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Title 37 C.F.R., Chapter II, § 201.14
Journal of Food Measurement and Characterization (2020) 14:31–38
https://doi.org/10.1007/s11694-019-00264-8

ORIGINAL PAPER

Purification of tea leaf (Camellia sinensis) polyphenol oxidase by using

affinity chromatography and investigation of its kinetic properties
Cansu Öztürk1 · Mine Aksoy1 · Ömer İrfan Küfrevioğlu1

Received: 26 February 2019 / Accepted: 4 September 2019 / Published online: 12 September 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Polyphenol oxidase (PPO) was purified from tea leaves (Camellia sinensis) via affinity chromatography for the first time
and the purified enzyme was characterized. The purity of enzyme and molecular weight was determined by SDS-PAGE
and non-denaturing PAGE (native PAGE). Single bands were observed with both electrophoretically methods. The PAGE
results indicated that the molecular weight of PPO from tea leaf was approximately 50 kDa. The pH, temperature, and
kinetic parameters were studied. K­ m values were 3.782 and 3.881 mM for catechol and 4-methylcatechol respectively. V ­ max
values for catechol and 4-methylcatechol were also determined as 1.676 and 1.912 µmol/L min respectively. Additionally,
the inhibition effects of sodium metabisulfite, sodium sulfite, ascorbic acid, glutathione, dithioerythritol were investigated
on the enzyme activity and ­IC50, ­Ki values were calculated for these substances.

Keywords  Tea leaf · Purification · Polyphenol oxidase · Characterization · Inhibition

Introduction they form melanin pigments polymerized with endogenous

Approximately 3.6 million tons of tea are produced annually
in the world. 78% of the consumed tea is fully-fermented
black tea, 20% is non-fermented green tea and 2% is semi-
fermented oolong tea. Turkey ranks the fifth in the world in
terms of tea production. Tea contains various compounds
such as teanin, polyphenols, theaflavins, purine alkaloids and
minerals [1–3]. Phenolic substances (polyphenols) are the
most important components of tea. Flavanols in tea structure
are important; together with catechins and gallocatechin,
they form their gallates [4]. The PPO enzyme, one of the
most important components of the tea plant, is found in the
leaf tissue and catalyzes the oxidation of catechins with oxy-
gen [5, 6]. The polyphenol oxidase enzyme catalyzes the
oxidation of phenolic compounds to o-quinones [7]. Since
these quinones are highly reactive electrophilic molecules,
* Ömer İrfan Küfrevioğlu
okufrevi@atauni.edu.tr
Cansu Öztürk
cansu_36_25@hotmail.com
Mine Aksoy
maksoy@atauni.edu.tr
1
Department of Chemistry, Faculty of Sciences, Atatürk
University, 25240 Erzurum, Turkey
amino acids and proteins. The complex brown pigments that
bring the enzymatic browning phenomenon are undesirable
during storage, post-harvest physiology, and processing of
fruits and vegetables [8]. Enzymatic browning is one of the
problems encountered in fruit and vegetable industries since
it is harmful to food quality in both sensory and nutritional
terms.[9]. Reduction or complete cease of the enzymatic
browning may be accomplished by removing one of the phe-
nolic materials, the molecular oxygen, or the polyphenol
oxidase enzyme. In addition, such browning reactions can be
prevented by the removal of substrates, reduction of the pH
of the medium, the addition of sodium sulfite and ascorbic
acid, or heat inactivation [10–12]. On the other hand, in the
black tea production process, approximately 75% of the cat-
echins in tea leaves are oxidized and partially polymerized
by the enzymatic transformation. The main enzyme involved
in these processes is tea leaf polyphenol oxidase [13].
In the presence of molecular oxygen, PPO catalyzes the
o-hydroxylation of monophenols to o-diphenols (monophe-
nolase activity) and oxidation of the o-diphenols to o-qui-
nones (diphenolase activity) [14]. The oxidation of tea cat-
echins to theaflavin and thearubigins has a significant impact
on black tea qualities such as flavor, color, and odor. This
oxidation is largely carried out by the PPO enzyme [15].
After studies using catechin and a chemical oxidizing agent

