Identification and characterization of α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae

  • Tomohiko MatsuzawaEmail author
  • Akihiko Kameyama
  • Katsuro Yaoi
Biotechnologically relevant enzymes and proteins


Aspergillus oryzae produces hydrolases involved in xyloglucan degradation and induces the expression of genes encoding xyloglucan oligosaccharide hydrolases in the presence of xyloglucan oligosaccharides. A gene encoding α-xylosidase (termed AxyA), which is induced in the presence of xyloglucan oligosaccharides, is identified and expressed in Pichia pastoris. AxyA is a member of the glycoside hydrolase family 31 (GH31). AxyA hydrolyzes isoprimeverose (α-d-xylopyranosyl-(1→6)-d-glucopyranose) into d-xylose and d-glucose and shows hydrolytic activity with other xyloglucan oligosaccharides such as XXXG (heptasaccharide, Glc4Xyl3) and XLLG (nonasaccharide, Glc4Xyl3Gal2). Isoprimeverose is a preferred AxyA substrate over other xyloglucan oligosaccharides. In the hydrolysis of XXXG, AxyA releases one molecule of d-xylose from one molecule of XXXG to yield GXXG (hexasaccharide, Glc4Xyl2). AxyA does not contain a signal peptide for secretion and remains within the cell. The intracellular localization of AxyA may help determine the order of hydrolases acting on xyloglucan oligosaccharides.


α-xylosidase Aspergillus oryzae Xyloglucan Glycoside hydrolase family 31 



We thank the National Research Institute of Brewing (Hiroshima, Japan) for the A. oryzae RIB40 strain.

Funding information

This study was supported by the Japan Society for the Promotion of Science KAKENHI (Grant-in-Aid for Scientific Research B, Grant No. 18H02132).

Compliance with ethical standards

Ethical approval

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

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

253_2019_10244_MOESM1_ESM.pdf (599 kb)
ESM 1 (PDF 599 kb)


