Name: NUR ANIS ATIKA BINTI ZAINAL ABIDIN

Class: M11N

Date : 19th October 2011

Tittle: ENZYME ACTIVITY

Objective

To investigate the effect of different temperature which is 5 0 C, 200C, 320C, 600C and 1000C the
rate of enzymatic reaction of diastase on starch given that it is immersed in water bath with the
corresponding temperature or ice for about 5 minute.

Introduction

Enzymes  are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at

the beginning of the process, called substrates, are converted into different molecules,
called products. Almost all chemical reactions in a biological cell need enzymes in order to occur
at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a
few reactions from among many possibilities, the set of enzymes made in a cell determines
which metabolic pathways occur in that cell.Like all catalysts, enzymes work by lowering
the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the reaction.
As a result, products are formed faster and reactions reach their equilibrium state more rapidly.
Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed
reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor
do they alter the equilibrium of these reactions. However, enzymes do differ from most other
catalysts in that they are highly specific for their substrates. The way enzymes work is called the
‘Lock and Key’ mechanism. The enzyme acts as the lock and the substrate acts as the key. The
two fit together in the active site of an enzyme, and are said to be complimentary. The
substrate and enzyme, when combined together is called an enzyme substrate complex.

Diastase is an enzyme that catalyses starch into maltose through hydrolysis process:

Starch+water diatase maltose

→

Another properties of enzymes is it can be denatured by heat 1. If starch is present thus the
colour of iodine will turn from brownish into dark blue.

1
http://en.wikipedia.org/wiki/Enzyme
Research Question

What is the effect of different temperature which is 50 C, 200C, 320C, 600C and 1000C the rate of
enzymatic reaction of diastase on starch given that it is immersed in water bath with
corresponding temperature or ice for about 5 minute?

Hypothesis

As the temperature increases, the rate of reaction will increase until the optimum temperature
is reached, then after this point the rate will decrease as the temperature continues to increase.

At low temperatures, the kinetic energy of the molecules is lower, so the rate of effective
collisions that can achieve the activation energy needed to start the reaction will be very low. In
order to convert substrate into product, enzymes must collide with and bind to the substrate at
the active site. Thus the rate of reaction will be very slow. When molecules collide, the kinetic
energy of the molecules can be converted into chemical potential energy of the molecules. If
the chemical potential energy of the molecules becomes high enough, the activation energy of
a reaction can be achieved and the reaction can occur. Thus the greater the kinetic energy of
the molecules in a system, the greater is the resulting chemical potential energy when two
molecules collide. As the temperature of the mixture of diastase and starch is increased, the
kinetic energy of the molecules increases and it is possible that more molecules per unit time
will collide and reach the activation energy. Thus the rate of the reaction will increase.
Increasing the temperature of a system will increase the number of collisions of diastase and
starch per unit time. Thus, within limits, the rate of the reaction will increase.

However, when the temperature reaches a certain point, the temperature becomes too high
for the enzymes, and the enzymes are denatured. As the temperature of the mixture of
diastase and starch is increased, the internal energy of the molecules in the mixture of diastase
and starch will increase. Some of this heat may be converted into chemical potential energy. If
this chemical potential energy increase is great enough some of the weak bonds that determine
the three dimensional shape of the active proteins will be broken. This could cause the protein
to denature and thus inactivate the protein. Thus too much heat can cause the rate of an
enzyme catalyzed reaction to decrease.
Variables

Variables Uni Range Possible effect on Methods on

t result manipulating the
variables
0
Independent Temperature C 50C,200C For 50C melted ice is used
,320C,60 .For 200C melted ice with
0
C,2000C some tap water is used
to get the 20 0C
temperature.
Water bath is used for
320C ,600C and the
temperature is adjusted
accordingly.For 200 0C
the water is heated until
boiled.
Dependent Time taken s 0-3600 s The iodine test for each
for the starch diastase-starch solution
to completely was done for at least an
hydrolysed hour at 5 minute
intervals.
Fixed Volume of ml 5ml Different volume 5ml of amylase solution
diastase may affect the is used for all the
results as more experiments, and the
enzymes will be volume measured using a
present to react 20ml measuring cylinder.
with starch thus
affecting the rate
of reaction.
Volume of ml 5ml Different volume 5ml of starch solution is
starch may affect the used for all the
solution results as more experiments, and the
substrates will be volume measured using a
present to react 20ml measuring cylinder.
with diastase
thus affecting the
rate of reaction.
Time interval min 5 min A fixed time The time interval for
interval is each iodine test is set at
necessary to be every 5 minute.
consistent in
performing the
iodine test.
Materials and apparatus

