Recalcitrant hemicellulose oligosaccharides from corn fiber: Structural insights and enzymatic strategies

Corn fiber is a major carbohydrate-rich by-product of the corn-processing industry, but its efficient utilization is hindered by the structural recalcitrance of hemicellulose. To analyze its resistance structure to enzymes, hemicellulose enzymatic oligosaccharides (HEOs) were generated by hydrolyzing corn fiber holocellulose with crude enzyme from Penicillium oxalicum MCAX. Integrated analyses of monosaccharide composition, glycosidic linkages, NMR, and MALDI-TOF-MS showed that HEOs retained glucuronic acid and acetyl substituents and contained key recalcitrant features, including T-α-Xyl-(1 → 3)-Ara, T-β-Xyl-(1 → 2)-Ara, T-β-Gal-(1 → 2)-Ara side chains, Ara-Ara linkages, and a previously unreported T-α-Gal-(1 → 3)-Ara motif in corn fiber hemicellulose. Based on these findings, structural models of HEOs were proposed, providing a framework for targeted enzyme selection strategies. Among seven GH31 α-xylosidases screened from diverse sources, only rAnXyl31A from Aspergillus niger showed activity toward HEOs and synergized with MCAX to increase the release of hemicellulose-derived monosaccharides. 2D NMR analysis of hydrolysates supported cleavage of the resistant T-α-Xyl-(1 → 3)-Ara motif by rAnXyl31A and its cooperative debranching effect with MCAX, resulting in a more extensive removal of terminal α-xylosyl and α-galactosyl substitutions. These results provide structural insights into corn fiber hemicellulose recalcitrance and offer practical guidance for the rational design of enzyme systems to achieve more efficient biomass conversion and utilization.