康氏木霉纤维素酶CBHI基因克隆及在大肠杆菌中的表达  

Cloning of CBHI gene of Trichoderma koningii cellulase and expression in Escherichia coli

作　　者：黄时海[1] 康超[1] 黄飞[2] 曹喜秀[1] 何鑫平[1] 汪晟[1] 吴孔阳[1] 曹普美[1] 梁智群[1] 李湘萍[3] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530005 [2]南宁新技术创业者中心,广西南宁530007 [3]广西大学动物繁殖研究所,广西南宁530005

出　　处：《中国酿造》2011年第2期76-80,共5页China Brewing

基　　金：广西区教委人才项目(桂教人200962);国家大学生创新基金(091059318);广西大学实验教改项目(092301)

摘　　要：将康氏木霉(Trichoderma koningii)总RNA反转录成cDNA第一链,并以之为模板进行RT-PCR,合成约1.5kb的纤维二糖水解酶Ⅰ(cbh I)基因。cbhI基因经测序确认后克隆到表达载体pET-30a(+)上,PCR和双酶切鉴定筛选阳性重组子;将阳性质粒转化大肠杆菌BL21(DE3)plysS,并用0.4mmol/L的IPTG诱导表达重组蛋白。实验结果:cbh I基因在BL21(DE3)plysS中胞内融合表达,重组蛋白pNPC酶活为15.6U/L,最适反应温度为45℃,最适pH值为5.0,Mn2+对酶活力有明显的促进作用,SDS-PAGE表明重组蛋白分子量约为70kDa。Cellobiase cbhI gene was cloned with RT-PCR by using Trichoderma Koningi cDNA as template. The cbhI gene was inserted directly into the temperature-inducible prokaryotic expression vector pET-30a（＋）and transformed into E. coli BL21 （DE3） plysS strain alter sequence. When induced with 4mmol/L IPTG for 4h, the BL21 strain produced a recombinant cellobiohydrolase. The results indicated that gene cbh I could expressed in strain BL21 （DE3） plysS. Enzyme activity of pNPC was 15.6U/L under the conditions of temperature 45℃ and pH value 5.0. Ion Mn2. significantly improves the activity of the enzyme. Molecular weight of the recombinant cellobiohydrolase was about 70 kDa by the SDS-PAGE analysis.

关 键 词：康氏木霉 纤维二糖水解酶I cbhI基因 克隆表达 

分 类 号：Q785[生物学—分子生物学]