Showing papers on "Melibiose published in 2009"

A novel α-galactosidase from Βifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression

Abstract: A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of approximately 243 kDa and a subunit size of approximately 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (approximately 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) > or = 3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization

Abstract: A novel α-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an α-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60°C and pH 4.0 and was stable at pH 3.0–12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K m values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial α-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant α-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.

Alkaline α-galactosidase degrades thylakoid membranes in the chloroplast during leaf senescence in rice

Abstract: Here, we studied the functional role of a chloroplast alkaline alpha-galactosidase (OsAkalphaGal) in the breakdown of thylakoid membranes during rice (Oryza sativa) leaf senescence. We assayed the enzyme activity of recombinant OsAkalphaGal with different natural substrates and examined the effect of ectopic OsAkalphaGal expression in rice plants. Recombinant OsAkalphaGal showed at least a two-fold greater substrate-binding affinity and a 10-fold greater turnover rate to galactolipid digalactosyl diacylglycerol than the raffinose family of oligosaccharides (verbascose, stachyose, raffinose) and melibiose. The OsAkalphaGal null mutant (osakalphagal) displayed a delayed leaf senescence phenotype. OsAkalphaGal complementation in osakalphagal recovered OsAkalphaGal expression and showed a senescence phenotype similar to that of wild-type plants. Transgenic plants overexpressing OsAkalphaGal (UbiP-OsAkalphaGal) exhibited retarded plant growth and development, and showed a pale-green phenotype coupled with a reduced chlorophyll content to 42% in newly unfolded leaves. UbiP-OsAkalphaGal leaves also showed a 29-fold increase in alkaline alpha-galactosidase activity compared with wild-type leaves. An ultrastructural study of Ubi-OsAkalphaGal chloroplasts in newly unfolded leaves revealed abnormal grana organization. Our findings strongly suggest that OsAkalphaGal is a thylakoid membrane-degrading enzyme involved in the degradation of digalactosyl diacylglycerol during rice leaf senescence.

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

Abstract: A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl-agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galbeta1-3GlcNAc) and type 2 (Galbeta1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k(ass) and k(diss) values are 5.14x10(4) M(-1) s(-1) and 2.9x10(-3) s(-1), respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.

Catabolism of Raffinose, Sucrose, and Melibiose in Erwinia chrysanthemi 3937

Abstract: Erwinia chrysanthemi (Dickeya dadantii) is a plant pathogenic bacterium that has a large capacity to degrade the plant cell wall polysaccharides. The present study reports the metabolic pathways used by E. chrysanthemi to assimilate the oligosaccharides sucrose and raffinose, which are particularly abundant plant sugars. E. chrysanthemi is able to use sucrose, raffinose, or melibiose as a sole carbon source for growth. The two gene clusters scrKYABR and rafRBA are necessary for their catabolism. The phenotypic analysis of scr and raf mutants revealed cross-links between the assimilation pathways of these oligosaccharides. Sucrose catabolism is mediated by the genes scrKYAB. While the raf cluster is sufficient to catabolize melibiose, it is incomplete for raffinose catabolism, which needs two additional steps that are provided by scrY and scrB. The scr and raf clusters are controlled by specific repressors, ScrR and RafR, respectively. Both clusters are controlled by the global activator of carbohydrate catabolism, the cyclic AMP receptor protein (CRP). E. chrysanthemi growth with lactose is possible only for mutants with a derepressed nonspecific lactose transport system, which was identified as RafB. RafR inactivation allows the bacteria to the assimilate the novel substrates lactose, lactulose, stachyose, and melibionic acid. The raf genes also are involved in the assimilation of α- and β-methyl-d-galactosides. Mutations in the raf or scr genes did not significantly affect E. chrysanthemi virulence. This could be explained by the large variety of carbon sources available in the plant tissue macerated by E. chrysanthemi.

Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs.

Abstract: A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80°C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, β-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-α- and methyl-β-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k ass) and dissociation rate constant (k diss) were determined for the lectin to be 4.3·105 M−1·sec−1 and 2.2·10−3 sec−1, respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

Debaryomyces hansenii UFV-1 intracellular α-galactosidase characterization and comparative studies with the extracellular enzyme.

Abstract: Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Selenomonas bovis sp. nov., isolated from yak rumen contents.

