Electrochemical investigations of lipase enzyme activity inhibition by methyl parathion pesticide: Voltammetric studies

Article abstract of DOI:10.1016/j.molliq.2012.12.032

Candida Rugosa Lipase mobilized sensor method was developed for the detection of organophosphorus pesticides like methyl parathion. Here p-Nitrophenyl Acetate was used as a substrate which releases p-Nitrophenol by the enzymatic hydrolysis and gives an an

Full text of DOI:10.1016/j.molliq.2012.12.032

Journal of Molecular Liquids 180 (2013) 26–30
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Electrochemical investigations of lipase enzyme activity inhibition by methyl
parathion pesticide: Voltammetric studies
b,
b
K. Gangadhara Reddy a, G. Madhavi a, , B.E. Kumara Swamy , Sathish Reddy ,
⁎
⁎
A.Vijaya Bhaskar Reddy a, V. Madhavi a
a
Dept. of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India
Dept. of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta-577 451, Shimoga, Karnataka, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2012
Received in revised form 18 December 2012
Accepted 20 December 2012
Available online 10 January 2013
Candida Rugosa Lipase mobilized sensor method was developed for the detection of organophosphorus pesti-
cides like methyl parathion. Here p-Nitrophenyl Acetate was used as a substrate which releases p-Nitrophenol
by the enzymatic hydrolysis and gives an anodic oxidation on electrochemical oxidation peak potential at
0.05 V versus SCE. Methyl parathion is a toxic substance and releases p-Nitrophenol when it is hydrolyzed
and it also has the property of inhibiting the enzymatic activity and this is taken as a basis for the inhibition
studies. The current response under the influence of pH, substrate concentration, enzyme concentration and
time variation effects on the hydrolysis process was studied for the reaction between enzyme and substrate
which releases p-Nitrophenol. A linear calibration for the methyl parathion was obtained in the concentration
range of 10–70 ppb with a correlation coefficient of 0.948 under the optimized conditions by following the incu-
bation time of 25 min. The detection limit was found as 26.32 ppb of methyl parathion with optimal conditions
of 750 μΜ substrate concentration, 125 U of enzyme, pH 7.0, 25 min of hydrolysis time and incubation time
of 25 min and the proposed method has a quantification limit of 87.72 ppb.
Keywords:
Candida Rugosa Lipase
Methyl parathion pesticide
p-Nitrophenol
p-Nitrophenyl Acetate
Voltammetric technique
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
to the tissues of the living organisms. Most of the traditional analytical
methods like High Performance Liquid Chromatography (HPLC), Gas
Organophosphorus (OP) pesticides (herbicides, fungicides, insec-
ticides) are the most toxic compounds and are widely used as pest
controllers in agriculture, medicine and industry. Most of the aquatic
eco-systems are contaminated by these pesticide residues due to
the heavy runoff rain water. The increasing concentrations of organo-
phosphorus pesticide residues are found to be present in many
sampled soils, aquatic ecosystems and waste-water streams. It has
generated the need to understand and evaluate the biological effects
of pollutants on aquatic ecosystems. In this sense, a large number
of studies have used biomarkers as functional tools to evaluate the
toxicity of such compounds for natural populations [1]. Methyl para-
thion is an organophosphorus insecticide (Table 1) and it is widely
used in the agriculture for the control of most of the pest verities.
The mistreatment of these pesticides results in contamination of
fields, crops, aquatic environment and air. Methyl parathion is rela-
tively insoluble in water, and readily soluble in most of the organic
solvents. The residues of these pesticides in air, aquatic environment,
soil and organisms in the environment will influence several chemical
and biological factors. Methyl parathion is readily absorbed via all
routes of exposure (oral, dermal, inhalation) and is rapidly distributed
Chromatography (GC), Liquid Chromatography (LC) coupled to sensi-
tive and specific detectors such as nitrogen–phosphorous detectors
(NPD) [2–4], flame ionization detectors (FID), ultraviolet detectors
(UV) or diode array detector (DAD) [5–7] and mass spectrometry
(MS) [8,9] are used for the quantification of environmental pollutants,
pesticide residues in water, soil and other sources. These sophisticated
techniques are time-consuming processes; require highly trained per-
sonnel, tedious extraction and clean-up procedures prior to instrumen-
tal analysis and therefore are not very suitable for routine analysis.
An electrochemical method of sol–gel immobilization acetylcho-
line esterase enzymatic biosensor method is developed for the deter-
mination of pesticide residues in environmental matrices due to their
good selectivity, sensitivity and rapid response towards pesticides
due to their eco-friendly nature [10]. An immobilizing potentiometric
biosensor [11] was developed by using Candida Rugosa Lipase enzyme
which shows an intrinsic capability to catalyze carboxylic ester bonds
to the corresponding alcohol and acid and the same enzyme was used
for the surface acoustic wave impedance sensor for the determination
of organophosphorus pesticides [12]. p-Nitrophenol is used as a starting
substrate for the synthesis of many fungicides, pesticides and pharma-
ceutical products. In particular p-Nitrophenol is a toxic derivative of
methyl parathion insecticide [13].
Based on electrochemical response of phenolic compounds [14]
towards carbon paste electrode, a simple voltammetric mobilized
⁎
Corresponding authors. Tel.: +91 8282 256225; fax: +91 8282 256255.
E-mail address: kumaraswamy21@yahoo.com (B.E.K. Swamy).
0167-7322/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molliq.2012.12.032