13
Vol.:(0123456789)

32
to synthesize theaflavins, theaflavins synthesis with polyphe-
noloxidase has become the mainstream. Nowadays there are
many studies in the literature on the enzymatic synthesis of
theaflavins and thearubigins [16–18].
In this paper, the purification of tea leaf PPO with Sepha-
rose 4B-L-tyrosine-p-aminobenzoic acid affinity column is
reported for the first time. By this chromatographic method,
the enzyme is purified a single step. The enzyme purity was
checked by sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS-PAGE) and non-denaturing PAGE (native
PAGE) was used to estimate the molecular mass of purified
PPO. In addition, various enzymatic properties including
substrate specificity, pH and temperature optima, kinetics,
and chemical inhibition were investigated.
Materials and methods
Chemicals
Tea leaves (Camellia Sinensis) used in this study were har-
vested from Rize (a province in Turkey) origin in Turkey.
The extract of tea leaves was prepared as quickly as possible
and stored deep-frozen at − 80 °C until used.
All chemicals used in this study were the best grade avail-
able. Enzyme assays were measured with the aid of Beck-
man Coulter Du 730 (UV–Vis) automated recording spectro-
photometer. Sepharose 4B-L-tyrosine-p-aminobenzoic acid
affinity gel used in this study was synthesized at Atatürk
University in Chemistry section laboratory.
Preparation of homogenates
All procedures were carried out at 4 °C. Tea leaves (Camel-
lia sinensis) (10 g) were placed in a Dewar flask under liquid
nitrogen for 10 min to decompose cell membranes. A 10 g
sample of tea leaves was homogenized using a homogenizer
for 2 min in 10 mL of 0.1 M acetate buffer (pH 6.0) con-
taining 10 mM ascorbic acid, 0.5% PEG (polyethylene gly-
col) and 1 mL Triton X-100. The crude extract was filtered
through four layers of cheesecloth and then the homogenate
was centrifuged at 20000xg for 30 min and the supernatant
was collected.
Affinity chromatography
The affinity gel was synthesized according to the method
synthesized by Arslan et al. [19]. In this method, the CNBr
activated Sepharose-4B matrix was covalently modified with
l-tyrosine to form the extension arm. p-aminobenzoic acid,
which is a specific inhibitor of PPO enzyme and which pro-
vides a high degree of purification of the enzyme by entering
the structure of the affinity gel, was chosen as the ligand. The
13
C. Öztürk et al.
gel obtained was used as a column filler in the purification
of PPO from tea plant leaves by affinity chromatography.
The homogenate prepared as described in Sect. 2.2 was
applied to the affinity column equilibrated with 0.05 M pH
6.0 phosphate buffer. Then the affinity gel was washed with
the same buffer. PPO was eluted with the solution of 0.1 M
Tris–HCl buffer/1 M NaCI (pH 8.5).
Protein determinations
Protein content was measured according to the Bradford
[20] method using bovine serum albumin as a standard. The
color of the reaction was measured spectrophotometrically
at 595 nm wavelength at 25 °C.
Assay of PPO activity
The measurement of enzyme activity is based on the
increase in absorbance at 420 nm during the conversion of
the phenolic substrate to quinones [21]. One unit of PPO was
defined as the amount of enzyme that causes an increase in
absorbance of 0.001.
Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis
SDS–PAGE was performed for electrophoretic analysis of
enzyme after purification [22]. The protein bands were visu-
lized using silver staining. The electrophoretic pattern was
photographed.
Native PAGE was performed similarly as SDS-PAGE
without the addition of sodium dodecyl sulfate and heating
[23]. The gel was stained with 10 mM l-Dopa.
The determination of optimum pH, temperature
and ionic strength
The optimum pH for PPO obtained from tea leaves was
determined using two varied substrates (0.1 M catechol and
0.1 M 4-methyl catechol). For the pH studies, acetate buffer
(for a pH range of 4.0–5.5), phosphate buffer (for a pH range
of 6.0–7.5), Tris–HCl buffer (for a pH range of 8.0–9.5) and
glycine–NaOH buffer (for a pH range of 10.0–11.0) were
used.
To determine the optimum temperature ranges of the
enzyme using catechol and 4-methyl catechol substrates,
enzyme activity at different temperatures in the range from
0 to 80 °C was measured.
The effect of ionic strength on the PPO enzyme was stud-
ied using 0.01–1 M concentrations of Tris–HCl buffer for
two substrates (catechol and 4-methyl catechol) at an opti-
mum pH.