Materials Quantity Volume/size

Starch solution - 15ml
Amylase - 15ml
Iodine solution 1 reagent bottle -
Ice cubes 1 basin -

Apparatus Quantity Volume/size

Measuring cylinder 1 20ml
White tile 1 -
Thermometer 1 -
Water bath 1 -
Dropper 2 -
Glass rods 3 -
Stopwatch 1 -
Test tubes 3 -

Method

1. 3 test tubes are labelled as Trial 1,Trial 2 and Trial 3 for 5 0C temperature.
2. 5ml of diastase solution is measured and added into each test tube.
3. The test tubes are placed into ice and the temperature is controlled for exactly 5
minutes.
4. 5ml of starch solution is measured and added to each test tube and mixed with a clean
glass rod. The temperature is monitored continuously.
5. At intervals of 5 minutes, each test tube is tested for the presence of starch. One drop of
diastase-starch mixture is withdrew, placed on a white tile with grooves, and a drop of
iodine solution is added.
6. The iodine test is continued for at least 1 hour.
7. Step 1 – 5 is repeated using a clean test tube for 200C,320C,600C and 2000C.
8. A complete record of the observations are made and presented in the table below

DATA COLLECTION

Quantitative data

Table of temperature and time taken for the iodine test to change the colour

Time taken Temperature ˚C (±0.5˚C)

 for the iodine 5˚C 20˚C 32˚C 60˚C 200˚C
test to change 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
the colour(s)
(±0.1s)
0.0 O O O O O O O O O O O O O O O
300.0 O O O O O O O O O O O O O O O
600.0 O O O O O O O O ̷ O O O O O O
900.0 O O O O O O ̷ ̷ ̷ O O O O O O
1200.0 O O O O O O ̷ ̷ ̷ O O O O O O
1500.0 O O O O O O ̷ ̷ ̷ O O O O O O
1800.0 O O O O O O ̷ ̷ ̷ O O O O O O
2100.0 O O O O O O ̷ ̷ ̷ O O O O O O
2400.0 O O O ̷ O O ̷ ̷ ̷ O O O O O O
2700.0 O O O ̷ ̷ ̷ ̷ ̷ ̷ O O O O O O
3000.0 O O ̷ ̷ ̷ ̷ ̷ ̷ ̷ O O O O O O
3300.0 ̷ ̷ ̷ ̷ ̷ ̷ ̷ ̷ ̷ O O O O O O
3600.0 ̷ ̷ ̷ ̷ ̷ ̷ ̷ ̷ ̷ O O O O O O
 ̷ -iodine colour remain unchange/brownish yellow
 O - iodine change from brownish yellow to dark blue

Qualitative data

1. The initial colour of the iodine is brownish yellow.

2. The colour of iodine change from brownish yellow to blue black if it is tested with
mixture of diastase and starch at certain time and temperature.
3. Some of the iodine remain unchanged after tested with diastase and starch at certain
time and temperature.
Data processing

1. Average time taken for the starch to be hydrolysed by the enzyme:

∑ of the time taken for theiodine colour ¿ remainbrownish ¿ for the 3 trials 3¿

Eg: For 50C

3300+ 3300+3000
¿
3
¿ 3200.0 s

2. Rate of enzymatic reaction of diastase on starch:

1
average time taken

Eg: For 50C±0.50C

1
¿
3200

¿ 0.0003125s-1

3. Uncertainty of rate of reaction, ∆R:

∆ time
∆ R= ( Rate of reaction)
averagetime taken(s)
Eg: For 50C±0.50C

0.1
∆ R= (0.00031)
3200.0 (s)
¿ ± 9.7 ×10 -9s-1

Temperature (0C) Average time taken Rate of reaction,R Uncertainty of

(±0.5°C) for starch to be (s-1) rate of reaction,
hydrolysed (s) (±0.1s) ∆ R, (s-1)
5.0 3200.0 0.000313 ± 9.69 ×10 -9
20.0 2300.0 0.000435 ± 1.87 ×10 -8
32.0 800.0 0.001250 ± 1.56× 10-7
60.0 ∞ 0 0
 100.0 ∞ 0 0

0
Rate of reaction (s-1)

0
0 1 2 3 4 5 6 7 8
0

Temperature (0C)
Graph of rate of enzymatic reaction
against the temperature
Discussion

1. The mixture of diastase and starch solution is immersed into the ice or water bath for 5 minutes
to ensure that the temperature is controlled to the specific temperature needed.
2. Iodine test is conducted to detect the presence of starch in the mixture of solution.Iodine’s
original colour is yellowish brown.When starch is present in the solution that is tested with
iodine ,the colour will change from yellowish brown to dark blue.If there is no change in colour
of iodine,this notate that starch is completely hydrolysed by the diastase into maltose.

