Functional Suitability of Soluble Peroxidases from Easily Available Plant Sources in Decolorization of Synthetic Dyes

With the growing commercial availability of dyes, their range and scope of application is also expanding. Consequently, a large amount of unused dyes are released in the industrial effluents. Ninety percent of textile dyes entering modern activated sludge sewage treatment plants pass through unchanged. There is an intense environmental concern about the fate of these unbound dyes. These discharged dyes form toxic products and their strong color causes turbidity which even at very low concentrations has a huge impact on the aquatic environment. These synthetic reactive dyes bond covalently with fabric and contain chromophoric groups like anthraquinone, azo, triarylmethane etc. along with reactive groups viz., vinyl sulphone, chlorotriazine, trichloropyrimidine etc. (Sumathi & Manju, 2000; Keharia & Madamvar, 2003). Disperse dyes and acid dyes have low solubility in water. They are mainly used in the dyeing of polyesters and find minor use in dyeing cellulose acetates and polyamides. Azo dyes constitute the largest group of colorants used in industry. These dyes can be precipitated or adsorbed only in small amounts, while under anaerobic conditions they are cleaved by microorganisms forming potentially carcinogenic aromatic amines (Chung & Cerniglia, 1992). Several studies on decolorization of textile dyes used in industries have been conducted. Some non-textile dyes commonly present in industrial effluents have been studied. Dye removal from wastewaters with traditional physicochemical processes, such as coagulation, adsorption and oxidation with ozone is expensive, can generate large volumes of sludge and usually requires the addition of environmentally hazardous chemical additives (Chen, 2006). On the other hand, most of the synthetic dyes are xenobiotic compounds which are poorly removed by the use of conventional biological aerobic treatments (Marco et al., 2007a, b). Although, biodegradation appears to be a promising technology, unfortunately the analysis of contaminated soil and water has shown persistence of toxic pollutants even in the presence of microorganisms (Robinson et al., 2001; Keharia and Madamvar, 2003). Decolorization of dye wastewater is an area where innovative treatment technologies need to be investigated. The focus in recent times has shifted towards enzyme based treatment of colored wastewater/industrial textile effluents. The peroxidase and polyphenol oxidases

participate in the degradation of a broad range of substrate even at very low concentration. Further, these peroxidases and polyphenol oxidases have been used for treatment of dyes but large scale exploitation has not been achieved due to their low enzymatic activity in biological materials and high cost of purification (Bhunia et al., 2001;Shaffiqu et al., 2002;Verma & Madamwar, 2002). Bioremediation is a viable tool for restoration of contaminated subsurface environments. It is gaining importance due to its cost effectiveness, environmental friendliness and production of less sludge as compared to chemical and physical decomposition processes. Here too microbial treatment has certain inherent limitations (Husain & Jan, 2000;Duran & Esposito, 2000;Torres et al., 2003). It has been shown that peroxidases catalyze a variety of oxidation reactions and importantly dyes recalcitrant to peroxidase shows significant decolorization in the presence of Redox mediators (Calcaterra et al., 2008). Redox mediated enzyme catalysis has wide application in degradation of polycyclic aromatic hydrocarbons which includes phenols, biphenyls, pesticides, insecticides etc. Calcaterra et al., 2008).

Peroxidases in dye decolorization with Redox mediators
Enzymatic approach has gained considerable interest in the decolorization/degradation of textile and other industrially important dyes present in wastewater. This strategy is ecofriendly and useful in comparison to conventional chemical, physical and biological treatments, which have inherent serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. Several enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases and azoreductases) have been evaluated for their potential in decolorization and degradation of dyes. Although, some recalcitrant dyes are not degraded/ decolorized in the presence of such enzymes, the addition of certain Redox mediator enhances the range of substrates and efficiency of degradation of the recalcitrant compounds. However, very few Redox mediators are frequently used which includes 1hydroxybenzotriazole (HOBT), veratryl alcohol, violuric acid (VA), 2-methoxyphenothiazone. The enzymes in soluble form cannot be exploited on large scale due to limitations of stability and reusability and consequently, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.

Dye decolorization with turnip (Brassica rapa) proteins
The potential of partially purified turnip (Brassica rapa) proteins have been studied by Matto and Husain, (2007) on decolorization of certain direct dyes like Direct Red 23, Direct Red 239, Direct Blue 80 and Direct Yellow 4. The turnip proteins showed enhanced decolorization in the presence of Redox mediators. Redox mediators explored for dye decolorization of these dyes were HOBT, alpha naphthol, vanillin, L-histidine, VA, catechol, quinol, bromophenol, 4-nitrophenol and gallic acid. Six out of 10 investigated compounds showed their potential in enhancing the decolorization of direct dyes. The performance of each was evaluated at different concentrations of mediator and enzyme. The decolorization of all tested direct dyes was maximum in the presence of 0.6 mM Redox mediator at pH 5.5 and 30°C. Complex mixtures of dyes decolorized maximally in the presence of 0.6 mM Redox mediator (HOBT/VA). In order to examine the operational stability of the enzyme preparation, the enzyme was exploited for the decolorization of mixtures of dyes for different times in a stirred batch process. There was no change in decolorization of an individual dye or their mixtures after 60 min of incubation; the enzyme caused more than 80% decolorization of all dyes in the presence of 1-hydroxybenzotriazole/violuric acid. However, there was no desirable increase in dye decolorization of the mixtures on overnight incubation. The treatment of such polluted water in the presence of Redox mediators caused the formation of insoluble precipitate, which could be removed by the process of centrifugation. Such catalyzed oxidative coupling reactions may be important for natural transformation pathways for dyes and indicate their potential use as an efficient means for removal of dyes color from waters and wastewaters.

