*3.5. Optimization of Biotransformation of Mangiferin by PgMA*

As the yield of the M1 compound from the biotransformation by *Pg*MA is too low to be easily purified, the glycosylation condition must be optimized. The glycosylation reaction was optimized with different sugar donors, concentrations of sugar donors, and reaction times. First, although the yield of the M1 using α-CD showed the best output (Figure 6a), maltodextrin was selected as the sugar donor for experiments due to its highly aqueous solubility at a much lower price.

Second, the GH enzymes contained both hydrolysis and transglycosylation activities. The transglycosylation activity of GH has been reported to increase under low water concentrations [43]. Thus, a solution with a higher sugar concentration and lower water concentration would increase its transglycosylation activity. The transglycosylation activity of *Pg*MA was indeed increased in 50% maltodextrin, the highest soluble concentration. When the maltodextrin concentration was increased from 1% to 50% (*w*/*v*), three compounds (M1, M2, and M3 in Figure 6b) were formed with higher yields (Figure 6c). The maximal yields of M1 and M2 reached 10% and 21%, respectively. The M3 compound was not completely separated with mangiferin, which is similar to the situation of the T3 compound with puerarin by *Bs*MA [34]. Therefore, only M1 and M2 were further studied.

**Figure 6.** *Cont.*

**Figure 6.** Effects of the sugar donor on the glycosylation of mangiferin by *Pg*MA. (**a**) Different sugar donors were used in the glycosylation of mangiferin. (**b**) HPLC analysis of the glycosylation mixture of mangiferin by *Pg*MA with 50% (*w*/*v*) maltodextrin. (**c**) Different maltodextrin concentrations were used in the glycosylation of mangiferin. The reaction was performed with 5.6 μg/mL *Pg*MA, 1 mg/mL mangiferin, and the tested sugar donors 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. The yield of the product was calculated by dividing the area of the product by that of mangiferin without an enzyme reaction (control reaction).

> Third, the yields of M1 and M2 in 50% maltodextrin by *Pg*MA were further determined under different time courses (Figure 7). The results showed that the yields plateau of M1 and M2 reached 13.2% at 168 h and 33.8% at 72 h, respectively.

**Figure 7.** Time course of the glycosylation of mangiferin by *Pg*MA. The reaction was conducted with 50% (*w*/*v*) maltodextrin, 5.6 μg/mL *Pg*MA, and 1 mg/mL mangiferin at 50 mM of PB (pH 7) and 65 ◦C. At the interval time, the reaction mixture was analyzed with HPLC. The conditions for HPLC and the calculation of the yield were the same as those described in the legend to Figure 6.
