- Pour amylase solution into a test tube to a depth of 2cm.
- Half fill another test tube with a 4% starch solution.
- Cool both test tubes and maintain at 10 degrees Celsius.
- 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 first 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 water to starch solution.
- Pour amylase solution in to the test tube of starch and shake quickly (measure temperature, try to maintain at 10 degrees Celsius).
- Repeat steps 4&5 (for the amylase, starch mixture) every 30 seconds until the blue/black colour no longer develops.
- Record the results in a table. Repeat steps 1-9 increasing the temperature by 5 degrees Celsius each time until the enzyme is denatured, this should be about 40 degrees Celsius.
To ensure that the tests are fair I will only vary two factors (at different stages of the experiment not simultaneously). All the quantities will have to be carefully measured since small variations in the amount of enzyme used can make significant variations in the results. In the experiment where temperature is investigated, 2cm of water is added. This is because the starch solution is neutral and in the previous investigation (for pH) 2cm of the appropriate buffer was added. If the 2cm of water were not added then it would not be a fair test since the volumes used in each part of the investigation would be different. If they were different then this would affect the results since the solution would be different concentrations and therefore one would react faster than the other would.
Apparatus:
I have chosen to use a measuring cylinder to measure the volumes of substances used since it is more accurate than a pipette. I will also use an electronic water bath for maintaining the mixture at a temperature above room temperature since the temperature is more accurate than a water bath above a Bunsen burner. I will have to use ice from a freezer to reduce the temperature of the mixture to reach 10 degrees Celsius. A 100 degrees Celsius thermometer will provide the temperature results of a sufficient accuracy (to 1 degrees Celsius). The pH buffer range is pre-prepared therefore I do not have to concern myself with measuring and maintaining pH levels.
Variables:
I have chosen to repeat the experiment 3 times this will allow me to calculate an average time. This will ensure that there are no abnormal results and it will increase the accuracy. I have decided to start the temperature at 10 degrees Celsius and increase by 5 degrees Celsius each time since it will allow me to see the increase and decrease of the enzymes activity. It should also de accurate enough for me to predict an optimum operating temperature to an accuracy of 5 degrees Celsius.
Enzymes:
Substances called catalysts speed up many chemical reactions. Catalysts called enzymes control the metabolic reactions in the body. Amylase is an enzyme; it is present in the digestive system of many animals. Amylase speeds up the breakdown of long chain starch molecules in 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 enzyme’s substrate to fit into (in the case of amylase this is starch). When a substrate molecules slots into the active site, the enzyme ‘tweaks’ the substrate molecule, pulling it out of shape and making it split into product molecules. High temperatures make enzymes inactive: this is because they are proteins, which are damaged by temperatures above about 40 degrees Celsius. Most enzymes work best at a pH of about pH7. This is also because they are proteins, which are damaged by very acidic or very alkaline conditions. Due to the enzyme’s unique site it can only convert one kind of substrate molecule into one kind of product.
Results
First of all the average time taken for the starch to be digested in each condition was calculated. First the temperature experiment results:
Graph 1
And the results for the pH experiment:
Graph 2
The results were then plotted on a graph (see Graphs 1&2) and the points joined together.
Form graph 1: I can see that as the temperature of the mixture increases, the time taken for the amylase to digest the starch decreases. This happens faster at 55 degrees Celsius, however if the temperature continues to rise then the time taken for the amylase to digest the starch rapidly increases. By the time 80 degrees Celsius is reached the amylase dose not digest the starch.
From graph 2: I can see that as the pH of the mixture increases, the time taken for the amylase to digest the starch decreases. This happens faster at pH5. At higher pH level than pH5 the time taken for the amylase to digest the starch increases.
In this investigation it was found that amylase operated fastest at 55 degrees Celsius but it 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 its shape has been changed denatured. For this information I can conclude that the optimum operating temperature for the enzyme amylase is 55 degrees Celsius.
Most enzymes work best at a pH of about 7. However in this experiment the amylase worked faster at pH5. This pH is lower than suggested above because the amylase is a bacterial enzyme, which has an optimum pH then the starch will on longer fit in the active site and therefore cannot be digested. By 80 degrees Celsius the amylase was completely between pH 4 and Ph 5.
Conclusion
In my design, I predicted that as the temperature increases, the speed of the reaction would increase. When a specific temperature was reached, I believed that the rate of the reaction would dramatically decrease. I believe this is the way most chemical reaction happen, e.g. faster when the temperature is higher. At higher temperatures molecules move around faster, which makes it easier for them to react together. Usually, rises of 10 degrees Celsius will double the rate of reaction. This is true for enzymes up to about 40 degrees Celsius. I predicted that the same would happen the further away the pH is from pH7. I believed this because the optimum pH for most enzymes is about pH7 therefore the further away the pH is from pH7 (either more alkaline or more acidic) the less effective the enzyme.
My results support most of the prediction since as the pH/temperature increases the rate of reaction increases until a point is reached and then the rate of reaction decreases. However, the optimum temperature was not 40 degrees Celsius but 55 degrees Celsius (unusually high) and the optimum pH was pH5 not pH7. I also predicted that a rise of 10 degrees Celsius would double the rate of reaction, I could not find a linear relationship between the temperature and the rate of reaction, however with enough results (from a very large range of temperatures) a parabola could be made on Graph 1, it would then be possible to find a mathematical formula for the construction of the parabola and hence find the relationship between the temperature and rate of reaction. Currently I do not have enough reliable evidence to draw any conclusions more specific than the rate of reaction is proportional to the temperature until 55 degrees Celsius is reached and after this point the temperature is inversely proportional to the rate of reaction.
Note: “Optimum operating temperature/pH” means the pH/temperature where the enzyme (amylase) operates the fastest.
I believe that the experiment was successful but some of the results were unexpected/unreliable. The time taken for the amylase to digest the starch at 40 degrees Celsius was far too fast (see Graph 1) it should have been between 4 and 4.5 minutes. All the other results seemed to fit into the trend on the graphs.
I believe that the experiment was designed well but there were a few problems. The optimum temperature for the amylase was too high. I believe that all the results were skewed because the enzyme was not given enough time at each particular temperature to be fully affected before it was added to the starch. They were only left in the water bath for 10 minutes before starting the experiment. However, they should have been in the water bath for about 30 minutes so that the amylase had been completely affected by the temperature before the experiment was started. I decided to conduct the experiment at 10 degrees Celsius intervals instead of 5 degrees Celsius because that would have made the graph too big. When the results were collected, I plotted them on a rough graph to find the optimum temperature and then conducted the experiment at this temperature to ensure it was the optimum temperature. I also conducted all three experiments for each condition at the same time to save time. Additional work, which could be carried out, is to repeat the experiment using, a wider range of temperatures and pH levels, and a range of different starch solution concentrations or using different enzymes such as protease with a protein.
Bibliography
G.C.S.E Biology second edition D.G. Mackean
ICS Biology Module A