Amylase is an enzyme that helps to digest starch into maltose

From this graph, one can see that the effectiveness of amylase peaks at about 37ºC. What I aim to do is to prove that amylase is most effective at helping to digest starch at about 37ºC by conducting a practical experiment. I predict that as I increase the temperature of the amylase & starch source from 10ºC to 50ºC, the rate of reaction of the enzyme will rise peaking when the temperature is about 37ºC, and fall after this point. Due to technical limitations, I will conduct this experiment only at the following temperatures: 10ºC, 20ºC, 30ºC, 40ºC, and 50ºC.

Method

In this experiment I will measure the time taken for starch to be digested with the aid of amylase, at different temperatures. I will do this my adding samples of the starch and enzyme to iodine solution, and then depending on the colour produced by the iodine, I will determine whether or not all of the starch in the mixture has been digested, as iodine changes from orange to blue/black if it is mixed with starch.

Pre-experimental set-up

Prepare 5 spotting tiles with one drop of iodine solution in each depression.

Section A: Experimental procedure for temperatures above and including 20°C

1. Heat water-bath W to temperature of 20°C, verifying with thermometer.
2. Place 2ml starch in test tube named S1
3. Place 4ml amylase in test tube named A1
4. Place both test tubes in water-bath W, and once the correct temperature of the starch and the amylase has been obtained and verified with a thermometer, proceed to step 5.
5. Combine contents of both test tubes in beaker B1. Start stopwatch. Place beaker B1 into water-bath W to maintain correct temperature.
6. Continue to step 1 in section C.

Section B: Experimental procedure for temperature of 10 °C

1. Mix 200ml of cold tap water, and about 10 ice cubes in large beaker B2.
2. Place 2ml starch in test tube named S2
3. Place 4ml amylase in test tube named A2
4. Place both test tubes in beaker B2, and once temperature of 10°C of starch and amylase has been obtained and verified with thermometer, proceed to step 5.
5. Combine contents of both test tubes in small beaker B3. Start stopwatch. Place and remove beaker B3 into and from beaker B2 to maintain temperature of 10°C, verifying with thermometer.
6. Continue to step 1 in section C.

Section C: Continuation of either section A or section B

1. After 30 seconds add two drops of mixture from either beaker B1 or beaker B2 depending on which temperature range of reaction is being investigated, to first depression on spotting tile.
2. Repeat step one every 30 seconds, adding mixture to following depression on spotting tile, until no blue/black colour is instantly observed in spotting depressions once mixture has been added to them. Record time (divisible by 30 seconds) taken to achieve this.
3. Repeat steps 1-2 two more times and calculate average time taken for reaction to take place.  
4. If investigating with temperatures above and including 20°C, repeat section C, but with temperature of starch and amylase increased by another 10°C.
5. Stop experiment once temperatures 10°C, 20°C, 30°C, 40°C, and 50°C have been investigated.

Variables

• My independent variable in this experiment is the temperature of the starch and enzyme.
• My dependant variable is the time taken for the starch to be digested.
• My controlled variable is starch at room temperature with no enzyme added.

Keeping a fair test

To maintain fairness throughout the experiment, I will make sure the following are exercised:

• The same volumes of starch and amylase will be used for each temperature and attempt.
• The same concentrations of starch and amylase will be used for each temperature and attempt.
• The same amount of iodine will be used for each test for the presence of starch.
• The test tubes and beakers will be all placed in the water-baths and ice-baths so all of their contents is warmed or cooled equally.
• The temperatures of the water-baths will always be constant, and not fluctuate.
• The mixtures of starch and amylase will be stirred the same amount for each temperature and attempt.

Safety

• Move slowly
• Wear goggles at all times
• Wear a lab coat at all times
• Do not ingest any lab chemicals

Equipment list

• Water bath
• Starch solution
• Amylase solution
• Spotting tiles x 6
• Test-tubes x 3
• Beaker
• Pipette
• Precision pipette
• Iodine solution

Results

These results can be displayed in a more clearly on the following two graphs:

Now to compare this graph to the graph I used in my hypothesis, I will plot 1/time against temperature/°C against taken for all starch to be digested, which produces a similar shaped curve.

Conclusion

From my experiment, I can conclude that as the temperature of the reaction increases approaching 40°C, the rate of reaction of the enzyme also increases. Moreover, past the temperature of 40°C, the rate of reaction of the enzyme decreases. This supports my prediction and hypothesis. This can also be seen on the second graph, where the rate of reaction of the enzyme peaks at 40°C. “Key Science” by D. Applin, published by Stanley Thornes publishers 1997 explains that as the temperature at which the enzyme is working increases (up to about 37°C), the rate of reaction of that enzyme also increases, due to more collisions of the starch substrate with the amylase, and then decreases because the enzyme begins to denature, which occurs since the three dimensional shape of the enzyme shakes, and the weak bonds holding it together break resulting in the enzyme itself shattering into more than one piece, rendering it more and more ineffective.

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Evaluation

My method proved to be faultless when carrying it out, and let me achieve the results I was hoping to achieve. My findings produced no unusual results, and for every temperature, the durations of time obtained in which all the starch was digested were very similar, which means that my method of data obtaining was not only stable, but also accurate and gave me data which was not spread out much. I feel that it is sufficiently conclusive to support my prediction. However, I can make a few changes that could improve this experiment. Namely, increasing the amount of water-baths at different temperatures, which would increase in 1°C increments and not 10°C. This would allow me to plot more precise curves and would also allow me to prove that indeed the optimum temperature of amylase is about 37°C and not 40°C, which is what I had to assume due to limitations in the equipment I had access to. Also the use of more accurate starch digestion detection methods could mean that it would be possible to obtain results that are more accurate than to the nearest ½ minute, as in my case.  To reinforce the idea that human enzymes work at an optimum temperature more, I could conduct the same experiment, but instead of using amylase to digest starch into maltose, I could use lipase to digest fat into fatty acids and glycerol. This experiment would also show that the optimum temperature for the rate of reaction of an enzyme in humans is around 37°C.