Purification of tea leaf (Camellia sinensis) polyphenol oxidase by using affinity…
The determination of stable pH
To determine pH stability of the enzyme, enzyme activi-
ties were measured in 0.05 M acetate buffer (pH 4.0–6.0),
0.05 M phosphate buffer (pH 5.0–7.5) and 0.1 M Tris–HCl
(pH 7.0–9.0) on a par with of purified enzyme sample and
buffers. The activity measurements were performed at 24 h
intervals for 5 days.
Kinetic studies and the determination of ­IC50 and ­Ki
values
At least five different substrate cuvette concentrations of
enzyme activity were measured to obtain K ­ m and V­ max val-
ues separately for catechol and 4-methylcatechol at optimum
pH and temperature. Then, Km and Vmax values were deter-
mined by drawing Lineweaver-Burke graphics.
Inhibitor effects on PPO activity were studied by using
the following inhibitors: sodium metabisulfite, sodium
sulfite, glutathione, l-ascorbic acid and dithiotretiol at five
concentrations of inhibitors with 100 mM catechol substrate
at Tris–HCl buffer, pH 9.0.
Activity %- [inhibitor] graphs were plotted to determine
­IC50 values by measuring enzyme activity at five different
inhibitor concentrations. Then using three constant inhibi-
tor concentrations, enzyme activities were measured at five
concentrations of the catechol. Lineweaver–Burk graphs
were drawn for each inhibitor and K ­ i (dissociation constant)
values were determined.
Results and discussion
Purification
Up to now, PPO has been purified from different sources
by the methods such as Triton X-100 extraction, dialy-
sis, ammonium sulfate precipitation, Phenyl-Sepharose
hydrophobic chromatography, Sephadex G-200 gel filtra-
tion chromatography, and affinity chromatography [17,
24–26]. Among them, affinity chromatography is an easy
technique to apply with ligand attachability. In this study,
Sepharose-4B-L-tyrosine-p-aminobenzoic acid affinity
Table 1  Summary of the PPO purification procedure
Purification steps Total Activity Protein (mg/mL) Total protein (mg) Total activity
volume (EU/
(mL) mL)
Homogenate 15 8000 0.3894
p-Aminobenzoic 2 1600 0.00394
acid affinity
chromotography
33
gel was synthesized using Arslan et al. [19] gel synthesis
method and PPO was purified from tea leaf in one step using
Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity chro-
matography. The purification profile of PPO is summarized
in Table 1. While the specific activity of the enzyme in the
homogenate was 20,544 EU/mg protein, the specific activ-
ity of the affinity purified PPO was 406,091 EU/mg pro-
tein. According to these results, the enzyme was purified
19.77-fold with 2.67% recovery. Using the same chroma-
tographic method previously, PPO was purified 13.9-fold
from mushroom (L.piperatus) [27], 74-fold from Mulberry
(Morus Alba L.) [19], 31.5-fold from pear (Pyrus elaegrifo-
lia) [28], and 43-fold from artichoke (Cynara scolymus L.)
[25]. Unlike ion exchange chromatography, affinity chroma-
tography, and gel filtration chromatography are commonly
used techniques for purifying PPO from a variety of sources.
Two PPO isoenzymes from the green cap of fresh basil were
purified 17-fold and 9.4-fold with the Sephadex G-100 col-
umn [29]. PPO was purified 2.7-fold and 2.2-fold purified by
ion exchange chromatography from the leaves and stems of
the ferrula plants [30]. The tea plant PPO was purified 3.32-
fold with 5.11% yield by DEAE-cellulose column [31]. In
another study with Camellia sinensis, two isozymes of PPO
were purified 48.94-fold via ammonium sulfate precipita-
tion, acetone, dialysis, gel column, ion exchange column
[18].
The determination of tea leaf PPO enzyme purity
and molecular weight
After purification by means of affinity chromatography, the
enzyme eluate was electrophoretically analyzed by both
native PAGE and SDS PAGE (Fig. 1). Single protein band
was detected under silver staining condition by using SDS-
PAGE method. NATIVE-PAGE studies of tea leaf PPO
showed a single band after staining with L-DOPA, too.
According to both PAGE results, the molecular weight of
PPO from tea leaf is approximately 50 kDa. It has been pre-
viously reported that the molecular weight of PPO varies
from different species. For example, the molecular weight
of PPO was found to be 65 kDa from Lactarius salmoni-
color, 35 kDa from wild pear, 39 kDa from cabbage, and
42 kDa from sunflower seed. Field bean PPO is constituted
Spesific Yield (%) Purification fold
(EU) activity
(EU/mg)
5.841 120,000 20,544 100 1
0.00788 3200 406,091 2.67 19.77
13

