Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet.
A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85 degrees C, respectively. AJ kept 85% of the initial activity after heating at 100 degrees C for 20 min on the zymogram gel. The Michaelis constant (K (m)) and K (cat) values of AJ towards alpha-casein were 0.38 mM and 19.73 s(-1), respectively. AJ cleaved the A alpha-chain of fibrinogen but did not affect the B beta- and gamma-chains, indicating that it is an alpha-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100 degrees C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.
Choi
Nack-Shick
NS
Enzyme Based Fusion Technology Research Team, Jeonbuk Branch Institute, KRIBB, Jeonbuk, 580-185, South Korea.
Song
Jae Jun
JJ
Chung
Dong-Min
DM
Kim
Young Jae
YJ
Maeng
Pil Jae
PJ
Kim
Seung-Ho
SH
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005285:Fermentation; D005337:Fibrin / Q000378:metabolism*; D005342:Fibrinolysis; D005399:Fishes / Q000382:microbiology*; D005516:Food Microbiology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007723:Korea; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D012965:Sodium Chloride; D005337:Fibrin