Purification and characterization of Ocimum basilicum L. polyphenol oxidase

Article abstract of DOI:10.1021/jf051646j

A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described. PPO in O. basilicum L. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and a Sepharose 4B-L-tyrosin

Full text of DOI:10.1021/jf051646j

10224 J. Agric. Food Chem. 2005, 53, 10224−10230
Purification and Characterization of Ocimum basilicum L.
Polyphenol Oxidase
SERAP DOGˇ AN,*,§ PıNAR TURAN,† MEHMET DOGˇ AN,† OKTAY ARSLAN,† AND
†
MAHIR ALKAN
Departments of Biology and Chemistry, Faculty of Science and Literature, University of Balikesir,
10100 Balikesir, Turkey
A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described.
PPO in O. basilicum L. was extracted and purified through (NH4)2SO4 precipitation, dialysis, and a
Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. The samples obtained from (NH4)2-
SO4 precipitation and dialysis were used for the characterization of PPO. At the end of purification
by affinity chromatography, 11.5-fold purification was achived. The purified enzyme exhibited a clear
single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of
the enzyme was estimated to be ∼54 kDa. The contents of total phenolic and protein of O. basilicum
L. extracts were determined. The total phenolic content of O. basilicum L. was determined
spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 280 mg
100 g-1 on a fresh weight basis. The protein content was determined according to the Bradford method.
The enzyme showed activity to 4-methylcatechol, catechol, and pyrogallol substrates, but not to
tyrosine. Therefore, of these three substrates, 4-methylcatecol was the best substrate due to the
highest Vmax/Km value, followed by pyrogallol and catechol. The optimum pH was at 6, 8, and 9 for
4-methylcatechol, catechol, and pyrogallol, respectively. The enzyme had an optimum temperature
of 20, 40, and 50 °C for 4-methylcatechol, catechol, and pyrogallol, respectively. It was found that
optimum temperature and pH were dependent on the substrates studied. The enzyme activity with
increasing temperature and inactivation time for 4-methylcatechol, catechol, and pyrogallol substrates
decreased due to heat denaturation of the enzyme.
KEYWORDS: Polyphenol oxidase; Ocimum basilicum L.; purification; substrate specificity; pH; temper-
ature; heat inactivation
INTRODUCTION
The presence of PPO in plant tissues is of concern to
processors and researchers. PPO catalyzes the formation of
highly active quinones that react with amino or sulfhydryl groups
in proteins or enzymes. These reactions lead to changes in
physical, chemical, or nutritional characteristics of proteins and,
in many cases, to inactivation of enzymes including PPO (2).
Quinones also lead to polymerization and condensation reactions
between proteins and polyphenols, forming brown pigments.
Quinones are very reactive compounds that strongly interact
with other molecules, leading to a large variety of dark-colored
compounds (4, 5). Because of the importance of this reaction
in the food industry, PPO has been intensively studied in several
plant tissues such as aubergine (6), Origanum (7), apricot (8),
Thymus species (9-11), SalVia (12), spinach (13), and tea leaves
(14). PPO has been found to differ in isoforms, latency, catalytic
behavior, molecular weight, isoeletric point, specificity, and
hydrophobicity (15). There is no investigation related to
polyphenol oxidase activity obtained from Ocimum basilicum
L. in the literature.
Surface discoloration, commonly called “browning” or “phe-
nolic browning”, is a serious problem in marketing and storage.
Tissue browning in fruits and vegetables damaged by mechan-
ical injury during harvesting, postharvest storage, or processing
is one of the main causes of quality loss and a great problem
for the food industry. This reaction, which is mainly catalyzed
by polyphenol oxidase (PPO), gives rise to the formation of
o-quinones, which subsequently polymerize, leading to the
appearance of brown pigments (1). Polyphenol oxidase (mono-
phenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC
1.14.18.1), also known and reported under various names
(tyrosinase, phenolase, catechol oxidase, catecholase, monophe-
nol oxidase, o-diphenol oxidase, and orthophenolase) based on
substrate specificity, is widely distributed in plants and fungi
(2, 3).
