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Decolorization of the Textile Dyes Using Purified Banana Pulp PolyphenolOxidaseUmesh U. Jadhava; Vishal V. Dawkara; Mital U. Jadhava; Sanjay P. Govindwara

a Department of Biochemistry, Shivaji University, Kolhapur, India

Accepted uncorrected manuscript posted online: 11 November 2010

First published on: 19 February 2011

To cite this Article Jadhav, Umesh U. , Dawkar, Vishal V. , Jadhav, Mital U. and Govindwar, Sanjay P.(2011)'Decolorization of the Textile Dyes Using Purified Banana Pulp Polyphenol Oxidase', International Journal ofPhytoremediation, 13: 4, 357 — 372, First published on: 19 February 2011 (iFirst)To link to this Article: DOI: 10.1080/15226514.2010.495142URL: http://dx.doi.org/10.1080/15226514.2010.495142

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International Journal of Phytoremediation, 13:357–372, 2011Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2010.495142

DECOLORIZATION OF THE TEXTILE DYES USINGPURIFIED BANANA PULP POLYPHENOL OXIDASE

Umesh U. Jadhav, Vishal V. Dawkar, Mital U. Jadhav,and Sanjay P. GovindwarDepartment of Biochemistry, Shivaji University, Kolhapur, India

Polyphenol oxidase (PPO) purified using DEAE-cellulose and Biogel P-100 column chro-matography from banana pulp showed 12.72-fold activity and 2.49% yield. The optimumtemperature and pH were found to be 30◦C and 7.0, respectively for its activity. Catecholwas found to be a suitable substrate for banana pulp PPO that showed Vmax, 0.041 mMmin−1 and Km, 1.6 mM. The enzyme activity was inhibited by sodium metabisulfite, citricacid, cysteine, and β-mercaptoethanol at 10 mM concentration. The purified enzyme coulddecolorize (90%) Direct Red 5B (160 µg mL−1) dye within 48 h and Direct Blue GLL(400 µg mL−1) dye up to 85% within 90 h. The GC-MS analysis indicated the presence of4-hydroxy-benzenesulfonic acid and Naphthalene-1,2,3,6-tetraol in the degradation prod-ucts of Direct Red 5B, and 5-(4-Diazenyl-naphthalene-1-ylazo)-8-hydroxy-naphthalene-2-sulfonic acid and 2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid in the degrada-tion products of Direct Blue GLL.

KEY WORDS: banana, dye decolorization, enzyme purification, polyphenol oxidase, reme-diation

INTRODUCTION

Polyphenol oxidase (PPO) is a group of copper proteins that are widely distributedfrom bacteria to mammals (Shi et al., 2001). Polyphenol oxidase is a mixed function ox-idase enzyme that catalyzes both the ortho hydroxylation of monophenols to o-diphenols(cresolase activity) and further oxidation of o-diphenols to o-quinones (catecholase activ-ity). The o-quinones are highly reactive compounds and can polymerize spontaneouslyto form melanin (a brown pigment), or react with amino acids and proteins (Cho andAhn, 1999). Properties of PPO isolated from various plants such as grapes, banana, plums,potato, tea, papaya, chickpea and peaches have been extensively studied (Kavrayan andAydemir, 2001). Browning of the edible part is due to the presence of PPO in fruits andvegetables, which is undesirable and can lead to unpleasant sensory qualities and losses inthe nutrient quality. PPO also plays an important role as an efficient reagent for cleaning ofthe wastewater-containing polyphenols (Shi et al., 2001).

Address correspondence to Sanjay P. Govindwar, Department of Biochemistry, Shivaji University, Kolhapur416 004, India. E-mail: spg biochem@unishivaji.ac.in

357

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358 U. U. JADHAV ET AL.

Textile industries release millions of liters of the treated effluents daily in the form ofwastewater into public drains that are finally discharged into rivers, causing serious healthproblems leading to enhanced biochemical oxygen demand (BOD) and chemical oxygendemand (COD) affecting the ecosystem drastically. These harmful chemicals not only addcolor to water but simultaneously produce extensive toxicity to aquatic and other formsof life (Senan and Abraham, 2004; Yesiladali et al., 2006). Hence, treatment of industrialeffluents containing aromatic compounds becomes necessary prior to their final dischargeinto the environment and drinking water sources. Conventional methods for the effectiveremoval of phenols, aromatic amines and dyes have been outdated because of the inherentlimitations (Husain and Jan, 2000; Khan and Husain 2007b).

