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Chandni Hindocha L6G

The Effect of Temperature on the Activity


of the Enzyme Amylase
Introduction
Enzymes are large globular proteins which are biological catalysts. They speed up the
rate of reaction without being used up themselves. They are found in cells and out of cells i.e. in
the blood plasma. Enzymes generally lower the activation energy of a reaction; the activation
energy is the amount of energy in joules needed to start the reaction. Amylase is the enzyme
which catalyses the reaction of the hydrolysis of starch into maltose, which is two glucose
molecules bonded together. The starch is broken down and water molecules are added.
Amylase is found in several sources like in saliva and in the pancreas. There are two
types of amylase, alpha amylase and beta amylase. Alpha amylase is the most common found in
the human body but can be found as fungal amylase. Beta amylase is less common and found in
bacteria, fungi and plants.1 I will be using amylase from a fungal source, it will be alpha amylase
and the source is Aspergillus species, which are a common group of filamentous fungi that are
readily recovered from soil, decaying vegetation, air and many other environments2 therefore
there are no ethical issues or environmental issues surrounding it. However there is an
environmental issue concerning the iodine because it destroys aquatic life; however I am only
using minute quantities.
Starch is a polysaccharide made up of 79% amylopectin and 21% amylose3 and is only
found in plants, its insoluble therefore good for storage as well as transport. Both amylopectin
and amylose are compact and so good for storage. When iodine is added to starch it is thought
that the iodine fits into the coils of amylose.4The diagram shows what starch breaks down into.5

Starch

Amylase

Maltose

Maltase

Energy for growth


Glucos
Cellulose for cell walls

There are two theories which explain how enzymes and substrates
work; the first is the Lock and Key Theory. In this theory the substrate,
starch, locks on to the active site on the enzyme, amylase. Amylase is
therefore specific to starch and only starch will fit into the active site of
the enzyme. The two shapes are said to be complimentary as the diagram
1
2
3
4
5

en.wikipedia.org/wiki/Amylase
http://www.microbelibrary.org/ASMOnly/details.asp?id=1978&Lang=
Bio Factsheet Number 159. www.curriculum-press.co.uk
http://en.wikipedia.org/wiki/Starch
Biology by Martin Rowland Page 484

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Chandni Hindocha L6G

shows6. Once an enzyme-substrate complex is formed a reaction takes place in which starch is
broken down into maltose. This is catabolism. Molecules of maltose are released (number
depends on size of starch molecule) and the enzyme can be reused.
The other theory is the Induced Fit Theory which states that when the substrate enters the
enzyme, the enzyme moulds into the shape of the substrate to accommodate the substrate. The
enzyme then breaks down the substrate and once the substrate leaves the enzyme returns to its
shape, rather like a glove on a hand.

Input Variables
I will be changing the temperature of amylase and starch in order to monitor the effects
on the activity of amylase breaking down starch. At extreme temperatures the enzyme is
denatured. Denaturation is any change in the structure of a protein which leaves it inactive7
Enzymes are 3-dimentional and the effects of extreme temperature, extreme pHs and hydrogen
ions means the enzyme becomes unfolded, losing its 3D
shape (tertiary and quaternary structures), hydrogen
bonds which hold the enzyme in its shape are easily
overcome by extreme temperature.8
Amylase, as well as all other enzymes works
best at around its specific optimum temperature, for
fungal amylase this is 20oC to 60oC. At the optimum the
substances have more kinetic energy and are more
likely to collide and therefore it is more likely for there
to be successful collisions. We expect low temperatures
to do the opposite; the enzymes have less kinetic energy
so it is less likely for a successful collision to occur. A graph to show how temperature affects the
activity of amylase is shown.9 I will be controlling this by using water baths and a thermometer.

