- Time taken for the starch to be broken down (output variable)
In this particular experiment we will only be changing one of the input variables – temperature. The rest will be controlled in order to make it a fair test. The output variable that we will measure will be the time taken for amylase to digest the polysaccharide – starch.
Prediction
I predict that the amylase will break down the starch most effectively at 40oC, and with decreasing efficiency towards 0oC, at which time the amylase would be unable to break down the starch at all. This is because body temperature is around 40oC, and enzymes are designed to work at this optimal temperature. The reason for enzymes behaving like this involves kinetic theory. Kinetic theory is the idea that, when a substance is heated e.g. enzymes are heated their particles gain more energy and are therefore more energetic and move quicker which results in more collisions per second so therefore the rate of reaction with the starch increases. When they are at a low temperature e.g. 3°c they will take a longer time to react because they the particles will have less energy and move slower which will result in less collisions per second and a slower rate of reaction. Therefore, at temperatures over 40oC, I predict, the amylase will begin to denature to an extent that, at temperatures much over 50oC, it will be totally ineffective. This is because enzymes denature at around 40-50°c because at higher temperatures the shape of the active site changes therefore prevents the reactant from fitting into the active site and so the reaction stops.
Preliminary study
.
The aim of my preliminary study was to investigate how long was needed to digest starch using enzymes at different pHs. I planned to determine the exact range of times to test and the volumes of starch and amylase to use after conducting the preliminary test for my final experiment.
My results showed that the enzymes at pH 7 digested the quickest. Therefore this is its optimum pH. At pH 1 and pH 14 there was hardly any reaction this is because the rate of reaction for very low pHs (acid) or very high pH (alkali) are very low and often result in no reaction at all.
After the preliminary test had been carried out, I decided to use 10cm3 starch solution and 5cm3 of amylase solution in the two initial test tubes for the main experiment. It proved easier to, before the experiment was started and while the starch and amylase were heating up, set up a tile with iodine drops already on it. A drop of the solution collected during the experiment could then simply be added to this. In order to decide at which point there was no more starch present, glucose solution was mixed with iodine in a beaker. This provided an indication of what colour the iodine would turn if there were no starch present, but there was glucose, produced by the enzyme. Based on the preliminary test results, the main experiment would be conducted at 10oC intervals between the temperatures in order to obtain an adequate number of sufficiently different results.
Risk assessment
Throughout the experiment, I took safety precautions to ensure that the experiment was conducted in a safe style: safety goggles were to be worn at all times. All parts of the experiment would be undertaken with care, to ensure that there was no spillage. Any spillages of iodine or starch would be washed thoroughly with soap and water. If any starch solution, iodine indicator or amylase were accidentally ingested, medical attention would be sought immediately. To make my experiment even safer I be used a water bath for all of the temperatures, water baths are a safer option than Bunsen burners and often more effective. Less equipment to deal with, heat not as extreme and no open flames equal less of a chance for accidents. They are often more effective because the temperature of the water can be controlled more easily and the heat gradually increases, unlike the Bunsen burner where it is very difficult to control the heat. The heat increase is almost instant and low temperatures are hard to achieve because of the severe concentration of heat.
Equipment List
- Water baths
- 2 test tubes
- Test tube holder
- 2 measuring cylinders
- 2 conical flasks
- Beaker 100ml
- Iodine solution
- Starch solution
- Amylase solution
- Dropping pipettes
- Spotting tiles
- Thermometer
- Stopwatch
- Goggles
Method
1. First collect and set up the equipment.
2. Put two drops of iodine into each spot on the dimple tray
3. Using one of the conical flasks fill it with water and add a few pieces of ice.
4. Using a blue measuring cylinder measure out 10cm of starch, pour it into a boiling tube and place in the beaker
5. Using a different clear measuring cylinder measure out 5cm of the enzyme solution amylase, pour into a separate boiling tube and place in the same conical flask.
6. Place the thermometer in the beaker and wait for both solutions to reach equilibrium of 10°C
7. Pour the starch solution into the amylase after ensuring that the temperature of both solutions is 10°C
8. Start timer straight away.
9. Withdraw 0.05 of the solution using the pipette and put into a dimple tray with the iodine, withdraw one drop every 30 seconds starting at 0 until the solution turns brown then stop timer and record result.
10. Fill one beaker with amylase and the other with starch. Place both beakers into the same water bath and wait until they reach both reach 20°C.
11. Measure out 10cm of the starch solution using the blue measuring cylinder (rinse it out first), then pour into a boiling tube.
12. Measure out 5cm of the amylase using the clear measuring cylinder (rinse out first), then pour it into the boiling tube with the starch.
