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

Abstract

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 ␣-amylase (CBM Amy ) to cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans. One fusion enzyme, CGT-CBM Amy , was constructed by fusing the CBM Amy to the C-terminal region of CGTase, and the other fusion enzyme, CGT⌬E-CBM Amy , was obtained by replacing the E domain of CGTase with CBM Amy . 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⌬E-CBM Amy and CGT-CBM Amy 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.

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

“…The wild-type CGTase produced the highest AA-2G titer at 36°C, which was also reported in our previous work (9-11) and was similar to the optimal temperature for ␣-CGTase-catalyzed AA-2G synthesis with ␤-cyclodextrin as the glycosyl donor (7). However, for all mutant CGTases, the optimal temperature for AA-2G synthesis was 28°C, which was the same as that of the chimeric enzyme constructed by the fusion of CGTase to the carbohydrate-binding module (CBM) of A. amylolytica (CGT-CBM Amy ) and of the enzyme constructed by replacing the E domain of CGTase with that of CBM Amy (CGT⌬E-CBM Amy ) (11). Figure 3 shows the influence of pH on AA-2G synthesis by the wild-type and mutant CGTases.…”

“…This outcome suggested that mutagenesis was effective for improving the specificity of CGTase toward maltodextrin by affecting the hydrogen bond interactions between the enzymes and linear substrates. In addition, we enhanced the transformation efficiency of soluble starch to AA-2G by fusing a carbohydrate-binding module from Alkalimonas amylolytica ␣-amylase with CGTase from P. macerans (11).…”

Fusion of Self-Assembling Amphipathic Oligopeptides with Cyclodextrin Glycosyltransferase Improves 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis with Soluble Starch as the Glycosyl Donor

“…In summary, the enhanced soluble starch specificity for AA-2G synthesis was achieved via fusion of self-assembling amphipathic peptides to the N terminus of CGTase from P. macerans. Similarly, soluble starch substrate specificity was improved by fusing the carbohydrate-binding module of A. amylolytica ␣-amylase with CGTase from P. macerans in a previous study (11). These outcomes suggest that protein fusion technology is an effective approach to the modification of CGTase for AA-2G production.…”

“…2A, the optimal temperature for AA-2G production with the wild-type CGTase was 36°C, which was also reported in our previous studies (8)(9)(10) and is similar to the optimal temperature for ␣-CGTase-catalyzed AA-2G synthesis with ␤-cyclodextrin as the glycosyl donor (6). However, SAP5-CGTase and SAP6-CGTase produced the highest AA-2G titer at 28°C, and this optimal temperature is the same as that of production with the chimeric enzymes CGT-CBM Amy and CGT⌬E-CBM Amy (11). Figure 2B shows the influence of pH on AA-2G synthesis with the wild-type CGTase, SAP5-CGTase, and SAP6-CGTase with soluble starch as the glycosyl donor.…”

“…For instance, we have enhanced the transformation efficiency of maltodextrin for AA-2G production through site-directed saturation mutagenesis of the Ϫ3 subsite (8), ϩ2 subsite (9), and Ϫ6 subsite residues (10) in the CGTase from Paenibacillus macerans. Furthermore, we improved the transformation efficiency of soluble starch to AA-2G by fusing the carbohydrate-binding module from Alkalimonas amylolytica ␣-amylase with CGTase from P. macerans (11).…”