Organic solvent enhanced spectrofluorimetric method for determination of laccase activity Original Research Article

A novel fluorimetric method for determination of laccase activity in organic solvent is proposed. It is based on the oxidation of o-phenylene diamine (1,2-diaminobenzene, OPDA) catalyzed by laccase during a fixed time period yielding the fluorescent product, 2,3-diaminophenazine, which was determined and enhanced fluorimetrically in organic media such as ethanol, 1,4-dioxane or acetone at λex/λem=464 nm/530 nm by a fixed time method. The optimal conditions for laccase determination in an organic medium are 55°C, pH 6.5, 1.0×10−2 mol l−1 OPDA, 1.25 ml of ethanol, 1.25 ml of 1,4-dioxane and 1.25 ml of acetone. The linear ranges of the method proposed in ethanol, 1,4-dioxane and acetone media were 0.4–19.3 U, 0.1–20.8 U, 0.4–21.0 U with detection limit (signal:background=3) of 0.09, 0.02 and 0.08 U, respectively. The proposed method has been applied to the determination of laccase activity in real samples with more accuracy and sensitivity than the previous method reported.