关键词: 绿色木霉, 纤维二糖水解酶Ⅱ, 编码基因, 酿酒酵母, 表达

Abstract:

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 ℃.

Key words: Trichoderma viride, cellobiohydrolase Ⅱ, encoding gene, Saccharomyces cerevisiae, expression