重组大肠杆菌细胞不对称还原4-氯乙酰乙酸乙酯合成(R)-(+)-4-氯-3-羟基丁酸乙酯

 从赭色掷孢酵母(Sporobolomyces salmonicolor ZJU0105)中克隆出NADPH依赖型醛基还原酶基因,构建了重组大肠杆菌E.coli BL21(pET28-ALR0105), 该工程菌可以高效地表达醛基还原酶. 将重组细胞用于催化4-氯乙酰乙酸乙酯不对称还原,合成出具有光学活性的(R)-(+)-4-氯-3-羟基丁酸乙酯. 实验发现,在加入适量辅酶及辅酶再生酶的条件下,利用重组细胞催化还原反应可以获得比使用赭色掷孢酵母更高的转化率、产率和ee值,得到了几乎是光学纯的(R)-(+)-型产物,从而解决了酵母细胞催化此类反应ee值较低的问题. 考察了辅酶及共底物的添加、底物和产物的浓度、pH值、温度以及菌体密度等因素对还原反应的影响. 结果表明,不对称还原反应必须在辅酶NADPH和辅酶再生酶系及共底物葡萄糖的参与下进行; 底物和高浓度的产物对还原反应有一定的抑制作用; 当pH＞6.0时,反应的转化率及产率都显著降低; 高密度重组细胞可以减小底物的抑制作用.

Asymmetric Reduction of Ethyl 4-Chloroacetoacetate to Ethyl (R)-(+)-4-Chloro-3-Hydroxybutyrate by Recombinant Escherichia coli

The NADPH-dependent aldehyde reductase gene was cloned from the genome of Sporobolomyces salmonicolor ZJU0105,and the recombinant E.coli BL21(pET28-ALR0105) strain was constructed.The asymmetric reduction of ethyl 4-chloroacetoacetate(COBE) to ethyl(R)-(+)-4-chloro-3-hydroxybutyrate((R)-CHBE) by the recombinant cells in an aqueous phase was investigated.The product is optically pure(R)-CHBE,and both the(R)-CHBE yield and stereoselectivity are higher than those in the reaction catalyzed by Sporobolomyces salmonicolor.The glucose dehydrogenase,NADPH and glucose are required for the recombinant cells to regenerate NADPH.The reaction is also inhibited by high concentration of COBE and CHBE,but high density of recombinant cells can decrease this inhibitory effect.A yield of 96.5% and ee of 99% for(R)-CHBE are obtained under the appropriate reaction conditions.