Several physical and chemical methods are being used for the removal of dyes, whichhave several disadvantages such as high cost, low efficiency, and in-applicability to a widevariety of hazardous dyes (Stephenson and Sheldon, 1996; De Morares et al., 2002). Thetextile finishing industry consumes proportionally 100 l of water to treat about 1 kg of textilematerial (Abadulla et al., 2000). New closed-loop technologies of reuse of the microbial orenzymatically treated dye effluents could help to reduce this enormous water consumption.Enzymatic decolorization and degradation is an eco-friendly, cost-competitive alternative tothe conventional chemical decomposition process. This approach is becoming an effectivealternative strategy to the routine chemical, physical as well as microbial treatments, whichhave operational and costing limitations (Duran and Esposito, 2000; Bhunia et al., 2001).Oxidoreductive enzymes mainly peroxidases and polyphenol oxidases have the potentialityfor the degradation of aromatic pollutants from various contaminated sites. These enzymescan act efficiently on a broad range of substrates and catalyze the degradation/removal oforganic pollutants present in very low concentrations at the contaminated sites (Husainand Jan, 2000). The procedure of dye decolorization by peroxidases adds additional costto the processes due to use of an expensive hydrogen peroxide as a co-substrate (Shaffiquet al., 2002). To date, microbial oxidases have been explored for the biodegradation oftextile dyes but plant polyphenol oxidases are cheaper alternatives for the decolorization ofaromatic pollutants. They are readily available in nature and utilize free molecular oxygenas an oxidant (Wada et al., 1995; Duran and Esposito, 2000). Enzymes from plant sourcesare safe and easy to use compared to microbes, eliminating any chance of the escapeof mutant microbes into the environment. In the present study, purified oxidase (PPO)from banana fruit pulp that is readily available throughout the year has been selected forthe decolorization of various textile dyes. Various parameters to achieve maximum dyedecolorization have been optimized and the intermediates formed during the degradationof textile dyes have been analyzed using HPLC and GC-MS techniques.

MATERIALS AND METHODS

Plant Material

Fruits of bananas were procured from a local commercial source (Musa paradisicaspecies cv Basrai) in Kolhapur (Maharashtra, India).

Reagents

Catechol, L-dopa, ascorbic acid, sodium metabisulfite, thiourea, and β-mercaptoethanol were obtained from Sigma Chemical Company (St. Louis, MO, USA).Sodium azide, ammonium sulfate, and L-tyrosine were purchased from Merck, India. All

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other chemicals used were of analytical grade. All dyes were collected from Manpasandtextile industry, Ichalkaranji, India.

Extraction of Banana Pulp PPO

Banana pulp from healthy fruits (20 g) was homogenized in 50 mL of 50 mMphosphate buffer (pH 7.0). The homogenate was filtered through two layers of cheesecloth.The filtrate was centrifuged at 14,000 × g for 15 min at 4◦C. The supernatant was collectedand used as a crude PPO enzyme source in the following experiments.

Column Chromatography

Extraction of PPO was carried out in 50 mM phosphate buffer (pH 7.0). The crudeenzyme preparation (50 mL) containing 1.6 U activity was loaded onto a DEAE-cellulosecolumn (1 × 16 cm), pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.0). Thecolumn was washed with 100 mL of 50 mM sodium phosphate buffer (pH 7.0). Boundproteins were eluted using stepwise gradient of NaCl (0–1 M) in fractions of 2 mL, eachhaving a flow rate of 0.5 mL min−l. Protein concentration of each fraction was determinedat 280 nm by spectrophotometric method using dual beam UV-Visible spectrophotometer(U2800; Hitachi, Tokyo, Japan) (Robyt and White, 1987). The fractions with enzymeactivity were pooled and dialyzed against 50 mM sodium phosphate buffer (pH 7.0) andloaded on a column of Biogel P-100 equilibrated with 50 mM sodium phosphate buffer(pH 7.0). The fractions containing PPO activity were pooled and used as a pure enzymefor further studies.

Measurement of PPO Activity

PPO activity was determined by measuring the initial rate of quinone formation asindicated by an increase in the absorbance at 420 nm (Kavrayan and Aydemir, 2001) usingHitachi U-2800 spectrophotometer. One unit of an enzyme activity was defined as a changein the absorbance unit min−1 mg of protein−1. The sample cuvette contained 1.90 mL of10 mM catechol solution in 50 mM phosphate buffer (pH 7.0) and 0.1 ml of the enzymesolution. The blank contained 2 mL of substrate solution. PPO activity was assayed intriplicate.

