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Properties of beta-glucan synthetase from Saccharomyces cerevisiae.

Properties of beta-glucan synthetase from S. cerevisiae were studied. The enzyme exhibited optimal activity at pH 6.7 and 24 C. Km for UDP-glucose was 0.12 mM. 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 beta-glucan synthetase. Acid proteases were neither stimulatory nor destructive. Thus it seems unlikely that beta-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, beta-glucan or the protease inhibitors PMSF, Antipain or Pepstatin A did not protect beta-glucan synthetase from inactivation. Glucan synthetase was not stimulated by addition of cellobiose or beta-glucans. The synthesis of beta-glucans was competitively inhibited by UDP (Ki = 0.45 mM). Glucono-delta-lactone, a known inhibitor of beta-glucosidases was a strong non-competitive inhibitor of beta-glucan synthetase.

Cell-Free System

Effect of brewers' yeast or beta-glucan derived from Saccharomyces cerevisiae on breast milk supply following preterm birth: the BLOOM randomised controlled trial.

OBJECTIVE: Breast milk is the optimal source of nutrition for preterm infants; however, low breast milk production is common following a preterm birth. This study aimed to determine if taking brewers' yeast or beta-glucan improves daily expressed breast milk volume. DESIGN: Randomised, blinded, parallel, placebo-controlled trial. SETTING: Three Australian tertiary-level neonatal units. PATIENTS: Mothers with a singleton or twin pregnancy who gave birth at <34 weeks' gestation. INTERVENTIONS: Mothers were randomised within 72 hours of birth into three parallel groups in a 1:1:1 ratio to receive either brewers' yeast, beta-glucan or placebo capsules for 7&#x2009;days. MAIN OUTCOME MEASURE: Total expressed breast milk volume over a 24-hour period on day 7 of intervention. RESULTS: A total of 105 mothers underwent randomisation between August 2022 and April 2024 (36 brewers' yeast, 35 beta-glucan and 34 placebo). The adjusted mean difference in daily expressed breast milk volume was 94&#x2009;mL/day (95%&#x2009;CI -51 mL/day to 239&#x2009;mL/day) between the brewers' yeast and placebo groups and -25&#x2009;mL/day (95%&#x2009;CI -173 mL/day to 123&#x2009;mL/day) between the beta-glucan and placebo groups. Maternal side effects were similar across groups. CONCLUSION: We found no clear effect of short-term administration of brewers' yeast or beta-glucan on breast-milk production following preterm birth; both interventions were well tolerated. Given the small sample size, these findings do not rule out the possibility of a clinically meaningful benefit of brewers' yeast and suggest further research with a larger sample size may be warranted to clarify the potential clinical impact. TRIAL REGISTRATION NUMBER: ACTRN12622000968774.

Intensive Care Units, Neonatal

Biosynthesis of beta-glucans by cell-free extracts from Saccharomyces cerevisiae.

Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U-14C]glucose into beta-1,3-glucans which contained a significant proportion of beta-1,6-glycosidic linkages. When GDP-[U-14C]glucose was used as substrate only trace amounts of glucose were incorporated. Activity of beta-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. Beta-glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of beta-1,6-glycosidic linkages respectively. A marked decrease in the activity of beta-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.

Cell Cycle

The formation of oligoglucans linked to lipid during synthesis of beta-glucan by characterized membrane fractions isolated from peas.

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble beta-glucans, which like the oligosaccharides contained beta(1leads to 3) and beta-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in beta-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.

Amylases

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414.

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.

Amino Acids

1,3-beta-D-glucanases from Pisum sativum seedlings. II. Substrate specificities and enzymic action patterns.

