2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid widely used in food, cosmetic, and pharmaceutical industries; however, its enzymatic production remains limited by high enzyme consumption and low catalytic efficiency. In this study, a marine-derived cyclodextrin glycosyltransferase (CGTase) was engineered using a semi-rational design strategy to enhance AA-2G synthesis with maltodextrin as a cost-effective glycosyl donor. Based on sequence alignment, two residues were selected for saturation mutagenesis, and beneficial variants (Y260F and A236P) were identified from mutant libraries through activity-based screening. Subsequently, the double mutant Y260F/A236P was constructed and characterized. Compared with the wild-type enzyme, the double mutant achieved an AA-2G concentration of 30.2 g/L, corresponding to an 11.8% increase in yield. Kinetic analysis revealed a 20.8% decrease in Km and a 1.48-fold increase in kcat/Km toward L-ascorbic acid. Under optimized conditions, the engineered system achieved high AA-2G yield with significantly reduced enzyme loading (120 U/g substrate), and the double mutant enabled rapid conversion, reaching high yield within 12 h. In addition, A236 was identified as a novel mutagenesis site in CGTases. These results provide an efficient and cost-effective strategy for AA-2G production and offer new insights into enzyme engineering.
| [1] |
Aerts D, Verhaeghe T, Joosten HJ, Vriend G, Soetaert W, Desmet T. Consensus engineering of sucrose phosphorylase: The outcome reflects the sequence input. Biotechnol Bioeng, 2013, 110(10): 2563-72.
|
| [2] |
Aga H, Yoneyama M, Sakai S, Yamamoto I. Synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus. Agric Biol Chem, 1991, 55(7): 1751-6.
|
| [3] |
Ali A, Riaz S, Khalid W, Fatima M, Mubeen U, Babar Q, Manzoor M, Khalid M, Madilo F. Potential of ascorbic acid in human health against different diseases: an updated narrative review. Int J Food Prop, 2024, 27(1): 493-515.
|
| [4] |
Amin N, Liu AD, Ramer S, Aehle W, Meijer D, Metin M, Wong S, Gualfetti P, Schellenberger V. Construction of stabilized proteins by combinatorial consensus mutagenesis. Protein Eng Des Sel, 2004, 17(11): 787-93.
|
| [5] |
Chen S, Xiong Y, Su L, Wang L, Wu J. Position 228 in Paenibacillus macerans cyclodextrin glycosyltransferase is critical for 2-O-D-glucopyranosyl-L-ascorbic acid synthesis. J Biotechnol, 2017, 247: 18-24.
|
| [6] |
Chen H, Liu Y, Ren X, Wang J, Zhu L, Lu Y, Chen X. Engineering of cyclodextrin glycosyltransferase through a size/polarity-guided triple-code strategy with enhanced α-glycosyl hesperidin synthesis ability. Appl Environ Microbiol, 2022, 88(17): e0102722.
|
| [7] |
Chen H, Ju L, Dong Y, Lu S, Bao Y, Zhu L, Chen X. Kinetics-guided engineering of cyclodextrin glycosyltransferase with enhanced intermolecular transglycosylation activity. AIChE J, 2024, 70: e18512.
|
| [8] |
Dauter Z, Dauter M, Brzozowski AM, Christensen S, Borchert TV, Beier L, Wilson KS, Davies GJ. X-ray structure of Novamyl, the five-domain maltogenic α-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7 Å resolution. Biochemistry, 1999, 38(26): 8385-92.
|
| [9] |
Eibaid A, Bashari M, Miao M, Musa A, Zhang T, Jiang B. Biosynthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid from maltose by cyclodextrin glucanotransferase from Bacillus sp. SK13.002. J Food Nutr Res, 2014, 2(4): 193-7.
|
| [10] |
Fujiwara S, Kakihara H, Sakaguchi K, Imanaka T. Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability. J Bacteriol, 1992, 174(22): 7478-81.
|
| [11] |
Gudiminchi RK, Towns A, Varalwar S, Nidetzky B. Enhanced synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid from α-cyclodextrin by a highly disproportionating CGTase. ACS Catal, 2016, 6(3): 1606-15.
|
| [12] |
Han R, Liu L, Li J, Du G, Chen J. Functions, applications and production of 2-O-D-glucopyranosyl-L-ascorbic acid. Appl Microbiol Biotechnol, 2012, 95(2): 313-20.
|
| [13] |
Han R, Liu L, Shin HD, Chen RR, Li J, Du G, Chen J. Systems engineering of tyrosine 195, tyrosine 260, and glutamine 265 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid synthesis. Appl Environ Microbiol, 2013, 79(2): 672-7.
|
| [14] |
Han R, Li J, Shin HD, Chen RR, Du G, Liu L, Chen J. Recent advances in discovery, heterologous expression and molecular engineering of cyclodextrin glycosyltransferase for versatile applications. Biotechnol Adv, 2014, 32(2): 415-28.
