Monday, July 30, 2007
P72
Fibrinolytic enzymes production by a filamentous fungus isolated from Las Yungas rainforest
J. I. Rovati1, L. I. Castellanos de Figueroa1, F. Siñeriz2, and J. I. Fariña1. (1) Fungal Biotechnology, PROIMI-CONICET, Av Belgrano y Pje Caseros, S.M. Tucumán, Argentina, (2) Environmental Microbiology, PROIMI-CONICET, Av Belgrano y Pje Caseros, S.M. Tucumán, Argentina
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, which 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 in 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 determined by the fibrin plate method with plasmin as positive control. Protein was measured according to 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 fibrinolytic enzymes by submerged cultures of the selected fungal isolate. Furthermore, preliminary assays made for its recovery and purification led to successful results. Data obtained in this work open new frontiers for microbial production at fermenter scale, in order to offer an alternative high added-value pharmacological product.
A wild fungus isolated from Las Yungas, selected because of its outstanding fibrinolytic activity in previous assays, was grown under submerged culture conditions in 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 determined by the fibrin plate method with plasmin as positive control. Protein was measured according to 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 fibrinolytic enzymes by submerged cultures of the selected fungal isolate. Furthermore, preliminary assays made for its recovery and purification led to successful results. Data obtained in this work open new frontiers for microbial production at fermenter scale, in order to offer an alternative high added-value pharmacological product.
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