A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris.
In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and 37 degrees , respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin alpha-chain followed by the gamma-gamma chains. It also hydrolyzed the beta-chain, but more slowly. The Aalpha, Bbeta, and gamma chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the additions of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it 's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.
Kim
Jae-Sung
JS
Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University, Gwangju 501-759, Republic of Korea.
Sapkota
Kumar
K
Park
Se-Eun
SE
Choi
Bong-Suk
BS
Kim
Seung
S
Nguyen
Thi Hiep
TH
Kim
Chun-Sung
CS
Choi
Han-Seok
HS
Kim
Myung-Kon
MK
Chun
Hong-Sung
HS
Park
Yeal
Y
Kim
Sung-Jun
SJ
D000595:Amino Acid Sequence; D002918:Chymotrypsin; D032661:Cordyceps / Q000201:enzymology* / Q000254:growth & development; D003470:Culture Media; D005337:Fibrin / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D048690:Fruiting Bodies, Fungal / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D003470:Culture Media; D005343:Fibrinolytic Agents; D005337:Fibrin; D012697:Serine Endopeptidases; D002918:Chymotrypsin