Influence of Igepal reverse micellar and micellar systems on activity and stability of heme peroxidases

The activities of horseradish peroxidase (HRP) and lactoperoxidase (LPO) entrapped in reverse micelles of Igepal CO-520 in cyclohexane were studied. When the molar ratio of water to surfactant, w ₀ was ≥13, the activity of HRP encapsulated in the water pool of the reverse micelle was comparable with that measured in buffer. For LPO, however, lower activity was observed after its incorporation into the same system.

The activity of the investigated peroxidases was also measured in an aqueous solution of Igepal CO-720 or after incubation with this surfactant. The enzymes became inactivated in an aqueous micellar solution of Igepal CO-720, although this process was reversible.

The stability of HRP and LPO at 37 or 50°C was lower in the micellar systems than in buffer with the exception for HRP in reverse micelles at 50°C.     

Effect of surfactants on peroxidase activity. III. Effect of nonionic surfactants

The effect of a series of nonionic surfactants on the initial rate of the peroxide oxidation of 5-aminosalicylic acid in solution catalyzed by horseradish peroxidase was studied. As the surfactant concentration increases, the peroxidation rate first increases, then decreases, and the increase/decrease cycle is repeated. The primary increase may be induced by a change in properties of the medium under the action of surfactants, and the following decrease, by the enzyme inhibition. The secondary increase may be explained by to a change in the enzyme conformation and an increase in the accessibility of its active site for the substrate due to the immobilization of the protein in the surfactant aggregates, whereas the secondary decrease, by a shielding of the protein with these aggregates. For communication II, see [1].     

Catalytic and interfacial aspects of enzymatic polymer synthesis in reversed micellar systems

The enzyme horseradish peroxidase, when encapsulated in reversed micelles, is capable of catalyzing the synthesis of phenolic and aromatic amine polymers. The synthesis of polyethylphenol is specifically considered in this article and is found to be extremely feasible in the micellar system. Polymer chain growth can be controlled to some degree by manipulating the ability of the solvent to sustain chain solubility; this is effectively done by adjusting the surfactant concentration. This results in a degree of control of polymer molecular weight. The synthesized polymer drops out of solution and can be easily recovered. (c) 1993 John Wiley & Sons, Inc.     

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.     

Calcium promotes activity and confers heat stability on plant peroxidases

In this paper we demonstrate how peroxidase (PO) activities and their heat stability correlate with the availability of free Ca²⁺ ions. Calcium ions work as a molecular switch for PO activity and exert a protective function, rendering POs heat stable. The concentration ranges of these two activities differ markedly. POs are activated by µM Ca²⁺ concentration ranges, whereas heat stabilization is observed in the nM range. This suggests the existence of different Ca²⁺ binding sites. The heat stability of POs depends on the source plant species. Terrestrial plants have POs that exhibit higher temperature stability than those POs from limnic and marine plants. Different POs from a single species can differ in terms of heat stability. The abundance of different POs within a plant is dependent on age and developmental stage. The heat stability of a PO does not necessarily correlate with the maximum temperature the source species is usually exposed to in its natural habitat. This raises questions on the role of POs in the heat tolerance of plants. Consequently, detailed investigations are needed to identify and characterize individual POs, with regard to their genetic origin, subcellular expression, tissue abundance, developmental emergence and their functions in innate and acquired heat tolerance.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Biooxidation of n-hexanol by alcohol oxidase and catalase in biphasic and micellar systems without solvent

Alcohol oxidase from Pichia pastoris together with catalase from bovine liver was used to oxidize n-hexanol to hexanal. For this purpose, an aqueous buffer solution was mixed with large amounts of hexanol by simple agitation, yielding a biphasic system, or by adding the nonionic surfactant Brij 35. Initial velocities and reaction yields after 24 h were measured as a function of various parameters such as the amounts of enzymes, hexanol, or surfactant. High enzymatic activity was determined for hexanol concentrations of between 20 mass% and 80 mass% without using any additional organic solvent. The homogenization of the biphasic systems with the help of Brij 35 did not yield a significant improvement of the bioconversion, which would justify the use of surfactants.     

Protein extraction by reverse micelles: studies on the recovery of horseradish peroxidase

Phase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 1.11.1.7) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in wellstirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80mM phosphate buffer and 1 M KCI. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH<4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1 MNaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25xC. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios. (c) 1994 John Wiley & Sons, Inc.     

