Yeast Glucan Research Articles

Immunopotentiating effect of fungal glucans as revealed by frequency limitation of postchemotherapy relapse in experimental mouse tuberculosis.

Two kinds of fungal glucan, particulate yeast glucan (PYG) and lentinan, were examined for their immunopotentiating effect illustrated by reduced frequency of post-chemotherapy relapse in experimental mouse tuberculosis. Infected mice were treated by intensive chemotherapy with a three-drug combination [Streptomycin (SM) + isoniazid (INH) + rifampicin (RFP), ethambutol (EB) + INH + RFP, or SM + INH + pyrazinamide (PZA)] for 5 months. After termination of chemotherapy, the mice of each treated group were divided into three subgroups to receive or not to receive glucan for 4 weeks and again for 4 weeks after a month interval. During this 3-month period and the succeeding 5 months, the mice were subjected to occasional sacrifice to examine the growth of latent tubercle bacilli in the lung and spleen by cultivation of tissue homogenates. The results indicated that the regimens with SM + INH + RFP and EB + INH + RFP were highly effective in eliminating persistent tubercle bacilli down to undetectable levels. However, later multiplication of the latent bacilli was observed during the advanced postchemotherapy period. The application of PYG was most effective in prevention of this type of relapse in the spleen, but not so in the lung. Lentinan effect was manifested in the lung, but not in the spleen.

Therapeutic and prophylactic effects of yeast glucan and related polysaccharidic immunomodulators in experimental leprosy

Therapeutic and prophylactic effects of yeast glucan and related polysaccharidic immunomodulators in experimental leprosy

Therapeutic effect of intravenously administered yeast glucan, in mice locally infected by Mycobacterium leprae.

The management of leprosy is still primarily based on chemotherapy and antibiotic therapy, e.g., on directly acting substances which are simply more toxic for the parasite than for the host cells. Little efforts have been devoted thus far to the evaluation of drug candidates whose indirect antiparasitic power involves stimulation of the natural mechanism of host-defense. The dramatic problem caused by rapid spreading of resistance to sulphones prompted us to correct that situation, namely by means of immunostimulating polysaccharides whose antiparasitic efficacy had already been established against experimental tumors (19). Particulate β 1–3 glucan extracted from the cell walls of Saccharomyces cerevisiae was first chosen, because of its ability to act on several strategic parameters of humoral and cellular immunity (1,5,8) leading to enhancement of nonspecific resistance in a first stage (6) followed by specific immunostimulation as a second stage. Especially relevant to the case of leprosy, in which macrophages are the host-cells to the parasitic mycobacteria, is that this glucan appears as a most potent and versatile reticuloendothelial (RE) system-stimulator (5) through both activation of preexisting macrophages and overproduction of fresh mononuclear phagocytes.

The extracellular system of beta-1,3-glucanases of Alternaria tenuissima and Aspergillus vesicolor.

During cultivation in a minimal medium with glucose Alternaria tenuissima and Aspergillus vesicolor produce constitutively alpha- and beta-glucanases. Fractions of beta-1,3-glucanases exhibiting affinity for laminarin were separated by means of gel filtration chromatography. Two neutral beta-1,3-glucanases with affinity for yeast glucan were isolated by affinity chromatography and further characterized.

Soluble and insoluble glucans from different cell types of the human pathogen Sporothrix schenckii

The structures of soluble and insoluble glucans from isolated cell walls of yeast, mycelial, and conidial forms of Sporothrix schenckii were compared. Methylation analysis showed the presence of linear structures and did not reveal any qualitative variation of glycosidic linkage type. Periodate oxidation methods demonstrated that soluble and insoluble glucans were chemically very similar in all cell types. Soluble glucans from S. schenckii yeast forms have proportions of 44, 28, and 28% of, (1→3), (1→6), and (1→4) linkages, respectively. Insoluble glucans from yeast forms also contain (1→3), (1→6), and (1→4) linkages but their proportions are 66, 29, and 5% respectively. Proportions of these linkages in the mycelial and conidial glucans were similar to those in yeast glucans. The β -configuration of the d -glucopyranose units was shown by the specific rotations of the soluble glucans or the methylated derivatives of the insoluble ones. It was also determined by enzymolysis of both types of glucan with β -glucanases. The disruptive action of these enzymes on the fibrillar network of walls from all cell types was observed by electron microscopy. 13 C nuclear magnetic resonance spectra of soluble glucans are consistent with the configuration and position of substitution of glycosidic linkages indicated by other methods.

Studies on microbial enzyme active in hydrolyzing yeast polysaccharides. II. Purification and some properties of endo-.BETA.-1,6-glucanase from Acinetobacter sp.

