Ala Eddine Derardja1,2, Matthias Pretzler1, Ioannis Kampatsikas1, Malika Barkat2, Annette Rompel1. 1. Universität Wien , Fakultät für Chemie, Institut für Biophysikalische Chemie, Althanstraße 14, 1090 Wien, Austria. 2. Laboratoire Bioqual, INATAA, Université des Frères Mentouri , Constantine 1, Route de Ain El-Bey, 25000 Constantine, Algeria.
Abstract
Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (KM = 2.0 mM) and chlorogenic acid (KM = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103 ) by means of peptide mass fingerprinting.
Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (KM = 2.0 mM) and chlorogenic acid (KM = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103 ) by means of peptide mass fingerprinting.
Entities:
Keywords:
activation; apricot; characterization; polyphenol oxidase; protein purification; tyrosinase
Apricot (Prunus
armeniaca L.) is one of the most
delicious and important crops in the Mediterranean region and is consumed
worldwide. Algeria is the leading apricot producer of Africa and the
fifth largest producer in the world with an annual production of 286 000
tons (FAO, 2017). Apricot is considered as an excellent source of
phenolic compounds, carotenoids, and vitamin C.[1] Apricot conservation is a challenge resulting in a limited
period of storage (2 weeks), during which the fruit rapidly loses
its attractive and pleasant orange color and becomes brownish,[2] a color which is not accepted by the market.
This loss is mainly caused by enzymatic reactions that lead to browning.[3] Different preservation methods such as freezing,
canning and drying are commonly applied to extend the availability
of the fruits.[4] However, browning remains
a problem in apricot handling and preservation and is believed to
be a major factor of quality loss during processing.[5]Most browning reactions in fruits are assumed to
be a direct consequence
of polyphenol oxidase (PPO) action on phenolic compounds. This reaction
produces quinones, highly reactive compounds that can polymerize spontaneously
to form brown pigments, which are responsible for the loss of quality
of fresh-cut fruit and vegetable products.[5]Polyphenol oxidases (catechol oxidases (EC 1.10.3.1) and tyrosinases
(EC 1.14.18.1)) are oxidoreductases that activate molecular oxygen
and catalyze the hydroxylation (tyrosinases only) and/or oxidation
(both tyrosinases and catechol oxidases) of phenolic compounds. Although
PPOs have been investigated intensively for many years (biochemically
as well as structurally), the reasons for their divergent substrate
specificity is still under debate[6,7] and the natural
substrates of nearly all PPOs are still unknown. PPOs are widely distributed
in plants, fungi, and bacteria.[8] Plant
PPOs are mainly associated with enzymatic browning reactions and with
the protection of organisms against biotic and abiotic stress.[8] Plant PPOs consist of three domains: a signal
peptide (minority of plant PPOs) or a transit peptide containing a
thylakoid transfer domain (majority of plant PPOs) (∼4–9
kDa), a catalytically active domain (∼40 kDa), and a C-terminal
domain (∼19 kDa). PPOs are expressed as ∼64–68
kDa pro-enzymes (latent form) in vivo.[9] Apricot contains a gene coding for a PPO precursor
polypeptide of 597 amino acids with a calculated molecular weight
of 67.1 kDa; the mature protein (latent form) has 496 amino acids
(Asp102 → Ser597) with a calculated molecular weight of 56.2
kDa.[10] The C-terminal domain shields the
PPO’s active site,[7,11,12] wherefore the enzymes possess either only very weak or even no enzymatic
activity in vitro, unless the enzymes are activated
artificially, for example, by proteases, acidic pH, fatty acids, or
detergents (e.g., SDS).[13]PPOs are
members of the type-3 copper enzyme family that contain
a dinuclear copper site, in which both copper atoms are coordinated
by three histidines each and can bind either molecular oxygen (side-on
in a μ–η2:η2-peroxo
geometry), hydroxide, or water molecules in a bridging mode as reviewed
in ref (14). The distance
between the copper atoms ranges from 2.8–4.6 Å, depending
on the state of the active site.[14]The characterization of PPO activity in apricot is of great interest
to the food industry. Chevalier et al. (1999) reported the purification
of (latent) apricotPPO to homogeneity, even if the biochemical characterization
done was minimal.[10] Partial purification
of a latent PPO from peach (Prunus persica L. Cv.
Catherina) has been reported.[15] A better
inhibition method against enzymatic browning may be developed through
the understanding of the chemistry, structure, and mechanism of action
of PPOs.[3] Thus, in this paper, we investigate
both the molecular and kinetic properties of latent and active apricot
(Prunus armeniaca L.) PPO.
Materials
and Methods
Plant Material
Fruits of Prunus armeniaca L. cv. Bulida were harvested from a local farm in the M’sila
region of Algeria at commercial maturity in June 2016. The fruits
were immediately transported to the laboratory and stored at 4 °C,
where they were processed the same day. Apricots were rinsed with
tap water, deseeded, frozen in liquid nitrogen, immediately broken
into small pieces with mortar and pestle, lyophilized, and stored
at −25 °C until use.
Extraction of Apricot PPO
ApricotPPO (PaPPO) was extracted as described
by Ünal and Şener[16] with
some modifications. Using a prechilled
blender, the lyophilized apricots (100 g) were homogenized for 2 min
in 500 mL of cold acetone (−25 °C) containing 4 g of poly(ethylene
glycol) (PEG 4000). The slurry was filtered through filter paper,
and the residue was re-extracted several times with 300 mL of cold
acetone until a white powder was obtained. The resultant acetone powder
was dried overnight at room temperature and was homogenized in 1 L
of cold sodium phosphate buffer (0.1 M) at pH 6.8 containing 30 mM
sodium ascorbate, 1% (w/v) polyvinylpolypyrrolidone (PVPP), 0.5% (v/v)
Triton X-100 and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate
was stirred for 45 min at 4 °C, then centrifuged at 30000 × g for 30 min at 4 °C. The supernatant was subjected
to (NH4)2SO4 precipitation (85% saturation).
