Biosynthesis and Characterization of a Novel Fibrinolytic Alkaline Serine Protease from Newly Isolated Bacillus flexus BF12 for Biomedical Applications

Background: Cardiovascular Diseases (CVDs) such as stroke, high blood pressure, peripheral vascular disease, ischemic heart disease and acute myocardial infarction are some of the leading causes of death. To treat CVDs, commercially available thrombolytic agents are widely used. However, these thrombolytic agents have various side effects. Alternatively, fibrinolytic enzymes from bacterial sources are highly safe and have direct blood clot lytic activity. Methods: A fibrinolytic enzyme producing bacterial strain, Bacillus flexus BF12, was isolated from a solar saltpan in Kanyakumari District, Tamilnadu, India. Enzyme production was improved by optimizing physical factors and nutritional factors. Results: A novel fibrinolytic enzyme was isolated from a strain of the studied B. flexus BF12. Enzyme production was enhanced significantly by optimizing process parameters. The critical physical factors (pH and salinity) and influencing nutritional factors (carbon, nitrogen and ions) were optimized by one variable at a time approach, followed by the statistical method. The strain BF12 was highly active at alkaline pH (>7.0) and between 4 and 6% NaCl concentration. The nutrients such as fructose (carbon source), beef extract (nitrogen source) and CaCl 2 significantly influenced enzyme production. Central composite design and response surface methodology improved 3.2-fold enzyme yield than unoptimized culture medium. Fibrinolytic protease was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography. Discussion: The molecular weight of an enzyme was found to be 23 kDa. It was active at a broad temperature (40-60 °C) and pH (7.0-9.0) ranges. Enzyme activity was enhanced by Ca 2+ and Co 2+ ions. The purified protease retained 100% enzyme activity in the presence of ethanol and acetone. Acetonitrile, butanol, DMSO, methanol and chloroform showed enzyme activity of 63%, 92.5%, 94.7%, 92.3% and 90.4%, respectively. The purified enzyme degraded 100% of human blood clot. Conclusion: The Bacillus flexus BF12 fibrinolytic enzyme shows promising potentials in nutraceutical and food fortification applications. The application of fibrinolytic enzymes could prevent CVDs.