Horseradish Peroxidase and Hematin as Biocatalysts for Alizarin

Mar 2, 2010 - Argentina, Instituto de InVestigaciones Fisicoquımicas Teóricas y ... Degradation of organic dyes from the textile industry is a matte...
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Ind. Eng. Chem. Res. 2010, 49, 6745–6752

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Horseradish Peroxidase and Hematin as Biocatalysts for Alizarin Degradation Using Hydrogen Peroxide Silvina Pirillo,*,† Fernando Sebastia´n Garcı´a Einschlag,‡ Elsa H. Rueda,† and Marı´a Luja´n Ferreira†,§ Departamento de Quı´mica, UniVersidad Nacional del Sur, AVenida Alem 1253, B8000CPB Bahı´a Blanca, Argentina, Instituto de InVestigaciones Fisicoquı´micas Teo´ricas y Aplicadas (INIFTA, UNLP, CCT La Plata-CONICET), Diag. 113 y 64, Sucursal 4, C.C. 16, B1900ZAA La Plata, Argentina, and Planta Piloto de Ingenierı´a Quı´mica (PLAPIQUI-UNS-CONICET), Camino a la Carrindanga km 7, B8000CPB Bahı´a Blanca, Argentina

Degradation of organic dyes from the textile industry is a matter of enormous environmental concern. The horseradish peroxidase enzyme is known for its capacity to remove phenolic compounds and aromatic amines from aqueous solutions and also to decolorize textile effluents. This study evaluates the potential of the both enzyme horseradish peroxidase (HRP) and its biomimetic hematin in the decolorization of alizarin. We describe an UV-visible study of alizarin elimination from aqueous solutions by polymerization using HRP or hematin as catalysts, hydrogen peroxide as oxidant, and HCl as coagulant. The effects of the initial dye concentration and the temperature on the decolorization efficiencies, attained with each catalyst after 2 h of reaction time, are reported. Alizarin removal with HRP/H2O2 achieved the highest elimination level (88%) when the dye concentration was 50 mg/L. At the same dye concentration a degradation of 97% was obtained using hematin as biocatalyst. A comparative analysis of time-resolved spectra and dissolved oxygen profiles is presented. The oxygen profiles found for hematin/H2O2/alizarin and HRP/H2O2/alizarin systems are clearly different, pointing to oxygen consumption for hematin and oxygen production for peroxidase. 1. Introduction The color of wastewater is the most apparent indicator of waste pollution. Even the presence of small amounts of dyes (below 1 ppm) is clearly visible and considerably influences the water environment. Therefore, dye concentration should be reduced before its drop to the environment. The removal of dyes from wastewaters is often more important than the removal of other colorless organic substances.1 The main disadvantage of the commonly used traditional methods of wastewater purification is the fact that they are not destructive but only transfer the contamination from one phase to other. Among the alternatives for water treatment are the advanced oxidation processes (AOPs),2-4 which may lead to the complete mineralization of organic pollutants. Bioremediation methods have also been applied. They have the potential to be less expensive, less invasive, and more environmentally friendly than many chemical or physical remediation options.5,6 The use of free enzymes in bioremediation methods is desirable because they can perform the same function as many harsher chemicals, such as solvents, but at neutral pH, at moderate temperatures, and without production of hazardous waste. Although enzymes tend to be expensive due to the extraction and purification costs, they can be very cost-effective because they minimize waste disposal and energy requirements, especially if they can be reused.7,8 The utilization of plant peroxidases in removal of phenolic pollutants from aqueous solution is well documented.9,10 For example, it has been demonstrated that horseradish peroxidase (HRP) can catalyze free-radical formation in the presence of * To whom correspondence should be addressed. Tel.: (+54) 2914595159. Fax: (+54) 291-4595160. E-mail: [email protected]. † Universidad Nacional del Sur. ‡ INIFTA, CONICET. § PLAPIQUI-UNS-CONICET.

