The variable that is not controlled:
Apparatus:
- 2.5% starch
- 0.25% fungal amylase
- Buffer solutions 1,3,4,5,6,7 & 9
- 9 (10 centimetre cubed) pipettes and pumpette
- Test tube
- Test tube rack
- Spotting tile
- Dropping pipette
- Iodine
- Stop clock
- Marker pen.
Justification of the apparatus used:
- Buffer solutions are used to change the pH of the solutions.
- Pipettes are used to measure out accurate amounts of amylase, starch and the buffer solutions.
- Pumpettes are used to draw the solutions into the pipette.
- Spotting tile is used so that the solution can be tested for starch.
- Iodine is used to test for starch.
- Stop clock is used for timing so the starch can be added after 2 minutes and the solution can be tested for starch every 30 seconds.
- The dropping pipette is used to draw the solution out of the test tube.
- Test tube rack is used to hold the test tubes.
Outline method:
The presence of the starch can be detected by using a solution of iodine dissolved in potassium iodide. The colour of this solution is reddish brown but it turns dark blue in the presence of starch.
Method:
- Using the marker pen write on the pipettes what each one is going to be used for i.e if a pipette is going to be used to measure out the amylase we should write on it “amylase” so that we could use the same pipette next time.
- Using the pipette and pumpette measure out the 10mls of the pH and transfer it into the test tube.
- Then measure out 0.5 mls of fungal amylase using a pipette and transfer it to the test tube with the pH.
- Place the test tube in the test tube rack and leave it for 2 minutes so that the pH and the enzyme have time to react.
- Then add 10mls of starch to the solution.
- Start the stop clock and test the solution for starch every 30 seconds.
- Repeat the same method for different pH values.
- Repeat the experiment 3 times.
I have decided to use 0.5mls of fungal amylase because when I did a pilot test using 5mls, 2.5mls and 1ml the reaction was over quickly so I couldn’t get a reliable result. Using 0.25ml of the fungal amylase meant the reaction time for pH7 took too long so I have decided to use 0.5mls of amylase. I have also decided to carry out my experiment at room temperature because this is easier than setting up the water bath and would save time. I have decided to time in seconds because then it would be easier to draw the graph and would not cause any confusion. I have decided to use 10mls of starch and I’m going to keep it constant through out my experiment.
Safety:
The iodine that is going to be used is toxic and it is also an irritant and will stain clothing. So it would be safer to wear lab coat so as not to stain any of my clothing. Safety goggles should also be worn because i am using buffer solutions and enzymes which if they get into my eyes could be harmful. I should also wash my eyes thoroughly with water if anything gets into the eye. I should wash my hands thoroughly if anything spills on my hands especially the enzyme as it breaks down substances. I should avoid direct contact or inhalation of the fungal amylase. And since I’m using a lot of glassware I should take every care as not to break any of them. Ethically I don’t there is anything to worry and the enzyme I used can be thrown down the drain because we are only using a small quantity and so I don’t think it will have any effect if we put it down the drain. And environmentally I don’t think I have anything to worry about either because I’m not using any living organism so I don’t need to worry about putting them in their natural habitat.
Prediction:
The time taken for the reaction will increase if the pH is too acidic or too alkaline because we are using fungal amylase and this only works if the pH is not too acidic (pH1) and not too alkaline (pH9).Changing the pH outside the limited range in which the enzyme works denatures the enzyme. If the pH is too acidic this alters the number of free hydrogen ions. This affects the charge on the amino acid residues that make up the enzyme and this leads to the change in shape of the active site and the enzyme denatures. If the pH is too alkaline (pH 9) this alters the number of hydroxyl ions in the enzyme. This in turn effects the ionization of the side groups in the enzyme’s amino acid residues and the overall shape of the active site changes. This means the substrate can no longer fit into the active site because the precise shape of the active site is important for the enzyme-substrate complex formation. But there is a pH between pH 2 and pH 8 at which the enzyme will function most efficiently. This is known as the optimum pH and at this pH the enzyme will work its best i.e the shortest time taken for the starch to be converted to maltose.
IMPLEMENTING:
Pilot tests:
In order to find the suitable concentrations of amylase at which the time for the reaction is measurable and to do it within the range of time that was given to me, I tried different concentrations of amylase but I decided to keep the concentrations of starch and the buffer solutions constant because I didn’t have sufficient time to vary these factors as well.
TEST 1
2.5mls of Fungal amylase
TEST 1
1mls of Fungal amylase
TEST 1
Procedure:
When I started the experiment I made sure that I had labelled all my pipettes carefully so that I could use them again without mixing them up. Then I placed my test tubes in the test tube rack so that I wouldn’t accidently drop any of them. I then measured out 10mls of the buffer solution using the pipette and the pumpette. I also made sure that I measured out the 10mls as accurately as I possibly could without letting any air bubbles into the pipette. Then I transferred the 10mls into the test tube and added 0.5mls of the fungal amylase that were freshly made just before I started the experiment. I also took the same precaution as I did when I measured out the buffer solution but I also made sure that I did not spill any of the amylase on my hands or on the table top. I left the test tube with the buffer solution and the amylase for 2 minutes so that the pH and the enzyme will have the time to react together. Then I added 10mls of starch to this solution and started the stop clock. Then I tested the solution for starch every 30 seconds and continued to test the solution for until 40 minutes and even if after this time the solution had a positive starch test then I moved onto my next experiment. Due to limitation of the time I could only test the solutions for starch for only 40 minutes. When I used the iodine I made sure that I didn’t spill any iodine on my clothing or the lab coat. I also made sure that I wore my goggles at all time during the experiment.
