Kannan Y, Koga Y, Inoue Y, Haruki M, Takagi M, Imanaka T, Morikawa M, Kanaya S
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Appl Environ Microbiol. 2001 Jun;67(6):2445-52. doi: 10.1128/AEM.67.6.2445-2452.2001.
The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.
编码嗜热栖热菌枯草杆菌蛋白酶样蛋白酶的基因是从嗜热古菌嗜热栖热菌KOD1中克隆得到的。嗜热栖热菌枯草杆菌蛋白酶是枯草杆菌蛋白酶家族的一员，由422个氨基酸残基组成，分子量为43783。它由一个假定的前导序列、前肽序列和催化结构域组成。与细菌枯草杆菌蛋白酶一样，嗜热栖热菌枯草杆菌蛋白酶在大肠杆菌中以包含假定前肽序列的形式在包涵体中过量表达，在8 M尿素存在下溶解，并重新折叠转化为活性分子。然而，与细菌枯草杆菌蛋白酶不同的是，细菌枯草杆菌蛋白酶在重新折叠时通过自加工从催化结构域中去除前肽序列，而嗜热栖热菌枯草杆菌蛋白酶则以带有假定前肽序列的形式重新折叠。这种由398个氨基酸残基（Gly(-82)至Gly(316)）组成的重组嗜热栖热菌枯草杆菌蛋白酶的重新折叠蛋白，经纯化后在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上呈现单一条带，并对其生化和酶学性质进行了表征。通过SDS-聚丙烯酰胺凝胶电泳（44000）和凝胶过滤（40000）估计的分子量之间的良好一致性表明嗜热栖热菌枯草杆菌蛋白酶以单体形式存在。嗜热栖热菌枯草杆菌蛋白酶仅在Ca(2+)离子存在下才能水解合成底物N-琥珀酰-Ala-Ala-Pro-Phe-对硝基苯胺，最佳pH和温度分别为pH 9.5和80℃。与细菌枯草杆菌蛋白酶一样，它表现出广泛的底物特异性，对芳香族或大的非极性P1底物残基有偏好。然而，它比细菌枯草杆菌蛋白酶对热失活更稳定，在80℃下半衰期>60分钟、90℃下20分钟、100℃下7分钟时失去活性。