Two purified pea 1,3-beta-D-glucanases (EC 3.2.1.6) hydrolyse laminarin (degree of polymerization 20), laminaridextrins (degree of polymerization 3--7), and their reduced 3H-derivatives, 1,3-beta-D-glucans which are partially substituted (carboxymethyl-pachyman) or crystalline (curdlan), and mixed-linkage beta-glucans. Enzyme kinetics and product-formation indicate endo-hydrolase activity with weak transglycosylase capacity. The enzymes do not hydrolyse beta-glucosides, the 1,3 linkage adjacent to the reducing end of chains, or cellulose and its derivatives. They degrade mixed-linkage beta-glucans, in a manner similar to Rhizopus arrhizus endo-1,3-beta-D-glucanase, to form the products expected from hydrolysis of linkages adjacent to 1,3-beta linkages. With respect to action patterns, glucanase I (from apical growing tissue) differs from glucanase II (from basal maturing tissue) in several respects: (a) on a molar basis, I generates reducing groups from all substances more rapidly than II; (b) lower laminaridextrins are hydrolysed by I at the non-reducing terminal linkage, while II preferentially hydrolyses internal linkages; (c) laminarin is hydrolysed to lower laminaridextrins by I more rapidly than II, but I takes longer than II to completely degrade laminarin chains; (d) the enzymes are differentially sensitive to different classes of non-competitive inhibitors. It is concluded that these beta-glucanases differ in such a way that I preferentially continues to degrade fragments produced by endo-hydrolytic attack on long chains ('multiple attack' action pattern), while II hydrolyses internal linkages of the longest chains available ('multi-chain attack').

Fabaceae

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography.

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl alpha-D-mannopyranoside and appeared to be alpha-mannan, while that which passed through the column was virtually all beta-glucan. Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the alpha-mannan contained predominantly alpha-(1 leads to 2)-linked side chains branching from an alpha-(1 leads to 6)-linked backbone at the (1 leads to 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The beta-glucan seemed to contain mainly (1 leads to 6)-linked side chains branching from a (1 leads to 3)-linked backbone at the (1 leads to 3,6)-linked glucopyranosyl residues.

Agaricales

Studies of the spore walls of Agaricus bisporus and Agaricus campestris.

The composition and ultrastructure of spore walls of Agaricus bisporus and Agaricus campestris were compared by chemical and enzymatic assays and electron microscopy. Polymers of N-acetylglucosamine (chitin) and glucosamine (chitosan) were the major carbohydrate wall components of both species. The chitin to chitosan ratio in the spore walls of A. bisporus was about 0.38, while in A. campestris it was about 2.8 These polymers were associated with proteins, lipids (readily extractable and bound), melanin, and a low content of beta-glucan. The quantity of protein, total lipid, melanin, or beta-glucan was similar in spore walls of both species. With the electron microscopy, the spore walls appeared as two well-defined layers which may correspond to microfibrils of chitin and chitosan in which the electron-dense melanin was located in the external layer.

Agaricales

Lysis of intact yeast cells and isolated cell walls by an inducible enzyme system of Arthrobacter GJM-1.

Bacterium Arthrobacter GJM-1 known in the literature as a good producer of alpha-mannanase was found to accumulate in the culture fluid lytic activities against viable yeast cells during growth on isolated cell walls or beta-glucan fractions of yeast. The accumulation of the lytic activities showed an inducible character. The lytic system produced in the medium containing baker's yeast cell walls was capable of complete solubiliaztion of cell wals in vitro. The system lysed viable cells of a number of yeast species and induced their conversion to protoplasts in an osmotically stabilized medium. The lytic system showed different pH and temperature optima when viable cells or isolated cell walls were used as substrates. The pH optimum of the lysis of isolated cell walls was identical with pH optimum of beta-glucanase activities in the crude system. The results pointed out that in the lysis of intact cells, in addition to beta-glucanases, some other factor is involved. Substantial differences in the nature of the outer and the inner surface of cell walls of Saccharomuces cerevisiae were confirmed in this paper based on the different susceptibility to lysis of the cell walls in vivo and in vitro.

Arthrobacter

An alkali-soluble polysaccharide from the cell walls of Coprinus lagopus.

An alkali-soluble polysaccharide was isolated from the purified mycelial walls of Coprinus lagopus. The hydrolysis products, optical rotation, and infrared spectrum indicate a beta-glucan. Hydrolysis of the glucan after permethylation gave only 2,3,4,6-tetra-, 2,4,6-tri-, and 2,4-di-O-methyl-D-glucose. These methylated sugars and their relative quantities reveal that the glucan is a polysaccharide containing beta-1,3-linked glucose units with about 14% of the sugars having 1,6-linked branch points. Partial hydrolysis of the product derived from Smith degradation of the glucan released laminaribiose and gentiobiose suggesting that the branches are generally longer than a single glucose unit.

