ISSN 00036838, Applied Biochemistry and Microbiology, 2014, Vol. 50, No. 2, pp. 214–218. © Pleiades Publishing, Inc., 2014.

Phenol Removal from Aqueous Solutions by Peroxidase Extracted from Horseradish1 ˆ

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S. R. Savica, *, S. M. Stojmenovica, M. Z. Petronijevicb, and Z. B. Petronijevica a

b

University of Nis, Faculty of Technology, Leskovac, 16000 Serbia University of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Novi Sad, 21000 Serbia *email: [email protected] Received July 22, 2013

Abstract—Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzy matic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant con centrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular poly ethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct mea suring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively. DOI: 10.1134/S0003683814020161 1Phenolic

compounds are group of chemicals of environmental concern, because they have toxic and/or carcinogenic properties and have a potential danger to human health [1–3]. As a consequence of their widespread applications, they are present as pol lutants in many industrial wastewater streams [2, 4, 5]. Today there are many conventional methods for removal of these compounds from wastewaters based on chemical, physical and biological processes, but they are not always suitable [1, 6]. Because of these reason enzymebased methods are developed for wastewater treatment and they include several enzymes such us peroxidases, laccases and polyphenol oxidases [2, 7, 8]. The use of horseradish peroxidase (HRP, EC 1.11.1.7) in the removal process of phenolic com pounds from aqueous solutions, was firstly proposed by Klibanov and collaborators [9]. Since then, the method has been in the focus of interest for many researchers. On the contrary to many other enzymes, HRP has a catalytic ability in wider range of pH, tem perature and contaminant concentrations [8, 10]. In the presence of hydrogen peroxide HRP has ability to catalyze the oxidation of different aromatic com pounds such as phenols, biphenols, aromatic amines (aniline, benzidine and other) as well as related het eroaromatic compounds to form phenoxy radicals [11]. The free phenoxy radical products spontaneously 1 The article is published in the original.

polymerize to form waterinsoluble polymers which can be readily separated from solution by sedimenta tion or filtration [12–15]. Unfortunately, conventional methods for wastewa ter treatment and enzymatic treatment process have limitations [10]. The biggest problems in the process of enzymatic removal of phenol are excessive treatment cost appeared due to the high cost of the purified enzyme and its inactivation by various undesirable side reactions, as well as the potential formation of residual products in the aqueous phase [10]. Disadvantages of these enzymes can be reduced by using of protective additives and potentially inexpensive enzymes sources [10, 12, 16]. Various theories about the mechanism of inactiva tion of HRP in the process of removing phenols from wastewater appeared during the past years. Klibanov claimed that phenoxy radicals formed during the pro cess, attack catalytic active center of HRP and lead to the reduction and/or elimination of its catalytic ability [9, 17]. On the other hand, Nakamoto and Machida [18] rendered the hypothesis that HRP inactivation is due to a lack of contact between the substrate and enzyme because HRP is captured in the newly formed polymer during the polymerization of phenol. In addi tion, they demonstrated that the lifetime of enzymes can significantly extend in the presence of highly hydrophilic additives that have a higher affinity for the hydroxyl groups of the emerging polymer compared to HRP. This implies that compounds such as PEG and

