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SUMMARY Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular “Velcro” for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications.

Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein--carbohydrate composite material. We describe the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (K(d), B(max)) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier transform infrared spectroscopy, circular dichroism, and atomic force microscopy with peroxidase cross-linked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine cross-links. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.

A series of four cellulose nanocrystal (CNC) suspensions were prepared from bleached softwood kraft pulp using different conditions of sulfuric acid hydrolysis. The CNCs were identical in size (95 nm in length × 5 nm in width) but had different surface charges corresponding to the harshness of the hydrolysis conditions. Consequently, it was possible to isolate the effects of surface charge on the self-assembly and viscosity of the CNC suspensions across surface charges ranging from 0.27%S to 0.89%S. The four suspensions (never-dried, free of added electrolyte) all underwent liquid crystalline phase separation, but the concentration onset for the emergence of the chiral nematic phase shifted to higher values with increasing surface charge. Similarly, suspension viscosity was also influenced by surface charge, with suspensions of lower surface charge CNCs more viscous and tending to gel at lower concentrations. The properties of the suspensions were interpreted in terms of the increase in effective diameter of the nanocrystals due to the surface electrostatic repulsion of the negative sulfate half-esters, as modified by the screening effects of the H counterions in the suspensions. The results suggest that there is a threshold surface charge density (∼0.3%S) above which effective volume considerations are dominant across the concentration range relevant to liquid crystalline phase formation. Above this threshold value, phase separation occurs at the same effective volume fraction of CNCs (∼10 vol %), with a corresponding increase in critical concentration due to the decrease in effective diameter that occurs with increasing surface charge. Below or near this threshold value, the formation of end-to-end aggregates may favor gelation and interfere with ordered phase formation.

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.

The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced. When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed. The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da. The open reading frame is preceded by a Gram-positive-type ribosome binding site. A signal peptide sequence of 28 amino acids is present at its N terminus. The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues. There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids. These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex.

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