Agaricales

Chemical analysis of the hyphal wall of Schizophyllum commune.

1. Purified hyphal wall fragments of Schizophyllum commune are analysed and shown to consist of glucose (67.6%), mannose (3.4%), xylose (0.2%), (N-acetyl)glucosamine (12.5%), amino acids (6.4%) and some lipid material (3.0%). 2. The previously proposed structures of two glucans located at the hyphal wall surface (Wessels et al. (1972) Biochim. Biophys. Acta 273, 346-358) were essentially confirmed using methylation analysis. The mucilaginous glucan consists of 1,3-linked beta-glucan chains with branches of single glucose units attached by beta-1,6 linkages on every third unit, on average, along the chain. The alkali soluble S-glucan is an exclusively 1,3-linked alpha-glucan. 3. The alkali-insoluble R-glucan, occurring in close association with chitin, in the inner wall layer, has been characterised by methylation analysis, X-ray diffraction, enzymatic hydrolysis with purified exo-beta-1,3-glucanase and Smith degradation. It appears to be a highly branched beta-1,3,beta-1,6-glucan and a model of this glucan is proposed. Certain parts of this highly insoluble R-glucan bear a close structural similarity to the mucilaginous glucan present at the outer wall surface and in the medium.

Agaricales

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae cavara. II. Purification and properties of a beta-glucosidase.

Three components (GA, GB-1, and GB-2) of beta-glucosidase were detected in the culture filtrate of Pyricularia oryzae grown in a cellulose or cellulose derivative medium. Among them, GB-1 was induced most strongly. Purified GB-1 was homogeneous on polyacrylamide gel electrophoresis and showed an approximately 1,400-fold increase of specific activity over the starting material. The molecular weight was determined to be 240,000 by sodium dodecyl sulfate-gel electrophoresis. A similar value was also obtained by sucrose density gradient centrifugation. The enzyme contained a high proportion of acidic amino acids and mannose, and the isoelectric point of the enzyme was pH 4.15. The enzyme had a pH optimum of 5.5 and a temperature optimum at 55 degrees C. beta-Glucosidase activity was inhibited by Mn2+, Cu2+, Hg2+, p-chloromercuribenzoate, and glucono-delta-lactone. The enzyme split off glucose units one by one from the nonreducing ends of not only beta-glucooligosaccharides but also some beta-glucans, such as carboxymethylcellulose, laminaran, pustulan, and zeagallan. The affinity for cello- and laminari-oligosaccharides tended to increase in parallel with the chain length.

Amino Acids

Saccharomyces cerevisiae mutant defective in exo-1,3-beta-glucanase production.

Saccharomyces cerevisiae S288C produced two laminarinases (1,3-beta-glucanases) which were separated by diethylaminoethyl-Sephadex column chromatography; one was an endo-1,3-beta-glucanase, and the other was an exo-1,3-beta-glucanase active not only on laminarin but also on pustulan (1,6-beta-glucan) and on p-nitrophenyl-beta-D-glucoside. A mutant defective in the production of this last enzyme was isolated, and the mutation was named exb1-1. The selection procedure was based on the capacity of exo-1,3-beta-glucanase to hydrolyze synthetic glucosides. The level of endo-1,3-beta-glucanase in cell extracts of the mutant was normal, but the exo-1,3-beta-glucanase could not be detected by column chromatographic analysis of these extracts. The mutant phenotype, recessive in heterozygous diploids, was stable through successive meioses and showed a Mendelian segregation, indicating that the mutation affected a single gene, which was named EXB1. The lack of production of exo-1,3-beta-glucanase persisted through all the phases of growth, but growth itself was not impaired by the enzyme deficiency.

Genes

[Release of "Saccharomyces cerevisiae" protoplasts: scanning electron microscope study (author's transl)].

In a previous study we described the minimal methodology used to obtain protoplasts from ascomycetous yeasts. Using a reducing agent associated with 1,3-beta-glucanase at 26 degrees C, protoplasts were invariably obtained. In the present study we localized the disruption spots of the cell wall using the two same reagents. The observations were made with the scanning electron microscope. The disruption site was always in the subterminal region, and this very simple structure (proteins with disulphide bridges and 1,3-beta-glucans) was opposite the birth-scar. The dissociation of the two reagents showed that a small part of the yeast population was able to release protoplasts with only glucanase. We believe these very sensitive yeasts (2 to 10% population) to be very young cells. These disruption sites seemed very different from budding-sites. They might be identical with elongation-sites, or with the opening in the ascus-wall during germinating ascospore release.