34 C. Öztürk et al.

Fig. 1  a Sodium dodecyl

sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE):
M indicates marker, Lane 2
and 3, purified enzyme from
p-aminobenzoic acid affin-
ity chromatography. b Native
PAGE stained with L-Dopa:
M indicates marker, Lane 1:
purified enzyme from p-amin-
obenzoic acid affinity chroma-
tography

of four subunits with a molecular weight of about 30 kDa
and has a molecular weight of about 120 kDa. Previously, in
a study of Indian tea leaves, substrate staining of the acetone
powder extract showed the presence of a maximum of three
isozymes of this enzyme. In the study, chromatographic
steps such as gel filtration, hydroqapatite, centricon-30,
FPLC were performed and the enzyme was determined to be
72 kDa by SDS-PAGE. In another study, molecular masses
of two PPO isozymes isolated from Camellia sinensis were
determined to be about 85 kDa and 42 kDa by SDS-PAGE
[18].
Effect of pH and temperature
Catechol and 4-methyl catechol substrates were used to
determine the optimum pH of polyphenol oxidase enzyme
purified from tea leaves (Fig.  2). The pH optimum for
enzyme-catalyzed oxidation of catechol in Tris/HCl buffer

Fig. 2  Effect of pH and tem-

perature on PPO activity

13
Purification of tea leaf (Camellia sinensis) polyphenol oxidase by using affinity…
was found to occur at pH 9.0. For the 4-methyl catechol
substrate, the optimum pH was found to be 8.5 in Tris–HCl
buffer. The effect of pH on the tea leaf PPO isozymes PPO1
and PPO2 were determined over a pH range of 4.0–8.0 [32].
Different optimum pH values for PPO obtained from differ-
ent sources are reported in the literature. The optimum pH
values are 8.0 for raspberry [33], 7.0 for Amasya apple [34],
6.0 for Pyrus elaegrolifia (PePPO) [28], 7.0 for mulberry
fruit [19].
In order to determine pH stability, the enzyme solution
was incubated in acetate buffer solutions ranging from 4.0
to 6.0, phosphate buffer solutions ranging from 5.0 to 7.5,
and Tris–HCl buffer solutions ranging from 7.0 to 9.0 at
4 °C. The experiments has been repeated for 5 days with a
spectrophotometric measurement using catechol as a sub-
strate in every 24 h. The pH stability profile for the enzyme
shows that 83% of the PPO activity was retained at pH 5.5
phosphate buffer. In the pH 6 phosphate buffer, the enzyme
activity decreased to 68% after 5 days. Interestingly, the
enzyme did not show the same stability in pH 5.5 and 6
acetate buffer, and the remaining activity at pH 5.5 was 75%,
while at pH 6 it was 47%. The enzyme is not stable above
pH 7.5.
The activity of PPO was measured at different tempera-
tures for catechol and 4-methyl catechol substrates at opti-
mum pH. The enzyme showed the highest activity at 10ºC