Hypothesis
I hypothesize that as the temperature of the amylase starch mixture increases, the rate of
reaction will increase and therefore the rate will be higher. I will calculate the rate of reaction by
using the formula 1/time(s) = rate. During the experiment I will log the time it takes for the
amylase to break down the starch and then I will put the results into the formula to work out the
rate. I hypothesize the amylase will work fastest at 40oC because this is the centre of the
optimum that I found (see input variable). I think that after the optimum it will take longer for
the amylase to digest the starch and the rate will decrease because the enzymes begin to
denature.
6
7
8
9

www.scienceclarified.com/El-Ex/Enzyme.html
http://class.fst.ohio-state.edu/FST822/lectures/Denat.htm
http://www.madsci.org/posts/archives/2000-01/949009933.Bc.r.html
www.chemsoc.org/networks/learnnet/cfb/enzymes.htm

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Chandni Hindocha L6G

Other Variables
pH: I will be keeping the pH the same by using buffer solutions in the amylase and starch
solutions. I will also be using the same iodine solution so that pH is kept the same. pH is the
measure of concentration of hydrogen ions10. Each enzyme works best at a specific pH, the pH
for the type of amylase I am using is pH 3 to pH 6. The pH of an enzyme effects the rate of
reaction because it changes the charge of the amino acid, therefore the substrate will be repelled
from the enzyme, furthermore the bonds are lost and at extreme pHs, the enzyme is denatured.
This has to be controlled because if the pH changes then the rate of reaction changes which then
affects my results. I will be using a solution of pH5 for the amylase.
Enzyme Concentration: I will control this by using only one
concentration of the enzyme amylase (0.1% fungal amylase), this
means that all my tests will be fair because there will be the same
volume (using a syringe) and same concentration of amylase in the
mixture. Increasing the concentration means increasing the number
of enzymes in a certain volume. The rate of a reaction is directly
proportional to enzyme activity so its important to keep it the
same. The more enzymes there are, the more likely of an enzymesubstrate complex forming. The enzyme concentration is an
important factor because when this increase the rate of reaction
increases, so needs to be kept constant. When the substrate is in excess the rate follows the
diagram shown (directly proportional)11 however when the substrate becomes a limiting factor
the rate stays the same because all the active sites are full at one point, therefore the graph round
off into a horizontal line(see graph). The enzyme is said
to be saturated.
Substrate concentration: I will control the substrate
concentration by using only one concentration of starch
solution (1% starch solution) and using the same
volume as well. The more substrate there is the more
likely an enzyme will collide with a starch molecule.
Therefore when all the active sites are full the rate of
reaction starts to level out to a constant level. This is
called maximum velocity or V.max. After the leveling off, the substrates must wait until they can
enter the enzyme. This is shown by the graph12

Diagram
10
11
12

http://antoine.frostburg.edu/chem/senese/101/acidbase/faq/what-is-pH.shtm
ghs.gresham.k12.or.us/.../factorsenzconc.htm
www.science.siu.edu/.../11-Proteins.html

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Apparatus
Amylase Solution 0.1% concentration using 5cm3 in each test tube
Starch Solution 1% concentration using 5cm3 in each test tube
Iodine Solution 2 drops at a time
Water Baths at 30oC, 40oC, 50oC, 60oC and 70oC
Test Tubes- two per test
20cm3 syringes- to accurately read 5cm3 in order to measure the amylase and starch. I will need
two, one for the amylase and 1 for starch
Glass pipettes- in order to add 2 drops of amylase-starch solution to two drops of iodine. I will
need 3, one for amylase, one for starch and the last for iodine
Test tube holder
Spotting tray- I will need several in order to contain the starch-amylase mixture
Stop watch
Thermometer
Deep Watch Glass

Justified apparatus list


Amylase solution: I will be using amylase as an enzyme because it is the only enzyme that will
break down starch into maltose.