Glucan Endo-1,3-beta-D-Glucosidase

Role of bioprocessing in modifying cardiometabolic outcomes of an oat-based dairy alternative in a randomised controlled clinical intervention.

BACKGROUND & AIMS: A healthy diet rich in fibre-containing foods such as oats supports cardiometabolic health. Bioprocessing methods, including fermentation and enzymatic treatment, may further enhance the health benefits of oat-based foods by altering their physicochemical properties. The aims of this study were to investigate the effects of consuming fermented and non-fermented oat-based products enriched with fibre and protein on cardiometabolic outcomes gastrointestinal symptoms, and to consider how assessed physicochemical and nutritional differences between the products might relate to any observed effects. METHODS: In a 12-week randomised crossover trial, 56 adults with mild metabolic deterioration consumed fermented (gurt) and non-fermented (porridge) oat-based products enriched with fibre and protein as part of their habitual diet for three weeks each. The study products were specifically developed and prepared for this study using identical ingredients. Primary cardiometabolic factors and gastrointestinal symptoms (GSRS) were measured at four time points, while secondary outcomes were assessed at baseline and after both product periods. Physicochemical and nutritional characterization of the study products included cereal &#x3b2;-glucan (BG) and protein molecular weight distribution, starch and sugar analysis, microscopy, acidity, and viscosity. RESULTS: During the gurt consumption, non-high-density lipoprotein (non-HDL) and low-density lipoprotein (LDL) cholesterol concentrations decreased (-0.15 &#xb1; 0.51 mmol/L, p = 0.028; and -0.12 &#xb1; 0.46 mmol/L, p = 0.047, respectively), with a minimal impact on blood pressure and GSRS scores. Additionally, ferritin was lower after the gurt compared with baseline (-4.00 [-16.50, 6.25] &#x3bc;g/L, p = 0.015). Similarly, ferritin levels were lower after the porridge period (-7.50 [-20.50, 4.25] &#x3bc;g/L), accompanied with a modest decrease in blood pressure and HbA1c. These effects, however, did not substantially differ between the product periods. Insulin showed a significant sequence effect (psequence&#x2217;time <0.05) and was analysed in sequence groups. Insulin levels significantly decreased during the gurt consumption in the group that started with the porridge (-2.22 &#xb1; 6.16 mU/L, p = 0.015). Fermentation and enzymatic treatment induced significant changes in BG MW, starch, and composition in the gurt, which may alongside with increased fibre intake during the intervention explain the observed results. CONCLUSION: Consuming a fermented, oat-based gurt as part of habitual diet may improve cholesterol metabolism, likely due to increased oat fibre intake rather than fermentation as such. Moreover, greater intake of oat-based products, regardless of processing, can reduce ferritin concentrations and marginally improve other cardiometabolic factors. The study was registered in ClinicalTrials.gov as NCT06393114.

Humans

In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research.

We developed an in vitro methodology to study trained immunity using murine bone-marrow-derived macrophages stimulated with &#x3b2;-glucan and lipopolysaccharide (LPS). Longitudinal analysis of interleukin (IL)-6 and tumor necrosis factor (TNF) production demonstrates that trained macrophages secrete higher cytokine levels following primary stimulation with &#x3b2;-glucan compared to unstimulated macrophages (step 1). After a resting period, trained macrophages return to basal levels of cytokine production (step 2) but rapidly produce enhanced levels of IL-6 and TNF after secondary stimulation with LPS, compared to macrophages individually stimulated with either &#x3b2;-glucan (step 3) or LPS (step 4) alone. The combined cytokine production of macrophages after single stimulation with &#x3b2;-glucan (stimulus 1) and LPS (stimulus 2) is significantly lower than the cytokine levels produced by trained macrophages sequentially stimulated with both &#x3b2;-glucan and LPS (stimulus 1 + 2) (step 5). These results experimentally reproduce the distinctive functional stages that macrophages undergo during the training process.

Animals