Polyphenol oxidase from yacon roots (Smallanthus sonchifolius)

Article abstract of DOI:10.1021/jf063148w

Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490 ± 3500 Da and K_m

Full text of DOI:10.1021/jf063148w

2424 J. Agric. Food Chem. 2007, 55, 2424−2430
Polyphenol Oxidase from Yacon Roots
(Smallanthus sonchifolius)
VALDIR AUGUSTO NEVES* AND MARAIZA APARECIDA DA SILVA
Laborato´rio de Bioqu´ımica de Alimentos, Departamento de Alimentos e Nutric¸a˜o, FCF-Ar,
UNESP-Universidade Estadual Paulista, Rodovia Araraquara-Jau´, km 01, CEP 14801-902,
Araraquara, Sa˜o Paulo, Brazil
Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius)
was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The
enzyme had a molecular weight of 45 490 ( 3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM
for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When
assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids,
catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6,
depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds.
p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011
mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 °C showed
the enzyme to be relatively stable at 60-70 °C, with progressive inactivation when incubated at 80
and 90 °C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose,
glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal
inactivation at 75 and 80 °C.
KEYWORDS: Yacon roots; Smallanthus sonchifolius; polyphenol oxidase; purification; kinetics; inhibi-
tors; thermal inactivation; sugar protection
INTRODUCTION
processed yacon products, such as air-dried tuber slices,
unrefined yacon syrup, which has the consistency of honey and
can be marketed as a dietetic sweetener, and other products (1,
2, 5). During the processing of these products, it is necessary
to control enzymatic browning by thermal treatment, addition
of antioxidant, or both.
Yacon [Smallanthus sonchifolius (Poepp. & Endl.) H. Rob-
inson] is an under-exploited root crop [fam. Asteraceae]
originally cultivated in Andean South America. Like the related
“Jerusalem artichoke”, Helianthus tuberosus, the plant produces
large tuberous roots similar to sweet potatoes in appearance,
with a rather sweeter taste due to an abundance of soluble
carbohydrates such as fructose, glucose, sucrose, and fructo-
sylsucrose (FOS, GF2-9) (1), but lacking starch, which makes
it potentially beneficial in the diet of diabetics (2). The roots of
yacon also contain considerable amounts of phenolic compounds
(2030 mg/kg) with a predominance of chlorogenic acid (2, 3)
and caffeic acid derivatives (4). As natural antioxidants, these
compounds have an increasing importance to human health, as
they may protect cell membranes against damage by oxygen
radicals and its consequences in cardiovascular disease and
cancer. Therefore, it is very important to avoid oxidation of
phenols in yacon to maintain the dietary value of these roots.
Yacon root is sometimes used in home cooking, but it is not a
common foodstuff because of its poor shelf life, and the rapid
browning of the juice or injured tissues. In many localities
throughout the Andes, the fresh root is eaten like a “fruit”, due
to its juiciness, sweet taste, and relatively low energy value;
but farmers in Peru, Brazil, and Japan produce a number of
Enzymatic browning of fruits and vegetables during post-
harvest handling and processing is one of the main causes of
quality losses (6, 7). Browning is primarily related to the
oxidation of phenolic endogenous compounds into highly
unstable o-quinones, which are later polymerized to brown, red,
and black pigments. The degree of browning depends on the
nature and amount of endogenous phenolic compounds, on the
presence of oxygen, reducing substances, metallic ions, pH, and
temperature, and on the endogenous activity of polyphenol
oxidase (PPO), the main enzyme involved in the reaction (8).
PPO (E.C. 1.14.18.1) is a copper-containing enzyme widely
distributed in fruits and vegetables, which catalyzes the oxidation
of monophenols, diphenols, and polyphenols to orthoquinones.
PPO has been studied extensively for many years with respect
to its physicochemical properties and response to inhibitors and
substrates in a number of fruits and vegetables (7, 8). PPO is a
plastid enzyme in the higher plants, generally located on the
thylakoid membrane in chloroplasts (7, 8), requiring therefore
a strong non-ionic detergent (Triton X-100) to achieve full
extraction, although a readily soluble form of the enzyme has
* Author to whom correspondence should be addressed [telephone +55-
16-33016935; fax +55-16-33016920; e-mail nevesva@fcfar.unesp.br].
