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Research has been conducted on the helical xylan polymer. The recognition of carbohydrate-binding module family 13 xylan by X-ray crystallography and its relationship with other beta-trefoil carbohydrate-binding proteins have been investigated and the results are discussed.

Research has been conducted on the carbohydrate-binding modules. The binding of these modules under osmotic conditions have been investigated with the specificity for the beta-1,3- and beta-(1,3)(1,4)-glucans and the effect of the amino acids on this binding activity has been examined and the results are reported.

Crystal structure analysis of the xylanase C-terminal module of the family 9 carbohydrate-binding module shows that it exhibits a beta-sandwich fold and three metal binding sites. Data indicate that the sugar-binding site containing two tryptophan residues binds exclusively to the reducing ends of oligosaccharides.

Results demonstrate that C-terminal family carbohydrate-binding module of xylanase binds to all forms of cellulose and binds specifically to the reducing ends of the soluble polysaccharides and cellulose. Data indicate that the binding is specific to two glucose units of the carbohydrates.

A family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A has been studied. The ligand-binding site, its specificity and affinity of substrate binding are discussed, as is the role of tryptophan residues.

The properties and activities of four cellulases of Cellulomonas fimi obtained from the purification of Escherichia coli were compared. Different types and amounts of soluble products resulted from the hydrolysis of phosphoric acid-swollen cellulose (PASC), although cellobiose was the major product obtained. The variability in the molecular size distributions data in both the PASC and the bacterial microcrystalline cellulose is influenced by their susceptibility to enzyme attacks.

A study was conducted to analyze the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of the Cellulomonas fimi endoglucanase cenC. Differential scanning calorimetry was utilized to measure stability and oligosaccharide-binding thermodynamics. Results revealed that the intramolecular link in the Cellulose fimi gene contributed to the stability of the endoglucanase cenC and a global two-dimensional minimization routine was utilized to regress the unfolding thermodynamics for the endoglucanase cenC-cellopentaose system.

The structure of the N-terminal cellulose-binding domain from Cellulomonas fimi 1,4-beta-glucanase CenC was determined by multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The results revealed 10 beta-strands which were folded into two antiparallel beta-sheets, forming a jelly-roll beta-sandwich. The strands of the protein surface were shorter than those found in the opposite side of the protein, resulting in a five-stranded binding cleft containing hydrophobic residues at the center and flanked by polar hydrogen-bonding groups.

The carbohydrate-binding specificity of the N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) was studied utilizing affinity electrophoresis, binding assays and microcalorimetry in parallel with NMR and difference ultraviolet absorbance spectroscopy. CBDN1 binds amorphous cellulose with high affinity, binds Avicel weakly and does not bind highly crystalline bacterial or tunicin cellulose. Also, CBDN1 binds soluble cellooligosaccharides and beta-1,4-linked oligomers of glucose such as hydroxyethylcellulose, soluble beta-1,3-1,5-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3, or beta-1,6-polymers of glucose.

The gene cenC of Cellulomonas fimi encodes beta-1,4-glucanase (CenC) which has both endo- and exoglucanase activity. The fully processed CenC enzyme is most active at pH 5.0 and 45 degree centigrade. The enzyme acts on soluble, fluorophoric and chromophoric glycosides. It also hydrolyzes carboxymethyl cellulose, barley beta-glucan, lichenan, and glucomannan. The main product of the enzymatic activity on soluble cellodextrins and insoluble cellulose is cellobiose.

The cellulose-binding domain (CBD) properties of a fungal family I and a bacterial family II were tested by replacing the family II CBD in an exoglucanase/xylanase (Cex) with a family I CBD from cellobiohydrolase I (CbhI). The hybrid protein contained the Cex catalytic domain with a CbhI CBD and had similar activities and substrate specificities as normal Cex. However, the hybrid was more active on soluble cellulose and xylan and less on bacterial microcrystalline cellulose than Cex.

A study was conducted to determine the solution structure of a 110 amino acid cellulose-binding domain from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi. Multidimensional, multinuclear nuclear magnetic resonance spectroscopy was used in tandem with dynamic simulated annealing to determine the structure. The structures computed had an average root-mean-square deviation about the mean structure of 0.41 angstrom for backbone atoms and 0.67 angstrom for heavy atoms when superimposed on the secondary structural elements.

Circular dichroism spectroscopy reveals that structures of the mutants of endoglucanase gene CenA from Cellulomonas fimi, prepared by the replacement of Asp 216, Asp252, Asp287 and Asp 392 by alanine and glutamate, are similar to those of wild-type enzymes. Mutation exerts no influence on the active site structure. Examination of the catalytic ability of these mutants reveals that catalysis does not require Asp 216, and Asp287 cooperates with Asp252 in acid catalysis.

Disruption and hydrolysis of cellulose fibers involve the synergistic action of the catalytic domain and nonhydrolytic cellulose-binding domain (CBD) of the endoglucanase A from Cellulomonas fimi. The CBDs are comparable to the C1 system while the catalytic domains are comparable to the Cx system. During hydrolysis, the CBD promotes proximity between the substrate and the catalytic domain and rearranges the substrate to permit easy access to the anchored catalytic domain.

The prolyl and threonyl residue linker cellular-binding domains of Cellulomonas fimi Cex (PT-CBDCex) exhibit increased microcrystalline cellulose affinity with the presence of glycans. Glycosylation occurs only in the PT-CBDCex containing a Pro-Thr linker and yields a heterogeneous product with about 10 mol of D-mannose equivalents. The glycans include 4 to 12 alpha-D-mannosyl and alpha-D-galactosyl residues and are O linked.