Digestion Self-Design Practical: The Effect of pH on Enzyme Activity

Digestion Self-design Practical: The effect of pH on enzyme activity

Rationale
Amylase is an enzyme involved in the digestion system which catalyses the breakdown of starch into sugars. It is not only present in human saliva but also in the pancreas, where it hydrolyses dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. pH has an effect on the activity of all enzymes, including amylase. As the pH level increases, the enzyme activity increases, up until an optimum pH level where enzyme activity plateaus. For amylase, this optimum pH levels is usually about pH7. The present study investigates this relationship between pH levels and enzyme activity.

Aim
The aim of the present study is to examine the effect of different pH levels on the rate of amylase activity.

Research Question
What is the effect of varying pH levels on the rate of amylase activity?

Hypothesis
The hypothesis of the present study is that increasing pH levels will increase the rate of amylase activity; pH levels at 7 or higher will show a fast rate of amylase activity.

Variables:
Independent Variable: The independent variable was the varied pH levels manipulated by using a buffer solution of different pH levels. The different pH levels were pH0, pH4, pH6, pH8 and pH10

Dependent Variable: The dependent variable is the amylase activity. Amylase activity was measured by recording the concentration of starch in each test tube at 0minutes, 5minutes, 10 minutes and after the weekend. This was done by adding 2 drops of iodine to each test solution at the end of each interval and recording the colour of the solution using a colour chart.

Controlled Variable: A controlled variable was the time of measurements, timed with a timer on a mobile phone. The measurements were taken at 0 minutes, 5 minutes and 10 minutes. This needed to be controlled in order for the rate of enzyme activity to be measured accurately.

Materials/Apparatus
100mL of starch
20mL of buffer pH 0
20mL of buffer pH 4
20mL of buffer pH 6
20mL of buffer pH 8
20mL of buffer pH 10 pipette 20 test tubes
100mL of amylase
Iodine solution measuring cylinder to measure 5mL
Colour Scale
Method
With the measuring cylinder, 5mL of each pH buffer solution was measured (5mL of pH0, 5mL of pH4, 5mL of pH6, 5mL of pH8 and 5mL of pH10) and poured into 5 different test tubes.
Using the measuring cylinder again, 5mL of starch was measured. 5mL of starch was poured into each test tube with each buffer solution.
2 drops of iodine was added to each test solution.
Using the measuring cylinder again, 5mL of amylase for each test solution was measured and, as simultaneously as possible, 5mL of amylase was poured into each test tube.
As soon as amylase was added, the timer was started, and the first colour measurements were taken at 0 minutes.
After 5 minutes on the timer, the colour of each test solution was measured again.
After 10 minutes on the timer, the colour of each test solution was measured again.
Out of interest, after the weekend (approximately 48 hours), the colour of each test solution was measured again.
This method was repeated for 4 trials.

Precautions
Precautions that were taken in order to attain the most accurate results were the control of the time intervals. Safety precautions included washing our hands after the experiment.

Data Collection and Processing

TRIAL 1 - Colour measurements of test solutions

|0 minutes |5 minutes |10 minutes |following weekend (approx. 48hrs) | |pH0 |black |black |black |very dark blue | |pH4 |black |black |black |dark blue | |pH6 |black |black |black |light blue | |pH8 |black |black |light blue |no colour | |pH10 |white |white |white |no colour | |

TRIAL 2 - Colour measurements of test solutions

|0 minutes |5 minutes |10 minutes |following weekend (approx. 48hrs) | |pH0 |black |black |black |very dark blue | |pH4 |black |black |black |dark blue | |pH6 |black |black |black |light blue | |pH8 |black |black |light blue |no colour | |pH10 |white |white |white |no colour | |
TRIAL 3 - Colour measurements of test solutions

|0 minutes |5 minutes |10 minutes |following weekend (approx. 48hrs) | |pH0 |black |black |black |very dark blue | |pH4 |black |black |black |dark blue | |pH6 |black |black |black |light blue | |pH8 |black |black |light blue |no colour | |pH10 |white |white |white |no colour | |
TRIAL 4 - Colour measurements of test solutions

|0 minutes |5 minutes |10 minutes |following weekend (approx. 48hrs) | |pH0 |black |black |black |very dark blue | |pH4 |black |black |black |dark blue | |pH6 |black |black |black |light blue | |pH8 |black |black |light blue |no colour | |pH10 |white |white |white |no colour | |Evaluation and Conclusion
The results show an obvious trend. Every trial had the exact same results. This shows that the experiment was quite well controlled as the process was repeated for 4 trials and the same results were achieved. For pH0, pH4 and pH6, enzyme activity was rather slow as the starch remained fully present in the solution for at least 10 minutes, as the colour did not change from black in all trials. This shows that at lower pH levels, enzyme activity is very slow. However, at pH10, the colour of the solution immediately became white in all trials, demonstrating an extremely rapid rate of amylase activity as starch was broken down immediately. This shows that at a higher pH level, amylase activity is very fast. At pH8, after 10 minutes the solution was light blue, meaning a rather fast amylase activity rate, and after the weekend, the starch had been completely broken down as there was no colour in the solution. This supports the theory that at pH levels of 7, being the optimum pH for amylase, or higher, the rate of activity of amylase would be high. It also supports the research hypothesis, that with increasing pH levels, the rate of amylase activity would increase. In addition, it is evident that the time intervals effected enzyme activity, as after 48 hours, the starch had been broken down, at least slightly, even at pH0.

Sources of Error and Limitations
The fact that all trials achieved the exact same results shows the strength of the method’s accuracy. Having said this, all 4 trials were done at the same time. This arrises a limitation of the study; it was difficult to add the amylase to each test tube exactly simultaneously with just two people as there were 20 test tubes, and therefore the time intervals may have varied slightly. However, this would have been only a few seconds and would probably have not affected the data in a major way, if in any way at all. Even so, in order to address this problem, it may have been more useful to conduct the trials at different time or have some more people in order for it to be possible for the amylase to be poured into the test tubes at the same time. Human error is of course another source of error, especially as the measurements being taken were colours, the perception of which may be subjective to the experimenter’s own interpretation. Hence a colour chart was used in order to achieve the most accurate measurements of colour.

Suggestions for Improvement
A suggestion for improvement would be to either conduct the trials at different times or have more people available to pour the amylase into the test tubes simultaneously. This would allow the amylase to begin activity at exactly the same time in all 5 conditions and would mean that the time intervals would be more accurate. Another suggestion for improvement would be to add another condition level; pH7, as this is the optimum pH level for amylase and would have been conducive to previous research supporting this. It would have also helped to strengthen the support for the research hypothesis of the study. To take this investigation further, one could add other pH levels, possibly some extreme high pH levels, in order to further develop the theory of the effect of pH levels on enzyme activity.

Conclusion
In this study, it has been demonstrated that the effect of pH levels on amylase activity is as follows; as pH levels increase, the rate of amylase activity increases. In addition, pH levels at 7 or higher have been shown to be optimum for the activity of amylase.