Purification and characterization of peroxidases from liquid endosperm of Cocos nucifera (L.): Biotransformation

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

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Purification and characterization of peroxidases from liquid endosperm of Cocos nucifera (L.): Biotransformation Murugesan Balasubramanian ∗ , Rathnam Boopathy Department of Biotechnology, School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641046, Tamil Nadu, India

a r t i c l e

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Article history: Received 2 July 2012 Received in revised form 14 January 2013 Accepted 14 January 2013 Available online 20 January 2013 Keywords: Plant peroxidase Cocos nucifera Isoenzymes o-Dianisidine dihydrochloride Tender coconut water

a b s t r a c t Peroxidases are ubiquitous oxidoreductase enzymes and find application in various physiological/biochemical reactions. In the present study, class III peroxidase enzymes from the liquid endosperm (TCWP – Tender Coconut Water Peroxidase) of Cocos nucifera were identified and purified using ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography resulting in 9.77-fold of purified enzymes to its apparent homogeneity. The purification profile of peroxidase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis had shown two protein bands corresponding to two isoenzymes (TCWP1 and TCWP2 ) with a molecular weight of ∼47 and 49 kDa respectively. The purified isoenzymes TCWP1 and TCWP2 exhibited its maximal activity (3.5 and 3.2 U/ml; respectively) at pH 4.5 and 5.0 with o-Dianisidine dihydrochloride (o-D) as substrate at 40 ◦ C. TCWP1 and TCWP2 isoenzymes were stable up to 50 ◦ C for 1 h. The stability of the enzyme at increased temperature may be attributed to the presence of Ca2+ ions to the enzyme. Addition of excess Ca2+ ions to the enzyme mixture enhanced the stability further to 55 ◦ C for both the isoenzymes. The excess addition of H2 O2 inhibited the peroxidase activity and the TCWP isoenzymes were stable up to 10 mM of H2 O2 concentration at 25 ◦ C. The Km for o-D was determined to be 1.63 mM and 4.0 mM for TCWP1 and TCWP2 , respectively. The TCWP activity was enhanced with the addition of carboxylic compound (sodium acetate) at 7 mM and Mn2+ at 1 mM. Sodium cyanide and phenyl hydrazine inhibited the enzyme competitively but sodium azide and hydroxylamine were uncompetitive and non-competitive inhibitor respectively. The TCWP enzyme had the potential to biotransform the genotoxic compound into non-genotoxic compound. The meager difference in biochemical characters of TCWP isoenzymes leads to identify its applications in various industries. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Peroxidases (EC 1.11.1.7) are widely distributed in nature and can be easily extracted [1] from most plant cells, some animal tissues and fungus [2–5]. Plant peroxidase is a heme-containing enzyme that catalyzes the one or two electron oxidation of various organic and inorganic substrates in the presence of hydrogen peroxide [6]. Plant peroxidases have been isolated and purified from several plant sources viz., Leucaena leucocephala, Viscum angulatum, Vigna mungo, Solanum melongena fruit juice, Beta vulgaris, Roystonea regia, Tamarix hispida, and Jatropha curcas leaves [7–14]. These enzymes are involved in a wide range of physiological processes [15–20]. Peroxidase enzymes have been studied for many years for their potential of degradation/transformation of aromatic compounds into other harmless products [21].

∗ Corresponding author. Tel.: +91 422 242 5716; fax: +91 422 2422387. E-mail addresses: [email protected], [email protected] (M. Balasubramanian). 1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2013.01.009

Higher plants usually contain a large number of peroxidase isoenzymes classified as acidic, neutral or basic based on their isoelectric points. It is difficult to assign specific functions to each isoenzyme of peroxidases because of their poor substrate specificity. Isoenzymes, which are different forms of the same enzyme, are proteins produced by RNA translation. They are therefore genetic markers, but given their low polymorphism in coconut and provided very little information for diversity studies. Cardena et al. [22] reported that the isoenzymes were used to make the difference between the coconut varieties of Rennell Island Tall and the West African Tall [22]. Tender Coconut Water (TCW or coconut liquid endosperm) contains major bioactive constituents like sugars, minerals, minor fractions of fat and nitrogenous substances [23]. These constituents attribute to the antioxidant property of TCW and they are exploited for its therapeutic value against hypertension in humans. In addition to this, the coconut liquid endosperm contains a meager amount of many bio-active enzymes such as acid phosphatase, catalase, dehydrogenase, RNA polymerases and peroxidase which aid in digestion and metabolism [24,25]. Among these enzymes, the

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M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

