Food and Nutrition Sciences, 2013, 4, 727-734
http://dx.doi.org/10.4236/fns.2013.47093 Published Online July 2013 (http://www.scirp.org/journal/fns)
Purification and Partial Characterization of Polyphenol
Oxidase from Sapodilla Plum (Achras sapota)
Jorge Tamayo Cortez1, Carlos Hernán Herrera Méndez2*, Enrique Sauri Duch1,
María de Lourdes Vargas y Vargas1, Sara Solís Pereira1
1Research and Postgraduate Division, Instituto Tecnológico de Mérida, Merida, Mexico; 2Agroindustrial Engineering Department,
Campus Celaya-Salvatierra, Universidad de Guanajuato, Salvatierra, Mexico.
Received April 29th, 2013; revised May 29th, 2013; accepted June 6th, 2013
Copyright © 2013 Jorge Tamayo Cortez et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The browning of fruits can be considered as an enzymatic oxidation which is believed to be one of the main causes of
quality loss during post-harvest handling. The enzymes responsible for this are the oxidoreductases; the polyphenol oxi-
dase (PPO) (monophenol, o-diphenol, oxygen oxidoreductase; EC 1.14.18.1) belongs to this group. This enzyme, which
is found in the sapodilla plum (Achras sapota), was purified using a phenylsepharose and a SephacrylS-200 columns.
The molecular weight of the purified enzyme was estimated to be about 66 kDa by gel filtration and 29 kDa by
SDS-PAGE. A single protein band was found using the latter system (SDS-PAGE), which shows that the PPO of the
pulp of the sapodilla plum may be composed of two protein subunits with similar molecular weight. The optimum pH
was 7.0 and the optimum temperature 60˚C. The most effective inhibitors tested were ascorbic acid, sodium metabisul-
fite and acetic acid.
Keywords: Enzymatic System; Oxidation; Polyphenol Oxidase; Browning Process; Nutritional Value
1. Introduction
For nearly a century, fruits and vegetables have been
recognized as a good source of vitamins, minerals and
fiber. They are considered so important for our nutrition
that five daily rations are suggested. Compared with peo-
ple who consume a diet with only small amounts of fruits
and vegetables, those who eat generous amounts of them
as part of a healthful diet are likely to have reduced risk
of chronic diseases, including stroke and perhaps other
cardiovascular diseases, as well as certain cancers.
The sapodilla plum (Achras sapota) is a tree from the
tropical regions of the American continent, mainly from
the South of Mexico and Central America. The fruit is
climacteric, reaches commercial maturity around 6 - 8
days after harvest and enters senescence two days later; it
is, therefore, a perishable fruit.
The browning process is one of the main causes of the
loss of quality and nutritional value in fruit and vegetable
preservation [1]. This phenomenon can be considered as
an enzymatic oxidation. Polyphenol oxidases (PPO) are
responsible for the browning of fruits and vegetables by
catalyzing the oxidation of phenolic compounds; these
enzymes can be inactivated by heat or by the elimination
of oxygen [2,3].
The sapodilla plum is a perishable fruit. Enzymatic
browning, which is mainly caused by polyphenol oxidase
(monophenol, o-diphenol, oxygen oxidoreductase; EC
1.14.18.1), makes its conservation and commercialization
a difficult thing [4]. This enzyme is widely distributed in
microorganisms, animals, and plants, being responsible
not only for browning in plants but also for melanisation
in animals. Previous studies have reported the purifica-
tion and characterization of PPO from different sources:
the enzyme from cabbage showed a molecular weight of
40 kDa, an optimal pH of 7.6 and a residual enzymatic
activity of 25% after the protein was subjected to a 10
min thermal treatment at 100˚C [5]; the polyphenol oxi-
dase from avocado was isolated and partially purified in
the presence of TX-114, showing monophenolase/di-
phenolase activity with an optimum pH of 7 for both [6];
the purification and characterization of polyphenol oxi-
dase from banana pulp [7], purification and characteriza-
tion of the polyphenol oxidase in beets [8]. The purifica-
tion of PPO from sapodilla plum has not been reported
*Corresponding author.

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (Achras sapota) 728
yet.