SDS-PAGE

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was per-formed on the vertical slab gel electrophoresis using acrylamide gel (10%). The sampleswere prepared by incubation at room temperature for 15 min in Tris–HCl buffer pH 6.8(containing 50% sucrose, 5.0% SDS, and 0.5% bromophenol blue). Boiling of the samplewas avoided because it caused the aggregation of proteins and loss of enzyme activity.The gels were stained with 0.1% Coomassie brilliant blue (R-250) solution prepared inmethanol–water–acetic acid (4:5:1) after electrophoresis, and destained in the same solu-tion without dye. The purity of the enzyme after DEAE cellulose column chromatography,its molecular weight and the nature of the enzyme were analyzed using electrophoresis(Ghodake et al., 2009a).

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Optimum pH and Temperature

Optimum pH of the purified PPO was examined using 10 mM catechol as a substratein a pH range 1.0–10.0 (KCl-HCl buffer; pH 2.0, glycine-HCl buffer; pH 3.0, sodium-acetate buffer; pH 4.0–5.0, sodium-phosphate buffer; pH 5.0–7.0, tris aminomethane-HClbuffer; pH 8.0–9.0, NaHCO3-NaOH buffer; pH 10.0). The optimum temperature for PPOactivity was determined in the temperature range of 20–70◦C, the reaction mixture wasincubated for 10 min before the addition of the enzyme.

Substrate Specificity and Enzyme Kinetics

Substrate specificity was determined using five different substrates (Catechol, 4-methyl catechol, L-tyrosine, L-dopa and Guaicol) and substrate specificity was monitoredat their respective λmax of the products, in a reaction mixture of 2.0 mL containing 10mM substrate solution and 50 mM phosphate buffer (pH 7.0). Michaelis constant (Km)and maximum rates (Vmax) were determined using catechol in the range of concentrations2.0–14.0 mM at pH 7.0 and 30◦C. The reaction was followed in a spectrophotometer(Hitachi U-2800) and data were plotted according to the method of Lineweaver-Burk(Lineweaver and Burk, 1934).

Effect of Various Chemicals on PPO Activity

The effects of several chemicals (sodium metabisulfite, citric acid, cysteine, β-mercaptoethanol, L-asorbic acid, ethylenediamine tetra acetic acid (EDTA), thiourea,BaCl2, MnCl2, ZnSO4, CuSO4, NaCl) were screened at the concentration 1 and 10 mMfor their effect on banana PPO activity. The assay mixture contained 10 mM catechol asa reference substrate and 0.2 mL (5.6 U) of an enzyme in 50 mM phosphate buffer (pH7.0) at 30◦C in the presence and absence of the chemicals listed earlier. The results werereported as percentage inhibition. The activity in the absence of an inhibitor was consideredas 100% activity.

Decolorization of Textile Dyes

Seven textile dyes used in the textile industry (Direct Red 5B, Direct Blue GLL,Direct Yellow 5GL, Reactive Blue HERD, Reactive Navy Blue, Blue 2RNL and DK Red2B) were selected for the present study. Each dye (40 µg mL−1) was incubated with bananaPPO (33.33 U) in 50 mM phosphate buffer, pH 7.0 at 30◦C. The disappearance of the colorby banana PPO activity was monitored at λmax of the respective dye solution. The percentdecolorization was calculated by taking the maximum absorbance of each untreated dyesolution as control (100%) (Saratale et al., 2006). The optical density was measured usingHitachi U-2800 spectrophotometer.

Decolorization and Degradation Studies

Decolorization was monitored by measuring the optical density at λmax using UV-Vis spectrophotometer (Hitachi U-2800). The reaction mixture was centrifuged at 5000× g for 15 min after decolorization of the dyes. The supernatant obtained was used toextract the metabolites with an equal volume of ethyl acetate. The extracts were dried over

