Fermentation Of Fibrinolytic Enzyme From Bacillus Subtilis D21

As thrombosis becomes one of the main factors that threatened humans health, an increasing numbers of researchers have concentrated on thrombolytic agents.In this thesis, a strategy to improve fibrin plate method was provided. Fibrin plates with fibrinolytic discses was made followed the method published by Astrup et al. Then these plates were stained by coomassie brilliant blue R250 for 2 min. Subsequently, stained plates were destained by solution (acetic acid: methanol:distilled water=1:3:6). Areas of fibrinolytic discses were calculated by AutoCAD. It was concluded that fibrinolytic discses on stained plates were clearer than discses on plates without stained. In addition, stained fibrin plates could be stored at room temperature for at least 3 days whereas plates without stained could only be preserved at 4℃.Results showed that fibrinolytic activity of initial media (5 g/L yeast extract,10 g/L tryptone,10 g/L NaCl, pH 7) was 278.89±11.48 U/mL Carbon sources very significantly improved fibrinolytic enzyme fermentation (P < 0.01) and the highest activity was observed in media added with glucose (1650.37±50.23 U/mL). Nitrogen sources (except gelatin) very significantly inhibited fibrinolytic enzyme fermentation (P< 0.01) and the lowest activity was observed in media added with NH4Cl(19.99±0.44 U/mL). CaCl2 and MgCl2 significantly increased fibrinolytic activity to 350.03±18.72 U/mL and 354.15 ±24.44 U/mL respectively (P< 0.05). However, CuCl2 and ZnCl2 very significantly decreased fibrinolytic activity to 9.98±0.518 U/mL and 16.60± 3.235 U/mL respectively (P< 0.01). Fermentation was improved at initial pH of 5 (347.68±8.74 U/mL) and inhibited at pH of 9 (0 U/mL). The optimal temperature was 34℃ and fibrinolytic activity was decreased to 83.15±21.99 U/mL at 42℃.As glucose was the optimal carbon source and media added with glucose (5 g/L yeast extract,10 g/L tryptone,10 g/L NaCl,40 g/L glucose, pH 7) showed the highest activity, further study was concentrated on fermentation in media added with glucose. Results showed that the highest activity (3488.09± 5.72 U/mL) was observed at 72 h in media added with glucose wherase only 161.36±5.72 U/mL in the initial media at the same time course. SDS-PAGE and MALDI-TOF showed that there were two major protease (neutral protease and alkaline serine protease) in media added with glucose and only one major protease (alkaline serine protease) with lower concentration in the initial media.Alkaline serine protease gene of B. subtilis D21 was cloned and submited to Genbank (Genbank:KM519994.1). Subsequently, this gene was successfully expressed in Escherichia coli BL21 (DE3) and the culture supernatant of recombinant E. coli showed fibrinolytic activity. Electrophoresis pure alkaline serine protease and neutral protease also showed fibrinolytic activity.According to results above, types and amounts of fibrinolytic enzyme involved in the improvement of fermentation by glucose at 72 h.