Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia.
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 degrees 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.
Park
Se-Eun
SE
Department of Biotechnology and BK21 Research Team for Protein Activity Control Chosun University, Gwangju 501-579, Republic of Korea.
Li
Mei-Hong
MH
Kim
Jae-Sung
JS
Sapkota
Kumar
K
Kim
Ji-Eun
JE
Choi
Bong-Suk
BS
Yoon
Yeon-Hee
YH
Lee
Jin-Cheol
JC
Lee
Hyun-Hwa
HH
Kim
Chun-Sung
CS
Kim
Sung-Jun
SJ
D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005342:Fibrinolysis; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature