Vwd 1260

The formation of tyramine and 2-phenylethylamine in the different tested conditions was monitored by collecting samples at a defined time (0, 24, 48, 72, and 96 h). The cell-free supernatants were analyzed using an HPLC Agilent Instrument 1260 Infinity with the automatic injector (G1329B ALS 1260, loop of 20 μl), equipped with a UV detector (G1314F VWD 1260) set at 254 nm. A C18 Waters Spherisorb ODS-2 (150 × 4.6 mm, 3 μm) column was used for the chromatographic separation. Before the injection, the samples were subjected to a dansyl chloride derivatization (Sigma Aldrich, Gallarate, Italy) according to the method of Martuscelli et al. (44 (link)). The BAs were expressed as mg/L with reference to the BAs standard calibration curve. All the samples were analyzed in triplicate.

Ten micrograms of the corresponding purified protein was incubated with substrate (5 mM) for 15 min at room temperature in assay‐buffer (50 mM Tris‐HCl pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT) in a final volume of 100 μl in a microtitreplate. The reaction was stopped by freezing samples at −20°C. Five microlitres reaction products were analysed by HPLC (Agilent Technologies 1200 Infinity series) at room temperature (22°C) with the following conditions. Column: organic acid resin, 250 × 8 mm (CS Chromatographie); detector: VWD 1260 (Agilent Technologies) at 210 nm; solvent: 2.5 mM sulfuric acid; flow rate: 1.0 ml min^–1.