Fibrinolytic enzymes production by a filamentous fungus isolated from Las Yungas rainforest

ROVATI, JI; FIGUEROA LIC; SIÑERIZ F; FARIÑA JI

Denver

Congreso; Annual Meeting and Exhibition 2007 of the Society for Industrial Microbiology (SIM); 2007

SIM

Fibrin accumulation in blood vessels increases thrombosis risks promoting myocardial infarction and cardiovascular diseases. Typical thrombolytic agents for therapeutic purposes are tissue plasminogen activators (tPAs) that induce the conversion of plasminogen to plasmin, and the latter lyses fibrin clot. Fibrinolytic enzymes occur in bacteria, earthworms and snake venoms, but up to now, little is known about their production by fungi. A wild fungus isolated from Las Yungas, selected because of its outstanding fibrinolytic activity in previous assays, was grown under submerged culture conditions at shake flask scale. Culture supernatant was saturated with ammonium sulfate (80%). Precipitate was resuspended in HEPES buffer pH 8.0 and dialyzed. Fibrinolytic activity was checked by the fibrin plate method with plasmin as positive control. Protein was measured according to the Folin-Lowry method. Effect of metals and proteases inhibitors was also tested. Fibrinolytic enzymes could be recovered from culture supernatant. Precipitation and dialysis gradually increased enzyme specific activity. Final dialysate showed higher activity than plasmin. Fibrinolytic activity was significantly inhibited by PMSF, suggesting a serine protease. Activity was not affected by other inhibitors of proteases and metal ions such as Cu2+, Ca2+, Mg2+, Zn2+, Hg2+ and Fe2+ exerted no significant inhibition. These results confirmed the possibility to produce a fibrinolytic enzyme by submerged cultures of a selected fungal isolate. Furthermore, preliminary assays for its recovery and purification led to successful results. That opens new frontiers for the microbial production at fermenter scale in order to offer an alternative high added-value pharmacological product.