Examination of thrombolytic and anticoagulant activities of purified fibrinolytic enzyme extracted from Cochliobolus hawaiiensis under solid state fermentation

Abstract

Cochliobolus hawaiiensis Alcorn AUMC 8606 was chosen from the screened twenty fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, Upon optimization of fibrinolytic enzyme production under solid state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugar cane bagasse. The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20 fold. The purification procedures of fibrinolytic enzyme caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35kDa. Maximal activity was recorded at pH 7 and 37oC. The enzyme showed the highest affinity towards Fibrin, with Vmax of 240 U/ml and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, while Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non metalloprotease due to the great suppression in its activity by using phenylmethylsulfonyl fluoride and thylenediamine-tetraacetat, respectively. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplaetlet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders