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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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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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.
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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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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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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.
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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
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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
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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].
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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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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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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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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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