Mechanistic study of a diazo dye degradation by Soybean Peroxidase
© Kalsoom et al.; licensee Chemistry Central Ltd. 2013
Received: 28 March 2013
Accepted: 16 May 2013
Published: 27 May 2013
Enzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published.
The present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway.
We report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue.
Textile dyes are aromatic compounds representing a major class of organic pollutants that are found in the waste effluent discharged by different industries such as textile, petroleum refining, paper and pulp, leather and plastics, wood preservation, etc. These compounds are toxic and potentially carcinogenic in nature and must be removed from industrial effluent before entering into the larger water bodies such as ponds, rivers and lakes [1–4]. Conventional methods of removing such pollutants such as adsorption, sedimentation, coagulation, and filtration result in a secondary waste which in itself is a problem to dispose [5–7]. Chemical methods, such as advanced oxidation techniques, have been adopted for handling such effluent , but have limited success so far at a commercial scale .
The process of removing the contaminants in the environment by biological methods using the metabolic potential of microorganisms to degrade a wide variety of pollutants has been explored with some success. The main advantage of bioremediation is its reduced cost compared to conventional techniques [10, 11]. Furthermore, the method is environmentally friendlier as it leaves the local ecosystem intact and does not add any variables to it. The bioremediation process can also deal with lower concentrations of contaminants, whereby the cleanup by physical or chemical methods would not be feasible.
Although a number of microorganisms including bacteria, fungi, and yeasts have been found to decolorize textile dyes, biocatalysts such as enzymes have also been used for the degradation and mineralization of dyes [12, 13]. Enzymes can specifically react with organic pollutants and remove them by transforming them into other products. The catalytic action of enzymes is generally very efficient and selective as compared to chemical catalysts due to their higher reaction rates, milder reaction conditions and greater stereo-specificity. They can catalyze reactions at relatively low temperature and in the entire aqueous phase pH range.
Since dye molecules display a high structural variety, they are degraded by only few enzymes. These biocatalysts have one common mechanistic feature, i.e., they are all redox-active molecules and thus exhibit relatively wide substrate specificities. The enzymes are responsible for generating highly reactive free radicals that undergo a complex series of spontaneous cleavage reactions. Some common examples of enzymes used for dye degradation are lignin peroxidase, laccases, horseradish peroxidase, tyrosinase, Manganese peroxidase etc. [14–16]. Peroxidases that have been used for treatment of aqueous aromatic contaminants and degradation of dyes include horseradish peroxidase (HRP), lignin peroxidase (LiP) and a number of other peroxidases from different sources. Although there are numerous reports on the use of peroxidases for the degradation of organo-pollutants, studies that show detailed mechanistic pathway of dye breakdown are very few.
In the present study, we report on the use of commercial soybean peroxidase (SBP) for the degradation of a di-azo dye, Trypan Blue. Effect of parameters such as aqueous phase pH, H2O2 and SBP concentration, contact time and application of immobilized SBP and dye concentration has been investigated to optimize the dye degradation. Furthermore, the performance of the dye removal process by SBP immobilized in polyacrylamide gel matrix was evaluated in order to study the enzyme’s reusability. Most importantly, we used LC/MS-MS to identify various intermediates and develop a detailed mechanism for the degradation of this di-azo compound by SBP. Such detailed mechanistic degradation schemes for peroxidase-mediated dye degradation are important to understand the peroxidase mechanism and are unfortunately not widely published.
Soybean Peroxidase (SBP) used in this study was obtained from a commercial supplier (Bio-Research Products, Inc, USA) and was used as supplied. The specific activity of the enzyme was 2,700 U/mg and was supplied as a 26 μM solution, which was stored at 4°C.
