Applied Microbiology and Biotechnology

, Volume 102, Issue 23, pp 10091–10102 | Cite as

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

  • Maria João Maurício da Fonseca
  • Edita Jurak
  • Kim Kataja
  • Emma R. Master
  • Jean-Guy Berrin
  • Ingeborg Stals
  • Tom Desmet
  • Anita Van Landschoot
  • Yves Briers
Biotechnologically relevant enzymes and proteins


Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.


α-L-arabinofuranosidases Substrate specificity DSA-FACE HPAEC-PAD Enzyme analysis 



We thank Mireille Haon (INRA, Aix Marseille Univ., BBF, Marseille, France) for the production and purification of the recombinant PaAbf62A.


The research has been financially supported by the research fund of the University College Ghent and Ghent University (B/13845/01 ‘HS Annotatie enzymen’).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9389_MOESM1_ESM.pdf (1.8 mb)
ESM 1 (PDF 1814 kb)


  1. Albrecht S, van Muiswinkel GC, Schols HA, Voragen AG, Gruppen H (2009) Introducing capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the characterization of konjac glucomannan oligosaccharides and their in vitro fermentation behavior. J Agric Food Chem 57:3867–3876. CrossRefPubMedGoogle Scholar
  2. Alvarez TM, Goldbeck R, dos Santos CR, Paixão DAA, Gonçalves TA, Franco Cairo JPL, Almeida RF, de Oliveira Pereira I, Jackson G, Cota J, Büchli F, Citadini AP, Ruller R, Polo CC, de Oliveira Neto M, Murakami MT, Squina FM (2013) Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome. PLoS One 8:e70014. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Borsenberger V, Dornez E, Desrousseaux M-L, Massou S, Tenkanen M, Courtin CM, Dumon C, O’Donohue MJ, Fauré R (2014) A 1H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates. Biochim Biophys Acta 1840:3106–3114. CrossRefPubMedGoogle Scholar
  4. Broberg A, Duus J, Thomsen KK, Ferre H (2000) A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley. Analysis of substrate preference and specificity by nano-probe NMR. Eur J Biochem 6641:6633–6664. CrossRefGoogle Scholar
  5. Cairo JPLF, Leonardo FC, Alvarez TM, Ribeiro DA, Büchli F, Costa-Leonardo AM, Carazzolle MF, Costa FF, Leme AFP, Pereira GAG, Squina FM (2011) Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi. Biotechnol Biofuels 4:50. CrossRefGoogle Scholar
  6. Callewaert N, Geysens S, Molemans F, Contreras R (2001) Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology 11:275–281. CrossRefPubMedGoogle Scholar
  7. Couturier M, Haon M, Coutinho PM, Henrissat B, Lesage-meessen L, Berrin J (2011) Podospora anserina hemicellulases potentiate the Trichoderma reesei secretome for saccharification of lignocellulosic biomass. Appl Environ Microbiol 77:237–246. CrossRefPubMedGoogle Scholar
  8. Couturier M, Tangthirasunun N, Ning X, Brun S, Gautier V, Bennati-Granier C, Silar P, Berrin J (2016) Plant biomass degrading ability of the coprophilic ascomycete fungus Podospora anserina. Biotechnol Adv 34:976–983. CrossRefPubMedGoogle Scholar
  9. Defrancq L, Callewaert N, Zhu J, Laroy W, Contreras R (2004) DSA-FACE: high-throughput analysis of the N-glycans of NS0-cell secreted antibodies. Bioprocess Int 2:60–68Google Scholar
  10. Duus J, Gotfredsen CH, Bock K (2000) Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations. Chem Rev 100:4589–4614. CrossRefPubMedGoogle Scholar
  11. Eda M, Ishimaru M, Tada T, Sakamoto T, Kotake T, Tsumuraya Y, Mort AJ, Gross KC (2014) Enzymatic activity and substrate specificity of the recombinant tomato β-galactosidase 1. J Plant Physiol 171:1454–1460. CrossRefPubMedGoogle Scholar
  12. Evangelista RA, Liu MS, Chen FTA (1995) Characterization of 9-aminopyrene-1, 4, 6-trisulfonate derivatized sugars by capillary electrophoresis with laser-induced fluorescence detection. Anal Chem 67:2239–2245. CrossRefGoogle Scholar
  13. Fauré R, Courtin CM, Delcour JA, Dumon C, Faulds CB, Fincher GB, Fort S, Fry SC, Halila S, Kabel MA, Pouvreau L, Quemener B, Rivet A, Saulnier L, Schols HA, Driguez H, O’Donohue MJ (2009) A brief and informationally rich naming system for oligosaccharide motifs of heteroxylans found in plant cell walls. Aust J Chem 62:533–537. CrossRefGoogle Scholar
