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

Abstract

In this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans to catalyze 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33-and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.

Enhanced Synthesis of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid from α-Cyclodextrin by a Highly Disproportionating CGTase

“…To replace cyclodextrin with a less expensive and in terms of glucosyl content more completely utilizable donor substrate (e.g., starch, maltodextrin, or maltose), other CGTases in native or engineered form were considered. Variants of P. macerans CGTase showed enhanced specificity toward these noncyclic substrates. − However, the AA-2G yields thus obtained were still much lower than the “benchmark” yield using cyclodextrin. ,,, Interestingly, the increased disproportionation activity of the CGTase variants proved to be beneficial for the yield of AA-2G from cyclodextrin. ,, However, the engineered CGTases often showed a substantially lowered efficiency in the reaction with l -AA as compared to that with the wild-type enzyme. Table indicates strong variation in the degree of utilization of l -AA in the different studies of AA-2G synthesis performed.…”

“…30,32,48,49 Interestingly, the increased disproportionation activity of the CGTase variants proved to be beneficial for the yield of AA-2G from cyclodextrin. 32,50,51 However, the engineered CGTases often showed a substantially lowered efficiency in the reaction with L-AA as compared to that with the wild-type enzyme. Table 1 indicates strong variation in the degree of utilization of L-AA in the different studies of AA-2G synthesis performed.…”

Biocatalytic synthesis of 2‐O‐α‐D‐glucopyranosyl‐L‐ascorbic acid using an extracellular expressed α‐glucosidase from Oryza sativa

“…[ 10 ] The strategies including engineering CGTase and creating the fusion enzyme have effectively improved the specificity towards maltose or soluble starch, which are cheap and can be easily dissolved in aqueous solution. [ 47,48 ] Sucrose phosphorylase is another candidate enzyme that catalyzes the formation of AA‐2G from sucrose and L‐AA, but the existence of by‐product 6‐ O ‐ α ‐glucopyranosyl‐L‐ascorbic acid inevitably increased difficulty of AA‐2G purification. [ 17 ] At low pH value of 5.2, the sucrose phosphorylase from B. longum was proved to be both efficient and site‐selective for AA‐2G synthesis.…”

Comprehensive study on transglycosylation of CGTase from various sources

“…Previous studies have found that maltodextrin specificity is enhanced by site-saturation engineering of lysine 47 [ 46 ], tyrosine 195, tyrosine 260, and glutamine 265 [ 48 ], or the iterative saturation mutagenesis of -6 sub site residues [ 47 ] in α-CGTase extracted from Paenibacillus macerans when used to synthesize 2-O-D-glucopyranosyl-L-ascorbic acid. Moreover, starch specificity can be enhanced by chimeric modification, including fusion of the carbohydrate-binding module to the carboxyl terminus of CGTase, fusion of self-assembling amphipathic oligopeptides with α-CGTase [ 45 ], or replacing the E domain of α-CGTase [ 43 ], to synthesize 2-O-D-glucopyranosyl-L-ascorbic acid. The donor's specificity improvement makes the glycosyl donors more soluble and the synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid cheaper [ 44 ].…”