* Author to whom correspondence should be addressed (e-mail
sdogan@balikesir.edu.tr).
O. basilicum L. is an important medicinal plant and culinary
herb. The common or sweet basil (O. basilicum L.) is one of
§ Department of Biology, University of Balikesir.
† Department of Chemistry, University of Balikesir.
10.1021/jf051646j CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/23/2005

Ocimum basilicum L. PPO
J. Agric. Food Chem., Vol. 53, No. 26, 2005 10225
seen in Table 1, finally, PPO was purified up to 11.5-fold for O.
basilicum L.
Table 1. Total Phenolic Contents of Some Vegetables
protein
content (mg)
Electrophoresis and Molecular Mass Determination. Affinity
chromatography was done according to the method of Arslan and
Erzengin (21). In this method, PPO was purified from O. basilicum L.
on a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) was carried out using the method of Laemmli (22). SDS-PAGE
was carried out using an SDS marker protein kit as standard (MBI
Fermentas). Samples were applied to 10% polyacrylamide gels. The
slab gels of 1.5 mm thickness were run at a constant current of 180
mV. Gels were stained for protein using a standard Coomassie Blue
method. The molecular mass of the purified enzyme was determined
by SDS-PAGE.
vegetable
ref
fresh turmeric
broccoli
tomato
yam
mint
black carrots
aonla
beet root
176
88
68
Kaur and Kapoor (30)
92
400
350
349
323
artichoke
425
Dogˇan et al. (26)
Determination of Total Phenolic Compound Content. Total
phenolics were determined using the Folin-Ciocalteu reagent (23).
Samples (2 g) were homogenized in 80% aqueous ethanol at room
temperature and centrifuged in the cold at 10000g for 15 min, and the
supernatant was saved. The residue was re-extracted twice with 80%
ethanol, and the supernatants were pooled, put into evaporating dishes,
and evaporated to dryness at room temperature. The residue was
dissolved in 5 mL of distilled water. One hundred microliters of this
extract was diluted to 3 mL of the water, and 0.5 mL of Folin-Ciocalteu
reagent was added. After 3 min, 2 mL of the 20% sodium carbonate
was added, and the contents were mixed thoroughly. The color was
developed and the absorbance measured at 650 nm in a Carry |1E|g
UV-visible spectrophotometer after 60 min using catechol as a
standard. The result was expressed as milligrams of catechol per 100
g of fresh weight material.
several aromatic herbs or seed plants, the basils, belonging to
the genus Ocimum and the mint family Lamiaceae. Species of
Lamiaceae are valued for their pharmaceutical properties; for
example, the aromatic oils produced in their leaves are used as
antioxidants (16). It is called as “feslegˇen” in Turkey. Feslegˇen
is widely cultivated for the production of essential oils and is
also marketed as an herb, either fresh, dried, or frozen. The
essential oil of O. basilicum L. possesses antifungal, insect-
repelling, and toxic activities (17). Sweet basil is grown
commercially as a cultivated herb plant in the United States,
the Mediterranean region, and many other parts of the world.
Crude extracts of fruits, herbs, vegetables, cereals, and other
plant materials rich in phenolics are increasingly of interest in
the food industry because they retard the oxidative degradation
of lipids and thereby improve the quality and nutritional value
of food. A number of phenolic compounds with strong anti-
oxidant activity have been identified in these plant extracts (18).
The aim of this paper was to (i) partially purifiy, kinetically
characterize, and determine the molecular mass of O. basilicum
L. PPO and (ii) to determine the protein and phenolic compound
contents of O. basilicum L. Therefore, characterization of PPO
from O. basilicum L. was studied in terms of substrate
specificity, optimum pH and temperature, and thermal inactiva-
tion in order to help to predict the behavior of the Ocimum PPO.
Determination of Protein Content. Protein content was determined
according to Bradford’s method using bovine serum albumin as standard
(24).
