Another experiment shown as an example in the Key Science Biology textbook showed that pepsin (an enzyme that catalyses the breakdown of proteins) works best at around 35ºC and denatures (is destroyed) at temperatures above 50ºC.
A final experiment I carried out using pepsin investigated the relationship between Ph and the breakdown of protein. It showed that pepsin although did work in neutral conditions was much more effective and efficient in low/ acidic Ph levels.
The results of these experiments allow me to be able to come to the conclusion that enzymes including those in yeast are affected by both heat and pH.
Method:
Equipment: 1x ice cream container
2x syringe
1x beaker of yeast and sucrose solution
1x stop watch
1x ‘Log it’ thermometer
1x beaker of lime water
Set up equipment as follows:
Testing for CO2:
- Fill syringe with 5ml solution and 5ml air.
- Place in beaker of Limewater
- Begin timing.
- Observe for cloudiness in limewater
- Stop timer once cloudiness is observed. (N.B- this will be the time to allow each solution to adjust to water temperature)
Testing rate of respiration:
1. Boil kettle of water, pour water into ice cream container.
2. Fill syringes with 5ml yeast and sugar solution and 5ml air in each.
3. Take and record temperature of water.
4. Place syringes in water.
5. Time for no. of minutes discovered in ‘5’ to allow solution and air to
adjust to the water temperature.
6. Time for another minute, this time counting the number of bubbles of
CO2 produced from each syringe.
7. Record results.
8. Add beaker of cold water, stir, retake temperature and record.
9. Place new syringes in container and time as before.
10. Count and record number of bubbles CO2 produced from each syringe.
11. Repeat steps 8 – 10 five times, adding more cold water each time and adding
ice in the last test to bring solution to lowest possible temperature.
N.B. The test at each temperature will be conducted with two syringes each time, for accuracy. If an extreme anomaly is found, repeat test at that temperature to find an accurate mean.
Safety:
- Tie hair back and tuck in ties.
- Use tongs when using boiling water to prevent scalding.
- Set up equipment in the middle of the table to prevent equipment being accidentally knocked or broken.
Control:
- Only have one variable – temperature.
- Control all other factors – pH of solution, concentration of solution, amount of air and solution in syringe.
- Repeat each experiment twice for accuracy.
- Use largest ice cream container possible to minimise lose of heat. (closest possible surface area:volume ratio to minimise lose of heat via diffusion)
Uncontrollable Factors:
- Slight lose of heat over the one timed minute- minimised by the use of a large container & the test only being timed for a short period.
- Solubility of CO2 – carbon dioxide is very slightly soluble, 0.1688g per 100g of water (at room pressure & temperature)
Results of the experiment:
(See Next sheet for line graph of results)
Analysis:
The results that were collected, shown on the graph on the following page, illustrate that my hypothesis was partly correct. The graph shows how the rate of respiration increased with temperature until, by 85ºC, it had slowed dramatically. This proves my theory, that the enzymes in yeast, like those in our bodies, e.g. amylase, are more active with higher temperatures, however are denatured if the temperature becomes too high. This is because higher temperature makes the molecules in the solution move/vibrate more, increasing the chances of the enzyme meeting a substrate molecule to break down. However, the enzyme has very weak bonds so when the temperature becomes too high, these bonds are destroyed and the active site becomes deformed and can no longer break down the substrate.
Our results prove this theory. It can clearly be seen, on the graph overleaf, that above 85ºC, the enzymes in the solution are beginning to denature. The active sites become deformed and stop working.
From the results we collected, we can come to the conclusion that the enzymes in the yeast become inactive/dormant at around 7ºC or below. The enzymes act in a similar way to bacteria, becoming not destroyed, but dormant at low temperatures. This is because cooler temperatures slow the molecules’ movement until, when the temperature becomes low enough, they stop moving, becoming a solid (freezing).
This slowing in movement means that the rate that the enzymes meet substrate molecules is less so, therefore, the rate of anaerobic respiration is less and less carbon dioxide is produced.
Evaluation:
The experiment was quite successful. We found no extreme anomalies, but had planned for this eventuality, by stating in our plan that if an anomaly arose, we would repeat the test a third time to get a more accurate mean result.
Our results did, indeed, prove that our hypothesis was correct. However, the results could not conclude an exact temperature at which the yeast enzymes are most active or the temperature at which they denature. Our results suggest that the enzymes are most active at approximately 58ºC. However, as we have no result for the amount of carbon dioxide produced at temperatures between 58 and 85ºC, we cannot be sure. The rate of respiration could have continued increasing until up to 60 or even 70ºC. This means that, although the results we collected are not entirely inaccurate, due to lack of data, we can not be sure that our line of best fit is not inaccurate.
e.g.
To solve this problem, if I repeated the experiment, I would carry out tests at at least one temperature, if not two or three, between 58 and 85ºC, to make the results much more accurate and allow me to conclude on a more exact temperature at which yeast is most active and subsequently denatures.
Although to discover the exact temperature at which the enzymes denature would require skills & equipment beyond our means.
Other factors that I would change if I repeated the experiments are that I would time each test for at least two minutes instead of one. This would allow us to collect more carbon dioxide so that there is enough gas produced to be measured in a measuring cylinder, rather than the inaccurate method of counting bubbles (we do not, in fact, know the size of the bubbles or whether this size is consistent throughout the tests).
One of the factors that affected our results that is more difficult to control is the change of temperature of the water bath during the experiments. I tried to control this or, at least, keep it to a minimum by using a large container with a close surface area to volume ratio in order to minimise the amount of heat lost by diffusion. However, this method can only help the situation and not solve it. On the first test that we did, we took the temperature at the end of the minute to test for accuracy in variation of temperature, only to discover that the temperature still changed by approximately 2ºC. The only way to really solve this would be to use an electrically operated, thermostatically controlled water bath. However, this would make controlling the rest of the experiment much more difficult as it is a large, cumbersome device and it would be difficult to set up the equipment effectively.
A final factor that I would change is a piece of equipment that we used- the syringe. Using tongs to hold the syringe in place under the water was necessary
To prevent scalding however it made controlling the angle the syringe was held at difficult. The design of the syringe meant that any slight tilt off of horizontal caused solution to spill out into the container. One way to correct this would be to use less solution in each syringe so that the level of solution was further from the opening in the end.
e.g.-
Conclusion:
Considering the means we had to carry out this experiment. ( i.e.- the equipment etc. ) The experiment went very well , however there are many areas of the method that could be improved upon to consequently get much more accurate results to not only be able to prove the hypothesis but also to be able to conclude on exact temperatures at which the enzymes become dormant, most active & finally denature.
By Louise Martin 10ME