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1.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361791

RESUMO

As a key enzyme regulating postprandial blood glucose, α-Glucosidase is considered to be an effective target for the treatment of diabetes mellitus. In this study, a simple, rapid, and effective method for enzyme inhibitors screening assay was established based on α-glucosidase catalyzes reactions in a personal glucose meter (PGM). α-glucosidase catalyzes the hydrolysis of maltose to produce glucose, which triggers the reduction of ferricyanide (K3[Fe(CN)6]) to ferrocyanide (K4[Fe(CN)6]) and generates the PGM detectable signals. When the α-glucosidase inhibitor (such as acarbose) is added, the yield of glucose and the readout of PGM decreased accordingly. This method can achieve the direct determination of α-glucosidase activity by the PGM as simple as the blood glucose tests. Under the optimal experimental conditions, the developed method was applied to evaluate the inhibitory activity of thirty-four small-molecule compounds and eighteen medicinal plants extracts on α-glucosidase. The results exhibit that lithospermic acid (52.5 ± 3.0%) and protocatechualdehyde (36.8 ± 2.8%) have higher inhibitory activity than that of positive control acarbose (31.5 ± 2.5%) at the same final concentration of 5.0 mM. Besides, the lemon extract has a good inhibitory effect on α-glucosidase with a percentage of inhibition of 43.3 ± 3.5%. Finally, the binding sites and modes of four active small-molecule compounds to α-glucosidase were investigated by molecular docking analysis. These results indicate that the PGM method is feasible to screening inhibitors from natural products with simple and rapid operations.


Assuntos
Benzaldeídos/farmacologia , Benzofuranos/farmacologia , Glicemia/análise , Catecóis/farmacologia , Depsídeos/farmacologia , Diabetes Mellitus Tipo 2/diagnóstico , Inibidores de Glicosídeo Hidrolases/farmacologia , Monitorização Ambulatorial/métodos , alfa-Glucosidases/sangue , Acarbose/química , Acarbose/farmacologia , Benzaldeídos/química , Benzaldeídos/isolamento & purificação , Benzofuranos/química , Benzofuranos/isolamento & purificação , Sítios de Ligação , Técnicas Biossensoriais/instrumentação , Catecóis/química , Catecóis/isolamento & purificação , Depsídeos/química , Depsídeos/isolamento & purificação , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores de Glicosídeo Hidrolases/química , Humanos , Hidrólise , Cinética , Maltose/metabolismo , Simulação de Acoplamento Molecular , Monitorização Ambulatorial/instrumentação , Extratos Vegetais/química , Plantas Medicinais , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Termodinâmica , Dispositivos Eletrônicos Vestíveis , alfa-Glucosidases/química
2.
Food Microbiol ; 98: 103644, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875226

RESUMO

The potential of yeasts isolated from traditional chichas as starter cultures, either for controlled production of the native beverage or for industrial beer production, has been investigated. Three S. cerevisiae strains and one T. delbrueckii strain isolated from four different Ecuadorian chichas were compared to ale and lager beer strains with respect to fermentation performance, sugar utilisation, phenolic off-flavour production, flocculation and growth at low temperature. Fermentations were performed in 15 °P all-malt wort and in a model chicha substrate at 12 °C and 20 °C. Tall-tube fermentations (1.5 L) were also performed with both substrates to assess yeast performance and beer quality. Among the strains tested, only one Ecuadorian S. cerevisiae strain was able to ferment the wort sugars maltose and maltotriose. Fermentations with all Ecuadorian strains were poor in wort at 12 °C relative to 20 °C, but were similar in model chicha substrate at both temperatures. The aromatic profile was different between species and strains. These results indicate the potential of yeasts derived from traditional Andean fermented beverages for commercial applications. One of the chicha strains demonstrated traits typical of domesticated brewery strains and could be suitable for ale fermentation, while the other strains may have potential for low-alcohol beer or chicha production.


