However, as the enzyme reaches about 40°C in the substance, the break down rate shall begin to decrease due to the enzyme denaturing. This process is when the enzyme reaches a certain temperature at which its structure cannot cope with and begins to break down itself. Therefore I predict that the rate of reaction or breakdown will increase initially as the particles have more energy, then as the enzyme reaches the upper boundary of it’s optimum rate it will begin to denature and the rate shall decrease and finally stop. Producing the prediction graph shown below.
I predict the graph shall look as follows: -
To conduct the experiment I shall be using the following apparatus and equipment: -
- Thermometer
- Amylase solution
- Starch solution
- Iodine solution
- Water bath
- Test tubes
- Pipette
- Dipple tray
- Measuring cylinder
- Stopwatch
- Glass rod
- Benedict’s solution
These apparatus shall be set up as shown in the diagram on the next page: -
In this experiment there are few hazards facing the scientists conducting the experiment. The main risks are spilling a solution E.g. Iodine, on your clothes and it staining and the hot water from the water bath could burn someone. To overcome these risks the only precaution you can take is to perform the experiment in full concentration and handle all apparatus with care.
For the experiment to be a fair test I will have to have the same amount of starch and amylase in each solution. And to ensure the substances are the correct temperature, place them in the temperature five minutes before conducting the experiment. I shall strictly keep the concentration, surface area and volume of the starch and amylase all at constants and maintain the temperature as a variable to ensure a fair test.
- Set up the apparatus as shown in the diagram, next put a drop of iodine in each of the dipples in the dipple tray.
- In the first of three test tubes put in 5ml of liquid starch, and in the second put 5 drops balls of amylase.
- And in the third test tube put the contents of the first and second test tube after having been prepared for the suitable temperature at which the experiment is being carried out under.
- After every minute, take a drop from the solution and place it on a dipple on the dipple tray using the glass rod.
- The colour of the droplet shall be determined on how much starch is left in the solution.
- Each colour shall be recorded and analysed. As the experiment continues, the colour of the droplet placed in the iodine changes as the starch is being broken down.
- Once the colour turns to a constant it indicates the starch has been fully broken down and the experiment has ended for that specific temperature.
Before carrying out this experiment I carried out a trial run as part of my preliminary work for this investigation. This was done to gain a range for the main experiment and background knowledge and experience for this investigation. Through doing the trail run I was able to collect results and data that indicated a suitable range for me to use for the proper experiment.
I shall conduct this experiment for every 20°C in my range, which is 0° to 100°C. This is my range as it is the range of temperature at which water is in a liquid state.
Results for the experiment being carried out at the temperature .
Results for the experiment being carried out at the temperature .
Results for the experiment being carried out at the temperature .
Results for the experiment being carried out at the temperature .
I conclude from the information that I have obtained through carrying out the experiment, my results table and graph that: -
- Iodine reacts with starch to change its colour to black.
- If amylase is present the starch will be broken down so the iodine will not turn black.
- The enzyme amylase is affected and influenced by temperature.
- If the temperature is too high the enzyme will denature and no longer be able to perform its breaking down of starch.
- The process of denaturing dramatically occurs at 0°C, 80°C and 100°C.
- At 20°C, 40°C and 60°Cthe amylase successfully breaks down the starch completely.
- This shows the amylases optimum temperature is between 20°C and 60°C.
- The amylase turns white when it’s denatured.
- The results from the benedict’s solution test also confirm this, and prove my hypothesis to be correct. An orangey or yellowy colour proves the enzyme to have worked, this type of colour was present in the test in the enzyme’s optimum temperatures 20°C to 60°C.
- The rate of reaction can be calculated by dividing the amount of starch by the time taken to react. This can show the breakdown of units per time i.e. the speed of the breakdown of starch.
