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To cite this article: Hlya Yagar (2004) Some Biochemical Properties of Polyphenol
Oxidase from Celery, Preparative Biochemistry and Biotechnology, 34:4, 387-397,
DOI: 10.1081/PB-200031054
To link to this article: http://dx.doi.org/10.1081/PB-200031054
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ABSTRACT
Polyphenol oxidase (PPO, EC 1.14.18.1) was extracted from celery roots
(Apium graveolens L.) with 0.1 M phosphate buffer, pH 7.0. The PPO was
partially purified by (NH4)2SO4 and dialysis. Substrate specificity experiments were carried out with catechol, pyrogallol, L -DOPA, p-cresol,
resorcinol, and tyrosine. The Km for pyrogallol, catechol, and L -DOPA
were 4.5, 8.3, and 6.2 mM, respectively, at 258C. Data for Vmax/Km
values, which represent catalytic efficiency, show that pyrogallol has
the highest value. The optimum pH and temperature were determined
with catechol, pyrogallol, and L -DOPA. Optimum pH was 7.0 for catechol and L -DOPA, and 7.5 for pyrogallol. Optimum temperatures for
maximum PPO activity were 258C for pyrogallol, 408C for catechol,
and 458C for L -DOPA. Heat inactivation studies showed a decrease in
enzymatic activity at temperatures above 608C. The order of inhibitor
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effectiveness was: L -cysteine . ascorbic acid . glycine . resorcinol .
NaCl.
Key Words: Polyphenol oxidase; Celery; Apium graveolens; Isolation;
Characterisation.
INTRODUCTION
Celery (Apium graveolens L.) is a popular herb and vegetable in Europe
and the Mediterranean region; particularly the roots are often used as a vegetable in Turkey. One of the main problems associated with preserving the
vegetable is the enzymatic browning, which is catalysed by polyphenol
oxidase (PPO). Polyphenoloxidases (EC.1.14.18.1), also referred to as catecholoxidases, are widely distributed in the plant kingdom[1,2] and are known
as the main class of enzymes involved in the browning of damaged fruits
and vegetables.[3] Because the enzymatic browning results in an unpleasant
appearance and concomitant development of off-flavours, it is an economic
problem for producers and consumers.
Polyphenoloxidase is a copper-containing enzyme which catalyses two
reactions. Cresolase reactions catalyse the oxidation of monohydric phenols,
such as tyrosine and o-cresol, to form a hydroxyl group at the ortho position.
Catecholase involves the removal of two hydrogen atoms from an o-diphenol
such as catechol, chlorogenic acid, or 3,4-dihydroxyphenylalanine to form the
corresponding o-quinone. It has been related to enzymatic browning in several
plant tissues, including banana,[4] apple,[5] artichoke,[6] kiwi fruit,[7] edible
burdock,[8] head lettuce,[9] pears,[10,11] palmito,[12] cocoa bean,[13] oil
bean,[14] apricot,[15] quince,[16] Starking apple,[17] cabbage,[18] and tea
leaf.[19]. Little has been reported about celery PPO. We observed that celery
roots have the highest rate of enzymatic browning.
Our objective in this study was to isolate PPO from celery roots and
to study the characteristics of the enzyme at various pHs and temperatures.
Substrate and inhibitor effects were also studied. The enzymatic activity in
the celery products was carried out in the presence of air/oxygen.
EXPERIMENTAL
Isolation of Polyphenoloxidase
Celery roots (Apium graveolens L.) were obtained from the local market
of Edirne, Turkey and were stored at 48C. For the preparation of the crude
389
extract, 100 g of celery was cut quickly into thin slices and homogenised with
100 mL of 0.1 M phosphate buffer, pH 7.0, containing 10 mM ascorbic acid
and 0.1% polyvinylpyrrolidone, in a Waring Blender for 3 min. The homogenate was filtered through glass wool and the filtrate was centrifuged at
20,000 g for 30 min at 48C by using a Sanio MS 60 ultracentrifuge. The
supernatant was brought to 20 80% (NH4)2SO4 saturation with solid
(NH4)2SO4, pH 7.0. The precipitated PPO was separated by centrifugation
at 20,000 g for 30 min. The precipitate was dissolved in a small volume
of 0.1 M phosphate buffer, pH 7.0, and dialysed at 48C in the same buffer
for 6 hr with three changes of buffer during dialysis.
