Application of Fe(III)-EDDS complexes and soybean peroxidase in photo-Fenton processes for organic pollutant removal: insights into possible synergistic effects.

Photo-Fenton processes activated by biodegradable Fe(III)-EDDS complexes have attracted huge attention from the scientific community, but the operative mechanism of the photo-activation of H2O2 in the presence of Fe(III)-EDDS has not been fully clarified yet. The application of the Fe(III)-EDDS complex in Fenton and photo-Fenton (mainly under UV-B light) processes, using 4-chlorophenol (4-CP) as a model pollutant was explored to give insights into the operative mechanism. Furthermore, the potential synergistic contribution of soybean peroxidase (SBP) was investigated, since it has been reported that upon irradiation of Fe(III)-EDDS the production of H2O2 can occur. SBP did not boost the 4-CP degradation, suggesting that the possibly produced H2O2 reacts immediately with the Fe(II) ion with a quick kinetics that does not allow the diffusion of H2O2 into the bulk of the solution (i.e., outside the solvent cage of the complex). So, a concerted mechanism in which the photochemically produced H2O2 and Fe(II) react inside the hydration sphere of the Fe(III)-EDDS complex is proposed.

Self-assembly of soybean peroxidase nanohybrid for activity enhancement and dye decolorization: experimental and computational studies.

The soybean peroxidase (SBP) mediated nanohybrid [SBP-Cu3(PO4)2·3H2O] synthesis was carried out in the present study. The scanning electron microscopy (SEM) analysis showed a characteristic flower-like hierarchical structure of the SBP-nanohybrid. The mechanism of SBP-nanohybrid formation was elucidated using computational approaches. The predicted Cu2+ binding sites followed by molecular docking studies showed the two lowest energy (-4.4 kcal/mol and -3.56 kcal/mol) Cu2+ binding sites. These two binding sites are located at the opposite position and might be involved in the formation of SBP-nanohybrid assemblies. Further, these sites are different than the catalytic active site pocket of SBP, and may facilitate more substrate catalysis. Obtained computational results were confirmed by in-vitro guaiacol oxidations studies using SBP-nanohybrid. The effect of various parameters on SBP-nanohybrid activity was studied. The pH 7.2 was found optimum for SBP-nanohybrid activity. The enzyme activity increased with an increase in temperature up to 50 °C temperature and then decreased with an increase in temperature. Around ∼138% enhanced activity was recorded using SBP-nanohybrid compared to crude SBP. Also, the SBP-nanohybrid showed around 95% decolorization of methylene blue (MB) in 1 h and the MB degradation was confirmed by high-pressure liquid chromatography analysis (HPLC).Communicated by Ramaswamy H. Sarma.

Immobilized Soybean Peroxidase Hybrid Biocatalysts for Efficient Degradation of Various Emerging Pollutants.

In the present study, soybean peroxidase (SBP) was covalently immobilized onto two functionalized photocatalytic supports (TiO2 and ZnO) to create novel hybrid biocatalysts (TiO2-SBP and ZnO-SBP). Immobilization caused a slight shift in the pH optima of SBP activity (pH 5.0 to 4.0), whereas the free and TiO2-immobilized SBP showed similar thermal stability profiles. The newly developed hybrid biocatalysts were used for the degradation of 21 emerging pollutants in the presence and absence of 1-hydroxy benzotriazole (HOBT) as a redox mediator. Notably, all the tested pollutants were not equally degraded by the SBP treatment and some of the tested pollutants were either partially degraded or appeared to be recalcitrant to enzymatic degradation. The presence of HOBT enhanced the degradation of the pollutants, while it also inhibited the degradation of some contaminants. Interestingly, TiO2 and ZnO-immobilized SBP displayed better degradation efficiency of a few emerging pollutants than the free enzyme. Furthermore, a combined enzyme-chemical oxidation remediation strategy was employed to degrade two recalcitrant pollutants, which suggest a novel application of these novel hybrid peroxidase-photocatalysts. Lastly, the reusability profile indicated that the TiO2-SBP hybrid biocatalyst retained up to 95% degradation efficiency of a model pollutant (2-mercaptobenzothiazole) after four consecutive degradation cycles.

