重组大肠杆菌不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇

通过对近平滑假丝酵母(R)-专一性羰基还原酶（rCR）进行氨基酸序列分析并根据其序列的保守性设计PCR引物，以近平滑假丝酵母基因组为模板利用PCR技术得到目的基因rcr。该基因全长1011bp，共编码336个氨基酸。将该基因与表达载体pET21c连接后构建重组质粒pETRCR，并转化入Escherichia coli BL21（DE3）中进行表达。重组大肠杆菌具有(R)-专一性羰基还原酶活性，可催化不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇。研究发现，在重组菌培养过程中，同添加IPTG诱导培养的情况相比，不添加IPTG诱导培养的酶活及不对称转化(R)-苯基乙二醇的效果较好。在反应过程中，转化48 h及在pH值 8～9的条件下更有利于不对称反应的进行。另外，较高的底物浓度会对反应产生抑制作用，通过提高重组菌细胞浓度可显著提高转化效果。在5 g/L的底物浓度条件下，利用0.3 g/mL重组菌细胞可还原2-羟基苯乙酮得到(R)-苯基乙二醇，产物光学纯度为95.5% e.e，产率为92.6%。

Synthesis of (R)-1-phenyl-1,2-ethanediol by stereospecific reduction of 2-hydroxyacetophenone using recombinant Escherichia coli expressing (R)-specific carbonyl reductase

Based on the amino acid sequence information of Candida parapsilosis(rCR) by tryptic digestion,and gene encoding,the enzyme(rcr) was cloned and sequenced.It was shown that the rcr comprised 1008 nucleotides and encoded a polypeptide of 35,977 Da.The deduced amino acid sequence of the enzyme performed high similarity with those of proteins of the zinc-containing medium-chain dehydrogenase/reductase(MDR) superfamily with the cofactor-binding motif Gly-x-Gly-x-x-Gly.The rcr was expressed in Escherichia coli BL21(DE3) by constructing the expression vector of pETRCR without the induction of IPTG and the recombinant E.coli expressing rCR possessed the capability of catalyzing asymmetric reduction of 2-hydroxyacetophenone to(R)-1-phenyl-1,2-ethanediol.After optimization of the reaction conditions,a feasible approach was developed to convert 2-hydroxyacetophenone to(R)-1-phenyl-1,2-ethanediol with high optical purity of 95.5% enantiomeric excess and yield of 92.6% by the recombinant E.coli bearing pETRCR as the catalyst with unusual stereospecificity.