International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Special Issue-5, Dec.

-2015

ISOLATION AND STUDY THE PROFILE OF POLYPHENOL OXIDASE

FROM MYANMAR TEA LEAF (CAMELLIA SPP.)
1
PYIE PHYO MAUNG, 2BO BO, 3AUNG KO KO OO, 4KYAW NYEIN AYE
1,2
Department of Biotechnology, Mandalay Technological University, Mandalay Division, The republic
of the Union of Myanmar
3
Department of Food-Biotechnology, Biotechnology and Material Science Research
4
Department, Ministry of Science and Technology,The republic of the Union of Myanmar
E-mail: [email protected], [email protected], [email protected], [email protected]
1

Abstract- Polyphenol oxidase (PPO) was extracted from Camellia spp. and analyzed the effect of pH, temperature and their
stability. Among the analyzing of PVP using in PPO extraction, 0.1 % of added PVP amount had the highest enzyme activity
between 0.1 % and 0.5%. For the test of finding optimal pH for polyphenol oxidase crude enzyme, pH 7 was the best pH for
PPO crude enzyme and PPO enzyme also had the highest stability in pH7. For polyphenol oxidase crude enzyme, catechol
substrate showed the highest enzyme activity than other kinds of substrates, Gallic acid and hydroquinone that using as
substrate in the analysis of PPO. During the analysis of optimal temperature for PPO, the enzyme activity of PPO between 40
˚C - 60˚C were not much different but the temperature was higher than 60˚C, the enzyme activity was decrease dramatically. In
the test of thermal stability for polyphenol oxidase enzyme between 40˚C to 100˚C during 5 to 60 minutes, 40˚C to 60˚C under
20 minutes had similar enzyme activity but the enzyme activity was drop after the 20 minutes and longer time. When the
temperature was higher 70˚C to 100˚C, the enzyme activity was decrease and decrease even in 5 minutes.

Index Terms- Polyphenol oxidase enzyme (PPO), Substrate, pH, Temperature

I. INTRODUCTION other molecules, leading to a large variety of dark

colored compounds [1]. In different parts of the plant,
Tea is one of the world's three beverage crops, the content of PPO are also different, in general, young
originated from China. Its efficacy is rich in sites in the growing season, PPO's content is generally
polyphenols, vitamins, caffeine, tea polysaccharide, higher than the mature part of, while the injured are a
the aflavins and other bioactive substances; it is both PPO content is higher. Dry et al study found,
edible and medicinal. In Myanmar, tea is divided into compared to a mature organization, in the growing of
green tea, yellow tea, black tea, oolong tea, black tea fruit, foliage PPO were significantly higher [3]. PPO is
and laphet (fermented pickled tea leaves) six a nuclear – encoded plastid enzyme, the enzyme
categories but mainly consumed black tea and laphet. having plastids through some characteristic, i.e., a
Myanmar also has a habit of eating and drinking tea. polypeptide containing the transfer. PPO precursor
In tea, raw materials and processing technology used peptide containing the first synthesized in cytoplasm
in various types of tea, so distinctive flavor, and this leader peptide, in which with the help of the membrane
flavor formation is tea inclusions interaction, changing into the plastid, and then leader peptide remove, so
the product. This change is the inclusion of a portion that the PPO become mature and stable molecular
of the reaction caused by automatic; called the enzyme weight enzyme [12].
catalyzed reaction.
Leader peptide is generally from 80 to 90 amino acids
By heat treatment, one can promote the formation of and usually connected with the N-terminal PPO. In the
flavor compounds in tea and reduce the moisture N-terminal leader peptide having a large number of
content; on the other hand can be inactivated tea hydrophilic amino acid residues, such asserine,
enzymes, making tea quality deterioration of the leucine, and the C-terminus and about 50%of
enzymatic reaction cannot be proceed. But the usual hydrophobic amino acids, including alanine, proline,
hot air drying and low thermal efficiency during the etc. Browning reaction in food processing and storage
drying process, the tea prolonged exposure to high condition caused the effect of the appearance of food,
temperatures, making the content of vitamins and nutrition and quality. Browning reaction of fruits and
heat-sensitive substances flavor and polyphenols, vegetables, mainly due to the inclusion of changes and
caffeine, etc., so the quality of tea will be poor. this change can be divided into enzymatic browning
Polyphenol oxidase (Polyphenol Oxidase, here in after and non-enzymatic browning. In the main
referred to as PPO) is the tea a very important enzyme, non-enzymatic browning, PPO played a key role in the
and is closely related to the quality of tea. Polyphenol oxidation and caused color changing [10]. The aim of
oxidase (PPO) is a cupric enzyme that catalyzes the this paper is to extract the PPO enzyme from Myanmar
hydroxylation of monophenols to o-diphenols, and the tea leaves (Camellia spp.) and to evaluate the effect of
oxidation of o-diphenols to o-quinones. Quinones are pH, temperature, etc. And this study would provide the
very reactive compounds which strongly interact with understanding for the browning reaction of tea leaves.

