摘要
通过对匍枝根霉(Rhizopus stolonifer)内切葡聚糖酶EGII进行结构功能分析,发现其活性中心入口处的色氨酸W129在水解纤维素的过程中起着关键作用。对该位点进行定点突变(W129A),并研究了它对EGII和催化区(CD)的影响。经原核表达及纯化,获得目的蛋白EGII-W129A和CD-W129A。酶学特性研究结果显示,重组蛋白的比酶活、kcat和kcat/Km均低于EGII和CD,比酶活分别下降了23.8%和35.7%,同时Km值均有所提高。W129A导致酶与底物的亲和力和催化效率下降,使酶活力降低的原因主要是其减弱了活性中心入口处纤维素链的"载入"效率,即Trp129在EGII水解纤维素过程中起到加载纤维素链的重要作用。
Structural and functional analysis of Rhizopus stolonifer endoglucanase（ EGII） showed that W129 at active-site tunnel entrance played a key role in hydrolysis of cellulose. Site-directed mutagenesis（ W129A） had been applied to study its impact on EGII and catalytic domain（ CD）. The proteins（ EGII-W129 A and CD-W129A） were obtained by prokaryotic expression and purification. The results of studies on enzymatic properties indicated that the specific activity of recombinant protein were lower than those of the EGII and CD（ decrease of 23. 8% and 35. 7%）,meanwhile,the Kmvalues were improved. W129 A reduced the efficiency of the cellulose chain loading on the entrance of active-site tunnel,leading to the decrease of enzyme activity and affinity between substrate and enzyme. It was Trp129 that played an important role of loading cellulose chains and starting hydrolysis in the degradation of cellulose.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2015年第4期17-21,共5页
Food and Fermentation Industries
基金
国家自然科学基金项目(31270135)
关键词
匍枝根霉
内切葡聚糖酶
色氨酸
结构与功能
定点突变
Rhizopus stolonifer
endoglucanase
tryptophan
functional analysis
site-directed mutagenesis
