Bidirectional Loop Engineering Uncovers a Structural Determinant for Product Specificity in GH46 Family Chitosanases

Chitooligosaccharides (COSs) are functional oligosaccharides whose bioactivities depend on their degree of polymerization (DP). Enzymatic production of well-defined COSs remains challenging due to the broad product profiles of most chitosanases. Here, we employed a bidirectional loop-engineering strategy to decipher the function of a conserved loop (XDXXP) in GH46 chitosanases. Deleting this loop from Csn-BJ reduced hydrolytic activity by 5% but shifted the maximal product DP from 2 to 4 (yielding 14.2% ± 1.0% of DP4). Conversely, inserting the corresponding loop segment into Csn-BAC enhanced activity by 36% and increased the maximal product DP from 3 to 4 (yielding 20.2% ± 2.5% of DP4). Structural modeling, molecular docking, and molecular dynamics simulations revealed that the loop modulates the geometry, electrostatics, and hydrophilicity of the substrate-binding cleft, thereby influencing substrate recognition and product specificity. This work provides a promising strategy for tailoring chitosanases to produce high-value, customized COSs for food applications.