purification of peroxidase enzyme has not been completely studied so far. This is the first report on the purification and characterization of peroxidases from Tender coconut water (TCWP) or liquid endosperm of Cocos nucifera [West Coast Tall variety (WCT)]. 2. Materials and methods 2.1. Chemicals Analytical grade chemicals namely carboxymethyl cellulose, octyl sepharose and sephadex G-75 were purchased from Pharmacia Biotech, Sweden, o-Dianisidine dihydrochloride from Sigma, St. Louis, USA and other chemicals were procured from Himedia laboratories Ltd, Mumbai, India. 2.2. Coconut water collection For each coconut tree (WCT), a single coconut was used (4–6 months of development). The coconuts were gently removed with sharp scissors. The coconuts were removed from the tree at 07:00–08:00 h and the coconut water was extracted under sterile conditions. A single transversal cut was made on top of the coconut and the water was then withdrawn with a 10 cm-long, 20 gage Teflon needle (Sigma) attached to a 60 ml syringe (BD Biosciences). [26]. 2.3. Protein determination and enzyme assay The protein content of the coconut water was quantified according to the Lowry method [27]. The peroxidase activity with o-dianisidine dihydrochloride (o-D) as reducing substrate was determined using a modified procedure of Rompel et al. [28] in brief, 1 ml reaction mixture containing aliquot of purified enzyme (0.2 U/ml), 20 mM sodium acetate buffer (pH 4.5 for TCWP1 and pH 5.0 for TCWP2 ), 0.4 mM o-D and 1 mM hydrogen peroxide (H2 O2 ). The oxidation of o-D was observed in terms of increase in absorbance at 460 nm [molar extinction coefficient (ε) of o-D at 460 nm = 11.3 × 103 mM cm−1 ]. One unit of the peroxidase activity is defined as the amount of enzyme that oxidizes 1 mmol of substrate per min at 25 ◦ C [28]. Kinetic characterization (Km and Vmax ) of o-D and H2 O2 was performed at concentrations ranging between 0.4 and 8 mM. 2.4. Ammonium sulfate precipitation Ammonium sulfate was added to TCW slowly up to 60% saturation with gentle stirring and set aside for 4 h at 4 ◦ C for complete precipitation. The precipitate was pelleted twice by centrifugation at 12,000 rpm for 15 min at 4 ◦ C in 20 mM sodium acetate buffer, pH 4.5. The supernatant was dialyzed against 5 mM of sodium acetate buffer, pH 4.5. 2.5. Purification of peroxidase 2.5.1. Ion exchange chromatography The dialysate of ammonium sulfate precipitate was subjected to ion exchange chromatography using carboxymethyl-cellulose (CMC) column (1.5 cm × 8.5 cm) pre-equilibrated with 20 mM sodium acetate buffer, pH 4.5. The column was washed with five bed volumes of equilibration buffer before loading the sample. The bound proteins were eluted with a linear gradient of 0 to 0.5 M NaCl in 20 mM sodium acetate buffer, pH 4.5. Fraction with high peroxidase activity were pooled and dialyzed against 5 mM Tris–HCl buffer, pH 8.5.

2.5.2. Hydrophobic interaction chromatography Hydrophobic interaction chromatography of the pooled active fractions from the CMC was carried out using octyl-sepharose column (1.5 cm × 8.5 cm). Ammonium sulfate at a final concentration of 1 M was added to the pooled active fraction before loading onto the column. The column was pre-equilibrated with 20 mM Tris–HCl, pH 8.5 containing 1 M ammonium sulfate. The column was washed with five bed volumes of equilibration buffer before the bound proteins were eluted with a linear gradient of equilibration buffer to 20 mM Tris–HCl, pH 8.5. Fractions with high activity were pooled, lyophilized and dialyzed against 5 mM sodium acetate buffer, pH 4.5. 2.5.3. Size exclusion chromatography One milliliter of the concentrated (2 mg) enzyme sample from the previous step was loaded onto a pre-equilibrated sephadex G75 column (2.5 cm × 90 cm) and eluted with equilibration buffer (20 mM sodium acetate buffer, pH 4.5, containing 0.1 M NaCl) at a flow rate of 6 ml/h. About 3 ml fractions were collected at regular time intervals, protein concentration was estimated at 280 nm in a UV–Vis Spectrophotometer and the peroxidase activity was monitored at regular time intervals. 2.6. Electrophoretic analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% acrylamide gel was performed as described by Laemmli [29]. The protein samples were dissolved in loading buffer without thiol-reducing agents. The presence of protein band was visualized by silver staining [30]. 2.7. Characterization of peroxidase 2.7.1. pH and temperature profile of peroxidase activity and residual peroxidase activity Optimum pH of purified peroxidases was determined at pH ranging from 3.5 to 9 at 25 ◦ C. pH stability was determined by incubating the purified isoenzymes (4.0 U/ml) in 1 ml of 20 mM respective buffers at 25 ◦ C for 1 h. Buffers at 20 mM concentration used were: sodium acetate for pH 3.5–5.5; phosphate buffer for pH 6–7; Tris–HCl to pH 8–9. All the experiments were carried out in triplicates. The stability of the enzyme was expressed in percentage residual activity by comparing with control enzyme. The thermal stability of purified TCWP was determined by incubating the enzyme at varying temperature (30–70 ◦ C) for 1 h. In brief, 1 ml of 20 mM acetate buffer, pH 4.5 containing 4.0 U/ml enzymes was incubated at appropriate temperatures. An aliquot of 0.05 ml was drawn after 1 h from each tube, cooled by immersing in ice and assayed for residual peroxidase activity. Optimum temperature of purified peroxidases was determined by enzyme assay in temperature controlled Spectrophotometer at 30–70 ◦ C. 2.7.2. Inhibitor kinetics on TCWP activity The inhibition pattern of purified TCWP isoenzymes against four concentrations of o-Dianisidine (2 mM–4 mM) were kinetically analyzed using various peroxidase inhibitors such as sodium azide, sodium cyanide, hydroxylamine hydrochloride and phenyl hydrazine at ranges from 0.01 to 10 mM. The isoenzymes (0.2 U/ml) were incubated with respective inhibitors for 30 min at 25 ◦ C and the residual peroxidase activity was compared with control. 2.7.3. Possible effectors and activators on TCWP activity The TCWP isoenzymes (0.2 U/ml) were incubated with different concentrations (0.1 ␮M–10 mM) of metals: nickel sulphate, magnesium chloride, manganous chloride, and zinc sulphate; radical scavenger: ascorbic acid; thiol compounds: ␤-mercaptoethanol

M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

35

Peroxidase activity (units/fraction)

3.0 TCWP1

2.5

2.0

1.5

1.0

TCWP2

0.5

0.0 0

5

10

15

20

25

30

Fraction number Fig. 1. SDS-PAGE (12.5%) of purified peroxidase enzyme from C. nucifera. Lane M: molecular weight marker (kDa); lane 1: TCWP1 ; lane 2: TCWP2 .

and dithiothreitol (DTT); carboxylic compounds (1–7 mM): oxalate, tartarate, citrate and acetate in 20 mM sodium acetate buffer, pH 4.5. The residual enzyme activity was analyzed and compared with the control. 2.7.4. Effect of hydrogen peroxide and calcium chloride on TCWP activity The TCWP isoenzymes (4.0 U/ml) were incubated with three concentrations of H2 O2 (5, 10 and 15 mM) or calcium chloride (CaCl2 – 1, 2, 4 and 5 mM) in 1 ml of 20 mM sodium acetate buffer, pH 4.5 for 60 min at 25 ◦ C and 60 ◦ C respectively. About 0.05 ml of enzyme was withdrawn from the incubation mixture at 10 min intervals, cooled by immersing in ice and assayed for residual peroxidase activity. The residual enzyme activity was compared with appropriate control.

