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A Novel Fibrinolytic Enzyme from Bacillus Sphaericus MTCC 3672: Optimization and Purification Studies

Devchand N. Avhad, Swapnil S. Vanjari, virendra Rathod


A novel extracellular fibrinolytic enzyme was produced from Bacillus sphaericus MTCC 3672 for dissolving blood clots. Optimized fermentation parameters achieved by one factor at a time approach had demonstrated 2.85 fold increase in fibrinolytic activity i.e. from 3.5*104 U/l (basal media) to 9.98*104 U/l after 24 h of incubation in submerged fermentation. Statistical screening of six independent nutritional variables such as, glucose, yeast extract, NaCl, MgCl2, MnCl2, CaCl2 was studied using Plackett-Burman design. Amongst six variables, yeast extract was found to be significant factor affecting yield of a fibrinolytic enzyme. Furthermore, growth kinetics of biomass formation, enzyme production, and substrate utilization was evaluated by unstructured kinetic models and various biokinetic parameters such as µmax (0.37 1/h), Pr (0.12 1/h), YP/S (7.74 U/g) and YX/S (3.32 g/g) were determined. In purification step, ultrafiltered broth was purified with DEAE Cellulose anion exchange chromatography and Sephadex G100 gel filtration chromatography with 10 fold purity. In vitro fibrin clot degradation study had revealed significant breakdown of fibrin clot. The fibrinolytic activity of purified enzyme (7.5 mm) was found to be matching with marketed Nattolife® (8.0 mm) (nattokinase) and superior to Thromboflux® (0 mm) (streptokinase) formulations. Hence, efficient formulation containing purified fibrinolytic agent can be used for medical emergencies viz. myocardial infarction and deep vein thrombosis.


Fibrinolytic enzyme; Bacillus sphaericus; Plackett-Burman design; Kinetic Models; DEAE Cellulose

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