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 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.
Original language | English |
---|---|
Pages (from-to) | 10091–10102 |
Journal | Applied Microbiology and Biotechnology |
Volume | 102 |
Issue number | 23 |
DOIs | |
Publication status | Published - Dec 2018 |
MoE publication type | A1 Journal article-refereed |
Keywords
- DSA-FACE
- Enzyme analysis
- HPAEC-PAD
- Substrate specificity
- α-L-arabinofuranosidases
Fingerprint
Dive into the research topics of 'Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis'. Together they form a unique fingerprint.Equipment
-
Bioeconomy Research Infrastructure
Seppälä, J. (Manager)
School of Chemical EngineeringFacility/equipment: Facility