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Turnip (Brassica rapa) Peroxidase: Purification and Characterization Sepideh Motamed, Farnoosh Ghaemmaghami, and Iran Alemzadeh* Department of Chemical & Petroleum Engineering, Sharif UniVersity of Technology, P.O. Box 11155-9465, Azadi AVenue, Tehran, Iran
Partial purification of plant peroxidase from turnip (Brassica rapa) was optimized. Aqueous two phase system and precipitation by ammonium sulfate (as two parallel purification methods) were used. Polyethylene glycol/ ammonium sulfate/NaCl (25:7:3%, w/v) system followed by gel-filtration chromatography gave a purification factor of about 36 fold. On the other hand, ammonium sulfate precipitation (60-80% deg) followed by gel filtration gave only about 13 purification fold. Hence, the aqueous two-phase system was more efficient and useful method as a primary purification step since it was less laborious, less time-consuming, and led to more purification factor. The partially purified turnip enzyme was stable at a pH range 2.6-6.0 and had an optimum pH at 4.0. The enzyme was stable up to 55 °C with an optimum activity at 35 °C. 1. Introduction Peroxidases (E.C.1.11.1.7) are ubiquitous heme-containing oxidoreductases which are very widespread in nature.1 They have the ability to catalyze the oxidation of a wide spectrum of organic and inorganic electron donor substrates through a reaction with hydrogen peroxide or organic hydroperoxides.2 Peroxidases are of wide interest because of their extensive potential applications in the clinical, biochemical, biotechnological, and industrial fields and in the synthesis of useful compounds (e.g., various aromatic chemicals).3 Because of their broader catalytic activity, these enzymes could be exploited for the detoxification and remediation of various aromatic pollutants such as phenols, aromatic amines, and dyes presented in wastewater/industrial effluents coming out from several industries such as textile, dyes, printing, and paper and pulp,3 as well as removal of peroxide from materials such as food stuffs and industrial wastes.4 Peroxidases have been found in a wide range of plant species with multiple molecular forms and a broad subcellular distribution.5 They are found not only in cytoplasm and cell organelles but are also associated with cell walls.6 It is observed that plant peroxidases differ with respect to thermal stability, pH optima, substrate specificity, and amino acid composition from one plant to another.7 Over the years, horseradish tubers (Armoracia rustanica) have been the only commercial source of peroxidase production. However, other cultivated species can also provide peroxidases with similar or better substrate specificities, stability, yield, and economic feasibility.8 Therefore, numerous investigations of peroxidases from different origins were being carried out4 in order to improve already existing analytical procedures and/or the development of new ones.5 The only source of commercial production of peroxidase, horseradish root, is cultivated in relatively cool climates, not in Iran.6 Hence, in this work we use turnip (Brassica rapa) as a source of peroxidase, which is rich in peroxidase and locally available in Iran. This could be an alternative commercial source of high activity peroxidase. Although a great deal of research has been carried out to demonstrate the effectiveness of peroxidase-catalyzed removal of phenolic pollutants either from synthetic aqueous mixtures9,10 * To whom correspondence should be addressed. E-mail:
[email protected].
