pustulan and laminaran

Marine bacteria catabolize carbohydrate polymers of algae, which synthesize these structurally diverse molecules in ocean surface waters. Although algal glycans are an abundant carbon and energy source in the ocean, the molecular details that enable specific recognition between algal glycans and bacterial degraders remain largely unknown. Here we characterized a surface protein, GMSusD from the planktonic Bacteroidetes-Gramella sp. MAR_2010_102 that thrives during algal blooms. Our biochemical and structural analyses show that GMSusD binds glucose polysaccharides such as branched laminarin and linear pustulan. The 1.8 Å crystal structure of GMSusD indicates that three tryptophan residues form the putative glycan-binding site. Mutagenesis studies confirmed that these residues are crucial for laminarin recognition. We queried metagenomes of global surface water datasets for the occurrence of SusD-like proteins and found sequences with the three structurally conserved residues in different locations in the ocean. The molecular selectivity of GMSusD underscores that specific interactions are required for laminarin recognition. In conclusion, our findings provide insight into the molecular details of β-glucan binding by GMSusD and our bioinformatic analysis reveals that this molecular interaction may contribute to glucan cycling in the surface ocean.

Topics: Amino Acid Sequence; Bacterial Proteins; Bacteroidetes; Binding Sites; Chlorophyta; Crystallography, X-Ray; Glucans; Models, Molecular; Polysaccharides; Protein Conformation; Sequence Homology; Substrate Specificity

In this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40(T) The gly5M gene was cloned and overexpressed in Escherichia coli Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry, Gly5M was identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes.. In this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40(T) Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.

Topics: Cellulase; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cloning, Molecular; Escherichia coli; Gammaproteobacteria; Gene Expression; Glucans; Hydrolysis; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity

Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

Topics: beta-Glucans; Cell Membrane; Cell Wall; Chitin; Glucans; Killer Factors, Yeast; Polysaccharides; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spheroplasts

(1-->3)-beta-D-Glucans, a cell wall component in most microfungi, are suggested to play a role in the development of respiratory and general symptoms in organic dust-related diseases. The mechanisms by which they induce these effects are, however, not clear. In the present study, mediator release and its potentiation by the (1-->3)-beta-D-glucan as well as by the (1-->6)-beta-D-glucan found in yeast and other fungi were therefore examined. Blood leucocytes from healthy volunteers and from patients allergic to house dust mite were incubated with (1-->3)-beta-D-glucans with increasing 1,6-branchings: curdlan [a linear (1-->3)-beta-D-glucan], laminarin and scleroglucan, and furthermore with pustulan, a linear (1-->6)-beta-D-glucan. Histamine release was not observed on exposure to the glucans only, but in the presence of anti-immunoglobulin E (IgE) antibody or specific antigens, all the glucans investigated led to an enhancement of the IgE-mediated histamine release. The glucans induced a significant potentiation of the mediator release when present at concentrations in the range of 2-5 x 10(-5) M. These results suggest that (1-->3)-beta-D-glucan as well as (1-->6)-beta-D-glucan aggravates IgE-mediated histamine release. Knowledge concerning the effects of glucans on immune responses may be of importance for understanding and treating inflammatory and allergic diseases.

Topics: Adult; Air Pollution, Indoor; Allergens; beta-Glucans; Cell Wall; Dust; Environmental Exposure; Fungi; Glucans; Histamine; Humans; Immunoglobulin E; In Vitro Techniques; Leukocytes; Middle Aged; Polysaccharides; Respiratory Hypersensitivity

To evaluate the in vitro microbicidal activity against Acanthamoeba castellanii of a murine monoclonal anti-idiotypic antibody (KTmAb) and a synthetic killer mimotope (KP), which mimic a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity through interaction with specific cell wall receptors, mainly constituted by beta-glucans.. Amoebicidal activity was investigated after incubation of trophozoites under different experimental conditions with laminarinase, KTmAb, KP and a scrambled decapeptide (SP). To confirm the specific interaction of KP with beta-glucans, the experiments were also carried out in the presence of laminarin (beta1-3-glucan) or pustulan (beta1-6-glucan); both glucan molecules were co-incubated with KP or SP.. KTmAb and KP exhibited a time-dependent killing activity, in comparison with SP or heat-inactivated KTmAb; this activity was completely abolished by pre-incubation with laminarin, but not by pustulan. Notably, in vitro amoebicidal activity was observed in the presence of laminarinase, an enzyme that specifically hydrolyses beta-glucans. Furthermore, KP specifically inhibited the growth of Acanthamoeba on infected contact lenses and the remaining adherent KP-treated trophozoites appeared strongly damaged.. The results indicate that the expression of beta1-3-glucan receptors in the cell membrane is probably modulated during cell growth of A. castellanii and is critical for the killing activity of KT-like molecules. Our data confirm the broad antimicrobial spectra of KTmAb and KP, emphasize the crucial role of beta1-3-glucan in microbial physiology and suggest the potential use of KTmAb and KP in the prevention and therapy of Acanthamoeba infections or in preventing Acanthamoeba contamination during storage of contact lenses.

