Investigation on factors affecting the enzyme Amylase

Results:

First of all the average time taken for the starch to be digested in each condition was calculated.

Temperature(degrees celius)

0

21

32

40

50

55

60

67

80&90

Average Time (min)

9.8

6.7

5.3

5.5

3.5

3.0

3.5

4.5

0 +

pH

8

7

6

5

3

2

Average Time (min)

9.5

6.7

4.5

2.8

4.7

9.2

The results were then plotted on a graph (see Graphs 1 & 2) and the points joined together.

From graph 1, I can see that as the temperature of the mixture increases, the time taken for the amylase to digest the starch decreases. This happens fastest at 55 degrees celius, however if the temperature continues to rise then the time taken for the amylase to digest the starch rapidly increases. By the time 80 OC is reached the amylase does not digest the starch. From graph 2, I can see that as the pH of the mixture increases, the time taken for the amylase to digest the starch decreases. This happens fastest at pH 5. At a higher pH level than pH 5 the time taken for the amylase to digest the starch increases.

Amylase speeds up the breakdown of long chain starch molecules into smaller chains of maltose. Enzyme molecules have a very precise three-dimensional shape. This includes a 'dent', which is called the active site. It is exactly the right size and shape for the enzyme's substrate to fit into (in the case of amylase this is starch). When a substrate molecule slots into the active site, the enzyme 'tweaks' the substrate molecule, pulling it out of shape and making it split into product molecules.

High temperatures make enzymes inactive: this is because they are proteins, which are damaged by temperatures above about 40 degrees celius. In this investigation it was found that amylase operated fastest at 55 OC and was damaged above this temperature. The enzyme is damaged because the molecules are moving faster, these bombard the active site of the enzyme changing its shape; when it's shape has been changed then the starch will no longer fit in the active site and therefore cannot be digested. This is 15 OC above the suggested typical temperature, see Evaluating Evidence for possible reasons why it is higher than expected. By 80 degrees celius the amylase was completely denatured. From this information I can conclude that the optimum operating temperature for the enzyme amylase is 55 degrees celius.

Most enzymes work best at a pH of about 7. This is also because they are proteins, which are damaged by very acidic or very alkaline conditions. However in this experiment the amylase worked fastest at pH 5. This pH is lower than suggested above because the amylase provided by the school was a bacterial enzyme which had an optimum pH between pH 4 and pH 5.

Conclusion:

In my design, I predicted that as the temperature increases, the speed of the reaction would increase. When a specific temperature was reached, I believed that the rate of reaction would dramatically decrease. I believed this because most chemical reactions happen faster when the temperature is higher. At higher temperatures molecules move around faster, which makes it easier for them to react together. Usually, a rise of 10 degrees celius will double the rate of reaction. This is true for enzymes up to about 40 degrees celius. However at 40 degrees celius the enzyme begins to be damaged, so the reaction slows down. By 60 degrees celius the enzyme is completely denatured. I predicted that the same would happen the further away the pH is from pH 7. I believed this because the optimum temperature for most enzymes is about pH 7 therefore the further away from pH 7 (either more alkaline or more acidic) the less effective the enzyme.

My results support most of the prediction since as the pH/temperature increases the rate of reaction increases until a point is reached and then the rate of reaction decreases. However, the optimum temperature was not 40 OC but 55 OC (unusually high) and the optimum pH was pH 5 not pH7. I also predicted that a rise of 10 OC would double the rate of reaction; this was not correct. As is evident in the table above, the time taken does not halve for every 10 degrees celius increase in temperature. I could not find a linear relationship between the temperature and rate of reaction, however with enough results (from a very large range of temperatures) a parabola could be made on Graph 1, it would then be possible to find a mathematical formula for the construction of the parabola and hence find the relationship between the temperature and rate of reaction. At current I do not have enough reliable evidence to draw any conclusions more specific than the rate of reaction is proportional to the temperature until 55 degrees celius is reached and after this point the temperature is inversely proportional to the rate of reaction.

Note: "Optimum operating temperature/pH" means this is the temperature/pH where the enzyme (amylase) operates the fastest.

I believe that the experiment was successful but some of the results were unexpected/unreliable. The time taken for the amylase to digest the starch at 40 OC was far too fast (see Graph 1) it should have been between 4 and 4.5 minutes. All the other results seemed to fit into the trend on the graphs.

I believe that the experiment was designed well but there were a few problems. The optimum temperature for the amylase was too high. I believe that all the results were skewed because the enzyme was not given enough time at each particular temperature to be fully affected before it was added to the starch. Because of time restraints, they were only left in the water bath for 10 minutes before starting the experiment. However, they should have been left in the water bath for about 30 minutes so that the amylase had been completely affected by the temperature before the experiment was started.

I decided to conduct the experiment at 10 OC intervals instead of 5 OC because there was not enough time. When the results were collected, I plotted them on a rough graph to find the optimum temperature and then conduct the experiment at this temperature to ensure it was the optimum temperature. I also conducted all three experiments for each condition at the same time to save time.

Additional work, which could be carried out, is to repeat the experiment using, a wider range of temperatures and pH levels, and a range of different starch solution concentrations or using different enzymes such as protease with a protein.