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

Maria Joao Mauricio da Fonseca, Edita Jurak, Kim Kataja, Emma R. Master, Jean-Guy Berrin, Ingeborg Stals, Tom Desmet, Anita Van Landschoot, Yves Briers*

*Corresponding author for this work

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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 -L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -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 -D-xylosyl residues, whereas a GH43 -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.

Original languageEnglish
Pages (from-to)1009-10102
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume102
Issue number23
DOIs
Publication statusPublished - Dec-2018
Externally publishedYes

Keywords

  • alpha-L-arabinofuranosidases
  • Substrate specificity
  • DSA-FACE
  • HPAEC-PAD
  • Enzyme analysis
  • ALPHA-L-ARABINOFURANOSIDASES
  • INDUCED FLUORESCENCE DETECTION
  • CAPILLARY-ELECTROPHORESIS
  • BIFIDOBACTERIUM-ADOLESCENTIS
  • (1,4)-BETA-D-ARABINOXYLAN ARABINOFURANOHYDROLASE
  • PODOSPORA-ANSERINA
  • OLIGOSACCHARIDES
  • ARABINOXYLAN
  • GH62
  • POLYSACCHARIDES

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