The thermodynamic stability of family 16 endo-b-1,3-glucanase (EC ) from the hyperthermophilic archaeon Pyrococcus furiosusis decreased upon single (D287A, E53A) and double (E53A⁄D287A) muta-tion of Asp287 and Glu53. In accordance with the homology model predic-tion, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator.