Analyzing the Substrate Specificity of a Class of Long‐Horned‐Beetle‐Derived Xylanases by Using Synthetic Arabinoxylan Oligo‐ and Polysaccharides

Xylophagous long‐horned beetles thrive in challenging environments. To access nutrients, they secrete plant‐cell‐wall‐degrading enzymes in their gut fluid; among them are cellulases of the subfamily 2 of glycoside hydrolase family 5 (GH5_2). Recently, we discovered that several beetle‐derived GH5_2s use xylan as a substrate instead of cellulose, which is unusual for this family of enzymes. Here, we analyze the substrate specificity of a GH5_2 xylanase from the beetle Apriona japonica (AJAGH5_2‐1) using commercially available substrates and synthetic arabinoxylan oligo‐ and polysaccharides. We demonstrate that AJAGH5_2‐1 processes arabinoxylan polysaccharides in a manner distinct from classical xylanase families such as GH10 and GH11. AJAGH5_2‐1 is active on long oligosaccharides and cleaves at the non‐reducing end of a substituted xylose residue (position +1) only if: 1) three xylose residues are present upstream and downstream of the cleavage site, and 2) xylose residues at positions −1, −2, +2 and +3 are not substituted.