Assay of PPO Activity. The enzyme activity was determined by
monitoring the formation of quinones spectrophotometrically with time
and at constant temperature. The desired temperatures were provided
by using a Tempette Junior TE-85 temperature controller attached to
the cell holder of the spectrophotometer. The activity was determined,
using different substrates, by measuring the increase in absorbance at
420 nm for 4-methylcatechol and catechol, at 484 nm for tyrosine, and
at 320 nm for pyrogallol with a spectrophotometer, respectively.
Typically, assay mixtures (3 mL total volume) contained the substrate,
phosphate buffer, and enzymatic extract solutions. Reference cuvettes
contained all of the components except the enzyme extract, with a final
volume of 3 mL. The reaction was initiated by adding aliquots of
enzymatic extract in the assay medium. The oxidation of phenolic
substrate was followed by monitoring the increase in absorbance
resulting from the oxidation of substrate in the presence of oxygen.
Activity was determined by measuring the maximal slope from the
linear part of the curve. Unless otherwise indicated, PPO was added
last. Enzyme activity data are averages of duplicate measurements.
Monophenolase activity was determined using tyrosine as a substrate.
Diphenol oxidase activity was determined using 4-methylcatechol
(0.42-3.33 mM), catechol (5.0-20.0 mM), and pyrogallol (1.67-
8.33mM) as substrates in 0.1 M sodium phosphate buffer (pH 6.5).
One unit of PPO activity was defined as the amount of enzyme that
caused an increase in absorbance of 0.001 per minute for 1 mL of
enzyme at 25 °C (6).
MATERIALS AND METHODS
Materials. O. basilicum L. used in this study was harvested directly
from a local garden in I˙zmir and Aydin cities (Turkey) and stored at
-70 °C until processed. The chemicals used were obtained from Sigma
Chemical Co. and Merck and were of the best grade available. Enzyme
assays were measured with the aid of a Cary |1E|g UV-visible
spectrophotometer (Varian).
Extraction of PPO. The extraction procedure was adapted from
that of Wesche-Ebeling and Montgomery (19). O. basilicum L. (10 g)
was placed in a Dewar flask under liquid nitrogen for 10 min to rupture
cell membranes. A 10 g sample was homogenized using a Waring
blender for 2 min in 100 mL of 0.1 M phosphate buffer (pH 6.5)
containing 10 mM ascorbic acid and 5% poly(ethylene glycol). The
0.1 M concentration was chosen to avoid the influence of enzymatic
extract ionic strength on PPO activity, as described by Angleton and
Flurkey (20). The crude extract was filtered, and the filtrate was
centrifuged at 15000g for 30 min at 4 °C. The supernatant was brought
to 80% (NH4)2SO4 saturation with solid (NH4)2SO4. Inactive proteins
were partially removed by ammonium sulfate precipitation. The
precipitated PPO was separated by centrifugation at 15000g for 60 min
at 4 °C. The pellet then was dissolved in a small volume of 0.1 M
phosphate buffer (pH 7.0) and dialyzed at 4 °C in the same buffer for
3 days with three changes of buffer to remove excess ammonium sulfate
ions. The dialyzed sample was used as the PPO enzyme source in the
following experiments (11). After dialysis, the active fraction was
purifed with affinity chromatography. The gel used was synthesized
according to the method of Arslan and Erzengin (21). The purification
procedures are summarized in Table 1 for O. basilicum L. PPO. As
Enzyme Kinetics and Substrate Specificity. The catalytic activity
of the enzyme preparation was measured as a function of the substrate.
PPO activity was determined by using 4-methylcatechol, catechol,
L-tyrosine, and pyrogallol as substrates, and the rate of the PPO reaction
was measured at various substrate concentrations in the standard
reaction mixture in terms of the increase in absorbance at the wavelength
of maximum absorption for the corresponding chromophore. A graphi-
cal evaluation of the results was obtained by inserting the data into the
Michaelis-Menten equation. To obtain an equation of a straight line
and a more reliable determination of Vmax and Km, the Michaelis-
Menten equation was transformed into the double-reciprocal form (the
Lineweaver-Burk plot). Substrate specificity (Vmax/Km) was calculated
by using the data obtained on a Lineweaver-Burk plot (25).

