biocatalysis for the pharmaceutical industry …€¦ · 1 enzymes and their synthetic...

BIOCATALYSIS FOR THEPHARMACEUTICAL INDUSTRYDiscovery, Development, andManufacturing

Junhua (Alex) Tao

Elevance Renewable Sciences, USA

Guo-Qiang Lin

Shanghai Institute of Organic Chemistry, CAS, China

Andreas Liese

Hamburg University of Technology, Germany

BIOCATALYSIS FOR THEPHARMACEUTICAL INDUSTRY

BIOCATALYSIS FOR THEPHARMACEUTICAL INDUSTRYDiscovery, Development, andManufacturing

Junhua (Alex) Tao


Guo-Qiang Lin

Shanghai Institute of Organic Chemistry, CAS, China

Andreas Liese


Copyright # 2009 John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop, # 02-01,Singapore 129809

Visit our Home Page on www.wiley.com

All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted inany form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except asexpressly permitted by law, without either the prior written permission of the Publisher, or authorization throughpayment of the appropriate photocopy fee to the Copyright Clearance Center. Requests for permission shouldbe addressed to the Publisher, John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop, # 02-01, Singapore 129809,tel: 65-64632400, fax: 65-64646912, email: [email protected].

Designations used by companies to distinguish their products are often claimed as trademarks. All brand names andproduct names used in this book are trade names, service marks, trademarks or registered trademarks of theirrespective owners. The Publisher is not associated with any product or vendor mentioned in this book. Alltrademarks referred to in the text of this publication are the property of their respective owners.

This publication is designed to provide accurate and authoritative information in regard to the subject mattercovered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. Ifprofessional advice or other expert assistance is required, the services of a competent professional should be sought.

Other Wiley Editorial Offices

John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA

Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA

Wiley-VCH Verlag GmbH, Boschstrasse 12, D-69469 Weinheim, Germany

John Wiley & Sons Australia Ltd, 42 McDougall Street, Milton, Queensland 4064, Australia

John Wiley & Sons Canada Ltd, 5353 Dundas Street West, Suite 400, Toronto, ONT, M9B 6H8, Canada

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not beavailable in electronic books.

Library of Congress Cataloging-in-Publication Data

Tao, Junhua.Biocatalysis for the pharmaceutical industry: discovery, development, and manufacturing/Junhua Tao,

Guo-Qiang Lin, Andreas Liese.p. cm.

Includes bibliographical references and index.ISBN 978-0-470-82314-9 (cloth)

1. Pharmaceutical biotechnology. 2. Enzymes–Biotechnology. I. Lin, Guo-Qiang. II. Liese, A. (Andreas)III. Title.RS380.T36 20096150.19–dc22 2008045613

ISBN 978-0-470-82314-9 (HB)

Typeset in 10/12pt Times by Thomson Digital, Noida, India.Printed and bound in Singapore by Markono Print Media Pte Ltd, Singapore.This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least twotrees are planted for each one used for paper production.

