epiglucan and lichenin

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley β-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-β-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; beta-Glucans; Cellobiose; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Cloning, Molecular; Endo-1,3(4)-beta-Glucanase; Escherichia coli; Gastrointestinal Microbiome; Gene Expression; Genetic Vectors; Glucans; Glucose; Hydrolysis; Kinetics; Recombinant Proteins; Rumen; Sequence Alignment; Sequence Homology, Amino Acid; Sheep; Substrate Specificity; Trioses

A novel chitosanase gene, designated as PbCsn8, was cloned from Paenibacillus barengoltzii. It shared the highest identity of 73% with the glycoside hydrolase (GH) family 8 chitosanase from Bacillus thuringiensis JAM-GG01. The gene was heterologously expressed in Bacillus subtilis as an extracellular protein, and the highest chitosanase yield of 1, 108 U/mL was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant chitosanase (PbCsn8) was purified to homogeneity and biochemically characterized. PbCsn8 was most active at pH 5.5 and 70 °C, respectively. It was stable in a wide pH range of 5.0-11.0 and up to 55 °C. PbCsn8 was a bifunctional enzyme, exhibiting both chitosanase and glucanase activities, with the highest specificity towards chitosan (360 U/mg), followed by barley β-glucan (72 U/mg) and lichenan (13 U/mg). It hydrolyzed chitosan to release mainly chitooligosaccharides (COSs) with degree of polymerization (DP) 2-3, while hydrolyzed barley β-glucan to yield mainly glucooligosaccharides with DP > 5. PbCsn8 was further applied in COS production, and the highest COS yield of 79.3% (w/w) was obtained. This is the first report on a GH family 8 chitosanase from P. barengoltzii. The high yield and remarkable hydrolysis properties may make PbCsn8 a good candidate in industrial application.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Glucans; Chitin; Chitosan; Cloning, Molecular; Glucans; Glycoside Hydrolases; Hydrolysis; Industrial Microbiology; Oligosaccharides; Paenibacillus; Recombinant Proteins; Substrate Specificity

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, β-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of β-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Topics: Bacterial Proteins; beta-Glucans; Cellulose; Cloning, Molecular; Galactose; Glucans; Glycoside Hydrolases; Hot Temperature; Hydrogen-Ion Concentration; Mannans; Planctomycetales; Planctomycetes; Substrate Specificity; Talaromyces; Xylans

A clone encoding carboxymethyl cellulase activity was isolated during functional screening of a human gut metagenomic library using Lactococcus lactis MG1363 as heterologous host. The insert carried a glycoside hydrolase family 9 (GH9) catalytic domain with sequence similarity to a gene from Coprococcus eutactus ART55/1. Genome surveys indicated a limited distribution of GH9 domains among dominant human colonic anaerobes. Genomes of C. eutactus-related strains harboured two GH9-encoding and four GH5-encoding genes, but the strains did not appear to degrade cellulose. Instead, they grew well on β-glucans and one of the strains also grew on galactomannan, galactan, glucomannan and starch. Coprococcus comes and Coprococcus catus strains did not harbour GH9 genes and were not able to grow on β-glucans. Gene expression and proteomic analysis of C. eutactus ART55/1 grown on cellobiose, β-glucan and lichenan revealed similar changes in expression in comparison to glucose. On β-glucan and lichenan only, one of the four GH5 genes was strongly upregulated. Growth on glucomannan led to a transcriptional response of many genes, in particular a strong upregulation of glycoside hydrolases involved in mannan degradation. Thus, β-glucans are a major growth substrate for species related to C. eutactus, with glucomannan and galactans alternative substrates for some strains.

Topics: Bacterial Proteins; beta-Glucans; Clostridiales; Gastrointestinal Microbiome; Gene Expression; Glucans; Glycoside Hydrolases; Humans; Proteomics

Topics: Archaea; beta-Glucans; Bioreactors; Carboxymethylcellulose Sodium; Cellulase; Galactans; Glucans; Hordeum; Hot Temperature; Hydrothermal Vents; Islands; Mannans; Mediterranean Region; Metagenome; Plant Gums

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of β-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley β-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed β-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of β-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.

