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

(2018) APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 102(23). p.10091-10102
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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.
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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Citation

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Chicago
João de Carvalho Maurício da Fonseca, Maria, Edita Jurak, Kim Kataja, Emma R Master, Jean-Guy Berrin, Ingeborg Stals, Tom Desmet, Anita Van Landschoot, and Yves Briers. 2018. “Analysis of the Substrate Specificity of α-L-arabinofuranosidases by DNA Sequencer-aided Fluorophore-assisted Carbohydrate Electrophoresis.” Applied Microbiology and Biotechnology 102 (23): 10091–10102.
APA
João de Carvalho Maurício da Fonseca, M., Jurak, E., Kataja, K., Master, E. R., Berrin, J.-G., Stals, I., Desmet, T., et al. (2018). Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 102(23), 10091–10102.
Vancouver
1.
João de Carvalho Maurício da Fonseca M, Jurak E, Kataja K, Master ER, Berrin J-G, Stals I, et al. Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2018;102(23):10091–102.
MLA
João de Carvalho Maurício da Fonseca, Maria, Edita Jurak, Kim Kataja, et al. “Analysis of the Substrate Specificity of α-L-arabinofuranosidases by DNA Sequencer-aided Fluorophore-assisted Carbohydrate Electrophoresis.” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 102.23 (2018): 10091–10102. Print.
@article{8575642,
  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.},
  author       = {João de Carvalho Maurício da Fonseca, Maria and Jurak, Edita and Kataja, Kim and Master, Emma R and Berrin, Jean-Guy and Stals, Ingeborg and Desmet, Tom and Van Landschoot, Anita and Briers, Yves},
  issn         = {0175-7598},
  journal      = {APPLIED MICROBIOLOGY AND BIOTECHNOLOGY},
  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},
  language     = {eng},
  number       = {23},
  pages        = {10091--10102},
  title        = {Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis},
  url          = {http://dx.doi.org/10.1007/s00253-018-9389-3},
  volume       = {102},
  year         = {2018},
}

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