K.G. Reddy et al. / Journal of Molecular Liquids 180 (2013) 26–30
27
Table 1
The structure and molecular formulae of methyl parathion.
Sl. no
Selected organophosphorus
pesticide
Molecular
formula
Chemical structure
01
Methyl parathion
C8H10O5NPS
mono-enzyme sensor for the indirect, sensitive and selective deter-
mination of methyl parathion is developed. In this work a differential
pulse voltammetric lipase enzyme mobilized method is also devel-
oped which can be applied for the determination of methyl parathion
in the environmental samples.
2. Experimental section
Fig. 1. Cyclic voltammograms of in situ generated p-Nitrophenol in 0.1 M phosphate
buffer, pH 7.0 a) blank; b) 750 μM substrate alone; and c) substrate in the presence
of 125 U of enzyme.
2.1. Reagents and chemicals
All chemicals were obtained from commercial sources and used
without further purification. Lipase from Candida Rugosa (EC
3.1.1.3, type VII, ≥700/mg) p-Nitrophenyl Acetate, methyl para-
thion was purchased from Sigma-Aldrich Chemicals. The pesticide
stock solutions were prepared by dissolving in acetone (GR grade
solution). The fine graphite powder was obtained from Merck
chemicals. Silicon oil, acetone (GR grade) was procured from Himedia
chemicals. Phosphate Buffer [0.1 M] was prepared by using 0.1 M
disodium hydrogen phosphate and 0.1 M sodium dihydrogen phos-
phate. All chemicals were of analytical grade and aqueous solutions
were prepared with double distilled water. The enzyme stock solution
was stored at −5 °C and all stock and working solutions were stored
at 5 °C.
absence of enzyme and substrate (blank) the electrode gave no re-
sponse and only a small background current was observed (peak a).
In the presence of only 750 μΜ substrate a little back ground peak
current was observed (peak b). When enzyme was added to 750 μΜ
of substrate after 25 min a relatively large anodic peak current at
a potential of 0.05 V (peak c) was obtained. The p-Nitrophenol mole-
cule readily undergoes electrooxidation. Primarily, it is oxidized to
nitro-phenoxy radical as shown in Eq. (1) according to literature
report [14].
C6H4NO2OH→C6H4NO2O• þ Hþ þ e−
ð1Þ
2.2. Apparatus
This radical intermediate subsequently undergoes polymerization
leading to the formation of a non-sticky thin film on the electrode
surface. For further studies the non-sticky polymeric film electrode
surface was removed by physically smoothing against a tissue paper
[14]. When the concentration of enzyme was gradually increased
from 25 U to 250 U there was a gradual increase in the peak current
response and this response was finally saturated at 125 U of enzyme
concentration where the current values observed were almost con-
stant (Fig. 2) and the optimized enzyme concentration is presented
(Table 2).
Cyclic voltammetric experiments were performed with a CHI
model 660c electrochemical work station with a connection to a per-
sonal computer that was used for electrochemical measurement and
treating of data. A conventional three electrode cell was employed
throughout the experiments, with a bare carbon paste electrode
(homemade cavity of 3.0 mm diameter) as a working electrode, satu-
rated calomel electrode (SCE) as a reference electrode and a platinum
wire as a counter electrode. All the experiments were carried out at
room temperature 25 2 °C.
2.3. Preparation of bare carbon paste electrode
The bare carbon paste electrode was prepared by hand mixing
of 70% graphite powder and 30% silicon oil in an agate mortar to
produce a homogenous carbon paste. The paste was packed into the
cavity of homemade PVC (3 mm in diameter) and then smoothed
on a weighing paper. The electrical contact was provided by copper
wire connected to the paste at the end of the tube [15].
3. Results and discussion
3.1. Cyclic voltammetric studies
The electrochemical behavior of in situ generated p-Nitrophenol
from the hydrolysis of p-Nitrophenyl Acetate substrate in the presence
of Candida Rugosa Lipase enzymatic hydrolysis was examined. Fig. 1
shows a cyclic voltammogram of the p-Nitrophenol in the absence
and presence of 125 units of Lipase enzyme with 750 μΜ substrate
in 0.1 M phosphate buffer (pH 7.0) at a scan rate of 30 mV/s. In the
Fig. 2. Response of increasing concentrations of Lipase enzyme in 0.1 M phosphate
buffer pH 7.0.

