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.     

Effects of β-glucan extracted from Saccharomyces cerevisiae on humoral and cellular immunity in weaned piglets

Abstract

Twenty-four barrows were used to investigate the effects of β-glucan on immune function in weaned piglets. Pigs (8.09 ± 0.20 kg, 28 d of age) were fed a diet without or with supplemented β-glucan (50 mg/kg feed). All pigs were injected with ovalbumin (OVA) on day 14 to investigate their humoral immune response. On day 28, lymphocytes were isolated from all pigs to determine the effects of β-glucan on cellular immunity of pigs in vitro. Lymphocytes from six pigs of each group were incubated with 16 μg lipopolysaccharide (LPS) per ml culture medium, the remainder with an equivalent volume of culture medium alone. Samples were collected at 0, 3, 6, 12, 18, 24, and 48 h after LPS addition for determination of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10). On day 31, six pigs of each group were injected with either LPS (25 μg/kg BW) or an equivalent amount of sterile saline. Blood samples were collected at 3 h after LPS injection for analysis of IL-6, TNF-α, and IL-10 in plasma. The results indicated that dietary β-glucan enhanced pig antibody response to OVA only in the first week after injection. In vitro, the increases of IL-6 and TNF-α in culture medium were partially dampened in pigs supplemented with β-glucan when their lymphocytes were incubated with LPS, whereas the increase of IL-10 was potentiated. In vivo, dietary β-glucan attenuated the increase of plasma IL-6 and TNF-α, and enhanced the increase of plasma IL-10 when pigs were challenged with LPS. These results demonstrate that β-glucan can improve the humoral immunity of pigs and modulate cellular immunity of pigs by mitigating the elevation of pro-inflammatory cytokines and enhancing the increase of anti-inflammatory cytokines after an immunological challenge.     

In vivo evaluation of the antimutagenic and antigenotoxic effects of β-glucan extracted from Saccharomyces cerevisiae in acute treatment with multiple doses

Ample evidence suggests that cancer is triggered by mutagenic damage and diets or supplements capable of reducing such incidences can be related to the prevention of neoplasy development or to an improvement in life quality of patients who undergo chemotherapy. This research aimed to evaluate the antimutagenic and antigenotoxic activity of β-glucan. We set up 8 experimental groups: control (Group 1), cyclophosphamide (Group 2), Groups 3–5 to assess the effect of β-glucan administration, and Groups 6–8 to evaluate the association between cyclophosphamide and β-glucan. The intraperitonial concentrations of β-glucan used were 100, 150 and 200 mg/kg. Micronucleus and comet assays showed that within the first week of treatment β-glucan presented a damage reduction rate between 100–62.04% and 94.34–59.52% for mutagenic and genotoxic damages, respectively. This activity decreased as the treatment was extended. During the sixth week of treatment antimutagenicity rates were reduced to 59.51–39.83% and antigenotoxicity was not effective. This leads to the conclusion that the efficacy of β-glucan in preventing DNA damage is limited when treatment is extended, and that its use as a chemotherapeutic adjuvant need to be better clarified.     

Endonuclease α from Saccharomyces cerevisiae shows increased activity on ultraviolet irradiated native DNA

Molecular Genetics and Genomics - Endonuclease α isolated from the nucleus of the yeast Saccharomyces cerevisiae is a DNA endonuclease which has been shown to act preferentially on denatured...     

Conjugation of protein antigen to microparticulate β-glucan from Saccharomyces cerevisiae: a new adjuvant for intradermal and oral immunizations

Immunostimulatory glucose polymers known as β-glucans have been studied for many years. Our laboratory has prepared and characterized a novel microparticulate β-glucan (MG) from the budding yeast Saccharomyces cerevisiae. Because MG particles are rapidly phagocytized by murine peritoneal macrophages and induce the expression of B7 costimulatory molecules, we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. Here, we describe a procedure for conjugating the test vaccine antigen bovine serum albumin (BSA) to MG via water-soluble carbodiimide linkage. Conjugates with up to 0.4 mg of BSA/mg MG were prepared. MG/BSA conjugates were still actively phagocytized by mouse peritoneal macrophages. When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund’s adjuvant. Although primary oral immunization with MG/BSA caused no increase in serum anti-BSA antibody titers, booster immunization elicited a significant anti-BSA antibody response. These results suggest that protein antigens can be conjugated to MG via a carbodiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Ethanol production from xylo-oligosaccharides by xylose-fermenting Saccharomyces cerevisiae expressing β-xylosidase

Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a β-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular β-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. β-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.     