or from real wastewaters,11-13 the implementation of this process on a large-scale is still primarily hampered by the high cost associated with the production of the enzyme.14 Actually, the purification of the enzyme is a major hurdle for the realization of its commercial potential.4 Many methods for plant peroxidase purification have been reported. After homogenizing the crude material with buffer or water, the homogenate is filtered and centrifuged, and the enzyme is purified by applying different chromatographic steps, depending on its intended utilization.15 Different strategies such as ammonium sulfate precipitation,4,6,7 ion exchange chromatography,4,6,7 gel-filtration chromatography,7,16 Fast protein liquid chromatography (FPLC),15 and affinity chromatography15 have been employed for peroxidase purification in different systems in the literature. To reduce the purification process costs, another possibility was reported that was based on partitioning in an aqueous two-phase system17 as a potential primary purification technique to reduce the volume of the processing stream.16 In wastewater treatment, in order to reduce the cost, fully purified enzyme is not used.14 Hence, more attention should be paid to primary purification since crude or partly purified enzyme would likely be used for the wastewater treatment.14,18 Partitioning in an aqueous two-phase system (ATPS) is a selective method used for biomolecule primary purification.3 Conventional initial purification steps such as ammonium sulfate precipitation and acetone fractionation are laborious and timeconsuming. ATPS offers many advantages including low process time, low energy consumption, and biocompatible environment to the biomolecule due to the presence of large amounts of water in the systems.16,19 The aim of this work is to optimize the purification of soluble peroxidase from turnip, an inexpensive and easily available source of peroxidase, in a form suitable for phenol removal. As reported in the literature, ATPS has been used for purification of different plant enzymes,20 plant peroxidases,17 for example, Ipomea palmetta3,16 and soybean.21 However, it is not used for peroxidase from turnip. This article reports the feasibility of primary purification of turnip peroxidase by ATPS, its best composition, and the trend of the enzyme in this system. Ammonium sulfate precipitation, a conventional method of peroxidase primary purification, was also utilized, optimized, and compared with the results of ATPS. Partially purified peroxidase was dialyzed and further purified by gel-filtration
10.1021/ie801997e CCC: $40.75 2009 American Chemical Society Published on Web 10/07/2009
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Table 1. Effect of Phase Composition on Partitioning of Peroxidase in PEG/Ammonium Sulfate System phase composition PEG (%, w/v)/amm. sulfate (%, w/v) 10.0/10.7 15.0/10.0 20.0/8.9 23.0/7.9 24.0/7.5 25.0/7.0 26.0/6.6 27.0/6.2 30.0/4.7
volume top/bottom phase (mL)
partition coefficient (m)
enzyme recovery (bottom phase, %)
total protein recovery (bottom phase, %)
33.0/67.0 48.5/51.5 56.5/43.5 57.5/42.5 61.0/39.0 63.5/36.5 68.0/32.0 75.0/25.0 85.0/15.0
0.83 0.76 0.68 0.56 0.50 0.46 0.42 0.40 0.38
69.62 57.17 52.08 55.80 55.04 54.48 51.82 44.58 31.10
63.29 34.23 28.18 18.60 17.87 17.74 17.02 16.69 16.03
chromatography in order to report the thermal and pH stabilities of turnip peroxidase. 2. Materials and Methods 2.1. Materials. Turnip was purchased from local market. Polyethylene glycol (mol wt 6000), ammonium sulfate, sodium chloride, Sephadex G-200, bovine serum albumin, phenol, 4-amino-antipyrine (4-AAP), and hydrogen peroxide were obtained from Merck, Germany. All other chemicals used in this study were of analytical grade and obtained from commercial sources. The chemicals and other reagents were used without any further purification. 2.2. Crude Extract. Low purity turnip peroxidase was obtained by passing washed turnip roots (40 g) through a commercial juicer. The obtained juice and fibrous material were diluted separately by phosphate buffer 100 mM (pH 7.4) to 200 mL and stored at 4 °C for 30 min. The extract was filtered using a cheese cloth (arranged in four layers) to remove suspended fibrous solid particles. The mixture was then centrifuged at 9000g for 15 min. The supernatant was collected and stored at 4 °C and used as source of crude soluble turnip enzyme.18 The enzyme was warmed to room temperature immediately prior to use to be purified by an aqueous two-phase system or ammonium sulfate precipitation. 