Plant-derived soybean peroxidase stimulates osteoblast collagen biosynthesis, matrix mineralization, and accelerates bone regeneration in a sheep model.

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current "gold standard" autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.

Development of an enzymatic biosensor to determine eugenol in dental samples.

A GCE/CRGO-ßCD's/ADA-SPE/AuNPs biosensor was successfully developed to determine eugenol in dental samples. The optimal conditions to construct the biosensor were obtained from an experimental design based on the response surfaces methodology. The GCE/CRGO-ßCD/ADA-SPE/AuNPs biosensor exhibited a very good analytical performance for the quantification of eugenol. Thus, it shows a linear range between 1.3 × 10-8 and 1 × 10-5 mol L-1, with a sensitivity of (5.3 ± 0.3) x 10-3 A mol-1 L. The limits of detection and quantification were 4 × 10-9 mol L-1 and 1.3 × 10-8 mol L-1, respectively. Biosensors had an intraday and inter day reproducibility of 5% and 8%, respectively. The repeatability was of 3%, and the stability was 21 days (a decrease of 30% in current responses was observed after the fourth week). Recovery studies were performed in order to validate the proposed method. Recovery percentages were between 94 and 108%. A value of the apparent Michaellis-Menten constant, KMapp, of 3.1 × 10-6 mol L-1 was obtained using both Lineweaver-Burk and Eadi-Hofstee methods.

LC-MSMS based screening of emerging pollutant degradation by different peroxidases.

BACKGROUND: The presence of a wide range of bioactive organic pollutants in wastewater and municipal water sources is raising concerns about their potential effects on humans. Not surprisingly, various approaches are being explored that can efficiently degrade these persistent organic pollutants. Use of peroxidases has recently been recognized as a novel remediation approach that may have potential advantages over conventional degradation techniques. However, testing the abilities of different peroxidases to degrade diverse emerging pollutants is tedious and cumbersome. RESULTS: In the present study, we present a rapid and robust approach to easily test the degradability of 21 different emerging pollutants by five different peroxidases (soybean peroxidase, chloroperoxidase, lactoperoxidase, manganese peroxidase, and horseradish peroxidase) using an LC-MSMS approach. Furthermore, this approach was also used to examine the role of a redox mediator in these enzymatic degradation assays. Our results show that some of the organic pollutants can be easily degraded by all five of the peroxidases tested, whereas others are only degraded by a specific peroxidase (or when a redox mediator was present) and there are some that are completely resistant to degradation by any of the peroxidases tested (even in the presence of a redox mediator). The degradation of furosemide and trimethoprim by soybean peroxidase and chloroperoxidase, respectively, was investigated in detail by examining the transformation products generated during their degradation. Some of the products generated during enzymatic breakdown of these pollutants have been previously reported by others, however, we report many new transformation products. CONCLUSIONS: LC-MSMS approaches, like the one described here, can be used to rapidly evaluate the potential of different peroxidases (and redox requirements) to be used as bioremediation agents. Our preliminary result shows peroxidases hold tremendous potential for being used in a final wastewater treatment step.

Radical addition polymerization: Enzymatic template-free synthesis of conjugated polymers and their nanostructure fabrication.

Conjugated polymers are attractive for many applications due to their unique properties. Their molecular structure can easily be tuned, making them suitable for an enormous number of specific applications. Conjugated polymers have the potential to achieve electrical properties similar to those of noncrystalline inorganic semiconductors; however, their chemical structure is much more complex and somewhat resembles that of biomacromolecules. The molecular conformation and interactions of conjugated polymers play an important role in their functionality. The use of enzymes has emerged as a highly valuable alternative method to synthesize these polymers and is very useful in the fabrication of their nanostructures. Here, we present established strategies for the synthesis of conjugated polymers in template-free systems that do not interfere with the preparation of their nanostructures. These strategies are based on the use of peroxidases (class III; EC 1.11.1.7, donor: hydrogen peroxide oxidoreductase), which are enzymes that have the ability to catalyze the oxidation of a number of compounds (including aromatics such as aniline, pyrrole, thiophene and some of their derivatives), in the presence of hydrogen peroxide, to obtain conjugated polymers.

