JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012

Characterization of Polyphenol Oxidase in Sweet Potato (Ipomoea Batatas (L.)). Deepaa Manohan and Wong Chen Wai Department of Biotechnology, Faculty of Applied Sciences, UCSI University, No. 1 Jalan Menara Gading, UCSI Heights, 56000 Kuala Lumpur, Malaysia. [email protected]; [email protected] Abstract Polyphenol oxidases (PPOs) from several plant species, including sweet potato, have been implicated in the undesirable brown discoloration of food products. The present work was undertaken to obtain crude PPO extract from sweet potato (Ipomoea Batatas (L.)) and to characterize it in terms of pH, temperature, enzyme kinetic, substrate specificity, thermal inactivation and inhibitors. Spectrophotometric method was used to assay the PPO activity, by measuring the initial rate of quinone formation, as indicated by an increase in absorbance. The enzyme exhibits the maximum activity at pH 7 and 30°C. The substrate specificity for PPO was: 4-methylcatechol> catechol >catechin>pyrogallol. Km and Vmax for 4-methylcatechol were found to be 50mM and 416.67 EU/min/ml, 90.9mM and 476.19 EU/min/ml for catechin, 357.14mM and 1000 EU/min/ml for catechol, and 384.61mM and 1111.11 EU/min/ml for pyrogallol respectively. Inhibitor studies indicated that sodium bisulphite was the most potent inhibitor for sweet potato PPO, followed by ascorbic acid, L-cysteine, sodium chloride and citric acid. As for the thermal inactivation studies, the PPO activity decreased with increasing temperature. Denaturation of this enzyme, measured by loss in activity, could be described as a first-order reaction with k values between 0.0075 and 0.0657min-1. Results suggested that PPO is a relatively thermostable enzyme with a Z-value of 14.1°C and Ea of 95kJmol-1. The Gibbs free energy ΔG value was 98.66kJmol-1 at 333-348°K. Keywords: Polyphenol oxidase, Ipomeoa batatas (L.), Characterization

1. INTRODUCTION Polyphenol oxidases (PPOs) are copper containing oxidoreductases that catalyze the hydroxylation and oxidation of phenolic compounds in the presence of molecular oxygen. Approximately, nearly 50% of tropical fruits are discarded due to quality defects resulting from enzymatic browning [73]. The browning is mainly catalyzed by the enzyme polyphenol oxidase [47].Because of the deleterious effect of enzymatic browning on food products, PPO has been extensively studied in a variety of tissues [69, 60]. Sweet potatoes are native to the tropical parts of the Americas, and were domesticated there at least 5000 years ago [30].The sweet potato is one of the world's most significant food crops with both the tubers and foliage finding their way into the traditional dishes of many countries. This root is susceptible to browning reactions that affect quality and consumer acceptance. Sweet potatoes discolor when cut or sliced, peeled and heat-processed, and the tissue damage caused by these processes results in activation of PPO and leads to discoloration of the product [64, 4].

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 In plants, PPOs are located mainly in thylakoid membrane of chloroplasts and mitochondria [10]. Tanning reactions, taking place after the distruption of tissues rich in phenols may cause binding of soluble polyphenol oxidase to a ‘particulate’ fraction. In the presence of atmospheric oxygen and PPO, monophenol is hydrooxylated to o-diphenol (monophenol oxidase activity), and diphenol can be oxidized to o-quinones (diphenol oxidase activity), which then undergoes polymerization to yield dark brown polymers. The aim of this study was to characterize PPO from sweet potato in terms of substrate specificity, pH, temperature, thermal inactivation, enzymes kinetics and inhibitors.

2. MATERIALS AND METHODS Plant material and chemicals The sweet potatos (Ipomea batatas (L.)) as shown in Figure 1 used in this study were purchased from a morning market in Taman Puchong Perdana. All chemicals used in this study were analytical grade and were used without further purification.

Figure 1. Sweet potato (Ipomea batatas (L.))

Preparation of crude enzyme extract Sweet potato (100g) was cut into small pieces and homogenized in 200ml of pre-chilled (4°C) 0.1M phosphate buffer using blender for 1 minute at maximum speed. The slurry was centrifuged at 9000 rpm at 4°C for 15 minutes. The supernatant obtained was filtered under vacuum from a buncher funnel containing Whatman® No. 1 filter paper and the filtrate was collected in a conical flask. Then, 100ml of the filtrate was pipette drop by drop into 200ml of cold acetone (-20°C) for the formation of the precipitates. The precipitates, crude PPO as separated by centrifugation at 9000 rpm at 4°C for 15 minutes. The resultant light brown coloured acetone precipitates was dried overnight at room temperature. The acetone powder that obtained was stored at -20°C. The enzyme extraction from acetone powder was conducted by mixing 0.1g acetone powder, 15 ml of pre-chilled 0.1M phosphate buffer, pH 6.8 and stirring for 1 hour at 4°C with a magnetic stirrer. The temperature was maintained by covering the beaker with aluminum foil and was enclosed with ice surrounding the beaker. The suspension was centrifuged at 7500 rpm for 30 minutes at 4°C. The supernatant was used as crude PPO.

