**4. Materials and Methods**

## *4.1. Chemicals and Recombinant Enzymes*

GAA was purchased from Baoji Herbest Bio-Tech (Xi-An, Shaanxi, China). UDP-glucose was obtained from Cayman Chemical (Ann Arbor, MI, USA). Recombinant BsGT enzymes (BsGT110, BsUGT398, BsUGT489, BsGT292, and BsGT296) were obtained from our previous studies [6,17]. The other reagents and solvents used were commercially available.

#### *4.2. Glycosylation of GAA by Recombinant Enzymes*

Glycosylation was performed in 0.1 mL reaction mixture containing 1 mg/mL GAA, 15 μg/mL the recombinant enzymes, 10 mM MgCl2, and 10 mM UDP-glucose at pH 5-6 (50 mM acetate buffer) or pH 6–8 (50 mM PB). The reaction was performed at 40 ◦C for 30 min. Afterward, the reaction mixture was analyzed with UPLC. For optimization experiments, the above reaction mixture was incubated with 50 mM acetate buffer (pH 6) at different temperatures or with different metal ions.

For the kinetic experiments, different concentrations of GAA were mixture with 15 μg purified BsGT110 protein, 10 mM UDP-glucose, 10 mM MgCl2, and 50 mM PB (pH 7.0) or acetate buffer (pH 6) in 1 mL reaction mixture and incubated at 40 ◦C for 20 min. During the incubation, samples from each reaction were removed every 2 min and analyzed by UPLC. The amount of GAA-26-*O*-β-glucoside production from the reaction was calculated from the peak area of UPLC analysis normalized with a standard curve. The rate of the reaction at each concentration of GAA was obtained from the slope of the plot of the amount of product over time. Kinetic parameters were obtained from the double-reciprocal plot of substrate GAA concentration versus the rate of reaction.

#### *4.3. Ultra-Performance Liquid Chromatography (UPLC)*

UPLC was performed with an Acquity® UPLC system (Waters, Milford, MA, USA). The stationary phase was a C18 column (Acquity UPLC BEH C18, 1.7 μm, 2.1 i.d. × 100 mm, Waters, MA, USA), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The linear gradient elution condition was 0 min with 36% B to 7 min with 81% B at a flow rate of 0.2 mL/min. The detection condition was set at 254 nm.
