RESUMO
Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.
Assuntos
Kluyveromyces/enzimologia , Saponinas/farmacologia , beta-Galactosidase/efeitos dos fármacos , Calorimetria , Dicroísmo Circular , Cinética , Nitrofenilgalactosídeos/metabolismo , Casca de Planta/química , Estrutura Secundária de Proteína , Quillaja/química , Espectrometria de Fluorescência , beta-Galactosidase/metabolismoRESUMO
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.
Assuntos
Kluyveromyces/enzimologia , Taninos/metabolismo , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Animais , Calorimetria/métodos , Dicroísmo Circular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Nitrofenilgalactosídeos/química , Nitrofenilgalactosídeos/metabolismo , Conformação Proteica , Espectrometria de Fluorescência , Taninos/química , TermodinâmicaRESUMO
The fungus Penicillium purpurogenum degrades plant cell walls by the action of cellulolytic, xylanolytic and pectinolytic enzymes. The α-D-galactosidase is one of the enzymes which may act on pectin degradation. This enzyme has several biotechnological and medical applications. The aim of this work was to better understand the molecular mechanism of α-D-galactosidase from P. purpurogenum (GALP1). For this purpose, a gene coding for the enzyme was identified from the fungal genome and heterologously expressed in Pichia pastoris. The enzyme belongs to glycosyl hydrolase family 27. The protein of 435 amino acids has an optimum pH and temperature for activity of 5.0 and 50 °C, respectively. The KM for p-nitrophenyl-α-D-galactopyranoside (GalαpNP) is 0.138 mM. The enzyme is inhibited by GalαpNP at concentrations higher than 1 mM, and by the product galactose. A kinetic analysis of product inhibition shows that it is of mixed type, suggesting the presence of an additional binding site in the enzyme. To confirm this hypothesis, a structural model for GALP1 was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The data suggest that galactose may bind to an enzyme alternative pocket promoting structural changes of the active site, thus explaining its inhibitory effect. In silico site-directed mutagenesis experiments highlighted key residues involved in the maintenance of the alternative binding site, and their mutations for Ala predict the formation of proteins which should not be inhibited by galactose. The availability of an α-galactosidase with different kinetic properties to the existent proteins may be of interest for biotechnological applications.
Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Galactose/metabolismo , Penicillium/enzimologia , alfa-Galactosidase/antagonistas & inibidores , alfa-Galactosidase/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutação , Nitrofenilgalactosídeos/metabolismo , Nitrofenilgalactosídeos/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Temperatura , alfa-Galactosidase/química , alfa-Galactosidase/genéticaRESUMO
Treatment of Aspergillus niveus with 30 µg tunicamycin/ml did not interfere with α-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, ~56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65°C). The K(m) and V(max) values of under- and fully-glycosylated forms of α-glucosidase were similar when assessed for hydrolysis of starch (~0.6 mg/ml, ~350 µmol glucose per min per ml), maltose (~0.54 µmol, ~330 µmol glucose per min per ml) and p-nitrophenyl-α-D: -glucopyranoside (~0.54 µmol, ~8.28 µmol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.
Assuntos
Aspergillus/efeitos dos fármacos , Aspergillus/enzimologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases , Tunicamicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Maltose/metabolismo , Peso Molecular , Nitrofenilgalactosídeos/metabolismo , Amido/metabolismo , Temperatura , alfa-Glucosidases/química , alfa-Glucosidases/isolamento & purificação , alfa-Glucosidases/metabolismoRESUMO
An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.
Assuntos
Ascomicetos/enzimologia , Glucose/farmacologia , Xilose/farmacologia , beta-Glucosidase/metabolismo , Celobiose/metabolismo , Dissacarídeos/metabolismo , Ativadores de Enzimas/farmacologia , Indução Enzimática , Estabilidade Enzimática , Glucosídeos/metabolismo , Glicosídeos/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Lactose/metabolismo , Peso Molecular , Nitrofenilgalactosídeos/metabolismo , Especificidade por Substrato , Temperatura , beta-Glucosidase/química , beta-Glucosidase/isolamento & purificaçãoRESUMO
During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not beta-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-beta-D-galactopyranoside (ONPG), indicating that beta-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that beta-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of beta-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed.