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

Abstract

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.

Keywords: Caldicellulosiruptor bescii; Carbohydrate-binding protein; Cellulase; Cellulose; Endoglucanase Cel5D; Isothermal titration calorimetry (ITC).