CHARACTERIZATION OF A DOUBLE CELLULOSE-BINDING DOMAIN - SYNERGISTIC HIGH-AFFINITY BINDING TO CRYSTALLINE CELLULOSE

Most cellulose-degrading enzymes have a two-domain structure that cons ists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains r epresent one of the key questions for the understanding of the functio n of these enzymes. The CBDs of fungal cellulases are small peptides f olding into a rigid, disulfide-stabilized structure that has a distinc t cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellula se linkers. After expression in Escherichia coli, the protein was puri fied from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the doubl e CBD and its single CBD components was investigated on different type s of cellulose substrates as well as chitin. Under saturating conditio ns, nearly 20 mu mol/g of the double CBD was bound onto microcrystalli ne cellulose. The double CBD exhibited much higher affinity on cellulo se than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding b ehavior of the double CBD. A similar interplay between the domains the the native enzyme is suggested for its binding to cellulase.