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gelatin can attack the polymer and enable the enzyme to remain active in the solution. Previous studies have shown that crude extract from horseradish roots and high purity enzymes have similar removal potential in reaction of total phenols removal [9, 17]. The aim of the study was to investigate the possibil ity of using the crude peroxidase, extracted from horseradish, for removal of phenol in the presence of hydrogen peroxide and PEG 300 from aqueous solu tions as well as the influence of PEG 300, phenol and hydrogen peroxide on HRP activity. At the first time the effect of PEG 300 on crude HRP was evaluated and compared with action of PEG 3350 on phenol removal by HRP, because previous tests [13] were shown that PEG 3350 was the most effective on phe nol removal and HRP stability. Results from this study could be beneficial for cheapening the process of phe nol removal by HRP, as well as for the environmental safety. Some catalytic properties, stability and aqueous phenol treatment efficiency of crude HRP were com pared with published data in order to elucidate the effect of its phenolic solutions treatment. MATERIALS AND METHODS Enzyme isolation. 2 g of horseradish root (Armora cia rusticana syn. Cochlearia armoracia, found in the local market) was cleaned, chopped, and extracted by stirring with 20 ml 100 mM phosphate buffer (pH 7.2) at room temperature (20°C) for 30 min. After extrac tion, the centrifugation was performed at 800 g for 15 min. The obtained supernatant was transferred to a test tube and the rest of the plant material was sub jected to extraction again. Stock enzyme solutions were stored at 4°C and warmed to room temperature immediately prior to use. The enzyme removal of phenol. Batch reactions for phenol removal were carried out in glass flasks at room temperature using 30 ml reaction volumes. Reaction mix tures contained 2.0–10.0 mM phenol, 100–600 mg/L PEG 300, 1.0–3.5 mM H2O2, and 1.2 U/mL of HRP in a 0.1 M phosphate buffer (pH 7.2). Reaction was initi ated by addition of H2O2 solution. At different time intervals (20, 40, 60, 80, 100 and 120 min), aliquots were pipetted into the tubes and HRP activity and phenol content were examined. The influence of phe nol, PEG 300 and H2O2, on HRP activity and phenol removal were determined at constant concentrations of two reactants and variable concentration of the third reactant, phenol, PEG 300 or H2O2, respectively. The initial concentrations of those three reactants, PEG 300, phenol and H2O2, in reactions when they were constant, were 100 mg/L, 4 mM and 2.5 mM, respectively. HRP activity assay. Peroxidase activity was deter mined by method of Soysal and Söylemez [19], with slight modifications. A mixture containing 2.1 mL of 100 mM acetate buffer (pH 6.0), 0.2 ml sample solu tion and 0.2 ml of 0.125% solution of o–dianisidine in APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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methanol, was vigorously vortexed, and reaction was started by addition of 0.5 ml 8.8 mM H2O2. Absor bance change was recorded as a function of time at 460 nm, and activity of HRP was calculated by using following equation: (1) A[U/ml] = tanα × R/ε, where tanα is a slope of the plot, R is the total ratio vol ume of the reaction mixture and the enzyme and ε is molar extinction coefficient (ε460 = 11.3 mM–1 cm–1). The blank contained all reagents except the hydro gen peroxide, which was replaced by aqua destillata. One unit of peroxidase activity was defined as the amount of enzyme that transformed 1 μmol of o–dia nisidine per min. Phenolic compound assays. Phenol content was determined as described by Nicell and collaborators [13], with slight modifications. Aliquots (2.5 mL) from batch reactions were pipetted into the tubes at different time intervals (20, 40, 60, 80, 100 and 120 min) and reaction was stopped by adding 2.5 mL of 96% ethanol. After that, samples were treated with 2.0 mL 2 mM KAl(SO4)2 ⋅ 12H2O and pH was adjusted to 6.3 to optimize precipitation, using stock solutions of 0.1 M NaOH and 0.1 M HCl. Samples were centrifuged for 10 min at 9000 g. Residual phenol concentrations were determined spectrophotometrically at OD269. RESULTS AND DISCUSSION Effects of PEG 300, phenol and hydrogen peroxide on HRP activity during the reaction of phenol removal by crude HRP in the presence PEG 300 and hydrogen peroxide, are presented on Fig. 1. All changes in HRP activity were calculated in relation to the initial activ ity (1.2 U/mL). The determination of peroxidase activity and phenol content was carried out at room temperature (20°C). The reactions of phenol removal by crude HRP were continued for 2 h, and the pH value of the solutions was in optimal range for HRP activity (6 ≤ pH ≤ 9) [20]. The obtained results showed a gradual decrease of HRP activity in the reaction of phenol removal (Fig. 1). Based on the results of influ ence of PEG 300 on HRP activity, when the concen tration of phenol and hydrogen peroxide were con stant (Fig. 1a), it can be concluded that PEG 300 has a positive effect on HRP activity, i.e., increasing con centration of PEG 300 decreases degradation of HRP during the reaction. Obtained results were expected and they are in accordance with previously reported data [1, 13]. On the other hand, results of influence of the phenol and hydrogen peroxide concentrations on the HRP activity (Fig. 1, b and c) provide a conclusion that with increasing concentrations of phenol and hydrogen peroxide there is a gradual decrease in HRP activity. In both cases the major reduction effect in HRP activity occurred in the reaction during the first 20 min, but later this effect was considerably smaller.

