【摘要】 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.更多还原

【Abstract】 (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> 99. 9%) and the yield of 61. 70% was obtained from DKTP( 10 g/L) at p H = 8. 0[0. 1 mol/L triethanolamine( TEA) buffer] and 30 ℃ after reaction for 6 h,by employing glucose( 120 g/L) as the cosubstrate and coenzyme NADP+( 0. 1 mmol/L) to drive the cofactor regeneration system. During the reaction process,the p H value decreased significantly due to the formation of gluconic acid from glucose for cofactor recycling. Then the yield of( S)-DHTP was increased to 67. 73% by controlling the reaction p H. Substrate inhibition was observed when investigating the conversion of DKTP under different concentrations,and the space time yield of 1. 33 g·L-1·h-1was achieved for DKTP( 10 g/L).更多还原