Protein Determination
Protein concentration was determined by the method of Lowry, using
bovine serum albumin as an internal standard.[20]
Enzyme Assay
PPO activity was determined by measuring the increase in absorbance at
420 nm (475 nm for L -DOPA) in a spectrophotometer (Shimadzu UV-160 A)
in the presence of air/oxygen. The reaction mixture contained 0.2 mL of
enzyme solution (5.306 mg protein/mL) and 2.8 mL of 0.02 M substrate
solution in 0.05 M phosphate buffer, pH 7.0, at 258C. The blank sample
contained only 3.0 mL of substrate solution. Reaction velocity was computed
from linear slopes of absorbance time curve.[21] One unit of PPO activity
was defined as the amount of enzyme that caused an increase in absorbance
of 0.001 per min.
Substrate Specificity
PPO activity was determined by using six potential substrates (catechol,
pyrogallol, L -DOPA, p-cresol, resorcinol, and tyrosine). All substrate solutions were prepared at 0.02 M in 0.05 M phosphate buffer, pH 7.0. Activity
assays were performed in duplicate measurements, employing the standard
reaction mixture but with different substrate.
Kms and Vmaxs were calculated with the Lineweaver and Burk plot, with
substrate concentration ranging from 0.002 to 0.01 M in the standard reaction
mixture. Substrates examined included catechol, pyrogallol, and L -DOPA.
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390
Effect of Inhibitors
PPO activity was measured by using five different inhibitors (ascorbic acid,
cysteine, resorcinol, glycine, and sodium chloride) at the different concentrations with catechol as the substrate. The reaction mixture contained 0.02 M
catechol, 0.1 mL of inhibitor solution at the concentrations that were indicated
(Figs. 5 and 6), and 0.2 mL of enzyme solution. Inhibitor and substrate
solutions were prepared in 0.05 M phosphate buffer, pH 7.0. The percentage
of relative inhibition for each compound was compared with that of the control
(100% activity). The rate of the reaction was computed from the linear portion of
the curve absorbancetime, excluding lag-phases when they occurred.
391
Activity
(units/mL)
Km
(mM)
Vmax
(units/mL)
Vmax/Km
290
560
185
NA
NA
NA
8.3
4.5
6.2
400.0
666.6
222.2
48.19
148.13
35.83
Catechol
Pyrogallol
L -DOPA
Tyrosine
Resorcinol
p-Cresole
Note: NA: no activity.
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392
Figure 2.
393
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394
Figure 3.
with the enzyme, have been reported, i.e., chelation of copper at the active site
and reduction of Cu to Cu.
As seen from Fig. 6, the PPO activity decreased as the concentration of
glycine and resorcinol increased. The inhibitor effect of glycine on PPO
activity is stronger than with resorcinol and NaCl. The inhibitory effect
of glycine could be explained in atleast two ways: by reacting with the
o-quinones and by chelating the copper at the active site of PPO.[24] Resorcinol added to concentrations as high as 100 mM was able to inhibit celery PPO
about 50%. Resorcinol resembles the structure of the substrates for PPO; these
types of inhibitors are typically competitive.
The activity of celery PPO decreased 10% for celery PPO, when using
0.8 M sodium chloride. No additional tests were done at the other NaCl concentrations because of the low inhibition achieved at the high concentration.
395
Figure 4. Thermal stability profile of celery PPO. The enzyme solution was incubated
for various time intervals (5 30 min) at the specified temperature (25 808C) and
rapidly cooled. The activity was measured at 258C, was taken as 100%, and activities
which were measured (35 808C) were compared with the activity measured at 258C.
Figure 5.
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396
Figure 6.
From the data, it may be concluded that the inhibitory effect of NaCl was not
satisfactory. It is believed that the action of NaCl is due to the formation of a
complex between the halide ion and copper in the enzyme. Several works have
been reported on the effect of NaCl on PPO, in which a high inhibitor concentration was necessary to achieve inhibition. It was shown that the inhibition of
eggplant by 0.99 M sodium chloride was 90%, and inhibition from avocado by
0.8 M sodium chloride was 37%.[25]
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Received April 24, 2004
Accepted May 31, 2004
Manuscript 7401