- Research article
- Open Access
Soybean peroxidase-mediated degradation of an azo dye– a detailed mechanistic study
© Ali et al.; licensee BioMed Central Ltd. 2013
Received: 27 July 2013
Accepted: 28 November 2013
Published: 5 December 2013
Peroxidases are emerging as an important class of enzymes that can be used for the efficient degradation of organic pollutants. However, detailed studies identifying the various intermediates produced and the mechanisms involved in the enzyme-mediated pollutant degradation are not widely published.
In the present study, the enzymatic degradation of an azo dye (Crystal Ponceau 6R, CP6R) was studied using commercially available soybean peroxidase (SBP) enzyme. Several operational parameters affecting the enzymatic degradation of dye were evaluated and optimized, such as initial dye concentration, H2O2 dosage, mediator amount and pH of the solution. Under optimized conditions, 40 ppm dye solution could be completely degraded in under one minute by SBP in the presence of H2O2 and a redox mediator. Dye degradation was also confirmed using HPLC and TOC analyses, which showed that most of the dye was being mineralized to CO2 in the process.
Detailed analysis of metabolites, based on LC/MS results, showed that the enzyme-based degradation of the CP6R dye proceeded in two different reaction pathways- via symmetric azo bond cleavage as well as asymmetric azo bond breakage in the dye molecule. In addition, various critical transformative and oxidative steps such as deamination, desulfonation, keto-oxidation are explained on an electronic level. Furthermore, LC/MS/MS analyses confirmed that the end products in both pathways were small chain aliphatic carboxylic acids.
Extensive use of synthetic dyes and their subsequent release in industrial wastewater is a growing environmental problem. It is estimated that as much as 12% of the dyestuff amounting to about 280,000 ton/ year is released to the ecosystem. These dyes are used in various industrial applications (total consumption is more than one million tons of dyes annually) and are engineered to be generally resistant to fading. They not only need to sustain alkaline or acidic environment but also need to withstand washing with soaps and bleaching agents and be resistant to light and ultraviolet irradiation.
It is well-established that some dyes are potentially carcinogenic and mutagenic, as well as genotoxic[3–5]. Furthermore, the presence of color in water bodies causes less sunlight to penetrate through water which results in reducing the photosynthetic capability of aquatic plants and microorganisms. Industrial effluents containing dyes are able to color water even in concentrations as low as 1 mg/liter, whereas in most cases, these wastewater streams typically contain a much higher amount of the dye content, about 10-200 mg/liter, which gives intense coloration. It is therefore not surprising that both international and national regulations for industrial wastewater require substantial elimination of the dyestuff content form the effluent[7, 8]. Numerous approaches have been developed to treat industrially generated wastewater, such as coagulation/flocculation, adsorption, use of activated carbon, and various forms of Advanced Oxidation Processes, e.g. ozonation, and photochemical approaches[9–16]. Most of these methods have a disadvantage of either formation of large amounts of sludge or production of possible toxic byproducts.
Removal of contaminants from the environment by biological methods using enzymes and microorganisms is considered to be closer to nature as it is an environmentally friendlier technique which leaves the ecosystem intact. The technology is scalable and can also be used to treat other organic impurities. A number of microorganisms including bacteria, fungi, and yeasts have been used to treat the dye contaminated wastewaters[19–22].
Azo dyes are electron-deficient xenobiotic components because of their azo linkage (–N=N), and in many cases, they have sulphonic (SO3−) or other electron-withdrawing groups, which generate an electron deficiency and make the dye less susceptible to degradation by microorganisms. However, under appropriate conditions, they can be degraded by azo reductase-releasing microbes.
Enzyme mediated decolorization is another newer alternative, where the enzyme can specifically react with organic pollutants by transforming them into low molecular weight products. The main advantage of using enzymes in degrading dye solutions is that they have higher reaction rates and can also work in milder reaction conditions. Some azo dyes have been decolorized by using certain peroxidases, such as soybean peroxidase (SBP), manganese peroxidase (MnP), lignin peroxidase (LiP), laccase and horseradish peroxidase (HRP)[23–26]. It has been suggested that these enzymatic treatments could oxidize the dye structures to form compounds with lower molecular weight and lower toxicity and may eventually mineralize the dyes.
Although a considerable amount of research has been published on the use of pure enzymes to degrade dyes, detailed analyses of the breakdown pathway are almost non-existent. Structures of the intermediates produced as well as the mechanisms involved in the dye degradation pathway are important to properly understand and further exploit this novel approach to pollutant-contaminated water remediation.