Topics: Argentina; beta-Glucans; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Enterobacter; Enzyme Stability; Glucans; Glucose; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides; Recombinant Proteins; Soil Microbiology; Substrate Specificity; Temperature

A novel endo-β-1,3(4)-glucanase gene, cel16A, was cloned from the fungus Humicola insolens Y1. The 988-bp full-length gene encoded a 286-residue polypeptide consisting of a putative signal peptide of 20 residues and a catalytic domain belonging to glycosyl hydrolase family 16. It was successfully overexpressed in Pichia pastoris GS115. The purified recombinant Cel16A exhibited highest specific activity toward barley β-glucan, followed by lichenan and laminarin, but not toward CMC-Na, birchwood xylan, Avicel and filter paper, indicating that Cel16A is an endo-β-1,3(4)-glucanases. Recombinant Cel16A had a pH optimum at 5.5 and a temperature optimum at 55 °C with a specific activity of 693 U/mg toward barley β-glucan. It exhibited good stability over pH 5.0-9.0 and at temperatures up to 50 °C, retaining over 80% maximum activity. The K

Topics: Amino Acid Sequence; beta-Glucans; Catalytic Domain; Chromatography, Affinity; Cloning, Molecular; Endo-1,3(4)-beta-Glucanase; Enzyme Assays; Enzyme Stability; Fungal Proteins; Gene Expression; Genetic Vectors; Glucans; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Pichia; Protein Sorting Signals; Recombinant Proteins; Sequence Alignment; Sordariales; Substrate Specificity

The newly isolated Paenibacillus sp. M33 from freshwater swamp forest soil in Thailand demonstrated its potential as a cellulose degrader. One of its endoglucanase genes from Paenibacillus sp., celP, was cloned to study the molecular characteristics of its gene product. The celP gene was recognized firstly by degenerate primer designed from Paenibacillus endoglucanase gene, and subsequently identified flanking region by inverse PCR technique. The celP gene consists of an open reading frame of 1707 bp encoding for 569 amino acids including 33-amino acids signal sequence. CelP is a member of glycoside hydrolase family 5 appended with a family 46 carbohydrate-binding module. CelP from recombinant Escherichia coli was purified by affinity chromatography. SDS-PAGE analysis of purified CelP showed a protein band at about 60 kDa. The purified enzyme gave a specific CMCase activity of 0.03 μmol min

Topics: Amino Acid Sequence; Bacterial Typing Techniques; Base Sequence; beta-Glucans; Cellulase; Chromatography, Affinity; Cloning, Molecular; Cluster Analysis; DNA, Bacterial; DNA, Ribosomal; Electrophoresis, Polyacrylamide Gel; Environmental Microbiology; Enzyme Stability; Gene Expression; Glucans; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Paenibacillus; Phylogeny; Protein Sorting Signals; Recombinant Proteins; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Substrate Specificity; Temperature; Thailand

The aim of this work was to study the contribution of the N-terminal structure to cellulase catalytic performance. A wild-type cellulase (BaCel5) of glycosyl hydrolase (GH) family 5 from Bispora antennata and two hybrid enzymes (BaCel5(127) and BaCel5(167)) with replacement of the N-terminal (βα)3 (127 residues) or (βα)4 (167 residues)-barrel with the corresponding sequences of TeEgl5A from Talaromyces emersonii were produced in Pichia pastoris and biochemically characterized. BaCel5 exhibited optimal activity at pH 5.0 and 50°C but had low catalytic efficiency (25.4±0.8mLs(-1)mg(-1)). In contrast, BaCel5(127) and BaCel5(167) showed similar enzymatic properties but improved catalytic performance. When using CMC-Na, barley β-glucan, lichenan, and cellooligosaccharides as substrates, BaCel5(127) and BaCel5(167) had increased specific activities and catalytic efficiencies by ∼1.8-6.7-fold and ∼1.0-4.7-fold, respectively. The catalytic efficiency of BaCel5(167) was even higher than that of parental proteins. The underlying mechanism was analyzed by molecular docking and molecular dynamic simulation.

