Production, Isolation, Purification And Characterization Of Laccase Of Trametes Sp. SYBC-L3 Under Solid-state Fermentation

Laccase (EC 1.10.3.2), a type of polyphenol oxidases, can combine a number of copper ions. It can oxidize a wide range of substrates, such as polyphenols, carboxylic acids, biological pigments and several aromatic compounds. It also has the ability to degrade lignin. The study on laccase has a certain degree of theoretical and practical significance.Laccase produced by Trametes sp. SYBC-L3 was studied under solid-state fermentation with different medial and fermenting conditions. Moreover, the purification, characterization and decolorizing role in several dyes of laccase were also investigated. The results were as follows:(1) The optimal solid-state fermentation medium was soybean meal and sawdust with a ratio of 1 : 1, maltose 1.5 g/L, NaNO3 0.71 mol/L, Cu2+ 0.3 g/L, initial medium moisture 65%. The optimized loaded sample on solid-state fermentation condition was 40 g solid-state medium in 250 mL flask, 30℃,incubated for 10 d in dark. The laccase activity reached 91.06 U/g dry substrate with about 8.78 folds of the original fermenting level (10.37 U/g dry substrate).(2) The laccase produced by strain Trametes sp. SYBC-L3 was purified via (NH4)2SO4 fractionation, DEAE-cellulose-52 ion exchange chromatography, and gel filtration with Sephadex G-100 column. Its purity and yield were 19.58 folds and 18.54% of the crude extract, respectively. The molecular mass of the purified laccase (Lac-T) was about 58.7 kDa on SDS-PAGE gel. The Km of Lac-T was 1.32 mmol/L, its Vmax was 1742.76 U/mg protein.(3) The optimal catalytic temperature of laccase (Lac-T) was 65℃, the optimal pH was 3.5. Lac-T was stable under different pH and with relative activity of about 80% at the pH range between 4.0 and 9.0, which indicated that it adapted to a wide range of pH. Lac-T could be activated by SO42-, Mg2+ and Cu2+, and inhibited by CO32- and K+, more significant inhibited by Fe2+. NO3-, Cl-, PO43-, Na+ didn't affect the activity of Lac-T in a certain extend.(4) The laccase produced by Trametes sp. SYBC-L3 could degrade textile dyes. Eighty eight percent of decolorization was achieved with laccase treatment on Mordant Red 5G, 76% on Weak Acid Blue 25 and 75% on Acid Blue 129 for 40 min, respectively, which indicating that the laccase had the potential ability for decolorization.