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Abstract
Thirteen bacterial strains were selectively screened for the production of extracellular fibrinolytic enzyme. Bacillus subtilis was chosen as the most promising producer. Optimal conditions for fibrinolytic enzyme activity were, temperature 37oC, pH 6.6,15 gL-1 glucose as carbon source, 3.0 gL-1 alkali- treated soybean as nitrogen source and 0.5gL-1 MgSO4 and 0.05 gL-1 FeSO4. The D10 value of the B. subtilis was calculated to be 2.5 kGy. Irradiation dose at 4.0 kGy increases the fibrinolytic enzyme activity about four fold than the parent strain. The crude fibrinolytic enzyme showed high efficacy for human blood clot resolution in vitro, where the complete lysis was recorded after 270 min. Maximal activity of the crude enzyme was at pH 7.4 and 43oC, the enzyme protein concentration and substrate concentration were 2.0 mg/ml and 8.0 mg/reaction respectively. The acetone (40-70 %) fractions showed the highest fibrinolytic activity, it was further purified by gel filtration on Sephadex G-75 followed by re-chromatography of the most active peak on DEAE- cellulose DE52. The purification resulted in 312-fold purification of the enzyme. The apparent molecular weight of the purified enzyme was estimated to be 32.83 kDa. The purified fibrinolytic enzyme showed its maximal activity at 45oC after 60 min reaction time and pH 7.4. Km value and Vmax. of the purified enzyme in reaction mixture were 0.5 mg/ml and 33.3 Umg-1 protein respectively. The suitable substrate concentration and enzyme protein concentration were 13 mg/reaction and 10 µg/ml respectively, the purified enzyme showed high specificity to human fibrin. Enzyme activity was enhanced by Mg2+ ions but inhibited by Cu2+, Fe2+, Fe3+ and Cd2+ ions, furthermore enzyme activity was enhanced by EDTA and ethanol but inhibited by aprotinin indicating that the enzyme is serine protease. The purified fibrinolytic enzyme rapidly hydrolyzed α- chain of fibrinogen within 30 min indicating that the purified enzyme is α- fibrinogenase. The efficiency of the purified fibrinolytic enzyme from irradiated B. subtilis to dissolve blood clot was observed within 80 min.