加强辅酶循环再生实现不对称合成(S)-1-苯基-1,2-乙二醇

Improving cofactor recycle for asymmetric synthesis of (S)-1-phenyl-1,2-ethanediol

以β-羟基苯乙酮为底物,利用羰基还原酶不对称合成(S)-1-苯基-1,2-乙二醇(PED)过程中,葡萄糖脱氢酶和6-磷酸葡萄糖脱氢酶催化NADPH再生反应与还原反应形成耦联,合成(S)-PED的浓度分别达到0.78 g.L-1和0.75 g.L-1,对映体过量值(e.e.%)分别为91.84%和63.96%;连续分批还原反应后其总转化数(TTN)为26和58;经硫酸铵盐析、离子交换层析DEAE Sepharose、苯基疏水层析Phenyl Sepharose和亲和层析Blue Sepharose后得到电泳纯的羰基还原酶,比活为99.9 U/mg,亚基分子量为29,800;纯酶耦联葡萄糖脱氢酶和6-磷酸葡萄糖脱氢酶合成(S)-PED的浓度达到了1.04 g.L-1和1.19 g.L-1,证明了辅酶循环再生的存在.同时发现在利用胞内酶自耦联再生NADPH过程中,以乙醇作为辅助底物时效果最好.

In the process of asymmetric synthesis of （S）-1-phenyl-1, 2-ethanediol from β-hydroxyacetophenone, glucose dehydrogenase and glucose-6-phosphate clehydrogenase were used to regenerate NADPH coupled with reduction reaction. The concentration of （ S ）-PED reached 0.78 g·L^-1 and 0.75 g·L^-1 respectively, enatiomeric excess （e. e. % ） reached 91.84% and 63.96%. Total turnover number were 26 and 58 in the continuous batch reduction reactions. Carbonyl reductase was purified to electrophoretic homogeneity by ammonium sulfate precipitation, DEAE Sepharose chromatography, Phenyl-Sepharose chromatography and Blue Sepharose chromatography. Specific enzyme activity was 99.9U and the molecular weight of the subunit by SDS-PAGE was 29,800. The concentration of （S）-PED were 1.04 g·L^-1and 1.19 g·L^-1 when purified carbonyl reductase was coupled to glucose dehydrogenase and glucose-6-phosphate dehydrogenase to synthesize （S）-PED. It was demonstrated that the cofactor recycle exists in the purified enzyme. It was found that in the synthesis of （ S）-PED coupled with other enzyme in cell that can regenerate NADPH, it makes the best effect when alcohol is added as the cosubstrate.