www.wiley.com

Contents

Preface xi

1 Enzymes and Their Synthetic Applications: An Overview 1

1.1 Introduction 1

1.2 Enzyme Families 1

1.3 Enzyme Discovery and Optimization 2

1.4 Enzyme Production 3

1.5 Enzymes and Synthetic Applications 3

1.5.1 Ketoreductases (EC 1.1.1.2) 4

1.5.2 Enoate Reductases or Ene Reductases (EC 1.3.1.16) 4

1.5.3 Oxygenases (EC. xxxx) 5

1.5.4 Alcohol Oxidases (EC 1.1.3.X) 6

1.5.5 Peroxidases (EC 1.11.1.X) 7

1.5.6 Halogenases (EC. xxxx) 8

1.5.7 Nitrilases (EC 3.5.5.1) 9

1.5.8 Nitrile Hydratases (EC 4.2.1.84) 9

1.5.9 Epoxide Hydrolases (EC 3.3.2.X) 10

1.5.10 !-Transaminases (EC 2.6.1.X) 10

1.5.11 Hydroxynitrile Lyases (EC 4.1.2.X) 10

1.5.12 Aldolases (EC. xxxx) 12

1.5.13 Glycosidases (EC. xxxx) 12

1.5.14 Glycosyltransferase (EC. xxxx) 14

1.6 Conclusions 15

2 Expression Hosts for Enzyme Discovery and Production 21

2.1 Introduction 21

2.2 How to Choose an Expression System 22

2.3 Prokaryotic Expression Systems 24

2.3.1 Posttranslational Modification in Prokaryotes 25

2.3.2 Escherichia coli 25

2.3.3 Bacilli 26

2.3.4 Pseudomonas fluorescens 27

2.3.5 Other Prokaryotic Expression Systems 28

2.4 Eukaryotic Expression Systems 29

2.4.1 Yeasts 29

2.4.2 Filamentous Fungi 32

2.4.3 Insect/Baculovirus System 33

2.4.4 Mammalian Cell Cultures 34

2.4.5 Other Expression Systems 35

2.5 Cell-Free Expression Systems 36

2.6 Conclusions 37

3 Directed Enzyme Evolution and High-Throughput Screening 45

3.1 Introduction 45

3.2 Directed Evolution Library Creation Strategies 46

3.2.1 Random and Semi-Rational Mutagenesis 47

3.2.2 Gene Shuffling 48

3.3 Directed Evolution Library Screening/Selection Methods 52

3.3.1 In Vivo Methods: Genetic Complementation 52

3.3.2 In Vivo Methods: Chemical Complementation 53

3.3.3 In Vivo Methods: Surface Display 53

3.3.4 In Vitro Methods: Lysate Assay 55

3.3.5 In Vitro Methods: Ribosome Display 55

3.3.6 In Vitro Methods: In Vitro Compartmentalization 55

3.3.7 Equipment/Automation 56

3.4 Selected Industrial Examples 56

3.4.1 Activity 56

3.4.2 Thermostability 58

3.4.3 Substrate Specificity 58

3.4.4 Product Specificity 59

3.4.5 Enantioselectivity 59

3.5 Conclusions and Future Directions 60

4 Applications of Reaction Engineering to Industrial Biotransformations 65

4.1 Introduction 65

4.2 Metabolic Bioconversion 66

4.3 Enzymatic Biotransformations 67

4.3.1 Cofactor Regeneration 67

4.3.2 Racemic Mixtures 69

4.3.3 Equilibrium Conversion 72

4.3.4 By-Product Formation 76

4.3.5 Substrate Inhibition 79

4.3.6 Low Solubility 82

4.4 Conclusions 83

5 Chiral Synthesis of Pharmaceutical Intermediates Using Oxynitrilases 89

5.1 Introduction 89

5.2 HNL 89

5.2.1 The Natural Function and Distribution of HNLs 89

vi Contents

5.2.2 Classification of HNLs 91

5.2.3 New HNLs and High-Throughput Screening 93

5.3. Reaction of HNLs 95

5.3.1 Reaction System 95

5.3.2 Immobilization of Enzyme 97

5.3.3 Continuous Reactors 98

5.3.4 Henry Reaction 98

5.4 Transformation of Cyanohydrins 99

5.4.1 Transformation of Hydroxyl Group 99

5.4.2 Transformation of Nitrile Group 100

5.4.3 Intramolecular Reaction 102

5.5 Summary 104

6 Expanding the Scope of Aldolases as Tools for Organic Synthesis 111

6.1 Directed Evolution and Rational Mutagenesis 111

6.2 Reaction Engineering 114

6.3 Broad Substrate Tolerance of Wild-Type Aldolases 116

6.4 Conclusions 118

7 Synthetic Applications of Ketoreductases and Alcohol Oxidases 121

7.1 Ketoreductases 121

7.1.1 Wild-Type Whole-Cell Biocatalysts 122

7.1.2 Recombinant Whole-Cell Biocatalysts Overexpressing

Catalytic Enzymes 125

7.1.3 Isolated Enzyme 129

7.2 Alcohol Oxidases 142

7.2.1 Primary Alcohol Oxidases 142

7.2.2 Secondary Alcohol Oxidases 144

8 Applications of Nitrile Hydratases and Nitrilases 153

8.1 Introduction 153

8.2 NHase 153

8.2.1 New NHases 153

8.2.2 Applications 154

8.3 Nitrilase 163

8.3.1 New Nitrilases 163

8.3.2 Applications 167

8.4 Conclusions 178

9 Biosynthesis of Drug Metabolites 183


9.2 Metabolite Synthesis Using Mammalian Bioreactors 184

9.2.1 Selection of In Vitro Systems 185

9.2.2 Reaction Condition Optimization 187

9.2.3 Large Scale Incubations 190

Contents vii

9.2.4 Examples with Mammalian Bioreactors 190

9.2.5 In Vivo Samples 192

9.3 Metabolite Synthesis Using Microbial Bioreactors 192

9.3.1 Microbial Bioreactors Used in Metabolite Structure Elucidation 193

9.3.2 Microbial Bioreactors Used in Synthesis of Key Metabolites 193

9.3.3 Strain Selection 194

9.3.4 Microbial Glycoside Conjugation 196

9.3.5 Large Scale Reactions 199

9.3.6 Examples for Metabolite Synthesis with a Microbial Bioreactor 200

9.4 Recombinant Enzyme Bioreactors 202

9.4.1 Advantages of Using CYP Enzymes for Producing

Drug Metabolites 202

9.4.2 Human Cytochrome Biocatalysts 205

9.4.3 Microbial CYP Enzymes 206

9.5 Summary 207

10 Application of Whole-Cell Biotransformation in the

Pharmaceutical Industry 213


10.1.1 Whole-Cell Biotransformation Processes Used in Commercial

Production of Pharmaceuticals 214

10.1.2 Application of Whole-Cell Biotransformation Process in the

Synthesis of Chiral Pharmaceutical Intermediates 214

10.2 Disadvantages of Whole-Cell Process Compared with the Isolated

Enzyme Process 217

10.2.1 Substrate Availability and Recovery of Products in Low

Concentrations 217

10.2.2 Undesirable Side Reactions 217

10.2.3 Toxicity of Substrate and Product 217

10.3 Advantages of Whole-Cell Process Compared with the Isolated

Enzyme Process 218

10.3.1 More Stable Sources than Isolated Enzymes 218

10.3.2 Regeneration of Cofactors and Multi-Enzymes Reactions 218

10.3.3 Diversity and Availability 218

10.3.4 Reactions with Non-Commercially Available Isolated Enzymes

for Preparative Scale Synthesis 218

10.3.5 Cost Effectiveness and Ease of Operation 219

10.4 Approaches to Address the Disadvantages of Whole-Cell

Biotransformation 220

10.4.1 Control of Substrate and Product Concentration by

Absorbing Resins 220

10.4.2 Immobilized-Cell Technology 221

10.4.3 Aqueous–Organic Two-Phase System 222

10.4.4 Genetic Engineering Approaches 223

10.5 Conclusions 224

viii Contents

11 Combinatorial Biosynthesis of Pharmaceutical Natural Products 229


11.2 Combinatorial Biosynthesis: The Natural Way for Structural Diversity 230

11.3 Examples of Combinatorial Biosynthesis of Pharmaceutical

Natural Products 234

11.3.1 Erythromycin (Polyketide Biosynthesis) 234

11.3.2 Daptomycin (Nonribosomal Peptide Biosynthesis) 236

11.3.3 Patellamide (Ribosomal Peptide Biosynthesis) 239

11.4 Summary and Perspectives 240

12 Metabolic Engineering for the Development and Manufacturingof Pharmaceuticals 247


12.2 Metabolic Engineering Tools 248

12.2.1 Tools for the Cellular Metabolic Network Analysis 248

12.2.2 Tools for Rational Genetic Modification 251

12.3 Metabolic Engineering for the Development and Production

of Polyketide Pharmaceuticals 253

12.3.1 Biosynthesis of Polyketides 253

12.3.2 Metabolic Engineering for Improved Erythromycin Production 253

12.3.3 Metabolic Engineering for Overproduction of 6dEB

in Heterologous Hosts 254

12.3.4 Metabolic Engineering of Other Polyketides 256

12.3.5 Development of Novel Polyketides for Drug Discovery 256

12.4 Metabolic Engineering for the Production of �-Lactam 257

12.5 Metabolic Engineering for Isoprenoid Production 258

12.5.1 Biosynthesis Pathway of Isoprenoids 259

12.5.2 Metabolic Engineering for Enhancing Precursor Supply for

Isoprenoid Production 260

12.5.3 Metabolic Engineering for Artemisinine Development

and Production 261

12.5.4 Metabolic Engineering for Carotenoid Production 262

12.5.5 Metabolic Engineering for Taxol Development

and Production 262


13 Multimodular Synthases and Supporting Enzymes

for Chemical Production 273


13.2 Background 274

13.2.1 Multimodular Synthase Architecture 274

13.2.2 Natural Product Biosynthetic Cycle 276

13.3 Metabolic Engineering of Megasynthases 277

13.3.1 Daptomycin: Metabolic Engineering by Domain Swap 278

