Biosynthesis of β-glucans in fungi

Glucans are the most abundant polysaccharides present in fungi. The present review provides updated information on the structure and synthesis of -glucans in fungal cells. Synthesis of these polymers made up of B1,3 chains with a variable degree of B1,6 branching involves several reactions: initiation, chain elongation and branching, of which the most studied one is the elongation step. This reaction, catalyzed by the so-called glucan synthetases, utilizes UDPG as sugar donor. Properties of glucan synthetases are extremely variable depending on the fungal species, and their developmental stage. Because of the importance of these polysaccharides it is anticipated that comprehension of their mechanism of synthesis, is important for the understanding of cell wall assembly and cell growth and morphogenesis, as well as for the design of specific antifungal drugs.Abreviations UDPG uridine-diphospho-glucose - GDPG guanosine-diphospho-glucose - ADPG adenosine-diphospho-glucose - MW molecular weight - mic minimal inhibitory concentration - d.p. degree of polymerization - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Characterization of cyclic β-glucans of Bradyrhizobium by MALDI-TOF mass spectrometry

Periplasmic, cyclic β-glucans isolated from Bradyrhizobium elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium yuanmingense strains have been investigated by means of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), 1D and 2D nuclear magnetic resonance (NMR), as well as standard chemical methods. These compounds are built of 10–13 d-glucose residues. The main fractions contain molecules assembled of 12 hexose units (M_w = 1945.363 Da). Glucose monomers are linked by β-(1→3) or β-(1→6) glycosidic bonds. The ratio of β-(1→3) to β-(1→6) linked glucose is approximately 1:2. Moreover, methylation analysis demonstrated the presence of terminal, non-reducing, as well as branched (i.e., 3- and 6-substituted) glucoses. Thus, the basic structure of the investigated compounds is similar to that of periplasmic oligosaccharides from Bradyrhizobium japonicum and Azorhizobium caulinodans strains. The analyzed cyclic β-glucans are substituted by phosphocholine (PC) (one or two residues per ring) and highly decorated with acetate and succinate. The substituents are arranged diversely in the population of cyclic β-glucan molecules. The concentrations of cyclic β-glucans in Bradyrhizobium periplasmic space are osmotically regulated and increase in response to a decrease of medium osmolarity.     

Preparation of carboxylic acid-bearing polysaccharide nanofiber made from euglenoid β-1,3-glucans

This paper introduces a new strategy for creating surface modified polysaccharide nanofibers. To demonstrate proof of principle, the synthesis, structure, and self-assembly behavior of a carboxylic acid-bearing polysaccharide made from paramylon (β-1,3-glucan) and succinic anhydride were investigated. Examination by a combination of NMR, FT-IR, and SEC-MALLS confirmed that successful preparation of the desired succinylated paramylon without significant depolymerization. NMR, SEC-MALLS, visible absorption and CD spectroscopic analyses indicated that the paramylon derivative forms the triplex structure in solutions. SEM observation revealed that succinylated paramylon forms a nanofiber that has carboxylic acid on the surface.     

Oxygen controlled batch cultivations of Schizophyllum commune for enhanced production of branched β-1,3-glucans

Oxygen and shear stress are the key factors for enhanced glucan production with Schizophyllum commune. During batch cultivation control of or (specific oxygen uptake rate) was achieved by variation of the impeller speed. Biomass was modelled by using the carbon and oxygen balance derived from exhaust data. At mycel growth a of 0.042 h^–1 presents just the border before oxygen limitation arises and is simultaneously the optimum operation condition for maximum glucan formation. Related to an overall cultivation time of 72 h a maximum of both productivity (4.3 kg m^–3 d^–1) and yield (13 kg m^–3) were obtained.List of Symbols C kg m^–3 concentration - k _L a h ^–1 volume related oxygen transfer coefficient - K _s mol m^–3 substrate saturation constant - N rpm impeller speed - % oxygen partial pressure of the liquid phase - kg m^–3h^–1 oxygen uptake rate - h^–1 specific oxygen uptake rate, kg O₂ (kg biomass h)^–1 - t h time - yield coefficient (biomass formed/oxygen consumed) Greek Symbols h^–1 specific growth rate Indices O ₂ oxygen - X biomass - L liquid phase - * gas/liquid interface - S substrate (glucose) Dedicated to the 65th birthday of Professor Fritz Wagner.This work was kindly supported in parts by B. Braun Biotech International. The authors are grateful to Prof. Dr. Fritz Wagner for scientific support and appreciate the technical assistance of Detlev Rasch     

Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.     

Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates

β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme–product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54 mM) in the early phase of the reaction.     