|
| [15] |
Hao J, Huang LP, Chen X, Sun J, Liu J, Wang W, Sun M. Identification, cloning and expression analysis of an α-CGTase produced by Bacillus sp. Y112. Protein Expr Purif, 2017, 140: 8-15.
|
| [16] |
Harata K, Haga K, Nakamura A, Aoyagi M, Yamane K. X-ray structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011. Comparison of two independent molecules at 1.8 Å resolution. Acta Crystallogr D, 1996, 52(6): 1136-45.
|
| [17] |
Jiang Y, Zhou J, Wu R, Xin F, Zhang W, Fang Y, Ma J, Dong W, Jiang M. Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production. BMC Biotechnol, 2018, 18(1): 53.
|
| [18] |
Kelly RM, Leemhuis H, Dijkhuizen L. Conversion of a cyclodextrin glucanotransferase into an α-amylase: assessment of directed evolution strategies. Biochemistry, 2007, 46(39): 11216-22.
|
| [19] |
Kelly RM, Dijkhuizen L, Leemhuis H. The evolution of cyclodextrin glucanotransferase product specificity. Appl Microbiol Biotechnol, 2009, 84(1): 119-33.
|
| [20] |
Klein C, Schulz GE. Structure of cyclodextrin glycosyltransferase refined at 2.0 Å resolution. J Mol Biol, 1991, 217(4): 737-50.
|
| [21] |
Kumar S, Stecher G, Suleski M, Sanderford M, Sharma S, Tamura K. MEGA12: molecular evolutionary genetics analysis version 12 for adaptive and green computing. Mol Biol Evol, 2024, 41(1): 1-9.
|
| [22] |
Lawson CL, van Montfort R, Strokopytov B, Rozeboom HJ, Kalk KH, de Vries GE, Penninga D, Dijkhuizen L, Dijkstra BW. Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J Mol Biol, 1994, 236(2): 590-600.
|
| [23] |
Le SQ, Gascuel O. An improved general amino acid replacement matrix. Mol Biol Evol, 2008, 25(7): 1307-20.
|
| [24] |
Leemhuis H, Dijkstra BW, Dijkhuizen L. Mutations converting cyclodextrin glycosyltransferase from a transglycosylase into a starch hydrolase. FEBS Lett, 2002, 514(2–3): 189-92.
|
| [25] |
Lejeune A, Sakaguchi K, Imanaka T. A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (α-cyclodextrin) glucanotransferase. Anal Biochem, 1989, 181(1): 6-11.
|
| [26] |
Li X, Sun J, Wang W, Guo J, Song K, Hao J. Site-saturation mutagenesis of proline 176 in cyclodextrin glucosyltransferase from Bacillus sp. Y112 affects product specificity and enzymatic properties. Process Biochem, 2020, 94: 180-9.
|
| [27] |
Liu L, Han R, Shin HD, Chen RR, Li J, Du G, Chen J. Biosynthesis of 2-O-D-glucopyranosyl-L-ascorbic acid from maltose by an engineered cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr Res, 2013, 382: 101-7.
|
| [28] |
Nakamura A, Haga K, Yamane K. Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics. Biochemistry, 1994, 33(33): 9929-36.
|
| [29] |
Palanisamy V, Sanphui P, Palanisamy K, Prakash M, Bansal AK. Design of ascorbic acid eutectic mixtures with sugars to inhibit oxidative degradation. Front Chem, 2022, 10: 754269.
|
| [30] |
Pei J, Kim BH, Grishin NV. PROMALS3D: a tool for multiple sequence and structure alignment. Nucleic Acids Res, 2008, 36(7): 2295-300.
|
| [31] |
Porebski BT, Buckle AM. Consensus protein design. Protein Eng Des Sel, 2016, 29(7): 245-51.
|
| [32] |
Reetz MT, Bocola M, Carballeira JD, Zha D, Vogel A. Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. Angew Chem Int Ed, 2005, 44(27): 4192-6.
|
| [33] |
Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res, 2014, 42(W1): W320-4.
|
| [34] |
Rojas AM, Gerschenson LN. Ascorbic acid destruction in sweet aqueous model systems. LWT Food Sci Technol, 1997, 30(6): 567-72.
|
| [35] |
Shen Y, Xia Y, Chen X. Research progress and application of enzymatic synthesis of glycosyl compounds. Appl Microbiol Biotechnol, 2023, 107(14): 5317-28.
|
| [36] |
Shen Y, Guo J, Xia Y, Wei L, Lin X, Liu Y, Chen Y, Bao Y, Yang H, Chen X. Production of 2-O-α-D-glucopyranosyl-L-ascorbic acid using sucrose phosphorylase by semi-rational design. Int J Biol Macromol, 2025, 284: 138213.
