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1.
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
2.
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
3.
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
4.
Carbohydr Res ; 488: 107904, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901816

RESUMO

Starch hydrolysis to maltose by nano-magnetic combined cross-linked enzyme aggregates of α-amylase and maltogenic amylase (NM-Combi-CLEAs) is an important step to open new perspectives for special food and pharmaceutic production. Improvement of mass transfer, thermostability, functional specificity, and reusability of combined enzymes was performed. The obtained results exhibited that, 1:9 ratio of α-amylase/maltogenic amylase, use of tert-butanol as precipitant, 2 mM glutardialdehyde, 1:0.75 ratios of combined enzymes to lysine, 20 h crosslinking at 3-4 °C are well-suited conditions. The dynamic light scattering (DLS) results implied that the nanomagnetites diameter was about 81.9-88.9 nm, with polydispersity index (PDI) of 0.242 and a Ȥ-potential of -21 mV. Moreover, the particle size, PDI, and Ȥ-potential of NM-Combi-CLEAs were around 99.6 nm, 0.088, and -32 mV respectively. The NM-Combi-CLEAs kept 80.4% of its original activity after 10 cycles, its Km value exhibited about 1.5 folds reduction with about 1.5 times enhance in thermostability at 95 °C than free one. Immobilization activity yield revealed about 84% of activity retaining by NM-Combi-CLEAs strategy. Accordingly, this efficacious nanobiocatalyst with high thermostability and reusability recommended for starch conversion to maltose.


Assuntos
Glicosídeo Hidrolases/metabolismo , Maltose/metabolismo , Amido/química , alfa-Amilases/metabolismo , Difusão Dinâmica da Luz , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , Nanopartículas de Magnetita , Tamanho da Partícula , Termodinâmica
5.
J Agric Food Chem ; 68(5): 1419-1426, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31888328

RESUMO

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.


Assuntos
Bebidas Alcoólicas/microbiologia , Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeos/metabolismo , alfa-Glucosidases/metabolismo , Bebidas Alcoólicas/análise , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Etanol/metabolismo , Fermentação , Proteínas Fúngicas/genética , Glicosídeos/química , Glicosilação , Maltose/metabolismo , Oryza/metabolismo , Oryza/microbiologia , alfa-Glucosidases/genética
6.
Prep Biochem Biotechnol ; 50(2): 172-180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31846387

RESUMO

The ß-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated ß-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of ß-amylase after the five-reaction cycle was found to decrease to ∼70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60 °C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated ß-amylase. The attempt has been made for the first time for the successful encapsulation of ß-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13 mg/mL).


Assuntos
Reatores Biológicos , Maltose/metabolismo , Amido/metabolismo , beta-Amilase/metabolismo , Óleos/química , Água/química
7.
Colloids Surf B Biointerfaces ; 185: 110600, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704608

RESUMO

In this communication, fenugreek ß-amylase was immobilized onto functionalized tungsten disulfide nanoparticles through cross-linker glutaraldehyde and successful immobilization was confirmed by SEM, AFM and FTIR spectroscopy. To make the process economical and efficient, optimization of independent variables was carried out using Box-Behnken design of response surface methodology. Approximately similar predicted (85.6%) and experimental (84.2%) immobilization efficiency revealed that the model is suitable for design of space. Optimum temperature was calculated to be 60 °C. After immobilization, an increased Km (2.12 times) and a decreased Vmax (0.58 times), indicated inaccessibility of active site residues to the substrate. The immobilized enzyme retained 77% relative activity after 10 uses whereas 40% residual activity was obtained after 120 days. An increased half-life with concomitantly decreased kinetic rate constant revealed that the immobilized enzyme is more stable at a higher temperature and the process followed first-order kinetics (R2 > 0.93). The limit of detection for maltose and sucrose fluorescence biosensor was found to be 0.052 and 0.096 mM, respectively. Thermodynamic parameters such as changes in Gibbs free energy (ΔG < 0), enthalpy (ΔH > 0) and entropy (ΔS >0) revealed that the process is spontaneous and endothermic, driven by hydrophobic interactions. Thermo-stability data at higher temperature for the immobilized enzyme makes it a suitable candidate for industrial applications in the production of maltose in food and pharmaceutical industries. Furthermore, fluorescence biosensor could be used to detect and quantify maltose and sucrose to maintain the quality of industrial products.


