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Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases in the decolorization of synthetic and natural dyes
Journal of Genetic Engineering and Biotechnology (2013) 11, 95–102
Academy of Scientific Research & Technology and National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology www.elsevier.com/locate/jgeb
ARTICLE
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases in the decolorization of synthetic and natural dyes Azza M. Abdel-Aty a,b,*, Mohamed Belal Hamed a, Afaf S. Fahmy a, Saleh A. Mohamed a,c a b c
Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt Clinical Lab. Department, Al-Gad International Colleges for Medical Science, Saudi Arabia Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Received 20 March 2013; revised 10 May 2013; accepted 20 July 2013 Available online 26 September 2013
KEYWORDS Peroxidases; Synthetic; Natural dyes; Decolorization; Redox mediators
Abstract The aim of this study was to compare the potential of Ficus sycomorus latex peroxidase (POL) and horseradish peroxidase (HRP) in the decolorization of a wide spectrum of eight synthetic dyes and two natural dyes, hibiscus flower color and pomegranate juice. We study for the first time the decolorization of natural dyes enzymatically. The highest decolorization percent was reported at 20 mg/l for all dyes treated with POL and HRP. Both the enzymes had lower decolorization % for azo-carmin (30–33%). During decolorization treatment, both natural dyes and titan yellow formed precipitates which settled down and were removed by centrifugation. The enhancement of the decolorization % of the most tested dyes by treatment with POL and HRP was reported in the presence of some redox mediators. The rate of decolorization was enhanced by increasing the time and the most significant changes were observed during the first 6 h of incubation. One hundred percent enhancement in decolorization was reported for azo-carmine in the presence of histidine and anaphthol as redox mediators. A few of redox mediators caused no significant effect or decreases the decolorization % for a little number of tested dyes. The decolorization of dyes by POL and HRP in the presence of redox mediators appeared without the formation of precipitate. A similar decolorization % for all the tested dyes by POL and HRP was detected. The data suggested that the
* Corresponding author at: Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt. E-mail address: [email protected] (A.M. Abdel-Aty). Peer review under responsibility of National Research Center, Egypt.
Production and hosting by Elsevier 1687-157X ª 2013 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology. http://dx.doi.org/10.1016/j.jgeb.2013.07.001
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A.M. Abdel-Aty et al. peroxidase/mediator system was an effective biocatalyst for the decolorization of synthetic and natural dyes, and POL could be used as a potential option for the application of dye decolorization. ª 2013 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
1. Introduction Synthetic dyes are extensively used in many industries. The problems associated with the discharge of colored effluents from various industries such as textile, paper, food, plastics and cosmetics have concerned both industrial and academic scientists [35]. Approximately, 100,000 different dyes and pigments are used industrially and over 0.7–0.8 million tons of synthetic dyes are produced annually worldwide [40,41,37,34]. Due to their chemical structure, dyes are resistant to fading on exposure to light, water and many chemicals [61,43,4]. During processing, up to 15% of the used dyestuffs discharged in wastewater cause extensive pollution. In addition to dye visual effect, some synthetic dyes cause allergy, dermatitis, skin irritation and they are toxic, mutagenic and carcinogenic in humans [34,55,14,39]. Anthocyanins, as natural dyes, are water-soluble phenolic compounds and the most diverse group of plant pigments derived from the phenylpropanoid pathway, ranging in color from red to violet and blue [53]. These pigments accumulate in the vacuoles and their stability and hue depend on conditions such as pH, enzymatic activity and formation of complexes with metal ions [29,49]. Very little information has been reported on the biodegradation of natural dyes of the fruit juice or pellet extract in vitro. Conventional chemical and physical methods of dye decolorization are outdated because costs are high and they consume high amounts of chemicals and energy, other disadvantages are sludge formation and biomass accumulation [6,57]. Biological degradation of dyes included properties such as water solubility, large molecular weight and fused aromatic ring structures, which inhibit permeation through biological cell membranes. Other limitations of using microbes for treating pollutants were high costs of production of microbial culture, slow process of decolorization of dyes and metabolic inhibition [20,38]. Enzymatic systems fall between the two traditional categories of chemical and biological processes, since they involve chemical reactions based on the action of biological catalysts [5]. This was mainly because, unlike the chemical catalysts, the enzymatic catalysis showed its merits to convert complex chemical structures under mild environmental conditions with high efficiency [17,19,30]. Enzymes can act on specific recalcitrant pollutants to remove them by precipitation or transformation to other products [1,45]. Isolated enzymes were often preferred over intact organisms containing the enzymes because the isolated enzymes offered several advantages such as greater specificity, better standardization, easy handle, store and no dependence on bacterial growth rates [19,21]. Peroxidases (EC 1.11.1.7) are oxidoreductases which efficiently catalyze the oxidation of phenolic compounds and have great potential in treating a wide spectrum colored compounds [17,19,56,2,7]. These enzymes convert a broad range of substrates into less toxic insoluble compounds, which can be easily removed out of waste by a mechanism involving the formation
of free radical followed by insoluble product [17,51]. Peroxidase based dye treatment still provides a reasonable basis for the development of biotechnological processes for continuous color and aromatic compound removal from various industrial effluents at a large scale [18]. Sometimes these enzymes cannot act on organic pollutants and dye effluents due to the recalcitrant nature of such compounds. These recalcitrant substrates get converted into less toxic forms in the presence of certain low molecular weight compounds that are known as redox mediators which enhanced the rate of enzyme-catalyzed reaction and increased the range of selection of their substrates [17,3,25]. The present study has been made to investigate the potential of Ficus sycomorus latex peroxidase (POL) as compared to horseradish peroxidase (HRP), which represents the commercial peroxidase, in the decolorization of a wide spectrum synthetic dyes and natural dyes (Hibiscus sabdariffa flower color and pomegranate Punica granatum juice). Evaluation of the role of various low molecular weight redox mediators in the decolorization of the selected dyes mediated by the action of peroxidases is the second goal. 2. Materials and methods 2.1. Materials Bromophenol blue, catechol, vanilline, a-naphthol, L-histidine, methyl green, methylene blue, methyl orange were obtained from Sigma Chemicals Co. Azo-carmine, naphthylamine– azo-benzene, titan yellow were products of Fluka Co. Calyces of roselle (Hibiscus sabdariffa L.) and pomegranate (Punica granatum L.) were obtained from the local market. All other chemicals and reagents employed were of analytical grade and were used without any further purification. 2.2. Peroxidases We previously purified and characterized the peroxidases from F. sycomorus latex (POL) [33] and from Japanese horseradish roots (HRP) [32]. 2.3. Extraction of hibiscus pigments Roselle extract was routinely prepared by boiling 3 g of dried roselle petals for 3 min with 100 ml of distilled water. The extract was rapidly filtered through a four layers of cheez cloth and kept at 4 C [52]. 2.4. Extraction of Punica granatum pigments The method consisted of manually peeling the fruits, separating the seeds and extracting the juice by electric juice mixer, extract was rapidly filtered through a four layers of cheez cloth and kept at 4 C [31].