10226 J. Agric. Food Chem., Vol. 53, No. 26, 2005
Table 2. Purification of O. basilicum L. PPO
Dogˇan et al.
specific
activity
(EU µg
vol of
extract
(mL)
protein
purifi-
cation
fold
-
1
concn
activity
(EU mL
-
1
-
1
type of extract
(
µ
g mL
)
)
of protein)
crude extract
(NH4)2SO4
75
12.2
693
2265
3695
7619
5.33
3.36
0.6
precipitation
dialysis
affinity
12.1
2.4
1831
9438
530
5.15
59.46
1.5
11.5
8.9
Effect of pH and Temperature. The pH and temperature optima
of O. basilicum L. PPO were determined using 4-methylcatechol,
catechol, and pyrogallol as substrates. All buffer concentrations were
0.1 M. The buffers used were 0.1 M acetate (pH 4.0-6.0) and 0.1 M
phosphate (pH 6.0-9.0) adjusted with 0.1 M NaOH and HNO3 (26).
For determining the optimum temperature values of the enzyme, PPO
activity was measured at different temperatures in the range of 10-60
°C. The effect of temperature on the activity of PPO was tested by
heating the standard reaction solutions (buffer and substrate) to the
appropriate temperatures before introduction of the enzyme. The desired
temperatures were provided by using a Tempette Junior TE-85
temperature controller attached to the cell holder of the spectropho-
tometer. Once temperature equilibrium was reached, enzyme was added
and the reaction was followed spectrophotometrically at constant
temperature at given time intervals. The reaction mixture contained
0.6 mL of substrate, 2.3 mL of 0.1 M buffer solution, and 0.1 mL of
enzyme solution. All assays were performed in duplicate (26).
Heat Inactivation of PPO. To determine the thermal stability of
the PPO, the partially purified enzyme was studied at 35, 55, and 75
°C. For the study, 1 mL of enzyme solution in a test tube was incubated
at the required temperature for fixed time intervals. At the end of the
required time interval, the enzyme was cooled in an ice bath and brought
to room temperature; 0.1 mL of heated enzyme extract was mixed with
substrate and buffer, and residual PPO activity was determined
spectrophotometrically. The percentage residual PPO activity was
calculated by comparison with unheated enzyme (26).
Figure 1. SDS-PAGE of O. basilicum L. PPO.
Table 3. Molecular Mass of PPOs Purified from Various Sources
source
molecular mass (kDa)
ref
Vicia faba L.
sago palm
mushroom
Lactuca sativa
tea leaf
sunflower seeds
apple
banana
59
53
58
56
72
42
65
Lanker et al. (38)
Onsa et al. (39)
Espin et al. (40)
Fujita and Tono (41)
Halder et al. (14)
Raymond et al. (42)
Murata et al. (43, 44)
Galeazzi et al. (45)
Fujita et al. (46)
41; 62
39
± 2
cabbage
field bean seed
artichoke
120
57
±
3
Paul and Gowda (47)
Dogˇan et al. (26)
Table 4. Substrate Specificity of O. basilicum L. PPO
Vmax
(EU min
Km
V
max/Km
optimum
temp (°C)
optimum
pH
-
1
-
-1
substrate
catechol
4-Me-catechol
pyrogallol
)
(mM) (EU min 1 mM
)
6941
11586
10369
2.77
1.62
3.42
2500
7143
3030
40
20
50
8
6
9
RESULTS AND DISCUSSION
Total Phenolic Content. Phenolic compounds are important
constituents of plants, vegetables, and fruits. They are easily
degraded (e.g., by oxidation or hydrolysis) and may also form
covalent products and non-covalent complexes with various
types of molecules (27). The total phenolic content is generally
a major contributing factor to the degree of browning and
therefore plays a major role in the browning of O. basilicum L.
