Screening of Endophytic Fungi with Antithrombotic Activity and Identification of a Bioactive Metabolite from the Endophytic Fungal Strain CPCC 480097

Abstract

Fermentation extracts from 1,075 endophytic fungi were screened by the Markwardt method. The endophytic strain CPCC 480097 had the strongest antithrombotic activity and was identified as Fusarium sp. based on morphologic tests and internal transcriptional spacer sequence analysis. The target of the antithrombotic agent from the endophytic strain CPCC 480097 was identified by analysis of the fibrinogen clotting time, amidolytic activity, and fibrinolytic assay. The results showed that this antithrombotic agent is a 28-kDa single-chain fibrinolytic enzyme. The identification of this fibrinolytic enzyme was performed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The two internal sequences of this enzyme were obtained, and these showed no homology with those of other known fibrinolytic enzymes.

Xylarinase: a novel clot busting enzyme from an endophytic fungusXylaria curta

“…However, these agents suffer from major drawbacks like short half-life, internal bleeding and cost effectiveness, therefore studies are now oriented towards safe and site specific thrombolytic strategies 26,36 . Fibrinolytic agents that can directly act on fibrin represent a viable alternative to the present arsenal of thrombolytic drugs.…”

“…Physiological saline was used as negative control whereas plasmin served as positive control. After 1 h, the residual thrombus was weighed and the dissolution ratio of the thrombus was calculated 12,36 .…”

“…Fibrinogen clotting time assay was performed according to the method described by Wu et al 36 with minor modifications. First, xylarinase (1 mg/ml) was incubated with 50 ml of 1% fibrinogen (20 mM Tris-HCl, pH 7.8) for 5 min at 37 C. Subsequently 30 ml of 5 U/ml of thrombin was added and clotting time was determined (a).…”

Untitled

“…Another endophytic Fusarium sp. CPCC 480097 was found to produce an antithrombotic substance, 28 kDa single-chain fibrinolytic enzymes, which could inhibit the transformation of fibrinogen to fibrin 97 .…”

Enhancement of the Thermostability of a Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51

“…The dissolution ratio of thrombus was calculated using the following equation: the dissolution ratio of thrombus (%)=(W0-W1)/W0×100, where W0 was the thrombotic weight formed in an eppendorf tube and W1 was the residual thrombotic weight [24].…”