High-throughput Screening,Genetic Selection and Fingerprinting

Edited byJean-Louis Reymond

Enzyme Assays

InnodataFile Attachment3527607218.jpg

Edited byJean-Louis Reymond

Enzyme Assays

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High-throughput Screening,Genetic Selection and Fingerprinting

Edited byJean-Louis Reymond

Enzyme Assays

Editor:

Prof. Dr. Jean-Louis ReymondUniversity of BerneDepartment of Chemistry & BiochemistryFreistrasse 33012 BerneSwitzerland

Cover illustrationThe cover picture shows a cpk model of calcein(upper left), a fluorescent sensor useful forhigh-throughput screening of acylases, amino-peptidases, and proteases, as discussed in theIntroduction.

The image on the right is a close-up viewof an agar plate with colonies expressing mutantmonoamine oxidases in the presence of (S)-alpha-methyl benzylamine as substrate and 3,3�-diamino-benzidine as sensor. Colony staining results fromchemical oxidation of 3,3�-diaminobenzidine by thehydrogen peroxide produced in the enzyme oxida-tion, as discussed in Chapter 5.

The bottom grid shows a fingerprint of activity(color intensity) and enantioselectivity (purple =R-enantioselectivity, green = S-enantioselectivity)of Bacillus thermocatenulatus lipase (BTL2) onchiral ester substrates using the assay describedin Chapter 1 and the color coding method inChapter 10. The cover was based on a prototypeby Peter Bernhardt.

All books published by Wiley-VCH are carefullyproduced. Nevertheless, authors, editors, and pub-lisher do not warrant the information containedin these books, including this book, to be free oferrors. Readers are advised to keep in mind thatstatements, data, illustrations, procedural detailsor other items may inadvertently be inaccurate.

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All rights reserved (including those of translationin other languages). No part of this book maybe reproduced in any form – by photoprinting,microfilm, or any other means – nor transmittedor translated into machine language withoutwritten permission from the publishers.Registered names, trademarks, etc. used in thisbook, even when not specifically marked as such,are not to be considered unprotected by law.

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� All books published by Wiley-VCH arecarefully produced. Nevertheless, authors,editors, and publisher do not warrant theinformation contained in these books,including this book, to be free of errors.Readers are advised to keep in mind thatstatements, data, illustrations, proceduraldetails or other items may inadvertentlybe inaccurate.

Preface XIII

List of Contributors XV

Introduction 1Renaud Sicard and Jean-Louis Reymond

Enzyme Assays 1Part I: The Chemistry of Enzyme Assays 7Part II: Enzyme Assays and Genetic Selection 7Part III: Enzyme Profiling 9Enzyme Assays in Other Areas 11How to Use this Book 11

Part I High-throughput Screening 15

1 Quantitative Assay of Hydrolases for Activity and SelectivityUsing Color Changes 17Romas J. Kazlauskas

1.1 Overview 171.2 Direct Assays Using Chromogenic Substrates 181.3 Indirect Assays Using Coupled Reactions – pH Indicators 191.3.1 Overview of Quantitative Use of pH Indicator Assay 211.3.2 Applications 241.3.2.1 Searching for an Active Hydrolase

(Testing Many Hydrolases Toward One Substrate) 241.3.2.2 Substrate Mapping of New Hydrolases

(Testing Many Substrates Toward Hydrolase) 251.3.3 Comparison with Other Methods 261.4 Estimating and Measuring Selectivity 271.4.1 Estimating Selectivity without a Reference Compound 281.4.2 Quantitative Measure of Selectivity Using a Reference Compound

(Quick E and Related Methods) 30

V

Contents

1.4.2.1 Chromogenic Substrate 321.4.2.2 pH Indicators 331.4.3 Application 331.4.3.1 Substrate Mapping of Hydrolases 331.4.3.2 Screening of Mutants in Directed Evolution 331.4.4 Advantages and Disadvantages 36

References 38

2 High-throughput Screening Systems for Assayingthe Enantioselectivity of Enzymes 41Manfred T. Reetz

2.1 Introduction 412.2 UV/Vis Spectroscopy-based Assays 422.2.1 Assay for Screening Lipases or Esterases in the Kinetic Resolution

of Chiral p-Nitrophenyl Esters 432.2.2 Enzyme-coupled UV/Vis-based Assay for Lipases and Esterases 452.2.3 Enzymatic Method for Determining Enantiomeric Excess

(EMDee) 462.2.4 UV/Vis-based Enzyme Immunoassay as a Means to Measure

Enantiomeric Excess 472.2.5 Other UV/Vis-based ee-Assays 482.3 Assays Using Fluorescence 482.3.1 Umbelliferone-based Systems 482.3.2 Fluorescence-based Assay Using DNA Microarrays 512.3.3 Other Fluorescence-based ee-Assays 532.4 Assays Based on Mass Spectrometry (MS) 532.4.1 MS-based Assay Using Isotope Labeling 532.5 Assays Based on Nuclear Magnetic Resonance Spectroscopy 582.6 Assay Based on Fourier Transform Infrared Spectroscopy

for Assaying Lipases or Esterases 622.7 Assays Based on Gas Chromatography 652.8 Assays Based on HPLC 682.9 Assays Based on Capillary Array Electrophoresis 692.10 Assays Based on Circular Dichroism (CD) 712.11 Assay Based on Surface-enhanced Resonance Raman Scattering 732.12 Conclusions 73

