RESUMEN
Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity of gut bacterial communities to cooperatively metabolize structural variants of this substrate remains largely unknown. Herein, the characterization of a pectin methylesterase, BpeM, from Bifidobacterium longum subsp. longum, is reported. The purified enzyme was able to remove methyl groups from highly methoxylated apple pectin, and the mathematical modelling of its activity enabled to tightly control the reaction conditions to achieve predefined final degrees of methyl-esterification in the resultant pectin. Demethylated pectin, generated by BpeM, exhibited differential fermentation patterns by gut microbial communities in in vitro mixed faecal cultures, promoting a stronger increase of bacterial genera associated with beneficial effects including Lactobacillus, Bifidobacterium and Collinsella. Our findings demonstrate that controlled pectin demethylation by the action of a B. longum esterase selectively modifies its prebiotic fermentation pattern, producing substrates that promote targeted bacterial groups more efficiently. This opens new possibilities to exploit biotechnological applications of enzymes from gut commensals to programme prebiotic properties.
Asunto(s)
Hidrolasas de Éster Carboxílico , Heces , Malus , Pectinas , Prebióticos , Malus/microbiología , Pectinas/metabolismo , Heces/microbiología , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/genética , Fermentación , Humanos , Bifidobacterium longum/metabolismo , Bifidobacterium longum/enzimología , Microbioma Gastrointestinal , Bifidobacterium/enzimología , Bifidobacterium/metabolismoRESUMEN
Multiple species of Bifidobacterium exhibit the ability to bioconvert conjugated fatty acids (CFAs), which is considered an important pathway for these strains to promote host health. However, there has been limited progress in understanding the enzymatic mechanism of CFA bioconversion by bifidobacteria, despite the increasing number of studies identifying CFA-producing strains. The protein responsible for polyunsaturated fatty acid (PUFA) isomerization in B. breve CCFM683 has recently been discovered and named BBI, providing a starting point for exploring Bifidobacterium isomerases (BIs). This study presents the sequence classification of membrane-bound isomerases from four common Bifidobacterium species that produce CFA. Heterologous expression, purification, and enzymatic studies of the typical sequences revealed that all possess a single c9, t11 isomer as the product and share common features in terms of enzymatic properties and catalytic kinetics. Using molecular docking and alanine scanning, Lys84, Tyr198, Asn202, and Leu245 located in the binding pocket were identified as critical to the catalytic activity, a finding further confirmed by site-directed mutagenesis-based screening assays. Overall, these findings provide insightful knowledge concerning the molecular mechanisms of BIs. This will open up additional opportunities for the use of bifidobacteria and CFAs in probiotic foods and precision nutrition.
Asunto(s)
Bifidobacterium , Ácidos Grasos Insaturados , Bifidobacterium/enzimología , Bifidobacterium/genética , Bifidobacterium/metabolismo , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Simulación del Acoplamiento Molecular , Isomerismo , Cinética , Secuencia de Aminoácidos , Mutagénesis Sitio-Dirigida , Probióticos/metabolismoRESUMEN
Arabinoxylan (AX) and arabinoxylooligosaccharides (AXOs) are carbohydrate sources utilized by Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we characterized two genes, BLLJ_1850 and BLLJ_1851, in the hemicellulose-degrading gene cluster (BLLJ_1836-BLLJ_1859) of B. longum subsp. longum JCM 1217. Both recombinant enzymes expressed in Escherichia coli exhibited exo-α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside. BlArafE (encoded by BLLJ_1850) contains the glycoside hydrolase family 43 (GH43), subfamily 22 (GH43_22), and GH43_34 domains. The BlArafE GH43_22 domain was demonstrated to release α1,3-linked Araf from AX, but the function of BlArafE GH43_34 could not be clearly identified in this study. BlArafD (encoded by BLLJ_1851) contains GH43 unclassified subfamily (GH43_UC) and GH43_26 domains. The BlArafD GH43_UC domain showed specificity for α1,2-linked Araf in α1,2- and α1,3-Araf double-substituted structures in AXOs, while BlArafD GH43_26 was shown to hydrolyze α1,5-linked Araf in the arabinan backbone. Co-incubation of BlArafD and BlArafE revealed that these two enzymes sequentially removed α1,2-Araf and α1,3-Araf from double-substituted AXOs in this order. B. longum strain lacking BLLJ_1850-BLLJ_1853 did not grow in the medium containing α1,2/3-Araf double-substituted AXOs, suggesting that BlArafE and BlArafD are important for the assimilation of AX. KEY POINTS: ⢠BlArafD GH43 unclassified subfamily domain is a novel α1,2-L-arabinofuranosidase. ⢠BlArafE GH43 subfamily 22 domain is an α1,3-L-arabinofuranosidase. ⢠BlArafD and BlArafE cooperatively degrade α1,2/3-Araf double-substituted arabinoxylan.