13.3.2 Avermectin: Metabolic Engineering by Directed Fermentation 281

Contents ix

13.4 Excised Domains for Chemical Transformations 284

13.4.1 Function of Individual Domains, Domain Autonomy 284

13.4.2 Cyclization 285

13.4.3 Halogenation 288

13.4.4 Heterocyclization/Aromatization 290

13.4.5 Methylation 292

13.4.6 Oxygenation 294

13.4.7 Glycosylation 295


14 Green Chemistry with Biocatalysis for Production of Pharmaceuticals 305


14.2 Enzymatic Resolutions: Higher Yields, Less Waste 309

14.3 Bioreductions: Greener Ligands, Renewable Hydride

Donors, No Metals 312

14.3.1 Enzymatic Oxidations: Clean, Highly Selective and Catalytic 314

14.4 C�C Bond Formations: Atom Efficiency at Its Best 316

14.5 Summary and Outlook 318

Index 323

x Contents

Preface

Biocatalysis is evolving to be a transformational technology as a result of a confluence of

factors, which include (1) large scale and ever increasingly cost-efficient DNA sequencing

technologies; (2) exponential growth in GenBank; (3) powerful directed enzyme evolution

and high-throughput screening technologies; (4) robust expression systems for enzyme

production; (5) deep understanding of the logic of natural product biosynthesis; (6) industrial

successes of metabolic engineering and pathway engineering.

Consequently, many successful stories and a number of reviews have been reported recently

in developing biocatalysis for the pharmaceutical industry, across drug discovery, development,

and manufacturing. The book is dedicated to these advances, and divided into four parts:

. Chapters 1–4 serve as an introduction to emerging biocatalysts, modern expression hosts,

state of the art of directed evolution, high-throughput screening, and bioprocess

engineering for industrial applications.. Chapters 5–8 are directed to emerging enzymes, which include oxynitrilases, aldolases,

ketoreductases, oxidases, nitrile hydratases, and nitrilases, and their recent applications

especially in synthesis of chiral drugs and intermediates.. Chapters 9 and 10 focus on synthesis of drug metabolites and intermediates catalyzed by

P450s or whole cells.. Chapters 11–13 are devoted to combinatorial biosynthesis, metabolic engineering, and

autonomous enzymes for the synthesis and development of complex medicinal molecules.. Chapter 14 discusses the recent impact of biocatalysis in green chemistry and chemical

development.

Our main goal is to come up with a concise but comprehensive, practical but insightful

book covering the topics discussed above. We hope you enjoy reading this book. Any

suggestions and comments are welcome.

Junhua (Alex) Tao


Email: [email protected]

Guo-Qiang Lin

Guo-Qiang Lin Shanghai Institute of Organic Chemistry, China


Andreas Liese



1

Enzymes and Their SyntheticApplications: An Overview

Junhua (Alex) Tao1 and Jian-He Xu2

1Elevance Renewable Sciences, 175 E. Crossroads Parkway, Bolingbrook, IL 60440, USA2East China University of Science and Technology (ECUST) 130 Meilong Road, Shanghai,

200237, PR China

1.1 Introduction

Whole-cell biocatalysis has been exploited for thousands of years; for example, in preparing

barley for beer brewing.While the chemical, economic and social advantages of biocatalysis

over traditional chemical approaches were recognized a long time ago, their applications for

the drug industry have been largely underexplored until the recent technological break-

throughs in large-scale DNA sequencing, robust protein expression systems, metabolic

engineering and directed evolution. In this chapter, emphasis will be directed to the

discussion of those isolated enzymes which are uniquely suited for the synthesis of

small-molecule pharmaceutical ingredients.

1.2 Enzyme Families

Based on reactions they catalyze, enzymes can be broadly classified into six major

categories (Table 1.1) [1]. It was estimated that about 60% of biotransformations currently

rely on the use of hydrolases, followed by 20% of oxidoreductases [2]. On the other hand,

some of the C�C bond-forming and oxygenation enzymes catalyze reactions with very high

reaction efficiency and very low waste generation, underlining the potential of emerging

enzymes.

Biocatalysis for the Pharmaceutical Industry edited by Junhua Tao, Guo-Qiang Lin, and Andreas Liese

� 2009 John Wiley & Sons Asia (Pte) Ltd

1.3 Enzyme Discovery and Optimization

Traditionally, enzymes are discovered through screening of environmental samples and culture

enrichment. As a result of recent technological breakthroughs in large-scale DNA sequencing

and high-throughput screening, both themetagenomic approach and sequence-based discovery

have drastically shortened the cycle of enzyme discovery.

In the metagenomic approach, DNA was directly extracted from uncultured samples

followed by cloning and expression [3]. For example, by combination of directed evolution

with the metagenome approach, an a-amylase mutant with optimal activity at pH 4.5

and optimal thermostability at 105 �C was discovered for starch liquefaction and EtOH

production [4].

Sequence-based discovery (genome hunting) is increasingly attractive, as the public

sequence bank is growing rapidly. In this approach, known sequences encoding an enzyme

of interest are used to search gene databases to uncover enzymes of homologous sequences.

For example, using this method, a library of deoxyribose-phosphate aldolases (DERAs) were

rapidly constructed and from them a novel DERAwas identified to catalyze a sequential aldol

reaction of a nonnative substrate with high throughput and excellent stereoselectivity for the

synthesis of statin side chains [5].

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their

properties often have to be improved. Oneway to achieve this is to optimize reaction conditions

such as pH, temperature, solvents, additives, etc. [6–9]. Another way is to modulate the

substrates without compromising the synthetic efficiency of the overall reaction [10]. In most

cases for commercial manufacturing, however, the protein sequences have to be altered to

enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial

enzymes worldwide have been engineered for industrial applications [11]. Precise prediction

of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution

Table 1.1 Enzyme classes

Enzyme class Examples Reaction catalyzed

Hydrolases lipase, protease, esterase hydrolysis reactions in H2O

nitrilase, nitrile hydratase

glycosidase, phosphatase

Oxidoreductases dehydrogenase, oxidase oxidation or reduction

oxygenase, peroxidase

Transferases transaminase, glycosyltransferase transfer of a group from one molecule

to anothertransaldolase

Lyases decarboxylase, dehydratase,

deoxyribose-phosphate aldolase

nonhydrolytic bond cleavage

Isomerases racemase, mutase intramolecular rearrangement

Ligases DNA ligase bond formation requiring triphosphate

2 Biocatalysis for the Pharmaceutical Industry

has been demonstrated to be a powerful and robust technology to improve the desired

properties [12,13]. Among them, the error-prone PCR method is probably the most popular

to create random mutants by changing polymerization reaction conditions [14]. Alternatively,

recombination of homologous sequences or DNA-shuffling methods can be used to introduce

mutants with improved properties [15]. The major challenge in directed evolution is not

generation of mutant libraries; rather, it is the availability of high-throughput assays [16]. In

most cases, it requires screening of tens of thousands of mutants, which is usually tedious and

time consuming. As more and more protein structures are available from the protein database,

focused directed evolution or semi-rational protein design is becoming more and more

popular [17]. In this approach, the three-dimensional (3-D) structure of a suboptimal enzyme

is constructed by a computer algorithm from a homologous enzymewith known 3-D structure.

