| In order to increase laccase yield and activity of Curvularia lunata, we mutated Curvularia lunata strains by physical method (ultraviolet ray-60Co-y ray, microwave irradiation) and chemical method (diethyl sulfate). The laccase activity was increased from the original 0.55 U/ml to 1.668 U/ml after the treatment of ultraviolet ray-60Co-y ray. Then the activity of laccase was increased from 1.668 U/ml to 1.914 U/ml and from 1.914 U/ml to 1.949 U/ml after the treatment of microwave irradiation and diethyl sulfate, respectively. The final lacasse activity was about 3.544 times higher than the original lacasse activity. There was a certain correlation between fatality rate and positive mutation rate of Curvularia lunata. The highest positive mutation rate was obtained when the fatality rate was between 80% and 90%.After the series treatment of mutagenesis, a Curvularia lunata stain VC-W-L3 with high level for laccase production was obtained. The fermentation conditions of strain VC-W-L3 was optimized using single factor and orthogonal design. The optimal fermentation conditions were as follows:cultivation temperature 30℃, fermentation time 72 h, initial pH 6.0,120 ml of culture medium in 250 ml flask, rotating speed 160 r/min. And the media components were as follows:potato 200 g/L, sucrose 30 g/L, peptone 5 g/L, KH2PO4 5 g/L, guaiacol 0.2 mmol/L, tween80 0.1 ml/L, Mg2+ 0.5 mmol/L, Cu2+ 1 mmol/L, Ca2+ 1 mmol/L, Mn2+ 0.25 mmol/L. The activity of laccase reached 2.306 U/ml, which was increased about 18.32% of the original activity after optimization. When industrial waste was used as a main component of medium, the optimal conditions medium was as follows:discard molasses 30 ml/L, soybean meal 15 g/L, KH2PO4 5 g/L, guaiacol 0.2 mmol/L, tween80 0.1 ml/L, Mg2+ 0.5 mmol/L, Cu2+ 1 mmol/L, Ca2+ 1 mmol/L, Mn2+ 0.25 mmol/L. Under this conditions, the final activity of laccase achieved 1.458 U/ml.Laccase from C. lunata was purified using ammonium sulfate precipitation, DEAE-Sepharose FF anion-exchange chromatography and Sephadex G-75 gel filtration. The purification fold was 3.73 and recovery of total laccase activity was 31.69%. The molecular weight of the purified laccase was 86.3 kDa as estimated by SDS-PAGE.The optimal pH of the purified laccase was 3.0 with ABTS as the substrate. High laccase activity was maintained at pH range from 2.8 to 3.5, and the enzyme was stable between pH 5.5 to 6.5, under which more than 87% of residual activity was retained after 1 h. The optimum temperature of the purified enzyme was observed at 30℃. The enzyme was stable for 1 h in the temperature from 20℃to 30℃. The Km value for ABTS was 0.064 mmol/L. The result of dye decolorization indicated that the purified laccase showed the highest efficiency for decolorizing malachite green in 24 h, followed by alizarin red, neutral red, congo red, methylene blue, and methyl orange. Decolorization of malachite green proceeded fast in the initial 12 h. Furthermore, the purified enzyme from C. lunata could also effieciently decolorize dye mixtures, which indicated that the purified laccase from C. lunata could be applied in the treatment of dye-containing wastewater.The decolorization ability of Curvularia lunata mycelial pellets, which were formed during the growth process, was tested using several synthetic dyes. The result showed that more than 80% of the tested dyes could be removed within 24h. The mycelial pellets demonstrated a good stability and could be used for 6 times.
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