Automated Syntheses of Xylan Oligosaccharides Containing β3-Linkages Enable Substrate Specificity Studies of Xylanases from Marine Bacteria

Marine polysaccharides, including β3-xylan and mixed-linkage xylan (MLX), play a central role in the ocean's carbon cycle. Using an optimized xylose donor, automated glycan assembly enabled the synthesis of β3- and MLX-oligosaccharides up to decasaccharides. Interrogation of these structurally defined substrates with the marine xylanases Xyl4, AlXyn26A, and MfXyn26A revealed a surprisingly broad substrate scope of MLXases toward both β3-xylan and MLX motifs, highlighting AGA-derived glycans as powerful probes for studying marine polysaccharide metabolism.