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1.
Food Chem ; 297: 125018, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253265

RESUMO

Different Ohmic heating conditions (OH, 10, 100, and 1000 Hz at 25 V; 45, 60, and 80 V at 60 Hz) were assessed to manufacture whey-raspberry flavored beverages. The inhibition of α-glucosidase, α-amylase, and angiotensin-converting I enzymes, antioxidant capacity, fatty acid profile, and volatile organic compounds (VOCs) were determined. OH treated samples presented lower anthocyanins content than the conventional treatment (2.91 ±â€¯0.23 mg/g), while the mild-intermediate conditions (10,100-Hz at 25 V and 45,60-V at 60 Hz) presented the highest chemical antioxidant activity when compared to the extreme processing conditions (1000 Hz-25 V and 80 V-60 Hz). OH led to an increase of 10% in both α-glucosidase (>99%) and α-amylase (≥70%). Among the VOCs, furfural and 5-hydroxymethylfurfural, a major intermediate Maillard reaction product was found in all treatments. Overall, OH can be used in the processing of whey-flavored raspberry beverages.


Assuntos
Bebidas/análise , Aromatizantes/química , Rubus/química , Soro do Leite/química , Antocianinas/análise , Antioxidantes/química , Culinária , Ácidos Graxos/análise , Cromatografia Gasosa-Espectrometria de Massas , Glucosidases/antagonistas & inibidores , Glucosidases/metabolismo , Ondas de Rádio , Temperatura Ambiente , Compostos Orgânicos Voláteis/análise , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismo
2.
Carbohydr Res ; 480: 42-53, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31174176

RESUMO

Sulfolobus solfataricus ß-glycosidase (SS-ßGly) belongs to Glycosyl Hydrolase family1 (GH1) with broad substrate specificity. SS-ßGly catalyzes both hydrolysis and transglycosylation reactions. SS-ßGly is commonly used to synthesize variety of galacto-oligosaccharides. A comparison of SS-ßGly with bacterial and eukaryotic homologs, using DALI search, revealed unique inserts. Free enzyme molecular dynamics (MD) simulation was performed under two different pH conditions (pH 6.5 and 2.5) at a constant temperature of 65 °C using GROMACS. A probable active-site loop (residues 331-364) in SS-ßGly was identified. Dynamics of substrate binding cavity revealed that it was buried and inaccessible during most timeframes at pH 6.5 whereas open and accessible at pH 2.5. New cavities identified during both simulations may act as probable water channel or product egress path. Analyses of docked complexes of 3D structures obtained at every 1ns interval with compounds, involved in hydrolysis and tranglycosylation reactions, demonstrated that conformational states sampled by SS-ßGly during free enzyme dynamics mimic the stages in enzyme catalysis thereby providing a mechanistic perspective. Current study revealed that conformational changes were conducive for hydrolysis at pH 6.5 and multiple cycles of transglycosylation at pH 2.5. Probable role of salt-bridge interactions in determining the type of reaction mechanism was also explored.


Assuntos
Biocatálise , Glucosidases/química , Glucosidases/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Domínio Catalítico , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Especificidade por Substrato
3.
Nat Commun ; 10(1): 2222, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110237

RESUMO

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.


Assuntos
Domínio Catalítico , Glucosidases/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Biocatálise , Cristalografia por Raios X , Ensaios Enzimáticos/métodos , Glucosidases/química , Glucosidases/isolamento & purificação , Glicosídeos/metabolismo , Hordeum/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/metabolismo , Especificidade por Substrato
4.
J Microbiol Biotechnol ; 29(4): 562-570, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30955258

RESUMO

ß-Glucosylglycerol (ß-GG) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, ß-GG and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae ß-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of ß-GGs [ß-D-glucopyranosyl-(1→1/3)-D-glycerol and ß-D-glucopyranosyl- (1→2)-D-glycerol]. In the second transglycosylation reaction, ß-D-glucopyranosyl-(1 → 1/3)-Dglycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of α-Dglucopyranosyl-( 1→4)-ß-D-glucopyranosyl-(1→1/3)-D-glycerol and 28% of α-D-maltopyranosyl- (1→4)-ß-D-glucopyranosyl-(1→1/3)-D-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of ß-GG was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.