Docking studies are then applied to search potential ‘hot spots’, which are then swapped with

other amino acids by site-saturationmutagenesis. In thisway, there are generally less than a few

thousand mutants to be screened, significantly shortening the cycle of enzyme development.

The fact thatmost beneficialmutations are proved to be near the active sitemakes this approach

even more attractive [18].

1.4 Enzyme Production

Although some enzymes are still extracted from animal or plant tissue, most of them are now

produced frommicroorganisms by fermentation. Bacteria and fungi are the most popular hosts

for producing industrial enzymes, due to easy handling and high productivity. They can also be

readily genetically engineered to improve their performance; for example, by incorporating

secretion systems to facilitate enzyme isolation and purification. Some of the most popular

expression hosts are Escherichia coli, Pichia pastoris, Pseudomonas fluorescens, Aspergillus

sp. and Bacillus sp. Mammalian or plant cells are used in special cases [19–21]. By regulation,

the production host should have GRAS status (Generally Regarded as Safe Status).

In a typical enzyme production procedure, cells containing genes encoding desired enzymes

are grown in an Erlenmeyer flask. At large scale, a computer-controlled fermenter or bioreactor

is required tomaintain an appropriate control of pH,O2,NH3 andCO2 tomaximize cell density.

The cells are harvested by centrifugation in a batch or continuous fashion. Alternatively, they

can be collected through membrane filtration devices. The cell membranes are then disrupted

by an ultrasonicator or French press at small scale. At a scale of over 5–10 L, a homogenizer is

usually used. After centrifugation to remove cell debris, the crude enzymes remain in the

supernatant and can be concentrated through precipitation by adding either inorganic salts (e.g.

ammonium sulfate) or organic solvents (e.g. acetone). The crude enzymes are then purified by

dialysis or a variety of chromatographic methods. The dry powder is usually obtained after

lyophilization under freeze-drying conditions [22,23].

1.5 Enzymes and Synthetic Applications

Historically, the most popular enzymes used for chemical synthesis are lipases, esterases,

proteases, acylases and amidases, among others. Recently, a number of recombinant bioca-

talysts have been discovered and isolated, significantly expanding the toolbox for biotrans-

formations. In this section, the focus will be on these new enzymes.

Enzymes and Their Synthetic Applications: An Overview 3

1.5.1 Ketoreductases (EC 1.1.1.2)

Ketoreductases (KREDs) catalyze the conversion of a wide range of ketones and some

aldehydes to chiral alcohols regio- and stereo-selectively in the presence of NADH or NADPH

(Figure 1.1) [24,25]. This powerful transformation has been demonstrated in a number of

industrial transformations using either isolated enzymes or whole cells. The use of isolated

enzymes is often preferred because of a higher volumetric productivity and the absence of side

reactions. A key to its success is the availability of efficient and cost-effective cofactor

regeneration methods by using a formate dehydrogenase to recycle NADþ or a glucose

dehydrogenase to recycle NADPþ (Figure 1.1) [26,27]. It shall be noted that some alcohol

dehydrogenases are also able to catalyze the oxidation of alcohols to ketones or aldehydes [28].

1.5.2 Enoate Reductases or Ene Reductases (EC 1.3.1.16)

Enoate reductase (ER) catalyzes NAD(P)H-dependent reduction of carbon–carbon double

bonds of nonactivated enoates, as well as of a,b-unsaturated aldehydes, ketones, nitros, and

nitriles (Figure 1.2). For example, the ER from Clostridium tyrobutyricum shows high

stereospecificity and regioselectivity and broad substrate specificity [29]. Alkanes with up

to two chiral centers can be directly produced by asymmetric reduction of electron-deficient

alkene enzymes from the ‘old yellow enzyme’ family at the expense ofNAD(P)H. The cofactor

can be regenerated in vitro using a formate dehydrogenase or glucose dehydrogenase.

Alternatively, a whole-cell system can be used to co-express ERs with redox enzymes for

NAD(P)H recycling [30].

R1

OH

R2

O

R3

R1

OH

O

OR2

Examples:Product

R1 R2

O

NADH

ketoreductase

R1 R2

OH

NAD+

* * R1

OH

OR2

O

R3

*

HCOONH2CO2 NH+ 3

*

R1 R2

O

NADPH

ketoreductase

R1 R2

OH

NADP+

glucosegluconate

*

dehydrogenaseformate

dehydrogenaseglucose

RegenerationCofactorandReduction

R1 R2

OH

*

Figure 1.1


1.5.3 Oxygenases (EC. xxxx)

Oxygenases catalyze direct incorporation of molecular oxygen into substrates to produce

oxygenated molecules [31,32]. They are categorized as either monooxygenases (MOs) or

dioxygenases, depending upon whether one or both atoms of dioxygen are inserted into a

substrate. The metal-dependent MOs, such as P450s, catalyze a wide range of reactions via

metal oxo species (e.g. hydroxylation of alkanes and aromatics; epoxidation of alkenes

(Figure 1.3)), while flavin adenine dinucleotide (FAD)-dependent MOs are found to catalyze

oxidation of heteroatoms (S, N, Se, P) and Baeyer–Villiger reactions via FAD-hydroperoxide

(FAD-OOH) species (Figure 1.4).