Assuntos
Bebidas Alcoólicas/microbiologia , Saccharomyces cerevisiae/metabolismo , Trissacarídeos/metabolismo , Zea mays/microbiologia , Cerveja/microbiologia , Equador , Fermentação , Aromatizantes/química , Aromatizantes/metabolismo , Microbiologia de Alimentos , Maltose/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Leveduras/classificação , Leveduras/genética , Leveduras/metabolismo , Zea mays/metabolismo
3.
Int J Biol Macromol ; 175: 254-261, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33561459

RESUMO

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the ß-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 ߠ→ α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 ߠ→ α showed catalytic efficiency (kcat/Km) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 ߠ→ α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 ߠ→ α may be more suitable than the wild-type for cyclodextrin production.


Assuntos
Bacillus/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Maltose/metabolismo , Bacillus/genética , Proteínas de Bactérias/química , Sítios de Ligação/genética , Ciclização/genética , Ciclodextrinas/metabolismo , Cinética , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Paenibacillus/genética , Especificidade por Substrato/genética , beta-Ciclodextrinas/química
4.
mBio ; 12(1)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33468692

RESUMO

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure.IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.


Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Metabolismo Energético/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Trealose/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Fatores Corda/metabolismo , Fatores Corda/farmacologia , Diarilquinolinas/farmacologia , Metabolismo Energético/genética , Galactanos/metabolismo , Galactanos/farmacologia , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Maltose/metabolismo , Maltose/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Ácidos Micólicos/farmacologia , Rifampina/farmacologia , Trealose/farmacologia
5.
Int J Biol Macromol ; 172: 503-514, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33454330

RESUMO

The study aimed to reveal the different mechanisms of delaying starch digestion by ECG, EGCG and Procyanidin based on the perspective of α-amylase-flavanol interaction and starch-flavanol interaction. The interaction characteristics of flavanols with α-amylase were studied from five aspects: enzyme inhibition, kinetics, fluorescence quenching, circular dichroism (CD) and computer simulation. The IC50 of flavanols (ECG, EGCG and Procyanidin) against α-amylase were 172.21 ± 0.22, 732.15 ± 0.13 and 504.45 ± 0.19 µg/mL according to the results of α-amylase inhibition experiment, respectively. ECG and Procyanidin showed mixed inhibition against α-amylase, while EGCG showed non-competition against α-amylase. However, thermodynamic parameters,computer-based docking and dynamic simulation proved that ECG and EGCG-α-amylase complexs were mainly driven by van der Waals and hydrogen bonds, while Procyanidin-α-amylase complexs was driven by hydrophobic interaction. In addition, it was indicated, by means of starch­iodine complex spectroscopy, that flavanols inhibited the digestion of starch not only through bind with α-amylase but also through bind with starch. Thus, flavanols as a starch-based food additive have the potential to be employed as adjuvant therapy for diabetes.


Assuntos
Biflavonoides/química , Catequina/análogos & derivados , Inibidores de Glicosídeo Hidrolases/química , Proantocianidinas/química , Amido/química , alfa-Amilases/química , Biflavonoides/metabolismo , Sítios de Ligação , Catequina/química , Catequina/metabolismo , Glucose/química , Inibidores de Glicosídeo Hidrolases/metabolismo , Hidrólise , Cinética , Maltose/química , Maltose/metabolismo , Simulação de Acoplamento Molecular , Proantocianidinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Amido/metabolismo , Especificidade por Substrato , Termodinâmica , Trissacarídeos/química , Trissacarídeos/metabolismo , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismo
6.
Int J Biol Macromol ; 171: 166-176, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33421464

RESUMO

Exploring new multifunctional enzymes and understanding the mechanisms of catalytic promiscuity will be of enormous industrial and academic values. In the present study, we reported the discovery and characterization of a multifunctional enzyme BSGH13 from Bacillus subtilis BS-5. Remarkably, BSGH13 possessed α-amylase, endoglucanase, and xylanase activities. To our knowledge, this was the first report on an amylase from Bacillus species having additional endoglucanase and xylanase activities. Subsequently, we analyzed the effects of aromatic residues substitution at each site of the active site architecture on ligand-binding affinity and catalytic specificity of BSGH13 by a combination of virtual mutation and site-directed mutagenesis approaches. Our results indicated that the introduction of aromatic amino acids Phe or Trp at the positions L182 and L183 altered the local interaction network of BSGH13 towards different substrates, thus changing the multifunctional properties of BSGH13. Moreover, we provided an expanded perspective on studies of multifunctional enzymes.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Celulase/química , Endo-1,4-beta-Xilanases/química , alfa-Amilases/química , Substituição de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Celulase/genética , Celulase/metabolismo , Celulose/análogos & derivados , Celulose/química , Celulose/metabolismo , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Maltose/análogos & derivados , Maltose/química , Maltose/metabolismo , Modelos Moleculares , Mutação , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Tetroses/química , Tetroses/metabolismo , Triptofano/química , Triptofano/genética , Triptofano/metabolismo , Xilanos/química , Xilanos/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo
7.
Food Microbiol ; 94: 103629, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33279061