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 Assay of PPO activity PPO enzyme activity was determined with a spectrophotometer by measuring the initial rate of quinone formation as indicated by an increase in absorbance at 410nm at 15 seconds intervals at 30°C by using catechol as substrate. The activity of PPO was determined by reaction mixture which contained of 0.1ml freshly prepared crude enzyme extract, 3.9ml of 100mM phosphate buffer (pH 7.0) and 1.0ml of 50mM catechol. PPO activity was assayed in triplicate and the results expressed as means. The initial velocity was calculated from the slope of the absorbance vs. time curve. One unit (U) of PPO activity was defined as the amount of the enzyme that increased the absorbance by 0.001 minute-1 under the conditions of the assay [10]. Optimum pH The PPO activity was determined in a pH range of 6.0 to 8.0 by using 100mM phosphate buffers at 30°C. The reaction mixture contained 0.1ml of crude PPO extract, 1.0ml of 50mM catechol and 3.9ml of phosphate buffer. PPO activity was determined in the form of percent residual PPO activity at the optimum pH. The optimum pH value obtained for this enzyme was used in all the other studies. Optimum temperature PPO activity was measured at different temperatures in the range from 25°C to 55°C using catechol as a substrate (50mM). The standard mixture, without the enzyme, was heated to the appropriate temperature in a water bath. After equilibration of the reaction mixture at the selected temperature, the enzyme was added. The reaction mixture contained 3.9ml of phosphate buffer (pH 7.0), 1.0ml of 50mM substrate and 0.1ml of crude PPO extract. The PPO activity was determined in the form of percent residual PPO activity at the optimum temperature. The optimum temperature obtained from this study was used in other studies. Inhibition of PPO Citric acid, L-cysteine, ascorbic acid, NaCI, and sodium bisulphate were used as PPO inhibitors in this study and the effects of these inhibitors on crude PPO were determined by using catechol as a substrate at 30°C. The PPO activities were determined without inhibitor and in the presence of inhibitors at three different concentrations (10, 15, and 20mM for citric acid, 0.02, 0.025, and 0.03mM for L-cysteine, 0.2, 0.5 and 0.8mM for ascorbic acid, 50, 75, and 100mM for NaCI, and 0.01, 0.03 and 0.05mM for sodium bisulphite) with 5mM catechol substrate at pH 7.0. The reaction mixture contained 1.0ml of 5mM catechol, 2.9ml of 100mM phosphate buffer (pH 7.0), 0.1ml of crude enzyme extract and 1.0ml of inhibitor solution. The percentage inhibition for each inhibitor was calculated as in equation 3.1. Equation (3.1): Inhibition (%) = [(Ao – AI) / Ao ] X 100 Where Ao is initial PPO activity (without inhibitor); AI is PPO activity with inhibitor. 16

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 Thermal inactivation Thermal inactivation studies were carried out in the temperature range of 60°C to 75°C for 20 minutes. 1.0ml of crude PPO extract was added to 3.0ml of 100mM of phosphate buffer (pH 7.0) for catechol that was previously preheated to the selected temperature for 15 minutes to ensure the buffer reached the desired temperature. The enzyme samples were removed from the water bath after 20 minutes and were immediately transferred to an ice bath to stop thermal inactivation [55, 74]. 3ml of the heated enzyme solution was mixed with 0.75ml of 50mM catechol, and the residual PPO activity (A) was determined spectrophotometrically. The percentage residual PPO activity was calculated by comparison with unheated enzyme which was used as blank (Ao). The rate constants k for first-order inactivation was determined from the slopes of the inactivation time courses according to the following equation. Equation (3.2): log (A/A0) = - (k/2.303) t Where A0 is the initial enzyme activity and A is the activity after heating for time t. A useful indication of the rate of a first-order chemical reaction is the half-life t1/2, of a substance, the time it takes for its concentration to fall to half the initial value. The time for [A] to decrease from [A]o to ½[A]o in a first-order reaction. The half-life of the enzyme (t1/2) calculated according to the following equation. Equation (3.3): t1/2= 0.693/k The main point to observe about this result is that, for a first-order reaction, the half-life of a reactant is independent on its initial concentration. In addition, decimal reduction time (D value) was estimated from the relationship between k and D according to the following equation. Equation (3.4): D = In (10) / k The Z value, which is the temperature increase required for a one-log 10 reduction (90% decreases) in D value was determined from a plot of log10D versus temperature. The slope of the graph is equal to the 1/Z value. The temperature of treatment and the rate constant in a denaturation process was related according to the Arrhenius equation [3]. Equation (3.5): k = Ae(-Ea/RT) Equation 3.5 can be transformed as in equation 3.6. Equation (3.6): ln k = ln A – Ea/R × T

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 Where R is the universal gas constant (8.314 J mol-1 K-1), k is the reaction rate constant value, A is the Arrhenius constant, Ea is the activation energy (energy required for the inactivation to occur), and T is the absolute temperature in Kelvin. Slopes were calculated by linear regression. The energy of activation of denaturation (Ea) was calculated from the slopes of these Arrhenius plots (natural logarithm of k (In k) values versus reciprocal of absolute temperatures (1/T) according to equation 3.5. When the ln k is plotted against the reciprocal of the absolute temperature, a linear relationship was observed in the temperature range studied. The slope of the line obtained permitted to calculate the activation energy and the ordinate intercept corresponds to lnA [19]. The values of the activation energy (Ea) and Arrhenius constant (A) allowed the determination of different thermodynamic parameters [17] such as variations in enthalpy, entropy and Gibbs free energy, Δ H, Δ S and Δ G, respectively, according to the following expressions [46]. Equation (3.7): Δ H# = Ea - RT Equation (3.8): Δ S# = R ( lnA - ln KB/ hP - ln T) Equation (3.9): Δ G# = ΔH# - T ΔS# Where KB is the Boltzmann constant (1.38 x 10-23 J/K), hP is the Planck constant (6.626 x 10-34 J.s), and T is the absolute temperature. Substrate specificity and enzyme kinetics For the determination of Michaelis constant (Km) and maximum velocity (Vmax), PPO activities were determined using various substrates which include catechol, catechin, 4methylpyrocatechol, and pyrogallol at various concentrations (10mM - 100mM). The reaction mixture for PPO activity contained 3.9ml of 100mM phosphate buffer (pH 7.0), 1.0ml of substrate, and 0.1ml of crude PPO extract. Km and Vmax values of the PPO, for each substrate, were calculated from the plots of 1/V versus 1/[S] according to the method of Lineweaver and Burk [42]. Statistical analysis Statistical analyses of all experimental data on PPO activity for different parameters were done with Microsoft Office Excel 2010. All assays were performed in three replicates and values were expressed as mean ± SD.

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 3.0 RESULTS AND DISCUSSION Preparation of crude enzyme extract Cold acetone played an important role in the process of enzyme precipitation. 3.20g ± 0.12 of acetone powder was obtained from 100g of sweet potato. Assay of enzyme activity 0.07

Absorbance (410 nm)

0.06 0.05 0.04 0.03 0.02 0.01 0 0

50

100

150

200

Reaction time (s) Figure 2. Assay of enzyme activity using catechol as a substrate at pH 6.8 (30°C). Error bars: standard deviations; results are means of three determinations.