Assuntos
Dissulfetos/química , Enzimas Imobilizadas/metabolismo , Maltose/metabolismo , Nanopartículas/química , Sacarose/metabolismo , Trigonella/enzimologia , Compostos de Tungstênio/química , beta-Amilase/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Cinética , Maltose/química , Sacarose/química , Temperatura , Termodinâmica , beta-Amilase/química
8.
Food Chem ; 305: 125447, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499289

RESUMO

A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.


Assuntos
Maltose/metabolismo , Rhizomucor/enzimologia , alfa-Amilases/metabolismo , Pão/análise , Indústria Alimentícia , Concentração de Íons de Hidrogênio , Hidrólise , Pichia/metabolismo , Amido/metabolismo , Especificidade por Substrato , Temperatura , alfa-Amilases/química , alfa-Amilases/genética
9.
Appl Microbiol Biotechnol ; 103(23-24): 9423-9432, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713673

RESUMO

α-Glucosidase, Agl2, from Xanthomonas campestris was successfully overexpressed in Escherichia coli BL21(DE3) cells and purified with Ni columns. The enzyme exhibits glycosylation abilities towards a wide range of phenolic substrates, including phenol, vanillin, and ethyl vanillin, with maltose as the glycosyl donor. The catalytic properties of the purified enzyme were further investigated. It was observed that the synthesized glycosides started to degrade with prolonged catalytic time, giving an "n"-shaped kinetic profile. To understand such catalytic behavior, the Agl2-catalyzed glycosylation process was investigated kinetically. Based on the obtained parameters, it was concluded that although the substrate conversions are thermodynamically restricted in a batch system, the glycosylation efficiency can be kinetically controlled by the glycosylation/hydrolysis selectivity. Glucose was produced by both glycosylation and hydrolysis, significantly impacting the glycosylation efficiency. This study provides a mechanistic understanding of the α-glucosidase-catalyzed glycosylation process in a water-based system. The developed kinetic model was successful in explaining and analyzing the catalytic process. It is suggested that when α-glucosidase is employed for glycosylation in a water-enriched environment, the catalytic efficiency is mainly impacted by the enzyme's glycosylation/hydrolysis selectivity and glucose content in the catalytic environment.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeos/metabolismo , alfa-Glucosidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Catálise , Expressão Gênica , Glucose/metabolismo , Glicosídeos/química , Glicosilação , Hidrólise , Cinética , Maltose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Água/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/isolamento & purificação
10.
J Microbiol Biotechnol ; 29(11): 1729-1738, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31635439

RESUMO

In sourdough fermentation, lactic acid bacteria perform important roles in the production of volatile and antimicrobial compounds, and exerting health-promoting effects. In this study, we report the probiotic properties and baking characteristics of Lactobacillus plantarum SPC-SNU 72-2 isolated from kimchi. This strain is safe to use in food fermentation as it does not carry genes for biogenic amine production (i.e., hdc, tdc, and ldc) and shows no ß-hemolytic activity against red blood cells. The strain is also stable under simulated human gastrointestinal conditions, showing tolerance to gastric acid and bile salt, and adheres well to colonic epithelial cells. Additionally, this strain prevents pathogen growth and activates mouse peritoneal macrophages by inducing cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-12. Furthermore, the strain possesses good baking properties, providing rich aroma during dough fermentation and contributing to the enhancement of bread texture. Taken together, L. plantarum SPC-SNU 72-2 has the properties of a good starter strain based on the observation that it improves bread flavor and texture while also providing probiotic effects comparable with commercial strains.


Assuntos
Pão/microbiologia , Alimentos e Bebidas Fermentados/microbiologia , Microbiologia de Alimentos , Lactobacillus plantarum/metabolismo , Probióticos/metabolismo , Animais , Antibiose , Aderência Bacteriana , Ácidos e Sais Biliares/metabolismo , Pão/análise , Células CACO-2 , Fermentação , Humanos , Imunomodulação , Lactobacillus plantarum/genética , Lactobacillus plantarum/fisiologia , Maltose/metabolismo , Camundongos , Viabilidade Microbiana , Probióticos/análise , Compostos Orgânicos Voláteis/análise
11.
Res Microbiol ; 170(8): 321-337, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31560984