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K.G. Reddy et al. / Journal of Molecular Liquids 180 (2013) 26–30
Table 2
Optimized study conditions for Candida Rugosa Lipase enzyme activity.
Sl. no
Name of the optimized parameters
Optimized value
01
02
03
04
Substrate concentration
Enzyme units
Hydrolysis time
Selected pH
750 μΜ
125 U
25 min
7.0 pH
3.2. The effect of scan rate on the peak currents of p-Nitrophenol
The effect of scan rate for p-Nitrophenol was studied at carbon
paste electrode. It showed an increase in the oxidation peak current
with an increase of scan rates from 10 to 400 mVs−1. The graph of
current (Ipa) vs square root of scan rate (ν1/2) was plotted. The result
of the graph obtained is with a good linearity (Fig. 3) which indicates
that the electrode reaction is a diffusion controlled process [16–18].
Fig. 4. The effect of pH on hydrolysis of 750 μM substrate in 0.1 M phosphate buffer.
3.3. The effect of pH on hydrolysis of substrate and oxidation of
p-Nitrophenol
from 125 μΜ to 750 μΜ, and then after that the hydrolysis was con-
tinuously decreased from 750 up to 1750 μΜ. The obtained optimized
concentration of substrate hydrolysis by Lipase enzyme is tabulated
(Table 2).
To establish optimum conditions the effect of pH on 750 μΜ of sub-
strate hydrolysis by mobilized Lipase enzyme is studied in (0.1 M)
phosphate buffer with pH 6.0–8.0. The resulting profile showed the
maximum hydrolysis of substrate by mobilized Lipase enzyme at
pH 7.0 (Fig. 4) [19]. The obtained optimized pH value of substrate
hydrolysis by Lipase enzyme is tabulated (Table 2). The potential dia-
gram was constructed by plotting the graph of anodic peak potential
Epa vs pH of the solution (Fig. 5). The pH dependence of oxidation
peak potential of in situ generated p-Nitrophenol reveals that there
is a potential shift towards positive and the Ep=0.5752+0.071 and
this graph is almost linear with a slope of 71 mV/pH, this behavior is
nearly obeying the Nernst equation for an equal number of electron
and proton transfer reaction [20,21].
3.5. The effect of time on the hydrolysis of substrate
The effect of time variation on p-Nitrophenyl Acetate (substrate)
hydrolysis by 125 U of mobilized Lipase enzyme is studied in 0.1 M
phosphate buffer with 750 μΜ substrate. The time vs substrate
hydrolysis product (Fig. 7) showed a gradual increase in current
from 5 min to 25 min of hydrolysis and this increase was continued
till a plateau level was reached and after that there was no significant
increment in the hydrolysis of substrate up to 1 h. The obtained opti-
mized time for the hydrolysis of the substrate by Lipase enzyme is
presented (Table 2).
Table 2 shows the optimized parameters for Lipase enzyme activity
on p-Nitrophenyl Acetate (substrate) in 0.1 M phosphate buffer solu-
tion. These optimized experimental parameters for Lipase enzyme
were applied for further pesticide inhibition studies.
3.4. The effect of substrate concentration on enzyme catalysis nature
The effect of the substrate concentration on 125 U of Lipase
enzyme in 0.1 M phosphate buffer was investigated. Choosing of a
proper substrate concentration is important for enzyme inhibition
studies. Fig. 6 shows various concentration levels of substrate hydro-
lysis by the enzyme. From this it can be inferred that the hydrolysis
of p-Nitrophenol Acetate to p-Nitrophenol was linearly increased
3.6. Pesticide study
The Candida Rugosa Lipase enzyme was used to carry out inhibi-
tion studies by incubating with pesticide solution up to 25 min to
obtain lower detection limits. Inhibitor was mixed in 1:1 ratio with
Fig. 3. The effect of square root of scan rate on anodic peak current for p-Nitrophenol in
Fig. 5. The effect of pH on anodic peak potential (Epa) of p-Nitrophenol in 0.1 M phos-
0.1 M phosphate buffer of pH 7.0.
phate buffer.

