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Analyzes of the mechanisms of thermophilic and alkalophilic properties of aqualysin I,a protease from an extreme thermophile

极端嗜热菌蛋白酶 aqualysin I 的嗜热和嗜碱特性机制分析

基本信息

批准号：
08456046
负责人：
MATSUZAWA Hiroshi
金额：
$ 0.7万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456046/
关键词：
protein engineering protease heat-stable protease aqualysin I metal ion site-directed mutagenesis mutant enzyme 耐熱性酵素

项目摘要

Aqualysin I is a thermophilic and alkalophilic protease ; in the presence of calcium ion, the optimum temperature for the proteolytic activity is 80ﾟC,and the enzyme is stable at 80ﾟC for 3h. The optimum pH for the enzyme activity is about 10, and the enzyme is stable at pH 12 and 4ﾟC for long time. In order to investigate the structural properties which gives these characteristics to the enzyme, we have done this study, and obtained the following resuits.(1) The effects of rare earth metals on the heat-stability of the enzyme were exzmined. Depending on the ionic radii (0.93 to 1.06 angstrom) and independent of the positive charges, some rare earth metals showed the effect like Ca^<2+> (0.98 angstrom). La^<3+> was more effective than Ca^<2+>. The binding constant of La^<3+> with the enzyme was about ten times larger than that of Ca^<2+>, suggesting that the binding constant of metals is important for the stabilization of the enzyme.By random mutagenesis with PCR,mutations were introduced in the coding region of aqualysin I,and Escherichia coli cells were transformed with recombinant plasmids carrying the gene for the enzyme and its mutants. Mutant Clones, which seemed to produce mutant enzymes showing proteolytic activity at low temperatures or at low pHs, were selected. Such clones were obtained at the frequency of 10^<-3> or less, but purified enzymes from the clones did not show the expected properties.Replacement of Asn219 with serine increased the catalytic defficiency (K_<cat/>K_m) about twice as much as that of the wild type at the temperature rage of 10 to 90ﾟC.Such a replacement resulted in a significant change of K_<cat/> rather K_<m'> indicating that the side chain in the vicinity of the catalytic residue Ser222 affects the catalytic rate constant.

Aqualysin I是一种嗜热、嗜碱蛋白酶，在钙离子存在下，最适蛋白水解温度为80 ℃，在80 ℃下稳定3 h。该酶的最适pH为10左右，在pH 12和4 ℃下酶能长期稳定。为了研究赋予酶这些特性的结构性质，我们进行了这项研究，并获得了以下结果。(1)研究了稀土金属对酶热稳定性的影响。根据离子半径（0.93 ~ 1.06埃）和独立于正电荷的情况，某些稀土金属显示出类似Ca^2+（0.98埃）的效应。La^<3+>比Ca^<2+>更有效。La^<3+>与该酶的结合常数比Ca^<2+>大10倍左右，表明金属离子的结合常数对该酶的稳定性有重要作用。通过PCR随机诱变，在aqualysin I的编码区引入突变体，并将携带该酶基因及其突变体的重组质粒转化大肠杆菌细胞。选择似乎产生在低温或低pH下显示蛋白水解活性的突变酶的突变克隆。在10 ~ 90 ℃温度<-3>范围内，用丝氨酸取代Asn 219使催化效率（K_<cat/>K_m）比野生型提高了约2倍，这种取代导致K_K而<cat/>不是K_m '的显著变化，表明催化残基Ser 222附近的侧链影响催化速率常数。