Purification, Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae, an effective medicinal fungus widely used in anthelmintic therapy. The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%. The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum reaction pH and temperature are 7.5 and 50°C, respectively. The protease activity is largely enhanced by Ca2+, but highly inhibited by tetrasodium ethylenediaminetetraacetate (EDTA), a metal-chelator, suggesting that the enzyme is a metalloprotease. The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml−1 and 21.32 μg·min−1·ml−1, respectively. In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae (L3) of Ascaris suum. Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L. mylittae.