The precipitated fraction was separated from the supernatant by centrifugation
at 30000 × g for 30 min at 4 °C. The precipitate
was dissolved in 250 mL of 10 mM sodium phosphate buffer (pH 6.8)
to obtain the crude extract.
Purification by Fast Protein Liquid Chromatography
The crude extract was dialyzed overnight at 4 °C against three
changes of Tris-HCl buffer (10 mM, pH 8). The dialyzed suspension
was centrifuged at 30000 × g for 30 min at 4
°C and filtered (through a 0.45 μm poly(ether sulfone)
membrane) before being applied to an ÄKTA fast protein liquid
chromatography system (FPLC). The protein solution was loaded onto
an anion exchange column (Q-Sepharose FF, 20 mL) pre-equilibrated
with 10 mM Tris-HCl, pH 8. Bound proteins were eluted with a linear
gradient of sodium chloride (0 to 1 M) at a flow rate of 5 mL/min
(Figure A). The fractions
were monitored for protein content (280 nm) as well as for enzymatic
activity (diphenolase activity). Fractions containing activity were
pooled and concentrated by ultrafiltration (30 kDa molecular weight
cutoff) driven by centrifugal force (3200 × g, 4 °C). The protein solution was then applied to a Mono S HR
5/50 GL column for cation exchange chromatography. The column was
pre-equilibrated with 10 mM sodium acetate buffer, pH 5, and eluted
with a linear gradient of sodium chloride (0 to 1 M) at a flow rate
of 1 mL/min (Figure B). The active fractions were pooled, washed with 10 mM Tris-HCl
buffer, pH 8, and concentrated by ultrafiltration. Enzyme activity
during the purification was monitored as described below, using catechol
as substrate, in 50 mM sodium citrate buffer (pH 6.5) containing 2
mM SDS as activator.
Figure 1
Chromatographic separation steps. (A) AEX chromatography
using
Q-Sepharose as the stationary phase. (B) CEX chromatography on MonoS.
Legend: (blue) UV absorbance at 280 nm (mAU), (orange) UV absorbance
at 345 nm (mAU), (red) PPO activity (U/mL), (green) elution gradient
(% buffer B), (brown) conductivity (mS/cm).
Chromatographic separation steps. (A) AEX chromatography
using
Q-Sepharose as the stationary phase. (B) CEX chromatography on MonoS.
Legend: (blue) UV absorbance at 280 nm (mAU), (orange) UV absorbance
at 345 nm (mAU), (red) PPO activity (U/mL), (green) elution gradient
(% buffer B), (brown) conductivity (mS/cm).
Enzyme Activity and Protein Concentration
PaPPO activity measurements were performed at 25 °C in 200 μL
of assay mixture by measuring the increase in absorbance at 410 nm
using a microplate reader (Infinite M200, Tecan). The standard reaction
mixture consisted of 0.5 μg of enzyme and 10 mM catechol in
50 mM sodium citrate buffer (pH 4.5). PaPPO activity
was determined from the slope of the initial linear part of the experimental
curves (absorbance vs time) and expressed as U/mL. One unit of enzymatic
activity (U) was defined as the amount of enzyme that catalyzed the
formation of 1 μmol of quinones per minute (1 U = 1 μmol/min).
The purified enzyme (L-PaPPO) of 63 kDa was found
to be activated in the reaction media with the tested diphenol without
the addition of an external activator (e.g., SDS). All assays were
performed in triplicate. Protein contents of the enzyme solutions
were determined according to the Bradford method[17] using bovine serum albumin as standard.
Gel Electrophoresis
Denaturing SDS-PAGE was performed
as described by Laemmli[18] in a mini gel
apparatus (Mini-PROTEAN Tetra Cell, Bio-Rad). Enzyme solutions were
denatured by heating at 99 °C (Thermomixer comfort, Eppendorf)
for 5 min; the samples were applied under reducing conditions to 5%
stacking and 9% resolving polyacrylamide gels, which were subsequently
run at 120 V. Gels were stained with Coomassie Brilliant Blue G-250.
Molecular weight was estimated by comparison to molecular weight markers
(Precision Plus Protein Standard Dual Color, Bio-Rad). Target proteins
(bands at 63 kDa (L-PaPPO) and 38 kDa (A-PaPPO)) were cut out and used for protein identification.
To permit substrate staining, 9% partially denaturing SDS-PAGE was
performed as described above with the two sole modifications of omitting
the β-mercaptoethanol in the loading buffer and the skipping
of the heating step prior to loading the samples on the gel. Activity
staining for PaPPO was done by immersing the gel
in 50 mM sodium citrate buffer (pH 6.5) containing 0.2 M catechol
at 25 °C. The in-gel activity staining was performed at pH 6.5
to avoid L-PaPPO activation at low pH (optimum pH
4.5). Photographs of the gels were taken after 20 min of incubation
on an orbital shaker.
Effect of pH
The PaPPO activity as
a function of pH was determined under the same assay conditions as
described above, over the pH range from 2.0 to 9.5 with two kinds
of buffer solutions: 50 mM sodium citrate buffer for the pH range
of 2.0 to 7.0 and 50 mM Tris-HCl buffer for the pH range of 7.0 to
9.5. PaPPO activity was calculated in the form of
percent residual PaPPO activity at the optimum pH.
The optimum pH obtained was used in all other studies (except for
in-gel activity staining).