Assuntos
Glucosídeos/biossíntese , Glucosiltransferases/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/biossíntese , Biotecnologia , Celobiose/metabolismo , Deinococcus/enzimologia , Deinococcus/genética , Escherichia coli/genética , Glucosidases/metabolismo , Glucosídeos/análise , Glucosídeos/química , Glucosiltransferases/genética , Glicerol/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeos/análise , Glicosídeos/química
5.
Physiol Plant ; 166(1): 105-119, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30834537

RESUMO

The maximum quantum yield of photosystem II (as reflected by variable to maximum chlorophyll a fluorescence, Fv /Fm ) is regarded as one of the most important photosynthetic parameters. The genetic basis underlying natural variation in Fv /Fm , which shows low level of variations in plants under non-stress conditions, is not easy to be exploited using the conventional gene cloning approaches. Thus, in order to answer this question, we have followed another strategy: we used genome-wide association study (GWAS) and transgenic analysis in a rice mini-core collection. We report here that four single-nucleotide polymorphisms, located in the promoter region of ß-glucosidase 5 (BGlu-5), are associated with observed variation in Fv /Fm . Indeed, our transgenic analysis showed a good correlation between BGlu-5 and Fv /Fm . Thus, our work demonstrates the feasibility of using GWAS to study natural variation in Fv /Fm , suggesting that cis-element polymorphism, affecting the BGlu-5 expression level, may, indirectly, contribute to Fv /Fm variation in rice through the gibberellin signaling pathway. Further research is needed to understand the mechanism of our novel observation.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Glucosidases/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Celulases/genética , Celulases/metabolismo , Giberelinas/metabolismo , Glucosidases/genética , Complexo de Proteína do Fotossistema II/genética , Polimorfismo de Nucleotídeo Único/genética
6.
Int J Mol Sci ; 20(3)2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678336

RESUMO

Four novel acylglycosides flavones (AGFs) including two quercetin acylglycosides and two kaempferol acylglycosides were isolated from Fuzhuan brick tea (FBT) as follows: quercetin 3-O-[α-l-rhamnopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [ß-d-glucopyranosyl (1→3)-α-l-rhamnopyranosyl (1→6)]-ß-d-galactoside was named as camelliquercetiside E (1), quercetin 3-O-[α-l-rhamnopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [α-l-rhamnopyranosyl (1→6)]-ß-d-galactoside was named as camelliquercetiside F (2), kaempferol 3-O-[α-l-arabinopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [ß-d-glucopyranosyl (1→3)-α-l-rhamnopyranosyl (1→6)]-ß-d-glucoside was named as camellikaempferoside D (3), kaempferol 3-O-[α-l-arabinopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [α-l-rhamnopyranosyl (1→6)]-ß-d-glucoside was named as camellikaempferoside E (4). Chemical structures of AGFs were identified by time-of-flight mass (TOF-MS) and NMR spectrometers (¹H NMR, 13C NMR, ¹H-¹H COSY, HMBC and HSQC), and the MS² fragmentation pathway of AGFs was further investigated. The inhibitory abilities of AGFs and their proposed metabolites on α-glucosidase and HMG-CoA reductase were analyzed by molecular docking simulation, and the results suggested that inhibitory activities of AGFs were significantly affected by acyl structure, number of glycosyl and conformation, and part of them had strong inhibitory activities on α-glucosidase and HMG-CoA reductase, suggesting that AGFs and their metabolites might be important ingredients that participate in the regulation of hypoglycemic and hypolipidemic effects. The results provided new AGFs and research directions for the practical study of FBT health functions in future.


