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Updated: Jun 8, 2026
08:10
Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
Screens for active and stereoselective hydrolytic enzymes
Dominique Böttcher1, Marlen Schmidt, Uwe T Bornscheuer
1Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Greifswald, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|September 11, 2010
Summary
A new high-throughput screening (HTS) method efficiently identifies active esterase clones. This assay enables rapid screening of thousands of enzyme variants daily for applications in organic synthesis.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Biocatalysis
Background:
- Esterases are crucial enzymes with broad applications in organic synthesis and biotechnology.
- Developing efficient screening methods is essential for discovering novel esterases with desired properties.
Purpose of the Study:
- To establish a high-throughput screening (HTS) procedure for identifying active esterase variants.
- To enable rapid assessment of enzyme activity and enantioselectivity (E) for enzyme engineering.
Main Methods:
- A multi-step assay involving agar plate pretesting, microtiter plate enzyme expression, and spectrophotometric quantification of NADH formation.
- Utilized acetates of secondary alcohols as model substrates, with released acetic acid driving an enzyme cascade to NADH.
Main Results:
- The HTS method allows for the screening of thousands of esterase mutants per day.
- Successfully identified an esterase mutant exhibiting an enantioselectivity (E) greater than 100 for a valuable organic synthesis building block.
Conclusions:
- The developed protocol provides an efficient HTS strategy for esterase discovery and engineering.
- The method is adaptable for screening lipases and other soluble hydrolases expressed in Escherichia coli.
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