The activity of ATAs towards selected amino acceptors was measured using the acetophenone assay, which measures the rate of acetophenone generation by measuring the UV-absorbance of the assay mixture at 245 nm. Assay mixtures contained 50 mM HEPES buffer (pH 7.5), 5 mM (S)-1-PEA, 20 µM PLP, and 5 mM pyruvate or selected carbohydrates containing 5 mM galactose. When oxidized carbohydrates were used as the amino acceptor, 29.8 µg/mL FgrGaOx, 12.8 µg/mL catalase, and 1.8 µg/mL HRP were also added. The assay mixtures were pre-oxidized at 37 °C for an hour, and transamination reactions were carried out on a UV-compatible 96-well microtiter plate at 200 µL volumes. For each reaction, 30 µg ATA was added (300 ng when using pyruvate as the amino acceptor), and the plate was moved immediately to a plate reader operating at 37 °C without shaking. The absorbance readings were taken at 245 nm in 30-s intervals for 30 min, and linear portions of the absorbance-vs-time graphs were used to calculate the rates of acetophenone formation. All experiments were conducted in 4 replicates (n = 4).
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