Effect of Temperature
The effect
of temperature on
purified PaPPO activity was estimated over a temperature
range of 5 to 90 °C, using a Shimadzu UV spectrophotometer (UV-1800)
connected to a thermostat (Julabo F25) through a circulating water
bath in order to control and maintain the desired temperature. The
optimum temperature for PaPPO activity was determined
in 600 μL of assay mixture, containing 1.5 μg of enzyme
and 10 mM catechol in 50 mM citrate buffer (pH 4.5). The substrate
and buffer were incubated for 10 min at the appropriate temperature
using a water bath (Julabo F25), prior to the addition of the enzyme
solution. PaPPO activity is presented in the form
of percent residual activity relative to the PaPPO
activity at the optimum temperature.
Thermal Inactivation
To study the thermal inactivation
of PaPPO, the enzyme (0.5 μg) was incubated
in 50 mM citrate buffer (pH 4.5), for various times (2, 5, 10, 20,
and 30 min) at temperatures between 50 and 100 °C in a thermomixer
(Thermomixer comfort, Eppendorf), rapidly cooled in an ice bath for
10 min, and warmed up to 25 °C, and then the residual enzyme
activity was analyzed with 10 mM of catechol in 200 μL of assay
mixture. The maximum activity at optimum temperature was set as 100%.
Substrate Specificity and Kinetic Parameters
The substrate
specificity of PaPPO was determined with different
substrates: monophenols (phenol and tyramine), diphenols (l-dopa, caffeic acid, chlorogenic acid, catechol, 4-methylcatechol,
dopamine), and a triphenol (pyrogallol) at 10 mM concentration, except
for l-dopa and caffeic acid (1 mM) due to low substrate water
solubility. The results are expressed relative to the activity on
catechol (considered as 100%). Substrates that showed high activity
were selected to calculate the kinetic parameters. In order to determine
the Michaelis constant (KM) and maximum
velocity (Vmax), PaPPO
activities were determined under the assay conditions described above
using catechol, 4-methylcatechol, chlorogenic acid, and pyrogallol
as substrates at various concentrations (0–160 mM). KM and Vmax values
of the enzyme were calculated by nonlinear regression. The maximal
turnover rate (kcat) was calculated by
dividing total substrate converted per min by total molecules of PaPPO in the reaction mixture. Molar absorption coefficients
in 50 mM sodium citrate at pH 4.5 were determined by quantitative
oxidation of small quantities of the respective diphenol by an excess
of sodium periodate as described by Muñoz et al.[19]
Effect of SDS, Inhibitors and Metal Ions
on Enzyme Activity
The effects of different concentrations
of SDS (0–10 mM)
on PaPPO activity were studied, as well as the effects
of a wide range of inhibitors (4-hexylresorcinol, ascorbic acid, benzoic
acid, citric acid, kojic acid, succinic acid, EDTA, glutathione (reduced), l-cysteine, and sodium metabisulfite) for inhibitor concentrations
of 0.1, 1, 5, and 10 mM. The effects of various metal ions (MgSO4, CaCl2, FeSO4, KCl, NaCl, ZnSO4, CuSO4, MnCl2, AlCl3, and
NiCl2) on enzyme activity were also investigated for two
concentrations of metal ions, 1 and 10 mM, respectively. The enzymatic
activity of the control mixture without SDS, inhibitors, and metal
ions was taken as 100%, and then compared to the other treatments.
The activity was determined as described above.
Storage Stability
of the Purified PaPPO
In order to assess
the behavior of PaPPO during
storage at 4 °C, enzyme activity, SDS-PAGE, and activity gels
were assayed weekly over 4 weeks. The enzyme activity was tested without
and with SDS (0.5 mM) and reported relative to the initial PaPPO activity without SDS (set as 100%).
Molecular
Mass Determination
Electrospray ionization
liquid chromatography mass spectrometry was performed in an ESI–LTQ–Orbitrap
Velos (Thermo Fisher Scientific Bremen, Germany) with a mass range
of 200–4000 m/z and a mass
accuracy close to 3 ppm with external calibration. Prior to MS measurements,
the purified PaPPO solution was ultrafiltered by
centrifugation, and the buffer system was changed to 5 mM ammonium
acetate, pH 5, in order to reduce the concentration of nonvolatile
salts to a minimum. Afterward a protein solution with a concentration
of approximately 0.53 g/L was diluted 100 times in a mixture of 80%
(v/v) acetonitrile and 0.1% (v/v) formic acid.
Protein Identification
and Sequence Confirmation
The
gel pieces containing the bands at 63 and 38 kDa obtained by denaturing
SDS-PAGE (20 and 4 μg of protein, respectively) were used. Tryptic
digestions of PaPPO were analyzed by nanoUHPLC–ESI–MS/MS
using a high-resolution Orbitrap mass spectrometer (Dionex Ultimate
3000 RSLCnano, Q Exactive Orbitrap, Thermo Scientific). The data analysis
was performed with Proteome Discoverer 1.4 by searching against the P. armeniaca entries from the UniProt database (containing
all listed proteins from P. mume, P. persica, and P. armeniaca). Peptide mass tolerance was
5 ppm, and the fragment mass tolerance was 0.5 Da. Variable modifications
allowed were oxidation of methionines and carbamidomethylation of
cysteines.
Statistical Analysis
All experiments
were carried out
in triplicate; arithmetic mean and standard deviation are reported.
