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

Applied microbiology and biotechnology, 2018-12, Vol.102 (23), p.10091-10102 [Peer Reviewed Journal]

Springer-Verlag GmbH Germany, part of Springer Nature 2018 ;Applied Microbiology and Biotechnology is a copyright of Springer, (2018). All Rights Reserved. ;ISSN: 0175-7598 ;EISSN: 1432-0614 ;DOI: 10.1007/s00253-018-9389-3 ;PMID: 30267127

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Title:
Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Author: Maurício da Fonseca, Maria João ; Jurak, Edita ; Kataja, Kim ; Master, Emma R. ; Berrin, Jean-Guy ; Stals, Ingeborg ; Desmet, Tom ; Van Landschoot, Anita ; Briers, Yves
Subjects: Arabinofuranosidase ; Aspergillus nidulans - enzymology ; Bifidobacterium adolescentis - enzymology ; Binding sites ; Biomedical and Life Sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Carbohydrates ; Carbohydrates - analysis ; Degree of polymerization ; Deoxyribonucleic acid ; DNA ; Electrophoresis ; Fluorescent Dyes ; Glycoside hydrolase ; Glycoside Hydrolases - metabolism ; L-Arabinofuranosidase ; Life Sciences ; Limit of Detection ; Metagenomics ; Microbial Genetics and Genomics ; Microbiology ; Monomers ; Nucleotide sequence ; Oligosaccharides ; Podospora - enzymology ; Polymerization ; Sensitivity analysis ; Sequence Analysis, DNA ; Substrate Specificity ; Substrates
Is Part Of: Applied microbiology and biotechnology, 2018-12, Vol.102 (23), p.10091-10102
Description: 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 ( An Abf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis ( Ba AXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. Pa Abf62A, 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.
Publisher: Berlin/Heidelberg: Springer Berlin Heidelberg
Language: English
Identifier: ISSN: 0175-7598
EISSN: 1432-0614
DOI: 10.1007/s00253-018-9389-3
PMID: 30267127
Source: AUTh Library subscriptions: ProQuest Central
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Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Springer-Verlag GmbH Germany, part of Springer Nature 2018 ;Applied Microbiology and Biotechnology is a copyright of Springer, (2018). All Rights Reserved. ;ISSN: 0175-7598 ;EISSN: 1432-0614 ;DOI: 10.1007/s00253-018-9389-3 ;PMID: 30267127

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