I predict that the shape of the graph to be a U shape as to start with it would take longer because there would be less energy for the particles. As the temperature increases from 20°c and 35°c the graph will continue to curve down as it gets closer to body temperature. I think the optimum of the graph will be around 35°c or 40°c. As the temperature increases from 40°c the graph will gradually curve up as the enzyme begins to denature.
I can back-up my ideas from a preliminary piece of evidence from the computer simulation enzyme lab. This experiment showed what happened as the temperature increased from 10°c-80°c to amylase α and how many arbitrary units of maltose were produced in three minutes. Similarly to my experiment this experiment showed how well amylase digests starch into maltose and how much was produced at each temperature. The results were:
This experiment shows that the optimum temperature was 40°c. It shows that anything over 40°c begins to denature and less maltose is produced. With anything under 40°c maltose levels go down so this shows that the particles have less kinetic energy to move around. The shape of the graph in this experiment would be an n shape as the optimum is how much maltose is produced and that would be high on a graph.
Equipment list:
- Bunsen burner
- Tripod
- Gauze
- Bench mat
- Thermometer
- Stop clock
- Two test tubes for each temperature
- 250ml beaker
- Dropping pipette
- 10ml syringe and 1ml syringe
- Dimple tile
Solutions required:
- Amylase solution
- Starch solution
- Iodine solution
Method:
- Place 200ml of water into a beaker.
- Label two test tubes 1 and 2
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With test tube 1, use a small syringe to put 5cm3 of starch solution into it.
- Put test tube 1 into the beaker of water.
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Using the syringe put 1cm3 of amylase solution into boiling tube 2.
- Place test tube 2 into the beaker of water.
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Warm the water to 20°c.
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When the water has reached 20°c, wait 5 minutes whilst keeping the water at 20°c.
- During the 5 minutes take a pipette and put a few drops of iodine solution into a dimple tile.
- After 5 minutes, take test tubes 1 and 2 out of the beaker. Add the contents of test tube 1 into test tube 2 and shake very thoroughly.
- Return boiling tube 2 to the beaker of water.
- Every 15 seconds test the contents of test tube 2 to see if there is any starch left in them.
- Do this by using a pipette to add a few drops from tube 2 into the iodine in the dimple tiles.
- If the solution remains dark blue/black then starch is still present.
- If the solution turns to an orange/red colour then no starch is present and the amylase has broken the starch molecules down into sugar molecules.
- Keep repeating it every 15 seconds until you decide there is no starch present in the test tube and the solution in the dimple tiles has turned orange/red.
- Record the time taken in the table of results.
- Repeat the experiment in exactly the same way except use different temperatures to warm the water up to.
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Use the temperatures 20°c, 30°c, 35°c, 40°c, 45°c, 50°c, 60°c and 70°c.
- Repeat for each temperature to make sure that the results are accurate.
- Find an average time for all temperatures.
Fair test:
In this experiment there are a few factors that I will have to take into account to make it a fair test. First I will have to make sure I use the same amount of starch for each temperature. If one has slightly more starch in it the amylase will have more to digest and the time will be longer. If one has less in it then the amylase will have less starch to digest and the time will be quicker. Also the amount of amylase is a factor. Too much amylase means there will be more to break down the starch and the time will be quicker. Too little means that there will be less to break down the starch and the time will be longer. The amount of time the solutions are left before testing is important as well to keep it a fair test. During those five minutes the temperature has to remain constant to keep it a fair test. If the Bunsen burner is left in too long the temperature may raise too high, therefore there will be more collisions and the rate of reaction will be quicker. The temperature needs to be exact to keep it a fair test. Also I needed to decide when it had completely changed and the starch had all been broken down into sugar. I had to decide on the same colour for each set of results otherwise some of them may not have completely changed. After I had heated the water and test tubes to the certain temperatures I had to shake the mixture of amylase and starch very thoroughly. This meant that the enzymes were well spread out and could get to all of the starch. The pH had to kept constant as well because amylase digests starch quicker at alkaline temperatures so a buffer could be added to keep pH the same. The only thing that I did change was the temperature and everything else had to be kept the same. All of these had to be considered before I started the experiment. If it wasn’t a fair test then the results may become inaccurate.
