Showing papers on "Melibiose published in 2018"

A novel α-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable protease-resistance and high hydrolytic activity.

Abstract: A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-d- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0-10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing.

Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium melibiose transporter MelB.

Abstract: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein. Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2–3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.

In vitro fermentation of raffinose by the human gut bacteria

Abstract: Raffinose has become a major focus of research interest and recent studies have shown that besides beneficial bifidobacteria and lactobacilli, Escherichia coli, Enterococcus faecium and Streptococcus pneumoniae can also utilize raffinose and raffinose might lead to flatulence in some hosts. Therefore, it is required to find out the raffinose-metabolizing bacteria in the gut and the bacteria responsible for the flatulence. The BLASTP search results showed that the homologous proteins of glycosidases related to raffinose utilization are widely distributed in 196 of the 528 gut bacterial strains. Fifty-nine bacterial strains belonging to nine species of five genera were isolated from human feces and were found to be capable of utilizing raffinose; of these species, Enterococcus avium and Streptococcus salivarius were reported for the first time. High-performance liquid chromatography (HPLC) analysis of the supernatants of the nine species revealed that the bacteria could utilize raffinose in different manners. Glucose and melibiose were detected in the supernatants of Enterococcus avium E5 and Streptococcus salivarius B5, respectively. However, no resulting saccharides of raffinose degradation were detected in the supernatants of other seven strains, indicating that they had different raffinose utilization types from Enterococcus avium E5 and Streptococcus salivarius B5. Gas was produced with raffinose utilization by Escherichia coli, Enterococcus faecium, Streptococcus macedonicus, Streptococcus pasteurianus and Enterococcus avium. Thus, more attention should be paid to the raffinose-utilizing bacteria besides bifidobacteria and further studies are required to reveal the mechanisms of raffinose utilization to clarify the relationship between raffinose and gut bacteria.

Gustatory response and longevity in Aphidius parasitoids and their hyperparasitoid Dendrocerus aphidum

Abstract: Aphid parasitoids are commonly used in the biological control of aphids. However, their success in biological control largely depends on the availability of carbohydrate-rich food as an energy source for maintenance and reproduction. Therefore, as these resources have become rare in modern agricultural systems, external sugar sources like flowering plants or artificial sugar solutions are more and more used to provide the biocontrol agents with the necessary sugars. When developing such artificial food sources, it is essential to carefully select the sugars that best support the target parasitoids without benefiting non-target insects, such as pest insects or hyperparasitoids. Here, we investigated the gustatory response and longevity of two commonly used aphid parasitoids (Aphidius colemani and Aphidius matricariae) and their hyperparasitoid Dendrocerus aphidum when provided with one of eight plant- and/or insect-derived sugars (fructose, galactose, glucose, melibiose, melezitose, rhamnose, sucrose and trehalose). Our results showed that the studied insect species consumed the largest amounts of sugars that are most commonly found in honeydew (sucrose, fructose, glucose and melezitose) and also survived best when feeding on these sugars. Both Aphidius spp. survived well on melibiose, whereas D. aphidum performed poorly on this sugar. When melibiose was offered in a mixture with glucose, a significant reduction in longevity was observed for D. aphidum when compared to glucose only, while this was less pronounced for Aphidius spp. This knowledge can be exploited in tailoring food sources to selectively support Aphidius parasitoids, enhancing the biological control of aphids.

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants.

Abstract: A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.