Isolation And Study The Profile Of Polyphenol Oxidase From Myanmar Tea Leaf (CAMELLIA SPP.)

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International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Special Issue-5, Dec.-2015

II. MATERIALS AND METHODS A 1 ml aliquot of enzyme solution was kept in the
under conditions of 40-100 ℃, incubated 5min,
A. Samples Collection 10min, 20min, 30min, 40min, 50min, 60min,and then
Fresh tea leaves were obtained from Pyin OO Lwin immediately placed in an ice bath to cool, followed by
Township (Elevation – 3510 ft / 1070m), Myanmar. the enzyme assay. The enzyme activity determined the
All other chemicals were analytical grade and thermal stability of PPO [8].
collected or purchased from local market. Distilled III. RESULTS AND DISSCUSSION
water was used in all laboratory process analysis.
B. Enzyme Assay A. Extraction of Polyphenol oxidase crude enzyme
Enzyme activity was measured using a digital Firstly, weight 10 g of frozen tea leaves were washed
photo-colorimeter. Firstly, prepare 0.05M phosphate by using distilled water to remove the surface dirty and
buffer (pH = 7.0, containing 0.1M catechol) 2.8ml and other stains. And then 0.1 M of sodium phosphate
PPO enzyme solutionwas added0.2 ml into a buffer were mixed with 10 g of tea leaves and kept at
tube;absorbance was measured at 400nm immediate 4˚C for pre – cooled. Then rapid centrifugation 12000
lyafter mixing. The one activity unit(U) is defined as, rpm 30minutes took places. After centrifugation, the
under assay conditions, 1min absorbance change samples were leached at 4˚C about 3 hours. Then
caused by the amount of enzyme needed to 0.001 10000 rpm of centrifuge were again 20 minutes at 4˚C.
[8]-[10]. Finally the precipitate was discarded and the
U = (K ÷ V) × 1000 supernatant was obtained for analysis.
K = Absorbance values
V = addedcuvetteenzymesolution volume, ml B. Determination of the Usage of PVP on PPO
Extraction
C. Extraction of Polyphenol Oxidase Polyphenol is mainly substances in tea, non-covalent
The polyphenol oxidase crude enzyme were extract bonds with protein binding and protein aggregation
freshly from tea leaves (Camelia spp..) that collected can easily to make. When the quinones formed from
from PyinOoLwin tea farm by using 0.1M sodium phenolic compounds by the PPO enzyme in oxidation
phosphate buffer. reaction, it reacts non-enzymatically to form color
D. Determination of Optimal PVP Concentration pigments [10].When the enzyme extract from plant
The various concentration of polyvinyl pyrolidone sources that need to give full attention to the impact of
(PVP) from 0.05 % to 0.5% were added into the crude phenolic substances. Thus, cross-linked polyvinyl
polyphenol oxidase enzyme and analyzed by using pyrrolidone (PVPP), polyvinyl pyrrolidone (PVP),
enzyme assay with digital photo-colorimeter [10]. bovine serum albumin and some chemicals used as a
E Determination of Optimal Substrate protective agent. Alves et al., 2003, used PVPP as a
50mM phosphate buffer (pH = 7.0) were dissolved protective agent in the extraction and purification of
with catechol, Gallic acid and hydroquinone with PPO from coffee fruits. Some of Chinese researcher
different concentration of 10mM, 50mM, 100mM and (Liukun, 2013) added polyvinyl pyrrolidone as
then there action substrate was measured by using a protective agent in the study of PPO.Therefore, this
digital photo-colorimeter with enzyme assay[8]-[10]. experiment analyzed the relationship between the
F Determination of Optimal pH of Tea PPO amount of PVP added and the crude enzyme extract.
Tea PPO extracts was dissolved in 50mM of The result showed that 0.1%of PVP added had the
phosphate buffer solution that include 0.1M catechol maximum enzymatic activity 1525 U/ (min*ml) and
within a different pH range 5.5 to 9.0. The enzyme added 0.2 to 0.5 % added PVP had lower activity than
activity was measured by using a digital 0.1%of PVP. According to these analyzing results,
photo-colorimeter with enzyme assay and determined 0.1% PVP added amount was the optimum amount
the activity at different pH [8]-[12]. and if we added more, the enzyme activity decreased
G Determination of pH Stability on Tea PPO and decreased. The detail analyzed results shown in
Configuring a concentration of 50mM, pH range5.5 to Fig.1.
9.0 of a buffer solution, 1 ml of enzyme solution
mixed with 4ml buffer solution and placed at 4 ℃ for
24h, and then measured PPO enzyme activity using
enzyme assay and pH stability of PPO was determined
[8].

