Characterization and Application of a Novel Gellan Carbohydrate Binding Module from the CBM96 Family

Abstract Gellan gum exhibits excellent gelation properties and is a valuable hydrocolloid in the food industry. Carbohydrate-binding modules (CBMs) are important tools for polysaccharide research, particularly as probes for in situ analysis. However, no gellan gum CBM has been reported. Herein, a CBM96 domain from a putative gellan gum lyase was recombinantly expressed. The recombinant protein ClCBM96 specifically binds to gellan gum with an association constant (Ka) of 1.5 × 105 M–1. The crystal structure of ClCBM96 was determined at a resolution of 1.81 Å, revealing a typical β-sandwich fold. Site-directed mutagenesis identified residues W28, K32, T37, and R39 as critical for gellan gum binding. A fluorescent probe constructed by fusing ClCBM96 with green fluorescent protein was validated for in situ visualization of gellan gum. In summary, this study reports the first identification and characterization of a gellan gum-specific CBM, providing a valuable tool for future research on gellan gum.