Effects of cereal β-glucans and enzyme inclusion on the porcine gastrointestinal tract microbiota

This study was designed to evaluate the effect barley-based diets vs. oats based diets on levels of Lactobacillus, Bifidobacterium and Enterobacterium in the porcine gastrointestinal tract (GIT). In addition the effect of enzyme supplementation in both diets was explored. Twenty-eight boars were used in a 2 × 2 factorial arrangement and were assigned to 1 of 4 dietary treatments: barley-based (B) diet; barley-based diet plus an enzyme supplement (B + ES); oat-based (O) diet or oat-based diet plus an enzyme supplement (O + ES). The enzyme supplement contained endo-1,3-β-glucanase and endo-1,4-β-xylanase. Faecal samples were collected from the pigs prior to initiations of the experiment and at slaughter. At slaughter digesta samples were collected from the stomach, ileum, caecum, proximal and distal colon. Alterations in Lactobacillus species composition in the gastrointestinal tract (GIT) were analysed by genus-specific PCR – denaturing gradient gel electrophoresis (DGGE). DGGE profiles indicated that cereal source provoked shifts in Lactobacillus population. The most diverse populations of lactobacilli emerged after feeding the O diets. Enzymes inclusion altered the composition of Lactobacillus species prevalent throughout the GIT in animals fed the B diet, causing a shift in the dominant lactobacilli present in the caecum and proximal colon. No such effect was evident in animals fed the enzyme supplemented O + ES diet. Microbial plate counts revealed that the O diets gave rise to higher counts of Lactobacillus in the caecum and colon and Bifidobacterium counts in the ileum, caecum and colon than the B diets. The O diet caused a 2 log increase in Enterobacterium counts in the proximal colon, no such effects were observed in animals fed the B, the B + ES or the O + ES diets. Overall both O diets had a more positive influence on the counts of the beneficial microorganisms and richness of the Lactobacillus population in the porcine GIT.     

Do cereal mixed-linked β-glucans possess immune-modulating activities?

β-glucans are known for their immune-modulating properties. However, the heterogeneity of these glucose polymers makes a distinction between the different sources and structures necessary-a fact that has been little allowed for in the literature. We have focused on β-glucans from cereals as they are already used as functional food ingredients due to their established cholesterol lowering effect. Cereal β-glucans have shown in vitro activity on cytokine secretion, phagocytic activity and cytotoxicity of isolated immune cells, and activation of the complement system. Animal studies suggest a possible protective effect against an intestinal parasite, against bacterial infection, and a synergistic effect in antibody-dependent cellular cytotoxicity. Animal studies have shown activity of orally applied cereal β-glucans indicating uptake or interaction with cells of the gastrointestinal tract. However, uptake is still debated, interaction with intestinal epithelial cells has been suggested but not clarified, and mechanisms of action remain largely unknown. So far, cereal β-glucans have not shown immune modulation in the few conducted human studies and further studies are needed to clarify their effect.     

Characterization of the type of action of β-1,3-glucanases from marine invertebrates

• 1.1. Ten species of marine invertebrates possessing the highest laminarinase activity have been investigated.
• 2.2. The following approaches were used in determining the type of action of partially purified β-1,3-glucanases: effect on modified substrates, comparative study of enzymolysis products by the Nelson's and glucose oxidase methods and stereochemistry of the enzymolysis products.
• 3.3. Experimental results indicate that the examined marine invertebrates contain β-1,3-glucanases with endo-type activity; exo-enzymes are apparently absent.

     

Screening of microalgae for primary metabolites including β-glucans and the influence of nitrate starvation and irradiance on β-glucan production

Βeta-glucans, widespread glucose polymers in mushrooms, yeasts, and bacteria, but rarely found in microalgae, have wide applications and high medicinal and economical potential. Some β-glucans like paramylon from algae-like Euglena gracilis are well investigated, but there is little information about the β-glucan content of microalgae. Therefore, more than 40 species of cultured microalgae have been investigated for composition of their biomass regarding to lipids, carbohydrates including β-glucans and proteins. Most of algae species showed a rather similar biomass composition of about 10 % lipids, 25 % carbohydrates, and 40 % proteins if they have been cultivated in a full medium, rather low light conditions of 50 μmol photons m^?2 s^?1, 12/12 h light/dark cycle under aeration and a temperature of 25?±?2 °C. The content of β-glucans varied between 1.7 and 24.2 % of dry weight, respectively. Two microalgae, Scenedesmus ovalternus SAG 52.80 and Porphyridium purpureum SAG 1380-1d with a yield of more than 20 % of dry weight were identified as the best β-glucan producers under standard cultivation conditions. Culture optimization experiments revealed that enhanced irradiance increased the β-glucan content of Scenedesmus obtusiusculus A 189, a novel green algae isolate, from 6.4 to 19.5 %, but the β-glucan content of the green alga S. ovalternus SAG 52.80 remained unaffected (24.2 vs. 23.3 %). Nitrate starvation enhanced the β-glucan content of S. obtusiusculus A 189 from 16 to 23 % and of S. ovalternus SAG 52.80 from 23.3 to 36.7 %.     

