differences.
Outline method
The presence of starch can be detected by a solution of iodine
dissolved in potassium iodide. This reddish-brown solution turns dark
UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 27
blue-black when starch is present. I know from previous experiments
that this is an obvious result but it is not always easy to time the
exact point when all the blue-black colour has disappeared.
Key variables
Starch concentration must be the same for all experiments
Amylase concentration will be the same for all experiments
Amylase concentration will be kept the same for all experiments
The activity of each enzyme after heat treatment will be tested at
the same temperature.
The same concentration and size of drop of iodine solution will be
used each time
Buffer solution will be used to eliminate small variations in pH
The end point will be judged against the original iodine solution
I will take 3 test tubes each containing 2 cm3 of 1% starch solution
made up in pH 7.2 buffer solution to prevent small pH changes
affecting my results and place in a water bath at 250C. I will then
take 3 test tubes containing 2cm3 of the selected amylase solution
and place it in a water bath for temperature treatment (between
400C and 700C) I will then return the treated amylase to the 250C
water bath to equilibrate at this temperature. When the amylase has
cooled to 250C I will mix pour each tube into a separate tube of
starch. As soon as I do this I will start the stop watch and each
minute I will take a small sample with a pipette and test it by mixing
with a drop of iodine solution on a white tile. I will continue to do
this until there is no change in the colour of iodine on the tile.
I intend to test each amylase at four different temperatures and
three different times (5, 10, 15 mins). I will repeat each reading as
many times as the time available for the experiment allows.
As a control experiment I intend to test the enzymes without any
heat treatment so that I can compare this with the effects of
different temperatures.
28 UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide
Risk Assessment
Amylase Enzymes
These enzymes are bought in powdered form. All enzymes have
biological activity and need to be treated with care.
In powdered form
Handle carefully USE EYE PROTECTION. Avoid inhaling powder
As 2% solutions
USE EYE PROTECTION
Wash any spillage from skin with water as soon as possible
Low risk
Iodine in potassium iodide
Can be toxic and irritant USE EYE PROTECTION
Wash any spillage from skin with water
Use only small drops
Rinse dropping tiles carefully under running water.
Use only in clearly labelled dropping bottles
Starch solution
Very low risk
Keep in clearly labelled container
Pilot experiment
In order to determine suitable concentrations that will give me a
measurable result within the time I have available, different
concentrations of starch and enzyme were tried. I was aiming to
adjust the concentrations to give me a reaction time of 1-2 mins
which I felt would allow me to take accurate readings but also allow
me to carry out a number of repeats in a reasonable time scale
UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 29
Results of pilot tests
Using 2 cm3 1% starch solution
Temperature
0C
Fungal
Amylase
Bacterial
Amylase
Time to end
point (s)
20 1% 2 cm3 400
20 1% 2 cm3 600+
25 1% 2cm3 300
25 1% 2cm3 550
Using 2cm3 0.5% starch solution
Temperature
0C
Fungal
Amylase
Bacterial
Amylase
Time to end
point (s)
25 1% 2 cm3 30
25 1% 2 cm3 120
I also did a single trial to determine the range of temperature
treatment that would give me sensible results. I initially thought that
treating the enzyme for 10 mins at 700C would be sufficient to cause
full denaturation. In my trial I found that at this temperature the
enzymes were only slightly affected so I extended the temperature
range to 900C.
As a result of these trials I decided to carry out my experiment at
250C using 0.5% starch solution over a range of 40-900C. It was
evident that the fungal amylase was more active than the bacterial
amylase at these concentrations but I decided to keep to the same
concentration for each rather than introduce another variable. I am
planning to calculate the relative effect of temperature on each
which should take into account the difference in the rates of each
one.
30 UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide
Method
I measured 2cm3 of 1% bacterial amylase solution with a small
pipette and placed this in a clean test tube. In a second test tube I
measured 2cm3 of 0.5% starch solution dissolved in buffer solution
of pH 7.2 and then placed it in a water bath at 250C.
I prepared a second water bath at 400C and placed the bacterial
amylase in this for exactly 5mins. I checked the temperature of the
amylase with a thermometer to ensure that it was maintained at the
correct temperature for 5 minutes.
Whilst this was being treated I prepared a spotting tile by placing
equal drops of iodine solution in each depression and marking them
in pencil with 30s intervals.
As soon as the 5 mins was completed I removed the amylase from
the water bath and quickly cooled it to 250C. When both the
amylase and the starch were equilibrated at this temperature I
mixed them together a started the stopwatch. At 30s interval I
withdrew a small sample of this mixture from the tube and mixed it
with one of the iodine drops on the spotting tile. I continued this
until I found that the colour of the iodine remained exactly the same
as one of the untreated drops. At this point I recorded the time
taken.
