Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant of Flammulina velutipes Mycelia
2007; Oxford University Press; Volume: 71; Issue: 9 Linguagem: Inglês
10.1271/bbb.70193
ISSN1347-6947
AutoresSe‐Eun Park, Ho‐Keung Ng, Jae‐Sung Kim, Kumar Sapkota, Ji‐Eun Kim, Bong-Suk Choi, Yeon-Hee Yoon, Jin-Cheol Lee, Hyun‐Hwa Lee, Chun‐Sung Kim, Sung-Jun Kim,
Tópico(s)Enzyme-mediated dye degradation
ResumoIn this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu2+, Fe2+ and Fe3+ ions, but was found to be enhanced by Mn2+ and Mg2+ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.
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