Topics: Ascomycota; beta-Glucans; Catalysis; Cellulase; Cloning, Molecular; Glucans; Kinetics; Molecular Docking Simulation; Oligosaccharides; Pichia; Protein Domains; Sequence Analysis, Protein; Substrate Specificity; Temperature

Biomass is the most abundant and short-term renewable natural resource on Earth whose recalcitrance toward enzymatic degradation represents significant challenge for a number of biotechnological applications. The not so abundant but critically necessary class of GH45 endoglucanases constitutes an essential component of tailored industrial enzyme cocktails because they randomly and internally cleave cellulose molecules. Moreover, GH45 glucanases are core constituents of major-brand detergent formulations as well as enzymatic aid components in the cotton processing industry, clipping unwanted cellulosic fibers from cotton (cellulosic)-based tissues. Here we report on a recombinant high-yield Neurospora crassa OR74A NcCel45A production system, a single-band GH45 endoglucanase purification, and a complete enzyme functional characterization. NcCel45A is a bimodular endoglucanase showing maximum activity at pH 6.0 and 60 °C, while most active against lichenan and β-glucans and lesser active toward filter paper, carboxymethylcellulose, and phosphoric acid-swollen cellulose. Gluco-oligosaccharide degradation fingerprinting experiments suggest cellopentaose as the minimal length substrate and ThermalFluor studies indicate that NcCel45A displays excellent stability at elevated temperatures up to 70 °C and pHs ranging from 5 to 9. Remarkably, we show that NcCel45A is uniquely resistant to a wide-range of organic solvents and small-angle X-ray scattering show a monkey-wrench molecular shape structure in solution, which indicates, unlike to other known cellulases, a non-fully extended conformation, thus conferring solvent protection. These NcCel45A unique enzymatic properties maybe key for specific industrial applications such as cotton fiber processing and detergent formulations.

Topics: Amino Acid Sequence; beta-Glucans; Cellulase; Cloning, Molecular; Enzyme Stability; Glucans; Hot Temperature; Hydrogen-Ion Concentration; Molecular Sequence Data; Neurospora crassa; Protein Conformation; Scattering, Small Angle; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; X-Ray Diffraction

A novel endoglucanase encoding gene was cloned from Alicyclobacillus vulcanalis and expressed in E. coli. The deduced amino acid sequence showed highest identity with α-L-arabinofuranosidase-like proteins from glycoside hydrolase family 51. The recombinant enzyme was purified by affinity chromatography and characterised in terms of its potential suitability for lignocellulose hydrolysis at high temperature in the production of bioethanol. The purified enzyme displayed maximum activity at 80 °C and pH 3.6-4.5. Tween 20 was found to have a beneficial effect on enzyme activity and thermal stability. When incubated in the presence of 0.1% Tween 20, the enzyme retained full activity after 72 h at 70 °C and 78% of original activity after 72 h at 75 °C. Maximum activity was observed on carboxymethyl cellulose, and the purified enzyme also hydrolysed lichenan, barley β-glucan and xylan. The purified enzyme decreased the viscosity of carboxymethyl cellulose when assessed at 70-85 °C and was capable of releasing reducing sugars from acid-pretreated straw at 70 and 75 °C. The results indicate the potential suitability of the enzyme for industrial application in the production of cellulosic bioethanol.