Assuntos
Quempferóis/farmacologia , Simulação de Acoplamento Molecular , Quercetina/farmacologia , Chá/química , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Camellia sinensis/química , Glucosidases/química , Glucosidases/metabolismo , Glicosídeos/química , Humanos , Quempferóis/química , Ligação Proteica , Quercetina/análogos & derivados
7.
Molecules ; 24(3)2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30682802

RESUMO

Desert soil is one of the most severe conditions which negatively affect the environment and crop growth production in arid land. The application of organic amendments with inorganic fertilizers is an economically viable and environmentally comprehensive method to develop sustainable agriculture. The aim of this study was to assess whether milk tea waste (TW) amendment combined with chemical fertilizer (F) application can be used to improve the biochemical properties of sandy soil and wheat growth. The treatments included control without amendment (T1), chemical fertilizers (T2), TW 2.5% + F (T3), TW 5% + F (T4) and TW 10% + F (T5). The results showed that the highest chlorophyll (a and b) and carotenoids, shoot and root dry biomass, and leaf area index (LAI) were significantly (p < 0.05) improved with all amendment treatments. However, the highest root total length, root surface area, root volume and diameter were recorded for T4 among all treatments. The greater uptake of N, P, and K contents for T4 increased for the shoot by 68.9, 58.3, and 57.1%, and for the root by 65.7, 34.3, and 47.4% compared to the control, respectively. Compared with the control, T5 treatment decreased the soil pH significantly (p < 0.05) and increased soil enzyme activities such as urease (95.2%), ß-glucosidase (81.6%) and dehydrogenase (97.2%), followed by T4, T3, and T2. Our findings suggested that the integrated use of milk tea waste and chemical fertilizers is a suitable amendment method for improving the growth and soil fertility status of sandy soils.


Assuntos
Fertilizantes/análise , Leite/química , Solo/química , Resíduos Sólidos , Chá/química , Triticum/crescimento & desenvolvimento , Agricultura , Animais , Biomassa , Carotenoides/metabolismo , Clorofila/metabolismo , Glucosidases/metabolismo , Nitrogênio/química , Nutrientes/química , Oxirredutases/metabolismo , Fósforo/química , Potássio/química , Urease/metabolismo
8.
Food Chem ; 274: 543-546, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30372976

RESUMO

Apiosidases are glycosidases relevant for aroma development during fermentation of wines and black tea. Reaction mechanism of apiosidase from Aspergillus aculeatus in commercial glycanase Viscozyme L was studied by 1H NMR technique. Study of hydrolysis of 4-nitrophenyl ß-D-apiofuranoside revealed that this reaction proceeds with inversion of hydroxyl group in the anomeric center, which confirms inverting mechanism of the enzyme and its inability to catalyze transapiosylation in syntheses of apiosides.


Assuntos
Aspergillus/enzimologia , Glucosidases/metabolismo , Fermentação , Glicosídeos/metabolismo , Hidrólise
9.
Food Microbiol ; 76: 382-389, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30166164

RESUMO

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-ß Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.


Assuntos
Proteínas de Bactérias/genética , Lactobacillus pentosus/genética , Lactobacillus pentosus/metabolismo , Olea/microbiologia , Fenóis/metabolismo , Proteínas de Bactérias/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Fermentação , Ácido Gálico/análogos & derivados , Ácido Gálico/metabolismo , Glucosidases/genética , Glucosidases/metabolismo , Lactobacillus pentosus/enzimologia , Olea/química , Olea/metabolismo
10.
BMC Complement Altern Med ; 18(1): 77, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29499679

