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High-throughput screening of activity and enantioselectivity of esterases

Nature Protocols volume 1, pages 2340–2343 (2006)Cite this article

Abstract

A procedure for the high-throughput screening of esterases is described. This includes enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E>100 toward an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains. This protocol can be completed in 3–4 days.

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Figure 1: Enzyme production in microtiter plates and principle of the screening of mutant libraries for altered enantioselectivity by adding (R)- and (S)-substrates to separate wells of a microtiter plate containing the same enzyme variant.
Figure 2
Figure 3: Initial rates determined by using optically pure (R)- or (S)-1-phenyl ethyl acetate (5 mg μl−1).

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Acknowledgements

We thank M. Baumann, E. Henke and M. Schmidt for their support in developing and applying the methodology.

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  1. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, Greifswald, D-17487, Germany

    Dominique Böttcher & Uwe T Bornscheuer

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  1. Dominique Böttcher
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  2. Uwe T Bornscheuer
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Correspondence to Uwe T Bornscheuer.

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Böttcher, D., Bornscheuer, U. High-throughput screening of activity and enantioselectivity of esterases. Nat Protoc 1, 2340–2343 (2006). https://doi.org/10.1038/nprot.2006.391

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