Production,Purification,Characterization And Primary Application Of The Laccase From Trametes Sp. SYBC-L3

Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of polyphenols and other lignin-derived aromatic compounds. Laccases can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials; they can thus be widely used in paper pulp industry, food industry, medication and sanitation, biosensor and environment protection.In this thesis, a strain of high laccase producing microorganism was obtained from kraurotic chump by plate screening. It was identified by morphological observation and 18S rDNA sequence analysis and termed as Trametes sp. SYBC-L3.The laccase production by Trametes sp. SYBC-L3 was studied under liquid state fermentation with different media and fermentation conditions. The optimized liquid state fermentation medium contained: maltose 12 g/L, soy bean meal 6 g/L, CuSO4 0.4 g/L, KH2PO4 1 g/L, Na2HPO4 0.2 g/L, MgSO4·7H2O 0.5 g/L, MnSO4 0.034 g/L, guaiacol 0.8 mmol/L, Tween-80 0.5 g/L. The optimized culture condition was: 80 mL liquid-state medium in 250 mL Erlenmeyer flasks, initial pH 4.0, 10% of inoculation concentration, 200 r/min, 30℃of the fermentation temperature and 8 day of the cultivation time. The highest laccase yield hit 16,130 U/L after 8 days'cultivation which was 42 times of previous conditions (1,432 U/L).The laccase produced by strain Trametes sp. SYBC-L3 in liquid-state fermentation was isolated and purified by a two-step process: ammonium sulfate precipitation, ion-exchange chromatography. After the two-step purification, we obtained a electrophoresis pure laccase, its purity and yield were 8.98 folds and 24.88% of the crude extract respectively. The molecular mass of the laccase was about 58 kDa, and the molecular configuration should be single or homologous oligomers. The apparent Km of DMP was 0.78 mmol/L, the Vmax was 10,000 U/mg protein.The optimal temperature and pH of the purified laccase was 55℃and 3.5 separately. The remaining activity is 80% respectively at 70℃in 1 h and at pH4～7.5 in 24 h, which indicated that it adapted to a wide range of pH and was stable in high temperature. Laccase could be activated by Mn2+ and Cu2+, and inhibited by Al3+, Mg2+, K+, Ca2+, Fe3+ and Fe2+.Primary study on the effect of the laccase on the decolorization of the woolen textile wastewater was conducted. 70% of decolorization was achieved with laccase treatment on wastewater for 20 min, indicating the potential ability of laccase for decolorization.