The Effect Of Temperature On Amylase Activity
Introduction
The purpose of this experiment is to investigate if temperature will affect the
amount of starch broken down as enzyme activity can change by different
temperature. This is because as temperature rises the rate of chemical reactions
increases due to the temperature increases the rate of the molecules’ motion. More
interactions will be existed between an enzyme and its substrate.
The enzyme used in this lab exercise is amylase, which is commonly found in saliva
and germinating seeds, catalyzes the breakdown of starch. It also reacts quickly
when heat is present during the process of it reaction. However, if the temperature
is higher than the optimum point, enzymes can be denatured and they can no longer
bind to a substrate and catalyze reactions. My hypothesis is therefore the amylase
activity would increase as the temperature rise, until a certain high temperature at
which the amylase would denature and be non-functional.
In this experiment, I will observe the activity of amylase by using iodine as iodine
reacts with starch to form a dark brown/purple color. After adding in iodine, when
amylase breaking down starch, less and less starch will be present and the color of
this solution will become lighter and lighter.
Equipments
• 0.1% Amylase 0.5 ml
• 1% Starch 5ml
• 10 Test tubes and rack
• Iodine solution
• Syringes
• Marker pen
• Electronic water bath set at 20,
• Dropper
30, 40, 50, 60 and 70ºC with
thermometers
• Thermometer
• Beakers
• Spot-plates
• Stopwatch
Controlled variables
• Same temperature of amylase and starch solution
This can ensure that amylase can react with starch solution in the same
temperature, a more accurate result can be received
• Same amount of amylase and starch solution in each time of the experiment
Fewer variables will be occurred as the solution was fixed in a certain volume,
which will lead to a reliable result
• Water bath that letting the temperature stay constant
The stable temperature can ensure a stable reaction in the amylase and starch
mixture, a more reliable result will be introduce
Method
1 Use a syringe to pump amylase into one test tube and starch solution
into another. 2 Place the two tubes to equilibrate for 5 min.
3 Mix the amylase and iodine solution together and return the test tube to the
water bath. Start the stopwatch immediately for 5 min.
4 Use droppers to drop 3 drops of iodine in each column of the
spot-plate
5 Use droppers to drop 1 drop of the mixture in a column
6 Time the time taken for the iodine
become light
6 Repeat this procedure for the other
temperatures.
The color change is going to be observed by using spot-plates as the diagram below.
Temperature
(()
1
20
Time taken for starch to be
digested (seconds+/- 1) Average
2
3
time
taken
Activity rate
30
40
50
60
70
I will take 3 repeats to gain reliable data.
The
rate
will
be
calculated
by
1/
average
time
taken.
Uncertainties
• There is insoluble particles are found in the spot-plate
Those particles may contain competitive inhibitors that may reduce amylases’
activity, affecting my results by decreasing the rate of reaction.
• Unknown substances in the syringes
Those substances may vary the concentration of the solution which pumped in, the
solution may be having a bit diluted, and so my result may have a bit varied.
A result table to show the activity rate of amylase
Temperature
(°C)
20
l
Time taken for starch to be
digested
2 (seconds+/- l)3
>60
0
>60
0
255
>600
Average
time
l95 *¹ >600 >600taken
Activity
rate
0.00l
7
30
5l0
>60
570
0.00l
0
8
40
l90
2l7
220.67
0.004
5
50
l30
l09
ll2
ll7
0.008
5
60
l87
l52
l47
l62
0.006
2
70
24l
202
l96
2l3
0.004
7 is an
*¹ In the third repeat first result of the time taken for starch to be digested, there
anomalies which the time taken is l95 seconds. As it is very different compare with
the first and second repeats, I redo this repeat.
The graph above support the hypothesis of my experiment (the reaction rate will
increase as temperature increased, until a certain point at which the amylase
would denature and no longer working) as the amount of time for the amylase to
digest all the starch at 20°C was more than 600 seconds and the rate is 0.0017, and
then the temperature was increased to 50°C the time decreased to 117 seconds
and the rate is 0.0085, this showing the reaction was occurring at a faster rate
when the temperature is at 50°C which show that 50°C is the optimum
temperature of this amylase . However, when the temperature rose to 60°C the
enzyme apparently denatured as we can see that the rate decreased.
Conclusion
In conclusion this experiment further confirmed that enzymes do increase/decrease
their rate of reaction due to change in temperature. Each enzyme has its own
individual optimum temperature, therefore if they are removed from these
environments they will not function as well or possibly denature if it the temperature
is too high. Although, if an enzyme’s optimal temperature is maintained then the
enzymes will catalyze reactions at optimum rates.
The purpose of this experiment is to observe if the different temperatures affect the
amount of starch broken down. My results had supported my hypothesis which is
the amylase activity will increase as the temperature rise until a certain high
temperature at which the amylase will denature and be non-functional.
Since enzymes are proteins, their secondary and tertiary structures will be easily
affected by temperature, plus enzyme activity is closely related to the structure of
an enzyme, therefore, changes in their secondary or tertiary structure will lead to
changes in enzyme activity. Heat allows enzymes to speed up the process. However,
if more heat is added on their optimum temperature, those enzymes will soon be
loss their function and become denatured. Iodine forms dark brown/purple color
with starch. This is due to amylase undergoes a process which broken down starch
into glucose, the dark brown/purple color of the iodine solution and so disappears
and form a lighter color. Therefore, loss of the dark brown/purple color can be used
to measure of the extent of starch.
Evaluation
From the irrelevant results, I found that limitations either in the environment or the
apparatus that I used had played an important role on affecting the results.
• use pipettes instead of syringes
Using pipettes are a more accurate way to measure a certain amount of liquid than
using syringes, this is because pipettes have smaller diameter than syringes which
can reduce the errors. Therefore, my result may not be the most accurate but still
can show a general pattern.
• equilibrate iodine solution as well
As I only placed amylase and starch solution into the water bath to equilibrate but
not iodine solution, the temperature differences of iodine solution may lower down
the temperature of amylase-starch- solution, the enzyme activity may lowered
down. This may vary my result and make it less accurate. To improve, I will
equilibrate iodine solution as well. Therefore, every solution will be at the same
emperature to give out more reliable result.