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.
出处
《中国酿造》
CAS
北大核心
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)
