定点突变改变近平滑假丝酵母SCRⅡ催化苯乙酮衍生物底物谱

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

【目的】通过定点突变技术,改变近平滑假丝酵母短链羰基还原酶Ⅱ(SCRⅡ)催化苯乙酮衍生物的功能,为数种手性芳香醇的生产提供一种高效、安全的新型制备方法。【方法】通过氨基酸序列和蛋白结构比对的方法,选择SCRⅡ的底物结合域中关键氨基酸位点E228实施突变,构建相应的突变株Escherichia coliBL21/pET28a-E228S;以苯乙酮衍生物为底物,对突变株的酶活和生物转化功能进行了分析。【结果】酶活测定结果表明:突变株E.coli BL21/pET28a-E228S催化原始底物2-羟基苯乙酮的酶活仅为原始酶活的25%左右;而催化苯乙酮、4'-甲基苯乙酮、4'-氯苯乙酮的酶活是突变前的7-20倍。突变株E.coli BL21/pET28a-E228S生物转化2-羟基苯乙酮,获得产物(S)-苯基乙二醇的得率不超过10%,而以苯乙酮、4'-甲基苯乙酮、4'-氯苯乙酮为底物时,生物转化产物光学纯度维持在99%,得率高达80%以上。【结论】对底物结合域中的关键氨基酸实施突变,提高了SCRⅡ催化苯乙酮衍生物的底物广谱性,拓展了该酶的生物功能,为理性改造短链羰基还原酶的不对称还原催化功能和手性芳香醇的制备提供了新型途径。

[Objective] The function for catalyzing acetophenone derivatives of short-chain carbonyl reductase Ⅱ（SCRⅡ） 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.