RESUMEN
The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.
Asunto(s)
Bacteroides , Glicósido Hidrolasas , Glicósido Hidrolasas/química , Humanos , Plantas/metabolismo , Polisacáridos/metabolismo , Azúcares/metabolismo , alfa-Manosidasa/metabolismoRESUMEN
Li-CO2 batteries are regarded as next-generation high-energy-density electrochemical devices. However, the greatest challenge arises from the formation of the discharge product, Li2 CO3 , which would accumulate and deactivate heterogenous catalysts to cause huge polarization. Herein, Ru(bpy)3 Cl2 was employed as a solution-phase catalyst for Li-CO2 batteries and proved to be the most effective one screened so far. Spectroscopy and electrochemical analyses elucidate that the RuII center could interact with both CO2 and amorphous Li2 C2 O4 intermediate, thus promoting electroreduction process and delaying carbonate transformation. As a result, the charge potential is reduced to 3.86â V and over 60 discharge/charge cycles are achieved with a fixed capacity of 1000â mAh g-1 at a current density of 300â mA g-1 . Our work provides a new avenue to improve the electrochemical performance of Li-CO2 batteries with efficient mobile catalysts.
RESUMEN
In the published version of the article, the title should have read 'Duganella rivi sp. nov., Duganella fentianensis sp. nov., Duganella qianjiadongensis sp. nov. and Massilia guangdongensis sp. nov., isolated from subtropical streams in China and reclassification of all species within genus Pseudoduganella'.
RESUMEN
Four Gram-stain-negative, catalase-positive, rod-shaped and motile strains (FT55WT, FT93WT, CY13WT and DS3T) were isolated from subtropical streams in China. Comparisons based on 16S rRNA gene sequences indicated that strains FT55WT, FT93WT and CY13WT take strain Pseudoduganella danionis E3/2T, and strain DS3T takes strain Pseudoduganella eburnea 10R5-21T as their closest neighbour, respectively. The genome sizes of strains FT55WT, FT93WT, CY13WT and DS3T were 6.15, 5.10, 5.31 and 5.72 Mbp with G+C contents of 61.7, 60.9, 60.6 and 64.0%, respectively. The reconstructed phylogenomic tree based on concatenated 92 core genes showed that strain FT55WT clusters closely with Duganella radicis KCTC 22382T and Duganella sacchari Sac-22T, strains FT93WT and CY13WT form a distinct clade with P. danionis DSM 103461T and this clade clusters with the clades of genus Duganella together, and strain DS3T forms a distinct clade with P. eburnea JCM 31587T and Pseudoduganella violaceinigra DSM 15887T and this clade clusters closely with the clades of genus Massilia, respectively. The calculated pairwise OrthoANIu values and digital DNA-DNA hybridization (DDH) values among strains FT55WT, FT93WT, CY13WT, DS3T and related strains were in the ranges of 75.6-94.2% and 20.6-56.2%, respectively. Q-8 was the sole respiratory quinone of these four strains. The major fatty acids were C16:1ω7c, C16:0 and C12:0. The polar lipids included phosphatidylglycerol, phosphatidylethanolamine and one unidentified phospholipid. Considering the similar fatty acids and polar lipids profiles of species within genus Pseudoduganella, Massilia and Duganella, there is currently no justification for assigning the species of genus Pseudoduganella into the Massilia and Duganella clades in the phylogenomic tree. It is reasonable to transfer P. violaceinigra and P. eburnea to the genus Massilia as Massilia violaceinigrum comb. nov. and Massilia eburnea comb. nov., and transfer P. danionis to the genus Duganella as Duganella danionis comb. nov. Considering phylogenomic analysis, OrthoANIu data, digital DDH data and a range of physiological and biochemical characteristics, strains FT55WT, FT93WT and CY13WT should be assigned to genus Duganella, and strain DS3T should be classified as a novel species within genus Massilia, for which the names Duganella rivus sp. nov. (type strain FT55WT = GDMCC 1.1675T = KACC 21467T), Duganella fentianensis sp. nov. (type strain FT93WT = GDMCC 1.1683T = KACC 21475T), Duganella qianjiadongensis sp. nov. (type strain CY13WT = GDMCC 1.1669T = KACC 21461T) and Massilia guangdongensis sp. nov. (type strain DS3T = GDMCC 1.1636T = KACC 21312T) are proposed.