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases Table 1
97
Degree of decolorization of synthetic and natural dyes at different concentrations by POL and HRP.
Dyes
k (nm)
Decolorization (%) At different conc. of dyes (mg/l) with 0.25 unit of POL
Bromophenol blue Methyl green Methylene blue Methyl orange Azo-carmine Naphthylamine –azo-benzene Titan yellow Diethyl-p-phenylenediamine hydrochloride Hibiscus Punica granatum
430 630 662 502 507 485 405 515 530 510
HRP
100
50
20
100
50
20
11 22 14 7 0 10 10 25 27 30
43 53 39 29 12 30 34 54 57 60
73 80 74 66 30 70 72 85 80 86
15 19 10 5 2 10 9 21 18 26
48 49 36 22 13 33 33 50 40 51
75 80 71 60 33 72 72 80 73 82
The dye compounds at different concentrations (100–20 mg/l) were treated with each peroxidase (0.25 unit/ml) for decolorization and the mixture included 50 mM sodium acetate buffer, pH 5.5 and 2 mM H2O2 and incubated for 6 h at room temperature. Each value represents the average of two experiments.
100
POL+Bromophenol blue
Color removal (%)
Color removal (%)
100 80 60
None V H N C
40 20 0
0
5
10
15
20
HRP+Bromophenol blue
80 60
None V H N C
40 20
25
0
0
5
10
Time (h) 100
80 60 Non
40
V H N C
20 0
0
5
10
15
20
80 60 None V H N C
40 20 0
25
0
5
60 None
40
V H N C
20 5
10
15
Time (h)
20
25
Color removal (%)
Color removal (%)
100
POL+Methylene blue
0
10
15
20
25
Time (h)
80
0
25
HRP+Methyl green
Time (h) 100
20
POLI+Methyl green
Color removal (%)
Color removal (%)
100
15
Time (h)
HRP+Methylene blue
80 60 None V H N C
40 20 0
0
5
10
15
Time (h)
20
25
Figure 1 Time course of decolorization of synthetic and natural dyes at concentration 20 mg/l by POL and HRP (0.25 U/ml) in the absence and presence of 0.6 mM redox mediators [vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C)]. The mixture included 50 mM sodium acetate buffer, pH 5.5, 2 mM H2O2 and incubated for different time intervals (1–24 h) at room temperature. Each value represents the average of two experiments.
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A.M. Abdel-Aty et al.
2.5. Peroxidase assay Peroxidase activity was carried out according to [59]. The reaction mixture contained in one ml: 8 mM H2O2, 40 mM guaiacol, 50 mM sodium acetate buffer, pH 5.5 and 0.25 unit of enzyme. Assays were carried out at room temperature. The change of absorbance at 470 nm due to guaiacol oxidation was followed at 30 s intervals. One unit of peroxidase activity is defined as the amount of enzyme which increases the O.D. 1.0 per min under standard assay conditions. 2.6. Dye decolorization by POL and HRP The selected dyes (bromophenol blue, methyl green, methylene blue, methyl orange, azo-carmine, naphthylamine–azo-benzene, titan yellow, diethyl-p-phenylenediamine hydrochloride, and natural dyes extracted from hibiscus and pomegranate) at concentration of 100–20 mg/l were prepared in distilled water. Each dye was incubated with 0.25 unit/ml of POL or HRP in 50 mM sodium acetate buffer, pH 5.5 and 2 mM H2O2 for different time intervals (1–24 h) at room temperature. Dye
60 None V H N C
40 20
0
5
The rate of decolorization of dyes by POL and HRP in the presence of 0.2–2 mM of each redox mediator (vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C)). was tested under the same conditions.
3. Results and discussion Previously, we reported that POL and HRP vary with respect to their potential to catalyze the oxidation of various electron donor substrates (e.g. phenolic compounds) by H2O2 [33,32].
100
POL+Methyl orange
80
0
2.7. Effect of redox mediators on rate of the decolorization
Color removal (%)
Color removal (%)
100
decolorization was monitored at the visible absorbance maximum of each dye (Table 1). Control experiments were performed using the same condition without enzymes. The color removal was reported as decolorization (%) = (A0 At)/A0 · 100. where A0 is the absorbance of untreated dye as control; At is the absorbance of treated dye with the enzyme.
