Biocatalysts and Enzyme Technology E-Book

Klaus Buchholz

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch
• Veröffentlichungsjahr: 2012

Seitenzahl: 1067

Beschreibung

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.

Leseprobe

Contents

Cover

Related Titles

Title Page

Copyright

Dedication

Preface to the Second Edition

Preface

To the First German Edition

To the First English Edition

Chapter 1: Introduction to Enzyme Technology

1.1 Introduction

1.2 Goals and Potential of Biotechnological Production Processes

1.3 Historical Highlights of Enzyme Technology/Applied Biocatalysis

1.4 Biotechnological Processes: The Use of Isolated or Intracellular Enzymes as Biocatalysts

1.5 Advantages and Disadvantages of Enzyme-Based Production Processes

1.6 Goals and Essential System Properties for New or Improved Enzyme Processes

Exercises

Literature:

References

Chapter 2: Basics of Enzymes as Biocatalysts

2.1 Introduction

2.2 Enzyme Classification

2.3 Enzyme Synthesis and Structure

2.4 Enzyme Function and Its General Mechanism

2.5 Free Energy Changes and the Specificity of Enzyme-Catalyzed Reactions

2.6 Equilibrium- and Kinetically Controlled Reactions Catalyzed by Enzymes

2.7 Kinetics of Enzyme-Catalyzed Reactions

2.8 End Points of Enzyme Processes and Amount of Enzyme Required to Reach the End Point in a Given Time

2.9 Enzyme-Catalyzed Processes with Slightly Soluble Products and Substrates

2.10 Stability, Denaturation, and Renaturation of Enzymes

2.11 Better Enzymes by Natural Evolution, In Vitro Evolution, or Rational Enzyme Engineering

Exercises

Literature

References

Chapter 3: Enzyme Discovery and Protein Engineering

3.1 Enzyme Discovery

3.2 Strategies for Protein Engineering

3.3 Computational Design of Enzymes

Exercises

References

Chapter 4: Enzymes in Organic Chemistry

4.1 Introduction

4.2 Examples

Exercises

Literature

References

Chapter 5: Cells Designed by Metabolic Engineering as Biocatalysts for Multienzyme Biotransformations

5.1 Introduction

5.2 A Short Introduction to Metabolic Engineering

5.3 Examples

Exercises

Literature

References

Chapter 6: Enzyme Production and Purification

6.1 Introduction

6.2 Enzyme Sources

6.3 Improving Enzyme Yield

6.4 Increasing the Yield of Periplasmic and Extracellular Enzymes

6.5 Downstream Processing of Enzymes

6.6 Regulations Based on Risk Assessments/Safety Criteria that Influence the Production of Enzymes and Their Use for Analytical, Pharmaceutical, Scientific, and Technical Purposes

Exercises

Literature

References

Chapter 7: Application of Enzymes in Solution: Soluble Enzymes and Enzyme Systems

7.1 Introduction and Areas of Application

7.2 Space–Time Yield and Productivity

7.3 Examples for the Application of Enzymes in Solution

7.4 Membrane Systems and Processes

Exercises

Literature

References

Chapter 8: Immobilization of Enzymes (Including Applications)

