Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu2+ centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50°C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, −20°C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit®C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25°C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.