Dye decolorization with fenugreek (Trigonella foenum-graecum) seed proteins
Peroxidase from fenugreek (Trigonella foenum-graecum) seeds is highly effective in the decolorization of textile effluent. The role of six Redox mediators has been investigated for effective decolorization by FSP (Fenugreek Seed Proteins). The maximum decolorization of textile effluent was observed in the presence of 1.0 mM 1-hydroxybenzotrizole, 0.7 mM H 2 O 2 , and 0.4 U/ml of FSP in the buffer of pH 5.0 at 40°C in 2.5 h. The decolorization of textile effluent in a batch process by peroxidase is 85% in 5 h, whereas the complete decolorization of textile effluent by membrane entrapped FSP was observed within 11 h of its operation. The absorption spectra of treated effluent exhibited a marked diminution in the absorbance at different wavelengths compared to untreated effluent. The removal of colored aromatic compounds from wastewater by peroxidases is well-known. These enzymes are currently being employed for the treatment of aromatic compounds Husain et al., 2009). Most of the studies on dye decolorization have been done in a defined media or synthetic wastewater where a single dye or their mixtures are usually present. However, industrial effluents are more complex due to the presence of other contaminating substances along with colored compounds; under such conditions, the treatment of these pollutants is a difficult problem (Husain et al., 2010). Textile effluents alone are recalcitrant to the action of FSP, but in the presence of Redox mediators, they decolorized significantly. Many other aromatic pollutants such as aromatic amines, dyes and bisphenol A have already been oxidized by peroxidases in the presence of Redox mediators (Karim & Husain, 2009;Matto & Husain, 2008). The oxidation of effluent in the presence 1.0 mM HOBT is quite low compared to the Redox mediators used in earlier studies to treat various colored pollutants (Rodriguez-Couto et al., 2005;Matto & Husain, 2008). The high concentrations of Redox mediators may not be appropriate for wastewater treatment because of high cost of mediators or possibility of creating negative impacts on effluent toxicity or in the environment upon their disposal into receiving waters (Kurniawati & Nicell, 2007). The maximum decolorization of textile effluent with FSP was at 0.7 mM concentration of H 2 O 2 . The concentration of H 2 O 2 >1.2 mM acted as an inhibitor of peroxidase activity possibly by causing irreversible oxidation of enzyme ferri-heme group which is essential for its activity (Vazquez-Duarte et al., 2001). The differences in time course of removal of various dyes might be due to the structural barrier and the electron localization among them (Jauregui et al., 2003). The maximum decolorization (68%) of textile effluent was obtained by 0.4 U/ml of FSP. FSP catalyzed the decolorization of effluent over a wide range of pH with an optimum at pH 5.0. The www.intechopen.com maximum decolorization of effluent was at a temperature of 40°C. FSP successfully removed more than 85% color of the effluent in 5 h. After 5 h, the increase in the rate of effluent decolorization has not been recorded. Complete removal of color has been studied by using enzyme in a membrane bag. This type of reactor would increase the efficiency of dye decolorization because enzyme can be reused as it did not mix with the rest of the treated solution. Some workers reported that Pseudomonas putida CCRC14365 peroxidase was capable of removing phenol in hollow-fiber membrane bioreactor. P. putida fully degraded 2,000 mg/L of phenol within 73 h; even at a level of 2,800 mg/L, phenol could be degraded by more than 90% after 95 h of operation (Chung et al., 2004). In another report, a novel tube membrane bioreactor has been used for the treatment of an industrially produced wastewater arising in the manufacture of 3-chloronitrobenzene and nitrobenzene; in 1.7 h, over 99% of each 3-chloronitrobenzene and nitrobenzene from the wastewater was degraded (Livingston, 2004). For the decolorization and removal of colored compounds from textile effluent, spectral analysis is an important aspect to demonstrate a loss in these compounds after treatment with enzyme. The decrease in absorbance peaks in the UV-visible region is a strong evidence for the decolorization of aromatic pollutants from wastewater. Some earlier spectral analyses of the enzymatically treated aromatic amines and their mixtures have shown a significant decrease in absorbance (Kurniawati & Nicell, 2007). There was 80% decolorization of the chromophoric groups and a significant reduction in the peak associated with the aromatic ring. The UV-visible absorbance spectrum for diluted textile effluent has been taken before and after treatment by FSP in the presence of various Redox mediators. The treatment of effluent by FSP in the presence of Redox mediator, HOBT, produces insoluble aggregates which can successfully be removed by centrifugation. The peroxidase from fenugreek seeds has potential in the remediation of hazardous aromatic pollutant and its use could be extended to the large-scale treatment of textile effluents and other related aromatic compounds by employing more effective and cheaper Redox mediators.