RESUMO

ATP-binding cassette (ABC) transporters use the energy of ATP hydrolysis to transport a large diversity of molecules actively across biological membranes. A combination of biochemical, biophysical, and structural studies has established the maltose transporter MalFGK2 as one of the best characterized proteins of the ABC family. MalF and MalG are the transmembrane domains, and two MalKs form a homodimer of nucleotide-binding domains. A periplasmic maltose-binding protein (MalE) delivers maltose and other maltodextrins to the transporter, and triggers its ATPase activity. Substrate import occurs in a unidirectional manner by ATP-driven conformational changes in MalK2 that allow alternating access of the substrate-binding site in MalF to each side of the membrane. In this review, we present an integrated molecular mechanism of the transport process considering all currently available information. Furthermore, we summarize remaining inconsistencies and outline possible future routes to decipher the full mechanistic details of transport by MalEFGK2 complex and that of related importer systems.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Maltose/metabolismo , Polissacarídeos/metabolismo , Sítios de Ligação , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Modelos Moleculares , Proteínas Periplásmicas de Ligação/metabolismo , Conformação Proteica
12.
J Agric Food Chem ; 67(33): 9314-9324, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352776

RESUMO

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Trealose/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Fermentação , Maltose/metabolismo , Engenharia Metabólica
13.
J Agric Food Chem ; 67(32): 8986-8993, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347835

RESUMO

Trehalose plays a crucial role in response to freezing stress in baker's yeast. MAL62, a gene involved in the adenosine diphosphoglucose-dependent trehalose synthesis pathway, can increase trehalose content. However, the difference between MAL62-related trehalose synthesis and traditional uridine diphosphoglucose-dependent trehalose synthesis is not well-understood. MAL62 overexpression showed less effect in enhancing intracellular trehalose compared to TPS1 overexpression. However, MAL62 overexpression elicited trehalose synthesis before fermentation with enhanced maltose metabolism and had a similar effect on cell viability after freezing. Furthermore, MAL62 and TPS1 overexpression in the NTH1 deletion background further strengthened freezing tolerance and improved leavening ability. Our results suggest that the enhancement in freezing tolerance by MAL62 overexpression may involve multiple pathways rather than simply enhancing trehalose synthesis. The results reveal valuable insights into the relationship between maltose metabolism and freezing tolerance and may help to develop better yeast strains for enhancing fermentation characteristics of frozen dough.


Assuntos
Glucosiltransferases/metabolismo , Maltose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , alfa-Glucosidases/metabolismo , Farinha/análise , Farinha/microbiologia , Congelamento , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Trealase/genética , Trealase/metabolismo , Trealose/metabolismo , alfa-Glucosidases/genética
14.
Biosci Biotechnol Biochem ; 83(8): 1385-1401, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31159661

RESUMO

The koji mold Aspergillus oryzae has been used in traditional Japanese food and beverage fermentation for over a thousand years. Amylolytic enzymes are important in sake fermentation, wherein production is induced by starch or malto-oligosaccharides. This inducible production requires at least two transcription activators, AmyR and MalR. Among amylolytic enzymes, glucoamylase GlaB is produced exclusively in solid-state culture and plays a critical role in sake fermentation owing to its contribution to glucose generation from starch. A recent study demonstrated that glaB gene expression is regulated by a novel transcription factor, FlbC, in addition to AmyR in solid-state culture. Amylolytic enzyme production is generally repressed by glucose due to carbon catabolite repression (CCR), which is mediated by the transcription factor CreA. Modifying CCR machinery, including CreA, can improve amylolytic enzyme production. This review focuses on the role of transcription factors in regulating A. oryzae amylolytic gene expression.


Assuntos
Aspergillus oryzae/genética , Regulação Fúngica da Expressão Gênica , Glucana 1,4-alfa-Glucosidase/metabolismo , Proteínas Fúngicas/genética , Maltose/metabolismo , Fatores de Transcrição/metabolismo
15.
PLoS Genet ; 15(4): e1007853, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946741

RESUMO

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Trissacarídeos/metabolismo , Cerveja/microbiologia , Proteínas de Transporte/química , Evolução Molecular Direcionada , Fermentação , Proteínas Fúngicas/química , Genes Fúngicos , Hibridização Genética , Maltose/metabolismo , Modelos Moleculares , Mutagênese , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Recombinação Genética , Saccharomyces/crescimento & desenvolvimento , Sequenciamento Completo do Genoma
16.
Biochemistry ; 58(17): 2208-2217, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30950267

