Production, Characterization And Primary Application Of The Laccase From Armillariella Tabescens

Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of substrate, which has wide industrial application potential. This experiment to Armillariella tabescens as the target strains. The fermentation medium and fermentation conditions were statistical optimized. Lastly purification, characterization and primary application of laccase was operated from Armillariella tabescens. The main contents were as follows.Taking laccase activity of Armillariella tabescens as measurement to study the optimum culture media and conditions in liquid fermentation through orthogonal test, the optimum culture components were:Glucose0.5%,Starch1.5%,(NH4)2SO40.2%,Yeast Extract1.5%, the activity of enzyme reached4.97U/ml.The optimum conditions were:75ml medium in250ml-flask, inoculation6%,mycelium age7d, culture time10d, the activity of enzyme reached5.35U/ml.In the optimum culture and conditions, the activity of enzyme reached7.84U/ml by adding inductors. To study the effects of chinese herbal medicine residue on growth development and the extracellular laccase activity in Armillariella tabescens. The single factor experiment and the orthogonal experiment were employed to optimize fermentation conditions for growth development and the extracellular laccase production in Armillariella tabescens. Fruit body was not found with chinese herbal medicine residue. Radix paeoniae alba residue was found to be a good carbon source of laccase production and the ratio of radix paeoniae alba and wheat bran was3:7.The optimum glucose dosage, moisture content of substrate, inoculation dosage, and culture temperature were0.9%,65%,11%and25℃, the activity of enzyme reached6956U/g. After comprehensive studying, Laccase activity of Armillariella tabescens was still very low in the optimum culture media and liquid conditions, The solid fermentation was more suitable for the Armillariella tabescens producing Laccase.Laccase in Armillariella tabescens solid-state fermentation liquid was isolated and purified by ammonium sulfate precipitation, dialysis, DEAE-sepharose Fast Flowaion-exchange chromatography, After the three-step purification, After the three-step purification, we obtained a electrophoresis pure laccase, its yield were26.7%of the crude extract. The molecular mass of the laccase was about55.4KD. The optimum reaction temperature and pH for this enzyme were50℃and4.0, respectively, and the Km value was0.02114mmol/L with ABTS as a substrate. This enzyme maintained high and stable activity at pH4.5. Various acid radical ions and metal ions had different effects on laccase activity.To explore the ability for catalyze degradation of chlorophenols using laccase from Armillariella tabescens. Results show that, under the optimal conditions, the maximum degradation rate of2,4-DCP,2-CP and4-CP reached95.6%、85.7、83%. the crude laccase from Armillariella tabescens was able to effectively degrade2,4-DCP,2-CP and4-CP, indicating its potential application value in phenolic pollutant control and environmental protection.