i need a rough draft.

669 © IWA Publishing 2012 Water Science & Technology | 65.4 | 2012

Obtention of plant peroxidase and its potential

for the decolorization of the reactive dye Remazol

Turquoise G 133%

Maria Cristina Silva, Juliana Arriel Torres, Angelita Duarte Corrêa,

Allana Maria Bernardes Junqueira, Maria Teresa Pessoa Amorim

and Custódio Donizete dos Santos

ABSTRACT

Peroxidases can be used in the decolorization process. There is a growing interest for new sources of

this enzyme and for obtaining economically viable processes. In this work, a low-cost vegetable

peroxidase extraction process is proposed; the resulting enzyme is characterized to determine its

optimum pH, temperature, and stability conditions, and it is then applied in the decolorization of

reactive dye Remazol Turquoise G 133%. The turnip peroxidase (TP) was utilized as an enzymatic

source. This enzyme exhibited maximum activity at pH 7.0, and it was active in the temperature

range of 30 to 50 WC, which favors its use in industrial processes. Acetone was the most efficient

solvent to induce precipitation. The removal of Remazol Turquoise G 133% was 56.0% complete after

50 min, while 41.0% of the same dye was removed with the commercial horseradish peroxidase

enzyme in 50 min. TP presents potential as a viable alternative in the decolorization of textile

wastewaters.

doi: 10.2166/wst.2012.892

Maria Cristina Silva Juliana Arriel Torres (corresponding author) Angelita Duarte Corrêa Allana Maria Bernardes Junqueira Custódio Donizete dos Santos Department of Chemistry, Federal University of Lavras, 37200-000, Lavras, MG, Brazil E-mail: julianaarriel@hotmail.com

Maria Teresa Pessoa Amorim Department of Textile Engineering, University of Minho, 4800-058 Guimarães, Portugal

Key words | decolorization, environmental biocatalysis, peroxidase, textile dyes, turnip

INTRODUCTION

The textile industry plays an important role in most countries, being one of the traditional industrial segments.

However, textile processes consume large quantities of water, chemical products and synthetic dyes, and they gener- ate large volumes of wastewater that contain a high organic load.

In general, it is believed that approximately 20% of the dye load is lost in the dyeing residues during textile proces- sing, which represents one of the great environmental

problems faced by the sector (Guarantini & Zanoni ). Considering that more than 700 thousand tons of dyes and pigments are produced annually in the world and that

Brazil is responsible for the consumption of about 2.6% (Zanoni & Carneiro ), the harmful effect of the liberation of dyes in the environment becomes quite significant.

When not treated appropriately, and when discharged in natural waters, the wastewaters originating from the dye industry or processes involving the dyeing of textile fibers

can modify the ecosystem, reducing the transparency of the water and the penetration of solar radiation, which

can reduce photosynthetic activity and the solubility of gases.

In general, the textile industry uses treatment systems based on physico-chemical and biological systems, which

in many cases are incompatible with the characteristics of the wastewater generated, mainly with respect to the removal of the color. From this point of view, the study of

new treatment alternatives is essential. The peroxidases (donor H2O2 oxidoreductase, E.C.

1.11.1.7) are enzymes that catalyze the reduction of hydro-

gen peroxide or another organic peroxide when an electron donor is oxidized. The reaction occurs in multiple stages, as shown below:

PeroxidaseþH2O2 ! Compound IþH2O (1)

Compound Iþ AH2 ! Compound IIþA� (2)

�

670 M. C. Silva et al. | Obtention of peroxidase and its potential for decolorization Water Science & Technology | 65.4 | 2012

Compound IIþAH2 ! PeroxidaseþAH þH2O (3)

In the first stage of the catalytic process, the reaction of the active site with hydrogen peroxide occurs. The hydrogen

peroxide is reduced, producing water and compound I, a reactive intermediate that has a higher oxidation state than the native enzyme. In the second stage of the reaction, com-

pound I oxidizes a substrate molecule (AH2), generating a substrate radical and compound II. Finally, a second sub- strate molecule reduces compound II, returning the enzyme to its initial form (Hiner et al. ).

This class of enzymes is widely distributed in the plant and animal kingdoms, and it is found in microorganisms, plants and mammals (Veitch ).

