Studies On Fibrinolytic Enzyme Production By A Bacillus Subtilis

Fibrinolytic enzyme was a kind of enzymes degrading fibrins in blood. Enzymes from biological source was very important thrombolysis medicines with low molecular mass, non-immunogenicity, high enzyme activity, non-causing bleeding, long half life and being absorbed easily. It was one of the most potential thrombolytic agents which were safe and cheap. To start with, we screened for mutants with high yield of fibrinolytic enzyme and the main researches were as followings:Strain B2 (fibrinolytic enzyme activity 470IU/mL) was preserved in our laboratory and mutated by ultraviolet and diethyl sulfate. A mutant strain SFF-B2 was selected finally by preliminary screening and secondary screening with enzyme activity of 790IU/mL, which was 1.68 amount of the initial. It was identificated as Bacillus subtilis by analysis of colony morphology,cell morphology,physiology and biochemistry quality and 16S rDNA(GenBank accesstion number: FJ612600).The culture medium and the fermentation conditions of fibrinolytic enzyme production by mutant strain SFF-B2 were optimized. The optimal culture medium for enzyme production were as follows(g/L): amidulin 60, soybean flour 18, corn steep liquor 2, CaCl2 0.5,MgSO4 0.5,K2HPO4 2,NaCl 0.4. The optimal fermentation conditions for fibrinolytic enzyme production were as follows: seed age 14h, the quantity of inoculation 5%, initial pH7.0, volume in shake flask 30mL/250mL, temperature 37℃, rotation speed of rocking bed 180r/min, the enzyme activity of mutant strain SFF-B2 was 1218IU/mL with the optimal culture medium and optimal fermentation conditions, with enzyme activity enhanced by 54%.After separation purification of fibrinolytic enzyme from broth was by ammonium sulfate precipitation,bag filter desalting,Sephadex G-25 gel filtration chromatography,DEAE Sepharose Fast Flow ion exchange chromatography,Sephadex G-75 gel filtration chromatography, The activity recovery and purification factor of the fibrinolytic enzyme were 48% and 28; the pure enzyme was single strap verification by SDS-PAGE identification and the relative molecular weight was 29.8 kDa.The fibrinolytic enzyme is stable under common temperature, and would be devitalized above 50℃; Its maximum activity was at 40℃, stable in KH2PO4-NaOH and phosphate buffer. The enzyme activity was strongly inhibited by Cu2+,Ag+,Al3+ (the correlation between them was positively significant), and advanced by Ca2+,Mg2+. To sum up, fibrinolytic enzyme produced by mutant strain SFF-B2 indicated stability of temperature and pH and potential to carry out research further.