Applied Microbiology and Biotechnology

, Volume 87, Issue 6, pp 2125–2135 | Cite as

Functional analysis of glycoside hydrolase family 8 xylanases shows narrow but distinct substrate specificities and biotechnological potential

  • Annick Pollet
  • Jan Schoepe
  • Emmie Dornez
  • Sergei V. Strelkov
  • Jan A. Delcour
  • Christophe M. Courtin
Biotechnologically Relevant Enzymes and Proteins


The potential of glycoside hydrolase family (GH) 8 xylanases in biotechnological applications is virtually unexplored. Therefore, the substrate preference and hydrolysis product profiles of two GH8 xylanases were evaluated to investigate their activities and substrate specificities. A GH8 xylanase from an uncultured bacterium (rXyn8) shows endo action but very selectively releases xylotriose from its substrates. It has a higher activity than the Pseudoalteromonas haloplanktis GH8 endo-xylanase (PhXyl) on xylononaose and smaller xylo-oligosaccharides. PhXyl preferably degrades xylan substrates with a high degree of polymerization. It is sterically more hindered by arabinose substituents than rXyn8, producing larger end hydrolysis products. The specificities of rXyn8 and PhXyl differ completely from these of the previously described GH8 xylanases from Bifidobacterium adolescentis (BaRexA) and Bacillus halodurans (BhRex). As reducing-end xylose-releasing exo-oligoxylanases, they selectively release xylose from the reducing end of small xylo-oligosaccharides. The findings of this study show that GH8 xylanases have a narrow substrate specificity, but also one that strongly varies between family members and is distinct from that of GH10 and GH11 xylanases. Structural comparison of rXyn8, PhXyl, BaRexA, and BhRex showed that subtle amino acid changes in the glycon as well as the aglycon subsites probably form the basis of the observed differences between GH8 xylanases. GH8 xylanases, therefore, are an interesting group of enzymes, with potential towards engineering and applications.


Xylanase Glycoside hydrolase family 8 Substrate specificity Arabinoxylan Xylo-oligosaccharides 



The authors like to thank Dr. Charles C. Lee of the Agricultural Research Service (Albany, CA, USA) for donation of plasmid DNA from rXyn8 and Prof. Anna Kulminskaya from the Russian Academy of Science (St. Petersburg, Russia) for the labeled XOS. Financial support for the SBO IMPAXOS project by the ‘Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen’ (I.W.T., Brussels, Belgium), for the post-doctoral fellowship of E.D. by the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’ (F.W.O, Brussels, Belgium) and from the Research Fund K.U.Leuven (GOA/03/10 and IDO/03/005) are gratefully acknowledged. S.V.S. thanks the Biomedical Sciences Group (K.U. Leuven) for a start-up grant. The study is part of the Methusalem program ‘Food for the Future’ at the K.U. Leuven.