I repeated this experiment for 10mins and 15 mins at each of the
temperatures tested (40,50,60,70,80 and 900C). I then began again
and repeated exactly the same procedure with the fungal amylase
solution. I found that the end point was not always clear so I
recorded three different iodine colours to help me analyse the data
later. These were, a definite blue/black colour, a dark brownish red
and a light reddish brown which matched the original iodine drops.
As a control experiment three tests were made in an identical
manner with fungal and bacterial amylase except no temperature
treatment was given to the enzymes before testing.
UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 31
RESULTS
BACTERIAL AMYLASE
Treatment
Temp 0C
Time of
treatment
(s)
30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630
40 300 + + +/- +/- +/- -
600 + + + + +/- +/- +/- -
900 + + + + + +/- +/- -
50 300 + + + + + + + +/- +/- +/- -
600 + + + + + + + + + + +/- -
900 + + + + + + + + + + +/- -
60 300 + + + + + +/- +/- -
600 + + + + + + + +/- +/- -
900 + + + + + + + + + +/- +/- -
70 300 + + + + + + +/- +/- -
600 + + + + + + + +/- +/- -
900 + + + + + + + + + +/- +/- -
80 300 + + + + +/- -
600 + + + + + + + +/- +/- -
900 + + + + + + + +/- +/- +/- -
90 300 + + + + + + + + + + + + + + + + + + +/- +/- +/-
600 + + + + + + + + + + + + + + + + + + + +/- +/-
900 + + + + + + + + + + + + + + + + + + + +/- +/-
Key + Significant blue/black colour +/- Dark brown - Matches original light reddish brown iodine colour
32 UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide
FUNGAL AMYLASE
Treatment
Temp
0C
Time of
treatment
(s)
30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630
40 300 -
600 -
900 -
50 300 -
600 -
900 -
60 300 -
600 -
900 -
70 300 + + + + + + + +/- +/- +/- +/- -
600 + + + + + + + + + + + + +/- +/- +/- +/- +/- +/- +/- +/- +/-
900 + + + + + + + + + + + + + + + +/- +/- +/- +/- +/- +/-
80 300 + + + + + + + + + + + + + + + + + + +/- +/- +/-
600 + + + + + + + + + + + + + + + + + +/- +/- +/- +/-
900 + + + + + + + + + + + + + + + + + + + +/- +/-
90 300 + + + + + + + + + + + + + + + + + + + + +/-
600 + + + + + + + + + + + + + + + + + + + + +
900 + + + + + + + + + + + + + + + + + + + + +
Key + significant blue/black colour +/- Dark brown - Matches original light reddish brown iodine colour
UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 33
Results of control experiments
Bacterial amylase
Test no Time taken for iodine colour
change (s)
1 180
2 210
3 150
Mean 180
Fungal amylase
Test
no
Time taken for iodine colour
change (s)
1 30
2 30
3 30
Mean 30
To enable me to compare the effects of temperature treatment on
each enzyme I calculated the overall rate of reaction for each using
the formula
___________1______________ X 103
Time taken for colour change (s)
I then expressed the rate of each reaction as a percentage of the
rate of the untreated enzyme.
The results are shown in the following summary table.
34 UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide
Summary results table
Treatment
Temp (0C)
Time of
treatment (s)
Time to reach
end point (s)
Fungal
Rate of
reaction
(Arb Units)
Fungal
Percentage of
control rate
Fungal
Time to reach
end point (s)
Bacterial
Rate of reaction
(Arb units)
Bacterial
Percentage of
control rate
Bacterial
40 300 30 33.33 100 180 5.55 100
600 30 33.33 100 240 4.16 75.0
900 30 33.33 100 240 4.16 75.0
50 300 30 33.33 100 330 3.03 54.6
600 30 33.33 100 360 2.77 49.9
900 30 33.33 100 360 2.77 49.9
60 300 30 33.33 100 240 4.16 75.0
600 30 33.33 100 300 3.33 60.0
900 30 33.33 100 360 3.03 54.6
70 300 360 3.03 9.1 270 3.70 66.7
600 630+ 1.59 4.8 300 3.33 60.0
900 630+ 1.59 4.8 360 3.03 54.6
80 300 630+ 1.59 4.8 180 5.55 100
600 630+ 1.59 4.8 300 3.33 60.0
900 630+ 1.59 4.8 330 3.03 54.6
90 300 630+ 1.59 4.8 630+ 1.59 28.6
600 630++ 0 0 630+ 1.59 28.6
900 630++ 0 0 630+ 1.59 28.6
UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 35
36 UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide
Conclusions
Main trends and patterns
The results show that there is a significant difference in the response
of bacterial and fungal amylase to different temperature
treatments.