References 74

3 High-throughput Screening Methods Developedfor Oxidoreductases 77Tyler W. Johannes, Ryan D. Woodyer, and Huimin Zhao

3.1 Introduction 773.2 High-throughput Methods for Various Oxidoreductases 783.2.1 Dehydrogenases 78

ContentsVI

3.2.1.1 Colorimetric Screen Based on NAD(P)H Generation 783.2.1.2 Screens Based on NAD(P)H Depletion 793.2.2 Oxidases 803.2.2.1 Galactose Oxidase 803.2.2.2 D-Amino Acid Oxidase 823.2.2.3 Peroxidases 823.2.3 Oxygenases 853.2.3.1 Assays Based on Optical Properties of Substrates and Products 853.2.3.2 Assays Based on Gibbs� Reagent and 4-Aminoantipyrine 863.2.3.3 para-Nitrophenoxy Analog (pNA) Assay 873.2.3.4 Horseradish Peroxidase-coupled Assay 883.2.3.5 Indole Assay 893.2.4 Laccases 893.2.4.1 ABTS Assay 903.2.4.2 Poly R-478 Assay 903.2.4.3 Other Assays 903.3 Conclusions 91

References 92

4 Industrial Perspectives on Assays 95Theo Sonke, Lucien Duchateau, Dick Schipper, Gert-Jan Euverink,Sjoerd van der Wal, Huub Henderickx, Roland Bezemer,and Aad Vollebregt

4.1 Introduction 954.2 Prerequisites for an Effective Biocatalyst Screening in Chemical

Custom Manufacturing 974.3 CCM Compliant Screening Methods Based on Optical Spectroscopy

(UV/Vis and Fluorescence) 1014.3.1 Optical Spectroscopic Methods Based on the Spectral Properties

of the Product Itself 1014.3.1.1 Example: Isolation of the D-p-Hydroxyphenylglycine

Aminotransferase Gene 1024.3.2 Optical Spectroscopic Methods Based on Follow-up Conversion

of Product 1044.3.2.1 Example: Fluorometric Detection of Amidase Activity

by o-Phthaldehyde/Sulfite Derivatization of Ammonia 1064.3.2.2 Example: Colorimetric Detection of Amidase Activity by Detection

of Ammonia via Glutamate Dehydrogenase-coupled Assay 1084.3.2.3 Example: Colorimetric Detection of Amino Amidase Activity

Using Cu2+ as Sensor for Amino Acids 1124.4 CCM Compliant Screening Methods Based on Generic Instrumental

Assays 1144.4.1 Flow-injection NMR as Analytical Tool in High-throughput Screening

for Enzymatic Activity 115

Contents VII

4.4.1.1 History 1154.4.1.2 Current Practice 1174.4.1.3 Practical Aspects 1194.4.1.4 Example: Screening of a Bacterial Expression Library

for Amidase-containing Clones 1224.4.1.5 Example: Identification of a Phenylpyruvate Decarboxylase

Clone 1244.4.1.6 Example: Identification of Amidase Mutants with Improved Activity

towards �-Methylphenylglycine Amide 1254.4.2 Fast LC/MS for High-throughput Screening of Enzymatic

Activity 1264.4.2.1 Example: Screening of a Bacterial Expression Library

for Amidase-containing Clones 1274.4.2.2 Example: Screening of Enzymatic Racemase Activity 1294.5 Conclusions 132

References 133

Part II Genetic Selection 137

5 Agar Plate-based Assays 139Nicholas J. Turner

5.1 Introduction 1395.1.1 Directed Evolution of Enzymes: Screening or Selection? 1395.1.2 General Features of Agar Plate-based Screens 1415.2 Facilitated Screening-based Methods 1435.2.1 Amidase 1435.2.2 Esterase 1445.2.3 Glycosynthase 1445.2.4 Galactose Oxidase 1465.2.5 Monoamine Oxidase 1475.2.6 P450 Monooxygenases 1505.2.7 Carotenoid Biosynthesis 1515.2.8 Biotin Ligase 1535.3 In vivo Selection-based Methods 1545.3.1 Glycosynthase 1545.3.2 Prephenate Dehydratase/Chorismate Mutase 1555.3.3 Terpene Cyclase 1575.3.4 Tryptophan Biosynthesis 1575.3.5 Ribitol Dehydrogenase 1575.3.6 Inteins 1585.3.7 Aminoacyl-tRNA Synthetase 1595.4 Conclusions and Future Prospects 159

References 160

ContentsVIII

6 High-throughput Screens and Selectionsof Enzyme-encoding Genes 163Amir Aharoni, Cintia Roodveldt, Andrew D. Griffiths,and Dan S. Tawfik

6.1 Introduction 1636.2 The Basics of High-throughput Screens and Selections 1646.3 High-throughput Selection of Enzymes Using Phage Display 1656.4 High-throughput Selection of Enzymes Using Cell Display 1686.5 In vivo Genetic Screens and Selections 1696.6 Screens for Heterologous Protein Expression and Stability 1696.6.1 Introduction 1696.6.2 Screening Methodologies for Heterologous Expression 1716.6.3 Directed Evolution for Heterologous Expression –

Recent Examples 1736.7 In vitro Compartmentalization 1746.8 IVC in Double Emulsions 1776.9 Concluding Remarks 179