Results and Discussion
Extraction and Purification of L-PaPPO
To extract L-PaPPO, lyophilized
apricot was subjected
to acetone extraction to eliminate most interfering compounds.[20] PVPP was used during extraction to avoid interaction
of phenolic compounds with PPO.[21] Triton
X-100 is widely used in the recovery of membrane-bound PPOs as it
provides a mild, nondenaturing solubilization of proteins.[21] Ascorbic acid was also included to reduce quinones
during PPO extraction and initial purification.[22] PMSF was added to the extraction buffer as a serine protease
inhibitor.[23] After centrifugation, the
supernatant was subjected to ammonium sulfate precipitation at 85%
saturation.After ammonium sulfate precipitation, the L-PaPPO was purified by a three-step procedure (SDS page shown
in Figure and summarized
in Table ) involving
dialysis, anion exchange chromatography (Figure a), and cation exchange chromatography (Figure b). The crude extract
was dialyzed against Tris-HCl-buffer, and the resulting protein solution
was applied to an anion-exchange column using a FPLC system (Figure A). Five major protein
peaks were eluted in which PaPPO activity was found.
The highest activity was present in the fractions that were recovered
from the second peak eluted at ∼9 mS/cm (1210–1390 mL)
(Figure A). Negligible PaPPO activity occurred in some of the other eluting fractions.
However, there was a little activity with the unbound proteins present
in the flow-through of the column (data not shown), which induced
a loss of activity after this step of purification (Table ). Fractions possessing activity
were pooled, concentrated, and then further purified by cation exchange
on a MonoS column, which eluted the protein into a single peak of
L-PaPPO activity at ∼18.5 mS/cm (78–96
mL) (Figure B). L-PaPPO was successfully purified 23.2-fold with 51.0% recovery
and a specific activity on 10 mM catechol of 459 U/mg (see Figure and Table ).
Figure 2
SDS–PAGE of L-PaPPO purification steps:
(1) crude extract, (2) dialysis, (3) anion exchange on Q-Sepharose,
(Mw) molecular weight marker (values are given in kDa),
(4) cation exchange on MonoS.
Table 1
Purification of Polyphenol Oxidase
from Apricot
purification stage
volume (mL)
total
protein (mg)
total activitya (units)
specific
activitya (units/mg protein)
purification (fold)
yield (%)
crude
extract: 85% (NH4)2SO4 precipitation
250
130
2570
19.8
1
100
dialysis against
Tris-HCl
300
69.0
2312
33.5
1.7
89.0
anion exchange: Q-Sepharose FF 20 mL column
1.23
12.1
1506
125
6.3
58.6
cation exchange: MonoS 5/50 GL
column
1.00
2.9
1311
459
23.2
51.0
Enzymatic activity was determined
on 10 mM catechol in 50 mM citrate buffer, pH 4.5.
SDS–PAGE of L-PaPPO purification steps:
(1) crude extract, (2) dialysis, (3) anion exchange on Q-Sepharose,
(Mw) molecular weight marker (values are given in kDa),
(4) cation exchange on MonoS.Enzymatic activity was determined
on 10 mM catechol in 50 mM citrate buffer, pH 4.5.
Effect of pH on PaPPO Activity
The
activity of purified PaPPO was measured at different
pH values, ranging from 2.0 to 9.5, using catechol as substrate (Figure a). The enzyme remains
active from pH 2.5 (38%) to pH 9 (17%) with a pH optimum of maximal
activity at pH 4.5 (100%). The enzymatic activity of PaPPO was reduced to < 50% at pH > 6 and no enzyme activity was
detected at pH 2 and below. The pH-optimum reported here for PaPPO is even lower than the one found by Ünal and
Şener[16] and Fraignier et al.[24] who reported the optimum pH for PaPPO at pH 5–5.8 and 5–5.5 using catechol and 4-methylcatechol
as substrate, respectively. Arslan et al.[25] reported an optimum activity at pH 8.5 for PPO from Malatya apricot.
Similarly low pH optima around pH 4.5 have been reported for PPOs
from peach (cultivar Catherina) using 4-tert-butylcatechol
as substrate.[15]
Figure 3
Effects of pH and temperature
on the activity of PaPPO. (a) pH optimum of PaPPO activity, (b) optimal
temperature for PaPPO activity, (c) thermal inactivation
of PaPPO activity. The enzyme activity was measured
using 10 mM catechol as substrate. The experiment was repeated three
times, and each value is given as the mean value ± standard deviation.
Effects of pH and temperature
on the activity of PaPPO. (a) pH optimum of PaPPO activity, (b) optimal
temperature for PaPPO activity, (c) thermal inactivation
of PaPPO activity. The enzyme activity was measured
using 10 mM catechol as substrate. The experiment was repeated three
times, and each value is given as the mean value ± standard deviation.
Effect of Temperature on PaPPO Activity
The temperature effect on the PaPPO activity was
studied over a range from 5 to 90 °C. The results are depicted
in Figure b. The enzyme
is active from 5 to 85 °C, showing maximum activity at 45 °C.
