A fibrinolytic enzyme has been purified from the fruiting bodies of Korean Cordyceps militaris. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fibrin-zymography, and gel filtration chromatography. The 15 amino acid residues of the N-terminal sequence of the enzyme were APVEQCDAPVGLARL, which is dissimilar to those of fibrinolytic enzymes from other mushrooms. Optimal pH and temperature values of the enzyme were 7.0 and 40 degrees C, respectively. The enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), TPCK, 1,10-phenanthroline, Cu(2+), and Ba(2+). It was also significantly inhibited by aprotinin, EDTA, and EGTA. The enzyme showed a higher specificity for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, exhibiting that it is a chymotrypsin-like serine metalloprotease. The enzyme preferentially hydrolyzed the fibrinogen A alpha-, followed by the B beta-chains and the gamma-chain. The alpha, beta, and gamma-gamma chains of fibrin were also degraded by the enzyme. (C) 2010 Elsevier Ltd. All rights reserved.