Dye decolorization with Horse Radish Peroxidase (HRP)
HRP is extracted from horse radish roots, and its performance has been evaluated in soluble and immobilized form by conducting batch experiments in the presence of H 2 O 2 (Vasantha et al., 2006). The oxidation of Direct Yellow 12 dye has been tested as a function of HRP at fixed concentration of H 2 O 2 , and at constant HRP activity (1.8 units/ml). The optimum contact time required for dye removal was 1 h 45 min for vials containing 5 ml of dye solution (10 mg/L), 1.8 units of enzyme 1.5 ml/L of H 2 O 2 were added and the reaction mixture (24°C, pH 4) was agitated for 2 h 15 min. After this period, dye removal was negligible up to remaining 1 h 35 min. The Direct Yellow 12 dye at varying aqueous-phase pH of the reaction mixture between 2 and 10 by keeping the dye concentration at 10 mg/L, enzyme concentration at 1.8 units, H 2 O 2 dose at 1.5 ml/L, reaction temperature at 24°C and the contact time (1 h 45 min) constant exhibited 70% of the dye removal at an aqueous-phase pH of 4. The dye removal dropped significantly from pH 5 to 8 and the same trend continued up to an aqueous phase of pH 10. Aqueous phase of pH 4 resulted in higher HRP activity compared to other pH ranges from 3 to 9. Hydrogen peroxide acts as a co-substrate to activate the enzymatic action of peroxide radical. It contributed in the catalytic cycle of peroxidase oxidizing the native enzyme to form an enzymatic intermediate which accepts the aromatic compound to carry out its oxidation to form a free radical form. Experiments were carried out to find out the optimum H 2 O 2 concentration required to bring out the conversion of dye by varying the H 2 O 2 dose from 1 to 3 ml/L in the reaction mixture by keeping all the other experimental conditions constant (dye concentration 10 mg/L; temperature 24°C; enzyme concentration 1.8 units; reaction time 1 h 45 min). Their findings indicate maximum dye removal at H 2 O 2 concentration of 2 ml/L and similar degradation with 2.5 and 3 ml/L; therefore, 2 ml/L was taken as the optimum H 2 O 2 dose for dye removal (Vasantha et al., 2006). The concentration of the substrate present in the aqueous phase significantly influences enzyme-mediated reaction. If the amount of enzyme concentration is kept constant and the substrate concentration is gradually increased the reaction will increase until it reaches maximum. After obtaining the equilibrium state, further addition of the substrate will not change the rate of reaction. Studies carried out at different concentrations of the dye, i.e. 5-40 mg/L, keeping the other parameters constant indicated a dye concentration of 25 mg/L to be the cut-off concentration of the dye for optimum removal at the specified experimental conditions along with retaining the activity of HRP enzyme and to protect the protein from denaturation. Application of free enzyme in industrial process is not economically viable, while immobilization/ entrapment of enzyme results in repeated application. Two types of polymeric materials-alginate and acryl amide-have been used to study their relative efficiency in dye removal for the entrapment of peroxidase. Normally, enzyme immobilization is expected to provide stabilization effect restricting the protein unfolding process as a result of the introduction of random intra-and intermolecular crosslinks. Zille et al. (2003) have reported less availability of the enzyme for interaction with anionic dyes due to the immobilization in a particular matrix. For the immobilization of HRP enzyme acryl amide gel was more efficient in dye removal compared to alginate matrix. About 78% of dye removal was observed with acryl amide gel-immobilized beads, while with alginate matrix it was only 52%. Gel-immobilized HRP was effective in dye removal compared to free HRP (69%), and alginate-immobilized HRP showed inferior performance. The effect of aqueous-phase pH on the enzyme-catalyzed degradation with alginate-and acryl amide-immobilized HRP enzyme suggests that after pH 4 there is a decrease in the dye removal capacity for both types of the entrapped matrices. About 78% and 54% of the dye removal was observed at an aqueous phase of pH 4 for acryl amide and alginate-entrapped beads respectively. The relative inferior performance of alginate-immobilized HRP compared to acryl amide may perhaps be due to lesser availability of the peroxidase structure to the dye molecule in the alginate mix compared to acryl amide. The effective performance of acryl amide-entrapped beads may be attributed to the nonionic nature of the beads, which results in minimum modification of the enzyme properties and unaffected nature of the charged substrate as well as product diffusion. The electron-withdrawing nature of the azo linkages obstructs the susceptibility of azo dye molecules to oxidative reactions. Only specialized azo dye-reducing enzymes could degrade azo dyes. In addition, when Direct Yellow 12 dye was reduced using the enzymatic method, the oxidation capacity increased with increasing concentrations of HRP and H 2 O 2 at pH 4. Gel/alginate based enzyme immobilization reduced the azo dye. The immobilized enzyme beads could further be used two-three times for the removal of the same dye with lower efficiency. The application of enzyme-based systems in waste treatment is unusual, given that many drawbacks are derived from their use, including low efficiency, high costs and easy deactivation of the enzyme. www.intechopen.com

