高选择性不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺的重组氧化还原酶催化性质及其酶促转化

Characterization of highly stereoselective oxidoreductase for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine

【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] To construct a biocatalytic system for enzymatic synthesis of（S）-N,N-dimethyl-3-hydroxy-3-（2-thienyl）-1-propanamine（DHTP）, several stereoselective oxidoreductases were expressed from recombinant strains and explored on their properties of catalyzing asymmetric reduction toward N,N-dimethyl-3-keto-3-（2-thienyl）-1-propanamine（DKTP）.[Methods] From available recombinant strains involving oxidoreductases, enzymes were purified by Ni-ion affinity chromatography and their catalytic activities and stereoselectivities were evaluated toward DKTP. Among them, CR2 was further characterized, which could catalyze highly stereospecific synthesis of（S）-DHTP. Then, the catalytic process of CR2 was studied for asymmetric reduction of DKTP under optimal conditions. [Results] Enzyme CR2 was obtained with high stereoselectivity and catalytic activity for（S）-DHTP production. Its kinetic parameters of Km and kcat/Km were determined as0.135 mmol/L and 3.689 L/（mmol·s）, respectively. For CR2, the optimal p H was p H 8.4（0.1 mol/L triethanolamine buffer） and the optimal reaction temperature was 35 °C. It was more stable at the temperatures ranging from 10 °C to 45 °C and at the p H ranging from 7.5 to 8.5. Zn2＋ improved the enzyme activity of CR2. When the reaction was carried out for about 6 h, the target product was achieved with the yield of 92.1% and the optical purity of 99.9%. [Conclusion] This work provides the research foundation for further improvement of the enzymatic conversion efficiency of asymmetric reduction of DKTP.