A novel source of fibrinolytic activity: Bionectria sp., an unconventional enzyme-producing fungus isolated from Las Yungas rainforest (Tucumán, Argentina)

Abstract

Fibrinolytic enzyme production was evaluated in fungal specimens isolated from the sub-tropical Las Yungas Pedemontana forest (Tucuman, Argentina). Proteolytic and fibrinolytic activities were evaluated in freeze-thaw crude extracts from 230 fungal isolates on 1% w/v skimmed-milk or 0.25% w/v fibrin-agar plates, respectively. Proteolytic activity was positive in 62% of the isolates, whilst only three of them were able to produce extracellular fibrinolytic enzymes on solid nutritive medium. Fibrinolytic-positive extracts were able to degrade fibrin clots in a direct plasminogen-independent way. Selected isolates were identified by sequencing the 26S rDNA D1/D2 domain. Isolates LY 4.1 and LY 4.4 showed a 99.9% similarity with Bionectria ochroleuca, while LY 4.2 showed a 99.9% identity with Cladosporium cladosporioides. Under submerged culture conditions, LY 4.1 and LY 4.4 were able to excrete fibrinolytic enzymes, reaching a maximum at 120 h of cultivation of 100.2 and 107.9 U/ml in plasmin-equivalent units, respectively. Fibrinolytic enzyme production could be scaled-up to fermenter scale reaching similar values. Fibrin zymography showed that fibrinolytic activity was associated with ~173-, 153- and 80-kDa protein fractions. Extracellular fibrinolytic enzymes from Bionectria species may be potentially related to pathogenesis mechanisms, as already demonstrated for serine-proteases from the nematicidal anamorph Clonostachys rosea. This work reveals the potential of Bionectria strains as an unconventional and unexplored production alternative to already known thrombolytic agents. The value of Las Yungas forests as a reservoir of fungal species with promising biotechnological value could be also highlighted.