Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.051
Filtrar
1.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361767

RESUMO

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Xilanos/metabolismo , Sequência de Aminoácidos , Arabinose/química , Arabinose/metabolismo , Sequência de Carboidratos , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Glucuronatos/química , Glucuronatos/metabolismo , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Talaromyces/química , Talaromyces/genética , Xilanos/química
2.
Front Cell Infect Microbiol ; 11: 698146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368016

RESUMO

L-arabinose inducible promoters are commonly used in gene expression analysis. However, nutrient source and availability also play a role in biofilm formation; therefore, L-arabinose metabolism could impact biofilm development. In this study we examined the impact of L-arabinose on Salmonella enterica serovar Typhimurium (S. Typhimurium) biofilm formation. Using mutants impaired for the transport and metabolism of L-arabinose, we showed that L-arabinose metabolism negatively impacts S. Typhimurium biofilm formation in vitro. When L-arabinose metabolism is abrogated, biofilm formation returned to baseline levels. However, without the ability to import extracellular L-arabinose, biofilm formation significantly increased. Using RNA-Seq we identified several gene families involved in these different phenotypes including curli expression, amino acid synthesis, and L-arabinose metabolism. Several individual candidate genes were tested for their involvement in the L-arabinose-mediated biofilm phenotypes, but most played no significant role. Interestingly, in the presence of L-arabinose the diguanylate cyclase gene adrA was downregulated in wild type S. Typhimurium. Meanwhile cyaA, encoding an adenylate cyclase, was downregulated in an L-arabinose transport mutant. Using an IPTG-inducible plasmid to deplete c-di-GMP via vieA expression, we were able to abolish the increased biofilm phenotype seen in the transport mutant. However, the mechanism by which the L-arabinose import mutant forms significantly larger biofilms remains to be determined. Regardless, these data suggest that L-arabinose metabolism influences intracellular c-di-GMP levels and therefore biofilm formation. These findings are important when considering the use of an L-arabinose inducible promoter in biofilm conditions.


Assuntos
Arabinose , Proteínas de Bactérias , Biofilmes , Salmonella typhimurium , Arabinose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
3.
Angew Chem Int Ed Engl ; 60(42): 22925-22932, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34428345

RESUMO

The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.


Assuntos
RNA/metabolismo , Arabinose/química , Cristalografia por Raios X , Primers do DNA/metabolismo , Óxido de Deutério/química , Imidazóis/química , Cinética , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA/química , Relação Estrutura-Atividade , Moldes Genéticos , Água/química
4.
Food Chem ; 362: 130167, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34087714

RESUMO

The mucilage extracted from the convection-dried cladodes of O. ficus-indica and O. joconostle, two species of economic importance, delivered three fractions after methanol precipitation. Two were composed of high molar mass polysaccharides, and one included water-soluble mono-, di-, and oligosaccharides. The large polysaccharides have a molar mass range of 4.0 × 103 to 8.0 × 105 g·mol-1 and are consistently composed of galactose, arabinose, xylose, and rhamnose; however, the content of galacturonic acid was different between both fractions and species. Their fermentability by selected probiotics was relatively low, 11-27 % compared to glucose, and decreased with increasing levels of galacturonic acid in the molecules. In the third fraction, previously unreported oligosaccharides were found. These include simple- and complex-structured galactooligosaccharides with arabinosyl-, xylosyl- and galacturonosyl acid residues. Their fermentability by prebiotic species can be ascribed more to their structural characteristics and monosaccharide composition than their molecular dimensions.


Assuntos
Opuntia/química , Mucilagem Vegetal/química , Polissacarídeos/química , Prebióticos , Arabinose/análise , Fermentação , Galactose/análise , Ácidos Hexurônicos/análise , Monossacarídeos/análise , Oligossacarídeos/análise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/análise , Polissacarídeos/metabolismo , Prebióticos/análise , Probióticos/metabolismo
5.
J Bacteriol ; 203(16): e0022421, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096777

RESUMO

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.


