Laccase Activity Determination by ABTS Oxidation

The laccase activity was determined by using ABTS as substrate and measuring the absorbance change caused by the action of enzyme on the substrate within 1 min using a Specord 200 spectrophotometer (Analytic Jena, Germany) and WinASPECT PLUS software [31 (link)]. Molar absorption coefficient (ε) for ABTS at 420 nm and for DMP at 470 nm was 36,000 M^-1 cm^-1 and 14,800 M^-1 cm^-1 [32 (link)]. The reaction mixture contained 250 μL of 10 mM ABTS or DMP, 740 μL of Mc Ilvaine Buffer (composed of 0.2 M dipotassium hydrogen phosphate and 0.1 M citric acid, buffered to pH 4.5), and 10 μL of enzyme extract (Jasińska et al. [33 (link)]. One unit of laccase activity (U) was defined as the concentration of the enzyme required to oxidize 1 μM of substrate per minute.
Enzyme activity was calculated according to the formula adopted from Leonowicz and Grzywnowicz [34 (link)] with slight modifications:
$\mathrm{L}\mathrm{a}\mathrm{c}\mathrm{c}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left[\mathrm{U}/\mathrm{L}\right]=(\mathrm{\Delta }\mathrm{A}\mathrm{b}\mathrm{s}\times \mathrm{V}\times {10}^6)/\left(36,000\times \mathrm{V}\mathrm{e}\times \mathrm{\Delta }\mathrm{t}\right),$
where ΔAbs is the difference in absorbance values at Abs₆₀ and Abs₀;
V is the total volume of the sample [mL]; Ve is the volume of the enzyme [mL]; and Δt is the time [min].
The protein concentration was determined using the BCA test (bicinchoninic acid assay) according to the Pierce^™ BCA Protein Assay Kit protocol (Thermo Fisher Scientific).