一种新型(S)-羰基还原酶的克隆及其功能表达

Gene cloning and expression of a novel (S)-specific carbonyl reductase

【目的】从近平滑假丝酵母(Candida parapsilosis CCTCC M203011)基因组中钓取新型(S)-羰基还原酶基因(scrⅡ),对其生物转化手性醇的功能进行了验证。【方法】采用PCR的方法,从C.parapsilosis基因组中扩增出一段可能的羰基还原酶基因scrⅡ。以构建的重组菌Escherichia coli BL21/pET28a-scrⅡ为生物催化剂,2-羟基苯乙酮为底物进行催化反应,经HPLC分析,计算终产物的光学纯度和产率,确定了转化反应的最适温度和pH值。【结果】scrⅡ基因全长为840bp,编码279个氨基酸,与已报道的(S)-羰基还原酶基因scr的一致性为85%。氨基酸序列分析表明SCRⅡ具有典型短链醇脱氢酶的功能域:辅酶结合区域Thr40-Gly41-(X)3-Gly45-X-Gly47和催化三联体结构Ser172-(X)n-Tyr187-(X)3-Lys191。在30℃,0.1mmol/LIPTG的诱导下,(S)-羰基还原酶(SCRⅡ)在E.coli中过量表达。以10%(w/v)的重组菌为催化剂,高浓度(6g/L)2-羟基苯乙酮为底物,在最适反应温度35℃和pH5.5的条件下,转化产物(S)-苯基乙二醇的光学纯度高达99.1%e.e.,产率为89.6%。与(S)-羰基还原酶SCR相比较,底物浓度提高了一倍,产物的光学纯度和产率分别提高了10%和28%。【结论】采用分子克隆技术分离出新型羰基还原酶SCRⅡ的编码基因,该酶的发现为手性醇的高效制备奠定了坚实的研究基础。

[ Objective] A novel （S）-specific carbonyl reductase gene （scr Ⅱ ） was cloned from the genome of Candida parapsilosis CCTCC M203011, and its catalytic function for the biotransformation of ehiral alcohol was verified. [ Methods] The possible carbonyl reductase gene scr Ⅱ was amplified by PCR method from the C. parapsilosis genome. Using the recombinant Escherichia coli BL21/pET28a-scr Ⅱ as the catalyst and 2-hydroxyacetophenone as the substrate, the biotransformation was carried out. The optical purity and yield of the final product were investigated by HPLC analysis. The optimal pH and temperature of the reaction were also determined. [ Results ] The gene scr Ⅱ coded 279 amino acids with an open reading frame of 837 bp. It shared 85% identity with the reported gene scr. By analysis, scrⅡ contained two typical motifs of the short-chain carbonyl reductase including a Rossmann-fold Thr40-Gly41-（X） 3-Gly45- X-Gly47 and a conserved catalytic triad Ser172-（X） n-Tyr187-（ X）3-Lysl91. SDS-PAGE results showed that SCR II was overexpressed at 30℃ after the induction of 0.1 mmol/L IPTG. When the concentration of 2-hydroxyacetophenone was 6 g/L, 10% （w/v） wet recombinant E. coli cells showed excellent performance to give （S）-l-phenyl-1,2-ethanediol with high optical purity of 99.1% enantiomeric excess in a yield of 89.6% under the optimal conditions of pH 5.5 and 35℃.scrⅡ catalyzed the transformation of （S）-l-phenyl-1,2-ethanediol more efficiently than SCR. When compared with SCR, its substrate concentration was increased by two-fold, and the optical purity and yield of （S）-l-phenyl-1,2- ethanediol were improved by 10% and 28% , respectively. [ Conclusion] The gene coding for novel carbonyl reductase SCR Ⅱ was isolated using the molecular cloning technique and its discovery supplied a solid research foundation for chiral alcohol preparation efficiently.