hydrogen peroxide (H2O2), followed by spontaneous polymerization of a variety of aromatic oligomerized compounds. Dyes recalcitrant to common chemical bleaches were successfully treated by using horseradish and soybean peroxidases.11 Several reports have pointed out the effective performance of HRP to degrade azo dyes in the aqueous phase.12-14 An interesting option is the replacement of peroxidases by biomimetic compounds such as hematin.15 Peroxidases include a heme group, a needed cofactor for the reaction to take place. Heme is the protoporphyrin IX complex of the ferrous ion (Fe2+). Hemin is the protoporphyrin IX complex of the ferric ion (Fe3+). Hematin is the hydroxylated hemin.16 HRP is very much more expensive than hematin; hence, to achieve high efficiency by using hematin instead of HRP would be very advantageous. The goal of this paper is to present a comparative analysis of the capabilities of the horseradish peroxidase enzyme (HRP) and its biomimetic hematin, both free or unsupported, in specific reactions with alizarin red or alizarin (common names of the same commercial available textile dye 1,2-dihydroxy-9,10anthraquinone). Alizarin was chosen because its plane structure and high electronic conjugation are commonly present in dyes of the textile industry (for example, in alizarin red S, the sodium salt of alizarinesulfonate or 9,10-dihydro-3,4-dihydroxy-9,10dioxo-2-anthracenesulfonic acid sodium salt). In this study, results about decolorization efficiency, oxygen consumption, and kinetics profiles were obtained with both catalysts in their solubilized forms (unsupported). Theoretical modeling was employed to justify the experimental results considering aggregation of the dye and its impact in the analysis of UV/visible spectra. In the near future, new studies will be carried out on the efficiency of the catalytic degradation process, using the HRP enzyme and hematin in the immobilized form.

10.1021/ie901528y  2010 American Chemical Society Published on Web 03/02/2010

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Figure 1. Structure of alizarin.

2. Experimental Section 2.1. Materials. All reagents used in this study were of an analytical grade. Horseradish peroxidase (molecular weight 41 000 Da) was kindly provided by Amano Inc. (U.S.) and was used without further purification. Hematin (molecular weight 633.5 g/mol) from Sigma Chemical Co. was employed as provided. Alizarin was provided by The British Drug Houses Ltd., BDH Laboratory Chemicals Group, Poole, England. The chemical structure of alizarin is shown in Figure 1. 2.2. Methods. 2.2.1. Decolorization Efficiency Studies. Owing to the large difference in the molar masses of HRP and hematin, comparative experiments were performed using equal masses of catalysts per volume unit. The experiments were carried out using different dye concentrations (25, 50, 75, 100, and 150 mg/L) at pH 7 ( 0.1 (buffer KH2PO4/NaOH). This pH value was chosen since, on the one hand, HRP activity drastically drops in alkaline media and, on the other hand, at acidic pH alizarin is scarcely soluble and precipitation of alizarin oligomers on the enzyme may lead to catalyst inactivation. The final reaction volume was 10 mL and the amount of catalyst added was 0.25 mg. In all experiments, 80 µL of hydrogen peroxide (30% v/v H2O2) was added in four steps at 5, 10, 15, and 20 min after the addition of HRP or hematin, with a final H2O2 concentration of 0.021 M. These conditions were selected in order to compare the biocatalyst with the biomimetic, although no attempts to minimize deactivation of HRP by hydrogen peroxide were made. The solutions were magnetically stirred at 25 °C. After 2.5 h, 200 µL of 37% HCl was added as flocculant to precipitate the resulting oligomers/polymers. UV-visible analyses were carried out using a Agilent 8453 UV-visible spectroscopy system from Hewlett-Packard. Spectra were recorded in the 300-900 nm range. The evaluation of the treatment effectiveness for alizarin was carried out through absorbance readings before hydrogen peroxide addition, after 2 h, and after 24 h. Since preliminary tests showed that by varying the pH from acidic to alkaline branch alizarin changes from yellow to violet and that UV/ visible measurements at pH 11 give the best absorbance/ concentration response, the respective aliquots were alkalinized to pH 11 by adding NaOH before recording the spectra. The decolorization efficiency or percentage of alizarin conversion at selected wavelengths was estimated as [(C0 - Cf)100]/C0, where C0 is the initial concentration and Cf is the final concentration of the dye at 2 h. Free HRP and hematin were tested at 25, 45, and 65 °C using an alizarin concentration of 100 mg/L. 2.2.2. Kinetics Studies. These studies were performed at both pH 7 and 8 (KH2PO4/NaOH buffer) at selected conditions using the UV/visible spectrophotometer in continuous mode for 4 h. In contrast with to experiments of section 2.2.1, the timeresolved spectra were recorded without previous alkalinization. The dye concentration was 50 mg/L. Hematin or peroxidase was added to reach a final concentration of 25 mg/L. To achieve

