Showing papers on "Melibiose published in 2016"

Crystal structure of MytiLec, a galactose-binding lectin from the mussel Mytilus galloprovincialis with cytotoxicity against certain cancer cell types

Abstract: MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Galα(1,4)Galβ(1,4)Glcα1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Galα(1,6)Glc and the trisaccharide globotriose, Galα(1,4)Galβ(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt’s lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the β-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects.

ISOLATION AND CHARACTERIZATION OF A MOLYBDENUM-REDUCING AND GLYPHOSATE-DEGRADING Klebsiella oxytoca STRAIN SAW-5 IN SOILS FROM SARAWAK

Abstract: Bioremediation of pollutants including heavy metals and xenobiotics is an economic and environmentally friendly process. A novel molyb-denum-reducing bacterium with the ability to utilize the pesticide glyphosate as a carbon source is reported. The characterization works were carried out utilizing bacterial resting cells in a microplate format. The bacterium reduces molybdate to Mo-blue optimally between pH 6.3 and 6.8 and at 34 o C. Glucose was the best elec-tron donor for supporting molybdate reduction followed by lactose, maltose, melibiose, raffinose, d-mannitol, d-xylose, l-rhamnose, l-arabinose, dulcitol, myo-inositol and glycerol in descending order. Other requirements include a phosphate concentration at 5.0 mM and a molybdate concentration between 20 and 30 mM. The molybdenum blue exhibited an absorption spec-trum resembling a reduced phospho-molybdate. Molybdenum reduction was inhibited by mercury, silver, cadmium and copper at 2 ppm by 45.5, 26.0, 18.5 and 16.3%, respectively. Biochemical analysis identified the bacterium as Klebsiella oxytoca strain Saw-5. To conclude, the capacity of this bacterium to reduce molybdenum into a less toxic form and to grow on glyphosate is novel and makes the bacterium an important instrument for bioremediation of these pollutants.

Novel Lactulose and Melibiose Targeting Autophagy to Reduce PolyQ Aggregation in Cell Models of Spinocerebellar Ataxia 3

Abstract: Trehalose, a chemical chaperone and mTOR-independent autophagy enhancer, has shown promise in models of Huntington's disease, Parkinson's disease and tauopathies. In this study, two trehalase analogs, lactulose and melibiose, were examined for their potentials in spinocerebellar ataxia treatment. Using a SCA3 ATXN3/Q75-GFP cell model, we found that the ATXN3/Q75 aggregation was significantly prohibited by lactulose and melibiose because of their abilities to up-regulate autophagy. Meanwhile, lactulose and melibiose reduced reactive oxygen species production in ATXN3/Q75 cells. Both of them further inhibited the ATXN3/Q75 aggregation in neuronally differentiated SH-SY5Y cells. These findings suggest the therapeutic applications of novel trehalose analogs in polyglutamine aggregation-associated neurodegenerative diseases.

Response surface methodology-based optimization of production media and purification of α-galactosidase in solid-state fermentation by Fusarium moniliforme NCIM 1099

Abstract: Response surface methodology was used to enhance the production of α-galactosidase from Fusarium moniliforme NCIM 1099 in solid-state fermentation. Plackett–Burman design was employed for selection of critical media constituents which were optimized by central composite rotatable design. Wheat bran, peptone and FeSO4·7H2O were identified as significant medium components using PB design. Further CCRD optimized medium components as wheat bran; 4.62 μg, peptone; 315.42 μg, FeSO4·7H2O; 9.04 μg. RSM methodological optimization increased the enzyme production from 13.17 to 207.33 U/g showing 15.74-fold enhancement. The α-galactosidase was purified by 70% fractionation followed by DEAE anion exchange column chromatography which yields 23.33% with 28.68-fold purification. The molecular weight of α-galactosidase was 57 kDa which was determined by SDS-PAGE analysis. Purified enzyme has optimum pH of 4.0 and was found to be stable in wide pH range of 3.0–9.0. Its optimum temperature was 50 °C, whereas its activity remains above 50% up to 2 h at 75 °C. Hg2+ was found to be a potent inhibitor and Mg2+ acted as an activator of enzyme. No significant change was observed in enzyme activity for galactose concentration, ranging from 1 to 100 mM. The Km values of enzyme for substrates p-nitrophenyl-α-d-galactopyranoside, melibiose and raffinose were 0.20, 1.36, and 3.66 mM, respectively. Low Km and stability to various physiological conditions of enzyme represents its potential which can be exploited in various industrial applications.