Fig. 2. Elution profiles of peroxidase after cation exchange chromatography. TCWP isolated from liquid endosperm were separated on CM-cellulose column. Bound proteins were eluted by NaCl gradient from 0 to 0.5 M. The flow rate was 1 ml per 20 min and fractions of 1.0 ml were collected. Bound peroxidase activities could be separated into two peaks.

and designated as TCWP1 and TCWP2 . The TCWP2 isoenzyme was eluted with high concentration of NaCl compared to TCWP1 . Individually, each isoenzyme was dialyzed against 5 mM Tris–HCl buffer, pH 8.5 and subjected to the octyl sepharose chromatography. TCWP fractions require additional gel filtration purification, because its final polishing steps in chromatography. TCWP was purified to its homogeneity of 9.77-fold purity with 12.8% of recovery (Table 1), contradictory to the purification 30.64-fold and 8.34-fold of orange and apple seeds peroxidases; respectively [25]. The enzyme thus obtained was used for further studies.

2.8. DNA adduct formation 3.2. Optimum pH for activity and stability of TCWP isoenzymes DNA was isolated from peripheral blood as previously described [31]. The purity of DNA was evaluated by the ratio of A260 /A280 and the DNA was quantified at A260 . The biotransformed product was prepared according to the modified method of Kireyko et al. [32]. To the 50 ␮l reaction mixture (containing 0.01–0.04 mg o-dianisidine (precursor) or its biotransformed product), human peripheral blood DNA (2 ␮g/25 ␮l) and 4 mM citrate phosphate buffer (pH 5.6) were added and incubated for 30 min at room temperature. The precursor/product excluded reaction mixture was used as a control. The genotoxic effect of the above compounds in DNA sample was analyzed in 0.8% agarose gel [33]. 3. Results and discussion 3.1. Purification of TCWP isoenzymes The tender coconut water consists of certain important enzymes and peroxidase is one among them. Peroxidase enzymes are widely used as a marker enzyme in food industry [34]. It encompasses the attention of researchers to emphasize for characterization of peroxidase from different sources for its uniqueness. Hence, the TCWP was purified from the liquid endosperm of C. nucifera. In Fig. 1 the purified TCWP isoenzymes were shown in lane 1 and 2 corresponding to ∼47 and 49 kDa respectively. As seen in the CMCelution profile (Fig. 2), two well-separated peaks were obtained

pH is a determining factor in the expression of enzymatic activity as it alters the ionization states of amino acid side chains or the ionization of the substrate [35] and pH values provide information concerning the identities of the prototropic groups at the active site [36]. The effect of pH on TCWP activity presented in Fig. 3. The optimum pH (Fig. 3a) for peroxidation reaction was found in sodium acetate buffer at 4.5 and 5.0 for TCWP1 and TCWP2 respectively. The optimum pH of the purified peroxidase was ranged between 4.5 and 6.5 [37]. For Horseradish Peroxidase (HRP) optimum pH was found to be 4–5 [12], pH 4.0 for Marula fruit, pH 5.0 for rice, A. sativum and S. melongena [38–41], pH 6.0 for strawberry [42] and pH 5–6 in red beet hairy root [12]. According to Lee and Lee [43], the rapid inactivation of the enzyme above pH 8.0 may be due to conformational changes in the enzyme at the alkaline condition and the enzyme may undergo Maillard reaction and Strecker degradation. The TCWP1 was stable between pH 4.0 and 5.5, whereas TCWP2 was stable between pH 4.5 and 6.0 (Fig. 3b). The TCWP1 and TCWP2 were stable up to 50–60% between pH 4.0 and 6.0. It proved retention of enzymatic potential of TCWP in the acidic environment with respect to alkaline conditions, due to low pH of TCW. Similarly, the pH stability of spinach (Spinacia oleracea) leaves peroxidase was reported in 5.5 [44], for HRP lies at 4–6 [12] and soybean (Glycine max) peroxidase was observed ranging from 2 to 10 [45], in contradictory to our result the red beet hairy root was ranged from 6 to 8 [12].

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M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

Table 1 Purification of TCWP isoenzymes. Fraction

Total protein (mg)

Total enzyme (U)

Specific activity (U/mg)

Purification fold

Recovery (%)

Crude 60% ammonium sulfate CM-Cellulose Octyl sepharose Sephadex G-75

246 16.5 9.05 4.32 3.20

322 98.3 18.4 32.2 41.1

1.31 5.96 2.02 7.45 12.8

1 4.55 1.54 5.69 9.77

– 30.53 5.71 10 12.8

(a)

Peroxidase activity (%)

The enzyme activity against temperature resistance depends on the source of the enzyme as well as on the assay conditions, especially pH and the nature of the substrate employed. The variability in the heat stability of peroxidase can be attributed largely to the particular enzyme structure [46]. The effect of temperature between 30 ◦ C and 70 ◦ C under optimum pH and buffer concentration on TCWP isoenzymes were assayed and the results are shown in Fig. 4a. The optimum temperature of TCWP1 and TCWP2 was found to be at 40 ◦ C and 45 ◦ C respectively. Apoplastic, cytosolic and soluble peroxidases of several plant tissues also exhibited temperature optima from 40 ◦ C to 60 ◦ C [47–50]. The enzyme stability has been attributed to non-covalent, electrostatic and hydrophobic interactions, as well as extra ion pairs, hydrogen bonds and the degree of glycosylation of individual isoenzymes [46]. It has also been shown that the thermal stability of peroxidase was due to the presence of a large number of cysteine residues in the polypeptide chain [51]. The thermal stability of TCWP1 and TCWP2 isoenzymes were similar (Fig. 4b), and retained 55% of peroxidase activity up to 55 ◦ C for 1 h. Followed by drop in percentage of residual activity at high temperatures (>60 ◦ C), it could be due to the unfolding of the enzyme [52]. The thermal stability of TCWP1 and TCWP2 enzyme was similar to the Arabian

date palm peroxidase, which was reported to be stable at 55 ◦ C for 1 h and differ with Yam ionic peroxidase; it retained 35% activity at 70 ◦ C for 1 h [53,54]. However horseradish peroxidase and cassia anionic peroxidase was stable (90%) at 65 ◦ C even after 20 h of incubation [55].

