Showing papers on "Melibiose published in 2011"

Caldicoprobacter algeriensis sp. nov. a New Thermophilic Anaerobic, Xylanolytic Bacterium Isolated from an Algerian Hot Spring

Abstract: A thermophilic anaerobic bacterium (strain TH7C1T) was isolated from the hydrothermal hot spring of Guelma in the northeast of Algeria. Strain TH7C1T stained Gram-positive, was a non-motile rod appearing singly, in pairs, or as long chains (0.7–1 × 2–6 μm2). Spores were never observed. It grew at temperatures between 55 and 75°C (optimum 65°C) and at pH between 6.2 and 8.3 (optimum 6.9). It did not require NaCl for growth, but tolerated it up to 5 g l−1. Strain TH7C1T is an obligatory heterotroph fermenting sugars including glucose, galactose, lactose, raffinose, fructose, ribose, xylose, arabinose, maltose, mannitol, cellobiose, mannose, melibiose, saccharose, but also xylan, and pyruvate. Fermentation of sugars only occurred in the presence of yeast extract (0.1%). The end-products from glucose fermentation were acetate, lactate, ethanol, CO2, and H2. Nitrate, nitrite, thiosulfate, elemental sulfur, sulfate, and sulfite were not used as electron acceptors. The G+C content of the genomic DNA was 44.7 mol% (HPLC techniques). Phylogenetic analysis of the small-subunit ribosomal RNA (rRNA) gene sequence indicated that strain TH7C1T was affiliated to Firmicutes, order Clostridiales, family Caldicoprobacteraceae, with Caldicoprobacteroshimai (98.5%) being its closest relative. Based on phenotypic, phylogenetic, and genetic characteristics, strain TH7C1T is proposed as a novel species of genus Caldicoprobacter, Caldicoprobacter algeriensis, sp. nov. (strain TH7C1T = DSM 22661T = JCM 16184T).

Structural aspects of binding of α-linked digalactosides to human galectin-1

Abstract: By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-π stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH-π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH-π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.

Purification and Characterization of Aspergillus terreus α-Galactosidases and Their Use for Hydrolysis of Soymilk Oligosaccharides

Abstract: α-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4-7. Maximal activities were determined at 60, 55, and 50 °C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50 °C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg²⁺, Ag⁺, and Cu²⁺. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes.

The Use of Disaccharides in Inhibiting Enzymatic Activity Loss and Secondary Structure Changes in Freeze-Dried β-Galactosidase during Storage

Abstract: The purpose of this study is to show how disaccharides differ in their ability to protect lyophilized β-galactosidase from enzymatic activity loss and secondary structure changes during storage. β-galactosidase was lyophilized with trehalose, sucrose, cellobiose or melibiose at 2:1, 20:1 and 40:1 excipient/protein weight ratios, and stored up to 90 days at 45°C. Protein enzymatic activity was studied using o-nitrophenyl-β-d-galactopyranoside cleavage test, and its secondary structure in lyophilizates analyzed using Fourier transform infrared spectroscopy. The crystallization tendencies, glass transition temperatures and water contents of lyophilizates were evaluated using x-ray powder diffractometry, differential scanning calorimetry and thermogravimetry, respectively. The enzymatic activity of β-galactosidase decreased more slowly in lyophilizates containing trehalose or melibiose at 2:1 excipient/protein weight ratio when compared to those containing sucrose or cellobiose. Similar behavior was observed when analyzing the protein’s secondary structure in lyophilizates. In 20:1 and 40:1 excipient/protein weight ratio lyophilizates the decrease of enzymatic activity was less dependent on the excipient, but activity was always amongst the highest in melibiose lyophilizates. Melibiose was shown to be effective in protecting lyophilized β-galactosidase during storage. The protein secondary structure was shown to change at comparable rate in lyophilizates as its enzymatic activity after rehydration.

