Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2007-09, Vol.71 (9), p.2214-2222
• Main Authors: Park, S.E.(Chosun Univ., Gwangju (Korea R.)), Li, M.H, Kim, J.S, Sapkota, K, Kim, J.E, Choi, B.S, Yoon, Y.H, Lee, J.C, Lee, H.H, Kim, C.S, Kim, S.J
• Format: Article
• Language: English
• Subjects: Agaricales - enzymology; Amino Acid Sequence; Biological and medical sciences; BLOOD COAGULATION; CELL CULTURE; Chromatography, Gel; COAGULACION SANGUINEA; COAGULATION SANGUINE; CULTIVO DE CELULAS; CULTURE DE CELLULE; CULTURE MEDIA; Endopeptidases - chemistry; Endopeptidases - isolation & purification; Endopeptidases - metabolism; Enzyme Activation - drug effects; fibrinogenolysis; Fibrinolysis; FLAMMULINA VELUTIPES; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; MEDIO DE CULTIVO; metalloprotease; Metals - pharmacology; MICELIO; MILIEU DE CULTURE; Molecular Sequence Data; Molecular Weight; mycelia; MYCELIUM; PROTEASAS; PROTEASE; PROTEASES; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Temperature
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.70193

In 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 alpha-chain over beta-and gamma-gamma chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 deg C. The protease activity was inhibited by Cusup(2+), Fesup(2+) and Fesup(3+) ions, but was found to be enhanced by Mnsup(2+) and Mgsup(2+) 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 chymotrypsinlike 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.