for both substrates (Fig. 2). In the literature, the maximum
activity of various PPO species, using catechol as substrate,
has been reported as being at 22 °C for potato [35], 40 °C for
Chinese cabbage [36], 12 °C for Ferula sp. [30], and 25 °C
for artichoke [37].
The Effect of ionic strength
In the study of ionic strength; a series of Tris–HCl buffer
was prepared at concentrations of 0.01–1 M. For each con-
centration, activity measurements were done at the optimum
pH of the catechol and 4-methyl catechol substrates. As a
result of this study, both substrates exhibited high activity at
0.05 M buffer concentration. However, since there are very
few differences in activity at different concentrations, it can
Fig. 3  Vmax and ­Km values of
Tea leaf PPO with catechol and
4-methyl catechol
35
be said that ionic strength is not a serious factor affecting
enzyme activity.
Kinetic parameters
Changes in the activity of PPO were determined as
depending on increased substrate (catechol) concentra-
tion. For determining V ­ max and ­Km values of PPO, catechol
(1–20 mM) and 4-methylcatechol (1–20 mM) were used as
substrates to measure enzyme activities in the optimized pH
and temperature conditions. A plot of 1/V versus 1/[S] was
drawn by the method of Lineweaver & Burk to calculate ­Km
and ­Vmax values of tea leaf PPO for each substrate (Fig. 3).
The ­Km and V ­ max values obtained from the plot analysis of
PPO were found as 3.782 mM and 1.676 µmol/L min for
catechol, 3.881 mM and 1.912 µmol/L min for 4-methylcat-
echol respectively.
In a study carried out by Jia et al. [38] ­Km value for PPO
from sour cherry pulp was found to be 3.5 mM, using cat-
echol. Duangmal and Apenten [39] reported the following
­Km values for taro PPO: 67.9 mM for catechol, 9.0 mM for
4-methylcatechol, and 89.9 mM for pyrogallol. The same
investigators reported the following K ­ m values for potato
PPO: 6.8 mM for catechol, 1.1 mM for 4-methylcatechol,
and 1.5 mM for pyrogallol, but K ­ m value for tomato PPO
is calculated 8.5 mM for L-dopa, 25 mM for catechol, and
1.5 mM for pyrogallol. As can be seen, the affinity of PPOs
from various sources for various substrates varies widely.
The effects of inhibitors
In the present study, we showed inhibitory impacts of
sodium metabisulfite, sodium sulfite, ascorbic acid, glu-
tathione, dithioerythritol on PPO enzyme activity. All of
these compounds inhibited the tea leaf PPO. The order of
inhibition is as follows: sodium metabisulphite ˃ sodium
sulphite ˃ ascorbic acid ˃ dithioerythritol = glutathione. The
­IC50 values for sodium metabisulfite, sodium sulfite, ascor-
bic acid, glutathione, dithioerythritol were 21.65, 23.90,
69.31, 99.08 and 99.02 µM, respectively. The results were
summarized in Table 2.
13