Cytotoxicity and Glycan-Binding Profile of a d -Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare ( Aplysia kurodai )

Abstract: A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the kass and kdiss values are 2.4 × 103 M−1 s−1 and 3.8 × 10−3 s−1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

Physiological Properties of a Microbial Community in Spontaneous Fermentation of Maize (Zea mays ) for Ogi Production

Abstract: Based on the previous knowledge of spontaneously fermenting maize for the preparation of Ogi, four growth media were used for the isolation of the different common group of organisms implicated. Yeasts and Molds were identified as Saccharomyces cerevisiae, Candida sp., Rhodotorula sp., Aspergillus niger and Penicillium sp. The bacteria were Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum, Streptococcus lactis, Enterococcus feacalis, Escherichia coli, Staphylococcus aureus, Enterobacter sp., Citrobacter sp., Klebseilla sp., Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Aerobacter sp., and Corynebacteria sp. A sharp decrease in pH of fermenting gruel from 5.7 to 3.5 was observed within 24 hours while titratable acidity increases within the first 48 hours with a further increase and decrease in pH and titratable acidity at 72 hours.These isolates were subjected to pH ranges of 3.5 – 6 and all were able to grow at optimum pH 6 with a reduction in the number of isolates as pH reduces. Growth in temperature ranging from 25 0 C to 50 0 C, salt (NaCl, MgS0 4, K 2HP0 4, CuS0 4 and ZnS0 4) and sugar (glucose, sucrose, lactose, melibiose and raffinose) concentrations of 0.2% to 1% were carried out on all the isolates. With increase in temperature and salt concentration, a reduction in the number of isolates that grew was observed. From this study, the optimum conditions that favours the growth of all the group of organisms was found to be pH 6, 30 0 C, 0.2% of the salts used and a significant population of all the group of organisms utilized glucose as their best carbon source.

A D-galactose-binding lectin purified from coronate moon turban, Turbo (Lunella) coreensis, with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids.

Abstract: A divalent, cation-independent d-galactose-binding lectin was purified from coronate moon turban Turbo (Lunella) coreensis. This lectin recognizes d-galactose and is a 38-kDa dimeric protein consisting disulphide-bonded 22-kDa polypeptides under non-reducing and reducing conditions of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Haemagglutination activity was inhibited by D-galactose, N-acetyl D-galactosamine, melibiose, lactose, porcine stomach mucin, asialofetuin and bovine submaxillary mucin. The lectin has tolerance for pH 5-11 and temperature until 50°C for 1h. The lectin strongly aggregated Gram-negative bacteria, such as Vibrio parahaemolyticus and Salmonella O7, but weakly Gram-positive strain as Staphylococcus aureus and Bacillus subtilis. The glycan-binding profile of this lectin was evaluated using frontal affinity chromatography technology and the lectin appeared to recognize oligosaccharides such as lacto-series glycosphingolipids contained in blood type A and H substances in addition to complex-type N-linked glycoproteins. Partial primary structures of 139 amino acid residues of this lectin were determined from N-terminus polypeptides and 8 peptides derived by cleavage with lysyl-endopeptidase. The primary structure was slightly similar to other known sequences of lectin; however, a repeating motif has been included.

Functional Characterization of a Eukaryotic Melibiose Transporter

Abstract: Pathogenic fungi drastically affect plant health and cause significant losses in crop yield and quality. In spite of their impact, little is known about the carbon sources used by these fungi in planta and about the fungal transporters importing sugars from the plant-fungus interface. Here, we report on the identification and characterization of MELIBIOSE TRANSPORTER1 (MBT1) from the hemibiotrophic fungus Colletotrichum graminicola (teleomorph Glomerella graminicola), the causal agent of leaf anthracnose and stalk rot disease in maize (Zea mays). Functional characterization of the MBT1 protein in baker’s yeast (Saccharomyces cerevisiae) expressing the MBT1 cDNA revealed that α-d-galactopyranosyl compounds such as melibiose, galactinol, and raffinose are substrates of MBT1, with melibiose most likely being the preferred substrate. α-d-Glucopyranosyl disaccharides like trehalose, isomaltose, or maltose are also accepted by MBT1, although with lower affinities. The MBT1 gene shows low and comparable expression levels in axenically grown C. graminicola and upon infection of maize leaves both during the initial biotrophic development of the fungus and during the subsequent necrotrophic phase. Despite these low levels of MBT1 expression, the MBT1 protein allows efficient growth of C. graminicola on melibiose as sole carbon source in axenic cultures. Although Δmbt1 mutants are unable to grow on melibiose, they do not show virulence defects on maize.