This relationship has been demonstrated in a number of plants,
and therefore total phenolic content has been used as a measure
of browning potential in several crops such as apples and grapes
(28, 29). Browning appears to be a complex process involving
several factors including substrate levels, enzyme activity, and
the presence of ascorbic acid and other inhibitors or promoters
influencing the browning reaction, in addition to tissue damage
(27). We found that the level of total phenolic compounds in
the O. basilicum L. extracts was ∼280 mg per 100 g of fresh
weight. The total phenolic contents of some vegetables have
been given in Table 1. As seen in Table 1, the total phenolic
contents of some vegetables such as fresh turmeric, broccoli,
tomato, and yam (30) are lower than those obtained in O.
basilicum L. On the other hand, the total phenolic contents of
vegetables such as mint, black carrots, aonla, and beet root (30)
and artichoke (26) were higher than those obtained in O.
basilicum L. Generally, it can be said that O. basilicum L. has
a rich phenolic compound content.
2, finally, PPO was purified up to 11.5-fold. The molecular mass
of PPO was estimated on SDS-PAGE with a single band of 54
kDa (Figure 1). Table 3 shows the molecular mass of PPO
purified from various sources. Our results indicate that the
molecular mass of O. basilicum L. was similar to those of V.
faba L., sago palm, mushroom, artichoke, and L. satiVa but
different from those of tea leaf, sunflower seeds, apple, banana,
cabbage, and field bean seed.
Enzyme Kinetics and Substrate Specificity. Mono- and
diphenolic substrates were tested for substrate specificity of O.
basilicum L. PPO. The diphenolic substrates such as 4-meth-
ylcatechol, catechol, and pyrogallol were oxidized significantly
by O. basilicum L. PPO, whereas the enzyme was unable to
oxidize L-tyrosine, which is a monophenolic substrate. Some
plant polyphenol oxidases, for example, mushroom, potato, and
broadbean, catalyze both the hydroxylation of monophenols and
the oxidation of o-diphenols. However, many PPOs lack
monophenol activity (31). Substrate specificities clearly show
that the observed activity is diphenolase as reported for PPOs
from other plant sources such as artichoke (6, 8, 26). Substrate
saturation curves for each diphenolic substrate indicated that
O. basilicum L. PPO follows simple Michaelis-Menten kinetics.
Regression coefficients are very close to 1 (Table 4). Lin-
eweaver-Burk plots for the kinetic analysis of the reaction rates,
at a series of concentrations for each substrate (Figure 2),
resulted in individual Vmax and Km values (Table 4). Substrate
specificities were evaluated by using the Vmax/Km ratio as
Molecular Mass Determination. Table 2 shows the puri-
fication procedure of O. basilicum L. PPO. As seen in Table

Ocimum basilicum L. PPO
J. Agric. Food Chem., Vol. 53, No. 26, 2005 10227
Figure 2. Lineweaver−Burk plots for O. basilicum L. PPO.
Figure 3. Effect of pH on PPO activity.
Table 6. Optimum pH Values for PPO Obtained from Various Sources
Table 5. Km Values of PPO Obtained from Various Sources
optimum
source
substrate
Km (mM)
ref
source
strawberry
substrate
pH
ref
Amasya apple
strawberry
4-Me-catechol
3.1
2.71
Oktay et al. (48)
Wesche-Ebeling and
4-Me-catechol
4.5
Wesche-Ebeling and
Montgomery (19)
Rocha et al. (49)
Dogˇan et al. (9)
Montgomery (19)
Dogˇan et al. (6)
Starking apple
Thymus
artichoke
4.0
9.8
11.6
aubergine
dog-rose
artichoke
6.0
8.5
5.0
S¸
akirogˇlu et al. (36)
Dogˇan et al. (26)
Dogˇan et al. (26)
Yali pear
apricot
Amasya apple
Starking apple
Thymus
catechol
5.5
6.6
34.0
180.0
18.0
682.5
20.0
Zhou and Feng (50)
Arslan et al. (8)
Oktay et al. (48)
dog-rose
Amasya apple
artichoke
catechol
7.0
8.6
8.0
S¸akirogˇlu et al. (36)
Oktay et al. (48)
Dogˇan et al. (26)
Rocha et al. (49)
strawberry
pyrogallol
5.5
Wesche-Ebeling and
Montgomery (19)
Lee et al. (53)
Oktay et al. (48)
Arslan and Tozlu (54)
Dogˇan et al. (6)
Dogˇan and Dogˇan (11)
Nagai and Suzuki (51)
Siddiq et al. (52)
cabbage
Stanley plum
DeChaunac grape
Amasya apple
Anethum graveolens L.