Dye color removal with Bitter Gourd (Momordica charantia) Peroxidase
Application of partially purified BGP (Bitter Gourd Peroxidase) in the decolorization of textile and other industrially important dyes has been explored. Simple ammonium sulphate precipitated proteins from bitter gourd were taken for the treatment of a number of dyes present in polluted wastewater (Akhtar et al., 2005a, b). Partially purified preparation of BGP was obtained by adding 20-80% ammonium sulphate and this preparation exhibited a specific activity of 99.0 EU (Enzyme units) of peroxidase/mg protein. Peroxidases from bitter gourd were highly stable against pH, heat, urea, water miscible organic solvents, detergents and proteolysis. The dye solution was stable upon exposure to H 2 O 2 or to the enzyme alone. The dye precipitation was due to H 2 O 2 -dependent enzymatic reaction, possibly involving free-radical formation followed by polymerization and precipitation.
Eight textile reactive dyes were tested with increasing concentration (0.133-0.339 EU of BGP/ml) of reaction volume for 2 h at 37°C. The decolorization of Reactive Red 120, Reactive Blue 4, Reactive Blue 160 and Reactive Blue 171 was continuously enhanced by adding increasing concentration of BGP. Reactive Blue 4 was completely decolorized with 0.399 EU of BGP/ml of reaction mixture in the absence of HOBT. Reactive Orange 4, Reactive Orange 86, Reactive Red 11 and Reactive Yellow 84 were recalcitrant to the BGP action. Four reactive textile dyes were treated by step-wise addition of enzyme, adding 0.133 EU of BGP/ml of reaction volume at each step after every 1 h, to the decolorizing solution. Each enzyme addition exhibited rapid disappearance of color. Reactive Blue 160 was completely decolorized after the third enzyme addition while Reactive Blue 4 was almost decolorized completely after the second enzyme addition. Reactive Blue 171 and Reactive Red 120 were decolorized to 56% and 39%, respectively after the third enzyme addition. However, Reactive Red 11, Reactive Orange 4, Reactive Orange 86 and Reactive Yellow 84 were recalcitrant to decolorization by BGP. Of the reactive dyes incubated with 0.266 EU of BGP/ml of reaction volume for increasing time period only four dyes decolorized on treatment with BGP for 1h at 37°C. Although more color disappeared on incubation for longer duration, the rate of decolorization was slow. All the four dyes were decolorized with varying percentages (30-90%). The decolorization of dyes with 0.266 EU of BGP/ml of reaction volume on incubation at 37°C for 4 h was 88% for Reactive Blue 4, 71% for Reactive Blue 160, 31% for Reactive Blue 171 and 28% for Reactive Red 120. Rest of the four dyes: Reactive Red 11, Reactive Orange 4, Reactive Orange 86 and Reactive Yellow 84 were fully recalcitrant to decolorization by BGP even after 4 h incubation with similar treatment. Thirteen different non-textile dyes were treated with 0.167 EU of BGP/ml of reaction volume at 37°C for 1h. Carmine, Methyl Orange, Methylene Blue, Coomassie Brilliant Blue G-250, Rhodamine 6G, Methyl Violet 6B, 1:2 naphthaquinone 4-sulphonic acid and Martius Yellow dyes were recalcitrant to decolorization by the BGP action or were slowly decolorized during the progress of the reaction. Maximum decolorization achieved by partially purified BGP was 53% for Naphthalene Black 12B, 57% for Coomassie Brilliant Blue R 250, 96% for Evans Blue, 51% for Eriochrome Black T and 86% for Celestine Blue. Twenty-one dyes used in this study were treated with BGP in the presence of 1.0 mM HOBT and 0.6 mM H 2 O 2 at 37°C. Presence of HOBT drastically enhanced the rate of decolorization of recalcitrant dyes. Reactive Orange 4, Reactive Red 11, Reactive Yellow 84 and Reactive Orange 86 were recalcitrant to decolorization in the absence of HOBT. However, these dyes were decolorized up to 98%, 80%, 70% and 78%, respectively by the action of 0.399 EU/ml of BGP in the presence of 1.0 mM HOBT at 37°C for 2 h. Non-textile dyes e.g., Carmine, Methyl Orange, Coomassie Brilliant Blue G250, Rhodamine 6G, Methylene Blue and Methyl Violet 6B, which were recalcitrant to decolorization by BGP in absence of HOBT, were almost completely decolorized in presence of 1.0 mM HOBT. However, 1:2-naphthaquinone 4-sulphonic acid was recalcitrant to decolorization even in the presence of HOBT. Decolorization of textile reactive dyes in the absence of HOBT occurred by the formation of precipitate, which settled down and removed by centrifugation. Several earlier investigators have shown that the treatment of phenols and aromatic amines by peroxidases and tyrosinases resulted in the formation of large insoluble aggregates. However, the decolorization of textile and other dyes by BGP in presence of 1.0 mM HOBT appeared without the formation of any precipitate. It suggested that the decolorization of dyes took place via degradation of aromatic ring of the compounds or by cleaving certain functional groups as reported elsewhere (Christian et al., 2003;Husain et al., 2009). HOBT could have a dual role, first as a mediator by increasing the substrate range of dyes for BGP and second enhancing the rate of oxidation. Complex mixtures of various reactive textile and other industrially important dyes have been studied by mixing three or four different dyes in equal proportions and incubated with 0.266 EU of BGP/ml in the presence of 1.0 mM HOBT and 0.6 mM H 2 O 2 for 1h at 37°C. Wave length maxima for each dye mixture were determined and decolorization of mixtures was monitored after the incubation period. All the mixtures decolorized by more than 80%. The decolorization rate of mixtures of dyes was slower than that of pure dye solution. This supports an earlier observation that the biodegradation of various phenols in the form of mixtures was quite slow compared to the independent phenol (Kahru et al., 2000). Bourbonnais and Paice (1990) described for the first time the use of Redox mediators by allowing laccase to oxidize non-phenolic compounds thereby expanding the range of substrates that can be oxidized by this enzyme. The mechanism of action of laccase mediator system has been extensively studied and it is used in the textile industry in the finishing process for indigo stained materials. Several workers have demonstrated that the use of Redox mediator system enhanced the rate of dye decolorization by several folds but these mediators were required in very high concentrations (5.7 mM violuric acid/laccase system, 11.6 mM of HOBT/laccase system) (Soares et al., 2001a,b;Claus et al., 2002). For the first time, it was shown that the decolorization of dyes by BGP was effective at very low concentration of HOBT (1.0 mM). The rate of non-textile dyes decolorization was also enhanced by 2-100 folds (Akhtar et al., 2005a). Several reports indicate the enhancement of laccase activity by free radical mediators; however the enhancement of peroxidase activity by a Redox mediator has been studied for the first time. Peroxidase/ Redox mediator system will prove a sensitive and an inexpensive procedure for the treatment of dyes present in complex mixtures/industrial effluent. Matto and Husain (2009) investigated the role of concanavalin A (Con A)-cellulose-bound tomato peroxidase for the decolorization of direct dyes. Cellulose was used as an inexpensive material for the preparation of bioaffinity support. Con A-cellulose-bound tomato peroxidase exhibited higher efficiency in terms of dye decolorization as compared to soluble enzyme under various experimental conditions. Both Direct Red 23 and Direct Blue 80 dyes were recalcitrant to the action of enzyme without a Redox mediator. Six compounds were investigated for Redox-mediating property. Immobilized peroxidase decolorized both www.intechopen.com dyes to different extent in the presence of all the used Redox mediators. However, 1hydroxybenzotriazole emerged as a potential Redox mediator for tomato peroxidase catalyzed decolorization of direct dyes. These dyes were maximally decolorized at pH 6.0 and 40°C by soluble and immobilized peroxidase. The absorption spectra of the untreated and treated dyes exhibited a marked difference in the absorption at various wavelengths. Immobilized tomato peroxidase showed a lower Michaelis constant than the free enzyme for both dyes. Soluble and immobilized tomato peroxidase exhibited significantly higher affinity for Direct Red 23 compared to Direct Blue 80.