A decrease of PaPPO activity was observed at temperatures
≥ 50 °C, and the
enzyme lost 45%, 86% and 92% of its maximal activity at 60, 70, and
80 °C, respectively, until all activity was lost at 90 °C,
due to thermal inactivation. Ünal and Şener[16] reported a lower value for the temperature optimum
of apricot (cultivar Alyanak) PPO activity (30–40 °C)
using catechol as substrate. Thus, PPOs from three apricot cultivars
(Kabaasi, Çataloglu and Hacihaliloglu) showed optimal activity
at 35, 40, and 45 °C respectively.[26] An identical temperature optimum at 45 °C has been reported
for litchi pericarp PPO using (−)-epicatechin as substrate.[27] The optimum temperature for PPO activity generally
ranged between 30 and 40 °C.[28,29] A PPOs temperature
optimum usually depends on the environmental conditions where the
fruit grows, the plant species and variety as well as the substrate
used in the assay.[13]
Thermal Inactivation
of PaPPO
The
thermal inactivation profile of purified PaPPO, presented
as the residual activity after preincubation at various temperatures
from 50 to 100 °C for 2 to 30 min, is shown in Figure c. PaPPO exhibited
a reduction in catalytic activity as the temperature and duration
of incubation increased.The enzyme retained 56%, 31%, 18%,
13%, 6%, and 0% of its original activity after 2 min of incubation
at 50, 60, 70, 80, 90, and 100 °C, respectively, and more than
90% of activity was lost after incubation at 70 °C for 10 min. PaPPO was completely inactivated following incubation at
80, 90, and 100 °C for 30, 10, and 5 min, respectively (Figure c). Our results are
close to those of Yemenicioǧlu and Cemeroǧlu,[26] who reported that apricotPPO (Kabaasi cultivar)
lost more than 95% of its activity after 10 min of heating at temperatures
≥ 80 °C, using catechol as substrate. However, they found
that the enzyme was quite stable at 70 °C. PPO from plum[30] showed similar thermal stabilities at 55 °C
where the enzyme retained only 50% of its activity after 5 min of
incubation. Also PPO from blueberry[31] showed
similar behavior, the PPO retained only 3% of its activity after 2
min of incubation at 85 °C. By comparison with PaPPO, PPO from strawberry[32] and medlar
fruits[33] were relatively thermostable as
the enzymes retained more than 60% of their activity after 30 min
of incubation at 50 °C. On the other hand, PPO from potato[34] has been found to be sensitive to heat treatment;
the enzyme was completely inactivated after heating at 70 °C
for 10 min.
Substrate Specificity and Enzyme Kinetics
Substrate
specificity for PaPPO was investigated using 9 different
substrates (Table ) at the enzyme’s pH optimum for catechol as the substrate
(pH 4.5). The purified enzyme (L-PaPPO) was found
to be activated in the reaction media in contact with substrates without
the addition of an external activator (like SDS). This activation
can be attributed to the low pH optimum (4.5) of PaPPO. The latent PPO activation in the reaction media at low pH has
been reported before.[15] Activities were
compared with the activity observed in the presence of catechol (set
as 100%). The highest activity was found using chlorogenic acid, followed
by 4-methylcatechol, caffeic acid, pyrogallol, and catechol.
Table 2
Substrate Specificity of Purified PaPPOa
substrate
concn (mM)
λ
(nm)
ε(λ) (M–1 cm–1)
relative activity (%)
Monophenols
phenol
10
426
4300
0.00 ± 0.0
tyramine
10
395
1500
0.00 ± 0.0b
Diphenols
chlorogenic acid
10
475
511
738 ± 5.4
4-methylcatechol
10
400
1720
378 ± 12
catechol
10
426
4300
100 ± 0.71
caffeic acid
1
481
591
336 ± 0.65
l-dopa
1
392
1240
5.47 ± 0.33
dopamine
10
395
1500
8.85 ± 0.80
Triphenol
pyrogallol
10
457
926
181 ± 3.9
All measurements
were performed
in triplicate. Data is presented as mean ± standard deviation.
After storage A-PaPPO showed activity (0.0724% ± 0.0010%) on 4 mM tyramine (sodium
citrate buffer, pH 4.5; Figure S2).
All measurements
were performed
in triplicate. Data is presented as mean ± standard deviation.After storage A-PaPPO showed activity (0.0724% ± 0.0010%) on 4 mM tyramine (sodium
citrate buffer, pH 4.5; Figure S2).A lower activity toward dopamine
and l-dopa (diphenols)
was recorded, and no activity was detected with monophenols (phenol
and tyramine). The slight monophenolase activity reported for PaPPO after storage using tyramine as substrate suggests
that PaPPO has a weak activity on monophenols compared
to the strong activity on diphenols. Furthermore, reliable determination
of monophenolase activity requires the use of a large amount of enzyme
and a substantially extended monitoring time. The results obtained
in this study are in agreement with those of Arslan et al.[25] (1998) and Yemenicioǧlu and Cemeroǧlu[26] (2003), who reported that apricotPPO had no
activity toward monophenols using hydroquinone and p-cresol as substrate at pH 6.8. PPOs lacking monophenolase activity
were reported for other fruits among them mamey,[35] borage,[36] and blueberry.[31] Chlorogenic acid and 4-methylcatechol have been
found to be the best substrates for coffee[37] and applePPO[38] and the here investigated PaPPO. Chlorogenic acid is the main phenolic compound in
apricot,[39] which could explain the highest
catalytic efficiency of PaPPO on this substrate.KM and kcat were calculated by nonlinear regression using the four best substrates
at various concentrations under optimal conditions and are shown in Table . Substrate specificities
were evaluated by using the kcat/KM ratio (catalytic efficiency). kcat/KM followed the order
of activity of PaPPO on the tested substrates: chlorogenic
acid > 4-methylcatechol > pyrogallol > catechol. The lowest KM was observed for 4-methylcatechol (2 mM),
followed by chlorogenic acid (2.7 mM), catechol (5.3 mM), and pyrogallol
(11 mM). The KM values reported for PPOs
from apricot are 6.6 mM (cultivar Malatya)[25] and 1 mM (cultivar Moorpark)[40] using
catechol and chlorogenic acid, respectively. Close KM values were reported for mango (3.1 mM)[41] using 4-methylcatechol and Yali Pear (1.5 mM)[42] using chlorogenic acid as substrate.