Mechanism of action of βadrenergic receptor blocking agents in angina pectoris: Comparison of action of propranolol with dexpropranolol and practolol

The effect on exercise tolerance of racemic propranolol has been assessed in eight angina pectoris patients and compared with that of dexpropranolol (the dextro isomer of propranolol), practolol (I.C.I. 50172), and saline. Dexpropranolol has the same local anaesthetic action as propranolol with negligible β-adrenergic receptor blocking activity, while practolol is a cardio-selective β-adrenergic blocking agent which does not have local anaesthetic activity.Saline and dexpropranolol had no significant effect on exercise time; racemic propranolol and practolol improved exercise tolerance in six subjects, the response to the two drugs being very similar in individual patients. It was concluded that the beneficial effect of propranolol in angina pectoris results from its action as a β-adrenergic receptor blocking agent and is not due to its local anaesthetic, or quinidine-like, activity.     

Fine-tuning of POPC liposomal leakage by the use of β-cyclodextrin and several hydrophobic guests

The effect of entrapped β-cyclodextrin (β-CD) on the stability of multilamellar vesicles (MLVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), prepared by the dehydration-rehydration method, was studied by monitoring the release of 5(6)-carboxyfluorescein encapsulated into the liposomes. Different hydrophobic guests, such as Fullerene C₆₀, have been incorporated into the POPC bilayer in order to modify the membrane composition. The kinetic results as well as ESI-MS measurements evidenced that the destabilizing activity of β-CD is due to the formation of β-CD inclusion complexes and the consequent removal of selected bilayer constituents from the liposomal membrane. Hence, when β-CD was added to the liposomes in the form of a strong, water-soluble 2:1 β-CD/C₆₀ inclusion complex, such a destabilizing effect was not observed. However, the same β-CD/C₆₀ inclusion complex does not form as a result of C₆₀ extraction from the bilayer. This may be attributed either to the overwhelming concentration of POPC with respect to C₆₀ and/or to the fact that C₆₀ is largely aggregated in the bilayer. Turbidimetric and fluorimetric determinations of lamellarity and entrapped volume of the studied MLVs provided further evidence of the alteration of the liposomal bilayer as a consequence of the addition of β-CD and/or the presence of the studied guests.     

Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.     

β-1,4-endoglucanases from Talaromyces amestolkiae: Production of glucooligosaccharides from different β-glucans

Abstract

This article presents the purification and characterization of two β-1,4-endoglucanases from Talaromyces amestolkiae. The cellulase activities secreted by this fungus were studied in the presence of different carbon sources, attaining the maximal levels in the presence of Avicel as carbon source. In these conditions, two glycosylated β-1,4-endoglucanases with molecular masses of 25,573?kDa (EG1) and 51,825?kDa (EG2), were purified. Both isoenzymes have acidic isoelectric points, 5.4 and 4.6, respectively. Their optimum pH and temperature, either in crudes or after purification, were in the range normally used for the simultaneous saccharification and fermentation in bioethanol production. In addition, the enzymatic hydrolysis of different β-glucans by both enzymes was studied. In the assayed conditions, both enzymes hydrolysed carboxymethylcellulose, a typical substrate for endoglucanases, although EG2 was much more efficient. However, EG1 was also able to hydrolyse lichenan and laminarin. These findings suggest the potential interest of EG2 for specific hydrolysis of cellulose, present in plant cell walls, to produce bioethanol, while the more promiscuous enzyme EG1 could be used for production of glucooligosaccharides.     

