Characterization of a Double Cellulose-binding Domain SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE*

Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide- stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant dou- ble CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli , the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD compo-nents was investigated on different types of cellulose substrates as well as chitin. Under saturating condi-tions, nearly 20 (cid:109) mol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhib- ited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase. A bifunctional domain organization, consisting of a catalytic domain and a substrate-binding domain that are connected by an extended linker region, is typical for enzymes degrading solid substrates. The properties and role in hydrolysis of the cellulose-binding domains (CBDs) 1