Trypan Blue (TB) stock solution of 2,000 ppm was prepared in a 250 mL flask by first dissolving an appropriate amount of the dye in deionized water. Necessary dilutions of this stock were further done as per the requirement of the experiment. Unless otherwise indicated, the working concentration of the TB dye was 40 ppm. Dye degradation reactions were carried out by adding H2O2 to a buffered dye solution containing the SBP enzyme. Spectrophotometric measurements were made either using a CARY 50 UV/Vis spectrophotometer (3 ml reaction volume) or a Tecan Sunrise microplate reader (200 μl reaction volume).
For effect of temperature on dye degradation experiments, a temperature-controlled plate reader (Perkin Elmer, Victor X) set at appropriate temperature was used.
The absorbance value obtained in each case was plotted against time to obtain the order of % degradation. The % degradation for the dye was calculated by observing the changes in λmax (595 nm) of the solution. The studies were carried out at 25°C otherwise indicated. For pH studies, the dye solution was prepared in 33.33 mM buffers adjusted to specific pH using 0.1M glycine/HCl, acetate, phosphate and Tris/HCl buffers. These buffers did not cause any change in the λmax of the dye.
Polyacrylamide (PA) entrapped SBP was prepared as follows: 4.25 ml of potassium phosphate buffer (0.1M, pH = 7.0) was mixed with 2.7 ml of acrylamide solution (3g acrylamide and 0.08g of bisacrylamide in 10 ml potassium phosphate buffer) and 80 μl of ammonium persulfate solution (10% ammonium per sulfate in potassium phosphate buffer) and the resulting mixture was mixed in 20 ml vial. Subsequently, 0.45 ml of SBP solution (containing 1,215 units) was added followed by 10 μl of TEMED (N,N,N,N-tetra methylethylenediamine) reagent and the mixture was gently mixed. The complete polymerization of acrylamide/bisacrylamide took about 30 minutes (at room temperature). Gel was transferred subsequently to vacuum filter system to remove the solution and subsequently washed with phosphate buffer. Gel was broken by aspiration using a sharp knife into small equal size pieces (~ 0.5 cm x 0.5 cm x 0.5 cm) and stored at 4°C prior to use.
High performance liquid chromatography (HPLC) and LC/MS analyses were carried out as previously described . Briefly, an Agilent HP 1100 liquid chromatography system, (Agilent, USA) with an Agilent Zorbax® SB-C18 column 150 mm x 4.6 mm packed with 5 μm particle size, coupled to a diode array detector (Agilent, USA) and an ion trap 6310 mass spectrometer (Agilent technologies) were used to monitor and indentify dye degradation metabolites. 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 from 0% B to 80% B in 40 minutes at the flow rate was 1 mL/min was used to obtain the chromatographs. The mass spectrometer was equipped with an electrospray ionization source and operated in positive polarity. The ESI conditions were as follows: capillary voltage: 3.5 kV, endplate offset was fixed at 500 V; skimmer at 40 V; trap drive at 53 V; the nebulizer pressure was 70 psi and drying temperature was 350°C. The mass range was from 50 to 1200 m/z. Tandem MS experiment was done using the Auto MSn mode wherein Helium gas was used as a collision gas.
Results and discussion
Degradation of the Trypan Blue solutions
where A0 is the initial absorbance of dye solution and At is the absorbance of the dye solution at any given time. The results are shown in Figure 1B. One can see from this figure that in the early stages, the % degradation was fast which became slow with time and almost leveled off around 30 minutes. The addition of SBP or H2O2 alone did not show any degradation of the dye.
