The equipment our experiment requires is a spotting dish, a measuring cylinder, test tubes, a pipette, an electrical water bath (this will be more accurate than using a Bunsen burner), a stopwatch and a thermometer.
The above diagram is a summary of our method; this is how we will conduct our experiment into how temperature affects the activity of amylase.
5.0ml of amylase solution and 5ml of starch solution will be measured and each poured into a separate test tube. Each will then be placed in a water bath; a thermometer placed in each test tube will indicate when the amylase and starch has reached the intended temperature. A spotting dish must be prepared with a drop of iodine in each dimple; a pipette will be used for this. With a glass rod a drop of starch solution from the test tube will be mixed with the first drop of iodine in the spotting dish - a blue/black colour should develop; this will serve as a control. The glass rod will then be washed. Then the amylase and starch solutions must be mixed together, in doing this they must be shaken to ensure that the spread of each is even throughout the test tube. A drop from this mixture will be lifted with the glass rod every 30 seconds and placed with one of the drops of iodine in the spotting dish (obviously the spotting dish will be used systematically – we will use the drops of iodine in lines so that we can see the gradual conversion of colour and thus of starch.) As this is done I will record the results in a table. When the colour matches that of iodine, or there is no further change in colour the starch has been broken down and the experiment can stop.
The latter experiment will be conducted for temperatures ranging from 30°C to 70°C with a 10°C rise between each (the water bath will be set at each of these temperatures). We shall also carry this out with the solutions at room temperature, for this the starch and amylase will simply be left to cool in a test tube rack until they have reached the same temperature as their surrounding
Prediction
I predict that as the temperature increases so will the rate of reaction (of the breakdown of starch), until it exceeds 60°C, at this point the amylase will no longer catalyse the breakdown of starch. This is due to the fact that amylase consists of protein molecules and therefore it is easily denatured by heat, this is when it loses its shape and is no longer able to combine with the starch (explained in planning). If we were going to use natural amylase I would predict that this denaturing would take place at approximately 40°C, but the amylase which will be used in our experiments will be commercially produced, and thus will denature at a higher temperature, approximately 60°C.
I think that up until this point the rate of reaction will increase, because the increase in temperature will supply the molecules with more energy to react. This kinetic theory – more energy means the molecules will be moving more quickly increasing the likelihood of collisions.
I predict that if the temperature is doubled, so will the activity of the amylase – the rate of reaction will double. This is because twice as much heat given to the reactant means that the particles will have twice as much energy.
Results
This result appeared to be very anomalous so we repeated this temperature; the results are shown in the next table. 0.001
These results as they are, would be difficult to analyse. In order to aid this I will express these results in a graph. To do this I must calculate the rate of reaction for amylase breaking down starch, when at each of the temperatures. I will achieve this by using this formula
Rate of reaction = 1
Time (this is the total amount of time that the starch took to break down)
When using this the time will always be in seconds.
Rm temp 1/250=0.0042 cm3 of glucose per second
30°C 1/180=0.0056 cm3 of glucose per second
40°C 1/150=0.0067 cm3 of glucose per second
50°C 1/120=0.0083 cm3 of glucose per second
60°C 1/90=0.011 cm3 of glucose per second
70°C 1/90=0.011 cm3 of glucose per second
The line of best fit is also drawn onto the graph.
Conclusion
From my graph the positive correlation shows me that as the temperature increases so does the rate of reaction. This agrees with what I stated in my prediction. The reason for this is, as stated earlier, due to collision theory. The higher the temperature, the higher probability of an amylase and a starch molecule colliding together to react. I also predicted that if the temperature is doubled then the rate of reaction would also double. This agrees with my result – the rate of reaction for amylase digesting starch at 30°C was 0.0056, at 60°C the rate of reaction is 0.011 – twice as fast as 00056.
When a specific temperature was reached I believed that the rate of reaction would decrease. My results do not agree with this precisely. A reading above 60°C, gave identical results to that of the experiment carried out at 60°C. This may be a levelling point before the rate of reaction begins drop, unfortunately our range of results did not spread further than this, so an efficient analysis cannot be made. However, I know the shape the graph should take from other sources (An introduction to advanced physics) and my own knowledge I have extrapolated the graph further, showing how the following results should lie on the graph (this is shown with a dotted line). The drop is steep, but why doesn’t it immediately fall at a specific temperature? The reason is that as the denaturing begins, the higher rate of collisions equalises the reaction rate. This is the reason for my readings at both 60C and 70C giving identical results.
Evaluation
Overall I felt the experiment was a success, however it did have many weaknesses, which may have made the results more unreliable.
Firstly our rage if results was not sufficient to investigate the predictions fully. I cannot comment fully on what happens to the amylase at temperatures over 60°C, as I only have one reading to analyse this. If I were to repeat the experiment I would increase my range, heating the amylase and starch solutions to 80°C and 90°C.
Secondly it was almost impossible to decide on an ‘end point’, by this I mean it was difficult to say when the amylase had digested the starch fully. Variations in colour were often subtle making it hard to determine when the colour was constant, or the colour change was slight enough to rule out any starch content in the previously amylase/starch mixture. The fact that each experiment was not carried out simultaneously, and that the drops of iodine were uneven in volume further extended this problem. It meant that we could not compare our decided ‘end points’, due to this they may have been considerably different. Conducting the experiment for each temperature simultaneously could have reduced this problem. Then we could compare the final colours of the iodine for more accurate results.
Another error in this method was relatively minor, but still may have affected the accuracy of results. It also contributed to the latter problem of deciding when to stop. This was that the drops of iodine, although placed in the spotting dish with the same pipette were never equal volumes. Similarly the samples of the amylase and starch solution mixture taken with the glass rod could never be the identical quantity. This will have affected the colour changes seen in the iodine making the results less precise. A solution to this may be to use a pipette that has measurements on it (the one used in our experiment did not). You could then measure a fixed quantity of iodine for each drop. This method could also be used for the drops taken from the amylase and starch mixture.
Another error was purely human, there is no guarantee that our measurements were 100% accurate. A more accurate measuring cylinder and thermometer could have been used.
After closely studying my results one extremely anomalous result was apparent. It is marked on the graph (circled), and I could clearly tell it was anomalous because it did not lie near to or on the line of best fit as the rest of my results do. The reason for this, we suspect was no fault of our own. We surmise that this is due to a different concentration of amylase, during allocated time the concentration of amylase was perfected to 0.8%, but prior to this some confusion was present in this concentration and a different, lower concentration was presented to us, without our knowledge.
If this concentration was lower it would result in the likelihood collisions between the starch molecules and the amylase molecules to decrease, thus significantly decreasing the rate of reaction. To remedy this we repeated the result with the correct concentration of amylase solution, this gave a far more accurate result (it fitted with the line of bet fit).
The investigation could be extended by using a wider range of temperatures, this would give a wider range of results to analyse. Other factors, which affect the rate that enzymes operate at, could also be investigated. For example differing pHs could be looked into, or the concentration of either the amylase or the starch.
Bibliography
An introduction to advanced physics, C.J.Clegg
www.bbc.co.uk/revision
www.s-cool.com(a-level section)
Collins GCSE Biology