Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus, microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

Effect of gallic acid polydisulfide on activity and stability of catalase in various media

The effect of the active bioantioxidant polydisulfide of gallic acid (PDSG) on the catalytic activity and operational and thermal stability of catalase was studied in three media: distilled water (pH approximately 5.6), phosphate buffer, pH 7.4, and reversed micelles of Aerosol OT (AOT) in heptane of varied hydration degree w0. PDSG inhibited the catalase-induced decomposition of H2O2 by the mixed or noncompetitive mechanism: in various media the inactivation constant Ki varied in the range of (0.63-2.32).10-5 M. PDSG nearly twofold decreased the rate constant of interaction of the complex I of catalase with H2O2 (k2, M-1.sec-1) in water and reversed micelles of AOT and 3-5 times increased the effective rate constant of catalase thermal inactivation, k*in, sec-1, depending on the reaction medium. PDSG significantly decreased the rate constant of catalase inactivation during the enzymatic reaction, kin, sec-1, and thus increased the enzyme operational stability in water and reversed AOT micelles in heptane. The interaction of PDSG with catalase in water and in phosphate buffer was accompanied by significant changes in CD spectra in the far UV-region that indicated disturbances in the secondary structure of catalase subunits induced by the bioantioxidant; the latter was suggested to initiate the reaction of thiol--disulfide exchange with the enzyme. The problem of the compatibility of catalase with disulfide bioantioxidants is discussed.     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.     

Determination of phenol using an enhanced chemiluminescent assay

Enhanced chemiluminescence (ECL) describes the phenomenon of increased light output in the luminol oxidation reaction catalysed by horseradish peroxidase (HRP) in the presence of certain compounds, such as para‐iodophenol. In this work, the effects of phenol on the para‐iodophenol‐enhanced HRP‐catalysed chemiluninescent reaction intensity in an aqueous buffer (Tris–HCl buffer, pH 8.5) and in a surfactant–water–octane mixture were compared. Preincubation of HRP at low phenol concentrations stimulated the chemiluminescent intensity in the assay performed in an aqueous buffer, but did not have significant effect in the sodium bis(2‐ethylhexyl)sulphosuccinate) (Aerosol OT, AOT) applied system. It was also observed that HRP preincubation with phenol concentration higher than 0.003 mg/mL produced an inhibitory effect on the enzyme activity for both assay systems. Only an inhibitory effect of phenol on the chemiluminescent intensity in the surfactant system in octane (as organic solvent) was observed. Three assays were developed to determine phenol concentration in water and in an organic solvent mixture. The detection limits were 0.006, 0.003 and 0.0005 mg/mL, respectively, for the buffer‐containing system, the AOT‐applied system with phenol standard solutions in water and for the AOT‐applied system with phenol standard solutions in octane. Copyright © 2002 John Wiley & Sons, Ltd.     

BEHAVIOUR OF HORSERADISH PEROXIDASE IN AOT REVERSED MICELLES

The activity and stability of horseradish peroxidase (HRP) solubilised in AOT reversed micelles in isooctane and decalin was studied using guaiacol (2-methoxyphenol) as the electron donor.

The activity of the enzyme in both reversed micellar systems increases with the water content until reaching a maximum value that remains fairly constant for water contents higher than 3.05% (v/v) in isooctane and 2.20% in decalin. The effect of pH on the activity profile was studied in the system AOT/isooctane. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.05% (v/v) but it was completely deactivated at pH 9. The effect of surfactant concentration on HRP activity was also investigated. At low water contents a strong dependence was observed, whilst no further activity increase was observed for water content values higher than 2.7% (v/v).

The stability of HRP was found to be strongly dependent on the water content of the system with higher levels of stability obtained for higher values of water content. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.     

Characteristics of antigen-antibody interaction in the reversed micelles of surface-active agents in heptane

The alterations in the catalytic activity of the horseradish peroxidase after its interaction with antibodies against this enzyme have been studied in buffered solution and in reversed Aerosol OT (AOT) micelles in heptane. The antibodies were obtained by immunizing the rabbits with electrophoretically homogeneous enzyme and were purified by affinity chromatography. In the AOT micelles and mixed micelles containing AOT and Triton X-45, the enzyme interacted with antibodies very rapidly (in less than 5 min), i.e. the micelles did not hinder effective interaction between the enzyme and antibodies. The decrease in the peroxidase catalytic activity upon its interaction with antibodies in a micellar medium was determined by [H2O]/[AOT] ratio, pH and molarity of polar nucleus, as well as by the initial concentration of antibody. In buffered solutions, the decrease n the peroxidase activity of the enzyme--antibody complex was only weakly dependent on pH and molarity of a buffer solution.     

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Unusual Behavior of Membrane Somatic Angiotensin-Converting Enzyme in a Reversed Micelle System

Properties of the membrane and soluble forms of somatic angiotensin-converting enzyme (ACE) were studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. The membrane enzyme with a hydrophobic peptide anchor was more sensitive to anions and to changes in pH and composition of the medium than the soluble enzyme without anchor. The activity of both forms of the enzyme in the reversed micelles significantly depended on the molarity of the buffer added to the medium (Mes-Tris-buffer, 50 mM NaCl). The maximum activity of the soluble ACE was recorded at buffer concentration of 20-50 mM, whereas the membrane enzyme was most active at 2-10 mM buffer. At buffer concentrations above 20 mM, the rate of hydrolysis of the substrate furylacryloyl-L-phenylalanyl-glycylglycine by both ACE forms was maximal at pH 7.5 both in the reversed micelles and in aqueous solutions. However, at lower concentrations of the buffer (2-10 mM), the membrane enzyme had activity optimum at pH 5.5. Therefore, it is suggested that two conformers of the membrane ACE with differing pH optima for activity and limiting values of catalytic constants should exist in the reversed micelle system with various medium compositions. The data suggest that the activity of the membrane-bound somatic ACE can be regulated by changes in the microenvironment.     

