The Study Of Myrothecium Verrucaria GH-01 Extended Laccase Culture、Purification And Electrochemistry

Myrothecium Verrucaria GH-01 as the experimental strains, studyed the optimum conditions of the production of laccase and obtain the purified extracellular laccase. Futhermore, the enzymatic properties and its electrochemical properties are surveyed.Based on several means of single factor effect on enzyme production, the ideal enzyme production conditions are determined:choose to full of spore plating, inoculating nine bacteria cake to the fluid of the seed, foster in oscillation incubator in 10 d under constant 28℃, with the amount of 2% inoculating into the fermented liquid medium to culture. The concentration of Cu2+ in seed liquid and fermented liquid is 0.2mmol/L. In applying the enzyme production conditions to the expanded training experiment, fermentation liquid in the fermentation tank reach the stationary phase only after 5 days, and the enzyme activity reach 10.53U/ml, the protein content reach 0.31mg/ml.Buffer system and elution salt system are determined based on analysis. After ammonium sulfate fractional precipitation, dialysis, DEAE-Sepharose FF ion exchange column chromatography, ultrafiltration concentration and SephadexG-75 gel, purified laccase protein is obtained. The pure protein after collection by SDS-PAGE and Native-PAGE is a molecular weight of 62kDa and has the activity of laccase fragments, and the protein is monomeric protein. The purification fold is 4.77, and the recovery rate is 29.34%.By using ABTS as substrate, the optimal reaction temperature for laccase activity is 40℃, and the optimum pH is 4.0. Besides, the laccase can better maintain the stability under the low temperature and alkaline conditions. The decolorization experiment of laccase on degradation four kinds of dyes shows that:the orange Ⅰ and alizarin red degradation rate is higher, the reaction after 1 hours, the degradation rate can reach above 80%, the decolorization rate of fuchsin is lower at 20%, the lowest decolorization rate of the four dyes is methylene blue. When the concentration of dyes is 50mg/L,three kinds of dyes decolorization rate is the best. The decolorization rate of orange Ⅰ significantly increase with the increase of the quantity of enzyme over a period of time.By using Nafion film immobilized laccase protein of GH-01, the Lac-Nafion/GP electrode, which can help direct electron transfer between the electrode and laccase, is obtained. The Lac-Nafion/GP electrode in buffer containing 3mM ABTS, get obvious reduction peaks of O2, visible GH-01 laccase catalytic reduction of O2 to water. The Lac-Nafion/GP electrode showes a good current response to H2O2, and the response current gradually increased with increase of the pH value and the concentration of H2O2. Applying Lac-Nafion/GP electrode to detect the concentration of H2O2, found that the concentration of H2O2 induction current exists certain linear relationship.