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Starch solution: I will be using this because it is the substrate which amylase breaks down into
maltose
Iodine solution: its the indicator which tests for the presence of starch in a mixture. No other
indicators test for the presence of starch
Water baths: I will be using a water bath because it keeps the temperature constant, I will also
check it by using a thermometer in it. Using a water bath means I can control the temperature
which is better than using a beaker with water in it which I heat with a Bunsen burner because it
will make my tests fairer
Test tubes: I am using these to contain the mixture, I am using test tubes rather than beakers or
conical flasks because they hold a small volume, and they have the right capacity for the
volumes I am using.
20cm3 syringes: these will be precise and its easy to push out liquid as well, I am using a
syringe rather than a pipette because I will be able to force out liquid as well without having air
bubbles.
Glass pipette: I can control how many drops I of iodine I put, I am using this rather than a
syringe because its easier to control the liquid coming out.
Test tube holder: to hold the test tubes if and when they are out of the water bath.
Spotting tray: so I can clearly see, against a white background, the colour of the liquid, I am
using this rather then test tubes or anything else because I only need a small volume of iodine
(around two drops) and amylase-starch mixture to see a colour change.
Stop watch: to time how long the mixtures are in the water bath and how long it takes for the
solution to turn iodine yellow. I will be using this to get a fair test because if I leave the test tubes
in for different times then it becomes unfair. I am using this rather than a wall clock or other time
measuring devices because it is the most precise.
Thermometer: I will use this in order to check the test tubes are the right temperature.
Deep Watch Glass: to put a volume of iodine in so I can measure against it. I am using this
rather then one of spots on a spotting tray because it needs to be kept completely separate and if
its not then it could be used as the iodine I am using to test the amylase-starch mixture

Method

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Chandni Hindocha L6G

Place 5cm3 of 0.1% amylase in one test tube and 5cm3 of 1% starch in another.
Put the two test tubes in a water bath for 5 minutes use a stop watch to time it.
Put two drops of iodine in each spot of a spotting tray. Use a long glass pipette. Dont leave the
iodine in the spotting tray too long before adding the mixture.
Add the starch to the amylase and mix the solution. The time at which they are mixed is zero.13
Every 20 seconds put two drops of the starch-amylase mixture into a spot reducing the time to 5
seconds when the spots of the tray get lighter. Time how long it takes for the iodine to turn pale
yellow/brown.
Iodine with starch is black
Iodine without is pale yellow or brown.
Repeat at each of the five temperature three times

Justified method

Place 5cm3 of amylase in one test tube and 5cm3 starch in another. Put the two test tubes
in a water bath for 3 minutes
The starch and the amylase need to reach the same temperature so they can equilibrate.
Put two drops of iodine in each spot of a spotting tray.
Iodine is the indicator which will tell us whether the starch has been digested or not because it
tests for the presence of starch. If there is starch it will turn black. If there isnt it will turn pale
yellow or brown
Add the starch to the amylase and mix.
The enzymes can now break down the starch.
Every 20 seconds put two drops of the starch-amylase mixture into a spot. Start reducing
the time to 5 seconds when the spots of the tray get lighter.
This is to time how long the amylase will take to digest the starch. To make it more accurate, we
reduce the time nearer the end to get closer to the actual time it takes rather than with at most a
19 second error bar.
Repeat three times in order to find an average
Some information in my plan is subject to change after preliminary trials. There is a possibility
of me changing the temperatures of the water baths, changing the concentration of amylase and
starch, changing the time which they are left for in the water bath, the number of repeats I do. I
will do my preliminary trials on the day of my practical. I will do this by checking the upper and
lower (extremes) of water bath and how the different concentrations of amylase work on starch at
those temperatures.

Preliminary results
Temperature(oC)

5cm3 Starch
with 5cm3

5cm3 Starch
with 2.5cm3

13

5cm3 Starch
with 1cm3

5cm3 Starch
with 0.5cm3

http://www.associatedcontent.com/article/8679/the_effect_of_temperature_on_amylase.ht
ml?page=2

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Chandni Hindocha L6G

Amylase
20 seconds for
the starch to be
digested too
fast
Enzyme
denatured.

30

70

Amylase
60 seconds for
starch to be
digested too
fast

Amylase
240 seconds for
starch to be
digested

Amylase
520 seconds for
starch to be
digested too
slow.

After doing preliminary experiments I decided to change my volumes as well as several other
pieces of equipment

5cm3 of 1% starch with 1cm3 of 0.1% amylase at pH5.


