CHARACTERIZATION OF THE CELLULOSE-BINDING DOMAIN OF THE CLOSTRIDIUM-CELLULOVORANS CELLULOSE-BINDING PROTEIN-A

Cellulose-binding protein A (CbpA), a component of the cellulase compl ex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA codi ng for this putative CBD was subcloned into pET-8c, an Escherichia col i expression vector. The protein produced under the direction of the r ecombinant plasmid, pET-CBD, had a high affinity for crystalline cellu lose. Affinity-purified CBD protein was used in equilibrium binding ex periments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crys talline cellulose samples (e.g., cotton) was greater than that of samp les of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of C BD per g. The measured dissociation constant was in the 1 muM range fo r all cellulose samples. The results suggest that the CBD binds specif ically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cel lulose. This result suggests that the CBD recognition site is larger t han a simple cellobiose unit or more complex than a repeating cellobio se moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an in dependently functional domain.