RESUMO

De novo sourdough cultures were here assessed for their potential as sources of yeast strains for low-alcohol beer brewing. NGS analysis revealed an abundance of ascomycete yeasts, with some influence of grain type on fungal community composition. Ten different ascomycete yeast species were isolated from different sourdough types (including wheat, rye, and barley) and seven of these were screened for a number of brewing-relevant phenotypes. All seven were maltose-negative and produced less than 1% (v/v) alcohol from a 12 °Plato wort in initial fermentation trials. Strains were further screened for their bioflavouring potential (production of volatile aromas and phenolic notes, reduction of wort aldehydes), stress tolerance (temperature extremes, osmotic stress and ethanol tolerance) and flocculence. Based on these criteria, two species (Kazachstania servazzii and Pichia fermentans) were selected for 10 L-scale fermentation trials and sensory analysis of beers. The latter species was considered particularly suitable for production of low-alcohol wheat beers due to its production of the spice/clove aroma 4-vinylguaiacol, while the former showed potential for lager-style beers due to its clean flavour profile and tolerance to low temperature conditions.


Assuntos
Álcoois/análise , Cerveja/microbiologia , Pão/microbiologia , Maltose/metabolismo , Pichia/metabolismo , Saccharomycetales/metabolismo , Álcoois/metabolismo , Cerveja/análise , Fermentação , Aromatizantes/análise , Aromatizantes/metabolismo , Hordeum/metabolismo , Hordeum/microbiologia , Odorantes , Secale/metabolismo , Secale/microbiologia , Triticum/metabolismo , Triticum/microbiologia
8.
Biomed Res Int ; 2020: 9494528, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33145362

RESUMO

The antioxidant function and metabolic profiles in mice after dietary supplementation with methionine were investigated. The results showed that methionine supplementation enhanced liver GSH-Px activity and upregulated Gpx1 expression in the liver and SOD1 and Gpx4 expressions in the jejunum. Nrf2/Keap1 is involved in oxidative stress, and the western blotting data exhibited that dietary methionine markedly increased Keap1 abundance, while failed to influence the Nrf2 signal. Metabolomics investigation showed that methionine administration increased 2-hydroxypyridine, salicin, and asparagine and reduced D-Talose, maltose, aminoisobutyric acid, and inosine 5'-monophosphate in the liver, which are widely reported to involve in oxidative stress, lipid metabolism, and nucleotides generation. In conclusion, our study provides insights into antioxidant function and liver metabolic profiles in response to dietary supplementation with methionine.


Assuntos
Suplementos Nutricionais , Regulação da Expressão Gênica/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Fígado/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Metionina/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Antioxidantes/metabolismo , Asparagina/metabolismo , Álcoois Benzílicos/metabolismo , Dieta/métodos , Feminino , Glucosídeos/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Inosina Monofosfato/metabolismo , Jejuno/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lactonas/metabolismo , Fígado/metabolismo , Maltose/metabolismo , Metaboloma/fisiologia , Metionina/administração & dosagem , Camundongos , Camundongos Endogâmicos ICR , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Piridonas/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
9.
Microbiology (Reading) ; 166(8): 759-776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32490790

RESUMO

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.


