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【目的】通过表达多种重组立体选择性氧化还原酶,分析其催化不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)的性质,从而构建酶促合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(DHTP)的反应体系。【方法】基于已有立体选择性氧化还原酶重组大肠杆菌,通过Ni离子亲和层析法纯化得到重组氧化还原酶,以DKTP为底物,考察不同重组氧化还原酶对DKTP的催化活性和选择性,进一步对高选择性酶促合成(S)-DHTP的重组酶CR2进行性质分析,并考察其在最适条件下不对称还原DKTP的过程。【结果】筛选获得产物构型为(S)-型的催化活性最高的酶为CR2,该酶米氏常数Km为0.135 mmol/L,kcat/Km为3.689 L/(mmol·s),最适p H 8.4(0.1 mol/L三乙醇胺缓冲液),最适反应温度为35°C,在10-45°C条件下和p H 7.5-8.5较为稳定,Zn2+离子对酶活有促进作用。CR2催化DKTP不对称还原反应6 h后,DHTP的产率达92.1%、光学纯度达99.9%。【结论】基于活性和选择性分析,获得不对称还原DKTP的目标酶CR2,其催化特性有利于高立体选择性还原DKTP生成度洛西汀中间体(S)-DHTP,从而为进一步提高酶促不对称还原DKTP的转化效率提供研究基础。
【Objective】 The purpose of this study was to investigate the catalytic asymmetric reduction of N, N-dimethyl-3-keto-3- (2-thiophene) -1-propylamine (DKTP) Thus, a reaction system for enzymatically synthesizing (S) -N, N-dimethyl-3-hydroxy-3- (2-thiophene) -1-propanamine (DHTP) was constructed. 【Method】 Recombinant redox enzymes were purified by Ni ion affinity chromatography based on the recombinant E. coli with stereoselective oxidoreductase. The catalytic activity and selection of different recombinant oxidoreductases on DKTP were investigated by using DKTP as a substrate To further characterize the recombinant enzyme CR2 highly selective for the enzymatic synthesis of (S) -DHTP, and investigate the process of asymmetric reduction of DKTP under the optimal conditions. 【Result】 The results showed that the highest catalytic activity was (CR2). The Km of the enzyme was 0.135 mmol / L, kcat / Km was 3.689 L / (mmol · s) pH 8.4 (0.1 mol / L triethanolamine buffer). The optimal reaction temperature was 35 ° C. Under the conditions of 10-45 ° C and pH 7.5-8.5, Zn 2+ was more stable. After 6 hours of asymmetric reduction of DKTP catalyzed DKTP, the yield of DHTP reached 92.1% and the optical purity reached 99.9%. 【Conclusion】 Based on the activity and selectivity analysis, the target enzyme CR2 obtained by asymmetric reduction of DKTP was obtained. Its catalytic properties were favorable for the high stereoselective reduction of DKTP to produce Duloxetine intermediate (S) -DHTP, Asymmetric reduction of DKTP conversion efficiency provides the basis for the study.