10
15
20
80 60 40
0
25
None V H N C
POL+Azo-Carmine
100 Color removal (%)
Color removal (%)
0
5
10
60 40
20
25
None V H N C
HRP+Azo-Carmine
80 60 40 20
20
0
0
5
10
15
20
25
0
5
10
100
POL+Naphthylamine-azo-benzene
80 60 None V H N C
40 20 0
0
5
10
15
20
25
Color removal (%)
100
15
20
25
Time (h)
Time (h)
Color removal (%)
15
Time (h)
80
0
None V H N C
20
Time (h) 100
HRP+Methyl orange
HRP+Naphthylamine-azo-benzene
80 60 None V H N C
40 20 0
0
Time (h)
5
10
15
Time (h)
Figure 1 (continued)
20
25
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases 100 Color removal (%)
Color removal (%)
100 POL+Titan yellow 80 60 40
None V H N C
20 0
0
5
10
15
20
HRP+Titan yellow
80 60 None V H N C
40 20 0
25
0
5
10
60 None V H N C
40 20 0
5
10
15
20
Color removal (%)
Color removal (%)
100
POL+Diethyl-p-phenylenediamine hydrochloride
80
0
POL+Punica granatum
60 None V H N C
40 20 0
0
5
10
20 5
10
15
20
Color removal (%)
Color removal (%)
None V H N c
40
60 None V H N C
40 20 0
0
5
60 None V H N C
40 20 0
0
5
10
15
20
25
Color removal (%)
Color removal (%)
100
80
25
80
25
HRP+Hibiscus
20
HRP+Punica granatum
10
15
Time (h)
Time (h)
100
15
Time (h)
100
60
0
25
80
25
80
0
20
HRP+Diethyl-p-phenylenediamine hydrochloride
Time (h) 100
15
Time (h)
Time (h)
100
99
20
25
POL+Hibiscus
80 60 None V H N C
40 20 0
0
5
10
15
20
25
Time (h)
Time (h) Figure 1 (continued)
In this study the ability of these enzymes to decolorize eight synthetic and two natural dyes was reported. The dyes used in this study including: firstly, synthetic dyes such as (i) triphenylmethane dyes (bromophenol blue and methyl green), (ii) heterocyclic dyes (methylene blue), (iii) azo dyes (methyl orange, azo-carmine, naphthylamine–azo-benzene and titan yellow), (iv) diethyl-p-phenylenediamine hydrochloride, and secondly, natural dyes extracted from hibiscus and pomegranate.
The effect of dye concentration (20–100 mg/l) on the rate of decolorization by POL or HRP is shown in Table 1. The highest decolorization percent ranging from 30% to 86% was reported at 20 mg/l for all tested dyes treated with POL and HRP. Both the enzymes had low decolorization % for azo-carmin (30–33%) at 20 mg/l of dye concentration. A similar decolorization % for the all tested dyes by POL and HRP was detected. In contrast, synthetic non-textile
100 Table 2
A.M. Abdel-Aty et al. Degree of decolorization of synthetic and natural dyes in the absence and presence of redox mediators by POL and HRP.
Dyes
Decolorization (%) POL
Bromophenol blue Methyl green Methylene blue Methyl orange Azo-Carmine Naphthylamine –azo-benzene Titan yellow Diethyl-p-phenylenediamine hydrochloride Hibiscus Punica granatum
HRP
None
V
H
N
C
None
V
H
N
C
73 80 74 66 30 70 72 85 80 86
73 87 80 75 45 77 64 86 77 86
84 81 60 73 61 71 71 80 76 86
80 92 55 80 65 70 83 84 76 86
70 81 91 83 44 66 56 91 91 93
75 80 71 60 33 72 72 80 73 82
77 84 78 70 44 61 60 83 72 82
88 82 55 70 60 61 73 83 73 83
81 84 50 75 62 72 82 84 71 84
74 87 85 70 40 66 66 88 84 88
Each dye compound (20 mg/l) was treated with each peroxidase (0.25 unit/ml) for decolorization in the absence and presence of mediating property by using 0.6 mM of each redox mediator [vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C)]. The mixture included 50 mM sodium acetate buffer, pH 5.5 and 2 mM H2O2 and incubated for 6 h at room temperature. Each value represents the average of two experiments.