From Figure 2, the enzyme activity calculated from the slope of absorbance versus time curve was 3720 EU/min/ml. The yield of PPO extracted from sweet potato was 558000EU/g of acetone powder. Optimum pH The pH dependencies of sweet potato PPO activities in a pH range of 6 to 8 were measured using catechol as a substrate. Assay of the maximum PPO activity of sweet potato was at pH 7.0 as seen in Figure 3. In general, most plants show maximum PPO activity at or near neutral pH values [11, 13, 14, 66, 76]. However, the enzyme has lower or higher pH optimum in some species such as strawberry (pH 4.5) [72] or apple (pH 9.0) PPO [54]. Different optimum pHs for PPO obtained from various sources were reported in the literatures. For example, it was reported that optimum pH values are 6.0 for DeChaunac grape [39], 7.0 for Amasya apple [54], aubergine[19], Yali pear [80], raspberry [27], AnethumgraveolensL.[6], 7.2 for guava [8], 7.5 for Allium sp. [6] and 8.5 for Dog rose. [62], using catechol as substrate. As for the comparison of tubers, the pH also varies depending on the sources. From the previous studies, it was reported that the optimum pHs for the PPO extracted from tubers were 4.6 for taro tuber (colocasiaesculenta) [36], 6.5 for rooster potato (solanumtuberosum cv rooster) [53], 6.8 for potato (solanumtuberosumvarromano) [36], 7.0 for Jerusalem artichoke (Helianthus tuberosus L.) [65], edible yam [65], 7.4 for mustard tuber [43], and 7.5 for cassava (manihotesculenta c.) [9] using catechol as substrate. Moreover, the pH values differ in different parts of the same [48, 17]. Lim [41] reported that sweet potato leaves shows higher value of pH 8.0 because plant PPO concentrated in plastids. 19

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012

120 Relative Activity (%)

100 80 60 40 20 0 pH 6

pH 6.5

pH 7

pH 7.5

pH 8

Figure 3. Effect of pH on PPO activity. Error bars: standard deviations; results are means of three determinations.

Concisely, Alyward and Haisman[1] reported that the optimum pH for maximum PPO activity in plants ranged from 4.0 to 7.0, depending on the purity of the enzyme, the type of buffer used and the substrates used for the assay. Moreover, the nature of phenolic substrates and extraction methods may also affect PPO activity [20].As soon as the enzyme extract was mixed with the substrate (50mM catechol), the solution immediately turned brown, which absorbed strongly at 410nm. The pH was increased from slightly acidic to neutral, the enzyme activity increased and started to decline after pH 7.0. Temperature optimum Temperature dependency of sweet potato PPO enzyme, activities was measured at different temperatures ranging from 25 to 55°C. Assay of PPO activity at different temperatures was shown and the maximum PPO activity of sweet potato was observed at 30°C as shown in Figure 4. Figure 4 shows the relative enzyme activity was reduced in a range of 55% - 80%. The relative enzyme activity decreased rapidly on increasing temperatures above 30°C. The decrease in activity at high temperature was possibly owing to the thermal denaturation of PPO. According to the theory, the intermolecular bonds holding the structure of the PPO in place were broken by heat. Hence when an enzyme was heated up, these bonds break and the active site specificity of the enzyme was lost. Hence it becomes denatured and cannot participate as a catalyst [1]. In this study, the lowest temperature used was 25°C, which exhibit the lowest activity among other temperature tested. At lower temperature, the PPO has less energy to move comparing with other temperatures tested and thus the rate of reaction was slower. The optimal temperature of PPO has been reported to be varied, depending on species and habitat temperature [28]. The majority of enzymes exhibit optimum temperature in the range of 30°C to 40°C. The optimum temperature of PPO varies for different plant sources and for different parts in same plants as well [48, 17]. Recent study by Lim [41] stated that optimum temperature for sweet potato leaves was 45 °C. The temperature also varies among tuber types such as cassava (manihotesculenta c.) [9], taro tuber (colocasiaesculenta) [36], edible yam [65] and rooster potato (solanumtuberosum cv. rooster) have temperature optimum of 30°C while 20

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 Jerusalem artichoke (Helianthus tuberosus L.) shows optimum temperature of 60°C [65] and Liu Zhu-Ming [43] reported 53°C as optimum temperature for mustard tuber. Other than tuber’s PPO, PPOs from apple [80], banana [77] and mango [59] have temperature optima of 30°C while PPOs from cocoa bean [40] and sunflower [75] show 45°C optima under the specified experimental conditions. 120

Relative Activity (%)

100 80 60 40 20 0 25°C 25ºC

30°C 30ºC

35°C 35ºC

40°C 40ºC

45°C 45ºC

50°C 50ºC

55°C 55ºC

Figure 4. Effect of temperature on PPO activity. Error bars: standard deviations; results are means of three determinations.

On the other hand, temperature optima as low as 20°C for bartlet pear and as high as 45°C for berry fruits PPO has been observed by Siddiq and Cash [66] and Wlazly and Targonski [75], respectively. Other than that, some of them reported that optimum temperature values 15°C for Amasya apple [54], 20°C for DeChaunac grape [39], 25°C for Dog rose [62], 30°C for aubergine [19], and 40°C chinese cabbage [52], using catechol as a substrate, 20°C for Dog rose [62], 30°C for aubergin e[19] and 56°C for Amasya apple [54], using 4-methylcatechol as a substrate, and 15°C for Dog rose [62] and 70°C for Amasya apple [54], using pyrogallol as a substrate. Effects of inhibitors Enzymatic browning of fruit may be delayed or eliminated by removing the reactants, such as oxygen and phenolic compounds, or by using PPO inhibitors [19]. The inhibition of enzymatic browning in plants can be the result of inactivation of PPO, elimination of one of the substrates (O2, polyphenols) for the reaction and the action of inhibitors on reaction products of enzyme action to inhibit the formation of coloured products in secondary reactions [8]. Complete elimination of oxygen from plants during drying is difficult because oxygen is ubiquitous [61]. There are a number of inhibitors, such as sodium metabisulphite [8, 39, 62], ascorbic acid [8, 39, 62, 77], sodium cyanide [39, 55], glutathione [31, 39, 55], tropolone [33, 19, 57], thiourea [39, 62, 80], sodium diethyldithiocarbamate [39, 55, 80, 77], myricetin [32], citric acid and acetic acid [77], L-cysteine, sodium azide, tannic acid, benzoic acid and β-mercaptoethanol [62] used by researchers to prevent enzymatic browning. In this study, citric acid, sodium bisulphite, Lcysteine, sodium chloride and ascorbic acid were selected as an inhibitor to prevent the enzymatic browning of sweet potato PPO. The lack of color development observed in the 21