Enzymatic preparation of melibiose and alkyl β-d-fructofuranosides by commercial lactase

Abstract: Lactozyme 3000L was found to possess β-fructofuranosidase activity and catalyzed hydrolysis of raffinose to melibiose and formation of alkyl β-d-fructofuranosides from sucrose. The total chemical yield of melibiose was 95.5%, yields of alkyl β-d-fructofuranosides ranged from 5.2 to 20.2% depending on the structure of the acceptor. Alcohols larger than butanol were not glycosylated.

Cloning and functional expression of α -galactosidase cDNA from Penicillium janczewskii zaleski

Abstract: In recent years, α-galactosidase has been attracting more and more attention because of its potential applications in many aspects. Using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, a full-length cDNA sequence composed of 2,439 bp was cloned from Penicillium janczewskii zaleski and was subcloned into pPICZαA and transformed into Pichia pastoris strain X-33. In a 10-L fermentor, the recombinant yeast expressed α-galactosidase with a yield of 254 U/mL by methanol induction for 120 h. The recombinant enzyme showed the optimal activity at 40°C and pH 5.2. The K m values of the recombinant enzyme using p-nitrophenyl-α-D-galactopyranoside (pNPG), melibiose, raffinose and stachyose as substrates were 1, 16, 17.8 and 5.3 mM, respectively. V max values were 227.3, 116.7, 104.8, and 80.6 μM/min using pNPG, melibiose, raffinose and stachyose as substrates, respectively. The α-galactosidase exhibited no sensitivity to various metal ions and ethylenediaminetetraacetic acid, and hydrolyzed melibiose, raffinose and stachyose with different levels of galactose release. The biochemical characteristics of the α-galactosidase suggest that the enzyme may have a prospective application in feed industry as an additive.

Purification and Identification of a Natural Lectin from the Seed of Peanut Arachis hypogaea

Abstract: A natural lectin from the seed of peanut Arachis hypogaea was purified by singlestep affinity chromatography using galactoside-coupled agarose. The native molecular mass of purified A. hypogaea lectin (PN-L) was 29 kDa. The lectin PN-L was detected for agglutinating activity, glycoinhibiting action and thermostability. The influence of pH on those activities was also tested. The results showed that PN-L could not agglutinate three kinds of human erythrocytes. But it showed a strong affinity to human A, B and O erythrocytes (RBC) treated by neuraminidase. Agglutinating activity of PN-L to neuraminidase treated human O erythrocytes was inhibited by lactose, raffinose, melibiose and D-galactose. The agglutinating activity of peanut seed lectin was stable up to 55°C and at pH 5.0-11.0. The results of MALDI-TOF- TOF analysis indicated that the protein PN-L showed highly homology with the Peanut Lectin Chain A protein (gi|1942899).

Enzymatic Synthesis and Identification of Galactosyl-β-cyclodextrin

Abstract: Galactosyl-β-cyclodextrin(Gal-β-CD) was synthesized under the catalysis of α-D-galactosidase derived from germinated coffee beans using melibiose as a donor and β-cyclodextrin as an acceptorThe optimum synthesis parameters were enzyme dose 20 U/g melibiose,β-CD-to-melibiose molar ratio 1:2,pH 65(50 mmol/L acetate buffer),reaction time 24 h and reaction temperature 40 ℃,respectivelyThe product obtained under the optimized conditions was purified by preparative HPLC,and the purified product was 6-O-α-D-galactosyl-β-cyclodextrin according to ESI-MS,IR,and NMR analyses

Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in Yersinia pestis strains of the main and nonmain subspecies

Abstract: Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and nonmain subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of nonmain subspecies (caucasica, altaica, hissarica and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence in strains of the main species of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of nonmain subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.

