大肠杆菌碱性磷酸酶突变体的构建及其结构与功能研究

CONSTRUCTION OF MUTANTS OF ALKALINE PHOSPHATASE AND REASERCH ON THEIR STRUCTURE AND FANCTION IN E.Coli

以大肠杆菌碱性磷酸酶（alkaline phosphatase,AP）高活性突变株（L138PG233S）为亲本,采用重叠延伸PCR的方法进行定点突变,成功获得了氨基酸替换突变体3个,并对它们的酶活性、特性、内源荧光发射峰等与亲本酶进行了比较分析.结果发现：与亲本酶相比,突变体酶的酶活性降低了18%-85%,Vmax值降低,Km增加,内源荧光发射峰λmax增大等.与亲本酶相同,Zn^2＋,Mn^2＋,Mg^2＋仍是它们的激活剂,Na3PO4是它们的抑制剂;这些实验结果说明突变体酶活性的降低直接与其构象变化有关,被替换的氨基酸在维持碱性磷酸酶的结构和功能方面具有特定的作用.

Alkaline phosphatase（AP）, which is a kind of hydrolase, catalyzes the nonspecific hydrolysis of phosphate monoester. In order to study the relationship between structure and funcation of AP of Eschierichia coli （E. coli）, Over-lap extension PCR is used to make site-directed mutagenesis of phoA gene mutant L138PG233S to construct three mutants, of which activities, properties and fluoresence spectrum are analyzed. The results show that these mutants have an activity 18-85% lower than the parent, the reason for the lower activity is that they have a lower Vmax value and higher Km than the parent, Similar to the parent, three metal ions including Zn^2＋. Mn^2＋, Mg^2＋ are effective activator of these mutants, and NaaPO4 is their effective inhibitor, Unlike above, their λmax is greater than the parent. The optimum reaction temperature and pH of these mutants are also determined. In general, our study indicates that the decrease of activity of these mutants is related to their conformation change; and these substituted amino acids play a key role in the structure and funcation of Alkaline phosphatase.