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DYE DECOLORIZATION BY BANANA PULP PPO 361

anhydrous Na2SO4 and evaporated to dryness in a rotary evaporator. The crystals obtainedwere dissolved in a small volume of high performance liquid chromatography (HPLC)grade methanol and used for HPLC analysis. HPLC analysis was carried out (watersmodel no.2690) on C18 column (symmetry, 4.6 × 250 mm) using gradient of methanoland acetonitrile (75:25) with the flow rate of 1 mL min−1 for 10 min and UV detector at254 nm. GCMS analysis of metabolites was carried out using a Shimadzu 2010 MS Engine,equipped with an integrated gas chromatograph with a HP1 column (60-m long, 0.25-mmid, nonpolar). Helium was used as carrier gas at a flow rate of 1-ml min−1. The injectortemperature was maintained at 280◦C with oven conditions as: 80◦C kept constant for 2min—increased up to 200◦C with 10◦C min−1—raised up to 280◦C with 20◦C min−1 rate.The compounds were identified on the basis of mass spectra and using the NIST library.

RESULTS AND DISCUSSION

Extraction and Purification of Polyphenol Oxidase

The results of PPO purification are summarized in Table 1. The enzyme was purifiedup to 12.72-fold enhancement in the activity and having 2.49% yield. PPO purification upto 2- to 8-fold has been reported from different sources (Park et al., 1980; Wisemann andMontgomery, 1985; Zawistowski et al., 1988; Socerkall and Soderhall, 1989). Polyphenoloxidase (PPO) from Broccoli florets was also extracted and purified through (NH4)2SO4

precipitation, ion-exchange and gel filtration chromatography (Gawlik-Dziki et al., 2007).Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried appleskin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammoniumbromide (DTAB). The purification fold and recovered activity of PPO were 12.6% and71%, respectively (Imm and Kim, 2009). Polyphenol oxidase (PPO) was also isolatedfrom butter lettuce (Lactuca sativa var. capitata L.) and its biochemical characteristic werestudied (Gawlik-Dziki et al., 2008).

Molecular Weight Determination and Spectral Properties

of the Purified PPO

The purified enzyme showed a single band on SDS-PAGE confirming homogeneousenzyme preparation. The molecular weight was estimated to be about 41 kDa by SDS-PAGE, similar to earlier report (Yang et al., 2000). The molecular weight of pulp PPO varieswith the banana species. The molecular weight of PPO obtained from Musa cavendishii L,var. Nanica was estimated to be about 62 kDa (Yang et al., 2001). The PPO enzyme showeda single band for the activity staining with L-Dopa on native PAGE. PPO showed a singleexcitation peak at 340 nm in the UV-Visible spectrum (Figure 1) as reported earlier (Shiet al., 2001; Xu et al., 2004).

Table 1 Purification of banana PPO

Purification Volume Total protein Total activity Specific activity Purification Yieldstep (mL) (mg) (U) (U mg−l) fold (%)

Crude 10 62.50 28.05 0.44 1 100DEAE cellulose 2 0.342 1.68 4.91 11.15 5.98Biogel P-100 1 0.125 0.70 5.60 12.72 2.49

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362 U. U. JADHAV ET AL.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

280 320 360 400 440 480 520 560 600 640 680 720 760 800

Wavelength (nm)

Ab

sorb

ance

PPO enzyme

Figure 1 Absorption spectrum (280–800) of banana pulp PPO in 50 mM sodium phosphate buffer (pH 7.0)showing characteristic single excitation peak at 340 nm.

Optimum Temperature and pH

As shown in Figure 2, the purified PPO enzyme had an optimum temperature at 30◦Csimilar to the report of Yang et al. (2000, 2001). In contrast, Palmer (1963) reported 37◦Coptimum temperature for banana PPO. Purified PPO from various sources also showed

0

5

10

15

20

25

30

35

20 30 40 50 60 70

1 2 3 4 5 6 7 8 9 10

Temperature oC/pH

pH

Temperature

En

zym

e ac

tivi

ty (

U m

in –l

mg

of

pro

tein

-l)

Figure 2 Effect of the temperature (20◦C to 70◦C) and pH (1–10) on PPO activity.

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DYE DECOLORIZATION BY BANANA PULP PPO 363

variations in the optimum temperature (Kavrayan and Aydemir, 2001). The pH activityprofile for the oxidation of catechol by purified PPO is presented in Figure 2 indicatingmaximum activity at pH 7, which was similar to other reports (Palmer, 1963; Yang et al.,2000). Differences in the optimum pH with several substrates have been reported for PPOfrom strawberries and other plant sources (Mayer and Harel, 1979; Wesche-Ebeling andMontgomery, 1990; Sojo et al., 1998, 2000).