2.3. Peroxidase Assay and Protein Concentration Determination. A calorimetric activity assay was determined by phenol and hydrogen peroxide as substrates and 4-AAP as chromogen at 25 °C, using a Spectronic UV-vis spectrophotometer.22 One ml enzyme solution was diluted by phosphate buffer (100 mM) to 10 mL. The assay mixture contained 0.40 mL of diluted enzyme solution, 1.50 mL of 50 mM phenol, 0.75 mL of 24 mM 4-AAP, 1.50 mL of 1 mM H2O2, and 1.85 mL of 100 mM phosphate buffer (pH ) 7.4).18 The generation of color by the reaction of phenol, 4-AAP, and H2O2 can be represented by the following total reaction:22 2H2O2 + 4-AAP + phenol f dye + 4H2O The rate of H2O2 consumption in the assay is calculated from the rate of the formation of the colored product, which absorbs light at a peak wavelength of 510 nm, with an extinction coefficient of 6.58 M-1 cm-1, for 2 min in which the substrate concentration is not significantly reduced. The concentration of active enzyme in the assay is proportional to the rate of H2O2 consumption.23 One unit of activity (U) is defined as the number of micromoles (µmol) of hydrogen peroxide converted per minute at pH 7.4 and 25 °C.18 Protein was determined by Lowry method, using bovine serum albumin as standard.24 2.4. Purification of Peroxidase. 2.4.1. Initial Purification. 2.4.1.1. Preparation of Aqueous Two-Phase System (ATPS). A mixture of ammonium sulfate and polyethylene glycol dissolved in water is turbid
above certain concentrations and the two phases are in equilibrium. ATPS was prepared by mixing the required quantities of polyethylene glycol and ammonium sulfate in the enzyme extract and adjusting the total volume of the system to 100 mL. In this experiment, various PEG concentrations of 10, 15, 20, 23, 24, 25, 26, 27, and 30 (%w/v) in 80 mL of crude enzyme were prepared. Ammonium sulfate was added gradually to the mixture. A homogeneous mixture was initially obtained, and after some amount of ammonium sulfate was added, an additional amount caused turbidity and a two-phase system arose. The addition of ammonium sulfate was continued to adjust the whole volume to 100 mL. After being mixed thoroughly, the system was allowed to separate into two phases in a 100-ml separating funnel. The lighter phase is enriched in PEG while the heavier is enriched in salt.21 Volumes of the separated phases were measured. Aliquots of the phases were taken for enzyme assay and determination of protein concentration. The partition coefficient of the enzyme (m) was determined from the equation m ) (Ct/Cb), where Ct and Cb are equilibrium concentrations of the enzyme in the top phase and bottom phase, respectively. The partition coefficient and specific activity of the enzyme were calculated. The phase composition with desired reduced bottom phase volume, selective partitioning, and reasonable enzyme recovery was selected. It is observed that adding NaCl to the ATPS has a significant effect on the selectivity of partitioning.16 Hence, various NaCl concentrations (1, 2, 3, and 4%) were added to the selected composition of PEG/ammonium sulfate. The phase composition with desired selective partitioning was selected. The salt-rich bottom phase was dialyzed against distilled water to remove salt and further concentrated to 5.0 mL by antidialysis against PEG (6000) solution with PEG concentration more than the PEG percent of the selected system.3,16 2.4.1.2. Ammonium Sulfate Precipitation. Ammonium sulfate precipitation was done by using the finely ground ammonium sulfate. The powder was weighed and added slowly by constant stirring to ensure complete solubility, and the solution was kept at 4 °C overnight for complete precipitation. Different degrees of saturation were achieved by progressively adding the specified quantity of ammonium sulfate according to the relevant saturation chart. After each saturation step the precipitate was collected by centrifuging the enzyme extract at 9000g for 20 min at 4 °C. The collected fractions (0-20%, 20-40%, 40-60%, 60-80%, 80-90%) were analyzed for enzyme activity and total protein content. The specific activity was calculated, and the values were expressed in terms of fold purification.4 The fraction with maximum specific activity was dialyzed for 48 h. The dialyzed fraction was used for further purification by gel-filtration chromatography. 2.4.2. Gel filtration on Sephadex G- 200 column. A 3 mL fraction of the enzyme sample from the previous step was loaded onto a Sephadex G-200 column. The column (2.7 × 40.0 cm)