A critical review on the impacts of β-glucans on gut microbiota and human health

The β-glucans are the glucose polymers present in the cells walls of yeast, fungi and cereals. β-Glucans are the major compositions of various nutritional diets such as oats, barley, seaweeds and mushrooms. Various biological activities of β-glucans have been reported such as anticancer, antidiabetic, anti-inflammatory and immune-modulating effects. The importance of β-glucans in food processing industries such as bread preparation, yogurt and pasta have been well elucidated. In recent findings on food science research gut microbiota plays a significant role and vastly studied for its intermediate role in regulating health. Several reports have suggested that β-glucans should have a significant impact on the gut microbiota changes and in turn on human health. The review was aimed to accumulate the evidence on types of β-glucans, their functional properties and the mechanism by how the β-glucans regulate the gut microbiota and human health. The various in vitro, in vivo and clinical studies, have been summarized, in particular, the changes happening upon the β-glucans supplementation on the gut microbiota. Overall, this review updates the recent studies on β-glucans and gut microbiota and also inputs the demanding questions to be addressed in β-glucans–microbiota research in the future.     

Biosynthesis of cyclic β-(1,2)-glucans inRhizobium leguminosarum biovarsviciae,phaseoli andtrifolii

Inner membranes of Rhizobium leguminosarum biovars viciae, phaseoli, and trifolii synthesized a heterogenous family of neutral cyclic -(1,2)-glucans in a reaction system that used oligosaccharide intermediates covalently bound to a large protein. This glucoprotein showed a slightly lower mobility on SDS-polyacrylamide gels (apparent mol. mass of 320 kDa) than the -(1,2)-glucan intermediate protein of Rhizobium meliloti. In vivo pulse-label experiments with growing cells of R. leguminosarum biovar trifolii RS800 using radioactive glucose showed that few species of cyclic -(1,2)-glucans were synthesized and up to 30% were substituted with charged non-glycosidic residues, probably sn-1-phosphoglycerol.     

Artificial mixed-linked β-glucans produced by glycosynthase-catalyzed polymerization: tuning morphology and degree of polymerization

The glycosynthase derived from Bacillus licheniformis 1,3-1,4-β-glucanase was able to polymerize glycosyl fluoride donors (G4)(m)G3GαF (m = 0-2, G = Glcβ) leading to artificial mixed-linked β-glucans with regular sequences and variable β1,3 to β1,4 linkage ratios. With the E134A glycosynthase mutant, polymers had average molecular masses (M(w)) of 10-15 kDa. Whereas polymer 2 ([4G4G3G](n)) was an amorphous precipitate, the water-insoluble polymers 1 ([4G3G](n)) and 3 ([4G4G4G3G](n)) formed spherulites of 10-20 μm diameter. With the more active E134S glycosynthase mutant, polymerization led to high molecular mass polysaccharides, where M(w) was linearly dependent on enzyme concentration. Remarkably, a homo-polysaccharide [4G4G4G3G](n) with M(w) as high as 30.5 kDa (n ≈ 47) was obtained, which contained a small fraction of products up to 70 kDa, a value that is in the range of the molecular masses of low viscosity cereal 1,3-1,4-β-glucans, and among the largest products produced by a glycosynthase. Access to a range of novel tailor-made β-glucans through the glycosynthase technology will allow to evaluate the implications of polysaccharide fine structures in their physicochemical properties and their applications as biomaterials, as well as to provide valuable tools for biochemical characterization of β-glucan degrading enzymes and binding modules.     

Innate recognition of cell wall β-glucans drives invariant natural killer T cell responses against fungi

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.     

Re-examination of cellular cyclic β-1,2-glucans of Rhizobiaceae: Distribution of ring sizes and degrees of glycerol-1-phosphate substitution

Gel-filtration and thin layer chromatography of low molecular weight carbohydrates from culture filtrates of Agrobacterium radiobacter, Isolate II, have shown, that next to the neutral -1,2-glucan fraction a major acidic fraction was present which was found to be glycerophosphorylated cyclic -1,2-glucans. Re-examination of cyclic -1,2-glucan preparations which had been obtained by extraction of Rhizobium cells with hot phenol-water also showed these acidic modified -1,2-glucans to be present. Cyclic -1,2-glucans from R. leguminosarum (9 strains) and of R. phaseoli (1 strain) had ring size distribution with degrees of polymerisation (DPs) of 19 and 20 as major ring sizes of which a minor part was glycerophosphorylated; -1,2-glucans of R. trifolii (3 strains) had ring sizes with DPs measuring 19–22 as prominent components which were largely unsubstituted, and R. meliloti (7 strains) had -1,2-glucans with ring size distributions extending to still higher DPs of 19–25 of which the major part appeared to be glycerophosphorylated.     

Ferulic acid esterase catalyses the solubilization of β-glucans and pentosans from the starchy endosperm cell walls of barley

Summary Two separate, highly purified ferulic acid esterases from a fungal and bacterial source are both capable of releasing -glucan and pentosans from the cell walls of the starchy endosperm of barley. This suggests that ester linkages involving ferulic acid contribute to the integrity of such walls.