Tae Jeong 11 Biology
Optimal pH for amylase
Tae Jeong 11L
Aim:
To find out the optimal pH for amylase
Background information:
Enzymes are sensitive to factors such as temperature and pH. Each enzyme has its specific optimal
temperature and pH. It means each enzyme can perform its job to catalyse its specific reaction in
optimal condition. If the temperature and pH become further away, the enzyme cannot do its job
well. (Anne Semple, 2006) This is because the enzyme denatured and
cannot hold the substrate molecules in further optimal pH. (S-cool,
2011)
Benedict’s solution is the solution which is used to figure out how
much sugar in a solution. It means it is the indicator of concentration
of sugar in a solution by looking at the colours of Benedict’s regent.
As the table beside shows, the colour of Benedict’s reagent changes
when the sugar concentration changes. (Anne Semple, 2006)
Fg1: the table which shows the colour of
Benedict’s reagent changes (Anne Semple, 2006)
Alpha amylase is the enzyme which breaks down starch into
sugar. It is present in almost all plants, animals and microorganisms. The example of starch which
Alpha amylase breaks down is amylose. (Marshall)
Banana is the fruit which has the highest amount of amylose. (HudsDirk)
The starch should be small to be efficient. Because the solution is transported from the surface of
starch to the centre, the surface area should be bigger and volume should be smaller to be efficient.
Thus the surface area should be bigger, but as a cell increases in size the volume increases more
rapidly than does the surface area, so starch should be small. (BlamireProfessor, 2001)
The optimal temperature of amylase is 30OC, and the optimal pH if amylase is 6.
(T. K. DUTTA, M. JANA, P. PAHARI, T. BHATTACHARYA, 2005)
Figure 2: The effect of temperature and pH on amylase activity. (T. K. DUTTA, M. JANA, P. PAHARI, T. BHATTACHARYA, 2005)
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In this experiment, the colours after reaction with benedict’s solution and the weights of banana
before reaction with amylase and after the reaction will be investigated. Then the relative activity will
be observed to get optimal pH value. Finally, the optimal pH value will be concluded.
Variable:
My experiment is to get an optimal pH value for amylase. To get an optimal value, I varied the pH of
amylase solution. Also, I controlled the temperature to make the experiment fair because the
enzyme performs its job to catalysis reaction differently if the temperature is different. To make the
experiment efficiently and fairly, I put all amylase solutions with banana in optimal temperature
(30oC).
Hypothesis:
Based on the background information, the reaction in pH6 would show the most enzyme activity.
Therefore, optimal pH would be 6 and that pH, the concentration of sugar would be the biggest.
Pilot test
Material:
-
5% stock pepsin solution
Thermometer
pH meter
boiled egg
test tube
carpenter
Dropper
150mL Beaker
-
5 50mL Beakers
Scale
HCl stock solution
Water bath
Paper towel
NaOH stock solution
Callipers
Label
Method:
1.
2.
3.
4.
5.
6.
7.
8.
9.
100ml of 5% stock pepsin was obtained.
1 hard-boiled egg was peeled away shell and cut in half to remove the yolk portion.
The egg white was cut into 20 cubes that each side measures 0.5 cm.
20 ml of pepsin solution was poured into each of the 50ml beakers.
The pH of the pepsin solution in each beaker was varied by adding HCl to make it
more acidic or NaOH to make the solution more basic (from pH 1 to pH 5).
Using a watch glass and the electronic balance, the mass of each 4 cubes of egg
white were recorded.
The 4 cubes of albumen were added to one test solution and placed test solution in
the water bath at 37°C for 2 days.
The egg whites were removed from the pepsin solutions, carefully blotted, dried and
recorded the new mass of the albumen.
The % of albumen digested was calculated to observe how much was digested by the
pepsin solution.
Tae Jeong 11 Biology
Results:
A
pH of Pepsin
solution
1
2
3
4
5
B
Initial mass of
albumen (g)
C
Final mass of
albumen (g)
D
Change in
albumen mass
D= B – C
E
% of albumen
digested
E = D/B x 100
4.60
4.84
4.77
4.66
4.62
0
0
0.07
0.25
0.20
4.60
4.84
4.70
4.41
4.42
100
100
98.5
94.6
95.7
Conclusion:
The results above show that the Pepsin solution in pH 1 and 2 digests albumen most efficiently.
Therefore, the optimal pH of Pepsin solution is between pH1 and pH2. It was the results I expected.
This is because the optimal pH of pepsin solution is around 2.
(Worthington Biochemical Corporation, 2012)
Modifications to method:
In this experiment, we got the results which are not clear. Because the % of albumen digested in pH
1 and 2 is same (100%), it was not possible to figure out the optimal pH of Pepsin solution. Because
the albumen was digested perfectly for 2 days, it would be better to place the solution for 1 day to
get an exact optimal pH. Also, by looking at the table above, the % of albumen digested in pH 4 and
5 is quite weird because the albumen in the pH value which is further from optimal pH should react
smaller. Therefore, it would be better to repeat this experiment more than 2 times.
Main test
Material:
-
5% stock amylase solution
Thermometer
pH meter
banana
test tube
carpenter
stop watch
50mL Benedict’s solution
Dropper
Clean wrap
-
150mL Beaker
5 50mL Beaker
Scale
HCl stock solution
Water bath
Paper towel
NaOH stock solution
Callipers
distilled water
Fg 3: sets of apparatus
Tae Jeong 11 Biology
Method:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
100mL of the 5% stock alpha amylase solution was placed in 150mL beaker.
