Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS.

A serine protease with preference for fibrin protein was purified and characterized from Bacillus
amyloliquefaciens MCC2606, isolated from dosa batter. The protease was purified using ammonium
sulfate precipitation, ion-exchange, and gel filtration chromatography. The degradation activity of the
protease toward the fibrin was significantly higher compared with other protein substrates in the study.
The molecular weight of the CFR15-protease was estimated to be 32 kDa based on SDS-PAGE. The
purified enzyme exhibited both fibrinolytic and fibrinogenolytic activity. The optimum pH and
temperature for the activity of the enzyme was found to be 10.5 and 45°C. A significant inhibition was
seen with the protease inhibitors phenyl methyl sulphonyl fluoride and ethylene diamine tetra acetic acid
and the activity of the enzyme was enhanced in presence of Mn2+. There was an observed increase in
vitro activated partial thromboplastin time and prothrombin time of both time and dose dependent
study. Among the four chains of fibrin, the β-chain of fibrin appears to be the primary component and
site susceptible for CFR15-protease in early action as indicated by MS/MS analysis of initial degradation
products. These results indicated that the CFR15-protease have the potential to be an effective
fibrinolytic agent.