Dye decolorization with Pointed Gourd (Trichosanthes dioica) Peroxidase
The effects of Trichosanthes dioica peroxidase along with Redox mediators on decolorization of water insoluble disperse dyes; Disperse Red 19 (DR19) and Disperse Black 9 (DB9) have been studied by Jamal et al., (2010). Nine different Redox mediators; bromophenol, 2, 4-dichlorophenol, guaiacol, 1-hydroxybenzotriazole, m-cresol, quinol, syringaldehyde, vanillin and violuric acid were evaluated [ Figure-1]. Among the chosen mediators, 1-hydroxybenzotriazole was most effective for decolorization with PGP (Pointed Gourd Peroxidase). At a concentration of 0.45U/mL the peroxidase could decolorize Disperse Red 19 to a maximum of 79% with O.2mM 1-hydroxybenzotriazole whereas Disperse Black 9 decolorized upto 60% with 0.5mM 1-hydroxybenzotriazole [ Figure-2]. The time, pH and temperature at which maximum decolorization were recorded was 60 min, 4 and 42°C. It has been observed that the dye solutions were recalcitrant upon exposure to HOBT, H 2 O 2 or to the enzyme alone but the enzyme in the presence of Redox mediators was much effective in performing the decolorization of the dyes, implying dye decolorization was a result of Redox mediated H 2 O 2 -dependent enzymatic reaction. It has already been reported that Redox mediators have the potential to mediate an oxidation reaction between a substrate and an enzyme.  [Jamal et al., 2010] www.intechopen.com Different Redox mediators have different mediation efficiency which is governed by Redox potential of the mediator and the oxidation mechanism of the substrate. Oxidation of substrate occurs by free radical formation by the mediator. The free radicals can be formed either by one-electron oxidation of substrate or by abstraction of a proton from the substrate. The pointed gourd peroxidase was effective in decolorizing the dyes at low concentrations of HOBT. Although the extent of decolorization of DR19 and DB9 increased with increasing concentrations of HOBT, the maximum decolorization was observed to be 79% and 60% with 0.2 mM and 0.5 mM for DR19 and DB9, respectively. Further addition of HOBT resulted in a slow decrease in decolorization of both the dyes. This inhibition could likely be due to the high reactivity of HOBT radical, which might undergo chemical reactions with side chains of aromatic amino acid by enzyme thereby; ina ct iv at ing i t . H enc e , the do sag e of R e dox me d iat or is an impor t ant fa ct or f or the enzyme-mediated decolorization. The enzyme reacted well to decolorize both the dyes in the presence of 0.8 mM H 2 O 2 [ Figure-3]. The maximum decolorization was obtained at 0.8mM H 2 O 2 which is slightly higher than reported for soybean peroxidase, bitter gourd peroxidase (BGP) and turnip peroxidase. The reaction temperature is an important parameter which effects the decolorization of dyes. The maximum decolorization for both the dyes DR19 and DB9 was at 42°C [ Figure-4]. It has been reported that BGP mediated disperse dye decolorization was optimal at 40°C.  It has earlier been reported that the degradation of industrially important dyes by enzymes such www.intechopen.com as horse radish peroxidase, polyphenol oxidase, BGP and laccase was also maximum in the buffers of acidic pH. DR19 and DB9 were maximally decolorized within 20 min of incubation [ Figure-6]. There was slow and gradual enhancement of decolorization upto 60 min of incubation. It was also evident from the observation that DR19 was decolorized to a greater extent within 20 min in the presence of only 0.2 mM HOBT. However, DB9 was decolorized maximally in the presence of 0.5 mM HOBT and decolorization rate was slow. This is consistent with reports that decolorization rate varies, depending upon the type of dye to be treated.

