Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 53
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Molecules ; 26(15)2021 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-34361714

RESUMEN

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.


Asunto(s)
Antracenos/química , Proteínas Fúngicas/antagonistas & inhibidores , Inhibidores de Glicósido Hidrolasas/química , Hipoglucemiantes/química , Perileno/análogos & derivados , alfa-Glucosidasas/química , Antracenos/metabolismo , Sitios de Unión , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Inhibidores de Glicósido Hidrolasas/metabolismo , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Hipoglucemiantes/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Perileno/química , Perileno/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/enzimología , Resonancia por Plasmón de Superficie , alfa-Glucosidasas/metabolismo
2.
Biochem Biophys Res Commun ; 515(1): 190-195, 2019 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-31133380

RESUMEN

Here we studied the effect of molecular crowding on the hydrolysis of ortho- and para-nitrophenyl-ß-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli ß-Galactosidase in the presence of 0-35%w/v 6kD polyethyleneglycol (PEG6000). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG6000] as well as for ONPG in the absence of PEG6000 so a Michaelian model was applied to calculate the kinetic parameters KM and kcat (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG6000, the two slopes visualized in Eadie-Hofstee plots leaded to apply a biphasic kinetic model to fit initial rate vs. [ONPG] plots hence calculating two apparent KM and two kcat values. Since the rate limiting-step of the enzymatic hydrolysis mechanism of ONPG, but not of PNPG, is the water-dependent one, the existence of several molecular water populations differing in their energy and/or their availability as reactants may explain the biphasic kinetics in the presence of PEG6000. With PNPG, KM as well as kcat varied with [PEG6000] like a parabola opening upward with a minimum at 15 %w/v [PEG6000]. In the case of ONPG, one of the components became constant while the other component exhibited a slight increasing tendency in kcat plus high and [PEG6000]-dependent increasing KM values. Sedimentation velocity analysis demonstrated that PEG6000 impaired the diffusion of ß-Gal but not that of substrates. In conjunction, kinetic data reflected complex combinations of PEG6000-induced effects on enzyme structure, water structure, thermodynamic activities of all the chemical species participating in the reaction and protein diffusion.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Sustancias Macromoleculares/metabolismo , Agua/metabolismo , beta-Galactosidasa/metabolismo , Algoritmos , Biocatálisis/efectos de los fármacos , Difusión , Hidrólisis/efectos de los fármacos , Cinética , Sustancias Macromoleculares/química , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Polietilenglicoles/metabolismo , Polietilenglicoles/farmacología , Solventes/química , Termodinámica , Agua/química , beta-Galactosidasa/química
3.
Food Chem ; 275: 346-353, 2019 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-30724206

RESUMEN

Tannins are compounds with antinutrient properties that hinder food digestibility, prejudicing human and animal nutrition. This work aimed to evaluate the negative effects of tannic acid on Kluyveromyces lactis ß-galactosidase catalytic activity and correlate these changes with the protein structure. ß-Galactosidase activity decreased in the presence of tannins, which caused changes to the structure of the enzyme, as demonstrated by circular dichroism. It was verified that tannin binds to the protein by a static mechanism. Additionally, isothermal titration calorimetry suggested that tannic acid modified the molecular interaction between ß-galactosidase and o-nitrophenyl-ß-d-galactoside, reducing their affinity and prejudicing the protein activity. This study helps to understand the effects of tannins on the ß-galactosidase structure and how they are related to the enzyme catalytic activity. The alterations in the conformation and activity of the enzyme should be taken into consideration when dairy products are consumed with tannin-rich food.


Asunto(s)
Kluyveromyces/enzimología , Taninos/metabolismo , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , Animales , Calorimetría/métodos , Dicroismo Circular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Conformación Proteica , Espectrometría de Fluorescencia , Taninos/química , Termodinámica
4.
J Biosci ; 43(5): 941-945, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30541954