Assuntos
Corynebacterium glutamicum/enzimologia , Lipoproteínas/metabolismo , Transferases/metabolismo , Acilação , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/metabolismo , Teste de Complementação Genética , Maltose/metabolismo , Mutação , Mycobacterium tuberculosis/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Transferases/genética
10.
Food Chem ; 330: 127316, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569933

RESUMO

Dynamics of microbial community and changes of metabolites during production of type Ι sourdough steamed bread made by retarded sponge-dough method (SSB) were studied. Lactobacillus sanfranciscensis and Lactobacillus pontis were the dominant bacterial species. Particularly, relative abundances of Lactobacillus sanfranciscensis were significantly higher than that of other sub-dominant bacterial species. The dominant fungal species were Saccharomyces cerevisiae and Kazachstania humilis, and the latter was the most predominant. A stable bacterial and fungal consortia was established in sponge dough retarded from 12 to 24 h and main dough proofed from 30 to 60 min. Metabolism preference for maltose of Lactobacillus sanfranciscensis favoured a mutualistic association with maltose-negative Kazachstania humilis, and hence contributing to their competitiveness and dominance. Volatile compounds became more abundant with much more esters as sponge retarding time extended. Probably, the accumulation of organic acids and ethanol contributed mostly to formation of ethyl esters in sponge dough during retarding.


Assuntos
Pão/microbiologia , Microbiota , Pão/análise , Fermentação , Microbiologia de Alimentos , Lactobacillus/genética , Lactobacillus/metabolismo , Maltose/metabolismo , Microbiota/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vapor , Simbiose , Compostos Orgânicos Voláteis/metabolismo
11.
J Agric Food Chem ; 68(30): 7974-7983, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32551626

RESUMO

Human sweet taste receptor (hSTR) recognizes a wide array of sweeteners, resulting in sweet taste perception. Maltitol and lactitol have been extensively used in place of sucrose due to their capability to prevent dental caries. Herein, several molecular modeling approaches were applied to investigate the structural and energetic properties of these two polyols/hSTR complexes. Triplicate 500 ns molecular dynamics (MD) simulations and molecular mechanics/generalized Born surface area (MM/GBSA)-based free energy calculations revealed that the TAS1R2 monomer is the preferential binding site for maltitol and lactitol rather than the TAS1R3 region. Several polar residues (D142, S144, Y215, D278, E302, R383, and especially N143) were involved in polyols binding through electrostatic attractions and H-bond formations. The molecular complexation process not only induced the stable form of ligands but also stimulated the conformational adaptation of the TAS1R2 monomer to become a close-packed structure through an induced-fit mechanism. Notably, the binding affinity of the maltitol/TAS1R2 complex (ΔGbind of -17.93 ± 1.49 kcal/mol) was significantly higher than that of the lactitol/TAS1R2 system (-8.53 ± 1.78 kcal/mol), in line with the experimental relative sweetness. These findings provide an in-depth understanding of the differences in the sweetness response between maltitol and lactitol, which could be helpful to design novel polyol derivatives with higher sweet taste perception.


Assuntos
Maltose/análogos & derivados , Receptores Acoplados a Proteínas G/metabolismo , Álcoois Açúcares/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Cinética , Maltose/química , Maltose/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Álcoois Açúcares/química
12.
Prep Biochem Biotechnol ; 50(9): 857-864, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32538270

RESUMO

Curdlan has wide potential application in the food and biomedical fields due to its unique thermal gel and biological activity. This study investigated the effect of six sugars including glucose, fructose, lactose, maltose, sucrose and xylose as carbon sources on production and properties of curdlan using Agrobacterium sp. DH-2. The maximum production (38.1 g/L and 37.4 g/L, respectively) and yield (0.58 g curdlan/g sucrose and 0.53 g curdlan/g maltose, respectively) of curdlan were achieved by sucrose and maltose, followed by glucose, fructose, lactose and xylose. Scanning electron micrographs showed that the surface of cells was smooth in strain growth phase, while cells were covered by curdlan matrix acted as a net in the curdlan synthesis phase. The highest glucosyltransferase activity (19.9 U/g biomass) corresponded to the maximum curdlan production using the sucrose medium. The molecular weight and gel strength of curdlan were influenced by the carbon sources. The curdlan from xylose medium resulted in a maximum molecular weight of 1.59 × 106 Da and the highest gel strength of 989.2 g/cm2, while the curdlan from sucrose medium resulted in a lowest molecular weight of 1.10 × 106 Da and gel strength of 672.8 g/cm2. The high molecular weight of curdlan had high gel strength.