Effects of microwaves (900 MHz) on peroxidase systems: A comparison between lactoperoxidase and horseradish peroxidase

ABSTRACT

This work shows the effects of exposure to an electromagnetic field at 900?MHz on the catalytic activity of the enzymes lactoperoxidase (LPO) and horseradish peroxidase (HRP). Experimental evidence that irradiation causes conformational changes of the active sites and influences the formation and stability of the intermediate free radicals is documented by measurements of enzyme kinetics, circular dichroism spectroscopy (CD) and cyclic voltammetry.     

Single step purification of lactoperoxidase from whey involving reverse micelles‐assisted extraction and its comparison with reverse micellar extraction

The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles‐assisted extraction and compared with reverse micellar extraction. The reverse micelles‐assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25‐fold, respectively. Whereas reverse micelles‐assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39‐fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profiles also evidenced that higher purification was obtained in reverse micelles‐assisted extraction as compared of reverse micellar extracted lactoperoxidase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

Peroxidase biocatalysis in water-soluble ionic liquids: activity,kinetic and thermal stability

Abstract

The activity and stability of commercial peroxidase was investigated in the presence of five 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) with either bromide or chloride anions: [C_xmim][X]. The peroxidase activity and stability were better for the shorter alkyl chain lengths of the ILs and peroxidase was more stable in the presence of the bromide anion, rather than chloride. The thermal inactivation profile was studied from 45 to 60 °C in [C₄mim][Cl] and [C₄mim][Br]. The activation energy was also determined. Kinetic analysis of the enzyme in the presence of the [C₄mim][Br] or control (buffer solution) showed that the KM value increased 5-fold and Vm decreased 13-fold in the presence of the IL. The increase in KM indicates that this IL can reduce the binding affinity between substrate and enzyme.     

非离子反胶束中木素过氧化物酶催化性能研究

木素过氧化物酶（LiP）在环己烷/Brij30/水反胶束体系中可体现催化活力,然而在水/醇/TritonX100/环己烷反胶束体系中却没有催化活力。对影响Brij30反胶束中LiP催化活力各主要因素进行了优化并测定了LiP在其中的时间稳定性;结果表明,20℃下,使LiP体现最佳活力的Brij30反胶束介质条件为：ω₀＝8.5,pH＝2.2,［Brij30］＝600mmol/L;在此条件下,LiP的半衰期可达到50h;与水介质相比,酶活力下降了,但稳定性却提高了。直链醇是TritonX100形成反胶束的必要组分,为揭示醇的作用,还考察了戊醇对Brij30 反胶束中LiP催化活力的影响,发现高浓度戊醇对LiP有失活作用。据此推测助表面活性剂醇可能是LiP在环己烷/TritonX100/戊醇/水反胶束中不能体现催化活力的主要原因。     

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio (W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters (V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter‐ion and iso‐propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04‐fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:845–855, 2014     

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system

The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.     

Protein extraction and activity in reverse micelles of a nonionic detergent

We describe, for the first time, the ability of a polyoxyethylene sorbitan trioleate-isopropanol microemulsion in hexane to solubilize pure proteins. The dependences of cytochrome c extraction and buffer solubilization by the reverse micellar system on ionic strength of the aqueous phase, detergent concentration, and cosurfactant concentration result in increased extraction. In addition, subtilisin (a serine protease) is shown to be active in this microemulsion. Further the activity of the enzyme can be regulated by the water content of the micelles, enabling control of enzyme activity by "solvent engineering."     

Kinetic studies on oxidation of aromatic donor molecules by horseradish peroxidase and lactoperoxidase

Based on kinetic evidence, it has been shown for the first time that the mode of binding of aromatic donor molecules is similar in horseradish peroxidase and lactoperoxidase; also that the nature of the heme plays an important role in the reaction with hydrogen peroxide, and has no effect on the reaction of the intermediate compound II with aromatic substrates.     

Activity and stability of catalase in nonionic micellar and reverse micellar systems

Catalase activity and stability in the presence of simple micelles of Brij 35 and entrapped in reverse micelles of Brij 30 have been studied. The enzyme retains full activity in aqueous micellar solution of Brij 35. Catalase exhibits "superactivity" in reverse micelles composed of 0.1 M Brij 30 in dodecane, n-heptane or isooctane, and significantly lowers the activity in decaline. The incorporation of catalase into Brij 30 reverse micelles enhances its stability at 50 degrees C. However, the stability of catalase incubated at 37 degrees C in micellar and reverse micellar solutions is lower than that in homogeneous aqueous solution.