烯酮还原酶基因的克隆与优化表达

Cloning and Optimizing Expression of Enoate Reductase Gene from Clostridium acetobutylicum in Escherichia coli

烯酮/酯还原酶可以专一性地还原烯酮/酯中的碳碳双键,并引入两个手性中心,是手性化合物生物催化合成的重要酶类之一;在使用烯酮/酯还原酶进行全细胞手性分子生物催化合成中,其他还原酶的存在常导致副反应的产生.为研究烯酮/酯还原酶的理化性质、底物的专一性、产物的手性特点等,需要经过纯化的烯酮/酯还原酶.为此,根据烯酮还原酶(Enoate reductase,ERs,EC 1.3.1.31)的基因序列设计引物,以丙酮丁醇梭菌(Clostridium acetobutylicum)ATCC824基因组DNA为模板,通过PCR扩增技术得到了目标酶的基因,目的片段全长为1 995 bp,共编码664个氨基酸,相对分子质量为73×103.将烯酮还原酶基因连接到pET-32a(+)上,构建表达质粒pET-32a(+)-ERs,并成功在Escherichia coli Rosetta(DE3)pLysS中表达.在摇床上优化的表达条件为:诱导温度为18℃,诱导起始时菌体D600 nm为0.6～0.8,转速为100 r/min,诱导剂IPTG浓度为0.1 mmol/L,诱导培养时间为12 h,表达的烯酮还原酶大部分以可溶性形式存在于菌体内.

Enone reductase is a class of biocatalysts which catalyze the reduction of C=C bond of enones and/or enoates.The gene which encodes enone reductase from Clostridium acetobutylicum ATCC824 was cloned.The cloned sequence consisted of 1 995 bp and encoded a protein of 664 amino acids,with a molecular weight of 73 ×103.The gene was cloned into the expression vector pET-32a（＋）,and the resulting plasmid DNA was transformed into the Escherichia coli Rosetta（DE3） pLysS strain.The heterologous expression was optimized and the best results were obtained for induction at an D600 nm of 0.6～0.8 with 0.1 mmol/L IPTG 12 h at 18 ℃.The SDS-PAGE analysis indicated that the ERs was expressed in the supernatant.