Research On Culture Conditions Of Tricholoma Populinum And Preliminary Isolation And Purification Of Its Laccase

Laccase can use many substrates and catalyze the oxidation of phenolic compounds and aromatic amines.And there are many substrates that can interact with laccase.Laccase can be divided into different types according to different methods.Among them,the distribution of plant laccase is extensive,and edible fungi are one of the producers of plant laccase.At present,laccase play an important role in wastewater treatment,beverage clarification,environmental protection and paper industry.By screening the laccase activity of the existing strains in the laboratory,the wild strain YK-1 with higher laccase activity was selected.Then,by analyzing the morphology and 18sDNA ITS sequence,the strain was identified as Tricholoma populinum.By optimizing the culture conditions of mycelium,and the optimum culture conditions of the strain YK-1 were as follows:the carbon source was glucose,the nitrogen source was yeast extract,the carbon and nitrogen ratio was 20:1,the opt:imum temperature was 28,the optimum pH value was 7.After optimization,the average growth rate of mycelium was 7.05mm/d.The results of enzyme production characteristics test showed that the laccase activity of the strain YK-1 was?199.72±5.32?U/mL,the lectin activity was slightly lower;the protease activity and a-galactosidase activity were lower,they were?0.86±0.04?U/mL and?0.04±0.01?U/mL,respectively.And no RNase activity.The crude enzyme was purified as a protein fraction by DEAE-Sepharoase anion exchange chromatography,which was D4.After CM-cellulose cation exchange chromatography,the protein fractions CM4 were purified.Finally,the purification fold was 3.34 and the recovery rate was 17.47%.The specific activity was 34.81 U/mg.After study on the enzymatic properties of purified laccase,laccase activity was highest at 50 ?.When temperature below 40 ? for 1h,laccase activity was hardly affected.At 60 ? for 1h,the enzyme activity almost disappeared.The optimum pH value of laccase was 4.0,and the enzyme activity was stable at pH value of 3.0-6.0.Mg²⁺ has little effect on laccase activity,but Fe²⁺ has a strong inhibitory effect on laccase;Low concentration of Cu²⁺ can promote the activity of laccase,and high concentration of Cu²⁺can inhibit the activity of laccase.Ascorbic acid,oxalic acid and EDTA had a strong inhibitory effect on laccase,but urea had no effect on laccase activity.In addition,under the condition of different concentrations of ABTS as substrate,the Km was 0.192mM,Vmax was 9.64?M min^-1.