The tomato leafminer Tuta absoluta (Meyrick) is one of the most important
pests of tomato crops (De Medeiros et al., 2005).
Many literatures were cited and deal with control of T.absoluta out
breaks (Antonio et al., 2011). However, studies
concerned with the pest enzyme systems such that of phenoloxidase are relatively
The phenoloxidase systems, which in insects leads to sclerotization and tanning
of the cuticle and acts as a defensive mechanism against pathogens has been
studied in several species (Ishaaya, 1972; Duvic
and Brehelin, 1998; Mullen et al., 2004;
Shelby and Poham, 2006; Amin and
Azazy, 2008; Dorrah, 2009). The phenoloxidases
system consists of two types of phenoloxidases: Ortho-diphenoloxidase (tyrosinase,
phenoloxidase, catecholoxidase, EC 220.127.116.11) and laccases (EC 18.104.22.168). The
former enzymes are very sensitive towords inhibitors, such as thiourea and able
to oxidize o-diphenols to o-quinones and being inactive towards p-diphenols.
The laccases will oxidize both o-and p-diphenols and they are insensitive towards
thioureas (Andersen, 1989).
Reviews about T. absoluta phenoloxidase system, to the best of our knowledge,
is lacking, so this report aimed to study some kinetic properties of phenoloxidases
in T. absoluta larvae.
MATERIALS AND METHODS
Insects: T. absoluta larvae were a laboratory strain maintained
in plant protection research institute, Dokki, Giza, Egypt. When the insects
were brought into the analysis laboratory as 4th larval instar, they were frozen
at -20°C until used for biochemical assay. Storage for 1 month was possible
without significant loss of the phenoloxidse activity.
Chemicals: The substrate; catechol and bovine serum albumin were purchased
from sigma chemical company (St.Louis). Methanol, ethanol and acetone were from
Fluka chemie Gmb (Switzerland). Chemicals used for preparing buffers were purchased
from local companies and were of high quality.
Apparatus: Insects were homogenized for biochemical analyses in a chilled
glass Teflon tissue homogenizer (ST-2 Mechanic-preczyina, Poland).
Double beam ultraviolet/visible spectrophotometer (spectronic 1201, Milton
Royco., USA) was used to measure absorbance of colored substances or metabolic
Preparation of insects for analysis: The larvae were homogenized in
distilled water (50 mg 1 mL-1).
Homogenates were centrifuged at 8000 r.p.m for 15 min at 5°C in a refrigerated
centrifuge. The deposits were discarded and the supernatants were kept in a
deep freezer till use.
Kinetic properties of phenoloxidases system: Phenoloxidase activity
was determined with modification of the method described by Ishaaya
(1971). The reaction mixture consisted of 0.5 mL phosphate buffer (0.1 M),
200 μL of catechol solutions as substrate and 200 μL of the sample.
Effect of pH, substrate concentration and reaction temperature was determined
to detect the optimum conditions of the reaction.
Prior to the initiation of the reaction, the substrate and other ingredients
were separately incubated at the optimum temperature of the reaction. The absorbance
of phenoloxidase activity was recorded for 10 min at 1 min interval against
sample blank as the zero adjustment at 405 nm. Phenoloxidase activity was expressed
as ΔO.D. unitsx103 min-1 mg protein-1.
Total protein of the sample was determined. in each sample by the method of
Bradford (1976) using bovine serum albumin as standard.
Activation and inhibition of phenoloxidases: The effect of various organic
solvents such as methanol, ethanol and acetone (Absolute, HPLC grade) on phenoloxidase
activity was determined. This to elucidate some characteristic properties of
the tomato leafminer phenoloxidase and to detect some potent inhibitors of this
Serial concentration of the solvents were prepared, then 200 μL of the
tested solvent were added to 200 μL of the sample and incubated for 5 min
at the optimum temperature of the reaction. The reaction was initiated by adding
the substrate solution and preceded at the optimum conditions which were found
experimentally. The results were compared to reaction mixture containing 200
μL of Δ H2O instead of solvent solution.
