Title: Investigating Enzyme Activity
Aim: To investigate the effect of temperature on
enzymatic rate of reaction
Enzyme molecular structure is affected by
temperature. To assess the effect of temperature
on enzyme activity, or reaction rate, the following
experiment is carried out. In this experiment
samples of amylase – a starch digesting enzyme –
are exposed to various temperatures for 5
minutes. The time required for each sample to
digest the same quantity of starch at the same
temperature is then estimated by using iodine
solution (which turns blue black in the presence of
starch) as an indicator. Try to make the method as
quantitative as possible so that you can say how
much faster the reaction occurs at one
temperature than another.
Requirements
 Test tube rack (2)
 Test tubes (10)
 Labels
 Stopclock
 Syringes (10cm³)
 White tile
 Glass rods
 Water baths maintained at 25°C, 40°C, 60°C and
100°C
 Thermometers
 Beakers
 Amylase solution
 Starch solution
 Iodine solution
 Ice
Procedure
1. Label 5 test tubes as follows: - Room
temperature (measure it!), 25°C, 40°C,
60°C and 100°C
2. Add 5cm³ of amylase solution to each test
tube.
3. Label an additional 5 test tubes as follows: S room temperature (measure it!), S25°C,
S40°C, S60°C and S100°C. Add 5cm³ of
starch to these test tubes.
You should now have a total of 10 test tubes in
front you, 5 with amylase solution and 5 with
starch solution.
4.
Keep the two room temperature test tubes
at room temperature, and place the other
test tubes into
the appropriate water
baths for exactly 5 minutes.
5. During this time, prepare a results sheet/
table with time as the first column, and an
additional 5 columns for each of the
temperatures.
Lower Six Biology – 2008
Make sure the starch and amylase solutions both
reach the temperature of the water baths they were
in before you mix them.
6. Starting with your test tubes at the lowest
temperature, add the 5cm³ of starch solution
to the appropriately labelled tube with
amylase solution and mix the resulting
solutions with a clean glass rod.
7. At intervals of 30 seconds (starting at 0
seconds), test the mixture for the presence of
starch by withdrawing one drop of the starchenzyme mixture, place it on a white tile, and
then add one drop of iodine solution.
8. Repeat steps 6 and 7 for each temperature.
Take care to use a separate glass rod for each
tube and a different one for the iodine solution.
9. Carefully record each result as it is obtained in
terms of a consistent colour scheme. Do not
continue your observations for longer than 10
minutes (600 seconds).
10. In a separate (2nd) summary table record how
long it took for the blue-black colour to cease
to be obtained when the iodine solution was
added to each reaction mixture, and convert
this time to a rate. (Note: you will have to
decide what colour represents the end point of
the reaction).
11. Plot a graph of the time taken to reach the
endpoint (as a rate) versus temperature, and
describe it.
For Consideration:
1. What are the components of starch and how do
they react with iodine in potassium iodide solution?
2. When starch is broken down by amylase, what
are the end point products?
3. In your experiment what was the colour of the
endpoint of the reaction that you observed?
4. Suggest how you might relate the colour of the
iodine to the proportion of starch which has been
broken down.
5. Why was amylase and starch kept separate
while being allowed to reach the same temperature,
before mixing?
6. On the basis of the data collected, describe and
compare the rates of amylase catalysed reaction at
different temperatures. (i.e. the effect of temperature
on rate of reaction - use your graph to aid you).
7. Give two reasons why the rate of reaction
increases with temperature up to a point.
8. How adequate is the colour change of iodine
solution as an indicator of endpoint of the reaction?
9. How
could
you
make
the
endpoint
measurements more precise?
Skills to be assessed: ORR and M&M
Effect of Temperature on Enzymatic Rate of Reaction: SKILLS ASSESSED
A: Observation, Recording and Reporting
Table 1 Observation of colour of iodine as an indication of breakdown of starch by amylase at 5 different
temperatures.
Time (seconds) Temperature (°C)
60
100
25
Room temp. (28)
40
0
30
60….
600
Table 2 Rate of hydrolysis of starch by amylase at different temperatures
Mean time taken for complete
Rate of reaction (min-1)
Temperature (°C)
hydrolysis of starch* (min)
25
28 (room temperature)
40
60
100
* Complete hydrolysis should be when the iodine does not produce any further colour change with starch/amylase mixture – it stays yellow
brown, or if time does not permit, it stays red violet.
Graph should have
 Title and Scale
 Labelled x axis (temperature °C) and y axis (rate of reaction min-1) [with units]
 Points with either a circled dot or an x
 Plotted points connected by straight lines
B: Manipulation and Measurement
Criteria
1. Working in groups - organisation
 Divide up and designate tasks within the group
 Read procedure and follow instructions successfully
 Performed at least 1 task with the group, not just standing and watching
2. Set up and maintain water bath at appropriate temperature, by manipulating
heat from Bunsen burner.
3. Use of thermometer - measurement
 Interpreting accuracy of scale,
 Examines thermometer first and does not require teacher’s assistance to
read thermometer
 Take reading while bulb is immersed, and at eye level
4. Use of thermometer – handling apparatus
 Immerse bulb completely in liquid
 Bulb does not make contact with container
 Stir liquid for even distribution of heat
5. Use of solutions
 Adding starch and amylase to test tubes to place in water bath before
mixing
 Adding iodine to starch/amylase mixture after mixing starch and amylase
 Economical use of solutions – does not waste iodine
 Cleans tile properly for each temperature to be tested
6. Work area (start and end)
 Arrange apparatus (test tubes, test tube holders and Bunsen burner) for
ease of use and in an uncluttered manner.
 Wash up all apparatus and wipe work area at the end of exercise.
TOTAL
Lower Six Biology – 2008
Marks
2
1
2
2
3
2
12