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【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。
【Objective】 The objective of this study was to optimize the optimal ratio of enzyme activity and to design a novel method for designing a new strain of Candida parapsilosis CCTCC M203011 which is compatible with Bacillus sp. YX-1 glucose dehydrogenase In E. coli co-expression system, the recombinant bacterium can efficiently catalyze 2-hydroxyacetophenone and synthesize (S) -phenylglycol. 【Method】 The (S) -carbonyl reductase Ⅱ and glucose dehydrogenase were purified from recombinant E. coli respectively. The optimal ratio of 2-hydroxyacetophenone co-catalyzed by the two enzymes was studied. The optimal catalytic temperature and pH, thereby constructing a co-expression system of (S) -carbonyl reductase II and glucose dehydrogenase. 【Result】 The specific activity of (S) -carbonyl reductase Ⅱ was 1.3 U / mg and the specific activity of glucose dehydrogenase was 13.5 U / mg. At the total enzyme activity of 1 U, the optimal ratio of the two enzymes was determined to be in the range of 1: 1 to 5: 1 (U / U) for the (S) -carbonyl reductase II and glucose dehydrogenase co- The optimum reaction temperature was 30 ℃ and the pH was 7.0. On this basis, a coexpression system of (S) -carbonyl reductase Ⅱ and glucose dehydrogenase gene ratio of 1: 1 was constructed and the recombinant bacteria were used to co-express (S) -carbonyl reductase Ⅱ and glucose The activities of hydrogenase were 0.76 U / mg and 0.73 U / mg respectively, the ratio of the two enzymes was 1: 1. Under the above optimum conditions, the optical purity and yield of 10 g / L 2-hydroxyacetophenone and (S) -benzenediol were both above 99%. Compared with recombinant Escherichia coli containing only (S) -carbonyl reductase Ⅱ, the yield of (S) -benzenediol was significantly increased and the conversion time was shortened from 24 h to 13 h. 【Conclusion】 In order to establish a chiral catalytic co-expression system of target enzyme and coenzyme regenerating enzyme, the optimal ratio of (S) -carbonyl reductase Ⅱ and glucose dehydrogenase was established to provide efficient preparation of chiral compounds The research foundation.