Structure-based mining of a chitosanase with distinctive degradation mode and product specificity.

Chitosan derivates with varying degrees of polymerization (DP) have attracted great concern due to their excellent biological activities. Increasing the abundance of chitosanases with different degradation modes contributes to revealing their catalytic mechanisms and facilitating the production of chitosan derivates. However, the identification of endo-chitosanases capable of producing chitobiose and D-glucosamine (GlcN) from chitosan substrates has remained elusive. Herein, an endo-chitosanase (CsnCA) belonging to the GH46 family was identified based on structural analysis in phylogenetic evolution. Moreover, we demonstrate that CsnCA acts in a random endo-acting manner, producing chitosan derivatives with DP ≤ 2. The in-depth analysis of CsnCA revealed that (GlcN)3 serves as the minimal substrate, undergoing cleavage in the mode that occupies the subsites - 2 to + 1, resulting in the release of GlcN. This study succeeded in discovering a chitosanase with distinctive degradation modes, which could facilitate the mechanistic understanding of chitosanases, further empowering the production of chitosan derivates with specific DP. KEY POINTS: • Structural docking and evolutionary analysis guide to mining the chitosanase. • The endo-chitosanase exhibits a unique GlcN-producing cleavage pattern. • The cleavage direction of chitosanase to produce GlcN was identified.