NAD(P)H

reductaseene

NAD(P)+

R2EWG

R1

R3

R2EWG

R1

R3

Examples:Product

* *

R2

R1

R3

* *

O

H R2NO2

R1

R3

* *R2

CN

R1

R3

* *

O

R1

R2

R' **

aldehydes,=EWGnitrilesnitros,ketones,

Figure 1.2

Fe5+

O

S

NNN

NFe2+

S

NNN

NO2

NAD(P)H

iron oxo species

HR2R3

R

R1OHR2

R3

R1

R

O

H OH

hydroxylation

hydroxylation

epoxidation

OP-450s, 2

OP-450s, 2

OP-450s, 2

Simplified Mechanism (P450s)

R-H

H2OR-OH

representing hemeN:representing cysteine residualS:

Examples:

Figure 1.3


Dioxygenases usually contain a tightly bound iron atom and catalyze hydroperoxidation of

allylic molecules or carboxylic acids, and dihydroxylation of aromatics (Figure 1.5) [33].

Currently, these oxygenation reactions are usually carried out in whole cells, the outcome of

which is often unpredictable. The discovery of novel oxygenases and efficient hosts for protein

expression remain keys to further expanding the applications of these enzymes in chemical

synthesis and drug metabolism studies [34–37].

1.5.4 Alcohol Oxidases (EC 1.1.3.X)

Alcohol oxidases (AOs) catalyze oxidationof alcohols to aldehydes or ketones in the presenceof

molecular oxygen, with hydrogen peroxide being the usual by-product (Figure 1.6). Some of the

most well-studied AOs are cholesterol oxidases, short-chain aliphatic alcohol oxidases,

aromatic alcohol oxidases, pyranose oxidases, glycolate oxidases, glucose oxidases, galactose

oxidases and nucleoside oxidases [38–40]. Cholesterol oxidases catalyze oxidation of allylic

alcohol in the cholesterol scaffolds [41]. The regeneration of cofactor FAD is relatively easier, as

it is tightly bound. Although these enzymes use oxygen, they can also be deactivated by oxygen

or the hydrogen peroxide by-product. It is to be noted that peroxidases and cholorperoxidases

can also catalyze the oxidation of alcohols using hydrogen peroxide (H2O2).

N

HN

NH

N O

O

OFAD-MOs, 2

NADPH NH

HN

NH

N O

OO

HO

XR XR OPSe,N,S,=X

R1 R2

O

R1 O

O

R2

Baeyer-Villiger

heteroatom oxidation

OFAD-MOs, 2

OFAD-MOs, 2

Simplified Mechanism (FAD-dependent MOs)

NH

HN

NH

N O

OO

H

R1 O

O

R2

R1 R2

OFAD-OOH

Examples:

Figure 1.4

dioxygenases

R

dioxygenases

dihydroxylation

hydroperoxidation R

OOH

OH

OH

ROH

O dioxygenases

hydroperoxidationR

OH

O

OOH

Figure 1.5


1.5.5 Peroxidases (EC 1.11.1.X)

Peroxidases utilizeH2O2 as the oxidant (Figure 1.7). The active site of peroxidasesmay involve

a heme unit (horseradish peroxidase), selenium (glutathione peroxidase), vanadium (bromo-

peroxidase) and manganese (manganese peroxidase). These enzymes catalyze a wide range of

oxidations, including hydroxylation of arenes, oligomerization of phenols and aromatic

amines, epoxidation and halogenation of olefins, oxygenation of heteroatoms and reduction

of hydroperoxides [42–44].

OHO

OH

OH

OH

HO

O2

OO

OH

OH

OH

HO

OHO

OH

O

OH

HO

P2O

GAOX

P2O = pyranose oxidaseGAOX = galactose oxidase

H+ 2O2

HO

R

O

Rcholesteroloxidase

R OH O2R O

arylalkyl,=R

Examples:

AOs

Figure 1.6

OH

MeO

R

HO

MeOR

OH

OMeR

OH

peroxidase

H2O2

peroxidase

H2O2

OH

OMe

peroxidase

H2O2

OH

OMe npolymer

R1 peroxidase

H2O2

R1

O

Figure 1.7


1.5.6 Halogenases (EC. xxxx)

Halogenases catalyze regio- and stereo-selective halogenation (Figure 1.8) [45,46]. For

electron-rich substrates, nature often uses flavin-dependent halogenases for chlorination,

bromination or iodination via FADH-OX (X¼ halide) as the halogenation agent (Figure 1.9).

For electron-deficient molecules such as alkanes, mononuclear iron halogenases are utilized

through a radical mechanism (Figure 1.9). Fluorinases adopt an SN2 nucleophilic substitution

NH

R2

R1 ClNH

R2

R1

tryptophan7-halogenase

R1 MeH,=R2 nitrilealkyl,amine,=

FADHNaCl, 2 O, 2

H2N

S

O

enzyme

chlorinaseH2N

S

O

enzyme

Cl

Cl-

ON

NN

N

NH2

S+HO2C

Me

NH2

OH OH

fluorinase

F- ON

NN

N

NH2

F

OH OH

Figure 1.8

HNN V OH

O

OO

vanadium haloperoxidase H2O2

NFeIII

N

N

N

heme-iron haloperoxidase H2O2

NH

N

NH

HN

R

O

Oflavin-dependent halogenase O2

OFeII

Cl

N

OH

HN

N

NH

-O2C

O

mononuclear iron halogenase R.

and Cl.

Cofactors and Oxidants of Halogenating Enzymes

Figure 1.9


mechanism in the presence of F� to introduce a fluorine atom [47].While it is still in its infancy,

the use of enzymatic halogenation has shown great promise, especially in whole-cell systems

(Figure 1.9). For example, tryptophan 7-halogenase was able to catalyze regioselective

halogenation of a wide range of indole derivatives and aromatic heterocycles [48]. It is to

be noted that halogenation can also be catalyzed by haloperoxidases, which often gives poor

regio- and stereo-selectivity, since the activated halogen source, (e.g. hypohalous acids) is

freely diffusible within and away from enzymes (Figure 1.8) [49].

1.5.7 Nitrilases (EC 3.5.5.1)

Nitrilases convert nitriles to the corresponding carboxylic acids and NH3 through a cysteine

residue in the active site [50]. Because of their high enantio- and regio-selectivity, nitrilases are

attractive as ‘green’ catalysts for the synthesis of a variety of carboxylic acids and derivatives

(Figure 1.10) [51,52]. Recently, a number of recombinant nitrilases have been cloned and

characterized heterologously for synthetic applications [50,53,54].

