催化学报

催化学报 ›› 2012, Vol. 33 ›› Issue (10): 1650-1660.DOI: 10.1016/S1872-2067(11)60436-1

• 研究论文 • 上一篇    下一篇

不对称还原制备光学纯 (R)-2-羟基-4-苯基丁酸乙酯的双酶共表达重组菌的构建

宿宇宁, 倪晔*, 王骏超, 徐志豪, 孙志浩   

  1. 江南大学生物工程学院, 工业生物技术教育部重点实验室, 江苏无锡 214122
  • 收稿日期:2012-04-26 修回日期:2012-06-24 出版日期:2012-09-28 发布日期:2012-09-28

Two-Enzyme Coexpressed Recombinant Strain for Asymmetric Synthesis of Ethyl (R)-2-Hydroxy-4-phenylbutyrate

SU Yuning, NI Ye*, WANG Junchao, XU Zhihao, SUN Zhihao   

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
  • Received:2012-04-26 Revised:2012-06-24 Online:2012-09-28 Published:2012-09-28

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摘要： 克隆了来自于枯草芽孢杆菌的羰基还原酶基因 IolS 和葡萄糖脱氢酶基因 GDH, 采用 Ni-NTA 镍亲和层析柱对重组蛋白 IolS 进行纯化, 并对纯酶进行了酶学性质研究. 结果表明, 该羰基还原酶的最适温度和 pH 值分别为 30 oC 和 6.0; 在 40 oC 以下具有较好的热稳定性; 在 pH 5.5-7.0 的偏酸性范围内能保持 75% 以上的酶活. 采用三种策略构建了 IolS 和 GDH 的共表达重组质粒, 结果发现, 采用双启动子的重组质粒能够实现羰基还原酶 IolS 的高效表达, 粗酶液中的 IolS 和 GDH 的比酶活均达到 1.5 U/mg. 运用该重组菌对 10 g/L 的 OPBE 进行不对称还原, 反应 15 h 后, 底物转化率大于 99%, 产物 (R)-2-羟基-4-苯基丁酸乙酯的 ee 值达到 99.5%.

关键词: 羰基还原酶, 葡萄糖脱氢酶, 共表达, (R)-2-羟基-4-苯基丁酸乙酯, 不对称还原

Abstract: (R)-2-Hydroxy-4-phenylbutyrate (HPBE) is an important chiral intermediate for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Asymmetric reduction of ethyl 2-oxo-4-phenyl-butyrate (OPBE) to (R)-HPBE using a recombinant strain can provide high enantioselectivity. Cofactor regeneration is a critical issue in the application of a recombinant strain. A carbonyl reductase gene (iolS) and a glucose dehydrogenase (GDH) gene from Bacillus subtilis were cloned. Recombinant IolS was purified using a Ni-NTA column and its enzyme activity properties were investigated. The purified IolS exhibited maximum activity at pH 6.0 and 30 oC, and the enzyme showed good thermostability below 40 oC. It retained over 75% of its activity in the acidic pH range of 5.5-7.0. Three coexpression strategies were used for the recombinant vectors. The recombinant E. coli strain containing polycistronic plasmid pET-G-T7-I showed excellent carbonyl reductase activity, and the specific activity of both IolS and GDH in the crude cell extract reached 1.5 U/mg. In the asymmetric reduction of OPBE by recombinant E. coli cells in aqueous system, the yield of (R)-HPBE reached over 99% with an enantiomeric excess of 99.5% at 10 g/L of OPBE within 15 h.

Key words: carbonyl reductase, glucose dehydrogenase, coexpression, (R)-2-hydroxy-4-phenylbutyrate, asymmetric reduction