The commercial potential of the Sapodilla plummight
be expanded if the enzymatic browning process is con-
trolled. Therefore, our objective was to purify and par-
tially characterize the polyphenol oxidase from sapodilla
plum. This study could help understand the role of poly-
phenol oxidase on the quality deterioration of the sapo-
dilla plum, which could be useful in the search of effect-
tive methods for inhibiting browning during storage.
2.Materials and Methods
2.1. Materials
Sapodilla plum (Achras sapota), harvested at commercial
maturity in the February-May season from an orchard in
the Cansahcab town of the state of Yucatan, Mexico,
stored at 4˚C.
2.2. Extraction Procedure
Unless otherwise stated, all extraction procedures were
carried out at 4˚C in order to reduce enzyme activity.
Ripe fresh fruit (50 g after peeling and coring) were
blended for 1 min with 100 ml of 0.1 M phosphate buffer,
pH 7, and 0.2 g of ascorbic acid, different concentrations
of polyvinylpolypyrrolidone (PVPP) and 0.1% of TX100
at 4˚C.
The raw enzymatic extract (PPO) was obtained from
the sapodilla plum using Triton TX100 detergent from
Sigma; this detergent has been used for the extraction
and solubilisation of polyphenol oxidase in several fruits
[6-8]; ascorbic acid (Sigma) was used as reducer agent of
endogenous phenolic compounds found in the sapodilla
plum; finally, PVPP from Sigma was used to prevent
quinone formation, as it reacts with the proteins that are
present in the enzymatic extract.
2.3. Monitoring of Polyvinylpolypyrrolidone
(PVPP) Activity in Collected Fractions, and
Purification Procedure
The presence of excessive quantities of impurities may
interfere with bioassays. Phenolic compounds, which are
widespread in plants and frequently occur in high con-
centrations, seem to be a major source of impurities and
to have possible inhibitory activity. Polyvinylpolypyr-
rolidone (PVPP) has been shown to be reasonably spe-
cific in separating a phenolic fraction from plant tissue
extracts by hydrogen bond formation [9].
In order to evaluate the effect of PVPP on the process
of obtaining the enzymatic extract, several PVPP concen-
trations were analysed: 0%, 1%, 2%, 3%, 4%, and 5% in
fresh fruit weight. We used 50 g of ripe fresh fruit, which
were suspended in 100 ml of 0.1 M phosphate buffer, pH
7, for each of the above-mentioned PVPP concentrations,
0.2 g of ascorbic acid and 0.1% of TX100 at 4˚C.
This mixture was homogenised for 1 min and filtered
through 6 gauze-layers right after it was filtered through
a N.1 filter-paper; it was then centrifuged at 10,000 g for
20 min at 4˚C. The sapodilla plums filtrate was treated
with solid ammonium sulphate with a saturation of 20% -
80%. The precipitate was collected by centrifugation at
1200 × g for 40 min and redissolved in buffer (0.01 M
phosphate buffer, pH 7.0). The fraction obtained through
precipitate with ammonium sulphate (Sigma) with the
highest activity of PPO was dialyzed overnight against 4
changes (4 × 1 L) of the same buffer, centrifuged and
later applied to a phenylsepharose column. The (6 cm × 1
cm) column, with a 4 ml packed volume, was equili-
brated with a 0.01 M phosphate buffer, pH 7, and eluted
with a decreasing ammonium sulphate gradient (0.3% to
0%). The first eluted fraction that maintained the enzy-
matic activity was introduced into another sephacryl S-
200 column, and the protein amount (280 nm) as well as
the PPO enzymatic activity (420 nm) were determined.
2.4. Protein Determination
Protein content was determined by the Bradford method
[10], using immunoglobulin G (Sigma) as standard. Brad-
ford reagent (1.5 ml) was added to 0.05 ml of sample.
Absorbance at 595 nm was determined [11]. The en-
richment process of the PPO fraction was determined
according to the protein concentration method [12].
2.5. Molecular Mass Determination by
Size-Exclusion Chromatography
The molecular weight of the native enzyme was deter-
mined by gel filtration with a Sephacryl S-200 (30 cm ×
1 cm) column, which was equilibrated with a 0.01 M
phosphate buffer, pH 7. The column was pre-calibrated
using molecular weight markers: cythocrome c (12.5
kDa), carbonic anhydrase (29 kDa), bovine albumin (66
kDa), alcohol dehydrogenase (150 kDa), alpha-amylase
(200 kDa), apoferritin (443 kDa), thyroglobuline (669
kDa) and blue dextran (2000 kDa). Filtration was carried
out following Andrews’s method [13].