Asunto(s)
Glicósido Hidrolasas , Xilanos , Bifidobacterium/enzimología , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Especificidad por Sustrato , Xilanos/metabolismoRESUMEN
Two α-L-arabinofuranosidases (BfdABF1 and BfdABF3) and a ß-D-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25°C. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 32-α-L-arabinofuranosyl-xylobiose (A3X), and 23-α-Larabinofuranosyl-xylotriose (A2XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo-ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-L-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 23,33-di-α-L-arabinofuranosyl-xylotriose (A2+3XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50°C, and it has the exclusively high activities on AXOS such as A3X and A2XX. BfdABF3 mainly catalyzes the removal of L-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55°C, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A3X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceulloseutilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce L-arabinose and D-xylose from various AOS, XOS, and AXOS.
Asunto(s)
Bifidobacterium/enzimología , Glicósido Hidrolasas , Escherichia coli/genética , Escherichia coli/metabolismo , Glicósido Hidrolasas/metabolismo , Hidrólisis , Oligosacáridos/química , Especificidad por Sustrato , Xilanos , XilosidasasRESUMEN
The extracellular secreted protein of Bifidobacterium longum (B. longum) plays an important role in maintaining the homeostasis of the human intestinal microenvironment. However, the mechanism(s) of interaction remain unclear. Lysozyme is a kind of antibacterial peptide. In this study, the amino acid sequence of a lysozyme-like protein of B. longum based on whole-genome data of an isolate from human gut feces was found. We further predicted functional domains from the amino acid sequence, purified the protein, and verified its bioactivity. The growth of some bacteria were significantly delayed by the 020402_LYZ M1 protein. In addition, the gut microbiota was analyzed via high-throughput sequencing of 16S rRNA genes and an in vitro fermentation model, and the fluctuations in the gut microbiota under the treatment of 020402_LYZ M1 protein were characterized. The 020402_LYZ M1 protein affected the composition of human gut microbiota significantly, implying that the protein is able to communicate with intestinal microbes as a regulatory factor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bifidobacterium/metabolismo , Microbioma Gastrointestinal , Interacciones Microbianas , Proteínas Bacterianas/química , Bifidobacterium/enzimología , Biología Computacional/métodos , Heces/microbiología , Humanos , Modelos Moleculares , Proteoma , Proteómica/métodos , Relación Estructura-ActividadRESUMEN
Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on ß-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of ß-D-Frup-(2â1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-ß-D-Frup (diheterolevulosan II) and ß-D-Fruf-(2â1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-ß-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with ß-D-Fruf and ß-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).
