Biology HL Internal Assessment

The effect of different temperature of homogeneous mixture consisting of distilled water and
Agaricus bisporus on enzyme activity.

Biology HL Internal Assessment

The effect of different temperature of homogeneous mixture consisting of distilled water and
Agaricus bisporus on enzyme activity?

School name: **********************

Candidate name: Agnes *********
Candidate number: *********

Candidate number: ******-006

Research question:
What is the effect of different temperature of homogeneous mixture consisting of
distilled water and Agaricus bisporus on enzyme activity?

Background information:

Enzymes are globular proteins, which speed up reaction without being changed
themselves. Without them, the reactions would be very slowly and nothing would control the
reactions. Enzymes are nearly always proteins, though they may contain non-protein
component.1 The main properties of enzymes are:

Work rapidly.

Work in both directions.

Speed up reactions.

Are dependent on pH, temperature and substrate concentration.

Can be reused.
With the increase of temperature, enzyme activity increases too. The phenomena

occurs until the reaction obtain the optimum point, where the temperature is so high, that
increased a little more will damage the enzymes structure. At this point, the enzyme ceases
to function; a phenomenon called enzyme or protein denaturation.
Extremes in acidity (pH) can also cause the protein structure of enzymes to denature.
Poisons often work by denaturing enzymes or occupying the enzymes active site so that it

Michael Roberts, Michael Reiss, advanced Biology, UK, Cheltenham, Nelson (2000).
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does not function. In some cases, enzymes will not function without cofactors, such as
vitamins or trace elements.2
One of the fastest enzyme is catalyse. This enzyme is found in number of organs and
tissues, including the liver where its job is to speed up decomposition of hydrogen peroxide
into oxygen and water:
2 H2O2 2 H2O + O2
Enzyme activity is the quantity of active enzyme present and is thus dependent on
conditions, which should be specified. Optimum temperature of enzyme is the temperature at
which enzyme activity is the fastest.
Agaricus bisporus is more known as white mushrooms and is the most popular type of
mushroom in the word. It has surprisingly high nutritional values and is excellent source of
protein and plenty of vitamins and minerals.3

Tracey Greenwood, Richard Allan, Senior Biology 1, UK, Edinburgh, BIOZONE

(2009).
3

http://en.wikipedia.org/wiki/Agaricus_bisporus

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Picture 14
Picture presenting the nutritional values in 100g of white mushrooms.

Hypothesis:

According to the background information, the temperature has a big influence on the
rate of reaction and therefore on enzyme activity. Increases the temperature of the
environment, will increase the rate of reaction. This pattern will continue, till the enzyme get
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http://en.wikipedia.org/wiki/Agaricus_bisporus

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its optimum point temperature at which it works the fastest. If temperature will increase
above the optimum temperature, protein will start to denature and the rate of the reaction will
decrease. Finally the protein will be damaged completely and no reaction will occur.

Variables:
Independent:

Temperature of the homogeneous solution. The temperatures will be as

follows:

and

Dependent:

High of the foam produced after adding 3% hydrogen peroxide into

homogeneous solution at different temperatures.

Controlled:

One source of Agaricus bisporus.

One source of distilled water.

The same size and type of test tubes. This will eliminate a source of error
during the process of measuring the high of the foam produced.

Once prepared homogeneous solution for all trials. This will eliminate a source
of error, because every trial will the same ratio of Agaricus bisporus to distilled
water.

One source of distilled water, distilled water from one bottle.

Careful timing. In this experiment, we wait just only for 5 seconds before
taking measurements. This requires focus and high precision. Good stopper
will be essential.

The same temperature. Conducting trials for one temperature of homogeneous

solution, before putting the next test tube to hot water bath, the temperature of
the bath must be checked. What is more, the use of polystyrene cup in which
beaker with hot water is placed, will decrease the change of temperature.

The same laboratory conditions, e.g. no change in light intensity, temperature.

The same source of 3% peroxide.

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Apparatus and materials:

300 cm3 beaker.
200cm3 beaker with lid.
Polystyrene cup for 300cm3 beaker.
Bursen burner.
Noncontact digital infrared thermometer laser with accuracy

Electronic balance with accuracy

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1cm pipette with accuracy

10g of Agaricus bisporus.
100g of distilled water.
Blender.
Marker.
50 the same type of test tubes.
Stopper.
50cm3 of 3% hydrogen peroxide in a bottle with tip.
Ruler with accuracy

Rubber gloves.
Tweezers.

