Oxidative enzymes during decolorization The oxidative enzymes were assayed spectrophotometrically in the cell free extract.
Laccase activity was determined in a reaction mixture of 2 ml containing 10% ABTS in 20 mM potassium phosphate buffer (pH 4.0) and increase in the optical density was measured at 420 nm. Molar extinction coefficient of ABTS was 0.036 mM/cm at 420 nm (21).
Lignin peroxidase activity was determined by monitoring the formation of propanaldehyde at 300 nm in a 2.5-ml reaction mixture containing 100 mM n-propanol, 250 mM tartaric acid, 10 mM H2O2 (6).
Veratryl alcohol oxidase activity was determined by using veratryl alcohol as a substrate (22).
The reaction mixture contained 1 mM veratryl alcohol in 0.1 M citrate phosphate buffer (pH 3.0) and 0.2 ml enzyme.
Total volume of 2 ml was used for the determination ofoxidase activity.
Oxidation of the substrate at room temperature was monitored by an absorbance increase at 310 nm due to the formation of veratraldehyde.
All enzyme assays were conducted in triplicate and the average rates were calculated to represent the enzyme activity. One unit of enzyme activity was defined as a change in absorbance Unit min/mg protein of the enzyme.