3.4. Determination of substrate kinetic constants for TCWP isoenzymes The TCWP1 and TCWP2 has 1.63 mM and 4.0 mM as Km value for o-D (Fig. 5a and c) and 1.65 mM and 1.23 mM as Km value for H2 O2 (Fig. 5b and d) respectively as determined by Lineweaver–Burk (LB) plot. This TCWP isoenzyme Km values were lower than those found for soluble peroxidase from kiwi fruit (7.4 mM), tomato peroxidase (10 mM) and garlic peroxidase (9.5 mM) as well as corn root plasma membrane peroxidase [47,56,8]. The B. vulgaris root peroxidase had Km value for o-D (2.14 mM) and H2 O2 (1.39 mM) [12]; it’s shown the difference with o-D and similarity with H2 O2 for TCWP isoenzymes. The Vmax values of TCWP1 and TCWP2 were determined to be 0.38 mmol min−1 ml−1 , 0.57 mmol min−1 ml−1 for o-D (Fig. 5a and c) and 0.29 mmol min−1 ml−1 , 0.23 mmol min−1 ml−1 for H2 O2 ; respectively (Fig. 5b and d). Interestingly, the TCWP1 had lower Km and Vmax with o-D and higher with H2 O2 when compared to TCWP2 .

(a)

100 80 60 40 20 0 3

4

5

6

7

8

Peroxidase activity (%)

3.3. Optimum temperature for activity and stability of TCWP isoenzymes

100 80 60 40 20 0

9

30

80 60 40 20 3

4

5

6

7

8

9

Residual activity (%)

Peroxidase activity (%)

(b)

100

0

50

Temperature

pH

(b)

40

60

70

( oC)

100 80 60 40 20 0

30

40

50

60

70

Temperature ( oC)

pH Fig. 3. Effect of pH on peroxidase activity of TCW. (a) The rate of o-D oxidation by TCWP1 () and TCWP2 () determined under the standard assay conditions except that 20 mM sodium acetate (pH 3.5–5.5), phosphate buffer (pH 6.0–7.0) and Tris–HCl buffer (pH 8.0–9.0) buffers were used. (b) The enzyme stability was analyzed by preincubation in 20 mM different buffers at various pH values at 25 ◦ C for 1 h and the resulting activity was measured. The relative activity of TCWP at pH 4.5 was taken as 100%. Each point represents the mean ± SD of the three independent experiments.

Fig. 4. Effect of temperature on peroxidase activity and stability of TCW. (a) To determine the optimal temperature, the enzyme activity was measured at different temperatures. The activity at 40 and 45 ◦ C was taken as 100%. (b) To determine the thermostability, the enzyme was incubated for 1 h at each temperature in optimum pH of 20 mM sodium acetate buffer, before the assay. The residual activity was determined at 25 ◦ C. The activity without pre-incubation was taken as 100%. Symbols: () TCWP1 ; () TCWP2 . Data presented are average values ± SD of three independent experiments.

M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

(a)

20

(b)

14

18

12

16 14

10

1/v (mmole/min/mg)

1/v (mmole/min/mg)

37

Vmax= 0.38 mM Km= 1.631 mM

8 6 4

12

Vmax= 0.29 mM Km= 1.65 mM

10 8 6 4

2

-1.5

-1.0

-0.5

0 0.0

2

0.5

1.0

1.5

2.0

-1.5

2.5

-1.0

-0.5

0 0.0

0.5

1.0

1.5

2.0

2.5

1/[H2O2] (mM)

1/[o-dianisidine] (mM)

(c)

(d)

12

18 16 14

1/v (mmole/min/mg)

1/v (mmole/min/mg)

10

Vmax= 0.57 mM Km= 4.01 mM

8 6 4

Vmax= 0.23 mM Km= 1.23 mM

12 10 8 6 4

2 2

-1.0

-0.5

0 0.0

0.5

1.0

1.5

2.0

2.5

1/[o-dianisidine] (mM)

-1.5

-1.0

-0.5

0 0.0

0.5

1.0

1.5

2.0

2.5

1/[H2O2] (mM)

Fig. 5. Determination of kinetic parameters for TCWP1 and TCWP2 . (a and c) Double reciprocal plot for o-dianisidine oxidation by the TCWP1 and TCWP2 : Assays were carried out at 4 mM H2 O2 in optimum pH of 20 mM sodium acetate buffer at 25 ◦ C. (b and d) Double reciprocal plot of activity vs H2 O2 concentration for the TCWP1 and TCWP2 : Assays were carried out at 2 mM o-dianisidine in 20 mM sodium phosphate buffer (pH 7.0) at 25 ◦ C.

