title: 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.
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 β-hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β-hydroxy-β-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 β-hydroxy-β-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 β-hydroxy-β-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 β-hydroxy-β-trifluoromethyl ketone.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich Heine University of Düsseldorf, Research Centre Jülich, Wilhelm-Johnen-Strasse, 52426, Jülich, Germany.|Faculty of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Dalia
authors.initials: D
authors.lastname: Bulut
authors.affiliation: Department of Chemistry, Cologne University, Greinstrasse 4, 50939, Köln, Germany.
authors.forename: Nongnaphat
authors.initials: N
authors.lastname: Duangdee
authors.affiliation: Faculty of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Gröger
authors.affiliation: Department of Chemistry, Cologne University, Greinstrasse 4, 50939, Köln, Germany.
authors.forename: Albrecht
authors.initials: A
authors.lastname: Berkessel
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich Heine University of Düsseldorf, Research Centre Jülich, Wilhelm-Johnen-Strasse, 52426, Jülich, Germany. werner.hummel@uni-bielefeld.de.|Faculty of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany. werner.hummel@uni-bielefeld.de.
authors.forename: Werner
authors.initials: W
authors.lastname: Hummel
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D010088:Oxidoreductases; D010843:Pichia / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007659:Ketones; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase
keywords: alcohol dehydrogenases; asymmetric reduction and oxidation; biocatalysis; diols; enantioselectivity; kinetic resolution