Purification,Characterization And Fermentation Condition Of Fibrinolytic Enzyme From Bacillus Sp.

With living conditions improved, thrombosis diseases have become great threatening to human beings. Tl~rombo1ytic therapy is one of the best ways to cure thrombosis diseases. Fibrinolytic enzymes have apparent significance in thrombosis therapy in human. At present, thrombolytic agents ( t-PA) was transformed, at the same time, there has been interest in search for new thrombolytic agents from various origins with particular reference to microbial sources. Bacillus sp.2, which produces a strongly fibrinolytic enzyme, was screened in our laboratory, to develop the enzyme for use as a thrombotic agent. In this paper, through single factors and homogenous design experiment, the suitable conditions were given to produce fibrinolytic enzyme from Bacillus sp.2(Bs-2FE). The enzyme was purified from the fermented broth of Bs-2 and characterized in substrate specificity, inhibitor characters, optimum temperature and pH, thermal stability, effects of metal ions. The results are as follows: The fermentation conditions in shaking flasks were investigated. By the method of homogenous design experiment, the suitable main medium was achived , which contained 2.5% corn starch, 4.0% soybean meal, 2.5% wheat bran, 0.5% MgSO4.7H20, 0.5% Na2HPO412H2O, 0.01% NP-40 and 0.03% KH2PO4,. The Bs-2 was 2 inoculated into 2Onil medium in 250m1 triangle flask, shaking in 22C at 170 rlmin for 72h.with initial pH7.0, the fibrinolytic enzyme activity amounted to 1152.6 tPA lU/mi, increased 10 fold. By ammonium sulphate fractional precipitation, Sephadex G-75 gel filtration, Q-Sepharose anion exchange chromatography and preparative PAGE , a fibrinolytic enzyme was purified from the fermented broth of Bs-2 . The purified Bs-2 FE consisted of three subunits with a molecular weight of approximately l9kD, 22kD, 23kD, respectively, and the molecular weight of the enzyme was about 64kD on the gel of SDS-PAGE. The isoelectric point was about 7.2 by analytical isoelectric focusing electrophorsis. The optimum temperature and pH of Bs-2FE was 35C and 9.0, respectively. Bs-2FE was stable under 45~C within 2h, and it rapidly declined by treating the enzyme at 50X2 for 2h , the metal ions Zn2~, Cu2~,Co2~,Hg2~ and Pb2~ showed strong inhibition. Serine protease inhibitor PMSF can strongly inhibit activity of Bs-2FE, it indicated that the enzyme belongs to senile protease. Bs-2FE activity was almost full inhibited by TPCK, and half inhibited by TLCK; the Bs-2FE can hydrolyze specific substrate of chymotrypsin and trypsin, it suggested that Bs-2FE possessed activity of chymotrypsin and trypsin0 On the fibrin plate and plasminogen-free fibrin plate, Bs-2FE showed the same fibrinolytic activity. The result indicated that the enzyme might contain both a fibninolytic enzyme which degraded fibrin directly and a plasminogen activator which degraded fibrin by activating plasminogen. Bs-2FE showed stronger activity against Chromozym P, Chromozym UK and S-2288, this result was similar to which showed on fibrin-agar plate.