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Indian Journal of Chemical Technology
Vol. 13, September 2006, pp. 459-463
Reversible inhibition of polyphenol oxidase from apple using L-cysteine
R N Gacchea*, A M Sheteb, N A Dholea & V S Gholeb
aSchool of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431 606, India
bDepartment of Biochemistry, University of Pune, Pune 411 007, India
*Email: rngacche@rediffmail.com
Received 24 October 2005; revised received 27 June 2006; accepted 7 July 2006
Alternate strategies for prevention of enzymatic browning of fruits and vegetables are required by food industries due
to restrictions and several negative health attributes of sulphites, which are effective inhibitors of enzymatic browning reac-
tion. These reactions result mostly from polyphenol oxidase (PPO). The present study was conducted to evaluate the anti-
oxidant and PPO inhibitory potential of L-cysteine. Studies have revealed that with increase in concentration of L-cysteine
there was corresponding decrease in the rate of browning reaction and the activity of PPO. The kinetic study indicates that
PPO inhibition by L-cysteine is non-competitive. The results suggest that the L-cysteine is a time bound inhibitor of PPO,
since its highest concentration (5 mM) gives protection up to 4 h having no further effect on the rate of browning reaction in
apple juice. The assessment of radical-scavenging ability of L-cysteine demonstrated that it has high affinity towards a sta-
ble free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•).
Keywords: Polyphenol oxiadse, Enzymatic browning, L-Cysteine, Antioxidant
IPC Code: C12N9/00
Undesirable quality changes in fruits and vegetables
during handling, processing and storage is a conse-
quence of oxidative browning mediated by PPO
(EC1.14.18.1). PPO catalyses the oxidation of variety
of phenolic compounds and generates reactive qui-
none which undergoes further reactions and results in
the formation of brown pigments (Fig. 1), a cause for
imparting undesirable sensory properties to raw fruits
and vegetables1. Apples and apple juice are highly
susceptible to oxidative browning due to the high
concentration of polyphenols and PPO2. Due to poten-
tial health risks posed by sulphite usage as an anti-
browning agent, several types of biocompatible and
non-toxic chemicals are used as an alternative to sul-
phites3. Some chemicals act directly as inhibitors of
PPO, other by rendering the medium inadequate for
the development of browning reaction, still some
other act by reacting with the products of the PPO
reaction before these can lead to the formation of dark
pigments. Besides the usual usage of ascorbic acid
and its derivatives4,5 and citric acid possessing the
acidulant and metal chelating property6, array of other
chemicals are recommended for the prevention of en-
zymatic browning. Inorganic halides7 and zinc chlo-
ride have been investigated as an antibrowning
agents, especially when used in combination with
CaCl2, vitamin C and citric acid8. Various sulphated
polysaccharides including carrageenans, amylose
sulphate, xylan sulphate are reported to possess anti-
browning activity for apple juice and diced apple9.
Aromatic carboxylic acids are considered to be better
inhibitors than aliphatic acids, especially those with
an electron-withdrawing group in the ‘4’ position on
the benzene ring10. Also several hydroxylated cou-
marins derivatives are reported to possess antioxidant
potential11. Natural substances like pine apple juice
which contains low MW compound, 3(methylthio)-
propionic acid methyl ester inhibits PPO12. Anacardic
acids cardols and 2-methyl-cardols are also reported
as inhibitors of oxidative browning13. The thiol (–SH)
containing compounds are reported as effective
reducing agents having antibrowning potential14.
Iyergar and McEvily15 have reported that the reducing
agents including sulphydryl-containing compounds
cause chemical reduction of o-quinones to colourless
o-diphenols.
In this investigation it was aimed to determine the
kinetic behaviour of PPO in the presence of
L-cysteine, and effect of L-cysteine as an antibrown-
ing agent and inhibitor of PPO. The affinity of
L-cysteine towards a stable free radical 2,2-diphenyl-
1-picrylhydrazyl (DPPH•) is also demonstrated in or-
der to evaluate its antioxidant potential.
INDIAN J. CHEM. TECHNOL., SEPTEMBER 2006
460
Experimental Procedure
Materials
Apples were purchased from the local market at
Nanded city. L-cysteine and L-DOPA (3,4-dihydroxy
phenyl L-alanine) a substrate for PPO16 were procured
from S. D. Fine Chemicals Ltd. Mumbai while
DPPH• was obtained from Sigma-Aldrich (St. Louis,
MO, USA). Other chemicals were of AR grade.
Kinetics of PPO inhibition using L-cysteine
To realize the kinetic behaviour of PPO, three con-
centrations of L-cysteine (1-3 mM) were used and
compared with the kinetic behaviour of a control set.
Here the enzyme quantity (50 μL, 0.045 mg protein)
and total volume of reaction mixture (3 mL) were
kept constant while substrate (L-DOPA) concentra-
tion was varied form 0.10 to 0.18 μM.