RESUMO

BACKGROUND: Extensive epidemiological and clinical studies revealed that Alzheimer's Disease (AD) and Type 2 Diabetes Mellitus (T2D) are most likely to appear simultaneously in aged people as T2D is a major risk factor for AD. Therefore, development of potential multifunctional agents for dual therapy of AD and T2D has received much attention. Buchanania axillaris, Hemidesmus indicus and Rhus mysorensis have been used extensively in popular medicine. The present study was aimed at phytochemical profiling and evaluating multifunctional ability of titled plants in the AD and T2D dual therapy. METHODS: Methanolic extracts and their derived fractions were evaluated for their inhibitory capacities against acetylcholinesterase (AChE) & butyrylcholinesterase (BuChE), and α- & ß-glucosidase besides kinetic analysis of inhibition using methods of Elmann and Shibano, respectively. Antioxidant potency of active fractions was assessed by their DPPH and ABTS radical scavenging activities. Active fractions were tested by the MTT assay to verify cytotoxicity and neuroprotective ability in human nueroblastoma cell lines. Phytochemical screening was done with the aid of spectrophotometric methods. RESULTS: All the methanolic extracts of test plants (BAM, HIM, RMM) showed concentration dependent inhibitory activities against AChE, BuChE, α- and ß-glucosidase enzymes. Subsequent fractionation and evaluation revealed that chloroform fractions BAC, HIC and RMC with IC50 values of 12.29±2.14, 9.94±2.14, 16.65±1.99 and 27.38±1.24; 28.14±0.9, 5.16±0.22, 11.03±0.5 and 87.64±15.41; 41.35±1.6, 15.86±7.3, 26.04±0.37 and 25.33±0.3 were most prominent with regard to inhibition potential against AChE, BuChE, α- and ß-glucosidase, respectively. Kinetic analysis of these active fractions proved that they disclosed mixed-type inhibition against AChE, BuChE, α- and ß-glucosidase enzymes. In the MTT assay, active fractions BAC, HIC, RMC showed significant cell viability at high concentrations (400 µg). Moreover, in MTT assay, the active fractions displayed excellent neuroprotective effects against oxidative stress induced cell death and significant cell viability in SK N SH cells at all concentrations. CONCLUSION: The strong anticholinesterase, antiglucosidase, antioxidant and neuroprotective activities of methanolic extracts and their derived chloroform fractions indicate the potential of Buchanania axillaris, Hemidesmus indicus and Rhus mysorensis as multifunctional therapeutic remedies for the dual therapy of T2D and AD.


Assuntos
Doença de Alzheimer/enzimologia , Antioxidantes/química , Inibidores da Colinesterase/química , Diabetes Mellitus Tipo 2/enzimologia , Fármacos Neuroprotetores/química , Extratos Vegetais/química , Plantas Medicinais/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Butirilcolinesterase/metabolismo , Linhagem Celular , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Avaliação Pré-Clínica de Medicamentos , Glucosidases/antagonistas & inibidores , Glucosidases/metabolismo , Humanos , Cinética , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia
11.
Int J Mol Sci ; 19(3)2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29543768

RESUMO

Onion (Allium cepa L.) is widely consumed as food or medicinal plant due to its well-defined health benefits. The antioxidant and antihyperlipidemic effects of onion and its extracts have been reported well. However, very limited information on anti-hyperglycemic effect is available in processed onion extracts. In our previous study, we reported that Amadori rearrangement compounds (ARCs) produced by heat-processing in Korean ginseng can reduce carbohydrate absorption by inhibiting intestinal carbohydrate hydrolyzing enzymes in both in vitro and in vivo animal models. To prove the enhancement of anti-hyperglycemic effect and ARCs content by heat-processing in onion extract, a correlation between the anti-hyperglycemic activity and the total content of ARCs of heat-processed onion extract (ONI) was investigated. ONI has a high content of ARCs and had high rat small intestinal sucrase inhibitory activity (0.34 ± 0.03 mg/mL, IC50) relevant for the potential management of postprandial hyperglycemia. The effect of ONI on the postprandial blood glucose increase was investigated in Sprague Dawley (SD) rats fed on sucrose or starch meals. The maximum blood glucose levels (Cmax) of heat-processed onion extract were significantly decreased by about 8.7% (from 188.60 ± 5.37 to 172.27 ± 3.96, p < 0.001) and 14.2% (from 204.04 ± 8.73 to 175.13 ± 14.09, p < 0.01) in sucrose and starch loading tests, respectively. These results indicate that ARCs in onion extract produced by heat-processing have anti-diabetic effect by suppressing carbohydrate absorption via inhibition of intestinal sucrase, thereby reducing the postprandial increase of blood glucose. Therefore, enhancement of ARCs in onion by heat-processing might be a good strategy for the development of the new product on the management of hyperglycemia.


