Internal Assessment

1. Investigate the effect of temperature on amylase activity
Introduction
Amylase is an enzyme that catalyses the breakdown of starch into sugars. Amylases are found
in almost all plants, animals and microorganisms. Large amounts of amylase occur in germinating
cereals, and in the pancreas and saliva of higher animals.
Aim
The aim of this experiment is to find out the rate of reaction between amylase and starch in a
range of different reaction temperatures.
Hypothesis
As the reaction temperature of amylase solution and starch solution increase, the reaction rate
of amylase and starch will increase. After reach the optimal temperature of amylase, the reaction
rate of amylase and starch will rapidly decrease.
The lock and key model explains my hypothesis. Most enzymes are very specific for a certain
substrate, the active site on the enzyme molecule forms a "keyhole" into which the substrate fits
like a key, the substrate molecule is then broken up into many smaller pieces. The higher reaction
temperature, the more kinetic energy the substrate molecules will have. It means that the
molecules can move faster and have a greater chance to collide and react. However, if the
reaction temperature is too high, enzyme may become denatured. Denaturation is a structural
change in a protein that alters its three-dimensional shape and causes the loss of its biological
properties1. As the temperature rises the amount of active enzyme progressively decreases, and
the rate of reaction is slowed. So there should be an optimal temperature for each enzyme.
Research Question
How does a range of different reaction temperature (0°C, room temperature during the
experiment, 40°C, 60°C, 80°C) effect on the rate of reaction between amylase and starch?
Variables
Dependent variables: the required time for the disappearance of blue-black color (color of Iodine
solution mix with starch solution ).
Independent variables: reaction temperature (0°C, room temperature during the experiment,

2. 40°C, 60°C, 80°C).
Controlled variables
Significance/effect if not controlled Method of control
Volume of amylase solution in each test tube The volume of amylase solution in
each test tube needs to be a constant number, because we do not know if this variable will affect
result. To ensure that the result is accurate, it is necessary to maintain same volume of amylase
solution in each test tube. So that the reaction temperature will be the only independent variable
influence the required time for the disappearance of iodine color. Using same measuring
cylinder and read accurately.
Volume of starch solution in each test tube The volume of starch solution in each test
tube should be the same. If not, the rate of reaction between amylase and starch will be affected.
Some test tubes which have more starch solution than others may decrease the time required to
reach the reaction temperature. And also, the rate of reaction between amylase and starch will
be affected because it may take more time to completely break down starch molecules if there is
more starch solution. The required time for the disappearance of blue-black color in test tubes
will become inaccurate. Using same measuring cylinder and read accurately.
pH value Change in pH can have a dramatic effect on the rate of amylase –catalyzed reaction.
This effect of pH is because the structure of a protein is maintained by various bonds within the
three-dimensional structure of the protein. A change of pH from the optimum values alters the
bonding patterns, progressively changing the shape of the molecules. If the molecules are
denatured, the reaction cannot happen, the result will become unreliable. pH value will be a new
independent variable if it is not controlled. Using pH test strips to test the pH value of amylase
solution and starch solution before the experiment.
Concentration of amylase solution in each test tube Concentration of amylase
solution in each test tube should be the same. If the volume of amylase solution is same in
each test tube, the lower concentration means that there are less amylase molecules in that tube.
If the amylase molecules are not enough to combine with starch molecules, starch cannot be
completely broken down, the mixed solution may still appear blue-black color. Therefore, it will
affect our result if it is not controlled. Using amylase solution in the concentration level.
Concentration of starch solution in each test tube Concentration of starch solution
is the substrate concentration. As the substrate concentration increases, the rate of reaction
increases until all active sites engaged in catalysis. To prevent Concentration of starch solution
from being a factor of changing the rate of reaction, it must be controlled. Using starch solution in
the concentration level.
Uncontrolled variables Significance/effect if not controlled Method of control
Microorganism Microorganism will slowly pollute the solutions and may cause some
unpredictable results. Sanitize laboratory before doing this experiment.
Light Sunlight in different stages carry different amount of heat. It may cause the room
temperature changes slightly. Avoiding sunlight shooting straight.

3. Apparatus
100cm3 beakers X2
Test tubes X10
50cm3 1% starch solution
50cm3 1% amylase solution
Thermometers X11
15cm3 Iodine solution
Pipette
Stopwatch X5
Test tube rack x3
Marker pen
10cm3 measuring cylinder X2
Water bath X3 (40°C, 60°C, 80°C)
150g crushed ice
pH test strips
Lab coat
Safety gloves
Safety goggles
Risk Assessment
1. Starch solution and Iodine solution are required for this experiment.
Wear safety goggles to prevent any starch solution or iodine solution getting in your eyes.

