CAJ | 학술논문

[目的]构建可降解纤维类固体废弃物的工程菌.[方法]采用RT-PER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44.1×103.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.
[목적]구건가강해섬유류고체폐기물적공정균.[방법]채용RT-PER방법극륭료록색목매(Trichoderma viride)AS313711적포취당내절매Ⅲ(EGⅢ)적cDNA,측서후구건도효모표체재체pESP-2상,병통과전격법장기전도효모감수태세포중거,득도효모표체전화자.통과DNS법측정해전화자재불동온도、불동pH치하매활력적대소.[결과]EGⅢ적cDNA개방열독광장도위1 257 bp,편마418개안기산,추측단백질분자량위44.1×103.재pH치위4.9、온도재60℃조건하,EGⅢ매활력최고,상대매활위100%.[결론]획득료고표체효솔적EGⅢ-T-pESP-2효모표체재체,기표체활성요비천연적매고출3～5배,지요조절호온도、pH치적관계,가제고섬유소포취당내절매적하유전화섬유소효솔,재대규모생산중생산출대량적포도당.
[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%.