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Table 2. Effect of NaCl Concentration on Partitioning of Peroxidase in PEG/Ammonium Sulfate System at 25.0/7.0%, w/v Composition NaC l(%)
volume top / bottom phase (mL)
partition coefficient (m)
enzyme recovery (bottom phase, %)
total protein recovery (bottom phase, %)
61.0/39.0 60.0/40.0 58.5/42.5 55.0/45.0
0.30 0.25 0.23 0.27
66.67 71.33 74.50 73.74
22.40 22.79 22.04 24.42
1.0 2.0 3.0 4.0
Table 3. Purification of Peroxidase from Turnip (ATPS + Gel Filtration) fraction crude extract ATPS (partition coefficient: m ) 0.23) top phase bottom phase G-200 column chromatography
total enzyme (U)
total protein (mg)
specific activity (U/mg)
purification factor
recovery (%)
11215
77.36
144.96
1.00
2645 8355 6153
58.00 17.00 1.19
45.60 491.47 5170.47
0.31 3.39 35.66
23.58 74.50 54.86
100
Table 4. Purification of Peroxidase from Turnip (Ammonium Sulfate + Gel Filtration) fraction
total enzyme (U)
total protein (mg)
specific activity (U/mg)
purification factor
recovery (%)
crude extract ammonium sulfate fractionation G-200 column chromatography
2803 2673 2307
19.34 11.77 1.27
144.93 227.10 1816.82
1.00 1.57 12.53
100 95.36 82.32
was equilibrated and eluted with phosphate buffer 100 mM, pH 6.0, at a flow rate of 12 mL/h. Fractions of 3 mL were collected throughout the elution. Absorbance at 280 nm and peroxidase activity were monitored.16 2.5. pH Optima for the Activity and Stability of Purified Enzyme. The optimum pH value for the activity of the turnip peroxidase was found by assaying enzyme activity at different pH levels. The assay was carried out by taking buffers of different pHs such as pH 2.6, 3.2, 3.6, 4.0, 4.5, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.2, 9.6, and 10 (pH 2.6-3.6, citratephosphate; pH 4-5, acetate; pH 6-8, phosphate; pH 9.2-10, borax-caustic soda) separately for diluting enzyme. For measuring the stability of enzyme over a range of pH, 1.0 mL of the purified enzyme was mixed with 1.0 mL of each of the different buffers such as pH 2.6, 3.2, 3.6, 4.0, 4.5, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.2, 9.6, and 10, separately. The mixture stayed at room temperature overnight (ca. 15 h). The activity was measured under standard assay conditions.4 2.6. Thermal Inactivation Study. The activities of turnip peroxidase were measured at various temperatures (25, 35, 45, 55, 65, and 75 °C) after incubating 1.0 mL of the enzyme in its optimum pH under standard assay conditions in order to investigate the optimum temperature value for the activity of peroxidase.2 Temperature effects on the purified enzyme were carried out by measuring the residual activity after incubating 1.0 mL of the enzyme in its optimum pH for 45 min at different temperatures (35, 45, 55, and 65 °C) in a water bath. Aliquots (0.1 mL) taken at different time intervals, were assayed immediately, and remaining activity was expressed as percent decrease from the original activity.4 3. Results and Discussion 3.1. Effect of Phase Composition. Table 1 depicts the results obtained by partitioning the enzyme at various phase compositions. Phase compositions were varied in such a way that the volume of the bottom phase is reduced. The preferential partitioning of turnip peroxidase to the bottom phase indicates that the characteristics of the enzyme could be different from other plant peroxidases, which have preferred the PEG-rich (top)
