Universität zu Köln

Bulut, Dalia, Duangdee, Nongnaphat, Groeger, Harald, Berkessel, Albrecht ORCID: 0000-0003-0470-7428 and Hummel, Werner (2016). Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic beta-Hydroxy-beta-trifluoromethyl Ketone. ChemBioChem, 17 (14). S. 1349 - 1359. WEINHEIM: WILEY-V C H VERLAG GMBH. ISSN 1439-7633

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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 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Bulut, Dalia UNSPECIFIED UNSPECIFIED UNSPECIFIED Duangdee, Nongnaphat UNSPECIFIED UNSPECIFIED UNSPECIFIED Groeger, Harald UNSPECIFIED UNSPECIFIED UNSPECIFIED Berkessel, Albrecht UNSPECIFIED orcid.org/0000-0003-0470-7428 UNSPECIFIED Hummel, Werner UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-269821
DOI:	10.1002/cbic.201600101
Journal or Publication Title:	ChemBioChem
Volume:	17
Number:	14
Page Range:	S. 1349 - 1359
Date:	2016
Publisher:	WILEY-V C H VERLAG GMBH
Place of Publication:	WEINHEIM
ISSN:	1439-7633
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Organic Chemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language GLUCONOBACTER-OXYDANS; COFACTOR REGENERATION; GENOME SEQUENCE; BIOCATALYSIS; TAG; NAD(+); MEDIA; NADH Multiple languages Biochemistry & Molecular Biology; Chemistry, Medicinal Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/26982