Topics: Acanthamoeba castellanii; Amebicides; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; beta-Glucans; Cell Wall; Contact Lenses; Glucans; Killer Factors, Yeast; Mice; Mycotoxins; Oligopeptides; Polysaccharides

Hydrolysis of plant cell wall polysaccharides, a process which is of intrinsic biological and biotechnological importance, requires the concerted action of an extensive repertoire of microbial cellulases and hemicellulases. Here, we report the identification of the gene cluster unk16A, regA and cel5B in the aerobic soil bacterium Cellvibrio mixtus, encoding a family 16 (CmUnk16A) glycoside hydrolase (GH), an AraC/XylS transcription activator (CmRegA) and a family 5 (CmCel5B) endo-glucanase, respectively. CmUnk16A is a modular enzyme comprising, in addition to the catalytic domain, two family 32 carbohydrate-binding modules (CBMs), termed CBM32-1 and CBM32-2, a CBM4 and a domain of unknown function. We show that CBM32-2 binds weakly to laminarin and pustulan. CmRegA is also a modular protein containing a highly hydrophobic N-terminal domain and a C-terminal DNA-binding domain of the AraC/XylS family. The role of the identified enzymes in the hydrolysis of cell wall polysaccharides by aerobic bacteria is discussed.

Topics: Amino Acid Sequence; Bacterial Proteins; Cell Wall; Cellulase; Cellvibrio; Cloning, Molecular; Glucans; Molecular Sequence Data; Multigene Family; Polysaccharides; Sequence Alignment

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source beta-1,3 and beta-1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The beta-(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.

Topics: Carboxylic Ester Hydrolases; Cell Wall; Culture Media; Glucan 1,3-beta-Glucosidase; Glucans; Glucose; Glycoside Hydrolases; Pectins; Polygalacturonase; Polysaccharides; Rhizoctonia; Solanum tuberosum; Substrate Specificity

Three extracellular (1-->3)-beta-glucanases were purified from the fungus Acremonium sp. IMI 383068. Higher activities were unexpectedly obtained with pustulan, a (1-->6)-beta-glucan as carbon source, than when grown with laminarin, a (1-->3)-beta-glucan. Preliminary evidence suggests that these enzymes are not constitutive, but are inducible, and that their synthesis is repressed by glucose. All three had the same molecular masses, similar pH and temperature optima and none were glycosylated. They all appeared to have an exo-hydrolytic mode of substrate attack. N-terminal amino acid sequence data indicate that substantial post-translational modification of these had occurred, and that while two may be encoded by the same gene, the third may be genetically different.

Topics: Acremonium; Amino Acid Sequence; Culture Media, Conditioned; Gene Expression Regulation, Fungal; Glucan 1,3-beta-Glucosidase; Glucans; Molecular Sequence Data; Polysaccharides

An engineered, killer decapeptide (KP) has been synthesized based on the sequence of a recombinant, single-chain anti-idiotypic antibody (KT-scFv) acting as a functional internal image of a yeast killer toxin. Killer decapeptide exerted a strong fungicidal activity against Candida albicans, which was attributed to peptide interaction with beta-glucan. As this polysaccharide is also a critical component of the cryptococcal cell wall, we wondered whether KP was also active against Cryptococcus neoformans, a human pathogen of increasing medical importance. We found that KP was able to kill both capsular and acapsular C. neoformans cells in vitro. Furthermore, KP impaired the production of specific C. neoformans virulence factors including protease and urease activity and capsule formation, rendering the fungus more susceptible to natural effector cells. In vivo treatment with KP significantly reduced fungal burden in mice with cryptococcosis and, importantly, protected the majority of immunosuppressed animals from an otherwise lethal infection. Given the relevance of cryptococcosis in immunocompromised individuals and the inability of conventional drugs to completely resolve the infection, the results of the present study indicate KP as an ideal candidate for further studies on novel anticryptococcal agents.