20mL of solution was poured into each of 50mL beaker
Banana was cut into 20 cubes that each side measures 0.5 cm.
The weight of banana cubes each was measured and recorded.
Using HCl, water and NaOH solution, the pH of amylase solution was varied (4, 5, 6, 7 and 8).
4 cubes were put into each of solutions and covered with clean wrap.
The temperature of each solution was measured 30oC in water bath.
Banana cubes were removed from beakers after 20 hours.
The banana cubes were dried and cleaned using paper towel.
The weight of banana cubes reacted was measured and recorded.
20 drops of benedict’s solution were dropped into the solution in beaker.
The colours of each solution were recorded.
Results:
the value of pH, pH
mass before reaction, g
mass after reaction, g
the relative activity, %
the percentage of change. %
Colour after reaction with benedict’s
The results table from experiment
3.99
1.63
1.72
-65.09
-5.52
green
5
2.04
2.01
17.33
1.47
green
6.03
1.65
1.51
100.00
8.48
green
6.99
1.76
1.63
87.15
7.39
green
8
2.23
2.08
79.36
6.73
green
Analysis:
By the result, the % of relative activity in pH 6 is the biggest. This graph below shows the pH with the
optimal pH value. By looking at the graph, the % of relative activity in pH 6 is the biggest and the
others which are smaller than in pH 6. By this we found the optimal pH value is 6. The % of relative
𝑚𝑎𝑠𝑠 𝑎𝑓𝑡𝑒𝑟 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛
activity and change were calculated by this. The % of change = (1 − 𝑚𝑎𝑠𝑠 𝑏𝑒𝑓𝑜𝑟𝑒 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛) x100%,
𝑡ℎ𝑒 % 𝑜𝑓 𝑐ℎ𝑎𝑛𝑔𝑒
The relative activity = 𝑡ℎ𝑒 % 𝑜𝑓 𝑐ℎ𝑎𝑛𝑔𝑒 𝑓𝑜𝑟 𝑜𝑝𝑡𝑖𝑚𝑎𝑙 𝑝𝐻 × 100%
Table which shows the relative activity changes against the pH value
Tae Jeong 11 Biology
Discussion:
In this experiment pH values were varied in optimal temperature to get an optimal pH value. By
looking at the results, the optimal pH of amylase is 6. Because the relative activity is the largest in pH
6, it is the optimum. Based on the background, the optimal pH is the best condition which can
perform its job to catalyse its specific reaction. This result is what was expected in hypothesis; based
on the background information, the reaction in pH6 would show the most enzyme activity.
Therefore, optimal pH would be 6 because the relative activity of amylase in pH 6 is the biggest. (T. K.
DUTTA, M. JANA, P. PAHARI, T. BHATTACHARYA, 2005)
However, by looking at the colour after reaction with benedict’s solution, there was no difference.
By looking at the background information, the colour of benedict’s solution changes into green when
the sugar concentration is 0.5% - 1.0%. It means the reaction time was too short to figure out the
difference. So it could be improved by putting solutions in water bath for 2 or 3 days.
Also, it would be better to dry bananas perfectly after reaction because my results show that the
banana absorbs the solution. Even though the amylase catalysed to digest banana, banana gains its
weight.
The sizes of each banana cubes were different. This means the experiment was not fair. Based on
the background, the smaller cubes are more efficient. (BlamireProfessor, 2001) Therefore, the
experiment could be improved by making the cubes exactly same.
Additionally, the results of this experiment would be more accurate by repeating more than 2 times.
This is because the error is reduced when the experiment is repeated. (JoyceHelen, 2003)
In this experiment, the theory could be proven that each enzyme has its specific optimal
temperature and pH and the enzyme cannot do its job well if the temperature and pH become
further away. It is easy to see this by looking at the results I got.
Conclusion:
The optimal pH of amylase is 6 based on the results.
Works Cited
Anne Semple. (2006). Nelson Bilology. South Mebourne: Eleanor Gregory.
Blamire, P. J. (2001). The Problem of Size. Retrieved February 22nd, 2012, from Exploring life:
http://www.brooklyn.cuny.edu/bc/ahp/LAD/C5/C5_ProbSize.html
Huds, D. (n.d.). Sources of Amylose in Diet. Retrieved 4 26, 2012, from eHow health:
http://www.ehow.com/list_6883683_sources-amylose-diet.html
Joyce, H. (2003, 11 24). Erasing experimental error . Retrieved 5 17, 2012, from Plus magazine:
http://plus.maths.org/content/erasing-experimental-error
Tae Jeong 11 Biology
Marshall. (n.d.). Alpha Amylase. Retrieved 4 26, 2012, from marshall.edu:
http://science.marshall.edu/murraye/alpha_amylase.htm
S-cool. (2011). Enzymes. Retrieved 5 16, 2012, from GCSE Rivision: http://www.scool.co.uk/gcse/biology/enzymes/revise-it/enzymes
T. K. DUTTA, M. JANA, P. PAHARI, T. BHATTACHARYA. (2005, 7 25). The effect of temperature (¡C) on
a-amylase. Retrieved 4 26, 2012, from The Effect of Temperature, pH, and Salt on Amylase:
http://journals.tubitak.gov.tr/zoology/issues/zoo-06-30-2/zoo-30-2-11-0507-14.pdf
Worthington Biochemical Corporation. (2012). Worthington Biochemical Corporation. Retrieved 5 11,
2012, from Effect of pH: http://www.worthingtonbiochem.com/introbiochem/effectsph.html