a total concentration of 0.021 M, 30% (v/v) H2O2 was added in four steps at 5, 10, 15, and 20 min after the addition of HRP or hematin. Time-resolved spectra were analyzed in order to develop a simple model to compare the main kinetic and mechanistic features associated with each biocatalyst. Multivariate selfmodeling curve resolution (MCR) can be applied to bilinear spectroscopic-kinetic data from a chemical reaction to provide information about composition changes and reaction kinetics.17 In the present work we have chosen the alternating least squares method (ALS).18 This algorithm can help to simultaneously estimate concentration and spectral profiles. ALS extracts useful information from the experimental absorbance matrix A(t × w) by the iterative use of the matrix product: A ) CST + E where C(t × n) is the matrix of the kinetic profiles; ST(n × w) is that containing the spectral profiles and E(t × w) represents the error matrix. The numbers t, n, and w denote the sampling times, minimum number of factors contributing to the absorbance, and recorded wavelengths, respectively.19 The value of n is usually unknown, and curve resolution methods cannot give a single solution due to rotational and scale ambiguities. Therefore, we applied factor analysis and singular value decomposition for the estimation of n. In addition, we used matrix augmentation strategy20 and we introduced some chemically relevant constraints such as nonnegativity (for both spectra and concentrations) and unimodality (for the kinetic profiles) to reduce rotational and scale ambiguities.17 The software including standard algorithms for multivariate analysis of the UV/visible spectra was developed in one of our laboratories. 2.2.3. Oxygen Consumption/Release. The oxygen consumption was recorded with a heavy-duty dissolved oxygen meter, a Model 407510 Extech instrument. The reactions were carried out at 25 °C with a concentration of 100 mg/L of alizarin for both catalysts. In the case of peroxidase 160 µL of 30% (v/v) hydrogen peroxide at reaction times of 5, 10, 15, and 20 min and the necessary amount of pH 7 ( 0.1 buffer (KH2PO4/NaOH) to reach a final volume of 50 mL were added with magnetic stirring. The final concentration for HRP was 10 mg/L, and the final [H2O2] was 8.6 × 10-3 M. For hematin at 5, 10, 15, and 20 min, 400 µL of 30% (v/v) hydrogen peroxide and the necessary amount of pH 7 ( 0.1 buffer (KH2PO4/NaOH) were added with magnetic stirring to reach a final volume of 50 mL. The final concentration for hematin was 25 mg/L, and the final [H2O2] was 2.14 × 10-2 M. 3. Results and Discussion 3.1. Effect of Initial Dye Concentration on the Amount of Dye Removed. Figure 2 shows the percentage of alizarin removed by peroxidase and hematin for different initial dye concentrations at 25 °C and pH 7. The percentages of dye removed, calculated as described in section 2.2.1, show that, when the concentration of alizarin is higher than 25 mg/L, hematin removes more dye than HRP at equal mass of catalyst. However, this does not imply a higher activity of hematin per mole of catalyst since its molar concentration is about 65 times greater than that of HRP. From a practical point of view, costs are related to weight and not to mole numbers per unit volume. In this sense, hematin is more efficient in a per milligram basis but it is not so in a molar basis. When the dye concentration increases, decreases in both

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Figure 2. Percentage of alizarin removed by HRP and hematin for different initial dye concentrations. Contact time 2 h, pH 7, and 25 °C. [H2O2] ) 0.021 M. (b) HRP. (9) Hematin. Table 1. Percentage of Dye Removed by Hematin and HRP at Different Temperaturesa temp (°C) % of dye removed by hematin % of dye removed by HRP 25 45 65 a

93 98 98

67 90 89

Initial alizarin concentration ) 100 mg/L; pH 7.