aubergine
Allium sp.
dog-rose
6.0
7.0
7.0
7.0
7.5
8.5
7.0
artichoke
Thymus
Yali pear
tea leaf
Amasya apple
cabbage
spinach
pyrogallol
5.2
5.5
2.6
17.8
27.0
15.4
15.7
Dogˇan et al. (26)
Dogˇan and Dogˇan (11)
Zhou and Feng (50)
Halder et al. (14)
Oktay et al. (48)
Nagai and Suzuki (51)
Golbeck and Cammarata (13)
Arslan et al. (55)
S
¸
akirogˇlu et al. (36)
artichoke
Dogˇan et al. (26)
and catechol, are very efficient substrates for diphenolases from
several sources (6, 7, 32).
catalytic efficiency (11). Substrate binding affinities were within
the range of 1.62-3.42 mM for the substrates examined. Table
5 shows the Km values of PPO obtained from various sources.
In this study, the values of Km for PPO obtained from O.
basilicum L. for the substrates assayed were similar to those of
Amasya apple, strawberry, and Starking apple for 4-methylcat-
echol as a substrate; for Yali pear and apricot using catecol as
a substrate; and for artichoke, Thymus, and Yali pear using
pyrogallol as a substrate. The Km value of O. basilicum L. PPO
is lower than those of Thymus and artichoke with 4-methylcat-
echol as a substrate; of Amasya apple, Starking apple, Thymus,
cabbage, and Stanley plum with catechol as a substrate; and of
tea leaf, Amasya apple, cabbage, and spinach with pyrogallol
as a substrate. 4-Methylcatechol was oxidized by O. basilicum
L. PPO at the highest rate with a much lower Km value.
4-Methylcatechol has a lower Km value by ∼2-fold compared
with that when pyrogallol was used as substrate. 4-Methylcat-
echol was clearly a good substrate for the enzymes, but,
surprisingly, catechol was a much poorer substrate. The catalytic
efficiency values, Vmax/Km, indicated that 4-methylcatechol was
the most suitable phenolic substrate for O. basilicum L. PPO
(Table 4). Similar results were found for aubergine (6) and
medlar fruits (32). These results are consistent with previous
studies indicating that o-diphenols, such as 4-methylcatechol
pH Optimum. The pH value of the medium affects signifi-
cantly the enzyme activity. The enzyme activity with increasing
pH values reaches a maximum value and then drops to zero in
the alkaline region. Optimum pH values for O. basilicum L.
PPO were determined in pH ranges of 4-9. The pH optimum
profile for O. basilicum L. PPO is given in Figure 3. Generally,
although the enzyme is active over a large pH range, in the
case of 4-methylcatechol, having a pH optimum of 6.0, the
enzyme catalyzes the oxidation of catechol and pyrogallol over
a wider pH range of 8 and 9 for each substrate, respectively. It
seems that O. basilicum L. PPO interacts with 4-methylcatechol
much more efficiently and catalyzes its oxidation at a much
lower pH value than the other two substrates. The optimum
pH, depending on the exraction method, the substrates used for
assay, and the localization of the enzyme in the plant cell for
PPO activity in the plants, differs among plant sources (33).