Asunto(s)
Proteínas Bacterianas/química , Bifidobacterium/enzimología , Glicósido Hidrolasas/química , Modelos Moleculares , Oligosacáridos/química , Cristalografía por Rayos X , Glicósido Hidrolasas/clasificaciónRESUMEN
Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1â3)-l-Ara and ß-l-Arap-(1â3)-l-Ara from gum arabic AGP and ß-l-Arap-(1â3)-l-Ara from larch AGP, and the α-d-Galp-(1â3)-l-Ara release activity was found to be 594-fold higher than that of ß-l-Arap-(1â3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1â3)-l-Ara in B. longum Notably, when α-d-Galp-(1â3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1â3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longumIMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1â3)-l-Ara and ß-l-Arap-(1â3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.
Asunto(s)
Proteínas Bacterianas/genética , Bifidobacterium/enzimología , Glicósido Hidrolasas/genética , Proteínas Bacterianas/metabolismo , Bifidobacterium/genética , Galactanos/metabolismo , Glicósido Hidrolasas/metabolismo , Goma ArábigaRESUMEN
Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly, Bifidobacterium bifidum and Bifidobacterium longum possess lacto-N-biosidases with unrelated structural folds to release the disaccharide lacto-N-biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting ß-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium. Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans.
Asunto(s)
Proteínas Bacterianas/química , Bifidobacterium/enzimología , Bifidobacterium/metabolismo , Metabolismo de los Hidratos de Carbono , Oligosacáridos/metabolismo , Conformación Proteica , Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Proteínas Bacterianas/metabolismo , Bifidobacterium/clasificación , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Oligosacáridos/química , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Bile salt hydrolase (BSH) activity is a desirable trait in putative probiotic bacteria, such as those belonging to the Bifidobacterium genus. On the one hand, bile salt hydrolysis is considered to represent a bile detoxification mechanism for gut commensal bacteria and thus the presence of this activity was believed to be a predictor of bile tolerance of putative probiotic strains. On the other hand, it has recently been revealed that chemical modifications of the bile acid pool performed by the gut microbiota strongly impact on host health. This explains the increasing interest to investigate the role played by bile-modifying enzymes of gut commensals on lowering cholesterol levels, on modulating gut inflammation or on influencing the development of cancer or metabolic disorders. This chapter compiles qualitative and quantitative methods to analyse BSH activity in bifidobacteria, though they could be adapted to other bacterial groups of interest.
Asunto(s)
Amidohidrolasas/metabolismo , Bifidobacterium/enzimología , Pruebas de Enzimas/métodos , Amidohidrolasas/análisis , Aminoácidos/análisis , Aminoácidos/metabolismo , Bifidobacterium/metabolismo , Ácidos y Sales Biliares/metabolismo , HidrólisisRESUMEN
Bivalve molluscan shellfish such as oysters are filter feeders and are able to accumulate human noroviruses (NoVs) largely due to the presence of human histo-blood group antigens (HBGAs)-like carbohydrates in their intestine. Since the fucose contents play a key role in the binding of NoVs to HBGAs, this study intended to investigate the influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated NoV binding. On the contrary to the expected, after a treatment of the oyster digestive tissue extracts with Bifidobacterium bifidum strain JCM 1254, the binding of human NoV GII.4 virus like particles (VLPs) to the oyster digestive tissue extracts enhanced significantly (OD450 from 1.15 ± 0.05 to 1.51 ± 0.02, P < 0.001) in an in vitro direct binding assay. The accumulation of human NoV GII·P16-GII.4 also enhanced significantly in the intestine of B. bifidum JCM 1254 treated oysters from 4.27 ± 0.25 log genomic copies/g oyster digestive tissue to 5.25 ± 0.29 log genomic copies/g oyster digestive tissue (P < 0.005) as observed in an in vivo test. Correspondingly, the type A antigenicity of the oyster digestive tissue extracts enhanced (OD450 from 0.77 ± 0.04 to 1.06 ± 0.05, P < 0.01) after the treatment with B. bifidum JCM 1254. These results could be explained by the substrate specificity of the B. bifidum JCM 1254 associated fucosidases. This study identified an indirect interaction possibly happening between the bacterial microbiota with human NoVs during their transmission in the food systems. We also supplied a potential strategy to mitigate the NoV contamination from shellfish, suppose bacterial strains with specified fucosidase production could be obtained in the future.
