cellulase 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

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library(1). We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

Topics: Alkaline Phosphatase; Base Sequence; Cellulase; Enzymes; Escherichia coli; Flow Cytometry; Gene Library; Glucosides; High-Throughput Screening Assays; Lipase; Metagenomics; Nitrophenols; Organophosphorus Compounds; Substrate Specificity

The hydrolysis of p-nitrophenyl-β-1,4-cellobioside (pNP-G2) by the catalytic domain of the retaining-family 5-2 endocellulase Cel5A from Thermobifida fusca (Cel5Acd) was studied. The dominant reaction pathway involves hydrolysis of the aglyconic bond, producing cellobiose (G2) and a 'reporter' species p-nitrophenol (pNP), which was monitored spectrophotometrically to track the reaction. We also detected the production of cellotriose (G3) and p-nitrophenyl-glucoside (pNP-G1), confirming the presence of a competing transglycosylation pathway. We use a mechanistic model of hydrolysis and transglycosylation to derive an expression for the rate of pNP-formation as a function of enzyme concentration, substrate concentration, and several lumped kinetics parameters. The derivation assumes that the quasi-steady-state assumption (QSSA) applies for three intermediate species in the mechanism; we determine conditions under which this assumption is rigorously justified. We integrate the rate expression and compare its integral form to pNP-versus-time data collected for a range of enzyme and substrate concentrations. The integral comparison gives a stringent test of the mechanistic model, and it serves to quantify the lumped kinetics parameters with good statistical precision, particularly a previously unidentified parameter that determines the selectivity of hydrolysis versus transglycosylation. The integrated rate expression accounts well for pNP-versus-time data under all circumstances we have investigated.

Topics: Actinomycetales; Catalytic Domain; Cellobiose; Cellulase; Enzyme Inhibitors; Glucose; Glucosides; Glycosylation; Hydrolysis; Kinetics; Substrate Specificity

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

Type C-4 strain of Trichoderma harzianum was isolated as a microorganism with high cellulolytic activity. Beta-glucosidase is involved in the last step of cellulose saccharification by degrading cellobiose to glucose, and plays an important role in the cellulase enzyme system with a synergic action with endoglucanase and cellobiohydrolase for cellulose degradation. Beta-glucosidase from T. harzianum type C-4 was purified to homogeneity through Sephacryl S-300, DEAE-Sephadex A-50, and Mono P column chromatographies. It was a single polypeptide with the molecular mass of 75,000 by SDS-PAGE. The enzyme was very active at pH 5.0 and 45 degrees C. No significant inhibition was observed in the presence of metal ions, thiol reagents, or EDTA. The enzyme was stable in the presence of 5% ox gall and digestive enzymes. p-Nitrophenyl-beta-D-cellobioside worked as a substrate for the enzyme as much as p-nitrophenyl-beta-glucopyranoside. Glucose and gluconolactone showed competitive inhibition with a Ki of 1 mM and 1.8 microM, respectively, while galactose, mannose, and xylose did not inhibit the enzyme significantly.

Topics: beta-Glucosidase; Cellulase; Cellulose; Chromatography, Liquid; Edetic Acid; Enzyme Inhibitors; Enzyme Stability; Gluconates; Glucose; Glucosides; Hydrogen-Ion Concentration; Lactones; Metals; Substrate Specificity; Trichoderma

A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.

Topics: Amino Acid Sequence; Cellulase; Cellulose; Glucosides; Mannans; Molecular Sequence Data; Peptide Mapping; Phanerochaete; Sequence Homology, Amino Acid; Substrate Specificity; 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 interaction of cellobiose dehydrogenase (CDH) with cellobiohydrolase I (CBH I) in cellulose-grown cultures of Phanerochaete chrysosporium was investigated to clarify the role of CDH in cellulose degradation. Decomposition of bacterial microcrystalline cellulose by CBH I was enhanced significantly in the presence of the CDH/ferricyanide redox-system compared with CBH I alone. To explain this phenomenon, a model system, using p-nitrophenyl-beta-D-cellobioside as a substrate, was elaborated for measurement of CBH I activity with and without the CDH redox-system. The activity of CBH I for hydrolysis of p-nitrophenyl-beta-D-cellobioside was also enhanced in the presence of the redox system. It was found that Km for hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I was lower in the presence than in the absence of the CDH/ferricyanide redox-system, 142 microM and 384 microM, respectively, while no significant difference was observed between the k(cat) values. These results indicate that cellulase activity is enhanced by an increased affinity for p-nitrophenyl-beta-D-cellobioside, rather than by an increased hydrolysis rate. This shows that cellobiose, the hydrolysis product, acts as a competitive inhibitor of the interaction between CBH I and p-nitrophenyl-beta-D-cellobioside. This was confirmed by addition of cellobiose, which was found to competitively inhibit hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I in the absence of the CDH redox system, and the Ki value for cellobiose inhibition was estimated to be 65 microM. However, this inhibition did not occur if cellobiose was incubated with CDH before addition of CBH I. It was concluded from these results that the reason for the enhancement of CBH I activity in the presence of the CDH redox system was that it relieves competitive inhibition of cellobiose by its oxidation to cellobionolactone.

Topics: Basidiomycota; Binding, Competitive; Carbohydrate Dehydrogenases; Cellobiose; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Ferricyanides; Glucosides; Hydrolysis; Kinetics; Oxidation-Reduction; Substrate Specificity

Bacillus sp. D04 secreted a bifunctional cellulase that had a molecular weight of 35,000. This cellulase degraded Cm-cellulose, cellotetraose, cellopentaose, p-nitrophenyl-beta-D-cellobioside, and avicel PH101. Based on the high performance liquid chromatography analysis of the degradation products, this cellulase randomly cleaved internal beta-1, 4-glycosidic bonds in cellotetraose and cellopentaose as an endoglucanase. It also hydrolyzed the aglycosidic bond in p-nitrophenyl-beta-D-cellobioside and cleaved avicel to cellobiose as an exoglucanase. Cellobiose competitively inhibited the p-nitrophenyl-beta-D-cellobioside degrading activity but not Cm-cellulose degrading activity. Ten mM p-chloromercuribenzoate inhibited p-nitrophenyl-beta-D-cellobioside degrading activity completely, but Cm-cellulose degrading activity incompletely. Cm-cellulose increased p-nitrophenyl-beta-D-cellobioside degrading activity, and vice versa, whereas methylumbelliferyl-beta-D-cellobiose strongly inhibited p-nitrophenyl-beta-D-cellobioside degrading activity. The cellulase gene (cel gene), 1461 base pairs, of Bacillus sp. D04 was cloned. The nucleotide sequence of the cel gene was highly homologous to those of Bacillus subtilis DLG and B. subtilis BSE616. The cel gene was overexpressed in Escherichia coli, and its product was purified. The substrate specificity and substrate competition pattern of the purified recombinant cellulase were the same as those of the purified cellulase from Bacillus sp. D04. These results suggest that a single polypeptide cellulase had both endo- and exoglucanase activities and each activity exists in a separate site.

Topics: Amino Acid Sequence; Bacillus; Base Sequence; beta-Glucosidase; Cellulase; Cloning, Molecular; Escherichia coli; Genes, Bacterial; Glucan 1,3-beta-Glucosidase; Glucosides; Kinetics; Molecular Sequence Data; Multienzyme Complexes; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity

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

A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.

Topics: beta-Glucosidase; Cellobiose; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Glucosidases; Glucosides; Glycoside Hydrolases; Glycosides; Kinetics; Methods