Purification And Characterization Of Antifungal Peptide From Isaria Felina And Antifungal Protein From Tricholoma Giganteum

Isaria felina was a contaminating fungus isolated from the substrate when cultivating edible mushroom Pholiota nameko. The fungus induced a broad inhibition zone when introduced to Rhizoctionia solani on potato dextrose agar medium. Through ethanol extraction, absorption on YPR-II macropore adsorption resin, ethyl acetate extraction, petroleum ether precipitation and recrystallization from ethyl acetate fine colourless crystal, isarfelin, was obtained from mycelia of I. felina. The inhibitory activity to R. solani was enhanced after each step purificated. Isarfelin was broad-spectrum, not only exhibited strong antifungal activity against several phytopathogens but also presented inhibition to Agrobacterium rhizogenes K.27 which caused crown gall of peach, whereas showed no inhibition to mycelia of several mushrooms. Isarfelin exhibited cytotoxicity to MBL, L1210 and hepG2 cell lines and inhibited HIV-1 reverse transcriptase. The inhibitory activity functioned stably on pH 4-9, and it was heat and light stable. Isarfelin was a mixture of isarfelin A and isarfelin B. The molecular weight of isarfelin A was 669.4178 with the molecular formula C₃₆H₅₅O₇N₅; the molecular weight of isarfelin B was 655.4028 with the molecular formula C35H53O7N5. Isarfelin A made up 41.2% and isarfelin B made up 58.8%. Isarfelin A only had a CH₂ more than isarfelin B and the structure of both was very similar. The scan electron microscopy showed isarfelin caused the abnormity of mycelia of Sclerotinia sclemtiorum which including excessive branching and expanded hyphal tip. Whereas, isarfelin exhibited no effect on the permeability of cell membrane and respiration of Sclerotinia sclerotiorum.There have been some reports on the bioactivity compound from Tricholoma giganteum and the antifungal protein resource will be widened through the purification of antifungal protein from Tricholoma giganteum. An antifungal protein, trichogin, was isolated from the mushroom Tricholoma giganteum. The protocol included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and gel filtration by fast protein liquid chromatography on Superdex 75. It was a monomeric protein with the molecular mass of 27 kDa. The N-terminal sequence of trichogin, QVHWPMF, was not found in any reported antifungal protein, such as Alveolarin, Eryngin, and Lyophyllum antifungal protein which were previously isolated from mushroom. The behavior of trichogin on the various ion exchange and affinity chromatographic media bnemployed in the isolation procedure is similar to that reported for other antifungal proteins. It was unabsorbed on DEAE-cellulose but was absorbed on Affi-gel blue gel and CM-cellulose. Trichogin exhibited antifungal activity against phytopathogenic fungus Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. The IC50 of the antifungal activity of trichogin toward M. arachidicola was estimated to be 3.8 ± 0.28 uM, and furthermore, trichogin inhibited HIV-1 reverse transcriptase with an IC₅₀ of 83 ±2.7 nM. However, it was devoid of ribonuclease and hemaggluting activities.