References 179

7 Chemical ComplementationScott Lefurgy and Virginia Cornish 183

7.1 Introduction 1837.2 Complementation Assays 1847.2.1 Introduction 1847.2.2 Early Complementation Assays 1847.2.3 Enzymology by Complementation 1867.2.4 Directed Evolution by Complementation 1887.3 Development of Chemical Complementation 1917.3.1 Introduction 1917.3.2 Three-hybrid Assay 1927.3.2.1 Original Yeast Three-hybrid System 1927.3.2.2 Dexamethasone–Methotrexate Yeast Three-hybrid System 1947.3.2.3 Technical Considerations 1967.3.2.4 Other Three-hybrid Systems 1987.3.3 Chemical Complementation 1987.3.3.1 Selection Scheme and Model Reaction 1997.3.3.2 Results 2027.3.3.3 General Considerations 2037.3.3.4 Related Methods 2037.4 Applications of Chemical Complementation 2047.4.1 Introduction 2047.4.2 Enzyme–Inhibitor Interactions 2047.4.2.1 Rationale 2057.4.2.2 Screen Strategy 205

Contents IX

7.4.2.3 Enzyme Library Screen 2087.4.2.4 General Considerations 2107.4.3 Glycosynthase Evolution 2107.4.3.1 Rationale 2117.4.3.2 Selection Scheme 2127.4.3.3 Glycosynthase Assay 2137.4.3.4 Directed Evolution 2157.4.3.5 General Considerations 2167.5 Conclusion 216

References 217

8 Molecular Approaches for the Screening of Novel EnzymesValéria Maia de Oliveira and Gilson Paulo Manfio 221

8.1 Introduction 2218.2 Use of Nucleic Acid Probes to Detect Enzyme-coding Genes

in Cultivated Microorganisms 2228.2.1 Current Knowledge and Applications 2238.2.2 Limitations of Probe Technology and the Need for Innovative

Approaches 2248.3 The Microbial Metagenome: a Resource of Novel Natural Products

and Enzymes 2268.3.1 Accessing the Uncultivated Biodiversity: the Community DNA

Concept 2268.3.2 Unravelling Metabolic Function: the BAC Strategy 2278.3.3 Analysis of Metagenomic Libraries: Activity versus Sequence-driven

Strategy, Enrichment for Specific Genomes and Applicationof High-throughput Screening Methods 230

8.3.4 Follow-up of the Metagenome Harvest 2338.4 Concluding Remarks 235

References 236

Part III Enzyme Fingerprinting 239

9 Fluorescent Probes for Lipolytic Enzymes 241Ruth Birner-Grünberger, Hannes Schmidinger, Alice Loidl,Hubert Scholze, and Albin Hermetter

9.1 Introduction 2419.2 Fluorogenic and Fluorescent Substrates for Enzyme Activity 2429.2.1 Triacylglycerol Lipase Activity Assay 2459.2.2 Diacylglycerol Lipase Activity Assay 2479.2.3 Cholesteryl Esterase Activity Assay 2499.2.4 Phospholipase Activity Assay 2509.2.5 Sphingomyelinase Activity Assay 2529.3 Fluorescent Inhibitors for Quantitative Analysis of Active Enzymes

and Functional Enzyme Fingerprinting 254

ContentsX

9.3.1 Lipase and Esterase Profiling 2549.3.1.1 Microbial Lipases and Esterases 2559.3.1.2 Porcine Pancreatic Lipase 2579.3.1.3 Hormone-sensitive Lipase 2579.3.2 Probing Biophysical Enzyme Properties 2599.3.3 Affinity-based Proteome Profiling (ABPP) 2629.3.3.1 Functionality-based Serine Hydrolase Profiling in Tissue Preparations

and Cell Lines 263References 267

10 Fingerprinting Methods for HydrolasesJohann Grognux and Jean-Louis Reymond 271

10.1 Introduction 27110.1.1 One Enzyme – One Substrate 27310.1.2 Enzyme Activity Profiles 27510.1.3 The APIZYM System for Microbial Strain Identification 27610.2 Hydrolase Fingerprinting 27810.2.1 Fingerprinting with Fluorogenic and Chromogenic Substrates 27910.2.2 Fingerprinting with Indirect Chromogenic Assays 28410.2.3 Cocktail Fingerprinting 28710.3 Classification from Fingerprinting Data 28910.3.1 Fingerprint Representation 29010.3.2 Data Normalization 29310.3.3 Hierarchical Clustering of Enzyme Fingerprints 29510.3.4 Analysis of Substrate Similarities 29710.4 Outlook 299

References 300

11 Protease Substrate ProfilingJennifer L. Harris 303

11.1 Introduction 30311.2 Functional Protease Profiling – Peptide Substrate Libraries 30411.2.1 Solution-based Peptide Substrate Libraries 30611.2.2 Solid Support-based Synthesis and Screening

of Peptide Libraries 31411.2.3 Genetic Approaches to Identifying Protease Substrate

Specificity 32011.3 Identification of Macromolecular Substrates 32211.3.1 Genetic Approach to the Identification of Macromolecular

Substrates 32311.3.2 Proteomic Approaches to Identifying Protease Substrates 32611.4 Conclusions 327

