A Fibrinolytic Enzyme from the Medicinal Mushroom Cordyceps militaris

  • Kim Jae-Sung (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Sapkota Kumar (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Park Se-Eun (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Choi Bong-Suk (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Kim Seung (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Hiep Nguyen Thi (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Kim Chun-Sung (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Choi Han-Seok (Department of Food Science and Technology, Chonbuk National University) ;
  • Kim Myung-Kon (Department of Industrial Crop Production and Processing, Iksan National college) ;
  • Chun Hong-Sung (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Park Yeal (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University) ;
  • Kim Sung-Jun (Department of Biotechnology, BK 21Research Team for Protein Activity Control, Chosun University)
  • Published : 2006.12.31


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 $37^{\circ}C$, 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 A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ 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.



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