phase.20 However, its trend is similar to the peroxidase from Ipomea palmetta.3,16 A phase system having a composition of PEG 25.0% and ammonium sulfate 7.0% w/v gave overall desired results in terms of reduced bottom phase volume (54.37% reduction) and selective partitioning (0.46) with reasonable enzyme recovery. Hence, this system was selected for further studies in optimizing the NaCl concentration. It may be noted that a further decrease in the bottom phase volume (81.25% reduction) in the phase system having the composition of PEG 30.0% and ammonium sulfate 4.7% resulted in very poor enzyme recovery (31.10). Hence, it was not considered for further studies. 3.2. Effect of NaCl Concentration. ATPSs with NaCl addition are more sensitive to protein surface hydrophobicity. In such systems, the surface hydrophobicity difference between target protein and host contaminants is crucial for selective partitioning. This factor has been exploited by attaching hydrophobic fusions to improve the selectivity of partitioning.25 The effect of NaCl concentration on the partitioning of peroxidase, studied only in a phase system having the composition of PEG 25.0% and ammonium sulfate 7.0%, is shown in Table 2. A low partition coefficient (0.23), high enzyme recovery (74.50%) and considerable reduction in the volume of bottom phase (46.87%) were observed at 3.0% NaCl concentration. Hence, the system with 3.0% NaCl was used in further purification protocols of peroxidase. 3.3. Ammonium Sulfate Fractionation. The crude enzyme extract was concentrated by progressive fractionation by ammonium sulfate precipitation from 0-20, 20-40, 40- 60, 60-80, 80-90%. The fraction obtained with 60-80% showed maximum specific activity. Hence, this fraction was dialyzed against water for 48 h. The dialyzed fraction was used for further purification by gel-filtration chromatography. 3.4. Purification of Peroxidase. The specific activity of the crude enzyme was 144.96 U/mg, which is more than the value reported in the literature for soluble turnip peroxidase.6 This is because we extracted soluble enzyme from the fibrous of the turnip roots in addition to its juice. ATPS, employed here as a primary purification step, achieved its main purpose of improving the degree of enzyme purification
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Figure 1. Gel filtration of turnip peroxidase on Sephadex G-200 column (2.7 cm × 40.0 cm): (9) [peroxidase (µ/mL)] × 10-3; and (2) absorbance at 280 nm (for ATPE + gel-filtration method).
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Figure 3. The pH stability after incubating the enzyme for 15 h in different pH values.
Figure 4. Thermostability of purified enzyme in a 45 min period for 4 temperatures. Figure 2. Optimum pH (temperature ) 25 °C, time of reaction ) 2 min).
(purification factor ) 3.39) while considerably reducing the volume of the crude sample (46.87%) that has to be further processed with a chromatography step. The purification conditions employed resulted in the enrichment of enzyme specific activity (491.47 U/mg), which is due to the differential partitioning of the desired enzyme and contaminating enzymes/ proteins to the opposite phases. Figure 1 shows the elution profile of the enzyme in Sephadex G-200 column. A total of 55 fractions were collected from gel column. Absorbance at 280 nm was observed only for fractions 15-48. Activity was assayed for these fractions and maximum activities were observed for fractions 25-34. By determining protein for these fractions, fractions 29-31, which had the maximum specific activity, were chosen as purified enzyme. G-200 column chromatography of antidialysate resulting from the salt-rich bottom phase of the ATPS markedly increased the degree of enzyme purification (about 36-fold). These purification protocols of the turnip peroxidase are summarized in Table 3. By the combination of methods, the enzyme was purified about 36 times with a recovery of 55%. The specific activity of the purified enzyme was 5170 U/mg. Similar work was done, in which the peroxidase was purified by the conventional method of ammonium sulfate precipitation and column chromatography. The purification factor after G-200 column chromatography was only about 13 (Table 4). Thus, it could be restated that ATPS could be an efficient purification step in the purification chain of certain biomolecules, and the present case of plant peroxidase is a useful example. Hence, we characterized purified enzyme from ATPS and gel filtration.
Figure 5. Optimum temperature of peroxidase (pH ) 4.0, time of reaction ) 2 min).
