Studies On The Fibrinolytic Enzyme Produced By Microorganism

A Asperillus oryzae strain, SA-6 was isolated from sauce propagation with the clarity circle method on plate and the activity of fibrinolytic enzyme was 25 U/mL. It was showed that fibrinogen and thrombin could be replaced with fibrin to determine the activity of fibrinolytic enzyme through experimentation, which can save much money among screening of many strains. The mutant was isolated after SA-6 was treated with UV,⁶⁰Co and NTG and the activity of firbrinolytic enzyme was enhanced to 63 U/mL which was 1.52 times higher than that of SA-6. Comparing with the initial strain, the fastigium of the mutant producing fibrinolytic enzyme was later about 10 hours.By single-factor experiments, the optimal carbon source of NA-25 producing fibrinolytic enzyme was bran and the optimal nitrogen source was peptone and NaNO₃. Plackett-Burman design was undertaken to evaluate the effects of different factors. By the statistical regression analysis, the significant factors affecting the fibrinolytic enzyme activity in fermentation liquor were determined as follows: NaNO₃, peptone, CaCl₂. In the second phase of the optimization process, a central composite design was used to optimize the above critical internal factors. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentration of the variables were determined as: 18.5g/L NaNO₃, 4.97g/L peptone, 1.63g/L CaCl₂. In the optimal medium conditions the activity of fibrinolytic enzyme in fermentation liquor was 98.13 U/mL, increased by 55.8%. Then other conditions were optimized: rotate speed 180 r/min, temperature 30℃. In the optimal fermentation conditions the activity of fibrinolytic enzyme in fermentation liquor was 123 U/mL, increased by 93.4%.By filtration, ammonium sulphate fractional precipitation, dialyse and DEAE-cellulose ion exchange chromatogram, the fibrinolytic enzyme was purified. The rate of recovery was 12.9% and the purification multiple was 7.5.After the fibrinolytic enzyme was purified simply, the optimum temperature was 37℃and the optimum pH was 9.0. In the pH range 6.0-9.0, the fibrinolytic enzyme was stable. The metal ions K+, Ca²⁺ showed activation and Cu²⁺, Mn²⁺ showed inhibition. On the fibrin plate and plasminogen-free fibrin plate, the fibrinolytic enzyme didn't show the same fibrinolytic activity. The result indicated that the enzyme might contain both a fibrinolytic enzyme which degraded fibrin directly and a plasminogen activator which degraded fibrin by activating plasminogen.