*2.1. Biotransformation of GAA by Recombinant BsGT110 from B. subtilis ATCC 6633*

Our previous study showed that *B. subtilis* ATCC 6633 can biotransform GAA primarily into one major compound (**1**), GAA-15-*O*-β-glucoside, and one unknown minor compound (**2**) (Figure 1) [17]. To obtain enough unknown compound (**2**) through in vitro enzymatic biotransformation and then identify that compound's chemical structure, we strived to identify corresponding GT enzymes from the *B. subtilis* ATCC 6633 strain. In our previous work, we selected five GT genes—BsGT110, BsGT292, BsGT296, BsUGT398, and BsUGT489—and successfully overexpressed and purified them in *Escherichia coli* [17]. However, none of them were found to catalyze the biotransformation of GAA into compound (**2**) under a general GT reaction condition: 10 mM Mg2<sup>+</sup>, 40 ◦C, and pH 8 [17]. We assayed the five recombinant BsGTs under different pH values and determined that BsGT110 produces a reasonable amount of compound (**2**) from the biotransformation of GAA under an acidic condition (pH 6), as shown in the solid line in Figure 2a. BsUGT398 and BsUGT489 produced only small amounts of compound (**2**) under the acidic condition (pH 6) (solid lines in Figure 2b,c). As expected, compound (**2**) was no longer produced from the biotransformation of GAA by any of the three GTs at pH 8 (dashed lines in Figure 2a–c). BsUGT398 and BsUGT489 produced large amounts of GAA-15-*O*-β-glucoside at pH 8. However, no metabolite was detected from the reactions with BsGT292 and BsGT296 at pH 6 or pH 8 (data not shown). We thus selected BsGT110 to produce compound (**2**) at pH 6 for further analysis. The amount of GAA-15-*O*-β-glucoside and compound (**2**) that can be catalyzed from GAA by BsGT110 at different pH values were indicated in Table 1. It is noted that the maximum amount of compound (**2**) was produced under 1 mg/mL GAA, 10 mM UDP-glucose, 15 μg/mL BsGT110, 10 mM MgCl2, and 50 mM acetate buffer at pH 6.

(**a**)

**Figure 2.** *Cont.*

**Figure 2.** Ultra-performance liquid chromatography (UPLC) analysis of the biotransformation of GAA by BsGT110 (**a**), BsUGT398 (**b**), and BsUGT489 (**c**) at pH 6 (solid line) and pH 8 (dashed line). The biotransformation mixture contained 15 μg/mL purified enzyme, 1 mg/mL GAA, 10 mM uridine diphosphate (UDP)-glucose, 10 mM MgCl2, and 50 mM acetate buffer at pH 6 or phosphate buffer (PB) at pH 8 and was incubated at 40 ◦C for 30 min. After incubation, the reaction was analyzed using UPLC. The UPLC operation procedure was described in the Materials and Methods section.


**Table 1.** Relative production <sup>a</sup> of GAA-15-*O*-β-glucoside and compound (**2**) catalyzed from GAA by BsGT110.

<sup>a</sup> Relative production was normalized to the UPLC area of the peak of compound (**2**) in an acetate buffer of pH 6. <sup>b</sup> 50 mM of acetate buffer. <sup>c</sup> 50 mM of PB.

To optimize the production of compound (**2**), a standard mixture was made of 1 mg/mL GAA, 10 mM UDP-glucose, 15 μg/mL BsGT110, and 50 mM acetate buffer at pH 6 under different temperature and metal ion conditions. After incubation, the amount of compound (**2**) produced was determined with UPLC (Figure 3). The results revealed that the optimal conditions for the production of compound (**2**) from GAA by the recombinant BsGT110 is pH 6, 40 ◦C, and 10 mM MgCl2. The relative production of GAA-15-*O*-β-glucoside was less than 5% for all testing conditions.

**Figure 3.** The production of compound (**2**) from GAA by BsGT110 under different temperature or metal ion conditions. The standard condition was set as 15 μg/mL purified enzyme, 1 mg/mL GAA, 10 mM MgCl2, and 10 mM UDP-glucose in 50 mM acetate buffer at pH 6.0 and 40 ◦C. The tests were carried out by changing only the temperature (**a**) or metal ions (**b**) and maintaining all other settings. Relative activities were obtained by dividing the area summation of the UPLC reaction peak of the test condition by that of the standard condition. The data are expressed as mean ± SD, *n* = 3.