Effect of SBP on dye degradation
The effect of SBP concentration on Trypan Blue degradation and the rate of dye degradation
% Dye Degradation
Initial Rate (min-1)
Effect of dye concentration
The concentration of the substrate present in the reaction mixture directly effects enzyme-mediated reactions. The rate of the reaction should increase and reach the maximum value if the amount of enzyme concentration is kept constant and the substrate concentration is gradually increased. Once the saturation is achieved (maximum enzyme velocity, νmax), any further addition of the substrate will not change the rate of reaction. Studies in this regard were carried out at different concentrations of the dye (10, 20, 40 and 80 ppm), while keeping all the other parameters constant (H2O2 = 64 μM; pH = 7; temperature = 25°C). With the increase in dye concentration, the removal was found to be most effective at the lowest dye concentration (10 ppm). Further increase in dye concentration up to 80 ppm resulted in relatively slower dye removal (Additional file 1: Figures S1) – at 80 ppm (92 μM TB) the dye degradation observed of about 60% was consistent with the H2O2:TB ratio of 2:3. A similar profile for studying the oxidation of 2,4-chlorophenol oxidation by horseradish peroxidase has also been reported in the literature .
Effect of H2O2addition
Effect of pH
(SH indicates a generic substrate)
The above mentioned reactions steps of the catalytic cycle are pH dependent and work best under acidic conditions. In the first step (2), the formation of compound I is favored by the presence of a network of hydrogen bonds between the Fe-heme/H2O2 adduct and the distal histidine and arginine side chains, whereas, in the other steps (3) and (4), the substrate oxidation depends on its protonation state .
Effect of temperature
In summary, SBP was found to be very effective for the degradation of TB, and under optimal conditions could degrade the dye very quickly. Similar results were also observed for another reactive dye namely Turquoise Blue where SBP was used for the degradation resulting in 95% degradation of the dye .
Degradation of dye with Polyacrylamide (PA)-entrapped SBP
HPLC-DAD-MS/MS analysis of dye degradation
Summary of the tandem mass spectrometry fragment analysis of the ten Trypan Blue degradation intermediates
327, 283, 259, 227, 177, 171, 133,
251, 248, 231, 203, 179, 139
200, 173, 155, 237, 122, 111
195, 185, 167, 155
192, 167, 136, 74
175, 165, 148
137, 121, 93
149, 140, 131, 123, 109, 57,
148, 135, 121, 107,89
145, 135, 121, 117, 107, 101, 73
It is also interesting to note that some of the resulting intermediates produced during Trypan Blue degradation are aromatic amines (compounds 1, 2, and 4), however, they are quickly degraded to smaller and less-aromatic compounds (as shown in Figure 9) and may eventually be mineralized to CO2 and ammonium ions. We plan to carry out studies on the toxicity of intermediate solutions in the near future as well as to confirm the actual mineralization of the dye, which are slightly outside the scope of the current study. Another surprising finding of our report is that peroxidase-H2O2 mediated degradation of phenolic compounds normally produces high molecular weight polymers, which were not observed here. Perhaps the complex structure of our dye (phenolic, amines and sulphonic groups) shifted the equilibrium in favor of smaller degradation products as opposed to higher molecular weight polymers.
In summary, the experimental results presented here showed the effectiveness of the peroxidase catalyzed enzymatic reaction in the degradation of Trypan Blue in aqueous phase. The performance of SBP catalyzed reaction for dye removal was found to be dependent upon the reaction time, dye concentration, enzyme concentration, H2O2 dose and pH value. The stepwise addition of H2O2 caused more than 90% degradation of the dye in less than 15 minutes. Immobilized SBP in polyacrylamide matrix also showed efficient dye degradation and could enable effective use of the same enzyme many times. HPLC-DAD analysis showed rapid formation of new products with different UV–vis spectra upon the addition of the enzyme. In addition to the above-mentioned optimization of various parameters for efficient dye degradation, our study employed extensive LC-MS and tandem MS-MS analyses to identify the intermediate metabolites produced in the process and propose a detailed mechanism of dye degradation involving symmetrical azo-bond.
This research was partially funded by UAEU/NRF Research Grant Program 27/11/2 to SSA and MAR. The authors are also thankful to Higher Education Commission (HEC) of Pakistan for providing financial assistance to Ms. Umme Kalsoom, under the Indigenous Ph.D. Fellowship Program.
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