Peroxidase has been used in biotechnology and several other areas of science for the establishment of clinical diag- noses, in the evaluation of pathological processes, in food

quality analysis, in the construction of biosensors for quali- tative and quantitative analysis of pharmaceutical and cosmetic formulas and in paper and cellulose manufactur-

ing. In the environment, the water pollution index can be evaluated through tests using peroxidases. Furthermore, these enzymes can be used in the decolorization process to decrease textile industry pollutant residues (Maciel et al. , ).

Besides the use in decolorization processes, peroxidases can be used for the removal of phenolic compounds by oxi-

dizing them to phenoxy radicals, which then react to create less soluble hydrophobic polymeric products (Li et al. ). horseradish peroxidase has been utilized for the removal of

halogenated phenols (Meizler et al. ) and pentachloro- phenol (Li et al. ). Other peroxidases, such as peroxidases from Allium sativum, Ipomoea batatas, Rapha- nus sativus, Sorghum bicolor and soybean peroxidase have

also been applied to phenol removal (Al-Ansari et al. ; Diao et al. ).

The limitation on the use on plant peroxidases is the low

yield and high production cost compared with bacterial or fungal enzymes. However, the production cost of these enzymes can be reduced by optimizing the extraction con-

ditions and by using plant material with high enzymatic activity that can be directly applied with the same efficiency as the purified enzyme (Dec & Bollag ).

The peroxidases occur in the soluble and bonded form, associated with the cell walls of plant cells and probably associated with certain organelles (Khan & Robinson ). The soluble fraction can be extracted with water or

with a low ionic strength buffer, the ionically bonded

fraction with a high ionic strength buffer containing NaCl

or CaCl2, and the covalently bonded form using cellulolytic or pectinolytic enzymes (Vámos-Vigyázó ).

The use of additives is advisable in the extraction of

plant enzymes. The chemical compounds that are usually used include buffers, whose function is the protection of the enzymes from acids liberated from the vacuoles after the rupture of the cell and the desorption of the enzyme

bonded to the cell wall; PEG and PVP (polyvinylpolypyrro- lidone), which protect against inactivation by phenols and their oxidation products and EDTA as a chelator (Doonan

). The most commonly used commercial peroxidase source

is horseradish (horseradish peroxidase), which is usually cul-

tivated and harvested in cold climate countries (Maciel et al. ). Several reports in the literature point to the use of horseradish peroxidase in the degradation of several dyes: (1) Mohan et al. () obtained 79% degradation of

acid black dye with the horseradish peroxidase immobilized in acrylamide gel, and 67% with the free enzyme; (2) Ferreira-Leitao et al. () studied the degradation of

methylene blue dye by horseradish peroxidase. In this work, only 4.7% of the dye remained in solution, for a 1:10 pro- portion of dye/H2O2; and (3) Bhunia et al. () showed

that horseradish peroxidase can be effective in the degra- dation and precipitation of important industrial azo dyes.

Due to the widespread use of peroxidases, mainly as an

environmental biocatalyst, there is a growing interest in new sources of this enzyme.

In this context, the objectives of this work included obtaining a new vegetable source rich in peroxidase; the

determination of the optimum pH, temperature and stability conditions of this enzyme, which are important parameters to evaluate the potential application of the enzyme in indus-

trial processes; the evaluation of its potential in the degradation of the reactive dye Remazol Turquoise G 133%; and a comparison with the degradation potential of

the commercial horseradish peroxidase enzyme.

METHODS

Vegetables, commercial enzyme and dye

The following vegetables were used to obtain the peroxi- dase: turnip (Brassica campestre ssp. rapifera), radish

(Raphanus sativus), zucchini squash (Curcubita pepo), gilo (Solanum gilo Raddi) and sweet potato (Ipomoea

671 M. C. Silva et al. | Obtention of peroxidase and its potential for decolorization Water Science & Technology | 65.4 | 2012

potatoes (L.) Lam.). The fruits of the squash and gilo, the

roots of the turnip and radish, the tuber of the sweet potato, and the leaves and the peels of all the vegetables were used.

The horseradish peroxidase enzyme (HPR II) was acquired from Sigma Aldrich and used in aqueous solution (30 mg of the commercial enzyme in 20 mL of 0.05 mol L�1

phosphate buffer, pH 6.5). After the preparation, the

enzyme was stored at an average temperature of 4 WC.

