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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

Abstract

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 3.2.1.55) 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.

Keywords

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

Notes

Acknowledgements

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

Funding

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)

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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

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