Location: Bioproducts ResearchTitle: Cloning of an a-L-arabinofuranosidase and characterization of its action on mono- and di-substituted xylopyranosyl units
Submitted to: Advances in Enzyme Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2022
Publication Date: 12/12/2022
Citation: Wong, D., Batt Throne, S.B. 2022. Cloning of an a-L-arabinofuranosidase and characterization of its action on mono- and di-substituted xylopyranosyl units. Advances in Enzyme Research. 10(4):75-82. https://doi.org/10.4236/aer.2022.104005.
Interpretive Summary: Arabinoxylans from cereal grains are highly branched. Those from wheat and barley endosperm contain considerable level of main chain Xylp residues singly or doubly substituted with arabinofuranosyl unit (Araf) attached to C2/C3 positions. The cooperative interactions between main chain enzymes and enzymes liberating side chain substituents (collectively known as accessory enzymes) are key to complete depolymerization of biomass xylan. Notably, endo-xylanase has been known to act synergistically with increased efficiency when arabinofuranosidase is used to remove Araf side groups. The present work reports the cloning and characterization of an '-L-arabinofuranosidase that catalyzes the release of arabinose from doubly substituted xylose units.
Technical Abstract: An alpha-L-arabinofuranosidase (XaARF) gene of 1503 bp was synthesized, subcloned into pET26b vector, and expressed in Escherichia coli. The enzyme purified in active form consisted of 500 amino acid residues, corresponding to 55 kD based on SDS-PAGE. The affinity purified protein was characterized using arabinofuranosyl xylooligosaccharides (AXOS) as substrates. The pH effect was investigated showing an optimum at pH 5.5. XaARF catalyzed the cleavage of arabinose at C3 of the xylopyranosyl unit efficiently if the arabinofuranosyl substitution was at the terminal compared to the internal xylose unit. The enzyme was able to act on di-substituted xylopyranosyl units with first cleavage at C3 followed by C2 linkages.