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

Abstract

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium celulovorans, 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 ,umol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 gmol of CBD per g. The measured dissociation constant was in the 1 FM 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 celiobiose 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.

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila

“…9B). The wild type protein showed the lowest K d (0.64 Ϯ 0.10 M) and highest B max (4.68 Ϯ 0.16 mol of protein/g of chitin), which is well within the range of binding constants reported for other chitin binding proteins (23,30,31). The apical domain showed an ϳ2-fold increase in K d (1.14 Ϯ 0.05 M) and reduction in B max (2.95 Ϯ 0.05 mol of protein/g).…”

“…The primary role of peritrophic membrane is to protect the underlying epithelium against bacterial pathogens and their toxins in the food of the larvae (47). A number of insecticidal proteins with chitinase activity are known; however, not all the chitin-binding proteins have catalytic activity (23,30,31); XnGroEL appears to belong to the second category. Another atypical chitin-binding protein has been described earlier in S. marcescens, which interacted with chitin through solvent-exposed polar side chains (23).…”

Adsorption of Bacillus subtilis endo‐β‐1,4‐glucanase to cellulosic materials

“…The practical maximum adsorption was about 20 mg/g of cellulose. This value was lower than that the reported for the CBD from Clostridium cellulovorans (21).…”

Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis

“…Hence, it is expected that the loss of cellulose from the tunic of sj mutants would cause malformation of the tunic. To examine the presence or absence of cellulose in sj mutants, we visualized cellulose microfibrils by using the cellulose-binding domain (CBD) peptide and its specific antibody (28,29). A clear difference in the signal appeared between sj mutants and normal larvae at the same age (Fig.…”