4. 2. Some water bathes are very hot.
Don’t directly touch the water inside the water bathes. Using a rack to put test tubes into the
water bathes.
3. Crushed ice can damage your hands.
Wear safety gloves to protect your hands from cold and stiff.
Method
1. Before start the experiment, lab coat, safety glasses and safety gloves must be worn. Clean
up your hands as well.
2. Measure the room temperature with a new thermometer. Use marker pen to name beaker 1
and beaker 2. Divide 10 test tubes into 2 groups, name them from 1-5 in both of the group. You
will need five stopwatches to record the time for the disappearance of blue-black color in test
tubes, name them from 1-5 as well.Stopwatch 1 is corresponding to the test tubes 1 in beaker
1(0°C ),stopwatch 5 is corresponding to the tst tubes 5 in 80°C water bath. Record the room
temperature.
3. Add crushed ice into beaker 1 and fill it with tape water. Beaker 2 only fills with tape water.
Put a thermometer in both of the beakers. After a few minutes, check the thermometers in
beaker 1 and beaker 2 to see if they have reached 0°C and the room temperature. Once the
temperatures reach our reaction temperatures, the experiment may start.
4. Make sure that the amylase solution and starch solution you used in this experiment must
have a , you may test the pH values by using pH test strips. Add about 3cm3 of amylase solution
into a 10cm3 measuring cylinder,add more drops or pour away more drops until it reaches the
3cm3 mark. And pour the solution into test-tubes 1-5 in group 1. Repeat the step to the starch
solutiona and pour them into test-tubes 1-5 in group 2.
5. Put test tubes 1 from group 1 and group 2 into beaker 1, and put test tubes 2 from group 1 and
group 2 into beaker 2. Place test tubes 3 in a test tube rack and put it into water bath(40°C ). Test
tubes 4 and 5 should be puttrd into water bath 60°C and 80°C with racks.
6. Check the temperature of solutions in each container every 30 seconds by using thermometers
to determine the current temperature of the solutions. Be careful not to mix thermometers up,
they are single-use items in this experiment.
7. If the two types of solution in one container have already reached the reaction
temperature, mix them up and start the corresponding stopwatch as fast as you can. Add a drop
of iodine solution immediately into the mixture of amylase solution and starch solution. The
solutions cannot be taken out of the beakers or water bath during your operation, it will prevent
the solutions loss of heat in air temperature. Observe the color change in test tube, stop the
stopwatch when the blue-black color completely disappears, record the time. Repeat it five times,
observation of color changes in the five test tubes can proceed at same time.
8. Repeat step 2-7 to get accurate results.
9. The experimental place and the tools should be cleaned up as the experiment is

5. completed.
Observation
There is no obvious color change in the five test tubes, the blue-black doesn't disappear. No other
phenomena occured. After waiting for 25 minutes, The test tubes appear blue-black color, there
is still no color change in any of the test tubes.
Conclusion
The experiment is basiclly not successful, the test result is not essential.
Evaluation of results
The result doesn't show a lot of valuable information to me. No any color changes,the test tubes
still appear blue-black color, it means that the experiment is a failure. If starch reacts with
amylase, the color should turn to brown-yellow(color of iodine solution). Only thing I received
from the result is there must be some mistakes in my method.
Evaluation of the method
I am not able to prove my hypothesis correct. I fell that my method could be improved
dramatically as stated below.
Weakness/limitation Reason Improvements
Too many drops of iodine solution. I didn't control the drops of iodine solution should be
added into each test tubes. Too many drops may darken the color, and it would become difficult
to see some small changes. Use a smaller pipette and only add one drop of iodine solution in
each test tube.
The heat has already broken down starch before mixing with amylase solution. Starch molecules
will be broken down by heat. I leave the starch solution in water bathes for too long, the heat
accelerated the decomposition of starch. And there is no starch anymore when I added it into
amylase solution. Therefore the reaction wouldn't occur, the color wouldn't disappear. Check
the temperature more frequent; don't leave the solutions in the water bathes for too long.
temperature of iodine solution. The iodine solution I used in this experiment is in air
temperature. When I added iodine solution, the reaction temperature between starch and
amylase would be greatly affected. The result will become unreliable. Prepare another 5 test
tubes for iodine solution, and put them in beakers and water bathes to achieve a same reaction
temperature with amylase solution and starch solution.
It may cause loss of heat when I open the water bath to check the temperature of the solutions.

6. Because the loss of heat of the solutions, the temperature may be lower than it should be. It
will increases the time of the solution soaking in hot water, which is not good for the result. Use
beaker and burner to instead water bath or use electronic temperature.
References
1. "Biology for IB Diploma" by C.J.Clegg, published by Hodder Education. 2007.
Bibliography
Ramasubbu, N.; Paloth, V.; Luo, Y.; Brayer, G. D.; Levine, M. J. (1996). "Structure of Human Salivary
α-Amylase at 1.6 Å Resolution: Implications for its Role in the Oral Cavity".
Campbell, Neil, J. Reece, and L. Mitchell.Biology 5th Edition. Menlo Park, CA.
Benjamin/Cummings, 1999.