Effect of L-cysteine concentration on rate of browning
reaction
Clear apple juice was prepared by blending chilled
(4°C) and peeled apples (100 g). The mixture was
diluted to 400 mL with distilled water and filtered
through 8 layered muslin cloth. The preparation was
re-filtered through Whatman’s filter paper to obtain
clear juice. Specific precautions were followed during
preparation of apple juice, which include: (I) Proper
peeling of apple skin (to prevent interference with
apple flesh); (II) Immediate immersion of peeled and
cut apples into previously (boiled and then) chilled
water (to minimize browning during process).
Individual concentrations (1-5 mM) of L-cysteine
were prepared from freshly prepared 50 mM stock
solution. Six containers containing 40 mL of juice
were used for studies. To these containers 10 mL of
L-cysteine with different concentrations were added
serially and 6th container was kept as a control
(without L-cysteine). After every hour the content of
the containers were observed spectrophotometrically
at 420 nm.
Isolation and activity measurements of PPO
To study the effect of different concentrations of
L-cysteine on the activity of PPO, a semi-pure
enzyme preparation was used. In brief, 2 × 100 g of
fresh apples (peeled pieces) were blended and
extracted in chilled phosphate buffer (pH 7.0, 20 mM)
to make the final volume 150 mL which was filtered
through 8 layers of cheesecloth. To achieve 50%
saturation of ammonium sulphate, 75 g of ammonium
sulphate was added to 150 mL of filtrate and stirred
continuously till it dissolves completely and then kept
for 2 h at -5°C for enzyme precipitation to occur.
The residue was collected by centrifugation and re-
dissolved in 5 mL of phosphate buffer to which
0.5 mL 100% glycerol was added and stored at –20°C
for further study. The protein determination was
carried out as per the reported method17.
The enzyme assay was carried out as per the
method described elsewhere18 with slight modifica-
tions16. The reaction mixture for activity measure-
Fig. 1⎯Reaction of PPO mediated browning
GACCHE et al.: REVERSIBLE INHIBITION OF POLYPHENOL OXIDASE FROM APPLE USING L-CYSTEINE
461
ments of PPO contained L-DOPA (1 mL, 4 mM),
enzyme solution (0.5 mL, 0.9 mg protein), 1 mL indi-
vidual concentration of L-cysteine and phosphate
buffer (pH 7.0, 20 mM) to a final volume of 3 mL.
One unit of enzyme activity was determined as the
amount of an enzyme which caused an increase in
absorbance of 0.001 min-1 at 470 nm at 25°C and pH
7.0 which corresponds to the formation 0.01 μM of
product (dopachrome).
Determination of reducing activity of different concentrations
of L-cysteine against the stable radical DPPH•
The reducing activity of different concentrations of
L-cysteine (1-5 mM in absolute ethanol) against
DPPH• radical (10-4 M in absolute ethanol) was de-
termined by mixing equal volume (1.5 mL each) of
individual concentration of L-cysteine (1.5 mM) and
DPPH (1 mM) solution. After 20 min the absorbance
at 517 nm was recorded on UV-Visible spectropho-
tometer. The difference in absorbance between indi-
vidual concentrations of L-cysteine and that of the
control was taken as the % reducing activity.
Results
The results of the kinetics of inhibition of PPO
using different concentrations (1-3 mM) of L-cysteine
were plotted as double reciprocal Lineweaver-Burk
plot (Fig. 2). It is clear from the results that the nature
of PPO inhibition by L-cysteine is non-competitive.
The calculated Vmax values for the control and 1-3 mM
concentrations of L-cysteine were 6.53, 4.54, 3.97 and
2.63 μM, respectively with a Ki = 0.28 μM. It is also
clear from the plot (Fig. 2) that the Km value (0.80
μM) remains constant while Vmax decreases (1/V0
increases) with increase in L-cysteine concentration.
The browning reaction was observed up to 4 h after
the preparation of apple juice and the results are
summarized in Fig. 3. It is evident from the results
shown in Fig. 3, that there was significant delay in
browning reaction over a time as compared to control
set. The amount of protein from 100 g apple was es-
timated to be 1.8 mg/mL. The results of the effect of
different concentrations of L-cysteine on PPO activity
(U/mg protein) are presented in Table 1. The results
show that there exists a negative correlation between
different concentrations of L-cysteine and enzyme
activity with a correlation coefficient (γ) = -0.33 indi-
cating that with increase in L-cysteine concentration
there is corresponding decrease in PPO activity.