Table 3
Kinetic Parameters of Purified PaPPO
substrate
KM (mM)
kcat (s–1)
kcat/KM (s–1 mM–1)
catechol
5.3 ± 0.60
210 ± 30
40 ± 10
chlorogenic acid
2.7 ± 0.16
1400 ± 210
500 ± 80
4-methylcatechol
2.0 ± 0.26
700 ± 110
340 ± 70
pyrogallol
11 ± 1.5
590 ± 90
50 ± 10
Effect of Inhibitors, Metal Ions, and SDS on PaPPO Activity
The effects of ten various inhibitors, among
them the most commonly used inhibitors of enzymatic browning, on PaPPO activity were investigated at four different inhibitor
concentrations (0.1, 1, 5, and 10 mM). The results are presented in Table . At 0.1 mM, 4-hexylresorcinol
was the most effective inhibitor with only 34% activity retained.
Increasing the concentration of inhibitors to ≥ 1 mM, sodium
metabisulfite, ascorbic acid, and l-cysteine were the most
effective inhibitors for PaPPO activity, followed
by reduced glutathione, 4-hexylresorcinol, kojic acid, and benzoic
acid. Complete inhibition was observed with sodium metabisulfite and
ascorbic acid at 1 mM, with l-cysteine and glutathione at
5 mM, and with 4-hexylresorcinol at 10 mM. Citric acid, EDTA, and
succinic acid were the weakest inhibitors with 81%, 87% and 89% of
residual activity, respectively, even at 10 mM concentration. All
of the inhibitors hinder the formation of melanin by preventing the
accumulation of o-quinones or by forming stable colorless
products.[13] Our results are in agreement
with those of Ünal and Şener[16] who detected a complete inhibition of apricotPPO with 1 mM of sodium
metabisulfite and ascorbic acid. Metabisulfite and ascorbic acid are
reducing agents, which inhibit enzymatic browning reactions by reacting
with quinones formed by PPO catalyzed oxidation of o-dihydroxy phenols, forming a stable, colorless product.[7] In addition metabisulfite can act on the enzyme
itself by irreversibly binding to the “met” and “oxy”
forms of binuclear copper at the active site.[7]
Table 4
Effect of Various Inhibitors on PaPPO Activitya
relative
activity (%)
inhibitor
0.1 mM
1 mM
5 mM
10 mM
4-hexylresorcinol
34 ± 1.4
11.0 ± 0.66
0.31 ± 0.05
0.0 ± 0.0
ascorbic acid
89 ± 1.1
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
benzoic acid
95 ± 2.2
41 ± 1.2
8.3 ± 0.11
5.4 ± 0.49
citric acid
100 ± 1.5
93 ± 1.5
88 ± 2.1
80.6 ± 0.87
kojic acid
65 ± 2.2
28 ± 1.1
7.3 ± 0.47
4.1 ± 0.52
succinic acid
99 ± 1.6
94 ± 1.6
90.9 ± 0.80
89.1 ± 0.53
EDTA
99.7 ± 0.45
93.7 ± 0.59
90.3 ± 0.43
88 ± 1.5
glutathione, reduced
84.3 ± 0.67
2.5 ± 0.21
0.0 ± 0.0
0.0 ± 0.0
l-cysteine
89.3 ± 0.45
0.65 ± 0.07
0.0 ± 0.0
0.0 ± 0.0
sodium metabisulfite
72 ± 2.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
All measurements were performed
in triplicate. Data is presented as mean ± standard deviation.
All measurements were performed
in triplicate. Data is presented as mean ± standard deviation.Ascorbic acid and l-cysteine were found to provide effective
inhibition of PPO in blueberry.[31] Sodium
metabisulfite was reported as the best inhibitor for PPOs from a wide
variety of species, among them pears,[43] potato,[34] grape,[44] mamey,[35] mango,[41] and blueberry.[31] Citric acid, succinic
acid, and EDTA have also been found not to be effective as inhibitors
for PPO from mango, even at 10 mM concentration, with 67%, 69% and
79% retained activity, respectively.[41]The effect of metal ions on PaPPO is shown in Table . The presence of
most of the tested metal ions did affect the enzymatic activity negatively.
Metal ions such as Al3+, Ni2+, Mn2+, Ca2+, and Fe2+ inhibited PPO activity markedly;
less than 35% of PPO activity was retained at a concentration of 10
mM. The exceptions were MgSO4, ZnSO4, and CuSO4; their presence stimulated the PaPPO activity.
Similar effects were observed in apple[29] where 50 mM of CuSO4 enhanced PPO activity to 145% and
3.69 mM of AlCl3 decreased it to 50%. Conversely PPO activity
in litchi was reduced in the presence of FeSO4 and NaCl
and stimulated in the presence of MnSO4 and CuSO4.[45] PPO of longan fruit was found to be
activated in the presence of MnSO4 and CuSO4.[46] The activities of PPO from mango[41] and Japanese honeysuckle[47] were found to be stimulated in the presence of Mg2+; however in contrast to our results, they noted that the activity
was decreased in the presence of CuSO4 and increased in
the presence of ZnSO4. According to Sun et al.,[48] metal ions like Ca2+ can cause the
repulsion of oxygen and therefore the inhibition of PPO activity.
However, some metal ions can promote the binding of the substrate
to the active site and enhance enzymatic activity by combining with
substrate or PPO.[35]
Table 5
Effect of Metal Ions on PaPPO Activitya
relative
activity (%)
salt
1 mM
10 mM
MgSO4
102 ± 1.7
107 ± 1.8
FeSO4
50 ± 2.6
34 ± 1.9
ZnSO4
104 ± 1.8
108 ± 1.9
CuSO4
108 ± 1.5
134 ± 1.1
KCl
87 ± 1.4
45 ± 2.8
NaCl
94 ± 1.5
56 ± 1.2
CaCl2
78 ± 2.5
23 ± 1.4
MnCl2
85 ± 0.99
23 ± 1.3
NiCl2
84 ± 2.3
15 ± 1.4
AlCl3
80 ± 2.0
3.67 ± 0.31
All measurements
were performed
in triplicate. Data is presented as mean ± standard deviation.