The raw data shows that bacterial amylase is significantly less active
in these conditions compared with fungal amylase.
Fungal amylase retains 100% of its activity when treated at
temperatures up to 600C. Above this temperature fungal amylase
activity rapidly decreases to less than 5% of its original rate but still
retains some limited activity even at when treated at 900C. Length of
time of treatment appeared to have little effect on this pattern since
there were only small variations at 700C and at 900C.
In contrast bacterial amylase activity was inhibited by treatment at
400C for 600s and 900s. It could be said therefore that the bacterial
amylase was more sensitive to temperature but although activity
was inhibited the bacterial enzyme was affected much less by
treatments in the range 50-900C. It can be seen from the graph that
the bacterial enzyme retained over 50% of its activity up to 800C. It
also appeared that length of time of treatment was also more
important to the activity of bacterial amylase as there was an
obvious relationship showing that the longer the treatment the
slower the reaction at three of the temperatures tested.
The results for bacterial amylase also showed a much greater degree
of variability. There were obvious anomalies at 60 and 800C for the
900s treatment although it was interesting to note that all of the
rates rose unexpectedly between 50 and 600C. This may be an
important trend or experimental error which would need further
investigation. It is possible that because of the much slower basic
rate of the bacterial amylase that experimental errors could have a
much more significant effect on the final results when converted
percentage differences.
Explanation of results
Enzymes are globular proteins made up of folded polypeptide
chains. The shape of the final protein is vital to the activity of an
enzyme because it forms an active site into which a substrate
molecule must fit to allow the enzyme to work. In large proteins this
UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 37
3-D shape is maintained by holding the folds of the polypeptide in
position by using cross links between them. These cross links are
mainly disulphide bridges and hydrogen bonding between amino
acids. Increases in temperature make it more likely that these cross
links become broken and as the polypeptide unfolds it is more and
more likely that the change in shape will affect the active site and
stop the enzyme working.
Whilst the active site of enzymes acting on the same substrate must
be very similar it is possible that variations in the protein structure of
different enzymes can differ meaning that different types and
numbers of cross links can be used to make the same shape. It is
possible therefore that the effect of increasing temperature can be
different.
In this case if the results for bacterial amylase were shown to be
correct it would be possible to speculate that as temperature
increased some cross links were broken which slowed the rate of
reaction but other stronger bonds remained to keep its activity over
a wide temperature range. The effect of increasing the time of
treatment of bacterial amylase especially at 60, 70, and 800C showed
an increased inhibition with longer treatment. This could support
the idea that the remaining links were stronger but prolonged
treatment at these higher temperatures broke them down too.
In the case of fungal amylase the cross links could resist change up
to 600C but at this point many similar links are broken and activity
falls quickly. It would be possible to investigate this idea by detailed
analysis of the protein structure of each enzyme.
Experimental limitations
The most important limitation to this experiment is the difficulty in
making an accurate judgement of the final end point. The colour of
the iodine changes slowly in some cases. In the slower reactions this
was particularly difficult. Whilst some effort was made to distinguish
between reactions where the dark brown colour remained after 630s
and those where the drops remained blue/black by counting the rate
of reaction as zero where there was no sign of change this remained
an inaccurate method.
The other major limitation involved temperature treatment.
Especially at higher temperatures it was difficult to determine the
exact time of temperature treatment. The solution itself was
checked but inevitably it took longer for the enzyme to reach these
higher temperatures so the exact time of treatment did vary.
Whilst exactly the same concentration of each enzyme was used the
powdered enzymes themselves were rather crude preparations and
it is likely that they contain different amounts of active ingredients.
This may have accounted for the big difference between the activity
of the two enzymes and makes direct comparison more difficult.
The overall reliability of the investigation was also limited by time
constraints. To cover the full range of temperatures and times of
treatment meant that 36 separate experiments were required. This
meant unfortunately there was not time to carry out repeat tests. It
is evident from the variability of the results especially for bacterial
amylase that this was an important limitation.
Overall it appeared that this investigation did provide some evidence
to support my hypothesis that bacterial amylase would work better
over a wider temperature range than fungal amylase.
Clearly repeating these tests at least three times would be the most
important type of further work which would be needed. If the
trends shown in the first set of data are consistent it would also be
important to investigate smaller increases in temperature between
50 and 700C.
If time was available it would also be profitable to investigate a
wider range of fungal and bacterial enzymes possibly from different
sources.