2.6. SDS-PAGE
Electrophoresis was carried out in accordance with the
Laemmli method [14] with the following characteristics:
a 10% acrylamide resolution gel and a 6 cm high and 1.5
mm thick buffer Tris, pH 8.8; for the sample we used the
Laemmli buffer 2×, and the run or electrode buffer was
Tris-glycine, pH 6.8, with sodium dodecyl sulphate (SDS)
from Sigma. Equal amounts of protein samples (10 mg)
were loaded onto each lane. Protein bands were visual-
ized by Coomassie blue staining according to the manu-

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (Achras sapota) 729
facturer’s protocol (Sigma).
2.7. Enzyme ActivityAssay
Enzymatic activity was measured spectrophotometrically
by the method described by Oktay [15] using pyrocate-
chol (J.T. Baker) as substrate. One unit of enzyme active-
ity was taken as the increase in absorbance at 420 nm at
30˚C. The standard reaction mixture consisted of 0.1 ml
of enzyme solution and 2.5 ml of pyrocatechol (0.06 M)
in 0.1 M phosphate buffer (pH 7). Activity measurements
were carried out in triplicate. In order to determine en-
zyme activity in the presence of inhibitors, the enzyme
was preincubated for about 15 min with these com-
pounds.
The kinetic characterization of the sapodilla plum’s
PPO was carried out using a 0.1 M phosphate buffer, pH
7, at an incubation temperature of 30˚C, a 200 rpm shak-
ing speed, 20 min enzyme-substrate reaction time, and,
as substrate, pyrocatechol at several concentration levels
(0.02, 0.04, 0.06, 0.08, 0.1 and 0.12 M).
2.8. Optimum pH
PPO activity as a function of pH was determined within a
pH range of 4.0 - 6.0 in 0.1 M acetate buffer and of 6.0 -
8.0 in 0.1 M phosphate buffer adjusted with 0.1 MNaOH
and HNO3 [16]. PPO activity was assayed using the
standard reaction mixture at 30˚C, but changing the
buffer. PPO activity was calculated as percent residual
activity at the optimum pH.
2.9. Effect of Temperature
In order to determine the optimum temperature values of
the enzyme, PPO activity was measured at different
temperatures in the range of 30˚C - 70˚C using pyro-
catechol as substrate. The effect of temperature on the
activity of PPO was tested by heating the standard reac-
tion solutions (buffer and substrate) to the appropriate
temperatures before introducing the enzyme. Once tem-
perature equilibrium was reached, the enzyme was added
and the reaction followed spectrophotometrically at a
constant temperature, given time intervals and a pH of 7.
The standard reaction mixture consisted of 0.1 ml of
enzyme solution and 2.5 ml of pyrocatechol in 0.1 M
phosphate buffer (pH 7) [17].
2.10. Monitoring the PPO Inhibitory Activity
The inhibitors examined were ascorbic acid (Sigma), so-
dium metabisulfite (Merk), sodium azide (Merk), acetic
acid (Fermont), citric acid (Sigma), tartaric acid (Baker),
oxalic acid (Baker) and honey. These inhibitors are re-
ducing agents that play a role in preventing enzymatic
browning either by reducing o-quinones to colorless di-
phenols or by reacting irreversibly with o-quinones to
form stable colorless products [18]. The reaction mixture
contained 2.8 ml of pyrocatechol at a final concentration
of 0.06 M in 0.1 M phosphate buffer (pH 7.0) and 0.2 ml
of the enzymatic solution, plus the inhibitors solution in
several concentration levels. Percentage inhibition was
calculated using the following equation: Inhibition (%) =
[(A0 − Ai)/A0)]100, where A0 is the initial PPO activity
(without inhibitor) and Ai is the PPO activity with in-
hibitor [19].
3. Results and Discussion
3.1. Extraction of the Sapodilla Plum’s PPO
It has been reported that some plant PPOs are mem-
brane-bound. Therefore, using detergents is required in
order to solubilise the enzyme [20]. Phenolic compounds
interfere with the purification of proteins from plants.
