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Enzyme Assays: High-throughput Screening, Genetic Selection and Fingerprinting

Enzyme Assays: High-throughput Screening, Genetic Selection and Fingerprinting

Jean-Louis Reymond (Editor)

ISBN: 978-3-527-60721-1

May 2006

386 pages

Select type: E-Book


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Edited by one of the leading experts in the field, this book fills the need for a book presenting the most important methods for high-throughput screenings and functional characterization of enzymes. It adopts an interdisciplinary approach, making it indispensable for all those involved in this expanding field, and reflects the major advances made over the past few years.
For biochemists, analytical, organic and catalytic chemists, and biotechnologists.

List of Contributors.

Introduction (Renaud Sicard and Jean-Louis Reymond).

Enzyme Assays.

Part I: The Chemistry of Enzyme Assays.

Part II: Enzyme Assays and Genetic Selection.

Part III: Enzyme Profiling.

Enzyme Assays in Other Areas.

How to Use this Book.

Part I High-throughput Screening.

1 Quantitative Assay of Hydrolases for Activity and Selectivity Using Color Changes (Romas J. Kazlauskas).

1.1 Overview.

1.2 Direct Assays Using Chromogenic Substrates.

1.3 Indirect Assays Using Coupled Reactions – pH Indicators.

1.4 Estimating and Measuring Selectivity.

2 High-throughput Screening Systems for Assaying the Enantioselectivity of Enzymes (Manfred T. Reetz).

2.1 Introduction.

2.2 UV/Vis Spectroscopy-based Assays.

2.3 Assays Using Fluorescence.

2.4 Assays Based on Mass Spectrometry (MS).

2.5 Assays Based on Nuclear Magnetic Resonance Spectroscopy.

2.6 Assay Based on Fourier Transform Infrared Spectroscopy for Assaying Lipases or Esterases.

2.7 Assays Based on Gas Chromatography.

2.8 Assays Based on HPLC.

2.9 Assays Based on Capillary Array Electrophoresis.

2.10 Assays Based on Circular Dichroism (CD).

2.11 Assay Based on Surface-enhanced Resonance Raman Scattering.

2.12 Conclusions.

3 High-throughput Screening Methods Developed for Oxidoreductases (Tyler W. Johannes, Ryan D. Woodyer, and Huimin Zhao).

3.1 Introduction 77

3.2 High-throughput Methods for Various Oxidoreductases.

3.3 Conclusions.

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

4.1 Introduction.

4.2 Prerequisites for an Effective Biocatalyst Screening in Chemical Custom Manufacturing

4.3 CCM Compliant Screening Methods Based on Optical Spectroscopy (UV/Vis and Fluorescence).

4.4 CCM Compliant Screening Methods Based on Generic Instrumental Assays.

4.5 Conclusions.

Part II Genetic Selection.

5 Agar Plate-based Assays (Nicholas J. Turner).

5.1 Introduction.

5.2 Facilitated Screening-based Methods.

5.3 In vivo Selection-based Methods.

5.4 Conclusions and Future Prospects.

6 High-throughput Screens and Selections of Enzyme-encoding Genes (Amir Aharoni, Cintia Roodveldt, Andrew D. Griffiths, and Dan S. Tawfik).

6.1 Introduction.

6.2 The Basics of High-throughput Screens and Selections.

6.3 High-throughput Selection of Enzymes Using Phage Display.

6.4 High-throughput Selection of Enzymes Using Cell Display.

6.5 In vivo Genetic Screens and Selections.

6.6 Screens for Heterologous Protein Expression and Stability.

6.7 In vitro Compartmentalization.

6.8 IVC in Double Emulsions.

6.9 Concluding Remarks.

7 Chemical Complementation (Scott Lefurgy and Virginia Cornish).

7.1 Introduction.

7.2 Complementation Assays.

7.3 Development of Chemical Complementation.

7.4 Applications of Chemical Complementation.

7.5 Conclusion.

8 Molecular Approaches for the Screening of Novel Enzymes (Valéria Maia de Oliveira and Gilson Paulo Manfio).

8.1 Introduction.

8.2 Use of Nucleic Acid Probes to Detect Enzyme-coding Genes in Cultivated Microorganisms.

8.3 The Microbial Metagenome: a Resource of Novel Natural Products and Enzymes.

8.4 Concluding Remarks.

Part III Enzyme Fingerprinting.

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

9.1 Introduction.

9.2 Fluorogenic and Fluorescent Substrates for Enzyme Activity.

9.3 Fluorescent Inhibitors for Quantitative Analysis of Active Enzymes and Functional Enzyme Fingerprinting.

10 Fingerprinting Methods for Hydrolases (Johann Grognux and Jean-Louis Reymond).

10.1 Introduction.

10.2 Hydrolase Fingerprinting.

10.3 Classification from Fingerprinting Data.

10.4 Outlook.

11 Protease Substrate Profiling (Jennifer L. Harris).

11.1 Introduction.

11.2 Functional Protease Profiling – Peptide Substrate Libraries.

11.3 Identification of Macromolecular Substrates.

11.4 Conclusions.

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

12.1 Introduction.

12.2 Immobilization Strategies.

12.3 Microarray-based Methods for Detection of Enzymatic Activity.

12.4 Conclusions.

Subject Index.

an excellent and thoughtful reflection on the advantage of screening for enzyme function rather than predicting function from structural screening." (Clinical Chemistry, January 2007)

" excellent overview presenting a unique exposition of all important developments, techniques and concepts in this rapidly advancing field. [...] In summary, this book gives an excellent overview of current and relevant approaches where there is a need for enzyme assay development and use, and should be of interest to a broad readership. It is an indispensable source of information and inspiration for everyone working in the field and can highly be recommended." (ChemBioChem)