Substrate Specificity and Enzyme Kinetics

The apparent Vmax and Km of purified banana PPO with catechol as a substratewere 0.041 mM min−1 and 1.6 mM, respectively. Banana PPO showed activity with allthe o-diphenolic substrates. Maximum activity was detected toward catechol, followed by4-methyl catechol and L-tyrosine (Table 2). It is generally known that the main phenoliccompounds contained in the plant serve as good substrates of the enzyme (Kimberlyand Lee, 1980) but L-tyrosine could not be oxidized by PPO obtained from peppermint(Kavrayan and Aydemir, 2001). The banana PPO indicated lower Km value than plum (20mM) and Concord grape PPO (67 mM) with catechol as a substrate (Tan and Li, 1984).

Effect of Various Chemicals

The effect of various chemicals on the catecholase activity of banana PPO is presentedin Table 3. The PPO activity was completely inhibited by sodium metabisulfite, (100%)however, it was partially inhibited by citric acid, cysteine and β-mercaptoethanol. Sodiummetabisulfite was a better reducing agent for o-benzoquinones. Enzymes could react withthe oxidized form of ascorbic acid (dehydroascorbic acid). Cysteine, on the other hand,may react directly with sulphydryl groups with the reduction of o-quinone (Kavrayan andAydemir, 2001).

Decolorization of Textile Dyes

Banana PPO could degrade 7 textile dyes (40 µg mL−1 each) as demonstrated inTable 4 which showed that the percent decolorization varied according to the structure ofthe dye. Differences in the time required for the decolorization of the dye indicated the effectof structural diversity of the dyes on their decolorization. Various chromophoric groupsare responsible for the color of the dye. These chromophoric groups could be responsiblefor accelerating or reducing the decolorization rate of dyes or may even cause completeinhibition of decolorization. All decolorized textile dyes by PPO, as shown in Table 4(Direct Red5B, Direct BlueGLL, Reactive Blue HERD, Reactive Navy Blue, and DK Red

Table 2 Substrate specificity of banana PPO

Substrates Enzyme activity (U)

Catechol 32.52 ± 0.0084-methyl Catechol 12.80 ± 0.030L-Tyrosine 4.62 ± 0.006L-Dopa 1.40 ± 0.002Guaicol 0.52 ± 0.003

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364 U. U. JADHAV ET AL.

Table 3 Effect of various compounds on PPO activity (32.52 U)

% Inhibition

Compound 1 mM 10 mM

None 0 0Sodium metabisulfite 30 100citric acid 20 85cysteine 30 80β-Mercaptoethanol 15 70L-asorbic acid 10 66EDTA 25 58Thiourea 5 40BaCl2 5 20MnCl2 0 5ZnSO4 0 2CuSO4 0 0NaCl 0 0

2B) have also been decolorized by different microorganisms (Dawkar et al., 2008; Jadhavet al., 2008a, 2008b).

The ortho position of carboxyl group in Methyl red has been shown to inhibitdecolorization by purified Orange II azo reductase (Zimmermann et al., 1982). Hsueh andChen (2007) showed the effect of the functional groups and molecular structures presentin model azo dyes on the color removal capability of Pseudomonas luteola. As methylorange (p-MO) and 4-(4′-dimethylaminophenylazobenzoic acid) sodium salt (denoted p-MR) contain sulfo group and carboxyl group respectively, at para positions to the azo bond,the decolorization rate for (p-MO) was higher, owing to the stronger electron withdrawingeffect of the sulfo group. For isomers, when the functional groups e.g., sulfo group of2-(4′-dimethylamino-phenylazo) benzene sulfonic acid sodium salt (o-MO) or carboxylgroup of methyl red (o-MR)) were ortho to azo bond, the decolorization rate significantlydecreased (e.g., p-MO > o-MO or p-MR>o-MR), which may be likely because of thesteric hindrance near azo linkage(s). Regarding the electronic effect, azo dyes with strongerelectron-withdrawing group (e.g., sulfo group) at specific positions (e.g., at para) could bemore easily decolorized than those with a carboxyl group.