References 328

Contents XI

12 Enzyme Assays on Chips 333Souvik Chattopadhaya and Shao Q. Yao

12.1 Introduction 33312.2 Immobilization Strategies 33512.2.1 Covalent versus Noncovalent Immobilization 33512.2.2 Site-specific versus Nonspecific Immobilization 33612.2.3 Site-specific Immobilization of Peptides/Small Molecules 33612.2.4 Site-specific Immobilization of Proteins 33712.2.4.1 Intein-mediated Protein Biotinylation Strategies 33812.2.4.2 Puromycin-mediated Protein Biotinylation 34312.2.4.3 Immobilization of N-terminal Cysteine-containing Proteins 34312.3 Microarray-based Methods for Detection of Enzymatic Activity 34412.3.1 Enzyme Assays Using Protein Arrays 34512.3.2 Enzyme Assays Using Peptide/Small Molecule

Substrate Arrays 34812.3.2.1 Proteases and Other Hydrolytic Enzymes 34812.3.2.2 Kinases 35112.3.2.3 Carbohydrate-modifying Enzymes 35512.3.2.4 Other Enzymes 35612.3.3 Enzyme Assays Using Other Types of Arrays 35612.4 Conclusions 357

References 359

Subject Index 363

ContentsXII

When I discuss an enzyme assay with a chemist, we spend our time devising aprocess that will turn an enzymatic reaction into a detectable signal. The chal-lenge lies in the synthesis of the molecular elements involved in the assay andwhether they will behave as expected. Enzyme assay design has elements of ra-tional drug design if it requires docking an unnatural substrate into an enzy-me’s active site. An enzyme assay may also offer a testbed for a supramolecularfunctional device, serving to demonstrate its utility. Eventually new principlesemerge that might change enzyme analytics altogether.

Then I turn to the biochemist or microbiologist, who sees the enzyme assayas one of many elements in a broader setup, such as the genetic selection of anactive enzyme, or the study of its function and mechanism. We usually settlefor a commercially available probe or couple the enzyme reaction to a biologicalsystem. Our attention focuses on the genetic design of the experiment or itsbiochemical interpretation. When it succeeds, we wonder with amazement atthe results which we only very partly understand.

Finally I meet the industrial researcher, who is hard pressed for preparativeperformance within a short time window. Our discussion is narrowed down bytight specifications bound to the goals and methods. Nevertheless, the unalteredpassion of the scientist keeps shining through. In addition, the products of in-dustrial research and development are remarkable and vindicate the efforts ofthe entire community.

Romas Kazlauskas, Manfred Reetz, Huimin Zhao, Theo Sonke, Nick Turner,Dan Tawfik, Andrew Griffiths, Virginia Cornish, Valéria Maia de Oliveira, GilsonPaulo Manfio, Albin Hermetter, Jennifer Harris, and Yao Qin Shao have agreed tojoin forces with me to compose a book on enzyme assays. These authors belong tothe world’s leading figures in this area. I thank them and their co-authors for theirtime and efforts, which were essential to the project. I also thank my co-authorsand students Johann Grognux and Renaud Sicard, and Elke Maase and RomyKirsten at Wiley-VCH, for their precious help in editing.

The field of enzyme assays is evolving rapidly and touches an ever increasingnumber of applications. The present volume captures what we as authorsbelieve is a fair coverage of the area at that point in time. We hope that thebook will prove a useful source of information, inspiration, and references forits readers across chemistry and biology.

Berne, October 2005 Jean-Louis Reymond

XIII

Preface

XV

List of Contributors

Amir AharoniThe Weizmann Institute of ScienceDepartment of Biological ChemistryRehovot 76100Israel

Roland BezemerDSM Food SpecialtiesAnalysisPO Box 12600 MA DelftThe Netherlands

Ruth Birner-GrünbergerGraz University of TechnologyDepartment of BiochemistryPetersgasse 12/28010 GrazAustria

Souvik ChattopadhayaNational University of SingaporeDepartment of Biological Science3 Science Drive 3Singapore 117543Republic of Singapore

Virginia CornishColumbia UniversityDepartment of Chemistry3000 Broadway, MC 3111New York, NY 10027USA

Lucien DuchateauDSM Pharma Chemicals –Advanced SynthesisCatalysis & DevelopmentPO Box 186160 MD GeleenThe Netherlands

Gert-Jan EuverinkUniversity of GroningenBioExploreGroningen Biomolecular Sciencesand Biotechnology InstitutePO Box 149750 AA HarenThe Netherlands

Andrew D. GriffithsUniversity Louis PasteurInstitut de Science et d’IngénierieSupramoleculaires (ISIS)CNRS UMR 70068 alleé Gaspard Monge, BP 7002867083 Strasbourg CedexFrance

Johann GrognuxUniversity of BerneDepartment of Chemistry &BiochemistryFreistrasse 33012 BerneSwitzerland

List of ContributorsXVI

Jennifer L. HarrisThe Genomics Institute of theNovartis Research Foundation10675 John Jay Hopkins DriveSan Diego, CA 92121USAandThe Scripps Research InstituteDepartment of Molecular BiologyThe Scripps Research Institute10550 North Torrey Pines RoadSan Diego, CA 92121USA

Huub HenderickxDSM ResolvePO Box 186160 MD GeleenThe Netherlands

Albin HermetterGraz University of TechnologyDepartment of BiochemistryPetersgasse 12/28010 GrazAustria

Tyler W. JohannesUniversity of IllinoisDepartment of Chemistry600 S. Mathews AveUrbana, IL 61801USA

Romas J. KazlauskasUniversity of MinnesotaDepartment of BiochemistryMolecular Biology and Biophysicsand The Biotechnology Institute1479 Gortner AvenueSaint Paul, MN 55108USA