Methodology:
I.

Label test tubes.

II.

Make a homogeneous solution by mixing 10g of Agaricus bisporus with 100cm 3

distilled water. To measure the volume of water, put the blender on the scale, tear the
scale and put into the blender 100g of water ( 1cm3 = 1g).

III.

Pour homogeneous solution into 200cm3 beaker and cover it to prevent water loss
from the mixture.

IV.

Using pipette pour 1ml of homogeneous solution into first tube.

V.

Pour 200g of water into 300 cm3 beaker and check the temperature using noncontact
digital infrared thermometer laser. We want the temperature to be

a) If it is too high place the beaker in freezer and keep checking the temperature

until it will be desired one.

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b) In a case, that the temperature is too low, heat water using Bursen burner to

obtain desired one.

VI.

Place beaker with water into polystyrene cup. This will help to slow the heat loss/
gain from environment. This will make experimental results more accurate.

VII.

Put the first test tube into water bath and wait for three minutes. Measure the time with
the stopper, to avoid mistakes.

VIII.

After 3 minutes, the homogeneous solution get the temperature of the water bath.
Immediately take out the test tube and add 3 drops of 3% hydrogen peroxide.

IX.

Wait 5 seconds and using ruler measure the height of produced foam.

X.

Record the data and again put 1 cm3of homogeneous solution into the next test tube.

XI.

Check the temperature of water bath and if the temperature changed and:
a) If it is too high place the beaker in freezer and keep checking the temperature until

it will be desired one.

b) In a case, that the temperature is too low, heat water using Bursen burner to obtain

desired one.
XII.

Place beaker with water into polystyrene cup. This will help to slow the heat loss/
gain from environment. This will make experimental results more accurate.

XIII.

Put the next test tube into water bath and wait for three minutes. Measure the time
with the stopper, to avoid mistakes.

XIV.

After 3 minutes, the homogeneous solution get the temperature of the water bath.
Immediately take out the test tube and add 3 drops of 3% hydrogen peroxide.

XV.

Wait 5 seconds and using ruler measure the height of produced foam.

XVI.

Record the data and repeat the steps X to XV for the rest 8 test tubes.

XVII.

Repeat the steps from IV to XVI for the next homogeneous solutions at temperature
and

and record outcomes.

Safety rules:

Wear lab coat and glasses.

Use Bursen burner carefully.

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Data collection:

Qualitative data:

In samples with low temperature of homogeneous mixture, the height of foam

smaller. The tallest foams were obtained from
temperature, e.g.

. However in the solutions with high

, the foam was absent, which is the result of denatured protein. This

led me to notice trend that with the increase of temperature, the foam height increases,
Contrary, in

and

mixtures, we could observe the decrease in height of foam

produced and no foam respectively.

Quantitative data:

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Data processing:
Mean:

Mean is the average of outcomes obtained from all trials for one type of
solution. It will be very useful in drawing conclusions. Considering the average value
for each type of medium, we will be able to see the pattern the relationship between
the concentration of ethanol and the rate of diffusion. The formula is:

,
where

are the respective values of

absorbance from one kind of solution.

Table 2.

Mean height in cm of foam produced by the reaction of hydrogen

peroxide with homogeneous mixture at different temperatures.
Temperature of
Mean height
homogenous solution
of foam
/ C 0.1
/ cm
100

0.0

40

1.50

30

3.51

20

1.81

10

0.76

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ANOVA Test:
ANOVA Test is the statistical test used to determine the difference between means of
multiple independent groups, which in this experiment are different temperatures of
homogeneous solutions. The purpose of this test is to check the validity of null hypothesis,
which is :

where is the value of group mean and k is number of groups5.

The null hypothesis states that means of all groups are equal and by conducting ANOVA test,
the hypothesis can be rejected or confirmed.

Picture 2.
Picture represents the results obtained from ANOVA test calculated in Excel.

From the picture 2 we can easily notice that in this case with critical
value of 0.05, F value equal to 572.620 is significantly greater than F critical
value equal to 2.579. What is more, the P value is very small, much below
0.05, what reflects ratio between F and F crit. This means that we can reject

https://statistics.laerd.com/statistical-guides/one-way-anova-statistical-guide.php

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null hypothesis and therefore conclude that there is huge difference between
groups.

Uncertainties:
Calculating uncertainties is very important for analyzing experimental data.
We have already calculated mean and standard deviation, but what we also need is the
uncertainties involving mediums.
1) Total percentage uncertainty

Table 3.

