Modulation of oligomerization profiles in chitosan degradation by key chitosanase residues via steric and hydrophilic effects

Chitosan, a marine-derived polysaccharide with immense industrial potential, serves as the precursor for chitooligosaccharides (COS). COS exhibit structure-dependent bioactivities critical for biomedical and nutraceutical applications, yet precise control of their degree of polymerization (DP) remains challenging. This study investigated GH46 family chitosanases to elucidate mechanisms governing product specificity. Phylogenetic analysis identified the chitosanase Csn-BJ, which contains a characteristic loop structure. Biochemical characterization revealed Csn-BJ as an endo-acting enzyme with optimal activity at 40 °C and pH 7.0, hydrolyzing chitosan to glucosamine (GlcN) and chitobiose, with chitotriose as the minimum hydrolyzable substrate. Site-directed mutagenesis of residues Q97 (subsite -1) and A134 (subsite +1) modulated product oligomerization profiles, shifting from exclusive DP1-DP2 generation in wild-type to DP1-DP3 mixtures in mutants. Molecular docking localized these residues within the substrate-binding cleft. Complementary molecular dynamics simulations and free energy calculations revealed that the Q97K mutation strengthened electrostatic and van der Waals interactions, optimizing the catalytic microenvironment, while the A134C mutation induced structural fluctuations that displaced the substrate. We propose a novel "steric-hydration dual-target regulatory model", providing an enzymatic engineering strategy for the tailored production of COS with specific DP.