I then decided to conduct the experiment as follows: Two test tubes would be taken, with equal amounts of starch and amylase solution. The contents of the two tubes would then be combined in a separate tube, and a timer started. Then, every minute, a sample of the mixture would be taken using a pipette, and placed on a ceramic tile. A drop of iodine would be added to each drop. The colour would be noted and a colorimeter used to measure the index of concentration. The index of concentration would then be noted in a table for each sample. I planned to determine the range of temperatures to test, the volumes of starch and amylase solutions to use and the concentrations of these solutions after conducting a pilot study.
After the pilot study had been carried out, I decided to use 10cm3 of 2% starch solution and 10cm3 of 0.01% 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 acclimatising, set up a tile with iodine drops already on it, in the depressions on the tile. A drop of the solution collected during the experiment could then simply be added to this and the colour noted. I also decided not to note the index of concentration, as this was impractical, and instead just to note down the time after which there was no more starch present in the solution. In order to decide at which point there was no more starch present, maltose solution was mixed with iodine in a beaker. This provided an indication of what colour the iodine would turn if there was no starch present, but there was maltose, produced by the enzyme. Based on the pilot study results, I decided to conduct the main experiment between temperatures of 20oC and 70oC inclusive. The pilot study showed that 20oC was the lowest temperature at which the enzyme could function at all effectively and that the enzyme would denature possibly at 70oC and definitely at temperatures above that. The main experiment would be conducted at 10oC intervals between these temperatures in order to obtain a sufficient number of sufficiently dissimilar results.
Throughout the experiment, safety precautions would taken to ensure that the experiment was conducted in a safe manner: safety goggles and a lab coat would 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 was accidentally ingested, medical attention would be sought immediately.
Obtaining Evidence
The following apparatus was used:
- Test tubes
- Thermometers
- Beaker
- Water baths
- Pipette
- Ceramic tile with depressions
- Stop clock
The apparatus was set up as follows:
Two test tubes were taken. One was filled with 10cm3 of 0.01% amylase solution and the other with 10cm3 of 2% starch solution, measured using a measuring tube. Both test tubes were left to acclimatise in a water bath at 20oC, with a thermometer in each test tube. The tubes were checked every minute and, when both test tubes had reached 20oC, their contents were combined in a separate, larger tube, which had also been left in the water bath. As soon as the two solutions were combined, a stop clock was started and a sample of the contents of the tube was taken using a pipette. A drop of the sample was dropped into one of the depressions on a ceramic tile, in which iodine had already been put. The colour of the iodine was noted in order to determine whether or not there was still starch present in solution in the test tube. A separate beaker was set up in which iodine solution was mixed with maltose. As maltose is produced when amylase digests starch, this would give an indication of the colour that the iodine would turn when there was no remaining starch in the test tube but just maltose. A sample was taken every minute and the same procedure repeated, until the iodine had reached the colour in the beaker, at which point it would be evident that there was no more starch. The time at which the starch had all been digested was recorded. This process was repeated twice for each of the temperatures 20oC, 30oC, 40oC, 50oC, 60oC and 70oC. Throughout the experiment, thermometers were used in each water bath to ensure that the temperatures remained constant.
The following results were obtained:
Analysing Evidence
In order to show the effectiveness of the enzyme rather than the time it takes to digest the starch, the inverse of the time taken to digest the starch was plotted on the y-axis, with temperature on the x-axis:
The graph shows that, between 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 proportional 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.
It was impossible to test the prediction that, with time, the starch concentration would decrease for each temperature tested, showing exponential decay so that, after every x minutes, the starch concentration would half and would therefore never be totally broken down. However, the results did clearly show that the starch concentration decreased with time for all temperatures except when the enzyme denatured. If a graph could have been drawn showing the concentration of starch over time for each temperature, it would probably have been an exponential decay curve.
The reason for this behaviour of the enzyme shown in the graph involves theory concerning enzymes in general, amylase itself and kinetic theory.
Kinetic theory is the idea that, when a substance is heated, its molecules, having been supplied with energy, move around faster. In this experiment, as the temperature increased, the enzyme and starch molecules collided more frequently (Brownian motion) and with more energy which caused them to react more efficiently. At low temperatures, the molecules did not collide as frequently and the starch was therefore not broken down as quickly. This is true of any reaction, whether or not it involves catalysts, biological or otherwise.
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.
Evaluating Evidence
The experiment worked well overall, proving beyond reasonable doubt that the optimum temperature of the amylase used in the experiment was around 40oC. Despite the erratic 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 colorimeter. This device provides an indication of how deep a colour is, and could have been used to measure the index of 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, 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 pipette between each reading, or using a new pipette each time, but this could not practically happen. There was always some solution left over in the pipette from the previous reason. 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. Maltose was mixed with iodine to give an indication of what colour the solution would turn when there was no remaining starch, and the resulting colour was yellow. However, with the samples collected, this colour was never reached and the readings were stopped after the solution turned a yellow/brown colour and stayed that colour. Had the experiment been undertaken with greater precision, it is likely that the yellow colour would have been reached. 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 using a narrower gauge measuring tube or by using a syringe.
An ideal solution would have been to automate the whole system, with a sample of the mixture being automatically taken every minute, or preferably more frequently, and the concentration of starch stored on computer. This would have overcome the inaccuracies of the timing, which could not always be exact using a stop clock and someone watching it, and would have eliminated the effect of human error from the experiment. If these steps had been taken, it is likely that a graph more similar in shape to that suggested in the prediction would have resulted.
In conclusion, the accuracy of the results was certainly good enough to make a sensible 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.