Purification and partial characterization of a low molecular fibrinolytic serine metalloprotease C142 from the culture supernatant of Bacillus subtilis C142

Novel serine metalloprotease-like enzyme, C142 was purified from the culture supernatant of Bacillus subtilis C142. The C142 was purified to homogeneity by a two-step procedure with a 20.7-fold increase in specific activity and 0.9% recovery. The molecular mass of C142 was approximately 23.5 kDa based on SDS-PAGE. The N-terminal amino acid sequence of the first 21 amino acids of C142 was AQSVPYGISQIKAPALHSQGY. Its optimum pH, optimum temperature, pH stability, and thermal stability were pH 6, 40 °C, pH 6-8, and 20-35 °C, respectively. C142 was strongly inhibited by PMSF and EGTA, suggesting that C142 was a serine metalloprotease-like enzyme. C142 showed the highest specificity toward the substrate for t-PA. The apparent Km, Vmax, and Kcat values of C142 toward H-d-Ile-Pro-Arg-pNA were determined as 0.34 mM, 0.25 mmol mg−1 min−1, and 46.83 s−1. C142 exhibited fibrinolytic activity, which is stronger than that of plasmin. C142 hydrolyzed Aα, and Bβ-chains of fibrinogen, but did not cleave γ-chains. C142 had antithrombotic effect in three animal models. C142 was devoid of hemorrhagic activity at a dose of 20,000 FU/kg. Taken together, our results indicate that B. subtilis C142 produces a serine metalloprotease-like enzyme/fibrinolytic enzyme and this enzyme might be used as a new thrombolytic agent.