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

COLIN RUPRECHT; FRANK SCHUMACHER; DEBORAH SENF; SEBASTIAN SIMONETTI; GSWINUS DE KRUIJFF; FABIAN PFRENGLE; PETER SEEBERGER

CHEMISTRY-A EUROPEAN JOURNAL

WILEY-V C H VERLAG GMBH

Lugar: Weinheim; Año: 2017

0947-6539

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as biofuel production. 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.