HRP efficiency and hematin/HRP molar ratio required to achieve the same dye removal are observed (see Figure 2b). This would indicate a saturation behavior at high substrate concentrations in the case of HRP. 3.2. Effect of Temperature. Table 1 shows the effect of the temperature in the removal of alizarin by hematin and HRP. In the case of hematin the percentage of alizarin removed is >90% for all the conditions tested. The temperature exerts an important effect on HRP activity when going from room temperature to 45 °C, although no significant change is observed with further temperature increase. With both catalysts the level of alizarin removed remains practically constant in the 45-65 °C range. Hematin is always more efficient than HRP at these selected conditions. 3.3. Kinetic Studies. To obtain preliminary information concerning reaction intermediates in the transformation process, kinetic studies were conducted to further characterize the reaction system. Although some papers use mass spectrometric and HPLC data to study their results of dye degradation using enzymes, there are several problems to consider: (a) the needed steps of enzyme separation from the reaction media (that also may be affecting the residual concentrations of reagents and products), (b) the lack of calibration standards for unknown degradation or oligomerization products for HPLC, and (c) the lack of enough information to elucidate the results obtained in mass spectrometry (due to the potential and probable presence of isomers with similar molecular weights or degraded nonseparated enzyme). On the other hand, the use of UV/visible techniques and the proper analysis of the kinetic results may give further information without the potential problems associated with these techniques, even when they can add more structural information. We consider that the combined use of the oxygen evolution/ consumption with the UV/visible kinetic data gives valuable information concerning the main reactions taking place during the transformation of the alizarin.

Figure 3. (a) Results obtained with the PM3 semiempirical method from Chem 3 D Ultra 5.0 (from Cambridge Soft) for a tetramer of alizarin. (b) Formation standard enthalpies for different alizarin aggregates.

We performed the kinetic studies at pH 7 and 8 using a concentration of alizarin of 50 mg/L. For this dye, one of the most important aspects to take into account is the tendency to form aggregates below pH 7.8 in distilled water.21 Dye aggregates may decrease the intensity of the characteristic bands or widen the bands with bathochromic shifts with respect to the monomer, and they are promoted by the presence of macromolecules.22 These conditions are present in the initial solutions of alizarin with hematin and alizarin with peroxidase, where the impact of an average molecular weight molecule (hematin) or of an enzyme protein can be analyzed. The aggregate formation would be the cause of the broadening and shift of the alizarin original band with the additive aggregation. Figure 3 shows the results obtained with the PM3 semiempirical method (parameterized model 3) from Chem 3 D Ultra 5.0 (from Cambridge Soft). Figure 3a shows the tetramer structure, even when the trimer has been found to be the most stable aggregation state. This figure shows how the aggregation takes place through strong H-bonding. With the object of clarifying the spectral analysis, the standard enthalpies of formation for alizarin aggregates were calculated until finding that the addition of a new alizarin molecule results in an endothermic reaction (Figure 3b). The PM3 method provides the ∆H°formation for the minimized MM2/PM3 configuration, and therefore it is possible to estimate the ∆H°reaction for the aggregation reactions. The formation until hexamers was evaluated. The aggregates of different number of alizarin molecules have demonstrated to be favorable from the theoretical point of view in gas phase. Probably, the aggregates are further stabilized in a polar environment such as buffer solution in an aqueous media. Time-resolved spectra, in the presence of either hematin or HRP, at pH 7 and 8, are shown in Figure 4. For the experiments performed at pH 7, the formation of aggregates is evident from the spectral shapes that show high absorption values in the

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Figure 4. Time-resolved spectra in the presence of either hematin or HRP at pH 7 and 8 with initial dye concentration of 50 mg/L and 25 °C.

650-900 nm wavelength range. On the other hand, the spectra recorded in the experiments carried out at pH 8 show negligible absorptions in the same spectral domain. It should be pointed out that complementary tests in the absence of H2O2 were performed showing that a decrease in solution pH below 7 induces spectral changes which are particularly noticeable in the 650-900 nm range; the same effect was observed by the addition of hematin or HRP (data not shown). Moreover, these differences between the spectra at pH 7 and 8 were also observed in the kinetic experiments before any H2O2 addition. Therefore, it can be concluded that aggregation is the main cause of the spectral behavior observed in the 650-900 nm wavelength range. This is in line with previously reported results.21 In addition, by taking into account the spectral information obtained in the aforementioned complementary tests, we corrected the time-resolved spectra obtained in the kinetic experiments at pH 7. Removing the disturbance that the aggregation effects produce in the spectra, no significant differences between the results at both pH values were found. The evolution of the absorption values around the main dye band suggests a decrease of at least 60% in alizarin concentration within the analyzed time scale for all conditions tested. However, in the presence of HRP the degradation/polymerization processes are much faster and the efficiencies somewhat higher than the ones observed in the presence of hematin at both pH values. Moreover, a smooth absorption decrease is observed in the presence of the biomimetic while in the presence of HRP a practically instantaneous disappearance of the dye band is followed by a much slower bleaching. The theoretical results help to explain the initial UV/visible spectra obtained with aqueous solutions of alizarin in the presence of hematin and peroxidase. The aggregation of alizarin

