Topic: Enzymes

Research question:

To what extent does pH 2,4,6,8,10 affect enzyme activity of catalase on hydrogen peroxide
measured by the time taken for a yeast bead to rise.

Prediction:

Enzymes have a limited range of pH at which it functions efficiently. The structure of the
enzyme and shape of active site is maintained by various bonds within 3-D structure of the
protein. A change in pH from the optimum value will shape of the molecule and disrupt
bonding.
The optimum pH level of catalase is between pH 4 and pH 6 (R;, Relationship between ph and
medium dissolved solids in terms of growth and metabolism of lactobacilli and saccharomyces
cerevisiae during ethanol production). When the pH decreases below the optimal value, the
enzyme activity is creased. When the pH value far increases above the optimal value, the
enzyme activity is said to be denatured which makes it inactive. If the pH is beyond this
level, the catalase may be denatured, increasing the time needed for catalase to break down
hydrogen peroxide. Therefore, I predict that the reaction happens the fastest at the pH level of
4 and 6.

Quantitative data

A yeast sphere is dropped into a test tube containing hydrogen peroxide. The sphere will sink
to the bottom of the cylinder. As the catalase produced by the yeast reacts with hydrogen
peroxide to form O2 gas bubbles around the sphere, the sphere will rise to the surface.

Raw data
Table 1: raw data
pH value of the buffer Time taken for yeast bead to Average time taken for a yeast
solution rise second(+/-0.5s) bead to rise(+/-0.5s)
2 58 47
47
44
38
46
4 35 38
37
43
37
38
6 35 39
37
43
43
37

1
 pH value of the buffer Time taken for yeast bead to Average time taken for a yeast
solution rise second(+/-0.5s) bead to rise(+/-0.5s)

8 43 41
38
39
44
41
10 34 35
37
32
35
38

Processed data

Table 2: processed data

Average time
taken for a yeast standard
33% of the mean Reliable?
bead to rise(+/- deviation
0.5s)
47.00 15.51 7.27 yes

38.00 12.54 3.00 yes

39.00 12.87 3.74 yes

37.00 12.21 1.87 yes

35.00 11.55 2.39 yes

2
 Average time taken for a yeast bead to rise(+/- The effect of pH on yeast activity
50
45
f(x) = − 1.05 x + 46.3
40 R² = 0.55125
35
30
25
0.5s)

20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11
pH value of buffer solution

Error bar=± 1 SD

The error bars had shown overlaps, suggesting statistical insignificance differences between
the enzyme activity in different pH solutions. The One-way ANOVA test was carried out
because there are more than two independent variables.

ANOVA test

The one-way ANOVA is used to determine if the means of three or more independent sets of
samples are significantly different from one and another. It tested the null and alternative
hypothesis at significance level of 0.05. The following hypothesis were considered:

Null hypothesis (H0): There is no significant difference between the time taken for a yeast
bead to rise in different pH solutions.

Alternative hypothesis (H1): There is a significant difference between the time take for a yeast
bead to rise in different pH solutions

One-way ANOVA of your  kk=5 independent treatments: 

Table 3: anova test

sum of  degrees of  mean square  F

source p-value 
squares SS freedom νν MS statistic

treatmen
385.3600 4 96.3400 5.6671 0.0032
t

error 340.0000 20 17.0000

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 sum of  degrees of  mean square  F
source p-value 
squares SS freedom νν MS statistic

total 725.3600 24

The p-value of one-way ANOVA is lower than 0.05, suggesting that the one or more
treatments are significantly different. The Tukey HSD test follows. This test would likely
identify which of the pairs of treatments are significantly different from each other.

Table 4: Tukey HSD results

treatments  Tukey HSD  Tukey HSD  Tukey HSD 
pair (pH) Q statistic p-value inferfence 

2 vs 4 4.6640 0.0263887 * p<0.05

2 vs 6 4.1217 0.0586408 insignificant

2 vs 8 5.2063 0.0114636 * p<0.05

2 vs 10 6.1825 0.0024402 ** p<0.01

4 vs 6 0.5423 0.8999947 insignificant

4 vs 8 0.5423 0.8999947 insignificant

4 vs 10 1.5185 0.7964840 insignificant

6 vs 8 1.0847 0.8999947 insignificant

6 vs 10 2.0608 0.5897060 insignificant

8 vs 10 0.9762 0.8999947 insignificant

It if concluded that only one pair of data (2 vs 10) is significantly different from each other as
p<0.01.

Conclusion

From the graph, it can be seen that the enzyme activity was low at pH 2. When the pH is
increased to 4 and 6, the reaction happened faster. The reaction rate decreased in pH 8 and
increased again in pH 10 where the enzyme was most active. The result obtained doesn’t
match my hypothesis which suggested possible errors in the investigations such as systematic
or random errors. This would be further elaborated in the evaluation part.

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The 33% of the mean and standard deviation were calculated. The results collected were
reliable as the 33% of the mean was greater than the standard deviation which indicated the
results were reliable

Strengths
1. Each trial was repeated for five times. The number of trials provide sufficient and
reliable data to identify the relationship between pH and enzyme activity
2. The standard deviation was lower than 33% of the mean. This means the standard
deviation is small which points out that the data was close to the mean and therefore
reliable.
3. The ANOVA test showed that P-value < 0.05 which shows the data is statically
significant.

Limitations
Source of error
Human reaction time error
when stopping the
stopwatch
There could be leakage of
oxygen gas from the system
Effect on result Possible improvements
Stopwatch may be stopped Repeat the trial 10 times
earlier or later due to instead of 5 times
reaction time delay. The
value obtained may be
higher or lower than the
desired value.
There is less oxygen present Use stoppers to prevent
in the test tubes. oxygen from escaping.

The pH values of buffer Check the buffer solutions

Didn’t check the pH of solutions were assumed to with a pH meter before
solutions before using them be 2,4,6,8,10. However, the doing the experiment.
values could be slightly
higher or lower.

Possible extension

Besides optimum pH, enzymes also have their optimum temperatures at which enzymes work
its best (Enzyme operating conditions - proteins - national 5 biology revision - BBC Bitesize)
. As the temperature increases, the enzymes become more active. Its activity is increased to a
maximum value at the optimum temperature. When the temperature exceeds the optimum
temperature, enzymes can no longer function, and denaturation occurs. Therefore, it is
essential to find out the optimum temperature for catalase to maximum the rate of reaction.

Citations

1. R;, N. N. V. P. (n.d.). Relationship between ph and medium dissolved solids in terms

of growth and metabolism of lactobacilli and saccharomyces cerevisiae during
ethanol production. Applied and environmental microbiology. Retrieved July 12,
2022, from https://pubmed.ncbi.nlm.nih.gov/15870306/ 

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 2. BBC. (n.d.). Enzyme operating conditions - proteins - national 5 biology revision -
BBC Bitesize. BBC News. Retrieved July 12, 2022, from
https://www.bbc.co.uk/bitesize/guides/zcr74qt/revision/4