| Laccases are multicopper phenoloxidases that has special catalyze capability and wide range of substrates, which interest in laccases has been fueled by their potential uses in paper processing, bio-bleaching, detoxicate of toxiferous compound, biological monitoring.In this dissertation, the mutagenic breeding of laccase by Ganoderma lucidum, solid state fermentation, chemical modification, immobilized laccase and its application in Waste Water of Papermaking Industry were investigated. The results were as follows:A mutant strain Ganoderma lucidum LYL263with higher production was mutagenized by ultraviolet, nitrosoguanidine, ultraviolet and nitrosoguanidine. The activity of laccase from the mutant strain was187.0%higher than primary strain Ganoderma lucidum.The solid state fermentation for Ganoderma lucidum LYL263was investigated. The cassava bran was proved to be a good substrate and the appropriate ratio of cassava bran to wheat bran was6:4. The optimum solid state fermentation medium was test the content of malt sugar, yeast extract and CuS4·5H2O were0.5%,2.5%,0.01%respectively, which the activity of laccase reached up to212336.1U/g.Laccase was modified with citraconic anhydride and L-Ascorbic acid/Fe3+in order to improve its stability against high temperature. The research indirected that Laccase was more stable and higher enzymatic recovery when using L-Ascorbic acid/Fe3+as chemical modification. The optimum conditions for L-Ascorbic acid/Fe3+were determined as follows:the concentration of enzyme was14g/L, modificate time was3.5h and pH5.0. The modified laccase can keep enzymatic recovery up to 89.95%, the half-life of enzyme activity at65℃was increased by50.16%(4.64h), and its amino modification rate was53.68%.Compared with sodium alginate-gelatin system and sodium alginate-chitosan system, the activity of laccase immobilized in sodium alginate-gelatin-chitosan system was showed2.14-folds and2.75-folds respectively. The alginate-gelatin-chitosan systems best concentration of materials were as follow:the concentration of sodium alginate was20g/L, the concentration of gelatin was10g/L, the concentration of chitosan was3g/L, the concentration of calcium chloride was360g/L. And the benzoic acid was used to entrapped immobilized laccase for the first time, the suitable content of benzoic acid can highly increased laccase activity after single factor experiments. The conditions of immobilization of laccase were optimized, which could be specified as:the concentration of benzoic acid was2mmol/L, the concentration of glutaraldehyde was0.32%for50min, and the concentration of enzyme was100g/L. In this way,635.7U/g of catalytic activity of immobilized enzyme was available. After5cycles of operation, the immobilized enzyme only31%of its initial activity. We also use sodium alginate-gelatin-chitosan system to immobilized unmodified laccase and modified laccase, our researchs clear reveal that the thermal stability of immobilized enzyme was increased compare the free enzyme. The immobilized enzyme which was modified increased the thermal stability compare the immobilized enzyme which was unmodified.Using COD and total phenol of pulping wastewater decreasing rates as targets. As a results:staying time of wastewater was5hours, COD and total phenolic removal rates were higher than others. The best react temperature of modified immobilized laccase was50℃, and the unmodified immobilized laccase was35℃. The best react pH of modified immobilized laccase was6.0, and the unmodified immobilized laccase was5.0. If the modified immobilized laccase dosage was15g/L, the total phenolic removal rate was58.7%. The dosage was20g/L, the COD removal rate was75.9%. If the unmodified immobilized laccase dosage was20g/L, the total phenolic removal rate was35.1%, the COD removal rate was69.9%. |
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