dyes were recalcitrant to decolorization by Bitter gourd peroxidase [4]. During decolorization treatments, both natural dyes and titan yellow formed precipitates which settled down and were removed by centrifugation. Several investigators have shown that the treatment of phenols and aromatic amines by peroxidases resulted in the formation of large insoluble aggregates [20,54,50,16]. Anthocyanins, as natural dyes, were also oxidized by peroxidases that they may undergo polymerization and thus go out of solution by forming precipitates [60]. Peroxidases are among the enzymes that can use anthocyanins as substrates [26,27]. The results showed a change in color of hibiscus and Punica granatum extractions from red to brown during the first min of incubation, then the color was removed gradually by increasing the time of incubation suggesting the involvement of peroxidases in change of color, in many instances becoming brownish, and the degradation of anthocyanins under oxidizing conditions provoked by exogenous application of H2O2 in such a way that they become colorless [60]. A peroxidase isolated from a Gamay grapevine cell culture degraded anthocyanins in solution in the grape fruit [10]. Peroxidases were also involved in loss of color in processed strawberries and with exogenous application of H2O2 on strawberry slices caused a more rapid decrease in anthocyanin concentration relative to non-treated slices [10]. In the present study, relatively low concentration of H2O2 (2 mM) was used in agreement with other studies using HRP [24], soybean peroxidase [12] and turnip peroxidase [15]. There were also many researches reporting that the negative effect of high H2O2 concentrations in the reaction mixture resulted in the inactivation of peroxidases [57,36,9,22,58]. Fig. 1 shows the effect of time course of decolorization on the selected dyes treated with POL or HRP. Our observations have suggested that the decolorization of the dyes was continuously increased during the first 6 h of incubation and no change in decolorization % was observed up to 24 h for the almost selected dyes. The drop in the rate of dye degradation after an initial fast reaction could be due to inactivation of the enzyme. In several studies, dye removal was found to be dependent upon the reaction time when treated with peroxi-
dases as 90% of Orange II was degraded during 36 h [47], Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green dyes were completely degraded within 8 h [46]. The decolorization of dyes by POL and HRP in the presence of varying concentrations of redox mediators (0.2–2 mM), vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C) was evaluated, where 0.6 mM gives the best result (Data not shown). This concentration was quite low and in agreement with that reported by other studies [4,44,23,28]. On the contrary, the redox mediators were used in very high concentrations at 5.7 mM as Violuric Acid (VLA)/laccase system, 11.0 mM 1-hydroxybenzotrizole (HOBT)/laccase system, 2.0 mM HOBT/laccase system and 2.0 mM HOBT/turnip peroxidase system [25,48,13]. The effect of redox mediators and incubation time on the decolorization of dyes treated with POL or HRP enzymes is also shown in Fig. 1. The decolorization of the dyes was also continuously increased during the first 6 h of incubation and no change in decolorization % up to 24 h was observed. The results at 6 h of incubation are summarized in Table 2. The enhancement of the decolorization of dyes by treatment with POL and HRP was reported in the presence of V (methyl green, methylene blue, methyl orange and azo-carmine), H (bromophenol blue, methyl orange and azo-carmine), N (bromophenol blue, methyl green, methyl orange, azo-carmine and titan yellow) and C (methylene blue, methyl orange, azo-carmine, diethyl-p-phenylenediamine hydrochloride, hibiscus and Punica granatum). However, POL and HRP enhanced the decolorization of naphthylamine–azo-benzene and methyl green in the presence of V and C, respectively. One hundred percent enhancement in decolorization was reported for azocarmine (from 30% to 60%) by using H and N treated with POL or HRP. This attributed to the diffusible redox mediators providing high redox potentials (>900 mV) to attack recalcitrant structural analogs and are able to migrate into the aromatic structure of the compound [8]. Nevertheless, C, N, H and V had no significant effect on the decolorization by POL and HRP for bromophenol blue, naphthylamine–azo-benzene, titan yellow and punica granatum, respectively. In contrast, the color removal effect by POL and HRP was decreased for
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases titan yellow, methylene blue and naphthylamine–azo-benzene, diethyl-p-phenylenediamine hydrochloride and hibiscus in the presence of some redox mediators. During the enzymatic treatment, no precipitates were formed in the presence of redox mediators. This was attributed to the degradation of the aromatic ring of the compounds or by cleaving certain functional groups [7,25]. The efficiency of each mediator depended on the type, molecular structure of dye to be treated and enzyme inactivation [13,42] as in case of turnip peroxidase, [28] have shown that after addition of the mediator there was no significant effect on the decolorization of some dyes such as DR 23 and DR 239, while in DB 80 decolorization was continuously decreased with increase in the concentration of mediator. Also, among the six types of dyes, only reactive blue 38 was resistant to enzyme–mediator treatment. Acid blue 74, reactive blue 19 and aniline blue were partially decolorized by the enzyme alone, although decolorization was much more efficient and rapid in the presence of mediators, whereas reactive black 5 and azure B could be decolorized only in the presence of mediators [11]. It has been reported that the mediators could play a dual role, as mediator because they increased the rate of recalcitrant dye decolorization and acted as inhibitor for enzyme activity. 4. Conclusion Here, it has been shown that POL would be effectively employed for the decolorization of industrially important synthetic dyes and, for the first time, natural dyes. Our results suggested that, the peroxidase from Ficus sycomorus latex has shown its potential in decolorization of all selected dyes. More than 90% of Punica granatum, Hibiscus, Diethyl-p-phenylenediamine hydrochloride, methylene blue and methyl green dyes were decolorized by employing more effective and cheaper redox mediators with POL. These findings suggested that the POL could be extended to the large-scale treatment of textile effluents as a potential option of dye decolorization. References [1] S. Akhtar, Q. Husain, Chemosphere 65 (2006) 1228–1235. [2] S. Akhtar, A.A. Khan, Q. Husain, J. Chem. Technol. Biotechnol. 96 (2005) 1804–1811. [3] S. Akhtar, A.A. Khan, Q. Husain, Bioresour. Technol. 96 (2005) 1804–1811. [4] S. Akhtar, A.A. Khan, Q. Husain, Chemosphere 60 (2005) 291– 301. [5] Y. Anjaneyulu, N.S. Chary, D.S.S. Raj, Rev. Environ. Sci. Biotechnol. 4 (2005) 245–273. [6] I.M. Banat, P. Nigm, D. Singh, R. Marchant, Bioresour. Technol. 58 (1996) 217–227. [7] A. Bhunia, S. Durani, P.P. Wangikar, Biotechnol. Bioeng. 72 (2001) 562–567. [8] R. Bourbonnais, M.G. Paice, FEBS Lett. 267 (1990) 99–102. [9] V. Brenda, A. Marcela, V. Rafael, Chem. Biol. 9 (2002) 555– 565. [10] A.A. Calderon, E. Garciaflorenciano, R. Munoz, A.R. Barcelo, VITIS 31 (1992) 139–147. [11] S. Camarero, D. Ibarra, M.J. Martınez, A.J. Martınez, Appl. Environ. Microbiol. 71 (2005) 1775–1784. [12] N. Caza, J.K. Bewtra, N. Biswas, K.E. Taylor, Water Res. 33 (1999) 3012–3018.