Decolorization of various textile dyes by soluble and Celite bound potato PPO wasalso observed by Khan and Husain (2007a). Duran and Esposito (2000) showed the useof PPO enzyme in wastewater and soil treatment. Earlier, workers have described the

Table 4 Percent decolorization of different textile dyes by Banana PPO

Sr. no. Name of dyes λmax (nm) Time (h) Decolorization (%)

1 Direct Red5B 512 24 902 Direct BlueGLL 570 26 803 Direct Yellow 5GL 501 30 504 Reactive Blue HERD 615 30 605 Reactive Navy Blue 510 30 806 Blue 2RNL 560 36 407 DK Red 2B 535 36 25

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DYE DECOLORIZATION BY BANANA PULP PPO 365

AU

0.00

0.10

0.20

0.30

0.40

0.50

Time (minutes)1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0 11.0

2.430

(a)

(b)

AU

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

Time (minutes) 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

1.686

2.3552.971

3.702

Figure 3 (a) HPLC analysis of the control DR5B dye and (b) degradation products of DR5B using PPO.

decolorization and effective removal of industrially important dyes using immobilizedperoxidases (Shaffiqu et al., 2002; Khan and Husain, 2007b). The application of peroxidasesin the wastewater treatment is not cost effective due to requirement of an expensive co-substrate, hydrogen peroxide. The use of stable and reusable PPOs could avoid the use ofH2O2, because these enzymes catalyze reactions in the presence of molecular O2 (Husainand Jan, 2000).

UV-Visible Spectra of DR5B and DBGLL

PPO enzyme showed 90 and 85% decolorization of DR5B and DBGLL within 24 and26 h, respectively. There was a remarkable decrease in the absorbance and shift of the peaktowards UV region. Earlier reports showed degradation of phenols, aromatic amines anddyes using peroxidases and polyphenol oxidases (Husain and Jan, 2000; Bhunia et al., 2001;

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366 U. U. JADHAV ET AL.

AU

0.00

0.02

0.04

0.06

0.08

0.10

Time (minutes)1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

1.560

(a)

(b)

AU

0.000

0.005

0.010

0.015

0.020

Time (minutes)

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

2.902 3.141

.

4.568

Figure 4 (a) HPLC analysis of the control DBGLL dye and (b) degradation products of DBGLL using PPO.

Akhtar et al., 2005). The treatment of phenols with the enzymes resulted in the formationof a large insoluble aggregate, which could be easily removed by simple centrifugation,sedimentation or filtration from the reaction mixture (Khan and Husain, 2007b). In thepresent study, we could not observe insoluble aggregates indicating the removal of dyesdue to degradation.

Degradation Analysis

HPLC analysis of extracted sample showed original dye DR5B at retention time2.430 min (Figure 3a). The peaks for the metabolites were at retention time 1.686, 2.355,

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SO3H N NO

N NOH

SO3HNH

SO3H OH + NH2

+

N N NOH

SO3HNH O

Direct Red 5B

MW =675

[MW (mz+2) =174]Unidentif ied product

SO3H N N OH +

NH O

N

SO3H

NH2

+

OH

NNH2

SO3H

OH

+

ONH

H

N2Desulfonation

OHO

OHOH

OH

4-hydroxy-benzenesulfonic acid

Naphthalene-1,2,3,6-tetraol, compound with m ethanol

Unidentified productUnidentified product

Unidentif ied product Unidentif ied product

[MW (mz+2) =208]

[1] [A]

[B] [C]

[D] [E]

[2]

Figure 5 Proposed pathway for the degradation of DR5B by purified banana pulp PPO. The compound representedby an alphabet in a bracket has not been found, but their existence is rationalized as necessary intermediates forthe final products found. The compound represented by Arabic number in a bracket has been found in reactionmixture.

2.971, and 3.702 min (Figure 3b) after complete decolorization. HPLC chromatogram ofDBGLL showed the retention time of 1.56 min (Figure 4a). The peaks for the metaboliteswere at the retention time of 2.902, 3.141, and 4.568 min respectively (Figure 4b) aftercomplete decolorization.

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368 U. U. JADHAV ET AL.

O

S OON N N N

SO

OO

N NS OOO

NH2

DBGLL DyeMW=842

S OOO

OH+

NNH2 N N N

SO

OO

NSO

OO

NH2Unknown product

Unknown product

Oxidative cleavage

NH2 N N N

SO

OO

OH

+NNH2

SO

OO

NH2

5-(4-Diazenyl-naphthalene-1-ylazo)-8-hydroxy-naphthalene-2-sulfonicacid anion

Unknown product

MW= 406

SO

OON N N NH2

2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid anion

MW= 390

+

SO

O OOH

SO

OO

N N

NH2

Unknown product

Oxidative cleavage

[A] [B]

[C]

[1]

[2][D]

Figure 6 Proposed pathway for the degradation of DBGLL by purified banana pulp PPO. The compoundrepresented by an alphabet in a bracket has not been found, but their existence is rationalized as necessaryintermediates for the final products found. The compound represented by Arabic number in a bracket has beenfound in reaction mixture.