I had to change the volumes because the reaction was happening too quickly at 30oC (the coolest
temperature) therefore at 50oc for example, the reaction would happen even faster and I would
have no real results.
Instead of using 20cm3 syringes I used a 5cm3 syringe for the starch and a 2cm3 syringe
for the amylase.
This was mainly for ease and also precision because the smaller the syringe the smaller the limits
of accuracy and therefore the more precise the results
Instead of using a glass pipette to put the amylase-starch solution into the iodine I used a
micropipette of 0.1cm3
I decided to use a micropipette because I found on the day of the experiment that it mattered how
much amylase-starch solution you put into the iodine. The more you put the more dilute the
mixture becomes and therefore the lighter to looks.
Because of these changes, my method changed slightly to accommodate these changes.

Results Table of Raw Data


Temperature(oC)
30
40
50
60
70**

First attempt
(s)
340.0
180.0
160.0
2280.0
0

Second
attempt (s)
360.0
200.0
120.0
1980.0
0

Third attempt
(s)
400.0
160.0
160.0
2100.0
0

This table corresponds to the following units on my graph.


Temperature(oC) First attempt Second
Third attempt
(s-1)
attempt (s-1)
(s-1)
30
0.0030
0.0033
0.00250
40
0.0055
0.0050
0.00625
50
0.00625
0.0083
0.00625
60
0.00043
0.0005
0.00047
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Average (s)
366.6
180.0
146.6
2120.0
0

Average rate
(s-1)
0.00290
0.00558
0.00693
0.00046

Average rate
(1/s)
1/366.6
1/180
1/146.6
1/2120
0

Chandni Hindocha L6G

70**

** I have not put any results for 70oC because after 2 hours and 30 minutes (9000 seconds) I
received no results. The enzyme would have denatured after this result because it is extreme to
the specific enzyme. the tolerance level of the enzyme was from 20oC -60oC so because 70oC is
out of this range the enzyme would have unraveled from its tertiary state and therefore could no
longer digest the starch because the active site would have lost its specific shape for starch. I
checked the 70oC water bath every 10 minutes and each spot in the tray was black showing that
starch was still present (so the amylase could not digest the starch because it was denatured). If I
had left it longer, for example over 5 days then there is a possibility I could have gotten some
results.

Overall Description of results


The results I have found follow a clear pattern. At 30oc
the rate is around 0.0029s-1 and this rises to the optimum of
0.00693s-1, it then rapidly drops to 0.00046s-1 at 60oc. After this
no results were obtained because after around 2 hours there
was no change. The rate at 70oC would, if calculated, be very
low or zero. The graph shows that the rate of reaction
increasing until 50oC and I found this on all the attempts apart
from the third attempt where the rate of reaction at 40oC and
50oC were the same. After it reaches this maximum it decreases
rapidly because the enzymes are denaturing.
My graph looks similar to the graph which shows how
temperature affects the rate of reaction and that it follows the trend I expected to find. My
research showed that amylase had a temperature tolerance of 20oC to 60oC and you can see this
in the graph I have drawn. Due to this tolerance level I expected to find the optimum of 40oC
because this is the centre of the tolerance.
30oC- 40oC
My preliminary trials were done at 30oC so I knew I expected to find the rate around 0.003s-1.
Between 30oC and 40oC the rate rises by 0.00268 units. This shows that as the temperature
increases the rate increases because the enzymes and substrates are given more kinetic energy
and are therefore more likely to collide. When this happens starch can be broken down into
maltose. The line of best fit doesnt actually go through the average but passes through 0.0058s-1
which is very close. At 40oC there is quite a large error bar ranging from 0.0055s-1 to 0.00625s-1
so a total variance of 0.00125.
40oC - 50oC

