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

da Fonseca, M. J. M., Jurak, E., Kataja, K., Master, E. R., Berrin, J-G., Stals, I., Desmet, T., Van Landschoot, A. & Briers, Y., Dec-2018, In : Applied Microbiology and Biotechnology. 102, 23, p. 1009-10102 12 p.

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

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
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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