RESUMEN

ß-Galactosidase is a metal-activated enzyme, which breaks down the glucosidic bond of lactose and produces glucose and galactose. Among several commercial applications, preparation of lactose-free milk has gained special attention. The present objective is to demonstrate the activity kinetics of ß-galactosidase purified from a non-pathogenic bacterium Arthrobacter oxydans SB. The enzyme was purified by DEAE-cellulose and Sephadex G-100 column chromatography. The purity of the protein was checked by high-performance liquid chromatography (HPLC). The purified enzyme of molecular weight ~95 kDa exhibited specific activity of 137.7 U mg-1 protein with a purification of 11.22-fold and yield 12.42%. The exact molecular weight (95.7 kDa) of the purified protein was determined by MALDI-TOF. Previously, most of the studies have used Mg+2 as a cofactor of ß-galactosidase. In this present investigation, we have checked the kinetic behavior of the purified ß-galactosidase in presence of several bivalent metals. Lowest Km with highest substrate (orthonitrophenyl- ß-galactoside or ONPG) affinity was measured in presence of Ca2+ (42.45 µM ONPG). However, our results demonstrated that Vmax was maximum in presence of Mn+2 (55.98 µM ONP produced mg-1 protein min-1), followed by Fe=2, Zn+2, Mg+2, Cu+2 and Ca+2. A large number of investigations reported Mg+2 as potential co factor for bgalacosidase. However, ß-galactosidase obtained from Arthrobacter oxydans SB has better activity in the presence of Mn+2 or Fe2+.


Asunto(s)
Arthrobacter/química , Proteínas Bacterianas/química , Coenzimas/química , Magnesio/química , Manganeso/química , beta-Galactosidasa/química , Arthrobacter/enzimología , Proteínas Bacterianas/aislamiento & purificación , Calcio/química , Cationes Bivalentes , Cobre/química , Pruebas de Enzimas , Galactosa/química , Glucosa/química , Hierro/química , Cinética , Lactosa/química , Peso Molecular , Nitrofenilgalactósidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Zinc/química , beta-Galactosidasa/aislamiento & purificación
5.
Acta Biochim Pol ; 65(3): 383-389, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30188963

RESUMEN

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 µM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.


Asunto(s)
Cannabis/enzimología , Rafinosa/aislamiento & purificación , Semillas/enzimología , alfa-Galactosidasa/química , Bromosuccinimida/química , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Metales Pesados/farmacología , Peso Molecular , Nitrofenilgalactósidos/química , Rafinosa/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triptófano/análisis , alfa-Galactosidasa/antagonistas & inhibidores , alfa-Galactosidasa/aislamiento & purificación
6.
PLoS One ; 13(5): e0197067, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29738566

RESUMEN

A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-d- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0-10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing.


Asunto(s)
Bacillus coagulans/enzimología , Péptido Hidrolasas/química , Probióticos/química , alfa-Galactosidasa/química , Estabilidad de Enzimas , Hidrólisis , Nitrofenilgalactósidos/química , Especificidad por Sustrato , alfa-Galactosidasa/aislamiento & purificación
7.
Proc Natl Acad Sci U S A ; 114(47): 12530-12535, 2017 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-29109294

RESUMEN

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic ß-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or ß-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-ß-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.


Asunto(s)
Proteínas Arqueales/química , Halorubrum/enzimología , Nitrofenilgalactósidos/química , beta-Galactosidasa/química , Sustitución de Aminoácidos , Regiones Antárticas , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Dominio Catalítico , Clonación Molecular , Frío , Cristalografía por Rayos X , Expresión Génica , Halobacterium/enzimología , Halobacterium/genética , Halorubrum/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nitrofenilgalactósidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Termodinámica , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
8.
Chembiochem ; 18(15): 1544-1550, 2017 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-28474804

RESUMEN

Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m-1 for the H85N variant, compared to 17 000 m-1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction.


Asunto(s)
Citidina Monofosfato/análogos & derivados , Histidina/química , Pasteurella/enzimología , Ácidos Siálicos/química , Sialiltransferasas/química , Asparagina/química , Dominio Catalítico , Citidina Monofosfato/química , Glicosilación , Histidina/genética , Hidrólisis , Cinética , Lactosa/química , Mutagénesis Sitio-Dirigida , Nitrofenilgalactósidos/química , Mutación Puntual , Sialiltransferasas/genética , Agua/química
9.
Antimicrob Agents Chemother ; 60(10): 5995-6002, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27458225

RESUMEN

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.


Asunto(s)
Pared Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Péptidos/farmacología , Peptidomiméticos/farmacología , beta-Galactosidasa/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Pared Celular/química , Pared Celular/metabolismo , Compuestos Cromogénicos/química , Compuestos Cromogénicos/metabolismo , Eritromicina/farmacología , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ácido Fusídico/farmacología , Expresión Génica , Hidrólisis , Pruebas de Sensibilidad Microbiana , Mutación , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Novobiocina/farmacología , Biblioteca de Péptidos , Péptidos/química , Peptidomiméticos/química , Permeabilidad/efectos de los fármacos , Rifampin/farmacología , beta-Galactosidasa/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
10.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 12): 2433-48, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26627651

RESUMEN

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 ß-galactosidase capable of hydrolyzing short ß-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Šresolution) and its catalytic mutant E323A (at 2.50 Šresolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/ß domain, and the smallest all-ß domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Šfrom each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Šat the wide opening and ∼5 Šat the small opening and ∼40 Šin length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.