Scott LefurgyColumbia UniversityDepartment of Chemistry3000 Broadway, MC 3153New York, NY 10027USA

Alice LoidlGraz University of TechnologyDepartment of BiochemistryPetersgasse 12/28010 GrazAustria

Valéria Maia de OliveiraCenter of Chemistry, Biologicaland Agricultural ResearchCPQBA/UNICAMP, CP 6171CEP 13081-970Campinas, SPBrazil

Gilson Paulo ManfioNatura Inovação e Tecnologiade Produtos Ltda.CEP 07750-000Cajamar, SPBrazil

Manfred T. ReetzMax-Planck-Institut fürKohlenforschungKaiser-Wilhelm-Platz 145470 Mülheim/RuhrGermany

List of Contributors XVII

Jean-Louis ReymondUniversity of BerneDepartment of Chemistry &BiochemistryFreistrasse 33012 BerneSwitzerland

Cintia RoodveldtThe Weizmann Institute of ScienceDepartment of Biological ChemistryRehovot 76100Israel

Dick SchipperDSM Food SpecialtiesAnalysisPO Box 12600 MA DelftThe Netherlands

Hannes SchmidingerGraz University of TechnologyDepartment of BiochemistryPetersgasse 12/28010 GrazAustria

Hubert ScholzeGraz University of TechnologyDepartment of BiochemistryPetersgasse 12/28010 GrazAustria

Renaud SicardUniversity of BerneDepartment of Chemistry &BiochemistryFreistrasse 33012 BerneSwitzerland

Theo SonkeDSM Pharma Chemicals –Advanced SynthesisCatalysis & DevelopmentPO Box 186160 MD GeleenThe Netherlands

Dan S. TawfikThe Weizmann Institute of ScienceDepartment of Biological ChemistryRehovot 76100Israel

Nicholas J. TurnerUniversity of ManchesterSchool of ChemistryOxford RoadManchester M13 9PLUK

Sjoerd van der WalDSM ResolvePO Box 186160 MD GeleenThe Netherlands

Aad VollebregtDSM Anti-InfectivesDAI InnovationPO Box 4252600 AK DelftThe Netherlands

Ryan D. WoodyerUniversity of IllinoisDepartment of Chemistry600 S. Mathews AvenueUrbana, IL 61801USA

List of ContributorsXVIII

Shao Q. YaoNational University of SingaporeDepartment of Biological Science andDepartment of Chemistry3 Science Drive 3Singapore 117543Republic of Singapore

Huimin ZhaoUniversity of IllinoisDepartment of Chemicaland Biomolecular Engineering600 S. Mathews AvenueUrbana, IL 61801USA

Renaud Sicard and Jean-Louis Reymond

An enzyme assay is a test for enzyme function. The enzyme assay probes thechemistry of a single catalytic step in an enzyme and makes it return an an-swer, which may be a light signal or color change in the sample, or a biologicalselection event, or both. How to achieve this is left to the experimenter, whocan, and usually must, combine various chemical insights and intuitions toarrive at a working assay system. It is a molecular game with plenty of degreesof freedom, but strict demands on efficacy. Ideally, the assay should be simpleand free of mistakes – that is, no false positives or false negatives. Success isalso rated in terms of which actual reaction is being assayed, some being moredifficult than others, and in terms of ease of implementation, which oftenreduces to the price and availability of the reagents necessary to perform theassay.

Fortunately, the design and utilization of enzyme assays serve a useful purpose.Enzyme assays are indispensable tools for enzyme discovery and enzyme charac-terization. The present book aims to reflect the tremendous developments thathave taken place in these areas over the last 10 years, particularly with regard tohigh-throughput screening assays and array experiments with multiple substrates.These developments have been discussed in several review articles [1].

The driving force for the invention of new enzyme assays comes in large partfrom the field of enzyme discovery and engineering [2]. In these areas of inves-tigation enzyme assays are used to identify active enzymes from microorganismcollections or randomly generated enzyme mutant libraries. This approach hasbeen found to be very practical for discovering industrially useful catalysts. En-zyme engineering has led to an increased acceptance and utilization of enzymesfor manufacturing, in particular in the area of fine chemicals synthesis [3].

Enzyme Assays

What an enzyme assay does to visualize enzyme function is equivalent to whatstructural analysis tools do for visualizing enzyme structure. However, while

1

Introduction

one understands structure intuitively through its three-dimensional representa-tion, there is no unified representation of function. Function can be describedas a list of qualitative statements, or as a series of values for suitably defined pa-rameters. For small molecules such as drugs, function correlates well withstructure, and predictive quantitative structure–activity relationship (QSAR)models allow one to reduce function to structural elements. For macromole-cules, however, the relationship between structure and function is blurred bycomplexity, and structural analysis delivers at best a crude insight into molecu-lar function. This is particularly true for enzymes, where insignificant altera-tions in either protein or substrate structure can produce dramatic changes atthe level of function, whether it is catalytic activity, selectivity or regulation ofthe enzyme. In this case the description of molecular function becomes largelyindependent of structure (Figure 1). Experience has shown that the functionalinformation delivered by an enzyme assay on thousands of mutants is muchmore useful in showing how an enzyme could be improved than a detailedstructure of a single enzyme.

