Optimization Of Phellinus Sp. SYBC-L2 Solid-state Fermentation For Laccase Production And The Enzyme Characterization

Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of polyphenols and other lignin-derived aromatic compounds. It can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials, so laccases can be widely applied in paper making industry, drinking processing and environment protection. So, the studies on laccase including the separation, purification were conducted. It was found that laccase isolated in this study possessed a certain theoretical and realistic significance.By the method of plate screening, a strain of laccase producing microorganism was obtained from Cinnamomum camphora in Qing Shan Wan campus of Jiangnan University. It was morphologically identified as Phellinus sp. and termed as Phellinus sp. SYBC-L2.The laccase production by Phellinus sp. SYBC-L2 was studied under solid-state fermentation with different media and fermentation conditions. The optimized solid-state fermentation medium contained(w/w): soybean meal and sawdust mixed ratio of 1:1, xylose 0.5%, ammonium tartrate 2.0%, CuSO4 0.04%, CaCl2 0.48%, KH2PO4 0.5%, MgSO4·7H2O 0.5%, medium moisture content 65%. The optimized solid-state fermentation condition was: 50 g solid-state medium in 250 mL Erlenmeyer flasks, thickness about 4.5 cm, initial pH 7.0, 10μmol/L tannins was added, 30℃of the fermentation temperature and 9 day of the cultivation time. After optimizing the solid-state fermentation medium and the culture conditions, the strain Phellinus sp. SYBC-L2 laccase activity reached 21.462 U/g wet substrate, increased about 10 folds than that of original fermentation (1.914U/g wet substrate).The laccase produced by strain Phellinus sp.SYBC-L2 in solid-state fermentationwas isolated and purified by a three-step process: ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. After the three-step purification, we obtained a electrophoresis pure laccase,its purity and yield were 28.27 folds and 14.76% of the crude extract respectively. The molecular mass of the laccase was about 64.3 KDa, and the molecular configuration should be single or homologous oligomers. The apparent Km of DMP was 1.18 mmol/L, the Vmax was 15.95 U/mg protein.The optimal temperature of the purified laccase was 60℃. The optimal pH value of the enzyme was 3.5, with relative activity of about 80% at the pH rang from 6.0 to 8.5, which indicated that it adapted to a wide range of pH. Laccase could be acti-vated by SO42-, CO32- and Cu2+, and inhibited by Cl-, Fe3+ and Fe2+.