紫杉木齿菌漆酶的分离纯化及其对木质素的降解分析-isolation and purification of laccase from chinese fir dentistry and its degradation of lignin.docxVIP

华 华 中 科 技 大 学 硕 士 毕 业 论 文 II II Abstract Echinodontium taxodii has high lignin-degrading ability which depends on its extracellular laccase. However, few studies focused on characteristics and catalysis of the laccase from E. taxodii. In this study, the fermentation condition of E. taxodii was optimized in order to obtain the maximum enzyme production. The laccase was purified and its characteristics were investigated. Besides, the lignin degradation with the laccase was studied. The optimum medium for laccase producing was as follows: solube starch and tryptone were the best carbon source and nitrogen source with final concentration of 1.5% and 0.3% respectively, and the C:N is 14:1. Veratryl alcohol with concentration of 4 mM was added in the culture after 4-day fermentation，and the maximum activity of laccase could reach 11.05 IU/mL after 8-day fermentation. After salting-out, HIC, and anion-exchange chromatography, a purified laccase was obtained from extracellular fermentation product of E. taxodii. The enzyme was purified 23.3-fold to a high specific activity of 30.8 IU/mg and relatively high yield of 38.8%. The molecular weight of the laccase was 55.5 kDa. The absorption spectrum showed that it had a typical structure of blue laccase. The enzyme also showed a much higher level of specific activity for ABTS than for DMP and guaiacol. The optimum pH and temperature for the purified laccase were 3.0 and 60℃ respectively. Most ions significantly inhibited the laccase activity at high concentration, others like Mn2+, Mg2+ didn’t affect the laccase activty, no metal ions we studied increased the activity of laccase. Sodium azide strongly inhibited the laccase activity. For the first time the extracelluar lignin degradation of laccase from E.taxodii was studied. The laccase showed a strong lignin degrading ability of breaking the aromatic ring, ether bond β-O-4 and methoxy group without any mediators, the hydroxylation of the lignin was also enhanced, which indicat