Studies On The Enzyme Production Of Ganoderma Lucidum LYL263 By Mixed Cultivation And Its Mechanism Of Action And Applications

Laccases are multicopper phenoloxidases. Laccasea have potential uses in dye decolorization, degradation of pulping wastewater and bio-pulping. Cellulase and xylanase have been utilized in waste paper deinking and pulp bleaching. Microbial mix-cultivation can be used to increase production of enzyme for the synergistic advantage of different strains. A lot of achievements have been achieved in this field.In order to screen the suitable strains of mix-culture, the mutagenic strain Ganoderma Lucidum LYL263 was cultured in liquid medium with Pleurotaus ostreat, Flammulina velutipes and Pleurotus eryngii, respectively. The results showed that Pleurotaus ostreat was the best compatible strain in the mix-cultural strains with Ganoderma Lucidum LYL263. The maximum activity of laccase reached 1346.4 U/mL by the co-cultivation of Ganoderma Lucidum LYL263 and Pleurotaus ostrea. Compared to the pure culture of Ganoderma Lucidum LYL263, the lacccase production of the co-cultivation of Ganoderma Lucidum LYL263 and Pleurotaus ostrea raised 35.7%. In contrast, cellulase and xylanase production in the mixed culture were lower than that of pure culture with Ganoderma Lucidum LYL263. The inoculation proportion of mixed cultivation of Ganoderma Lucidum LYL263 and Pleurotaus ostreat was optimized and the overall activity of laccase reached the maximum when the inoculation proportion was 4:2. The synchronous inoculation order in the co-cultivation of Ganoderma Lucidum LYL263 and Pleurotaus ostrea was optimal.The optimization of fermentation medium for the co-cultivation of Ganoderma Lucidum LYL263 and Pleurotaus ostrea was investigated. The optimum medium composition was as follows:glucose 25g/L, peptone 1.5g/L, Tween-801.0?L/L and rice straw 0.1 g/L. With this optimal medium, the maximum activities of laccase, cellulase and xylanase reached 2743.8U/mL,92,3U/mL and 55.1U/mL, respectively.Culture temperature and pH had no effects on enzyme properties whether to the pure culture or to the co-cultivation. The optimal temperature and pH for laccase activity were 50? and pH 4.5. In the acidic environment and the temperature below 30?, the laccase showed high stability. The optimal temperature and pH for cellulase and xylanase activity were 50? and pH6.0-7.2, respectively. In the neutral or weak acidity environment and the temperature below 30?, the cellulase and xylanase showed better stability.On the plate cultivation of Ganoderma Lucidum LYL263 with Pleurotaus ostrea, their hyphae interweaved each other on their border and secreted yellow pigment after a few days growth. The fermentation liquor of Pleurotaus ostrea filtered by 0.45?m membrane before use could stimulate Ganoderma Lucidum LYL263 to produce more laccasees. The staining results of Native-PAGE showed that the types of laccase isozymes had no change in the co-culture, but the concentration of laccase isozymes increased.The decolorization of methyl orange, reactive brilliant blue, malachite green, bromphenol blue and crystal violet by crude enzyme from co-cultivation of Ganoderma Lucidum LYL263 and Pleurotaus ostrea was also studied. The optimal conditions of methyl orange decolorization were as follows:pH 4.0, temperature 40?, laccase activity 100U/mL. Under these conditions, the decolorization rate of methyl orange reached about 73.2% in 4 hours. The optimal decolorization conditions of reactive brilliant blue were as follows:pH 4.0, temperature 40?, laccase activity 100U/mL. The decolorization rate of reactive brilliant blue reached about 96.6% in 4 hours under these conditions. The optimal decolorization conditions of malachite green were as follows:pH 4.0, temperature 30?, laccase activity 100U/mL. The decolorization rate of malachite green reached about 96.6% in 4 hours accordingly. The optimal decolorization conditions of bromphenol blue were as follows:pH 3.0, temperature 40?, laccase activity 100U/mL. The decolorization rate of bromphenol blue reached about 77.5% in 4 hours accordingly. The optimal decolorization conditions of crystal violet were as follows:pH 4.0, temperature 30?, laccase activity 75U/mL. The decolorization rate of crystal violet reached about 88.5% in 7 hours accordingly.The degradation of pulping wastewater by crude enzyme from co-cultivation and pure cultivation of Ganoderma Lucidum LYL263 was studied, respectively. COD and chroma removal rates of pulping wastewater in co-cultivation were higher than that of pure cultivation. The optimal conditions for chroma degradation were as follows:pH 5.0, temperature 40?, laccase activity 100U/mL. Under these conditions, the removal rate of chroma reached about 47.6%. The optimal conditions of COD degradation were as follows:pH 5.0, temperature 50?, laccase activity 125U/mL. Under these conditions, the removal rate of COD reached about 63.8%.