Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis.
Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis.
复制标题
牙龈拟杆菌培养上清液中蛋白酶的分离和表征。
DOI：
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发表时间：
1987
影响因子：
3.5
通讯作者：
R. Nakamura
中科院分区：
文献类型：
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作者：
M. Otsuka;J. Endo;D. Hinode;A. Nagata;R. Maehara;M. Sato;R. Nakamura
采用丙酮分离、Sepharose CL-6B凝胶过滤、0.8% 1- o - n -辛基-β- d -葡萄糖苷溶解、精氨酸-Sepharose 4B亲和层析等方法，从牙龈拟杆菌381培养上清中分离纯化酪蛋白水解蛋白酶。通过亲和层析将蛋白酶分离成3个同工酶，其中1个不吸附在精氨酸- sepharose 4B上，而另外2个在不同精氨酸浓度的缓冲液中被吸附和洗脱。后两者的溶酪蛋白活性高度依赖于二硫苏糖醇，并被巯基阻断剂和一些微生物蛋白酶抑制剂抑制；但在前同工酶中没有发现这种抑制作用。酶对精氨酸- sepharose的亲和力越高，对硫醇的依赖性越强，对这些抑制剂的抑制作用也越强。磷酰胺和四乙酸乙二胺对三个峰的活性均无影响，说明该酶不是金属蛋白酶。除了酶解胰蛋白酶的合成底物如BApNA、TAME外，还水解了胰凝乳蛋白酶和钾激肽。唾液溶菌酶和蛋白溶菌酶也被该酶降解。这些结果提示，牙龈芽孢杆菌产生的蛋白酶不仅可以直接破坏牙周组织，还可以削弱口腔抗菌机制。
Caseinolytic protease was isolated and purified, with high yield, from culture supernatant of Bacteroides gingivalis 381 by procedures including acetone fractionation, gel filtration on Sepharose CL-6B, solubilization with 0.8% 1-O-N-octyl-β-D-glucopyranoside and affinity chromatography on arginine-Sepharose 4B. By the affinity chromatography, the protease was separated into three isoenzymes, one of which was unadsorbed on the arginine-Sepharose 4B, but the other two of which were adsorbed and eluted with the buffer at a different concentration of arginine. Caseinolytic activities of the latter two were highly dependent on dithiothreitol and were inhibited by both thiol-blocking reagents and some microbial protease inhibitors; but such inhibition was not found in the former isoenzyme. The higher the affinity of the enzyme to arginine-Sepharose, the stronger the thiol dependency and the inhibition by these inhibitors. Phosphoramidone and ethylenediamine tetraacetate had no effect on the activity of any of the three peaks, indicating that the enzyme is not a metaloprotease. Besides the enzymehydrolyzed synthetic substrates of trypsin, such as BApNA, TAME, substrates of chymotrypsin and kallikrein were also hydrolyzed. The salivary and egg-white lysozymes were also degraded by this enzyme. These findings suggest that the protease produced by B. gingivalis may play a role as a periodontopathogen not only in directly destroying periodontal tissues but also in weakening the oral antibacterial mechanism.