Enzymatic treatment for removal of hazardous aqueous arylamines, 4,4'-methylenedianiline and 4,4'-thiodianiline.

The search for an effective and sustainable treatment method to remove the recalcitrant atom-bridged bis-anilino compounds, 4,4'-methylenedianiline (MDA) and 4,4'-thiodianiline (TDA) from water is a major challenge and focus of this study. The escalating discharge of these two toxic and carcinogenic pollutants from industrial sources may pose a serious threat to the environment. Crude soybean peroxidase (SBP), isolated from soybean seed hulls (coats), catalyzes the oxidative polymerization of these aqueous pollutants in the presence of hydrogen peroxide. The effects of several process parameters, i.e., pH, hydrogen peroxide-to-substrate concentration ratio and SBP concentration, were investigated to optimize the performance of enzymatic treatment. The minimum effective SBP concentration required for removal of MDA was 0.70 U/mL, which was higher than that of TDA (0.15 U/mL). The reaction time course to achieve ≥95% removal of these compounds from water was determined under those optimum conditions. Identification of the transformed products was performed by means of high-resolution electrospray ionization mass spectrometry. The products generally observed were protonated oxidized oxidative dimers and higher oligomers (most commonly azo-coupled products). Michaelis constant, KM, and maximum reaction velocity, Vmax, obtained from the Michaelis-Menten (M-M) model revealed that TDA had a 65-fold lower KM than MDA (indicating TDA's higher affinity for SBP), and almost 5-fold higher Vmax than MDA. A pro-forma cost analysis is presented to assess the possibility of commercialization of enzymatic treatment as an alternative to conventional/traditional treatment methods.

Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays.

Enzyme labeling of an antigen or an antibody helps to visualize and amplify the signal and is an important reagent used in immunoassays for the detection of a target of interest. In this research, soybean peroxidase (SBP), a less commonly used enzyme reporter, was compared in immunoassays with the two most commonly used reagents, horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The enzyme-antibody conjugates were evaluated by their performance in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and in an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) for ractopamine (RAC). The results revealed that the more affordable SBP offers a long-lasting chemiluminescent signal, which outperformed ALP and HRP. SBP-antibody conjugate (SBP-Ab) based immunoassays produced lower limits of detection (LODs) and better accuracy in the detection of RAC in animal urine samples. Additionally, SBP-Ab has advantages in being more resistant to heat, acid and organic solvents. These results suggest that SBP could be a potentially excellent alternative to HRP and ALP for the development of immunoassay in food safety field.

Determination of kinetic parameters of the enzymatic reaction between soybean peroxidase and natural antioxidants using chemometric tools.

The oxidation of eugenol, isoeugenol and vanillin natural antioxidants catalyzed by the soybean peroxidase enzyme was studied using uv-vis spectroscopy. An experimental design was used to optimize the different variables. The multivariate curve resolution method was used to obtain the profiles of antioxidant absorbance's as a function of time due to uv-vis absorption bands of both antioxidants and the enzymatic reaction product/s show a strong overlap. From these results, apparent Michaelis-Menten constants as well as the kinetic parameters k1 and k3 involved in the catalytic cycle of peroxidases were calculated. The antioxidant apparent acidity constants were also determined at different pH's from uv-vis spectrophotometric measurements. Values of k1 were (0.6 ±â€¯0.1) × 105 M-1 s-1, (2.0 ±â€¯0.2) × 105 M-1 s-1 and (7.0 ±â€¯0.5) × 106 M-1 s-1 and k3 (4.0 ±â€¯0.2) × 105 M-1 s-1, (6.0 ±â€¯0.6) × 105 M-1 s-1 and (6.0 ±â€¯0.9) × 106 M-1 s-1 for eugenol, isoeugenol and vanillin, respectively.

Development of a third-generation biosensor to determine sterigmatocystin mycotoxin: An early warning system to detect aflatoxin B1.

