Digestion and Enzymes

Digestion and Enzymes – APP.
Hypothesis:
The enzyme, Amylase which is used to break down carbohydrates will work the best when heated at 40°C. Also, as the temperature increases the reaction rate of amylase increases too. However, the reaction rate of amylase will start decreasing when the temperature reaches the enzyme’s optimal temperature.

Many enzymes are specific for a certain substrate. For example, lipase is a specific enzyme for fat substrates and protease, a specific enzyme for protein substrates. In the same way amylase is a specific enzyme used for carbohydrate substrates. In our experiment, our substrate is starch and so our enzyme is amylase. I think that amylase will not work properly when heated at 10°C because enzymes need heat for the particles to gain kinetic energy and collide with each other. At10°C, enzymes don’t move as fast as they can if the temperature is increased, and thus the collision and the reaction don’t take place. However, if the temperature is increased over its optimal temperature then the enzymes become denatured. The breaking of a substrate happens, when a substrate molecule fits inside its specific enzyme molecule, so when an enzyme molecule becomes denatured, the 3D shape of the enzyme molecule changes and so the substrate can no longer inside the active site of the enzyme molecule due to its denatured shape.

So I think the amylase might become denatured when heated at 80°C and thus it won’t be able to work. This tells me that amylase will work the best at 40°C as it might be amylase’s optimal temperature.

Variables:

I will change the temperature, at which the solution is heated. I will measure the time it takes for the colour of starch and iodine mixture to disappear. The independent variable in this experiment will be the temperature and the dependent variable time taken between the mixing of amylase and starch solutions and the end of the test. The control variables will be: Volume of amylase solution in each test tube –
The volume of amylase solution in each test tube needs to be a constant number, because if there is different amount of amylase in each test tube would make it an unfair test as the test tube with more or less amylase might react different to different temperatures. So a specific amount of amylase should be used in each of the test tubes.

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 changes 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 can be helpful.

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.

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.

Water level in bath

Amount of iodine.

Equipment:

Test Tubes x6
Beaker
Pipette
Cavity Tile
Ice cubes
Water Bath
Goggles
Gloves
Lab Coat
Measuring Cylinder
Stop Watch
Iodine
Amylase solution
Starch.
Universal Indicator.
Bunsen Burner

Method:

1. Take 5ml of amylase and add it into a test tube and add 5ml of starch solution into another test tube.
2. Make sure that the amylase solution and starch solution you used in this experiment must have a neutral pH, you may test the pH values by using pH universal indicator. This can be done when before
3. Take a water bath and add water and wait till the temperature reaches 40 C.
4. Once the temperature is 40 C, place the two test tubes in a water bath for two minutes.
5. Meanwhile take a cavity tile and add a drop of iodine in each cavity.
6. Pour the amylase solution into the tube of starch solution. Stir well using a pipette.
7. Using the pipette, remove a small amount of the mixture and add a drop to the iodine solution in the cavity tile
8. Repeat the iodine test at intervals of two minutes, washing the pipette with distilled water between each test until the mixture fails to give a blue/black colour with iodine.
9. Record the total time taken between the mixing of amylase and starch solutions and the end of the test. This is the time taken for all the starch to be digested by amylase at 40 C
10. Repeat the whole experiment at other temperatures (10 C and 60 C)

For temperatures higher than the room temperature using a water bath and when the required temperature is reached remove the Bunsen burner. Gentle heating at intervals may be necessary to maintain the temperature of the water bath.
For the temperatures lower than the room temperature, use ice cubes to bring the temperature down.

It is important that for each of these experiments the amylase and the starch solutions are warmed or cooled to the required temperature before mixing them.
I will test 10 C, 40 C and 60 C and I will repeat my experiment three times in order to get fair and reliable results.

Risk Assessment:

Risk
Hazard
How to prevent it?
Starch solution or iodine.
May get into your eyes.
Wear goggles.
Some water bathes are very hot.
Can burn your hand or any other parts of your body.
Don’t directly touch the water inside the water bathes. Using a rack to put test tubes into the water bathes.
Water and electricity.
If water is spilled on a nearby switch, it can electrocute the person.
Keep beakers, test tubes and water bath away from any electrical equipment.
Breaking a test tube.
Glass pieces can be harmful to people and to you.
Keep the equipment in the middle of the desk and not near the edges of the table. Also make sure to stand and do the experiment.
Universal indicator.
If comes in contact with eye, may cause eye irritation. If comes in contact with skin, may cause skin irritation. If swallowed, can cause blindness and nausea.
Wear gloves while holding the universal indicator. Wear goggles to stop the contact with your eyes.
Report the teacher immediately if accidently swallowed.
Hair.
It can get dipped into chemicals and may damage your hair. It makes it difficult to see, making accurate measurements and observations almost impossible.
Tie your hair back.
Spilling chemical over your body.
Can damage your clothes and may possibly come in contact with your skin which can be very harmful and may damage your skin.
Wear Lab coat.
Not cleaning up the the area once the experiment is finished. Coming in contact with chemicals.
It may be harmful for someone who is not aware of the experiment and may unknowingly come in contact with harmful chemicals or may trip over the equipment causing fatal injuries.
Clean up the experimental place and wash your hands to avoid others getting any further contact with chemical germs.
Crushed ice can damage your hands.

Injure your hand.
Wear safety gloves.