Soybean Peroxidase Research Articles

Enhanced Stability of Enzymes Adsorbed onto Nanoparticles

We have discovered that the highly curved surface of C60 fullerenes enhances enzyme stability in strongly denaturing environments to a greater extent than flat supports. The half-life of a model enzyme, soybean peroxidase, adsorbed onto fullerenes at 95 degrees C was 117 min, ca. 2.5-fold higher than that of the enzyme adsorbed onto graphite flakes and ca. 13-fold higher than that of the native enzyme. Furthermore, this phenomenon is not unique to fullerenes, but can also be extended to other nanoscale supports including silica and gold nanoparticles. The enhanced stability was exploited in the preparation of highly active and stable polymer-nanocomposite films. The ability to enhance protein stability by interfacing them with nanomaterials may impact numerous fields ranging from the design of diagnostics, sensors, and nanocomposites to drug delivery.

Covalent Attachment of Ferrocene to Soybean Peroxidase Glycans: Electron Transfer Mediation to Redox Enzymes

The electrochemical properties of native soybean peroxidase (SBP) and of SBP modified with covalently attached ferrocene electron-transfer mediators within microcavities etched at the tip of 25 mum radius platinum microelectrodes are reported. The microcavities incorporate approximately 50 fmol of SBP. In the microcavity, native SBP undergoes a relatively fast reduction but a very slow oxidation. Ferrocene mediators have been attached to the SBP glycans (Fc-SBP) (approximately 1.5 ferrocene mediators per SBP molecule). Cyclic voltammetry reveals that these centers are capable of mediating the reduction of oxidized SBP and increase the rate of heterogeneous electron transfer between the enzyme and the electrode by >10-fold. Micromolar concentrations of H2O2 chemically oxidize the SBP and Fc-SBP systems leading to an electrocatalytic reduction at approximately -0.1 V. Successive additions of 2.5 micromol of H2O2 at a constant applied potential of -0.1 V gave a steady-state constant current of approximately 60 nA within 20 s. The steady-state current increased linearly with peroxide concentration for 2.5 <or= [H2O2] <or= 42 microM. Ferrocene-modified SBP shows an approximately 3-fold increase in the sensitivity of the steady-state current response to successive additions of hydrogen peroxide compared to the native enzyme. This observation indicates increased turnover of the redox enzyme per unit time in the presence of the covalently attached ferrocene mediators.

A simple preparation method of crystals of soybean hull peroxidase

Soybean hull peroxidase (SHP) was crystallised from an enzyme solution with low purity by a simple method. The enzyme solution was purified by cooperation salting out of acetone and ammonium sulphate, and lumpy crystals were obtained with the size of about 40 x 30 mum when ammonium sulphate was quickly added to the enzyme solution. The crystal was examined and confirmed to be an SHP crystal by the method of activity test. The result shows that, though the purity of the enzyme solution was not high, crystals could be formed when the enzyme solution rapidly reached to a degree of supersaturation, which was different from the traditional methods of protein crystallisation. Additionally, a purification method of acetone and ammonium sulphate fractional salting out was also studied, in which the procedure was simplified, and a satisfactory purification effect was obtained.

Removal of dinitrotoluenes from water via reduction with iron and peroxidase-catalyzed oxidative polymerization: A comparison between Arthromyces ramosus peroxidase and soybean peroxidase

A two-step process for the removal of dinitrotoluene from water is presented: zero-valent iron reduction is coupled with peroxidase-catalyzed polymerization of the resulting diaminotoluenes (DAT). The effect of pH was examined in the reduction step: at pH 6 the reaction occurred much more rapidly than at pH 8. In the second step, optimal pH and substrate ratio, minimal enzyme concentration and effect of polyethylene glycol (PEG) as an additive for greater than 95% conversion of DAT, over a 3h reaction period were determined using high performance liquid chromatography. Two enzymes were investigated and compared: Arthromyces ramosus peroxidase (ARP) and soybean peroxidase (SBP). The optimal pH values were 5.4 and 5.2 for ARP and SBP, respectively, but SBP was more resistant to mild acid whereas ARP was more stable in neutral solutions. SBP was found to have a greater hydrogen peroxide demand (optimal peroxide/DAT molar ratio for SBP: 2.0 and 3.0 for 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT), respectively; for ARP: 1.5 and 2.75 for 2,4-DAT and 2,6-DAT, respectively) but required significantly less enzyme (0.01 and 0.1 U ml(-1) for 2,4-DAT and 2,6-DAT, respectively) to convert the DAT than ARP (0.4 and 1.5 U ml(-1) for 2,4-DAT and 2,6-DAT, respectively). PEG was shown to have no effect upon the degree of substrate conversion for either enzyme.

