康氏木霉纤维素酶CBHI基因克隆及在大肠杆菌中的表达

摘要

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 transsformed into E. coli BL21 (DE3) plysS strain after 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 ofpNPC 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.%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降到表达载体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:cbhI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.