8.1 Principles

8.2 Carriers

8.3 Binding Methods

8.4 Examples: Application of Immobilized Enzymes

Exercises

Literature

References

Chapter 9: Immobilization of Microorganisms and Cells

9.1 Introduction

9.2 Fundamental Aspects

9.3 Immobilization by Aggregation/Flocculation

9.4 Immobilization by Entrapment

9.5 Adsorption

9.6 Adhesion

9.7 Perspectives

References

Chapter 10: Characterization of Immobilized Biocatalysts

10.1 Introduction

10.2 Factors Influencing the Space–Time Yield of Immobilized Biocatalysts

10.3 Effectiveness Factors for Immobilized Biocatalysts

10.4 Mass Transfer and Reaction

10.5 Space–Time Yields and Effectiveness Factors for Different Reactors

10.6 Determination of Essential Properties of Immobilized Biocatalysts

10.7 Comparison of Calculated and Experimental Data for Immobilized Biocatalysts

10.8 Application of Immobilized Biocatalysts for Enzyme Processes in Aqueous Suspensions

10.9 Improving the Performance of Immobilized Biocatalysts

Exercises

References

Chapter 11: Reactors and Process Technology

11.1 General Aspects, Biochemical Engineering, and Process Sustainability

11.2 Types of Reactors

11.3 Residence Time Distribution, Mixing, Pressure Drop, and Mass Transfer in Reactors

11.4 Process Technology

Exercises

Literature

References

Chapter 12: Case Studies

12.1 Starch Processing and Glucose Isomerization

12.2 Biofuels from Biomass

12.3 Case Study: the One-Step Enzymatic Process to Produce 7-ACA from Cephalosporin C

12.4 Case Study: Biocatalytic Process for the Synthesis of the Lipitor Side Chain

Exercises

References

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

Related Titles

Bisswanger, H.Practical Enzymology2011ISBN: 978-3-527-32076-9

Whittall, J., Sutton, P.Practical Methods for Biocatalysis and Biotransformations2010ISBN: 978-0-470-51927-1

Tao, J., Lin, G.-Q., Liese, A.Biocatalysis for the Pharmaceutical IndustryDiscovery, Development, and Manufacturing2008ISBN: 978-0-470-82314-9

Grogan, G.Practical BiotransformationsA Beginner's Guide2009ISBN: 978-1-4051-7125-0

Behme, S.Manufacturing of Pharmaceutical ProteinsFrom Technology to Economy2009ISBN: 978-3-527-32444-6

Fessner, W.-D., Anthonsen, T. (eds.)Modern BiocatalysisStereoselective and Environmentally Friendly Reactions2009ISBN: 978-3-527-32071-4

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Preface to the Second Edition

We have been very pleased by the success of the first English edition of our book. We are especially grateful that it serves as primary source for teaching courses in Biocatalysis and Enzyme Technology at many universities around the world. We would also like to thank the readers who pointed out corrections and to those who made useful suggestions for this second edition.

More than 7 years have passed since the first English edition was published and we have observed substantial and exciting developments in all areas of biocatalysis. Hence, we did not simply update the first edition with new references and add singular sentences, but substantially expanded and reorganized the book. For instance, the importance of enzyme discovery and protein engineering is now treated in a separate new section (Chapter 3). Although biocatalysis primarily refers to the use of isolated (and immobilized) enzymes, we decided to cover also the use of designed whole cells for biotransformations in the new Chapter 5 to allow the reader to get a glimpse on the emerging field of metabolic engineering, where the understanding and biochemical characterization of enzymes is of course an important aspect. Furthermore, we have included several new case studies in Chapter 12, to exemplify how biocatalysis can be performed on the large scale and which criteria are important to establish a novel process. These include starch processing and glucose isomerization, biofuels from biomass, and the production of 7-ACA by direct hydrolysis of cephalosporin C as examples for current as well as potential industrial processes. In addition, enzymatic routes for the synthesis of advanced pharmaceutical intermediates for the drug Lipitor are covered.

Furthermore, we have added a few new sections on topics of current interest: process sustainability and ecological considerations, process integration, biofilm catalysis, microbial fuel cells, and regulations that influence the production and use of enzymes.

We have also expanded and updated the exercises and decided that also the solutions be directly given in the book – in Appendix B – so that the students can first try to answer the questions by themselves and then look up the solutions.

Finally, we would like to thank Ulrich Behrendt, Sonja Berensmeier, Matthias Höhne, Zoya Ignatova, Hans-Joachim Jördening, Burghard König, Sven Pedersen, Ralf Pörtner, Klaus Sauber, and Antje Spiess for valuable discussions, revisions, and suggestions while preparing this book.

