DETERMINATION OF OPTIMAL PH, TEMPERATURE AND SUBSTRATE CONCENTRATION IN THE QUANTIFICATION OF LACCASE ENZYME OBTAINED FROM Pleurotus ostreatus.

Pleurotus ostreatus are cosmopolitan basidiomycete fungi that live as saprophytes in dead and decaying woods, therefore, secrete enzymes involved in the degradation of these substrates as the laccases. These enzymes catalyze the oxidation of a wide range of phenolic and aromatic amines and they are a promising alternative for some activities with the potential environmental impact such as cellulose pulp bleaching, detoxification of polycyclic aromatic effluents and oxidation of hydrocarbons. Laccases can also be applied in the pharmaceutical, food, beverage, cosmetic, synthetic chemical and nanobiotechnology industries. Since they have a broad biotechnological potential, whenever possible new strains should be studied. The objective of this work was to determine the optimal pH, temperature and concentration of 2,6-dimethoxyphenol (DMP) in the activity of the laccase produced by the fungus P. ostreatus. For this, the microorganisms were inoculated and cultured in Vogel medium supplemented with 1% of glucose and 4% of yeast extract for 7 days at 28 ± 2 °C, under shaking 180 rpm. The crude enzymatic extract (EE) was obtained after centrifugation at 6000 rpm for 15 minutes and this cell-free supernatant with their respective incubation systems (IS) were subjected to spectrophotometric analysis to measure laccase enzyme activity at from the quantification of the oxidation product of DMP (ε468nm 10000 M-1.cm-1). Evaluation of the optimum pH was obtained by varying the pH of the McIlvaine buffer (0.05 M citric acid/0.1 M Na2HPO4) between 3 and 7. The influence of the optimum temperature was evaluated by varying the incubation temperature between 30 and 70 °C. In the experiments, the IS was composed of 150 μL of DMP (10 mM), 150 μL of buffer, 200 μL of deionized water and 500 μL of EE, totaling 1 mL. For the evaluation of the optimum concentration of the substrate, its quantity was altered in IS in 50, 100, 150, 200 and 250 μL of DMP and 1 mL volume supplemented with deionized water. The experiments were performed in triplicate and the enzymatic activity was expressed in International Units (IU). The results indicated that the best conditions for the enzymatic quantification were pH 4, a temperature of 50 °C and 150 μL of DMP, with laccase enzyme activity of 10.32 IU. It was possible to conclude that the optimization of the analyzed parameters was effective and although new parameters as the prospection of the potential of biotechnological application should be analyzed and tested.