Inhibition of Peroxidase and Catalase Activities and Modulation of Hydrogen Peroxide Level by Inositol Phosphoglycan-like Compounds

Inositol phosphoglycan-like compounds are produced by the hydrolysis of the membrane bound glycosyl phosphoinositides. Besides being short term mediators of insulin action, they inhibit peroxidases and catalase, increasing the concentration of cellular hydrogen peroxide. Although high concentrations of hydrogen peroxide are toxic, moderate increases of its basal level are signals for different metabolic pathways. The inhibitor, localized in the cytosol of the cell, acts on peroxidases and catalase of the same tissue (homologous action) and of other tissues or organisms (heterologous action). The inositol phosphoglycan-like compound inhibits peroxidases with different prosthetic groups, i.e. containing iron such as: thyroid peroxidase, lactoperoxidase, horseradish peroxidase, soy bean peroxidase; and containing selenium such as glutathione peroxidase and 2-cys peroxiredoxin with no prosthetic group. Besides peroxidases, the inositol phosphoglycan-like compound inhibits catalase, another heme enzyme. The inhibition kinetics demonstrates a noncompetitive effect. The site of action is not the prosthetic group, given that the inhibitor does not produce any effect on the peak in the Soret region in the presence or absence of hydrogen peroxide. In conclusion, the inositol phosphoglycan-like compound is the general inhibitor of peroxidases and catalase involved in the modulation of hydrogen peroxide level that acts in different metabolic pathways as a signal transducer.

Electric field effects on adsorption/desorption of proteins and colloidal particles on a gold film observed using surface plasmon resonance

We present results for the enhancement or retardation of the attachment of the protein soybean peroxidase to a gold substrate using electric fields. We detect the influence of the electric fields on the proteins using the surface plasmon resonance phenomenon. The gold surface on which the surface plasmon resonance is generated also acts as one of the electrodes required to apply the electric field. The second electrode has a semi-insulating layer that limits current flow, and limits electrolysis effects. The results show that at pH 7, when the soybean peroxidase is negatively charged, a greatly enhanced deposition of protein is obtained when a positive potential is applied to the gold electrode. A negative potential can inhibit protein attachment, or reduce the amount of protein attached; however, after repeated applications of a positive potential, a negative potential has little effect. Results are presented of experiments using colloidal silica that has negatively charged particles of similar size to soybean peroxidase, showing the attraction and repulsion of negatively charged particles by the gold surface.

Elimination of 4-chlorophenol by soybean peroxidase and hydrogen peroxide: Kinetic model and intrinsic parameters

A kinetic model for the initial rate reaction, based on the Ping-Pong Bi-Bi mechanism, has been assumed for the removal of 4-chlorophenol with soybean peroxidase and hydrogen peroxide. Several sets of experimental data, obtained in a batch reactor at different concentrations of hydrogen peroxide and 4-chorophenol, were used to estimate the kinetic parameters. A new method for determining the intrinsic parameters of the reaction in processes involving a high reaction rate was developed. The method consists of determining several sets of apparent parameters at different times and then extrapolating these to zero time.

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.

Experimental behaviour and design model of a fluidized bed reactor with immobilized peroxidase for phenol removal

Abstract In this work, immobilized derivatives of soybean peroxidase, covalently bound to glass supports with different surface areas, were used in a laboratory scale fluidized bed reactor to study their viability for use in phenol removal. The influence of the different operational variables on the process was also studied. When derivatives immobilized on supports with the highest surface area were used, 80% removal was achieved. Since knowledge of the removal process in the fluidized bed reactor and its simulation is vital before a continuous industrial scale process can be proposed, a reactor model based on the experimental results that predicts the system's behaviour both in steady and transient state was developed. The model considers the fluidized bed reactor as a plug flow reactor in series with an ideal mixer and follows a kinetic law based on the observed external mass transfer resistances in order to work out the process rate. The values of the model parameters were obtained by fitting phenol conversion values obtained experimentally to the model, using the CurveExpert, V 1.3, software. The good agreement obtained between the experimental and calculated values of phenol conversion demonstrates that the model is valid as a predictive model for using this reactor configuration.

Dramatic Solvent and Hydration Effects on the Transition State of Soybean Peroxidase

Enzymes are shown to function in nonaqueous media; however, relatively little information is available on the influence of the organic solvent as well as its associated water content on the properties of the enzymatic transition states. A better understanding of these effects will be useful in developing kinetic models that can then be used to predict optimal solvent and substrate choices for enzymatic reactions in organic media. The influence of the reaction media on soybean peroxidase-catalyzed oxidation of para-substituted phenols was studied using Hammett analysis for several organic solvent systems. The catalytic activity and substrate specificity of the enzyme are influenced by the nature of the solvent and its associated hydration. These findings may allow one to draw conclusions about the reaction mechanism and the roles of solvent and solvent hydration on enzyme function.

