RESUMEN
Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.
Asunto(s)
Escherichia coli/genética , Peroxidasas/biosíntesis , Peroxidasas/genética , Peroxidasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Clonación Molecular , Cuerpos de Inclusión/enzimología , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Significant conformational differences between native and recombinant horseradish peroxidase have been shown by tritium planigraphy, which includes a method of thermal activation of tritium followed by amino acid analysis of the protein preparation. Comparison of radioactivity distribution among the amino acid residues with the theoretical (calculated) accessibility shows that the recombinant enzyme is characterized by high hydrophobicity and compactness of folding. The protective role of oligosaccharides in native enzyme has been confirmed. An unexpected result of the study is a finding on high accessibility of a catalytic histidine residue in solution. An effect of low dose (3 Gy) of irradiation on the accessibility of amino acid residues has been unequivocally demonstrated. The data can be interpreted as swelling of the compact folding and increase in the surface hydrophilicity of the recombinant enzyme. In the case of native enzyme, irradiation does not cause remarkable changes in the accessibility of amino acid residues indicating the possible extensive radical modification of the native enzyme in the life-course of the cell. The catalytic histidine is an exception. It becomes inaccessible after the enzyme irradiation, while its accessibility in the recombinant enzyme increases. An additional observation of a 5-fold decrease in the rate constant towards hydrogen peroxide points to the destructive effect of irradiation on the hydrogen bond network in the distal domain of the native enzyme molecule and partial collapse of the active site pocket.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Proteínas Recombinantes/química , Aminoácidos/análisis , Estabilidad de Enzimas/efectos de la radiación , Rayos gamma , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/aislamiento & purificación , Peroxidasa de Rábano Silvestre/efectos de la radiación , Conformación Proteica/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/efectos de la radiación , TritioRESUMEN
A quantitative approach for estimation of the non-enzymatic interaction between ammonium 2,2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation product and a poorly oxidized substrate was developed using a system including tobacco peroxidase, a mediator substrate (ABTS), and a second substrate. The approach is based on the establishment of a pseudo-steady-state concentration of the ABTS oxidation product in the course of co-oxidation with a poor substrate. A mathematical description of the experimental curve shape has been proposed to linearize the kinetic data and estimate the rate constant for such non-enzymatic interaction. The rate constants calculated from the steady-state kinetics for the non-enzymatic interaction of ABTS oxidation product with phenol and resorcinol were 360 +/- 40 and 770 +/- 60 M(-1).sec(-1), respectively. The values obtained have the same order of magnitude as the rate constant for ABTS oxidation product interaction with veratryl alcohol, calculated from electrochemical measurements (170 M(-1).sec(-1)) by Donal Leech's group. However, the kinetic curves for co-oxidation of ABTS and veratryl alcohol catalyzed by tobacco peroxidase exhibit a pronounced lag-period, which either points to the high rate of the non-enzymatic interaction between ABTS oxidation product and veratryl alcohol and thus, contradicts the electrochemical calculations, or indicates an enzymatic nature of the co-oxidation phenomenon in this particular case.
Asunto(s)
Nicotiana/enzimología , Peroxidasa/metabolismo , Alcoholes/metabolismo , Benzotiazoles , Catálisis/efectos de los fármacos , Cinética , Fenol/farmacología , Resorcinoles/metabolismo , Ácidos Sulfónicos/química , Ácidos Sulfónicos/metabolismoRESUMEN
Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.
Asunto(s)
Peroxidasa de Rábano Silvestre/metabolismo , Triptófano/química , Catálisis , Estabilidad de Enzimas , Peroxidasa de Rábano Silvestre/química , Concentración de Iones de Hidrógeno , Cinética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The effect of extremely acidic pH on the stability of tobacco peroxidase and lignin peroxidase holoenzymes has been studied. Stabilization of tobacco peroxidase holoenzyme in the presence of calcium cations at pH < 2 and stabilization of lignin peroxidase at pH > 2 in the presence of veratryl alcohol have been shown. The dependence of the reaction rate constant for hydrogen peroxide interaction with tobacco peroxidase on pH suggests that the reaction rate is under control of a group with pK of 2.5. A tobacco peroxidase model structure has been created by means of homology modeling on the basis of the tobacco peroxidase sequence and the coordinates of peanut peroxidase crystal structure. The model structure demonstrates the presence of the negatively charged Glu-141 at the entrance to the active site and its electrostatic repulsion from heme propionates and triad of Asp-76, -79, and -80 residues. The results on tobacco holoperoxidase stabilization at pH 1.8 in the presence of calcium cations and drop in reaction rate constant for the enzyme interaction with hydrogen peroxide are explained by a hypothetical formation of ionic bonds between Glu-141 and the triad of aspartic acid residues via calcium cation lowering the accessibility of the active site and stabilizing the holoenzyme.
Asunto(s)
Nicotiana/enzimología , Peroxidasa/metabolismo , Plantas Tóxicas , Secuencia de Aminoácidos , Arachis/enzimología , Arachis/genética , Calcio/metabolismo , Dominio Catalítico , Estabilidad de Enzimas , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Peroxidasa/química , Peroxidasa/genética , Peroxidasas/química , Peroxidasas/genética , Peroxidasas/metabolismo , Conformación Proteica , Homología de Secuencia de Aminoácido , Electricidad Estática , Nicotiana/genéticaRESUMEN
The purification and refolding of the recombinant horseradish peroxidase produce by E. coli transformants are described. The recombinant enzyme is of 34 kDa and has an isozyme spectrum similar to Sigma type VI horseradish peroxidase. The specific activity of the refolded peroxidase is of about 2000 U/mg with ABTS as a substrate. The recombinant and native enzyme are similar with respect to their catalytic properties in the reaction of enhanced chemiluminescence. Operational and thermal stability of the refolded peroxidase is two to three times lower than for the native one.
Asunto(s)
Peroxidasa de Rábano Silvestre/metabolismo , Isoenzimas/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/genética , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/genética , Cinética , Mediciones Luminiscentes , Peso Molecular , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformación GenéticaRESUMEN
Conditions were determined for preparation of spheroplasts from E. coli under the action of lysozyme in the presence of EDTA. The preparation took from 10 to 15 min. The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm. The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose. At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent. The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.
Asunto(s)
Amoníaco-Liasas/análisis , Aspartato Amoníaco-Liasa/análisis , Escherichia coli/enzimología , Esferoplastos/aislamiento & purificación , Escherichia coli/ultraestructura , Esferoplastos/enzimologíaRESUMEN
The present review systematizes the literature data on localization of enzymes in cells of Gram-negative bacteria. It centres round the methods used for studying the enzyme localization. The criteria according to which one can locate an enzyme in bacterial cells are discussed.
Asunto(s)
Enzimas/aislamiento & purificación , Bacterias Gramnegativas/enzimología , Técnicas Bacteriológicas , Membrana Celular/enzimología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Centrifugación , Citoplasma/enzimología , Bacterias Gramnegativas/efectos de los fármacos , Histocitoquímica , Sustancias Macromoleculares , Mutación , Presión Osmótica , Esferoplastos/enzimologíaRESUMEN
Experiments were carried out to investigate the process of a continuous enzymatic synthesis of L-aspartic acid from ammonium fumarate in uniform filling flow reactors. Escherichia coli (Soviet strain 85) cells immobilized in polyacrylamide gel granules reinforced by a solid carrier were used as biocatalysts. The conditions, under which a high aspartase activity of the biocatalyst and a stable hydrodynamic performance of the reactor were maintained, were determined. The main kinetic characteristics of a continuous performance of the reactor for 150 days were obtained.