Calcium modulates activity and stability in a novel halotolerant endo-mannanase

β-Mannanases are involved in the hydrolysis of mannan, a major component of hemicellulose. The genome of Glutamicibacter halophytocola sp., an endophytic bacterium isolated from Crithmum maritimum, a halophilic plant from the Island of Crete, encodes a putatively secreted β-mannanase (Gh_GH26) belonging to glycoside hydrolase 26 family (GH26). This sequence occupies a poorly explored region of the GH26 sequence space. Gh_GH26 consists of three domains, including a C-terminal carbohydrate-binding module 23 (CBM23), which is uncommon among GH26s and has a peculiar amino acidic composition at the -3/-4 substrate subsites. Gh_GH26 displays halophilic behavior, retaining activity up to 2.5 M NaCl, and exhibits endo-mannanase activity. Functional analyses combined with structural predictions suggest that Gh_GH26 coordinates Ca²⁺ ions through a single binding site located within the CBM23 domain. The residues involved in Ca²⁺ binding are conserved in the CBMs of other characterized GH26s, despite their low similarity with Gh_GH26. Site-directed mutagenesis shows that Ca²⁺ ions contribute to both enzyme activity and stability. These results suggest that Ca²⁺ may play an evolutionary conserved activation role, modulating CBM flexibility and its orientation relative to the catalytic domain. Overall, this work expands our understanding of the functional and structural diversity of the GH26 family, highlighting its potential for application in the degradation of mannan-containing biomass under saline conditions.