A kind of endo-β-1, 6-glucanase has been purified from the culture filtrate of Acinetobacter sp. grown in the medium containing baker’s yeast cells as a carbon source. A 100-fold purified preparation was obtained by DEAE-Sephadex A–50 column chromatography. The enzyme hydrolyzed pustulan giving a series of gentio-oligosaccharides and glucose. Gentiotriose and gentiotetraose were hydrolyzed by this enzyme yielding glucose and gentiobiose, and glucose, gentiobiose and gentiotriose, respectively. Gentiobiose was not hydrolyzed. Baker’s yeast glucans obtained from the isolated cell walls were also hydrolyzed by this enzyme giving a series of oligosaccharides and glucose. From the action patterns on these carbohydrates, we concluded the present enzyme being endo-β-1, 6-glucanase.

Host-Pathogen Interactions

A beta-glucan isolated from the mycelial walls of Phytophthora megasperma var. sojae and a glucan purified from yeast extract stimulate the accumulation of phytoalexins in red kidney bean, Phaseolus vulgaris, and stimulate the accumulation of the phytoalexin, rishitin, in potato tubers, Solanum tuberosum. These glucans have previously been shown to be potent elicitors of glyceollin accumulation in soybean, Glycine max.Treatment of kidney bean cotyledons with the glucan elicitors resulted in the accumulation of at least five fungistatic compounds. These compounds migrate during thin layer chromatography identically to the fungistatic compounds which accumulate in kidney beans which have been inoculated with Colletotrichum lindemuthianum, a fungal pathogen of kidney beans.Potatoes accumulate as much as 29 micrograms of rishitin per gram fresh weight following exposure to the glucan from Phytophthora megasperma var. sojae and as much as 19.5 micrograms of rishitin per gram fresh weight following exposure to yeast glucan. Potatoes accumulated 28 micrograms of rishitin per gram fresh weight following inoculation with live Phytophthora megasperma var. sojae.

Production and Ecological Significance of Yeast Cell Wall-Degrading Enzymes from Oerskovia

Motile actinomycetes capable of degrading walls of viable yeast cells were isolated from soil and identified as Oerskovia xanthineolytica. A lytic assay based on susceptibility of enzyme-treated cells to osmotic shock was developed, and 10 of 15 strains of O. xanthineolytica, Oerskovia turbata, and nonmotile Oerskovia- like organisms from other collections were found to possess yeast lytic activities. All lytic strains produced laminaranase and alpha-mannanase, but the amounts, determined by reducing group assays, were not proportional to the observed lytic activities. The Oerskovia isolates demonstrated chemotactic, predatory activity against various yeast strains and killed yeasts in mixed cultures. Of 15 carbon sources tested for production of lytic enzyme, purified yeast cell walls elicited the highest activity. Glucose repressed enzyme production and caused cells to remain in the microfilamentous and motile rod stages of the Oerskovia cell cycle. Crude lytic activity was optimal at pH 5.6 to 7.0 and inactivated by heating for 6 min at 50 degrees C. Partial purification by isoelectric focusing showed that all lytic activity was associated with four beta-(1-->3)-glucanases. The absence of protein disulfide reductase, N-acetyl-beta-d-hexosaminidase, and phosphomannanase in crude preparations indicated that the principal enzyme responsible for yeast wall lysis was a beta-(1-->3)-glucanase that produced relatively little reducing sugar from yeast glucan.

Substrate specificity and mode of action of a cellulase from Aspergillus niger.

The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.

Concerted induction of .BETA.-glucanases of Bacillus circulans WL 12 in response to various yeast glucans.

Bacillus circulans WL 12 was grown on cells of six species of yeasts as inducer substrates, and the courses of progress of β-glucanase production were compared. Also, the isoelectric focusing patterns of β-1,3 and β-1,6 glucanases were determined. Conspicuous differences were observed among the isoelectric focusing patterns of the crude enzymes produced with different yeast species as inducer substrates. Lyses of the cell walls of Pyricularia oryzae P2 and several species of yeasts by the enzyme systems were compared. The lysis of the cell walls of Debaryomyces hansenii was stronger in the order of the complexity (number of major peaks etc.) of the β-1,3 glucanases in isoelectric focusing patterns.

Mode of increase in cell wall polysaccharides in synchronously growing Saccharomyces cerevisiae

The mode of increase in cell wall polysaccharides of yeast (glucan and mannan) during cell cycle was analyzed using cell wall samples obtained from a synchronous culture. The increase in mannan and total glucan proceeded almost linearly throughout the cell cycle except for a short period of their leveling off at the time of cell division. However, the constituents of glucan behaved characteristically: Alkalisoluble glucan and insoluble glucan increased mainly in the former and the later half of the cell cycle, respectively.