|
| [37] |
Shim JH, Kim YW, Kim TJ, Chae HY, Park JH, Cha H, Kim JW, Kim YR, Schaefer T, Spendler T, Moon TW, Park KH. Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR. Protein Eng Des Sel, 2004, 17(3): 205-11.
|
| [38] |
Song K, Sun J, Wang W, Hao J. Heterologous expression of cyclodextrin glycosyltransferase My20 in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production. Front Microbiol, 2021, 12: 664339.
|
| [39] |
Sun T, Wang W, Sun J, Jiang C, Hao J. Expression and enzymatic properties of domain-deleted mutants of cyclodextrin glucosyltransferase My20. Food Sci (in Chinese), 2024, 45(20): 154-60.
|
| [40] |
Tao X, Wang T, Su L, Wu J. Enhanced 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis through iterative saturation mutagenesis of acceptor subsite residues in Bacillus stearothermophilus NO2 cyclodextrin glycosyltransferase. J Agric Food Chem, 2018, 66(34): 9052-60.
|
| [41] |
Tao X, Su L, Wu J. Current studies on the enzymatic preparation of 2-O-α-D-glucopyranosyl-L-ascorbic acid with cyclodextrin glycosyltransferase. Crit Rev Biotechnol, 2019, 39(2): 249-57.
|
| [42] |
Tao X, Kong D, Zhang H, Su L, Chen S, Rao D, Wei B, Wu J, Wang L. Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose. Bioprocess Biosyst Eng, 2023, 46(6): 903-11.
|
| [43] |
Tao X, Su L, Chen S, Wang L, Wu J. Producing 2-O-α-D-glucopyranosyl-L-ascorbic acid by modified cyclodextrin glucosyltransferase and isoamylase. Appl Microbiol Biotechnol, 2023, 107(4): 1233-41.
|
| [44] |
Teze D, Zhao J, Wiemann M, Kazi ZGA, Lupo R, Zeuner B, Vuillemin M, Rønne ME, Carlström G, Duus JØ, Sanejouand YH, O’Donohue MJ, Karlsson EN, Fauré R, Stålbrand H, Svensson B. Rational enzyme design without structural knowledge: A sequence-based approach for efficient generation of transglycosylases. Chemistry-A Eur J, 2021, 27(40): 10323-34.
|
| [45] |
Uitdehaag JCM, Mosi R, Kalk KH, van der Veen BA, Dijkhuizen L, Withers SG, Dijkstra BW. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat Struct Biol, 1999, 6(5): 432-6.
|
| [46] |
van der Veen BA, van Alebeek GJ, Uitdehaag JCM, Dijkstra BW, Dijkhuizen L. The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms. Eur J Biochem, 2000, 267(3): 658-65.
|
| [47] |
van der Veen BA, Leemhuis H, Kralj S, Uitdehaag JCM, Dijkstra BW, Dijkhuizen L. Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin glycosyltransferase. J Biol Chem, 2001, 276(48): 44557-62.
|
| [48] |
Wind RD, Uitdehaag JCM, Buitelaar RMB, Dijkstra BW, Dijkhuizen L. Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. J Biol Chem, 1998, 273(10): 5771-9.
|
| [49] |
Yamamoto I, Muto N, Murakami K, Suga S, Yamaguchi H. L-ascorbic acid alpha-glucoside formed by regioselective transglucosylation with rat intestinal and rice seed alpha-glucosidases: its improved stability and structure determination. Chem Pharm Bull (Tokyo), 1990, 38(11): 3020-3.
|
| [50] |
Yin X, Chen K, Cheng H, Chen X, Feng S, Song Y, Liang L. Chemical stability of ascorbic acid integrated into commercial products: a review on bioactivity and delivery technology. Antioxidants, 2022, 11: 153.
|
| [51] |
Zhang Z, Li J, Liu L, Wang F, Du G, Chen J. Enzymatic transformation of 2-O-α-D-glucopyranosyl-L-ascorbic acid by α-cyclodextrin glucanotransferase from recombinant Escherichia coli. Biotechnol Bioprocess Eng, 2011, 16(1): 107-13.
|
| [52] |
Zhang W, Huang Q, Yang R, Zhao W, Hua X. 2-O-D-glucopyranosyl-L-ascorbic acid: properties, production and potential application as a substitute for L-ascorbic acid. J Funct Foods, 2021, 82: 104481.
|
| [53] |
Zheng S, Zhou W, Lin X, Li F, Xie C, Liu D, Yao D. Increased yield of 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by α-glucosidase using rational design regulating the ground state of enzyme–substrate complex. Biotechnol J, 2023, 18(10): e202300122.
|
Funding
Central Public-interest Scientific Institution Basal Research Fund, CAFS
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Jiangnan University
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