Production, extraction and characterization of a serine protease with fibrinolytic, fibrinogenolytic and thrombolytic activity obtained by Paenibacillus graminis

This study provides optimized parameters for producing and purifying a microbial serine protease by Paenibacillus graminis in submerged fermentation and the innovative use of endosperm of Gliricidia sepium as substrate. Biphasic aqueous systems were the method of choice for the partial purification of this protease. After extracting the enzyme, its biochemical characterization and fibrinolytic, fibrinogenolytic and thrombolytic activity were performed. The highest protease activity (16.25 U/mL) was obtained in the PEG-rich phase in the composing assay of MPEG (8000 g/mol), CPEG (12.5 g/mol), CPHO (10%) and pH 8.0 (optimum pH). Recoveries greater than 80% were obtained with a purification factor of 5.21, a partition coefficient of 1.13 and a yield of 145.52%. The fibrinolytic protease (50 kDa) purified achieved a 46.96% reduction of thrombus in vitro and managed to fully degrade the B beta-chain of human fibrinogen and the gamma-chain of bovine fibrinogen. The enzyme activity was enhanced by Cu2+, Zn2+, Na+, Ca2+ and K+. These results suggest that the protease purified by ATPS can potentially be used as a future agent thrombolytic.