All measurements
were performed
in triplicate. Data is presented as mean ± standard deviation.PPO exists in plant tissues
in its active form and its latent form
where full activity can only be observed by adding detergents or fatty
acids or by treatment with proteases.[49] For this reason the enzymatic activity of the purified PaPPO was determined under normal assaying conditions using catechol
as substrate with various concentrations of SDS. The PPO activity
without SDS was taken as 100%. As displayed in Figure , SDS at lower concentrations (0.1, 0.5,
1, and 2 mM) enhanced the enzymatic activity, with the maximum activity
(155%) at 0.5 mM, whereas at higher concentrations of SDS (5 and 10
mM) PaPPO activity diminished markedly to 80% and
55%, respectively. The activation of the enzyme with SDS suggests
that purified PaPPO is present in its latent form.
Activation of latent PPO by SDS was reported in many organisms, among
them banana,[50] coffee,[37] peach,[15] mushroom,[51] and petals of C. grandiflora.[52] The effect of SDS (3.5 mM) on PPO
activity of five Prunus species (peach, almond, cherry,
plum, and apricot) was investigated by Fraignier et al.[24] They noted that the activity of PPO in the presence
of SDS was found to be 1.5 and 2 times higher than the activity without
SDS in plum and cherry, respectively, and a slight increase in activity
was noticed in almond and apricot. In peach, however, they found a
little decrease in activity due to SDS. The increase of PPO activity
in the presence of SDS might be due to the activation of latent PPO
through conformational changes.[53] Thus,
the activation of PPOs is observed at low concentrations of SDS, and
higher concentrations can cause the opposite effect and decrease the
enzyme activity instead of increasing it,[50] which is also what we observed for PaPPO.
Figure 4
Effect of SDS
on PaPPO activity.
Effect of SDS
on PaPPO activity.
Storage Stability of the Purified PaPPO
In order to determine the effect of storage at 4 °C (in 10 mM
Tris-HCl buffer at pH 8) on stability of the purified PaPPO, we monitored the enzymatic activity with and without SDS over
4 weeks, where PaPPO activity without SDS at day
one was taken as 100%. The results are shown in Figure A; PaPPO activity assayed
in the presence of SDS diminished by 50% after one month of storage
from 155% activity at day one to 78% activity at week 4. On the other
hand just a small decrease (13%) in PaPPO activity
was observed in the tests without SDS from 100% to 87% activity. Furthermore,
starting from the third week, SDS switched from being an activator
to acting as an inhibitor of the enzymatic activity, where we noted
a decrease of 10% of activity at week 4 from 87% in the absence of
SDS to 77% in the presence of SDS. Loss of PPO activity during storage
at 4 °C is a common observation in studies on PPOs.[28] However, the peculiar behavior of PaPPO during storage noted in this study, in the presence and absence
of SDS was not reported in earlier works. For that and to better understand
this behavior, denaturing SDS-PAGE (reduced samples) and partially
denaturing SDS-PAGE were performed over the storage period (Figure B,C).
Figure 5
Effect of storage at
4 °C on PaPPO. (A) activity
of PaPPO on catechol in the absence (SDS −)
and presence (SDS +) of 0.5 mM SDS, (B) denaturing SDS–PAGE
gels (reduced sample) of purified PaPPO, (C) activity
gels of purified PaPPO. (Mw) molecular
weight marker (values given in kDa), (D1) freshly purified protein,
(W1) 1 week of storage, (W2) 2 weeks of storage, (W3) 3 weeks of storage,
(W4) 4 weeks of storage. The enzyme was stored at 4 °C in Tris-HCl
buffer (pH 8).
Effect of storage at
4 °C on PaPPO. (A) activity
of PaPPO on catechol in the absence (SDS −)
and presence (SDS +) of 0.5 mM SDS, (B) denaturing SDS–PAGE
gels (reduced sample) of purified PaPPO, (C) activity
gels of purified PaPPO. (Mw) molecular
weight marker (values given in kDa), (D1) freshly purified protein,
(W1) 1 week of storage, (W2) 2 weeks of storage, (W3) 3 weeks of storage,
(W4) 4 weeks of storage. The enzyme was stored at 4 °C in Tris-HCl
buffer (pH 8).