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This part of the graph again rises, but it rises to an optimum of 50oC which I mentioned earlier
that I did not expect to find this as my hypothesis shows I expected the optimum to be 40oC for
this specific type of fungal amylase. Though my graph shows the maximum at 50oC it could be
anywhere between 45oC and 55oC because I didnt measure the rate at those temperatures I do
not know that it is not one of these. With more points on the graph the pattern would be clearer
and if I did the experiment again I would do more. 50oC has a great error bar from 0.0083 to
0.00625 (which is the maximum point of the error bar at 40oC) this is a difference of 0.00205
which leads to me believe that the point at 0.0083 is an anomaly. The raw data shows that two
results were the same (repeat 1 and 3) and repeat two was very different.
50oC - 60oC
Here, the graph shows a very large drop from 0.00693s-1 to 0.00046s-1. This is a drop of 0.00647
arbitrary units. This is an enormous drop and clearly shows the activity of amylase decreasing
greatly as well as rapidly. It took an average of 2120 seconds (around 35 minutes) for the starch
to be digested by the amylase at 60oC. The rapid decrease is because amylase is being denatured,
each enzyme is being destroyed by the high temperature. The bonds which hold each enzyme in
its secondary and tertiary state will break and this will change the shape of the enzyme. The
repeats are very close together at 60oC showing little variance.
60oC - 70oC
The enzyme completely denatures between these two temperatures and after 2 hours there was
no change at 70oC. However the starch could oxidize over several days. There is a decrease of
0.0046 arbitrary units. The enzyme can no longer work as an enzyme because all the hydrogen
bonds are broken so it just becomes a coil with no secondary or tertiary shape. This means that
starch molecule cannot be broken down because the active site has changed.

Conclusions
The activity of amylase changes over a range of temperatures. Its pattern over these
temperatures follows the classic bell shape. This pattern can be seen in my graph clearly as a
steady rise to an optimum and a rapid drop as the enzyme denatures. This happens because the
enzyme and substrate are given energy so have more kinetic energy so can move faster leading to
a higher rate of reaction. From this I can clearly say that temperature greatly affects the activity
of amylase because after 50oC the enzymes bonding is destroyed so the secondary and tertiary
structures are lost. The results do prove the prediction that I made, but since this is just one
experiment we do not know that the investigation always produces this result. I would need to
repeat the whole investigation to make my results more accurate.

Evaluation

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From looking at my investigation I can see that there is a lot of variability in my results
for 40 C and 50oC, where I would consider the second repeat at 50oC an anomaly because it is
quite far from my average as well as quite far from my line of best fit. I would also consider this
as an anomaly because repeat 1 and 3 are the same (0.00625) and repeat 2 is much higher. The
error bar at 30oC is short and so is the error bar at 60oC therefore these two results are more
accurate because there is less variance from the average.
o

The error bar at 40oC is quite large as well but I do not consider this an anomalous result,
in order to improve these results I would have to repeat them again, until I had a clear set of data
for 40oC. The more times I repeat the results the more reliable they become because my mean
(average) gets smaller and more reliable. It would be more reliable if other people did the
experiment as well to show that the predicted results would occur regardless of the person
conducting the experiment.
The error bar at 50oC is my largest error bar. It ranges from 0.0083s-1 to 0.00625s-1 which
shows a total variance of 0.00205. This is my fastest result which only took 120 seconds. Due to
this result it pulls my average for 50oC upwards making the average has a higher rate as well.
This anomaly could have occurred for several reasons. Firstly the environmental conditions
could have changed so the temperature of the water bath changed. If the temperature of the room
got slightly warmer then the water bath may have changed temperature.
Another possible reason for this anomaly is that the iodine could have oxidised in the
spotting tray because it had been left open, or in warm weather in direct sunlight, therefore it
would have produced a lighter colour. So when the amylase-starch mixture was dropped into the
iodine, it would have seemed digested when it wasnt. Moreover we were using different bottles
of iodine because it ran out quickly. These would be slightly different colours because of where
they were kept and if they had been left open or if they had been left in the same conditions as
the iodine that I used at the beginning of the experiment. Having so much variance in this part of
the graph obviously reduces the reliability of the results and it would need to be repeated over
again in order to make them more reliable and to prove that this set of results was not a
coincidence. If repeated I think the real result would be close to 0.00625s-1 as this was the result
of the other two repeats.
There are other factors that could increase the variability of the results. The precision of
the apparatus and the method are very important factors. I used a 5cm3 syringe and a 2cm3
syringe in my apparatus. Each of the repeats I did could have had different volumes of liquid
because the syringe was not as precise at exactly 5cm3, but maybe 5cm3 0.1cm3. However I
was using the 5cm3 syringe to measure out 5cm3 so this is more likely to be precise then if I used
a 20cm3 syringe to measure out 5cm3. I used a 2cm3 for the amylase (measuring out 1cm3) and
this syringe is more precise then the 5cm3 syringe because the smaller the syringe the smaller the

