Production & purification of a fibrinolytic enzyme (thrombinase) from Bacillus sphaericus

Treatment of thromboembolic vascular disease has relied on anticoagulants. However, recognition that lysis of preformed fibrin could be accomplished in vivo by a process involving the conversion of inactive plasminogen to active plasmin enzyme led to an alternative enzyme-based approach. The drugs used for this therapy are called the fibrinolytic enzymes. In this study we attempted the production, purification and characterization of fibrinolytic enzyme from Bacillus sphaericus.

The seed was prepared in nutrient yeast salt medium (NYSM) in shake flask and organism was produced in 100 l pilot fermentor. Biomass was separated by centrifugation and crude protein was prepared by ammonium sulphate precipitation. Purification was done by ion exchange chromatography using Q sepharose followed by gel filtration chromatography using Sephacryl S- 300. Molecular weight was determined through HPLC. Fibrinolytic activity was assayed by fibrin plate method.

The production method yielded 64 mg/l of the crude enzyme and after purification it was 6.3 mg/l. The molecular weight of the compound was 18.6 kDa.

The enzyme exhibited similar fibrinolytic activity as that of streptokinase, on fibrin plates that were devoid of plasminogen, suggesting that its fibrinolytic action is independent of plasminogen and it is not a plasminogen activator.