Assuntos
Arabinose/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/metabolismo , Ramnose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Repressão Catabólica , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Regulon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
Int J Biol Macromol ; 183: 2074-2087, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34097961

RESUMO

Lycium barbarum polysaccharides (LBPs) are known for their beneficial effects on diabetes, NAFLD and related chronic metabolic diseases induced by high-fat diet (HFD). However, the relevant researches are mainly about the whole crude polysaccharides, the specific active ingredient of LBPs and its bioactivity have been rarely explored. Herein, a homogeneous polysaccharide (LBP-W) was isolated and purified from crude LBPs. Structure characterizations indicated that LBP-W contained a main chain consisting of a repeated unit of →6)-ß-Galp(1 â†’ residues with branches composed of α-Araf, ß-Galp and α-Rhap residues at position C-3. The objective of this study was to evaluate the anti-obesogenic effect of LBP-W and figure out the underlying mechanisms. In vivo efficacy trial illustrated that LBP-W supplements can alleviate HFD-induced mice obesity significantly. Gut microbiota analysis showed that LBP-W not only improved community diversity of intestinal flora, but also regulated their specific genera. Moreover, LBP-W can increase the content of short-chain fatty acids (SCFAs), a metabolite of the intestinal flora. In summary, all these results demonstrated that the homogeneous polysaccharide purified from L. barbarum could be used as a prebiotic agent to improve obesity by modulating the composition of intestinal flora and the metabolism of SCFAs.


Assuntos
Fármacos Antiobesidade/farmacologia , Bactérias/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Prebióticos , Animais , Fármacos Antiobesidade/química , Arabinose/química , Arabinose/farmacologia , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Disbiose , Ácidos Graxos/sangue , Galactose/química , Galactose/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/sangue , Obesidade/microbiologia , Ramnose/química , Ramnose/farmacologia , Relação Estrutura-Atividade
7.
Carbohydr Polym ; 265: 118081, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33966845

RESUMO

The polysaccharide (AP1-b) of molecular weight 6.59 × 105 Da was isolated from lignified okra (Abelmoschus esculentus (L.) Moench) by hot-water extraction, 40 % ethanol precipitation and purified by DEAE Cellulose chromatography, respectively. The structure and anti-inflammatory activity of AP1-b were investigated. AP1-b was composed of galactose, rhamnose, gluctose, arabinose and galacturonic acid in a molar ratio of 1.98:1.00:0.15:0.32:0.29. The structural features showed that the AP1-b consisted of →2)-α-d-Rhap-(1→, →4)-ß-d-Galp-(1→, →4)-α-d-GalpA-(1→, →6)-ß-d-Galp-(1→, ß-d-Glcp-(1→ and α-l-Araf-(1→. AP1-b could observably improve the inflammatory injury of LPS-induced RAW 264.7 cells by inhibiting the secretion of NO and decreasing the levels of pro-inflammatory factors (IL-1ß, iNOS and TNF-α). AP1-b also inhibited the phosphorylation levels of IκB and p65 proteins, manifesting the anti-inflammatory activity of AP1-b may associated with inhibition of NF-κB signaling pathway. Therefore, AP1-b had potential value in treating inflammatory injury.


Assuntos
Abelmoschus/química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Arabinose/química , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Galactose/química , Ácidos Hexurônicos/química , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Peso Molecular , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Células RAW 264.7 , Ramnose/química
8.
Food Funct ; 12(12): 5451-5464, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-33997873

RESUMO

A novel heteropolysaccharide fraction (BBP-24-3) with a relative molecular weight of 145.1 kDa was isolated from blackberry fruits. The BBP-24-3 was mainly composed of arabinose, glucose, and galacturonic acid with a ratio of 5.30 : 3.60 : 91.10 mol%. Structural analysis showed that BBP-24-3 possessed a 1,6-linked ß-d-Glcp, 1, 2, 3, 5 linked α-l-Araf, and 1, 4 linked α-d-GalpA backbone with branches substituted at the C-2 and C-5 positions of arabinose units, which included 1, 2, 3, 4 linked ß-d-Glcp and T-linked ß-d-GalpA. The conformation analysis indicated that BBP-24-3 exhibited a solid spherical structure with a uniform distribution in 0.1 M NaCl solution. The BBP-24-3 exhibited excellent α-glucosidase inhibitory activity with an IC50 value of 3.70 mg mL-1, which was due to the structural change, including α-helix and random coil of α-glucosidase caused by BBP-24-3. The current work suggests the potential utilization of BBP-24-3 as an α-glucosidase inhibitor in healthy food for reducing the postprandial blood glucose level.