Analysis of the graph

1. Initially,a curve showing positive correlation is obtained however at certain point it will drop
and shows negative correlation between the rate of enzymatic reaction and the
temperature. The graph shows that as the temperature increases, the rate of reaction
increases. The highest point on the curve is the assumed the optimum temperature for the
diastase enzyme to catalyse the reaction of starch into maltose, as this is the point where
the rate of reaction is at maximum.The graph shows that the rate of reaction initially
increases slowly but between 200C and 320C the rate of enzymatic reaction increases
steeply.
2. At 5.0± 0.5 °C, the rate of reaction is at lowest point because at low temperatures,
the kinetic energy of the molecules are low, thus the rate of effective collisions that
can achieve the activation energy needed to start the reaction will be very low. In
order to convert substrate into product, enzymes must collide with and bind to the
substrate at the active site. At this low temperature, the rate of collisions will be
very low, therefore, the rate of reaction will be very slow.Therefore the time taken
for the starch to be hydrolysed is longer for 50C± 0.5 °C.
3. At 20± 0.5 °C the rate of reaction increases more than 50C± 0.5 °C because the kinetic
energy of the molecules are slightly higher,thus the rate of effective collisions that
can achieve the activation energy needed to start the reaction will be slightly higher.
At 20± 0.5 °C the rate of collision will be slightly higher .Therefore the time taken for
the starch to be hydrolysed is shorter than that of 50C± 0.5 °C and the rate of
reaction of diastase on starch in 20± 0.5 °C is higher than 50C± 0.5 °C.
4. At 32± 0.5 °C temperature the time taken for the iodine colour to remain
unchanged is the lowest thus it indicates that the rate of reaction is the highest
because at this temperature,the temperature is assumed the most optimal for the
enzyme to work as the rate of reaction is the highest.The kinetic energy of the
molecules are the highest, thus the rate of effective collisions that can achieve the
activation energy needed to start the reaction will be very high.
5. At 600C to 1000C there is no reaction occurring.It is assumed that the temperature is
too high for the activity of enzyme.Denaturation might occur that cause the enzyme
to dysfunction and become inactive thus it will change the shape of the active site of the
enzyme. Starch can no longer be hydrolysed as it cannot fit into the active site of the
enzyme.Therefore throughout the experiment the colour of iodine will always turn from
yellowish brown to dark blue.

Evaluation

Limitations Suggestion
The heat from our hand might affect the Hold the beaker using test tube holder when
temperature of the mixture of starch and conducting the experiment.
diastase in the test tube.
 Parallax error might occur while taking the
measurement of starch solution and diastase
solution.
Iodine might be oxidized if left open during
the experiment
The dropper used to drop the iodine might
be contaminated with the mixture of
diastase-starch solution
Impurities in the apparatus might affect the
result.
Ensure that the eyes is parallel to the
meniscus point so that precise reading can
be taken.
Close the iodine reagent bottle with it’s cap
and only open it when needed.
Rinse it after every iodine test is conducted.
Rinse all the apparatus using distilled water
before it is used.

Conclusion
It can be concluded that as the temperature increases, the rate of reaction of diastase
(enzyme) increases, until the optimum temperature which is about 32 0C; after the
optimum temperature, the rate of reaction decreases as the temperature continues to
increase and at one point the reaction will stop because the too high of temperature
causes the enzyme to denature. This is because as the temperature increases, the
kinetic energy of the molecules increase, and the molecules collide more frequently
with high energy, so more molecules can achieve the activation energy needed for the
reaction, hence increasing the number of reactions per second, which is the rate of
reaction. However, this apply only till a certain point, where highest rate of reaction is
reached. This is the optimum temperature, and after this point the gradient of the graph
will slope downwards, as the rate of reaction decreases, because the temperature is too
high and the diastase are denatured, losing its three-dimensional form, becoming
inactive and unable to catalyse the reaction of starch to maltose.

References

1. Biology for the IB Diploma, CJ Clegg, Hodder Education, London, 2007.

2. Biology IB Study Guide, Oxford University Press, 2007.
3. Wikipedia
(http://en.wikipedia.org/wiki/Enzyme)