扩展青霉碱性脂肪酶的克隆、表达及表达产物的活性分析  

Cloning and Expression of Structural Gene Encoding Alkaline Lipase from Penicillium expansum

作　　者：陈丹[1] 卢士英[1] 柳增善[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《河北师范大学学报（自然科学版）》2008年第2期234-238,共5页Journal of Hebei Normal University：Natural Science

基　　金：国家自然科学基金(30371059)

摘　　要：以扩展青霉为出发菌株,用Biospin RNA Simply P试剂盒提取其总RNA,反转录-聚合酶链式反应(RT-PCR)扩增出扩展青霉碱性脂肪酶结构基因.构建真核重组表达质粒pEL-pIC9,电转化His4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.重组子发酵液经SDS-PAGE分析显示获得了分子量约28 ku的1条特异条带,用橄榄油检验板法和NaOH滴定法测其活性,酶活可达225 U/mL.结果表明,扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中能高效表达为有脂肪酶活性的高活力酶.Alkaline lipase gene cDNA fragment from Penicilliun, expansum （PEL）was amplified by RTPCR method. Expression vector PEL-PIC9 containing alkaline lipase gene was constructed and the gene was expressed in His4 mutant yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression products of PEL gene was analysis by SDS-PAGE, and molecular weight of the expressed protein was estimated to be 28 ku. The activity of lipase was up to 225 u/mL determined by the method of olive oil plate and titration of NaOH. The result indicated that PEL gene was functionally overexpressed in Pichia pastor.

关 键 词：扩展青霉 碱性脂肪酶 毕赤酵母 基因表达 

分 类 号：Q78[生物学—分子生物学]