2.6 Transglycosylation catalyzed by rAGL
The transglycosylation reaction was performed as previously described with modification [18]. Briefly, the reaction mixture (0.3 mL) consisted of 267 mM maltose, 178 mM ascorbic acid sodium salt (AA·Na), 13 mM thiourea, 1.12 U of rAGL (in 150 μL culture supernatant) , and sodium acetate buffer (100 mM, pH 4.0), and was incubated in the dark at 37 °C for 1–6 h, unless otherwise specified. The samples taken every hour were mixed with 600 μL of 1.06% (w/v) metaphosphoric acid to stop the reaction, and the solutions were centrifuged at 12000 × g for 10 min. The supernatant was collected and stored at -20 °C waiting for high performance liquid chromatography (HPLC) analysis. On the basis of the initial reaction condition, that is, the reaction last for 6 h at 37 °C, pH 4.0, with the molar ratio of maltose to AA·Na set to 3:2, the influence of reaction temperature (30, 37, 40, 45, and 50 °C), pH (100 mM sodium citrate buffer at pH 3.0–3.5 and 100 mM sodium acetate buffer at pH 4.0–5.5), and maltose/AA·Na molar ratio (1:1, 3:1 and 3:2) on the synthesis of AA-2G catalyzed by rAGL was investigated. Besides, the influence of enzyme concentrations on AA-2G synthesis was also investigated. The reaction mixture was scaled up to 10 mL, containing 267 mM maltose, 267 mM AA·Na, 13 mM thiourea, various concentrations of rAGL (1, 2, 3 and 4 U/mL) , and sodium acetate buffer (100 mM, pH 4.0), and was incubated in the dark with shaking at 37 °C for 1–8 h. All samples (200 μL) collected each hour were mixed with 600 μL of 1.06% (w/v) metaphosphoric acid to stop the reaction. Other treatments to the samples were the same as the above.