Prediction:
I know that all enzymes have different optimum temperatures at which they work best and after that they continue to work but soon become denatured. This occurs when the bonds within the amylase are shaken apart collapsing the 3D structure of the protein destabilises. Also enzymes have a structure which they hold best at their optimum temperature so at the optimum which I expect that the enzyme will work better as it holds this shape. Enzymes that work better at higher temperatures tend to have a more rigid structure. I know that most enzymes work best at around 40 degrees Celsius so I predict that as the temperature increases up to 40 degrees the enzyme will work better as it will have more energy causing more collisions with the substrate. After this temperature I expect that the enzyme will denature and collapse therefore not yielding any glucose after this optimum temperature.
Apparatus:
Beaker x 4 (to heat boiling tubes at two different temperatures and to store amylase and starch)
Test tube x 5 (will hold enzymes to be heated in water bath)
Bunsen burner/gauze/tripod x2 (to heat enzymes)
Thermometer x 2 (to ensure listed temperatures are used)
Starch Solution (2.5%) 0.2dm3 (substrate, concentrations explained earlier)
Amylase (1%) 0.2dm3 (enzyme, concentrations explained earlier)
Measuring Cylinder (measure out volumes of enzyme/substrate needed for each test)
Pipette (used to add benedict’s reagent to enzyme/substrate complex)
Stopwatch (used to ensure each test for glucose is being carried out at the allocated time)
Benedict’s reagent (used to test if enzyme has broken substrate down into glucose)
Distilled Water (used to rinse out equipment prior to use)
Fair Test:
The following measures were taken to ensure that the experiment was a fair test:
- All equipment used was rinsed with distilled water to avoid the affect of any remaining or left over chemicals for example may have on the beakers
- The variable to be changed was changed in the same proportion for every test. What this means is that for every test the temperature was increased by the same amount as it was in the previous test, simply by heating and checking the temperature using a thermometer.
- In this experiment there is no need to take extra measures to ensure other variables such as ph and concentration are kept constant as so long as the method is followed accurately they will not influence the results.
- Each test was repeated 3 times and an average was used to obtain a representative and fair result. This was done so any anomalies that may have been present in individual tests will not show up on the final results table and on the graphs. The error bars on the graphs however give some idea as to the range of results recorded.
Safety:
The obvious safety precautions in this experiment involve the use of goggles and an apron as a Bunsen is in constant use, obviously caution was taken with this apparatus and as one beaker will contain boiling water
Caution was taken when working with Amylase; although not very dangerous it can be if inhaled excessively or up taken in some other way so caution must be take to avoid this.
When using Benedict’s reagent it is also advised to be cautious, ideally goggles should be worn, if excessively inhaled or swallowed then it is dangerous so generally precautions and caution needs to be taken when handling the Benedict’s.
Method:
Firstly the starch was diluted by adding 0.1dm3 of water to the 0.1dm3 of 5% starch solution to get it to the desired concentration of 2.5%
The amylase was then diluted from pure 100% amylase down to 1% amylase by taking 1cm3 of the pour solution and diluting it with 100cm3 of water.
2cm3 of amylase was placed into 6 test tubes that were placed inside a water bath and heated to 250C. Once they had reached this temperature they were removed one by one and exposed to the starch solution for fixed lengths of time, (the time was measured using a stop watch as mentioned in the apparatus) starting with 30 seconds and increasing in 30 second intervals up until 3 minutes. Once the time had elapsed the enzyme substrate was placed in boiling water and benedict’s was added to test for glucose. These tests produced a set of 6 results showing weather how long it takes for the Amylase to break down the starch if it is heated to 250C
These tests were repeated only the temperature was increased by 10 degrees each time, I continued to increase the temperature by 10 degrees until no positive test for glucose was achieved within the 3 minute testing time. At which point it was assumed that the enzyme has denatured
Once this had been completed now a full set of 5 temperature ranges tested across a time of 3 minutes had been recorded
The experiment was now complete but this was repeated twice over to gather a set of three results from which an average was obtained. The temperature is that of the enzyme before it was the substrate was exposed to it and the time in seconds shows how long it took for the enzyme to break down the substrate into glucose.
Results:
Test 1:
Test 2:
Test 3:
Average table:
To show the effectiveness of the Amylase rather than the time taken for glucose to be produced two graphs were made one which does show the time taken and a rate time graph which shows the effectiveness of the enzyme, firstly the rate had to be calculated; this is simply the inverse of the time:
Working out the temperature coefficient of the enzyme will give me an idea as to how the enzyme changes its effectiveness with each increase in temperature. The temperature coefficient or Q10 is worked out using the following formula where X is the rate of reaction:
Q10 = X + 100C/X
The following table shows the temperature coefficient for each temperature change in my experiment:
The table shows that until the optimum temperature is reached the rate doubles increases as the coefficient decreases, this supports my prediction and also the coefficient increases after the optimum temperature meaning the rate must have decreased, this again supports my prediction.
Analysis:
From the results I can clearly see that the optimum temperature of the Amylase used was roughly 45 degrees Celsius.
