title: Improvement of operational stability of Ogataea minuta carbonyl reductase for chiral alcohol production.
abstract: Directed evolution of enantio-selective carbonyl reductase from Ogataea minuta was conducted to improve the operational stability of the enzyme. A mutant library was constructed by an error-prone PCR and screened using a newly developed colorimetric assay. The stability of a mutant with two amino acid substitutions was significantly higher than that of the wild type at 50°C in the presence of dimethyl sulfoxide. Site-directed mutagenesis analysis showed that the improved stability of the enzyme can be attributed to the amino acid substitution of V166A. The half-lives of the V166A mutant were 11- and 6.1-times longer than those of the wild type at 50°C in the presence and absence, respectively, of 20% (v/v) dimethyl sulfoxide. No significant differences in the substrate specificity and enantio-selectivity of the enzyme were observed. The mutant enzyme converted 60 mM 2,2,2-trifluoroacetophenone to (R)-(-)-α-(trifluoromethyl)benzyl alcohol in a molar yield of 71% whereas the conversion yield with an equivalent concentration of the wild-type enzyme was 27%.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: honda@bio.eng.osaka-u.ac.jp.
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Mizuha
authors.initials: M
authors.lastname: Inoue
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ono
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Okano
authors.affiliation: Mitsubishi Chemical Group Science and Technology Research Center, Inc., 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Dekishima
authors.affiliation: Mitsubishi Chemical Group Science and Technology Research Center, Inc., 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawabata
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)
keywords: Carbonyl reductase; Directed evolution; Ogataea minuta; Organic-solvent tolerance; Thermal stability