Abstract
A procedure for the high-throughput screening (HTS) of esterases is described. This includes a pretest for discrimination of active and inactive clones using an agar plate overlay assay, the 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 towards 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.
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References
Bornscheuer, U.T. and Kazlauskas, R.J. (2006) Hydrolases in Organic Synthesis – Regio- and Stereoselective Biotransformations, 2nd Edn. Wiley-VCH, Weinheim.
Cherry, J.R. and Fidantsef, A.L. (2003) Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol 14, 438–443.
Turner, N.J. (2003) Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol 21, 474–478.
Schmidt, M., Baumann, M., Henke, E., Konarzycka-Bessler, M., and Bornscheuer, U.T. (2004) Directed evolution of lipases and esterases. Methods Enzymol 388, 199–207.
Lorenz, P., Liebeton, K., Niehaus, F., and Eck, J. (2002) Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space. Curr Opin Biotechnol 13, 572–577.
Handelsman, J. (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68, 669–685.
Kourist, R., Krishna, S.H., Patel, J.S., Bartnek, F., Weiner, D.W., Hitchman, T., et al. (2007) Identification of a metagenome-derived esterase with high enantioselectivity in the kinetic resolution of arylaliphatic tertiary alcohols. Org Biomol Chem 5, 3310–3313.
Wahler, D. and Reymond, J.-L. (2001) High-throughput screening for biocatalysts. Curr Opin Chem Biol 12, 535–544.
Reetz, M.T. (2004) Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramnifications. Proc Natl Acad Sci USA 101, 5716–5722.
Goddard, J.P. and Reymond, J.-L. (2004) Recent advances in enzyme assays. Trends Biotechnol 22, 363–370.
Reymond, J.-L. (2005) Enzyme Assays. Wiley-VCH, Weinheim.
Schmidt, M. and Bornscheuer, U.T. (2005) High-throughput assays for lipases and esterases. Biomol Eng 22, 51–56.
Reetz, M.T. and Jaeger, K.E. (2000)Enantioselec-tive enzymes for organic synthesis created by directed evolution. Chem Eur J 6, 407–412.
Schmidt, M., Hasenpusch, D., Kähler, M., Kirchner, U., Wiggenhorn, K., Langel, W., et al. (2006) Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block. ChemBioChem 7, 805–809.
Baumann, M., Stürmer, R., and Bornscheuer, U.T. (2001) A high-throughput-screening method for the identification of active and enantioselective hydrolases. Angew Chem Int Ed Engl 40, 4201–4204.
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Böttcher, D., Schmidt, M., Bornscheuer, U.T. (2010). Screens for Active and Stereoselective Hydrolytic Enzymes. In: Streit, W., Daniel, R. (eds) Metagenomics. Methods in Molecular Biology, vol 668. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-823-2_11
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DOI: https://doi.org/10.1007/978-1-60761-823-2_11
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