کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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4753382 | 1416553 | 2017 | 6 صفحه PDF | دانلود رایگان |
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)-(â)-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%.
Journal: Journal of Bioscience and Bioengineering - Volume 123, Issue 6, June 2017, Pages 673-678