Fermentation Optimization And Purification Of The Food-Grade Laccase

Laccase is an oxidoreductase belonging to the multinuclear copper-containing oxidase and it can be investigated for a variety of applications including detoxification of harmful substances, bleaching of paper pulp, decolorization of textile dyes, transformation of lignins or their derivatives and improvement of food properties. At present, there is much study of laccase, but these laccases can’t be used for food industry. In this study, author screened into a strain of producing laccase from the author’s laboratory in a high yield. The determination of laccase activity and the fermentation medium and condition for laccase production were optimized. Laccase produced by Lentinus tigrinus were purified and characterized. The results were as follows:A Lentinus tigrinus, which produced laccase, was isolated using PDA screening medium and shaking culture.The fermentation process for laccase production by Lentinus tigrinus in submerged fermentation was studied by using some culture that can be used in food industry. The obtained optimal parameters were fructose 48 g/L, corn flour 12 g/L, tryptone 9 g/L, NH4H2PO41 g/L, KH2PO42 g/L, MgSO4·7H2O 0.25 g/L, CuSO4·5H2O 1 mmol/L, temperature 30℃, pH 5.7, seed age 3 d, inoculum size 6%, loaded liquid 150 mL/500 mL, shaking speed 150 r/min in 1-3 d and 200 r/min in 4-11 d. Under these optimum conditions, the laccase activity reached at 462.59 U/mL after 11 days. Based on previous results, the fermentation process was scaled up to 7 L bioreactor and the laccase yield was 376.11 U/mL after 13 d.Laccase were purified by using (NH4)2SO4 precipitation, dialysis, DEAE-Sepharose Fast Flow anion-exchange chromatography. The molecular mass of laccase is 62.12 kDa, and it contained 31.30% carbohydrates. The uv-vis spectrum of the purified laccase had qualitative characteristic absorption peaks at 614 nm and showed a broad shoulder at 330 nm.The enzymatic properties of laccase were also studied. The laccase relative activity was above 40% between 20-80℃ and the optimal temperature was 65℃. The laccase relative activity was above 40% between pH 2.2-4.0 and the optimal pH was 2.2. The laccase had good stabilities toward pH and temperature. Metal ions had inhibiting effect on laccase activity except sodium. The Km value of Laccase was 0.2 mmol/L for ABTS as substrate.