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.

Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria

“…First of all, it must be said that all showed results are activities not of the native but the His 10 -tagged proteins. In former studies, the influences of various His-tags on the N-and C-termini were investigated [42][43][44][45][46][47][48][49]. On basis of those research works, no uniform statement can be done about the influence of His-tags.…”

Expanding the Application Range of Microbial Oxidoreductases by an Alcohol Dehydrogenase from Comamonas testosteroni with a Broad Substrate Spectrum and pH Profile

“…Oxidoreductases have a wide substrate scope and can act on organic substrates including alcohols, ketones/aldehydes, amines and inorganic substrates including small ions such as sulphite [1][2][3][4]. Therefore, they are a valuable class of enzymes to synthesize complex chiral products under mild reaction conditions and with high chemo-, regio-and stereoselectivity [5,6].…”

“…(iii) The spectrum of substrates accepted by ADHs is rather broad ranging-for example, from small alcohols such as isopropanol [18] via sugars [12] to complex steroids [19,20]-and is often accompanied by a significant and synthetically attractive enantioselectivity. Accordingly, in case an important reaction step in asymmetric synthesis is accomplished, rather than mutating a well-known ADH, it is often more promising to screen for a novel ADH activity with the required profile [1]. With the growing number of annotated but not further characterized ADHs revealed by genome sequencing, it becomes increasingly attractive to perform such screening efforts in silico [21].…”