Abstract: Two strictly anaerobic, crescent-shaped bacterial strains, designated WG T and Ycb08, were isolated from a cellulose-degrading mixed culture enriched from yak rumen contents. The strains were Gram-negative, non-spore-forming and motile, with four to six flagella situated at the centre of the concave side of the cell. The cells were 0.9–1.14–6 mm. Growth was observed at 27– 46 6C (optimum 39 6C) and pH 4.2–8.3 (optimum pH 7.0–7.2). Arabinose, glucose, mannose, cellobiose, lactose, sucrose, trehalose, melibiose, raffinose, salicin and aesculin were fermented. The end products of glucose fermentation were acetate, propionate and CO2. The G+C contents of strains WG T and Ycb08 were respectively 63.9±0.2 and 62.5±0.2 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains were related to the genera Mitsuokella and Selenomonas at similarity levels below 97%; however, they differed from members of the genus Mitsuokella in their flagellar arrangement. On the basis of phenotypic, genotypic and physiological evidence, strains WG T and Ycb08 are identified as members of a novel species of the genus Selenomonas, for which the name Selenomonas bovis sp. nov. is proposed. The type strain is WG T (5CGMCC 1.5073 T 5JCM 15470 T ).

NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris.

Abstract: The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14 500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains α and γ, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using 1H–15N HSQC spectra as a function of increasing concentrations of lactose, melibiose, d-galactose, methyl α-d-galactopyranoside and methyl β-d-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain α had a slow or intermediate exchange regime on the chemical-shift timescale (Kd = 10−2 to 10−1 mm), whereas that in subdomain γ had a fast exchange regime for these sugars (Kd = 2–6 mm). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.

Specificity of cellular interaction in Streptococcus mitis ATCC 903. Inhibition of aggregation by carbohydrates.

Abstract: To demonstrate the specificity of cellular interaction in the spontaneous aggregation of Streptococcus mitis ATCC 903 the inhibitory effect of various sugars was studied. More than 90% inhibition was recorded in 0.1 M concentrations of D-glucosamine, D-galactosamine and D-mannosamine. A reduction of the inhibition by more than 50% was observed when the N-acetylated derivatives of the hexosamines were tested. Polymers containing hexosamines such as hyaluronic acid, heparin and fetuin were potent inhibitors, in contrast to dextrans of different molecular weights. Inhibition was less than 20% in lactose, melibiose, cellobiose, methyl beta-D-galactopyranoside and a number of other sugars tested in 0.1 M concentration. The bacteria retained their ability to aggregate after removal of the inhibitory sugars by washing in phosphate buffer. These findings support the hypothesis that the specific nature of the spontaneous aggregation of S.mitis ATCC 903 depends on a lectin-ligand type interaction.

Microbial Biosensors for Selective Detection of Disaccharides

Abstract: Seven microbial strains were screened for their ability to detect disaccharides as components of Clark-type oxygen biosensors. Sensors responded to varying degrees to maltose, cellobiose, sucrose, and melibiose, but none responded strongly to lactose. Although microbial sensors are relatively nonspecific, it is possible to obtain differential measurements of specific substrates using multiple sensors with different relative specificities. For example, Escherichia coli strain K-802 oxidized maltose but had low activity against sucrose, while Bacillus subtilis strain VKM B-434 responded more strongly to sucrose than maltose. Furthermore, signals from these two sensors were additive for selected samples. Results suggest that a two-component biosensor utilizing these strains could be used for differential detection of sucrose and maltose.

Determination of Monosaccharides and Soybean Oligosaccharides in White Vinegar and Tofu Water by Ion Chromatography and Pulsed Amperometric Detection

Abstract: A chromatographic method was developed for the determination of galactose, glucose, fructose, sucrose, melibiose, raffinose and stachyose in white vinegar and tofu water. These seven saccharides can be separated well in 30 min by CarboPac PA10 high performance anion exchange column(HPAEC) with sodium hydroxide gradient elution. The detection limits of galactose, glucose, fructose, sucrose, melibiose, raffinose and stachyose (25 mu L injection, S/N = 3) using Pulsed Amperometric Detection(PAD) are 3, 2, 3, 6, 4, 7 and 6 mu g/L, respectively. Moreover, all analytes have wide linearity range(0.02 - 20 mg/L), good relative standard deviations(0.2% - 3.2%) and satisfying recoveries(92% - 104%). This method is quite suitable for the determination of common saccharides in soybeans and soybean products for its simple and good separating performance as well as high sensitivity. No derivation is needed in the process of determination.

Method of producing melibiose

Abstract: PROBLEM TO BE SOLVED: To provide a method of producing melibiose capable of selectively producing melibiose from glucose and galactose as inexpensive raw materials. SOLUTION: The method of producing melibiose using glucose and galactose as raw materials, uses α-galactosidase derived from a stock belonging to Geobacillus thermocatenulatus which can selectively synthesize the melibiose. COPYRIGHT: (C)2009,JPO&INPIT