is a phenomenon that affects the UV/visible spectra, and as far as we know, this has not been properly considered in the available literature. With the aim of extracting the main spectral and kinetic features from the spectrophotometric measurements, numerical techniques of multivariate regression were used (see Experimental Section). In order to assess the number of factors required to reproduce the experimental results, singular value decomposition of time-resolved-spectra matrixes was used. Singular values suggested that the experimental results can be accurately described by using the contribution of three independent factors. Figure 5 shows the kinetic profiles obtained using the constrained alternating least squares method. The concentration profiles calculated at pH 7 and 8 are fairly similar, suggesting that the previously discussed formation of aggregates by pH effect (in the pH 7-8 range) has a minor effect on the kinetic behavior of these systems. Thus, the radical formation is not affected by the aggregation phenomena. In addition, the comparison of the time scales observed in the presence of each catalyst confirms that dye consumptions at both pH values are practically instantaneous in the presence of HRP while the hematin-catalyzed reactions span much higher half-lives. 3.4. Oxygen Consumption/Release. Figure 6 shows the oxygen consumption for alizarin with HRP and hematin. In the case of HRP, the native enzyme (E) is oxidized by H2O2 to an active intermediate enzymatic form called compound I (E1). Compound I accepts alizarin (AH2) into its active site and carries out its oxidation. A free radical AH• is produced and released into solution, leaving the enzyme in the compound II (E2) state. Compound II oxidizes a second molecule of AH2, releasing another free radical and returning the enzyme to its native state, thereby completing the cycle.16 It should be pointed

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Figure 5. Kinetic profiles obtained for alizarin using the constrained alternating least squares method. Table 2. Comparison of the Main Compounds/Radicals Proposed To Be Present after a Few Seconds/Minutes of H2O2 Addition, with HRP and Hematin as Biocatalysts for Alizarin (AH2) Elimination at pH 7 H2O2

Figure 6. Oxygen evolution for alizarin. (9) Hematin. (b) Peroxidase. [Dye] ) 100 mg/L. [Hematin] ) 25 mg/L. [Peroxidase] ) 10 mg/L.

out that, since the pKa value of alizarin is 5.25, at pH 7 the molar ratio of the ionized species AH- related to the nonionized one AH2 is 56.2. Thus, the main form present at pH 7 would be AH- and not AH2. Then this AH-/AH2 ratio must be taken into account in analysis results. In order to understand the oxygen evolution, several additional steps should be taken into account. Radicals from the dyes may be generated and decomposed spontaneously or react with O2 in different ways.16-23 In addition to the reactions that involve organic substrates, the catalase-like activity of peroxidase may play a decisive role in the concentration profiles recorded for dissolved oxygen: 2H2O2 f O2 + 2H2O

(1)

The probable species formed in the system HRP/H2O2 with the dye in the different reaction steps are shown in Table 2. In contrast to HRP, hematin mainly generates HO• and HOO• radicals (see Table 2).23 In addition, the main reaction

HRP/H2O2

O2

hematin/H2O2

HOO•, O2•-, HO•, OH-

AH2 AH , (AH)2-, (AH)2•-, AHO-OHA AH•-, AHOH•, AH (OH)2•, AOO• •-

of hematin consuming O2 is the formation of the superoxide anion: hematin(Fe2+) + O2 f hematin(Fe3+) + O2•-

(2)

Figure 6 shows that the net reaction of alizarin in H2O2/ catalyst systems at selected conditions involves consumption of O2 for hematin and evolution of O2 with HRP. Catalase-like activitysand the O2 evolutionsis very important for the HRP/ H2O2 system, but it is not for the hematin/H2O2 system. Although Kalyanaraman et al.23 reported several reactions of H2O2 and radicals with hematin involving O2 release, our results in the alizarin/H2O2/hematin system indicate that the reactions of oxygen consumption are the main reactions in the presence of hematin as catalyst. 3.5. Theoretical and Mechanistic Analysis of the UV/ Visible Deconvolution Results. There are two main pathways to consider in alizarin reactions with hematin or HRP and H2O2: (a) degradation because of the hydrogen peroxide radicals attack to organic neutral compounds and radicals, or (b) oligomerization due to the coupling of radicals. The first step in the case of (b) should be the formation of 3,3′-bializarin (1,1′,2,2′-tetrahy-