Hydrolysis of oligosaccharides by a fungal α‐galactosidase from fruiting bodies of a wild mushroom Leucopaxillus tricolor

Abstract: A novel acidic α-galactosidase (EC 32122) designated as Leucopaxillus tricolor α-galactosidase (LTG) has been purified to homogeneity from the fruiting bodies of L tricolor to 855-fold with a specific activity of 956 U mg-1 by the application of chromatography and gel filtration The molecular mass of LTG was estimated to be 60 kDa as determined by both SDS-PAGE and by gel filtration The purified enzyme was identified by LC-MS/MS and four inner amino acid sequences were obtained When 4-nitrophenyl α-D-glucopyranoside (pNPGal) was used as substrate, the optimal pH and optimal temperature of LTG were pH 50 and 50 °C, respectively The enzyme activity was strongly inhibited by Hg2+ , Fe3 , Cu2+ , Cd2+ , and Mn2+ ions The chemical modification agent N-bromosuccinimide (NBS) completely inhibited the enzyme activity of LTG, indicating the paramount importance of tryptophan residue(s) to its enzymatic activity Besides, LTG displayed wide substrate diversity with activity toward a variety of substrates such as stachyose, raffinose, melibiose, locust bean gum, and guar gum Given the good ability of degrading the non-digestible and flatulence-causing oligosaccharides, this fungus may become a useful source of α-galactosidase for multiple applications

Galacto-oligosaccharide hydrolysis by genetically-engineered alpha-galactosidase-producing Pseudomonas chlororaphis strains

Abstract: Various Pseudomonas chlororaphis strains have been shown to produce rhamnolipid, poly(hydroxyalkanoate), and antifungal compounds for plants. Ability to metabolize galacto-oligosaccharides would allow P. chlororaphis to use soy molasses as a low-cost fermentation feedstock. In this study, genetically engineered P. chlororaphis strains expressing a Streptomyces coelicolor α-galactosidase (α-gal) were constructed. In recombinant P. chlororaphis [chr::AG], the α-gal was integrated into the chromosome. P. chlororaphis [pBS-dAG], however, contains a truncated α-gal (coding for the N-terminal catalytic domain of the enzyme) on an expression vector. Real-time RT-qPCR showed 1,438-fold higher α-gal gene expression in [pBS-dAG] than [chr::AG]. In agreement with qPCR study, the results of an enzyme assay using p-nitrophenyl-α-galactopyranoside (p-NP-α-Gal) as a chromogenic substrate also showed that the cell extracts of [pBS-dAG] contained ca. 8-times higher p-NP-α-Gal-hydrolyzing activity than that of [chr::AG]. The cell extracts of [pBS-dAG] were also demonstrated to hydrolyze raffinose (32.7 ± 4.1% of the initial amount remained in the reaction mixture) > melibiose (65.4 ± 7.9%) > stachyose (72.8 ± 11.9%). The incubation of an EDTA-permeabilized (1.5 μM, 28 °C, 200 rpm shaking, 20 min) P. chlororaphis [pBS-dAG] whole-cell preparation with 0.5% (w/v) raffinose in a Medium E* for 7 days resulted in the reduction of the carbon source to 0.14% (w/v), or 28% relative to the initially added amount, and the biomass reached a value of 0.46 g CDW (cell dry weight)/l. In contrast, EDTA-permeabilized wild-type P. chlororaphis did not hydrolyze the 0.5% (w/v) raffinose in the medium, and the final biomass yield was 0.26 g CDW/l.

Preparation method of oligosaccharide for promoting proliferation of intestinal probiotics

Abstract: The invention discloses a preparation method of oligosaccharide for promoting the proliferation of intestinal probiotics. According to the method, based on the selection specificity of enzymes on different substrates, lactose, cellobiose, melibiose, trehalose and other general disaccharide oligomers are respectively used as mixed substrates, a transglycosylation reaction is catalyzed with beta-galactosidase derived from Lactobacillus plantarum 70810, and the obtained products are subjected to classification purification through active carbon beta-diatomaceous earth adsorption chromatography soas to obtain various iso galactooligosaccharide mixtures. According to the present invention, the preparation method has characteristics of simple process and low cost; and the content of the oligosaccharides comprising more than three molecules in the oligosaccharides prepared by using the method is more than 95%; and the prepared oligosaccharide provides the significant proliferation activity to intestinal probiotics, can inhibit the growth of harmful bacteria, contains almost no monosaccharides and disaccharides, has the expanded application population and the expanded application field, has high safety, and can be directly added to various foods.