Assuntos
Frutas/química , Hipoglicemiantes/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Rubus/química , Arabinose , Glicemia , Glucose , Inibidores de Glicosídeo Hidrolases/química , Ácidos Hexurônicos , Conformação Molecular , Peso Molecular , Polissacarídeos/isolamento & purificação , alfa-Glucosidases/química
9.
FEMS Yeast Res ; 21(4)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33890624

RESUMO

The capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.


Assuntos
Proteínas Fúngicas/metabolismo , Kluyveromyces/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Pentoses/metabolismo , Arabinose/metabolismo , Transporte Biológico , Xilose/metabolismo
10.
Biochem Biophys Res Commun ; 556: 163-170, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33845307

RESUMO

Although efficient methods of gene silencing have been established in eukaryotes, many different techniques are still used in bacteria due to the lack of a standardized tool. Here, we developed a convenient and efficient method to downregulate the expression of a specific gene using ∼140 nucleotide RNA with a 24-nucleotide antisense region from an arabinose-inducible expression plasmid by taking Escherichia coli lacZ and phoA genes encoding ß-galactosidase and alkaline phosphatase, respectively, as target genes to evaluate the model. We examined the antisense RNA (asRNA) design, including targeting position, uORF stability elements at the 5'-end, and Hfq-binding module at the 3'-end, and inducer amount required to obtain effective experimental conditions for gene silencing. Furthermore, we constructed multiplexed dual-acting asRNA genes in the plasmid, which were transcribed as polycistronic RNA and were able to knockdown multiple target genes simultaneously. We observed the highest inhibition level of 98.6% when lacZ was targeted using the pMKN104 asRNA expression plasmid, containing a five times stronger PBAD -10 promoter sequence with no requirement of the Hfq protein for repression. These features allow the system to be utilized as an asRNA expression platform in many bacteria, besides E. coli, for gene regulation.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Inativação Gênica , Genes/genética , RNA Antissenso/genética , Arabinose/metabolismo , Arabinose/farmacologia , Sequência de Bases , Códon de Iniciação/genética , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes/efeitos dos fármacos , Genes Reporter , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Antissenso/biossíntese
11.
Carbohydr Res ; 503: 108311, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33866267

RESUMO

A series of oleanolic acid derivatives bearing acetyl-substituted l-arabinose moiety has been synthesized and screened in vitro for cytotoxicity against ten cancer cell lines and four normal cell lines. The antiproliferative evaluation indicated that synthetic derivatives showed excellent selectivity, as they were toxic against only A431 cell line. Among them, the compound 6 possesses the best inhibitory activity. A series of pharmacology experiments showed that compound 6 significantly induced A431 cells apoptosis and cell cycle arrest, which could serve as a promising lead candidate for further study.


Assuntos
Antineoplásicos/farmacologia , Arabinose/farmacologia , Ácido Oleanólico/farmacologia , Saponinas/farmacologia , Acetilação , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Arabinose/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Conformação Molecular , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Saponinas/síntese química , Saponinas/química
12.
Carbohydr Polym ; 264: 118038, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33910721

RESUMO

Cassava is one of the three major potato crops due to the high starch content in its tubers. Unlike most current studies on the utilization of cassava tubers, our research is mainly focused on the stem of cassava plant. Through nuclear magnetic resonance (NMR), fourier transform infrared spectrometer (FTIR) and other methods, we found that cassava stalk hemicellulose consists of ß-1,4 glycosidic bond-linked xylan backbone with a tetrasaccharide reducing end and decorated with methylated glucuronic acid, acetyl groups and a high degree of arabinose substitutions. Hemicellulose content gradually increased from the upper to the lower parts of the stem. The apical part of cassava stalk contained more branched and heterogeneous glycans than the middle and basal parts, and the molecular weight of hemicellulose increased from top to bottom. Our findings will be helpful in understanding of structural variations of cassava hemicellulose during xylogenesis, as well as in better utilization of cassava plant waste in industry.


