Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone

Bulut D, Duangdee N, Gröger H, Berkessel A, Hummel W (2016)
Chembiochem 17(14): 1349-1358.

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Abstract
The stereoselective synthesis of chiral 1,3-diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of beta-hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a beta-hydroxy-beta-trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2-trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the beta-hydroxy-beta-trifluoromethyl ketone was identified after purification and subsequent MALDI-TOF mass spectrometric analysis. As a result, a new NADP(+) -dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the beta-hydroxy-beta-trifluoromethyl ketone to its corresponding 1,3-diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N- or C-terminal His-tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N-terminal His-tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the beta-hydroxy-beta-trifluoromethyl ketone. 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Bulut D, Duangdee N, Gröger H, Berkessel A, Hummel W. Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone. Chembiochem. 2016;17(14):1349-1358.
Bulut, D., Duangdee, N., Gröger, H., Berkessel, A., & Hummel, W. (2016). Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone. Chembiochem, 17(14), 1349-1358.
Bulut, D., Duangdee, N., Gröger, H., Berkessel, A., and Hummel, W. (2016). Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone. Chembiochem 17, 1349-1358.
Bulut, D., et al., 2016. Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone. Chembiochem, 17(14), p 1349-1358.
D. Bulut, et al., “Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone”, Chembiochem, vol. 17, 2016, pp. 1349-1358.
Bulut, D., Duangdee, N., Gröger, H., Berkessel, A., Hummel, W.: Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone. Chembiochem. 17, 1349-1358 (2016).
Bulut, Dalia, Duangdee, Nongnaphat, Gröger, Harald, Berkessel, Albrecht, and Hummel, Werner. “Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone”. Chembiochem 17.14 (2016): 1349-1358.
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