Screening Of Fibrinolytic Enzyme-Producing Strain Dc-C4 From Chinese Douchi

Five strains with high fibrinolysis activity were isolated from traditional Chinese food Douchi with the improved fibrin plate. The fibrinolysis activity of the zymotic fluid was determined and analyzed in the thrombolysis experiment in vitro. The strain DC-C4 showed the highest fibrinolysis activity which hits 280IU/mL. Comparative 16S-23S rDNA sequence analyses revealed that strain DC-C4 was most related to the strain Bacillus cereus ATCC14579. And the biological features of strain DC-C4 was very similar to that of Bacillus cereus. Based on the results obtained, strain DC-C4 was identified as Bacillus cereus.The optimal fermentation conditions for producing DouChi fibrinolytic enzyme (DFE) by strain DC-C4 were determined and the results are as follows. The compositions of fermentation medium are: peptone from soya 3.0%, yeast extract 0.3%, starch soluble 2.0%, K2HPO4 0.3%, KH2PO4 0.1%, MgSO4-7H2O 0.05%, CaCl2-2H2O 0.02%. The optimal cultivation temperature is 35℃, the optimal shaking speed is 200 r/min when 500mL flask contains 200mL medium, and the fermentation time is 72h. Under these conditions the fibrinolytic activity of the supernatant can reach 880 urokinase units per milliliter broth.The optimum temperature and pH of unpurified DFE were 45℃ and 7.5,respectively; and the DFE was stable in the pH range of 6.0-11.0 or temperature under 45℃. DFE can directly degrade the fibrin, but not activate plasminogen into plasmin and hydrolyze blood cells. PMSF(phenylmethylsulfonyl fluoride) can completely inhibit its fibrinolytic activity, which indicated that DFE is a subtilisin-like serine protease. The protein content of the fermented broth was estimated by Coomassie brilliant blue staining and the specific activity of the fermented broth was 514 IU/mg. The research will provide experimental evidence for developing new thrombolytic remedy and fibrinolytic functional food.