乙醛还原酶工程菌的构建以及与葡萄糖脱氢酶工程菌偶联还原制备R-CHBE  

Production of R-CHBE using two recombinants carrying aldehyde reductase coupling GDH gene

作　　者：金永琴[1] 徐娴[1] 毛政[2] 何冰芳[1] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京210009 [2]浙江省食品药品监督管理局培训中心,浙江杭州310012

出　　处：《化工进展》2010年第6期1120-1124,共5页Chemical Industry and Engineering Progress

基　　金：国家973计划资助项目(2009CB724700)

摘　　要：R-4-氯-3-羟基丁酸乙酯(R-CHBE)是多种药物的手性中间体,可由4-氯乙酰乙酸乙酯(COBE)立体选择性还原制备,而辅酶高效再生是绝大多数氧化还原酶实际应用的瓶颈。本研究从赭色掷抱酵母细胞ZJU010中克隆出NADPH依赖型乙醛还原酶ARI基因(alr),构建重组大肠杆菌E.coliBL21/pET-alr,并与葡萄糖脱氢酶的基因工程菌(BL21/pET-gdh)进行偶联转化COBE。合理调整双菌双酶比例,当BL21/pET-alr和BL21/pET-gdh的湿菌重比为6∶1时,CHBE的产量达到最高,无需添加辅酶,转化22h摩尔转化率达到95.88%,产物R-CHBE的对映体过量值ee为96%,较好地实现了双酶偶联制备R-CHBE。R-Ethyl-4-chloro-3-hydroxybutanoate（R-CHBE）is used as a chiral building block for the synthesis of pharmaceutical target compounds. R-CHBE can be produced by the asymmetric reduction of ethyl 4-chloro-3-oxobutanoat（eCOBE）. However,effective cofactor regeneration is a key step for the application of most oxidoreductases. In this research,the NADPH-dependent aldehyde reductase gene（alr）was cloned from the genome of Sporobolomyces salmonicolor ZJU010,and the recombinant E.coli BL21/pET-alr strain was constructed. Asymmetric reduction of COBE to CHBE was performed using the recombinant E.coli BL21/pET-alr coupling with E.coli BL21/pET-gdh. The highest reduction activity was obtained by recombinant resting cells at 6∶1 weight ratio of E.coli BL21/pET-alr with E.coli BL21/pET-gdh. Yield and the enantiomeric excess（ee）of R-CHBE reached to 95.88% and 96%,respectively,after 22h bioconversion without adding coenzyme NADPH or NADP＋. R-CHBE could be effectly produced using the recombinant E.coli BL21/pET-alr coupling with E.coli BL21/pET-gdh for NADPH regenerantion.

关 键 词：NADPH再生 葡萄糖脱氢酶 乙醛还原酶 R-4-氯-3-羟基丁酸乙酯 

分 类 号：Q55[生物学—生物化学] Q78