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Name: MD SHAHEEN KABIR Date: 17 November, 2023

Student Id: 2656494 Group: 06
Section: Wednesday

Enzymes

1. Introduction: Enzymes are a class of molecules usually proteins that catalyze a

chemical reaction by increasing the rate of reaction specifically in biological systems.
Some factors contribute to the enzyme activity – temperature, pH, substrate
concentration, enzyme concentration, co-factors and co-enzymes, and inhibitors
(Scanlon et al., 2018) . Every chemical reaction to occur must exceed a minimum
energy which is called activation energy. The enzymes usually decrease the activation
energy of that particular reaction and thus facilitate the reaction rate
(Gardner et al., 2019)
. As the temperature increases the molecules of the enzymes and substrate move
rapidly and increase the rate of collision, but at a very high temperature, the proteins
get denatured and lose their catalytic activity
(Alberts, Bruce; Johnson, A. Lewis, 2014).
Every catalyst has its optimum temperature and pH to work properly. The
ionization state of the residues of amino acids in the active site of an enzyme can
change in response to changes in pH and, therefore can impact catalysis and substrate
binding (Hardin et al., n.d.). Besides, when the concentration of substrate increases, the
enzymes get more substrate to catalyze and can catalyze a large number of substrates
simultaneously thus increasing the rate of reaction. However, at a given point in time,
all enzyme’s active sites get occupied by substrates and there is no empty active site
when the rate stabilizes (Hardin et al., n.d.). Furthermore, inhibitors bind in the active
site of the substrate and interrupt the activities of enzymes (Engel, 2020) . In this
experiment, the main objective is to examine and calculate the impact of temperature,
pH, substrate concentration, and enzyme concentration on the enzyme activity and rate
of reaction.

2. Methods:

2.1 : Detect Starch and Hydrolysis:

1. In the spot plate, 30 circles were drawn for the series of time – 30 minutes.
The spot plate was placed on white paper so that the color change was
observed easily.
2. One drop of iodine was placed in each circle on the spot plate.
3. Using a micropipette, 5 ml (5000 μl ) of starch 1% solution was taken in a
test tube. Then 500 μl of amylase 1% was added to the test tube.
4. Using a pasture pipette, one drop of the starch + amylase solution was
dropped in the first drop of iodine on the spot plate instantly (0 minutes).
5. The color turned to blue indicating that starch was present in the solution
and the reaction was not complete. The procedure 4 was repeated after
each minute interval.
6. After 29 minutes, all 30 circles were filled with starch-amylase mixture.
The color change was recorded in Table 3.1.1
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7. At one point in which the color remains yellow, the color of iodine, it was
assumed that the reaction ended.
2.2 Effect of Temperature on the Rate of Hydrolysis:
1. In three spot plates, 30 circles on each were drawn for a series of time – 30
minutes. The spot plate was placed on a sheet of white paper so that the
color change could be observed easily.
2. One drop of iodine was placed on each circle.
3. 5 ml of starch suspension was taken into three different test tubes and
marked as 4° C, 25° C, and 100° C.
4. The test tube of 100° C was immersed in the water bath, 25° C tube was
placed on the benchtop, and 4° C tube was placed on ice. They were left in
the water bath for 10 minutes.
5. A drop of solution was placed in the negative control to verify the presence
of starch.
6. After 10 minutes, 0.5 ml of amylase was added to the starch-containing test
tubes and mixed by vortexing. A drop of amylase + starch from each tube
was placed in the first circle of each plate 1 instantly.
7. The tubes were then placed in the water bath, benchtop, and ice again.
8. Procedure 2.1 was done in 1-minute intervals.
9. The color change at time intervals was recorded in Table 3.2.1

2.3 Effect of Ph:

2.3.1 In three spot plates, 10 circles on each plate were drawn for a series of
times – 10 minutes. The spot plate was placed on a sheet of white paper so
that the color change could be observed easily.
2.3.2 One drop of iodine was placed on each circle.
2.3.3 5 ml of starch suspension was taken into three different test tubes and
marked as pH 4, pH 7, and pH 11.
2.3.4 A drop of solution and a drop of buffer solution were placed in the
negative control to verify the presence of starch.
2.3.5 0.5 ml of amylase and buffer solution were taken to 3 different test tubes
and mixed by vortexing.
2.3.6 0.5 ml of this buffer + amylase solution was added to those marked test
tubes starting with the pH 4 tube.
2.3.7 Procedure 2.1 was done in 1-minute intervals.
2.3.8 Color changes over minute intervals were recorded in Table 3.3.1

2.4 Effect of Substrate Concentration:

2.4.1 Four test tubes were marked for the following concentrations: 50%, 25%,
10%, and 5%.
2.4.2 In the test tubes, the following starch dilution was prepared:
Dilution (%) Starch (ml) Water (ml)
50 5 5
25 2.5 7.5
10 1 9
5 0.5 9.5

2.4.3 0.1 ml of Amylase was added to each tube and mixed well by vortexing.
2.4.4 Procedure 2.1 was done in 1-minute intervals to determine the time to
complete the reaction for each concentration.
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2.4.5 All data was recorded in Table 3.4.1

