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Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly.

Authors
  • Senf, Deborah1, 2
  • Ruprecht, Colin1
  • de Kruijff, Goswinus H M1, 2, 3
  • Simonetti, Sebastian O1, 4
  • Frank Schuhmacher1, 2
  • Seeberger, Peter H1, 2
  • Pfrengle, Fabian1, 2
Type
Published Article
Journal
Chemistry - A European Journal
Publisher
Wiley (John Wiley & Sons)
Publication Date
Mar 02, 2017
Volume
23
Issue
13
Pages
3197–3205
Identifiers
DOI: 10.1002/chem.201605902
PMID: 28092124
Source
Medline
Keywords
License
Unknown

Abstract

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.

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