RESULTS
0.5mls of Fungal amylase
TEST 1
TEST 2
TEST 3
Analysis:
SUMMARY TABLE.
I have only used pH 3, 4, 5, 6 and 7 to find the rate of the reaction and I have decided not to use pH 1 and 9 because the test for starch is positive even after 40minutes. The rate of the reaction can be found by using the formula:
Rate = 1 ÷ Time
I drew the rate graph because this shows the optimum pH of the fungal amylase at which it works the best. By looking at the rate graph it is possible to tell that the fungal amylase works better in acidic conditions because the rate of the reaction is faster in the pH 3 to 6 range but the activity of the enzyme starts to decrease at pH 7.
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Main trends and patterns:
As the pH increased or became too acidic or too alkaline the starch is not broken down to maltose. The reaction time for pH 1 is 2400 seconds but even at that time the test for starch is positive. This shows that the starch has not been broken down by the enzyme. It is the same for pH 9- even after 2400 seconds the test for starch is positive. This shows that the enzyme has been denatured because the starch is still present after 2400 seconds. This also shows that my hypothesis is right and that the enzyme doesn’t work if the pH is too high or too low. The enzyme doesn’t work at this pH because the shape of the active site is changed by this extreme pH. This result also suggests that the enzyme will have an optimum pH somewhere between pH 2 and pH 7. At pH 3 the time taken for the reaction is 470 seconds, pH 4 is 210 seconds, pH 5 is 150 seconds, pH 6 is 130 seconds and Ph 7 is 2400 seconds. Till pH 6 the time taken for the reaction keeps decreasing but after pH 6 the time taken for the reaction to be completed increases again. This results show that the optimum pH for fungal amylase is pH 6 because it is at this pH that the time taken for the reaction is the least. At the optimum pH the enzyme will function its best and the products are produced really fast compared to the other pH at which that enzyme will work. This theory can be supported by looking at the rate graph. The rate graph shows that the rate of the reaction is at the maximum at pH 6.
Looking at the graph with the error bars shows that the result I got for pH 7 is not very accurate because the error bar is quiet big. This graph is drawn so that any anomalous points could be picked out because the graph drawn on the computer without the error bars doesn’t show any anomalous result when we look at it. From the graph it shows that the most accurate reading I got is for pH 5 and that of pH 6, 3 and I are also quiet accurate.
Evaluation:
The results I obtained maybe due to factors such as the concentration of amylase used. If the concentration of amylase is increased the rate of the reaction will increase and the reaction will be over quickly because there will be more active site available for the starch substrate to combine with. The opposite happens if the concentration of amylase is decreased- the rate of the reaction decreases because the number of active site is greatly reduced so there is less active for the substrate to combine with. The overall time for the reaction will therefore depend on the concentration of the fungal amylase used.
Looking at the graph with the error bars it shows that my result for pH 7 appears to be anomalous because the error bar is big. The anomalous result maybe due to a number of reasons. One of them might be because we carried out the experiment on three different days. When I measured the temperature of the room in which I carried out the experiment on the first day it was 23 degrees Celsius , the second day it was 25 degrees Celsius and on the third day the temperature was 22 degrees Celsius. The temperature of 25 degrees Celsius was because i carried the experiment near an open window so air kept drifting in and out of the room and so the temperature kept fluctuating. Although the temperature change is only very small this still might have an effect on the results I got. Even though I took the temperature of the room at the beginning of the experiment the temperature still might have changed after I started the experiment but this is only going to be a small change so it is not going to have such a big effect on the results.
Another reason why I might have got an anomalous result is because the fungal amylase was freshly made just as I was about to start the experiment. The fungal amylase works really quickly when it is freshly made but as time goes by the reactivity of the amylase slows down and so the time taken for the reaction to be completed increases. So the pH that were tested when the amylase was still fresh could have a faster reaction time than those that were tested after some time had elapsed. The only way to get over this limitation is to keep making fresh fungal amylase each time a pH is t be tested but this would be a waste of the amylase that I hadn’t used. Also when I did the experiment I added the pH and the enzyme and left the mixture for some time before I added the starch. But if I had been given more time then I would have tried adding the starch and the pH before adding the amylase. Then I could compare the results get for this with the results that I already have to see if this would cause any difference.
The most important limitation to this was in making an accurate judgement of the final end point of the reaction. After the end of some reaction the iodine appeared to have a dark brown colour and at the end of some it had a light brown colour so the exact end point was hard to judge. When I did the experiment I only started timing after I had completely added my 10mls of starch and this is not very accurate because the enzyme would have started reacting with the starch as soon as the first drop was added. So I can’t completely say that the time I got for the experiment is very accurate because some of the starch would have already been converted to maltose even before I started the stop clock.
Since there was only a limited amount of time given to me to do the experiment , I only did pH 1,3,4,5,6,7 and 9 but if I had been given more time then I could have tested a wide range of pH eg: pH 4.5, 6.5,8 etc to see exactly at which point the amylase works best. I could have also change the concentration of the starch if I had more time.
But overall with the work I have done and by looking at my results I can draw a firm conclusion that my hypothesis is right and that the enzyme does get denatured if the pH is too acidic or too alkaline and the time taken for the reaction to be completed increases. The experiment also shows that there is a certain pH at which enzymes will work but if the pH is too extreme the enzyme gets denatured. But clearly repeating my experiment would further give me more reliable results.
