(R)与(S)-羰基还原酶偶联一步法制备(S)-苯乙二醇

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

【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OD6000.6～0.8时,添加终浓度1mmol/L IPTG,30℃诱导蛋白表达10h。【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37kDa和30kDa。重组菌生物转化结果表明:在pH7.0的磷酸缓冲液中,添加5mmol/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。

[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 pETDuet^ TM -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 OD_ 600 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 C 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 Zn^ 2＋ 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 75.9% 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 supply a new procedure in chiral alcohols preparation.