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 presence of the inhibitors in this study was due to their ability to reduce quinones formed by enzymatic oxidative reaction back to colorlesso-dihydroxyphenols [69]. The effects of various inhibitors on PPO from sweet potato were shown in Table 1. Sodium metabisulfite was found to be the most effective inhibitor among others, followed by ascorbic acid, L-cysteine, sodium chloride and citric acid. The most potent inhibitors were ascorbic acid and sodium metabisulfite since these compounds induced a high degree of inhibition, even at the lowest concentration used. Table 1 Effects of inhibitors

Inhibitor

Concentration (mM)

Inhibition (%)

10 15 20

25 ± 0.57 31 ± 0.15 38 ± 0.05

Sodium bisulphite

0.01 0.03 0.05

84 ± 0.02 94 ± 0.11 97 ± 0.23

L-cysteine

0.02 0.025 0.03

72 ± 0.24 85 ± 0.63 90 ± 0.51

Sodium chloride

50 75 100

25 ± 0.04 38 ± 0.05 56 ± 0.05

Ascorbic acid

0.2 0.5 0.8

88 ± 0.01 90 ± 0.05 94 ± 0.12

Citric acid

Sulfiting agents were utilized broadly in the fruit and vegetable industry as anti-browning agents because of their effectiveness and low price [23, 66]. It is believed that sulfites not only simply act as a reducing agent but also have ability to directly inhibit PPO. They also interact with quinones preventing their further participation in forming brown pigments [5]. However, due to safety concerns, their use in fresh fruit and vegetables was banned by the Food and Drug Administration [63, 49]. They are still allowed for use on shrimp to delay the formation of black spot and maintain the quality during storage or processing [37]. Sodium bisulphite possibly inhibits the enzyme or can react directly with the quinones to reduce them to original phenols. In the earlier studies Kavrayan and Aydemir [34], Siddiq and Cash [13], Paul and Gowda [56] reported sodium metabisulfite as the most effective inhibitor against peppermint, pears, field bean and pineapple fruit PPO, respectively. A strong inhibitory effect of sodium bisulphite on sweet potato PPO at was also observed in this study, which was 84%, 94% and 97% of inhibition by using 0.01mM, 0.03mM and 0.05mM sodium bisulphite respectively. Similarly, Kiattisak and Richard [36] reported that sodium metabisulphite was effective inhibitor followed by ascorbic acid for taro tuber (colocasiaesculenta), edible yam (dioscoreaopposita) and for mushroom [25]. 22

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012

Ascorbic acid is also known to inhibit browning due to its capacity of reducing o-quinones to corresponding o-diphenols. In recent reports by [31, 26, 52], Jiang et al . [31], Gomez-Lopez [70], Nagai and Suzuki [52] have observed strong inhibitory effect of ascorbic acid against potato, avocado, Chinese cabbage and banana PPO. In this study, ascorbic acid of 0.2mM, 0.5mM and 0.8mM inhibited the PPO activity by 88%, 90% and 94%, respectively. Beyond 0.8mM of ascorbic acid, there was almost no further inhibition of sweet potato PPO. In a similar study for Fuji apple PPO, Wakayama [70] reported 64.0%, 44.0%, 19.5% and 14.10% retention at pH 5.0 and, 87.5%, 62.6%, 9.9% and 2.4% retention in PPO activity at pH 3.0 with 0.028mM, 0.085mM, 0.142mM and 0.179mM of ascorbic acid. Ming [51] also reported 69% inhibition of lichi PPO with 1mM ascorbic acid. Nevertheless, L-cysteine was also one of the most effective PPO inhibitor where it shows 72, 85 and 90% of inhibition for 0.02mM, 0.025mM, and 0.03mM. Liu Zhu-Ming [43] reported that L-cysteine was the strongest inhibitor for mustard tuber. On the other hand, sodium chloride and citric acid were observed to be the weakest PPO inhibitor in several plant tissues [34, 77, 75]. Similar results were obtained in this study, whereby citric acid and sodium chloride were only able to inhibit 25% - 56% (10mM- 100mM). Thermal Inactivation The thermal inactivation parameters of PPO between 60°C and 75°C were presented in Table 2. Table 2 Thermal inactivation parameters of sweet potato.

Temperatures (°C)

k (10-2 min-1)

t1/2 (min)

D (min)

60 65

0.75 1.25

92.0 55.4

305.7 184.2

-

70

6.09

11.4

37.8

-

75

6.57 -

10.5 -

35.0 -

14.1

Z (°C)

The enzyme activity decreased with increasing temperatures. PPO of sweet potato lost 62% of its activity at 60°C for 10 minutes and almost completely inactivated after 10 minutes at 70°C. Approximately, 38 % of activity was retained after heating at 60°C, 35 % remained when heated to 65°C, 14% at 70°C and 12% at75°C, respectively. PPO enzyme showed a typical temperaturedependent inactivation profile in the presence of the substrate used. At higher temperature, the enzyme most likely underwent denaturation and lost its activity. Stauffer [67] states that denaturation is the heat induced spontaneous, irreversible breakdown of the secondary and tertiary structure of the enzyme protein such that the enzyme will no longer function and cannot re-activate. The results of the heat inactivation studies suggest that PPO belongs to the group of extremely heat-stable enzymes. As indicated by results of heat inactivation of PPO from various sources, short exposures to temperatures of 70-90°C are generally sufficient for partial or total inactivation of the enzyme. It has been noted that heat stability of the enzyme may be related to ripeness of the fruit and in some cases it is also dependent on pH. In addition, different molecular forms from the same source may have different thermostabilities [39, 8].