While structural determination methods use physical principles, enzyme as-says are mostly born out of chemical principles. Enzyme assay technologybuilds on classical bioorganic chemistry, and starts with a detailed analysis andunderstanding of an enzyme’s reaction mechanism and the chemical propertiesof substrates and products. Engineering of substrate structure or the use ofchemical sensors then allows the catalytic reaction to be translated into an ob-servable signal. The assay design largely depends on intuition to formulate foreach enzyme a working principle capable of turning enzymatic turnover into asignal.

In enzyme discovery and engineering the assay is used to select improved en-zyme variants from pools of enzymes or enzyme mutants. The assay is criticalin these experiments because “you get what you screen for”. This adage sum-marizes the outcome of many experiments: the product of a selection procedureis only as good as the selection principle used. The detailed chemistry of the as-say involved is therefore a key parameter for ensuring success in isolating thedesired enzyme.

Introduction2

Fig. 1 Enzyme assays as tools for functional analysis.

The principles and applications of enzyme assays are reviewed in this book.In Part I the chemistry of enzyme assays is discussed, in Part II the assays usedin the context of genetic selection are covered, and in Part III multisubstrate as-says for biochemical characterization of enzymes are discussed. Before evenstarting into these applications one should remember that positive hits fromhigh-throughput screening assays must always be confirmed by an independentmethod before concluding that a new enzyme has been discovered (Figure 2).

There are many ways to connect the conversion of a substrate into a productwith an observable signal (Figure 3). Enzyme activity can often be detected bythe action of the enzyme on its natural substrate. An enzyme activity might leadto heat production if the reaction is exothermic, or induce a macroscopic changein the reaction medium, such as the clearing of an insoluble polymer substrate,or the precipitation of a reaction product. It is also possible to follow reactionturnover using standard analytical methods such as chromatography (gas chro-matography or high-performance liquid chromatography) and mass spectrome-try, or by nuclear magnetic resonance (NMR) spectroscopy. Several applicationexamples of such methods, in particular with respect to assays for measuringenantioselectivity, are discussed by Manfred Reetz in Chapter 2 and by TheoSonke and the DSM group in Chapter 4. Direct high-throughput screening as-says for enantioselectivity are particularly important in the context of fine chem-ical synthesis because enantioselectivity is almost always the property being pur-sued in the course of developing a new catalyst. Electrochemical monitoring ofenzyme activity is typically used for glucose-sensing mediated by glucose oxi-dase [4], and has recently been applied for the lipase cutinase using a hydroqui-

Enzyme Assays 3

Fig. 2 Principles of high-throughput screening enzyme assays. The signalproduced by the assay on the test sample must be checked against a blanksample (medium only) and against an active enzyme as positive control.A positive identification must be repeated, and then confirmed by anindependent method.

none monobutyrate ester substrate covalently linked to the surface of a goldelectrode [5]. Microbial growth as a signal allows one to pick active coloniesgrowing on substrates as carbon source, and also occurs upon genetic selection(Part II).

The largest group of enzyme assays are those that induce recordable changesin light absorbency or fluorescence in the assay medium. The simplestapproach relies on colorimetric or fluorimetric chemosensors that respond toproduct formation or substrate consumption, such as pH indicators (Chapter 1).Such assays are particularly useful because they allow one to work with the sub-strate of synthetic interest. There are a number of strategies for inducing sig-nals indirectly upon enzymatic turnover, as illustrated by the following exam-ples.

In the copper-calcein assay in Figure 4 [6], an amidase releases a free aminoacid as reaction product from the corresponding amide as substrate. Aminoacids are strong chelators for metal ions, in particular Cu2+ ions, while aminoacid amides are not. The assay is based on a complex of Cu2+ and the commer-cially available fluorescein derivative calcein (3), in which the calcein fluoro-phore is quenched by the metal ion. The deacetylation of N-acetyl-l-methionine(1) by acylase I induces a fluorescence increase because the free amino acid re-action product l-methionine (2) chelates Cu2+, which releases free calcein,which regains its fluorescence. The copper-calcein assay can also be used to as-say aminopeptidases and proteases using as substrate amino acid amides andbovine serum albumin, respectively. Following a similar principle, indirect prod-uct detection in a chemical transformation can also be realized by means of an

Introduction4

Fig. 3 Signals for enzyme assays producedfrom enzymatic reactions. Note that obser-vable spectral changes may occur eitherdirectly due to structural differences betweensubstrate and product (e.g. Chapters 2, 4, 5,9, 11 and 12), or indirectly, for example

through an chemical indicator system(e.g. Chapter 1), by processing of thereaction product by secondary enzymes orreagents (e.g. Chapters 3, 6 and 10), or viathe induction of gene expression by thereaction product (Chapter 7).

immunoassay using an antibody capable of differentiating product from sub-strate [7]. In these immunoassays the product-selective antibody plays the samerole as the Cu2+ ion in the amidase assay above.

Another elegant indirect assay by Matile and coworkers is based on vesiclescontaining a concentrated, autoquenched solution of fluorescein (Figure 5) [8].The vesicles are equipped with synthetic pores for fluorescein. The pores areplugged by the enzyme substrate, but not by the reaction product. Reaction pro-gress results in unplugging of the pore, which leads to diffusion of fluoresceinoutside the vesicles and an increase in fluorescence. The assay has been demon-strated for fructose bis-phosphate aldolase, alkaline phosphatase, galactosyltrans-ferase, DNA exonuclease III, DNA polymerase I, RNase A, apyrase, heparinaseI, hyaluronidase, papain, ficin, elastase, subtilisin, and pronase.

