羰基还原酶工程菌构建及其应用于还原制备S-CHBE  

Construction of recombinant strain with carbonyl reductase gene for production of ethyl S-4-chloro-3-hydroxybutanoate

作　　者：张志斌[1,2] 吴小芳[1] 杨慧林[1,3] 颜日明[1] 曾庆桂[1] 汪涯[3] 朱笃[1,3] 

机构地区：[1]江西师范大学生命科学学院江西省亚热带植物资源保护与利用重点实验室,江西南昌330022 [2]国家单糖化学合成工程技术研究中心,江西南昌330027 [3]江西科技师范大学生命科学学院江西省生物加工过程重点实验室,江西南昌330013

出　　处：《食品与发酵工业》2013年第12期24-29,共6页Food and Fermentation Industries

基　　金：国家科技支撑计划(2009BAI175B02);2011年江西省重大科技专项及江西省亚热带植物资源保护与利用重点实验室开放基金资助项目

摘　　要：S-4-氯-3-羟基丁酸乙酯(S-CHBE)是多种药物的手性中间体,可由4-氯乙酰乙酸乙酯(COBE)不对称还原合成。该研究对来自Candida magnoliae AKU4643的S1羰基还原酶基因进行E.coli密码子优化,获得的S1'基因序列并进行人工合成,利用该序列构建了基因工程菌BL21(DE3)/pET28a-S1',提高了羰基还原酶表达效率,经诱导条件优化后羰基还原酶活力达11.49 U/mg蛋白;将BL21(DE3)/pET28a-S1'与E.coli BL21(DE3)/pET28a-gdh进行偶联,在单一水相中未添加辅酶条件下转化10 h,产物的摩尔转化率和对映体过量值(e.e.)分别达92.1%和100%。S-Ethyl-4-chloro-3-hydroxybutanoate （S-CHBE） is used as a chiral building block for the synthesis of pharmaceutical target compounds. S-CHBE can be produced by the asymmetric reduction of ethyl 4-chloro-3-oxobu- tanoate （COBE）. In present study, the Sl＇gene was delivered from the NADPH-dependent carbonyl reduetase gene （SI） of Candida magnoliae AKU4643 followed artificially synthesizing and codon optimizing for E. coli host. The re- combinant strain BL21 （DE3）/pET28a-S1＂ with S1＂ gene showed more effective expression of carbonyl reduetase gene. The highest activity of carbonyl reductase was achieved 11.49U/mg protein after induction conditions optimiza- tion. Furthermore, the asymmetric reduction of COBE to CHBE was performed using the recombinant E. coli BL21 （DE3）/pET28a-S1＂ coupling with E. coli BL21/pET28a-gdh in an aqueous system and the enantiomeric excess （e. e. ）of S-CHBE was arrived to 92. 1% and 100% respectively after 10h bioconversion without adding coenzyme NADPH or NADP＋.

关 键 词：生物催化 羰基还原酶 密码子优化 诱导表达 S-4-氯-3-羟基丁酸乙酯 

分 类 号：Q814[生物学—生物工程]