Trichosanthes dioica proteins in decolorizing industrially important textile, nontextile dyes and dye mixtures
In one of my recent works, the competency of Trichosanthes dioica proteins in decolorizing industrially important textile, non-textile dyes and dye mixtures in the presence of Redox mediators under varying experimental conditions of pH, temperature, time intervals and enzyme concentration on the basis of one-factor-at-a-time (OFAT) method has been studied. All the textile dyes and non-textile dyes, ammonium sulphate, and Tween-20 were p r o c u r e d f r o m S i g m a C h e m i c a l C o . ( S t . L o uis, MO, USA). Redox mediator's viz., 1-hydroxybenzotriazole (HOBT) and vanillin were obtained from SRL Chemicals (Mumbai, India). All other chemicals were of analytical grade. The pointed gourds were purchased from the local market. The samples were aseptically transferred into sterilized plastic bags. Briefly, 100 g of pointed gourd was homogenized in 180 ml of 100 mM sodium acetate buffer, pH 5.6. The homogenate was filtered through multi-layers of cheese cloth and then centrifuged at the speed of 10,000 × g on a Remi C-24 cooling centrifuge for 30 min at 4°C. The clear solution thus obtained was used for salt fractionation by adding 10% to 80% (w/v) (NH 4 ) 2 SO 4 . The proteins were precipitated by continuously stirring at 4°C overnight. The precipitate was collected by centrifugation at 10,000 × g on a Remi C-24 cooling centrifuge, dissolved in 100 mM sodium acetate buffer, pH 5.6 and dialyzed against the assay buffer (0.1 M glycine HCl buffer, pH 4.0) (Akhtar et al., 2005a). Protein concentration was estimated by taking BSA as a standard protein and following the procedure of Lowry et al (1951). Peroxidase activity was determined by a change in the optical density (A 460 nm) at 37°C by measuring the initial rate of oxidation of 6.0 mM o-dianisidine HCl in the presence of 18.0 mM H 2 O 2 in 0.1 M glycine-HCl buffer, pH 4.0, for 20 min at 37°C. One unit of activity was defined as the amount of enzyme that transformed 1 mol of o-dianisidine HCl as substrate per min. The dyes (45-210 mg/l) were solubilized in 100 mM glycine HCl buffer, pH 4.0. Each dye was independently incubated with pointed gourd peroxidase (PGP) (0.45 EU/mL) in 100 mM glycine HCl buffer, pH 4.0 in the presence of 0.80 mM H 2 O 2 for varying times at 37°C. The reaction was stopped by boiling at 100°C for 7 min. Dye decolorization was monitored by measuring the difference at the maximum absorbance for each dye as compared with control experiments without enzyme on UV-visible spectrophotometer (JASCO V-550, Japan). Untreated dye solution (inclusive of all reagents except the enzymes) was used as control (100%) for the calculation of percent decolorization. The dye decolorization was calculated as the ratio of the difference of absorbance of treated and untreated dye to that of treated dye and converted in terms of percentage.

Effect of Redox mediators on decolorization profile of textile and non-textile dyes
The effect of different Redox mediators on the dye decolorization by PGP is shown in Table-1. Out of the two different Redox mediators studied for dye decolorization, HOBT was more effective in decolorizing the dyes under study. The extent of decolorization in the presence of HOBT was in the range of 98.6% to 69.8% for the reactive dyes whereas the disperse dyes studied exhibited decolorization in the range of 79.2% to 61.2%. The effective HOBT concentrations were 1.0 mM and 0.2 mM for reactive and disperse dyes, respectively. The dye decolorization with vanillin was 71.2% to 60.2% for reactive dyes and 55.3% to 34.5% for disperse dyes at 1.0 mM concentration. Table-1 shows the effect of increasing concentrations of HOBT and vanillin (0.05 to 1.5 mM) on textile and other dyes. With increasing concentration of HOBT or vanillin there was an increase in the extent of decolorization of both dyes. However, the decolorization of each textile dye was lower at each of the varying concentration of vanillin than HOBT. The non-textile dyes were effectively decolorized at 1.0 mM of HOBT and vanillin independently but here too, influence of HOBT was substantial. There was not much effect in percent decolorization of the dyes above these concentrations of HOBT or vanillin.  -7b]. The decolorization profile for all the dyes significantly increased after the first increase in peroxidase concentration in a period of 2 h. There was no significant change on dye decolorization either on increasing peroxidase concentration or the incubation time.  Table-1 for max of each dye. Figure-8 shows that the percent decolorization improved with the increasing concentration of H 2 O 2 and the maximum decolorization was observed at a concentration of 0.8 mM and 1.0 mM of H 2 O 2 for disperse and reactive dyes respectively which remained substantially unaffected till 1.2 mM H 2 O 2 .  Table-1 for max of each dye.  Table-1 for max of each dye.

H 2 O 2 and pH activity profile of decolorization of textile dyes
To find out the range of pH in which significant decolorization was observed; buffers in the range of pH 2.0 to pH 10.0 were used. The percent decolorization is shown in [ Figure-9]. An acidic range of pH (3.0 to 6.0) was better suited for dye decolorization. Maximum www.intechopen.com decolorization was observed at pH 4.0 and pH 5.0 at fixed concentration of T. dioica peroxidase and HOBT for disperse and reactive dyes respectively. There was significant decrease in the extent of decolorization in an alkaline medium and at pH 10.0 the decolorization action of the enzyme was almost insignificant / lost. Fig. 9. Percent Dye Decolorization of Textile Dyes in the presence of fixed concentration of HOBT, T. dioica peroxidase enzyme (EU/ml) and varying pH. Please see Table-1 for max of each dye.

