- Enzymatic transformation of 2-O-α-d-glucopyranosyl-L-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli
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This work aims to produce 2-O-α-d-glucopyranosyl-L-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl2 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.
- Zhang, Zichen,Li, Jianghua,Liu, Long,Sun, Jun,Hua, Zhaozhe,Du, Guocheng,Chen, Jian
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- Walking a Fine Line with Sucrose Phosphorylase: Efficient Single-Step Biocatalytic Production of l-Ascorbic Acid 2-Glucoside from Sucrose
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The 2-O-α-d-glucoside of l-ascorbic acid (AA-2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA-2G is currently produced through biocatalytic glucosylation of l-ascorbic acid from starch-derived oligosaccharides. Sucrose would be an ideal substrate for AA-2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8–6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2-O-α-glucosylation of l-ascorbic acid from sucrose with high efficiency and perfect site-selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L?1; 460 mm), from which pure AA-2G was readily recovered in ～50 % overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a “reverse-protonation” mechanism in which the catalytic base Glu232 on the glucosyl–enzyme intermediate must be protonated for attack on the anomeric carbon from the 2-hydroxyl of the ionized l-ascorbate substrate.
- Gudiminchi, Rama Krishna,Nidetzky, Bernd
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- Enhanced Synthesis of 2-O-α- d -Glucopyranosyl- l -ascorbic Acid from α-Cyclodextrin by a Highly Disproportionating CGTase
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2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an industrially important derivative of vitamin C [l-ascorbic 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.
- Gudiminchi, Rama Krishna,Towns, Andrew,Varalwar, Subhash,Nidetzky, Bernd
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- METHOD FOR PRODUCING 2-O- ALPHA-D-GLUCOSYL-L-ASCORBIC ACID ANHYDROUS CRYSTAL-CONTAINING POWDER
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[Object] An object of the present invention is to provide a process for enabling the production of a particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside that significantly, more hardly cakes, even when the production yield of ascorbic acid 2-glucoside does not reach 35% by weight. [Construction] The above object is solved by providing a process for producing a particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside, which comprises allowing a CGTase to act on a solution containing either liquefied starch or dextrin and L-ascorbic acid and then allowing a glucoamylase to act on the resulting solution to obtain a solution with an ascorbic acid 2-glucoside production yield of at least 27%, purifying the obtained solution to increase the ascorbic acid 2-glucoside content to a level of over 86% by weight, precipitating anhydrous crystalline ascorbic acid 2-glucoside by a controlled cooling method or pseudo-controlled cooling method, collecting the precipitated anhydrous crystalline ascorbic acid 2-glucoside, and ageing and drying the collected anhydrous crystalline ascorbic acid 2-glucoside.
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Paragraph 0125; 0126; 0127; 0128
(2013/11/05)
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- Highly efficient and regioselective production of an erythorbic acid glucoside using cyclodextrin glucanotransferase from Thermoanaerobacter sp. and amyloglucosidase
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In order to continuously supply erythorbic acid (EA) for long-term cell cultures, we synthesized a stable EA derivative, 2-O-α-d-glucopyranosyl-d- erythorbic acid (EA-2G), as a useful tool for analyzing the biological function of EA. The specific and effi
- Tai, Akihiro,Iwaoka, Yuji,Ito, Hideyuki
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- Synthesis of 2-O-α-D-Glucopyranosyl L-Ascorbic Acid by Cyclomaltodextrin Glucanotransferase from Bacillus stearothermophilus
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Cyclomaltodextrin glucanotransferase (CGTase) was found to catalyze the transglycosylation from α-cyclodextrin (α-CD) to L-ascorbic acid (AA).A main product formed by this reaction was identified as 2-O-α-D-glucopyranosyl L-ascorbic acid (AA-2G) with the enzymatic hydrolysis and ultraviolet absorption spectra using standard AA-2G.A series of maltooligosaccharide substituted 2-O-derivatives of AA were also detected by HPLC.These were thoroughly hydrolyzed to AA-2G and glucose by treatment with glucoamylase .A large amount of AA-2G was prepared with 500 g AA and 1000 g of α-CD.Two hundred and fifty grams of purified AA-2G, the content of which was 97percent, was obtained through enzyme reactions and purification with HPLC using ion-exchange resin.
- Aga, Hajime,Yoneyama, Masaru,Sakai, Shuzo,Yamamoto, Itaru
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p. 1751 - 1756
(2007/10/02)
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