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Universal Journal of Pharmaceutical Research 2021; 6(6):60-62
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Volume 6, Issue 6, 2021
RESEARCH ARTICLE
CHARACTERIZATION OF PARTIALLY PURIFIED TYROSINASE ISOLATED
FROM BITTER KOLA (GARCINIA KOLA) TYROSINASE
B.O. Itakorode , O.E. Agboola , M.B. Adeboye , C.C. Benedict, K.N. Terkula ,
M.F. Aregbesola, M.O. Ajayi
Department of Chemical Sciences, Oduduwa University Ipetumodu, Ile-Ife, Osun State, Nigeria.
ABSTRACT
Objective: Tyrosinase is a glycosylated, copper-containing oxidase that catalyzes the first two steps of mammalian melanogenesis
as well as enzymatic browning events in damaged fruits during post-harvest handling and processing. Human skin
hyperpigmentation and enzymatic browning in fruits are both undesirable. In this study, the properties and inhibitory effect of
some compounds on bitter kola tyrosinase were investigated.
Methods: Bitter kola tyrosinase was isolated and characterized using standard protocols. Partial purification was carried out on
Sephadex G-100 loaded column chromatography.
Results: Bitter kola tyrosinase was purified with a specific activity of 3.5 U/mg protein, purification fold of 2.4 and a yield of
34%. The optimum pH value was found to be 6.0 while the optimum temperature value for maximum enzyme activity was
observed at 60°C. The enzyme was stable at 40oC for 20 minutes. Metals such as NaCl, KCl, MgCl2 and CaCl2 had inhibitory
effect on the activity; though MgCl2 and CaCl2 had minimal effect. Also, EDTA, β-marcaptoethanol and glutathione greatly
inhibited the enzyme activity at all the tested concentration.
Conclusion: The properties of bitter kola tyrosinase compare very well with the tyrosinase from other sources. Also, the study was
able to establish the inhibitory effect of some compounds and this could be applied in food processing industries.
Keywords: bitter kola, inhibitors, melanin, specific activity, Tyrosinase.
Article Info: Received 2 November 2021; Revised 11 December; Accepted 25 December, Available online 15 January 2022
Cite this articleItakorode BO, Agboola OE, Adeboye MB, Benedict CC, Terkula KN, Aregbesola MF, Ajayi MO, Benedict
CC. Characterization of partially purified tyrosinase isolated from bitter Kola (Garcinia kola) tyrosinase.
Universal Journal of Pharmaceutical Research 2021; 6(6):60-62.
DOI: https://doi.org/-/ujpr.v6i6.700
Address for Correspondence:
BO Itakorode, Department of Chemical Sciences, Oduduwa University Ipetumodu, Ile-Ife, Osun State, Nigeria. Tel-; E-mail:-
INTRODUCTION
Tyrosinase (also called monophenol monooxygenase,
EC 1.14.18.1) is a binuclear copper containing enzyme
that catalyzes the conversion of monophenols
(tyrosine) into o-diphenols followed by the oxidation
of o-diphenol to the
corresponding o-quinone
derivative1,2. It is the only copper-containing enzyme
known to catalyze both of these processes; the related
catechol oxidases only catalyze the second reaction,
which uses o-diphenols as a substrate3,4. For the first
step, tyrosinases attract molecular oxygen as a cosubstrate5. The phenolic ring incorporates one of the
oxygen atoms, while the other is reduced to a water
molecule. The first stage of this catalytic cycle
necessitates the use of a reducing agent to provide two
electrons. These two electrons originate from the
second step of the reaction. The final product of the
tyrosinase-catalyzed reaction is DOPA-quinone, which
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is a precursor for the synthesis of melanin6. Melanins
are pigments that play important roles in the survival of
the organisms ranging from bacteria, plants, to
mammals7-9. In mammals, including humans, melanin
is mostly found in the skin, where it functions in photo
protection against UV radiation10. Plants employ oquinones to modify and harden the protective exterior
layer and as an agent against invasive organisms.
Function also occurs in fruits and potato, and in the
fruit bodies of fungi. Because of the importance of
enzymatic browning in post-harvest physiology and
food technology, numerous fruits and vegetables have
had their plant tyrosinase extracted, purified, and
characterized11. Numerous studies have been conducted
on its role in the change of fruits and vegetables during
processing and storage of processed foods. Although
some tyrosinase inhibitors have been discovered and
reviewed12,13; the need for more inhibitors cannot be
overemphasized. Tyrosinases have been isolated and
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Itakorode et al.,
Universal Journal of Pharmaceutical Research 2021; 6(6):60-62
characterized from different sources. In this study, we
focused on extracting tyrosinase from bitter kola and to
investigate the effect of pH, temperature and the
inhibitory effect of some selected compounds on the
enzyme.
percentage yield of tyrosinase from different sources
have been reported. The results obtained in this study is
similar to the one reported by other researchers. The
activity of bitter kola tyrosinase was found to be
optimum at pH 6.0 while the optimum temperature for
activity was 60oC (Figure 1 and Figure 2).
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Enzyme purification
The partially purified enzyme had a specific activity of
3.50 micromole per mg of protein (U/mg), a
purification fold of 2.4 and a percentage yield of
34.3%. Specific activity, purification fold and
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Activity (U/ml)
-
Figure 1: pH effect on bitter cola tyrosinase
activity.
