【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。更多还原

【Abstract】 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.更多还原