Assuntos
Antioxidantes/farmacologia , Restrição Calórica , Hipoglicemiantes/farmacologia , Cebolas/química , Extratos Vegetais/farmacologia , Animais , Glicemia/metabolismo , Glucosidases/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Sacarase/metabolismo
12.
J Gen Appl Microbiol ; 63(6): 355-361, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-29187680

RESUMO

Resistant starch is not digestible in the small intestine and is fermented by lactic acid bacteria in the large intestine into short chain fatty acids, such as acetate, propionate and butyrate, which result in several health benefits in analogy with dietary fibre components. The mode and mechanism of resistant starch degradation by lactic acid bacteria is still not understood. In the present study, we have purified α-D-glucosidase from Lactobacillus fermentum NCDC 156 by employing three sequential steps i.e. ultra filtration, DEAE-cellulose and Sephadex G-100 chromatographies. It was found to be a monomeric protein (~50 kDa). The optimum pH and temperature of this enzyme were found to be 5.5 and 37°C, respectively. Under optimised conditions with p-nitrophenyl-D-glucopyranoside as the substrate, the enzyme exhibited a Km of 0.97 mM. Its activity was inhibited by Hg2+ and oxalic acid. N-terminal blocked purified enzyme was subjected to lysyl endopeptidase digestion and the resultant peptides were subjected to BLAST analysis to understand their homology with other α-D-glucosidases from lactobacillus species.


Assuntos
Glucosidases/isolamento & purificação , Glucosidases/metabolismo , Lactobacillus fermentum/enzimologia , Amido/metabolismo , Metabolismo dos Carboidratos , Ativação Enzimática/efeitos dos fármacos , Glucosidases/antagonistas & inibidores , Glucosidases/química , Concentração de Íons de Hidrogênio , Cinética , Mercúrio/farmacologia , Peso Molecular , Ácido Oxálico , Alinhamento de Sequência , Especificidade por Substrato , Temperatura Ambiente
13.
Eur J Nutr ; 57(1): 231-242, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27722779

RESUMO

PURPOSE: Orange juice (OJ) flavanones undergo limited absorption in the upper gastrointestinal tract and reach the colon where they are transformed by the microbiota prior to absorption. This study investigated the ability of two probiotic bacteria, Bifidobacterium longum R0175 and Lactobacillus rhamnosus subsp. Rhamnosus NCTC 10302 to catabolise OJ flavanones. METHODS: The bacteria were incubated with hesperetin-7-O-rutinoside, naringenin-7-O-rutinoside, hesperetin and naringenin, and the culture medium and intracellular cell extracts were collected at intervals over a 48 h of incubation period. The flavanones and their phenolic acid catabolites were identified and quantified by HPLC-HR-MS. RESULTS: Both probiotics were able to subject hesperetin to ring fission yielding 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid which was subsequently demethylated producing 3-(3',4'-dihydroxyphenyl)propionic acid and then via successive dehydroxylations converted to 3-(3'-hydroxyphenyl)propionic acid and 3-(phenyl)propionic acid. Incubation of both bacteria with naringenin resulted in its conversion to 3-(4'-hydroxyphenyl)propionic acid which underwent dehydroxylation yielding 3-(phenyl)propionic acid. In addition, only L. rhamnosus exhibited rhamnosidase and glucosidase activity and unlike B. longum, which was able to convert hesperetin-7-O-rutinoside and naringenin-7-O-rutinoside to their respective aglycones. The aglycones were then subjected to ring fission and further catabolised in a similar manner to that described above. The flavanones and their catabolites were found in the culture medium but not accumulated in the bacterial cells. CONCLUSIONS: These findings demonstrate the enzymatic potential of single strains of bifidobacterium and lactobacillus which may be involved in the colonic catabolism of OJ flavanones in vivo.


Assuntos
Bifidobacterium longum/metabolismo , Citrus sinensis , Flavanonas/metabolismo , Frutas/química , Lactobacillus rhamnosus/metabolismo , Probióticos/metabolismo , Bifidobacterium longum/enzimologia , Bifidobacterium longum/crescimento & desenvolvimento , Flavanonas/farmacocinética , Sucos de Frutas e Vegetais/análise , Glucosidases/metabolismo , Hesperidina/metabolismo , Absorção Intestinal , Lactobacillus rhamnosus/enzimologia , Lactobacillus rhamnosus/crescimento & desenvolvimento
14.
Ecotoxicol Environ Saf ; 150: 104-115, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29272714