Temperature activity profile of decolorization of textile dyes
The percent decolorization was plotted as a function of temperature and the results are shown in [ Figure-10]. Among the textile dyes the reactive dyes exhibited maximum decolorization at 50°C whereas the disperse dyes showed maximum decolorization at 40°C in the presence of 1.0 mM and 0.2 mM HOBT. Reactive Blue 15 (96.1%), Reactive Orange 15 (94.4%), Reactive Red 4 (85.2%) whereas disperse dyes under study decolorized in the range of 61.2% to 79%.

Time activity profile of decolorization of textile dyes
The extent of decolorization of textile dyes as a function of time is shown in [ Figure-11]. Maximum decolorization of reactive and disperse dyes were observed within 1 h of incubation at 50°C and 40°C with 0.45 EU/ml of PGP and 1.0 mM and 0.2 mM HOBT respectively. However, further decolorization of these dyes progressed slowly upto 4 h, although no effective increase was observed even when the dyes were further incubated for longer times.   Table-1 for max of each dye.  Table-1 for   max of each dye. www.intechopen.com

Decolorization profile of non-textile dyes by T. dioica peroxidase as a function of enzyme concentration and time
Nine different non-textile dyes were studied. These dyes were treated with different amount of T. dioica peroxidase in the range of 0.065 EU/ml to 0.50 EU/ml and incubated for varying time interval with and without the Redox mediator HOBT (1.0 mM) at 37°C as shown in [

