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 CaCl2 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 Ca2+ and Co2+ 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.