RESUMO

We studied the extracellular enzyme activity (EEA) in the riverbed sediment along a 518km gradient of the Ganga River receiving carbon and nutrient load from varied human sources. Also, we tested, together with substrate-driven stimulation, if the heavy metal accumulated in the sediment inhibits enzyme activities. Because pristine values are not available, we considered Dev Prayag, a least polluted site located 624km upstream to main study stretch, as a reference site. There were distinct increases in enzyme activities in the sediment along the study gradient from Dev Prayag, however, between-site differences were in concordance with sediment carbon(C), nitrogen (N) and phosphorus (P). Fluorescein diacetate hydrolysis (FDAase), ß-glucosidase (Glu) and protease activities showed positive correlation with C, N and P while alkaline phosphatase was found negatively correlated with P. Enzyme activities were found negatively correlated with heavy metal, although ecological risk index (ERi) varied with site and metal species. Dynamic fit curves showed significant positive correlation between heavy metal and microbial metabolic quotient (qCO2) indicating a decrease in microbial activity in response to increasing heavy metal concentrations. This study forms the first report linking microbial enzyme activities to regional scale sediment heavy metal accumulation in the Ganga River, suggests that the microbial enzyme activities in the riverbed sediment were well associated with the proportion of C, N and P and appeared to be a sensitive indicator of C, N and P accumulation in the river. Heavy metal accumulated in the sediment inhibits enzyme activities, although C rich sediment showed relatively low toxicity due probably to reduced bioavailability of the metal. The study has relevance from ecotoxicological as well as from biomonitoring perspectives.


Assuntos
Monitoramento Ambiental/métodos , Sedimentos Geológicos/microbiologia , Hidrolases/metabolismo , Metais Pesados/toxicidade , Rios/microbiologia , Poluentes Químicos da Água/toxicidade , Fosfatase Alcalina/metabolismo , Ecotoxicologia , Sedimentos Geológicos/química , Glucosidases/metabolismo , Índia , Metais Pesados/análise , Peptídeo Hidrolases/metabolismo , Rios/química , Poluentes Químicos da Água/análise
15.
Planta ; 247(3): 625-634, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29147812

RESUMO

MAIN CONCLUSION: A Catharanthus roseus mutant accumulates high levels of ajmalicine at the expense of catharanthine and vindoline. The altered chemistry depends on increased expression and biochemical activities of strictosidine ß-glucosidase and ajmalicine synthase activities and reduced expression and biochemical activity of geissoschizine synthase. The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is a valuable source for several monoterpenoid indole alkaloids (MIAs), such as the powerful antihypertensive drug ajmalicine and the antineoplastic agents, vinblastine and vincristine. While biosynthesis of the common MIA precursor strictosidine and its reactive aglycones has been elucidated, the branch point steps leading to the formation of different classes of MIAs remain poorly characterized. Screening of 3600 ethyl methyl sulfonate mutagenized C. roseus plants using a simple thin-layer chromatography screen yielded a mutant (M2-0754) accumulating high levels of ajmalicine together with significantly lower levels of catharanthine and vindoline. Comparative bioinformatic analyses, virus-induced gene silencing, and biochemical characterization identified geissoschizine synthase, the gateway enzyme that controls flux for the formation of iboga and aspidosperma MIAs. The reduction of geissoschizine synthase transcripts in this high ajmalicine mutant, together with increased transcripts and enzyme activities of strictosidine ß-glucosidase and of heteroyohimbine synthase, explains the preferential formation of ajmalicine in the mutant instead of catharanthine and vindoline that accumulates in the wild-type parent. Reciprocal crosses established that that the high ajmalicine phenotype is inherited as a Mendelian recessive trait.


Assuntos
Carbolinas/metabolismo , Catharanthus/metabolismo , Proteínas de Plantas/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Catharanthus/enzimologia , Catharanthus/genética , Glucosidases/metabolismo , Complexos Multienzimáticos/metabolismo , Mutação/genética , Vimblastina/análogos & derivados , Vimblastina/metabolismo , beta-Glucosidase/metabolismo
16.
FEBS J ; 284(22): 3931-3953, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28975708

