Purification and characterization of a novel fibrinolytic protease from Fusarium sp. CPCC 480097.
A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45 degrees C and 8.5, respectively. Fu-P cleaved the alpha-chain of fibrin (ogen) with high efficiency, and the beta-chain and gamma-gamma (gamma-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S-S-G-T-P-A-T-I-R-V-L-V-V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.
Wu
Bin
B
Department of Biological Pharmacy, Shanghai Institute of Pharmaceutical Industry, 200040, Shanghai, China.
Wu
Licheng
L
Chen
Daijie
D
Yang
Zhijun
Z
Luo
Minyu
M
D000595:Amino Acid Sequence; D001709:Biotechnology; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology* / Q000254:growth & development; D006801:Humans; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D003470:Culture Media; D005656:Fungal Proteins; D005337:Fibrin; D045726:Metalloproteases; D012697:Serine Endopeptidases