Glycosyltransferase activity assays

The reaction mixture contained 0.5 mM sugar donors (1.0 mM for di-glycosylation), 0.25 mM aglycons, 50 mM Tris-HCl (pH 7.4), and 50‒100 μg of purified AbCGT in a final volume of 100 μL. Due to the instability of some acceptors in the buffer of pH 11.0, the reactions were performed under the physiological conditions (pH 7.4, 30 °C) to compare the catalytic activities of AbGT27 with different substrates under the same conditions. The reactions were performed at 30 °C for up to 12 h and terminated by the addition of 200 μL of ice-cold methanol. Subsequently, samples were centrifuged at 15,000 × g for 30 min to collect the supernatant, and aliquots were analyzed by HPLC-UV/ESIMS. HPLC analysis was performed on a Shiseido CAPCELL PAK C18 MG III column (250 mm × 4.6 mm I.D., 5 μm, Shiseido Co., Ltd., Japan) at a flow rate of 1 mL min^‒1. The mobile phase was a gradient elution of solvents A (0.1% formic acid aqueous solution) and B (methanol). The gradient programs were used for the analysis of the reactions (Supplementary Table ⁶). The conversion rates of the enzyme reactions were calculated from peak areas of glycosylated products and substrates as analyzed by HPLC. To facilitate inferential statistical analysis, three parallel assays were routinely performed; the means ± SD from triplicate analyses are reported here.