  1. Attia MA, Brumer H (2016) Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan. Curr Opin Struct Biol 40:43–53PubMedCrossRefGoogle Scholar
  2. Bauer S, Vasu P, Mort AJ, Somerville CR (2005) Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans. Carbohydr Res 340:2590–2597PubMedCrossRefGoogle Scholar
  3. Bauer S, Vasu P, Persson S, Mort AJ, Somerville CR (2006) Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc Natl Acad Sci U S A 103:11417–11422PubMedPubMedCentralCrossRefGoogle Scholar
  4. Carpita NC, Gibeaut DM (1993) Structure models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3:1–30PubMedCrossRefGoogle Scholar
  5. Chaillou S, Lokman BC, Leer RJ, Posthuma C, Postma PW, Pouwels PH (1998) Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus. J Bacteriol 180:2312–2320PubMedPubMedCentralGoogle Scholar
  6. Crombie HJ, Chengappa S, Hellyer A, Reid JS (1998) A xyloglucan oligosaccharide-active, transglycosylating β-d-glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings—purification, properties and characterization of a cDNA clone. Plant J 15:27–38PubMedCrossRefGoogle Scholar
  7. Déjean G, Tauzin AS, Bennett SW, Creagh AL, Brumer H (2019) Adaptation of syntenic xyloglucan utilization loci of human gut Bacteroidetes to polysaccharide side chain diversity. Appl Environ Microbiol.
  8. Edwards M, Dea IC, Bulpin PV, Reid JS (1986) Purification and properties of a novel xyloglucan-specific endo-(1→4)-β-d-glucanase from germinated nasturtium seeds (Tropaeolum majus L.). J Biol Chem 261:9489–9494PubMedGoogle Scholar
  9. Edwards M, Bowman YJ, Dea IC, Reid JS (1988) A β-d-galactosidase from nasturtium (Tropaeolum majus L.) cotyledons. Purification, properties, and demonstration that xyloglucan is the natural substrate. J Biol Chem 263:4333–4337PubMedGoogle Scholar
  10. Fry SC, York WS, Albersheim P, Darvill A, Hayashi T, Joseleau J-P, Kato Y, Lorences EP, Maclachlan GA, McNeil M, Mort AJ, Grant Reid JS, Seitz HU, Selvendran RR, Voragen AGJ, White AR (1993) An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol Plant 89:1–3CrossRefGoogle Scholar
  11. Grishutin SG, Gusakov AV, Markov AV, Ustinov BB, Semenova MV, Sinitsyn AP (2004) Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochim Biophys Acta 1674:268–281PubMedCrossRefGoogle Scholar
  12. Hemsworth GR, Thompson AJ, Stepper J, Sobala ŁF, Coyle T, Larsbrink J, Spadiut O, Goddard-Borger ED, Stubbs KA, Brumer H, Davies GJ (2016) Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut. Open Biol 6:160142PubMedPubMedCentralCrossRefGoogle Scholar
  13. Ito Y, Sasaki T, Kitamoto K, Kumagai C, Takahashi K, Gomi K, Tamura G (2002) Cloning, nucleotide sequencing, and expression of the β-galactosidase-encoding gene (lacA) from Aspergillus oryzae. J Gen Appl Microbiol 48:135–142PubMedCrossRefGoogle Scholar
  14. Jabbour D, Borrusch MS, Banerjee G, Walton JD (2013) Enhancement of fermentable sugar yields by α-xylosidase supplementation of commercial cellulases. Biotechnol Biofuels 6:58PubMedPubMedCentralCrossRefGoogle Scholar
  15. Kameyama A, Thet Tin WW, Toyoda M, Sakaguchi M (2019) A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase. Biochem Biophys Res Commun 513:186–192PubMedCrossRefGoogle Scholar
  16. Kato Y, Matsushita J, Kubodera T, Matsuda K (1985) A novel enzyme producing isoprimeverose from oligoxyloglucans of Aspergillus oryzae. J Biochem 97:801–810PubMedCrossRefGoogle Scholar
  17. Kudo K, Watanabe A, Ujiie S, Shintani T, Gomi K (2015) Purification and enzymatic characterization of secretory glycoside hydrolase family 3 (GH3) aryl β-glucosidases screened from Aspergillus oryzae genome. J Biosci Bioeng 120:614–623PubMedCrossRefGoogle Scholar
  18. Larsbrink J, Izumi A, Ibatullin FM, Nakhai A, Gilbert HJ, Davies GJ, Brumer H (2011) Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification. Biochem J 436:567–580PubMedCrossRefGoogle Scholar
  19. Larsbrink J, Rogers TE, Hemsworth GR, McKee LS, Tauzin AS, Spadiut O, Klinter S, Pudlo NA, Urs K, Koropatkin NM, Creagh AL, Haynes CA, Kelly AG, Cederholm SN, Davies GJ, Martens EC, Brumer H (2014a) A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroides. Nature 506:498–502PubMedPubMedCentralCrossRefGoogle Scholar
  20. Larsbrink J, Thompson AJ, Lundqvist M, Gardner JG, Davies GJ, Brumer H (2014b) A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus. Mol Microbiol 94:418–433PubMedPubMedCentralCrossRefGoogle Scholar
  21. Léonard R, Pabst M, Bondili JS, Chambat G, Veit C, Strasser R, Altmann F (2008) Identification of an Arabidopsis gene encoding a GH95 α1,2-fucosidase active on xyloglucan oligo- and polysaccharides. Phytochemistry 69:1983–1988PubMedCrossRefGoogle Scholar
  22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  23. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495PubMedCrossRefGoogle Scholar
  24. Lovering AL, Lee SS, Kim YW, Withers SG, Strynadka NC (2005) Mechanistic and structural analysis of a family 31 α-glycosidase and its glycosyl-enzyme intermediate. J Biol Chem 280:2105–2115PubMedCrossRefGoogle Scholar
  25. Mark P, Baumann MJ, Eklöf JM, Gullfot F, Michel G, Kallas AM, Teeri TT, Brumer H, Czjzek M (2009) Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo-transglycosylases and endo-hydrolases. Proteins 75:820–836PubMedCrossRefGoogle Scholar
  26. Marui J, Tanaka A, Mimura S, de Graaff LH, Visser J, Kitamoto N, Kato M, Kobayashi T, Tsukagoshi N (2002) A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae. Fungal Genet Biol 35:157–169PubMedCrossRefGoogle Scholar
