Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A

M. A. Goldstein, M. Takagi, S. Hashida, O. Shoseyov, R. H. Doi*, I. H. Segel

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

147 Scopus citations

Abstract

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 μmol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 μmol of CBD per g. The measured dissociation constant was in the 1 μM range for all cellulose samples. The results suggest that the CBD binds specifically 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 cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.

Original languageAmerican English
Pages (from-to)5762-5768
Number of pages7
JournalJournal of Bacteriology
Volume175
Issue number18
DOIs
StatePublished - 1993
Externally publishedYes

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