The studies carried out to determine the DPPH free
radical scavenging activity of L-cysteine are
Fig. 2⎯A double reciprocal Lineweaver-Burk plot showing non-
competitive PPO inhibition by L-cysteine (1-3 mM) with L-Dopa
(100 μL, 0.10-0.18 M), enzyme (50 μL, 0.045 mg protein) in
phosphate buffer (20 mM, pH 7.0) to total volume of 3 mL
Fig. 3⎯Rate of browning in control and treated apple juice with
different concentrations of L-cysteine. The results shown here are
the mean ± Standard deviation (n=2)
Table 1⎯Effect of treatment of different concentrations of
L-cysteine on PPO activity and DPPH• reduction
Inhibitor
(mM)a
Activity
(U/mg)b
% DPPHc
0 968 (±1.41) 0 (±0.0)
1 320 (±1.98) 34.2 (±1.10)
2 291 (±2.12) 48.3 (±0.75)
3 264 (±1.43) 59.1 (±1.14)
4 180 (±1.87) 71.4 (±0.85)
5 136 (±0.98) 82.2 (±0.73)
The results presented herein are the mean ± Standard devia-
tion (n=2) with coefficient of correlation (γ = -0.33) between
a & b and γ = +0.98 between a & c.
INDIAN J. CHEM. TECHNOL., SEPTEMBER 2006
462
expressed as % DPPH scavenging activity. It is clear
from the results summarized in Table 1, that there is a
positive correlation between different concentrations
of L-cysteine and % DPPH scavenging activity with a
correlation coefficient (γ) = +0.98 indicating that
there was corresponding increase in DPPH scaveng-
ing activity with increase in L-cysteine concentra-
tions.
Discussion
PPO inhibitors are broadly classified into two cate-
gories. First category inhibitors are reported to inter-
act with the copper site (present in the active site of
PPO) and second category inhibitors show interfer-
ence with the site for phenolic substances. The former
and latter show competitive and non-competitive
mode of inhibition, respectively19. In the present study
the nature of PPO inhibition by L-cysteine was found
to be non-competitive. This type of inhibition is re-
versible and cannot be overcome by increasing sub-
strate concentration20. The calculated Km value for
PPO was 0.80 µM. Km value is an indicator of the af-
finity of enzyme towards its substrate; the greater the
value of Km, lesser is the affinity21. The binding of –
SH group of L-cysteine to a very specific site of PPO
or PPO-substrate complex may be the probable reason
for PPO inactivation. Alternatively, PPO inhibition
may be due to the interaction of L-cysteine with PPO
generated quinones converting it into a stable colour-
less compounds. Glutathione (reduced form), a mem-
ber of –SH containing compound has been investi-
gated for inhibition of apple PPO. It was reported to
inhibit PPO by non-competitive mode with a Km value
of 6.66 mM and Vmax of 0.29 mM for 5 mM glu-
tathione as compared to control (0.65 mM) 22.
In the present study it was observed that L-cysteine
can be efficiently used as a time bound inhibitor of
PPO since the highest concentration (5 mM) of it,
gives protection up to 4 h having no significant effect
on further browning reaction (Fig. 3). The kinetic
studies of PPO inhibition can be attributed to the rate
of browning reaction wherein with increase in the
concentration of L-cysteine there was corresponding
significant reduction in Vmax values (Fig. 2), the same
holds true for the rate of PPO mediated browning
reaction (Fig. 3). The Vmax for 5 mM concentration of
L-cysteine was calculated to be 2.63 μM as compared
to control (6.53 μM). After 4 h it was observed that
there was no significant antibrowning effect even at
5 mM L-cysteine concentration. Oxidation of
L-cysteine (-SH) by atmospheric oxygen to L-cystine
(-S-S-) may be resulting in reactivation of PPO, which
lead to browning. There exists a negative correlation
(γ = -0.33) between different concentrations of
L-cysteine and activity of PPO suggesting the concen-
tration dependent antibrowning effect of L-cysteine.
The DPPH• assay was carried out to assess the
antioxidant potential of different concentrations of
L-cysteine. This assay is based on the measurement of
the scavenging ability of antioxidant towards the
stable radical DPPH•. The free radical DPPH• is
reduced to corresponding hydrazine when it reacts
with hydrogen donors23. In general on health grounds,
compounds with reducing ability and superoxide
anion scavenging effect would be a good candidate
for inhibition of cycloxygenase: a key enzyme
involved in recruiting inflammation process24. It has
been presented in Table 1, that the % reducing ability
of L-cysteine increases with corresponding increase in
its concentrations with a (γ) = +0.98.
Conclusion
It is concluded from the present studies that
L-cysteine can be efficiently used as a time bound
inhibitor of oxidative browning for a considerable
period of time (4 h). It will be beneficial to minimize
the browning if L-cysteine is used right from the
beginning of the juice making process, and packing
the juice under oxygen free condition will supplement
in further minimizing the brown pigmentation. Anti-
oxidants are important for food stability and foods
rich in antioxidants are known to have positive effects
in humans25. Considering L-cysteine as a natural, non-
toxic and antioxidant bioactive compound its
addition in apple juice will increase the acceptance of
the products such as apple juice, pulp etc. by the
consumers.
Acknowledgements
Authors acknowledge Dr. (Mrs.) Suniti Pathak, Mr.
Bhanu Chander for their critical comments. Thanks
are also due to Ms. Rashmi Bagul and Ms. Susan Ro-
sylyn Abraham for their help in carrying out some
initial experiments. The financial assistance from
UGC, India is gratefully acknowledged.
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