Topics: Animals; Antifungal Agents; beta-Glucans; Candida albicans; Cryptococcosis; Cryptococcus neoformans; Dose-Response Relationship, Drug; Female; Glucans; Humans; Macrophages; Melanins; Mice; Mice, Inbred BALB C; Neutrophils; Peptides; Polysaccharides; Survival Rate; Virulence Factors

Agaricus bisporus H 25 produced extracellular endo-1, 3-beta-glucanase when grown in a static culture at 25 degrees C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3-beta-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against beta-1,3- linkages and, to a lesser extent, against beta-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40 degrees and 50 degrees C for laminarin, and between 30 degrees and 50 degrees C for pustulan. The optimum pH and temperature were 4.5 and 45 degrees C for both substrates.

Topics: Agaricus; Fungal Proteins; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Hydrogen-Ion Concentration; Polysaccharides; Temperature

The use of a novel monoclonal antibody (mAb) that reacts with (1,6)-beta-glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)-beta-glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)-beta-glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)-beta-glucan in the cell wall of C. albicans has also been studied.

Topics: Antibodies, Monoclonal; beta-Glucans; Binding, Competitive; Candida albicans; Cell Wall; Chromatography, Affinity; Epitopes; Fluorescent Antibody Technique; Fungal Proteins; Glucans; Mannans; Membrane Glycoproteins; Polysaccharides; Tunicamycin

Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained beta(1----3)- and beta(1----6)-chains but no mixed intra-chain beta(1----3)/(1----6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67.0% to 76.6%) of beta(1----6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% beta(1----3) and 43.3% to 53.2% beta(1----6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% beta(1----3) and 14% beta(1----6) linkages. Branch points accounted for 6.7%, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.

Topics: Candida albicans; Cell Wall; Chromatography, Gas; Glucans; Magnetic Resonance Spectroscopy; Methylation; Polysaccharides

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl-beta-D-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A50 has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different beta-glucanases located in the periplasmic space is presented.

Topics: Ascomycota; Cell-Free System; Chromatography, DEAE-Cellulose; Chromatography, Gel; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glucose; Glycoside Hydrolases; Molecular Weight; Pichia; Polysaccharides; Protoplasts

Tanaka, Hirosato (University of California, Davis), and Herman J. Phaff. Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J. Bacteriol. 89:1570-1580. 1965.-A number of microorganisms, able to decompose and grow on yeast cell walls, were isolated from soil. These isolates demonstrated various types of attack on yeast walls. A bacterium, identified as Bacillus circulans, and a species of Streptomyces produced clear, lysed zones when grown on an agar medium containing baker's yeast cell walls. The streptomycete formed glucanase, mannanase, and protease, but B. circulans produced only glucanases. Purified mannan could be prepared from the culture fluid of B. circulans grown on baker's yeast cell walls. In a liquid, mineral medium, extracellular lytic enzyme production by B. circulans was optimal after 3 days of aerobic growth at 30 C with 0.5% baker's yeast cell walls as the carbon source. Twelve other carbon sources were ineffective as inducers. Among a number of polysaccharides tested, the crude enzymes of B. circulans hydrolyzed only beta-1-->3 glucan (laminarin) and beta-1-->6 glucan (pustulan), both by a random mechanism, to a mixture of dimer and glucose. The beta-1-->3 and beta-1-->6 glucanases were separated from each other by diethylaminoethyl cellulose column chromatography. Water-soluble oat glucan, which contains in the linear chain both beta-1-->3 and beta-1-->4 bonds, was also hydrolyzed by the bacterial beta-1-->3 glucanase. The products of this reaction indicated that this enzyme hydrolyzes beta-1-->3 or beta-1-->4 glucosidic linkages, provided the beta-glucopyranosyl units composing these bonds are substituted in the 3 position by another glucose unit.

Topics: Bacillus; Cell Wall; Chemical Phenomena; Chemistry; Chromatography; Culture Media; DEAE-Cellulose; Glucans; Glucosidases; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides; Peptide Hydrolases; Polysaccharides; Research; Saccharomyces; Saccharomyces cerevisiae; Soil Microbiology; Streptomyces; Yeasts