10.1021/jf063148w CCC: $37.00
© 2007 American Chemical Society
Published on Web 02/23/2007

Polyphenol Oxidase from Yacon Roots
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2425
Protein Determination. Protein concentration was determined by
the dye-binding method of Bradford (14) using bovine serum albumin
as standard.
pH Optimum and pH Stability. PPO activity was determined as a
function of pH with 4-methylcatechol, catechol, chlorogenic, and caffeic
acid as substrates. Assays were run at the appropriate temperature for
each substrate, in McIlvaine’s buffer at various pH values (pH 3-8),
with partially purified enzyme. All of the assays were performed in
triplicate.
also been reported (8-10). Yoruk and Marshall (8) reported
that the apparent presence of multiple forms of the enzyme in
purification studies of various plant sources is due, at least in
part, to artifacts of the extraction procedures, where the
simultaneous presence of the phenolic substrate and enzyme
can give rise to aggregated forms as a consequence of the
browning reaction. Thus, the minimization of o-quinone forma-
tion during the extraction procedure is very important, to avoid
these artifacts, and this can be achieved by adding phenol-
scavengers such as polyethylene-glycol (PEG), insoluble poly-
vinyl(poly)pyrrolidone (PVPP), or one of several reducing
agents as ascorbic acid, 2-mercaptoethanol, cysteine, and so on
(11, 12).
In particular, the yacon root darkens rapidly on storage,
cutting, or during processing, and this tendency could be related
to its phenolic content, especially to the levels of chlorogenic
and caffeic acids (2, 3), and the endogenous PPO activity.
Although the presence of PPO in yacon roots has been
demonstrated (13), there is no published study on the properties
of the enzyme from this source.
Optimum Temperature and Stability. PPO activity was determined
as a function of temperature from 10 to 60 °C for various substrates.
Heat inactivation studies were performed over the range of 60-90 °C.
After being heated for a given period in the absence of substrate, the
enzyme aliquot was cooled and immediately assayed as described above.
To study the effect of additives on heat stability, PPO was incubated
in the presence of various sugars, and the remaining activity was
determined. The rate constant (k) for inactivation was calculated from
the slope of the time course of denaturation, using the equation: log-
(A/Ao) ) -(k/2.303)t or ln(A/Ao) ) -kt, where Ao is the initial enzyme
activity and A is the activity measured at time t. The slopes of these
plots were determined by linear regression, and the rate constants
calculated were replotted. The apparent activation energies (Ea) were
calculated from the slopes of the Arrhenius plots: ln k × 1/T, by
applying the equation ln k ) -Ea/RT, where R is the gas constant (8.314
J mol-1 K-1), and T is the temperature in kelvin. Slopes were calculated
by linear regression. All of the assays were performed in triplicate.
Substrate Specificity and Kinetics Studies. The Michaelis-Menten
constant (Km) and maximum velocity (Vmax) of yacon PPO were
determined for various substrates (catechol, 4-methylcatechol, caffeic,
and chlorogenic acid). For each substrate, data were plotted as 1/V ×
1/S, where V is the reaction rate and S is the substrate concentration,
according to the method of Lineweaver-Burk (15). All of the assays
were performed in triplicate.
Thus, the objective of this work was to investigate the
properties of PPO from yacon root that catalyze the browning
reaction during storage and handling following harvesting.
MATERIALS AND METHODS
Material. Yacon roots were obtained from the local market. They
were washed with distilled water, dried on filter paper, and stored at 4
°C until use. The fresh roots were weighed and used for the preparation
of the enzyme extracts.
Reagents. Catechol, 4-methylcatechol, L-ascorbic acid, 2-mercap-
toethanol, DL-DOPA, 1,4-dithioerythritol (DTE), L-tyrosine, Triton
X-100, chlorogenic, cinnamic, p-coumaric, ferulic, caffeic, protocat-
echuic, and benzoic acids, resorcinol, and pyrogallol were obtained
from Sigma Chemical Co. (St. Louis, MO). All other chemicals used
were analytical reagent grade.
Effect of Inhibitors on PPO Activity. The effect of several
inhibitors on yacon PPO activity was measured in the standard reaction
medium in the presence and absence of various concentrations of
inhibitors. Using two distinct concentrations of substrates, PPO activity
was assayed at various concentrations of the inhibitors [i]. Values of
1/V × [i] were plotted, and the Ki was determined by the method of
Dixon (16). All of the assays were performed in triplicate.
Molecular Weight Determination. Molecular weight was deter-
mined by gel filtration on a Sephadex G-100 column, as described by
Whitaker (17). The column (60 × 2.5 cm) was calibrated for molecular
weight with standard proteins: cytochrome c (12.4 kDa), soybean
trypsin inhibitor (21.5 kDa), ovalbumin (45 kDa), and bovine serum
albumin (67 kDa).
Extraction and Partial Purification of PPO from Yacon Root.
Enzyme extracts were prepared so that PPO activity was as high as
possible. Yacon roots (100 g) were peeled, cut into small pieces, and
homogenized in an Ultra-turrax homogenizer (type TP 18-10, from
Jankle & Kunkel, IKA, Germany) with 200 mL of 0.05 M phosphate
buffer (pH 6.0) containing 5 mM ascorbic acid, 0.35 M KCl, 0.5%
Triton X-100, and Polyclar aT (PVPP) (0.1 g/g), as phenolic scavenger.
The homogenate was filtered through two layers of cheesecloth and
centrifuged at 10 000g for 40 min at 4 °C, and the supernatant was
collected. Solid (NH4)2SO4 was added to the supernatant to obtain 20%
saturation with gentle stirring. The resulting suspension was centrifuged
at 10 000g for 40 min at 4 °C. The supernatant was then treated further
with (NH4)2SO4, and the protein fraction precipitating between 20%
and 80% saturation after centrifugation was pooled. The precipitate
was dissolved in 0.05 M phosphate buffer (pH 7.0) containing 3 mM
ascorbic acid and desalted on a column of Sephadex G-25 eluted with
the same buffer. The desalted sample was applied on Sephadex G-100
column (60 × 2.5 cm) equilibrated with 0.05 M phosphate buffer (pH
7.0), and fractions of 5.2 mL were collected. The PPO-active fractions
were pooled and used in further experiments.
RESULTS AND DISCUSSION
Extraction and Partial Purification. Several buffer com-
positions were employed for extracting PPO from yacon, and
Polyclar aT, Triton X-100, and ascorbic acid in the extraction
buffer gave the desired result. PEG-8000 (poly ethylene glycol),
SDS (sodium dodecylsulphate), EDTA, L-cysteine, and 2-mer-
captoethanol, at several concentrations in the buffer, alone or
together with others, were not effective in the extraction of yacon
PPO. The PPO extract was partially purified in various steps,
including ammonium sulfate fractionation and gel filtration on
Sephadex G-100. Only one peak with PPO activity was obtained
from gel chromatography (Figure 1). From the total activity
and protein applied to the column, 6.04% and 0.76% were
recovered, respectively, in the peak. The enzyme after am-
monium sulfate precipitation and elution on Sephadex G-25 was
applied to a DEAE-cellulose column, revealing only one PPO
form (data not shown). A summary of protein and activity data
for each purification stage is given in Table 1. Polyacrylamide
gel electrophoresis of the peak PPO activity eluted from the
Sephadex G-100 column revealed only one band of enzyme
activity with 4-methylcatechol as substrate (data not shown).
PPO Assay. PPO activity was determined by measuring the initial
linear rate of quinone formation at 30 °C as indicated by an increase
in the absorbance at 420 nm for 4-methylcatechol (4-methyl-1,2-
benzenediol), catechol, and caffeic acid, and 410 nm for chlorogenic
acid (11). An Ultrospec 2000 (Amersham Pharmacia Biotech, Upsala,
Sweden) spectrophotometer was employed throughout. The reaction
mixture contained the substrates in 0.05 M phosphate buffer, pH 6.0,
and enzyme solution, for a total volume of 3 mL in the cuvette. The
initial rate of the enzyme-catalyzed reaction was linear with time for 3
min. In all determinations, PPO activity was assayed in triplicate, and
one unit was defined as a change of 0.001 absorbance per min at the
appropriate wavelength for each substrate.

2426 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Neves and da Silva
Figure 1. Chromatography on Sephadex G-100 for yacon PPO. Enzyme
from (NH4)2SO4 fractionation was applied on the Sephadex G-100 column
Figure 2. pH activity profile for yacon PPO with various substrates. The
PPO activity was measured at 30
pH values.
