基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生

【目的】构建己糖激酶与葡萄糖-6-磷酸脱氢酶的大肠杆菌共表达体系,以葡萄糖为底物实现辅酶NADPH的高效再生。【方法】通过分子生物学方法,克隆己糖激酶HKgs、HKpp基因,并于Escherichia coli BL21(DE3)中表达,再将己糖激酶HKgs、HKpp分别与葡萄糖-6-磷酸脱氢酶Gpd PP共表达,实现NADPH的原位再生。比较两个共表达工程菌的辅酶再生效果,并针对催化活力较高的工程菌BL21(HKgs+Gpd PP)进行表达条件优化。【结果】NADPH再生活力达到856 U/L。该辅酶再生体系与醇脱氢酶Adh R联合催化,使不对称还原4-氯乙酰乙酸乙酯的催化活力提高至原始值的2.5倍。【结论】通过己糖激酶与葡萄糖-6-磷酸脱氢酶在大肠杆菌中的共表达,构建了一个新的NADPH高效再生体系,并用于醇脱氢酶催化的不对称还原反应。

Efficient NADPH regeneration based on co-expression of hexokinase and glucose-6-phosphate dehydrogenase

Objective] This study aimed to construct a co-expression system of hexokinase and glucose-6-phosphate dehydrogenase in Escherichia coli to regenerate NADPH with glucose as substrate efficiently. Methods] Hexokinase genes hkgs and hkpp were cloned and expressed in E. coli BL21(DE3), and then the co-expression system of hexokinase and glucose-6-phosphate dehydrogenase was established to achieve efficient in-situ regeneration of NADPH. Among the two co-expression systems, BL21(HKgs+GpdPP) was better, so the expression condition of BL21(HKgs+GpdPP) was optimized. Results] The catalytic activity of NADPH regeneration reached 856 U/L. The coupling catalysis was performed with this co-enzyme regeneration system and alcohol dehydrogenase AdhR, the catalytic activity of asymmetric reduction of ethyl 4-chloro-3-oxobutanoate was enhanced up to 2.5 times. Conclusion] Through co-expression system of hexokinase and glucose-6-phosphate dehydrogenase in Escherichia coli, new effective NADPH regeneration system was constructed, and success for a symmetric reduction by alcohol dehydrogenase.