Table of results:
The time at which the rate of reaction is the quickest was 60°c.
I worked out an average because if one of my results was anomalous and the other result accurate it would reduce the margin of error and my results would be more accurate.
The optimum temperature was 60° (75 seconds on average) and the slowest time taken was at 20° (352.5 seconds on average).
Analysis:
The shape of my graph is a U shape. When the temperature was at 20°c the time taken was the longest out of all the temperatures (352.5 seconds on average). The reaction time went down gradually until 40°c. This occurred because the particles were gaining more kinetic energy and therefore there were more collisions between the enzyme and starch particles. This meant the particles were reacting more. At 45°c the reaction time was slightly longer than at 40°c. The reaction time then went down at 50°c and 60°c and 60°c was the optimum. This is because with more kinetic energy the particles were colliding more. At 70° the graph began to rise as the enzymes began to denature and reaction time started to get longer. Again with more kinetic energy the collisions between active site and substrate were greater. However this caused the enzymes to vibrate more, it broke their bonds and the enzymes began to change shape and denature. This meant it was more difficult for the substrate to fit into the enzyme and be broken down.
The optimum temperature on my graph was 60°c. My prediction for optimum was between 35°c and 40°c which is near body temperature. My prediction was wrong because my prediction was based on salivary amylase which is produced in the salivary glands and is adapted to the conditions of body temperature. We were using bacterial enzymes which although are chemically similar to salivary enzymes are adapted to different conditions. They may be a slightly different shape to salivary enzymes and this could affect the temperature that they work best at. If we had been using salivary enzymes I would expect the optimum temperature to be around 37°c as this is body temperature.
Evaluation:
Some of my results may be slightly inaccurate so I will have to evaluate the reliability of my results. To make the results more accurate I could have done more tests to get a better average. The problem with doing only two tests was that if one of those results was wrong it would have greatly affected the average. If I had done about six sets of results then I would have had a more accurate average and I would have also known which results were anomalous. Anomalous results are results which are inaccurate and do not fit with the line of best fit. Another factor which would have affected the reliability of my results was to keep the temperature constant. If I had been using a water bath the temperature would have remained constant and the temperature wouldn’t have risen into the next interval. We were using Bunsen burners and thermometers and so when the Bunsen burner was removed sometimes the temperature continued to rise. This is because the water’s particles were still moving with kinetic energy. This meant that the temperature sometimes went up into the next interval. Also I could have done another temperature above 70°c just to check that the enzyme had denatured. My result at 70°c could have been anomalous so I could have tried 80°c to make sure the enzyme was denaturing.
My result at 45°c was anomalous. I would have expected the average result to be around 150 seconds whereas my results were 187.5 seconds. To make this result more accurate I could have done more sets of results to get a more accurate average. My other results were quite accurate although the results at 50°c didn’t fit on the line of best fit. But again I could have done another set of results to find a more accurate average and this would have shown which results were anomalous.
There are a few improvements I could have made to make my results more precise. I could have used closer time intervals than 15 second to check when the starch had completely changed. When I did it every 15 seconds it could have changed during those 15 seconds and doing it more frequently would give me a more accurate time. Also I could have used a colorimeter which is a device used to measure colour change. This would have helped to check when the starch had completely changed into maltose. One of the problems I had was that it was difficult to find an exact colour and you had to use that same exact for all of the temperatures. A colorimeter would have made sure that the starch had completely changed each time. Another thing that would have made the results more accurate would have been to use closer temperatures to the optimum. My optimum was 60°c but this was not that precise as the actual optimum could have been anywhere between 51°c and 69°c. If I had done intervals of every 2°c then I would have had a wider span of results and I may have had a different optimum. Also I could have used a water bath (thermostat) to keep the temperature constant.
A Further experiment I could do about enzymes is to do the same experiment with salivary enzymes. I would expect to get an optimum of around 37°c as this is body temperature. Also I could do the effects of pH on amylase do see what conditions it works best in. I would expect amylase to work best in alkaline conditions as saliva is alkaline. Amylase is also found in the small intestines and to make the conditions alkaline, secretions are added. These secretions are found in the pancreas and liver and are called pancreatic secretions and bile salts. Other experiments I could do are to see at what temperatures protein and fats are digested best at.