The most frequently used enzyme assays involve fluorogenic and chromo-genic substrates. A synthetic substrate is designed such that the enzyme turns anonfluorescent or colorless appendage of the substrate into a fluorescent or co-lored product. Thus, a color or fluorescent signal is created out of a dark or col-orless solution by the direct action of the enzyme. This principle is realized bysubstrates with cleavable ethers or esters of electron-poor conjugated aromaticphenols. The conjugate bases of these phenols show very strong color and fluo-rescence properties not present in the protonated, alkylated or acylated deriva-tives. These include the well-known yellow nitrophenolate (4), the blue fluores-

Enzyme Assays 5

Fig. 4 An indirect fluorogenic assay for acylase I. Calcein (3) is acommercially available inexpensive fluorescein derivative. The assay is alsosuitable for other amino acid-releasing enzymes, such as aminopeptidasesand proteases, when using the appropriate substrate.

cent umbelliferone anion (5), the red fluorescent resorufin anion (6) and thegreen fluorescent fluorescein anion (7) (Figure 6). Many fluorogenic and chro-mogenic enzyme substrates are commercially available and serve as referencesubstrates for hydrolytic enzymes (see Chapter 1).

Introduction6

Fig. 5 A general fluorescence enzyme assay using synthetic pores.Product turnover unplugs the pores, which allow fluorescein to diffuseto the outside of the vesicles and become fluorescent.

Fig. 6 Acidic conjugated electron-poor phenols used in fluorogenic andchromogenic enzyme substrates. The corresponding neutral phenols aregenerally colorless and nonfluorescent.

Part I: High-throughput Screening

The significance of an enzyme assay and its successful application depends onits chemical and analytical design. Part I discusses enzyme assays tailored tothe problem of enzyme discovery, which requires high-throughput screening po-tential for relevant chemical transformations. In the context of fine chemicalsynthesis this means the ability to screen for enantio- and stereoselectivity ofthe targeted reactions.

In Chapter 1, Romas J. Kazlauskas describes the use of a colorimetric pH in-dicator together with reference fluorogenic substrates to carry out efficient high-throughput screening of esterolytic enzymes with chiral substrates. The methodallows stereoselectivity information to be obtained directly from high-through-put screening with any substrate of synthetic interest.

In Chapter 2, Manfred T. Reetz reviews enzyme assays for screening enantio-selective reactions. Analysis of isotopically labeled pseudo-enantiomeric mixturesby MS and NMR provides a practical approach for screening kinetic resolutionsof racemic mixtures or the deracemization of prochiral substrates. For the caseof asymmetric induction where a chiral product is formed from an achiral andnonprochiral substrate, the situation is more complex and requires indirectsensing of product chirality by enantioselective sensors.

In Chapter 3, Tyler W. Johannes, Ryan D. Woodyer, and Huimin Zhao reviewfluorogenic and chromogenic systems for redox enzymes. These assays are criti-cal because redox enzymes have a particularly important and yet largely un-tapped potential for industrial applications. For example alkane monoxygenasescan perform selective hydroxylation reactions on hydrocarbons that are simplynot accessible at all to chemical catalysts [9]. In addition many chemical redoxreagents are expensive, toxic, and difficult to handle, implying that economicalenzyme replacements should be possible in almost all cases.

The best test bed for enzyme assays occurs in an industrial context, wherepractical catalysts need to be developed rapidly and applied in large-scale pro-duction. In Chapter 4, Theo Sonke, Lucien Duchateau, Dick Schipper, Gert-JanEuverink, Sjoerd van der Wal, Huub Henderickx, Roland Bezemer, and AadVollebregt report their own experiences at the Dutch company DSM, where in-direct colorimetric assays and high-throughput direct analyses such as HPLCand NMR have been used. This industrial contribution highlights the impor-tance of screening for enantioselectivity, as also discussed in Chapters 1 and 2.

Part II: Genetic Selection

Enzyme assays play a central role in the context of microbial screening and di-rected evolution experiments. In this field the catalysis signal is used as the se-lection criterion to accept or reject single genes or microbial colonies in thehope of isolating enzyme mutants with desirable catalytic properties. The genet-ic diversity undergoing selection through the enzyme assay consists either in

Part II: Genetic Selection 7

enzyme mutants generated artificially, or in biodiversity collections, such asgene libraries from the metagenome or microorganism collections (Figure 7).

Screening preferentially describes experiments in which the assay signal isused to direct an external device to pick individual active enzymes or enzyme-producing genes, such as manual picking from agar plates or microtiter platesor the use of fluorescence-activated cell sorting. The term “genetic selection”best describes systems where the expression of an active enzyme is linked tocell survival without external signal processing.

In Chapter 5, Nicholas J. Turner reviews the design and application of enzymeassays in the context of selecting active enzymes by colony picking on agar, whichis the most common screen used for microbial cultures. The chapter discusseshow much can be achieved quickly by implementing straightforward chemical re-action principles in a microbiological context. A critical overview of genetic selec-tion methods used to isolate active enzymes is also presented.