June 2012

Klaus Buchholz/BraunschweigVolker Kasche/BremenUwe T. Bornscheuer/Greifswald

Preface

To the First German Edition

Biotechnology is the technical application of biological systems or parts thereof to provide products and services to meet human needs. It can, besides other techniques, contribute towards doing this in a sustainable manner. Since, in the majority of cases, renewable raw materials and biological systems are used in biotechno-logical processes, these processes can – and should – be performed practically without waste, as all of the byproducts can be recycled.

The development of natural and engineering science fundamentals for the design of such processes remains a challenge to biotechnology – a field that originated from the overlapping areas of biology, chemistry, and process engineering.

The requisite education for a career in biotechnology consists, in addition to a basic knowledge of each of these fields, of further biotechnological aspects which must provide an overview over the entire field and a deeper insight into different areas of biotechnology. The biotechnological production of various materials is performed either in fermenters using living cells (technical microbiology), or with enzymes – either in an isolated form or contained in cells – as biocatalysts. Indeed, the latter aspect has developed during recent years to form that area of biotechnology known as enzyme technology, or applied biocatalysis.

The aim of the present textbook is to provide a deeper insight into the fundamentals of enzyme technology and applied biocatalysis. It especially stresses the following inter-relationships: A thorough understanding of enzymes as biocatalysts and the integration of knowledge of the natural sciences of biology (especially biochemistry), cell and molecular biology; physico-chemical aspects of catalysis and molecular interactions in solutions; heterogeneous systems and interphase boundaries; and the physics of mass transfer processes. The same applies to the inter-relations between enzyme technology and chemical and process engineering, which are based on the above natural sciences.

In less than a century since the start of industrial enzyme production, enzyme technology and its products have steadily gained increasing importance. In the industrial production of materials to meet the demands of everyday life, enzymes play an important role – and one which is often barely recognized. Their application ranges from the production of processed foods such as bread, cheese, juice and beer, to pharmaceuticals and fine chemicals, to the processing of leather and textiles, as process aids in detergents, and also in environmental engineering.

Meeting the demand for these new products – which increasingly include newly developed and/or sterically pure pharmaceuticals and fine chemicals – has become an important incentive for the further development of biocatalysts and enzyme technology. Of similar importance is the development of new sustainable production processes for existing products, and this is detailed in Chapter 1, which forms an Introduction.

Enzymes as catalysts are of key importance in biotechnology, similar to the role of nucleic acids as carriers of genetic information. Their application as isolated catalysts justifies detailed examination of the fundamentals of enzymes as biocatalysts, and this topic is covered in Chapter 2. Enzymes can also be analyzed on a molecular level, and their kinetics described mathematically. This is essential for an analytical description and the rational design of enzyme processes. Enzymes can also catalyze a reaction in both directions – a property which may be applied in enzyme technology to achieve a reaction end-point both rapidly and with a high product yield. The thermodynamics of the catalyzed reaction must also be considered, as well as the properties of the enzyme. The amount of enzyme required for a given conversion of substrate per unit time must be calculated in order to estimate enzyme costs, and in turn the economic feasibility of a process. Thus, the quantitative treatment of bioca-talysis is also highlighted in Chapter 2.

When the enzyme costs are too high, they can be reduced by improving the production of enzymes, and this subject is reviewed in Chapter 3 (Chapter 4 in the present book).

In Chapter 4 (here Chapter 5), applied biocatalysis with free enzymes is described, together with examples of relevant enzyme processes. When single enzyme use is economically unfavorable, the enzymes can be either reused or used for continuous processes in membrane reactors (Chapter 4; here Chapter 5) or by immobilization (Chapters 5 and 6; here Chapters 6 and 7). The immobilization of isolated enzymes is described in detail in Chapter 5, while the immobilization of microorganisms and cells, with special reference to environmental technology, is detailed in Chapter 6.

In order to describe analytically the processes associated with immobilized biocatalysts that are required for rational process design, the coupling of reaction and diffusion in these systems must be considered. To characterize immobilized biocatalysts, methods which were developed previously for analogous biological and process engineering (heterogeneous catalysis) systems can be used (Chapter 7; here Chapter 8).