°C in McIlvaine’s buffer at the indicated
(60
× 2.5 cm). Elution was carried out with 5 mM potassium phosphate
-
1
-
buffer, pH 7.5. Activity: A420nm min mL 1. Protein: A280nm
.
Table 1. Summarya of the Purification Stages of Yacon Root PPO
purification
step
volume
(mL)
activity
protein
specific purification
(Units/mL/min) (mg/mL) activityb
fold
crude extract
ammonium sulfate
182
26
80 990
101 400
61.6
42.1
1314.2
2408.5
1
1.8
precipitation
(20
−80%)
Sephadex G-100
4.8
4896
0.47
10 417.0
7.9
a Mean of four replicates. b Units/mg protein.
Thus, the procedure adopted provided a partially purified
enzyme preparation. The molecular weight for yacon PPO
determined from the Sephadex G-100 column was found to be
45.49 ( 3.50 kDa. The elution of the enzyme in a single peak
indicated the presence of only one molecular species. While
there can be uncertainty in the number, size, forms, and
composition of PPOs from many plants, as a function of the
development stage of the tissue, cellular localization, ripening,
storage conditions, presence of proteases, and so on, the isolation
and purification procedures can also produce artifacts that result
in the modification of the native form of the enzyme (7, 8).
Thus, the extraction, isolation, and purification of yacon PPO
were achieved in conditions that avoided the browning due to
the oxidation of the phenolic compounds in the tissue and the
possible attachment of its oxidation products to the native
enzyme. Even so, the PPOs from many plants have been
reported to vary in molecular mass mostly within the range of
35-100 kDa (7, 10, 18, 19).
Effect of pH on PPO Activity. The pH optima of yacon
PPO for various substrates are shown in Figure 2. When the
enzyme was assayed with 4-methylcatechol as substrate, it
displayed high activity between pH 6.0 and 6.5 and was
optimally active at pH 6.2 with a rapid loss of activity above
pH 6.8. With caffeic acid and catechol, the enzyme showed
optimal activity at pH 6.6, more than 90% of this activity
remaining between pH 6.0 and 7.0. It was also observed that
approximately 85% of the optimal activity was retained at pH
4.5 and 5.8, with a maximum at pH 5.0, when chlorogenic acid
was the substrate. The optimum pH of PPO varies with the
source of enzyme and also depends on the phenolic substrate
chosen for the assay, so that a range of values of maximum
PPO activity have been reported in the literature between pH
4.0 and 7.0 for the enzyme from several sources with various
substrates. It is reported that the optimum pH values of PPO
are 5.0 and 5.7 for Victoria grape and egg-plant (9, 20), 4.5-
Figure 3. Temperature activity profiles for yacon PPO in McIlvaine’s buffer
with various substrates.
6.5 for sweet potato (10), 4.5-6.6 for palm-heart (11), 5.0-
7.0 for globe artichoke heads (19), 6.6-6.8 for potato (18, 21),
and 6.0-9.0 for sweet basil (Ocimum basillicum L.) (22). The
optimum pH reported here for all substrates tested is within
these reported values.
Effect of Temperature on PPO Activity. The temperature
dependence of the activity of the partially purified PPO from
yacon roots for various substrates is presented in Figure 3.
Optimum temperature for the enzyme was near 30 °C, for all
four substrates, and for caffeic acid and catechol there was a
rapid decline up to 50-55 °C. PPO from Victoria grape (9),
sweet potato (10), palm-heart (11), artichoke heads (19), potato
(18, 21), egg-plant (20), and persimmon fruits (23) has optimum
temperatures between 10 and 60 °C, which depend on the
substrate used. It is clear that in general the optimum temper-
atures for PPO are quite species- and substrate-dependent (7,
8).
Substrate Specificity and Enzyme Kinetics. The PPO from
yacon roots showed activity with various o-diphenols. The
enzyme was unable to oxidize L-tyrosine, suggesting a possible
absence of monophenol monooxygenase (cresolase) activity.
Types and relative concentrations of natural phenols vary widely
in different plant sources. Thus, it appears that substrate
specificity of PPO similarly depends on the species and cultivar.