They cross-link proteins by hydrogen bonds and covalent
interactions. Furthermore, the homogenization of the
plant tissues initiates enzymatic browning, which results
in the formation of quinones. Quinones may also form
covalent linkages that may be irreversible. Phenol- ab-
sorbing polymers such as PVPP and reducing agents
such as ascorbic acid are commonly used in order to
overcome these problems [20]. During the extraction of
the sapodilla plum’s PPO we noticed (Figure 1) that
when the polyvinylpolypyrrolidone (PVPP) concentra-
tion increased from 0% to 5%, PPO activity in the extract
increased; this can be a result of the PVPP effect on the
sapodilla plum’s endogenous polyphenols; this means a
greater enzymatic activity per mg protein: between 0.097
and 0.091 mg/ml in the analyzed conditions. It was also
discovered that a PVPP concentration of 3% allowed to
preserve a maximum activity of PPO. Values near 2.5%
Figure 1. PVPP, the effect on polyphenol oxidase activity.
PVPP concentrations of 0%, 1%, 2%, 3%, 4% and 5% of
fresh fruit weight were analyzed. The reaction medium at
30˚C contained 0.1 M phosphate buffer, pH 7 for each
PVPP concentration, 0.2 g of ascorbic acid and 0.1% of
TX100. (■) Enzymatic activity; (●) specific activity.

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (Achras sapota) 730
were found when PPO was extracted from apple tissue
[21].
3.2. Enrichment of the Sapodilla Plum’s PPO
Enzymatic Extract
One of the most commonly used methods for protein
enrichment and concentration is fractional precipitation
with ammonium sulphate [22]. This method was applied
to the sapodilla plum extract.
Table 1 presents a summary of the results of PPO ex-
traction and purification from Sapodilla plums and shows
that PPO activity increased in the precipitate with the
highest saturation percentage of ammonium sulphate in
the sapodilla plum extract. We can also observe that
when there is an ammonium sulphate saturation concen-
tration of 80% (Table 1), the sapodilla plum’s PPO pre-
cipitation is almost total, due to the fact that under these
conditions a purification factor of approximately 4.8 was
obtained, and, therefore, a specific activity factor of
762.7 UE factor was found. On the other hand, a recov-
ery factor of almost 3 was reported by Jharna R. [23] in
the purification and characterization of the pineapple’s
PPO, and a similar value of 4.5 was obtained in the par-
tial purification of persimmon by Nuñez, Sojo, García
and Sánchez [24].
3.3. Purification of the PPO Enzyme with
Phenylsepharose and Sephacryl S-200
Chromatography
The extract precipitate at 80% was resuspended in 1 ml
of buffer; dialysed and passed through a phenylsepharose
column. The first eluted fraction that maintained the en-
zymatic activity was introduced into another sephacryl
S-200 column, and the protein amount as well as the PPO
enzymatic activity was determined. Two major fractions
were obtained from the column (16 and 17 fractions),
and one peak with PPO activity was separated by
sephacrylS-200 chromatography (Figure 2). The major
peak corresponded to a 51-fold increase in specific activ-
ity over the crude extract and a recovery of 6.8%.
Hydrophobic interaction chromatography has been
utilized in the purification of PPO from various fruits,
including peaches [25], strawberries [26] and pineapples
[27]. Purification factors, elution profiles, and the per-
centage of enzyme recovery varied between studies. Re-
ported purification factors were 120-fold for PPO from
Delicious cortex [28] and 13.8-fold for PPO from apricot
[29]. Enzyme recoveries of 80% for PPO from apricot
[29] and 40% for PPO from apple peel [30] have been
reported.
SDS-PAGE was performed in order to check the ho-
mogeneity of the purified PPO. These results, according
to the molecular weight patterns and the number of frac-
tions 16 and 17, suggest that the native molecular weight
of the sapodilla plum’s PPO is approximately 66 kDa,
reaching a purification factor of approximately 38 times
for the phenylsepharose column, and 51 times for the
sephacryl S-200 column. A molecular weigh of 57 kDa
was found in the characterization and purification of the
artichoke’s polyphenoloxidase (Cynarascolymus L.)
[31].
