Сarbohydrate binding module CBM28 of endoglucanase Cel5D from Caldicellulosiruptor bescii recognizes crystalline cellulose.

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·104 and 2.2·106M-1 for cellotetraose and cellohexaose, respectively. The interaction between CbСВМ28 and amorphous cellulose is best described by a two-binding-site model with the binding constants of 1.5·105 and 1.9·105M-1. In a competitive binding assay in the presence of a 10-fold excess of cellohexaose the binding constant of CbСВМ28 to amorphous cellulose was 1.9·105M-1. A two-binding-site model also better approximates the binding to Avicel with the binding constants of 8.3·105 and 3.2·104M-1; while in the presence of cellohexaose, the binding is described by a single-binding-site model with the binding constant of 2.3·104M-1. With CbСВМ28 binding to bacterial crystalline cellulose with a constant of 7.4·104M-1, this is the first report of such a strong binding to crystalline cellulose for a module of family 28.