A third-generation enzymatic biosensor was developed to quantify sterigmatocystin (STEH). It was based on a glassy carbon electrode modified with a composite of the soybean peroxidase enzyme (SPE) and chemically reduced graphene oxide. The optimal conditions to construct the biosensor were obtained through an experimental design based on the response surfaces methodology. The experiments were performed in 0.1 mol L-1 phosphate buffer solution, pH 5. Amperometric measurements were carried out at - 0.09 V vs Ag/AgCl (3 mol L-1 NaCl). The biosensor showed a lineal response in the concentration range from 6.9 × 10-9 to 5.0 × 10-7 mol L-1. The limit of detection was 2.3 × 10-9 mol L-1 for a signal: noise ratio of 3: 1. Values of the apparent Michaellis-Menten constant, KMapp, obtained by using both Lineweaver-Burk and Eadi-Hofstee methods were (1.5 ± 0.2) × 10-6 and (1.2 ± 0.2) × 10-6 mol L-1, respectively. STEH was analyzed in corn samples spiked with STEH, with an average recovery of 96.5%. The biosensor was also used to determine STEH in corn samples inoculated with the Aspergillus flavus fungus, which is an aflatoxins producer. Considering that STEH is a precursor of aflatoxin B1 (AFB1) in its biological transformation, its decrease over time was related to the production of AFB1. The STEH concentration determined using the biosensor was in very good agreement with that determined by HPLC.

Two-step enzymatic strategy for the synthesis of a smart phenolic polymer and further immobilization of a ß-galactosidase able to catalyze transglycosydation reaction.

A rapid and efficient enzymatic procedure for the preparation of an immobilized ß-galactosidase has been described. In a first step, soybean peroxidase was used to catalyze the polymerization of a strategically activated phenol (N-Succinimidyl 3-(4-hydroxyphenyl)propionate, known as Bolton-Hunter reagent). The phenolic support was directly employed for immobilizing ß-galactosidase from Bacillus circulans (ATCC 31382, ß-Gal-3), giving rise to a new biocatalyst subsequently applied in the synthesis of a ß-galatodisaccharide (Gal-ß(1-3)-GlcNAc and Gal-ß(1-3)-GalNAc). The reaction proceeded with high conversion rates and total regioselectivity. Reusability assays were performed with the same reaction conditions finding that the immobilized enzyme retains about 55% of its activity after eight batches. Finally and based on our results, the two-step enzymatic procedure presented here is a good and green alternative to the preparation of carbohydrates with biological activities.

Electrochemiluminescent detection of cardiac troponin I by using soybean peroxidase labeled-antibody as signal amplifier.

This work proposed an electrochemiluminescence (ECL) immunosensor for quantitative monitoring of cardiac troponin I (cTnI) in plasma with soybean peroxidase (SBP) labeled-antibody as signal amplifier. The ECL sandwich immunosensor was constructed by covalent binding anti-cTnI capture antibody (Ab1) to polyethylenimine-functionalized graphene matrix, which was obtained by a simple hydrothermal reaction between polyethylenimine (PEI) and graphene oxide (GO). After that, the SBP-labeled detection antibody (SBP-Ab2), synthesized by NaIO4 method, was immobilized on the surface of electrode through sandwich immunoreaction. The SBP on electrode surface displayed strong and stable ECL signal of luminol in the presence of H2O2, which could be used for cTnI detection with a concentration range of 5-30,000pg/mL and a detection limit of 3.3pg/mL. This proposed SBP-modified immunosensor displayed high sensitivity, selectivity and accuracy, and was expected not only to detect cTnI in practical human plasma sample but also to be used in other biomarkers detection.

Differential Degradation and Detoxification of an Aromatic Pollutant by Two Different Peroxidases.