RESUMO

6-phospho-ß-glucosidases and 6-phospho-ß-galactosidases are enzymes that hydrolyze the ß-glycosidic bond between a terminal non-reducing glucose-6-phosphate (Glc6P) or galactose-6-phosphate (Gal6P), respectively, and other organic molecules. Gan1D, a glycoside hydrolase (GH) belonging to the GH1 family, has recently been identified in a newly characterized galactan-utilization gene cluster in the bacterium Geobacillus stearothermophilus T-1. Gan1D has been shown to exhibit bifunctional activity, possessing both 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities. We report herein the complete 3D crystal structure of Gan1D, together with its acid/base catalytic mutant Gan1D-E170Q. The tertiary structure of Gan1D conforms well to the (ß/α)8 TIM-barrel fold commonly observed in GH enzymes, and its quaternary structure adopts a dimeric assembly, confirmed by gel-filtration and small-angle X-ray scattering results. We present also the structures of Gan1D in complex with the putative substrate cellobiose-6-phosphate (Cell6P) and the degradation products Glc6P and Gal6P. These complexes reveal the specific enzyme-substrate and enzyme-product binding interactions of Gan1D, and the residues involved in its glycone, aglycone, and phosphate binding sites. We show that the different ligands trapped in the active sites adopt different binding modes to the protein, providing a structural basis for the dual galactosidase/glucosidase activity observed for this enzyme. Based on this information, specific mutations were performed on one of the active site residues (W433), shifting the enzyme specificity from dual activity to a significant preference toward 6-phospho-ß-glucosidase activity. These data and their comparison with structural data of related glucosidases and galactosidases are used for a more general discussion on the structure-function relationships in this sub-group of GH1 enzymes. DATABASES: Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively.


Assuntos
Geobacillus stearothermophilus/enzimologia , Glucosidases/química , Glucosidases/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Galactosefosfatos/metabolismo , Modelos Moleculares , Fosfatos/metabolismo , Conformação Proteica , Homologia de Sequência , Especificidade por Substrato
17.
Hum Mol Genet ; 26(21): 4190-4202, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973524

RESUMO

Mutations in the PRKCSH, SEC63 and LRP5 genes cause autosomal dominant polycystic liver disease (ADPLD). The proteins products of PRKCSH (alias GIIB) and SEC63 function in protein quality control and processing in the endoplasmic reticulum (ER), while LRP5 is implicated in Wnt/ß-catenin signaling. To identify common denominators in the PLD pathogenesis, we mapped the PLD interactome by affinity proteomics, employing both HEK293T cells and H69 cholangiocytes. Identification of known complex members, such as glucosidase IIA (GIIA) for PRKCSH, and SEC61A1 and SEC61B for SEC63, confirmed the specificity of the analysis. GANAB, encoding GIIA, was very recently identified as an ADPLD gene. The presence of GIIA in the LRP5 complex pinpoints a potential functional connection with PRKCSH. Interestingly, all three PLD-associated protein complexes included filamin A (FLNA), a multifunctional protein described to play a role in ciliogenesis as well as canonical Wnt signalling. As ciliary dysfunction may also contribute to hereditary liver cyst formation, we evaluated the requirement of PRKCSH and SEC63 for ciliogenesis and Wnt signaling. By CRISPR/Cas9 induced knockdown of both ADPLD genes in HEK293T cells and H69 cholangiocytes, we identified that their depletion results in defective ciliogenesis. However, only H69 knockouts displayed reduced Wnt3a activation. Our results suggest that loss of PRKCSH and SEC63 leads to general defects in ciliogenesis, while quenching of the Wnt signaling cascade is cholangiocyte-restricted. Interactions of all three PLD-associated protein complexes with FLNA may mark a common link between the ADPLD proteins and the cystogenic processes driving this disease.