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Academy of Scientific Research & Technology and National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology www.elsevier.com/locate/jgeb
ARTICLE
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases in the decolorization of synthetic and natural dyes Azza M. Abdel-Aty a,b,*, Mohamed Belal Hamed a, Afaf S. Fahmy a, Saleh A. Mohamed a,c a b c
Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt Clinical Lab. Department, Al-Gad International Colleges for Medical Science, Saudi Arabia Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Received 20 March 2013; revised 10 May 2013; accepted 20 July 2013 Available online 26 September 2013
KEYWORDS Peroxidases; Synthetic; Natural dyes; Decolorization; Redox mediators
Abstract The aim of this study was to compare the potential of Ficus sycomorus latex peroxidase (POL) and horseradish peroxidase (HRP) in the decolorization of a wide spectrum of eight synthetic dyes and two natural dyes, hibiscus flower color and pomegranate juice. We study for the first time the decolorization of natural dyes enzymatically. The highest decolorization percent was reported at 20 mg/l for all dyes treated with POL and HRP. Both the enzymes had lower decolorization % for azo-carmin (30–33%). During decolorization treatment, both natural dyes and titan yellow formed precipitates which settled down and were removed by centrifugation. The enhancement of the decolorization % of the most tested dyes by treatment with POL and HRP was reported in the presence of some redox mediators. The rate of decolorization was enhanced by increasing the time and the most significant changes were observed during the first 6 h of incubation. One hundred percent enhancement in decolorization was reported for azo-carmine in the presence of histidine and anaphthol as redox mediators. A few of redox mediators caused no significant effect or decreases the decolorization % for a little number of tested dyes. The decolorization of dyes by POL and HRP in the presence of redox mediators appeared without the formation of precipitate. A similar decolorization % for all the tested dyes by POL and HRP was detected. The data suggested that the
* Corresponding author at: Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt. E-mail address: [email protected] (A.M. Abdel-Aty). Peer review under responsibility of National Research Center, Egypt.
Production and hosting by Elsevier 1687-157X ª 2013 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology. http://dx.doi.org/10.1016/j.jgeb.2013.07.001
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A.M. Abdel-Aty et al. peroxidase/mediator system was an effective biocatalyst for the decolorization of synthetic and natural dyes, and POL could be used as a potential option for the application of dye decolorization. ª 2013 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
1. Introduction Synthetic dyes are extensively used in many industries. The problems associated with the discharge of colored effluents from various industries such as textile, paper, food, plastics and cosmetics have concerned both industrial and academic scientists [35]. Approximately, 100,000 different dyes and pigments are used industrially and over 0.7–0.8 million tons of synthetic dyes are produced annually worldwide [40,41,37,34]. Due to their chemical structure, dyes are resistant to fading on exposure to light, water and many chemicals [61,43,4]. During processing, up to 15% of the used dyestuffs discharged in wastewater cause extensive pollution. In addition to dye visual effect, some synthetic dyes cause allergy, dermatitis, skin irritation and they are toxic, mutagenic and carcinogenic in humans [34,55,14,39]. Anthocyanins, as natural dyes, are water-soluble phenolic compounds and the most diverse group of plant pigments derived from the phenylpropanoid pathway, ranging in color from red to violet and blue [53]. These pigments accumulate in the vacuoles and their stability and hue depend on conditions such as pH, enzymatic activity and formation of complexes with metal ions [29,49]. Very little information has been reported on the biodegradation of natural dyes of the fruit juice or pellet extract in vitro. Conventional chemical and physical methods of dye decolorization are outdated because costs are high and they consume high amounts of chemicals and energy, other disadvantages are sludge formation and biomass accumulation [6,57]. Biological degradation of dyes included properties such as water solubility, large molecular weight and fused aromatic ring structures, which inhibit permeation through biological cell membranes. Other limitations of using microbes for treating pollutants were high costs of production of microbial culture, slow process of decolorization of dyes and metabolic inhibition [20,38]. Enzymatic systems fall between the two traditional categories of chemical and biological processes, since they involve chemical reactions based on the action of biological catalysts [5]. This was mainly because, unlike the chemical catalysts, the enzymatic catalysis showed its merits to convert complex chemical structures under mild environmental conditions with high efficiency [17,19,30]. Enzymes can act on specific recalcitrant pollutants to remove them by precipitation or transformation to other products [1,45]. Isolated enzymes were often preferred over intact organisms containing the enzymes because the isolated enzymes offered several advantages such as greater specificity, better standardization, easy handle, store and no dependence on bacterial growth rates [19,21]. Peroxidases (EC 1.11.1.7) are oxidoreductases which efficiently catalyze the oxidation of phenolic compounds and have great potential in treating a wide spectrum colored compounds [17,19,56,2,7]. These enzymes convert a broad range of substrates into less toxic insoluble compounds, which can be easily removed out of waste by a mechanism involving the formation
of free radical followed by insoluble product [17,51]. Peroxidase based dye treatment still provides a reasonable basis for the development of biotechnological processes for continuous color and aromatic compound removal from various industrial effluents at a large scale [18]. Sometimes these enzymes cannot act on organic pollutants and dye effluents due to the recalcitrant nature of such compounds. These recalcitrant substrates get converted into less toxic forms in the presence of certain low molecular weight compounds that are known as redox mediators which enhanced the rate of enzyme-catalyzed reaction and increased the range of selection of their substrates [17,3,25]. The present study has been made to investigate the potential of Ficus sycomorus latex peroxidase (POL) as compared to horseradish peroxidase (HRP), which represents the commercial peroxidase, in the decolorization of a wide spectrum synthetic dyes and natural dyes (Hibiscus sabdariffa flower color and pomegranate Punica granatum juice). Evaluation of the role of various low molecular weight redox mediators in the decolorization of the selected dyes mediated by the action of peroxidases is the second goal. 2. Materials and methods 2.1. Materials Bromophenol blue, catechol, vanilline, a-naphthol, L-histidine, methyl green, methylene blue, methyl orange were obtained from Sigma Chemicals Co. Azo-carmine, naphthylamine– azo-benzene, titan yellow were products of Fluka Co. Calyces of roselle (Hibiscus sabdariffa L.) and pomegranate (Punica granatum L.) were obtained from the local market. All other chemicals and reagents employed were of analytical grade and were used without any further purification. 2.2. Peroxidases We previously purified and characterized the peroxidases from F. sycomorus latex (POL) [33] and from Japanese horseradish roots (HRP) [32]. 2.3. Extraction of hibiscus pigments Roselle extract was routinely prepared by boiling 3 g of dried roselle petals for 3 min with 100 ml of distilled water. The extract was rapidly filtered through a four layers of cheez cloth and kept at 4 C [52]. 2.4. Extraction of Punica granatum pigments The method consisted of manually peeling the fruits, separating the seeds and extracting the juice by electric juice mixer, extract was rapidly filtered through a four layers of cheez cloth and kept at 4 C [31].
