通过定点诱变法研究耐热碱性磷酸酯酶的活性位点、耐热性和亲热性  

Studies of the Active Site, Thermostability and Thermophilicity of the Thermostable Alkaline Phosphatase by Site-directed Mutagenesis

作　　者：季朝能[1] 张冰 姜涛[1] 盛小禹[1] 毛裕民[1] 

机构地区：[1]复旦大学生命科学院遗传所

出　　处：《Acta Genetica Sinica》2000年第12期1100-1107,共8页

基　　金：国家自然科学基金项目!(批准号:39870402)

摘　　要：通过对耐热碱性磷酸酯酶TAPND27活性位点S（69）两侧的E（68）和S（70）的定点突变，得到了3个突变子E68S、S70A和E68SS70A。在蛋白纯化的基础上测定了3个变体的一些酶学性质，与TAPND27相比，E68S的比活力上升了8倍，Tm下降了3℃，最适反应温度上升了20℃；S70A的比活力上升了1部，Tm下降了2℃，最适反应温度上升了5℃；E68SS70A的比活力下降了50%，Tm下降了19℃，最适反应温度上升了5℃。说明活性位点S（69）两侧的氨基酸残基不仅和酶蛋白的催化能力，而且与酶的热稳定性和亲热性有很大的关系。为提高该酶的比活力和研究其热稳定性及亲热性提供了突变方向。：Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained. Their enzymatic characteristics was determined. It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20℃ and its Tm descended 3℃; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5℃ and its Tm descended 2℃; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5℃ and its Tm descended 19℃. These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity. The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.

关 键 词：耐热碱性磷酸酯酶 定点诱变 热稳定性 亲热性 

分 类 号：Q55[生物学—生物化学]