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.
Mycorrhizae involving Boletus edulis and Pinus massoniana have been successfully established and their morphological characteristics described.Optimal conditions for mycorrhizal synthesis were determined using orthogonal testing of three parameters:substrate,age of host plant and inoculation procedure.Following integration analysis of plant survival rates,diameter of seedling rootstock,height of seedling shoot,number of mycorrhizae,ratio of shoot/root biomass,number of mycorrhizae,and the ratio of infected/non-infected root tips,we concluded that a double-layered growth substrate that simulated natural soil structure was superior for mycorrhizal synthesis compared with the more commonly used single-layered peat moss and vermiculite substrate,especially when sepiolite powder was also added.
FU Shaochun TAN Qi CHEN Mingjie SHANG Xiaodong CAI Lingyi ZHANG Meiyan
Bioactive proteins represent an important group of functional agents in medicinal mushrooms.Trametes versicolor(L.) Lloyd is a mushroom frequently used in traditional Chinese medicine for its anti-tumor and immunomodulatory activities.A new immunomodulatory protein from T.versicolor,named TVC,was purified by ammonium sulfate precipitation,ion-exchange chromatography and gel filtration chromatography.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed a single band with a molecular weight of 15.0 kD.Native polyacrylamide gel analysis revealed a band at 30 kD,indicating that TVC exists in solution as a homodimer.Isoelectric focusing showed that TVC was an acidic protein with an isoelectric point of 4.0.TVC was found to lack carbohydrate modifications(based on periodic acid/Schiff staining) and it does not agglutinate mouse red blood cells,suggesting that TVC is not a lectin-like protein.Biological activity assays dem-onstrated that TVC can enhance the proliferation of splenocytes,while it has no stimulatory effects on CD4+and CD8+T cells. TVC markedly increases the proliferation of human peripheral blood lymphocytes in a dose-dependent manner and enhances the production of both nitric oxide and tumor necrosis factor-alpha by lipopolysaccharide-induced murine macrophages.The results indicate that TVC is an immunostimulant that can boost immune response.Comparison of the N-terminal amino acid residues and mass spectrometry results with the protein database revealed no homologous proteins.
