Soybean Peroxidase Research Articles

Soybean Peroxidase-Catalyzed Oxidation of Luminol by Hydrogen Peroxide

Anionic soybean peroxidase Glycine max (SbP) is shown to efficiently catalyze luminol oxidation by hydrogen peroxide. Contrary to horseradish peroxidase, the presence of p-iodophenol in the reaction medium affects slightly the efficiency of SbP catalysis. A maximal intensity of chemiluminescence, produced through this enzymatic reaction, was detected at pH 8.4-8.6. Contrary to anionic palm tree peroxidase, in the presence of SbP, chemiluminescence intensity increases with the reaction buffer concentration. The detection limit of SbP in the reaction of luminol oxidation is 0.3 x 10(-12) M. Therefore, high sensitivity in combination with the long-term chemiluminescent signal is indicative of good prospects for application of this enzyme in enzyme immunoassay with chemiluminescent detection.

Enzymatic Biobleaching of Two Recalcitrant Paper Dyes with Horseradish and Soybean Peroxidase

A stilbene dye (Direct Yellow 11) and a methine dye, Basazol 46L, recalcitrant to common chemical bleaches, were treated with horseradish and soybean peroxidases. Both enzymes were effective at chromophore removal. When compared to laccase in combination with a mediator (ABTS), soybean peroxidase was more effective at oxidative dye removal, especially for the methine dye.

Peroxidase‐Catalyzed Coupling of Solid‐Supported ortho‐Methoxyphenols

AbstractEnzyme‐catalyzed CC bond formation has been performed on solid‐supported phenols, thereby expanding the repertoire of enzymatic catalysis on resin‐bound substrates. In the presence of hydroquinone bound to various commercial resins, soybean peroxidase catalyzed the coupling of apocynin (acetovanillone) to give dimers, trimers, and oligomers with structures similar to what is obtained in solution‐phase reactions. In addition to phenolic coupling, peroxidase catalyzed a Fries rearrangement presumably due to rearrangement of the acyl phenolic radicals generated on the solid phase through peroxidase catalysis. This represents the first enzymatic Fries rearrangement reported to date. Together with the solid‐phase biocatalytic phenolic coupling, these results provide a route to the further expansion of enzymatic catalysis in combinatorial library synthesis.

Synthesis of polycardanol from a renewable resource using a fungal peroxidase from Coprinus cinereus

A fungal peroxidase from Coprinus cinereus (CiP) was successfully used for oxidative polymerization of cardanol in water–organic solvent mixtures. Cardanol is a phenol derivative from a renewable resource having the meta-substituent of a C15 unsaturated hydrocarbon chain mainly with one to three double bonds. So far, only uneconomic plant peroxidases, such as soybean peroxidase (SBP), have been used to polymerize cardanol. The fungal peroxidase used was easily produced by cultivating C. cinereus, and was purified by ultrafiltration and size exclusion chromatography. The purified peroxidase had a specific activity of 4960 U/mg. The CiP-catalyzed polymerization of cardanol was carried out in aqueous/organic solvents. Microbial CiP catalyzed the cardanol polymerization as efficiently as SBP. The structure and molecular weight of the polycardanol produced by CiP were comparable to those produced by SBP. A low reaction temperature of 10 and 15 °C produced polycardanol in high yield and the hydrogen peroxide feed rate was found to affect the initial reaction rate and the final conversion. From a practical point of view, it is believed that microbial CiP will be found more useful for the synthesis of a range of polyphenols from renewable resources than plant peroxidases.

Template-free enzymatic synthesis of electrically conducting polyaniline using soybean peroxidase

Synthesis of polyaniline (PANI) catalyzed by soybean peroxidase at 1 °C in either aqueous or partially organic media, was studied as a function of pH and reaction media. Kinetic studies indicated that, unlike chemical polymerization, enzymatic polymerization of aniline showed neither induction period nor auto acceleration. The redox reversibility and chemical structure of the synthesized PANI was strongly dependent on the starting pH of the reaction medium. UV–vis, FT-IR, WAXD and TGA analysis are used to explain how the enzymatic reaction conditions influence both the chemical structure and physical properties of the PANI. Optimal reaction conditions are outlined for the direct enzymatic synthesis of electrically conductive emeraldine salt with yield as high as 71%.

Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes

We have examined the structure and function of two enzymes, alpha-chymotrypsin (CT) and soybean peroxidase (SBP), adsorbed onto single-walled carbon nanotubes (SWNTs). SBP retained up to 30% of its native activity upon adsorption, while the adsorbed CT retained only 1% of its native activity. Analysis of the secondary structure of the proteins via FT-IR spectroscopy revealed that both enzymes undergo structural changes upon adsorption, with substantial secondary structural perturbation observed for CT. Consistent with these results, AFM images of the adsorbed enzymes indicated that SBP retains its native three-dimensional shape while CT appears to unfold on the SWNT surface. This study represents the first in depth investigation of protein structure and function on carbon nanotubes, which is critical in designing optimal carbon nanotube-protein conjugates.

