*2.2. Biotransformation of 8-OHDe by Recombinant DgAS Protein*

The purified DgAS were used to catalyze the biotransformation of 8-OHDe. The reaction contained 0.1 mg/mL of 8-OHDe, 12.5 μg/mL of DgAS, and 20 mM of sucrose as the sugar donor. The reaction mixtures at 0 min (the dashed line) and after 30-min incubation were analyzed with ultra-performance liquid chromatography (UPLC). One major product with a retention time (RT) of 3.1 min was observed (Figure 2).

**Figure 2.** Biotransformation of 8-OHDe with DgAS. The reaction mixtures at 0 min (dashed line) and 30 min (solid line) were analyzed with UPLC. The UPLC operation conditions are described in Materials and Methods.

To optimize compound (**1**) production for further analysis, a standard mixture with 1 mg/mL of 8-OHDe and 125 μg/mL of DgAS was carried out at different temperatures, pH levels, and serial concentrations of sucrose for 30-min incubation. After incubation, the production conversion of the major product was determined with UPLC (Figure 3). The results revealed the optimal condition for the production of compound (**1**) from 8-OHDe by the recombinant DgAS is pH 7, 40 ◦C, and 300 mM of sucrose.

**Figure 3.** Optimal condition for the production of compound (**1**) from 8-OHDe by DgAS. (**a**) 125 μg of the purified DgAS and 1 mg/mL of 8-OHDe were incubated at different temperatures, (**b**) serial concentrations of sucrose, and (**c**) pH levels for 30 min. After incubation, the reaction was analyzed with UPLC. The conversion was calculated by dividing the amount of the produced compound (**1**) in each reaction by the theoretical production value (1.6 mg) for 100% conversion. The mean (*n* = 3) is shown, and the standard deviations are represented by error bars.
