Laccase Production By Solid Fermentation With Spent Compost Of Pleurotus Eryngii And Its Application In Dye Decolorization

Edible fungi production increased rapidly due to its nutrition and delicacy, but it caused considerable spent mushroom substrate, which has become a mushroom industry challenge. Laccase is an important lignocellulosic enzyme in edible fungi. Study the synthesis of laccase by edible fungus residue can not only reduce laccase production cost, but also make high use of spent mushroom substrate, which meets the green chemical requirements.The aim of this paper is to investigate the feasibility of laccase production by spent substrate from Pleurotus eryngii, the optimum conditions for laccase production, microbial community structure changes, the enzymatic properties of laccase and its application in decolorization of dyes. The main results are as follows:1) The laccase-producing medium optimized by the traditional one-factor-a-time strategy were like these: 6% bagasse?w/w? as carbon source, 1.2% ammonium nitrate as nitrogen source, 1% alkaline lignin?w/w? inducer and 150%?v/v? inorganic salt solution, the composition of inorganic salt solution(g·L^-1) were as follows: KH₂PO₄ 0.8,Na₂HPO₄·7H₂O 0.75,Zn SO₄·7H₂O 0.002,Mg SO₄·7H₂O 0.5,Fe SO₄·7H₂O 0.005,Cu SO₄·7H₂O 0.02,Ca Cl₂·2H₂O 0.06,Mn SO₄·H₂O 0.05. And the optimal culture conditions were as follows: p H value of culture medium was 7, culture temperature was 28?, culture time was 6 days. Under the optimum conditions, the activity of laccase reached 434.9 U·gds^-1, which was 35.3 times of the initial enzyme activity 12.3 U·gds^-1.2) Through the sthudy of the effects of microbial flora contained in SMS on the production of Laccase by Pleurotus eryngii by constructing molecular library, high-throughput sequencing and bioinformatics analysis, it's found that the bacterial community during fermentation has no effect on laccase synthesis. The abundance of the Ascomycetes community was higher than before within the genus of Chaetomycetes, Penicillium, Curvularia and Aspergillus, which means some fungi may promote the laccase synthesis of Pleurotus eryngii SMS. The mycelia of Pleurotus eryngii and bagasse were observed to have great changes during the fermentation process by scanning electron microscopy.3) The purified enzyme properties were studied, which was obtained through ammonium sulfate precipitation, ion exchange chromatography and molecular sieve chromatography purification process. Based on the SDS-PAGE results, it was confirmed that laccase was produced by Pleurotus eryngii through mass spectrometry, and its size was calculated to be 65.18 k Da, The enzyme has a high activity at p H 2⁶ and was stable under the conditions of p H value 5⁸. The relative enzyme activity was higher than 70% at 20⁷0?, and the enzyme would be inactivated when the temperature was higher than 60?, i.e., the optimum p H and temperature for DMP were 4 and 60?, for ABTS were 4 and 40?, respectively. 0.1 and 1 mmol·L^-1 metal ions had no obvious inhibitory effect on laccase activity, and 10 mmol·L^-1 metal ions and Na N3 at all tested concentrations had significant inhibitory effect on laccase activity.4) Four common synthetic dyes were decolorized with crude enzyme. The decolorization rate of reactive brilliant blue K3 R, acid blue 209 and reactive brilliant blue KNR were all more than 65% within 3 hours without the presence of the mediators, the decolorization rate were all more than 80%?methyl red untested? when added mediators.