H. Determination of Optimal Temperature of Tea

Polyphenol Oxidase
The substrate buffer under the condition of 40-100 ℃
insulation 20min, and then measured by using the
enzyme assay. The enzyme activity was determined
the optimum reaction temperature of PPO [8]-[12].
I. Determination Thermal Stability of Tea PPO Fig. 1 The effect of usage PVP on the PPO extraction

Isolation And Study The Profile Of Polyphenol Oxidase From Myanmar Tea Leaf (CAMELLIA SPP.)

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International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Special Issue-5, Dec.-2015

C. Optimal Substract for PPO Enzyme dropped over 50% loss and the enzyme activity loss
The affinities of enzyme changing depend on the using about 70% between the pH ranges 6.5 – 7.5. The
of substrates. In this experiment, the enzyme activity results are shown in Fig. 4.
was tested with 3 kinds of substrates (catechol, Gallic
acid, and hydroquinone) with different concentration.
According to the analyzing results, the PPO enzyme
from tea had the highest activity with catechol 0.1M
and the lowest activity with Gallic acid that used as a
substrate. And this analysis showed that the 0.1 M
concentration had higher activity than other
concentration (0.05 M and0.01M). Thus PPO had the
greatest affinity towards catechol [2]-[6].The results
were shown in Fig. 2.

Fig.3 Determination of optimal pH

Fig. 2 The analysis of optimal substrate for PPO enzyme

D. Optimizing pH and Stability of Polyphenol

Oxidase Activity
The enzymatic activity of PPO extract was analyzed
between the pH ranges of 5.5 to 9.0 for the Fig. 4 Analysis of the pH stability for PPO enzyme activity
determination of optimum pH by using phosphate
buffer (50mM) containing 0.1M catechol as a E. Determination of Optimal temperature and Stability
substrate. According to the analysis results, pH 7 was The optimum temperature is important for enzyme to
found the optimum pH for the PPO crude extract from achieve maximum activity. Avoiding the proper
tea leaves. As shown in the figure 3, the tea temperature of reaction is an efficient way to inhibit
polyphenol oxidase had good activity in the neutral enzyme activity [6]. Therefore, the effect of
and also in alkaline pH but the activity was sharply temperature on tea PPO activity was measured
declined in the acidic pH. In generally, the PPO between the pH range 40 - 100˚C and the analyzed
activity from vegetable and fruits have the highest results showed in figure 5. As shown in figure 6, the
activity at near neutral but nay vary with the sources of optimum temperature for tea PPO’s activity was 40˚C
enzyme and substrate within a relatively wide range of but 50 ˚C and 60˚C also had similar enzymatic activity
pH [6]-[9]. Queriroz et al., 2012 found that the with 40˚C’s activity. And then, the enzymatic activity
optimum pH for Cashew apple was 6.5. And Unit Unal started to decrease gradually. According to this
et al., 2011 found that the optimal pH was 6.02 in the analysis, the optimum temperature of PPO depends on
study of PPO from tea leaf (Cammeliasinensis). the condition to which the fruit develops, for example,
the in tropical regions, higher temperature may be
Analysis on the pH stability test, the enzymatic required to achieve the maximum activity [6].
activity of tea PPO extracts was measured after 1 day During the analysis the thermal stability of PPO
kept in 4˚C by using colorimeter. According to the enzyme, the enzyme activity was measured in
analysis results, the PPO activity was nearly stable in different temperature range 40˚C-100˚C within the
acidic condition. But the enzymatic activity of PPO different time 5 minutes –60minutes. In this analysis,
extracts were decline near neutral and alkaline pH the differences between the enzyme activities with
condition. The activity of tea PPO was nearly stable different temperature were not much different in
under pH 6.0 and lost under 20% activity. But above 5.After 20 minutes later, the enzyme activity started to
pH 6.0, the enzyme activity showed that the activity decline. Therefore this analysis showed that the

Isolation And Study The Profile Of Polyphenol Oxidase From Myanmar Tea Leaf (CAMELLIA SPP.)

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International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Special Issue-5, Dec.-2015

enzyme didnot have the stability in higher temperature will be decreased. And this study would be helpful to
with long time. The detail analysis results showed in understand the browning properties of tea.
Fig. 6.
ACKNOWLEDGEMENT

The author would like to thank Dr. Kyaw Nyein Aye

and Dr. Bo Bo for their supervising, tireless advices
and their encouragement. And also thanks to Dr. Aung
Ko Ko Oo for his helpful advice and support in
creating favorable working environment at the
beginning of this research.

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Isolation And Study The Profile Of Polyphenol Oxidase From Myanmar Tea Leaf (CAMELLIA SPP.)

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