* Rinse the glass rod.
* Add 2cm3 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.
To investigate the effect of temperature 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.
* Cool both test tubes and maintain at 10OC
* 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 2cm3 of water to starch solution (see below).
* Pour amylase solution into the test tube of starch and shake quickly (measure temperature, try to maintain at 10OC).
* Repeat steps 4 & 5 (for the amylase & starch solution mixture) every 30 seconds until a blue/black colour no longer develops.
* Record the results in a table. Repeat steps 1-9 increasing the temperature by 5OC each time until the enzyme is denatured, this should be around 40OC (see part 6 on enzymes.)
To ensure that the test is 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 section where temperature is investigated, 2cm3 of water is added. This is because the starch solution is neutral and in the previous investigation (for pH) 2cm3 of the appropriate buffer was added. If the 2cm3 of water was 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 of 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 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 the freezer to reduce the temperature of the mixture to 10OC. A 100OC thermometer will provide temperature results of a sufficient accuracy (to 1OC). The pH buffer range will be 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 because it therefore allows me to calculate an average time. This will ensure that there are no abnormal results and it will increase accuracy. I have decided to start the temperature at 10OC and increase by 5OC each time since it will allow me to see the increase and decrease of the enzyme activity. It should also be accurate enough for me to predict an optimum operating temperature to an accuracy of 5OC. I have no control over which pH buffers I will use since the school prior to the experiment will provide them.
* 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 systems 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 molecule 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 40OC. Most enzymes work best at a pH of about 7. This is also because they are proteins, which are damaged by very acidic or very alkaline conditions. Due to the enzyme's unique active site it can only convert one kind of substrate molecule into one kind of product.
First of all the average time taken for the starch to be digested in each condition was calculated.
Temperature(OC) 10 21 32 40 50 55 60 67 80&90
Average Time (min) 9.8 6.7 5.3 5.5 3.5 3.0 3.5 4.5 10 +
pH 8 7 6 5 3 2
Average Time (min) 9.5 6.7 4.5 2.8 4.7 9.2
The results were then plotted on a graph (see Graphs 1 & 2) and the points joined together.
From 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 fastest at 55OC, however if the temperature continues to rise then the time taken for the amylase to digest the starch rapidly increases. By the time 80 OC is reached the amylase does 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 fastest at pH 5. At a higher pH level than pH 5 the time taken for the amylase to digest the starch increases.
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.
High temperatures make enzymes inactive: this is because they are proteins, which are damaged by temperatures above about 40OC. In this investigation it was found that amylase operated fastest at 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. This is 15 OC above the suggested typical temperature, see Evaluating Evidence for possible reasons why it is higher than expected. By 80OC the amylase was completely denatured. From this information I can conclude that the optimum operating temperature for the enzyme amylase is 55OC.
Most enzymes work best at a pH of about 7. This is also because they are proteins, which are damaged by very acidic or very alkaline conditions. However in this experiment the amylase worked fastest at pH 5. This pH is lower than suggested above because the amylase provided by the school was a bacterial enzyme which had an optimum pH between pH 4 and pH 5.
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 reaction would dramatically decrease. I believed this because most chemical reactions happen faster when the temperature is higher. At higher temperatures molecules move around faster, which makes it easier for them to react together. Usually, a rise of 10OC will double the rate of reaction. This is true for enzymes up to about 40OC. However at 40OC the enzyme begins to be damaged, so the reaction slows down. By 60OC the enzyme is completely denatured. I predicted that the same would happen the further away the pH is from pH 7. I believed this because the optimum temperature for most enzymes is about pH 7 therefore the further away from pH 7 (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 OC but 55 OC (unusually high) and the optimum pH was pH 5 not pH7. I also predicted that a rise of 10 OC would double the rate of reaction, this was not correct. As is evident in the table above, the time taken does not halve for every 10OC increase in temperature. I could not find a linear relationship between the temperature and 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. At current I do not have enough reliable evidence to draw any conclusions more specific than the rate of reaction is proportional to the temperature until 55OC is reached and after this point the temperature is inversely proportional to the rate of reaction.
Note: "Optimum operating temperature/pH" means this is the temperature/pH where the enzyme (amylase) operates the fastest.Evaluating Evidence
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 OC 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. Because of time restraints they were only left in the water bath for 10 minutes before starting the experiment. However, they should have been left 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 OC intervals instead of 5 OC because there was not enough time. When the results were collected I plotted them on a rough graph to find the optimum temperature and then conduct 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, a range of different starch solution concentrations or using different enzymes such as protease with a protein.