  1. Biely P, Vrsanska M, Tenkanen M, Kluepfel D (1997) Endo-β-1, 4-xylanase families: differences in catalytic properties. J Biotechnol 57:151–166CrossRefGoogle Scholar
  2. Brennan Y, Callen WN, Christoffersen L, Dupree P, Goubet F, Healey S, Hernandez M, Keller M, Li K, Palackal N, Sittenfeld A, Tamayo G, Wells S, Hazlewood GP, Mathur EJ, Short JM, Robertson DE, Steer BA (2004) Unusual microbial xylanases from insect guts. Appl Environ Microbiol 70:3609–3617CrossRefGoogle Scholar
  3. Broekaert WF, Courtin CA, Verbeke K, Van de Wiele T, Verstraete W, Delcour JA (2010) Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides and xylo-oligosaccharides. Crit Rev Food Sci Nutr, Accepted for publicationGoogle Scholar
  4. Buchert J, Tenkanen M, Kantelinen A, Viikari L (1994) Application of xylanases in the pulp and paper industry. Bioresour Technol 50:65–72CrossRefGoogle Scholar
  5. Butt MS, Tahir-Nadeem M, Ahmad Z, Sultan MT (2008) Xylanases and their applications in baking industry. Food Technol Biotechnol 46:22–31Google Scholar
  6. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238CrossRefGoogle Scholar
  7. Collins T, Meuwis MA, Stals I, Claeyssens M, Feller G, Gerday C (2002) A novel family 8 xylanase, functional and physicochemical characterization. J Biol Chem 277:35133–35139CrossRefGoogle Scholar
  8. Collins T, Hoyoux A, Dutron A, Georis J, Genot B, Dauvrin T, Arnaut F, Gerday C, Feller G (2006) Use of glycoside hydrolase family 8 xylanases in baking. J Cereal Sci 43:79–84CrossRefGoogle Scholar
  9. Cosgrove DJ (1997) Assembly and enlargement of the primary cell wall in plants. Annu Rev Cell Dev Biol 13:171–201CrossRefGoogle Scholar
  10. Courtin CM, Delcour JA (2002) Arabinoxylans and endoxylanases in wheat flour bread-making. J Cereal Sci 35:225–243CrossRefGoogle Scholar
  11. De Vos D, Collins T, Nerinckx W, Savvides SN, Claeyssens M, Gerday C, Feller G, Van Beeumen J (2006) Oligosaccharide binding in family 8 glycosidases: Crystal structures of active-site mutants of the β-1, 4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product. Biochemistry 45:4797–4807CrossRefGoogle Scholar
  12. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132CrossRefGoogle Scholar
  13. Eneyskaya EV, Brumer H, Backinowsky LV, Ivanen DR, Kulminskaya AA, Shabalin KA, Neustroev KN (2003) Enzymatic synthesis of β-xylanase substrates: transglycosylation reactions of the β-xylosidase from Aspergillus sp. Carbohydr Res 338:313–325CrossRefGoogle Scholar
  14. Frederix SA, Courtin CA, Delcour JA (2004a) Substrate selectivity and inhibitor sensitivity affect xylanase functionality in wheat flour gluten-starch separation. J Cereal Sci 40:41–49CrossRefGoogle Scholar
  15. Frederix SA, Van Hoeymissen KE, Courtin CM, Delcour JA (2004b) Water-extractable and water-unextractable arabinoxylans affect gluten agglomeration behavior during wheat flour gluten-starch separation. J Agric Food Chem 52:7950–7956CrossRefGoogle Scholar
  16. Fushinobu S, Hidaka M, Honda Y, Wakagi T, Shoun H, Kitaoka M (2005) Structural basis for the specificity of the reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125. J Biol Chem 280:17180–17186CrossRefGoogle Scholar
  17. Honda Y, Kitaoka M (2004) A family 8 glycoside hydrolase from Bacillus halodurans C-125 (BH2105) is a reducing end xylose-releasing exo-oligoxylanase. J Biol Chem 279:55097–55103CrossRefGoogle Scholar
  18. Kadam KL, Chin CY, Brown LW (2008) Flexible biorefinery for producing fermentation sugars, lignin and pulp from corn stover. J Ind Microbiol Biotechnol 35:331–341CrossRefGoogle Scholar
  19. Kenealy WR, Jeffries TW (2003) Enzyme processes for pulp and paper: a review of recent developments. Wood Deterioration and Preservation 845:210–239CrossRefGoogle Scholar
  20. Lagaert S, Van Campenhout S, Pollet A, Bourgois TM, Delcour JA, Courtin CM, Volckaert G (2007) Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis. Appl Environ Microbiol 73:5374–5377CrossRefGoogle Scholar
  21. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefGoogle Scholar
  22. Lee CC, Kibblewhite-Accinelli RE, Wagschal K, Robertson GH, Wong DWS (2006) Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library. Extremophiles 10:295–300CrossRefGoogle Scholar
  23. Li Y, Lu J, Gu GX (2005) Control of arabinoxylan solubilization and hydrolysis in mashing. Food Chem 90:101–108CrossRefGoogle Scholar
