Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004

Abstract

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.

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

“…The current armamentarium of thrombolytic drugs comprises of PAs or t-PA which are considered as one of the most effective agents in thrombolytic therapy 9,35 . 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 .…”

“…Briefly, 250 ml of 1% fibrinogen in 20 mM Tris-HCl buffer (pH 7.8) was incubated with 50 ml (1 mg/ml) of purified enzyme at 37 C. At different time intervals, 20 ml fraction was withdrawn and analysed by SDS-PAGE-commassie staining 9,35 .…”

“…The pH stability was DOI: 10.3109/14756366.2016.1151013 determined by incubating the purified enzyme in the respective buffer for overnight at 4 C 26,35 . The optimal temperature for xylarinase activity was determined by measuring the residual activity after incubating the enzyme (1 mg/ml, 10 ml) with 90 ml of 20 mM Tris-HCl buffer at different temperatures from 25 to 70 C for 1 h. The residual fibrinolytic activity was assessed by fibrin plate method 26,35 .…”

“…Effect of metal ions on xylarinase activity was evaluated using C. The effect of metal ions on xylarinase activity was then analysed by fibrin plate assays as described earlier and was expressed as relative fibrinolytic activity 25,35 .…”

“…The residual fibrinolytic activity was analysed by fibrin plate assay and expressed in terms of relative fibrinolytic activity 25,35 .…”