Enzymatic Removal of Phenol and Chlorophenols Using Soybean Seed Hulls

AbstractSoybean peroxidase (SBP), (EC 1.11.1.7) can be readily extracted from soybean seed hulls. This study reports on the direct use of soybean seed‐hull extracts for the bioremediation of phenolic wastes. The crude SBP extract from the hulls, like pure soybean peroxidase, is catalytically active in a broad range of pH and temperatures. As SBP is gradually released into the aqueous solution from seed hulls, the direct use of soybean seed hulls can reduce SBP inactivation by H2O2 and enhance the utilization efficiency of SBP through the slow release of the enzyme from the seed hulls. However, large doses of soybean seed hulls were found to be ineffective in phenol removal. Gradual additions of H2O2 in combination with the SBP released from the hulls were applied to optimize the bioremediation. Since the crude extract contains a mixture of multiple soybean proteins, soybean seed hull slurry required a higher concentration of H2O2 to remove the phenolic substrates than did the purified enzyme. Under the experimental conditions, 80 % of phenol (10.6 mM), 96 % of 2‐chlorophenol (3.9 mM), 95 % of 2,4‐dichlorophenol (3.1 mM), and 94 % of mixed phenol and chlorophenols were removed using soybean seed hulls in a single batch reactor. These results demonstrate that soybean seed hulls, compared to purified SBP, may be a more cost‐effective alternative in the enzymatic removal of phenolic compounds through polymerization reactions.

Novel surface modification of high‐density polyethylene films by using enzymatic catalysis

AbstractThe surface of high‐density polyethylene (HDPE) films was modified by an enzyme, soybean peroxidase (SBP). The enzymatic surface modification was performed using a peroxidase as catalyst and hydrogen peroxide as oxidizing agent. The chemical composition and morphology of HDPE surfaces were characterized by X‐ray photoelectron spectroscopy, infrared spectroscopy, and scanning electron microscopy. Results showed that after enzymatic treatment, the O/C atomic ratio of HDPE surfaces increased, and new functional groups such as –CO– appeared. Moreover, the surface of treated HDPE films became rougher than untreated surfaces. The hydrophilicity of treated and untreated HDPE films was analyzed by UV–vis spectroscopy and contact angle measurements. The decreased contact angle of the HDPE with water and increased adsorption ability of the surface to a water‐soluble dye clearly indicated that enzymatic treatment can significantly increase the hydrophilicity of the surfaces of HDPE films. The catalytic mechanism of SBP was also discussed. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 3673–3678, 2004

Horseradish peroxidase-catalyzed polymerization of cardanol in the presence of redox mediators.

Horseradish peroxidase-catalyzed polymerization of cardanol in aqueous organic solvent was investigated in the presence of a redox mediator. Cardanol is a phenol derivative from a renewable resource mainly having a C15 unsaturated hydrocarbon chain with mostly 1-3 double bonds at a meta position. Unlike soybean peroxidase (SBP), it has been shown that horseradish peroxidase (HRP) is not able to perform oxidative polymerization of phenol derivatives having a bulky meta substituent such as cardanol. For the first time, redox mediators have been applied to enable horseradish peroxidase to polymerize cardanol. Veratryl alcohol, N-ethyl phenothiazine, and phenothiazine-10-propionic acid were tested as a mediator. It is surprising that the horseradish peroxidase-catalyzed polymerization of cardanol took place in the presence of N-ethyl phenothiazine or phenothiazine-10-propionic acid. However, veratryl alcohol showed no effect. FT-IR and GPC analysis of the product revealed that the structure and properties of polycardanol formed by HRP with a mediator were similar to those by SBP. This is the first work to apply a redox mediator to enzyme-catalyzed oxidative polymerization. Our new finding that oxidative polymerization of a poor substrate, which the enzyme is not active with, can take place in the presence of an appropriate mediator will present more opportunities for the application of enzyme-catalyzed polymerization.

Activity, stability and conformational flexibility of seed coat soybean peroxidase

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that includes the classical peroxidase, namely horseradish peroxidase (HRP). We have measured the catalytic activity ( k cat) and catalytic efficiency ( k cat/ K M) of SBP and that of HRP-C for the oxidation of ABTS [2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonate)] by hydrogen peroxide at 25 °C. We observed that the k cat and k cat/ K M values for SBP are much higher than those for HRP-C at all pH values, rendering SBP a more potent peroxidase. This is attributed to the relatively more solvent exposed δ- meso heme edge in SBP. We observed that the maximum catalytic activity and conformational stability of SBP is at pH ∼5.5. A pH maximum of 5.0 for the catalytic activity of SBP has recently been reported. Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of β-strands and β-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein.

