49P（del）点突变提高中性纤维素内切酶EGV热稳定性的初步研究

目的：探讨利用点突变方法改善EGV热稳定性的可能性和有效性。方法：对来源于Melanocarpus albomyces endoglucanase的耐热性纤维素酶maEG进行同源建模和序列比较，删除49位脯氨酸49P（del）进行定点突变，并将得到的突变体在毕氏酵母X33中表达，对表达产物进行酶活性和热稳定性检测。结果：突变酶49P（del）在70℃处理120min，热稳定性比EGV提高了21．6％，且突变酶其他性质与野生型酶基本相似。结论：通过对中性纤维素内切酶EGV的定点突变，提高了该酶的热稳定性，并为进一步研究其结构和功能提供了材料。结果同时表明利用生物信息学和分子模拟技术，缩短表面环区对于酶的热稳定性有一定的作用。

Improvement of Cellulase EGV Thermostability by Site-directed Mutagenesis at 49P（del）

Objective： To explore the possibility and effects of improving enzyme thermostability by site-directed mutagenesis. Methods： Homology models and sequence alignment were conducted for Melanocarpus albomyces endoglucanase maEG. Then 49P（del） mutation was introduced into wide type EGV by site-directed mutagenesis. Both EGV and the mutant 49P （del） were expressed in Pichia pastoris. Their enzymatic properties were determined. Results： The result revealed that the thermostability of49P （del） was 34.5% higher than that of EGV at 70℃ for 180 min. The optimum temperature of 49P（del ） was increased by 10℃ in mutant enzymes than that in natural phytase. Additionally, the other enzymatic properties of mutant were similar to EGV. Conclusions： The mutant 49P（del） is a good materials for further research on structure and function of Cellulase EGV.