长枝木霉eg1基因的克隆及表达

Cloning and Expression of eg1 Gene from Trichoderma longibrachiatum

从长枝木霉3.1029基因组中克隆了内切葡聚糖酶EGI基因,该基因全长1 566 bp,由3个外显子2个内含子组成,编码461个氨基酸,编码蛋白的N端为22aa组成的信号肽。采用重叠PCR法获得无内含子的内切葡聚糖酶基因eg1,构建成pYE-Leg1重组质粒;同时将其成熟肽编码序列插入酿酒酵母分泌型表达载体pYEα中,构建成pYEα-Leg1重组质粒;分别转化酿酒酵母。重组转化子经β-半乳糖诱导,检测表达产物的酶活,结果表明,pYE-Leg1转化子无明显胞外酶活;而pYEα-Leg1转化子在刚果红平板上可产生明显的水解圈,酶活检测显示pYEα载体可有效地将该基因在酿酒酵母中表达并分泌到胞外,发酵液中的酶活在培养96 h达到最高1.16 U/mL,最适酶解温度为50℃,最适pH值为5.6。以上研究将为利用酿酒酵母生产胞外纤维素酶提供依据。

A gene （ egl ） coding for an endoglucanase I （EGI） was cloned from Trichoderma longibrachiatum stain 3. 1029. The gene is composed of 1 566 bp, interrupted by 2 introns, and coding for 461 amino acid residues which revealed a 22aa signal peptide from the N-terminus. The egl gene with no introns was obtained by overlapping PCR and the recombinant plasmid pYE-Legl was constructed, pYEα-Legl was also constructed by inserting the sequence encoding the mature peptide into the S. cerevisiae secretion vector pYEa. The two plasmids, pYE-Legl and pYEα-Legl were then transformed into S. cerevisiae respectively. After induction of galactose, there is no clear halo around pYE-Legl transformants. However, pYEα-Legl transformants secreted a recombinant EGI that had higher enzymatic properties detected by congo red assay and enzyme activity assay. Extracellular enzyme activity of pYEα-Legl transformants approached the highest level （ 1.16 U/mL） when cultured for 96 hours. The optimum temperature and pH were 50 ℃ and 5.6 respectively. This study would provide a basis for producing secretory cellulase by S. cerevisiae.