Purification and characterization of a novel anticoagulant and fibrinolytic enzyme produced by endophytic bacterium Paenibacillus polymyxa EJS-3.
BACKGROUND
Endophytes may become a new source of thrombolytic agents for thrombosis treatment.


METHODS
A novel fibrinolytic enzyme from Paenibacillus polymyxa EJS-3 (PPFE-I) was purified with ammonium sulfate precipitation, hydrophobic chromatography, ion exchange and gel filtration chromatography. The characterization of the enzyme was investigated by means of fibrinolysis plate, hydrolysis of fibrinogen and anticoagulant effect in vitro.


RESULTS
The fibrinolytic enzyme is purified to homogeneity with a purification of 14.5 fold and a recovery of 3.3%. The enzyme was shown to have a molecular mass of 63.3kDa by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The optimum temperature and pH value were 37°C and 7.5, respectively. Results from the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chains. It also hydrolyzed the γ-chains, but more slowly. It was activated by metal ions such as Zn(2+), Mg(2+), and Fe(2+), but inhibited by Ca(2+) and Cu(2+). Furthermore, PPFE-I activity was inhibited strongly by PMSF, and it was found to exhibit a higher specificity for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA for chymotrypsin, indicating that the enzyme is a chymotrypsin-like serine protease. Additionly, PPFE-I showed a significant anticoagulant effect in vitro.


CONCLUSIONS
The fibrinolytic enzyme PPFE-I from endophytic bacterium Paenibacillu polymyxa EJS-3 exhibits a profound fibrinolytic activity.
Lu
Fengxia
F
Lu
Zhaoxin
Z
Bie
Xiaomei
X
Yao
Zhengying
Z
Wang
Yufeng
Y
Lu
Yaping
Y
Guo
Yao
Y
D000925:Anticoagulants / Q000302:isolation & purification / Q000494:pharmacology; D004795:Enzyme Stability; D005337:Fibrin / Q000378:metabolism; D050299:Fibrin Modulating Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D056507:Paenibacillus / Q000737:chemistry / Q000201:enzymology*; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000494:pharmacology
D000925:Anticoagulants; D050299:Fibrin Modulating Agents; D005343:Fibrinolytic Agents; D011480:Protease Inhibitors; D005337:Fibrin