Asunto(s)
Oxalobacteraceae/clasificación , Oxalobacteraceae/aislamiento & purificación , Filogenia , Ríos/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , China , ADN Bacteriano/genética , Ácidos Grasos/química , Genoma Bacteriano , Hibridación de Ácido Nucleico , Oxalobacteraceae/genética , Oxalobacteraceae/fisiología , Fosfatidiletanolaminas , Fosfolípidos/química , ARN Ribosómico 16S/genéticaRESUMEN
An unstudied ß-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The ß-N-acetylhexosaminidase was able to hydrolyze ß1,2-linked, ß1,3-linked, ß1,4-linked, and ß1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the ß-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.
Asunto(s)
Actinomycetales/metabolismo , Disacáridos/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Aminoácidos/metabolismo , Quitina/metabolismo , Quitinasas/metabolismo , Escherichia coli/metabolismo , Oximas/metabolismo , Fenilcarbamatos/metabolismo , Microbiología del SueloRESUMEN
A putative GH35 ß-galactosidase gene from the mucin-degrading bacterium Akkermansia muciniphila was successfully cloned and further investigated. The recombinant enzyme with the molecular mass of 74 kDa was purified to homogeneity and biochemically characterised. The optimum temperature of the enzyme was 42 °C, and the optimum pH was determined to be pH 3.5. The addition of sodium dodecyl sulphate (SDS) reduced the enzyme's activity significantly. The addition of Mg2+-ions decreased the activity of the ß-galactosidase, whereas other metal ions or EDTA showed no inhibitory effect. The enzyme catalysed the hydrolysis of ß1,3- and ß1,6- linked galactose residues from various substrates, whereas only negligible amounts of ß1,4-galactose were hydrolysed. The present study describes the first functional characterisation of a ß-galactosidase from this human gut symbiont.
Asunto(s)
Proteínas Bacterianas/metabolismo , Verrucomicrobia/enzimología , beta-Galactosidasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Estabilidad de Enzimas , Galactosa/análogos & derivados , Galactosa/metabolismo , Magnesio/química , Dodecil Sulfato de Sodio/química , Especificidad por Sustrato , Verrucomicrobia/genética , beta-Galactosidasa/química , beta-Galactosidasa/genéticaRESUMEN
Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived ß-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.
Asunto(s)
Galactosiltransferasas/metabolismo , Glicoconjugados/biosíntesis , Ostreidae/enzimología , UDPglucosa 4-Epimerasa/metabolismo , Animales , Uridina Difosfato Galactosa/metabolismoRESUMEN
BACKGROUND: Mannoside phosphorylases are frequently found in bacteria and play an important role in carbohydrate processing. These enzymes catalyze the reversible conversion of ß-1,2- or ß-1,4-mannosides to mannose and mannose-1-phosphate in the presence of inorganic phosphate. METHODS: The biochemical parameters of this recombinantly expressed novel mannose phosphorylase were obtained. Furthermore purified reaction products were subjected to ESI- and MALDI-TOF mass spectrometry and detailed NMR analysis to verify this novel type of ß-1,3-mannose linkage. RESULTS: We describe the first example of a phosphorylase specifically targeting ß-1,3-mannoside linkages. In addition to mannose, this phosphorylase originating from the bacterium Zobellia galactanivorans could add ß-1,3-linked mannose to various other monosaccharides and anomerically modified 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars). CONCLUSIONS: An unique bacterial phosphorylase specifically targeting ß-1,3-mannoside linkages was discovered. GENERAL SIGNIFICANCE: Functional extension of glycoside hydrolase family 130.
Asunto(s)
Flavobacteriaceae/enzimología , Manosa/metabolismo , Manósidos/biosíntesis , Fosforilasas/aislamiento & purificación , Catálisis , Fosforilasas/química , Fosforilasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
We herein report the use of 1,3-di(2-pyridyl)-1,3-propanedione (DPPD) as a fluorogenic labeling reagent for sugars. Reaction of DPPD with the anomeric carbon affords a fluorescent 2-pyridylfuran (2-PF) moiety that permits the sensitive HPLC-based detection of monosaccharides. 2-PF-labeled monosaccharides can be easily separated and analyzed from mixtures thereof, and the reported protocol compares favorably with established labeling reagents such as 2-aminobenzoic acid (2-AA) and 1-phenyl-3-methyl-5-pyrazolone (PMP), ultimately allowing subfemtomole detection of the galactose-derived product. Furthermore, we demonstrate the application of DPPD in the labeling of monosaccharides in complex biological matrices such as blood and milk samples. We envisage that DPPD will prove to be an excellent choice of labeling reagent in monosaccharide and carbohydrate analysis.
