Study On Production Optimization, Purification And Partial Characteristics Of Laccase From Ganoderma Lucidum

Production conditions of laccase from Ganoderma lucidum were investigated, and the optimization condition: the optimum carbon resource and its concentration, the optimum nitrogen resource and its concentration, the optimum initial pH, the effective metal ion and its optimal concentration, were ascertained. Taking the four culture conditions as experiment factors, orthogonal design experiment were carried by dividing each factor into five levers , the optimum ingredients of culture medium were ascertained as follows: glucose 3%; yeast extract 0.1%; KH₂PO₄ 0.3%; MgSO₄·7H₂O 0.1%; Cu²⁺ 2.854mmol/L; VB₁ 0.01%; pH 2.60 after sterilization.The laccase from G. lucidum was purified by the following methods: vaccum filtering, （NH₄）₂SO₄ fractionating, dialyzing, polyethylene glycol 6000 （PEG6000） concentrating, and DEAE-cellulose ion exchange chromatography （1.6cm×40cm） with phosphate buffer （0.01mol/L, pH5.8） as the mobile phase. And the 280nm absorption value of the eluate was tested by Ultraviolet Spectrometer. Then, the laccase activity of the eluate at absorption peak was tested by ABTS, the result indicated that the phosphate buffer with 0.2mol/L NaCl was the best concentration to obtain the purified laccase. On this basis, the purity of the laccase was identified by SDS-PAGE electrophoresis, the result indicated that purification multiple was 3.6 and the activity reclamation rate was 49.1%.Partial enzymology properties of purified laccase from G lucidum were determined. The molecular weight of the purified laccase is 45KDa. The optimum reaction temperature of purified laccase is 75℃. The laccase is stable within 40℃-65℃and is sensitive to temperature higher than 70℃. The optimum reaction pH value of purified laccase is 3.0-3.6 and its stability is within 3.0-5.0. The higher the pH is, the less stable the laccase is. Metal ions have very important influence over purified laccase. As the concentration of different metal ions was 50mmol/L, Na⁺ and Zn²⁺ had no obviously effect on laccase activity, whereas Cu²⁺, Mn²⁺and Mg²⁺ evidently promoted the enzyme reaction, while Ag⁺ and Fe²⁺ inhibited the laccase activity strongly. Among Cu²⁺, Mn²⁺and Mg²⁺, Cu²⁺ had the most effective activation on laccase and Ag⁺ was the strongest inhibitor to the laccase. At 30℃, the Michaelis constant of the laccase is 186μmol/L when using ABTS as the substrate.