Assuntos
Manihot/química , Caules de Planta/química , Polissacarídeos/química , Arabinose/química , Produtos Agrícolas/química , Espectroscopia de Ressonância Magnética/métodos , Peso Molecular , Desenvolvimento Vegetal , Tubérculos/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Amido/química , Açúcares/química , Termogravimetria/métodos , Xilanos/química
13.
Nat Chem Biol ; 17(7): 817-827, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33903769

RESUMO

The L-arabinose-responsive AraC and its cognate PBAD promoter underlie one of the most often used chemically inducible prokaryotic gene expression systems in microbiology and synthetic biology. Here, we change the sensing capability of AraC from L-arabinose to blue light, making its dimerization and the resulting PBAD activation light-inducible. We engineer an entire family of blue light-inducible AraC dimers in Escherichia coli (BLADE) to control gene expression in space and time. We show that BLADE can be used with pre-existing L-arabinose-responsive plasmids and strains, enabling optogenetic experiments without the need to clone. Furthermore, we apply BLADE to control, with light, the catabolism of L-arabinose, thus externally steering bacterial growth with a simple transformation step. Our work establishes BLADE as a highly practical and effective optogenetic tool with plug-and-play functionality-features that we hope will accelerate the broader adoption of optogenetics and the realization of its vast potential in microbiology, synthetic biology and biotechnology.


Assuntos
Fator de Transcrição AraC/genética , Arabinose/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Luz , Fator de Transcrição AraC/metabolismo , Arabinose/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo
14.
J Biosci Bioeng ; 131(6): 647-654, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33676868

RESUMO

Xylan is the major hemicellulose present in sugarcane stem secondary cell walls. Xylan is composed of xylose backbone with a high degree of substitutions, which affects its properties. In the present study, the xylan from sugarcane tops (SCT) was extracted and characterized. Compositional analysis of xylan extracted from SCT (SCTx) displayed the presence of 74% of d-xylose residues, 16% of d-glucuronic acid residues and 10% of l-arabinose. High performance size exclusion chromatographic analysis of SCTx displayed a single peak corresponding to a molecular mass of ∼57 kDa. The Fourier transform infrared spectroscopic analysis of SCTx displayed the peaks corresponding to those obtained from commercial xylan. FESEM analysis of SCTx showed the granular and porous surface structure. Differential thermogravimetric analysis (DTG) of SCTx displayed two thermal degradation temperatures (Td) of 228°C, due to breakdown of the side chains of glucuronic acid and arabinose and 275°C, due to breakdown of xylan back bone. The presence of arabinose and glucuronic acid as a side chains was confirmed by the DTG and thermogravimetric analysis. The CHNS analysis of SCTx showed the presence of only carbon and hydrogen supporting its purity. The recombinant xylanase (CtXyn11A) from Clostridium thermocellum displayed a specific activity of 1394 ± 51 U/mg with SCTx, which was higher than those with commercial xylans. The thin layer chromatography and electrospray ionization mass spectroscopy analyses of CtXyn11A hydrolysed SCTx contained a series of linear xylo-oligosaccharides ranging from degree of polymerization 2-6 and no substituted xylo-oligosaccharides because of the endolytic activity of enzyme. The extracted xylan from SCT can be used as an alternative commercial substrate and for oligo-saccharide production.


Assuntos
Saccharum/química , Xilanos/isolamento & purificação , Arabinose/isolamento & purificação , Arabinose/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Comércio , Indústria Alimentícia , Ácido Glucurônico/isolamento & purificação , Ácido Glucurônico/metabolismo , Hidrólise , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/metabolismo , Componentes Aéreos da Planta/química , Componentes Aéreos da Planta/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Saccharum/metabolismo , Xilanos/química , Xilanos/metabolismo , Xilose/isolamento & purificação , Xilose/metabolismo
15.
J Agric Food Chem ; 69(12): 3638-3646, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33734700