The objective of the present study was to study in detail the mechanism of an azo-dye degradation by soybean peroxidase enzyme (SBP). In addition to identifying the various intermediates produced and proposing possible pathways, factors affecting dye degradation such as initial dye concentration, H2O2 concentration, pH, and presence of redox mediator were also evaluated. The present study is one of the very few studies that show in detail the various intermediates produced during peroxidase-mediated degradation of an azo-dye and possible electron-level mechanisms involved.
The azo dye namely Crystal Ponceau 6R (C.I name = Acid Red 44, C.I number = 16250, Molecular Formula = C20H12N2O7S2Na2, FW = 502.446 g mol−1), herein abbreviated as CP6R was used as a model dye. The dye was procured from Sigma-Aldrich Chemicals and used as such. All the other chemicals used in this work were obtained from Sigma-Aldrich and were of high purity (< 98%).
Dye degradation studies
Stock solution (2,000 ppm) of the dye was prepared in a 250 mL flask by first dissolving an appropriate amount in deionized water. Further dilutions from this stock were done as per the requirement of the experiment. Unless otherwise indicated, the working concentration of the CP6R dye was 40 ppm. Dye degradation reactions were carried out by adding H2O2 to a buffered dye solution containing SBP enzyme. Spectrophotometric measurements were made using a CARY 50 UV/Vis spectrophotometer. The absorbance value obtained in each case was plotted against time to obtain the % degradation. The % degradation for the dye was calculated by observing the changes in λmax (510 nm) of the solution. The studies were carried out at 25°C otherwise indicated. For pH studies, the dye solution were prepared in 33.33 mM universal buffers (citrate-phosphate) adjusted to specific pH value.
Where A 0 is the initial absorbance of dye solution and A t is the absorbance of the dye solution at any given time.
Total Organic Carbon (TOC) analyses
TOC analyses were carried out using GE Sievers InnoVox TOC analyzer properly calibrated with fresh standards. The CP6R samples tested were 0% degradation sample which consisted of 400 ppm CP6R in 33 mM citrate-phosphate buffer, pH 4, 0.78 μM SBP, 0.1 mM HOBT and 100% degradation sample which was exactly the same as the 0% sample but contained 1 mM H2O2. Analyses were carried out in triplicates and the data is presented as TOC values normalized to 0% CP6R degradation sample.
HPLC and LC/MS experiments
High performance liquid chromatography (HPLC) and LC/MS analyses were carried out similar to as previously described. Briefly, samples were analyzed on an Acquity UPLC system, (Waters Corporation, Milford, MA, USA) with an Acquity UPLC BEH C18 column with 1.7 μm particle size (2.1 mm I.D. × 100 mm length, Waters Corporation, Milford, MA, USA) maintained at 35°C, coupled to Acquity tunable ultraviolet/visible detector (Waters Corporation, Milford, MA, USA) and an Acquity Tandem quadruple mass spectrometer (Waters Corporation, Milford, MA, USA). The mobile phase consisted of solution A (0.1 M ammonium formate (pH 6.7)) and solution B (1:1 acetonitrile/methanol) and a gradient of 0% B to 80% B in 13.80 minutes at the flow rate was 0.2 mL/min was used to obtain the chromatographs. The mass spectrometer was equipped with an electrospray ionization source operated in negative ion mode. The ESI conditions were as follows: capillary voltage: 3.0 kV, Cone voltage 30 V, collision energy 50 V, desolvation gas (Nitrogen at 500 L/Hr), Cone gas (Nitrogen at 2 L/Hr), desolvation temperature was set at 350°C and source temperature was 150°C. The mass range was detected from 50 to 700 m/z. Tandem MS experiment was done using Waters Masslynx V 4.1, wherein Argon gas was used as the collision gas.
Results and discussion
Optimizing the enzymatic dye degradation reaction conditions
As expected, the degradation of dye was found to be very much affected by the initial amount of dye content in solution. Studies carried out at different concentrations of the dye showed the optimum CP6R under our chosen conditions was 40 ppm (data not shown). Optimizations of other parameters are described below:
Requirement of HOBT for SBP-mediated degradation of CP6R
Initial experiments using only SBP and H2O2 showed that unlike other dyes, CP6R was unable to be degraded by SBP/H2O2 alone (data not shown). It is well known that the presence of redox mediators such as 1-hydroxybenzotriazole (HOBT), veratryl alcohol, violuric acid, 2- methoxyphenothiazone, etc. can dramatically increase the rate of dye degradation[27–30]. The mechanism involved is well known, wherein, the substrate initially undergoes an one-electron oxidation in the presence of a redox mediator and transforms into a radical cation followed by abstraction of a H-atom from the substrate by the mediator and converting it into a radical, which can then cause the substrate to co-oxidize.