RESUMO

In cellular environments, proteins not only interact with their specific partners but also encounter a high concentration of bystander macromolecules, or crowders. Nonspecific interactions with macromolecular crowders modulate the activities of proteins, but our knowledge about the rules of nonspecific interactions is still very limited. In previous work, we presented experimental evidence that macromolecular crowders acted competitively in inhibiting the binding of maltose binding protein (MBP) with its ligand maltose. Competition between a ligand and an inhibitor may result from binding to either the same site or different conformations of the protein. Maltose binds to the cleft between two lobes of MBP, and in a series of mutants, the affinities increased with an increase in the extent of lobe closure. Here we investigated whether macromolecular crowders also have a conformational or site preference when binding to MBP. The affinities of a polymer crowder, Ficoll70, measured by monitoring tryptophan fluorescence were 3-6-fold higher for closure mutants than for wild-type MBP. Competition between the ligand and crowder, as indicated by fitting of titration data and directly by nuclear magnetic resonance spectroscopy, and their similar preferences for closed MBP conformations further suggest the scenario in which the crowder, like maltose, preferentially binds to the interlobe cleft of MBP. Similar observations were made for bovine serum albumin as a protein crowder. Conformational and site preferences in MBP-crowder binding allude to the paradigm that nonspecific interactions can possess hallmarks of molecular recognition, which may be essential for intracellular organizations including colocalization of proteins and liquid-liquid phase separation.


Assuntos
Substâncias Macromoleculares/química , Proteínas Ligantes de Maltose/química , Conformação Proteica , Ligantes , Substâncias Macromoleculares/metabolismo , Espectroscopia de Ressonância Magnética , Maltose/química , Maltose/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica
17.
Food Chem ; 288: 268-275, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902292

RESUMO

The allergenicity suppression of tropomyosin (TM) from Exopalaemon modestus by glycation with saccharides of different molecular sizes (glucose, maltose, maltotriose, maltopentaose and maltoheptaose) was investigated using immunoblotting, human colon epithelial cell line (Caco-2) and human basophil cell line (KU812). Glycation of TM by glucose, maltotriose, maltopentaose and maltoheptaose significantly destructed and masked TM epitopes to obtain lower allergenicity, while glycation of TM by maltose had insignificant suppression on TM allergenicity. In addition, the glycated TM by glucose, maltotriose, maltopentaose and maltoheptaose inhibited the proliferation and IL-8 secretion of Caco-2, and the CD63 and CD203c expression, MAPK signaling of KU812 basophils, while the glycated TM by maltose had insignificant suppression on the allergy reactivities of Caco-2 cells and KU812 basophils. Glycation of TM by saccharides with larger molecular sizes (such as maltoheptaose) could provide new insight into the desensitization of shrimp-induced food allergy.


Assuntos
Alérgenos/imunologia , Oligossacarídeos/metabolismo , Palaemonidae/metabolismo , Tropomiosina/metabolismo , Adolescente , Animais , Células CACO-2 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Pré-Escolar , Feminino , Glicosilação , Humanos , Imunoglobulina E/sangue , Interleucina-8/metabolismo , Masculino , Maltose/metabolismo , Pessoa de Meia-Idade , Trissacarídeos/metabolismo , Tropomiosina/imunologia , Tropomiosina/farmacologia
18.
Food Chem ; 275: 265-272, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30724196

RESUMO

Glucose oxidase (GOD) is an enzyme widely used in glucose monitoring systems owing to its high specificity towards glucose. However, in our previous work maltose was found to show significant interaction with GOD and based on this observation, a novel microplate-based method was developed to assess α-amylase inhibitory activity (GOD method). Concerns regarding the interaction of GOD with maltose has limited the widespread use of the GOD method in assessing α-amylase activity. The present paper provides answers to concerns regarding the interaction of GOD with maltose using HPLC studies and application of the GOD method in assessing α-amylase activity. According to the results, the newly developed GOD method can be considered as a well-suited method for the determination of α-amylase activity and as an easy method to do kinetic studies compared to other available methods.