Random mutagenesis protocols such as gene shuffling [10], error-prone poly-merase chain reaction (PCR) [11], and the later improvements or variations ofthese methods [12] readily allow on the order of 1012 mutants of a given en-zyme to be generated in a single experiment. However, high-throughput screen-ing experiments in microtiter plates or even on agar plate can only test a fewtens of thousands of mutants for catalytic activity. In recent years several groupshave invented methods to allow efficient screening of such large numbers ofmutants.

In Chapter 6, Amir Aharoni, Cintia Roodveldt, Andrew D. Griffiths, and DanS. Tawfik provide a general overview of screening methods applicable in thecontext of both functional genomics and directed evolution. The authors discussthe critical problem of choosing the right expression system for a given enzyme,and the implementation of selection pressure that is able to distinguish between

Introduction8

Fig. 7 Selection of active enzymes from genetic libraries and biodiversity.

protein expression levels and protein function. A variety of high-throughputscreening approaches for enzymes, such as phage-display and fluorescence-acti-vated cell sorting, are reviewed, including the author’s own elegant emulsion-based compartimentalization system for screening large genetic libraries.

In Chapter 7, Scott Lefurgy and Virginia Cornish review high-throughput se-lection methods by chemical complementation. The chapter includes an excel-lent review of genetic selection experiments that can be used to perform direct-ed evolution, and emphasizes chemical complementation by the yeast three-hybrid system. In this system a synthetic chemical inducer of dimerization(CID) acts as a tether between a DNA-binding domain and a transcription acti-vation domain. The CID either serves as a substrate cleavable by the enzyme, oris the product of an enzyme coupling. The experiment is set up such that acti-vation or deactivation of gene expression in the presence of an enzyme cleavingor forming the CID is conditional for cell survival, allowing genetic selection totake place. Chemical complementation is demonstrated by various examples, in-cluding �-lactamases and glycosynthase enzymes.

In recent years microbiologists studying biodiversity have come to realize thatthe natural environment, in particular biotopes under extreme conditions, har-bor a very large number of diverse microbes. In Chapter 8, Valéria Maia de Oli-veira and Gilson Paulo Manfio present an overview of screening methods in thecontext of exploiting the genetic biodiversity available in microbial collectionsand in environmental DNA. Environmental DNA is recovered by direct PCRamplification and includes genetic material from noncultivable microbes, whichis considered to be the vast majority (>99%), and is collectively called the meta-genome [13]. In addition to screening for expressed enzyme activity in such li-braries, it is also possible to analyze gene sequences for conserved sequence pat-terns indicative of certain enzyme activities.

Part III: Enzyme Fingerprinting

An enzyme assay is a tool designed to visualize enzyme function. In its sim-plest expression, the resulting picture of enzyme activity is a single pixel in twocolors (e.g. white = no activity, black= activity, with respect to the assay being per-formed). The picture can adopt higher levels of definition if the number of pix-els is augmented, or if a color shading is allowed for each pixel. This can be re-alized by combining several different assays for the same enzyme into an array,and by obtaining quantitative rather than qualitative data from each assay. Theresulting pictures of enzyme function are called activity profiles, or fingerprints(Figure 8).

The notion of fingerprint is associated with the possibility of using the activityprofile as an identification mark for an enzyme or enzyme-containing sample,which is the prerequisite for all diagnostic applications of enzyme assays. Anydevice capable of recording an enzyme fingerprint is the equivalent of a camerafor taking pictures of enzyme function. As for screening, enzyme assays for fin-

Part III: Enzyme Fingerprinting 9

gerprinting must be applicable in high-throughput. The goal here is to collectthe enzyme activity data simultaneously on many different substrates or inmany different reaction conditions.

In Chapter 9, Ruth Birner-Grünberger, Hannes Schmidinger, Alice Loidl,Hubert Scholze, and Albin Hermetter discuss assays used for the identificationand biochemical study of lipases and esterases. These include fluorogenic sub-strates specifically designed for targeted hydrolases. The authors also review ac-tive-site labeling probes, which are used to covalently tag active enzyme for lateridentification by gel electrophoresis and mass spectrometry. Such active-site la-beling probes have established themselves as useful reagents for the discoveryof new disease-related enzymes.

In Chapter 10, Johann Grognux and Jean-Louis Reymond report a series ofpractical methods for recording activity fingerprints of enzymes, mostly in thecase of hydrolytic enzymes such as lipases, esterases, and proteases. Enzymefingerprinting involves recording a reproducible image of the reactivity profileof an enzyme, such that the images obtained from different enzymes can beused for functional classification. The principle derives from multi-enzyme pro-filing as used for phenotyping in microbiology and medical diagnostics. Meth-ods of fingerprinting include arrays of indirect fluorogenic substrates acting bya common mechanism of fluorescence release, and substrate cocktail reagents,which allow recording of an activity fingerprint in a single experiment. Dataacquisition and statistical analysis techniques leading to functional classificationof enzymes are presented.

In Chapter 11, Jennifer L. Harris reviews the application of fluorogenic pep-tide substrate libraries for large-scale profiling of proteases, a method which isused to define the substrate specificities and the actual natural substrates of pro-teases. The chapter reviews a number of protease profiling methods and experi-ments. Protease profiling has proven to be a indispensable tool for the biochem-ical study of these enzymes. The concept of positional scanning peptide librariesis central to surveying the entire sequence space of peptide substrates within areasonable experimental effort.

The promise of fingerprinting lies not only in multiparametric analysis forstudying enzymes, but also in possible applications in the area of diagnostics

Introduction10

Fig. 8 Enzyme activity fingerprinting.