K.G. Reddy et al. / Journal of Molecular Liquids 180 (2013) 26–30
29
Fig. 8. Differential pulse voltammograms of (c) substrate alone, (b) enzyme inhibition
in the presence of pesticide, and (a) complete hydrolysis of substrate in the presence of
enzyme.
Fig. 6. The effect of substrate concentration on enzymatic hydrolysis.
125 U of enzyme stock solution and incubated at a room temperature
of 25 2 °C. To obtain an inhibition plot for methyl parathion the
percentage of inhibition method was studied. The detection was
based on the measurement of initial (Ii) steady state current response
for the product obtained due to complete hydrolysis of liquid phase
mobilized enzyme towards the selected concentration of 750 μΜ
substrate in 0.1 M phosphate buffer solution at (pH 7.0). The 1:1
ratio of enzyme and pesticide solution was incubated for 25 min,
followed by the transfer of the inhibited enzyme into the electro-
chemical cell for the measurement of final (If) steady state current
response in the presence of inhibited enzyme towards hydrolysis
of 750 μΜ substrate. The rate of inhibition (%) and residual enzyme
activity was determined according to the following Eqs. (2) and (3)
[10,22].
pesticide which is evidenced by the reduction in current response.
(Peak a) represents the complete hydrolysis of substrate in the presence
of enzyme.
Calibration plots based on the dependence of the percentage of in-
hibition on concentration are linear (Fig. 9), the detection limit and
limit of quantification values were 26.32 ppb and 87.72 ppb respec-
tively for methyl parathion. Various concentrations ranging from 10
to 70 ppb were tested in terms of their effect on enzyme activity
at different incubation times (5, 10, 15, 20 and 25 min) in pesticide
solutions. When the concentration of methyl parathion increased
the residual enzyme activity of the enzyme was decreased with time
as shown (Fig. 10). Determination of detection limit (DL) and quanti-
fication limit (QL) was carried by using the following formulae (4)
and (5) [23–25].
Inhibition % ðI %Þ ¼ ½ðIi−If Þ=If ꢀ ꢁ 100
ð2Þ
ð3Þ
Residual enzyme activity % ðREA %Þ ¼ ½If =Iiꢀ ꢁ 100
DL ¼ 3 Sb=S
ð4Þ
ð5Þ
Methyl parathion pesticide is known to inhibit the catalytic nature
of lipase enzyme towards substrate therefore toxic reference to test
the lipase enzyme activity was chosen for inhibition studies. Fig. 8
shows the differential pulse voltammogram of substrate alone (peak
c) indicating the presence of little amount of p-Nitrophenol, a starting
substance for the synthesis of p-Nitrophenyl Acetate, (peak b) indi-
cates the inhibition of enzyme catalytic activity in the presence of
QL ¼ 10 Sb=S
where Sb is standard deviation, S is the slope of the working curve, DL
is the detection limit and QL is the quantification limit. Table 3 shows
the various parameters determined for methyl parathion.
Fig. 9. Inhibition plots of methyl parathion after 25 min incubation time with the
Fig. 7. Time variation on enzymatic hydrolysis.
measurement condition in 0.1 M phosphate buffer.

30
K.G. Reddy et al. / Journal of Molecular Liquids 180 (2013) 26–30
rendering it suitable for electroanalysis by enzyme inhibition method.
The proposed detection method is simple, cost effective, eco-friendly
and can be safely used for the determination of methyl parathion in
environmental samples.
Acknowledgment
The authors are thankful to the University Grants Commission,
New Delhi Government of India for providing the financial assistance
through major research project F. no. 39 744/2010 (SR).
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Fig. 10. The effect of incubation time at various inhibitor concentrations on the ac-
tivity of mobilized Candida Rugosa Lipase enzyme in 0.1 M phosphate buffer pH 7.0
for methyl parathion: (a) 10 ppb, (b) 20 ppb, (c) 30 ppb, (d) 40 ppb, (e) 50 ppb,
(f) 60 ppb, and (g) 70 ppb.
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Table 3
The various parameters determined for methyl parathion.
Sl. no
Parameters
Methyl parathion
1
2
3
4
5
6
7
Incubation time (min)
Response time
Linear range (ppb)
Correlation coefficient
Standard deviation
Detection limit (DL) (ppb)
Quantification limit (QL) (ppb)
25 min
05 min
10–70
0.948
12.1607
26.32
87.72
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Sensors 2 (2012) 1–7.
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4. Conclusions
The present study described the preparation of lipase based mobi-
lized electrochemical sensor within the electrochemical cell to deter-
mine the concentration of organophosphorus pesticides. This method
is based on the study of inhibition percentage of lipase enzyme activity.
Electroanalytical investigation of methyl parathion is achieved down to
10 ppb (correlation coefficient=0.948 and slope=1.3862) at pH 7.0.
The plot obtained with concentration of methyl parathion vs inhibition
percentage yields a straight line almost passing through the origin
[25] G. Madhavi, J. Damodar, S.K. Mohan, S.J. Reddy, Bulletin of Electrochemistry
15 (1999) 535–538.