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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 50oC,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 -1and 21.32μg·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.
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 50oC, 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 substrates 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 that that the proteolysis of larvae protein s caused by this protease may relate to the anthelmintic activity of L.mylittae.