Statistical analysis

The variance analysis of the results, when applicable, was made using SISVAR software (Ferreira ) and, when sig-

nificant, the Scott–Knott test, to 5% of probability, was applied for comparison of the averages.

Obtention of the raw enzymatic extract

The fruits, roots and tubers of the vegetables were properly washed in running water and distilled water. Soon after-

wards, 25 g of the peeled vegetable tissue was cut up and homogenized in a blender with 100 mL of 0.05 mol L�1

phosphate buffer (pH 6.5) for 30 s. The homogenate was fil-

tered through organza cloth and centrifuged at 10,000g for 15 min, at 4 WC. The supernatant solution was stored at 4 WC and used as the enzymatic source of the peroxidase

(Fatibello-Filho & Vieira ). The peels and leaves of the vegetables were washed in running water and distilled, cut into small uniform pieces, and subjected to the same extrac- tion procedure described previously.

The experimental design used was completely random- ized with 15 treatments and three repetitions of three vegetables.

Determination of the enzymatic activity

The activity was determined according to Khan & Robinson (), using the following reaction medium: 1.5 mL guaia- col (Vetec; 97%) 1% (v/v), 0.4 mL of H2O2 (Vetec, PA,

0.3% (v/v)), 0.1 mL enzyme (maintained in an ice bath) and 1.2 mL of 0.05 mol L�1 phosphate buffer (pH 6.5). The reaction was incubated for 5 min at 30 WC in a Spectrovision spectrophotometer that was coupled to a

thermostatic bath. One unit of peroxidase activity represents the oxidation

of 1 μmol of guaiacol in 1 min in the assay conditions, and

it was calculated using data relative to the linear portion of the curve.

Influence of additives on the enzymatic activity

The additives NaCl (0.2 mol L�1), KCl (0.2 mol L�1), PEG 4000 (polyethylene glycol, 2% (p/v)), PVP (insoluble polyvi-

nylpyrrolidone, 2% (p/v)) and EDTA (10 mmol L�1) were added separately into the extrator solution to evaluate their influence on the enzymatic activity (Holschuh ). The control was the activity in the absence of any additives.

The experimental design we adopted was completely randomized with five treatments and three repetitions.

Influence of pH on enzymatic activity and thermal stability

The optimum pH was determined by varying the pH of the buffer solutions from 2.0 to 9.0 in intervals of one pH unit. The buffers used were citrate buffer (0.1 mol L�1, pH 2.0 to 6.0) and Tris-HCl buffer (0.1 mol L�1, pH 7.0 to 9.0).

The experimental design that was adopted was comple- tely randomized with eight treatments and three repetitions.

The thermal stability of the peroxidase was evaluated by

incubating the enzyme for 1, 2 and 4 h, and by varying the temperature from 30 to 90 WC at intervals of 10 WC. After the thermal treatment, the samples were cooled in an ice

bath, and the residual activity was determined. As a control, the raw enzymatic extract activity without thermal treat- ment was determined. Three repetitions for each treatment

were carried out.

Precipitation of proteins

Precipitation by acetone

Cold acetone was added to the raw enzymatic extract until it

reached a concentration of 65% (v/v). After incubating for 12 to 14 h, at �18 WC, the homogenate was centrifuged at 11,000g for 15 min at 4 WC. The supernatant was discarded.

The precipitate containing the peroxidase was submitted to the removal of the acetone by evaporation in an ice bath for 3 h. The precipitate was re-suspended in 10 mL of

sodium phosphate buffer (pH 6.5), and the resulting suspen- sion was used for the determination of the enzymatic activity. The procedure was repeated three times.

Precipitation by ammonium sulfate

The precipitation by ammonium sulfate was performed

according to literature precedent (Zeraik et al. ), with some modifications. In the first stage, solid ammonium

Table 1 | Peroxidase activity of raw extracts from different plant tissues

Sample Plant tissues

Activity (U mL�1)*

Activity (U g �1

plant tissue)

Zucchini squash (Curcubita pepo)

Fruit 0.223 c 0.892 Peel 1.253 g 5.012 Leaves 0.845 e 3.380

Sweet potato (Ipomoea potatoes (L.) Lam.)

