绿色木霉CBHⅡ基因的克隆及在酿酒酵母中的表达

Cloning of Cellobiohydrolase Ⅱ Encoding Gene from Trichoderma viride and Its Expression in Saccharomyces cerevisiae

采用反转录聚合酶链式反应的方法从绿色木霉中克隆得到纤维二糖水解酶Ⅱ(CBHⅡ)的编码基因cbh2.将该基因片段接入酿酒酵母整合型表达载体pScIKP中,得到重组质粒pScIKPc-bh2.电转化酿酒酵母菌株,通过G418浓度梯度筛选出高抗转化子.采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测CBHⅡ蛋白的表达,并采用羧甲基纤维素糖化力法检测重组CBHⅡ的酶活.实验结果表明:该基因大小为1 416 bp,编码有472个氨基酸残基,并已将序列提交GenBank,登录号为DQ864992.SDS-PAGE分析发现重组CBHⅡ成功分泌到了胞外,其相对分子质量约为70 000.培养液中的酶活最高可达7.71U/mL,其作用的最适pH值为5.0,最适反应温度为65℃,且具有较好的热稳定性.

Cellobiohydrolase Ⅱ （ CBH Ⅱ ） encoding gene cbh2 was obtained from Trichoderma viride by using the reverse transcription polymerase chain reaction （RT-PCR） method, and was then cloned into the integrative expression vector pSclKP of Saccharomyces cerevisiae to generate a recombinant plasmid pScIKP-cbh2. Afterwards, the plasmid was transformed by electroporation into Saccharomyces cerevisiae, and the transformants were screened and selected via G418 concentration gradient. Moreover, the protein expression of CBHⅡ was tested by means of SDSPAGE, and the enzyme activity of recombinant CBH Ⅱ was measured via CMC （ Carboxymethylcellulose Sodium） saccharification. Experimental results indicate that cbh2 gene coding 472 amino acid residues is composed of 1416 nucleotides, and that it submits the sequence to GenBank with an accession number of DQ864992. SDS-PAGE results show that the transformants result in a successful exocytosis of the recombinant CBHⅡ with a relative molecular mass of 70000. According to the CMC saccharification results, it is also found that the recombinant CBHⅡ with excellent thermal stability is of an activity up to 7.71 U/mL, and that it exhibits optimum catalytic activity at pH 5.0 and 65 ℃.