3.5. Inhibition kinetics of TCWP isoenzymes Sodium azide, potassium cyanide and phenyl hydrazine are typical peroxidase inhibitors [56,57]. Hence, its inhibition effect on TCWP isoenzymes activity was investigated and expressed in LB plot (Figs. 6 and 7 for TCWP1 and TCWP2 respectively). In Figs. 6a and 7a, sodium azide showed uncompetitive mode of inhibition and it suggested that azide preferentially binds to peroxo-enzyme intermediate rather than to the enzyme itself. The Km (1.11, 0.37, 0.22, 0.17 mM) and Vmax (0.08, 0.03, 0.02, 0.01 mmol min−1 ml−1 ) values for TCWP1 and Km (0.81, 0.49, 0.29, 0.20 mM) and Vmax (0.67, 0.41, 0.25, 0.17 mmol min−1 ml−1 ) values for TCWP2 were obtained from Figs. 6a and 7a; respectively. Both Km and Vmax values decreased with increasing o-D concentration reveals the uncompetitive mode of inhibition. The peroxidases from S. melongena, maize and sunflower also found to be inhibited by azide [11,56]. The inhibition pattern of sodium cyanide on TCWP1 , TCWP2 isoenzymes were shown as competitive for o-D in Figs. 6 and 7. The Km (0.48, 1.01, 1.56, 2.04 mM) and Vmax (0.68 mmol min−1 ml−1 ) values for TCWP1 and Km (0.33, 0.65, 0.91, 1.19 mM) and Vmax (0.32 mmol min−1 ml−1 ) values for TCWP2 were obtained

from Figs. 6b and 7b; respectively. The Km increased, as the result of increasing cyanide concentration and the value for Vmax was not altered. Inhibition at low concentration is conceivable because cyanide, in contrast to the other inhibitor compounds, does react with the native enzyme rather than peroxoenzyme complex. The cyanide was reported as competitive inhibitor for tomato, sweet potato, tea and turnip [58–61]. The inhibition effect of hydroxylamine on TCWP1 and TCWP2 isoenzyme activity was shown in Figs. 6c and 7c. The Km (1.85 mM) and Vmax (6.25, 4.35, 2.63, 1.92 mmol min−1 ml−1 ) values for TCWP1 and Km (0.69 mM) and Vmax (6.67, 8.33, 3.45, 2.38 mmol min−1 ml−1 ) values for TCWP2 were obtained from Figs. 6c and 7c; respectively. When different hydroxylamine concentrations were used, Km value was unaffected and Vmax decreased with increasing o-D concentration, suggesting a non-competitive type of inhibition. The non-competitive type of inhibition observed with hydroxylamine suggests the existence of a binding site for hydroxylamine; it was structurally dissimilar to o-D. On contrary to the present study, hydroxylamine was reported to have mixed types of inhibition (competitive and uncompetitive) with vanadium chloroperoxidase [62].

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M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

175

1 mM 2 mM 5 mM 7.5 mM

(a)

10 20 30 40

(b)

150

18

m m m m

16 14 1/v mmole/min/ml)

1/v (mmole/min/mg)

125 100 75 50

12 10 8 6 4

25

-7

-6

-5

-4

-3

-2

-1

0

2 0

1

2

3

4

5

-3

-2

-1

5.5

2.5 (mM) 5 (mM) 7.5 (mM) 10 (mM)

(d)

5.0 4.5

3.5 3.0 2.5 2.0 1.5 1.0 0.5

-3

-2

-1

0.0

1

2

3

4

5

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -3.0-2.5-2.0-1.5-1.0-0.50.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.2 mM 0.4 mM 0.6 mM 0.8 mM

1/v (mmole/min/ml)

1/v (mmole/min/ml)

4.0

-4

0

1/[O-dianisidine] (mM)

1/[O-dianisidine] (mM)

(c)

0

0

1

2

3

4

5

1/[O-dianisidine] (mM)

1/[O-dianisidine] (mM)

Fig. 6. Effect of various inhibitors on the peroxidase activity of TCWP1 . Type of inhibition is obtained from plotting 1/v against the various inhibitor concentration at various o-dianisidine concentrations at a fixed concentration of H2 O2. The inhibitors used were (a) sodium azide, (b) sodium cyanide, (c) hydroxylamine and (d) phenyl hydrazine.

Phenyl hydrazine also revealed the competitive type of inhibition with o-D and completely inhibited the isoenzymes activity at 1 mM (Figs. 6d and 7d). The Km (0.98, 1.69, 2.13, 2.78 mM) and Vmax (0.19 mmol min−1 ml−1 ) values for TCWP1 and Km (0.47, 0.72, 1.03, 1.47 mM) and Vmax (0.15 mmol min−1 ml−1 ) values for TCWP2 were obtained from Figs. 6d and 7d; respectively. The Phenyl hydrazine was reported as a competitive inhibitor of S. melongena peroxidase activity [11]. The TCWP isoenzymes were followed the similar inactivation mechanism of lacrimal gland peroxidases. Thus, the inactivation was due to the involvement of free radical species generated by enzymatic oxidation of phenyl hydrazine [63]. 3.6. Effect of possible inhibitors and activators on the TCWP isoenzymes activity A wide variety of proteins and enzymes harbor metal ions or metal complexes into their overall structure and which triggers enhancement of their activity. Among the different metal ions tested, TCWP activity was activated to 116% and 121% in the presence of 1 mM concentration of Mn2+ (Tables 2a and 2b), whereas, the other metal ions such as Mg2+ , Zn2+ and Ni2+ were drastically reduced the TCWP activity varying between 76 to 84% at

10 mM. Similarly, Mg2+ was showed peroxidase activity inhibition at 5.0 and 3.0 mM concentration for Golden delicious HP (38%) and Red delicious (55%) apple varieties. But, Mn2+ stimulated the Royal delicious apple variety peroxidase activity to 12% at 1.0 mM concentration [64], which was similar to the activation of TCWP isoenzymes activity. Previous studies have also shown that Ca2+ or Mn2+ enhances the peroxidase activity in vitro, because these two metals are involved in the maintenance of protein conformation and in the regulatory process of the enzyme [65]. Using 1 mM concentration of Ascorbic acid, ␤-mercaptoethanol and DTT, ∼90% of inhibition was observed in TCWP isoenzymes activity (Tables 2a and 2b). Ascorbate was a scavenger for peroxy radicals and therefore inhibits the formation of hydroperoxides [66]; hence both the TCWP isoenzyme activity was drastically decreased at 1 mM concentration of ascorbate. In contrast, Crocus sativus peroxidase activity (50%) was observed even at 15 ␮M concentration [67] and inhibitory effect was reported in soybean root peroxidase activity [68,69]. The inhibition effect of ␤-mercaptoethanol and DTT on TCWP activity reveals that cysteine residue(s) may have significant effect on the structure and activity of the enzyme. The TCWP isoenzymes activity was inhibited (∼90%) at a 1 mM concentration of the above three chemicals, similarly,