Rate = Amount of starch
Time taken
0.25g ÷ 0 mins = 0.25g/min (at 0°C)
(No starch broken down at 0°C)
0.25g ÷ 16 mins = 0.0156g/min (at 20°C)
0.25g ÷ 17 mins = 0.0147g/min (at 40°C)
0.25g ÷ 12 mins = 0.0208g/min (at 60°C)
0.25g ÷ 11mins = 0.0227g/min (at 80°C)
0.25g ÷ 0 mins = 0.25g/min (at 100°C)
(No starch broken down at 100°C)
- I have calculated the rates of reactions to provide further evidence to my analysis and conclusion that the optimum temperature range is around 55°C. This is proved by my calculations, as the rate of reaction for 60°C is the highest calculated.
- My prediction that the enzyme will be at its optimum rate around human body temperature was correct, but the experiment showed that the enzyme could work between 20°C and 60°C with some success.
- Amylase speeds up the breakdown of long chain starch molecules into smaller chains of maltose. Enzyme molecules have a very precise three-dimensional shape. This includes a ‘dent’, which is called the active site. It is exactly the right size and shape for the enzyme’s substrate to fit into (in the case of amylase this is starch). When a substrate molecule slots into the active site, the enzyme ‘tweaks’ the substrate molecule, pulling it out of shape and making it split into product molecules.
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High temperatures make enzymes inactive: this is because they are proteins, which are damaged by temperatures above about 40 degrees Celsius. In this investigation it was found that amylase operated fastest at about 55 OC and was damaged above this temperature. The enzyme is damaged because the molecules are moving faster, these bombard the active site of the enzyme changing its shape; when it’s shape has been changed then the starch will no longer fit in the active site and therefore cannot be digested. By 80 degrees Celsius the amylase was completely denatured. From this information I can conclude that the optimum operating temperature for the enzyme amylase is around 55 degrees Celsius.
There were few strange results obtained during my experiment. Apart from when carrying out the experiment at 0°C the temperature rose to 3°C due to the ice melting. The colour of the iodine’s solution after 10 minutes was a strange result, as it did not follow the trend of a dark brown colour. At 10 minutes the iodine colour turned a lighter brown due to the enzyme working more efficiently as it was less denatured.
There are many improvements that can be made to the experiment, these are listed below: -
- A higher quality water bath would achieve more accurate temperatures giving more accurate readings. This would also solve the problem of using ice to measure the experiment at 0°C, therefore reducing the risk of and achieving strange results.
- More time for preparation of the experiment would help me to achieve more accurate results. There was a time limitation to the experiment due to my lesson being 1 hour long. More time would enable longer time to prepare the starch and amylase solutions beforehand, therefore having the solutions at accurate temperatures enabling me to achieve more accurate results.
I will modify and expand the original experiment in a number of ways. Firstly I must decide what I’m going to investigate.
I am going to investigate the effect of pH on the activity of the enzyme amylase.
I predict that the pH level of the solution will affect the rate of reaction in which the breakdown of starch occurs. With a change of pH the weak bonds holding the enzyme together will break. This will denature the enzyme, thus deactivating the active site. As enzymes normally exist within cells where the pH is 7, the most favourable pH is obviously 7. In other words, the more acidic or alkali, or the further away from a neutral pH the solution, the quicker the rate of reaction due to the weaker bonds.
Method to investigate the effect of pH on the activity of the enzyme amylase:
- Pour amylase solution into a test tube to a depth of 2cm.
- Half fill another test tube with a 4% starch solution.
- With a pipette place a drop of iodine into each dimple in a dimple tray.
- With a glass rod lift a drop of the starch solution from the test tube and mix it with the first drop of iodine in the first dimple in the tray. A blue/black colour should develop; this will be used as the control.
- Rinse the glass rod.
- Add 2cm of the appropriate pH buffer to the starch solution and shake.
- Pour amylase solution into the test tube of starch and shake quickly.
- Repeat steps 4 & 5 (for the amylase, starch & pH buffer mixture) every 30 seconds until a blue/black colour no longer develops.
- Record the results in a table. Repeat steps 1-8 for each pH buffer range until all the provided pH buffers have been used.