A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography

A new screening method for β-(1,3–1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.     

Properties of β-glucan synthetase from Saccharomyces cerevisiae

Properties of -glucan synthetase from S. cerevisiae were studied. The enzyme exhibited optimal activity at pH 6.7 and 24 C. Km for UDP-glucose was 0.12mm. Addition of Mg⁺⁺ or Mn⁺⁺ stimulated its activity by 60% and 21% respectively. High concentrations of EDTA and hydroxyquinoline were inhibitory. Glucan synthetase was fully active in cell-free extracts. Small concentrations of trypsin or subtilopeptidase A from Bacillus subtilis, caused only a slight increase in glucosyl transferase activity, but larger concentrations destroyed -glucan synthetase. Acid proteases were neither stimulatory nordestructive. Thus it seemsunlikelythat -glucan synthetase exists in a zymogen form. Glucan synthetase was unstable. It was inactivated more rapidly at 28 C than at 0 C. The presence of substrate, -glucan or the protease inhibitors PMSF, Antipain or Pepstatin A did not protect -glucan synthetase from inactivation. Glucan synthetase was not stimulated by addition of cellobiose or -glucans. The synthesis of -glucans was competitively inhibited by UDP (K_i=0.45mm). Glucono--lactone, a known inhibitor of -glucosidases was a strong non-competitive inhibitor of -glucan synthetase.This work was supported by grants PNCB 00071 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.     

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0–5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.     

Degradation of barley β-glucan by endoglucanase C of Clostridium thermocellum

Summary Endoglucanase C encoded by the celC gene of Clostridium thermocellum has been purified to homogeneity from a recombinant Escherichia coli strain. It was found that this enzyme is highly efficient in degrading glucans with alternating -1,4- and -1,3-linkages but lacks activity on unmodified cellulosic substrates. The properties of endoglucanase C were compared to those of Bacillus subtilis -glucanase, an enzyme used in the brewing industry for -glucan degradation. Both enzyme cause a rapid decrease of the viscosity of barley -glucan as a result of internal chain cleavage. Endoglucanase C hydrolyses non-specifically -1,3- and -1,4-bonds adjacent to unsubstituted or 4-O-substituted cellobiose units. Due to its lower pH optimum and increased thermostability endoglucanase C compares favourably with B. subtilis -glucanase and seems suitable for use in the mashing process of beer brewing.     

Oxidation of oat β-glucan in aqueous solutions during processing

The study investigated carbonyl group formation along the chain and the chain cleavage of cereal β-glucan during heat treatments, high pressure homogenisation, cold storage and ascorbic acid treatment of aqueous solutions of this soluble dietary fibre. The carbonyl group content and its distribution along the chain were simultaneously determined with the chain cleavage using a HPSEC/labelling method, originally developed for water-insoluble cellulose. Ascorbic acid treatment resulted in a relatively high degree of carbonyl content and extensive degradation of β-glucan, even in concentrations typically found in foods. The thermal oxidation of the β-glucan was considerable at 120 °C in a β-glucan solution with co-extracted compounds from oat ingredient, and in the highly purified solutions in presence of ferrous ions. Oxidation also probably contributed to the molecular properties during high pressure homogenisation, even thou the main degradation mechanism is the hydrolysis caused by mechanical energy. In addition to the cleavage of the β-glucan chain, the formation of compact, high molar mass species or molecule clusters were obtained in the study after ascorbic acid, heat (120 °C) and homogenisation treatments.     

Physiological activities of a β-glucan produced by Panebacillus polymyxa

In vitro bioactivities of a beta-glucan produced by Panebacillus polymyxa JB115 were investigated. Nitric oxide production by RAW 264.7 macrophage cells pre-treated with beta-glucan JB115 (from 0.1 to 1 mg ml(-1)) was significantly increased, compared to that in untreated cells (P < 0.001). The beta-glucan JB115 increased superoxide radical-scavenging activity by 66% at 1 mg ml(-1). It also suppressed hyaluronidase (32%) and collagenase (33%) activities and, additionally, displayed antitumor activity, blocking the growth of Sarcoma 180 cells in a concentration-dependent manner. The immune-stimulatory, antioxidant, collagenase inhibitory and hyaluronidase inhibitory effects of the beta-glucan support its potential role in the prevention of bacterial disease against fish and in the protection of skin against aging.     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Activation of members of a MAPK module in β-glucan elicitor-mediated non-host resistance of soybean