The potential substrates resorcinol (400 nm), DL-DOPA (475
nm), pyrogallol (470 nm), protocatechuic (410 nm), p-coumaric
(410 nm), ferulic (410 nm), and cinnamic acids (410 nm),
catechin, and quercetin were not oxidized by yacon PPO,
although these compounds have been shown to be substrates

Polyphenol Oxidase from Yacon Roots
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2427
Table 2. Kinetic Constantsa (Km and Vmax) for Various Substrates of
Yacon Polyphenoloxidase
Table 3. Effect of Various Inhibitor Substances on Yacon PPO
Activitya
Km
(mM)
Vmax
(Units/min)
V
max/Km
% inhibition (substrates)
concentration
in the assay
substrate
(Units/min/mM)
R2
chlorogenic
caffeic
acid
4-methyl
catechol
substance
control
p-coumaric acid
cinnamic acid
ferulic acid
pyrogallol
protocatechuic acid
resorcinol
(
µ
mol)
acid
chlorogenic acid
4-methylcatechol
caffeic acid
1.14
1.34
0.23
5.00
1428.6
525.3
494.4
666.0
1253.2
392.0
1890.4
133.2
0.989
0.993
0.994
0.991
0
0
0
0.5
0.5
0.5
2.0
2.0
2.0
2.0
95.00
76.25
68.75
62.50
41.25
36.25
13.75
24.75
21.00
42.45
9.80
3.28
1.60
5.02
3.30
60.72
18.00
catechol
a Calculated by the method of Lineweaver
−Burk from a plot of 1/V versus 1/[S].
22.04
31.70
43.50
for other plant PPO (8). The PPO extracted from various sources
has shown varying substrate specificity in published reports (11,
19, 24).
phenylalanine
a Chlorogenic, caffeic acids, and 4-methylcatechol were used as substrates as
described in Material and Methods.
The enzyme kinetic constants, Km, and Vmax for yacon PPO
with various substrates are listed in Table 2. The Km for caffeic
acid was 0.23 mM, a value lower than those reported for potato
(21) and sweet potato enzymes (10) at 2.3 and 5 mM,
respectively, and comparable to the value of 0.590 mM for palm-
heart PPO (11), indicating a high affinity of yacon PPO for
this substrate. The Km for 4-methylcatechol was lower than those
reported for grape (9), artichoke heads (19), egg-plant (20), and
pear (25). Lee et al. (26) found Km values of 15.9 and 24.6
mM for DeChaumac grape with caffeic and 4-methylcatechol
as substrate, respectively. Duangmal and Owusu-Apenten (21)
reported that 4-methylcatechol was the preferred substrate for
PPO from taro and potato with Km values of 9.0 and 1.1 mM,
respectively. When using catechol as substrate, the Km for yacon
PPO is close to the values of 6.25, 6.8, and 7.51 mM reported
for peppermint (27), potato (21), and Victoria grape (9).
Maximum activity was observed with chlorogenic acid,
followed by catechol, 4-methylcatechol, and caffeic acid;
however, this does not define the order of substrate specificity,
because the best substrate for each enzyme depends on two
factors: strong substrate binding, indicated by a low Km, and
high catalytic efficiency, as expressed by a high Vmax value.
Thus, the criterion for choosing the best substrate is the highest
Table 4. Effect of Inhibitor Substances on Yacon PPO Activitya
% inhibition (substrates)
concentration
in the assay
chlorogenic
caffeic
acid
4-methyl
substance
control
(
µ
mol)
acid
catechol
0
0
0
0
salicylic
1.2
3.5
23.8
oxalic acid
1.2
2.0
62.2
81.3
100
7.5
21.5
32.3
16.9
62.3
6.6
44.6
100
34.6
58.4
38.7
73.1
8.4
metabisulphite
cysteine
0.15
0.30
0.15
0.30
0.15
0.30
0.06
0.30
0.60
2.0
100
56.5
73.1
48.2
81.3
10.4
19.2
31.1
38.8
43.0
DTE
23.3
8.5
ascorbic acid
22.5
28.7
23.4
23.8
30.3
77.3
0
benzoic acid
3.0
4.20
a Chlorogenic, caffeic acids, and 4-methylcatechol were used as substrates as
described in Material and Methods.