Micellar catalysis of polyphenol oxidase in AOT/cyclohexane

The catalytic behaviour of mushroom polyphenol oxidase has been studied in dioctylsulphosuccinate (AOT)/cyclohexane reverse micelles. The steady-state conditions were accomplished up to 20 min and 17 μg protein in the assay towards 4-methylcatechol and no loss of specific activity was observed relative to aqueous medium. The pH activity profile of the enzyme was kept in reverse micelles as in water, showing a plateau between 5 and 6.5. The stability of polyphenol oxidase to pH was also studied and about 20% inactivation was found in reverse micelles relative to aqueous medium at neutral pHs. Moreover there was a decrease of stability at acidic pHs. The optimum W_o obtained was 20 and the enzyme was nearly independent of the surfactant concentration at constant W_o.

Kinetic studies of polyphenol oxidase towards several substrates showed that the substrate inhibition by p-cresol and 4-methylcatechol observed in buffer was not kept in AOT/cyclohexane reverse micelles. Moreover, the K_m increased and the catalytic efficiency (V/K_m) of the enzyme decreased as the hydrophobicity of substrates was increased.     

Stabilization of diluted aqueous solutions of horseradish peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.     

Glutathione S-transferase from the diamond back moth (Plutella xylostella linnaeus)

Glutathione S-transferase was present in all the developmental stages of Plutella xylostella. The enzyme levels increased rapidly and reached a maximum at the pupal stage and then declined towards adulthood. The resistant strain was found to contain between 3–4 times more glutathione S-transferase than the susceptible strain. However, the enzyme from both the strains had similar K_m values for GSH and DNCB, respectively. The crude enzyme had an optimum pH of 8.3 and its activity was affected by buffer molarity. The enzyme was completely inactivated on dialysis and the stability of the enzyme in the crude supernatant could be maintained in the presence of 1 mM concentrations of either GSH, 2-mercaptoethanol or cysteine. Metal ions had no effect on the stability of the enzyme. Data from Arrhenius plots, column chromatographic techniques and isoelectric focusing suggested the presence of a single form of the enzyme. The enzyme had an isoelectric point of 9.26 and a molecular weight of 36,400.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30-40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Studies on the extraction and back-extraction of a recombinant cutinase in a reversed micellar extraction process

This work deals with the extraction and back-extraction of a recombinant cutinase using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration and temperature on the extraction and back-extraction of the cutinase was investigated. High extraction (97%) of the cutinase was achieved at pH 7.0 with a 50 mM Tris-HCl buffer solution containing 100 mM KCl, but a low activity was detected in the reversed micellar phase. At pH 9.0, cutinase was extracted (75%) to the reversed micelles with higher activity. Cutinase was recovered (50%) from a reversed micellar phase (100 mM AOT/isooctane) into a 50 mM Tris-HCl buffered solution at pH 9.0 with 100 mM KCl, and 20°C. Protein and cutinase activity global yields of 38 and 45%, respectively, were obtained for the global process, extraction and back-extraction steps, using low ionic strength, pH 9.0, 100 mM AOT and 20°C.Maria das Graças Carneiro da Cunha acknowledges a Ph.D. fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisas Aggeu Magalhães, Brasil. This work was partly financed by the BRIDGE Programme (Contract BIOT-CT91-0274(DTEE)).     

Addition of reducing agents to the peroxidase-o-phenylenediamine buffer reduces background of enzyme immunoassays

The concentrations of o-phenylenediamine (OPD), H2O2, citrate and H+ in a substrate buffer for peroxidase immunoassays were optimized for minimal background. The background was reduced 2-3 fold with 5.5 mM OPD, 3 mM H2O2, 150 mM citric acid/sodium citrate, pH 4.8, and the reproducibility interassay was increased. A further 3-5 fold reduction of the background was obtained by the addition of 1.5 mM acetanilide, 0.14 mM beta-mercaptoethanol and 5 mM nitrilotriacetic acid to the substrate buffer. This low-background substrate buffer allows increased sensitivity and lowers the interassay variation coefficient. It has been used successfully in peroxidase immunoassays of human C-reactive protein, human antiestreptolysin and human rheumatoid factor.     

Purification and properties of adenosinetriphosphatase from Zea mays seedling microsomes

A simple method was developed for selective solubilization of membrane ATPase from etiolated corn seedlings using 0.01% Triton X100 and 0.01% deoxycholate containing 200 mM KI. An 81-fold enriched enzyme preparation, with specific activity of 133 μmol Pi/mg protein/hr, was obtained. The enzyme stored in 25 mM Tris-HCl buffer (pH 7.5) at 4° showed rapid loss of activity. The enzyme was stabilized by 1 mM EDTA with addition of 1.2 mM Mg²⁺°. Mg²⁺ and Ca²⁺ (1.2 mM) increased enzymatic activity by 12 and 10.8% respectively, whereas Na⁺ and K⁺ brought about a 20% increase in ATP-hydrolysis. The effect of combined mono- and di-valent ions was neither synergistic nor additive. Ouabain exerted no effect on enzyme activity. The enzyme showed two pH optima (6.0 and 7.5) in the presence of Na⁺ and K⁺, and one optimum at pH 6.5 in the absence of these ions. On polyacrylamide gel the enzyme was resolved into two protein bands, both exhibiting ATPase activity. It is suggested that the soluble enzyme from the microsomal fraction of corn seedlings contains two ATP-hydrolyzing enzymes, one of them being stimulated by Na⁺ and K⁺ ions.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.