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 Exposure time and the temperature necessary for the inactivation of the enzyme are relatively variable among different plant sources. For example, the studies on thermal stability of a grape PPO indicated that the enzyme shows about a 50% reduction in activity at 65°C after 20 minutes and complete inactivation can be achieved at 75°C after 15 minutes [68]. At 60°C, PPO from apple has a half-life of 30 minutes [80]. PPOs from lettuce [29] and cocoa bean [40] are relatively heat stable. Heat treatment up to 70°C for 5 minutes did not affect lettuce PPO activity while at 90°C no activity remained after 5 min [29]. PPO in mango skin is also relatively thermo stable, requiring more than 15 minutes at 80°C for 50% loss of activity [59]. Thermal stability of PPO may also be influenced by nature of phenolic substrate used during determination [55, 72]. Moreover, Lourenco et al. [44], while working on PPO from sweet potato variety Norin 1, found that sucrose and salts in the reaction environment functions as protective agents for the enzyme against thermal denaturation [39]. The thermal inactivation of sweet potato PPO between 60°C and 75°C followed first-order kinetics and the k value which is the first-order inactivation constants are depicted in Table 2. Thermal inactivation of PPOs from several sources was shown to follow first-order kinetics [74, 72, 59, 78]. However, loss in PPO activity from sunflower seeds did not follow first-order kinetics at temperatures of 80°C and l00°C but at a lower temperature of 65°C [75]. According to the results presented in the Table 2, it was clear that the enzyme was less thermo stable at higher temperatures since a higher rate constant, k, means that the enzyme is less thermo stable [45]. The half-life (t1/2) is another parameter that plays an important role in the characterization of enzyme stability [7]. Based on the results shown in Table 2, the t1/2 values in the temperatures ranging between 60°C and 75°C varied between 92.0 minutes and 10.5 minutes. The increasing temperature from 60°C to 75°C resulted in a decrease in t1/2 values. Heat- stability was reported to differ among cultivars and multiple form of PPO from the same source, as well as between fruit tissues homogenates and their respective juices [79]. Example of the reported PPO t1/2 values include between 25.6-91.2 minutes at 68°C and 2.4-4.3 minutes at 78 °C for PPO of various apple cultivars [78], 18.8 minutes at 60°C and 8.5 minutes at 70°C for mango kernel PPO [7], 4.5 and 31.6 minutes at 75°C for Ravat and Niagara grapes [74], respectively. In order to establish the link between treatment time and enzyme activity, the D-values were calculated [58]. The decimal reduction time (D value) was calculated according to equation 3.3. D value is the time, at a given temperature and pressure, needed for 90% reduction of the initial activity. The D values in the temperature between 60°C and 75°C obtained were in the range from 305.7 minutes to 35 minutes (Table 2). Some of the reported D include between 30.3-56.6 minutes at 73°C and 8.1-14.4 minutes at 78°C for various apple cultivars [78]. The energy of activation of denaturation, Ea was calculated from the Arrhenius plot and it was 95kJ mol-1. Ea values reflect a greater sensitivity of the enzyme to temperature change. Some of the reported Ea values were 87.8 kJ mol-1 for taro PPO [77], and 37.8-49.2 kJ mol-1 for two cherry laurel cultivars [81]. Yemenicioglu et al. [78] revealed that the Ea values PPO in Amasya (Ea= 255.6 kJ.mol-1) was the least heat-stable and starking delicious (Ea=240.6 kJ.mol-1) was the most heat- stable.

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 The Z value of the sweet potato PPO is calculated from the graph log10D versus temperature. The parameter Z is obtained from intercept point at 1/T = 0 [19]. The calculated value for Z was 14.1°C. The Z value found in this study compares well with the Z value of 13.02 °C for grapes [71]. In general, low Z-values are thought to indicate greater sensitivity to heat [9]. Differences in the kinetics of heat activation of PPO for different products may result from differences in their composition, which is reflective of their variety or the agronomic and climatic conditions under which they were grown [15]. The average values of ΔH, ΔS and ΔG were 94.87 kJ mol-1, -252.8 J mol-1 k-1 and 98.66 kJ mol1 ,respectively. The ΔH value of PPO suggested that the numbers of non-covalent bonds broken in forming a transition state for enzyme inactivation were similar. The high values of change in enthalpy obtained for the different treatment temperatures indicate that enzyme undergoes a considerable change in conformation during denaturation. Positive values of ΔH indicate the endothermic nature of the oxidation reaction. The Δ H obtained in this study was smaller than that of potato PPO which was 98.02 kJ mol-1 as reported by Duangmal and Owusu [20] However, the value was much higher than that of Lepistanuda (13 kJ mol-1) and Hypholomafasciculare (36 kJ mol-1) reported by Yang and Wang [77]. PPO from sweet potato of this study was more heat-resistant as compared to Lepistanuda and Hypholomafasciculare, apparently as a result of the larger Δ H value for inactivation. The negative values observed for the variation in entropy (ΔS) indicated that there are no significant processes of aggregation, since, if this would happen, the values of entropy would be positive [2]. The free energy of PPO increased slightly with increasing temperature. At all temperatures, it was positive and revealed the fact that oxidation reaction was not spontaneous. Based on the results obtained for thermal inactivation, it is concluded that thermal inactivation of PPO could be described by a first-order kinetic model. D, Z, k values and the high values obtained for activation energy, Ea and change in enthalpy indicated that a high amount of energy was needed to initiate denaturation of PPO, most likely due to its stable molecular conformation. Substrate specificity and enzyme kinetics There are several phenolic compounds serve as substrates for PPO. Types of natural phenols vary widely for different plant sources. There are a number of compounds such as dopamine [52, 62], catechol [7, 19, 39, 52, 54, 62, 72], chlorogenic acid [39, 72], pyrogallol [52, 39, 54], caffeic acid [39, 72], p-cresol [39, 62, 72], tyrosine [39, 62], 4-methylcatechol [19, 54, 62, 72] used as substrates for polyphenol oxidase in the literature. Table 3 Km, Vmax and Vmax/Km values for sweet potato PPO.

Substrates

Vmax/Km (min-1)

Km (mM)

Vmax (EU/min/ml)

