Thermomyces lanuginosus ZJB09222脂肪酶基因克隆及在大肠杆菌中的表达  

Gene Cloning of Lipase from Thermomyces lanuginosus ZJB09222 and Expression in Escherichia coli

作　　者：雷丽华[1] 郑仁朝[1] 柳志强[1] 黎小军[1] 郑裕国[1] 

机构地区：[1]浙江工业大学生物与环境工程学院,浙江杭州310014

出　　处：《食品与发酵工业》2012年第5期56-60,共5页Food and Fermentation Industries

摘　　要：Thermomyces lanuginosus脂肪酶(简称TLL)是具有重要商业应用价值的脂肪酶之一。运用RT-PCR技术,从Thermomyces lanuginosus ZJB09222基因组中克隆得到885 bp脂肪酶基因cDNA序列,其结构基因编码蛋白包含292个氨基酸。将脂肪酶基因cDNA序列开放阅读框克隆到大肠杆菌表达载体pET-28b中,转化大肠杆菌BL21(DE3),构建了基因工程菌E.coli BL21/pET28b-TLL。诱导表达后SDS-PAGE电泳显示该脂肪酶分子量约为32 ku。筛选获得廉价重组菌培养基,表达条件优化结果表明,当OD600约为0.6～0.8时,加入IPTG至终浓度为0.1 mmol/L,在28℃诱导培养7 h,酶活达到41 U/mL。Thermomyces lanuginosus lipase is one of the most important industrial enzymes. Based on published DNA sequence, the full length cDNA of the lipase was cloned using RT-PCR method. It revealed that the cDNA of the T. lanuginosus lipase gene encoded a protein lipase of 292 amino acid residues. The open reading frame of the cDNA was cloned into the plasmid pET-28b, which was then transformed into E. coil BL21 （DE3） strain. The Mr of the expression product was 32 ku by SDS-PAGE. The recombinant E. coli BL21/pET28b-TLL exhibited highest activi-ty of 41 U/mL at 28℃ for 7 h induction with 0. 1 mmol/L IPTG after screening of the best medium.

关 键 词：脂肪酶 THERMOMYCES LANUGINOSUS 克隆 大肠杆菌 诱导表达 

分 类 号：TS201.25[轻工技术与工程—食品科学]