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Figure 7. First steps of the degradation mechanism of alizarin.

droxy[3,3′-bianthracene]-9,9′,10,10′-tetrone), a dimer of alizarin. This reaction has been published as catalyzed by peroxidases purified from Senna angustifolia and horseradish.24 With the differences found in the UV/visible spectra (and with the available literature on HRP and hematin), we can propose that by using HRP we are going by the oligomerization step, whereas by using hematin the pathway would be the degradation step. We think that the evidence of the oxygen concentration determinations support our proposal. It has been reported that the first step in the oxidation of alizarin is the cleavage of the aromatic ring in the C-C bond near the CdO group to form small carbonylic species and colorless intermediates, mainly phthalic acid. Finally, these intermediates are mineralized to carbon dioxide in the case of photoassisted degradation.25-27 Figure 7 considers the first steps of the degradation mechanism of alizarin following the work of Xue et al.28 The role of enzymes such as HRP or extracellular enzymes or fungal enzymes in the degradation of phenolic pollutants through polymerization has been frequently reported.29,30 Minard et al.31 showed that oligomers ranging from dimers to pentamers of 2,4-dichlorophenol with phenol oxidase were formed, through C-C and C-O coupling. In a study from Weber and Huang32 phenanthrene undergoes direct chemical incorporation into phenolic oligomers during peroxidase-mediated oxidative polymerization of phenol. Xu et al.33 proposed that 1-naphthol is initially transformed to free radicals or naphthoquinones by HRP followed by the self-coupling or cross-coupling with previously generated polymerization products through C-C and C-O bond formation. According to these authors naphthol polymerization occurs preferentially through C-C bonding with the production of oligomers with nonreactive OH groups. Based on the mechanism proposed by Xu et al.33 for naphthol, a similar mechanism for alizarin oligomerization may be proposed. The main initial anion radicals formed by the action of HRP/H2O2 on alizarin at pH 7 are shown in Figure 8a. The results of the MM2/PM3 calculation for the ∆Η°f for different dimers formed by radical condensation are included in Figure 8b. Figure 8c shows the three-dimensional view of some of the different dimers of alizarin formed by condensation of different

radicals. The ∆Η°f is clearly more negative when the negative charges on the enolic oxygen atom of the dimer are far from each other. The kind of radicals formed from ionized alizarin at pH 7 is similar for hematin/H2O2 and HRP/H2O2. The main radicals formed initially are those obtained by H abstraction from AHspecies. HRP is a very efficient catalyst and the rate of radical AH• (or AH•-) formation is very high. Therefore, the potential for radical coupling instead of degradation is more favorable. This proposed mechanism is supported by previously published results.16,28 In the case of HRP, the catalytic reaction is the main involvement of H2O2. In the case of hematin OH•, HO2•, O2•-, and OH- are generated in high amounts.23 Although the organic radicals initially generated by the alizarin reaction are probably similar, the kind of reactive radicals present in the reaction media leads to two different pathways: degradation and oligomerization. The kinetics and simulation results are in agreement with this proposal, especially because the alizarin consumption at pH 7 and 8 is very high with HRP whereas it displays a much higher half-life with hematin. 4. Conclusions Hematin/H2O2 and HRP/H2O2 are efficient catalytic systems for the elimination of alizarin at pH 7. At an excess of hydrogen peroxide, both eliminate 85% of 25 mg/L alizarin at 25 °C. At the highest concentration evaluated (150 mg of alizarin/L), hematin degrades almost 75% whereas HRP degrades near 45%. The formation of aggregates at pH 7 does not affect the catalytic efficiency. The kinetic data obtained at pH 7 and 8 are similar in terms of the total amount removed, even when the UV/visible profiles at pH 7 are compatible with the presence of aggregates. The oxygen profiles found for hematin/H2O2/alizarin and HRP/ H2O2/alizarin systems are clearly different, pointing to oxygen consumption for hematin and oxygen production for peroxidase. Hematin was demonstrated to be a potential, useful, and efficient biomimetic of HRP. Under selected and appropriate conditions, hematin is more temperature resistant and more active than HRP in a per milligram basis, reaching 90% of dye removal at alizarin concentration e100 mg/L. Further work

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Figure 8. (a) Main alizarin radicals in the presence of HRP/H2O2 at pH 7. (b) Main dimeric species at pH 7 from radicals shown in (a). (c) Threedimensional view of dimeric species 1, 2, and 3. The order of stability considering the ∆Η°f for the conformer is included.