50

416.7

8.3

Catechin

90.9

476.2

5.2

Catechol

357.1

1000.0

2.8

Pyrogallol

384.6

1111.1

2.9

4-Methyl Catechol

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 In this study, 4-methylcatechol, catechol, pyrogallol and catechin were used as substrates. Vmax, Km and Vmax/Km for each substrate were shown in Table 3.Michaelis–Menten constants (Km), maximum velocities of the reaction (Vmax) were determined using these substrates at various concentrations (10mM – 100mM) at pH 7.0 at 30°C as shown in Table 3. From the LineweaverBurk plots of this enzyme showed that Km values of 50.0mM for 4-methylcatechol, 90.9mM for catechin, 357.1mM for catechol and 384.6mM for pyrogallol. Vmax values 416.7 EU/min/ml, 476.2 EU/min/ml, 1000.0 EU/min/ml and 1111.1 EU/min/ml, were determined for 4methylcatechol, catechin, catechol and pyrogallol, respectively. The best substrate for each enzyme depends on 2 factors which is strong substrate binding or high affinity (low Km value) and high catalytic efficiency (high Vmax value) for a fixed enzyme concentration. The Vmax/Km ratio, referred to as “catalytic power”, can identify the most effective substrate [60]. As seen from Vmax/Km values in Table 3, the enzyme had a relatively high affinity for 4-methylcatechol, which was a better substrate as compared to other type of substrates in this study. The affinity of sweet potato PPO for 4-methylcatechol was the highest followed by catechin, catechol and pyrogallol. This result was consistent with those reported by Dincer [18], whereby 4-metylcatechol was usually the best substrate for PPOs. The lowest activity for sweet potato PPO was obtained with pyrogallol as substrate. The substrate affinity of PPO generally changes depending on the source of the enzyme. For example, Amasya apple PPO [12] has more affinity for 4-methylcatechol than for the other substrates. In an earlier work, [16] found 40mM for the Km value of the PPO of cocoyam tubers with catechol as substrate. Barthet and Veronique [9] reported that cassava (manihotesculenta c.) has Km values of 28.1mM and 5.27mM for catechol and catechin respectively. Whereas, using 4-methylcatechol and catechol as substrates, Lourenco et al. [44] reported Km values of 26.0mM and 96.0mM, respectively, for PPO purified from sweet potato variety Norin 1. Catechin, epicatechin and caffeic acid derivatives are believed to be common natural substrates of several other fruit PPOs. In addition, PPO isoforms in a tissue of interest may toward monophenols and o-diphenols also exhibit differential substrate specificities and variations in their relative activities [12, 55]. 4. CONCLUSION This study has reported the characterization of polyphenol oxidase (PPO) obtained from sweet potato root (Ipomoea batatas (L.)). In the present study, the best conditions to measure PPO activity for catechol were at pH 7.0 at 30°C. For thermal inactivation study, the enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature from 60°C to 75°C. From the outcome, it is clear that PPO belongs to the group of heat-stable enzyme. As for the substrate specificity, it is found that sweet potato PPO was very effective towards 4methylcatechol as substrate, followed by catechin, catechol and pyrogallol. Besides, the sweet potato PPO activity was very sensitive to some of the general PPO inhibitors, especially to sodium bisulphite, ascorbic acid and followed by L-cysteine were proven to be capable of inhibiting the PPO activity more than 70%. These findings evidenced the hypothesis that PPO was responsible for brown discoloration of the fruit tissue when damaged or exposed to molecular oxygen during storage and processing. As for the future studies, PPO characterization should be done on different varieties of sweet potato such as beauregard and also comparative studies should be carried out among sweet potato varieties.

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 7. REFERENCES [1] Alyward, F., and Haisman, D.R., 1969. Oxidation system in fruits and vegetables-their relation to the quality of pressured products. Advances in Food Research, 17, 1-76. [2] Anema, S.G., and McKenna, A.B., 1996. Reaction kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk. Journal of Agricultural and Food Chemistry, 44, 422-428. [3] Arrhenius, S., 1889. Quantitative relationship between the rate a reaction proceeds and its temperature. Journal of Biology and Chemistry, 4, 226-248. [4] Arthur, J.C., and McLemore, T.A., 1956. Properties of polypohenolases causing discoloration of sweet potato during processing. Journal of Agriculture and Food Chemistry, 4, 553-555. [5] Ashie, I.N.A., Simpson, B.K., and Smith, J.P., 1996. Mechanisms for controlling enzymatic reactions in foods. Critical Review in Food Science and Nutrition, 36, 1-30. [6] Arslan, O., Temur, A., and Tozlu, I., 1997. Polyphenol oxidase from Allium sp. Journal of Agriculture and Food Chemistry, 45, 2861-2863. [7] Arogba, S.S., Ajiboye, O.L., Ugboko, L.A., Essienette, S.Y., and Afikabi, P.O., 1998. Properties of polyphenol oxidase in mango (Mangiferaindica) kernel. Journal of Science and Food Agriculture, 77, 459-462. [8] Austin, S., Bingham, E.T., Matthews, D., Shahan, M., Will, J., and Burgess, R.R., 1995. Production and field performance of transgenic alfalfa expressing alpha-amylase and manganese-dependent lignin peroxidase. Journal of Euphytica, 85, 381-393. [9] Barthet, V.J., and Veronique., 1997. Polyphenol oxidases from cassava (Manihotesculenta C.) root: extraction, purification and characterization. Journal of Food Science and Agricultural Chemistry, 4, 179-198. [10] Benjamin, N., and Montgomery, M.W., 1973. Polyphenol oxidase of royal ann cherries: purification and characterization. Journal of Food Science, 38, 799-806. [11] Betrosian, K., Steinburg, M.P., and Nelson, A.I., 1960. Effect of borates and other inhibitors on enzymic browning in apple tissue. Journal of Mechanism in Food Technology, 41, 480499. [12] Bouchilloux, S., Mcmahhil, P., and Mason, H.S., 1963. The multiple forms of mushroom tyrosinase. Journal of Biology and Chemistry, 238, 1699-1707. [13] Cash, J.N., Sistrunk, W.A., and Stutte, C.A., 1976. Characteristics of Concord grape polyphenoloxidase involved in juice color loss. Journal of Food Science, 41, 1398-1402. [14] Chan, H.T., and Yand, H.Y., 1971. Identification and characterization of some oxidizing enzymes of the McFarlin cranberry. Journal of Food Science, 35, 169-172. [15] Chutintrasri, B., and Noomhorm, A., 2006. Thermal inactivation of polyphenoloxidase in pineapple puree. Journal of Lebensmittel-Wissenschaft, 39, 492-495. [16] Colak, A., Ozen, A., Dincer, B., Guner, S., and Ayaz, F.A., 2005.Diphenols from two cultivars of cherry laurel (LaurocerasusofficinalisRoem.) fruits at an early stage of maturation. Journal of Food Chemistry, 90, 801-807. [17] Corsini, D.L., Pavek, J.J., and Dean, B., 1992. Differences in free and protein-bound tyrosine among potato genotypes and their relationship to internal blackspot resistance. Journal of the American chemical society, 69, 423-434. [18] Dincer, B., Colak, A., Aydin, N., Kadioglu, A., and Guner, S., 2002. Characterization of polyphenoloxidase from medlar fruits (Mespilusgermanica L. Rosaceae). Journal of Food Chemistry, 77, 1-7.

