Search

The temperatures needed to denature enzymes from thermophilic organisms have been measured by melting the two analogous protein domains E2(sub cd) and CenA(sub P30). The former is the isolated catalytic domain of cellulase E2 of Thermomonospora fusca while the latter is the analogous domain of the cellulase CenA from Cellulomonas fumi. Results indicate that T. fusca has a melting temperature (Tm) of 72.2 C, a van't Hoff enthalpy of unfolding (deltaH(sub VH)) of 190 kcal/mol and an entropy of unfolding (deltaS(sub u)) of 0.55 kcal/mol*K. C. fumi has a Tm of 56.4 C, a deltaH(sub VH) of 107 kcal/mol and a deltaS(sub u) of 0.32 kcal/mol*K

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.

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.

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.

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.

Determination of the high-resolution crystal structure of the Cex catalytic domain in Cellulomonas fimi reveals that the crystal structure contains 2400 protein atoms, 0.217 R-factor and 45 water molecules at a resolution of 1.8 Angstrom units. The folding of the protein into an eight-parallel-stranded alpha/beta-barrel is new for the microbial beta-glycanase Cex. While the active site exists as an open cleft on the alpha/beta-barrel carboxy-terminal, the hydrogen bonding network in the domain stabilizes the ionization states of vital residues for regulating the equilibrium of the reactions.

CenA and Cex are β-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C.fimi-type cellulose-binding domains. CenA and Cex belong to families and F, respectively, in the classification of β-1,4-glucanases and β-l,4-xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 β-l,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km for hydrolysis of the heterosidic bond of p-nitrophenyl-β-D-xylobiosideis 14385 min-1 · mM-1 at 25 °C; the corresponding kcat/Km for p-nitrophenyl-β-D-cellobioside hydrolysis is 296 min-1 · mM-1. [ABSTRACT FROM AUTHOR]

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA), Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA catalytic domain are closely related to those of CBH II, an exocellobiohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesei. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family. [ABSTRACT FROM AUTHOR]