Biochemical characterization of recombinant SiUGTs. (A) Substrates used for UGT assay. LC–UV chromatograms of the reaction mixture of the in vitro assay using the purified recombinant SiUGT1 (B) or SiUGT2 (C). The assay was conducted using hydroxytyrosol (left), tyrosol (middle) or phenethyl alcohol (right) as a substrate. Chromatograms show the absorbance at 280 and 254 nm. Note that the amount of protein used in the assay was 0.875 μg for SiUGT1 and 3 μg for SiUGT2. (D) Specific activity of the recombinant SiUGT1–5 toward C6–C2 compounds. The means ± standard error (n = 3, technical replicates) are shown. (E) Relative activities of SiUGT1–5 toward C6–C2 and C6–C3 compounds. The activity toward the compound with the highest specific activity among the target compounds is taken to be 100%. Abbreviation: tr., trace level.