Assuntos
Agrobacterium/metabolismo , Microbiologia Industrial , beta-Glucanas/metabolismo , Agrobacterium/enzimologia , Carbono/metabolismo , Meios de Cultura/metabolismo , Glucosiltransferases/metabolismo , Maltose/metabolismo , Sacarose/metabolismo
13.
Acta Crystallogr D Struct Biol ; 76(Pt 5): 418-427, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32355038

RESUMO

Mycoplasma hyopneumoniae is a prokaryotic pathogen that colonizes the respiratory ciliated epithelial cells in swine. Infected animals suffer respiratory lesions, causing major economic losses in the porcine industry. Characterization of the immunodominant membrane-associated proteins from M. hyopneumoniae may be instrumental in the development of new therapeutic approaches. Here, the crystal structure of P46, one of the main surface-antigen proteins, from M. hyopneumoniae is presented and shows N- and C-terminal α/ß domains connected by a hinge. The structures solved in this work include a ligand-free open form of P46 (3.1 Šresolution) and two ligand-bound structures of P46 with maltose (2.5 Šresolution) and xylose (3.5 Šresolution) in open and closed conformations, respectively. The ligand-binding site is buried in the cleft between the domains at the hinge region. The two domains of P46 can rotate with respect to each other, giving open or closed alternative conformations. In agreement with this structural information, sequence analyses show similarities to substrate-binding members of the ABC transporter superfamily, with P46 facing the extracellular side as a functional subunit. In the structure with xylose, P46 was also bound to a high-affinity (Kd = 29 nM) Fab fragment from a monoclonal antibody, allowing the characterization of a structural epitope in P46 that exclusively involves residues from the C-terminal domain. The Fab structure in the complex with P46 shows only small conformational rearrangements in the six complementarity-determining regions (CDRs) with respect to the unbound Fab (the structure of which is also determined in this work at 1.95 Šresolution). The structural information that is now available should contribute to a better understanding of sugar nutrient intake by M. hyopneumoniae. This information will also allow the design of protocols and strategies for the generation of new vaccines against this important swine pathogen.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Maltose/metabolismo , Mycoplasma hyopneumoniae/imunologia , Xilose/metabolismo , Sítios de Ligação , Ligação Proteica , Domínios Proteicos
14.
Elife ; 92020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32420869

RESUMO

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics' droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Maltose/metabolismo , RNA-Seq/métodos , Análise de Célula Única/métodos , Carbono/metabolismo , Metabolismo Energético/fisiologia , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Esferoplastos , Transcrição Genética/genética , Transcriptoma/genética
15.
Prep Biochem Biotechnol ; 50(8): 820-826, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32282271

RESUMO

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45 U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.


Assuntos
Glicosiltransferases/metabolismo , Lactobacillus reuteri/enzimologia , Oligossacarídeos/metabolismo , Biocatálise , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosiltransferases/genética , Microbiologia Industrial , Lactobacillus reuteri/genética , Lactobacillus reuteri/metabolismo , Maltose/metabolismo , Modelos Biológicos , Sacarose/metabolismo
16.
PLoS One ; 15(3): e0229734, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32126122