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Fig. 1. Timedependent change of HRP activity in reac tion of phenol removal by crude HRP in the presence of PEG 300 and hydrogen peroxide. Concentration effect of PEG 300, phenol and hydrogen peroxide on HRP activity is shown in a, b and c, respectively. a: 1–100; 2–200; 3– 300; 4–400; 5–500 and 6–600 mg/L of PEG 300; b: 1–2; 2–4; 3–5; 4–6; 5–8 and 6–10 mM phenol; c: 1–1.0; 2– 1.5; 3–2.0; 4–2.5; 5–3.0 and 6–3.5 mM H2O2.

Based on previous studies it is known that in the absence of stabilizing additions inhibition effect of phenol and hydrogen peroxide was complete in 5 min [21]. Results in this study are different and that is most probably because the presence of PEG 300 has a pro tective effect on HRP activity. On the other side, hydrogen peroxide effect on HRP activity can be explained by the fact that H2O2 taken at low concentrations cause reversible inhibition of HRP but with further increasing of hydrogen perox ide concentrations irreversible inhibition becomes dominant [21]. Results from the study of the PEG 300, phenol and hydrogen peroxide effect on phenol removal by HRP in the presence of PEG 300 and hydrogen peroxide are presented on Fig. 2. Phenol removal from the reaction mixture was not observed when HRP or hydrogen per oxide were absent. It should be mentioned, that at the end of the reaction, even in the case when HRP, H2O2 and PEG 300 were present in reaction mixture, it showed absorption at a wavelength that is characteris tic for phenolic compounds. The occurrence of this phenomenon is expected and it is in accordance with previously results published by Nicell and collabora tors [13]. In addition, these authors showed that PEG 300 did not absorb in the wavelength at which the measurement was performed [13]. It can be concluded from Fig. 2 that during the reaction of phenol removal in all cases there is a reduc tion in the phenol amount. When the concentration of PEG 300 was a variable, and concentrations of phenol and hydrogen peroxide were constant (Fig. 2a), the highest phenol removal (almost 95% of the initial phe nol amount) was obtained when the concentration of PEG 300 was 300 mg/L. It was obtained after 2 h of the reaction. This result is in a good agreement with obtained results of investigation influence of PEG 300 on purified HRP activity [1]. Comparing the results for phenol removal with previously published results [1, 13], it can be concluded that PEG 300 compared to PEG 3350 has a better effect on phenol removal. The influence of phenol concentration, when the concen trations of hydrogen peroxide and PEG 300 were con stant (Fig. 2b), showed that the highest phenol removal was achieved when its concentration was 2 mM. These results can be explained by the fact that phenol has a inhibition effect on HRP [21], and at low concentrations of phenol inactivation of HRP is lower and the removal of phenol is higher. Figure 2c shows that increasing of hydrogen perox ide at constant concentrations of PEG 300 and the phenol, decreases phenol amount. The results indicate that at initial concentration of hydrogen peroxide of 3.0 mM, the phenol removal after 20 min is over 65%, and the highest phenol removal was achieved after 2 h of incubation at the initial concentration of hydrogen peroxide 2.5 mM. The results obtained in the reaction of phenol removal by HRP revealed a good agreement with previous results when the initial concentration of

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hydrogen peroxide was in the range from 1.5 to 3.0 mM [13, 22, 23]. In this study we demonstrated that crude peroxi dase extracred from horseradish root can be used for the phenol removal from aqueous solution. Presence of PEG 300 shown a stabilizing effect on HRP activity and compared to PEG 3350. In addition, crude HRP used in the study compared to previous reports had a greater resistance to phenol and hydrogen peroxide inactivation which provided a higher phenol removal. The highest phenol removal was achieved when the concentrations of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 mM and 2.5 mM, respectively. Based on these study, it should be noted that these results can provide a basis for a future research in order to reduce costs in the process of phe nol removal.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Educa tion and Science of the Republic of Serbia under Project No. TR–34012 and OI–172044.

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Fig. 2. Timedependent change of phenol amount in reac tion of phenol removal by crude HRP in the presence of PEG 300 and hydrogen peroxide. Concentration effect of PEG 300, phenol and hydrogen peroxide on phenol removal is shown in a, b and c, respectively. The labels of the curves 1–6 in a, b and c are the same as in Fig. 1. APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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