Asunto(s)
Bifidobacterium/enzimología , Antígenos de Grupos Sanguíneos/metabolismo , Norovirus/metabolismo , Ostreidae/virología , Mariscos/virología , alfa-L-Fucosidasa/metabolismo , Animales , Anticuerpos Monoclonales , Bifidobacterium/fisiología , Antígenos de Grupos Sanguíneos/inmunología , Humanos , Intestinos/inmunología , Intestinos/virología , Ostreidae/inmunologíaRESUMEN
Short-chain cello-oligosaccharides (COS; degree of polymerization, DP ≤ 6) are promising water-soluble dietary fibers. An efficient approach to their bottom-up synthesis is from sucrose and glucose using glycoside phosphorylases. Here, we show the intensification and scale up (20 mL; gram scale) of COS production to 93 g/L product and in 82 mol % yield from sucrose (0.5 M). The COS were comprised of DP 3 (33 wt %), DP 4 (34 wt %), DP 5 (24 wt %), and DP 6 (9 wt %) and involved minimal loss (≤10 mol %) to insoluble fractions. After isolation (≥95% purity; ≥90% yield), the COS were examined for growth promotion of probiotic strains. Benchmarked against inulin, trans-galacto-oligosaccharides, and cellobiose, COS showed up to 4.1-fold stimulation of cell density for Clostridium butyricum, Lactococcus lactis subsp. lactis, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus but were less efficient with Bifidobacterium sp. This study shows the COS as selectively functional carbohydrates with prebiotic potential and demonstrates their efficient enzymatic production.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bifidobacterium/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Lactobacillus/metabolismo , Oligosacáridos/metabolismo , Fosforilasas/metabolismo , Probióticos/metabolismo , Bifidobacterium/enzimología , Bifidobacterium/crecimiento & desarrollo , Lactobacillus/enzimología , Lactobacillus/crecimiento & desarrollo , Lacticaseibacillus rhamnosus/enzimología , Lacticaseibacillus rhamnosus/crecimiento & desarrollo , Oligosacáridos/química , Prebióticos/análisis , Sacarosa/metabolismoRESUMEN
Resistant starch (RS) is a complex prebiotic carbohydrate beneficial to the human gut. In the present study, four genes encoding for putative amylolytic enzymes, likely to be responsible for RS-degradation, were identified in the genome of Bifidobacterium adolescentis P2P3 by comparative genomic analysis. Our results showed that only three enzymes (RSD1, RSD2, and RSD3) exhibited non-gelatinized high amylose corn starch (HACS)-degrading activity in addition to typical α-amylase activity. These three RS-degrading enzymes (RSD) were composed of multiple domains, including signal peptide, catalytic domain, carbohydrate binding domains, and putative cell wall-anchoring domains. Typical catalytic domains were conserved by exhibiting seven typical conserved regions (I-VII) found mostly in α-amylases. Analysis of enzymatic activity revealed that RSD2 displayed stronger activity toward HACS-granules than RSD1 and RSD3. Comparative genomics in combination with enzymatic experiments confirmed that RSDs might be the key enzymes used by RS-degrading bifidobacteria to degrade RS in a particular ecological niche, such as the human gut.
Asunto(s)
Amilasas/metabolismo , Bifidobacterium adolescentis/enzimología , Microbioma Gastrointestinal , Almidón Resistente/metabolismo , Secuencia de Aminoácidos , Amilasas/química , Bifidobacterium/clasificación , Bifidobacterium/enzimología , Bifidobacterium/genética , Bifidobacterium adolescentis/clasificación , Bifidobacterium adolescentis/genética , Biología Computacional/métodos , Genoma Bacteriano , Humanos , Hidrólisis , FilogeniaRESUMEN
Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas, blas, bpas, and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0-6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries.
