羰基还原酶CR2重组酶体系不对称合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺

构建了羰基还原酶CR2重组酶体系,并优化了相关的酶促催化反应条件.通过在催化体系中添加辅酶NADP+(0.1 mmol/L)和辅底物葡萄糖(120 g/L),在30℃及p H=8.0的条件下反应4 h,CR2重组酶体系不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP,10 g/L),合成了高光学纯度(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(S)-DHTP,e.e.值99.9%],产率为62%.在酶促催化过程中,由于辅酶循环生成葡萄糖酸导致反应体系p H值下降而影响催化效率.通过调控反应体系p H值,(S)-DHTP的产率提高到68%.不同浓度底物的反应过程表明底物对CR2酶促反应具有抑制作用,且在10 g/L底物浓度下反应的时空产率可达1.3 g·L-1·h-1.

Asymmetric Synthesis of ( S)-N,N-Dimethyl-3-hydroxy-3-( 2-thienyl)-1-propanamine by Cell-free System of Carbonyl Reductase CR2

( S )-N, N-Dimethyl-3-hydroxy-3-( 2-thienyl )-1-propanamine ( S )-DHTP ] is an important intermediate for the production of ( S)-duloxetine, which is a new and effective antidepressant drug. Since the efficiency of biosynthesis of ( S)-DHTP is yet limited, development of suitable biocatalytic system is necessary for ( S )-DHTP production from asymmetric reduction of N,N-dimethyl-3-keto-3-( 2-thienyl)-1-propanamine (DKTP). In this study, the cell-free biocatalytic system involving recombinant carbonyl reductase CR2 was constructed and the corresponding reaction conditions were optimized. (S)-DHTP with high optical purity(e.e. value>999％) and the yield of 61. 70％ was obtained from DKTP ( 10 g/L ) at pH=80 01 mol/L triethanolamine(TEA) buffer] and 30 ℃ after reaction for 6 h, by employing glucose(120 g/L) as the co-substrate and coenzyme NADP+(01 mmol/L) to drive the cofactor regeneration system. During the reaction process , the pH value decreased significantly due to the formation of gluconic acid from glucose for cofactor re-cycling. Then the yield of (S)-DHTP was increased to 6773％ by controlling the reaction pH. Substrate inhi-bition was observed when investigating the conversion of DKTP under different concentrations, and the space time yield of 133 g·L-1·h-1 was achieved for DKTP(10 g/L).