A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley - Analysis of substrate preference and specificity by nano-probe NMR

Henrik Ferré, Anders Broberg, Jens Øllgaard Duus, Karl K. Thomsen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of approximate to 60 and approximate to 66 kDa, a pI of 4.55 and almost identical N-terminal amino-acid sequences. With p-nitrophenyl alpha -L-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a K-m of 0.5 mm and a V-max of 6.7 mu mol.min(-1).(mg of protein)(-1). Maximum activity was displayed at pH 4.2 and 60 degreesC, and, under these conditions, the half-life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p-nitrophenyl alpha -L-arabinopyranoside or p-nitrophenyl beta -D-xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released arabinose from high-molecular-mass arabinoxylan and arabinoxylan-derived oligosaccharides but was inactive against linear or branched-chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo-oligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substituents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-O-linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non-reducing terminal xylose, the 3-O-linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p-nitrophenyl alpha -L-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.
Original languageEnglish
JournalEuropean Journal of Biochemistry
Volume267
Issue number22
Pages (from-to)6633-6641
ISSN0014-2956
DOIs
Publication statusPublished - 2000
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Carbohydrate Sequence
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Enzyme Stability
  • Glycoside Hydrolases
  • Hordeum
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Nuclear Magnetic Resonance, Biomolecular
  • Oligosaccharides
  • Peptide Fragments
  • Protein Subunits
  • Substrate Specificity
  • Thermodynamics
  • EC 3.2.1.- Glycoside Hydrolases
  • EC 3.2.1.55 alpha-N-arabinofuranosidase
  • BIOCHEMISTRY
  • ALPHA-L-ARABINOFURANOSIDASE
  • NUCLEAR-MAGNETIC-RESONANCE
  • FERULIC ACID-ESTERS
  • DIRECT LINEAR PLOT
  • HORDEUM-VULGARE
  • CELL-WALLS
  • ASPERGILLUS-AWAMORI
  • (1->4)-BETA-XYLAN ENDOHYDROLASES
  • H-1-NMR SPECTROSCOPY
  • HYDROLYZING ENZYMES
  • arabinofuranosidase
  • arabinoxylan arabinofuranohydrolase
  • barley
  • nano-probe NMR
  • xylo-oligosaccharides
  • Monocotyledones Angiospermae Spermatophyta Plantae (Angiosperms, Monocots, Plants, Spermatophytes, Vascular Plants) - Gramineae [25305] Hordeum vulgare cultivar-Maud
  • arabinoxylan 9040-27-1 enzyme substrate
  • arabinoxylan arabinofuranohydrolase purification, substrate specificity
  • oligosaccharide enzyme substrate
  • p-nitrophenyl alpha-L-arabinofuranoside 6892-58-6 enzyme substrate
  • xylo-oligosaccharide enzyme substrate
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 51518, Plant physiology - Enzymes
  • Biochemistry and Molecular Biophysics
  • nano-probe NMR analytical method
  • TLC thin layer chromatography analytical method
  • Enzymology

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