（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联，实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta，获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时，添加终浓度1 mmol/L IPTG，30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达，它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中，添加5 mmol/L Zn2+时，获得产物(S)-苯乙二醇，产物光学纯度为91.3% e.e.，产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能，实现了(S)- 苯乙二醇的一步法转化，为简化手性醇制备途径提供了一条崭新的思路。

Construction of an enzyme-coupled system consisting of (R)- and (S)- specific carbonyl reductases for one-step preparation of (S)-1-phenyl-1,2-ethanediol

[Objective] To prepare (S)-1-phenyl-1,2-ethanediol by a one-step method, we constructed an enzyme coupled system consisting of (R)- and (S)- specific carbonyl reductases in Escherichia coli. [Methods] The genes coding (R)- and (S)- specific carbonyl reductases from Candida parapsilosis were inserted into a co-expression vector pETDuetTM-1. The positive plasmid was transformed into codon optimized E. coli Rosetta and an enzyme-coupled recombinant strain E. coli Rosetta /pETDuet-rcr-fdh was constructed. When the OD600 value of the culture reached 0.6-0.8, IPTG with a final concentration of 1 mmol/L was added to induce both target proteins at 30 oC for 10 h. [Results] SDS-PAGE analysis showed that two target enzymes were expressed simultaneously with the relative molecular weights of 37 kDa and 30 kDa. When 5 mol/L Zn2+ was added into a phosphate buffer (pH7.0), the biotransformation results showed that the product (S)-1-phenyl-1,2-ethanediol was produced with the optical purity of 91.3% enantiomeric excess and a yield of 75.8% by E. coli Rosetta/pETDuet-rcr-scr. [Conclusion] The functions of two redox enzymes were integrated by molecular recombinant technique and a one-step method for preparation of (S)-1-phenyl-1,2-ethanediol was developed through an enzyme-coupled system. The method may simplify procedure in chiral alcohols preparation.