Data analysis: All obtained values wore pooled from Triplicate. Using
costat statistical software (Cohort software, Brekley), means and standard deviation
were obtained and data in Table 1 were analysed by completely
randomized one way ANOVA. The means were separated using the Duncan,s
multiple range test (p<0.01).
RESULTS AND DISCUSSION
Optimum condition for phenoloxidase activity: Effect of pH, temperature,
time and substrate concentration on T. absoluta phenoloxidase
system-catalysed reactions was determined to detect some kinetic properties
of such enzymes. Therefore, optimum conditions were determined for each factor
separately, all other factors being at the optimum.
The phenoloxidases system activity towards catechol was studied at seven pH
values ranging from 4-8 (Fig. 1) The enzyme activity increased
sharply between pH 5 and 5.5 (optimal pH 5) and began to decline reaching to
the lowest value at pH 8.
Effect of temperature on the oxidation of catechol by phenoloxidase was studied
at temperatures ranged between 20 and 55°C (Fig. 2). The
activity increased gradually from 15°C to the optimal temperature; 35°C
at 40 and 50°C, the activity decreased by 11.63 and 37.3%, respectively,
as compared to that of the optimal temperature. This indicates that the enzymes
is somewhat thermostable. However, it losts its most activity at 55°C where
the activity decreased by 60.5% less than the activity at 35°C.
||Activation and inhibition of phenoloxidase system from T.
|Data are presented as the Mean±SD, Values with different
letter are significantly different at p<0.01 using Duncans multiple
||Effect of pH of reaction on phenoloxidase activity in T.
absoluta larvae, reaction time was 2 min at 35°C, Each point represents
the mean of three determinations vertical bars indicate standard deviation
of the mean
||Effect of temperature of reaction on phenoloxidase activity
in T. absoluta larvae, Reaction time was 2 min at pH 5.5, Each point
represents the mean of three determinations vertical bars indicate standard
deviation of the mean
||Reaction time of phenoloxidase activity in T. absoluta
larvae, Temperature of the reaction was 35°C, pH was 5.5, Each point
represents the mean of three determinations vertical bars indicate standard
deviation of the mean
Ten minutes were allowed for phenoloxidase reaction to determine the suitable
time allowed for phenoloxidase reaction (Fig. 3).
A period of only 2 min for tomato leafminer phenoloxidase activity was found
to fit well whithin the linear part of the enzyme activity curve.
Effect of substrate concentration on the phenoloxidase activity was studied
by measuring the activity at seven concentrations ranged between 3.6x10-8
and 10-2 M (Fig. 4). The oxidation had linear
dependence on the substrate concentration up to 10-4, where the peak
is reached. The reaction rate decreased as the concentration of the substrate
was further increased, indicating substrate inhibition (Bell
and Bell, 1988).
Activation and inhibition of phenoloxidase system: Some investigators
have shown that various organic solvents such as acetone, ethanol or methanol
can induce phenol oxidase activity (Preston and Taybr,
||Effect of substrate concentration of oxidation of catechol
by phenoloxidase from T. absoluta larvae, Each point represents the
mean of three determinations vertical bars indicate standard deviation of
||Double reciprocal (Lineweaver-Burk) plot of 1/V versus 1/(S)
for reaction catalyzed by phenoloxidases for T. absoluta larvae at
pH 5.5, 35°C
Table 1 indicate that enzyme pretreatment with methanol strongly
enhanced phenoloxidase activity. The efficiency of activation depends on the
methanol concentration in the reaction mixture. An enzyme treatment with 5%
methanol has the highest reaction rate of about 1.34 folds of the untreated
Inhibition of phenol oxidase activity occurred when methanol concentration
was above 5%, reaching to 40% decrease in activity when the concentration was
20%. Ethanol or acetone showed no stimulatory effect at any of the concentration
tested. A significant inhibition of enzymatic activity was obtained with ethanol
at concentrations above 5% and of acetone above 2.5%. However, ethanol caused
more inhibition than acetone when the concentration reached to 20%.