  24. Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Cα geometry: ϕ, ψ and Cβ deviation. Proteins Struct Funct Bioinformat 50:437–450CrossRefGoogle Scholar
  25. Pollet A, Beliën T, Fierens K, Delcour JA, Courtin CM (2009a) Fusarium graminearum xylanases show different functional stabilities, substrate specificities and inhibition sensitivities. Enzyme Microb Technol 44:189–195CrossRefGoogle Scholar
  26. Pollet A, Vandermarliere E, Lammertyn J, Strelkov SV, Delcour JA, Courtin CM (2009b) Crystallographic and activity-based evidence for thumb flexibility and its relevance in glycoside hydrolase family 11 xylanases. Proteins Struct Funct Bioinformat 77:395–403CrossRefGoogle Scholar
  27. Pollet A, Delcour JA, Courtin CM (2010a) Structural determinants of the substrate specificities of xylanases from different glycoside hydrolase families. Crit Rev Biotechnol. doi: 10.3109/07388551003645599 Google Scholar
  28. Pollet A, Lagaert S, Eneyskaya E, Kulminskaya A, Delcour JA, Courtin CM (2010b) Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases. Biochim Biophys Acta Proteins Proteomics 1804:977–985CrossRefGoogle Scholar
  29. Reilly PJ (1981) Xylanases: structure and function. In: Hollaender A (ed) Trends in the biology of fermentation for fuels and chemicals, basic life sciences. Plenum, New York, pp 111–129Google Scholar
  30. Saha BC (2003) Hemicellulose bioconversion. J Ind Microbiol Biotechnol 30:279–291CrossRefGoogle Scholar
  31. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815CrossRefGoogle Scholar
  32. Swennen K, Courtin CM, Lindemans GCJE, Delcour JA (2006) Large-scale production and characterisation of wheat bran arabinoxylooligosaccharides. J Sci Food Agric 86:1722–1731CrossRefGoogle Scholar
  33. Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N, Kuhara S, Horikoshi K (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317–4331CrossRefGoogle Scholar
  34. Trogh I, Courtin CM, Andersson AAM, Aman P, Sorensen JF, Delcour JA (2004a) The combined use of hull-less barley flour and xylanase as a strategy for wheat/hull-less barley flour breads with increased arabinoxylan and (1–3, 1–4)-β-d-glucan levels. J Cereal Sci 40:257–267CrossRefGoogle Scholar
  35. Trogh I, Courtin CM, Delcour JA (2004b) Isolation and characterization of water-extractable arabinoxylan from hull-less barley flours. Cereal Chem 81:576–581CrossRefGoogle Scholar
  36. Trogh I, Courtin CM, Goesaert H, Delcour JA, Andersson AAM, Aman P, Fredriksson H, Pyle DL, Sorensen JF (2005) From hull-less barley and wheat to soluble dietary fiber-enriched bread. Cereal Foods World 50:253–260Google Scholar
  37. Van Craeyveld V, Swennen K, Dornez E, Van de Wiele T, Marzorati M, Verstraete W, Delaedt Y, Onagbesan O, Decuypere E, Buyse J, De Ketelaere B, Broekaert WF, Delcour JA, Courtin CM (2008) Structurally different wheat-derived arabinoxylooligosaccharides have different prebiotic and fermentation properties in rats. J Nutr 138:2348–2355CrossRefGoogle Scholar
  38. van den Broek LAM, Lloyd RM, Beldman G, Verdoes JC, McCleary BV, Voragen AGJ (2005) Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083. Appl Microbiol Biotechnol 67:641–647CrossRefGoogle Scholar
  39. Van Petegem F, Collins T, Meuwis MA, Gerday C, Feller G, Van Beeumen J (2003) The structure of a cold-adapted family 8 xylanase at 1.3Å—Structural adaptations to cold and investigation of the active site. J Biol Chem 278:7531–7539CrossRefGoogle Scholar
  40. Vazquez MJ, Alonso JL, Dominguez H, Parajo JC (2000) Xylooligosaccharides: manufacture and applications. Trends Food Sci Technol 11:387–393CrossRefGoogle Scholar
  41. Yoon KH, Yun HN, Jung KH (1998) Molecular cloning of a Bacillus sp. KK-1 xylanase gene and characterization of the gene product. Biochem Mol Biol Int 45:337–347Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Annick Pollet
    • 1
  • Jan Schoepe
    • 2
  • Emmie Dornez
    • 1
  • Sergei V. Strelkov
    • 2
  • Jan A. Delcour
    • 1
  • Christophe M. Courtin
    • 1
  1. 1.Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe)Katholieke Universiteit LeuvenLeuvenBelgium
  2. 2.Laboratory for BiocrystallographyKatholieke Universiteit LeuvenLeuvenBelgium

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