Method for producing manninotriose and melibiose by fermenting soy molasses

Abstract: The invention relates to a method for producing manninotriose and melibiose by fermenting soy molasses, which comprises the following steps: (1) preparing a spore fungus suspension; (2) inoculating the spore fungus suspension into a sterilized liquid seed culture medium; (3) inoculating the primary liquid seed into a sterilized 5L seeding tank, and fermenting and culturing to obtain secondary liquid seed; (4) inoculating the secondary liquid seed into a sterilized fermentation tank, and fermenting and culturing by using soy molasses dilution as the culture medium; and (5) centrifugating the fermentation liquid to remove thalli, and carrying out chromatographic separation by using active carbon columns, thereby further obtaining manninotriose and melibiose crystals. The method is used for producing the manninotriose and the melibiose by fermenting the cheap material soy molasses, thereby effectively solving the problem of utilization of waste molasses; and the invention has the advantages of simple technique, high yield, good quality and low cost, does not need complicated equipment, and is beneficial to development and application of the manninotriose and the melibiose.

Enzymatic Synthesis of α-Galactosyl-β-cyclodextrin by α-galactosidase

Abstract: Parameters affecting the synthesis of α-galactosyl-β-cyclodextrin by α-galactosidase of germinating coffee beans were systematically studied by Box-Behnken design.The optimized conditions were as follows,enzyme concentration,73 nkat;Reaction time,28 h;pH,6.3;Reaction temperature,40 ℃;Oscillatory speed,75 r/min;β-cyclodextrin,0.4 mol/L;melibiose,0.8mol/L;and acetate buffer(50 mmol/L),2 mL,The yield of α-galactosyl-β-cyclodextrin was 28.4% under these conditions,which was 41.3% higher than conditions before optimization.

Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells

Abstract: Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50 kDa possessed the responsive features of RCG1 binding and lactose competition.

Role of s. grantii lectin in detection of the surface carbohydra tes of rbc of norm al and cancer patients

Abstract: Several studies have shown that the antigenic determinants on the surface of RBC are carried by glycoproteins and glycolipids.Since interaction of lectins with the carbohydrate moiety of glycoproteins on cell membranes is highly specific, lectins have been frequently used to study membrane changes in malignant transformation, as compared to normal. In the present study qualitative changes in sugar specificit y of the normal, and various types of cancer were investigated. The haemagglutination inhibition reaction of S. Grantii lectin showed that galactose, lactose, melibiose & raffinose were present in 82% controls but were absent in breast, stomach, tongue & chick cancer. In breast cancer 65.5% cases showed presence of glucose and mannose but these sugars were absent in controls. In 90% cervix cancer, maximum number of sugars were present on cell surface viz. glucose, fructose, fucose, arabinose, cellobiose, galactose, lactose, melibiose, mannose, ribose and xylose. In 50% chick cancer cases, fucose and xylose were present but these were absent in controls. Qualitative analysis re veals that with help of lectin, one can pin point the sugar appearance or due to . It is relevant to record that one speculates the susceptibilit y to cancer by testing it for glycoproteins. This is convenient method than taking the biopsy sample in certain cases.

Substrate Specificities of α-Galactosidase from Rice

Abstract: The α-galactosidase from rice cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose and stachyose, but also liberated the terminal galactosyl residues attached O-6 position of the reducing-end mannosyl residue in mannobiose and mannotriose. In addition, the enzyme tore off the stubbed galactosyl residues attached inner-mannosyl residues in mannopentaose. It also could catalyze efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.