  27. Matsuno YK, Saito T, Gotoh M, Narimatsu H, Kameyama A (2009) Supported molecular matrix electrophoresis: a new tool for characterization of glycoproteins. Anal Chem 81:3816–3823PubMedCrossRefGoogle Scholar
  28. Matsuzawa T (2019) The metagenomic approach: a new resource for glucosidases. Trends Glycosci Glycotechnol 31:E15–E20CrossRefGoogle Scholar
  29. Matsuzawa T, Yaoi K (2016) GH74 xyloglucanases: Structures and modes of activity. Trends Glycosci Glycotechnol 28:E63–E70CrossRefGoogle Scholar
  30. Matsuzawa T, Saito Y, Yaoi K (2014) Key amino acid residues for the endo-processive activity of GH74 xyloglucanase. FEBS Lett 588:1731–1738PubMedCrossRefGoogle Scholar
  31. Matsuzawa T, Kaneko S, Yaoi K (2015) Screening, identification, and characterization of a GH43 family β-xylosidase/α-arabinofuranosidase from a compost microbial metagenome. Appl Microbiol Biotechnol 99:8943–8954PubMedCrossRefGoogle Scholar
  32. Matsuzawa T, Kimura N, Suenaga H, Yaoi K (2016a) Screening, identification, and characterization of α-xylosidase from a soil metagenome. J Biosci Bioeng 122:393–399PubMedCrossRefGoogle Scholar
  33. Matsuzawa T, Mitsuishi Y, Kameyama A, Yaoi K (2016b) Identification of the gene encoding isoprimeverose-producing oligoxyloglucan hydrolase in Aspergillus oryzae. J Biol Chem 291:5080–5087PubMedPubMedCentralCrossRefGoogle Scholar
  34. Matsuzawa T, Watanabe M, Kameda T, Kameyama A, Yaoi K (2019a) Cooperation between β-galactosidase and an isoprimeverose-producing oligoxyloglucan hydrolase is key for xyloglucan degradation in Aspergillus oryzae. FEBS J 286:3182–3193PubMedCrossRefGoogle Scholar
  35. Matsuzawa T, Watanabe M, Nakamichi Y, Fujimoto Z, Yaoi K (2019b) Crystal structure and substrate recognition mechanism of Aspergillus oryzae isoprimeverose-producing enzyme. J Struct Biol 205:84–90PubMedCrossRefGoogle Scholar
  36. Moracci M, Cobucci Ponzano B, Trincone A, Fusco S, De Rose M, van Der Oost J, Sensen CW, Charlebois RL, Rossi M (2000) Identification and molecular characterization of the first α-xylosidase from an Archaeon. J Biol Chem 275:22082–22089PubMedCrossRefGoogle Scholar
  37. Nakai H, Tanizawa S, Ito T, Kamiya K, Kim YM, Yamamoto T, Matsubara K, Sakai M, Sato H, Imbe T, Okuyama M, Mori H, Sano Y, Chiba S, Kimura A (2007) Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are α-glucosidase and α-xylosidase. J Biochem 142:491–500PubMedCrossRefGoogle Scholar
  38. Nelson CE, Attia MA, Rogowski A, Morland C, Brumer H, Gardner JG (2017) Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification. Environ Microbiol 19:5025–5039PubMedPubMedCentralCrossRefGoogle Scholar
  39. Noguchi Y, Sano M, Kanamaru K, Ko T, Takeuchi M, Kato M, Kobayashi T (2009) Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae. Appl Microbiol Biotechnol 85:141–154PubMedCrossRefGoogle Scholar
  40. Okuyama M, Mori H, Chiba S, Kimura A (2004) Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli. Protein Expr Purif 37:170–179PubMedCrossRefGoogle Scholar
  41. Pauly M, Keegstra K (2016) Biosynthesis of the plant cell wall matrix polysaccharide xyloglucan. Annu Rev Plant Biol 67:235–259PubMedCrossRefGoogle Scholar
  42. Sampedro J, Sieiro C, Revilla G, González-Villa T, Zarra I (2001) Cloning and expression pattern of a gene encoding an α-xylosidase active against xyloglucan oligosaccharides from Arabidopsis. Plant Physiol 126:910–920PubMedPubMedCentralCrossRefGoogle Scholar
  43. Scott-Craig JS, Borrusch MS, Banerjee G, Harvey CM, Walton JD (2011) Biochemical and molecular characterization of secreted α-xylosidase from Aspergillus niger. J Biol Chem 286:42848–42854PubMedPubMedCentralCrossRefGoogle Scholar
  44. Shigeyama T, Watanabe A, Tokuchi K, Toh S, Sakurai N, Shibuya N, Kawakami N (2016) α-Xylosidase plays essential roles in xyloglucan remodeling, maintenance of cell wall integrity, and seed germination in Arabidopsis thaliana. J Exp Bot 67:5615–5629PubMedPubMedCentralCrossRefGoogle Scholar
  45. Silipo A, Larsbrink J, Marchetti R, Lanzetta R, Brumer H, Molinaro A (2012) NMR spectroscopic analysis reveals extensive binding interactions of complex xyloglucan oligosaccharides with the Cellvibrio japonicus glycoside hydrolase family 31 α-xylosidase. Chemistry 18:13395–13404PubMedCrossRefGoogle Scholar
  46. Tuomivaara ST, Yaoi K, O’Neill MA, York WS (2015) Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature. Carbohydr Res 402:56–66PubMedCrossRefGoogle Scholar
  47. Watanabe A, Suzuki M, Ujiie S, Gomi K (2015) Purification and enzymatic characterization of a novel β-1,6-glucosidase from Aspergillus oryzae. J Biosci Bioeng 121:259–264PubMedCrossRefGoogle Scholar
  48. Yaoi K, Mitsuishi Y (2002) Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase. J Biol Chem 277:48276–48281PubMedCrossRefGoogle Scholar
  49. Yaoi K, Mitsuishi Y (2004) Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128. FEBS Lett 560:45–50PubMedCrossRefGoogle Scholar
  50. Yaoi K, Nakai T, Kameda Y, Hiyoshi A, Mitsuishi Y (2005) Cloning and characterization of two xyloglucanases from Paenibacillus sp. strain KM21. Appl Environ Microbiol 71:7670–7678PubMedPubMedCentralCrossRefGoogle Scholar
  51. Yoshikawa K, Yamamoto K, Okada S (1994) Classification of some α-glucosidases and α-xylosidases on the basis of substrate specificity. Biosci Biotechnol Biochem 58:1392–1398PubMedCrossRefGoogle Scholar
  52. Zabotina OA (2012) Xyloglucan and its biosynthesis. Front Plant Sci 3:134PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)IbarakiJapan
  2. 2.Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST)IbarakiJapan

Personalised recommendations