Explore the vast range of titles available.

Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker’s yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes ( suc2 , mel1 , and fsy1 ) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of d-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s−1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M−1 s−1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides d-(+)-galactose, the purified enzyme also acted against d-(+)-raffinose, α-d-(+)-melibiose, and methyl-α-d-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

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.

MytiLec-1, a 17 kDa lectin with β-trefoil folding that was isolated from the Mediterranean mussel (Mytilus galloprovincialis) bound to the disaccharide melibiose, Galα(1,6) Glc, and the trisaccharide globotriose, Galα(1,4) Galβ(1,4) Glc. Toxicity of the lectin was found to be low with an LC50 value of 384.53 μg/mL, determined using the Artemia nauplii lethality assay. A fluorescence assay was carried out to evaluate the glycan-dependent binding of MytiLec-1 to Artemia nauplii. The lectin strongly agglutinated Ehrlich ascites carcinoma (EAC) cells cultured in vivo in Swiss albino mice. When injected intraperitoneally to the mice at doses of 1.0 mg/kg/day and 2.0 mg/kg/day for five consecutive days, MytiLec-1 inhibited 27.62% and 48.57% of cancer cell growth, respectively. Antiproliferative activity of the lectin against U937 and HeLa cells was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro in RPMI-1640 medium. MytiLec-1 internalized into U937 cells and 50 μg/mL of the lectin inhibited their growth of to 62.70% whereas 53.59% cell growth inhibition was observed against EAC cells when incubated for 24 h. Cell morphological study and expression of apoptosis-related genes (p53, Bax, Bcl-X, and NF-κB) showed that the lectin possibly triggered apoptosis in these cells.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra (SN) and proteinaceous α-synuclein-positive Lewy bodies and Lewy neuritis. As a chemical chaperone to promote protein stability and an autophagy inducer to clear aggregate-prone proteins, a disaccharide trehalose has been reported to alleviate neurodegeneration in PD cells and mouse models. Its trehalase-indigestible analogs, lactulose and melibiose, also demonstrated potentials to reduce abnormal protein aggregation in spinocerebellar ataxia cell models. In this study, we showed the potential of lactulose and melibiose to inhibit α-synuclein aggregation using biochemical thioflavin T fluorescence, cryogenic transmission electron microscopy (cryo-TEM) and prokaryotic split Venus complementation assays. Lactulose and melibiose further reduced α-synuclein aggregation and associated oxidative stress, as well as protected cells against α-synuclein-induced neurotoxicity by up-regulating autophagy and nuclear factor, erythroid 2 like 2 (NRF2) pathway in DAergic neurons derived from SH-SY5Y cells over-expressing α-synuclein. Our findings strongly indicate the potential of lactulose and melibiose for mitigating PD neurodegeneration, offering new drug candidates for PD treatment.

The melibiose carrier from Escherichia coli (MeIB) couples the accumulation of the disaccharide melibiose to the downhill entry of H⁺, Na⁺, or Li⁺. In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MeIB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MeIB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na⁺ binding. Secondly, though Asp124 is not essential for Na⁺ binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na⁺-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na⁺ binding but it is a melibiose ligand. The location of these residues in two independent threading models of MeIB is consistent with their proposed role.

Infrared difference spectroscopy analysis of the purified melibiose permease of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na+, melibiose) in either H2O or in D2O media. Essentially, the data first show that addition of Na+ induces appearance of peaks assigned to changes in the environment and/or orientation of α-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na+ produces peaks corresponding to additional changes of α-helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+)cm−1/1576 (−)cm−1) appearing in the Na+-induced difference spectrum is assigned to the antisymmetric stretching of COO− groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na+ coordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (−)cm−1/1567 (+)cm−1), appear in the melibiose-induced difference spectra, and may again be assigned to COO− groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding.

The alpha-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus thermophilus was over 40%. The alpha-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 degrees C and pH 7. The enzyme hydrolyzed p-nitrophenyl-alpha-D -galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.Guan and Hariharan report two crystal structures of melibiose transporter MelB in complex with substrate analogs, nitrophenyl-galactoside, and dodecyl-melibioside. Both structures revealed similar specific site for sugar recognition and resolved the cation-binding pocket, advancing the understanding of MelB and related transporters.