3.5. pH Optima for Activity and Stability. When checked for pH optima, the purified enzyme showed maximum activity at pH 4.0 (Figure 2), which is near to the reported value for turnip peroxidase in the literature.6,26 Turnip peroxidase has an advantage, in comparison with horseradish peroxidase, to be utilized in acidic environment, in which horseradish peroxidase cannot operate properly (pH optimum for horseradish peroxidase is 7.023). The enzyme was stable over a range of pH from 2.6 to 5.0 after 15 h (Figure 3). Hence, this enzyme can retain its activity in acidic environment. 3.6. Enzyme Thermostability. The thermostability of the purified enzyme is reported as the percent decrease of activity as a function of four different temperatures during a period of 45 min. Enzyme showed negligible inactivation up to 45 °C. It
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has slight decrease in activity at 65 °C. However, it retained more than 60% activity even after 20 min (Figure 4), which is similar to the values for turnip peroxidase in the literature.26,27 This enzyme retains its activity in high temperatures better than horseradish peroxidase, which lost its activity at 65 °C after 11 min.4 The enzyme was stable up to 55 °C. The enzyme activity showed negligible change with temperature from 25 to 45 °C with optimum activity at 35 °C under the assay conditions employed (Figure 5). This temperature is near to the reported value in the literature for turnip peroxidase.26 4. Conclusions Peroxidase from turnip was investigated in this study as an alternative commercial source of high activity peroxidase, other than horseradish. The peroxidase obtained from turnip was purified by two different purification processes, an aqueous twophase system and ammonium sulfate precipitation. Both of the mentioned methods were followed by gel-filtration chromatography. The aqueous two-phase system was demonstrated to be an efficient primary purification step for the plant peroxidase in comparison with ammonium sulfate precipitation. The potential advantages of ATPS as compared to ammonium sulfate precipitation is mainly associated to several factors; shorter process period, ease of utilizing the process, independency from equipments, less energy consumption, environmentally benign, and higher purification factor. Hence, ATPS is more promising to be utilized for partial purification of peroxidase to be used for phenol removal from industrial wastewaters. On the basis of optimum pH, turnip peroxidase could be a better option than horseradish peroxidase, when the environment is acidic. Moreover, although turnip has its best activity in low temperatures, it retains its activity in high temperatures better than horseradish peroxidase. Hence, it could be a better option in high temperatures than the commercial peroxidase. Literature Cited (1) Duarte-Vazquez, M. A.; Garcia-Almendarez, B. E.; Rojo-Dominguez, A.; Whitaker, J. R.; Arroyave-Hernandez, C.; Regalado, C. Monosaccharide Composition and Properties of a Deglycosylated Turnip Peroxidase Isozyme. Phytochemistry 2003, 62, 5. (2) Kulshrestha, Y.; Husain, Q. Bioaffinity-Based an Inexpensive and High Yield Procedure for the Immobilization of Turnip (Brassica Rapa) Peroxidase. Biomol. Eng. 2006, 23, 291. (3) Srinivas, N. D.; Barhate, R. S.; Raghavarao, K. S. M. S. Aqueous Two-Phase Extraction in Combination with Ultrafiltration for Downstream Processing of Ipomoea Peroxidase. J. Food Eng. 2002, 54, 1. (4) Rudrappa, T.; Lakshmanan, V.; Kaunain, R.; Singara, N. M.; Neelwarne, B. Purification and Characterization of an Intracellular Peroxidase from Genetically Transformed Roots of Red Beet (Beta Vulgaris L.). Food Chem. 2007, 105, 1312. (5) Leon, J. C.; Alpeeva, I. S.; Chubar, T. A.; Galaev, I. Yu.; Csoregi, E.; Sakharov, I. Yu. Purification and Substrate Specificity of Peroxidase from Sweet Potato Tubers. Plant Sci. 2002, 163, 1011. (6) Hamed, R. R.; Maharem, T. M.; Abdel Tatah, M. M.; Ataya, F. Sh. Purification of Peroxidase Isoenzymes from Turnip Roots. Phytochemistry 1998, 48, 1291. (7) Forsyth, J. L.; Robinson, D. S. Purification of Brussels Sprout Isoperoxidases. Food Chem. 1998, 63, 227.
