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Fibrinolytic protease from Bacillus cereus S46: Purification, characterization, and evaluation of its in vitro
Desrie H D'Souza1, Sourav Bhattacharya1, Arijit Das1
1Department of Microbiology, School of Sciences, JAIN (Deemed-to-be University), Bangalore, Karnataka, India.
Abstract:
Intravascular thrombosis is a prime cause of cardiac complications worldwide. Microbial fibrinolytic proteases are of clinical significance in thrombosis treatment. The present study discusses the purification and characterization of a protease from Bacillus cereus S46, ascertaining its in vitro thrombolytic activity against a blood clot. By the three-step purification involving precipitation, dialysis, and diethylaminoethyl-cellulose ion-exchange chromatography, a 12.37-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the protease was 30 kDa, as found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was observed at pH 8.0 and 40°C. The enzyme retained an 82.19% residual activity at pH 8.0 and 40°C for 1 h. The Km and Vmax values of the protease with casein were 0.0027 mM and 9.712 µmol/min, respectively. In an in vitro assay, the purified protease resulted in 97.02% lysis of the blood clot. The fibrinolytic potential of the enzyme, together with its characteristics of being active and stable under near-physiological conditions, may suggest its application as a therapeutic agent.
Insights
A novel protease from Bacillus cereus demonstrated significant fibrinolytic activity, effectively dissolving blood clots. This microbial enzyme shows potential as a therapeutic agent for treating thrombosis due to its stability and efficacy.
Area of Science:
- Microbiology
- Biochemistry
- Biotechnology
Background:
- Intravascular thrombosis is a major global health concern, leading to severe cardiac complications.
- Microbial fibrinolytic proteases offer promising therapeutic avenues for thrombosis management.
Purpose of the Study:
- To purify and characterize a fibrinolytic protease from Bacillus cereus S46.
- To evaluate the in vitro thrombolytic activity and stability of the purified enzyme.
Main Methods:
- Protease purification using precipitation, dialysis, and ion-exchange chromatography.
- Enzyme characterization including molecular mass determination (SDS-PAGE), optimal activity conditions (pH, temperature), and kinetic analysis (Km, Vmax).
- In vitro thrombolytic assay to assess blood clot lysis efficacy.
Main Results:
- A protease was purified 12.37-fold to homogeneity with an apparent molecular mass of 30 kDa.
- Optimal enzyme activity was observed at pH 8.0 and 40°C, with high residual activity (82.19%) maintained for 1 hour.
- The purified protease achieved 97.02% lysis of blood clots in vitro, exhibiting Km of 0.0027 mM and Vmax of 9.712 µmol/min with casein.
Conclusions:
- The Bacillus cereus protease exhibits potent fibrinolytic activity and stability under near-physiological conditions.
- Its characteristics suggest potential application as a novel therapeutic agent for intravascular thrombosis treatment.
