谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

Co-expression of glutamate dehydrogenase and alcohol dehydrogenase for preparation of (R)-2-chloro-1-phenylethanol

摘要:

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇，但由于自身再生辅酶NADH的能力不足，需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物，再生辅酶NAD(P)H，具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA，构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系，提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA，并在大肠杆菌(Escherichia coli) BL21(DE3)中表达，分析辅酶再生活力；再与醇脱氢酶AdhS共表达，优化表达条件；分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后，制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较，GdhA协助再生辅酶NADH，可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶，与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。

Abstract:

[Background] Alcohol dehydrogenase AdhS can produce (R)-2-chloro-1-phenylethanol by catalyzing asymmetric reduction. However, due to its insufficient ability to regenerate coenzyme NADH, coenzyme regenerator is needed to assist NADH regeneration. Glutamate dehydrogenase can regenerate coenzyme NAD(P)H with glutamate as substrate, and has the potential of coenzyme regeneration. [Objective] This study aimed to clone and express glutamate dehydrogenase gene gdhA, and construct the co-expression system of glutamate dehydrogenase GdhA and alcohol dehydrogenase AdhS in Escherichia coli, for improving conversion efficiency of (R)-2-chloro-1-phenylethanol preparation by AdhS. [Methods] GdhA was cloned from Bacillus subtilis 168 and expressed in Escherichia coli BL21(DE3) for the analysis of coenzyme regeneration activity. Then it was co-expressed with alcohol dehydrogenase AdhS to optimize the co-expression condition. Finally, the influence of different coenzyme regeneration schemes on conversion efficiency for (R)-2-chloro-1-phenylethanol preparation was studied. [Results] Specific activity of glutamate dehydrogenase GdhA to regenerate NADH was 694 U/g. The conversion efficiency for (R)-2-chloro-1-phenylethanol preparation reached 465 U/L through co-expression of AdhS and GdhA and optimization of expression conditions. By comparison, the conversion efficiency for (R)-2-chloro-1-phenylethanol preparation by AdhS was improved to about 3 times with NADH regeneration assisted by GdhA. [Conclusion] Glutamate dehydrogenase GdhA can be used for highly efficient regeneration of NADH, and through its co-expression with alcohol dehydrogenase AdhS, the conversion efficiency for (R)-2-chloro-1- phenylethanol preparation by AdhS have been enhanced significantly.