Agilent 1290 diode array detector

The total GA was detected using the HPLC method according to the previously described method [33 (link)]. Briefly, dried mycelia (0.05 g) were weighed and fully ground and then added to 2 mL of a 10% (w/v) KOH-75% (v/v) ethanol solution. The samples were left for 2 h for saponification and the mixture was extracted 3 times with 2 mL hexane. Samples of the hexane layer were collected and vaporized under nitrogen gas until dry. The residue was dissolved in 0.5 mL of acetonitrile. An Agilent 1290 Infinity UPLC system (Agilent Technologies, Santa Clara, CA, USA) was used with an Agilent 1290 diode array detector and an Agilent Zorbax Eclipse Pluss C18 Rapid Resolution HD 18-Micron column (2.1 ×100 mm) (Agilent, Tokyo, Japan).

Reaction stoichiometry was determined by correlating the amounts of O₂ consumed and vanillin produced using an oxygraph and HPLC-based assays. The sample was prepared by reacting 50 μM lignostilbene or DCA-S with ∼0.1 μΜ LSD4 in air-saturated 40 mM HEPES (I = 0.1 M, pH 7.5) at 25 °C. The reaction was quenched upon the consumption of ∼50 μM O₂ with 1% (v/v) glacial acetic acid. The protein was removed by centrifugation and passage through 0.22 μm filter. Vanillin produced from the LSD4-catalyzed reaction was quantified using an Agilent 1290 series UPLC system equipped with a Luna 2.5 um C18(2)-HST 100 Å column (Phenomenex) and a gradient of acetonitrile in 0.16% formic acid. Vanillin was detected at 265 nm using an Agilent 1290 diode array detector. Peak areas were converted to concentrations using a calibration curve established with authentic vanillin (R² > 0.995).