Thermostable Fibrinolytic Serine Metalloproteases from Bacillus subtilis and Bacillus thuringiensis: Optimized Production, Partial Purification, and Enzymatic Characterization of Thrombolytic Activity".
TL;DR
The findings suggest that fibrinolytic enzymes from both strains were potent, stable, and promising candidates for thrombolytic drug development.
Abstract
Fibrinolytic enzymes of microbial origin are gaining attention as safer alternatives to conventional thrombolytic drugs. In this study, fibrinolytic enzymes were isolated and characterized from Bacillus subtilis and Bacillus thuringiensis derived from soil samples. Process optimization revealed maximum enzyme production at 37 °C and pH 7.0 after 24 hrs of incubation, with inoculum sizes of 1.5% (B. subtilis) and 1% (B. thuringiensis) in media supplemented with glucose/sucrose and yeast extract. Partial purification yielded specific activities of 191.15 U/mg (B. subtilis) and 218.24 U/mg (B. thuringiensis) with recovery yields of 14.39% and 13.89%, respectively. Both enzymes exhibited stability over broad ranges of pH (5.0-9.0) and temperature (10-60 °C). Enzyme activity was enhanced by Mg2+ and Ca2+ but completely inhibited by EDTA and PMSF, confirming that the enzymes are serine metalloproteases. Amidolytic assays demonstrated high activity with a chymotrypsin substrate, while hemolytic activity remained below 5%, indicating therapeutic safety. SDS-PAGE revealed molecular masses of 35 kDa (B. subtilis) and 33 kDa (B. thuringiensis). Kinetic analysis showed Km values of 3.488 mg/mL and 3.125 mg/mL with Vmax values of 232.56 U/mL and 178.57 U/mL, respectively. These findings suggest that fibrinolytic enzymes from both strains were potent, stable, and promising candidates for thrombolytic drug development.