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Enhancing 2-O-α-D-glucopyranosyl-l-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose

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Abstract

2-O-α-D-glucopyranosyl-l-ascorbic acid (AA-2G) is a stable derivative of l-ascorbic acid (l-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with l-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased l-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

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References

  1. Yamamoto I, Muto N, Murakami K, Suga S, Yamaguchi H (1990) 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 38:3020–3023. https://doi.org/10.1248/cpb.38.3020

    Article  CAS  Google Scholar 

  2. Wenbin Z, Qicheng H, Ruijin Y, Wei Z, Xiao H (2021) 2-O-D-glucopyranosyl-L-ascorbic acid: properties, production, and potential application as a substitute for L-ascorbic acid. J Funct Foods 82:104481. https://doi.org/10.1016/j.jff.2021.104481

    Article  CAS  Google Scholar 

  3. Song K, Sun J, Wang W, Hao J (2021) Heterologous expression of cyclodextrin glycosyltransferase my20 in Escherichia coli and its application in 2-O-alpha-d-glucopyranosyl-L-ascorbic acid production. Front Microbiol 12:664339. https://doi.org/10.3389/fmicb.2021.664339

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lee SB, Nam KC, Lee SJ, Lee JH, Inouye K, Park KH (2004) Antioxidative effects of glycosyl-ascorbic acids synthesized by maltogenic amylase to reduce lipid oxidation and volatiles—production in cooked chicken meat. Biosci Biotechnol Biochem 68:36–43. https://doi.org/10.1271/bbb.68.36

    Article  CAS  PubMed  Google Scholar 

  5. Zhou Y, Gan T, Jiang R, Chen H, Ma Z, Lu Y, Zhu L, Chen X (2022) Whole-cell catalytic synthesis of 2-O-alpha-glucopyranosyl-L-ascorbic acid by sucrose phosphorylase from Bifidobacterium breve via a batch-feeding strategy. Process Biochem 112:27–34. https://doi.org/10.1016/j.procbio.2021.11.023

    Article  CAS  Google Scholar 

  6. Mukai K, Tsusaki K, Kubota M, Fukuda S, Miyake T (2014) Process for producing 2-O-alpha-D-glucopyranosyl-L-ascorbic acid

  7. Tao X, Su L, Wu J (2019) Current studies on the enzymatic preparation 2-O-alpha-D-glucopyranosyl-L-ascorbic acid with cyclodextrin glycosyltransferase. Crit Rev Biotechnol 39:249–257. https://doi.org/10.1080/07388551.2018.1531823

    Article  CAS  PubMed  Google Scholar 

  8. Gudiminchi RK, Towns A, Varalwar S, Nidetzky B (2016) Enhanced synthesis of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid from alpha-cyclodextrin by a highly disproportionating CGTase. Acs Catal 6:1606–1615. https://doi.org/10.1021/acscatal.5b02108

    Article  CAS  Google Scholar 

  9. van der Veen BA, van Alebeek G-JWM, Uitdehaag JCM, Dijkstra BW, Dijkhuizen L (2000) The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms. Eur J Biochem 267:658–665. https://doi.org/10.1046/j.1432-1327.2000.01031.x

    Article  PubMed  Google Scholar 

  10. Leemhuis H, Dijkstra BW, Dijkhuizen L (2003) Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase—mechanism and kinetics of inhibition by acarbose and cyclodextrins. Eur J Biochem 270:155–162. https://doi.org/10.1046/j.1432-1033.2003.03376.x

    Article  CAS  PubMed  Google Scholar 

  11. Gaston JAR, Costa H, Rossi AL, Krymkiewicz N, Ferrarotti SA (2012) Maltooligosaccharides production catalysed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R in batch and continuous operation. Process Biochem 47:2562–2565. https://doi.org/10.1016/j.procbio.2012.08.008

    Article  CAS  Google Scholar 

  12. Zhang Z, Li J, Liu L, Sun J, Hua Z, Du G, Chen J (2011) Enzymatic transformation of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid by alpha-cyclodextrin glucanotransferase from recombinant Escherichia coli. Biotechnol Bioprocess Eng 16:107–113. https://doi.org/10.1007/s12257-010-0161-5

    Article  CAS  Google Scholar 

  13. Xiong Y, Su L, Wang L, Wu J, Chen S (2015) Anchorage of cyclodextrin glycosyltransferase on outer membrane of Saccharomyces cerevisiae to produce 2-O-alpha-D-glucopyranosyl-L-ascorbic acid. Acta Microbiol Sin 55:1305–1313

