Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis

Abstract

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.