Related Experiment Video
Updated: Nov 29, 2025
Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus
Published on: June 2, 2023
Biosynthesis and Characterization of a Novel Fibrinolytic Alkaline Serine Protease from Newly Isolated Bacillus
T Sterlin Raj1, S Athimoolam1, P Vijayaraghavan2
1Department of Physics, University College of Engineering, Anna University Constituent College, Nagercoil, Konnam 629 004, Tamilnadu, India.
Insights
A novel bacterial fibrinolytic enzyme from Bacillus flexus BF12 effectively degrades blood clots. This safe, enzyme-based approach shows potential for preventing cardiovascular diseases (CVDs) and in food applications.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Cardiovascular diseases (CVDs) are leading causes of death globally.
- Current thrombolytic agents for CVDs have side effects.
- Bacterial fibrinolytic enzymes offer a safer alternative with direct clot-lytic activity.
Purpose of the Study:
- Isolate and characterize a novel fibrinolytic enzyme from Bacillus flexus BF12.
- Optimize enzyme production through physical and nutritional factor manipulation.
- Evaluate the enzyme's potential in food and nutraceutical applications.
Main Methods:
- Isolation of Bacillus flexus BF12 from a solar saltpan.
- Optimization of physical (pH, salinity) and nutritional (carbon, nitrogen, ions) factors for enzyme production.
- Purification of fibrinolytic protease using ammonium sulphate precipitation, dialysis, and gel filtration chromatography.
- Characterization of enzyme properties including molecular weight, temperature/pH stability, and solvent tolerance.
Main Results:
- A novel fibrinolytic enzyme was successfully isolated from Bacillus flexus BF12.
- Enzyme production was significantly enhanced (3.2-fold) using optimized conditions and statistical methods (response surface methodology).
- The purified enzyme has a molecular weight of 23 kDa, broad temperature (40-60 °C) and pH (7.0-9.0) stability, and retains activity in various organic solvents.
- The enzyme completely degraded human blood clots and showed enhanced activity with Ca2+ and Co2+ ions.
Conclusions:
- The characterized Bacillus flexus BF12 fibrinolytic enzyme demonstrates potent clot-degrading capabilities.
- Optimized production and purification yielded a stable and active fibrinolytic protease.
- This enzyme holds significant promise for nutraceuticals, food fortification, and the prevention of cardiovascular diseases.
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.