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases Table 1
97
Degree of decolorization of synthetic and natural dyes at different concentrations by POL and HRP.
Dyes
k (nm)
Decolorization (%) At different conc. of dyes (mg/l) with 0.25 unit of POL
Bromophenol blue Methyl green Methylene blue Methyl orange Azo-carmine Naphthylamine –azo-benzene Titan yellow Diethyl-p-phenylenediamine hydrochloride Hibiscus Punica granatum
430 630 662 502 507 485 405 515 530 510
HRP
100
50
20
100
50
20
11 22 14 7 0 10 10 25 27 30
43 53 39 29 12 30 34 54 57 60
73 80 74 66 30 70 72 85 80 86
15 19 10 5 2 10 9 21 18 26
48 49 36 22 13 33 33 50 40 51
75 80 71 60 33 72 72 80 73 82
The dye compounds at different concentrations (100–20 mg/l) were treated with each peroxidase (0.25 unit/ml) for decolorization and the mixture included 50 mM sodium acetate buffer, pH 5.5 and 2 mM H2O2 and incubated for 6 h at room temperature. Each value represents the average of two experiments.
100
POL+Bromophenol blue
Color removal (%)
Color removal (%)
100 80 60
None V H N C
40 20 0
0
5
10
15
20
HRP+Bromophenol blue
80 60
None V H N C
40 20
25
0
0
5
10
Time (h) 100
80 60 Non
40
V H N C
20 0
0
5
10
15
20
80 60 None V H N C
40 20 0
25
0
5
60 None
40
V H N C
20 5
10
15
Time (h)
20
25
Color removal (%)
Color removal (%)
100
POL+Methylene blue
0
10
15
20
25
Time (h)
80
0
25
HRP+Methyl green
Time (h) 100
20
POLI+Methyl green
Color removal (%)
Color removal (%)
100
15
Time (h)
HRP+Methylene blue
80 60 None V H N C
40 20 0
0
5
10
15
Time (h)
20
25
Figure 1 Time course of decolorization of synthetic and natural dyes at concentration 20 mg/l by POL and HRP (0.25 U/ml) in the absence and presence of 0.6 mM redox mediators [vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C)]. The mixture included 50 mM sodium acetate buffer, pH 5.5, 2 mM H2O2 and incubated for different time intervals (1–24 h) at room temperature. Each value represents the average of two experiments.
98
A.M. Abdel-Aty et al.
2.5. Peroxidase assay Peroxidase activity was carried out according to [59]. The reaction mixture contained in one ml: 8 mM H2O2, 40 mM guaiacol, 50 mM sodium acetate buffer, pH 5.5 and 0.25 unit of enzyme. Assays were carried out at room temperature. The change of absorbance at 470 nm due to guaiacol oxidation was followed at 30 s intervals. One unit of peroxidase activity is defined as the amount of enzyme which increases the O.D. 1.0 per min under standard assay conditions. 2.6. Dye decolorization by POL and HRP The selected dyes (bromophenol blue, methyl green, methylene blue, methyl orange, azo-carmine, naphthylamine–azo-benzene, titan yellow, diethyl-p-phenylenediamine hydrochloride, and natural dyes extracted from hibiscus and pomegranate) at concentration of 100–20 mg/l were prepared in distilled water. Each dye was incubated with 0.25 unit/ml of POL or HRP in 50 mM sodium acetate buffer, pH 5.5 and 2 mM H2O2 for different time intervals (1–24 h) at room temperature. Dye
60 None V H N C
40 20
0
5
The rate of decolorization of dyes by POL and HRP in the presence of 0.2–2 mM of each redox mediator (vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C)). was tested under the same conditions.
3. Results and discussion Previously, we reported that POL and HRP vary with respect to their potential to catalyze the oxidation of various electron donor substrates (e.g. phenolic compounds) by H2O2 [33,32].
100
POL+Methyl orange
80
0
2.7. Effect of redox mediators on rate of the decolorization
Color removal (%)
Color removal (%)
100
decolorization was monitored at the visible absorbance maximum of each dye (Table 1). Control experiments were performed using the same condition without enzymes. The color removal was reported as decolorization (%) = (A0 At)/A0 · 100. where A0 is the absorbance of untreated dye as control; At is the absorbance of treated dye with the enzyme.