27

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 [19] Dogan, M., Arslan, O., and Dogan, S., 2002. Substrate specificity, heat inactivation and inhibition of polyphenol oxidase from different aubergine cultivars. International Journal of Food Science and Technology, 37, 415-423. [20] Duangmal, K., and Owusu, A.R., 1999. A comparative study of polyphenoloxidases from taro (Colocasiaesculenta) and potato (Solanumtuberosum var. Romano). Journal of Food and Chemistry, 64, 351-359. [21] Eskin, N.M., 1990. Enzymatic browning in Biochemistry of Foods. Journal of Biochemistry, 6, 401-432. [22] Eskin, N.M., Henderson, H.M., and Townsend, R.J., 1971. Browning reactions in foods. Journal of Biochemistry, 7, 69-83. [23] Espin, J.C., Trujano, M.F., Tudela, J., and Garcia-Canovas, F., 1995. Monophenolase activity of polyphenol oxidase from Haas Avocado. Journal of Agriculture and Food Chemistry, 45, 1091-1096. [24] Galani, D., and Apenten, R.K.O., 1997. The comparative heat stability of bovine βlactoglobulin in buffer and complex media. Journal of Science and Food Agriculture, 74, 89-98. [25] Golan-Goldhirsh, A., Osuga, D.T., Chen, A.O., and Whitaker, J.R., 1992. Effect of ascorbic acid and copper on proteins. Journal of Bioorganic Chemistry of Enzymatic Catalysis, 8, 61-76. [26] Gomez Lopez, V.M., 2002. Some biochemical properties of polyphenol oxidase from two varieties of avocado. Journal of Food Chemistry, 77, 163-159. [27] Gonzalez, B., De Ancos, M.P., and Cano, 1999. Partial characterization of polyphenol oxidase activity in raspberry fruits. Journal of Agriculture and Food Chemistry, 4, 4068– 4072. [28] Hasegawa, S., and Maier, V.P., 1980. Polyphenol oxidases of dates. Journal of Agricultural and Food Chemistry, 28, 891-893. [29] Heimdal, H., Larsen, L.M., and Poll, L., 1994. Characterization of polyphenol oxidase from photosynthetic and vascular lettuce tissues (Lactucu saliva). Journal of Agriculture and Food Chemistry, 42, 1428–1433. [30] International Potato Center, CIP., 1996. CIP sweetpotato facts. A compendium of key figures and analysis for 33 important sweetpotato-producing countries. [31] Jiang, Y., Fu, J., Zauberman, G., and Fuchs, Y., 1999. Purification of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid. Journal of Food Science and Agriculture, 79, 950-954. [32] Jimenez, M., and Garcia-Carmona, F., 1999. Oxidation of the flavonolquercetin by polyphenol oxidase. Journal of Agriculture and Food Chemistry, 47, 56-60. [33] Kahn, V., and Andrawis, A., 1985. Inhibition of mushroom tyrosinase by tropolone. Journal of Phytochemistry, 24, 905-908. [34] Kavrayan, D., and Aydemir, T., 2001. Partial purification and characterization of polyphenol oxidase from peppermint (Menthapeperita). Journal of Food Chemistry, 74, 147-154. [35] Keha, E., and Kufrevioglu, I.O., 1997. Antioxidant constituents in some sweet pepper (Capsicum annuum L.). Journal of Food Science and Technology, 40, 97-147. [36] Kiattisak, D., Richard, K., and Owusu, A., 1999. A comparative study of polyphenol oxidases from taro (Colocasiaesculenta) and potato (Solanumtuberosum var. Romano). Journal of Food Chemistry, 64, 351-359. 28

JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 [37] Kim, J., Marshall, M.R., and Wei, C., 2000. Polyphenoloxidase. In Seafood Enzymes Utilization and Influence on Postharvest Seafood Quality. 2nd Ed. New York: Marcel Dekker, pg 271-315. [38] Kim, J.Y., Sea, Y.S., Kim, J.E., Sung, S.K., Song, K.J., and An, G., 2001. Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple. Journal of Plant Science, 161, 1145-1152. [39] Lee, C.Y., Smith, N.L., and Pennesi, A.P., 1983. Polyphenoloxidase from de Chaunac grapes. Journal of the Science of Food and Agriculture, 34, 987-991. [40] Lee, P.M., Lee, K., and Karim, M.I.A., 1991. Biochemical studies of cocoa bean polyphenol oxidase. Journal of Science and Food Agriculture, 55, 251-260. [41] Lim M.Y., 2011. Characterization of polyphenol oxidase from sweet potato leaves. Thesis. Malaysia: UCSI University. [42] Lineweaver, H., and Burk, D., 1934. The determination of enzyme dissociation constants.Journal of the American Chemical Society, 56, 658-66. [43] Liu Zhu, M., 2005.Characterization of polyphenol oxidase from mustard tuber. Journal of Hubei University, 4, 625-712. [44] Lourenço, E.J., Neves, V.A., and Da Silva, M.A., 1992. Polyphenoloxidase from sweet potato: Purification and properties. Journal of Agricultural and Food Chemistry, 40, 23692373. [45] Marangoni, A.G., 2002. Enzyme Kinetics: A Modern Approach. 2nd Ed. New York: John Wiley and Sons, pg 248-250. [46] Marin, E., Sanchez, L., Perez, M.D., Puyol, P., and Calvo, M., 2003. Effect of heat treatment on bovine lactoperoxidase activity in skim milk: kinetic and thermodynamic analysis. Journal of Food Science, 68, 89-93. [47] Marshall, M.R., Kim, J., and Wei, C.I., 2000. Enzymatic browning in fruits, vegetable and seafoods. Journal of Food and Agriculture Organization, 41, 259-312. [48] Matheis, G., and Whitaker, J.R., 1987. Modification of proteins by polyphenol oxidase and peroxidase and their products. Journal of Food Biochemistry, 8, 137-162. [49] Martinez, V.M., and Whitaker, J.R., 1995. The biochemistry and control of enzymatic browning. Journal of Food Science Technology, 6, 195-200. [50] Mayer, A.M., and Harel, E., 1979. Phenoloxidase in plants. Journal of Phytochemistry, 18, 193-215. [51] Ming, J.Y., Zauberman, G., and Fuchs, Y., 1997. Partial purification and some properties of polyphenol oxidase extracted from litchi fruit pericarp. Journal of Post-harvest Biology and Technology, 10, 221-228. [52] Nagai, T., and Suzuki, N., 2001. Partial purification of polyphenol oxidase from Chinese cabbage (Brassica rapa L). Journal of Agriculture and Food Chemistry, 49, 3922-3926. [53] Ni Eidhin, D.M., Murphy, E., and O’Beirne, D., 2008. Polyphenol oxidase from apple (MalusdomesticaBorkh. cvBramley’s seedling): purification strategies and characterization. Journal of Food Science, 71, 51-58. [54] Oktay, M., Kufrevioglu, I., Kocacalıskan, I., and Sakiroglu, H., 1995. Polyphenol oxidase from Amasya apple. Journal of Food Science, 60, 494-496. [55] Park, E.Y., and Luh, B.S., 1985. Polyphenol oxidase of kiwifruit. Journal of Food Science, 50, 679-684.

