Abstract:[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 as 0.135 mmol/L and 3.689 L/(mmol·s), respectively. For CR2, the optimal pH was pH 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 pH 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.
