*3.4. Determination of Transglycosylation Activity by Recombinant PgMA*

Transglycosylation activity is an important property of MAs for biotechnology applications. To clarify the transglycosylation activity of recombinant *Pg*MA, 17 different molecules, including mangiferin (Table S2), belonging to triterpenoids, saponins, flavonoids, flavonoid glycosides, or xanthone glycoside, were used as sugar acceptors with 1% (*w*/*v*) β-CD (as the sugar donor) for activity. The reaction mixture was then analyzed with HPLC. The results showed that only puerarin (Figure 5a) and mangiferin (Figure 5b) could be glycosylated by *Pg*MA.

**Figure 5.** High-performance liquid chromatography (HPLC) analysis of the biotransformation products of puerarin (**a**) and mangiferin (**b**) by *Pg*MA. The reaction was performed with 1% (*w*/*v*) β-CD, 5.6 μg/mL of *Pg*MA, and 1 mg/mL of puerarin or mangiferin at 50 mM of PB (pH 7) and 65 ◦C for 24 h. After the reaction, the reaction mixture was analyzed with HPLC. The conditions for HPLC are described in Section 2.

Except mangiferin and puerarin, the other four tested triterpenoids, two triterpenoids saponins, nine flavonoid aglycones, and glycosides could not act as the sugar acceptors in the transglycosylation of *Pg*MA. *Pg*MA could transglycosylate puerarin, which has the isoflavone-8-*C*-glucosdie structure. However, *Pg*MA could not transglycosylate isoflavone-7-*O*-glucoside (8-hydroxydaidzein-7-*α*-*O*-glucoside) or flavone-8-*C*-glucoside (vitexin). The results imply that *Pg*MA has a narrow and/or specific substrate range. Nevertheless, the

main finding is that *Pg*MA can glycosylate mangiferin, which will expand the biotechnological applications of MAs in the future. MAs have been proven to glycosylate some small molecules, such as hydroquinone [29], caffeic acid [30], ascorbic acid [31], puerarin [32–34], genistin [35], neohesperidin [36], and naringin [37]. Our results also showed that *Pg*MA glycosylated puerarin to three major products, P1, P2, and P3 (Figure 5a). These three major products were not identified in advance because the glycosylation of puerarin has been studied based only on known MAs [32,34]. Li et al. (2004) reported that *Bs*MA glycosylated puerarin to three products (T1, T2, and T3), two of which were identified as maltosyl-α-(1→6)-puerarin (T1) and glucosyl-α-(1→6)-puerarin (T2), while T3 was not identified [34]. Li et al. (2011) further reported that a maltogenic amylase (*Tf*MA) from the archaeon *T. pendens* glycosylated puerarin to a series of products containing glucosyl puerarin and maltosyl puerarin, although they did not identify the exact chemical structures of the products [32]. From the results of the two studies, the P1–P3 products might contain glucosyl and maltosyl puerarin.

The results revealed that *Pg*MA glycosylates mangiferin to produce low amounts of the M1 compound with a yield of 2.3% (Figure 5b). This is the first study to report that MA could glycosylate mangiferin, of which mangiferin glycoside may have better aqueous solubility for different applications. Therefore, we mainly focused on the unknown mangiferin glycoside by *Pg*MA in the following assays.
