Study On The Laccase From White-rot Fungus Pleurotus Ostreatus And Its Decolorization For Anthraquinone Dye And Alkali Lignin

In this dissertation, lignolytic enzyme producing strains are screened from a large number of white-rot fungi for improving enzyme production. Lignolytic enzyme is isolated and purified by modern bio-chromatography, then the characteristics of the enzyme and its decolorization to anthraquinone dye SN4R are researched. Meanwhile the abilities of degradation and decolorization of alkali lignin by white-rot fungi are studied under shake condition.When white-rot fungi grow in N-limited medium, lignin peroxidase(LiP) and manganese peroxidase(MnP) are not detected in the supernatant, laccase is the only ligninolytic activity detected. Of which, the laccase production ability of Pleurotus ostreatus 324 is highest, that of Pleurotus ostreatus QH, Pleurotus eryngii and Pleurotus ostreatus 5526 is medium, and that of Lentinus edodes 9680, Pleurotus ostreatus 792, G1,G5 and BY is lower. Polyporus versicolor, Fomes lignosus and Votvariella volvcea do not produce laccase under this condition. When white-rot fungi grow in N-rich medium, lignin peroxidase(LiP) is not detected in the supernatant, laccase and manganese peroxidase(MnP) are main ligninolytic enzymes. Laccase production in N-limited cultivation is superior to that in N-rich cultivation, and laccase production is inhibited by high concentration nitrogen.The laccase activity from Pleurotus ostreatus 324 in static cultivation is higher than that in agitated cultivation .The optimal conditions of laccase production are obtained as follows: temperature 30℃, pH 5.0 and volume 20ml/250ml. Among tested carbon source, cellobiose and arabinose is the best for laccase production. When using organic nitrogen as nitrogen source, a higher activity level is obtained. The initial C/N ratio more than 10 is of benefit to laccase production. Adding 0.5mmol/L of various metal ions in culture meduim , Cu2+ and Ca2+have a positive effect on laccase production. ABTS (0.5-lmM/L) is shown to be the best inducer of laccase production, reaching maximum values of about 410 U/ml. The white-rot fungus Pleurotus ostreatus 324 is an excellent producer of the industrially important enzyme laccase.Laccase from white-rot fungus Pleurotus ostreatus 324 has been purified by three chromatography steps with an overall yield of 38.3%. The molecular weight of purified laccase is about 56.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis(PAGE). The laccase has very acidic optimal pH values 3.5 in sodium acetate buffer, and the optimal temperature of the laccase is 65℃. The laccase is stable in sodium acetate buffer(pH5.0) when treated with the temperature below 50℃. The laccase can oxidize ABTS and phenolic compounds, especially p-,o-methoxyphenol, hydroxylphenol second, aminophenol, ferulic acid,pyrogallol are last. The laccase activity is strongly inhibited by sodium azide. Compared with other fungus laccases, the laccase has different molecular weight and enzyme characteristics. We may conclude that the laccase from white-rot fungus Pleurotus ostreatus 324 is a new enzyme.Laccases are copper-containing polyphenoloxidases, which are widely distributed among white-rot fungi. Laccases catalyze the oxidation of a broad number of phenolic compounds and aromatic amines. The oxidized substrates by laccase can be extended in the presence of small molecular mediators. Among textile dyes, anthraquinone dye SN4R could be directly decolourized by crude laccase(30U/ml), the optimal pH is 4.0, the optimal temperature is 40 ℃, the decolorization rate is 55% after 12 hour treated . The enzyme activity is not propertional to the decolorization rate . Through a series of comparative experiments, we find that anthraquinone dye SN4R could not be decolourized by pure laccase or small molecule mediator ABTS. When adding small molecule mediator ABTS in pure laccase, anthraquinone dye SN4R can be decolourized. The small molecule mediators mediate the oxidation between laccases and nonsubstrate dyes, so do the oxidation of anthraquinone dye SN4R by the laccase. The small molecule mediators are conducive to improving...