Total percentage uncertainty of foam produced by the reaction of hydrogen

peroxide with homogeneous mixture at different temperatures.
Temperature of
Total percentage
homogenous solution
uncertainty of foam
/ C 0.1
/ %
100
0.00
40

6.67

30

2.76

20

5.50

10

13.64

2) Absolute uncertainty

Table 4.

Absolute uncertainty of foam produced by the reaction of hydrogen peroxide

with homogeneous mixture at different temperatures.
Absolute
Temperature of
uncertainty of foam
homogenous solution
/ cm
/ C 0.1
100

0.0

40

0.1

30

0.1

20

0.1

10

0.1

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Data presentation:
Now, as we collected and processed all data and calculated uncertainties, we can
present them together in one table and then translate into graph.
Table 5.

Mean height of foam with absolute uncertainty at different temperatures of

homogenous solution
Temperature of
homogenous solution
/ C 0.1

Mean height of foam

with absolute uncertainty
/ cm

100

0.000.0

40

1.500.1

30

3.510.1

20

1.810.1

10

0.760.1

I displayed results on the graph, but I was not able to make a trend line, because in my
investigation I did not find optimum temperature and its value.

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Conclusion:
Having my knowledge and going on background information, I can conclude that the
experimental results are reasonable and justifiable. During the experiment I confirmed that
my hypothesis was correct, because as we could notice with the increase the enzyme activity
expresses as the height of foam, increases with the increase of temperature. At low
temperature, enzymes work much slower, because the movement of particles is too slow, they
do not catalyze and therefore are not able to speed up the reaction. As temperature increases,
the rate of reaction increases to the optimum point, which in my experiment is little above
30C. We can see that at

, the height of foam rapidly decrease, slowing down

chemical reactions in Agarius biporus. This shows that the reaction starts to becoming
denatured and that this process is much faster than the increasing the enzyme activity. At the
end, at 100 C , the enzyme becomes denatured, the protein changes its shape and are useless
(inactive in biological reaction) and stop working. Points plotted on the graph 1, proves that
the slope of enzyme activity increases slower with increase the increase of temperature, than
decreases after reaching optimum temperature. It means that even a little higher temperature
than optimum, makes a significant damage to the enzyme.

Evaluation:
During this experiment a certain number of systematic errors were made while reading
the height of foam. I used ruler and my sight to measure those values. It could be improved by
use of a professional equipment, or by use of test tubes with scale on their walls. This would
to some extend decrease human error.
What is more, as I was conducting trials, homogenous mixture was kept in one place
and with time I noticed that at the bottom of beaker, the solution was a little bit more dense
than on the top. This could significantly affect my results because as I was taking solution
samples for the next trials, they might have different Agaricus bisporus concentration and
give less accurate results. The way to avoid it, is to stir the solution before taking a sample.
Moreover during heating homogenous substances test tubes were not covered, same
water evaporated from test tubes as well as from water bath. I did not replace the later lost
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from water bath before starting the next trials. This might affect, however slightly, but still
could change outcomes.
Timing 5 second is very difficult, I had to be really focus and despite my most effort
to measure the height of foam after exactly 5 second, I am painfully aware that it was unlikely
to detect result exactly at the same moment.
Using different way of measurement foam height will eliminate the major errors and
give much more accurate results. What is more, to avoid lose of temperature and evaporation
of water, test tubes during heating should be covered and conducting experiment for
when the rate of evaporation is high, water replacement in water bath might be good idea.
In my experiment I used different temperatures with 10C difference. I concluded that
the optimum temperature is little above 30C and was not able to give more specific result.
Having professional equipment, I would changed the scale with 5C difference.

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Bibliography:
Books:
1. Atkins P., de Paula J., 2006. Physical chemistry for the Life Sciences, UK, Oxford,

Oxford University Press.

2. Greenwood T. , Allan R., 2009. Senior Biology 2, UK, Edinburgh, Biozone.
3. Roberts M., Reiss M., 2000. Advanced Biology, UK, Cheltenham, Nelson.

Webs:

1. Lard statistics, One-way ANOVA. Available on: https://statistics.laerd.com/statistical-

guides/one-way-anova-statistical-guide.php. [Access: 09.01.2014]

2. Wikipedia, Agaricus bisporus. Available on:

http://en.wikipedia.org/wiki/Agaricus_bisporus. [Access: 09.01.2014]

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