米黑根毛霉脂肪酶基因的克隆、表达及活性分析  

Cloning,Expression and Activity Analysis of Lipase Gene from Rhizomucor miehei

作　　者：苗长林[1] 罗文[1] 刘姝娜[1] 吕鹏梅[1] 李惠文[1] 杨玲梅[1] 袁振宏[1] 蒋剑春[2] 

机构地区：[1]中国科学院广州能源研究所中国科学院可再生能源与天然气水合物重点实验室,广州510640 [2]中国林业科学研究院林产化学工业研究所,南京210042

出　　处：《生物技术通报》2013年第6期122-127,共6页Biotechnology Bulletin

基　　金：“十二五”农村领域国家科技计划课题(2011BAD22B05);中科院广州能源所博士启动基金项目(y107rb1001)

摘　　要：以米黑根毛霉(Rhizomucor miehei)为出发菌株,采用UNIQ-10柱式Trizol总RNA抽提试剂盒提取其总RNA,反转录-聚合酶链式反应(RT-PCR)扩增出米黑根毛霉脂肪酶(RML)结构基因。构建真核重组表达质粒RML-pPIC9K,电转化His+缺陷型巴斯德毕赤酵母(Pichia pastoris)GS115,利用MD-MM平板及PCR方法筛选和鉴定出重组子,进行甲醇诱导表达。结果表明,RML基因能够在巴斯德毕赤酵母中实现高效表达。重组子发酵液经SDS-PAGE分析显示获得了与预期大小(约39 kD)一致的蛋白表达特异条带,用NaOH滴定法测其活性,酶活可达84 U/mL。Lipase gene cDNA fragment from Rhizomucor miehei（RML）was amplified by RT-PCR method.Expression vector RMLpPIC9K containing lipase gene was constructed and the gene was expressed in His4 mutant Pichia pastoris yeast GS115.Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR.The expression product of RML gene was analysis by SDS-PAGE,and molecular weight of the expressed protein was estimated to be 39 kD.The activity of lipase was up to 84 U/mL determined by the method of titration of NaOH.The result indicated that RML gene was functionally overexpressed inPichia pastoris.

关 键 词：米黑根毛霉 脂肪酶 毕赤酵母 基因克隆 基因表达 

分 类 号：Q78[生物学—分子生物学]