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  

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

作　　者：宿宇宁[1] 倪晔[1] 王骏超[1] 徐志豪[1] 孙志浩[1] 

机构地区：[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

出　　处：《催化学报》2012年第10期1650-1660,共11页

基　　金：国家重点基础研究发展计划(973计划;2011CB710800);新世纪优秀人才支持计划(NCET-11-0658);江苏省自然科学基金(BK2011150);高等学校学科创新引智计划(111计划;111-2-06);江苏高校优势学科建设工程~~

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30oC和6.0;在40oC以下具有较好的热稳定性;在pH5.57.0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1.5U/mg.运用该重组菌对10g/L的OPBE进行不对称还原,反应15h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%.（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.

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 

分 类 号：TQ245.24[化学工程—有机化工]