Enzymatic degradation of organic pollutants is a new and promising remediation approach. Peroxidases are one of the most commonly used classes of enzymes to degrade organic pollutants. However, it is generally assumed that all peroxidases behave similarly and produce similar degradation products. In this study, we conducted detailed studies of the degradation of a model aromatic pollutant, Sulforhodamine B dye (SRB dye), using two peroxidases-soybean peroxidase (SBP) and chloroperoxidase (CPO). Our results show that these two related enzymes had different optimum conditions (pH, temperature, H2O2 concentration, etc.) for efficiently degrading SRB dye. High-performance liquid chromatography and liquid chromatography -mass spectrometry analyses confirmed that both SBP and CPO transformed the SRB dye into low molecular weight intermediates. While most of the intermediates produced by the two enzymes were the same, the CPO treatment produced at least one different intermediate. Furthermore, toxicological evaluation using lettuce (Lactuca sativa) seeds demonstrated that the SBP-based treatment was able to eliminate the phytotoxicity of SRB dye, but the CPO-based treatment did not. Our results show, for the first time, that while both of these related enzymes can be used to efficiently degrade organic pollutants, they have different optimum reaction conditions and may not be equally efficient in detoxification of organic pollutants.

The effect of dissolved organic matter on soybean peroxidase-mediated removal of triclosan in water.

Dissolved organic matter (DOM) is ubiquitous in water and involved in numerous important chemical processes in aqueous systems, enabling it a unique challenge for a variety of water treatment processes. Soybean peroxidase (SBP)-based enzymatic process, as a promising treatment technique, has been successfully applied to remove pollutants in wastewaters such as coal-tar and refinery wastewater. In this study, the effect of DOM on the removal of polychlorinated aromatic antimicrobials triclosan (TCS) by SBP was investigated. Our results suggested that DOM significantly suppressed the catalytic performance of SBP to TCS, presumably resulting from the competition of the phenolic moiety in DOM structure as the active substrate of SBP via the analysis of excitation emission matrix (EEM) spectra of DOM. Although the product species of TCS in SBP-mediated system with DOM has no change compared with the system without DOM, the yields of self-coupling products relative to total transformed TCS were remarkably reduced in the presence of DOM, suggesting that DOM participated in the oxidative coupling reactions. Cross-coupling between TCS and DOM was also verified using guaiacol as a model DOM constituent. Moreover, the products including self-coupling products and co-polymers in SBP-mediated TCS reaction system with DOM were innocuous through growth inhibition assay of S. obliquus.

Degradation of orange dyes and carbamazepine by soybean peroxidase immobilized on silica monoliths and titanium dioxide.

In this paper, the removal of three common dyes (orange I, orange II, and methylorange) and of the anticonvulsant drug carbamazepine from aqueous solutions by means of enzymatic and photocatalytic treatment was studied. Soybean peroxidase (SBP) was used as biocatalyst, both free in solution and immobilized on silica monoliths, and titanium dioxide as photocatalyst. The combination of the two catalysts led to a faster (about two to four times) removal of all the orange dyes compared to the single systems. All the dyes were completely removed within 2 h, also in the presence of immobilized SBP. As for carbamazepine, photocatalytic treatment prevails on the enzymatic degradation, but the synergistic effect of two catalysts led to a more efficient degradation; carbamazepine's complete disappearance was achieved within 60 min with combined system, while up to 2 h is required with TiO2 only.

Additive Effect on Soybean Peroxidase-Catalyzed Removal of Anilines from Water.

Soybean peroxidase has been shown to be effective in removal of aromatic compounds from wastewater, while the use of additives effectively reduces enzyme concentration requirement, hence overall treatment cost. Enzymatic treatment, an oxidative polymerization, was successful in removal of over 95% of both aniline and o-anisidine. The originality of this study lies in the findings that the additives, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), Triton X-100, and sodium dodecanoate (SDOD), reduced enzyme concentration requirement, while polyethylene glycol (PEG, average molar mass of 3350 g/mol) had no effect on the required enzyme concentration. In addition, the presence of SDS also enhanced treatment by improving precipitation and color removal. These results are enabling advancement of soybean peroxidase-catalyzed treatment of anilines found in wastewaters as a new sustainable method.

Removal of triclosan via peroxidases-mediated reactions in water: Reaction kinetics, products and detoxification.

