Enzyme activity assay

Laccase was freshly prepared and assayed for activity before each experiment. A colorimetric assay was used to quantify the activity of the laccase enzyme, in which ABTS was oxidized by laccase catalysis to ABTS^•+ which strong absorbance at 414 nm was measured for quantification (Cary®50, Varian, Inc.)³⁹. The enzyme activity assay medium was made up of 1.9 mL sodium acetate buffer (10 mM, pH 4.6), 100 μL fresh laccase solution, and 1 mL ABTS (1 mM). One enzyme unit corresponds to the amount of laccase that oxidizes 1 μmol ABTS per min.

The assay protocol described above was also modified and used to explore the influence of labetalol on the conversion of ABTS^•+ (see Fig. S1 of SI). The experiments were performed in 25 mL conical flasks as batch reactors under room temperature. Each reactor contained 10 mL solution containing 5 μM ABTS and 0.1 U mL⁻¹ laccase buffered at pH 7.0. At the end of 30 min incubation, 5 mL methanol was added to the solution to inactivate laccase⁴⁰, after which a 3 mL sample was transferred to a 1-cm quartz cuvette and then 30 μL labetalol (5 μM, final concentration) was added to the reaction solution and mixed. At each of the following time intervals: 0, 2, 4, 6, 10, 14, 20, 24, 30 min, the UV absorbance spectrum of the solution in the cuvette was scanned using a Cary®50 spectrophotometer. A 0.5 mL aliquot of the reaction solution was sampled at each of the reaction times 0 and 30 min. Labetalol concentration remaining in the solution was analyzed using HPLC. Details of the HPLC method for labetalol analysis are shown in SI.