10
15
20
80 60 40
0
25
None V H N C
POL+Azo-Carmine
100 Color removal (%)
Color removal (%)
0
5
10
60 40
20
25
None V H N C
HRP+Azo-Carmine
80 60 40 20
20
0
0
5
10
15
20
25
0
5
10
100
POL+Naphthylamine-azo-benzene
80 60 None V H N C
40 20 0
0
5
10
15
20
25
Color removal (%)
100
15
20
25
Time (h)
Time (h)
Color removal (%)
15
Time (h)
80
0
None V H N C
20
Time (h) 100
HRP+Methyl orange
HRP+Naphthylamine-azo-benzene
80 60 None V H N C
40 20 0
0
Time (h)
5
10
15
Time (h)
Figure 1 (continued)
20
25
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases 100 Color removal (%)
Color removal (%)
100 POL+Titan yellow 80 60 40
None V H N C
20 0
0
5
10
15
20
HRP+Titan yellow
80 60 None V H N C
40 20 0
25
0
5
10
60 None V H N C
40 20 0
5
10
15
20
Color removal (%)
Color removal (%)
100
POL+Diethyl-p-phenylenediamine hydrochloride
80
0
POL+Punica granatum
60 None V H N C
40 20 0
0
5
10
20 5
10
15
20
Color removal (%)
Color removal (%)
None V H N c
40
60 None V H N C
40 20 0
0
5
60 None V H N C
40 20 0
0
5
10
15
20
25
Color removal (%)
Color removal (%)
100
80
25
80
25
HRP+Hibiscus
20
HRP+Punica granatum
10
15
Time (h)
Time (h)
100
15
Time (h)
100
60
0
25
80
25
80
0
20
HRP+Diethyl-p-phenylenediamine hydrochloride
Time (h) 100
15
Time (h)
Time (h)
100
99
20
25
POL+Hibiscus
80 60 None V H N C
40 20 0
0
5
10
15
20
25
Time (h)
Time (h) Figure 1 (continued)
In this study the ability of these enzymes to decolorize eight synthetic and two natural dyes was reported. The dyes used in this study including: firstly, synthetic dyes such as (i) triphenylmethane dyes (bromophenol blue and methyl green), (ii) heterocyclic dyes (methylene blue), (iii) azo dyes (methyl orange, azo-carmine, naphthylamine–azo-benzene and titan yellow), (iv) diethyl-p-phenylenediamine hydrochloride, and secondly, natural dyes extracted from hibiscus and pomegranate.
The effect of dye concentration (20–100 mg/l) on the rate of decolorization by POL or HRP is shown in Table 1. The highest decolorization percent ranging from 30% to 86% was reported at 20 mg/l for all tested dyes treated with POL and HRP. Both the enzymes had low decolorization % for azo-carmin (30–33%) at 20 mg/l of dye concentration. A similar decolorization % for the all tested dyes by POL and HRP was detected. In contrast, synthetic non-textile
100 Table 2
A.M. Abdel-Aty et al. Degree of decolorization of synthetic and natural dyes in the absence and presence of redox mediators by POL and HRP.
Dyes
Decolorization (%) POL
Bromophenol blue Methyl green Methylene blue Methyl orange Azo-Carmine Naphthylamine –azo-benzene Titan yellow Diethyl-p-phenylenediamine hydrochloride Hibiscus Punica granatum
HRP
None
V
H
N
C
None
V
H
N
C
73 80 74 66 30 70 72 85 80 86
73 87 80 75 45 77 64 86 77 86
84 81 60 73 61 71 71 80 76 86
80 92 55 80 65 70 83 84 76 86
70 81 91 83 44 66 56 91 91 93
75 80 71 60 33 72 72 80 73 82
77 84 78 70 44 61 60 83 72 82
88 82 55 70 60 61 73 83 73 83
81 84 50 75 62 72 82 84 71 84
74 87 85 70 40 66 66 88 84 88
Each dye compound (20 mg/l) was treated with each peroxidase (0.25 unit/ml) for decolorization in the absence and presence of mediating property by using 0.6 mM of each redox mediator [vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C)]. The mixture included 50 mM sodium acetate buffer, pH 5.5 and 2 mM H2O2 and incubated for 6 h at room temperature. Each value represents the average of two experiments.
