Purification And Characters Of Laccase From Amanita Verna And Decolourization Of Azo Dyes By The Enzyme

Firstly,In the liquid culture system,the influence on the production of laccase by Amanita verna in different fermentation conditions has been studied.Through a series of orthogonal experiments,effects of carbon source,nitrogen source and other factors on laccase production from strain Amanita verna were studied.The liquid medium used for laccase production contained(per liter)200g potato extract with 20g dextrose, 5g yeast extract,20g bran,3g KH₂PO₄,1.5g MgSO₄,pH5.0-5.4.Shaking cultivation was performed at 25℃on a rotary shaker at 120 rpm.Under the optimized fermentation process,the laccase production by strain Amanita verna was increased from the 5th day,reached the highest production on the 8th day.The laccase was purified by a three-step procedure involving salting out,ion exchange,size exclusion chromatography.A typical procedure provided 22.03-fold purification,with a yield of 22.02%,using o-tolidine as substrate.The molecular weight of the purified laccase was 63 kDa as estimated by 12%(w/v)SDS-PAGE gel.The enzyme's pH optimum for o-tolidine was 4.6 and optimal activity was at 20℃.After pre-incubation at different pH values for 3.5h and different temperatures for 70 min,more than 60%of the initial laccase activity was retained between pH 3.6 to 5.2 and a relative activity of 19.3%was observed at 60℃.The Km value of laccase for o-Tolidine was 66.7μmol/L at pH4.6.The effect of different chemicals on laccase activity was also tested.This laccase was strongly inhibited by L-cysteine,β-mercaptoethanol even at a low concentration,and slightly inhibited by EDTA.The combination of thermotolerance with low pH optima for aromatic compounds substrates suggests that the laccase possesses potential for use in biotechnological applications.The laccase from Amanita verna could degradate 6 dyes efficiently under keep on ventilating condition.The laccase showed maximal decolorization rates of both dyes at 50℃and optimal decolorization rates at pH 5.5 and 3.5 for direct black G and neutral dark yellow GL,respectively.The higher the pollutant primary concentration and laccase activity,the higher decoloration rate,when the primary concentration and laccase activity were below 20mg/L and 10 U/mL,respectively.The decoloration rate of direct black G was 80%,while it was 60%to neutral dark yellow GL.Laccase was immobilized by the sodium alginate entrapment method and the sodium alginate-chitosan entrapment-crosslinking method.The immobilization conditions and the characterizations of the immobilized and free laccase were investigated.The results showed that the optimal conditions of immobilized laccase by the entrapment method and the entrapment-crosslinking method were 3%sodium alginate,1.5%CaCl₂ and 2%sodium alginate,2%CaCl₂,1.5%chitosan,1% glutaradehyde,respectively.These two kinds of immobilized laccase exhibited the same optimal pH value and optimal action temperature which were 5.0 and 30℃, however the free enzyme's optimal pH value and optimal action temperature were 4.6 and 20℃.The ability of decolourizing azo dyes in shaky situation by sodium alginate-immobilized laccase approached to the free enzyme,and there was no loss of enzyme activity during 2 repeated batch reactions.Compare with entrapment method, entrapment-crosslinking method has a better mechanical strength and operation stability,but the decolorization rate was low because of chitosan' adsorption,so we should optimize accessories of entrapment-crosslinking immobilized laccase used for dye degradation.