Studies on enzyme lysing yeast cell from Oerskovia sp. CK. VI. The synergistic effects among .BETA.-1,3-glucanases from Oerskovia sp. CK on lysis of viable yeast cells.

Oerskovia sp. CK produced three types of β-1,3-glucanases designated as F-L, F-0 and F-2. F-L showed high lytic activity to viable yeast cells and weak activity to yeast glucan. F-0 and F-2 had little or no lytic activity and strong β-1,3-gIucanase activity.F-0 or F-2 showed high lytic activities to yeast cells pretreated with small amounts of F-L which did not lysed the cells. Lytic activity of F-0 or F-2 also increased when cells were treated with alkaline pH or with both reducing agents and pH.From these results, it is supposed that the ineffectiveness of F-0 or F-2 on the lysis of yeast cells might be attributed to a spatial inaccessibility of enzymes to the yeast glucan layer. However, the treatment of F-L, alkaline pH and reducing agents would bring about a modification of cells to give F-0 or F-2 access to the wall glucan and consequently the lysis of cells would occur.

Therapeutic effect of yeast glucan in mice infected with Mycobacterium leprae [proceedings

Therapeutic effect of yeast glucan in mice infected with Mycobacterium leprae [proceedings

Influence of dosage on intensity and evolution of the granulomatous response in normal mouse liver and spleen, after intravenous administration of yeast glucan [proceedings

Influence of dosage on intensity and evolution of the granulomatous response in normal mouse liver and spleen, after intravenous administration of yeast glucan [proceedings

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12.

Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.

On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study.

The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.

Further studies on the heterogeneity of the lytic activity for isolated yeast cell wall of the components of an Arthrobacter glucanase system: Properties of the two components of a .BETA.-1,3 glucanase.

Isolation and some properties of the two components of an Arthrobacter β-1,3 glucanase (glucanase II–1 and glucanase II–2) are described. Both of these glucanases hydrolyzed various β-1,3 glucans, accumulating as final products, glucose and laminaribiose. They resembled each other in instability at acidic pH values, lability at temperature above 55°C, having an optimal pH range of 5.5-6.5 and requirement of the presence of glucanase I for the effective hydrolysis of yeast glucan. Glucanase II–1 showed a high lytic activity for isolated yeast cell wall, while the activity of glucanase II–2 was low. Culture fluids of early cultures contained glucanase II–1 predominantly, while those of late cultures contained glucanase II–2, predominantly.

Purification, crystallization, and properties of an endo .BETA.-1,3-glucanase from Rhizopus chinensis R-69.

An endo β-1, 3-glucanase was purified in crystalline form from a culture filtrate of Rhizopus chinensis R-69. Molecular weight of the enzyme was determined to be 23, 000 by molecular sieve chromatography and the mode of action of the enzyme was suggested to be a less random type of β-1, 3-glucanase. Km and Vmax of the enzyme for laminarin are 3.4g/liter and 154 I. U., respectively. The enzyme does not decompose the cell walls of living yeast; it decomposes, however, the preparation of yeast glucan.

Purification and properties of a specific, inducible β-glucanase, succinoglucan depolymerase from Flavobacterium

Succinoglucan depolymerase from the culture fluid of Flavobacterium sp. Strain M64 was purified by fractionation with (NH 4) 2SO 4, followed by chromatographies on DEAE-cellulose and Sephadex G-200. 90-fold purification was achieved and the final preparation showed about 90% purity on polyacrylamide gel disc electrophoresis. The enzyme has a molecular weight of 180 000. The optimal pH for enzyme activity is 5.8 and the enzyme is stable from pH 4.5 to 10.0. It is stable up to 35 °C, but rapidly loses activity above 35 °C. The K m values for succinoglucan and desuccinylated succinoglucan are 1.7 mg/ml and 1.2 mg/ml, respectively. Succinoglucan is depolymerized to yield a polymer with a degree of polymerization of 12 and the product was suggested to be a structural unit of succinoglucan. β-Glucans and β-oligosaccharides, including schizophyllan, kefiran, laminaran, lutean, luteose, carboxymethylcellulose, curdlan, β-1,6- and β-1,3-oligosaccharides, cellobiose, 6-O- laminaritriosylglucose and lactose are not hydrolyzed while yeast glucan and pachyman are hydrolyzed only slightly. Succinoglucan depolymerase is only formed when succinoglucan or desuccinylated succinoglucan is present as the sole carbon source or with another carbon compound such as xylose. Compounds such as glucose and succinic acid repress depolymerase formation induced by succinoglucan. On partial acid hydrolysis or enzymolysis of succinoglucan, most of its inductive effect is lost.