Assuntos
Cílios/patologia , Cistos/metabolismo , Cistos/patologia , Glucosidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Proteínas de Membrana/metabolismo , Cílios/genética , Cílios/metabolismo , Cistos/genética , Retículo Endoplasmático/patologia , Técnicas de Inativação de Genes , Glucosidases/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/metabolismo , Fígado/patologia , Hepatopatias/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana/genética , Via de Sinalização Wnt , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , alfa-Glucosidases/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
18.
Food Chem Toxicol ; 109(Pt 1): 356-362, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28919409

RESUMO

Antifungal activity of some natural molecules can be abated or blocked by efflux pumps in Candida albicans, which restricts the discovery of potential antifungal agents. Here we found that the steroidal alkaloid solasodine is active against C. albicans efflux pump-deficient strains but inert towards the wild type. However, the glucosylated solasodine-3-O-ß-d-glucopyranoside exhibits antifungal activity towards the wild type strain. Further investigation revealed that the entry of solasodine into C. albicans cells is blocked by efflux pumps. Glucosylation provides an alternative access not disturbed by efflux pumps. Once inside cells, the carried glucosylated solasodine is cleaved into the active molecule solasodine by the glucosidase, which is located in cytoplasm membrane and exhibits selective activity against hydrolyzing glucosyl natural products but not against other monosaccharide-substituted products. This glucosidase is not encoded by orf19.4031, considered homologous to steryl-ß-glucosidase encoded by the gene EGH1 in Saccharomyces cerevisiae. Our study reveals that glucosylation is an alternative approach for introducing potential antifungal activity into C. albicans cells and overcoming the drug-resistance resulting from hyperactivation of efflux pumps.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Glucosidases/metabolismo , Glucosídeos/farmacologia , Alcaloides de Solanáceas/metabolismo , Alcaloides de Solanáceas/farmacologia , Candida albicans/enzimologia , Farmacorresistência Fúngica , Glucosídeos/metabolismo , Glicosilação
19.
J Ind Microbiol Biotechnol ; 44(12): 1643-1651, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28942503

RESUMO

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, ß-D-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular ß-D-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted ß-D-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable ß-D-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that ß-D-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.


Assuntos
Celulose/metabolismo , Clostridiales/genética , Clostridiales/metabolismo , Glucosidases/genética , Glucosidases/metabolismo , Proteoma/metabolismo , Actinomycetales/enzimologia , Actinomycetales/genética , Celobiose/metabolismo , Celulose/química , Clostridiales/crescimento & desenvolvimento , Estabilidade Enzimática , Fermentação
20.
Cell Oncol (Dordr) ; 40(6): 579-591, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28929344

RESUMO

PURPOSE: Glucosidase II plays a major role in regulating the post-translational modification of N-linked glycoproteins. Previously, we found that the beta subunit of glucosidase II (GluIIß) levels are significantly increased in lung carcinoma tissues, indicating a potential role in lung tumorigenesis. Here, we investigated the role of GluIIß in the regulation of autophagy and apoptosis in lung carcinoma- and immortalized human bronchial epithelial-derived cells. METHODS: A selective glucosidase II inhibitor, bromoconduritol, was used to inhibit GluII enzyme activity and a siRNA-based technology was used to suppress the expression of the GluIIß encoding gene PRKCSH in lung carcinoma cells differing in p53 status. Cell viability was assessed using a MTT assay, cell cycle progression was assessed using flow cytometry, autophagy was assessed using Western blotting and apoptosis was assessed using an annexin V-FITC/PI double labeling method. RESULTS: We found that GluIIß inhibition resulted in the induction of autophagy in all cell lines tested, but apoptosis in only wild-type p53 cells. We also found that GluIIß inhibition dose-dependently decreased activation of the EGFR/RTK and PI3K/AKT signaling pathways. Although the apoptosis inducing effect of GluIIß inhibition appeared to be p53-dependent, we found that a combined treatment with lysosomal inhibitors to block autophagy enhanced the apoptotic effect of GluIIß inhibition in both wild-type p53 and p53-null cells. CONCLUSIONS: Our data indicate that GluIIß inhibition results in autophagy and apoptosis in lung carcinoma-derived cells, supporting the hypothesis that this enzyme may play a role in blocking these two tumor suppressive processes. Since blocking autophagy by lysosomal inhibitors enhanced the apoptosis-inducing effect of bromoconduritol, independent of p53 status, their combined use may hold promise for the treatment of cancer, particularly lung cancer.


Assuntos
Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , alfa-Glucosidases/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cicloexenos/farmacologia , Glucosidases/genética , Glucosidases/metabolismo , Humanos , Inositol/análogos & derivados , Inositol/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais/efeitos dos fármacos
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