V
max/Km ratio. From the apparent Vmax/Km values, caffeic and
Only resorcinol exerted stronger inhibition with 4-methylcat-
echol, while phenylalanine showed strong inhibition with caffeic
acid. The inhibition by p-coumaric and cinnamic acids with
chlorogenic acid as substrate was shown to be competitive, with
Ki values of 0.017 and 0.011 mM, respectively, calculated from
Dixon plots (16). Ferulic acid was the phenolic acid that
inhibited both chlorogenic and caffeic acids efficiently and
showed uncompetitive inhibition with chlorogenic acid. Oxalic
and benzoic acids showed relatively poor inhibition (Table 4),
while the thiol reagents dithioerythritol (DTE), sodium met-
abisulfite, and cysteine at the concentrations indicated were
effective inhibitors of yacon PPO, in contrast to ascorbic acid,
which showed low inhibition for all substrates. The thiol
reagents and ascorbic acid produced a lag period, before any
changes in absorbance were observed during the measurements
of PPO activity. This effect increased with inhibitor concentra-
tion for all substrates in a variable manner between 2 and 5
min. This apparent lag has also been observed by other authors
(7, 9, 10) for thiols and ascorbic acid with PPO from other
sources. The lack of color development observed at the start of
the reaction in the presence of these inhibitors is attributed to
the inhibitor being involved in reducing the quinones formed
back to the colorless o-dihydroxyphenols.
chlorogenic acids (Table 2) were the best substrates for yacon
PPO, while catechol was the worst. Rapeanu et al. (9) reported
that Victoria grape PPO showed higher values of substrate
specificity for chlorogenic, catechin, and 4-methylcatechol, in
this order, while Duangmal et al. (21) showed the order for
substrate specificity was 4-methylcatechol, caffeic acid, catechol,
and chlorogenic acid for potato PPO. However, until now there
are no data for yacon PPO in the literature.
It has been found that yacon contains considerable amounts
of phenolic compounds (3); for that reason, it represents a rich
source of phenolic acids and other radical scavenging com-
pounds (4). Simonovoska et al. (28) detected chlorogenic,
ferulic, and caffeic acids, quercetin, and unidentified flavonoids
in yacon roots by thin-layer chromatography and HPLC/MS.
Effect of Inhibitors on PPO Activity. The behavior of yacon
PPO toward inhibitors was examined with various compounds,
including analogues of the substrates. The effect of these
inhibitors on enzyme activity with chlorogenic acid, caffeic acid,
and 4-methylcatechol as substrates is presented in Tables 3 and
4. The inhibition study appears to show stronger inhibition for
yacon PPO by the compounds tested when using chlorogenic
and caffeic acids as substrates as compared to 4-methylcatechol.
It is important to note that the former are natural substrates
present in yacon tissue (2, 3, 28). p-Coumaric, cinnamic, and
ferulic acids strongly inhibited the enzyme with chlorogenic acid
as substrate, as compared to caffeic acid and 4-methylcatechol.
Thermal Inactivation Kinetics of Yacon PPO. From the
log-linear plots of residual yacon PPO activity against inactiva-
tion time at constant temperature, it can be concluded that the
thermal inactivation of the enzyme is adequately described by

2428 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Neves and da Silva
Figure 4. Heat inactivation plots of yacon PPO at different temperatures.
Enzyme aliquots in 5 mM potassium phosphate buffer, pH 7.5, were
incubated at the indicated temperatures. The remaining activity was
determined with chlorogenic acid as substrate. Ao and A, initial and residual
activity at the time measured, respectively. The rate constants (k) for
inactivation were determined from the slopes of the logarithmic plot of
activity against time: log(A/Ao×100) ) −(k/2.303)t.
Figure 5. Heat inactivation of yacon PPO. The apparent activation energy
(Ea) was calculated from the slope of the Arrhenius plot according to the
equation ln k ) −Ea/RT, where k is the rate of inactivation at T, R is the
-
1
-
gas constant (8.314 J mol
K
1), and T is the temperature in kelvin.
Slopes were calculated by linear regression.
a first-order model in the temperature range tested (Figure 4).