dyes were recalcitrant to decolorization by Bitter gourd peroxidase [4]. During decolorization treatments, both natural dyes and titan yellow formed precipitates which settled down and were removed by centrifugation. Several investigators have shown that the treatment of phenols and aromatic amines by peroxidases resulted in the formation of large insoluble aggregates [20,54,50,16]. Anthocyanins, as natural dyes, were also oxidized by peroxidases that they may undergo polymerization and thus go out of solution by forming precipitates [60]. Peroxidases are among the enzymes that can use anthocyanins as substrates [26,27]. The results showed a change in color of hibiscus and Punica granatum extractions from red to brown during the first min of incubation, then the color was removed gradually by increasing the time of incubation suggesting the involvement of peroxidases in change of color, in many instances becoming brownish, and the degradation of anthocyanins under oxidizing conditions provoked by exogenous application of H2O2 in such a way that they become colorless [60]. A peroxidase isolated from a Gamay grapevine cell culture degraded anthocyanins in solution in the grape fruit [10]. Peroxidases were also involved in loss of color in processed strawberries and with exogenous application of H2O2 on strawberry slices caused a more rapid decrease in anthocyanin concentration relative to non-treated slices [10]. In the present study, relatively low concentration of H2O2 (2 mM) was used in agreement with other studies using HRP [24], soybean peroxidase [12] and turnip peroxidase [15]. There were also many researches reporting that the negative effect of high H2O2 concentrations in the reaction mixture resulted in the inactivation of peroxidases [57,36,9,22,58]. Fig. 1 shows the effect of time course of decolorization on the selected dyes treated with POL or HRP. Our observations have suggested that the decolorization of the dyes was continuously increased during the first 6 h of incubation and no change in decolorization % was observed up to 24 h for the almost selected dyes. The drop in the rate of dye degradation after an initial fast reaction could be due to inactivation of the enzyme. In several studies, dye removal was found to be dependent upon the reaction time when treated with peroxi-
dases as 90% of Orange II was degraded during 36 h [47], Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green dyes were completely degraded within 8 h [46]. The decolorization of dyes by POL and HRP in the presence of varying concentrations of redox mediators (0.2–2 mM), vanilline (V), L-histidine (H), a-naphthol (N) and catechol (C) was evaluated, where 0.6 mM gives the best result (Data not shown). This concentration was quite low and in agreement with that reported by other studies [4,44,23,28]. On the contrary, the redox mediators were used in very high concentrations at 5.7 mM as Violuric Acid (VLA)/laccase system, 11.0 mM 1-hydroxybenzotrizole (HOBT)/laccase system, 2.0 mM HOBT/laccase system and 2.0 mM HOBT/turnip peroxidase system [25,48,13]. The effect of redox mediators and incubation time on the decolorization of dyes treated with POL or HRP enzymes is also shown in Fig. 1. The decolorization of the dyes was also continuously increased during the first 6 h of incubation and no change in decolorization % up to 24 h was observed. The results at 6 h of incubation are summarized in Table 2. The enhancement of the decolorization of dyes by treatment with POL and HRP was reported in the presence of V (methyl green, methylene blue, methyl orange and azo-carmine), H (bromophenol blue, methyl orange and azo-carmine), N (bromophenol blue, methyl green, methyl orange, azo-carmine and titan yellow) and C (methylene blue, methyl orange, azo-carmine, diethyl-p-phenylenediamine hydrochloride, hibiscus and Punica granatum). However, POL and HRP enhanced the decolorization of naphthylamine–azo-benzene and methyl green in the presence of V and C, respectively. One hundred percent enhancement in decolorization was reported for azocarmine (from 30% to 60%) by using H and N treated with POL or HRP. This attributed to the diffusible redox mediators providing high redox potentials (>900 mV) to attack recalcitrant structural analogs and are able to migrate into the aromatic structure of the compound [8]. Nevertheless, C, N, H and V had no significant effect on the decolorization by POL and HRP for bromophenol blue, naphthylamine–azo-benzene, titan yellow and punica granatum, respectively. In contrast, the color removal effect by POL and HRP was decreased for
Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases titan yellow, methylene blue and naphthylamine–azo-benzene, diethyl-p-phenylenediamine hydrochloride and hibiscus in the presence of some redox mediators. During the enzymatic treatment, no precipitates were formed in the presence of redox mediators. This was attributed to the degradation of the aromatic ring of the compounds or by cleaving certain functional groups [7,25]. The efficiency of each mediator depended on the type, molecular structure of dye to be treated and enzyme inactivation [13,42] as in case of turnip peroxidase, [28] have shown that after addition of the mediator there was no significant effect on the decolorization of some dyes such as DR 23 and DR 239, while in DB 80 decolorization was continuously decreased with increase in the concentration of mediator. Also, among the six types of dyes, only reactive blue 38 was resistant to enzyme–mediator treatment. Acid blue 74, reactive blue 19 and aniline blue were partially decolorized by the enzyme alone, although decolorization was much more efficient and rapid in the presence of mediators, whereas reactive black 5 and azure B could be decolorized only in the presence of mediators [11]. It has been reported that the mediators could play a dual role, as mediator because they increased the rate of recalcitrant dye decolorization and acted as inhibitor for enzyme activity. 4. Conclusion Here, it has been shown that POL would be effectively employed for the decolorization of industrially important synthetic dyes and, for the first time, natural dyes. Our results suggested that, the peroxidase from Ficus sycomorus latex has shown its potential in decolorization of all selected dyes. More than 90% of Punica granatum, Hibiscus, Diethyl-p-phenylenediamine hydrochloride, methylene blue and methyl green dyes were decolorized by employing more effective and cheaper redox mediators with POL. These findings suggested that the POL could be extended to the large-scale treatment of textile effluents as a potential option of dye decolorization. References [1] S. Akhtar, Q. Husain, Chemosphere 65 (2006) 1228–1235. [2] S. Akhtar, A.A. Khan, Q. Husain, J. Chem. Technol. Biotechnol. 96 (2005) 1804–1811. [3] S. Akhtar, A.A. Khan, Q. Husain, Bioresour. Technol. 96 (2005) 1804–1811. [4] S. Akhtar, A.A. Khan, Q. Husain, Chemosphere 60 (2005) 291– 301. [5] Y. Anjaneyulu, N.S. Chary, D.S.S. Raj, Rev. Environ. Sci. Biotechnol. 4 (2005) 245–273. [6] I.M. Banat, P. Nigm, D. Singh, R. Marchant, Bioresour. Technol. 58 (1996) 217–227. [7] A. Bhunia, S. Durani, P.P. Wangikar, Biotechnol. Bioeng. 72 (2001) 562–567. [8] R. Bourbonnais, M.G. Paice, FEBS Lett. 267 (1990) 99–102. [9] V. Brenda, A. Marcela, V. Rafael, Chem. Biol. 9 (2002) 555– 565. [10] A.A. Calderon, E. Garciaflorenciano, R. Munoz, A.R. Barcelo, VITIS 31 (1992) 139–147. [11] S. Camarero, D. Ibarra, M.J. Martınez, A.J. Martınez, Appl. Environ. Microbiol. 71 (2005) 1775–1784. [12] N. Caza, J.K. Bewtra, N. Biswas, K.E. Taylor, Water Res. 33 (1999) 3012–3018.
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