1.5.8 Nitrile Hydratases (EC 4.2.1.84)

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides (Figure 1.11) and has

been used for production of acrylamide and nicotinamide at large scale. NHases are roughly

R N R S-enzyme

NH2

OH

nitrilase

*R OH

O

R = aryl, alkenyl, alkyl

OH

O

N n

Product Examples:

R1

O

OH

R2

*

Mechanism:

*R OH

O

H2O

Figure 1.10

NH2

O

N n

Product Examples:

R1

O

NH2

R2

*

R NR OH

NHnitrile

hydratase

*R NH2

O

aryl, alkenyl, alkyl=R

M3+R N M3+

H2O

Mechanism:

*R NH2

O

Figure 1.11


classified into iron and cobalt types according to the metal involved in the active

site [55,56]. Recent elucidation of the catalytic mechanism and characterization of a

number of NHases have led to a wide range of applications in both biotransformation and

bioremediation [53].

1.5.9 Epoxide Hydrolases (EC 3.3.2.X)

Epoxide hydrolases (EHs) catalyze the hydrolysis of a wide range of epoxides (Figure 1.12).

They are cofactor independent and robust for the synthesis of enantiopure epoxides, diols and

their derivatives [57–59]. There are over 100 epoxide hydrolase gene sequences, and the X-ray

structures are available for fungal, bacterial and mammalian epoxide hydrolases [60]. Hetero-

logous expression in E. coli and other hosts has also been successful. In addition, several

efficient high-throughput screening methods have been developed, allowing the improvement

of EHs through site-directed mutagenesis and directed evolution [61,62]. EHs also play

important roles in the detoxification of genotoxic compounds and drug metabolism [63].

1.5.10 v-Transaminases (EC 2.6.1.X)

v-Transaminases catalyze the conversion of a ketone group to an amine in the presence of the

cofactor pyridoxal-50-phosphate (PLP), which is tightly bound to the active site. The catalysis

starts with the formation of an imine between the cofactor and an amine donor (Figure 1.13)

[64]. Since this is a one-step method to prepare (R)- or (S)-amines from ketones, it has huge

potential (Figure 1.14) [65,66]. For example, a high-throughput biocatalytic process to

(S)-methoxyisopropylamine, a moiety common to the two important chloroacetamide herbi-

cides metolachlor and dimethenamid, has been reported by enzymatic transamination of

methoxyacetone using isopropylamine as the donor [67].

1.5.11 Hydroxynitrile Lyases (EC 4.1.2.X)

Hydroxynitrile lyases (HNLs or oxynitrilases) catalyze C�C bond-forming reactions between

an aldehyde or ketone and cyanide to form enantiopure cyanohydrins (Figure 1.15), which are

versatile building blocks for the chiral synthesis of amino acids, hydroxy ketones, hydroxy

acids, amines and so on [68]. Screening of natural sources has led to the discovery of both

enzyme

O O-

O

enzyme

O O

OH

O H2O

epoxidehydrolase H2O

OH

OH

Examples:

O

R

O

R1

R2O

R1

R2O

R1

R2R3

O

R1

R2R3

R4

Mechanism:

Figure 1.12


N

CHO

HOPO3

2-

R' R"

NH2(amine donor) N

HOPO3

2-

R1 R2

N

N

HOPO3

2-

H2N

R' R"

O

R1 R2

O

+

N

HOPO3

2-

R1 R2

N

R1 R2

NH2

*

N

CHO

HOPO3

2-

Mechanism:

(amine acceptor)

Figure 1.13

R1 R2

O(S)-transaminases

R1 R2

NH2

R1

NH2

OR2

O

R3

R1

O

OR2

PLP

Other product examples:

R1 R2

O(R)-transaminases

R1 R2

NH2

PLP

(R)-amines

(S)-amines

NH2

* *

Examples:

Figure 1.14

R H

O(R)-oxynitrilase

R CN

OH

R H

O(S)-oxynitrilase

R CN

OH

aromatic, alkyl, heterocyclic=R

R

OH

NH2*

R

OH

R'*

O

R

OH

OH

O*

R

OH

NH2

O*

-CN

-CN

Figure 1.15


(R)- and (S)-selective HNLs. A number of recombinant HNLs have also been expressed in E.

coli, Saccharomyces cerevisiae, and Pichia pastoris. Recently, protein engineering has been

successfully applied to the development of a tailor-made HNL for large-scale production of

specific cyanohydrins [69,70].

1.5.12 Aldolases (EC. xxxx)

Aldolases catalyze asymmetric aldol reactions via either Schiff base formation (type I aldolase)

or activation by Zn2þ (type II aldolase) (Figure 1.16). The most common natural donors of

aldoalses are dihydroxyacetone phosphate (DHAP), pyruvate/phosphoenolpyruvate (PEP),

acetaldehyde and glycine (Figure 1.17) [71]. When acetaldehyde is used as the donor,

2-deoxyribose-5-phosphate aldolases (DERAs) are able to catalyze a sequential aldol reaction

to form 2,4-didexoyhexoses [72,73]. Aldolases have been used to synthesize a variety of

carbohydrates and derivatives, such as azasugars, cyclitols and densely functionalized chiral

linear or cyclic molecules [74,75].

1.5.13 Glycosidases (EC. xxxx)

Glycosidases catalyze the hydrolysis of glycosidic linkages via an oxonium intermediate or

transition state similarly to acid-catalyzed hydrolysis of glycosides under either a retention of

the configuration at the anomeric center (Mechanism, Figure 1.18) or less common inversion.

The oxonium is presumably stabilized by a carboxylate group such as glutamic acid, a common

structural motif in the active site of glycosidases [76]. Glycosidase-catalyzed synthesis of

glycosides can be achieved under either equilibrium-controlled conditions or kinetic-controlled

conditions [77–79]. In the former case, the reaction is established to shift the equilibrium towarda

product; for example, by adding organic solvents. In the latter case, activated glycosyl donors are

XR1

OEnzyme-NH2 X

R1

NEnzyme

XR1

HNEnzyme

R H

O

aldol reaction

R R1

N

X

OHEnzyme

R R1

O

X

OH

Schiff-base Mechanism

X OH

O

X O

O

H

Zn2+

X O

O

H

Zn2+

R H

O

aldol reaction

Zn2+ chelation Mechanism

X O

O Zn2+

OHR

X OH

O

OHR

Zn2+

enzyme

Figure 1.16


OH

O

OP

O-O

O-

+H

Z

O

X

Y

**

O

OP

O-O

O-Z

OH

X

Y

**

OHDHAP

-O2C

O+

HZ

O

X

Y

** -O2C

O

Z

OH

X

Y

**

pyruvate

H

O+

HZ

O

X

Y

** H

O

Z

OH

X

Y

**

acetaldehyde

OH

O+

H R

O

HO

O

R

OH

NH2 NH2

NO,H,=ZY,X,

arylalkyl,=R

glycine

H

O+

H R

O

acetaldehyde

OR

OH

OHDERAs

alkyl=R

Examples:

Figure 1.17

OHO

OH

OR

OHOH

OHO

OH

OR'

OHOH

+ R'OHβ-galactosidase

R = aryl, oligosaccharides, etc.

