Thermostable fibrinolytic enzymes from Bacillus subtilis and Bacillus thuringiensis: Optimized production, partial...

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 h 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, suggestive of serine metalloproteases classification. Amidolytic assays demonstrated high activity with a chymotrypsin substrate, while hemolytic activity remained below 5%, indicating the possibility of initial therapeutic safety. SDS-PAGE revealed molecular masses of 35 kDa (B. subtilis) and 33 kDa (B. thuringiensis). Kinetic analysis showed K_m values of 3.488 mg/mL and 3.125 mg/mL with V_max values of 232.56 U/mL and 178.57 U/mL, respectively. These findings suggest that fibrinolytic enzymes from both strains exhibit promising activity and stability profiles and provide a basis for future studies to determine their potential role in thrombolytic drug development..