Studies on Fibrinolytic agent(s) from endophytic fungi

Abstract

The present study was oriented towards exploration of potential endophytic fungi isolated from conserved rain forests of India for their fibrinolytic activity. Of the 211 isolates of endophytic fungi screened, culture filtrates of 67 isolates exhibited in vitro proteolytic activity while only 37 isolates displayed in vitro fibrinolytic activity. However, the direct in vitro fibrinolytic activity was only found in culture filtrates of six isolates. The endophytic fungal isolate #37 CRSTBRT exhibited maximum in vitro proteolytic as well as in vitro fibrinolytic activity. Using morphological as well as molecular taxonomic studies, #37 CRSTBRT was identified as Xylaria curta. The fibrinolytic moiety expressed in the culture filtrate of Xylaria curta was isolated using chromatographic techniques. Further, its purity, molecular mass and enzymatic activity was ascertained by using electrophoretic techniques, HPLC and MALDI–ToF MS analysis. Xylarinase is a non–toxic, bi–functional monomeric fibrinolytic metalloprotease with a molecular mass of 33.76 kDa. The enzyme displayed both plasmin like and plasminogen activator like activity under in vitro conditions. It hydrolyses both Aα and Bβ chains of fibrin(ogen) displaying αβ fibrinogenase activity. Optimal fibrinolytic activity of xylarinase was observed at 35 °C, pH 8. EDTA and EGTA were the potent inhibitors of xylarinase suggesting that the enzyme was a metalloprotease having calcium dependence. However, Fe2+ and Zn2+ strongly inhibited the in vitro fibrinolytic activity of xylarinase. The Km and Vmax of xylarinase was 246 µM and 1.22 µM/min. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Further, xylarinase was found to prolong the APTT and PT. Xylarinase exhibited no cytotoxic effect over RAW 264.7 murine macrophage cell. The production yield of xylarinase was enhanced (8.5 % from 0.09 %) by producing it on rice chaff using solid state fermentation. The purified enzyme exhibited similar biochemical properties as that of xylarinase. The present study thus establishes endophytic fungi as a potential bio–resource of fibrinolytic agents. Xylarinase stands out as a potential candidate for further pre-clinical studies using animal models for development into a thrombolytic agent.