Assuntos
Bioquímica/métodos , Glucose Oxidase/metabolismo , Extratos Vegetais/farmacologia , alfa-Amilases/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Farinha , Glucose/metabolismo , Hidrólise , Cinética , Maltose/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Amido/metabolismo , alfa-Amilases/antagonistas & inibidores
19.
Chem Senses ; 44(4): 249-256, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30753419

RESUMO

Starch-related sweet taste perception plays an important role as a part of the dietary nutrient sensing mechanisms in the oral cavity. However, the release of sugars from starchy foods eliciting sweetness has been less studied in humans than in laboratory rodents. Thus, 28 respondents were recruited and evaluated for their starch-related sweet taste perception, salivary alpha-amylase (sAA) activity, oral release of reducing sugars, and salivary leptin. The results demonstrated that a 2-min oral mastication of starchy chewing gum produced an oral concentration of maltose above the sweet taste threshold and revealed that the total amount of maltose equivalent reducing sugars produced was positively correlated with the sAA activity. In addition, respondents who consistently identified the starch-related sweet taste in two sessions (test and retest) generated a higher maltose equivalent reducing sugar concentration compared to respondents who could not detect starch-related sweet taste at all (51.52 ± 2.85 and 29.96 ± 15.58 mM, respectively). In our study, salivary leptin levels were not correlated with starch-related sweet taste perception. The data contribute to the overall understanding of oral nutrient sensing and potentially to the control of food intake in humans. The results provide insight on how starchy foods without added glucose can elicit variable sweet taste perception in humans after mastication as a result of the maltose generated. The data contribute to the overall understanding of oral sensing of simple and complex carbohydrates in humans.


Assuntos
Glucose/metabolismo , Maltose/metabolismo , Saliva/enzimologia , alfa-Amilases Salivares/metabolismo , Amido/metabolismo , Sacarose/metabolismo , Percepção Gustatória , Adulto , Feminino , Preferências Alimentares , Glucose/análise , Humanos , Masculino , Maltose/análise , Amido/química , Sacarose/análise
20.
Poult Sci ; 98(6): 2570-2576, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30753716

RESUMO

Intestinal glucose absorption varies with growth; however, the dynamics of these variations has not been yet fully elucidated in broiler chickens. The present study aimed to compare jejunal glucose uptake and maltose digestion in broilers of 2 different ages, i.e., 1- vs. 5 wk old. Oral D-maltose gavage, everted sac, and Ussing chamber experiments were carried out to investigate intestinal glucose absorption and mRNA expression of glucose-transport-related genes as well as jejunal maltase activity. Upon gavage, glucose concentrations peaked at 10 min post-administration in 1-wk-old chicks, while they peaked at 40 min in 5-wk-old chickens. Glucose concentrations at 10 min were significantly higher in the 1-wk-old chicks (P = 0.010). Using the everted sacs experimental setup, 5 intestinal regions i.e., duodenum, proximal jejunum, distal jejunum, proximal ileum, and distal ileum, were targeted to examine D-maltose digestion and glucose transport across the intestinal mucosa. In the distal and proximal ileum, glucose concentrations were found to be significantly higher in the serosal compartment of the 1-wk-old chicks upon incubation with D-maltose (25 mM) (P < 0.05), while in the mucosal compartment the levels were significantly higher in the 5-wk-old chickens (P < 0.05). An Ussing chamber setup was employed to measure glucose-induced short-circuit current (ΔIsc) in the mucosal epithelium of the jejunum. In response to the addition of D-maltose (10 mM) into the mucosal compartment, ΔIsc was significantly higher in the 1-wk-old chicks (P = 0.018). Furthermore, no variations in jejunal maltase activity were observed between the 2 age groups. While jejunal glucose absorption was lower in the 5-wk-old chickens, the mRNA expression levels of jejunal SGLT1, GLUT2, and Na+/K+-ATPase did not show any significant differences between the 2 age groups. Our results suggest that the active transport of glucose across the jejunal epithelium decreases upon growth in broiler chickens but is not accompanied by any variations in maltase activity or in the expression of glucose-absorption-related genes.


Assuntos
Galinhas/metabolismo , Glucose/metabolismo , Jejuno/metabolismo , Maltose/metabolismo , Fatores Etários , Fenômenos Fisiológicos da Nutrição Animal , Animais , Transporte Biológico Ativo , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Digestão/fisiologia , Epitélio/metabolismo , Expressão Gênica , Mucosa Intestinal/metabolismo , Masculino , RNA Mensageiro
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