Topics: Alicyclobacillus; beta-Glucans; Biofuels; Carboxymethylcellulose Sodium; Cellulase; Chromatography, Affinity; Cloning, Molecular; Enzyme Activators; Enzyme Stability; Escherichia coli; Ethanol; Glucans; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Plant Stems; Polysorbates; Recombinant Proteins; Substrate Specificity; Xylans

A novel alkaline β-1,3-1,4-glucanase (McLic1) from a thermophilic fungus, Malbranchea cinnamomea, was purified and biochemically characterized. McLic1 was purified to homogeneity with a purification fold of 3.1 and a recovery yield of 3.7 %. The purified enzyme was most active at pH 10.0 and 55 °C, and exhibited a wide range of pH stability (pH 4.0-10.0). McLic1 displayed strict substrate specificity for barley β-glucan, oat β-glucan and lichenan, but did not show activity towards other tested polysaccharides and synthetic p-nitrophenyl derivates, suggesting that it is a specific β-1,3-1,4-glucanase. The K m values for barley β-glucan, oat β-glucan and lichenan were determined to be 0.69, 1.11 and 0.63 mg mL(-1), respectively. Moreover, the enzyme was stable in various non ionic surfactants, oxidizing agents and several commercial detergents. Thus, the alkaline β-1,3-1,4-glucanase may have potential in industrial applications, such as detergent, paper and pulp industries.

Topics: Amino Acid Sequence; Ascomycota; beta-Glucans; Detergents; Enzyme Stability; Fungal Proteins; Glucans; Glycoside Hydrolases; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Substrate Specificity

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-β-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley β-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at 50°C, with Azo-barley-glucan as the substrate. The metal ions Mn(2+), Mg(2+), Ca(2+), and Fe(2+) enhanced the enzymatic activity, whereas the Cu(2+) and Zn(2+) ions inhibited the enzymatic activity. The Km and Vmax values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

Topics: Bacillus; beta-Glucans; Cloning, Molecular; Enzyme Activators; Enzyme Inhibitors; Enzyme Stability; Escherichia coli; Gene Expression; Gene Library; Glucans; Glycoside Hydrolases; Hydrogen-Ion Concentration; Kinetics; Metagenomics; Metals; Molecular Sequence Data; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Soil Microbiology; Substrate Specificity; Temperature

A novel endo-1,3(4)-β-D-glucanase gene (bgl16C1) from Penicillium pinophilum C1 was cloned and sequenced. The 945-bp full-length gene encoded a 315-residue polypeptide consisting of a putative signal peptide of 18 residues and a catalytic domain belonging to glycosyl hydrolase family 16. The deduced amino acid sequence showed the highest identity (82%) with the putative endo-1,3(4)-β-glucanase from Talaromyces stipitatus ATCC 10500 and 60% identity with the characterized β-1,3(4)-glucanase from Paecilomyces sp. FLH30. The gene was successfully overexpressed in Pichia pastoris. Recombinant Bgl16C1 constituted 95% of total secreted proteins (2.61 g l⁻¹) with activity of 28,721 U ml⁻¹ in a 15-l fermentor. The purified recombinant Bgl16C1 had higher specific activity toward barley β-glucan (12,622 U mg⁻¹) than all known glucanases and also showed activity against lichenan and laminarin. The enzyme was optimally active at pH 5.0 and 55°C and exhibited good stability over a broad acid and alkaline pH range (>85% activity at pH 3.0-7.0 and even 30% at pH 11.0). All these favorable enzymatic properties make it attractive for potential applications in various industries.

Topics: Amino Acid Sequence; beta-Glucans; Bioengineering; Cloning, Molecular; Endo-1,3(4)-beta-Glucanase; Glucans; Molecular Sequence Data; Paecilomyces; Penicillium; Pichia; Polysaccharides; Recombinant Proteins; Sequence Alignment

In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.

Topics: Bacterial Proteins; beta-Glucans; Escherichia coli; Gene Targeting; Glucans; Glycoside Hydrolases; Hordeum; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Osmotic Pressure; Oxygen; Photosynthesis; Phylogeny; Recombinant Proteins; Salts; Sodium Bicarbonate; Substrate Specificity; Synechocystis