Tuber 0.105 b 0.420 Peel 0.812 e 3.248 Leaves 0.316 d 1.264

Gilo (Solanum gilo Raddi)

Fruit 0.275 d 1.100 Peel 0.159 c 0.636 Leaves 0.189 c 0.756

Turnip (Brassica campestre ssp. rapifera)

Root 1.080 f 4.320 Peel 1.487 h 5.948 Leaves 0.024 a 0.096

Radish (Armoracia rusticana)

Root 0.225 c 0.900 Peel 1.039 f 4.156

*Averages followed by the same letter in column do not differ among themselves by

Scott–Knott test at 5% probability.

672 M. C. Silva et al. | Obtention of peroxidase and its potential for decolorization Water Science & Technology | 65.4 | 2012

sulfate was added to the extract, so as to obtain a 40% satur-

ation. At this point, a clarification of the extract solution is observed, originating from the precipitation of the cyto- plasmic and nuclear materials and/or proteins present.

That solution was maintained at 4 WC for 20 h and centri- fuged at 8,000g for 10 min, at 4 WC. The precipitate was discarded, and more solid ammonium sulfate was added to the supernatant to reach an 85% saturation. The solution

was maintained at 4 WC for an additional 20 h. The super- natant was separated from the precipitate by centrifugation at 8,000g for 10 min at 4 WC, and at this stage, the super-

natant was discarded, and the precipitate was re- suspended in 5 mL of pH 6.5 sodium phosphate buffer.

The resulting suspension was dialyzed against a pH 6.5

sodium phosphate buffer for 24 h (32 mm benzoylated dialy- sis tubing with a cutoff range of 2 kDa, five daily changes, in a refrigerator and subjected to magnetic agitation) for the removal of the ammonium sulfate. The resulting suspension

was submitted for enzymatic activity determination. The procedure was conducted in triplicate.

Decolorization assays

Based on the methodology described by Khan & Robinson () with modifications, the enzymatic oxidation reactions of the textile dyes were conducted at 30 WC in 1.2 mL phos- phate buffer (0.05 mol L�1, pH 7.0) containing 0.4 mL H2O2,

1.5 mL Remazol Turquoise G 133%, and 0.1 mL of enzymatic solution. The final volume of reaction medium was 3.2 mL. The H2O2, enzyme and dye concentrations at middle were

100 μmol L�1, 20.3 U mL�1 and 50 mg L�1, respectively. The plant enzymatic extract that is considered most

appropriate for use in the oxidation of the dyes (which pre-

sents higher enzymatic activity) was treated with acetone to induce precipitation as described, and used in the decolori- zation assays.

The reaction mixture was incubated in a spectropho- tometer that was coupled to a thermostatic bath, and the absorbance of the dye was measured at different times during the experiments. Monitoring of the oxidation was done at

624 nm, the maximum wavelength for Remazol Turquoise G was 133%. The calculation to determine the percentage of color removal of the dyeswas done according to the equation:

absorbancyinitial � absorbancyfinal absorbancyinitial

× 100

To evaluate the dye adsorption by the enzymatic extract, the reaction medium containing 1.6 mL phosphate buffer

(0.05 mol L�1 pH 7.0), 1.5 mL of the Remazol Turquoise G

133% dye (50 mg L�1) and 0.1 mL of the enzymatic extract (12.18 U mL�1) was incubated in a spectrophotometer at 30 WC for 2 h and the dye removal was analyzed at 624 nm.

RESULTS AND DISCUSSION

Obtention of a vegetal source rich in peroxidase

Among the vegetable sources that were analyzed, the turnip peel (Brassica campestre ssp. rapifera) provided higher enzy- matic activity, while the turnip (Brassica campestre ssp.

rapifera) and radish leaves (Armoracia rusticana) presented lower activity (Table 1). It was also observed that the turnip root constitutes a rich peroxidase source; therefore, the

turnip was adopted (peel and root) as the main source of the enzyme. All of the subsequent assays were conducted using this enzymatic source.

Influence of additives on peroxidase activity

The influence of the additives on the peroxidase activity was investigated. There was significant variation in the activity of

the peroxidase obtained from the turnip extract (PET) under the influence of additives (Table 2). The extractor solution containing NaCl (0.2 mol L�1) induced a higher increase of the enzymatic activity, followed by KCl (0.2 mol L�1)

and EDTA (10 mmol L�1), while 2% (p/v) PEG and 2% (p/v) PVP resulted in a decrease in the activity.

