Purification And Characterization Of Laccase From Irpex Lacteus

The lignin oxidative system of 8 fungi species were analyzed qualitatively by two methods besides using syringaldazine as the substrate and the decolorization of aniline blue in plate cultivation. The result showed that 6 species had three enzymes activity of Lacese,Lip and Mnp, and 1 species had two. Another 1 species had none. By the decolorization assay, it was found that all of this 6 species have efficient and broad-spectrum discolored function to dye and Irpex lacteus had most excellent capability. The effect of the decolorization of simple mix dyes by Irpex lacteus is better than the others.Laccase from Irpex lacteus was purified using (NH₄)₂SO₄ salting-out, dialyzing, ultrafiltration, anion-exchange chromatography and gel filtration. The purification fold was 4.46 and recovery of total laccase activity was 31.41%. The molecular mass of the enzyme was 62.5 kDa as determined by SDS-PAGE. The pH profile for laccase activity against ABTS showed a peak of maximum activity at pH 3.0. The enzyme was stable at 25℃for 1 h in the pH range from 2.0 to 5.0. The optimum temperature of the purified enzyme was observed at 50℃. There was little loss of activity during preincubation of the enzyme for 1 h at temperature up to 40℃, but the activity decreased rapidly beyond 50℃. The purified enzyme was strongly inhibited by 0.1 mmol/L sodium azide, 1 mmol/L L-cysteine and DTT, whereas the metal ion chelator EDTA showed only a slight inhibitory effect. The metal ion Zn²⁺,Mg²⁺ and Ca²⁺ could activate the laccase activities.In the present study, the purified laccase from Irpex lacteus was able to decolorize Remazol Brilliant Blue R (RBBR) and Congo red efficiently in the absence of any redox mediators; it was able to decolorize about 90% of Congo red and RBBR in only 60 min with an enzyme activity of 10 U/mL. The decolorization ability of I. lacteus laccase demonstrated its potential applications in dye decolorization.