The carbonyl reductase CpCR from Candida parapsilosis ATCC 7330 was modified to improve its ability for catalyzing the synthesis of 2-phenylethanol (2-PE). A mutant library was constructed by error-prone PCR, and positive clones were screened by 2,4-dinitrophenylhydrazine staining, and amino acid mutation sites of the positive clones were determined by DNA sequencing. Then, virtual saturation mutagenesis was performed by protein semi-rational design, and site-directed mutagenesis was used for construction and evaluation of the efficient mutants. The mutant T171F has better catalytic ability and thermal stability. The effects of catalytic time, temperature, pH and substrate addition on the synthesis of 2-phenylethanol catalyzed by wtCpCR and T171F were further investigated. The results showed that the optimum temperature and pH of mutant T171F for the enzyme-catalyzed synthesis of 2-PE was 30 ℃ and pH 6.5. When the concentration of phenylacetaldehyde was 1 000 mg/L, the yield of T171F was 91.24%, 2.8 times that of wtCpCR. In addition, the catalytic time of mutant T171F was reduced from 10 h to 4 h compared with wtCpCR, and the catalytic efficiency was greatly improved. This study provides a scientific basis for the catalytic synthesis of 2-phenylethanol by carbonyl reductase, and has certain application value.
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