中文摘要：

【目的】通过优化获得最佳酶活配比,设计近平滑假丝酵母（Candida parapsilosis）CCTCC M203011的（S）-羰基还原酶Ⅱ与枯草芽孢杆菌（Bacillus sp.）YX-1葡萄糖脱氢酶在大肠杆菌中的共表达体系,实现重组菌高效催化2-羟基苯乙酮,合成（S）-苯乙二醇。【方法】分别从重组大肠杆菌中纯化了（S）-羰基还原酶Ⅱ和葡萄糖脱氢酶,研究了2种酶共催化2-羟基苯乙酮的最佳酶活比例,最适催化温度和pH,由此构建（S）-羰基还原酶Ⅱ和葡萄糖脱氢酶的共表达体系。【结果】（S）-羰基还原酶Ⅱ的比酶活力为1.3 U/mg,葡萄糖脱氢酶的比酶活力为13.5 U/mg。在总酶活力为1 U时,（S）-羰基还原酶Ⅱ和葡萄糖脱氢酶共催化体系中,确定了2种酶的最佳比例在1∶1到5∶1（U/U）之间,最适反应温度为30℃,pH为7.0。在此基础上构建了（S）-羰基还原酶Ⅱ和葡萄糖脱氢酶基因比为1∶1的共表达体系,共表达重组菌破碎上清液中（S）-羰基还原酶Ⅱ和葡萄糖脱氢酶酶活分别为0.76 U/mg和0.73 U/mg,两者的酶活比例为1∶1。在上述确定的最适催化条件下,其催化10 g/L 2-羟基苯乙酮,产物（S）-苯乙二醇的光学纯度和得率均高达99%以上。与仅含有（S）-羰基还原酶Ⅱ的重组大肠杆菌相比,共表达体系转化产物（S）-苯乙二醇的得率明显提高,且转化时间由原来的24 h缩短为13 h。【结论】通过确定（S）-羰基还原酶Ⅱ和葡萄糖脱氢酶最佳酶活配比,为构建手性催化的靶酶和辅酶再生酶共表达体系,为实现手性化合物的高效制备提供了研究基础。

英文摘要：

[Objective] To realize efficient biosynthesis of 2-hydroxyacetophenone to （S）-1-phenyl-1,2-ethanediol, we designed a co-expression system containing Candida parapsilosis CCTCC M203011 （S）-carbonyl reductase II （SCR Ⅱ ） and Bacillus sp. YX-1 glucose dehydrogenase （GDH） in Escherichia coli BL21（DE3）, based on the optimal ratio between the specific activities of the two enzymes. [Methods] The enzymes SCR II and GDH were purified from their corresponding recombinant E. coli strains. When the purified SCR Ⅱ and GDH were used for the reduction of 2-hydroxyacetophenone to （S）-l-phenyl-1,2-ethanediol, the optimal ratio between their specific activities, the optimal temperature and pH were determined. Based on above results, a co-expression system E. coli BL21 （DE3）/S- SD-AS-G harboring SCR Ⅱ and GDH was constructed. [Results] SCR Ⅱ and GDH exhibited specific activities of 1.3 U/mg and 13.5 U/mg. When the total enzyme activity was 1 U, the optimal ratio of their activities is between 1：1 and 5：1, and the optimal temperature and pH are 30 ℃ and 7.0, respectively. So we designed a co-expression system E. coli BL21/S-SD-AS-G, in which the ratio of the SCR II and GDH genes is 1：1. The specific activities of SCR II and GDH are 0.76 U/mg and 0.73 U/mg in the cell-free extracts of E. coli BL21 （DE3）/S-SD-AS-G, respectively. The ratio between SCR II and GDH activity is 1：1. Under the optimal conditions, the system showed excellent performance to produce （S）-1-phenyl-1,2-ethanediol with an optical purity and a yield both over 99% during the reduction of 2- hydroxyacetophenone. With respect to the recombinant E. coli BL21 （DE3）/pET-SCR II, the co-expression system obviously improved the yield of （S）-1-phenyl-1,2-ethanediol and reduced biotransformation time from 24 h to 13 h. [Conclusion] This work provides the research foundation on the construction of a co-expression system containing a target chiral catalyst and a cofactor-regeneration enzyme for efficient chiral bios