Gel Electrophoresis and
Mass Determination
Reported
molecular weights of PPO, range from very low masses like 25 kDa for
pineapplePPO[54] to relatively high molecular
masses, for example, 120 kDa for field bean PPO.[55] In our study, the purified L-PaPPO collected
from cation exchange chromatography appeared as a single protein band
on an SDS-PAGE gel with an apparent molecular mass of 63 kDa (Figure ). This mass is identical
to the molecular weight of the latent PPO detected in apricot and
four other species of Prunus.[24] Chevalier et al.[10] also reported
a similar mass of 60 kDa. Our apparent mass is higher than the molecular
weight of the mature protein (56.2 kDa) calculated by Chevalier et
al.[10] and the mass of the purified apricotPPO (57 kDa) detected by Ünal and Şener.[16]Denaturing SDS-PAGE (reduced samples)
stained with Coomassie brilliant blue G-250 was performed to monitor
storage behavior of L-PaPPO, and the respective gels
are presented in Figure B. The results show the formation of a new band at 38 kDa (A-PaPPO) during storage, which is apparent starting from the
first week of storage and becomes thicker over the weeks, and in parallel
the original band at 63 kDa (L-PaPPO) becoming thinner
until it almost totally disappears after the fourth week (Figure B). Under partially
denaturing SDS-PAGE, a single band was detected by in-gel enzymatic
activity staining with catechol as substrate (Figure C). The band appeared at 38 kDa in the same
position as the band observed when stained with Coomassie blue (A-PaPPO of Figure B). From these results, it is concluded that the band at 38
kDa represents the active form of apricotPaPPO,
a mass that is close to the molecular weight (43 kDa) detected by
activity staining previously reported by Fraignier et al.[24]The mass spectrum (ESI-LTQ) of the purified
enzyme (after storage)
is given in Figure S3 along with a zoomed-in
section of this spectrum, indicating the presence of one major protein
species. Twelve distinct peaks were used, and assuming that the positive
charge states are solely caused by the attachment of protons, the
average molecular mass can be assessed as 37 455.6 ± 0.9
Da. Those results confirm the results obtained by SDS-PAGE and indicate
that the latent apricotPPO (L-PaPPO) was practically
quantitatively converted to its active form after one month of storage
at 4 °C in Tris-HCl buffer (pH 8).PPOs are believed to
be stored in latent form in plants.[13] Latent
PPO could exist in a completely latent
or a partially active form,[56] as in the
case of L-PaPPO. The C-terminal domain is thought
to shield access of phenolic substrates to the active site.[57] From that, it can be concluded that following
harvest PaPPO is present in the fruit in the latent
form. Our results give a strong indication that latent PaPPO can also be activated spontaneously during storage at 4 °C.
Protein Identification and Sequence Confirmation
The
mature protein of apricotPPO has 496 amino acids (Asp102 →
Ser597) with a calculated molecular weight of 56.2 kDa.[10] The mass for A-PaPPO (37 455.6
Da) determined by mass spectrometry (ESI-LTQ) matches perfectly to
the mass of the polypeptide Asp102 → Leu429 with one thioether
bridge[58] and two closed disulfide bridges
containing all five cysteines present in the peptide chain (Figure ). This is additionally
supported by the results of enzymatic digestion. UHPLC-ESI-MS/MS identified
the purified latent PPO (L-PaPPO) at 63 kDa as apricotPPO (UniProt O81103) yielding a sequence coverage of 48.87% (PaPPO
(Asp102 → Ser597)) and a total of 36 identified peptides (Table S1), 16 peptides from the main domain and
20 peptides from the C-terminal part defining the enzyme latent form,
including peptides that cover the C-terminus of the protein. The peptides
are underlined in blue in Figure and listed in Table S1.
For A-PaPPO, we had a sequence coverage of 41.4%
(PaPPO (Asp102 → Leu429)), and all the peptides
that have been identified (12 peptides) are in the main domain region.
No tryptic peptides were detected and identified in the C-terminal
part beyond Arg430. The presence of Arg430 in the last peptide identified
can provide an indication that the removal of the enzyme’s
C-terminal domain proceeds via more than one cleavage site, as it
was also reported in recent research.[59] The identified peptides are underlined in crimson in Figure and listed in Table S2. PaPPO (Uniprot O81103) shows 96.65%
and 94.14% sequence identity to PPO from japanese apricot (Prunus mume; GenBank 645236994) and peach (Prunus persica; UniProt I1U4K7), respectively.
Figure 6
Sequence of PaPPO (UniProt O81103). (Peach
shading: signal peptide domain. Green shading: main domain (A-PaPPO). Yellow shading: C-terminal domain. Square brackets
indicate the start and the end of L-PaPPO. Pink vertical
line indicates the cleavage position (the end of A-PaPPO and the start of C-terminal domain) as deduced from matching
the amino acid sequence with the molecular mass determined for A-PaPPO by ESI LTQ Orbitrap Velos MS. The peptides identified
by UHPLC-ESI-MS/MS for L-PaPPO and A-PaPPO are underlined in blue and crimson, respectively.
Sequence of PaPPO (UniProt O81103). (Peach
shading: signal peptide domain. Green shading: main domain (A-PaPPO). Yellow shading: C-terminal domain. Square brackets
indicate the start and the end of L-PaPPO. Pink vertical
line indicates the cleavage position (the end of A-PaPPO and the start of C-terminal domain) as deduced from matching
the amino acid sequence with the molecular mass determined for A-PaPPO by ESI LTQ Orbitrap Velos MS. The peptides identified
by UHPLC-ESI-MS/MS for L-PaPPO and A-PaPPO are underlined in blue and crimson, respectively.
Authors: Edward I Solomon; David E Heppner; Esther M Johnston; Jake W Ginsbach; Jordi Cirera; Munzarin Qayyum; Matthew T Kieber-Emmons; Christian H Kjaergaard; Ryan G Hadt; Li Tian Journal: Chem Rev Date: 2014-03-03 Impact factor: 60.622
Authors: T Chevalier; D de Rigal; D Mbéguié-A-Mbéguié; F Gauillard; F Richard-Forget; B R Fils-Lycaon Journal: Plant Physiol Date: 1999-04 Impact factor: 8.340
Authors: J L Muñoz; F García-Molina; R Varón; J N Rodriguez-Lopez; F García-Cánovas; J Tudela Journal: Anal Biochem Date: 2006-01-26 Impact factor: 3.365
Authors: Ioannis Kampatsikas; Aleksandar Bijelic; Matthias Pretzler; Annette Rompel Journal: Angew Chem Int Ed Engl Date: 2019-04-17 Impact factor: 15.336
Authors: M A Daugavet; M I Dobrynina; T G Shaposhnikova; A I Solovyeva; A G Mittenberg; S V Shabelnikov; I Yu Babkina; A V Grinchenko; D V Ilyaskina; O I Podgornaya Journal: Sci Rep Date: 2022-08-22 Impact factor: 4.996
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