    CAS  Google Scholar 

  14. Xu Q, Han R, Li J, Du G, Liu L, Chen J (2014) Improving maltodextrin specificity by site-saturation engineering of subsite +1 in cyclodextrin glycosyltransferase from Paenibacillus macerans. Chin J Biotechnol 30:98–108

    CAS  Google Scholar 

  15. Han R, Liu L, Shin H-d, Chen RR, Li J, Du G, Chen J (2013) 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 79:672–677. https://doi.org/10.1128/aem.02883-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu L, Xu Q, Han R, Shin H-d, Chen RR, Li J, Du G, Chen J (2013) Improving maltodextrin specificity for enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans. J Biotechnol 166:198–205. https://doi.org/10.1016/j.jbiotec.2013.05.005

    Article  CAS  PubMed  Google Scholar 

  17. Han R, Liu L, Shin H-d, Chen RR, Li J, Du G, Chen J (2013) 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. Appl Environ Microbiol 79:7562–7568. https://doi.org/10.1128/aem.02918-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tao X, Wang T, Su L, Wu J (2018) Enhanced 2-O-alpha-D-glucopyranosyl-L-ascorbic acid synthesis through iterative saturation mutagenesis of acceptor subsite residues in Bacillus stearothermophilus NO2 cyclodextrin glycosyltransferase. J Agric Food Chem 66:9052–9060. https://doi.org/10.1021/acs.jafc.8b03080

    Article  CAS  PubMed  Google Scholar 

  19. Chen S, Xiong Y, Su L, Wang L, Wu J (2017) Position 228 in Paenibacillus macerans cyclodextrin glycosyltransferase is critical for 2-O-D-glucopyranosyl-L-ascorbic acid synthesis. J Biotechnol 247:18–24. https://doi.org/10.1016/j.jbiotec.2017.02.011

    Article  CAS  PubMed  Google Scholar 

  20. Nakamura A, Haga K, Yamane K (1994) 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 33:9929–9936. https://doi.org/10.1021/bi00199a015

    Article  CAS  PubMed  Google Scholar 

  21. Kanai R, Haga K, Akiba T, Yamane K, Harata K (2004) Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011: substrate binding and arrangement of the catalytic site. Protein Sci 13:457–465. https://doi.org/10.1110/ps.03408504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang Y, Wang L, Chen S, Wu J (2014) Optimization of beta-cyclodextrin production by recombinant beta-cyclodextrin glycosyltransferase. Biotechnol Bull 0:175–181

    Google Scholar 

  23. Li Z, Li B, Gu Z, Du G, Wu J, Chen J (2010) Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr Res 345:886–892. https://doi.org/10.1016/j.carres.2010.02.002

    Article  CAS  PubMed  Google Scholar 

  24. Tao X, Su L, Wang L, Chen X, Wu J (2020) Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution. Appl Microbiol Biotechnol 104:173–185. https://doi.org/10.1007/s00253-019-10249-8

    Article  CAS  PubMed  Google Scholar 

  25. Penninga D, Strokopytov B, Rozeboom HJ, Lawson CL, Dijkstra BW, Bergsma J, Dijkhuizen L (1995) Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity. Biochemistry 34:3368–3376. https://doi.org/10.1021/bi00010a028

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (32001637, 31972032 and 31771916), the Natural Science Foundation of Jiangsu Province (BK20221536), the Agricultural independent innovation fund of Jiangsu Province (CX (21) 3039), and the Jiangnan University Basic Research Program (JUSRP122010).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Xiumei Tao, Demin Kong, Lingqia Su, Sheng Chen, Deming Rao, Beibei Wei, Jing Wu & Lei Wang

  2. School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Demin Kong, Huihu Zhang, Lingqia Su, Sheng Chen, Deming Rao, Beibei Wei, Jing Wu & Lei Wang

  3. International Joint Laboratory On Food Safety, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Xiumei Tao, Demin Kong, Lingqia Su, Sheng Chen, Deming Rao, Beibei Wei, Jing Wu & Lei Wang

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  1. Xiumei Tao
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  2. Demin Kong
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  3. Huihu Zhang
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  9. Lei Wang
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Contributions

XT: conceptualization, methodology, data curation, visualization, writing-original draft; DK: methodology, visualization; HZ: formal analysis, writing—review and editing; LS: methodology, funding acquisition; SC: methodology, funding acquisition; DR: methodology; BW: methodology; JW: conceptualization, writing-review and editing; LW: conceptualization, supervision, writing-review and editing, funding acquisition.

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Correspondence to Lei Wang.

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Tao, X., Kong, D., Zhang, H. et al. Enhancing 2-O-α-D-glucopyranosyl-l-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose. Bioprocess Biosyst Eng 46, 903–911 (2023). https://doi.org/10.1007/s00449-023-02875-4

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