Decolorization profile of different dye mixtures by T. dioica peroxidase
To simulate the decolorization of dyes from industrial effluent, complex mixtures of textile dyes including reactive, disperse and non-textile dyes were prepared by mixing four different dyes in equal proportions and incubated with 0.45 EU/ml of T. dioica peroxidase in the presence of 1.0 mM HOBT and 1.0 mM H 2 O 2 for 2 h at 40°C. The decolorization was recorded at wave length maxima of each mixture determined spectrophotometrically. The combinations of different dyes showed decolorization by more than 82% [ Figure-12]. The rate of decolorization of dye mixture was slower in comparison to that of individual dyes both in the presence and absence of HOBT. However, the HOBT mediated dye decolorization was more effective. We have earlier reported the potential of a novel T. dioica plant proteins in the decolorization of disperse dyes (Jamal et al., 2010). In this paper we intended to widen the spectrum of industrially important textile dyes and included some well known non-textile dyes to study the extent of decolorization in the presence of Redox mediators. The cost of an enzyme depends on its degree of purity hence we opted to use ammonium sulphate precipitated proteins from T. dioica to study dye decolorization/degradation. The PGP was partially purified using 10% to 80% ammonium sulphate which retained a specific activity of 96 U/mg of protein. The experiments were performed at different enzyme concentration, pH, temperature, incubation period and Redox mediators namely, HOBT and vanillin. An interesting data profile was obtained for the assessment of this enzyme suitability to treat wastewater contaminated with these dyes. The reactive dyes underwent decolorization by the formation of precipitate which disappeared in the presence of 1.0 mM HOBT. This finding supports earlier reports that treatment of phenols and aromatic amines by peroxidases resulted in formation of large insoluble aggregates (Wada et al., 1995;Tatsumi et al., 1996;Husain & Jan, 2000;Duran & Esposito, 2000). The other dyes studied showed no formation of precipitate during decolorization in the presence of HOBT. This observation supports earlier view that decolorization of dyes took place via degradation of aromatic ring of the compounds or by cleaving certain functional groups (Akhtar et al, 2005a,b;Satar and Husain, 2009). All the other textile dyes showed insignificant or no decolorization with T. dioica peroxidase alone when studied under optimum conditions of dye decolorization in the absence of HOBT (data not shown).
The decolorization profile for all the dyes increased significantly at HOBT concentration of 0.2 mM [ Table-1]. Upon increasing the HOBT concentration further to 1.5 mM decolorization increased marginally. The reactive dyes under study exhibited decolorization maximally at 1.0 mM HOBT whereas disperse dyes showed maximum decolorization at lower values of HOBT suggesting that reactive dyes are comparatively more resistant to degradation. Vanillin was able to decolorize both the reactive and disperse dyes at 1.0 mM concentration but the extent of decolorization was sufficiently poor in comparison to HOBT for certain dyes like Reactive blue 15, Reactive orange 15 and most of the disperse dyes studied. Disperse Yellow 7 decolorized poorly (37.9%) with T. dioica peroxidase in the presence of relatively higher concentration of vanillin. The non textile dyes exhibited remarkable decolorization in presence of both the Redox mediators at 1.0 mM concentration. There was no significant change on dye decolorization of non textile dyes at concentrations above 1.0 mM. It has already been reported that Redox mediators have the potential to mediate oxidation reaction between a substrate and an enzyme (d' Acunzo et al., 2006). Different Redox mediators have different mediation efficiency which is governed by Redox potential of the mediator and the oxidation mechanism of the substrate (Baiocco et al., 2003). Oxidation of substrate occurs by free radical formation by the mediator. The free radicals can be formed either by one-electron oxidation of substrate or by abstraction of a proton from the substrate (Xu et al., 2001;Fabbrini et al., 2002). In this study, Redox-mediating property of two different compounds as peroxidase mediators was evaluated and extensive study on textile, non-textile and dye mixtures has been performed with HOBT.
Laccase have been used with Redox mediators to oxidize non-phenolic compounds (Bourbonnais & Paice, 1990). The mechanism of action of laccase mediator system has been extensively studied and it is used in the textile industry in the finishing process for indigo stained materials. Several workers have demonstrated that the use of Redox mediator system enhanced the rate of dye decolorization by several folds but these mediators were required in very high concentrations (Soares et al., 2001a,b;Claus et al., 2002). In the present study we have shown the decolorization of both textile and non-textile dyes as well as dye mixtures mediated by T. dioica peroxidase under the influence of low concentration of Redox mediators. The peroxidase reacted well to decolorize at concentration of 0.8 mM and 1.0 mM of H 2 O 2 for disperse and reactive dyes respectively and remained substantially unaffected till 1.2 mM [ Figure-8]. Although the concentration of H 2 O 2 greater than 0.75 mM acted as an inhibitor of peroxidase activity by irreversibly oxidizing the enzyme ferri-heme group essential for peroxidase activity (Satar and Husain, 2009) (Camarero et al., 2005).The enzyme could function better in an acidic medium of pH range 3-6, whereas its decolorizing/degrading activity was adversely affected in alkaline medium [ Figure-9]. This finding supports the earlier view of disperse dye decolorization in acidic medium by T. dioica and reports further that acid medium favours catalysis of reactive dyes too by this peroxidase. It has earlier been reported that the degradation of industrially important dyes by enzymes such as horse radish peroxidase, polyphenol oxidase, BGP and laccase was also maximum in the buffers of acidic pH (Galindo et al., 2000).
The reaction temperature is an important parameter which effects the decolorization of dyes. The maximum decolorization for both the reactive and disperse dyes were in the temperature range of 40°C to 50°C in the presence of fixed concentration of HOBT [ Figure-10]. The extent of decolorization was remarkable for Reactive Blue 15, Reactive Orange 15, Reactive Red 4 at 50°C whereas disperse dyes could be decolorized in the range of 61.2% to 79% at 40°C. The decolorization varies with the nature of the dyes but Redox mediated decolorization with T. dioica peroxidase was a better solution for effective decolorization of recalcitrant compounds. The rate of decolorization varied with time and maximum decolorization in the presence of HOBT was observed within one hour of incubation for Reactive Blue15 and Disperse Red 19 [ Figure-11]. However, for other dyes the extent of decolorization progressed slowly and reached a plateau after 4 h of incubation. This data is consistent with reports that decolorization rate varies, depending upon the type of dye to be treated (Camarero et al., 2005). The non-textile dyes studied for decolorization with T. dioica peroxidase exhibited enhanced decolorization in the presence of 1.0 mM HOBT, whereas in the absence of Redox mediator decolorization was much slower. Coomassie Brilliant Blue R250, Methylene Blue, Eriochrome Black T, Martius Yellow, Methyl Orange, Naphthol Blue Black and Rhodamine were extensively decolorized by T. dioica peroxidase under the influence HOBT [ Table-2].The decolorization of Evans blue was not significantly affected by the presence of Redox mediator, although in the presence of HOBT decolorization achieved was higher. The performance of this system was maximal during 160 min of incubation. Celestine blue was decolorized to 79.5% in the presence of HOBT at higher concentration of enzyme as well as longer incubation time. The data in [ Table-2] is suggestive of T. dioica peroxidase in conjunction with low concentration of HOBT to be wonderful decolorization/degradation system for non-textile dyes as well. Further, dye mixtures simulating industrial effluents exhibited more than 82% decolorization with 1-hydroxybenzotriazole [ Figure-12]. The rate of decolorization of dye mixture was slower in comparison to that of individual dyes both in the presence and absence of HOBT. The application of inexpensive peroxidase from easily available source can overcome the limitations in current wastewater treatment strategies. The use of peroxidases can be extended to large-scale treatment of wide spectrum of structural dyes by using immobilized PGP along with relatively cheaper Redox mediators. Thus, the study demonstrated that the peroxidase enzyme isolated from T. dioica can be coupled with Redox mediator into a system that can serve as an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dyeing and printing industries.

Future perspective
Dye wastewater discharged from textile and dyestuff industries needs to be treated due to their impact on water bodies and to address growing public concern over their toxicity and carcinogenicity. Many different and complicated molecular structures of dyes make dye wastewater treatment difficult by conventional biological and physico-chemical processes. Therefore innovative treatment technologies need to be investigated. The studies performed using peroxidases from different sources indicates that novel enzyme systems can be created to decolorize wide spectrum of textile, non-textile dyes and dye mixtures under varying set of conditions. The efficacy of decolorization drastically improves with Redox mediators and dyes/dye mixtures recalcitrant to peroxidase exhibited remarkable decolorization. The application of inexpensive peroxidases from easily available sources can www.intechopen.com overcome the limitations in current wastewater treatment strategies. The use of peroxidases can be extended to large-scale treatment of wide spectrum of structural dyes by using immobilized peroxidases along with relatively cheaper Redox mediators. The treatment of textile wet processing effluent to meet stringent governmental regulations is a complex and continually evolving process. Treatment methods that were perfectly acceptable in the past may not be suitable today or in the future. This book provides new ideas and processes to assist the textile industry in meeting the challenging requirements of treating textile effluent.