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

dc.contributorAalto-yliopistofi
dc.contributorAalto Universityen
dc.contributor.authorMaurício da Fonseca, Maria João
dc.contributor.authorJurak, Edita
dc.contributor.authorKataja, Kim
dc.contributor.authorMaster, Emma R.
dc.contributor.authorBerrin, Jean Guy
dc.contributor.authorStals, Ingeborg
dc.contributor.authorDesmet, Tom
dc.contributor.authorVan Landschoot, Anita
dc.contributor.authorBriers, Yves
dc.contributor.departmentGhent University
dc.contributor.departmentDepartment of Bioproducts and Biosystems
dc.contributor.departmentINRAE
dc.date.accessioned2019-01-14T09:21:18Z
dc.date.available2019-01-14T09:21:18Z
dc.date.embargoinfo:eu-repo/date/embargoEnd/2019-12-31
dc.date.issued2018-12
dc.descriptionThe research has been financially supported by the research fund of the University College Ghent and Ghent University (B/13845/ 01 ‘ HS Annotatie enzymen
dc.description.abstractCarbohydrate-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.en
dc.description.versionPeer revieweden
dc.format.extent10091–10102
dc.format.mimetypeapplication/pdf
dc.identifier.citationMaurício da Fonseca , M J , Jurak , E , Kataja , K , Master , E R , Berrin , J G , Stals , I , Desmet , T , Van Landschoot , A & Briers , Y 2018 , ' Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis ' , Applied Microbiology and Biotechnology , vol. 102 , no. 23 , pp. 10091–10102 . https://doi.org/10.1007/s00253-018-9389-3en
dc.identifier.doi10.1007/s00253-018-9389-3
dc.identifier.issn0175-7598
dc.identifier.issn1432-0614
dc.identifier.otherPURE UUID: 599f28fe-7b35-4535-b06d-f684c619f440
dc.identifier.otherPURE ITEMURL: https://research.aalto.fi/en/publications/599f28fe-7b35-4535-b06d-f684c619f440
dc.identifier.otherPURE LINK: http://www.scopus.com/inward/record.url?scp=85054188828&partnerID=8YFLogxK
dc.identifier.otherPURE FILEURL: https://research.aalto.fi/files/31095056/CHEM_Master_daFonseca_et_al_Analysis_of_the_substrate_2018_FAM.pdf
dc.identifier.urihttps://aaltodoc.aalto.fi/handle/123456789/35957
dc.identifier.urnURN:NBN:fi:aalto-201901141140
dc.language.isoenen
dc.relation.ispartofseriesApplied Microbiology and Biotechnologyen
dc.relation.ispartofseriesVolume 102, issue 23en
dc.rightsopenAccessen
dc.subject.keywordDSA-FACE
dc.subject.keywordEnzyme analysis
dc.subject.keywordHPAEC-PAD
dc.subject.keywordSubstrate specificity
dc.subject.keywordα-L-arabinofuranosidases
dc.titleAnalysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresisen
dc.typeA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessäfi
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