2.5 Effect of Enzyme Concentration:

2.5.1 Four test tubes were marked for the following concentrations: 100%, 50%,
20%, and 10%.
2.5.2 In the test tubes, the following starch dilution was prepared:
Dilution (%) Starch (ml) Water (ml)
100 1 0
50 0.5 0.5
20 0.2 0.8
10 0.1 0.9

2.5.3 5 ml of starch solution was added to each tube and mixed well by
vortexing.
2.5.4 Procedure 2.1 was done in 1-minute intervals to determine the time to
complete the reaction for each concentration.
2.5.5 All data was recorded in Table 3.5.1

3. Result: Since amylase catalyzes the breakdown of starch to maltose and glucose,
several factors like temperature, pH, the concentration of substrate and enzymes,
regulators or inhibitors, etc. affect the rate of this catalytic effect. The experiments
done above determined the effect clearly and presented below.

3.1 Enzyme Activity:

Table 3.1.1: Enzyme activity on the hydrolysis of starch. 1% amylase enzyme

was added to 1% starch solution. The iodine spot test was performed at 1-minute
intervals. When the color change was observed, the time interval was given as
hydrolysis time.
Procedure No. Hydrolysis Time
2.1 24 minutes.

As stated in Table 3.1.1, In experiment 2.1, the observed color changed to blue
until 24 minutes from the beginning of the reaction. So, it is exerted that the total
time for hydrolysis with the aid of amylase is 24 minutes.

3.2 Effect of Temperature:

Table 3.2.1: Temperature effect on Enzyme Activity. 1% amylase enzyme was

added to 1% starch solution. Three different test tubes containing amylase + starch
solution were placed in ice, benchtop, and water bath. The iodine spot test was
performed at 1-minute intervals. When the color change was observed, the time
interval was given as hydrolysis time.
Temperature (° C ) Reaction Time (Min) Reaction Rate (1/Min)
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4 >30 minutes 1/30

25 24 minutes 1/24
100 >30 minutes 1/30

Figure 3.2.1: Line graph of Temperature effect on enzyme activity of starch

hydrolysis. X axis is defined in temperature ( ° C ¿ and the Y axis is defined in
Reaction Rate (1/minutes).

As shown in Figure 3.2.1, at low temperatures (4° C ), the reaction time was 30
1
minutes, thus reaction rate was or 0.033 per minute. As the temperature
30
increased to 25° C , the collision of the substrate to enzymes increased, thus the
1
reaction time decreased to 24 minutes with reaction rate of or 0.042 per
24
minute. However, when the temperature increased to 100 ° C , the reaction lasted
more than 30 minutes because, at a higher temperature than the optimum
temperature, the enzyme got denatured and lost catalytic activity.

3.3 Effect of Ph:

Table 3.3.1: pH effect on Enzyme Activity. 1% amylase enzyme was added to

1% starch solution. Three different test tubes containing amylase + starch solution
were added to a buffer of pH 4, 7, and 11 to see the effect of pH on enzyme
reaction rate. The iodine spot test was performed at 1-minute intervals. When the
color change was observed, the time interval was given as hydrolysis time.
pH Reaction Time Reaction Rate
4 1 minute 1
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7 4 minutes 1/4
11 >30 minutes 1/30

Figure 3.3.1: Line graph of pH effect on enzyme activity of starch hydrolysis.

The X axis is defined in pH and the Y axis is defined in Reaction Rate
(1/minutes).

As shown in Figure 3.3.1, at low pH (pH 4) in, an acidic environment, the

reaction time was so low that only 1 minute was needed to complete the reaction.
As the pH increased, the rate of reaction started to decrease and in the basic
environment, at pH 11, the rate of reaction became nearly Zero.

3.4 Effect of Substrate Concentration:

Table 3.4.1: Effect of Substrate Concentration on Enzyme Activity. 1%

amylase enzyme was added to 1% starch solution. Four different test tubes
containing amylase + starch solution with different concentrations of starch were
examined to see the effect of substrate concentration on enzyme reaction rate. The
iodine spot test was performed at 1-minute intervals. When the color change was
observed, the time interval was given as hydrolysis time.
Substrate Reaction Time Reaction Rate
Concentration (%)
50 15 minutes 1/15
25 8 minutes 1/8
10 3 minutes 1/3
5 1 minute 1
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Figure 3.4.1: Line graph showing the effect of substrate concentration on

Enzyme Reaction Rate. The X axis is defined in Substrate Concentration (%)
and the Y axis is defined in Reaction Rate (1/minutes).

From Figure 3.4.1, it is evident that the relation between substrate concentration
and the reaction rate is disproportional. While the concentration of substrate
increased, the rate of reaction decreased.

3.5 Effect of Enzyme Concentration:

Table 3.5.1: Effect of Enzyme Concentration on Enzyme Activity. 1% amylase

enzyme was added to 1% starch solution. Four different test tubes containing
amylase + starch solution with different concentrations of amylase were examined
to see the effect of enzyme concentration on enzyme reaction rate. The iodine spot
test was performed at 1-minute intervals. When the color change was observed,
the time interval was given as hydrolysis time.
Enzyme Concentration Reaction Time Reaction Rate
(%)
100 17 minutes 1/17
50 22 minutes 1/22
20 30 minutes 1/30
10 30 minutes 1/30
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Figure 3.5.1: Line graph showing the effect of enzyme concentration on

Enzyme Reaction Rate. The X axis is defined in Enzyme Concentration (%) and
the Y axis is defined in Reaction Rate (1/minutes).