Analysis of bacterial community shifts in the gastrointestinal tract of pigs fed diets supplemented with β-glucan from Laminaria digitata,Laminaria hyperborea and Saccharomyces cerevisiae

This study was designed to evaluate the effects of algal and yeast β-glucans on the porcine gastrointestinal microbiota, specifically the community of Lactobacillus, Bifidobacterium and coliforms. A total of 48 pigs were fed four diets over a 28-day period to determine the effect that each had on these communities. The control diet consisted of wheat and soya bean meal. The remaining three diets contained wheat and soya bean meal supplemented with β-glucan at 250 g/tonne from Laminaria digitata, Laminaria hyperborea or Saccharomyces cerevisiae. Faecal samples were collected from animals before feeding each diet and after the feeding period. The animals were slaughtered the following day and samples were collected from the stomach, ileum, caecum, proximal colon and distal colon. Alterations in Lactobacillus in the gastrointestinal tract (GIT) were analysed using denaturing gradient gel electrophoresis (DGGE) profiles generated by group-specific 16S rRNA gene PCR amplicons. Plate count analysis was also performed to quantify total coliforms. DGGE profiles indicated that all β-glucan diets provoked the emergence of a richer community of Lactobacillus. The richest community of lactobacilli emerged after feeding L. digitata (LD β-glucan). Plate count analysis revealed that the L. hyperborea (LH β-glucan) diet had a statistically significant effect on the coliform counts in the proximal colon in comparison with the control diet. β-glucan from L. digitata and S. cerevisiae also generally reduced coliforms but to a lesser extent. Nevertheless, the β-glucan diets did not significantly reduce levels of Lactobacillus or Bifidobacterium. DGGE analysis of GIT samples indicated that the three β-glucan diets generally promoted the establishment of a more varied range of Lactobacillus species in the caecum, proximal and distal colon. The LH β-glucan had the most profound reducing effect on coliform counts when compared with the control diet and diets supplemented with L. digitata and S. cerevisiae β-glucans.     

Expression and Secretion of Bovine β-Lactoglobulin in Saccharomyces cerevisiae

A bovine β-lactoglobulin (β-LG) was expressed in Saccharomyces cerevisiae carrying bovine pre-β-LG cDNA and secreted into its growth medium. The expression plasmid was constructed by inserting the whole coding region of the cDNA encoding pre-β-LG between the promoter and terminator of the yeast glyceraldehyde 3-phosphate dehydrogenase gene of pYG100, a yeast expression vector. In the supernatant of the yeast growth medium, β-LG with a native conformation was detected by sandwich ELISA, and its amount was estimated to be 1.1 mg/l. A Western-immunoblotting analysis revealed that β-LG secrected in the growth medium had the same mobility as that of authentic bovine β-LG. The N-terminal sequence was also identical with that of authentic mature bovine β-LG.     

Mechanochemical Phosphorylation and Solubilisation of β-D-Glucan from Yeast Saccharomyces cerevisiae and Its Biological Activities

To obtain a water-soluble β-D-glucan derivative cleanly and conveniently, a highly efficient mechanochemical method, planetary ball milling, was used to phosphorylate β-D-glucan isolated from yeast Saccharomyces cerevisiae in solid state. Soluble β-D-glucan phosphate (GP) with a high degree of substitution (0.77–2.09) and an apparent PEAK molecular weight of 6.6–10.0 kDa was produced when β-D-glucan was co-milled with sodium hexametaphosphate at 139.5–186.0 rad/s for 12–20 min. The energy transferred was 3.03–11.98 KJ/g. The phosphorylation of GPs was demonstrated by Fourier transform infrared spectroscopy and ¹³C and ³¹P Nuclear magnetic resonance spectroscopy. Three GP products with different degree of substitution (DS) and degree of polymerisation (DP) were able to upregulate the functional events mediated by activated murine macrophage RAW264.7 cells, among which GP-2 with a DS of 1.24 and DP of 30.5 exerted the highest immunostimulating activity. Our results indicate that mechanochemical processing is an efficient method for preparing water-soluble and biologically active GP with high DS.     

