RESUMO
The Y509E mutant of ß-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g-1, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2Y509E immobilization.
RESUMO
Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 ß-xylosidase from Geobacillus stearothermophilus with dual activity of ß-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original ß-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K m value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Reagentes de Ligações Cruzadas/química , Geobacillus stearothermophilus/enzimologia , Glutaral/química , Glicosídeo Hidrolases/química , Agregados Proteicos , Proteínas de Bactérias/genética , Geobacillus stearothermophilus/genética , Glicosídeo Hidrolases/genética , Mutação de Sentido IncorretoRESUMO
The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family (ß-α-ß-ß-ß-α) at its N-terminal region (residues 1100), whereas the C terminus (residues 100197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.
Assuntos
Proteínas de Bactérias/química , Proteínas Ribossômicas/química , Ribossomos/química , Synechocystis/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Soluções , Synechocystis/metabolismo , TermodinâmicaRESUMO
It is possible to analyze peroxidase (POD) from different vegetable sources by electrophoresis. Zymography, i.e., a SDS-PAGE method to detect enzyme activity, is used to specifically detect POD activity and to visualize the total protein profile. For this purpose, we describe how a radish homogenate is prepared and submitted first to electrophoresis, and then, the POD activity present in the gel is reactivated and selectively stained using guaiacol as substrate. After scanning the gel, the same gel is further stained with Coomassie blue to determine the whole protein profile of the sample.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Peroxidase/análise , Raphanus/enzimologia , Corantes/análise , Guaiacol/metabolismo , Peroxidase/metabolismo , Raphanus/metabolismo , Corantes de Rosanilina/análise , Coloração e Rotulagem/métodosRESUMO
Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copolymerized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside time and resource saving.
Assuntos
Bacillus/enzimologia , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Lipase/análise , Peptídeo Hidrolases/análise , Bacillus/metabolismo , Eletroforese em Gel de Poliacrilamida/instrumentação , Ensaios Enzimáticos/instrumentação , Desenho de Equipamento , Indicadores e Reagentes/análise , Lipase/metabolismo , Peptídeo Hidrolases/metabolismo , Corantes de Rosanilina/análise , Prata/análise , Coloração e Rotulagem/métodosRESUMO
Lipases are esterases that occur widely in nature, yet those with commercial relevance are exclusively from microbial origin. Glycerol and long-chain fatty acids are the products after hydrolysis of esters bonds in saponifiable lipids catalyzed by lipases. In this work, we describe lipase/esterase activity contained in cell-free fractions from thermophilic bacteria, cultured in medium containing olive oil. Analysis of the cell-free fractions by electrotransference zymography, using tributyrin as substrate, revealed bands corresponding to lipase activity. The method is simple, fast, and inexpensive.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Esterases/metabolismo , Geobacillus/enzimologia , Lipase/metabolismo , Técnicas de Cultura de Células/métodos , Geobacillus/metabolismo , Hidrólise , Especificidade por Substrato , Triglicerídeos/metabolismoRESUMO
This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically detect peroxidase activity and furthermore, to analyze the total protein profile. After the assay, students may estimate the apparent molecular mass of the enzyme and discuss its structure. After the 4-h experiment, students gain knowledge concerning biological sample preparation, gel preparation, electrophoresis, and the importance of specific staining procedures for the detection of enzymatic activity.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Peroxidase/metabolismo , Ensino/métodos , Bioquímica/educação , Bioquímica/métodos , Humanos , Modelos Moleculares , Peso Molecular , Peroxidase/química , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Estudantes , UniversidadesRESUMO
Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.
Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Dicroísmo Circular/métodos , Dimerização , Concentração de Íons de Hidrogênio , Estrutura Quaternária de Proteína/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
The isolation, purification, biochemical and biophysical characterization of the first reported beta-xylosidase from Geobacillus pallidus are described. The protein has an optimum pH close to 8 and an optimum temperature of 70 degrees C. These biochemical properties agree with those obtained by spectroscopic techniques, namely, circular dichroism (CD), infrared (FTIR) and fluorescence measurements. Thermal denaturation, followed by CD and FTIR, showed an apparent thermal denaturation midpoint close to 80 degrees C. The protein was probably a hydrated trimer in solution with, an elongated shape, as shown by gel filtration experiments. FTIR deconvolution spectra indicated that the protein contains a high percentage of alpha-helix (44%) and beta-sheet (40%). The sequencing of the N terminus and the biochemical features indicate that this new member of beta-xylosidases belongs to the GH52 family. Since there are no reported structural studies of any member of this family, our studies provide the first clue for the full conformational characterization of this protein family.
Assuntos
Bacillaceae/enzimologia , Xilosidases/isolamento & purificação , Cromatografia em Gel , Dicroísmo Circular , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Desnaturação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por Substrato , Xilosidases/química , Xilosidases/metabolismoRESUMO
In Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans, no significant differences were observed in the phospholipid species of both morphological phases. The species observed were phosphatidylcholine (PC, 30-40%), phosphatidylethanolamine (PE, 27-28%), phosphatidylserine (16-19%), phosphatidylinositol (13-17%) and sphingomyelin (3-5%). The main fatty acids found in the yeast (Y) phase were palmitate (56%), linoleate (18%) and oleate (15%), while linoleate predominated (61%) in the mycelial (M) phase, followed by palmitate (27%) and oleate (7%). In the Y phase the main free sterol was ergosta-5,22-dien-3 beta-ol (82%) plus some lanosterol (12%) and ergosterol (6%), while in the M phase, the latter predominated (88%), followed by low levels of ergosta-5,22-dien-3 beta-ol (12%). Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a platelet aggregation inhibitor derived from garlic, induced alterations in phospholipid and fatty acid proportions such that PC was reduced to about 18% in both phases and PE increased to 38% (Y phase) or 44% (M phase), suggesting inhibition of PC synthesis. Ajoene also reduced saturated fatty acids (16:0 and 18:0) from 67 to 35% in the Y phase, with a corresponding increase in the unsaturated components. This effect was not seen in the M phase.