The Generation, Purification And Enzymatic Properties Of Laccase Produced By Fomes Fomentarius

In this study, the lignin oxidative system, laccase activities and decolorization assay of the 27 fungi were analyzed, which were preserved in the Laboratory of Microbiology and Immunology, Northeast Forestry University. From the results, Fomes fomentariu had been chosen as the strain produced laccase with its excellent capability and researched detailedly in fermentation conditions, purification and enzymatic properties on extracellular laccase which it produced. Main results as followed:(1) The lignin oxidative system of 27 fungi species were analyzed qualitatively by four methods besides using syringaldazine as the substrate and the decolorization of aniline blue in plate cultivation. The result showed that 5 species had three enzymes activity of Lacese, Lip and Mnp, and 5 species had two. Another 8 species only had one enzymes activity. There were also 12 species produced laccase activities. The 5 fungi, which grew quickly and produced high laccase activity, were selected to study how the cultivation mode and inducer could affect the production of laccase activity. The laccase activities was produced by Fomes fomentarius reaching a peak of 9496 U/ml on induction and static cultivation, which was obviously higher than other fungi. It proved that shaking cultivation could be taken place by static cultivation to produce and prepare laccase in the follow up work for Fomes fomentarius. By the decolorization assay, it was found that all of this 5 species have efficient and broad-spectrum discolored function to 4 head kinds of dye and Fomes fomentarius had most excellent capability.(2) Utilizing the orthogonal design experiment method, the best response system of media (g/L): starch 20, veratryl alcohol 2.5, peptone 2.5, Tween80 0.15 %, CuSO₄ 0.1 mM (pH4.0, 30℃).(3) Laccase from Fomes fomentarius was purified using DEAE-Sepharose FF anion-exchange chromatography and Sephadex G-75 gel filtration. The purification fold was 11.93 and recovery of total laccase activity was 20.18%.(4) The pH for laccase activity against ABTS showed a peak of maximum activity at pH 2.8. The enzyme was stable at pH range from 2.0 to 5.0. The optimum temperature of the purified enzyme was observed at 60℃and was stable at range from 20℃to 65℃. But the activity decreased rapidly beyond 65℃. The purified laccase showed activity against various substrates. The highest Km value (0.068 mmol/L) was found for ABTS. The purified enzyme was strongly inhibited by sodium azide, 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.(5) In the dye degradation assay, it was found that the co-culture system of Fomes fomentarius and dye had high degradation effect as the laccase purified on the congo red, crystal violet, methylene blue and alizarin red, which were belong azo compounds, triphenylmethane, heterocyclic and anthraquinones, respectively. This result showed that the laccase produced by Fomes fomentarius had industrial application potential on the dye degradation.