3.4. Electrophoresis of the Purified Fractions
(Phenylsepharose and Sephacryl S-200) in
Polyacrylamide gel (PAGE)
A denaturing gel electrophoresis (Figure 3) was per-
formedin order to examine the purification degree of the
PPO protein in the phenylsepharose and sephacryl S-200
columns. Figure 3 shows a typical molecular weight
profile of fractions obtained at each purification step. A
single band with the same molecular weight was noticed
for each type of chromatography, suggesting that the
fractions with the most activity and the most protein cor-
respond to a single protein molecule. This protein band
migrated, similarly to a protein with a molecular weight
of 29 kDa, to a carbonic anhydrase molecular weight
pattern. This suggests that the denaturalized molecular
weight of the sapodilla plum’s PPO is close to 29 kDa.
The results found by Jharna R. [23] in the purification
and characterization of the Indian pineapple’s PPO showed
that the molecular weight was 105 kDa with a Sephadex
G-150 column; however, the SDS-PAGE gel indicated a
single 25 kDa polypeptide band, suggesting a tetramer of
identical units. Applying this principle to the sapodilla
plum’s PPO, we think that, in this case, the polypeptide
is a dimmer of identical units. The molecular weights of
PPOs have been reported before: cabbage (39 kDa) [5],
banana (41 kDa) [7]. Our results are in agreement with
those reports in terms of the molecular weight of PPO.
This purification process was repeated 3 times.
Table 1. Activity of the sapodilla plum’s (Achras sapota) PPO, in precipitated fractions with ammonium sulphate.
Satura.
(%)
Volume
(ml)
Total Activity
(units)
Total Prot.
(mg/ml)
Specific activity
(units/mg protein)
Purification factor
(fold)
Recovery
(%)
Crude extract 16 45.5 0.286 159.25 1 100
80% (NH2SO4) 13 104 0.137 762.7 4.8 4.8
Sephacryl S-200 2.5 159 0.0196 8112.24 51 6.8

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (Achras sapota) 731
Figure 2. Elution profile of the sapodilla plum’s (Achras-
sapota) polyphenoloxidase in Sephacryl S-200 (Δ protein, □
activity).
Figure 3. Denaturing SDS-PAGE (10% gel) from PPO frac-
tions. Lane 1 shows prestained molecular mass markers
(kDa); lane 2 shows proteins after 80% (NH4)2SO4 frac-
tionation; lane 3 and 4 show the PPO fraction from Phenyl-
Sepharose, and lane 8 the PPO fraction from Sephacryl
S-200.
3.5. Some Characteristics of the Purified Enzyme
The isolated isoenzymes of superior plants and fruits can
oxidize a wide range of monophenols and ortho-diphe-
nols with highly variable kinetic parameters, including
maximum velocity (Vm) and the Michaelis-Menten con-
stant (Km). The Km is generally interpreted as a measure
of the affinity of an enzyme for its substrate. The affinity
of plant’s PPO is generally low. This means that they
have high Km values [32].
The sapodilla plum’s Km value was 12.4 mM and its
Vm was 69.49 UE/min·ml (results not shown); this is a
high value for the sapodilla plum; meaning that the en-
zyme has little affinity for the substrate. Similar values
have been reported by Vamos and Gazago [33] for a va-
riety of Jonathan and Starking apples; they are within a
range of 2 - 13 and 3 - 38 mM for 4-methylcathechol.
Another similar value, of 12.52 mM, was reported by
Halder, Tamuli and Bhaduri [34] for Indian tea leaves
(CameliaSinensis) with catechol as substrate, although-
much lower values have been reported, such as the one
that was published by Ridgway and Tucker [35], who
found a Km. of 3.6 mM for 4-methylcatechol.
3.6. Effects of Temperature on PPO Enzymatic
Activity
The effects of temperature are very complex and depend
on several factors; very high temperatures can alter the
rupture speed of the enzyme-substrate complex, the pH
values of functional groups taking part in the enzymatic
reaction, the affinity of the enzyme towards inhibitors,
activators and auxiliary systems, such as oxygen, which
can be the reaction’s substrate. In addition, it is possible
that high temperatures can inactivate the enzyme.
Figure 4 shows that the sapodilla plum’s PPO is active
within a wide temperature range, due to the fact that it
has an optimal activity at 60˚C. This can be due to the
environmental conditions present in the season of peak
production (April-May), when the temperature in the
region is around 40˚C.
On the other hand, the enzyme’s activity in high tem-
peratures has been demonstrated by the studies of Aroba
et al. [36]. This study found that the beginning of thermal
inactivation started at 70˚C, reaching a 50% decrease at
18.8 minutes of thermal treatment. Also, Núñez, Serrano,
Pérez and López [37] found that the activity range of the
PPO of table grapes was between 30˚C and 60˚C, but a
fast inactivation occurred after 70˚C.