The result of heat stability study is shown in Figure 3.
Research has shown tyrosinases with L-dopa as
substrate to have maximum activity at different
temperature. Balakrishnan and Kalirajan16, reported an
optimum temperature of 25oC for tubers of
Amorphophallus paeoniifoliu tyrosinase.
0.3
Activity (U/ml)
Bitter kola used in this study was obtained at Akinola
market in Ipetumodu, Ile Ife, Osun State Nigeria. All
used chemicals were provided by Sigma Chemical
Company, St Louis, USA.
Preparation of bitter kola homogenate
The Bitter kola was peeled and 10 g of it was
homogenized in 50 mM phosphate buffer at 4 oC. The
30% homogenate was centrifuged at 10,000×g for 30
min at 4oC in order to obtain clear crude supernatants.
The supernatant obtained was assayed for protein and
tyrosinase activity.
Tyrosinase activity assay
L-3,4-dihydroxyphenylalanine (L-DOPA) was used as
a substrate to assess tyrosinase activity in the partially
purified sample. The modified Lerch and Etlinger14
approach was used. In a spectrophotometer, the change
in absorbance of the assay mixture was monitored. A
final concentration of 50 mM sodium phosphate buffer
pH 6.5, 1 mM L-DOPA, and an aliquot of enzyme
concentration were contained in a 1 ml assay mixture
in a cuvette. The blank was a complete assay mixture
in which the enzyme was replaced with distilled water.
For 2 minutes, absorbance was measured at 475 nm at
10-second intervals, and the initial rate of reaction was
computed as the change in absorbance per minute. One
unit of enzyme activity was defined as the amount of
enzyme that catalyzed the formation of 1 µmole of
product (o-dopaquinone) per minute at 475 nm.
Determination of protein concentration
The protein concentrations in the crude homogenates,
partially purified or purified tyrosinase were
determined as described by Bradford15 using bovine
serum albumin (BSA) as the standard protein.
Effect of pH on activity tyrosinase
At pH levels ranging from 3 to 10, the effect of pH on
the activities of tyrosinase from bitter kola was studied.
At the pH ranges mentioned, the following buffer
systems were used: 50 mM citrate buffer (pH 3.0–6.0);
50 mM phosphate buffer (pH 7–8); 50 M borate buffer
(pH 9.0–10)
Effect of temperature on activity tyrosinase
The effect of temperature on activities of tyrosinase
from bitter kola was carried out by incubating the
reaction mixtures containing 50 mM phosphate buffer,
pH 6.0 and 1.5 mM L-DOPA at temperatures ranging
from 10 to 90oC for 4 min. The residual tyrosinase
activity was plotted against the different temperatures.
0.20
0.2
0.1
0.0
20
30
40
50
60
70
80
90
100
Temperature oC
Figure 2: Effect of Temperature on bitter cola
tyrosinase activity.
Also, Salah et al., 17 reported an optimum temperature
range of 37-40oC for Aspergillus terreus tyrosinase.
The result in this study corroborates the result obtained
in literature. The activity of most plant tyrosinases
peaks at or near neutral pH. The optimum pH for
maximum tyrosinase activity in plants varies,
depending on the extraction method, assay substrates,
and enzyme localization in the plant cell. In this
investigation, the optimal pH was found to be 6.0. The
best pH for potato tyrosinase activity was 6.4, while the
optimum pH for other fruits tyrosinase was 7.0.
Effect of salts on bitter kola tyrosinase activity
The results of the effect of salts show that the activity
of the enzyme was inhibited by NaCl, KCl, MgCl2 and
CaCl2 at high concentration; though the divalent metals
had little effect on the enzyme (Figure 4). Also, the
result of inhibitory effect EDTA, Glutathione and βmercaptoethanol is presented in Figure 6. A gradual
loss of activity was noticed up to the highest
concentration of EDTA (10 mM) that was used. At this
highest concentration, about 50 % activity had been
lost. β-Mercaptoethanol was found to be a potent
inhibitor for the enzyme. At concentration of 8 mM, βMercaptoethanol abolish the activity of the enzyme.
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Itakorode et al.,
Universal Journal of Pharmaceutical Research 2021; 6(6):60-62
AUTHOR’S CONTRIBUTIONS
The manuscript was carried out, written and approved
in collaboration with all authors
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Figure 3: Heat stability study; graph of % residual
activity against incubation time for the
determination of heat stability of tyrosinase.
100
10 mM
5 mM
Residual Activity
-
C
l
l2
K
aC
C
N
M
gC
aC
l2
l
20
Metals
Figure 4: Effect of some selected metal ions on
bitter cola tyrosinase activity.
Concentrations of 10 mM glutathione also had great
inhibitory effect on the enzyme. The result obtained in
this study is in line with what was obtained in previous
studies12,13.
Residual Activity
100
EDTA
beta-MCAP
glutathione
-
4
6
8
10
Concentration (mM)
Figure 5: Inhibitory effect of some compounds on
bitter cola tyrosinase activity.
CONCLUSION
The properties of bitter kola tyrosinase compare very
well with the tyrosinase from other sources. Also, the
study was able to establish the inhibitory effect of some
compounds and this could be of used in food
processing industries.
CONFLICT OF INTEREST
The authors declare that they have no conflict of
interests.
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