From Figure 3.5.1, it is evident that the relation between enzyme concentration
and the reaction rate is proportional. Since the concentration of enzyme increased,
the rate of reaction increased.
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4. Discussion: In order to observe the effects of different factors on Enzyme Activity,

we, group 6 performed procedures 2.1, 2.3 in pH 7, 2.4 in 25% concentration, and 2.5
in 50% concentration. The result we got is stated in Table 4.1

Table 4.1: Result of the procedures carried out by group 6.

Procedure Temperature pH Substrate Enzyme Reaction Reaction
Concentration Concentration Time Rate
2.1 >30 min. 1/30
2.2
2.3 7 4 min. 1/4
2.4 25 % 7 min. 1/7
2.5 50 % 27 min. 1/27

Figure 4.1: Fluctuation of our result from the average result for each procedure. In this
bar graph, the bar in color green is indicated as our obtained result and in red bar presents the
average result of the other groups. In the Y axis, reaction time is stated for the convenience of
interpreting.

From Figure 4.1, it is observed that the reaction time of amylase enzyme under
normal conditions (in the graph, “Enzyme Activity”) is surprisingly higher than the
average value of other groups. In the pH procedure, we got exactly the same result as
the average. In the substrate and enzyme concentration procedure, we got a slightly
higher and a slightly lower reaction time respectively than the average The reasons for
not showing the exact value of the average are some errors. These errors include
fluctuations in temperature, pH, contamination, inconsistent mixing, equipment
calibration errors, etc.

The optimum temperature and pH of amylase derived from Aspergillus oryzae is 50

℃ and 5.5 respectively (Bhanja Dey & Banerjee, 2015). They work well within the
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range of 50-60℃ and pH 4 – 7. In higher temperatures, the enzyme molecules move

faster and collide with the substrate thus increasing enzyme activity (Vats, n.d.).
However excess heat can denature the enzyme like more than 100 ℃ . On the other
hand, the ionization state of amino acid residues in the active site of an enzyme is
influenced by pH, which in turn impacts the enzyme's capacity to bind to the
substrate. Since many biological systems have pH ranges between slightly acidic and
neutral, amylases often function optimally in these settings (Vats, n.d.).

If the concentration of starch increases, the enzyme molecule should get more
substrate to catalyze and thus increase the reaction rate. However, it didn’t happen in
the above experiment. A reason for this is that though the concentration of substrate
increased, there was a limited amount of amylase molecule and the active sites were
all occupied by the substrate molecule. So, the reaction rate didn’t increase. However,
when the enzyme concentration increased (Figure 3.5.1), the active site increased and
catalyzed a large number of substrate at one time thus increased the reaction rate.

5. References:
Alberts, Bruce; Johnson, A. Lewis, J. (2014). Molecular Biology of the Cell. 1464.
https://books.google.com/books/about/Molecular_Biology_of_the_Cell.html?
hl=tr&id=2xIwDwAAQBAJ
Bhanja Dey, T., & Banerjee, R. (2015). Purification, biochemical characterization, and application
of α-amylase produced by Aspergillus oryzae IFO-30103. Biocatalysis and Agricultural
Biotechnology, 4(1), 83–90. https://doi.org/10.1016/J.BCAB.2014.10.002
Engel, P. (2020). Enzymes: A Very Short Introduction. Enzymes: A Very Short Introduction.
https://doi.org/10.1093/ACTRADE/9780198824985.001.0001
Gardner, A., Duprez, W., Stauffer, S., Ungu, D. A. K., & Clauson-Kaas, F. (2019). Introduction to
Biological Macromolecules. Labster Virtual Lab Experiments: Basic Biochemistry, 19–41.
https://doi.org/10.1007/978-3-662-58499-6_2
Hardin, J., Harlow, P., London, E. •, New, •, Boston, Y. •, San, •, Toronto, F. •, Dubai, S. •,
Singapore, •, Kong, H., Seoul, T. •, Taipei, •, Cape, D. •, Sao, T. •, Mexico, P. •, Madrid, C. •,
Amsterdam, •, Munich, •, Paris, •, … Authors, A. (n.d.). Becker’s World of the Cell NINTH
EDITION The Plant Cell ©.
Scanlon, M. G., Henrich, A. W., & Whitaker, J. R. (2018). Factors affecting enzyme activity in food
processing. Proteins in Food Processing, Second Edition, 337–365.
https://doi.org/10.1016/B978-0-08-100722-8.00014-0
Vats, S. (n.d.). A laboratory Text book of Biochemistry, Molecular Biology and Microbiology.
Retrieved November 9, 2023, from
https://books.google.com/books/about/A_laboratory_Text_book_of_Biochemistry_M.html?
hl=tr&id=Eh1uCAAAQBAJ