ESR Spectroscopy Investigation of the Denaturation Process of Soybean Peroxidase Induced by Guanidine Hydrochloride, DMSO or Heat

The involvement of protein denaturation and/or misfolding processes in the insurgence of several diseases raises the interest in structural dynamic studies of proteins. The use of nitroxide spin labels with electron paramagnetic resonance is a powerful tool for detecting structural changes in proteins. In the present study, we apply this strategy to soybean peroxidase (SBP), a protein characterised by high thermal and structural stability, and we propose a simple method to analyse the anisotropy changes of the protein system and to relate them with the structural changes induced by protein unfolding. We examined the effect of temperature, guanidine hydrochloride and dimethylsulfoxide on the stability of SBP and looked for correlations between the ESR results and the experimental findings obtained by other techniques, reported in the literature. The agreement between data obtained through different strategies supports the validity and reliability of the ESR approach to protein unfolding.

Chip-Based Polyketide Biosynthesis and Functionalization

We demonstrate construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microfluidic platform. THNS immobilized to Ni-NTA agarose beads is prepacked into a microfluidic channel, while SBP is covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result is a tandem, two-step biochip that enables the synthesis of novel polyketide derivatives. The first microchannel, consisting of THNS, results in the conversion of malonyl-CoA to flaviolin in yields up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and malonyl-CoA (extender substrate) feed rates, high yields of the pyrone products could be achieved, which is further structurally diversified from self- and cross-coupling in the SBP microchannel. The ability to rapidly evaluate the effects of reaction conditions and synthetic multienzyme pathways on a microfludic platform provides a new paradigm for performing metabolic pathway engineering, namely, the reconstruction of pathways for use in new compound discovery.

Removal of 4-chlorophenol by soybean peroxidase and hydrogen peroxide in a discontinuous tank reactor

A discontinuous tank reactor was used to eliminate 4-chlorophenol using soybean peroxidase (SBP) and hydrogen peroxide in an attempt to reduce the concentrations of buffer solutions used and to avoid adding polyethylene glycol. The stirred-tank reactor was operated in different operating conditions, and the influence of enzyme and substrate concentrations on the elimination of 4-chlorophenol was determined. Most of the assays provided good elimination percentages, which reached almost 90% for an enzyme concentration of 7.5·10 –3 mg cm –3 and a stoichiometric ratio of 1:1 for both substrates, when concentrations of 1.5 mmol dm –3 or less were used. This work is a first step in the study of the SBP/4-chlorophenol system in order to observe the influence of the reagents concentrations when no polyethylene glycol is added.

Inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices by peroxidase systems

To study the bactericidal properties of the lactoperoxidase (LPER)-thiocyanate and soybean peroxidase (SBP)-thiocyanate systems at low pH, their efficiency for inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices, their effect on colour stability of the juices and interaction with ascorbic acid. Three-strain cocktails of E. coli and Shigella spp. in selected juices were supplemented with the LPER or SBP system. Within 24 h at 20 degrees C, the LPER system inactivated both cocktails by > or = 5 log10 units in apple, 2-5 log10 units in orange and < or = 1 log10 unit in tomato juices. In the presence of SBP, browning was significant in apple juice and white grape juice, slight in pink grape juice and absent in orange or tomato juice. Ascorbic acid protected E. coli and Shigella against inactivation by the LPER system, and peroxidase systems significantly reduced the ascorbic acid content of juices. Our results suggest a different specificity of LPER and SBP for SCN-, phenolic substrates of browning and ascorbic acid in acidic juices. The LPER system appeared a more appropriate candidate than the SBP system for biopreservation of juices. This work may open perspectives towards the development of LPER or other peroxidases as biopreservatives in acidic foods.

Use of Soybean Peroxidase in Chemiluminescent Enzyme-Linked Immunosorbent Assay

A procedure for the production of conjugates of soybean peroxidase (SbP) oxidized by sodium periodate and anti-mouse IgG antibody (Ab) was optimized. A sandwich chemiluminescent enzyme-linked immunosorbent assay (ELISA) for determination of mouse IgG using SbP and specific Ab was developed, and SbP-catalyzed oxidation of luminol was carried out in the absence of any enhancer. Comparison of conjugates produced by labeling Ab by soybean and horseradish peroxidases in the chemiluminescent ELISA showed that in the case of SbP a rate of emission decay formed through luminol oxidation was significantly lower. Application of the soya enzyme allowed the development of the immunoassay with improved sensitivity and a wider linear range.