Michaelis-menten kinetics of phenoloxidase system: The kinetics of the
phenoloxidases from the tomato leafminer, T. absouta larvae were detected.
Maximum velocity of the reaction (Vmax) and substrate concentration
(Km) where the velocity of the reaction is one half of maximum velocity
were determined using Lineweaver-Burk plot (Fig. 5) When the
linear reciprocal plot is extrapolated, it intersects the negative portion of
the abscissa at -77 mM, which equal to 1/km. Thus, km
of reaction catalysed by phenoloxidases from T. absoluta larvae was 12.98x10-6
M and Vmax was 0.862 O.D. units/min/mg proteins.
The optimal temperature (35°C) of T. absoluta phenoloxidase activity
is usual, as compared with those in other insect. A temperature of 40°C
for phenol oxidase obtained from the California red scale and of about 37°C
for enzyme activity from the Florida red scale, was found to be the optimum
for enzymatic activity (Ishaaya, 1971). Takuji
et al. (1986) found that latent phenoloxidase from Musca domestica
was stable at temperature between 0 and 40°C. However, it was fairly unstable
at temperatures higher than 50°C and lost 80% of its activity at 60°C
. In the present study, the larval phenoloxidases lost 60.5% of its activity
as compared to that at the optimal temperature. In comparison to other enzymes
such as esterases, phenoloxidases considered to be less thermostable. Zhu
and Brindley (1990) found that in periplaneta Americana and Lymantria dispar,
the optimal temperature range 50-55°C has been reported for esterases. Moreover,
a temperature up to 100°C for 30 min without losing any esterolytic activity
was found in Triatoma infestans (De Malkenson
et al., 1984).
Study on effect of pH on phenoloxidase activity showed that the optimal pH
of tomato leafminer phenoloxidases was 5.5. This means that T. absoluta
phenoloxidase tend to be activated in acidic medium.
Many of phenoloxidases of other insect species had a slight acidic or neutral
pH, for example the optimal pH of Hyalophora cecropia equals to 6.5 (Andersson
et al., 1998); Pieris rapae, pH7 (Xue
et al., 2006); Parasarcophaga surcoufi, pH 6.2 and 6.6 (Ayaad
et al., 2001). However, there are some insects which have phenoloxidases
that act in acidic pH such as California and Florida red scales, pH 5.5 (Ishaaya,
Some investigators have shown that various organic solvents, can activate in
some cases insect phenoloxidases. Inactivation of Calliphora phenoloxidase
by alchols or some proteolytic enzymes such as subtilisin has been reported
previously (Cottrell, 1962). The present study showed
that methanol up to 5% in the reaction mixture activated phenol oxidases from
tomato leafminer. It was suggested that phenoloxidase activation by some solvents
results from aggregation of subunits which form the active enzyme (Mitchell
and Webber, 1965). In addition to solvents, acids such as kojic acid might
act as a specific inhibitor of phenoloxidase (Shelby and
Poham, 2006). The inhibition of phenoloxidases is of a significant importance
since it might lead to suppression of insect immunity. Amin
and Azazy (2008) found that inhibition of the red palm weevil phenoloxidase
by phenylthiourea led the weevil to be susceptible to infection by nematodes.
The Michaelis-menten kinetic constant (Km) of the phenoloxidases
from T. absoluta larvae is relatively low (12.98x10-6), compared
with those from other insect species:for example, Km of parasarcophaga
hertipes larvae is 4.3x10-4 M (Dorrah, 2009).
The small Km of tomato leafminer phenoloxidases indicates that they
can hydrolyze catechol efficiently, even at very low concentration.
Finally, 1 mL of the reaction mixture consists of about 5 μg sample protein
and 0.1 mM catechol in 0.1 M phosphate buffer (pH 5.5) at 25°C for 2 min,
represents the optimum condition for T. absoluta phenoloxidases system