Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004.
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.
The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, Jiangsu Normal University, Xuzhou, 221116, China.
Xiuyun
X
Ju
Xiaoying
X
Cao
Yong
Y
Sun
Zhe
Z
Wang
Chengliang
C
Cao
Jinjuan
J
Liu
Jihong
J
Jiang
D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D005343:Fibrinolytic Agents; D005656:Fungal Proteins; D005340:Fibrinogen