A pathway has been proposed for the degradation of DR5B and DBGLL by bananaPPO (Figures 5 and 6). All the molecules showed in the degradation pathways (repre-sented in Arabic numbers) of the dyes have been identified by GC-MS. Different enzymessuch as PPO, peroxidases and laccases have been known to have a profound role in thebiodegradation of dyes. Oxidative biodegradation of azo dyes primarily involves the lignin

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DYE DECOLORIZATION BY BANANA PULP PPO 369

Table 5 GC-mass spectral data of degraded products of DR5B and DBGLL

Relative abundance in mass spectrum:Dye Rt (min) m/z (% relative intensity) Proposed compound

DR5B 6.4 206 ± 2 45 (100) 4-hydroxy-benzenesulfonic acid16.80 174 ± 2 57 (30), 191 (100), 206 (20) Naphthalene-1,2,3,6-tetraol, compound

with methanolDBGLL 20.558 406 76 (5),149 (100), 223 (5) 5-(4-Diazenyl-naphthalene-1-ylazo)-8-

hydroxy-naphthalene-2-sulfonic acidanion

16.383 390 55 (20), 57 (100), 71 (80), 85 (50), 99(20), 113 (5)

2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acidanion

degrading enzyme system in the decolorization process. The lignin-degrading system con-tains a number of peroxidases that can catalyze the depolymerization of lignin as well asinitial oxidation of a wide range of other compounds. Azo dyes can be cleaved symmetri-cally or asymmetrically, with an active site available for an enzyme to excite the molecule(Pasti-Grigsby et al., 1992; Kalme et al., 2007; Ghodake et al., 2009b). In addition, azodyes can be degraded by oxidative cleavage (Chivukula and Renganathan, 1995). Two pos-sible mechanisms have been proposed for the degradation of DR5B dye. The first oxidativecleavage by PPO resulted in the formation of 4-hydroxy-benzenesulfonic acid and thesecond oxidative cleavage resulted in the formation of Naphthalene-1,2,3,6-tetraol, com-pound with methanol. For the degradation of DBGLL dye, we have proposed two possiblemechanisms. The first oxidative cleavage yielded 5-(4-Diazenyl-naphthalene-1 ylazo)-8-hydroxy-naphthalene-2-sulfonic acid anion, while the second resulted in the formation of2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid anion. The structure of detectedcompounds was assigned from the m/z value obtained (Table 5).

In our previous study, GC-MS analysis of the biodegraded products of DR5B andDBGLL by Comamonas sp UVS revealed the presence of 7-benzoylamino-3-diazenyl-4-hydroxy-naphthalene-2-sulfonic acid, and 3-(Naphthalene-1-ylazo)-naphthlene-1,5-disulfonic acid in the degradation products of DR5B and DBGLL respectively (Jadhavet al., 2008, 2009). These products are different from the products obtained using bananaPPO enzyme. The results indicated the different degradation pattern of DR5B and DBGLLdyes by PPO enzyme than Comamonas sp UVS. This might be due to the presence of otheroxidative enzymes in Comamonas sp UVS. The degradation of azo dyes by reductionresulted into the formation of aromatic amines. These aromatic amines are potentiallycarcinogenic (McCann and Ames, 1975; Chung and Cerniglia, 1992; Chivukula andRenganathan, 1995). Oxidative cleavage plays an important role in the detoxification ofazo dyes because this reaction releases azo linkages as molecular nitrogen, which prohibitsaromatic amine formation.

CONCLUSIONS

The banana pulp PPO has a great potential for the decolorization of textile dyes anddoes not need any redox mediators, saving the cost of the process. PPO enzyme carries outoxidative cleavage of the dyes, leaving no formation of amines which themselves are toxic

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370 U. U. JADHAV ET AL.

and mutagenic. The use of polyphenol oxidase may be extendable to the effluents from thetextile industries.

ACKNOWLEDGMENTS

We are thankful to Dr. Rajaram P. Patil for his fruitful suggestions and Mr. Nilesh V.Pawar for his technical assistance.

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