Peroxidase Activity in Soybeans Following Inoculation with Phytophthora sojae

The effects of race-specific resistance as conditioned by Rps genes (rps, Rps1-k, Rps2, Rps3, Rps6) in two genetic backgrounds (Williams & Harosoy) on accumulation of soluble peroxidases were determined by a soybean peroxidase capture assay (SPCA) after inoculation with P. sojae races 2, 7, or 25. Peroxidase activity increased in all isolines during the 72 h after inoculation, but reactions varied depending on time after inoculation, genetic background, Rps gene and P. sojae race. Peroxidase activity was higher in race-specific resistant than in susceptible reactions at 72 h. after inoculation, except for plants with the Rps2 gene which confers a unique form of root resistance in addition to the whole plant race-specific resistance. Williams isolines had larger increases in peroxidase activity than Harosoy isolines when data were averaged across Rps genes, and was most evident when plants were inoculated with race 2. When soybeans were inoculated with race 7 Rps1-k resistant plants had the highest increase in peroxidase activity, but Rps2 susceptible plants had a significantly higher peroxidase activity than plants with rps, Rps3, and Rps6 that were also susceptible. Results from inoculations with race 25 were somewhat different, Rps2 resistant plants had the highest increase in peroxidase activity; however, plants with the Rps3 or Rps6 gene that were also resistant did not have a significantly higher peroxidase activity than susceptible plants with the rps or Rps1-k gene.

Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation

Plant peroxidases, as typified by horseradish peroxidase (HRP), primarily catalyze the one-electron oxidation of phenols and other low oxidation potential substrates. In contrast, the mammalian homologues such as lactoperoxidase (LPO) and myeloperoxidase primarily oxidize halides and pseudohalides to the corresponding hypohalides (e.g., Br − to HOBr, Cl − to HOCl). A further feature that distinguishes the mammalian from the plant and fungal enzymes is the presence of two or more covalent bonds between the heme and the protein only in the mammalian enzymes. The functional roles of these covalent links in mammalian peroxidases remain uncertain. We have previously reported that HRP can oxidize chloride and bromide ions, but during oxidation of these ions undergoes autocatalytic modification of its heme vinyl groups that virtually inactivates the enzyme. We report here that autocatalytic heme modification during halide oxidation is not unique to HRP but is a general feature of the oxidation of halide ions by fungal and plant peroxidases, as illustrated by studies with Arthromyces ramosus and soybean peroxidases. In contrast, LPO, a prototypical mammalian peroxidase, is protected from heme modification and its heme remains intact during the oxidation of halide ions. These results support the hypothesis that the covalent heme-protein links in the mammalian peroxidases protect the heme from modification during the oxidation of halide ions.

Infrared spectroscopy used to evaluate glycosylation of proteins

Infrared (IR) spectroscopy is used for studying the carbohydrate moieties of glycosylated proteins. IR spectra of mono- and disaccharides in the fingerprint region are specific to each sugar and to the environment of the sugar molecules (i.e., aqueous solution or anhydrous glass phase). The IR spectra of glycosylated proteins (mucin, soybean peroxidase, collagen IV, and avidin) were compared with those of the constituent sugars and cytochrome c (a protein with no glycosylation). Our results demonstrate that the IR absorption spectra of glycosylated proteins show distinct absorption bands for the sugar moiety, the protein amide group, and water. Therefore, IR can be used to detect glycosylation.

Protein-coated Microcrystals for use in Organic Solvents: Application to Oxidoreductases

Protein-coated microcrystals (PCMC), a biocatalyst preparation previously demonstrated to display substantially increased transesterification activity of proteases and lipases in organic solvents when compared to their as received counterparts [Kreiner M, Moore BD, Parker MC (2001) Chem. Commun. 12:1096--1097], was applied to oxidoreductases. Horse liver alcohol dehydrogenase (HLADH), catalase (CAT), soybean peroxidase and horseradish peroxidase were immobilised onto K(2)SO(4) crystals and dehydrated in a 1-step process, resulting in PCMC. These PCMC preparations showed enhanced activity in different organic solvents in most types of reactions tested. The highest activation was observed with HLADH (50-fold as active as enzyme as received) and CAT (25-fold).

Comparison of commercial peroxidases for removing phenol from water solutions

This paper describes a comparison between horseradish peroxidase (HRP) and soybean peroxidase (SBP), the two most widely used commercial peroxidases for the removal of phenol from wastewater. Both enzymes achieve maximal removal efficiency in a neutral pH medium although they are still quite active in a pH range of between 6.0 and 8.0. The fact that both enzymes show similar phenol elimination levels at any temperature between 25 and 40 °C is also of interest. Studies were carried out in the absence and presence of different concentrations of a stabilisation additive, polyethylene glycol (PEG), in an attempt to optimise the phenol elimination procedure. The final choice of peroxidase will depend on the characteristics of the effluent, operational requirements and on economic considerations. Our findings show that HRP acts faster than SBP but is more susceptible to inactivation, although it is better protected by PEG. In consequence HRP will be the most suitable choice but the addition of a sufficient amount of PEG is critical.