Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia.

In this study we purified and characterized a fibrinolytic protease from the mycelia of Perenniporia fraxinea. The apparent molecular mass of the purified enzyme was estimated to be 42kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin zymography and size exclusion using fast protein liquid chromatography (FPLC). The first 20 amino acid residues of the N-terminal sequence were ASYRVLPITKELLPPEFFVA, which shows a high degree of similarity with a fungalysin metallopeptidase from Coprinopsis cinerea. The optimal reaction pH value and temperature were pH 6.0 and 35-40 degrees C, respectively. Results for the fibrinolysis pattern showed that the protease rapidly hydrolyzed the fibrin alpha-chain followed by the beta-chain. The gamma-gamma chains were also hydrolyzed, but more slowly. The purified protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chains of fibrinogen, followed by Bbeta- and gamma-chains. We found that protease activity was inhibited by Cu(2+), Fe(3+), and Zn(2+), but enhanced by the additions of Mn(2+), Mg(2+) and Ca(2+) metal ions. Furthermore, the protease activity was inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The mycelia of P. fraxinea may thus represent a source of new therapeutic agents to treat thrombosis.