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Proteínas Bacterianas/metabolismo , Bifidobacterium/enzimología , Glucanos/metabolismo , Glucosiltransferasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Clonación Molecular , Disacáridos/metabolismo , Estabilidad de Enzimas , Glucosiltransferasas/química , Glucosiltransferasas/clasificación , Glucosiltransferasas/genética , Calor , Homología de Secuencia , Especificidad por SustratoRESUMEN
The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.
Asunto(s)
Bifidobacterium/clasificación , Glutamato-Amoníaco Ligasa/genética , Filogenia , Técnicas de Tipificación Bacteriana , Bifidobacterium/enzimología , Cartilla de ADN , ADN Bacteriano/genética , Genes Bacterianos , Marcadores Genéticos , Genotipo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
α-Galactosidases are potent industrial glycoside hydrolases which are relatively less explored for their transglycosylation potential, especially from Lactobacillus genera. A GH36 α-galactosidase from Lactobacillus plantarum WCFS1 was cloned and over expressed in Hi-control Escherichia coli BL21(DE3). Ni-NTA affinity gel chromatography resulted in purified α-galactosidase (LpαG; specific activity 3077.35 U mg-1) having a monomeric weight of ~80 kDa with 29.3% yield. Size exclusion chromatography of LpαG showed native molecular mass of ~240.5 kDa. LpαG displayed optimum activity at pH 6 and 37 °C. The Km,Vmax and kcat/Km of LpαG towards pNPαGal were found to be 0.93 mM and 714.3 µmol ml-1 min-1 and 12,075 s-1 mM-1, respectively. LpαG displayed maximum transglycosylation activity towards melibiose substrate (as both donor and acceptor) and synthesized majorly a trisaccharide with 0.26 mg ml-1 yield. Nuclear magnetic resonance (NMR) characterization revealed that trisaccharide consist of only single species of α-linked galactooligosaccharide (manninotriose; α-d-Galp-(1 â 6)-α-d-Galp-(1 â 6)-d-Glcp) with α-(1 â 6) regioselectivity. Manninotriose displayed prebiotic property by supporting the growth of probiotic L. plantarum WCFS1 and Bifidobacteria adolescentis DSM 20083.
Asunto(s)
Bifidobacterium/enzimología , Lactobacillus plantarum/enzimología , Oligosacáridos/metabolismo , Prebióticos , alfa-Galactosidasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bifidobacterium/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Galactósidos/metabolismo , Glicosilación , Lactobacillus plantarum/genética , Trisacáridos/metabolismo , alfa-Galactosidasa/genéticaRESUMEN
Many effects of Coprinus comatus are attributed to its polysaccharide components. Therefore, the aim of this article is to take Coprinus comatus polysaccharides as the research topic to estimate the difference between the polysaccharides of Coprinus comatus fruiting bodies (CBPs) and the intracellular polysaccharides of liquid fermentation (ICPs). The total carbohydrate contents, monosaccharide compositions, molecular weights, functional groups, microstructures and functional properties of the two prepared polysaccharides were evaluated. At the same time, the influences of the two polysaccharides on the proliferation of lactobacillus and Bifidobacterium in vitro were compared. The structural analysis exhibited that there were slight differences in the two prepared polysaccharides. However, both ICPs and CBPs could be utilized by these two strains. Furthermore, the effects of the two prepared polysaccharides on the proliferation of the selected probiotics were dose-dependent manners within the scope of the experiment, and the ICPs group and CBPs group had no significant difference (P > 0.05). Therefore, this work demonstrates that ICPs could be an equivalent replacer for CBPs.