RESUMO

Gliadins are the main cause of wheat allergies, and the prevalence of gliadin allergy has increased in many countries. l-Arabinose, a kind of plant-specific five-carbon aldose, possesses beneficial effects on food allergy to gliadins. This study investigated the antiallergic activities and underlying mechanisms of l-arabinose in a wheat gliadin-sensitized mouse model. BALB/c mice were sensitized to gliadin by intraperitoneal injections with gliadin followed by being given a gliadin challenge. l-arabinose-treated mice exhibited a marked reduction in the productions of total immunoglobulin E (IgE), gliadin-specific IgE, gliadin-specific IgG1, and histamine, with an increase in IgG2a level as compared with gliadin-sensitized mice. Beside that, a significant decrease in Th2-related cytokine level, IL-4, and an increase in Th1-related cytokine level, IFN-γ, in the serum and splenocytes were observed after treatment with l-arabinose. l-Arabinose treatment also improved the imbalance of Th1/Th2 immune response on the basis of the expression levels of related cytokines and key transcription factors in the small intestine and spleen of sensitized mice. In addition, gliadin-induced intestinal barrier impairment was blocked by l-arabinose treatment via regulation of TJ proteins and suppression of p38 MAPK and p65 NF-κB inflammation signaling pathways. Notably, the results confirmed that l-arabinose treatment increased CD4+ Foxp3+ T cell populations and Treg-related factors associated with increased expression of IL-2 and activation of STAT5 in gliadin-sensitized mice. In conclusion, l-arabinose attenuated the gliadin-induced allergic symptoms via maintenance of Th1/Th2 immune balance and regulation of Treg cells in a gliadin-induced mouse model, suggesting l-arabinose could be used as a promising agent to alleviate gliadin allergy.


Assuntos
Hipersensibilidade Alimentar , Gliadina , Animais , Arabinose , Citocinas/genética , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores , Células Th1 , Células Th2 , Regulação para Cima
16.
Int J Biol Macromol ; 180: 152-160, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33741368

RESUMO

Sedum sarmentosum Bunge (SS) is clinically used as Chinese medicine for hepatitis related diseases treatment. The purpose of this study was to explore the chemical structures of polysaccharides from this plant. A neutral polysaccharide (SSWP) was isolated and purified by ion-exchange chromatography and Superdex-75 column. The obtained SSWP was a homogenous one with a molecular weight of 21.5 kDa according to the high-performance gel permeation chromatography. The major monosaccharide composition of SSWP was arabinose, glucose and galactose in a molar ratio of 2.4:1:1.8. The methylation analysis showed that SSWP consists mainly of Araf-(1→, →5)-Araf-(1→, →3,5)-Araf-(1→, →4)-Galp-(1→, →4)-Glcp-(1→. The NMR result and enzymatic digestion data comprehensively indicated that SSWP was a novel arabinogalactoglucan-type structure. The anticancer assay in vitro exhibited that SSWP could effectively inhibit 48.9% of Huh-7 cells growth at 50 µg/mL and arrest cells at S-phase, and induce tumor cells apoptosis. Together, polysaccharide from S. sarmentosum Bunge could be a potential natural antitumor agent.


Assuntos
Antineoplásicos/química , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Glucanos/química , Glucanos/farmacologia , Neoplasias Hepáticas/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Sedum/química , Apoptose/efeitos dos fármacos , Arabinose/análise , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Galactose/análise , Humanos , Neoplasias Hepáticas/patologia , Peso Molecular
17.
Microb Cell Fact ; 20(1): 27, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33522916

RESUMO

BACKGROUND: Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with L-arabinose in a newly constructed Escherichia coli expression host BL21-AI, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. RESULTS: Here, we show that BL21-AI is able to precisely regulate protein production rates on a cellular level in an L-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to L-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting. CONCLUSIONS: In all cases tested, BL21-AI outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.


Assuntos
Arabinose/farmacologia , Bacteriófago T7/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Engenharia Genética , Proteínas de Fluorescência Verde/metabolismo , Isopropiltiogalactosídeo/farmacologia , Cinética , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Carbohydr Polym ; 257: 117667, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33541670

