Abstract: To investigate the effect of cyclodextrin glucosyltransferase (CGTase) produced by the recombinant strain pET-28a(+)-cgt-T1/BL21 (DE3) on the synthesis of 2-O-α-D-glucoside (AA-2G), the recombinant protein CGTase-T1 was expressed in the fermentation medium LB, purified by affinity chromatography and concentrated, followed by the determination of β-cyclization and disproportionation activity. AA-2G was synthesized using vitamin C and β-cyclodextrin as substrates. The effects of different glycan donors, substrate concentration, pH, temperature, substrate ratio, protein concentration and reaction time on AA-2G yield were further explored by single-factor optimization experiments, and the kinetics of CGTase were also analyzed. The results showed that CGTase could synthesize AA-2G, and the yield of AA-2G was 0.67 g/L before optimization of reaction conditions. Considering low cost and economic benefits, soluble starch and maltodextrin were selected as sugar donors in reaction condition optimization. When soluble starch was used as glycan donor, the yield of AA-2G catalyzed by the recombinant CGTase could reach up to 12.68 g/L under the condition as follows: The concentration of soluble starch at 70 g/L, the amount of V_C: glycosyl at 3:3, CGTase-T1 used at 5.0 mg/mL, pH4.5 at 37 ℃ for 42 h reaction. While maltodextrin was used as glycan donor, the yield of AA-2G catalyzed by the recombinant CGTase could reach up to 4.96 g/L under the condition as follows: the concentration of soluble starch at 30 g/L, the amount of V_C: glycosyl at 4:2, CGTase-T1 used at 5.0 mg/mL, pH5.0 at 37 ℃ for 30 h reaction. Under the optimal conditions, the yield of AA-2G was 18.93 times and 7.40 times higher than those of the unoptimized reaction conditions respectively. Through the kinetic analysis of CGTase-T1, it was found that the catalytic efficiency of soluble starch as sugar donor was higher than that of maltodextrin. Therefore, soluble starch as sugar donor was better than maltodextrin, resulting in a higher yield of AA-2G synthesis. In this study, the recombinant CGTase-T1 was successfully applied in the synthesis of AA-2G. By optimizing the reaction conditions of enzymatic catalysis, the output of AA-2G was greatly increased, providing a reference for the industrial production of AA-2G.