羰基还原酶的克隆、异源表达及其在(R)-2-羟基-4-苯基丁酸乙酯合成中的应用  

Cloning and heterologous expression of carbonyl reductase for the synthesis of ethyl(R)-2-hydroxy-4-phenylbutyrate

作　　者：庞凯南 王鑫情 朱佳妮 来婉琪 刘锐华 邵飞鸿 黄一帆 罗希 PANG Kainan;WANG Xinqing;ZHU Jiani;LAI Wanqi;LIU Ruihua;SHAO Feihong;HUANG Yifan;LUO Xi(Institute of Biomass Resources,Taizhou University,Taizhou 318000,China)

机构地区：[1]台州学院生物质资源研究所,浙江台州318000

出　　处：《生物加工过程》2021年第5期539-546,共8页Chinese Journal of Bioprocess Engineering

基　　金：浙江省自然科学基金青年基金(LQ19B060003);台州学院大学生创新创业训练计划(201910350144)。

摘　　要：(R)-2-羟基-4-苯基丁酸乙酯是合成血管紧张素转换酶抑制剂类降压药的关键手性中间体。筛选了1株具有不对称还原2-氧代-4-苯基丁酸乙酯合成(R)-2-羟基-4-苯基丁酸乙酯活性的野生菌DY99,经鉴定此菌为Kazachstania naganishii。从该菌基因组序列中挖掘并克隆了发挥催化活性的羰基还原酶基因,构建了重组大肠杆菌E.coli-pET28-kncr,对其诱导表达条件和催化反应条件进行了优化,并在最适条件下进行了催化反应。结果发现:在最优条件(当OD 600达到0.8时,添加乳糖6 g/L,在28℃、150 r/min诱导12 h)下,重组菌酶活达到330.61 U/L发酵液。确定了全细胞催化不对称还原2-氧代-4-苯基丁酸乙酯制备(R)-2-羟基-4-苯基丁酸乙酯的最适反应条件为30℃,pH7.5,底物、葡萄糖、细胞生物量分别为15、15和20 g/L(以干质量计),在最适反应条件下,产物累积量、时空产率分别为11.2 g/L和26.88 g/(L·h),产物的对映体过量值(e.e.)>99%,显示了良好的应用前景。Ethyl(R)-2-hydroxy-4-phenylbutyrate is a key chiral building block for the synthesis of angiotensin-converting enzyme inhibitors.A wild type microbial stain DY99 with the activity of asymmetric reducing ethyl 2-oxy-4-phenylbutyrate to ethyl(R)-2-hydroxy-4-phenylbutyrate was isolated,and identified as Kazachstania naganishii.A carbonyl reductase gene was extracted and cloned from the genome sequence of this strain,and a recombinant bacteria Escherichia coli-pET28-kncr has been constructed.The induction conditions were optimized,under the optimal conditions(when OD 600 reached 0.8,lactose was added as inducer to the final concentration of 6.0 g/L and cultivated at 28℃,150 r/min for 12 h),the activity reached 330.61 U/L fermentation broth.The optimum conditions of preparing ethyl(R)-2-hydroxyl-4-phenylbutyrate using whole cells of E.coli-pET28-kncr were determined.Under the optimal reaction conditions(30℃,pH7.5,ethyl 2-oxy-4-phenylbutyrate,glucose and cells added at concentration of 15 g/L,15 g/L and 20 g/L,respectively),ethyl(R)-2-hydroxyl-4-phenylbutyrate was accumulated up to 11.2 g/L,with e.e.>99%,the space-time yield reached 26.88 g/(L·h),showing a good application prospect.

关 键 词：(R)-2-羟基-4-苯基丁酸乙酯 羰基还原酶 不对称还原 手性中间体 绿色合成 

分 类 号：Q814.9[生物学—生物工程]