Asunto(s)
Carbohidratos/análisis , Colorantes Fluorescentes/química , Furanos/química , Piridinas/química , Animales , Análisis Químico de la Sangre , Bovinos , Cromatografía Líquida de Alta Presión , Colorantes Fluorescentes/síntesis química , Galactosa/química , Humanos , Leche/química , Leche Humana/química , Monosacáridos/análisisRESUMEN
Metal-organic frameworks (MOFs) with well-defined porous structures and highly active frameworks are considered as promising electrode materials for sodium-ion batteries (SIBs). However, the structure pulverization upon sodiation/desodiation impacts on their practical application in SIBs. To address this issue, poly(p-phenylenediamine) (PPA) was uniformly coated onto the surface of MIL-88A, a typical Fe-based MOF through in situ polymerization initiated by the metal ions (Fe3+) of MIL-88A. Used as an anode material for SIBs, the PPA-coated MIL-88A, denoted as PPA@MIL-88A, showed significantly improved electrochemical performance. A reversible capacity as high as 230 mAh g-1 was achieved at 0.2 A g-1 even after 500 cycles. MIL-88A constructed with electrochemically active Fe3+ and fumaric acid ligands guarantees the high specific capacity, while the PPA polymer coating effectively inhibits the pulverization of MIL-88A. This work provides an efficient strategy for improving the structure and cycling stability of MOFs-based electrode materials.
RESUMEN
A rock-salt-structured Li-conducting high entropy oxide was prepared and utilized as an active filler in a polyethylene oxide (PEO)-based solid-state composite electrolyte. X-ray diffraction and high-resolution transmission electron microscopy were adopted to analyze the crystal structure of the high entropy oxide containing 20% of Li ions (HL20). The HL20 was crystallized in the Fm3Ì m space group with Li+ ions located at the center of the MO6 octahedra. The ionic conductivity of the composite membrane at 30 °C reaches 3.44 × 10-5 S cm-1. The inflection point of activation energy of the membrane with HL20 decreases by 5 °C compared with that of the pure PEO membrane. In the galvanostatic plating/stripping test, the Li||Li symmetric batteries could be cycled at a current density of 200 µA cm-2 for over 1200 h with an overpotential of 140 mV. The Li||LiFePO4 full battery could be charged/discharged at 0.5 C for 100 circles with a high capacity retention rate of 91%. Excellent rate performance is also achieved at lower temperatures and higher rates, showing the superiority of HL20 as an active filler. This work sheds light on the development of high entropy oxide as a new type of fast ionic conductor, promoting the practical application of all-solid-state batteries at a lower temperature.
RESUMEN
The vibrational spectra of ethyl hexanoate were calculated by the density functional theory (DFT) with B3LYP complex function, diffuse function and polarization function added to heavy atoms and light atoms. On the base of this, the normal Raman spectrum (NRS) and the infrared spectrum (IR) were assigned in detail in the present paper. Comparing the calculated results with the experimental data, the calculated results are in good agreement with the experimental results. The comparison of the experimental Raman and infrared spectra shows that in the experimental Raman spectrum, the strongest bands appear at the frequencies of 2600-3100 cm(-1), while the strongest band is not 1734 cm(-1) but 1444 cm(-1) at the frequencies of 400-2000 cm(-1). The band 1734 cm(-1) attributed to the C=O stretch vibration is the distinctive mark of organic ester compounds, and the band 1444 cm(-1) is related to the symmetric and anti-symmetric scissors vibration of C-H. In the experimental infrared spectrum, the strongest vibrational band is 1739 cm(-1), which is related to C=O stretch vibration; At the frequencies of 400-2000 cm(-1), the relative intensity of the infrared spectrum is distinctively stronger than that of the Raman spectrum, but the relative intensity of infrared spectrum is weaker than that of the Raman spectrum at the frequencies of 2600-3100 cm(-1). In the frequencies of 2600-2800 cm(-1), the vibrational bands 2762 and 2732 cm(-1) do not appear in the experimental spectra, which may originate from two reasons: (1) the weak interaction of molecules. Also, the relative intensity of these vibrational bands is very weak in the experimental spectra, and this may testify that the interaction of molecules is rather weak; (2) the vibrational bands may belong to second order vibrational mode at the frequencies of 2600-2800 cm(-1). The relative intensity of infrared bands is weaker than that of the Raman bands at the frequencies of 2600-2800 cm(-1). At the end, the stronger bands appearing in Raman and infrared experimental spectra are assigned as characteristic marks, respectively. The study on vibrational spectra of ethyl hexanoate molecule may have great application value in detection of liquor flavor, chemical industry and biology fields, providing important reference value for the related basic research field.
