Broader substrate specificity of Candida parapsilosis SCR Ⅱ for catalyzing acetophenone derivatives by site-directed mutagenesis

Abstract:

Abstract:［Objective］ The function for catalyzing acetophenone derivatives of short-chain carbonyl reductase Ⅱ ( SCR Ⅱ) from Candida parapsilosis was modified by site-directed mutagenesis． ［Methods］ An important site ( E228 ) was selected for mutagenesis through amino acid sequence and protein structure alignment，and the corresponding variant E228S was constructed in E． coli． Using the acetophenone derivatives as substrates，we determined specific activities and biotransformation function of the variant． ［Results］The specific activity of the variant E228S was reduced to 25% of the wild type for 2-hydroxyacetophenone reduction． However，it increased approximately 7-20 times for acetophenone，4'- methylacetophenone and 4'-chloroacetophenone． The biotransformation results showed that the variant catalyzed the transformation of ( S) - 1-phenyl-1，2-ethanediol in a yield of less than 10% ，while exhibited excellent performance to afford ( R ) - 1-phenethylethanol， ( R ) - 1-( 4-methylpheyl ) ethanol and ( R ) - 1-( 4-chlorophenyl ) ethanol from acetophenone，4'-methylacetophenone and 4'-chloroacetophenone with high optical purity of 99% in a yield of above 80% ． ［Conclusion］We broadened the substrate specificity and catalytic function of SCRⅡ by replacement of the critical amino acid ( E228 ) inside the substrate binding pocket，which provided a novel approach for rational modification of short-chain carbonyl reductases，making it as a potential tool for asymmetric reduction and chiral alcohol preparation．