Effect of hydrogen peroxide concentration on dye degradation
Since the peroxidase enzymes use H2O2 as a co-reactant, if the concentration of hydrogen peroxide used is too low, the enzyme activity becomes low; however, a very high peroxide concentration can irreversibly oxidize the enzyme and cause its inactivation. In this regard, experiments were carried out to optimize the H2O2 concentration while keeping the other parameters constant. The results obtained are shown in Additional file2: Figure S2, which showed the optimum H2O2 concentration to be 0.175 mM. Additionally, it can also be seen that at very high H2O2 concentrations, a significant reduction in dye degradation is observed (due to H2O2-mediated inactivation of SBP).
Effect of SBP enzyme concentration on dye degradation
Degradation of dye depends on the amount of catalyst added and the contact time. Thus experiments were also done to optimize the concentration of the enzyme in dye solution which was varied in the range of 0 to 2.7 μM while keeping the other parameters constant. The results are shown in Additional file3: Figure S3 and it can be noted that the enzyme dose had a significant effect on dye decoloration. At lower SBP concentrations, the dye degradation was not very significant, whereas at very high SBP concentration, the dye degraded very quickly in a very short time (almost 30% in a few seconds). Based on these data, an optimized concentration of 0.27 μM SBP was chosen for all subsequent reactions.
Effect of pH
Enzymatic driven reactions are known to be pH dependent[28, 29]. Thus experiments were done to optimize this parameter as well. SBP mediated dye degradation was studied at different pH values (from 2 to 9), while keeping the other conditions constant. The results are shown in Additional file4: Figure S4, which shows the dramatic effect of pH on SBP-mediated degradation of CP6R, with the enzyme being most active in the pH 3-5 range. A pH value of 5 was used for all the subsequent experiments. This role of pH on the peroxidase driven reactions has been reported in the literature for different dye degradation along-with its mechanism[17, 23].
Total Organic Carbon analysis
Analysis of product formation using HPLC/MS
Proposed mechanism of enzymatic degradation
The generation of CP6R radical by SBP in the presence of a mediator (HOBT) consists of four major steps. In the first step, SBP enzyme reacts with H2O2 to become an oxyl-ferric (Fe4+) cation radical, compound I (via loss of two electrons). The second step involves the abstraction of hydrogen from HOBT resulting in the formation of HOBT radical and compound II. In the third step, a second radical of HOBT is formed by the transfer of another hydrogen to compound II, leading to the regeneration of the original reduced (Fe3+) SBP enzyme and a water molecule. In the final step, HOBT radical attacks CP6R and abstracts a hydrogen, resulting in the formation of CP6R radical. Similar reactions have been previously well-documented.
Proposed mechanistic pathway of CP6R degradation by SBP-H2O2
Mass of all intermediate in asymmetrical degradation
2-carboxyvinyl -4,6disulfobenzonic acid.
3-methylhexa 2,4-diene dionic acid.
Mass of all intermediate in symmetrical degradation
2-carboxyvinyl -4,6disulfobenzonic acid.
3-methylhexa 2,4-diene dionic acid.
Asymmetrical azo bond cleavage of CP6R
Symmetrical azo bond cleavage of CP6R
Detailed electronic-level mechanism of asymmetrical cleavage of the azo bond
Oxidation of diketo
The oxidation of diketo to carboxylic acid has been observed in several photolytic degradation studies. The ring is initially opened by the attack of OH radical on alpha diketones resulting in the formation of dicarboxilic acid intermediate (A2). This is shown in the Additional file5: Figure S5.
Deamination and removal of azo group
Oxidation of phenol
Formation of amine in symmetrical cleavage
De-amination in symmetrical cleavage
In summary, we show here efficient degradation of an azo-dye, Crystal Ponceau 6R (CP6R), using the Soybean peroxidase/HOBT/H2O2 system. Under optimized conditions it was found that SBP could degrade 100% of the dye in under a minute. Dye mineralization was confirmed using TOC and HPLC experiment, and most importantly, extensive LC/MS/MS experiments were used to identify the various metabolites formed during the degradation process. Lastly, based on the LC/MS data and known radical-based reactions, we were able to develop detailed mechanisms for the various steps in the dye degradation. Our results show that the azo dye degraded via two different pathways, namely symmetric and asymmetricazo bond cleavage followed by diketo oxidation to carboxylic acids, desulfonation, deamination, and phenolic oxidation reactions.
This research was partially funded by UAEU/NRF Research Grant Program 27/11/2 (21S039 & 31S072) to SSA and MAR.
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