Table 2 | Influence of additives on the activity of peroxidase, obtained from the raw

extract of radish*

Additives Activity (U mL�1)*

Activity (U g�1 plant tissue)

PEG (2%) 0.248 a 0.992

PVP (2%) 0.262 a 1.048

Control (without additives)

0.330 b 1.320

EDTA (10 mmol L�1) 0.398 c 1.592

KCl (0.2 mol L�1) 0.473 d 1.892

NaCl (0.2 mol L�1) 0.776 e 3.104

*Averages followed by the same letter in the column do not differ among themselves by

Scott–Knott test at 5% probability.

Figure 1 | Effect of pH on peroxidase activity obtained from the crude extract of turnip.

Figure 2 | Influence of temperature and incubation time on the stability of peroxidase

obtained from the crude extract of turnip.

673 M. C. Silva et al. | Obtention of peroxidase and its potential for decolorization Water Science & Technology | 65.4 | 2012

Several studies describe a significant improvement in the activityand stabilityof enzymeswhen theextractionprocedure is accomplished in the presence of additives. Additives have been used for protection from enzyme inactivation, retention

of the water layer around the biocatalyst and as enzyme molecule dispersers and mass transport facilitators. The inter- actionof the additivewith the enzymecanpresent antagonistic

behavior; that is, the interaction can present a negative effect in the reaction of interest, decreasing the efficiency of enzyme, as was observed for the 2% (p/v) PEG and 2% (p/v)

PVP additives. Soares et al. () affirms that not all of the additives are efficient stabilizers, and the influence of the addi- tive on the enzymatic activity still has not been totally clarified.

Influence of pH on enzyme activity and thermal stability

The PET activity showed significant variation as a function

of the pH. PET presented high activity at pH 7.0 (Figure 1). In acidic pH, the peroxidase presented an activity

decrease. This activity decrease might have occurred

mainly by ionic alterations of the enzyme that alter the form of the enzyme and consequently the active site. The activity decrease can also be observed at pH 9.

The pH for the maximum peroxidase activity varies with the enzyme source, the isoenzyme composition, the donor substrate, and the buffer used for the analysis

(Vámos-Vigyázó ). pH values in the range of 5.7 to 5.9 were found for the

carom peroxidase, using guaiacol as the substrate (Holschuh ). The peroxidase of the leaves of Copaifera langsdorffii (Diesel Tree) presented high activity in the pH range of 5.5 to 6.0 (Maciel et al. ).

The study of the effect of temperature on the PET stability

showed that the enzyme is thermostable up to 40 WC after 1, 2 and 4 h of incubation (Figure 2). At 50 WC, a small activity

decreasewas observed after 2 and 4 h of incubation. The enzy-

matic activitywas lost completely at temperatures above 70 WC. The peroxidase stability is very important for its use in

various areas of the sciences. The higher the stability and

enzymatic activity, the better the enzyme application capacity in diverse methods such as application as biocatalysts (Maciel et al. ). According to the results obtained in a temperature range of 30 to 50 WC, the resulting

enzyme did not present a significant decrease in the enzy- matic activity, which favors its use in industrial processes.

Precipitation of the proteins

The most efficient precipitating agent was acetone, which led to a 94.48± 0.62% recovery in terms of the enzyme activity,

while theammoniumsulfate gaveonly 64.85± 6.49%recovery of activity. Besides providing high recovery, the precipitation

674 M. C. Silva et al. | Obtention of peroxidase and its potential for decolorization Water Science & Technology | 65.4 | 2012

with acetone does not require dialysis, thus reducing the dur-

ation and cost of the process, which yields the enzyme through aneconomically simple and viable process. Therefore, precipitationwith acetone to yield the enzymewas performed.

Figure 3 | Removal of the Remazol Turquoise G 133% dye catalyzed by the commercial

enzyme horseradish peroxidase (HPR) and by peroxidase of Brassica cam-

pestris ssp. rapifera (PET).

Decolorization of Remazol Turquoise G 133% enzymatic dye catalyzed by horseradish peroxidase (HPR) and turnip peroxidase

The Remazol Turquoise G 133% dye is widely used by tex- tile industries. It belongs to the class of reactive dyes and

contains a monofunctional group and vinyl sulfone as the reactive group.