13. Put the boiling tube into the water bath and start the timer.
14.Repeat steps 9 – 13 using the temperatures 40°C, 60°C, and 80°C ensuring use that you rinse each piece of equipment after use. Record each result in a table.
Conclusion
In conclusion, my graph shows that, at my starting temperature of 20°C the graph line rose quite steadily until at the temperature of 30°C when the steepness of the line rose much more considerably. But with temperatures over 40°the speed at which the starch was turned to glucose began to slow down until after 60°C at which the enzymes in the amylase denatured and failed to work completely, consequently stopping the reaction between the starch and amylase. Therefore I believe that temperatures of 20oC and 40oC, the efficiency of the enzyme increases with temperature. However, the graph between these points is a curve so the efficiency of the enzyme is not equal to the temperature. Between 40oC and 60oC, the efficiency of the enzyme decreases with temperature, mirroring the first part of the graph. The graph shows that the optimum temperature of the amylase tested was 40oC.
The graph supports my prediction that the optimum temperature of the enzyme would be around 40oC, and would have decreasing efficiency towards 0oC, at which the amylase would be unable to break down the starch at all. However, the results did not support the prediction that, at temperatures over 40oC, the enzyme would begin to denature to an extent that, at temperatures much over 50oC, it would be totally ineffective. The reason why the enzyme seems to have been able to survive without denaturing at higher temperatures is that the enzyme used was a bacterial enzyme, and bacterial enzymes do not necessarily behave in the same way as enzymes from the human body. It therefore could have had an optimum temperature of slightly above 40oC, and did not fully denature until the temperature was raised to 70oC, as shown in the results table above.
The enzyme was most effective at 40oC because this is body temperature, at which is it most used to working. In order for it to function most efficiently in the body, amylase must have an optimum temperature of 40oC. The reason why the amylase was less effective at higher temperatures was that it had started to denature. All enzymes start to denature at temperatures above their optimum temperatures, which renders them unable of catalysing reactions.
Evaluation
The experiment worked well overall, proving that the optimum temperature of the amylase used in the experiment was around 40oC. Despite the nature of the experiment, the results were sufficiently accurate that they were aligned almost perfectly on a curve, and were taken at intervals far enough apart that the readings were clearly distinguishable from each other. However, the results were not totally accurate. For example, the first time the experiment was carried out at 70oC, the starch disappeared in six minutes, while the second time the enzyme denatured quickly. At 60oC, the second time the experiment was conducted at this temperature, the start took twice as long (12 minutes) to disappear. The graph was not a perfectly smooth curve, and this was due to several different factors.
The apparatus used could have been improved in many ways. The water baths used were not all at the exact temperatures required, and each contained a different amount of water. If better quality water baths had been used, and there was time to ensure that each had exactly the same amount of water and was at the exact temperature required, anomalous results could have been eliminated. This could also have been achieved by repeating the experiment for each temperature more than twice, and also by performing the experiment at intervals smaller than 10oC. Another problem with the experiment was the use of iodine. Although iodine is a good indicator of whether or not starch is present, it does not provide accurate information about the concentration of starch present. It would have been more useful to obtain this information so that it could be plotted, to analyse how the concentration changes over time rather than at what single time there is no more starch. This quantitative approach could have been achieved by using a colour meter. This could of provided an indication of how deep the colour was, and could have been used to measure the concentration of the samples throughout the experiment. Values for concentration of starch in the samples could have been obtained by first recording a reading for known concentrations, and then comparing these readings with those obtained with the samples collected during the experiment
Additionally, the pipettes used were another area of error. More accurate results could have been obtained by cleaning the pipettes between each reading, or using a new pipette each time because there was always some solution left over in the pipette. Another problem with the pipettes was that there was time for the amylase to act on the starch while the solution was in the pipette, making the timings recorded slightly too small. However, this effect was lessened with most of the temperatures as the mixture was cooling down to room temperature in the pipette. The method by which we tested to see if there was starch remaining did not work entirely satisfactorily. Finally, the procedure of preparing the solutions of amylase and starch for the experiment could have been improved. It is likely that there was some solution left over from the previous repetition of the experiment, making the starch/amylase ratio different each time. This could have been overcome by washing out the test tubes between readings. The volumes of each solution could have been made more accurate by measuring the solutions by using a syringe.
Basic improvements I would make if I re-did my experiment would be I would: Use electronic water baths to measure the temperature more precisely, perform the experiment at intervals smaller than 20°C, use a colour meter, repeat each experiment for every temperature more than twice and clean the pipettes and test tubes between each reading.
In conclusion, the accuracy of the results was certainly good enough to make a good conclusion. If the experiment had been conducted under more strict conditions and with more advanced instruments, the conclusion would not have been different although the individual results might have been more accurate and the graph might have looked very slightly different.