Details of reactors and process engineering techniques in enzyme technology are provided in Chapter 8 (here Chapter 9), while the analytical applications of free and immobilized enzymes is treated in Chapter 10 (not covered in the present book).

Within each chapter an introductory survey is provided, together with exercises and references to more general literature and original papers citing or relating the content of that chapter.

This textbook is designed to address both advanced and graduate students in biology, chemistry and biochemical, chemical and process engineering, as well as scientists in industry, research institutes and universities. It should provide a solid foundation that covers all relevant aspects of research and development in applied biocatalysis/enzyme technology. It should be remembered that these topics are not of equal importance in all cases, and therefore selective use of the book – depending on the individual reader's requirements – might be the best approach to its use.

In addition to a balanced methodological basis, we have also tried to present extensive data and examples of new processes, in order to stress the relevance of these in industrial practice.

From our point of view it is also important to stress the interactions, which exist beyond the scientific and engineering context within our society and environment. The importance and necessity of these interactions for a sustainable development has been realized during the past two decades, and this has resulted in new economic and political boundary conditions for scientific and engineering development. Problems such as allergic responses to enzymes in detergents and, more recently, to enzymes produced in recombinant organisms, have direct influences on enzyme technology/applied biocatalysis. Therefore, an integrated process design must also consider its environmental impact, from the supply and efficient use of the raw materials to the minimization and recycling of the byproducts and waste. Political boundary conditions derived from the concept of sustainability, when expressed in laws and other regulations, necessitates due consideration in research and development. The design of sustainable processes is therefore an important challenge for applied biocatalysis/enzyme technology. Ethical aspects must also be considered when gene technology is applied, and this is an increasing consideration in the production of technical and pharmaceutical enzymes. The many interactions between research and development and economic and political boundary conditions must be considered for all applications of natural and engineering sciences. Most importantly, this must be appreciated during the early phases of any development, with subsequent evaluation and selection of the best alternative production processes to meet a variety of human needs, as is illustrated in the following scheme:

This book has been developed from our lecture notes and materials, and we also thank all those who provided valuable help and recommendations for the book's production. In particular, we thank Dipl.-Ing. Klaus Gollembiewsky, Dr. Lieker, Dr. Noll-Borchers, and Dipl. Chem. André Rieks.

Klaus BuchholzVolker Kasche

To the First English Edition

The basic philosophy of the previous German edition is retained, but the contents have been revised and updated to account for the considerable development in enzyme technology/applied biocatalysis since the German edition was prepared some 10 years ago. Hence, a new chapter (Chapter 3) has been added to account for the increasing importance of enzymes as biocatalysts in organic chemistry. Recent progress in protein design (by rational means and directed evolution) has been considerably expanded in Chapter 2. The final chapter has been amended with more detailed case studies to illustrate the problems that must be solved in the design of enzyme processes. An appendix on information retrieval using library and internet resources has also been added, and we thank Thomas Hapke (Subject Librarian for Chemical Engineering at the Library of the Technical University Hamburg-Harburg) for help in the preparation of this material. The chapter on enzymes for analytical purposes has been removed in this English edition as it now is beyond the scope of this textbook.

We thank Prof. Dr. L. Jaenicke and Prof. Dr. J.K.P. Weder for their very constructive suggestions for corrections and improvements of the German edition.

The authors of this edition thank Prof. Dr. Andreas Bommarius, Dr. Aurelio Hidalgo, Dr. Janne Kerovuo, Dr. Tanja Kummer, Dr. Dieter Krämer, Dr. Brian Morgan, Sven Pedersen, Poul Poulsen, Prof. Dr. Peter Reilly, Dr. Klaus Sauber, Dr. Wilhelm Tischer, and Dr. David Weiner for valuable discussions, revisions and suggestions while preparing this book.

January 2005

Klaus BuchholzVolker KascheUwe T. Bornscheuer

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/orthe 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.