Plants recognize microbial pathogens by discriminating pathogen-associated molecular patterns from self-structures. We study the non-host disease resistance of soybean (Glycine max L.) to the oomycete, Phytophthora sojae. Soybean senses a specific molecular pattern consisting of a branched heptaglucoside that is present in the oomycetal cell walls. Recognition of this elicitor may be achieved through a β-glucan-binding protein, which forms part of a proposed receptor complex. Subsequently, soybean mounts a complex defense response, which includes the increase of the cytosolic calcium concentration, the production of reactive oxygen species, and the activation of genes responsible for the synthesis of phytoalexins. We now report the identification of two mitogen-activated protein kinases (MAPKs) and one MAPK kinase (MAPKK) that may function as signaling elements in triggering the resistance response. The use of specific antisera enabled the identification of GmMPKs 3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. Elicitor specificity of MAPK activation as well as the sensitivity against inhibitors suggested these kinases as part of the β-glucan signal transduction pathway. An upstream GmMKK1 was identified based on sequence similarity to other plant MAPKKs and its interaction with the MAPKs was analyzed. Recombinant GmMKK1 interacted predominantly with GmMPK6, with concomitant phosphorylation of the MAPK protein. Moreover, a preferential physical interaction between GmMKK1 and GmMPK6 was demonstrated in yeast. These results suggest a role of a MAPK cascade in mediating β-glucan signal transduction in soybean, similar to other triggers that activate MAPKs during innate immune responses in plants. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. The nucleotide sequences encoding the MAPKs and MAPKK1 from soybean can be accessed through the GenBank database under GenBank accession numbers AF104247, AF329506, and AY070230.     

Contribution of surface β-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-β-D-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of β-glucan. To evaluate this hypothesis, gtfF genes of three β-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of β-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of β-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of β-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, β-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.     

Isolation of Robillarda sp. producing high β-1,4-glucan glucanohydrolase

Summary A fungus, Robillarda, sp. Y-20, which produces mainly, -1,4-glucan glucanohydrolase (Cx-enzyme) in the culture filtrate, was isolated from soil. The specific activity of the Cx-enzyme was up to 4.9 times higher than that of Trichoderma reesei QM 9414.     

A hemicellulosic β-glucan from the hypocotyls of Phaseolus aureus

A glucan of $\text{DP}$_nca 80 has been isolated from the hypocotyls of mung bean plants (Phaseolus aureus). Methylation analysis and periodate oxidation studies showed that the glucan has (1 → 3) and (1 → 4) linked d-glucopyranosyl residues in the molar ratio 1·0:1·7. Oligosaccharides containing both β(1 → 3) and β(1 → 4) linked residues were isolated from partial hydrolysates.     

Effect of oxygen on the rate of β-1,3-glucan microbial exopolysaccharide production

Summary Examination of the relationship between the rate of oxygen transfer and the rate of polymer production revealed an unexpectedly high requirement for oxygen. At a cell density of about 3 g (dry wt)/L, the threshhold value for OTR for optimal synthesis of polymer is about 50 mmoles O₂/L.hr. Whereas Rushton turbines are effecient at transfering oxygen to solution, their use reduces the quality of the recovered polymer. Although better quality polymer can be produced in a reactor employing an agitator which causes less shear stress, the productivity can be compromised due to the inefficiency in OTR. The present study describes operating conditions for the provision of sufficient OTR in a system compatible with the production of high-quality polymer whereby turbine impellers were replaced with a marine-type propeller and mass transfer was assisted by means of a gas dispersion device.     

Heteronuclear NMR studies of 13C-labeled yeast cell wall β-glucan oligosaccharides

Summary The structures of uniformly ¹³C-labeled -glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear (13) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a (16) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain (16) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain (16) linkages. Since isolated fungal membranes only synthesize linear (13) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.     