Application of comparative proteome analysis to reveal influence of cultivation conditions on asymmetric bioreduction of β-keto ester by Saccharomyces cerevisiae

Industrial bakers' yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and D: -arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.     

Immunomodulatory effect of dietary Saccharomyces cerevisiae, β-glucan and laminaran in mercuric chloride treated Nile tilapia (Oreochromis niloticus) and experimentally infected with Aeromonas hydrophila

Six hundred and forty Nile tilapia (Oreochromis niloticus) weighing 80–100 g were randomly allocated into eight equal groups (80 each). The first group acts as control. Groups S, B and L were fed on a ration supplemented with Saccharomyces cerevisiae, β-glucans and laminaran, respectively for 21 days. Groups M, MS, MB and ML were subjected throughout the experiment to sublethal concentration of mercuric chloride (0.05 ppm). Gps. MS, MB and ML were fed on a ration containing S. cerevisiae, β-glucan and laminaran respectively for 21 days. Fish were challenged with Aeromonas hydrophila (0.4 × 10⁷ cells mL^?1) via intra-peritoneal injection and the mortality rate was recorded up to 10 day post-challenge. The non-specific defense mechanisms, cellular and humoral immunity, beside the total and differential leukocytic count were determined.Lymphocyte transformation index, phagocytic activity percent, phagocytic index, total lymphocyte count, serum bactericidal activity and nitric oxide as well as the survival rate were insignificantly changed after 21 day in gps. MS&ML, when compared with mercuric chloride immune depressed group M. These parameters as well as the neutrophil adhesion, serum nitric oxide and survival rate were significantly increased in gp. MB when compared with gp. M. Meanwhile the cellular and humoral immunity beside the survival rate were significantly increased in groups S, B, L when compared with control group.It could be concluded that the whole yeast S. cerevisiae, β-glucan and laminaran can be used as immunostimulants for the farmed Nile tilapia. The β-glucans could be used in farmed Nile tilapia, under immune depressive stressful condition to increase their resistance to diseases.     

Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.     

Aided-efflux and high production of β-amyrin realized by β-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae

As a plant-derived pentacyclic triterpenoid, β-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β-cyclodextrin (β-CD) has been previously employed to aid the efflux of β-amyrin out of the cells. Nevertheless, the supplemented β-CD in the medium is not consistent with β-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of β-CD was developed in this study to enhance the biosynthesis of β-amyrin and its efflux. The in situ synthesis of β-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β-CD could capture 16% of the intracellular β-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of β-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.     

The Saccharomyces cerevisiae Ncw2 protein works on the chitin/β-glucan organisation of the cell wall

Antonie van Leeuwenhoek - The NCW2 gene was recently described as encoding a GPI-bounded protein that assists in the re-modelling of the Saccharomyces cerevisiae cell wall (CW) and in the repair of...     

The effect of ligand affinity on integrins’ lateral diffusion in cultured cells

The role of ligand affinity in altering αPS2CβPS integrins’ lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22 % and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins’ increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75 % slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99 % for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.     

Protective effects of β-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes

β-Glucans (BGs) are polysaccharides that are found in the cell walls of organisms such as bacteria, fungi, and some cereals. The objective of the present study was to investigate the genotoxic and antigenotoxic effects of BG extracted from the mushroom Agaricus brasiliensis (= Agaricus blazei Murrill ss. Heinemann). The mutagenic activity of BG was tested in single-cell gel electrophoresis assays with human peripheral lymphocytes. In addition, the protective effects against the cooked food mutagen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and (+/−)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), which is the main metabolite of B[a]P, and against ROS (H₂O₂)-induced DNA damage, were studied. The results showed that the compound itself was devoid of mutagenic activity, and that a significant dose-dependent protective effect against damage induced by hydrogen peroxide and Trp-P-2 occurred in the dose range 20–80 μg/ml. To investigate the prevention of Trp-P-2-induced DNA damage, a binding assay was carried out to determine whether BG inactivates the amine via direct binding. Since no such interactions were observed, it is likely that BG interacts with enzymes involved in the metabolism of the amine.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.