Carbohydrate metabolism in Oenococcus oeni : a genomic insight

Abstract: Oenococcus oeni is the bacterial species that drives malolactic fermentation in most wines. Several studies have described a high intraspecific diversity regarding carbohydrate degradation abilities but the link between the phenotypes and the genes and metabolic pathways has been poorly described. A collection of 41 strains whose genomic sequences were available and representative of the species genomic diversity was analyzed for growth on 18 carbohydrates relevant in wine. The most frequently used substrates (more than 75% of the strains) were glucose, trehalose, ribose, cellobiose, mannose and melibiose. Fructose and L-arabinose were used by about half the strains studied, sucrose, maltose, xylose, galactose and raffinose were used by less than 25% of the strains and lactose, L-sorbose, L-rhamnose, sorbitol and mannitol were not used by any of the studied strains. To identify genes and pathways associated with carbohydrate catabolic abilities, gene-trait matching and a careful analysis of gene mutations and putative complementation phenomena were performed. For most consumed sugars, we were able to propose putatively associated metabolic pathways. Most associated genes belong to the core genome. O. oeni appears as a highly specialized species, ideally suited to fermented fruit juice and more specifically to wine for a subgroup of strains.

Synthesis and structural characterization of raffinosyl-oligofructosides upon transfructosylation by Lactobacillus gasseri DSM 20604 inulosucrase

Abstract: A new process based on enzymatic synthesis of a series of raffinose-derived oligosaccharides or raffinosyl-oligofructosides (RFOS) with degree of polymerization (DP) from 4 to 8 was developed in the presence of raffinose. This process involves a transfructosylation reaction catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The main synthesized RFOS were structurally characterized by nuclear magnetic resonance (NMR). According to the elucidated structures, RFOS consist of β-2,1-linked fructose unit(s) to raffinose: α-d-galactopyranosyl-(1 → 6)-α-d-glucopyranosyl-(1↔2)-β-d-fructofuranosyl-((1 ← 2)-β-d-fructofuranoside)n (where n refers to the number of transferred fructose moieties). The maximum yield of RFOS was 33.4 % (in weight respect to the initial amount of raffinose) and was obtained at the time interval of 8–24 h of transfructosylation reaction initiated with 50 % (w/v) of raffinose. Results revealed the high acceptor and donor affinity of IS towards raffinose, being fairly comparable with that of sucrose for the production of fructooligosaccharides (FOS), including when both carbohydrates coexisted (sucrose/raffinose mixture, 250 g L−1 each). The production of RFOS was also attempted in the presence of sucrose/melibiose mixtures; in this case, the predominant acceptor-product formed was raffinose followed by a minor production of a series of oligosaccharides with varying DP. The easiness of RFOS synthesis and the structural similarities with both raffinose and fructan series of oligosaccharides warrant the further study of the potential bioactive properties of these unexplored oligosaccharides.

Efficient and regioselective synthesis of globotriose by a novel α-galactosidase from Bacteroides fragilis.