The carbohydrate-binding site of Bacillus macerans 1,3-1, 4-beta-D-glucan 4-glucanohydrolase has been analyzed through a mutational analysis to probe the role of protein-carbohydrate interactions defining substrate specificity. Amino acid residues involved in substrate binding were proposed on the basis of a modeled enzyme-substrate complex [Hahn, M., Keitel, T., and Heinemann, U. (1995) Eur. J. Biochem. 232, 849-859]. The effects of the mutations at 15 selected residues on catalysis and binding were determined by steady-state kinetics using a series of chromogenic substrates of different degree of polymerization to assign the individual H-bond and hydrophobic contributions to individual subsites in the binding site cleft. The glucopyranose rings at subsites -III and -II are tightly bound by a number of H-bond interactions to Glu61, Asn24, Tyr92, and Asn180. From k(cat)/K(M) values, single H-bonds account for 1.8-2.2 kcal mol(-)(1) transition-state (TS) stabilization, and a charged H-bond contributes up to 3.5 kcal mol(-)(1). Glu61 forms a bidentated H-bond in subsites -III and -II, and provides up to 6.5 kcal mol(-)(1) TS stabilization. With a disaccharide substrate that fills subsites -I and -II, activation kinetics were observed for the wild-type and mutant enzymes except for mutations on Glu61, pointing to an important role of the bidentate interaction of Glu61 in two subsites. Whereas removal of the hydroxyl group of Tyr121, initially proposed to hydrogen-bond with the 2OH of Glcp-I, has essentially no effect (Y121F mutant), side-chain removal (Y121A mutant) gave a 100-fold reduction in k(cat)/K(M) and a 10-fold lower K(I) value with a competitive inhibitor. In subsite -IV, only a stacking interaction with Tyr22 (0.7 kcal mol(-)(1) TS stabilization) is observed.

Topics: Amino Acid Sequence; Bacillus; beta-Glucans; Binding, Competitive; Disaccharides; DNA Mutational Analysis; Enzyme Activation; Enzyme Inhibitors; Glucans; Glycoside Hydrolases; Glycosides; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Plant Proteins; Substrate Specificity; Thermodynamics

Derivatives of curdlan and lichenan, linear (1----3)-beta-D- and (1----3/1----4)-beta-D-glucans, respectively, have been synthesised having alpha-L-arabinofuranosyl, alpha-L-rhamnosyl, beta-D-glucosyl, and beta-gentiobiosyl side chains attached at positions 6. These water-soluble derivatives, obtained by condensation of the 2,4- and 2,4-/2,3-di-O-phenylcarbamoyl derivatives of curdlan and lichenan, respectively, with appropriate ortho esters followed by saponification, were characterised by methylation analysis, g.p.c., and interaction with Congo Red. The curdlan derivatives and the lichenan derivative with few glucosyl branches were active against the Sarcoma 180.

Topics: Animals; Antineoplastic Agents; beta-Glucans; Female; Glucans; Mice; Sarcoma 180

2-Sulfoethyl, 3-sulfopropyl, and 4-sulfobutyl derivatives of the (1----3)-beta-D-glucan curdlan and the (1----3/1----4)-beta-D-glucan lichenan have been synthesised. The substituents are located mainly at positions 6. The curdlan derivatives strongly inhibited the growth of the Sarcoma 180 tumour, whereas the lichenan derivatives were inactive, indicating that a (1----3)-linked beta-D-glucan backbone is essential for activity.

Topics: Animals; Antineoplastic Agents; beta-Glucans; Female; Glucans; Mice; Sarcoma 180

By separating Limulus amebocyte lysate by cation-exchange chromatography with an SP-Toyopearl 650C column, a fraction insensitive to endotoxin, yet specifically sensitive to beta-glucan, was successfully obtained in the unadsorbed portion. This fraction showed beta-glucan dose-dependent clotting enzyme activity, although no sensitivity to endotoxin. This beta-glucan-dependent reaction showed no interference in the presence of endotoxin, with the fraction also showing sensitivity towards various kinds of beta-glucan, i.e. curdlan, pachyman, laminaran and lichenan. The sensitivity towards curdlan was approximately 10(-10) g/ml.

Topics: Animals; Bacterial Toxins; beta-Glucans; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Glucans; Horseshoe Crabs; Polysaccharides; Polysaccharides, Bacterial; Sensitivity and Specificity; Spectrophotometry, Ultraviolet