M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

13

1 mM 2 mM 5 mM 7.5 mM

-4

-3

10 20 30 40

12 11 1/v (mmole/min/mg)

-5

(b)

-2

-1

24

M M M M

22 20

10

18

9

16

1/v (mmole/min/ml)

(a)

8 7 6 5 4

1

2

0

1

2

3

4

5

-3

-2

-1

-2

1

2

3

4

5

3

4

5

60 50 1/v (mmole/min/ml)

2.5 2.0 1.5 1.0

0.0

0

70

0.2 mM 0.4 mM 0.6 mM 0.8 mM

3.0

-1

0

1/ [O-dianisidine] (mM)

3.5

1/v (mmole/min/ml)

-3

8 4

40 30 20 10

0.5

-4

10

2

(d)

2.5 (mM) 5 (mM) 7.5 (mM) 10 (mM)

12

6

1/[O-dianisidine] (mM)

(c)

14

3

0

39

0

1

2

3

4

5

-3

-2

-1

0

0

1

2

1/[O-dianisidine] (mM)

1/[O-dianisidine] (mM)

Fig. 7. Effect of various inhibitors on the peroxidase activity of TCWP2 . Type of inhibition is obtained from plotting 1/v against the various inhibitor concentration at various o-dianisidine concentrations and fixed concentration of H2 O2. The inhibitors used were (a) sodium azide, (b) sodium cyanide, (c) hydroxylamine and (d) phenyl hydrazine.

manganese peroxidase activity was reported to completely inhibit at 2 mM concentration [70]. The effect of carboxylic compounds on TCWP activity was given in Fig. 8. The sodium acetate enhances both the TCWP isoenzyme activity to 120%, whereas the oxalate and citrate reduces 72–85% of its original activity. The tartarate treated enzymes

retained 87% of its activity even at 7 mM concentration. The role of sodium acetate in TCWP isoenzymes activity imitates the function of acetate ions in halogenation reaction and the oxidation of amino to nitro groups of non-heme haloperoxidases [71]. In contrast, the highest manganese peroxidase activity was obtained by oxalate than acetate and citrate [72,73]. It revealed that the

Table 2a Effect of metals, radical scavenger and thiol group on TCWP1 . Metals or effectors

Residual activity (%) Concentration (mM)

MgCl2 MnCl2 NiSO4 ZnSO4 ␤-Mercaptoethanol DTT Ascorbic acid

0.001

0.01

0.1

21.1 ± 0.02 72.2 ± 0.04 63.6 ± 0.04 44.3 ± 0.02 97.2 ± 0.08 88.2 ± 0.003 N.D

17.5 ± 0.02 83.2 ± 0.02 68.9 ± 0.04 40.0 ± 0.03 93.3 ± 0.04 86.6 ± 0.07 N.D

17.5 92.2 59.1 35.1 38.8 66.3 55.8

1 ± ± ± ± ± ± ±

0.01 0.04 0.02 0.03 0.03 0.09 2.4

15.8 116 43.4 30.2 3.90 3.31 0.11

10 ± ± ± ± ± ± ±

0.01 0.05 0.03 0.02 0.04 0.02 0.02

14.9 ± 0.02 114 ± 0.02 37.2 ± 0.03 26.8 ± 0.02 0 0 0

N.D., not determined. The TCWP1 was pre-incubated in 20 mM sodium acetate buffer (pH 4.5) with each metal ion or chemical reagent at different concentrations at 30 ◦ C for 10 min, and then activities were determined under the standard assay condition. Data are given as mean ± SD of three independent experiments.

40

M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

Table 2b Effect of metals, radical scavenger and thiol group on TCWP2 . Metals or effectors

Residual activity (%) Concentration (mM)

MgCl2 MnCl2 NiSO4 ZnSO4 ␤-Mercaptoethanol DTT Ascorbic acid

0.001

0.01

0.1

39.1 ± 1.3 77.6 ± 0.6 68.7 ± 2.5 40.3 ± 0.09 81.1 ± 2.4 79.2 ± 1.1 N.D

26.5 ± 1.7 85.8 ± 1.3 62.2 ± 1.2 38.2 ± 1.3 77.8 ± 1.7 72.3 ± 2.2 N.D

21.5 94.2 56.1 32.1 54.2 64.8 63.8

1 ± ± ± ± ± ± ±

2.2 2.5 1.9 0.02 2.3 3.8 0.09

19.8 121 47.4 27.2 17.1 12.7 0.32

10 ± ± ± ± ± ± ±

11.9 ± 0.9 119 ± 1.6 27.2 ± 3.1 18.8 ± 2.7 0 0 0

0.02 2.4 2.4 0.5 2.4 4.2 0.04

N.D., not determined. The TCWP2 was pre-incubated in 20 mM sodium acetate buffer (pH 5.0) with each metal ion or chemical reagent at different concentrations at 30 ◦ C for 10 min, and then activities were determined under the standard assay condition. Data are given as mean ± SD of three independent experiments.