Xylem Sap Lectin, XSP30, Recognizes GlcNAc Sugar Chains of Glycoproteins in Cucumber Leaves.

The lectin activity of 30-kDa xylem sap protein (XSP30) found in cucumber (Cucumis sativas L.) xylem sap was analyzed by lectin blot coupled with immunological detection of XSP30. XSP30 is homologous to galactose-binding lectin, but it had only weak binding activity against asialofetuin, an animal glycoprotein with a terminal galactose. This reaction was not reduced by galactosidase treatement of asialofetuin. XSP30 bound strongly to soybean agglutinin, whose sugar chains consist solely of mannose and N-acetylglucosamine (GlcNAc), but bound only weakly to soybean peroxidase and γ-globulin, whose GlcNAc is fucosylated. The binding activity was inhibited by tri-N-acetylchitotriose (GlcNAc)3. Leaf parenchyma cells were stained with XSP30 and aniti-XSP30 antibody; this staining was reduced by proteinase treatment of the sections, and XSP30 bound to proteins in leaf particulate fraction. These results suggest that XSP30 transported from root via xylem sap binds to chitobiose, GlcNAc-GlcNAc, in N-linked glycans of leaf glycoproteins.

Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A-Agarose

Variables involved in adsorption and elution of horseradish and soybean seed peroxidases from a concanavalin A-Agarose matrix were studied. The effect of pH, ion strength and Ca 2+/Mg 2+ concentration on maximum capacity and dissociation constant was assessed through adsorption isotherms. The effect of flow rate and peroxidase concentration on dynamic capacity was assessed through breakthrough curves. For the elution step, NaCl and α- d-methylmannopyranoside concentrations were optimised. The best conditions for soybean peroxidase adsorption were pH 5.0 and 1 mM Ca 2+/Mg 2+ in the absence of salt, at a flow rate up to 3.2 cm/min and 3 mg/ml peroxidase concentration. Elution required the addition of 0.48 M α- d-methylmannopyranoside and 0.97 M NaCl. Under these conditions, the dynamic capacity was 16.9 mg/ml matrix and purification yield, 84.3%. In the case of horseradish peroxidase, the best adsorption conditions were pH 7.0, 5 mM ions and 0.75 M NaCl, at a flow rate up to 1.5 cm/min and a 4 mg/ml peroxidase concentration. Dynamic capacity was 9.6 mg/ml matrix, and elution required 0.36 M α- d-methylmannopyranoside to yield 75% of enzyme. The dynamic-to-static capacity ratios were 0.71 and 0.62 for horseradish and soybean peroxidases, respectively.

Thermal Unfolding of Soybean Peroxidase: APPROPRIATE HIGH DENATURANT CONCENTRATIONS INDUCE COOPERATIVITY ALLOWING THE CORRECT MEASUREMENT OF THERMODYNAMIC PARAMETERS

We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8-6.9 M), cooperative behavior is observed, which allowed the analysis by the two-state model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of DeltaH(m) on T(m) to the T(m) corresponding to 0 m GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.

Thermal and conformational stability of seed coat soybean peroxidase.

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.

Removal of aqueous phenol catalysed by a low purity soybean peroxidase

AbstractThe application of a low purity soybean peroxidase (LP‐SBP), obtained from wasted seed hulls, as catalyst for phenol polymerisation in aqueous solution in the presence of hydrogen peroxide is described. The polymers formed precipitate out from solution and may be readily separated by physico‐chemical techniques. LP‐SBP offers the advantage of reduced cost compared with horseradish peroxidase (HRP). The SBP activity in fresh hulls was greater than in aged hulls and was preserved at −10 °C. There was a linear correlation between initial phenol concentration (1, 2, 5 and 10 mmol dm−3) and the minimum dosage of LP‐SBP required to precipitate 95% of the phenolic polymers. Polyethylene glycol (molecular weight, 6000) at 1000 mg dm−3 did not extend LP‐SBP lifetime. At all phenol concentrations tested, a retention time of about 100 min was sufficient to achieve yields of 95%.© 2002 Society of Chemical Industry

Removal of nitroaromatics from synthetic wastewater using two-step zero-valent iron reduction and peroxidase-catalyzed oxidative polymerization.

Degradation of nitroaromatics, which are significant environmental pollutants, is difficult to achieve. Zero-valent iron reduction of nitroaromatics coupled with peroxidase-catalyzed capture of the resulting anilines as a two-step strategy for removing nitroaromatics from wastewater and process water is investigated here. The concentration range of nitroaromatics studied was that which would be present in industrial wastewater streams. Studies were done in continuous-flow columns. The enzymatic treatment following zero-valent iron reduction was carried out in a plug-flow reactor using a crude preparation of the enzyme soybean peroxidase extracted from soybean hulls. The complete reaction time for the two steps was 5 to 5.5 hours. Operating parameters including pH, peroxide/substrate ratio, enzyme concentration, and alum concentration were optimized. Optimum conditions obtained were approximately neutral pH with a hydrogen peroxide/substrate molar ratio of 1.5 for all of the nitroaromatics tested. Alum concentrations between 50 and 100 mg/L were useful in removing the apparent color from the treated water.