Demonstration of β-glucan receptors in the skin of aquatic mammals—a preliminary report

Using immunohistochemistry, the study clearly demonstrates three important β-glucan receptors (Ficolin/P35, MBL, Dectin-1; members of the lectin-complement pathway of innate immunity) in the integument of six marine and freshwater aquatic mammals (Northern fur seal, Common seal, Walrus, Coypu, Capybara, Otter), but only weakly in two dolphin species. Most of the non-dolphin mammals exhibited strong reactions, especially with regard to the skin glands (tubular apocrine glands, sebaceous glands), for L-Ficolin/P35 and MBL. Distinct reaction staining could also be observed in the epidermis and the outer epithelial sheath of primary hair follicles. Positive Dectin-1 staining was limited to secretory cells of the apocrine tubular glands, and to peripheral and central cells of sebaceous glands of the seals. The Capybara was the only animal to show a clear Dectin reaction in the epidermis (stratum granulosum). The findings are discussed with regard to the constant and high microbial challenge of the skin in the aquatic medium, and variations in hair density of the animals.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

The influence of β-glucan on the growth and cell wall architecture of Aspergillus spp

β-1,3-glucan is a major component of fungal cell walls with various biological activities, including effects on the production of inflammatory mediators in vivo and in vitro. However, few reports have examined its influence on the fungal cell itself. In this study, the influences of β-1,3-glucan on the growth and cell wall structure of fungi was examined. Aspergillus fumigatus was cultured with a synthetic medium, C-limiting medium, in the presence or absence of β-1,3-glucan. Hyphal growth was promoted in liquid and solid-cultures by adding β-1,3-glucan. Glucose and dextran did not induce growth. The influence on cell wall structure of the β-glucan-added cultures was examined by enzymolysis and NMR spectroscopy and the amount of β-1,3-glucan found to be changed. β-1,3-glucan has been widely detected in the environment. In this study, it was demonstrated that β-1,3-glucan causes promotion of the growth, and a change in the cell wall architecture, of Aspergillus. Unregulated distribution of β-1,3-glucan would be strongly related to the incidence of infectious diseases and allergy caused by Aspergillus spp.     

Application of β-1,3-glucan in production of ceramics-based elastic composite for bone repair

Background

Unsatisfactory surgical handiness is a commonly known disadvantage of implantable granular bioceramics. To overcome this problem, β-1,3-glucan, biotechnologically derived polysaccharide, has been proposed as a joining agent to combine granular ceramics into novel compact and elastic composite. Hydroxyapatite/glucan elastic material was processed and evaluated as a potential bone void filler.

Methodology

The procedure of composite formation was based on gelling properties of glucan. Its properties were studied using X-ray microtomography, SEM-EDS, FTIR spectroscopy, compression test and ultrasonic method. Sorption index was determined in phosphate buffered saline; bioactivity in simulated body fluid; sterility in growth broth and human blood plasma; implantation procedure in dog model.

Results

HAp/glucan composite is sterilizable, flexible and self-adapting to defect shape. It exhibits bioactivity, good surgical handiness, high sorption index and profitable mechanical properties, resembling those of spongy bone. Results of pilot clinical experiment on animal (dog) patients of a local clinic of animal surgery suggested good healing properties of the composite and its transformation into new bone tissue within critical-size defect.

Conclusions

The results obtained in this study confirm that flexible HAp/glucan composite has potential as a bone-substituting material. Promising results of pilot clinical experiment suggest that further in vivo experiments should be performed.     

High-affinity binding of fungal β-glucan elicitors to cell membranes of species of the plant family Fabaceae

