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

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

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

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.