36 C. Öztürk et al.

Table 2  IC50, ­Ki values and inhibition types for Tea leaf PPO histidine residues liganded to the copper of the active site
Inhibitors IC50 (µM) Ki (µM) Inhibition type
of PPO and/or completely remove the copper from the
enzyme [47, 48]. In addition, PPO inhibitors can be divided
Sodium metabisulfite 21.65 19.30 ± 0.005 Noncompetitive into two classes as binding of copper side and interference
Sodium sulfite 23.90 21.20 ± 0.005 Competitive with the side for phenolic substances. While the category
Ascorbic acid 69.31 32.67 ± 0.016 Competitive of interact with copper side shows competitive inhibition,
Glutathione 99.08 118.71 ± 0.024 Competitive the other category shows non-competitive inhibition [49].
Dithioerythritol 99.02 192.01 ± 0.097 Competitive In this study, competitive-type inhibition was obtained with
sodium sulfite, ascorbic acid, glutathione, dithioerythritol
inhibitors using catechol as substrate. Sodium metabisul-
Catechol was used as a substrate for the detection of phite inhibitor showed non-competitive type inhibition.
­Ki constants of these inhibitors. ­Ki constants and inhibi- Sodium bisulfite and ascorbic acid on PPO from round
tion types for these compounds were determined from brinjal showed competitive and non-competitive inhibition
Lineweaver–Burk graphs (Fig. 4). The lowest ­Ki constant respectively [50]. Competitive inhibition was found for bor-
was found for sodium metabisulphite (19.30 µM) followed age PPO with l-cysteine and sodium metabisulphite inhibi-
by sodium sulphite (21.20 µM), ascorbic acid (32.67 µM), tors using 4-methylcatecholas substrate. On the other hand,
glutathione (118.71 µM) and dithioerythritol (192.01 µM) ascorbic acid showed an uncompetitive inhibition with bor-
respectively. age PPO [51].
There are many studies reported to prevent enzymatic
browning. For example, the effect of glutathione and
L-cysteine on peach pulp PPO was investigated and the I­ C50 Conclusion
values were found to be 0.22 and 0.06 mM, respectively
[40]. Ascorbic acid on PPO from Cape gooseberry, 89.9% Polyphenol oxidase has received considerable, since it is
inhibition occurred at 0.1 mM concentration [41]. ­Ki values the key enzyme in melanin biosynthesis. Therefore, it is
of citric acid and l-cysteine were determined as 0.93 and important to understand the kinetic parameters of polyphe-
0.72 for the inhibition of Chinese parsley PPO [42]. nol oxidase (PPO), which is a food quality related enzyme.
The ability of thiol groups to inhibit PPO has been exten- This work reported the purification through sepharose
sively described [33–46]. Possible inhibition mechanisms 4B-L-tyrosine-p-aminobenzoic acid affinity chromatogra-
of thiol compounds can be divided into three types: (a) cop- phy for the first time. After purification, the enzyme elu-
per-nitrogen bond cleavage; (b) oxidation–reduction reac- ate was electrophoretically analyzed by natural PAGE and
tions; and (c) nucleophilic addition reactions [47, 48]. The SDS-PAGE and the molecular weight was determined as
first type involves PPO directly. It postulates that because 50 kDa. The inhibition effect of some sulphite group inhibi-
SH groups have a strong affinity for copper, they displace tors on enzyme activity was also investigated. Sodium

Fig. 4  Lineweaver–Burk graph with five different substrate (catechol) concentrations and three different inhibitors concentrations for determina-
tion of ­Ki

13
Purification of tea leaf (Camellia sinensis) polyphenol oxidase by using affinity…
metabisulphite was found to have the highest inhibitory
effects on the purified enzyme. The inhibition mechanisms
of sodium metabisulphit was non-competitive, while sodium
sulfite, ascorbic acid, glutathione, dithioerythritol inhibitors
were competitive.
Acknowledgements  The authors declare that there is no financial or
material support.
Author contributions  MA, OIK: Designed the experiments. MA and
CO: Performed the experiments. MA, CO and OIK: Analyzed the data.
OIK: Contributed reagents/materials. MA, CO and OIK: Wrote the
paper. The final manuscript was read and approved by all authors.
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