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

Tutkimustuotos: Lehtiartikkelivertaisarvioitu

Tutkijat

  • Maria João Maurício da Fonseca
  • Edita Jurak
  • Kim Kataja
  • Emma Master

  • Jean Guy Berrin
  • Ingeborg Stals
  • Tom Desmet
  • Anita Van Landschoot
  • Yves Briers

Organisaatiot

  • Ghent University
  • University of Toronto
  • Institut National de la Recherche Agronomique

Kuvaus

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.

Yksityiskohdat

AlkuperäiskieliEnglanti
Sivut10091–10102
JulkaisuApplied Microbiology and Biotechnology
Vuosikerta102
Numero23
TilaJulkaistu - joulukuuta 2018
OKM-julkaisutyyppiA1 Julkaistu artikkeli, soviteltu

ID: 28704629