As expected, at higher temperatures, inactivation proceeded
faster. It can be seen in Figure 4 that the heat inactivation of
PPO between 80 and 90 °C was dramatically rapid. The enzyme
activity determined with chlorogenic acid as substrate at pH
5.0 was reasonably stable at 60 °C; however, an increase from
60 to 70 °C inactivated PPO rather more quickly, with 50%
reduction after 20 min of heating at 70 °C. Above 75 °C, the
loss of activity became very rapid, whereas 2 min at 90 °C fully
inactivated the enzyme. In contrast to peroxidases, PPO is not
a heat-stable enzyme (7, 29, 30). The half-life of yacon PPO at
70 °C was similar to ones from other sources, such as grape
(9), taro, and potato (18, 21), and the enzyme was less resistant
to heating than the PPO from sweet potato (10), palm-heart (11),
persimmon (23), and peppermint (27).
Figure 6. Heat inactivation curves of PPO in the presence of sugars at
75 °C. Enzyme aliquots in 5 mM potassium phosphate buffer, pH 7.5,
were incubated in the presence of sugars at 10% (A), 20% (B), and 30%
(C) concentration (w/w %), and the remaining activity was determined
with chlorogenic acid as substrate. Ao and A: initial and residual activity
at the time measured, respectively.
important for the effective control of these undesirable reactions
in processed fruits and vegetables; moreover, POD is an
oxidative enzyme with an apparently higher resistance to heating
than PPO, demonstrated by its higher values of Ea for inactiva-
tion in a number of plant sources (7, 30, 31).
Effect of Sugars on Heat Stability. It is well established
that polyhydric compounds are among the most useful molecules
for stabilizing the native conformation of globular proteins in
solution, when added in high concentrations (33). Several
theories into the mechanism of action for these solvent additives,
also named cosolvents, appear to indicate that they are
preferentially excluded from the surface of the native protein
and cause a preferential protein-water interaction as a function
of the cosolvent concentration in the system (34). Arakawa and
The temperature dependence of the rate constant (k) for
thermal inactivation of partially purified yacon PPO was
evaluated from the Arrhenius equation, as shown by Figure 5.
The calculated Ea (activation energy) for enzyme inactivation
was 93.69 kJ mol-1, higher than for PPO from potato (21) and
lower than Ea for taro, Victoria grape, and DeChaunac grape
(9, 21, 26). The possible involvement of peroxidase (POD) in
the enzymatic browning has been reported (32), and this fact is

Polyphenol Oxidase from Yacon Roots
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2429
to the control without sugar (Figure 7). Values of 67%, 71%,
51%, and 58% of the original enzyme activity were obtained
for 30% (w/w) of trehalose, glucose, fructose, and sucrose in
the medium, after 90 s, respectively, as compared to 16% for
the control (Figure 7B).
Sola-Pena and Meyer-Fernandes (36) also observed a stronger
stabilizing effect for some enzymes with trehalose relative to
other sugars such as maltose, sucrose, glucose, and fructose.
The authors suggested that the large hydrated volume of
trehalose as compared to other sugars has a more pronounced
size-exclusion effect and that all sugars have similar effects if
the concentrations are corrected for the volume occupied by
each one. These observations corroborate the idea of the
preferential hydration of proteins, as suggested by Timasheff
(34).
Various oxidative enzymes, such as plant peroxidases, have
been shown to be less susceptible to thermal inactivation when
heated in sugar solutions at various concentrations, and this
variable susceptibility was reflected in alterations of Ea for
thermal inactivation (29, 30). According to our results and those
of other authors, enzymes from different sources appear to
behave differently in the presence of sugars on heating.
Kavrayan and Aydemir (27) observed, for peppermint PPO,
results contrary to those for yacon PPO, in which the sucrose
solutions at 20% and 40% (w/w) did not protect the enzyme
against heat inactivation. Lourenc¸o et al. (10) reported a higher
stability for sweet potato PPO in the presence of 20% and 40%
(w/w) sucrose. However, there are only few data on the
activation energy in the presence of sugars for inactivation of
PPO from other sources.
Figure 7. Heat inactivation curves of PPO in the presence of sugars at
80 °C. Enzyme aliquots in 5 mM potassium phosphate buffer, pH 7.5,
were incubated in the presence of sugars at 20% (A) and 30% (B)
concentration (w/w %), and the remaining activity was determined with
chlorogenic acid as substrate. Ao and A: initial and residual activity at
the time measured, respectively.
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Received for review November 1, 2006. Revised manuscript received
January 4, 2007. Accepted January 25, 2007. This research was
supported by the Faculty of Pharmaceutical Sciences-UNESP Research
Commission Program (PADC-FCF-UNESP).
JF063148W