If I analyse the rate/temperature graph I can see that there is a steady increase from temperatures of 25 degrees through till 35, at that point there is a sharp increase and 45 degrees clearly seems to be the optimum as it continues to drop after that. Both graphs are curves showing that in neither case are the time or the rate proportional to the temperature, it varies throughout.
The rate time graph shows how enzymes function and that is by firstly increasing their efficiency as temperature is increased, this is done because the enzyme and the substrate have more kinetic energy leading to a higher chance of collision and consequently reaction. The peak shows the enzymes optimum temperature at which point the enzyme is at its most effective and increasing the temperature after that quickly drops the efficiency of the enzyme until it has denatured because the substrate will no longer fit its active site.
In my prediction I stated that I expected the optimum temperature of the amylase to be 40 degrees Celsius, from my results I can see that I was slightly of as the actual optimum is 45 degrees.
I also predicted that once the amylase had reached this optimum temperature it would denature and discontinue to break down the starch into glucose, this also was not the case, the optimum may have been 45 but from my results I can see that the enzyme was able to continue working up till temperatures of 65 degrees where no results was obtained within the 3 minute testing time that was set, I can only assume that the amylase denatured somewhere between 55 degrees and 65 degrees Celsius.
Conclusion and Evaluation
As each test produced similar results it is possible that they were all incorrect and the procedure used was inadequate however I can conclude that the test was reasonably accurate because the results that were achieved are in accordance with scientific theory, the fact that the amylase had an optimum of higher than body temperature could be down to two reasons, either that this is a bacterial amylase and they are known to be able to withstand higher temperatures thus having a higher overall optimum temperature. It could however be that the optimum was indeed body temperature as this was not specifically tested. The range of temperatures started at 35 and went up in 10’s; it is therefore entirely possible that the optimum was somewhere in between 35 and 45 degrees Celsius and not at 45 itself.
The experiment did not go entirely as initially planned, as refinements had to be made with the range of temperatures and the concentrations of amylase and starch solution to be used
Firstly from the onset the idea was to use a range of 10 temperatures but it quickly became apparent that this would not be possible given the time constraints. The time of 10hrs needed for this practical were not enough to collect viable data as 3 tests using a range of only 5 temperatures are only enough to give an indication of the enzyme activity and not to accurately plot out its efficiency over time. Had I have been able to carry out 10 temperature tests instead of 5 it is entirely possible that I would have found the optimum temperature to be body temperature of 40 degrees and not 45. These results would have been more accurate and therefore more reliable however it was not possible to carry out this investigation with that much detail.
Secondly the plan was to use 5% starch solution with 3% amylase and to use the preliminary work to work out what volumes of each to use per test, however it quickly became apparent form the preliminary work that using these concentrations would not allow me to collect a range of results as they were producing glucose from the starch after only having 20 seconds to react and this was at room temperature, a good 20 degrees below its optimum. Upon this discovery it was quickly decided that the concentrations would have to be weakened to allow for a range of temperatures to be used and results to be yielded from them. Upon further dilution it was decided that 1% amylase and 2.5% starch would be used as this took 3 minutes to break down the starch into glucose at room temperature in a preliminary test.
The procedure used could have been improved so that given the time restraints the results collected were more accurate by:
- The enzymes were heated to the desired temperature and then one by one each was allowed to mix with the starch before being boiled with Benedict reagent, during this time it is inevitable that the temperature of the enzyme in the first test will have been higher than that of the last test as each was done one by one thereby hindering the accuracy and consequently the reliability of the results
- Carrying out more tests, repeating the experiment more times or even using a larger range or time or temperatures, for this obviously more time will be needed but by doing so more results will create a clearer picture of how the enzyme changes its efficiency as temperature increases.
- It would have significantly improved the experiment if I had used a colorimeter to test the concentration of glucose present, if I had done this along with using a larger range of temperatures then it would have been possible to note when the glucose concentration began to decrease instead of when there was no glucose. If I had time I could have carried out tests using colorimeters in my preliminary work and recorded known concentrations to test the ones obtained in the experiment against.
- It is probable that in each test tube/measuring cylinder there was some remaining amylase or starch from previous tests and washing this out each time would have increased the accuracy and reliability of the experiment.
- Certain stages of the experiment could have been computer controlled i.e. the timings and the concentrations and mixing; this would remove the imperfections associated with doing things like timing as soon as the amylase is dropped into the starch.
The equipment used could have been improved if:
- A volumetric or graduated pipette was used to measure out the amounts of amylase and starch to be used
- The water baths that were used all were at the exact same temperature, it was impossible to ensure this I used a manual water bath using a large beaker filled with water and a Bunsen burner to save time instead of using the water baths with set temperature controllers on them.
- As the experiment was carried out over a period of 2 weeks, at 10 different intervals it was very difficult to ensure that the same equipment was used every time this in turn will have hindered the reliability of the experiment.
In conclusion the experiment was of reasonable accuracy as the results obtained are consistent and coherent and in accordance with scientific theory however there are clearly several ways that the validity of this experiment could have been improved
To conclude the investigation it is clear that temperature does have an affect on amylase and I have found that the optimum temperature for amylase from my tests was 45 degrees and it denatured at 65 degrees Celsius.
Bibliography:
Micheal Kent, Advanced Biology