orabase and laminaran

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6-10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.

Topics: Bacillus subtilis; beta-Glucans; Carboxymethylcellulose Sodium; Cellulose; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucans; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Polysaccharides; Substrate Specificity; Temperature

A new method for extracting soil enzymes is described and a microplate method for assaying soil beta-1,4-glucanases (cellulases) and beta-1,3-glucanases (laminarinases). Soil samples were mechanically disrupted to produce crude enzyme extracts, and diluted preps incubated in microplates containing either carboxymethyl cellulose (CMC) to determine cellulase activity or laminarin substrate to determine laminarinase activity. The resulting glucose was measured using the fluorometric Amplex Red glucose assay. The method was reproducible, could be completed in 1 day and measured twice as much enzyme activity than the standard passive soil enzyme extraction procedure. The method described herein facilitates the development of high-throughput soil multiplex enzymatic assays from several soil samples at one time, and is well suited to the study of functional microbial ecology.

Topics: Carboxymethylcellulose Sodium; Cellulase; Cellulases; Glucans; Glucose; Polysaccharides; Reproducibility of Results; Sensitivity and Specificity; Soil

Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol 6 culture medium. The molecular weights of EG A and EG B were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60 degrees C for EG A and 50 degrees C for EG B. EG A was thermostable at 60 degrees C and remained active after 1 h at 70 degrees C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and beta-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg2+, Co2+, and Mn2+.

Topics: beta-Glucans; Carboxymethylcellulose Sodium; Cations, Divalent; Cellulase; Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucans; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Molecular Weight; Mutation; Penicillium; Polysaccharides; Substrate Specificity; Temperature; Xylans

In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-beta-D-glucanases (EG V-VII) against a range of soluble beta-linked glucans. Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict beta-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-beta-D-glucans. Time-course hydrolysis studies of 1,4-beta-D-glucan (carboxymethylcellulose; CMC), 1,3;1,4-beta-D-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-beta-D-glucan substrates. The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-beta-glucan (laminaran), in contrast to EG V. Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time. Kinetic constants (Km, Vmax, kcat and kcat/Km) determined for EG V-VII with BBG as substrate yielded similar Km and Vmax values for EG V and EG VI. EG VII exhibited highest affinity for BBG (Km value of 9.1 mg ml(-1)) and the highest catalytic efficiency (kcat/Km of 12.63 s(-1) mg(-1) ml).

Topics: Carbohydrate Conformation; Carboxymethylcellulose Sodium; Catalysis; Cellulase; Chromatography, Ion Exchange; Glucans; Glycoside Hydrolases; Hordeum; Kinetics; Polysaccharides; Solubility; Substrate Specificity; Talaromyces; Viscosity

Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.

Topics: beta-Glucans; Binding Sites; Binding, Competitive; Carboxymethylcellulose Sodium; Cell Adhesion Molecules; Cell Membrane; Glucans; Humans; Lipoproteins, LDL; Monocytes; Poly C; Poly I; Polysaccharides; Receptors, Immunologic; Receptors, LDL; Receptors, Scavenger; Sizofiran; U937 Cells

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

Comparative investigations were carried out concerning substrate specificity and action patterns of seven Bacillus-endo-beta-glucanases produced by the species, B. subtilis, B. macerans, B. amyloliquefaciens, B. circulans, B. laterosporus, B. pumilus and B. polymyxa. All enzymes with the exception of beta-glucanase from B. macerans hydrolyze lichenan and barley-beta-glucan only and were without action on laminaran and CM-cellulose. It was suggested that hydrolysis products of beta-glucanase produced by B. macerans were markedly different from the products of the other enzymes. We conclude that B. macerans enzyme, which cleaves laminaran and beta-1,3-1,4-glucans, represents "laminarinase" type (1-3-beta-D-glucan glucanohydrolase, E.C. 3.2.1.6). On the other hand the glucanases produced by the other Bacillus strains belong to "licheninases" 1-3,1-4-beta-D-glucan glucanohydrolases, E.C. 3.2.1.73).

Topics: Bacillus; Bacillus subtilis; Carboxymethylcellulose Sodium; Glucans; Glycoside Hydrolases; Polysaccharides; Substrate Specificity