The concentrations of 5‐HMF, FFA, FFCA and FDCA were determined using HPLC (JASCO, Tokyo, Japan) equipped with a fast acid analysis chromatographic column connected to a guard column (Biorad, Richmond, CA, USA), refractive index detector (ERC, Kawaguchi, Japan), a JASCO UV detector operating at 215 nm and a JASCO intelligent autosampler. The column temperature was maintained at 65°C in a chromatographic oven (Shimadzu, Tokyo, Japan). Samples were diluted with Milli‐Q quality water and mixed with 20% (v/v) H2SO4 (20 µl ml−1 sample) and then filtered. A 40 µl aliquot was injected in 0.5 mM H2SO4 mobile phase flowing at a rate of 0.4 ml min−1. The peaks for the different compounds were confirmed and quantified using the corresponding external standards.
To determine the FAD bound to the enzyme, 500 µl of 1 mg ml−1 of pure MycspAAO‐WT was filtered using 30 kDa cut‐off and washed twice using 500 µl of 0.1 M phosphate buffer pH 8, and then denatured at 100°C for 15 min and separated by centrifugation at 15 000 g for 5 min. The released FAD was detected at 450 nm in a spectrophotometer (Boateng et al., 2015 (link), Dishisha et al., 2019 (link)), and the concentration was calculated based on a standard curve.
To determine the FAD bound to the enzyme, 500 µl of 1 mg ml−1 of pure MycspAAO‐WT was filtered using 30 kDa cut‐off and washed twice using 500 µl of 0.1 M phosphate buffer pH 8, and then denatured at 100°C for 15 min and separated by centrifugation at 15 000 g for 5 min. The released FAD was detected at 450 nm in a spectrophotometer (Boateng et al., 2015 (link), Dishisha et al., 2019 (link)), and the concentration was calculated based on a standard curve.
