In this study, site saturation mutagenesis was performed on the - 3 (R44, D86, S90, and D192) and - 6 subsite (Y163, G175, G176, and N189) of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase to enhance its specificity for the donor substrate maltodextrin for 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) preparation. The AA-2G yields produced by the mutants S90D, G176H, and S90D/G176H were 181, 171, and 185 g/L, respectively. Our previous study found that the mutant K228R/M230L also increased the AA-2G yield. Therefore, the mutants S90D, G176H, S90D/G176H, and K228R/M230L were further used to generate combinatorial mutants. Among these mutants, the highest AA-2G yield (217 g/L) was produced by S90D/K228R/M230L with 500 g/L maltodextrin as the glucosyl donor, which was 56 g/L higher than that produced by wild-type CGTase. In addition, AA-2G was prepared by adding isoamylase to hydrolyze α-1,6 glucosidic linkages in maltodextrin that could not be utilized by CGTase to improve the utilization rate of maltodextrin. The addition of isoamylase reduced the concentration of maltodextrin from 500 to 350 g/L, while the AA-2G yield remained high (208 g/L). The preparation of AA-2G by complexing isoamylase with mutant S90D/K228R/M230L reduced the maltodextrin concentration by 150 g/L, while the AA-2G yield increased by 47 g/L than preparation with wild-type CGTase alone, which laid a foundation for the large-scale preparation of AA-2G. KEY POINTS: • Mutants exhibited improved maltodextrin specificity. • Mutant S90D/K228R/M230L produced high yield of AA-2G with maltodextrin as substrate. • AA-2G was first synthesized by a combination of isoamylase and CGTase.