3.7. Effect of H2 O2 and CaCl2 The spontaneous generation of extracellular H2 O2 was necessary for plant defense and growth. Release of H2 O2 into the apoplast by growing plant tissues has been reported in germinating radish seeds and roots [74]. Rodriguez et al.[75] also reported that the H2 O2 was generated in the expanding region of maize seedling leaves and excess release of H2 O2 diminishes the enzyme activity [75]. Though, the maximum residual activity was obtained only with 1 mM of H2 O2 , the TCWP activity was stable at above 80% even up to 10 mM of H2 O2 for 1 h (Fig. 9). But the residual activity was suddenly reduced to 2% of its original activity after 10 min of incubation at 15 mM of H2 O2 . Similarly the Raphanus saticus peroxidase activity was reported at 10 mM of H2 O2 after 1 h of incubation [76]. Earlier reports revealed that the peroxidase activity was enhanced and stabilized in the presence of CaCl2 [65]. The TCWP activity was also stable in the presence of 5 mM CaCl2 at 55 ◦ C for 1 h (Fig. 10). The protection of TCWP against irreversible thermal denaturation by CaCl2 was similar to that found in other plant

[77,78] and fungal peroxidases [79]. The stability of the enzyme was due to the susceptibility of cationic peroxidases by Ca2+ ions [80,81]. The HRP and Cassia acidic peroxidase showed higher enzyme stability at 55 ◦ C after 20 h of incubation and Sorghum grain peroxidase (SPC4) kept full activity up to 65 ◦ C for 90 min incubation [55,82]. 3.8. Effect of biotransformed and precursor on human genomic DNA The biotransformed product was prepared using TCWP isoenzyme and its effect on human peripheral DNA shown in Fig. 11. The possibility of DNA damage by the peroxidation products of o-D was proved using an agarose gel electrophoresis. This technique allows separation of native DNA and its modified forms resulting from the interaction with peroxidation products of xenobiotics, causing the formation of intra- and inter-chain covalent links [83]. When high molecular weight double helix DNA from human blood was exposed to increasing concentration of the precursor (0.01–0.04 mg), the DNA fluorescence intensity decreased proportionally. On the other hand, the same concentration of o-D biotransformed product treated DNA samples did not show any

(a)

Residual activity (%)

acetate was essential for plants rather than fungal peroxidase activity.

100 80 60 40 20

0 0

10

20

30

40

50

60

(b)

Residual activity (%)

Time (min) 100 80 60 40 20 0 0

10

20

30

40

50

60

Time (min) Fig. 8. Effect of carboxylic acid on enzyme activity. In the standard assay mixture, four different concentrations (() 0 mM; ( ) 1 mM; ( ) 3 mM; ( ) 5 mM; ( ) 7 mM) of four carboxylic compounds (tartarate, oxalate, citrate and acetate) were separately added. The residual activity was compared with control (devoid of carboxylic compound). Data presented are average values ± SD of three independent experiments.

Fig. 9. Effect of hydrogen peroxide on enzyme activity. The TCWP1 (a) and TCWP2 (b) was pre-incubated with 5 mM (♦), 10 mM () and 15 mM () of hydrogen peroxide in 20 mM sodium acetate buffer (pH 4.5 & 5.0 respectively) for 1 h. 0.05 ml of enzyme was drawn from the incubation mixture at 10 min interval and placed on ice until the start of the residual activity was determined at 25 ◦ C. The activity without pre-incubation was taken as 100%. Data presented are average values ± SD of three independent experiments.

(a)

Residual activity (%)

M. Balasubramanian, R. Boopathy / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 33–42

120 100 80 60 40 20 0

0

20

40

60

80

100

41

The characteristics features of purified isoenzymes were stable up to 50 ◦ C for 1 h; activity was enhanced with Ca2+ ions at 55 ◦ C; similar to other plant peroxidases TCWP isoenzymes were competitively inhibited by sodium cyanide and phenyl hydrazine and sodium acetate is essential for peroxidase activity. The characeterized TCWP isoenzymes biotransform the genotoxic compound into non-genotoxic compound. This unique character of TCWP isoenzyme find application in many industries.

(b)

Residual activity (%)

Time (min) Acknowledgements

120 100 80 60 40 20 0

0

20

40

60

80

100

Time (min) Fig. 10. Effect of CaCl2 on enzyme activity. Heat stability curves of (a) TCWP1 and (b) TCWP2 incubated at 55 ◦ C in 20 mM sodium acetate, pH 4.5, with 1 mM (♦), 2 mM (×), 4 mM () and 5 mM () Ca2+ . Samples were removed at the times indicated and incubated at 4 ◦ C for 1 h before residual peroxidase activity was assayed according to the standard assay protocol. The activity without pre-incubation was taken as 100%. Data presented are average values ± SD of three independent experiments.

Fig. 11. Genotoxicity of precursor and biotransformed o-D product on human peripheral blood DNA. Agarose gel electrophoresis of high molecular weight DNA from human blood (∼2 ␮g per lane) exposed to reaction medium (4 mM citrate phosphate buffer pH 5.6; 0.1 mg/ml DNA) for 30 min at room temperature: Lane 1: DNA; lanes 2–5: increasing concentration of precursor [0.01–0.04 mg respectively]; lanes 6–8: various concentrations of o-D byproduct [0.01 mg (6); 0.03 mg (7); 0.04 mg (8)].

difference in fluorescence intensity. The decreasing of fluorescence intensity in the precursor (o-D) treated DNA may be due to cross-linking of precursor with the DNA molecule. Feng et al. [84] reported that the biotransformed product of 4-Aminobiphenyl was formed adducts with DNA and it may induce mutations and initiate carcinogenesis [84]. This result can be further confirmed by using Amest test, which is widely used method to determine the carcinogenicity or genotoxicity of any compounds. 4. Conclusion Coconut water has been extensively studied since its introduction to the scientific community. In natural form, it is a refreshing and nutritious beverage, alternative for oral rehydration, also offer protection against myocardial infarction and play a vital role in aiding the human antioxidant system. In this work the peroxides isoenzymes were isolated and purified to its homogeneity.