Different optimum pH values for PPO obtained from vairous
sources are reported in the literature (Table 6). It is clear, from
previous papers, that the pH optimum for PPOs is highly
dependent on the enzyme source and the nature of the substrate
used (34).
Optimum Temperature. Temperature-activity profiles of
O. basilicum L. PPO for different substrates are presented in
Figure 4. It is clear that the enzyme possesses activity at

10228 J. Agric. Food Chem., Vol. 53, No. 26, 2005
Dogˇan et al.
Figure 4. Effect of temperature on PPO activity.
temperatures ranging from 10 to 60 °C. The enzyme showed a
temperature optimum at 20, 40, and 50 °C for 4-methylcatechol,
catechol, and pyrogallol substrates, respectively. Similar tem-
perature optima were reported for mulberry (35) and for dog-
rose (36) using 4-methylcatechol as a substrate and for Thymus
(11) using catechol as a substrate. It is clear that the optimum
temperatures for PPO are substrate-dependent. It was previously
reported that the greatest activity of medlar PPO was observed
at 35 °C for 4-methylcatechol and at 55 °C for catechol (32)
and that of dog-rose PPO at 25 °C for catechol (36).
Thermal Inactivation. O. basilicum L. PPO showed a typical
temperature-dependent inactivation profile in the presence of
4-methylcatechol, catechol, and pyrogallol as substrates (Figure
5). The enzyme was incubated at different temperatures for 60
min at pH 6.5, and after cooling, the residual enzyme activity
was measured using 4-methylcatechol, catechol, and pyrogallol
as substrates. PPO showed similar behavior for 4-methylcat-
echol, catechol, and pyrogallol substrates. The enzyme activity
decreased due to heat denaturation of the enzyme with increasing
temperature and inactivation time for 4-methylcatechol, catechol,
and pyrogallol substrates. The enzyme was quite stable at the
examined time range at 35 °C, and it retained its activity at 35
°C within this period. The enzyme activity for three substrates
dramatically decreased with time at 55 and 75 °C. Enzyme
activity decreased to about 50% at the end of 30 min of
incubation at 55 °C and to about 55, 80, and 70% for
4-methylcatechol, catechol, and pyrogallol substrates at 75 °C,
respectively. This indicated that the enzyme was rapidly
denatured at higher temperatures. The additional time of
incubation affected the thermal stability of the enzyme at 55
and 75 °C. It is clear that at temperatures >55 °C, heat
denaturation of the enzyme occurred with incubation time, and
the diphenolase activities were reduced at higher inactivation
temperatures because of heat denaturation of the enzyme or
some conformational changes in the tertiary structure due to
the increasing temperatures (34). It is obvious from these data
that O. basilicum L. PPO is heat-sensitive. It has been previously
reported that heat sensitivity of PPOs may depend on ripeness
of the plant or different molecular forms of enzyme from the
same plant source (37).
Figure 5. Change of PPO activity as a function of temperature and time.
4-methylcatechol, followed by pyrogallol and catechol. The
Michaelis-Menten constants, Km, for 4-methylcatecol, catechol,
and pyrogallol were 1.62, 2.77, and 3.42 mM, respectively.
Apparent pH optima were 6.0 for 4-methylcatechol, 8.0 for
catechol, and 9.0 for pyrogallol as substrates. The enzyme
showed a temperature optimum at 20, 40, and 50 °C for
4-methylcatechol, catechol, and pyrogallol substrate, respec-
tively. The enzyme activity decreased due to heat denaturation
of the enzyme with increasing temperature and inactivation time.
LITERATURE CITED
Conclusions. The characterization and purification of O.
basilicum L. PPO is reported for the first time. O. basilicum L.
had a rich phenolic compound content. PPO from O. basilicum
L. was purified to 11.5-fold to apparent homogeneity using a
three-step procedure. The enzyme existed as a single isoform
of Mr 54 kDa. We found that O. basilicum L. PPO was a
diphenol oxidase and that the most effective substrate was
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