CHAPTER 3 RESULTS AND DISCUSSION

Pesticide is a molecule that binds to an enzyme and decreases its activity. Since blocking an
enzyme’s activity can kill a pathogen or correct a matabolic imbalance , many drugs are enzyme
inhibitors such as pesticides biochemistry and molecular biology. Pesticides and other
chemicals can enter rain water, food, irrigation water or rivers in many cases, and may be
hazardous for living systems. Many chemical substances including fungicides, pesticides,
drugs and metal ions influence metabolism at very low concentrations by altering enzyme
activities. To detemine the enzyme activity in the pesenc of pesticide, So in the research project
following objectives was designed:
4.1 Effect of pesticide on functional characterization of amylase.
4.2Effect of pesticide on structural properties.
4.3Molecular modelling to observe the binding effect of enzyme and pesticide.
4.1.1Screening of the pesticides effect on enzyme activity:
The pesticides are designed to affect only specific target organisms or processes,they may have
toxic effects by interacting with biotic factors of soil ecosystem. Assay of soil enzymes like
amylase, cellulose, dehydrogenase, invertase, phosphate, urease indicates the importance of
their role in the complement of chemical and microbial anaylsis. The effect of pesticides on
amylase activity is either stimulating or inhibitory depend upon the type of pesticide used.so
determine the enzyme activity different pesticides (Table.4.1.1) are used:
Table 4.1.1 : screening of the pesticide effect on enzyme activity;
Sr Name of the chemical formula %Inhibition
no pesticide

1 Oxadiargyl C15H14Cl2N203 Nil

2 Sulfosulfuron C16H18N607O2 71%

3 Tebuconazole C16H22ClN3O Nil

There are three pesticides oxadiargyl, Sulfosulfuron, Tebuconazole are used to predict the
enzyme inhibition by agar plate method. Hydrolysis of insoluble blue starch polymer by a-
amylase from B. subtilis is shown in Fig.4.1. The substrate in the form of water insoluble cross-
linked and coloured blue starch polymer particles was mixed into warm agar, which after
cooling, resulted in a homogeneous agar-blue starch polymer layer. The size of the hydro-
lyzed large circular area on a given plate corresponds to the activity of a-amylase, with the
smaller areas corresponding to a concentration of pesticide and the larger areas corresponding
to a higher concentration of amylase enzyme. But oxadiargyl and tebuconazole pesticide has
no inhibitory effect on the amylase activity in fig( 4.2 and 4.3).

TEBUCONAZOLE SULFOSULFURON OXADIARGYL

Fig 4.1 showing the no zone of enzyme inhibition with pesticide

4.1.2Effect of pesticide concentration on enzyme activity:
The enzyme activity in the presence of pesticide was determined by standard DNSA method.
Effect of sub lethal concentrations of sulfosulfuron pesticide on the activity of enzyme Amylase
activity. The pesticide blocks the active sites of substrate and enzyme and itself binds with
enzyme, so data revealed that the pesticides reduced the activity of Amylase with increasing
concentrations but maximum reduction was found in the concentration 1000µm i.e.74%. The
results suggested that had great inhibitory action on amylase activity. The order of inhibitory
effect of pesticides on Amylase activity was found as follows -
Table 4.1.2 The activity of alpha amylase in presence of different conc of
pesticide*:
S. No Inhibitors con (µM) OD at 540 nm % of Enzyme % of
activity Inhibition

1 No inhibitors 0.846 100 ---

2 10 0.532 62 38
3 20 0.538 63 36

4 30 0.543 64 36
5 40 0.522 61 38

6 50 0.502 59 40

7 60 0.489 57 46
8 70 0.463 54 48
9 80 0.437 51 48.4

10 90 0.404 47 52
11 100 0.388 45 54

12 200 0.347 41 59

13 400 0.308 36 63

14 600 0.267 31 69
15 800 0.247 29 70

16 1000 (1mM) 0.228 26 74

17 1250 0.227 26 74

*Time of pesticide treatment 30 minutes Temp 50֯ C

4.1.2 Interaction time of pesticide with enzyme

Interaction time of pesticide on the inhibition of the α-amylase by DNSA method was tested at
50֯ C For 30 minutes i.e. 74%. Also, the inhibitory rate affected as the interaction time of
pesticide and enzyme was changed. In addition, the optimum temperature, if the interaction
time varies the rate of inhibition was increased but after optimum time rate of inhibition
becomes constant.

Table 4.1.3 Interaction time of pesticide with enzyme

S. No Incubation time Enzyme activity % Inhibitions

(minutes)
1 1 44 55
2 5 42 58
3 10 74 61
4 15 37 63
5 20 36 64
6 25 31 69
7 30 26 74
8 35 26 74

4.1.4 Effect of substrate concentration on the enzyme activity in presence on pesticide:

Different concentrations of starch from 0.1 ,0.2,0.3 ,0.4 … 1% was taken, 700 µl starch
solution from each concentration 300µl buffer solution, 500µl of pesticide and enzyme was
added in different test tubes. Activity of amylase in presence of pesticide measured by standard
method. If substrate concentration gradually increased, the reaction velocity will increase until
reaches maximum. But addition of pesticide, it will block the competing site of enzyme and
substrate and supress the velocity of reaction.
Table 4.1.4 Activity of amylase with pesticide in different concentrations of substrate.
S. Sub conc (%) Enzyme Activity(IU) Inhibitor activity(IU)
No
1 0.1 0.011 0.005
2 0.2 0.025 0.009
3 0.3 0.031 0.014
4 0.4 0.042 0.027
5 0.5 0.053 0.033
6 0.6 0.061 0.046
 7 0.7 0.075 0.050
8 0.8 0.079 0.059
9 0.9 0.111 0.075
10 1 0.113 0.078
11 1.10 0.125 0.082
12 1.20 0.125 0.082

0.14 0.09
Enzyme Activity (IU)

Inhibitor Activity (IU)

0.12 0.08
0.1 0.07
0.06
0.08 0.05 Inhibitor
0.06 0.04 activity
0.03
0.04 0.02
0.02 0.01
0
0
0 0.5 1 1.5
0 0.5 1 1.5
Sub conc (%)
Sub Conc (%)

Fig4.1 Amylase activity in different Fig 4.2 Inhibitor activity in different

concentration of substrate. Concentration of substrate

4.1.5Effect of pesticides on the kinetic parameters:

It was investigated that, The Michaelis-Menten constant (Kmax) for the hydrolysis of substrate
was found to be Kmax 0.9% and the Vmax, was 0.111 gmol/min/mg protein. The Kmax and
Vmax were both decreased by increased pesticide concentration. From the plots indicated that
the nature of the inhibition was of the pure uncompetitive type with Kmax is 1% and Vmax is
0.078 value estimated. The Kmax decreased by an increased concentration of pesticide.

Table 4.5 Determination of kinetic parameters:

S. No Kinetic parameters Enzyme activity(IU)
No inhibitor With inhibitor
1 Kmax 0.9 0.8
2 Vmax 0.111

3 Kcat
3.6Effect of pesticides on the structural properties:
3.6.1