Microsomal preparations of six species of the plant family Fabaceae were screened for high-affinity binding of branched (1 3), (1 6)--glucans. Oligoglucosides of this type are specific elicitors of phytoalexin accumulation in soybean (Glycine max L.), a member of this family. The species studied were alfalfa (Medicago sativa L.), broadbean (Vicia faba L.), chickpea (Cicer arietinum L.), french bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and white lupin (Lupinus albus L.). A ¹²⁵I-labeled 4-(2-aminophenyl)ethylamine conjugate of a (1 3), (1 6)--glucan fraction with an average degree of polymerization (DP) of 18, obtained from mycelial walls of Phytophthora sojae, was used as radioligand for initial screening. The structural complexity of this fraction allowed the identification of binding sites with affinities for isomeric structures other than the (1 3), (1 6) hepta--glucoside for which soybean binding sites display highest affinity. Radioligand competition experiments against unlabeled fungal -glucan resulted in the identification of high-affinity binding in alfalfa, bean, lupin, and pea. Half-maximal competition concentrations (IC₅₀) for fungal -glucan in these species were between 5 and 30 nM. Pseudoheterologous radioligand competition by unlabeled hepta--glucoside showed that for alfalfa, lupin and pea the IC₅₀ values for this structure (4 to 16 nM) were similar to those of soybean (7.7 nM). Bean microsomes, however, displayed an IC₅₀ significantly higher than soybean (68 nM) suggesting that the structural motif recognized by its binding sites is not identical to that of soybean or the other three species. Radioligand saturation assays with alfalfa, lupin and pea microsomes using an ₁₂₅I-labeled aminophenylethylamine hepta--glucoside conjugate gave dissociation constants (K_d) of 5.3, 3.7, and 1.8 nM, respectively. The affinity of these sites for hepta- glucoside was in the same range as that of soybean (K_d 1–3 nM), whereas the affinity of the binding sites of bean for the same ligand was significantly lower (K_d = 33 nM). Good correlation was found between the presence of high-affinity binding and the accumulation of isoflavonoid phytoalexins in roots of alfalfa, bean, chickpea and pea seedlings after exposure to fungal -glucan. Lupin displayed a strong wound-induced accumulation of prenylated isoflavones which was independent of the presence of -glucan, making it impossible to determine phytoalexin induction in response to elicitor. No specific binding or phytoalexin accumulation in response to glucans was observed in broadbean. This is the first report on the existence of possibly homologous elicitor-binding sites within a plant taxonomic family and may provide preliminary evidence for putative evolutionary relationships in pathogen perception mechanisms in plants.Abbreviations DP degree of polymerization - EC₅₀ concentration of elicitor necessary to obtain a half-maximal biological response - HG synthetic (1 3), (1 6)-hepta--glucoside phytoalexin elicitor - HG-APEA 1-[4-(2-aminophenyl)ethylamino-1-hexaglucosyl]deoxyglucitol - IC₅₀ ligand concentration necessary to obtain half-maximal displacement of radioligand in competition binding assays - K_d dissociation constant - OS branched (1 3), (1 6)--glucan obtained by hydrolysis of mycelial walls of Phytophthora sojae - OS-APEA 1-[4-(2-armnophenyl)ethylamino-1-oligoglucosyl]deoxyglucitol conjugate of OS This work was supported by the Comision Interministerial de Ciencia y Tecnologia grant BI091-0366 (E.G.C.), the Volkswagen-Stiftung (E.G.C. and J.E.), the Deutsche Forschungsgemeinschaft, SFB-369 (J.E.), the Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (J.E.), Fonds der Chemischen Industrie (J.E.) and the EU Human Capital and Mobility Program (J.E. and E.G.C.).     

Rheological and microstructural investigation of oat β-glucan isolates varying in molecular weight

The rheological properties and microstructure of aqueous oat β-glucan solutions varying in molecular weight were investigated. The structural features and molecular weights (MW) were characterized by ¹³C NMR spectroscopy and high performance size-exclusion chromatography (HPSEC), respectively. The microstructure of the β-glucans dispersions was also examined by atomic force microscopy (AFM). The samples with β-glucan content between 78 and 86% on a dry weight basis had MW, intrinsic viscosity ([η]) and critical concentration (c*) in the range of 142-2800 × 10³ g/mol, 1.7-7.2 dl/g and 0.25-1.10 g/dl, respectively. The flow and viscoelastic behaviour was highly dependent on MW and on the concentration of the β-glucans dispersions. Pseudoplastic behaviour was exhibited at high concentrations and Newtonian behaviour was evident at low concentrations. At the same concentration, the viscosity was higher for higher MW samples. The Cox-Merz rule was applicable for the lower molecular weight samples at higher concentrations whereas the high molecular weight sample deviated at concentrations greater than 1.0%, w/v. The mechanical spectra with variation of both MW and concentration were typical of entangled biopolymer solutions. AFM images revealed the formation of clusters or aggregates linked via individual polymer chains scattered heterogeneously throughout the system. The aggregate size increased with the molecular weight of the samples investigated and has been linked to the rheological behaviour of the samples.