The decolorization of the dye in aqueous solution catalyzed by horseradish peroxidase (22 U mL�1) was 41%

after 50 min in the presence of the enzyme, while when catalyzed by the peroxidase of Brassica campestre ssp. rapifera (20.3 U mL�1), 56% of the dye was degraded

under the same assay conditions (Figure 3). Similar results were found by Souza et al. () who

obtained59%ofRemazolTurquoiseG133%dyedecolorization

in aqueous solution with HPR under the following conditions: a dye concentration of 100 mg mL�1, 29.85 U mL�1 of HPR, 2 μmol L�1 of H2O2 and a pH between 4.0 and 5.0 at 30 WC.

There was no adsorption of the dye by the enzymatic

extract because in the absence of hydrogen peroxide, there was no dye reduction. This situation indicates that the deco- lorization occurs exclusively as a function of the catalytic

activity of the enzyme. Considering the kinetics of biodegradation of dye

Remazol Turquoise G 133% by turnip peroxidase and horse-

radish peroxidase enzymes, the pseudo-constant kinetics were calculated for both enzymatic reactions, in accordance with Barreto and coworkers ().

Data obtained (Table 3) suggested that both enzymatic reactions (Table 1), follow a pseudo-second-order kinetics. The results also showed that the reaction catalyzed by turnip peroxidase presented the higher pseudo-constant kin-

etic when compared with horseradish peroxidase.

Table 3 | Kinetic parameters calculated for biodegradation of the Remazol Turquoise G 133%

Enzyme Pseudo-order

Turnip peroxidase (TP) Zero Pseudo-first order Pseudo-second order

Horseradish peroxidase (HRP) Zero Pseudo-first order Pseudo-second order

Similar results were found by Barreto and coworkers (). The degradation of Procion Yellow and Procion Blue dyes by Ganoderma sp. resulted in the following pseudo-

constant kinetics: 0.31 and 0.43 × 10�3 h�1, respectively. The peroxidase of Brassica campestre ssp. rapifera turnip

peroxidase presented a degradation potential for this dye that

was superior to HPR without the need for purification. The elimination of purification steps decreases the cost to obtain the enzyme, enabling it to be used as an economically

viable alternative in the treatment of textile wastewaters. Many treatments can be efficient in the decolorization,

but it is essential to know if there is formation of toxic

products during the process. Previous studies showed that there was an increase in toxicity after enzymatic decoloriza- tion of Remazol Turquoise G 133% by horseradish peroxidase horseradish peroxidase (Forgiarini ). These

data suggest the formation of degradation products more toxic than the parental molecule. The increased toxicity may also be ascribed to the release of the Cu2þ ions of the

dye structure (Forgiarini ). Therefore, this fact shows the importance of toxicological evaluation after enzymatic treatment. In this case, when the metabolites formed are

dye by TP and HRP

Equation R2 K/10�3 h�1

y¼�15.957� 0.2805x 0.864 280.5 y¼�0.3697� 0.0103x 0.909 10.3 y¼ 0.0084þ 0.0004x 0.946 0.4

y¼�7.491� 0.2861x 0.964 286.1 y¼�0.1496� 0.0082x 0.9812 8.2 y¼ 0.0028þ 0.0002x 0.9928 0.2

675 M. C. Silva et al. | Obtention of peroxidase and its potential for decolorization Water Science & Technology | 65.4 | 2012

more toxic than the parent molecule, the enzymatic treat-

ment should be considered as pre-treatment.

CONCLUSIONS

The enzyme obtained presented optimum activity at pH 7.0

and proved thermally stable. The extraction solution contain- ing NaCl 0.2 mol L�1 provided an increase in enzyme activity.

The turnip peroxidase was capable of removing up to

56% of the Remazol Turquoise G 133% dye in aqueous sol- ution after 50 min of reaction. While the decolorization obtained for horseradish peroxidase was 41% under the

same conditions. The efficiency of turnip peroxidase in the oxidation of the

dye was comparable with horseradish peroxidase, a commer-

cial enzyme generally utilized in discoloration processes.

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First received 30 June 2011; accepted in revised form 3 October 2011

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