RESUMO

While gut bacteria have different abilities to utilize dietary fibers, the degree of fiber structural alignment to bacteria species is not well understood. Corn bran arabinoxylan (CAX) was used to investigate how minor polymer fine structural differences at the genotype × environment level influences the human gut microbiota. CAXs were extracted from 4 corn genotypes × 3 growing years and used in in vitro fecal fermentations. CAXs from different genotypes had varied contents of arabinose/xylose ratio (0.46-0.54), galactose (58-101 mg/g), glucuronic acid (18-32 mg/g). There was genotype- but not environment-specific differences in fine structures. After 24 h fermentation, CAX showed different acetate (71-86 mM), propionate (35-44 mM), butyrate (7-10 mM), and total short chain fatty acid (SCFA) (117-137 mM) production. SCFA profiles and gut microbiota both shifted in a genotype-specific way. In conclusion, the study reveals a very high specificity of fiber structure to gut bacteria use and SCFA production.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Xilanos/química , Zea mays/genética , Acetatos/química , Arabinose/química , Butiratos/química , Fibras na Dieta/análise , Ácidos Graxos Voláteis/química , Fezes , Fermentação , Galactose/química , Genótipo , Ácido Glucurônico/química , Humanos , Polímeros/química , Propionatos/química , Xilose/química
19.
Curr Microbiol ; 78(3): 944-953, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33638002

RESUMO

Pedobacter are a representative genus of soil-associated bacteria. Here we have provided the complete genome sequence of Pedobacter sp. PAMC26386 isolated from Antarctic soil, and functionally annotated the genome, describing the unique features of carbohydrate active enzymes (CAZymes) and α-L-arabinofuranosidase (α-L-ABF). The genome of Pedobacter sp. PAMC26386 is circular and comprises 4,796,773 bp, with a 38.2% GC content. The genome encodes 4,175 genes, including 7 rRNA and 44 tRNA genes. We identified 172 genes (8 auxiliary activities, 8 carbohydrate binding modules, 23 carbohydrate esterases, 86 glycoside hydrolases, 42 glycosyl transferases, and 5 polysaccharide lyases) related to CAZymes using the dbCAN2 tool. We checked enzyme activity on 11 substrates using the AZCL assay and obtained strong activity for arabinooligosaccharide and hemicellulose. This includes information regarding α-L-ABF, which is active at low temperatures, based on the annotation results. Our findings on Pedobacter sp. PAMC26386 provide the basis for research in the future. The favorable properties of Pedobacter sp. PAMC26386 make it a good candidate for industrial applications involving low temperatures.


Assuntos
Pedobacter , Regiões Antárticas , Arabinose , DNA Bacteriano/genética , Ácidos Graxos , Pedobacter/genética , Filogenia , Polissacarídeos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Temperatura
20.
Microbiology (Reading) ; 167(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33555250

RESUMO

l-Arabinose, a major constituent pentose of plant cell-wall polysaccharides, has been suggested to be a less preferred carbon source for fungi but to be a potential signalling molecule that can cause distinct genome-wide transcriptional changes in fungal cells. Here, we explore the possibility that this unique pentose influences the morphological characteristics of the phytopathogenic fungus Bipolaris maydis strain HITO7711. When grown on plate media under different sugar conditions, the mycelial dry weight of cultures on l-arabinose was as low as that with no sugar, suggesting that l-arabinose does not substantially contribute to vegetative growth. However, the intensity of conidiation on l-arabinose was comparable to or even higher than that on d-glucose and on d-xylose, in contrast to the poor conidiation under the no-sugar condition. To explore the physiological basis of the passive growth and active conidiation on l-arabinose, we next investigated cellular responses of the fungus to these sugar conditions. Transcriptional analysis of genes related to carbohydrate metabolism showed that l-arabinose stimulates carbohydrate utilization through the hexose monophosphate shunt (HMP shunt), a catabolic pathway parallel to glycolysis and which participates in the generation of the reducing agent NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). Then, the HMP shunt was impaired by disrupting the related gene BmZwf1, which encodes glucose-6-phosphate dehydrogenase in this fungus. The resulting mutants on l-arabinose showed remarkably decreased conidiation, but a conversely increased mycelial dry weight compared with the wild-type. Our study demonstrates that l-arabinose acts to enhance resource allocation to asexual reproduction in B. maydis HITO7711 at the cost of vegetative growth, and suggests that this is mediated by the concomitant stimulation of the HMP shunt.


Assuntos
Arabinose/metabolismo , Bipolaris/crescimento & desenvolvimento , Bipolaris/metabolismo , Bipolaris/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Mutação , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Reprodução Assexuada , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...