  14. Guttman A, Herrick S (1996) Effect of the quantity and linkage position of mannose (alpha 1,2) residues in capillary gel electrophoresis of high-mannose-type. Anal Biochem 239:236–239. CrossRefGoogle Scholar
  15. Hilz H, de Jong LE, Kabel MA, Schols HA, Voragen AG (2006) A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants. J Chromatogr A 1133:275–286. CrossRefPubMedGoogle Scholar
  16. Kabel MA, Heijnis WH, Bakx EJ, Kuijpers R, Voragen AGJ, Schols HA (2006) Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides. J Chromatogr A 1137:119–126. CrossRefPubMedGoogle Scholar
  17. Kormelink FJM, Searle-Van Leeuwen MJF, Wood TM, Voragen AGJ (1991a) Purification and characterization of a (1,4)-β-D-arabinoxylan arabinofuranohydrolase from Aspergillus awamori. Appl Microbiol Biotechnol 35:753–754. CrossRefGoogle Scholar
  18. Kormelink FJM, Searle-Van Leeuwen MJF, Wood TM, Voragen AGJ (1991b) (1,4)-β-D-Arabinoxylan arabinofuranohydrolase: a novel enzyme in the bioconversion of arabinoxylan. Appl Microbiol Biotechnol 35:231–232. CrossRefGoogle Scholar
  19. Koutaniemi S, Tenkanen M (2016) Action of three GH51 and one GH54 α-arabinofuranosidases on internally and terminally located arabinofuranosyl branches. J Biotechnol 229:22–30. CrossRefPubMedGoogle Scholar
  20. Lagaert S, Pollet A, Delcour JA, Lavigne R, Courtin CM, Volckaert G (2010) Substrate specificity of three recombinant α-L-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides. Biochem Biophys Res Commun 402:644–650. CrossRefPubMedGoogle Scholar
  21. Li X, Jackson P, Rubtsov DV, Faria-Blanc N, Mortimer JC, Turner SR, Krogh KB, Johansen KS, Dupree P (2013) Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes. Biotechnol Biofuels 6(1):94. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B (2013) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Mantovani V, Galeotti F, Maccari F, Volpi N (2018) Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides, and polysaccharides. Electrophoresis 39:179–189. CrossRefPubMedGoogle Scholar
  24. Mccleary BV, Mckie VA, Draga A, Rooney E, Mangan D, Larkin J (2015) Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-L-arabinofuranosidase and β-xylosidase. Carbohydr Res 407:79–96. CrossRefPubMedGoogle Scholar
  25. Mewis K, Lenfant N, Lombard V, Henrissat B (2016) Dividing the large glycoside hydrolase family 43 into subfamilies: a motivation for detailed enzyme characterization. Appl Environ Microbiol 82:1686–1692. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mittermayr S, Guttman A (2012) Influence of molecular configuration and conformation on the electromigration of oligosaccharides in narrow bore capillaries. Electrophoresis 33:1000–1007. CrossRefPubMedGoogle Scholar
  27. Ndeh D, Rogowski A, Cartmell A, Luis AS, Baslé A, Gray J, Venditto I, Briggs J, Zhang X, Labourel A, Terrapon N, Buffetto F, Nepogodiev S, Xiao Y, Field RA, Zhu Y, O’Neill MA, Urbanowicz BR, York WS, Davies GJ, Abbott DW, Ralet MC, Martens EC, Henrissat B, Gilbert HJ (2017) Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature 544:65–70. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Pastell H, Tuomainen P, Virkki L, Tenkanen M (2008) Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches. Carbohydr Res 343:3049–3057. CrossRefPubMedGoogle Scholar
  29. Pitson SM, Voragen AG, Beldman G (1996) Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases. FEBS Lett 398:7–11. CrossRefPubMedGoogle Scholar
  30. Pouvreau L, Joosten R, Hinz SW, Gruppen H, Schols HA (2011) Chrysosporium lucknowense C1 arabinofuranosidases are selective in releasing arabinose from either single or double substituted xylose residues in arabinoxylans. Enzym Microb Technol 48:397–403. CrossRefGoogle Scholar
  31. Rantanen H, Virkki L, Tuomainen P, Kabel M, Schols H, Tenkanen M (2007) Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-β-D-xylanase. Carbohydr Polym 68:350–359. CrossRefGoogle Scholar
  32. Saha BC (2000) Alpha-L-arabinofuranosidases: biochemistry, molecular biology and application in biotechnology. Biotechnol Adv 18:403–423. CrossRefPubMedGoogle Scholar
