Molecular mechanism, protein engineering, and application of a thermophilic and halophilic enzyme, thermolysin

嗜热嗜盐酶嗜热菌蛋白酶的分子机制、蛋白质工程和应用

基本信息

批准号：
11460040
负责人：
INOUYE Kuniyo
金额：
$ 3.39万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Effect of chemical modification of Tyr, COOH, and Lys residues of thermolysin (E) on its activity and halophilicity was examined. The activity was decreased by the modification of Tyr and Lys residues, while that was not change by that of carboxyls. The decrease in the halophilicity was observed with decreasing the charges on the surface of E.Lys residues were modified by polyethylene glycol (PEG). The activity of E was increased with the degree of the modification, and was 6-times higher when 6 Lys residues were modified. E was stabilized with the PEG modification. The effect of PEG was similar to that of the addition of salts. Two E varinats (T1 and T2) were isolated from mutants of the E-producing bacteria. As with T1, Val-140 of E was converted to Ala, and with T2, Ala-73 was converted to Val. The protease activity of T1 was 150% in comparison with that of the native E, and the peptidase activity was 35% On the other hand, the protease activity of T2 was 10% of that of the native E … More , but the peptidase activity was increased remarkably, to 1,200%. The both residues were shown to have critical influence in determining of the substrate-specificity of E.Introduction of a bulky group into the place of Asp-73 depressed strongly the protease activity, and turned E to a peptidase. The A73V enzyme has 10-times higher activity of synthesis of the aspartame precurser. The active-site zinc was replaced with cobal, and the Co-E was shown to have 5-times higher activity than the native E.Co-E exhibited halophilicity, as the parent E does, and the activity at 4 M NaCl was 15-times higher than that of the native one. The activity-and-structure of E was compared with that of matrilysin. It showed also strong halophilicity, and higher thermal stability. The structure was found to be similar to that of the catalytic domain of E.It was also observed that the molecular mechanism of of halophilicity of E and matrilysin is different ; namely that the halophilicity of E was derived by the stabilizing the transition-state of the substratre, whereas that of matrilysin was by stabilizing its ground-state of the substrate. Less

研究了Tyr，COOH和LYS保留热蛋白（E）的化学修饰对其活性和卤素的影响。 Tyr和Lys的修饰会降低活性，而羧基的变化并没有改变。通过聚乙烯乙二醇（PEG）修饰了E.透析表面上的电荷的降低，观察到卤素的降低。随着修饰的程度，E的活性增加了，当6个LYS幸存下来时，E的活性高6倍。 E通过PEG修饰稳定。 PEG的作用与添加盐的作用相似。从e产生细菌的突变体中分离出两个E varinats（T1和T2）。与T1一样，E的Val-140转换为ALA，使用T2，ALA-73转换为Val。与天然E相比，T1的蛋白酶活性为150％，另一方面，肽酶活性为35％，T2的蛋白酶活性为天然E的蛋白酶活性的10％，但肽酶的活性显着增加，至1,200％。两种残差都显示在确定笨重基团进入ASP-73的位置的底物特异性方面具有关键影响，并强烈抑制了蛋白酶活性，并将E变成了肽酶。 A73V酶的合成活性高10倍。活跃位点的锌被钴替代，并且表现出比本机E.Co-e暴露的卤素的活性高5倍，就像父e一样，4 M NaCl的活性比天然的活性高15倍。 E将E的活性和结构与基质素的活性进行了比较。它也显示出强的卤素和较高的热稳定性。发现该结构与E的催化结构域相似，也观察到E和基质素的卤素性分子机制不同。也就是说，E的卤代性是通过稳定底物的过渡状态得出的，而基质蛋白的卤代性是通过稳定其底物的地面而得出的。较少的