This study investigated and compared reaction kinetics, product characterization, and toxicity variation of triclosan (TCS) removal mediated by soybean peroxidase (SBP), a recognized potential peroxidase for removing phenolic pollutants, and the commonly used horseradish peroxidase (HRP) with the goal of assessing the technical feasibility of SBP-catalyzed removal of TCS. Reaction conditions such as pH, H2O2 concentration and enzyme dosage were found to have a strong influence on the removal efficiency of TCS. SBP can retain its catalytic ability to remove TCS over broad ranges of pH and H2O2 concentration, while the optimal pH and H2O2 concentration were 7.0 and 8µM, respectively. 98% TCS was removed with only 0.1UmL(-1) SBP in 30min reaction time, while an HRP dose of 0.3UmL(-1) was required to achieve the similar conversion. The catalytic performance of SBP towards TCS was more efficient than that of HRP, which can be explained by catalytic rate constant (KCAT) and catalytic efficiency (KCAT/KM) for the two enzymes. MS analysis in combination with quantum chemistry computation showed that the polymerization products were generated via CC and CO coupling pathways. The polymers were proved to be nontoxic through growth inhibition of green alga (Scenedesmus obliquus). Taking into consideration of the enzymatic treatment cost, SBP may be a better alternative to HRP upon the removal and detoxification of TCS in water/wastewater treatment.

A portable chemiluminescence imaging immunoassay for simultaneous detection of different isoforms of prostate specific antigen in serum.

A multianalyte chemiluminescence (CL) imaging immunoassay strategy for sensitive detection of different isoforms of prostate specific antigen (PSA) was developed. The microtiter plates were fabricated by simultaneously immobilizing of free-PSA (f-PSA) and total-PSA (t-PSA) capture antibody on nitrocellulose (NC) membrane. Each of the array were spotted in replicates of six spots within a spacing of 2mm. 16 or 48 detection wells were integrated on a single NC membrane and each well could be used as a microreactor and microanalysis chamber. Under a sandwiched immunoassay, the CL signals on each sensing site were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging. Soybean peroxidase (SBP) was used to label f-PSA or t-PSA monoclonal antibody. With the amplification effects of two enhancers, 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP), the CL intensity could significantly enhanced, which improved the sensing sensitivity and detection limit. Under the optimal conditions, the linear response to the analyte concentration ranged from 0.01-36.7ng/mL and 0.02-125ng/mL for f-PSA and t-PSA, respectively. The results for the detection of forty serum samples from prostate cancer patients and cancer-free patients showed good agreement with the clinical data, suggesting that the proposed assay had acceptable accuracy. The proposed CL imaging immunoassay possess high throughput and acceptable reproducibility, stability and accuracy, which made it great potential to available to distinguish different isoforms of PSA in serum samples.

Combined experimental and theoretical study on the removal of pollutant compounds by peroxidases: affinity and reactivity toward a bioremediation catalyst.

Water pollution is a significant and growing problem throughout the world, especially in developing countries. In order to minimize environmental problems, catalysts have increasingly been designed to remove pollutants from the water. In an attempt to innovate by the creation of new low-cost alternatives to efficiently remove pollutants, the enzymatic treatment has been intensely studied for this purpose. Reactions catalyzed by enzymes are able to perform specific treatments, commonly with high rates of the final products. With this, the enzyme, peroxidase, is a promising candidate as a bioremediation catalyst. The efficiency of oxidoreductive enzymes, such as horseradish peroxidase (HRP) and soybean peroxidase (SP) have been studied, given that their performance depends on the substrate. In this investigation, experimental techniques and theoretical calculations have been employed in order to investigate the oxidative process for the ferulic acid and bromophenol blue dyes, performed by HRP and SP. Both enzymes showed a comparable behavior with respect to ferulic acid substrate. On the other hand, by utilizing bromophenol blue dye as a substrate, the behavior of the employed catalysts was significantly different. Experimental data have shown that HRP was more active toward bromophenol blue when compared to ferulic acid, being more rapidly degraded by the HRP enzyme. This tendency was confirmed by our theoretical docking, PM6 semi-empirical method, and DFT calculation results, in which the interaction, binding energies, and transition states were determined.