Journal
CHEMBIOCHEM
Volume 17, Issue 14, Pages 1349-1358Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201600101
Keywords
alcohol dehydrogenases; asymmetric reduction and oxidation; biocatalysis; diols; enantioselectivity; kinetic resolution
Funding
- Federal Ministry of Education and Research, Germany (Bundesministerium fur Bildung und Forschung, BMBF) [031A184]
Ask authors/readers for more resources
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 Histag 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available