Optimisation of soybean peroxidase treatment of 2, 4-dichlorophenol

In the presence of hydrogen peroxide (H2O2), peroxidase enzymes (PE) catalyse the oxidation of various chlorinated phenols to free radicals, which then combine to form insoluble polymers that precipitate out of solution. This study systematically characterises the treatment of 2, 4-dichlorophenol (2, 4-DCP) using soybean peroxidase (SBP) as an oxidising catalyst. The effects of pH, SBP concentration, polyethylene glycol (PEG) additive and initial chlorophenol concentration on 2, 4-DCP treatments are reported. Optimum pH for removal of 2, 4-DCP without PEG was pH 8.2. The pH operating range of SBP was from 2.5 to 9.4 which is wider than reported for horseradish peroxidase (HRP). A general equation is presented that describes the units of SBP required (without PEG) to treat a given amount of 2, 4-DCP at the optimum pH of 8.2. Addition of PEG increased the effectiveness of SBP by factors of 10 and 50 for PEG-3350 and PEG-8000 respectively. A new pH optimum of 6.2 was also found when SBP was used with PEG. Batch and semi -batch enzyme delivery has also been identified as a crucial parameter for the SBP treatment process. The most effective addition scheme was based on five equal concentrations of SBP and H2O2 over 15 min and 30 min intervals respectively compared to a single batch addition. This protocol was the most effective as it took advantage of limiting the amount of SBP and H2O2 available at each step. This reduces the possible chance of SBP inactivation by excessive H2O2 when using a single batch concentration. Average 2, 4-DCP removals achieved were 83.5%, 75.5% and 71.5% for 100, 200 and 300 mg/l 2, 4-DCP concentrations respectively compared to 62%, 52% and 58% for the single batch addition control. WaterSA Vol.28(2) 2002: 149-158

New Polymers from Natural Phenols Using Horseradish or Soybean Peroxidase

Full Paper: The flavonol quercetin, its glycoside rutin, the flavanol catechin, the isoflavone daidzein and 5,6,4'trihydroxyisoflavone can be oxidatively polymerized via a radical polyrecombination mechanism catalyzed by horse- radish or soybean peroxidase in a mixture of phosphate buffer (pH 7) and 1,4-dioxane. The average molecular weights of the polymers were found to be in the range 4 000-12 000 g/mol. Daidzein derivatives with a methoxy (formononetin) or nitro group or a hydrogen atom at C-4' could not be polymerized under these conditions. These results and molecular modeling studies suggest that polymerization preferentially occurs via the electron-rich ring B of the isoflavonoids. The polymers were characterized by means of FT-IR and UV spectroscopy, and their redox behavior was analyzed using cyclic voltammetry.

New Polymers from Natural Phenols Using Horseradish or Soybean Peroxidase

The flavonol quercetin, its glycoside rutin, the flavanol catechin, the isoflavone daidzein and 5,6,4′-trihydroxyisoflavone can be oxidatively polymerized via a radical polyrecombination mechanism catalyzed by horseradish or soybean peroxidase in a mixture of phosphate buffer (pH 7) and 1,4-dioxane. The average molecular weights of the polymers were found to be in the range 4 000–12 000 g/mol. Daidzein derivatives with a methoxy (formononetin) or nitro group or a hydrogen atom at C-4′ could not be polymerized under these conditions. These results and molecular modeling studies suggest that polymerization preferentially occurs via the electron-rich ring B of the isoflavonoids. The polymers were characterized by means of FT-IR and UV spectroscopy, and their redox behavior was analyzed using cyclic voltammetry.

Oxidative enzymes possess catalytic activity in systems with ionic liquids

Oxidative enzymes, laccase C from Trametes sp. and horseradish and soybean peroxidases, catalyzed oxidation reactions in systems with ionic liquids whose content varied from several volume percent to almost total non-aqueous ionic liquids. Similar to the effects produced by standard organic solvents used in non-aqueous enzymology, catalytic activity of the enzymes was decreased by adding a water-miscible ionic liquid, 4-methyl-N-butylpyridinium tetrafluoroborate, or by suspending the enzyme in a water-immiscible ionic liquid, 1-butyl-3-methylimdizaolium hexafluorophosphate. For the oxidation of anthracene, catalyzed by laccase C and assisted by a number of mediators, addition of 4-methyl-N-butylpyridinium tetrafluoroborate, instead of tert-butanol, increased the yield of the oxidation product several-fold.