Fermentation, Purification And Characteristics Of A Novel Fibrinolytic Enzyme SPFE-Ⅲ

The incidence of thrombotic disorder including cerebral stroke, myocardial infarction, and venous thromboembolism are rapidly increasing throughout the world. A tremendous amount of research has been done in the area of prevention and the treatment of the diseases. An extracellar fibrinolytic strain, isolated from fermented shrimp paste, a popular seasoning in Asian countries, was shown to have a strong fibrinolytic activity. It was named as Bacillus sp. nov. SK006 by CCTCC. The strain was proved to produce four fibrinolytic isoenzymes. In this study, one fibrinolytic enzyme (SPFE-â…¢) from Bacillus sp. nov. SK006 was intensively investigated.The medium composition and fermentation conditions for fibrinolytic enzyme production were optimized in the research. The maximal yield of fibrinolytic enzyme (2.63 U/mL) could be obtained in the medium with concentrations of 20 g/L glucose, 30g/L tryptone, 15 g/L Na₂HPO₄·12H₂O, 1.3 g/L NaH₂PO₄·2H₂O, 0.5 g/L MgSO₄·7H₂O, 0.1 g/L CaCl₂, respectively, under the optimized cultivation conditions of pH 7.0, rotation speed 180 r/min and inoculation size 3% in 50 mL of culture medium in 250 mL shake flasks at 37℃for 24 h. The fibrinolytic enzyme production of Bacillus sp. nov. SK006 was 3.76 times of the control.A fibrinolytic enzyme (SPFE-â…¢) which produced from Bacillus sp. nov. SK006, was isolated using a combination of ethanol precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and Superdex 75 gel filtration chromatography. The purified SPFE-â…¢that has a molecular weight of 42.8 kDa was assessed homogeneous by SDS-PAGE. The specific activity was determined to be 7.1 U/mg using plasmin as a standard. SPFE-â…¢effectively degraded fibrin clots by direct fibrinolysis. During the degradation process of fibrinogen,α-subunits of fibrinogen were cleaved first, followed by slower release of theγ-chains.β-chains were resistant to the enzyme digestion.The enzyme had an optimal pH of 7.0-8.0. It had an optimal reaction temperature of 30℃-40℃, and kept 60% and 30% of the initial activity after heating at 40℃and 50℃for 1 hour. In addition, barium ion stimulated the enzyme activity whereas Zn²⁺, Ca²⁺, Fe³⁺, Mn²⁺ and Cu²⁺ caused deactivation. SPFE-â…¢was completely inhibited either by 10 mmol/L EDTA or 1 mmol/L 2-mercaptoethanol. The fibrinolytic activity was partially inhibited by other protease inhibitor.SPFE-â…¢had the highest affinity for N-Succ-Ala-Ala-Pro-Phe-pNA, which is a well-known substrate for subtilisin or chymotrypsin. SPFE-â…¢also degraded D-Val-Leu-Lys-pNA (for plasmin). However, it showed a lower activity for D-Val-Leu-Arg-pNA (for kallikrein) compared with other investigated fibrinolytic enzymes. K_m and k_cat for N-Succ-Ala-Ala-Pro-Phe-pNA were 0.239 mmol/L and 475.5 s^-1, respectively. In conclusion, SPFE-â…¢, a new type of fibrinolytic enzyme, has some potential for practical application as a source of functional foods and new therapeutic agents to treat thrombosis.