Asunto(s)
Proteínas Bacterianas/química , Galactosa/química , Geobacillus stearothermophilus/química , Oligosacáridos/química , Subunidades de Proteína/química , beta-Galactosidasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosa/metabolismo , Expresión Génica , Geobacillus stearothermophilus/enzimología , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Nitrofenilgalactósidos/química , Oligosacáridos/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Relación Estructura-Actividad , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
11.
Analyst ; 140(15): 4991-6, 2015 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-26042607

RESUMEN

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacterial detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles were released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1 h) with high sensitivity (10(2) CFU mL(-1)).


Asunto(s)
Técnicas Biosensibles/métodos , Agua Potable/microbiología , Escherichia coli/aislamiento & purificación , Oro/química , Nanopartículas del Metal/química , Staphylococcus aureus/aislamiento & purificación , beta-Galactosidasa/química , Técnicas Biosensibles/economía , Técnicas Electroquímicas/economía , Técnicas Electroquímicas/métodos , Enzimas Inmovilizadas/química , Límite de Detección , Nitrofenilgalactósidos/química
12.
Chembiochem ; 16(1): 126-39, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25407851

RESUMEN

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N-linked-lactosides and of eight O-linked N-acetyllactosamines, each substituted with a 1,2,3-triazole unit, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The totally regioselective ß-D-(1 → 4) galactosylation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor provided efficient access to the N-acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface-presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth-regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5-fold enhancement for prototype proteins, with further increases for galectins-3 and -4. Moreover, the availability of (15)N-labeled proteins and full assignments enabled (1)H, (15)N HSQC-based measurements for hu- man galectins-1, -3, and -7 against p-nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin-3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure-activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.


Asunto(s)
Amino Azúcares/química , Galectina 1/química , Galectina 3/química , Galectinas/química , Glicósidos/química , Acetilglucosamina/química , Alquinos/química , Amino Azúcares/síntesis química , Azidas/química , Proteínas Sanguíneas , Secuencia de Carbohidratos , Catálisis , Reacción de Cicloadición , Galectina 1/antagonistas & inhibidores , Galectina 3/antagonistas & inhibidores , Galectinas/antagonistas & inhibidores , Glicósidos/síntesis química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Nitrofenilgalactósidos/química , Unión Proteica , Relación Estructura-Actividad , Triazoles/química
13.
J Microbiol Biotechnol ; 25(1): 44-9, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25179901

RESUMEN

Thermostable enzymes derived from Thermotoga maritima have attracted worldwide interest for their potential industrial applications. Structural analysis and docking studies were preformed on T. maritima ß-glucosidase enzyme with cellobiose and pNP-linked substrates. The 3D structure of the thermostable ß-glucosidase was downloaded from the Protein Data Bank database. Substrates were downloaded from the PubCehm database and were minimized using MOE software. Docking of BglA and substrates was carried out using MOE software. After analyzing docked enzyme/substrate complexes, it was found that Glu residues were mainly involved in the reaction, and other important residues such as Asn, Ser, Tyr, Trp, and His were involved in hydrogen bonding with pNP-linked substrates. By determining the substrate recognition pattern, a more suitable ß-glucosidase enzyme could be developed, enhancing its industrial potential.


Asunto(s)
Simulación del Acoplamiento Molecular , Thermotoga maritima/enzimología , beta-Glucosidasa/metabolismo , Secuencia de Aminoácidos , Alcoholes Bencílicos/química , Alcoholes Bencílicos/metabolismo , Celobiosa/química , Celobiosa/metabolismo , Glucósidos/química , Glucósidos/metabolismo , Enlace de Hidrógeno , Himecromona/análogos & derivados , Himecromona/química , Himecromona/metabolismo , Cinética , Conformación Molecular , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Programas Informáticos , Especificidad por Sustrato , beta-Glucosidasa/química
14.
Methods Mol Biol ; 1200: 165-71, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25117234

RESUMEN

Equilibrium dialysis has been used to examine the binding affinity of ligands to proteins. It is a simple and reliable method, which requires only inexpensive equipment. For analysis of lectin-sugar interactions, the lectin and sugar are placed in the individual chambers separated by the membrane to allow the sugar to diffuse into the lectin chamber. After equilibrium has been reached, the concentrations of the sugar in both chambers are determined to evaluate the sugar-binding affinity of lectin. In this chapter, an example of the equilibrium dialysis experiment using the chromophoric derivatives of galactose and N-acetylgalactosamine is demonstrated, which reveals the difference in the affinity as well as specificities of two different carbohydrate-binding sites present in the B-chains of the plant lectin ricin.