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 [56] Paul, B., Gowda, L.R., 2000. Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab). Journal of Agricultural and Food Chemistry, 48, 3839-3846. [57] Perez-Gilabert, M., and Garcia-Carmona, F., 2000. Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase. Journal of Agriculture and Food Chemistry, 48, 695-700. [58] Riener, J., Noci, F., Cronin, D.A., Morgan, D.J., and Lyng, J.G., 2008. Combined effect of temperature and pulsed electric fields on apple juice peroxidase and polyphenoloxidase inactivation. Journal of Food Chemistry, 109, 402-407. [59] Robinson, S.P., and Dry, I.B., 1992. Broad bean leaf polyphenol oxidase is a 60 kilodalton protein, susceptible to proteolytic cleavage. Journal of Plant Physiology, 99, 317-323. [60] Rolle, R.S., Marshall, M.R., Wei, C.I., and Chen, J.S., 1990. Phenoloxidase forms of the Florida Spiny lobster: Immunological and spectropolarimetric characterization. Journal of Biochemistry and Physiology, 97, 483-489. [61] Roudsari, M.H., Signoset, A., and Crovzet, J., 1981. Eggplant polyphenol oxidase: Purification, characterization and properties. Journal of Food Chemistry, 7, 227-235. [62] Sakiroglu, H., Kufrevioglu, O.I., Kocacaliskan, I., Oktay, M., and Onganer, Y., 1996. Purification and characterization of Dog-rose (Rosa dumlisRechst.) polyphenol oxidase. Journal of Agriculture and Food Chemistry, 44, 2982-2986. [63] Sapers, G.M., 1993. Browning of foods: control by sulfites, antioxidants and other means. Journal of Food Technology, 47, 75-84. [64] Scott, L.A., and Kattan, A.A., 1957. Varietal differences in catechol oxidase content of sweet potato root. Journal of Horticulture Science, 69, 436-442. [65] Shuji Fujita, Yun-Zhen, H., and Tomoko, M., 2006.Purification and Characterization of polyphenol oxidase from edible yam (Dioscorea opposite). Journal of Food Science and Technology Research, 12, 235-239. [66] Siddiq, M., Sinha, N.K., and Cash, J.N., 1992. Characterization of polyphenol oxidase from Stanley plums. Journal of Food Science, 57, 1177-1179. [67] Stauffer, C.E., 1989. Enzyme assays for food scientists. 1st Ed. New York: Van Nostrand Reinhold, pg 67-78. [68] Valero, E., and Garcia-Carmona, F., 1998. pH-dependent effect of sodium chloride on latent grape polyphenol oxidase. Journal of Agriculture and Food Chemistry, 46, 2447-2451. [69] Vamos-Vigyazo, L., 1981. Polyphenoloxidase and peroxidase in fruits and vegetables. Critical Review in Food Science and Nutrition, 15, 49-127. [70] Wakayama, T., 1995. Polyphenol oxidase activity in Japanese apples.Differences among cultivars and prevention by heat, ascorbic acid and reduced oxygen. Journal of Agriculture and Food Chemistry, 600, 251-266. [71] Weemaes, C., Ludikhuyze, L.R., Van Den Broeck, I., Hendrickx, M., and Tobback, PP., 1998. Activity, electrophoretic characteristics and heat inactivation of polyphenoloxidases from apples, avocados, grapes, pears and plums. Journal of Lebensm -Wiss Technology, 31, 44-49. [72] Wesche-Ebeling, P., and Montgomery, M.W., 1990. Strawberry polyphenol oxidase: extraction and partial characterization. Journal of Food Science, 55, 1320–1325. [73] Whitaker, JR., 1972. Principles of enzymology for the food sciences.2nd Ed. New York: Prentice-Hall, pg 24-28.

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JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012 [74] Wissemann, K.W., Lee, C.Y., 1980. Purification of grape polyphenol oxidase with hydrophobic chromatography. Journal of Chromatography, 192, 232-235. [75] Wlazly, A., and Targonski, Z., 2000. Polyphenoloxidase and beta-glucosidase in selected berry fruits. Journal of Zywnosc, 7, 122-132. [76] Wong T.C., Luh, B.S., and Whitaker, J.R., 1971. Isolation and characterization of polyphenol oxidase isoenzyme of Clingstone peach. Journal of Plant Physiology, 48, 19-23. [77] Yang, C.P., Fujita, S., Kohno, K., Kusaboyashi, A., Ashafuzzaman, M.A., and Hayashi N., 2001. Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel. Journal of Agriculture and Food Chemistry, 49, 1446-1449. [78] Yemenicioglu, A., Ozkan, M., and Cemeroglu, B., 1999.Some characteristics of polyphenol oxidase and peroxidase from taro (Colocasiaantiquorum). Turkish Journal of Agriculture and Forestry, 23, 425-430. [79] Zawistowski, J., Billiaderis, C.G., and Murphy, E.D., 1988. Purification and characterization of Jerusalem artichoke polyphenol oxidase. Journal of Food Biochemistry, 12, 1-22. [80] Zhao, H., Moore, J.C., Volkov A.A., and Arnold F,H., 2005. Methods for Optimizing Industrial Enzymes by Directed Evolution. 2nd Ed. Washington: ASM Press, pg 597-604.

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