(8) Sakharov, I. Yu.; Vesga, B. M. K.; Galaev, I. Y.; Sakharova, I. V.; Pletjushkina, O. Yu. Peroxidase from Leaves of Royal Palm Tree Roystonea Regia: Purification and Some Properties. Plant Sci. 2001, 161, 853. (9) Klibanov, A. M.; Alberti, B. N.; Morris, E. D.; Felshin, L. M. Enzymatic Removal of Toxic Phenols and Anilines from Waste Waters. J. Appl. Biochem. 1980, 2, 414. (10) Zhang, G.; Nicell, J. A. Treatment of Aqueous Pentachlorophenol by Horseradish Peroxidase and Hydrogen Peroxide. Water Res. 2000, 34, 1629. (11) Klibanov, A. M.; Tu, T. M.; Scott, K. P. Peroxidase-Catalyzed Removal of Phenols from Coal-Conversion Waste Water. Science 1983, 221, 259. (12) Cooper, V. A.; Nicell, J. A. Removal of Phenols from a Foundry Wastewater Using Horseradish Peroxidase. Water Res. 1996, 30, 954. (13) Wagner, M.; Nicell, J. A. Treatment of a Foul Condensate from Kraft Pulping with Horseradish Peroxidase and Hydrogen Peroxide. Water Res. 2001, 35, 485. (14) Ikehata, K.; Buchanan, I. D.; Pickard, M. A.; Smith, D. W. Purification, Characterization, and Evaluation of Extracellular Peroxidase from Two Coprinus Species for Aqueous Phenol Treatment. Bioresour. Technol. 2005, 96, 1758. (15) Miranda, M. V.; Magri, M. L.; Navarro del Can˜izo, A. A.; Cascone, O. Study of Variables Involved in Horseradish and Soybean Peroxidase Purification by Affinity Chromatography on Concanavalin A-Agarose. Process Biochem. 2002, 38, 537. (16) Srinivas, N. D.; Rashmi, K. R.; Raghavarao, K. S. M. S. Extraction and Purification of a Plant Peroxidase by Aqueous Two-Phase Extraction Coupled with Gel Filtration. Process Biochem. 1999, 35, 43. (17) Miranda, M. V.; Fernandez-Lahore, H. M.; Dobrecky, J.; Cascone, O. The Extractive Purification of Peroxidase from Plant Raw Materials in Aqueous Two-Phase Systems. Acta Biotechnol. 1998, 18, 179. (18) Stanisavljevic´, M.; Nedic´, L. Removal of Phenol from Industrial Wastewaters by Horseradish (Cochlearia Armoracia L) Peroxidase. Work. LiVing EnViron. Prot. 2004, 4, 345. (19) Anadharamakrishnan, C.; Raghavendra, S. N.; Barhate, R. S.; Hanumesh, U.; Raghavarao, K. S. M. S. Aqueous Two-Phase Extraction for Recovery of Proteins from Cheese Whey. Food Bioprod. Process. 2005, 83, 191. (20) Vilter, H. Aqueous Two-Phase Extraction of Plant Enzymes from Source Containing Large Amounts of Tannins and Anionic Mucilages. Bioseparation 1990, 1, 283. (21) Da Silva, M. E.; Franco, T. T. Purification of Soybean Peroxidase (Glycine max) by Metal Affinity Partitioning in Aqueous Two-Phase Systems. J. Chromatogr., B 2000, 743, 287. (22) Nicell, J. A.; Harold, W. A Model of Peroxidase Activity with Inhibition by Hydrogen Peroxide. Enzyme Microb. Technol. 1997, 21, 302. (23) Saraiva, J. A.; Nunes, C. S.; Coimbra, M. A. Purification and Characterization of Olive (Olea europaea L.) PeroxidasesEvidence for the Occurrence of a Pectin Binding Peroxidase. Food Chem. 2007, 101, 1571. (24) Lowry, O. H.; Rosenberg, N. J.; Farr, A. L.; Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265. (25) Gu, Zh.; Glatz, C. E. Aqueous Two-Phase Extraction for Protein Recovery from Corn Extracts. J. Chromatogr., B 2007, 845, 38. (26) Matto, M.; Husain, Q. Decolorization of Direct Dyes by Immobilized Turnip Peroxidase in Batch and Continuous Processes. Ecotoxicol. EnViron. Saf. 2009, 72, 965. (27) Quintanilla-Guerrero, F.; Duarte-Vazquez, M. A.; Garcia-Almendarez, B. E.; Tinoco, R.; Vazquez-Duhalt, R.; Regalado, C. Polyethylene Glycol Improves Phenol Removal by Immobilized Turnip Peroxidase. Bioresour. Technol. 2008, 99, 8605.
ReceiVed for reView December 27, 2008 ReVised manuscript receiVed September 14, 2009 Accepted September 25, 2009 IE801997E
