摘要
Objective The aim was to construct bioengineering strains that could degrade the cellulosic solid waste. Method The cDNA of endo-β-glucanase III of Trichoderma vi ride AS313711 was cloned by RT-PCR method. After sequenced, this gene was constructed to expression vector pESP-2, and then the plasmid was transformed into competent cell of cerevisiae fermentum by electric shock, the transformant was then obtained. The enzyme activity of this transformant at the different temperatures and pH was measured by DNS method. Result The length of ORF of EG III was 1 257 bp, encoding 418 amino acids, while the deduced molecular weight was 44.1 × 103 kD. Conclusion The enzyme activity of EG III was the highest when it was at PH 4.9 and tempeture was of 60℃. Then the corresponding enzyme activity was about 100%.
[目的]构建可降解纤维类固体废弃物的工程菌。[方法]采用RT-PCR方法克隆了绿色木霉（Trichoderma viride）AS313711的葡聚糖内切酶Ⅲ（EGⅢ）的cDNA,测序后构建到酵母表达载体pESP-2上,并通过电击法将其转到酵母感受态细胞中去,得到酵母表达转化子。通过DNS法测定该转化子在不同温度、不同pH值下酶活力的大小。[结果]EGⅢ的cDNA开放阅读框长度为1 257 bp,编码418个氨基酸,推测蛋白质分子量为44.1×10~3。在pH值为4.9、温度在60℃条件下,EGⅢ酶活力最高,相对酶活为100%。[结论]获得了高表达效率的EGⅢ-T-pESP-2酵母表达载体,其表达活性要比天然的酶高出3～5倍,只要调节好温度、pH值的关系,可提高纤维素葡聚糖内切酶的下游转化纤维素效率,在大规模生产中生产出大量的葡萄糖。
