Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from <Emphasis Type="Italic">Staphylococcus</Emphasis> sp. strain AJ isolated from Korean salt-fermented Anchovy-<Emphasis Type="Italic">joet</Emphasis> |
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Authors: | Nack-Shick Choi Jae Jun Song Dong-Min Chung Young Jae Kim Pil Jae Maeng Seung-Ho Kim |
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Institution: | (1) Enzyme Based Fusion Technology Research Team, Jeonbuk Branch Institute, KRIBB, Jeonbuk, 580-185, South Korea;(2) Systemic Proteomics Research Center, KRIBB, Daejon, 305-333, South Korea;(3) Department of Microbiology, Changwon National University, Sarim-dong, Changwon, Kyungnam, South Korea;(4) Department of Microbiology, Chungnam National University, Daejon, 305-764, South Korea |
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Abstract: | 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°C, respectively.
AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K
m) and K
cat values of AJ towards α-casein were 0.38 mM and 19.73 s−1, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-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°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. |
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Keywords: | Fibrinolytic enzyme Staphylococcus sp strain AJ Anchovy-jeot Thermoacid-stable Fibrin zymography |
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