Carbohydrate-Binding Module-Cyclodextrin Glycosyltransferase Fusion Enables Efficient Synthesis of 2-O-D-Glucopyranosyl-L-Ascorbic Acid with Soluble Starch as the Glycosyl Donor

In this study, we achieved the efficient synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) from soluble starch by fusing a carbohydrate-binding module (CBM) from Alkalimonas amylolytica alpha-amylase (CBMAmy) to cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans. One fusion enzyme, CGT-CBMAmy, was constructed by fusing the CBMAmy to the C-terminal region of CGTase, and the other fusion enzyme, CGT Delta E-CBMAmy, was obtained by replacing the E domain of CGTase with CBMAmy. The two fusion enzymes were then used to synthesize AA-2G from soluble starch as a cheap and easily soluble glycosyl donor. Under the optimal conditions, the AA-2G yields produced using CGT Delta E-CBMAmy and CGT-CBMAmy were 2.01 g/liter and 3.03 g/liter, respectively, which were 3.94- and 5.94-fold of the yield from the wild-type CGTase (0.51 g/liter). The reaction kinetics of the two fusion enzymes were analyzed and modeled to confirm the enhanced specificity toward soluble starch. It was also found that, compared to the wild-type CGTase, the two fusion enzymes had relatively high hydrolysis and disproportionation activities, factors that favor AA-2G synthesis. Finally, it was speculated that the enhancement of soluble starch specificity may be related to the changes of substrate binding ability and the substrate binding sites between the CBM and the starch granule.