瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达

采用刚果红染色法从瑞氏木霉cDNA文库中分离到一株具有CMCase活性的阳性克隆 ,测序结果显示该基因所编码的蛋白质为瑞氏木霉内切葡聚糖酶Ⅲ (EGⅢ )。对重组酿酒酵母所产生的EGⅢ进行了酶学性质分析 ,其最适pH为 5 0 ,最适温度为 60℃。检测了酿酒酵母蛋白质分泌系统组分SSO2和SEB1对EGⅢ分泌的影响。结果表明 ,在可过量表达蛋白质分泌系统组分SSO2的酿酒酵母H837中 ,EGⅢ的分泌量最高。由此分析 ,酿酒酵母SSO2蛋白可能在EGⅢ的分泌中 ,是一个限速步骤。通过PCR方法删除EGⅢ基因 5′端非翻译区的 98bp核苷酸序列使EGⅢ表达量提高了 5 3倍。这提示我们 ,瑞氏木霉的EGⅢ基因在酿酒酵母细胞中表达时 ,其mRNA 5′端先导序列中可能存在影响该基因表达水平的调控序列。

CLONING AND EXPRESSION OF TRICHODERMA REESEI ENDOGLUCANASE

Using Congo red-staining method, one positive clone with CMCase activity was isolated from the Trichoderma ressei cDNA gene bank constructed in Saccharomyces cerevasiae. Sequencing result showed that the 1.5 kb-length DNA fragment inserted in the recombinant plasmid encoded EG III gene from T. reesei. Enzymatic characterization of the EG III produced by recombinant S. cerevasiae was analyzed. The experimental results indicated that the optimum pH and temperature for EG III are 5.0 and 60 degrees C, respectively. The effects of secretory system components SSO 2 and SEB1 of S. cerevisiae on EG III secretion were examined. The results indicated that the amount of EG III secreted by the strain with SSO 2-overexpression was highest among the different recombinant S. cerevisiae strains, showed that SSO 2 is a rate-limiting component of the secretory machinery in the process of EG III secretion. Furthermore, the EG III expression level was increased 5.3 times by deletion. Furthermore, the EG III expression level was increased 5.3 times by deletion of the 98 bp in 5' untranslated region of eg3 mRNA sequence. This result suggested that the regulation region could exist in the 5' untranslated region of EG III mRNA, which is recognized by the gene expression related factors of S. cerevasiae.