RESUMEN
Two genes encoding the ß-N-acetylhexosaminidases Am2301 and Am2446 were cloned successfully from the mucin-degrading bacterium Akkermansia muciniphila. The recombinant enzymes with molecular masses of 61â¯kDa and 78â¯kDa were isolated and biochemically characterised. The optimum temperature of both enzymes was 37⯰C, and the optimum pH was determined to be pH 5.0 for Am2301 and pH 6.5 for Am2446. The addition of sodium dodecyl sulphate (SDS) reduced the enzymes' activity significantly. Cu2+-ions decreased the activity of Am2301 by 70%, while the activity of Am2446 was significantly reduced by Fe3+-ions. PugNAc strongly inhibited both enzymes already in the sub-micromolar concentration range. The enzymes catalysed the hydrolysis of ß1,4-linked N-acetylgalactosamine and ß1,6-linked N-acetylglucosamine from glycan standards, as well as ß1,2-linked N-acetylglucosamine units from the non-reducing end of N-glycans. The present study describes the first functional characterisation of ß-N-acetylhexosaminidases from this human gut symbiont.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Mucinas/metabolismo , Verrucomicrobia/enzimología , beta-N-Acetilhexosaminidasas/metabolismo , Intestinos/microbiología , Especificidad por SustratoRESUMEN
Recent progress in the relationship between carbohydrate cross-reactive determinants (CCDs) and allergic response highlights the importance of carbohydrate moieties in the innate immune system. Previous research pointed out that the protein allergen in Ginkgo biloba seeds is glycosylated, and the oligosaccharides conjugated to these proteins might also contribute to the allergy. The aim of this study was to analyze carbohydrate moieties, especially N-linked glycans, of glycoproteins from Ginkgo seeds originating from different places for detailed structures, to enable further research on the role played by N-glycans in Ginkgo-caused allergy. Results of monosaccharide composition and immunoblotting assays indicated the existence of N-glycans. Detailed structural elucidation of the N-glycans was further carried out by means of hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 14 out of 16 structures detected by UPLC were confirmed by MALDI-TOF-MS and tandem mass spectrometry, among which complex-type N-glycans bearing Lewis A determinants and high-mannose-type N-glycans were identified from Ginkgo seeds for the first time. Precise quantification of N-glycans was performed by use of an external standard, and both the absolute amount of each N-glycan and the percentage of different types of N-glycan showed significant diversity among the samples without any pattern of geographic variation.
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Ginkgo biloba/metabolismo , Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Semillas/química , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Ginkgo biloba/química , Glicoproteínas/química , Datos de Secuencia Molecular , Proteínas de Plantas/química , Polisacáridos/química , Polisacáridos/metabolismo , Semillas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Microvirin (MVN) is a carbohydrate-binding protein which shows high specificity for high-mannose type N-glycan structures. In the present study, we tried to identify whether MVN could bind to high-mannose containing hepatitis C virus (HCV) envelope glycoproteins, which are heavily decorated high-mannose glycans. In addition, recombinantly expressed MVN oligomers in di-, tri- and tetrameric form were evaluated for their viral inhibition. MVN oligomers bound more efficiently to HCV virions, and displayed in comparison with the MVN monomer a higher neutralization potency against HCV infection. The antiviral effect was furthermore affected by the peptide linker sequence connecting the MVN monomers. The results indicate that MVN oligomers such as trimers and tetramers may be used as future neutralization agents against HCV infections.