RESUMO

European honeybee, Apis mellifera, produces α-glucosidase (HBGase) that catalyzes the cleavage of an α-glycosidic bond of the non-reducing end of polysaccharides and has potential applications for malt hydrolysis in brewing industry. Characterized by their substrate specificities, HBGases have three isoforms including HBGase II, which prefers maltose to sucrose as a substrate. Previous study found that the catalytic efficiency of maltose hydrolysis of N226P mutant of HBGase II was higher than that of the wild type (WT), and the catalytic efficiency of maltose hydrolysis of WT was higher than those of H227Y and N226P-H227Y mutants. We hypothesized that N226P mutation probably caused maltose to bind with better affinity and position/orientation for hydrolysis than WT, while H227Y and N226P-H227Y mutations caused maltose to bind with worse affinity and position/orientation for hydrolysis than WT. Using this hypothesis, we performed molecular dynamics on the catalytically competent binding conformations of maltose/WT, maltose/N226P, maltose/H227Y, and maltose/N226P-H227Y complexes to elucidate effects of N226P and H227Y mutations on maltose binding in HBGase II active site. Our results reasonably support this hypothesis because the N226P mutant had better binding affinity, higher number of important binding residues, strong and medium hydrogen bonds as well as shorter distance between atoms necessary for hydrolysis than WT, while the H227Y and N226P-H227Y mutants had worse binding affinities, lower number of important binding residues and strong hydrogen bonds as well as longer distances between atoms necessary for hydrolysis than WT. Moreover, results of binding free energy and hydrogen bond interaction of residue 227 support the role of H227 as a maltose preference residue, as proposed by previous studies. Our study provides important and novel insight into how N226P and H227Y mutations affect maltose binding in HBGase II active site. This knowledge could potentially be used to engineer HBGase II to improve its efficiency.


Assuntos
Abelhas/enzimologia , Domínio Catalítico/genética , Proteínas de Insetos/genética , Maltose/metabolismo , alfa-Glucosidases/genética , Substituição de Aminoácidos , Animais , Abelhas/genética , Proteínas de Insetos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética , alfa-Glucosidases/metabolismo
17.
Food Res Int ; 130: 108844, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32156340

RESUMO

Understanding lipid oxidation mechanisms in low moisture foods is necessary to develop antioxidant strategies to increase shelf life and/or to improve nutritional quality by increasing polyunsaturated fatty acid concentrations. In this study, we examined the influence of water activity (aw), sugars (glucose, maltose, maltodextrin, and cyclodextrin), and proteins (casein and gluten) on the lipid hydroperoxide and hexanal lag phases of model crackers. Oxidative stability of crackers was in an order: aw 0.7 > aw 0.4 > aw 0.2 > aw 0.05. Higher water activities resulted in bigger differences between hydroperoxide lag phases and hexanal lag phases. Compared to non-reducing cyclodextrin and no added sugar controls, reducing sugars including glucose, maltose, and maltodextrin at the same dextrose equivalence increased both hydroperoxide and hexanal lag phases. At the same dextrose equivalence, oxidative stability was in the order of maltose > maltodextrin > glucose > control (no sugar added). The antioxidant effectiveness of maltose, a low sweetness profile sugar, increased with increasing concentrations from 1.1 to 13.8%. Increasing aw increased the antioxidant activity of maltose. For example, 1.1% maltose increased both hydroperoxides and hexanal lag phases by 9 days at an aw of 0.2, but increased hydroperoxide lag phase by 24 days and hexanal lag phase by 15 days at an aw of 0.7. Gluten was able to inhibit lipid oxidation with activity increasing with increasing aw while casein showed minimal antioxidant impact. Antioxidant activity of gluten decreased when its sulfhydryl groups were blocked by N-ethylmaleimide suggesting that cysteine was an important antioxidant component of gluten. Adjusting water activity and addition of reducing sugars and gluten could be strategies to increase oxidative stability of low moisture crackers.


Assuntos
Grão Comestível/química , Análise de Alimentos/métodos , Metabolismo dos Lipídeos/fisiologia , Proteínas/metabolismo , Açúcares/metabolismo , Água/química , Antioxidantes/metabolismo , Caseínas/metabolismo , Ciclodextrinas/metabolismo , Glucose/metabolismo , Glutens/metabolismo , Maltose/metabolismo , Oxirredução , Polissacarídeos/metabolismo
18.
Food Funct ; 11(2): 1672-1683, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32031198

