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

Abstract

2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an industrially important derivative of vitamin C [Lascorbic acid (L-AA)]. A useful synthetic route toward AA-2G is the selective glucosylation of L-AA by cyclodextrin glucanotransferase (CGTase). However, the cyclodextrin donor substrate is utilized rather inefficiently, because only one of its constituent glucosyl residues is coupled to the L-AA acceptor. A CGTase catalyzing disproportionation of the linear maltooligosaccharide chain formed in the initial coupling reaction might utilize a greater portion of the substrate for L-AA glucosylation and thus boost the AA-2G yield of cyclodextrin conversion. We present here a detailed characterization of the transfer reactions involved in the formation of AA-2G from α-cyclodextrin by a commercial CGTase preparation from Thermoanaerobacter sp. (Toruzyme 3.0L). We demonstrate that besides coupling, disproportionation constitutes a major route of glucosylation of L-AA by this enzyme. L-AA glucosides with oligoglucosyl chains between 1 and 12 units long were produced in the reaction. After chain-trimming hydrolysis with glucoamylase, however, AA-2G was recovered as the sole product of the enzymatic transglucosylation. The molar yield of AA-2G from cyclodextrin was 1.4, thus clearly exceeding the maximal yield of 1 for the coupling reaction. Using conditions optimized for transfer efficiency and productivity, we obtained AA-2G at the highest concentration (143 g/L, 424 mM) so far reported from an enzymatic glucosylation of L-AA. The synthetic yield was 30% based on L-AA (250 g/L, 1420 mM) offered in ≤4.6-fold molar excess over α-cyclodextrin.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase

“…It is worth emphasizing that the α-linkage between glucose and pterostilbene is resistant to the action of amyloglucosidase. This resistance was also reported in the case of ascorbic acid [ 43 ] and hydroquinone [ 29 ], but not with genistein α-glucoside [ 31 ].…”

“…Although the CGTase displays a low hydrolytic activity on such linkages [ 41 ], we incorporated a glycosidase to accelerate the hydrolytic process and thus increase the yield of monoglucoside. A similar strategy was successfully employed for the glucosylation of inositol [ 42 ], genistein [ 31 ], ascorbic acid [ 43 ] or hydroquinone [ 29 ]. In the case of pterostilbene, after 10 h reaction with CGTase, we incubated the mixture with a recombinant glucoamylase from S. cerevisiae (var.…”

Walking a Fine Line with Sucrose Phosphorylase: Efficient Single‐Step Biocatalytic Production of l‐Ascorbic Acid 2‐Glucoside from Sucrose

“…AA‐2G is synthesized by a biocatalytic transglucosylation in which starch‐derived cyclic or linear oligosaccharides are reacted with l ‐AA by a glucanotransferase . In spite of the absolute stereoselectivity and the usually high site‐selectivity of the enzymes used, the glucanotransferase process inherently suffers from low yield with the oligosaccharide substrates utilized . Another limitation is that the enzyme attaches oligoglucosyl chains to l ‐AA, and these have to be trimmed to a single glucosyl unit, unless the enzyme has hydrolase side‐activity.…”

“…The synthesis of 1 was performed on the gram scale (Supporting Information), and the product (∼6 g, purity ≥98 %, yield≥50 %) was recovered from the reaction mixture by a two‐step chromatographic work‐up . The expected product structure was confirmed by 1 H and 13 C NMR (Figure S5).…”

Comprehensive study on transglycosylation of CGTase from various sources

“…(CGTase) 2-O-α-D-glucopyranosyl-d-erythorbic acid 4.0 40 °C 49.1 [ 154 ] L-ascorbic acid (Vitamin C) α-CD Thermoanaerobacter sp . (CGTase) 2-O-α-D-Glucopyrano syl-L-ascorbic acid 4.5 50 °C 30 [ 41 ] L-ascorbic acid (Vitamin C) α-maltosyl fluoride (aG2F) engineered cyclodextrin glucanotransferase 3-O-α-maltosyl-L-ascorbate 7.5 25 °C 29 [ 6 ] Diterpenoid Rubusoside Soluble starch Bacillus circulans (CGTase) Tri and tetra-glucosylated rubusoside 5.4 40 °C - [ 119 ] Stevioside Malto dextrin Alkalophilic Bacillus sp . BL-12 (CGTase) mono-, di-, and oligo-glycosyl stevioside 9.0 40 °C 76 [ 62 ] Taxol (paclitaxel) Soluble starch Bacillus macerans (CGTase) 7-Glycolylpaclitaxel 2″-O-α--glucobioside 7.0 40 °C 17 [ 139 ] 7-glycolylpaclitaxel 2′-O-α-glucotrioside 14 7-glycolylpaclitaxel 2′-O-α-glucotetraoside 11 7-glycolylpaclitaxel 2′-O-α-glucopentaoside 7 Stevioside Corn starch Paneibacillus macerans JFB05-01 (CGTase) Monoglucosylated stevioside 7.0 60 °C 77.11 [ 89 ] ...…”