orabase and 4-nitrophenyl-beta-cellobioside

An endo-β-1,4-glucanase gene,

Topics: Amino Acid Sequence; Bacillus; Bacterial Proteins; beta-Glucans; Biophysical Phenomena; Carboxymethylcellulose Sodium; Catalytic Domain; Cellulase; Cloning, Molecular; Enzyme Assays; Enzyme Stability; Glucosides; Hordeum; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Weight; Oryza; Protein Sorting Signals; Receptors, Cell Surface; Sequence Alignment; Substrate Specificity; Temperature

An extracellular 1,3-1,4-β-glucanase-producing strain S2 was isolated from booklice and identified as Bacillus methylotrophicus. Furthermore, a homogeneous extracellular 1,3-1,4-β-glucanase GCS2 was purified by ammonium sulfate precipitation and cation-exchange chromatography. The gene for the 1,3-1,4-β-glucanase was cloned, and the nucleotide sequence was determined. Characterization of the purified enzyme revealed the molecular mass of 26 kDa and the optimum activity at pH 7.5, 55°C. The purified enzyme can highly hydrolyze carboxymethylcellulose including oat gum, barley β-glucan, CMC and lichenan, while low activity on avicel, cellobiose, filter paper, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-glucoside, but no activity against microcrystalline cellulose or salicin. The enzyme was stable at wide range of pHs 5-10 and still maintained above 60% activity at 70°C. The enzyme activity was stimulated by Trixon X-100. The property of the enzyme GCS2 makes this enzyme a broad prospect in brewing and commercial detergent industry. To our knowledge, this is the first report of a 1,3-1,4-β-glucanase from microbes associated with booklice.

Topics: Animals; Bacillus; beta-Glucosidase; Carboxymethylcellulose Sodium; Cellobiose; Cellulose; Chromatography, Ion Exchange; Cloning, Molecular; Glucans; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; Phthiraptera; Sequence Analysis, DNA; Substrate Specificity; Temperature

A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.

Topics: Ammonium Sulfate; Animals; Anions; Blotting, Western; Bromides; Carboxymethylcellulose Sodium; Cellulase; Cellulases; Chlorides; DEAE-Dextran; Electrophoresis, Polyacrylamide Gel; Endo-1,4-beta Xylanases; Enzyme Activation; Enzyme Stability; Fluorides; Gastric Juice; Glucan 1,4-beta-Glucosidase; Glucosides; Glycosylation; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Liver; Molecular Weight; Mollusca; Nitrates; Spectrometry, Fluorescence; Starch; Substrate Specificity; Temperature; Xylans

Endocellulase E2 from the thermophilic bacterium Thermomonospora fusca is a member of glycosyl-hydrolase family 6 and is active from pH 4 to 10. Enzymes in this family hydrolyze beta-1,4-glycosidic bonds with inversion of the stereochemistry at the anomeric carbon. The X-ray crystal structures of two family 6 enzymes have been determined, and four conserved aspartic acid residues are found in or near the active sites of both. These residues have been mutated in another family 6 enzyme, Cellulomonas fimi CenA, and evidence was found for both a catalytic acid and a catalytic base. The corresponding residues in E2 (D79, D117, D156, and D265) were mutated, and the mutant genes were expressed in Streptomyces lividans. The mutant enzymes were purified and assayed for activity on three cellulosic substrates and 2, 4-dinitrophenyl-beta-D-cellobioside. Activity on phosphoric acid-swollen cellulose was measured as a function of pH for selected mutant enzymes. Binding affinities for each mutant enzyme were measured for two fluorescent ligands and cellotriose, and circular dichroism spectra were recorded. The results show that the roles of D117 and D156 are the same as those for the corresponding residues in CenA; D117 is the catalytic acid, and D156 raises the pK(a) of D117. No specific function was assigned to the CenA residue corresponding to D79, but in E2, this residue also assists in raising the pK(a) of D117 and is important for catalytic activity. The D265N mutant retained 7% of the wild-type activity, indicating that this residue is not playing the role of the catalytic base. Experiments were conducted to rule out contamination of the D265 enzymes by either wild-type E2 or an endogenous S. lividans CMCase.

Topics: Actinomycetales; Binding Sites; Carboxymethylcellulose Sodium; Cellobiose; Cellulase; Circular Dichroism; Deuterium Oxide; Filtration; Glucosides; Hydrogen-Ion Concentration; Hymecromone; Mutagenesis, Site-Directed; Paper; Phosphoric Acids; Solvents; Trisaccharides

The activity of four Clavibacter michiganensis subsp. sepedonicus strains against various cellulose substrates was investigated. Sixty-seven Clavibacter michiganensis subsp. sepedonicus strains grew well on media amended with carboxymethylcellulose, 64 strains produced zones of hydrolysis. Endoglucanase activity was optimal at 37 degrees C and pH 6.0 against carboxymethylcellulose incorporated in plate assays. Zymogram and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein band corresponding to the cellulolytic activity in the molecular weight (MW) range of approximately 28,000. Protein bands in the same range were detected in five Clavibacter michiganensis subsp. sepedonicus strains. Studies on crude enzyme extracts of Clavibacter michiganensis subsp. sepedonicus strain N-1-1 revealed that p-nitrophenyl beta-D-cellobioside (pNPC) was hydrolyzed, with optimal activity at 37 degrees C and pH 7.0.

Topics: Carboxymethylcellulose Sodium; Cellulase; Glucosides; Gram-Positive Asporogenous Rods; Molecular Weight; Solanum tuberosum

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.

Topics: Bacteroides; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genes, Bacterial; Glucans; Glucosides; Hydrogen-Ion Concentration; Molecular Weight; Polysaccharides; Xylans