Production and distribution of endoglucanase, cellobiohydrolase, and beta-glucosidase components of the cellulolytic system of Volvariella volvacea, the edible straw mushroom

The edible straw mushroom, Volvariella volvacea, produces a multicomponent enzyme system consisting of endo-1,4-beta-glucanase, cellobiohydrolase, and beta-glucosidase for the conversion of cellulose to glucose. The highest l evels of endoglucanase and cellobiohydrolase were recorded in cultures cont aining microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulos e, cotton wool, xylitol, or salicin, Biochemical analyses of different cult ure fractions in cultures exhibiting peak enzyme production revealed that m ost of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining afte r centrifugation of homogenized mycelia (32.6%), Cellobiohydrolase exhibite d a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversel y, most beta-glucosidase activity (63.9% of the total) was present in extra cts of fungal mycelia whereas only 9.4% was detected in culture filtrates, The endoglucanase and beta-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling, Endog lucanase was shown to be largely cell wall associated or located extracellu larly, with the highest concentrations being present in a region 1 to 2 mu m wide immediately adjacent to the outer surface of (and possibly including ) the hyphal wall and extending 60 to 70 mu m from the hyphal tip. Immunofl uorescence patterns indicated little if any intracellular endoglucanase. Mo st beta-glucosidase was located intracellularly in the epical area extendin g 60 to 70 mu m below the hyphal tip, although enzyme was also evident in t he extracellular region extending approximately 15 mu m all around the hyph al tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular beta-glucosidase, and the enzyme appears to be associated al most exclusively with, or located on the outside surface of, the hyphal wal l.