耐热碱性磷酸酶突变体耐热性变化机理研究

从耐热碱性磷酸酶（TAP）200多个随机突变体的克隆库中选出耐热性明显下降的4株突变体，进行全序列及表达产物的最高耐受温度和最适反应温度测定和酶分子高级结构的模拟，分析突变位点、高级结构和耐热性表现三者的关系，探讨引起耐热性变化的机理。结构模拟显示所有突变位点都仅能引起细微的、局部的结构变化，除T320→I外都未直接触及酶的活性中心；结构上的细微改变虽然对最适反应温度影响不明显，但却使最高耐受温度降低了10℃左右；T320→I靠近酶的活性中心，尽管未能引起结构的较大变化，但却使最高耐受温度和最适反应温度同时显著降低。可见，多数点突变对高级结构的影响都不剧烈，但对耐热性尤其是最高耐受温度的影响却比较明显，一般地，在非活性区的突变通常只能引起最高耐受温度的降低，靠近活性区的突变则能同时引起最适反应温度和最高耐受温度的降低。

Studies on the Mechanism of Thermostability and Thermophilicity Change of Thermostable Alkaline Phosphatase and Its Mutants

The relationship among the substituted amino acids, the 3D structure simulated on PC through CPHmodels Server ( http://www.cbs.dtu. dk/services/CPHmodels/) and the thermostable performance of 4 thermostable alkaline phosphatase(TAP) mutants selected from a clone bank of more than 200 mutants were analyzed to explore the mechanism of thermostability change. These mutants are TAP(A410T) (A410-->T), TAP(P396S) (P396-->S), TAP2(N100S T320-->I) and TAP4(N100-->S P396-->S A410 -->V P490-->S). TAP and the mutants' thermostable performance was evaluated by measuring the highest tolerable temperature (T1/2) and the optimal reaction temperature (Topt). The 3D structure neighboring the substituted amino acids was simulated by Swiss-PDBViewer to observe the relationship between the structure change and the thermostable performance of TAP and its mutants. The results displayed that all these amino acid substitutions except the T320-->I mutant brought about only a little local change on TAP's 3D structure and very little effect on their optimal reaction temperature, but a significant decrease (nearly 10 degrees C) on their highest tolerable temperature. However, the T320-->I mutation due to close to TAP's active sites did bring about a significant descendents of the mutant in both the highest tolerable temperature and the optimal reaction temperature. Thus, it seems to be able to conclude that most of the amino acid substitutions, no matter where they locate and what structure change they may make, can cause TAP's highest tolerable temperature reduced significantly. What's more, if the mutation occurring near or in the active sites, it can also cause TAP's optimal reaction temperature reduced significantly at the same time.