Asunto(s)
Acetilgalactosamina/análogos & derivados , Diálisis/métodos , Nitrofenilgalactósidos/química , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Ligandos , Nitrofenilgalactósidos/metabolismo , Unión Proteica , Ricina/metabolismo
15.
J Biosci Bioeng ; 117(6): 706-10, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24462527

RESUMEN

ß-Galactosidase (lactase), which catalyzes the hydrolysis of lactose into glucose and galactose, is one of the most important enzymes used in dairy processing. In this study, a gene that encoded an extremely thermostable ß-galactosidase from Pyrococcus furiosus (Pflactase) was cloned and expressed in Escherichia coli BL21. The recombinant enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The enzyme displayed optimal activity at 90°C and pH 7.0 in phosphate buffer. The specific activity of the recombinant enzyme on o-nitrophenyl-ß-d-galactopyranoside was 10.2 U/mg at 0°C and 130.0dU/mg at 90°C. The half-lives of the enzyme were 31423.4, 8168.3, 4017.7, 547.4, 309.6, and 203.5 min at 70°C, 80°C, 85°C, 90°C, 95°C, and 100°C, respectively. The recombinant enzyme exhibited both ß-galactosidase and ß-glucosidase activity. The active inclusion bodies of ß-galactosidase were easily isolated by nonionic detergent treatment and directly used for lactose conversion in a repetitive batch mode. More than 54% (90°C) or 88% (10°C) of the original enzyme activity was retained after 10 conversion cycles under optimum conditions. These results suggest that the recombinant thermostable ß-galactosidase may be suitable for the hydrolysis of lactose in milk processing.


Asunto(s)
Proteínas Arqueales/química , Lactosa/química , Pyrococcus furiosus/enzimología , beta-Galactosidasa/química , Animales , Proteínas Arqueales/aislamiento & purificación , Estabilidad de Enzimas , Escherichia coli , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Leche/química , Leche/metabolismo , Nitrofenilgalactósidos/química , Pyrococcus furiosus/genética , beta-Galactosidasa/aislamiento & purificación
16.
Appl Biochem Biotechnol ; 172(6): 2787-99, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24435763

RESUMEN

A ß-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-ß-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L(-1) in which the target protein (6.24 g L(-1)) occupied approximately 86 %. The recombinant ß-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the ß-galactosidase may have a good application potential in whey and milk industries.


Asunto(s)
Aspergillus oryzae/enzimología , Proteínas Fúngicas/química , Lactosa/química , Leche/química , beta-Galactosidasa/química , Animales , Aspergillus oryzae/química , Secuencia de Bases , Codón , Fermentación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Expresión Génica , Ingeniería Genética , Glucosa/metabolismo , Glicerol/metabolismo , Hidrólisis , Cinética , Datos de Secuencia Molecular , Nitrofenilgalactósidos/química , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes , Especificidad por Sustrato , Temperatura , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genética
17.
Z Naturforsch C J Biosci ; 69(11-12): 479-87, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25854768

RESUMEN

The two genes lacZ1 and lacZ2 from Klebsiella pneumoniae 285, encoding ß-galactosidase isoenzymes II and III (KpBGase-II and -III), were each cloned downstream of a T7 promoter for expression in Escherichia coli BL21(DE3), and the resulting recombinant enzymes were characterized in detail. The optimum temperature and pH value of KpBGase-II were 40 °C and 7.5, and those of KpBGase-III were 50 °C and 8.0, respectively. KpBGase-III was more stable than KpBGase-II at higher temperature (>60°C). Both ß-galactosidases were more active towards o-nitrophenyl-ß- D-galactopyranoside as compared to lactose. The enzymatic synthesis of lactulose and 1-lactulose catalyzed by KpBGase-II and KpBGase-III was investigated. Using 400 g/L lactose and 200 g/L fructose as substrates, the resulting lactulose and 1-lactulose yields with KpBGase-II were 6.2 and 42.3 g/L, while those with KpBGase-III were 5.1 and 23.8 g/L, respectively. KpBGase-II has a potential for the production of 1-lactulose from lactose and fructose. Like other ß-galactosidases, the two isozymes catalyze the transgalactosylation in the presence of fructose establishing the ß-(1→1) linkage.


