诺卡氏菌腈水合酶突变基因在重组大肠杆菌中的高活性表达  

High level expression of a mutated nitrile hydratase gene of Nocardia sp.in recombinant Escherichia coli

作　　者：史悦[1] 于慧敏[1] 罗晖[2] 田卓玲[1] 沈忠耀[1] 

机构地区：[1]清华大学化学工程系,北京100084 [2]北京科技大学环境工程系,北京100083

出　　处：《清华大学学报（自然科学版）》2007年第12期2176-2179,共4页Journal of Tsinghua University(Science and Technology)

基　　金：国家自然科学基金资助项目(20206014);全国优秀博士学位论文作者专项资金资助项目(200345)

摘　　要：为了提高腈水合酶基因的重组表达水平,提出了3种基因策略:在重组大肠杆菌中共表达激活子序列、在重组毕赤酵母中表达以及对α亚基的起始密码子进行定点突变。结果表明:对α亚基的起始密码子进行定点突变的基因策略为最佳方案,突变后的腈水合酶基因在重组E.coli XL1-Blue(pUC18-NHBAM)中表达时,腈水合酶的比酶活(以干菌质量计)提高到51 U/mg。进一步以pET28a为载体,插入突变后的腈水合酶基因,构建重组菌株E.coli BL21(DE3)/pETNHM。对优选菌株进行培养条件和诱导条件的优化,腈水合酶的最高比酶活达到450 U/mg。Three approaches were developed to improve active expression of the nitrile hydratase （NHase） gene from Nocardia sp. The approaches are co-expression of the downstream activator sequence of the NHase gene in E. coli, expression in a Pichia pastoris system and site-directed mutagenesis of the start codon of the NHase a subunit with expression in E. coll. The results show that the third strategy is the best with a superior strain, E. coli XL1-Blue （pUC18-NHase^M） successfully constructed and selected. Its NHase activity reached 51 U/mg dry ceil. The mutated NHase gene was further ligated into plasmid pET28a, and a new recombinant strain of E. coli BL21（DE3）/pETNH^M was selected. The conditions for cell cultivation and NHase induction are optimized to increase the specific activity of the recombinant strain to 450 U/mg with lactose as the inducer.

关 键 词：腈水合酶 重组大肠杆菌 重组毕赤酵母 定点突变 比酶活 

分 类 号：TQ920.6[轻工技术与工程—发酵工程] Q789[生物学—分子生物学]