Abstract: Globotriose (Galα1–4Galβ1–4Glc) is an important cell surface epitope that acts as the receptor for Shiga-like toxins, and it is also the core structure of Globo H and SSEA4 that are tumor-associated glycans. Hence, the enzymatic synthesis of globotriose would be necessary for the development of carbohydrate-based therapeutics for bacterial infections and cancers. Here, a novel GH27 α-galactosidase gene (agaBf3S), a 1521-bp DNA encoding 506 amino acids with a calculated molecular mass of 57.7 kDa, from Bacteroides fragilis NCTC9343 was cloned and heterogeneously expressed in Escherichia coli. The recombinant enzyme AgaBf3S preferentially hydrolyzed p-nitrophenyl-α-D-galactopyranoside (pNPαGal) in all tested nitrophenyl glycosides. It showed maximum activity at pH 4.5 and 40 °C, and it was stable at pH 4.0–11.0 below 40 °C and metal-independent. The K m and k cat values for pNPαGal, melibiose, and globotriose were 1.27 mM and 172.97 S−1, 62.76 mM and 17.74 S−1, and 4.62 mM and 388.45 S−1, respectively. AgaBf3S could transfer galactosyl residue from pNPαGal to lactose (Galβ1–4Glc) with high efficiency and strict α1–4 regioselectivity. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation reaction catalyzed by AgaBf3S were studied in detail. AgaBf3S could synthesize globotriose as a single transglycosylation product with a maximum yield of 32.4 % from 20 mM pNPαGal and 500 mM lactose (pH 4.5) at 40 °C for 30 min. This new one-enzyme one-step synthetic reaction is simple, fast, and low cost, which provides a promising alternative to the current synthetic methods for access to pharmaceutically important Galα1–4-linked oligosaccharides.

Purification of thermostable α-galactosidase from Irpex lacteus and its use for hydrolysis of oligosaccharides

Abstract: A monomeric α-galactosidase (ILGI) from the mushroom Irpex lacteus was purified 94.19-fold to electrophoretic homogeneity. ILGI exhibited a specific activity of 18.36 U mg(-1) and demonstrated a molecular mass of 60 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ILGI was optimally active at 80 °C and pH 5.0, and it was stable over a temperature range of 4-70 °C and a wide pH range of 2.0-12.0. ILGI was completely inactivated by Ag(+) and Hg(2+) ions and N-bromosuccinimide (NBS). Moreover, ILGI exhibited good resistance to proteases. Galactose acted as a noncompetitive inhibitor with Ki and Kis of 3.34 and 0.29 mM, respectively. The α-galactosidase presented a broad substrate specificity, which included p-nitrophenyl α-D-galactopyranoside (pNPGal), melibiose, stachyose, and raffinose with Km values of 1.27, 3.24, 7.1, and 22.12 mM, correspondingly. ILGI exhibited efficient and complete hydrolysis to raffinose and stachyose. The aforementioned features of this enzyme suggest its potential value in food and feed industries.

Melibiose, a Nondigestible Disaccharide, Promotes Absorption of Quercetin Glycosides in Rat Small Intestine

Abstract: We demonstrated that melibiose, a nondigestible disaccharide composed of galactose and glucose with α-1,6 glycoside linkage, promotes the absorption of water-soluble quercetin glycosides in ligated small intestinal loop of anesthetized rats Water-soluble quercetin glycoside, a quercetin-3-O-glucoside mixture (Q3GM), includes quercetin-3-O-glucoside (Q3G, 319%), mono (212%) and di (171%), glucose adducts with α-1,4 linkages After instillation of Q3GM into the intestinal loop with or without melibiose, the plasma concentration of quercetin derivatives in the portal blood was considerably higher in the melibiose group at 60 min Furthermore, we evaluated the hydrolytic rate of Q3G by the mucosal homogenate of the small intestine with six different disaccharides Melibiose and isomaltose, which have α-1,6 glycoside linkage, were found to promote Q3G hydrolysis to aglycone These results suggest that melibiose promotes quercetin glycoside absorption in rats by increasing glycoside hydrolysis in the intest

Effects of ten naturally occurring sugars on the reproductive success of the green lacewing, Chrysoperla carnea

Abstract: The longevity and reproduction of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) when fed on ten naturally occurring sugars and on an artificial diet composed by honey and pollen (1:1) were evaluated. The effect of these diets differed between sexes. However insects of both sexes lived longer when fed on fructose than on sucrose, melezitose or raffinose. None of the females oviposited when offered raffinose, galactose or mannose. Moreover, lifetime fecundity of females fed on trehalose was not different from the artificial diet. The intrinsic rate of natural increase did not differ between trehalose, maltose, melibiose, fructose, glucose and artificial diet, being significantly lower for individuals fed on melezitose and sucrose. The importance of these results for the selection of natural sugar sources that enhance C. carnea effectiveness as a biological control agent against crop pests is discussed.

Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase, and properties of the products.

Abstract: Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.

Truncation of Gal4p explains the inactivation of the GAL/MEL regulon in both Saccharomyces bayanus and some Saccharomyces cerevisiae wine strains.

Abstract: In the past, the galactose-negative (Gal(-)) phenotype was a key physiological character used to distinguish Saccharomyces bayanus from S. cerevisiae In this work, we investigated the inactivation of GAL gene networks in S. bayanus, which is an S. uvarum/S. eubayanus hybrid, and in S. cerevisiae wine strains erroneously labelled 'S. bayanus'. We made an inventory of their GAL genes using genomes that were either available publicly, re-sequenced by us, or assembled from public data and completed with targeted sequencing. In the S. eubayanus/S. uvarum CBS 380(T) hybrid, the GAL/MEL network is composed of genes from both parents: from S. uvarum, an otherwise complete set that lacks GAL4, and from S. eubayanus, a truncated version of GAL4 and an additional copy of GAL3 and GAL80 Similarly, two different truncated GAL4 alleles were found in S. cerevisiae wine strains EC1118 and LalvinQA23. The lack of GAL4 activity in these strains was corrected by introducing a full-length copy of S. cerevisiae GAL4 on a CEN4/ARS plasmid. Transformation with this plasmid restored galactose utilisation in Gal(-) strains, and melibiose fermentation in strain CBS 380(T) The melibiose fermentation phenotype, formerly regarded as characteristic of S. uvarum, turned out to be widespread among Saccharomyces species.

Molecular Characterization of the α-Galactosidase SCO0284 from Streptomyces coelicolor A3(2), a Family 27 Glycosyl Hydrolase.

Abstract: The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40°C and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters Vmax and Km for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.

Production of α-galactosidase from Aspergillus foetidus MTCC 6322 by solid state fermentation and its application in soymilk hydrolysis

Abstract: The production of α-galactosidase from the wild fungal strain Aspergillus foetidus MTCC 6322 using solid state fermentation (SSF), its characterization, and its efficacy in the hydrolysis of soymilk using response surface methodology were studied. The optimum conditions for production of α-galactosidase by SSF were: wheat bran (10 g), moisture content (64%), inoculum volume (1.0 mL; 6 x 10(7) spores/mL) with a yield of 4.1 x 10(3) units per gram dry substrate (U/gds) at 96 h. The enzyme showed optimum activity at 6.0, temperature 40 degrees C, pH stability between 5.0-8.0, and temperature stability between 30-40 degrees C. The enzyme was stable in the presence of trypsin, lipase, and collagenase and it showed susceptibility of the substrates such as raffinose, melibiose, guar gum and soymilk to hydrolysis in varying degrees. The optimized conditions for soymilk hydrolysis were: soymilk (10 mL) from defatted soybean meal (1.5%), α-galactosidase (0.15 UmL(-1) at 30 degrees C, pH 6.0 and duration of 1 h.

Saccharomyces cerevisiae capable of being co-fermented by multiple carbon sources and application thereof

Abstract: The invention discloses saccharomyces cerevisiae capable of being co-fermented by a plurality of carbon sources and an application thereof, belonging to the technical fields of microbes and fermentation engineering. The saccharomyces cerevisiae namely a CCTCC M 2014463 strain is separated from Chinese Maotai-flavor liquor wine brewing fermented grains. The strain can adopt glucose, galactose, xylose, allulose, cane sugar, maltose, melibiose, turanose, mycose and melitose, and can adopt glucose and other sugars synchronously. Meanwhile, the saccharomyces cerevisiae can be used for resisting high temperature, high ethanol and high acid, and also can be used for producing acids, alcohols, esters and various flavor substances such as benzeneacetaldehyde, acetophenone 4-vinyl guaiacol and the like. The saccharomyces cerevisiae disclosed by the invention can be directly applied to the production of beverage wines, the reinforcement of wine brewing fermented grains in beverage wine production, and the production of fuel ethanol which is used as a carbon source substrate after starch and cellulose saccharification, thereby significantly improving the carbon source utilization rate and the ethanol yield.