O

O

OHOHHO

NO2

+ O

OH OH

OCH3OH

HO α-fucosidase

O OHOHHO

O

OH OH

OCH3O

HO

OHO

OR

OHOH

OHO

OH

Nu

OHOH

NuH

HO

O-O

Enzyme

O+

HO

OHOH

HOOO-

Enzymeenzyme-bound

oxonium

NuH = nucleophile

Mechanism:

Examples:

Figure 1.18


used, which include di- or oligo-saccharides, aryl glycosides, glycosyl fluorides and so on [80].

Owing to greater promiscuity toward donors and wide availability, these enzymes were found to

have a wide range of applications in preparation of carbohydrates and derivatives [81–83].

1.5.14 Glycosyltransferase (EC. xxxx)

Glycosyltransferases (Gtfs) accept activated sugars such as uridine diphosphate (UDP)

nucleotide sugars or glycosyl phosphates as monosaccharide donors under either a retention

or inversion of the configuration at the anomeric center (Mechanism, Figure 1.19; only

inversion scenario shown). Recently, a number of Gtfs have been discovered to be quite

promiscuous, and used to synthesize many oligosaccharides, their derivatives or glycosylated

natural products, which are otherwise difficult to obtain [84–87]. For large-scale applications,

the main issue to be overcome is to recycle released nucleotide monophosphate (NMP) or

nucleotide diphosphate (NDP), which are expensive. Several methods have been re-

ported [88,89], and one utilizes sugar nucleotide pyrophosphorylase, which transfers the

sugarmoiety of a sugar phosphate to a free uridine triphosphate (UTP) to regenerate the desired

UTP-sugar (Figure 1.19) [90]. The availability of a wide range of Gtfs and sugar donors also

provides a general strategy to synthesize glycosylated molecules in vivo through pathway

engineering and combinatorial biosynthesis. For example, novelmacrocyclic polyketides have

been produced by applying a promiscuous Gtf from picromycin biosynthesis, which accepts a

wide range of sugar donors (Figure 1.19) [91,92].

OHO

OH

Nu

OHOH

OO

Enzyme

scenario):(inversionMechanism

OHO

OHOH

OUDPHO

OOH

OH

HONHAc

HO+

β-1,4-galactosyltransferase O

HO

OHOH

HO

OOH

OH

HONHAc

O

O

O

O

+ Sugar

O

OH

HO

O

O

OO

O

HO

O

OHHO

OHO

O

NHMe2O

OH

OHglucose

OHO O

Me

OH

OHNHAc OH

HO

glycosyltransferase

O

NHAcHO

sugar =sugar

OHO

OHOH

OHOLG

H

H-Nu

LG = leaving group: phosphate, UTP, TDP, etc.NuH = nucleophile: alcohol, sulfide, amine, etc.

Examples:

Figure 1.19


1.6 Conclusions

Biocatalysis has been practiced historically mostly by whole-cell systems, which limits its

applications due to low throughput and complex in vivo pathways. As a result of recent

advances in genomics and high-throughput screening, more and more diverse recombinant

enzymes are available in catalogs. Subsequently, a number of them have been successfully

applied to the commercial production of nonnatural molecules. More recently, biocatalysis is

emerging to be one of the greenest technologies for chemical synthesis [93,94]. Specifically,

biocatalysis can prevent waste generation by using catalytic processes with high stereo- and

region-selectivity, prevent or limit the use of hazardous organic reagents by using water as the

green solvent, design processes with higher energy efficiency and safer chemistry by con-

ducting reactions at room temperature under ambient atmosphere, and increase atom economy

by avoiding extensive protection and deprotection to maximize the use of renewable feedstock

designed for degradation.

Enzymes can catalyze transformations which are difficult to achieve by traditional chemical

methods. To truly realize the promise of this emerging technology for the pharmaceutical

industry, it is essential to integrate biocatalysis into drug discovery, development and

manufacturing. For drug production, the key relies on integration of enzymatic transformations

with modern chemical research and development at the retrosynthetic level to deliver efficient

and practical synthetic sequences with fewer synthetic steps and significantly reduced waste

streams [95,96].

References

[1] Tipton, K. and Boyce, S. (2000) History of the enzyme nomenclature system. Bioinformatics, 16, 34–40.

[2] Straathof, A.J., Panke, S. and Schmid,A. (2002) The production offine chemicals by biotransformations.Current

Opinion in Biotechnology, 13, 548–556.

[3] Langer, M., Gabor, E.M., Liebeton, K. et al. (2006) Metagenomics: an inexhaustible access to nature�s diversity.Biotechnology Journal, 1, 815–821.

[4] Mathur, E.J., Toledo, G., Green, B.D. et al. (2005) A biodiversity-based approach to development of performance

enzymes: applied metagenomics and directed evolution. Industrial Biotechnology, 1, 283–287.

[5] Hu, S., Tao, J. and Xie, Z. Chemoenzymic synthesis of atorvastatin. WO2006134482.

[6] Liu, Y.-Y., Xu, J.-H., Xu, Q.-G. and Hu, Y. (1999) Significant enhancement of lipase enantioselectivity toward

(S)-ketoprofen ester at pH 2. Biotechnology Letters, 21, 143–146.

[7] Xu, T.-W., Xu, J.-H., Yu, W. and Zhong, J.-H. (2006) Investigating pH and Cu(II) effects on lipase activity and

enantioselectivity via kinetic and spectroscopic methods. Biotechnology Journal, 1, 1293–1301.

[8] Liu, Y.-Y., Xu, J.-H. and Hu, Y. (2000) Enhancing effect of Tween-80 on lipase performance in enantioselective

hydrolysis of ketoprofen ester. Journal of Molecular Catalysis B: Enzymatic, 10, 523–529.

[9] Gong, P.-F., Xu, J.-H, Tang, Y.-F. andWu, H.-Y. (2003) Improved catalytic performance of Bacillus megaterium

epoxide hydrolase in a medium containing Tween-80. Biotechnology Progress, 19, 652–654.

[10] Tao, J., Zhao, L. and Ran, N. (2007) Recent advances in developing chemoenzymatic processes for active

pharmaceutical ingredients. Organic Process Research Development, 11, 259–267.