Asunto(s)
Isoenzimas/biosíntesis , Klebsiella pneumoniae/enzimología , Lactulosa/biosíntesis , beta-Galactosidasa/biosíntesis , Escherichia coli , Fructosa/química , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Klebsiella pneumoniae/genética , Lactosa/química , Lactulosa/química , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Temperatura , beta-Galactosidasa/genética
18.
ACS Appl Mater Interfaces ; 4(8): 3773-8, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22823570

RESUMEN

A new fluorescent biosensor has been designed to screen α-glucosidase inhibitors (AGIs) sensitively by utilizing signal amplification effect of conjugated polymers. The fluorescence of cationic poly(fluorenylene phenylene) (PFP) was quenched in the presence of para-nitrophenyl-α-d-glucopyranoside and α-glucosidase, and turned on upon addition of AGIs. Thus, a new method was developed for AGIs screening based on the fluorescence turn-off/turn-on. The IC(50) values obtained for inhibitors were compared with that reported using absorption spectroscopy. All results present the new method is more sensitive and promising in screening AGIs and inhibitors of other enzymes whose hydrolysis product is 4-nitrophenol.


Asunto(s)
Técnicas Biosensibles , Fluorenos/química , Colorantes Fluorescentes/química , Compuestos de Amonio Cuaternario/química , alfa-Glucosidasas/química , Cationes , Diabetes Mellitus/metabolismo , Glucósidos/química , Humanos , Hidrólisis , Concentración 50 Inhibidora , Cinética , Nitrofenoles/química , Nitrofenilgalactósidos/química , Polímeros/química , Espectrofotometría/métodos
19.
J Gen Appl Microbiol ; 58(1): 11-7, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22449746

RESUMEN

Lactobacillus gasseri ATCC33323(T) expresses four enzymes showing phospho-ß-galactosidase activity (LacG1, LacG2, Pbg1 and Pbg2). We previously reported the purification and characterization of two phospho-ß-galactosidases (Pbg1 and Pbg2) from Lactobacillus gasseri JCM1031 cultured in lactose medium. Here we aimed to characterize LacG1 and LacG2, and classify the four enzymes into 'phospho-ß-galactosidase' or 'phospho-ß-glucosidase.' LacG1 and recombinant LacG2 (rLacG2), from Lb. gasseri ATCC33323(T), were purified to homogeneity using column chromatography. Kinetic experiments were performed using sugar substrates, o-nitrophenyl-ß-D-galactopyranoside 6-phosphate (ONPGal-6P) and o-nitrophenyl-ß-D-glucopyranoside 6-phosphate (ONPGlc-6P), synthesized in our laboratory. LacG1 and rLacG2 exhibited high k(cat)/K(m) values for ONPGal-6P as compared with Pbg1 and Pbg2. The V(max) values for ONPGal-6P were higher than phospho-ß-galactosidases previously purified and characterized from several lactic acid bacteria. A phylogenetic tree analysis showed that LacG1 and LacG2 belong to the phospho-ß-galactosidase cluster and Pbg1 and Pbg2 belong to the phospho-ß-glucosidase cluster. Our data suggest two phospho-ß-galactosidase, LacG1 and LacG2, are the primary enzymes for lactose utilization in Lb. gasseri ATCC33323(T). We propose a reclassification of Pbg1 and Pbg2 as phospho-ß-glucosidase.


Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/aislamiento & purificación , Lactobacillus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Dominio Catalítico , Análisis por Conglomerados , Medios de Cultivo/química , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Pruebas de Enzimas , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Cinética , Lactobacillus/genética , Lactosa/química , Nitrofenilgalactósidos/síntesis química , Nitrofenilgalactósidos/química , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato
20.
Prikl Biokhim Mikrobiol ; 48(6): 668-72, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23330395

RESUMEN

Beta-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-beta-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of beta-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, beta-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the beta-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular beta-galactosidase activity in E. coli Tuner (DE3) expressing -galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of beta-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.


Asunto(s)
Bioensayo , Escherichia coli/enzimología , Nitrofenilgalactósidos/metabolismo , beta-Galactosidasa/análisis , Calibración , Permeabilidad de la Membrana Celular , Colorimetría , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Lactosa/metabolismo , Nitrofenilgalactósidos/química , Sensibilidad y Especificidad , Espectrofotometría , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...