Enzymatic Assays for TYO and HpaBC

The activity assay of TYO was performed at 37 °C by monitoring the absorbance increase at 240 nm which indicates the formation of H₂O₂^{47 (link)}. A reaction mixture (200 μL) for the assay contained 100 mM phosphate buffer (pH 7.0), 0.75 mM dopamine and 0.002 mg mL⁻¹ purified TYO protein. Reaction was performed at 37 °C for 10 min. The H₂O₂ formation was monitored at 240 nm, with a SynergyMx Multi-Mode Microplate Reader (BioTek, USA). One unit of activity was defined as the amount of enzyme that catalyzes the production of 1 μM of H₂O₂ per min.
The HpaBC activity was assayed by quantifying the product formation with HPLC. One unit of activity was defined as the amount of enzyme catalyzing the conversion of 1 mM tyrosine per minute under the assay conditions^{19 (link)}.