Asunto(s)
Bifidobacterium/enzimología , Coprinus/fisiología , Lactobacillus/crecimiento & desarrollo , Bifidobacterium/efectos de los fármacos , Secuencia de Carbohidratos , Coprinus/química , Relación Dosis-Respuesta a Droga , Fermentación , Polisacáridos Fúngicos/química , Polisacáridos Fúngicos/farmacología , Lactobacillus/efectos de los fármacos , Micelio , PrebióticosRESUMEN
The gene encoding ß-galactosidase was cloned from Bifidobacterium longum RD47 with combinations of several bifidobacterial promoters and expressed in B. bifidum BGN4. Among the recombinant bifidobacteria, BGN4+G1 showed the highest ß-galactosidase level, for which the hydrolytic activity was continuously 2.5 to 4.2 times higher than that of BGN4 and 4.3 to 9.6 times higher than that of RD47. The ß-galactosidase activity of BGN4+G1 was exceedingly superior to that of any of the other 35 lactic acid bacteria. When commercial whole milk and BGN4+G1 were reacted, BGN4+G1 removed nearly 50% of the lactose in the milk by the 63-h time point, and a final 61% at 93 h. These figures are about twice the lactose removal rate of conventional fermented milk. As for the reaction of commercial whole milk and crude enzyme extract from BGN4+G1, the ß-galactosidase of BGN4+G1 eliminated 51% of the lactose in milk in 2 h. As shown below, we also compared the strengths and characteristics of the strong bifidobacterial promoters reported by previous studies.
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Proteínas Bacterianas/genética , Bifidobacterium/genética , Bifidobacterium/metabolismo , beta-Galactosidasa/genética , Animales , Proteínas Bacterianas/metabolismo , Bifidobacterium/enzimología , Clonación Molecular , Fermentación , Expresión Génica , Hidrólisis , Lactosa/metabolismo , Leche/química , Leche/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Arabinogalactan proteins (AGPs) are complex plant proteoglycans that function as dietary fiber utilized by human intestinal bacteria such as Bifidobacterium and Bacteroides species. However, the degradative mechanism is unknown because of the complexity of sugar chains of AGPs as well as variation among plant species and organs. Recently, AGP degradative enzymes have been characterized in Bifidobacterium and Bacteroides species. In this review, we summarize the characteristics and functions of AGP degradative enzymes in human intestinal bacteria.
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Bacterias/enzimología , Intestinos/microbiología , Mucoproteínas/metabolismo , Plantas/metabolismo , Bacteroides/enzimología , Bifidobacterium/enzimología , Pared Celular/metabolismo , Fibras de la Dieta/metabolismo , Humanos , Proteínas de Plantas/metabolismoRESUMEN
Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-ß, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.
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Autofagia , Bifidobacterium/fisiología , Señalización del Calcio , Interacciones Microbiota-Huesped , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Animales , Bifidobacterium/enzimología , Femenino , Microbioma Gastrointestinal , Vida Libre de Gérmenes , Células Caliciformes/fisiología , Factor 4 Similar a Kruppel , Masculino , Ratones , Mucina 2/genética , Mucinas/metabolismo , Mucinas/farmacología , Polisacáridos/metabolismoRESUMEN
10-hydroxy-cis-12 octadecenoic acid (10-HOE) is a type of octadecenoic acid with a hydroxyl on the C10 carbon. It is generated from linoleic acid (LA) catalyzed by linoleate hydratase in lactobacilli, which was initially named as myosin-cross-reactive antigen (MCRA). In lactobacilli, 10-HOE is the first intermediate in the production of conjugated LA (CLA). Although MCRA from bifidobacteria can generate 10-HOE, the precise role of 10-HOE in CLA production in bifidobacteria remains unknown. In the current work, 10-HOE and LA were added to the medium as the substrate both separately and synchronously to analyze their influence on CLA production. Using 10-HOE as the substrate, bifidobacteria were able to generate CLA by first converting it to LA, followed by CLA accumulation. Recombinant MCRA catalyzed the conversion of 10-HOE to LA, indicating that bifidobacterial MCRA can account for the reversible conversion between LA and 10-HOE. This is the first report to demonstrate the precise role of 10-HOE in the process of CLA production among bifidobacteria.