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using supported hematin and HRP would clarify the potential of hematin from a practical viewpoint in the degradation of alizarin. Acknowledgment M.L.F., F.S.G.E., and S.P. acknowledge CONICET for financial support. The authors acknowledge financial support from the PGI 24/Q022 Universidad Nacional del Sur (Bahı´a Blanca, Argentina), CONICET, and the SeCyt (Argentina). Supporting Information Available: Table listing parameters considering dye concentration and molar relationships between catalysts and dye. This material is available free of charge via the Internet at http://pubs.acs.org. Literature Cited (1) Grzechulska, J.; Morawski, A. W. Photocatalytic Decomposition of Azo-Dye Acid Black 1 in Water Over Modified Titanium Dioxide. Appl. Catal. B: EnViron. 2002, 36, 45. (2) Tseng, J.; Huang, C. P. Photocatalytic Oxidation Process for the Treatment of Organic Wastes. In Proceedings of the First International Symposium Chemical Oxidation Technologies for the Nineties; Vanderbilt University: Nashville, TN, 1991; Vol. 1, pp 262-276. (3) Herrmann, J.-M. Heterogeneous Photocatalysis: Fundamentals and Applications to the Removal of Various Types of Aqueous Pollutants. Catal. Today 1999, 53, 115. (4) Andreozzi, R.; Caprio, V.; Insola, A.; Marotta, R. Advanced Oxidation Processes (AOP) for Water Purification and Recovery. Catal. Today 1999, 53, 51. (5) Baker, K. H.; Herson, D. S. Bioremediation; McGraw-Hill: New York, 1994. (6) Seah, S. Y. K.; Labbe´, G.; Kaschabek, S. R.; Reifenrath, F.; Eltis, L. D. Comparative Specificities of Two Evolutionarily Divergent Hydrolases Involved in Microbial Degradation of Polychlorinated Biphenyls. J. Bacteriol. 2001, 183, 1511. (7) Gianfreda, L.; Rao, M. A. Potential of Extra Cellular Enzymes in Remediation: A Review. Enzyme Microb. Technol. 2004, 35, 339. (8) Godfrey, T.; Reichelt, J. Industrial Enzymology: The Applications of Enzymes in Industry; Nature: New York, 1996. (9) Aitken, M. D. Waste Treatment Applications of Enzymes: Opportunities and Obstacles. Chem. Eng. J. 1993, 52, B49. (10) Klibanov, A. M.; Tu, T. M.; Scott, K. P. Peroxidase-Catalyzed Removal of Phenols from Coal Conversion Waste Waters. Science 1983, 221, 259. (11) Knutson, K.; Kirzan, S.; Ragauskas, A. Enzymatic Biobleaching of Two Recalcitrant Paper Dyes with Horseradish and Soybean Peroxidase. Biotechnol. Lett. 2005, 27, 753. (12) Venkat Mohan, S.; Krishna Prasad, K.; Chandrasekhara Rao, N.; Sarma, P. N. Acid Azo Dye Degradation by Free and Immobilized Horseradish Peroxidase (HRP) Catalyzed Process. Chemosphere 2005, 58, 1097. (13) Laxmi Maddhinni, V.; Bindu Vurimindi, H.; Yerramilli, A. Degradation of Azo Dye with Horseradish Peroxidase (HRP). J. Indian Inst. Sci. 2006, 86, 507. (14) Krishna Prasad, K.; Venkat Mohan, S.; Sarma, P. N. Enzyme Catalyzed Degradation of Azo Dye to Enhance Biodegradability. In Water and EnVironment. Wastewater Treatment and Waste Management; Singh, V. P., Narayan Yadavo, R., Eds.; Allied Publishers Pvt. Ltd.: Mumbai, India, 2003; pp 205-213. (15) Rueda, E. H.; Saidman, S.; Ferreira, M. L. Activity of Free Peroxidases, Hematin, Magnetite Supported Peroxidases and Magnetite

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ReceiVed for reView September 29, 2009 ReVised manuscript receiVed February 15, 2010 Accepted February 18, 2010 IE901528Y