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inaccuracy. There was also a problem of getting air bubbles into the syringe which would reduce
the volume in the syringe.
The micropipette I used to put the amylase-starch solution into the iodine was my most
precise piece of equipment and is the only one I could trust to give me accurate results. I found
during my experiment that the volume of iodine used could affect the results. I was using a glass
pipette in order to put iodine into a spot. Even though I put two drops in, this is not precise
because each drop could be a different size depending on the pressure exerted on the syringe and
the volume of liquid in the syringe. The glass pipette limits my results greatly and makes the
quite unreliable.
Another piece of equipment which can be called into
Water bath
Actual
question is the water baths I used. The table shows the
temperature
temperature
actual temperatures of the water baths compared to what
o
(oC)
they are supposed to be. These were the results of the water ( C)
30
28
baths collected at the beginning of the experiment and
40
42
these could have fluctuated during the experiment. It also
50
49
means my results are not that reliable because the amylase
60
61
and starch were being raised to a different temperature to
70
70
what I thought they would be at. These may not be large
differences but they certainly put limits into my experiment and help explain the variability of
my results. For example the rate at 40oC was not actually the rate at 40oC but the rate at 42oC
which would be higher than that at 40oC.
I also found problems and limitations with my method. I found it very difficult to mix the
amylase and starch and also take a sample all at 0 seconds which my method said I should do. It
was difficult and I found myself taking a sample at 5 seconds and then again at 20 seconds, and
this is a 15 second gap rather than a 20 second gap. If I could do the experiment again I would
not test at 0 seconds but start to test at 20 seconds giving me time to mix the amylase and starch
thoroughly and then test the solution. I also had a problem with defining the colour of the
digested starch in iodine. You can never really tell when the starch is digested, no matter how
many times you test it because whilst this happened the iodine gets oxidised as well which
changes the colour.
The final problem I encountered was that in the amylase solution the concentration is different at
different parts of the beaker unless the beaker is swirled often. My beaker was not swirled often
enough. At the bottom of the beaker the concentration would be higher than that at the top of the
solution. So when I took out amylase solution with my syringe, it is possible that the
concentration would be different for each attempt.

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Improvements
To improve my results the first thing I would do is amend my apparatus, I would use a
micropipette for putting the iodine into the spotting tray as well as the one I used for the amylase
starch solution. This would make each result a lot more precise. Furthermore to test the iodine I
would use a colour wheel to measure the colour of the amylase-starch solution in the iodine. I
used deep watch glasses to test it against but this was not effective in deciding if the starch was
digested. I would choose a colour and always use that colour to test by. This would give me more
reliable results because we have to rely less on the experimenters personal view so the
experiment becomes much more objective. Moreover I would swirl the beaker of amylase for
each use and test from the same depth each time. I would also do many more repeat tests of 40oc
and 50oc because it would make my mean more accurate, rather then it being over a large range.
Furthermore I would also make sure the water baths stayed at a constant temperature rather than
fluctuating. I would do this by using a more advanced water bath in an area where the
environmental factors wont affect the temperature of the water bath. I would also change my
method so that I didnt have to mix and test the solution all at 0 seconds. I would mix the
amylase and starch at 0 seconds and then test at 10 seconds and then test every 20 seconds after
this. It makes my method slightly more complicated but also makes it slower and calmer which
is better for the person conducting the experiment and will lead to fewer mistakes. Above all, I
would repeat the experiment another 3 or 4 more times.

Further Investigations
Other investigations that could be done in order to increase my understanding of amylase
are firstly doing these experiments at 10oC and 20oC in order to get a fuller picture of the graph.
Therefore the plotting graph would look more like the predicted graph on page two and it would
give a fuller picture of how temperature affects the activity of amylase. Another investigation I
could do is changing the sources of amylase that I use. The type I was using in this experiment
was alpha amylase but I could use beta amylase, which is synthesized by fungi, bacteria and
plants.14 Furthermore I could use salivary and pancreatic amylase which has an optimum of 37oc.
however with salivary amylase there are ethical limitations.

14

en.wikipedia.org/wiki/Amylase

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