[11] Eggleston, G. and Vercellotti, J.R. (eds) (2007) Industrial Applications of Enzymes on Carbohydrate-based

Materials, ACS Symposium Series 6, Oxford University Press.

[12] Bloom, J.D., Meyer, M.M., Meinhold, P. et al. (2005) Evolving strategies for enzyme engineering. Current

Opinion in Structural Biology, 15, 447–452.

[13] Reetz, M.T. (2007) Directed evolution of enzymes for asymmetric syntheses, in Asymmetric Synthesis (eds

Christmann M. and Braese S.), Wiley–VCH, pp. 207–211.

[14] Eggert, T., Jaeger, K.-E. and Reetz, M.T. (2004) Directed evolution of randommutagenesis: a critical evaluation,

in Enzyme Functionality (ed. A. Svendsen), Marcel Dekker, Inc., New York, pp. 375–390.


[15] Kagami, O., Baik, S.-H. and Harayama, S. (2004) Effective DNA shuffling methods for enzyme evolution, in

Enzyme Functionality (ed. A. Svendsen), Marcel Dekker, Inc., New York, pp. 425–441.

[16] Arnold, F.H. and Georgiou, G. (eds) (2003) Directed Enzyme Evolution: Screening and Selection Methods,

Methods in Molecular Biology, vol. 230, Humana Press, Inc., Clifton, NJ.

[17] Lutz, S. and Patrick,W.M. (2004)Novelmethods for directed evolution of enzymes: quality, not quantity.Current

Opinion in Biotechnology, 15, 291–297.

[18] Morley,K.L. andKazlauskas,R.J. (2005) Improving enzymeproperties:when are closermutations better?Trends

in Biotechnology, 23, 231–237.

[19] Macauley-Patrick, S., Fazenda,M.L.,McNeil, B. andHarvey, L.M. (2005)Heterologous protein productionusing

the Pichia pastoris expression system. Yeast, 22, 249–270.

[20] Sorensen, H.P. and Mortensen, K.K. (2005) Advanced genetic strategies for recombinant protein expression in

Escherichia coli. Journal of Biotechnology, 115, 113–128.

[21] Nohr, J., Kristiansen, K. and Krogsdam, A.-M. (2003) Expression of Recombinant Proteins: An Introduction, in

Methods in Molecular Biology, vol. 232, Humana Press, Inc., Clifton, NJ, pp. 93–101.

[22] Gellissen, G. (ed.) (2005) Production of Recombinant Proteins, Wiley–VCH.

[23] Scopes, R.K. (ed.) (1994) Protein Purification, 3rd edn, Springer-Verlag.

[24] Goldberg, K., Schroer, K., Luetz, S. and Liese, A. (2007) Biocatalytic ketone reduction – a powerful tool for the

production of chiral alcohols – part I: processes with isolated enzymes.AppliedMicrobiology andBiotechnology,

76, 237–248.

[25] Yang, W., Xu, J.-H., Xie, Y. et al. (2006) Asymmetric reduction of ketones by employing Rhodotorula sp.

AS2.2241 and synthesis of the b-blocker (R)-nifenalol. Tetrahedron: Asymmetry, 17, 1769–1774.

[26] Husain, M. and Husain, Q. (2008) Applications of redox mediators in the treatment of organic pollutants by

using oxidoreductive enzymes: a review. Critical Reviews in Environmental Science and Technology, 38,

1–42.

[27] Goldberg, K., Schroer, K., Luetz, S. and Liese, A. (2007) Biocatalytic ketone reduction – a powerful tool for the

production of chiral alcohols – part II: whole-cell reductions. Applied Microbiology and Biotechnology, 76,

249–255.

[28] Kroutil, W., Mang, H., Edegger, K. and Faber, K. (2004) Biocatalytic oxidation of primary and secondary

alcohols. Advanced Synthesis & Catalysis, 346, 125–142.

[29] Steinbacher, S., Stumpf, M., Weinkauf, S. et al. (2002) Enoate reductase family. In Flavins and Flavoproteins

(2002), Proceedings of the 14th International Symposium, Cambridge, United Kingdom, July 14–18,

pp. 941–949.

[30] Stuermer, R., Hauer, B., Hall, M. and Faber, K. (2007) Asymmetric bioreduction of activated C:C bonds using

enoate reductases from the old yellow enzyme family. Current Opinion in Chemical Biology, 11, 203–213.

[31] Holland H.L. (1999) C�H activation. Current Opinion in Chemical Biology, 3, 22–27.

[32] Que, L. Jr. (2000) One motif–many different reactions. Nature Structural Biology, 7, 182–184.

[33] Emerson, J.P., Farquhar, E.R. and Que, L. Jr. (2007) Structural ‘snapshots’ along reaction pathways of non-heme

iron enzymes. Angewandte Chemie, International Edition, 46, 8553–8556.

[34] Yun, C.-H., Yim, S.-K., Kim, D.-H. and Ahn, T. (2006) Functional expression of human cytochrome P450

enzymes in Escherichia coli. Current Drug Metabolism, 7, 411–429.

[35] Celik, A., Flitsch, S.L. and Turner, N.J. (2005) Efficient terpene hydroxylation catalysts based upon P450

enzymes derived from actinomycetes. Organic & Biomolecular Chemistry, 3, 2930–2934.

[36] Fujita, K.-I. and Kamataki, T. (2002) Genetically engineered bacterial cells co-expressing human cytochrome

P450with NADPH-cytochrome P450 reductase: prediction of metabolism and toxicity of drugs in humans.Drug

Metabolism and Pharmacokinetics, 17, 1–22.

[37] Uno,T.,Nakao,A.,Masuda, S. et al. (2006)Modification of smallmolecules byusing cytochromeP450 expressed

in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 33, 1043–1050.

[38] Kroutil, W., Mang, H., Edegger, K. and Faber, K. (2004) Biocatalytic oxidation of primary and secondary

alcohols. Advanced Synthesis & Catalysis, 346, 125–142.

[39] Kumar, A.K. and Goswami, P. (2006) Functional characterization of alcohol oxidases from Aspergillus terreus

MTCC 6324. Applied Microbiology and Biotechnology, 72, 906–911.

[40] Overhage, J., Steinbuechel, A. and Priefert, H. (2006) Harnessing eugenol as a substrate for production of

aromatic compounds with recombinant strains of Amycolatopsis sp. HR167. Journal of Biotechnology, 125,

369–376.


biocatalysis for the pharmaceutical industry …€¦ · 1 enzymes and their synthetic...

Documents