The authors acknowledge gratefully to the Department of Atomic Energy-Board of Research in Nuclear Sciences (DAE–BRNS), Govt. of India, for the financial support and Bharathiar University for lab facility. Balasubramanian renders gratitude to his seniors Mr. V. Shunmugam, Dr. K.M. Ramkumar, and juniors Ms. L. Chitra, Ms. V. Laskshmi, Ms. Swapna Merlin David and Ms. V. Gayathri for valuable suggestions and support to complete this manuscript. References [1] N.D. Srinivas, R.S. Barhate, K.S.M.S. Raghavarao, J. Food Eng. 54 (2002) 1–6. [2] A.M. Azevedo, V.C. Martins, D.M.F. Prazeres, V. Vojinovie, J.M.S. Cabral, L.P. Fonseca, Biotechnol. Ann. Rev. 9 (2003) 1387–2656. [3] N. Veitch, Phytochemistry 65 (2004) 249–259. [4] R.A. Sunde, K.M. Thompson, J. Trace Elem. Med. Biol. 23 (2009) 132–137. [5] A. Conesa, P.J. Punt, C.A.M.J.J. Hondel, J. Biotechnol. 93 (2002) 143–158. [6] G.G. Bozzo, K.G. Raghothama, W.C. Plaxton, Biochem. J. 377 (2004) 419–428. [7] V.P. Pandey, U.N. Dwivedi, J. Mol. Catal. B: Enzym. 68 (2011) 168–173. [8] M.K. Das, R.S. Sharma, V. Mishra, J. Mol. Catal.B: Enzym. 71 (2011) 63–70. [9] C.M. Ajila, U.J.S. Prasada Rao, J. Mol. Catal. B: Enzym. 60 (2009) 36–44. [10] S.K. Vernwal, R.S. Yadav, K.D. Yadav, Ind. J. Biochem. Biophys. 43 (2006) 239–243. [11] T. Rudrappa, V. Lakshmanan, R. Kaunain, N.M. Singara, B. Neelwarne, Food Chem. 105 (2007) 1312–1320. [12] L. Watanabe, A.S. Nascimento, L.S. Zamorano, V.L. Shnyrov, I. Polikarpov, Acta Crystallogr. F 63 (2007) 780–783. [13] X.-H. Guo, Y.-D. Lv, J. Jiang, H.-Y. Li, G.-F. Liu, Afr. J. Biotechnol. 11 (2012) 1540–1544. [14] F. Cai, C. OuYang, P. Duan, S. Gao, Y. Xu, F. Chen, J. Mol. Catal. B: Enzym. 77 (2012) 59–66. [15] S. Shigeoka, T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta, K. Yoshimura, J. Exp. Bot. 53 (2002) 1305–1319. [16] A. Liszkay, B. Kenk, P. Schopfer, Planta 217 (2003) 658–667. [17] S.D. Allison, J. Schultz, J. Chem. Ecol. 30 (2004) 1369–1379. [18] F. Passardi, C. Penel, C. Dunand, Trends Plant Sci. 9 (2004) 534–540. [19] L.V. Bindschedler, J. Dewdney, K.A. Blee, J.M. Stone, T. Asai, J. Plotnikov, C. Denoux, T.I. Hayes, C. Gerrish, R. Davies, F.M. Ausubel, G.P. Bolwell, Plant J. 47 (2006) 851–863. [20] R. Huang, R. Xia, L. Hu, Y. Lu, M. Wang, Sci. Hortic. 113 (2007) 166–172. [21] Q. Hussain, Rev. Environ. Sci. Biotechnol. 9 (2009) 117–140. [22] R. Cardena, C. Oropeza, D. Zizumbo, Euphytica 102 (1998) 81–86. [23] P. Rethinam, in: K.I. Vasu, P.K. Thampan (Eds.), Coconut for Rural Prosperity, APCC, Jakarta, Indonesia, 2003, pp. 299–305. [24] J. Arditti, Micropropagation of Orchids, vol. 2, second ed., Blackwell Publishing, Oxford, UK, 2008. [25] M. Anjum Zia, M. Kousar, I. Ahmed, H. Muhammad Nasir Iqbal, R. Zahid Abbas, Afr. J. Biotechnol. 10 (2011) 6300–6303. [26] J.W.H. Yong, L. Ge, Y.F. Ng, S.N. Tan, Molecules 14 (2009) 5144–5164. [27] O.H. Lowry, A.L. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. [28] A. Rompel, M. Albers, J.I. Naseri, C. Gerdemann, K. Buldt-Karentzopoulos, B. Jasper, B. Krebs, Biochim. Biophys. Acta 1774 (2007) 1422–1430. [29] U.K. Laemmli, Nature 227 (1970) 680–685. [30] H. Blum, H. Beier, H.J. Gross, Electrophoresis 8 (1987) 93–99. [31] S.A. Miller, D.D. Dykes, H.F. Polesky, Nucleic Acids Res. 16 (1988) 1215–1218. [32] A.V. Kireyko, I.A. Veselova, T.N. Shekhovtsova, Russ. J. Bioorg. Chem. 32 (2006) 71–77. [33] L.A. Osterman, Techniques for the Investigation of Proteins and Nucleic Acids. Electrophoresis and Ultracentrifugation, Nauka, Moscow, 1981, p. 288. [34] J.-Y. Ciou, H.-H. Lin, P.-Y. Chiang, C.-C. Wangd, A.L. Charles, Food Chem. 127 (2011) 523–527. [35] D. Voet, J.G. Voet, Biochemistry, Wiley, USA, 2003. [36] J.R. Whitaker, Principles of Enzymology for the Food Science, second ed., Marcel Dekker, New York, 1994, pp. 271–300. [37] T. Thongsook, M. Barrett, J. Agric. Food Chem. 53 (2005) 3206–3214. [38] K.M. Mdluli, Food Chem. 92 (2005) 311–323. [39] H. Ito, N. Hiraoka, A. Ohbayashi, Y. Ohashi, Agric. Biol. Chem. 55 (1991) 2445–2454.

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