Figure 4. The optimum temperature of the enzyme was
between 30˚C and 70˚C, as determined by an activity assay
at 30˚C. The reaction medium contained 0.6 ml of the sub-
strate, 2.3 ml of 0.1 M buffer solution, and 0.1 ml of enzyme
solution. Standard deviations of all data were less than
10%.

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (Achras sapota) 732
3.7. Optimal pH of the Sapodilla Plum’s
Polyphenol Oxidase Enzyme
There is an optimal pH at which enzymes are most active.
The optimum pH for the activity of the sapodilla plum’s
PPO enzyme was determined using pyrocatechol as sub-
strate; we can also see that there is a wide activity range
at different pH values from 5 to 8. The optimum pH for
the activity of PPO from sapodilla plum was found to be
7 (Figure 5). Optimal values of pH 6 - 7, with catechol
as substrate, have also been reported for various fruits,
including apple [10], pineapple [27], avocado, grape and
pear [38]. However, at a pH below 4 the enzyme activity
is low, suggesting that the enzyme can be controlled
through the pH; similar values were found by Kavrayan
et al. [39] in the purification and partial characterization
of mint’s (Menthapiperita) polyphenoloxidase, with an
optimal value of pH 7, using cathechol as substrate; low
activity was found at pH values below 5.
3.8. Inhibition
The browning process of fruits and vegetables can be
prevented by removing the reactants, such as oxygen and
the internal phenolic compounds, or by using PPO in-
hibitors. The complete elimination of oxygen during fruit
processing is very difficult, due to the fact that it is found
in the environment.
Several compounds were studied in order to detect the
inhibitive action against the sapodilla plum’s PPO; Table
2 shows the values that were obtained with several com-
pounds used as inhibitors and pyrocathechol as substrate.
Of the inhibitors used, ascorbic acid was found to be the
most effective, followed by sodium metabisulfite, so-
dium azide, acetic acid and honey (Table 2). Honey was
Figure 5. The optimum pH for polyphenoloxidase activity in
the sapodilla plum. The buffers used were 0.1 M acetate
(pH 4.0 - 6.0) and 0.1 M phosphate (pH 6.0 - 8.0) adjusted
with 0.1 MNaOH and HNO3. Standard deviations of all
data were less than 10%.
Table 2. Effect of some inhibitors on the PPO activity from
sapodilla plum (Achras sapota).
Compound Concentration (%) Inhibition (%)
Ascorbic acid 0.02 98
Sodium metabisulfite 0.050 97
Sodium azide 0.002 95
Acetic acid 0.2 93
Honey 0.2 56
the weakest inhibitor. Ascorbic acid and sodium metabi-
sulfite have been shown to be strong inhibitors of PPO
for Monroe apple peel and Stanley plum [30,40]. The
citric, tartaric and oxalic acids did not show any inhibi-
tive action against PPO in the sapodilla plum; however,
ascorbic acid can be satisfactorily used to control the
darkening of this fruit during processing as long as it is in
its reduced form.
4. Conclusions
This work describes a method for the purification of PPO
from sapodilla plum. Polyphenol Oxidase (PPO) was
extracted from commercially ripe sapodilla plums that
were gathered during the February-May season from an
orchard in the Cansahcab town in the state of Yucatan,
Mexico. This was the first report on the purification of
PPO from sapodilla plum.
It can be concluded that the apparent molecular weight
of the polyphenoloxidase from sapodilla plum pulp is
approximately 60 kD, with a molecular weight (PAGE)
of 29 kD; because of we can say that the polypeptide of
the sapodilla plum’s PPO is a dimer of identical units.
In this study, we achieved a purification factor of ap-
proximately 38 times with the Phenylsepharose column,
and of 52 with the Sephacryl S-200 column; an optimal
reaction pH of 7 and an optimal temperature of approxi-
mately 60˚C using pyrocatechol as substrate; the Km
value for the sapodilla plum was 12.4 mM, and the H.S.
was 69.49 U/min·ml. The PPO inhibitors, ascorbic acid,
sodium metabisulfite, sodium azide, acetic acid and honey
were particularly effective. Therefore, this study provides
useful information about the sapodilla plum.
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