in a sustainable manner. Their application covers the production of desired products for all human material needs (e.g., food, animal feed, pharmaceuticals, bulk and fine chemicals, detergents, fibers for clothing, hygiene, and environmental technology), as well as for a wide range of analytical purposes, especially in diagnostics. In fact, during the past 50 years the rapid increase in our knowledge of enzymes – as well as their biosynthesis and molecular biology – now allows their rational use as biocatalysts in many processes, and in addition their modification and optimization for new synthetic schemes and the solution of analytical problems.

This introductory chapter outlines the technical and economic potential of enzyme technology as part of biotechnology. Briefly, it describes the historical background of enzymes, as well as their advantages and disadvantages, and compares these to alternative production processes. In addition, the current and potential importance and the problems to consider in the rational design of enzyme processes are also outlined.

1.1.2 Bio- and Chemocatalysts – Similarities and Differences

Berzelius, in 1835, conceived the pioneering concept of catalysis, including both chemo- and biocatalysis, by inorganic acids, metals such as platinum, and enzymes (Berzelius, 1835). It was based on experimental studies on both bio- and chemocatalytic reactions. The biocatalytic system he studied was starch hydrolysis by diastase (a mixture of amylases). In both systems, the catalyst accelerates the reaction, but is not consumed. Thus, bio- and chemocatalysis have phenomenological similarities. The main differences are the sources and characteristics of these catalysts. Chemocatalysts are designed and synthesized by chemists, and are in general low molecular weight substances, metal catalysts, complexes of metals with low molecular weight organic ligands, such as Ziegler-Natta and metallocene catalysts, and organocatalysts (Fonseca and List, 2004). In contrast, biocatalysts are selected by evolution and synthesized in living systems. Furthermore, enzymes (including ribonucleic acid-based biocatalysts) are macromolecules, their highly sophisticated structure being essential for their function, and notably for their regio-, chemo-, and enantioselectivity.

Due to development of gene and recombinant technologies in the past 40 years, enzymes that previously only could be obtained in limited amounts from microorganisms and tissues can now be synthesized in nearly unlimited quantities in suitable microorganisms. Further, based on the development in biochemistry, bioinformatics, and micro- and molecular biology, new tools have been developed to improve the properties of enzymes for their use in biocatalytic processes. They are rational protein design and in vitro evolution in combination with high-throughput screening tools. Very recently, also the de novo computational design of enzymes was described, but so far these show little activity in the same range as catalytic antibodies (Jiang et al., 2008; Röthlisberger et al., 2008).

Until the first oil crisis of 1973, the development and application of bio- and chemocatalysis occurred in – at that time – nonoverlapping fields. Biocatalysis was mainly studied by biochemists, biochemical engineers, microbiologists, physiologists, and some physical organic chemists (Jencks, 1969). It was mainly applied in the food, fine chemical, and pharmaceutical industries and medicine (see Section 1.3). Chemocatalysis was mainly studied by chemical engineers and chemists. It was applied in the production of bulk chemicals such as acids and bases, and products derived from coal and oil (fuel, plastics, etc.). This resulted for a long time in a small exchange of fundamental results between those who studied and applied bio- and chemocatalysis. The analytical description of heterogeneous catalysis, where the catalyst is located only in a part of the system, was first developed and verified experimentally for living systems in the 1920s by biochemists. Contrary to homogeneous catalysis, this description involves the coupling of the reaction with mass transfer. This applies also for heterogeneous chemocatalysis. The same description as for living systems was derived independently by chemical engineers in the end of the 1930s (see Chapter 10).

The detailed mechanism of the catalyzed reactions has now been determined for many bio- and chemocatalysts. This knowledge that is continuously increasing yields information that can be used to design improved bio- and chemocatalysts. This, however, requires a closer cooperation of those working with these catalysts. Fortunately, due to the increasing use of enzymes by organic chemists in the past decades, this cooperation has increased markedly.

1.2 Goals and Potential of Biotechnological Production Processes

Biomass – that is, renewable raw materials – has been and will continue to be a sustainable resource that is required to meet a variety of human material needs. In developed countries such as Germany, biomass covers ≈30% of the raw material need – equivalent to ~7000 kg per person per year. The consumption of biomass for different human demands is shown schematically in Figure 1.1. This distribution of the consumption is representative for a developed country in the regions that have a high energy consumption during the winter. However, the consumption of energy (expressed as tons of oil equivalent per capita in 2007) showed a wide range, from 8 in the United States to 4 in Germany and the United Kingdom, 1.5 i...

Table of contents

• Cover
• Related Titles
• Title Page
• Copyright
• Dedication
• Preface to the Second Edition
• Preface
• Chapter 1: Introduction to Enzyme Technology
• Chapter 2: Basics of Enzymes as Biocatalysts
• Chapter 3: Enzyme Discovery and Protein Engineering
• Chapter 4: Enzymes in Organic Chemistry
• Chapter 5: Cells Designed by Metabolic Engineering as Biocatalysts for Multienzyme Biotransformations
• Chapter 6: Enzyme Production and Purification
• Chapter 7: Application of Enzymes in Solution: Soluble Enzymes and Enzyme Systems
• Chapter 8: Immobilization of Enzymes (Including Applications)
• Chapter 9: Immobilization of Microorganisms and Cells
• Chapter 10: Characterization of Immobilized Biocatalysts
• Chapter 11: Reactors and Process Technology
• Chapter 12: Case Studies
• Appendix A: The World of Biotechnology Information: Seven Points for Reflecting on Your Information Behavior
• Appendix B: Solutions to Exercises
• Appendix C: Symbols and Abreviations
• Index