Characterization of a Previously Uncharacterized GH43 Arabinofuranosidase with Activity toward O -2 and O -3 Disubstituted Arabinoxylan Oligosaccharides

Arabinoxylans can be converted by endoxylanases into arabinoxylo-oligosaccharides (AXOS), which require debranching by arabinofuranosidases to yield xylo-oligosaccharides (XOS). However, production of XOS is limited by doubly substituted xylosyl residues which are difficult to debranch. In this study, we identified a previously uncharacterized arabinofuranosidase AsAbf43A (GH43_28) from Alkalitalea saponilacus. Notably, AsAbf43A can remove both the O-2- and O-3-linked arabinosyl substituents from doubly substituted xylosyl residues at the nonreducing ends of arabinoxylo-oligosaccharides (AXOS), a capability rarely reported among characterized GH43 enzymes. The kcat/Km of AsAbf43A was 175.03 ± 37.50 min–1·mM–1 for A2XX and 0.43 ± 0.08 min–1·mM–1 for A2,3XX. Comparison of the AlphaFold model of AsAbf43A with monosubstitution specific BsAXH-m2,3 from Bacillus subtilis suggested an additional pocket adjacent to the −1 subsite which may accommodate the second arabinosyl substituent. This provides a possible structural hypothesis for recognition of disubstituted AXOS.