  33. Sakamoto T, Inui M, Yasui K, Hosokawa S, Ihara H (2013) Substrate specificity and gene expression of two Penicillium chrysogenum α-L-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54. Appl Microbiol Biotechnol 97:1121–1130. CrossRefPubMedGoogle Scholar
  34. Schaeper JP, Sepaniak MJ (2000) Parameters affecting reproducibility in capillary electrophoresis. Electrophoresis 21:1421–1429.<1421::AID-ELPS1421>3.0.CO;2-7 CrossRefPubMedGoogle Scholar
  35. Shrivastava A, Gupta V (2011) Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chronicles Young Sci 2:21–25. CrossRefGoogle Scholar
  36. Siguier B, Haon M, Nahoum V, Marcellin M, Burlet-Schiltz O, Coutinho PM, Henrissat B, Mourey L, O’Donohue MJ, Berrin J-G, Tranier S, Dumon C (2014) First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies. J Biol Chem 289:5261–5273. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Sørensen HR, Jørgensen CT, Hansen CH, Jørgensen CI, Pedersen S, Meyer AS (2006) A novel GH43 α-L-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-L-arabinofuranosidases on wheat arabinoxylan. Appl Microbiol Biotechnol 73:850–861. CrossRefPubMedGoogle Scholar
  38. Van den Broek LA, Lloyd RM, Beldman G, Verdoes JC, McCleary BV, Voragen AG (2005) Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083. Appl Microbiol Biotechnol 67:641–647. CrossRefPubMedGoogle Scholar
  39. Van Laere KMJ, Beldman G, Voragen AGJ (1997) A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan. Appl Microbiol Biotechnol 43:231–235. CrossRefGoogle Scholar
  40. Van Laere KMJ, Voragen CHL, Kroef T, Van den Broek LAM, Beldman G, Voragen AGJ (1999) Purification and mode of action of two different arabinoxylan arabinofuranohydrolases from Bifidobacterium adolescentis DSM 20083. Appl Microbiol Biotechnol 51:606–613. CrossRefGoogle Scholar
  41. Wang H, Squina F, Segato F, Mort A, Lee D, Pappan K, Prade R (2011) High-temperature enzymatic breakdown of cellulose. Appl Environ Microbiol 77:5199–5206. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Wang W, Andric N, Sarch C, Silva BT, Tenkanen M, Master ER (2017) Constructing arabinofuranosidases for dual arabinoxylan debranching activity. Biotechnol Bioeng 115:41–49. CrossRefPubMedGoogle Scholar
  43. Westphal Y, Kühnel S, Schols HA, Voragen AG, Gruppen H (2010a) LC/CE–MS tools for the analysis of complex arabino-oligosaccharides. Carbohydr Res 345:2239–2251. CrossRefPubMedGoogle Scholar
  44. Westphal Y, Kühnel S, de Waard P, Hinz SW, Schols HA, Voragen AG, Gruppen H (2010b) Branched arabino-oligosaccharides isolated from sugar beet arabinan. Carbohydr Res 345:1180–1189. CrossRefPubMedGoogle Scholar
  45. Wilkens C, Andersen S, Petersen BO, Li A, Busse-Wicher M, Birch J, Cockburn D, Nakai H, Christensen HE, Kragelund BB, Dupree P, McCleary B, Hindsgaul O, Hachem MA, Svensson B (2016) An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site. Appl Microbiol Biotechnol 100:6265–6277. CrossRefPubMedGoogle Scholar
  46. Wilkens C, Andersen S, Dumon C, Berrin J, Svensson B (2017) GH62 arabinofuranosidases: structure, function and applications. Biotechnol Adv 35:792–804. CrossRefPubMedGoogle Scholar
  47. Wong MT, Wang W, Couturier M, Razeq FM, Lombard V, Lapebie P, Edwards EA, Terrapon N, Henrissat B, Master ER (2017) Comparative metagenomics of cellulose- and poplar hydrolysate-degrading microcosms from gut microflora of the Canadian Beaver (Castor canadensis) and North American moose (Alces americanus) after long-term enrichment. Front Microbiol 8:1–14. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Maria João Maurício da Fonseca
    • 1
  • Edita Jurak
    • 2
  • Kim Kataja
    • 2
  • Emma R. Master
    • 2
    • 3
  • Jean-Guy Berrin
    • 4
  • Ingeborg Stals
    • 5
  • Tom Desmet
    • 1
  • Anita Van Landschoot
    • 1
  • Yves Briers
    • 1
  1. 1.Department of BiotechnologyGhent UniversityGhentBelgium
  2. 2.Department of Biotechnology and Chemical TechnologyAalto UniversityEspooFinland
  3. 3.Department of Chemical Engineering and Applied ChemistryUniversity of TorontoTorontoCanada
  4. 4.INRA, Aix Marseille Université, UMR1163 BBFMarseilleFrance
  5. 5.Department of Materials, Textiles and Chemical EngineeringGhent UniversityGhentBelgium

Personalised recommendations