Purification and characterization of a fibrinogen-degrading protease in Bacteroides fragilis strain YCH46.

A novel fibrinogenolytic protease was purified from Bacteroides fragilis strain YCH46. The protease was extracted from cells by ultrasonic treatment and was purified 425-fold with a recovery of 2.1% by sequential procedures using azocasein as a substrate. The purified protease showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 100 kDa, which was consistent with the value obtained by gel filtration, indicating a monomeric native structure. Its optimal pH, Km, and Vmax for azocasein were 7.5, 0.2%, and 286 U/min/mg, respectively. The protease activity was completely inhibited by addition of 1 mM Hg2+, Cu2+, Zn2+, diisopropyl fluorophosphate, N-ethylmaleimide or p-chloromercuribenzoate but not by the inhibitors of metalloprotease or aspartic protease, suggesting that the enzyme is a serine-thiol-like protease. The protease hydrolyzed azocasein, casein, fibrinogen, gelatin, and azocoll, but not bovine serum albumin, ovalbumin, fibrin, fibronectin, immunoglobulins, transferrin, hemoglobin or types I, III, and IV collagen. The enzyme also hydrolyzed the chromogenic substrates alanyl-alanine p-nitroanilide, L-valyl-alanine p-nitroanilide, alanyl-alanyl-valyl-alanine p-nitroanilide, and glycyl-proline p-nitroanilide, but was inert toward L-alanine p-nitroanilide, alanyl-alanyl-alanine p-nitroanilide, and N-alpha-benzoyl-DL-arginine p-nitroanilide. The protease completely hydrolyzed the alpha-chain of fibrinogen at 37 C within 10 hr and at the same time the time required for clotting of protease-treated fibrinogen by thrombin was prolonged. The fibrinogenolytic activity of a crude extract of B. fragilis was stronger than that of other species of the Bacteroides fragilis group tested: B. ovatus, B. distasonis, B. eggerthii, B. uniformis, and B. thetaiotaomicron. These results suggest that the fibrinogenolytic protease is an important biological factor in Bacteroides infection.