Biochemical Characterization of a Family GH18 Specific-Domain Chitinase: Chitin-Binding Domain Modulates the Reaction...

Chitinase is an essential tool for the high-value utilization of chitin and the production of N-acetyl chito-oligosaccharides (N-acetyl COSs). The reaction specificity of chitinase is a key determinant of product composition. Previous studies have shown that carbohydrate-binding modules (CBMs) may influence the reaction specificity of glycoside hydrolases, though few studies have focused on this aspect in chitinases. Here, we identified a chitinase ChiZg from Zooshikella ganghwensis, characterized by the spatial separation of the chitin-binding domain (ChBD) from the catalytic domain (CD). ChiZg modulated product specificity for (GlcNAc)₂ in an atypical exo-mode, and the (GlcNAc)₂ yield ultimately maintained a relative balance as the substrate concentration and enzyme amount changed. Additionally, we found that the ChBD in ChiZg could modulate the enzyme's reaction specificity. A ChBD-truncated mutant exhibited additional N-acetylglucosaminidase activity, hydrolyzing (GlcNAc)₂ to GlcNAc. We also engineered a mutant by translocating the ChBD from the N-terminus to the C-terminus, which aligned with the CD spatial configuration. It enhanced product specificity for (GlcNAc)₃ with minimal GlcNAc production. This work expands the understanding of the ChBD-mediated reaction specificity in chitinases, providing an effective catalytic tool for the efficient degradation of chitin and the production of N-acetyl COSs with specific configurations.