RESUMO

Novel α-glucooligosaccharides were synthesized by the acceptor reaction of Leuconostoc citreum SK24.002 glucansucrase with maltose and sucrose. The impact of synthesis conditions, including the ratio of sucrose to maltose and the substrate concentration, on the formation of α-glucooligosaccharides was evaluated. Under the optimized experimental conditions, the yield of a mixture of α-glucooligosaccharides with DP 3-5 reached approximately 56.4% with a concentration of 170.7 mg mL-1. Each of these α-glucooligosaccharides was purified, and the structures were assigned as follows: α-D-Glcp-(1,6)-α-D-Glcp-(1,4)-D-Glcp (DP3), α-D-Glcp-(1,3)-α-D-Glcp-(1,6)-α-D-Glcp-(1,4)-D-Glcp (DP4), and α-D-Glcp-(1,6)-α-D-Glcp-(1,3)-α-D-Glcp-(1,6)-α-D-Glcp-(1,4)-D-Glcp (DP5), respectively. For these three structurally different oligosaccharides, the fermentation selectivity by fecal bacteria was determined in anaerobic batch culture. Fructooligosaccharide (FOS) was used as a positive prebiotic control. Similar to FOS, all three α-glucooligosaccharides selectively stimulated the proliferation of Bifidobacteria and Lactobacilli compared with the control. DP3 exhibited the strongest prebiotic ability to increase the Bifidobacterium and Lactobacillus population, whereas DP5 produced the most short-chain fatty acids. In addition, DP4 produced the highest butyrate concentration and resulted in the lowest acetate : propionate ratio. These results suggested that the enzymatically synthesized α-glucooligosaccharides were potential prebiotics, underlining correlations between the structural features of oligosaccharides and their impact on the metabolism of fecal microbiota.


Assuntos
Fermentação/fisiologia , Glicosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Prebióticos , Proteínas de Bactérias/metabolismo , Bifidobacterium/metabolismo , Fezes/microbiologia , Humanos , Lactobacillus/metabolismo , Leuconostoc/enzimologia , Maltose/metabolismo , Sacarose/metabolismo
19.
Biochem Biophys Res Commun ; 523(3): 651-657, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31948759

RESUMO

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.


Assuntos
Dextranase/metabolismo , Glucosiltransferases/metabolismo , Leuconostoc mesenteroides/enzimologia , Maltose/metabolismo , Oligossacarídeos/metabolismo , Dextranos/química , Dextranos/metabolismo , Hidrólise , Leuconostoc mesenteroides/química , Leuconostoc mesenteroides/metabolismo , Oligossacarídeos/química , Especificidade por Substrato
20.
Biomacromolecules ; 21(2): 955-965, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31917581

RESUMO

Soft nanoparticles are interesting materials due to their size, deformability, and ability to host guest molecules. Surface properties play an essential role in determining the fate of the particles in biological medium, and coating of the nanoparticles (and polymers) with carbohydrates has been found to be an efficient strategy for increasing their biocompatibility and fine-tuning other important properties such as aqueous solubility. In this work, soft nanogels of poly(N-vinylcaprolactam), PNVCL, were surface-functionalized with different glucose and maltose ligands, and the colloidal properties of the gels were analyzed. The PNVCL nanogels were first prepared via semibatch precipitation polymerization, where a comonomer, propargyl acrylate (PA), was added after preparticle formation. The aim was to synthesize "clickable" nanogels with alkyne groups on their surfaces. The nanogels were then functionalized with two separate azido-glucosides and azido-maltosides (containing different linkers) through a copper-catalyzed azide-alkyne cycloaddition (CuAAc) click reaction. The glucose and maltose bearing nanogels were thermoresponsive and shrank upon heating. Compared to the PNVCL-PA nanogel, the carbohydrate bearing ones were larger, more hydrophilic, had volume phase transitions at higher temperatures, and were more stable against salt-induced precipitation. In addition to investigating the colloidal properties of the nanogels, the carbohydrate recognition was addressed by studying the interactions with a model lectin, concanavalin A (Con A). The binding efficiency was not affected by the temperature, which indicates that the carbohydrate moieties are located on the gel surfaces, and are capable of interacting with other biomolecules independent of temperature. Thus, the synthesis produces nanogels, which have surface functions capable of biorelevant interactions and a thermoresponsive structure. These types of particles can be used for drug delivery.


Assuntos
Caprolactama/análogos & derivados , Glucose/química , Maltose/química , Nanogéis/química , Polímeros/química , Caprolactama/química , Caprolactama/metabolismo , Coloides/química , Coloides/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Glucose/metabolismo , Maltose/metabolismo , Polímeros/metabolismo , Propriedades de Superfície , Temperatura
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