Kevin Flores

Laboratory 8:
Effect of Temperature and
pH on Amylase Activity

Partners: Hector Flores, Frances Hatzopoulos

November 25, 2015

Abstract:
Essentially, this experiment was conducted to determine what effects pH has on
the activity level of enzymes, by observing %transmittance in a starch and amylase
mixture with varieties of buffer. Prior to conducting the lab, it was critical to know that
iodine easily stains starch and that amylase, the enzymes, hydrolyze starch. The harder
the enzymes are working, the less starch that will be present in a given solution.
Therefore, the lighter the coloring of the solution is, the more active the enzymes had to
be. During the study, the pH 3 solution had absolutely no increase in %transmittance,
while pH 7 had the greatest increase. On the other hand, pH 5 and 9 were similar in
%transmittance, although pH 9 was slightly higher. Thus, it was concluded that, pH 3
leads to minimal activity of the enzymes, pH 7 with the maximum activity and both pH 5
and 9 fall somewhere in between, with pH 9 resulting in slightly more reactivity. This
leads to the conclusion that enzymes are denatured and rendered inactive when placed
in solutions that deviate from pH 7.

Introduction:
The primary purpose of this experiment was to determine how pH affects the
activity of enzymes, specifically amylase, by measuring the %transmittance of starch.
Enzymes are proteins that help make chemical reactions more efficient. In order for this
to occur, a substrate must bind to the active site of an enzyme, however the bond is
quite specific as the substrate must match the active site perfectly. Any slight change to
the structure of the enzyme may hinder the reaction. In this study, the substrate
experimented with was starch, which happens to be easily stained by iodine. By staining
the starch first, adding amylase to it and proceeding to place the mixture into a
spectrophotometer, its %transmittance can easily be determined. The reason the
%transmittance of the mixture would change at all is because when the reaction
between the enzymes and the starch is successful, the starch is decomposed, and so
the coloring of the stain weakens; there is less starch to be stained. It is therefore the
case that the activity of the enzymes was directly related to the coloring of the mixture.
Prior to conducting the study, it was predicted that the more the pH deviated from
pH7, the less reactive the enzymes would be, as the amylase utilized normally functions
in pH 7 environments.
Materials and Methods:
To begin the experiment, starch was added to each of four flasks containing pH
3, 5, 7, and 9. A 4 mL sample was taken from each mixture and placed in separate
cuvettes, to which 3 drops of iodine were added. Each cuvette was then placed in the a
spectrophotometer, making sure the substances were mixed properly by sealing them
with parafilm and swirling the solution. The value was recorded and a different set of
cuvettes were prepared every 5 minutes, however, after the first trial, 1 mL of amylase

was added to all four flasks. The spectrophotometer was set to a wavelength of 560
nanometers during the entire experiment.
Results:
Starting with the lowest pH studied, pH 3, There was no change in
%transmittance at any point; the spectrophotometer always read 0.0% after every 5
minutes. The %transmittance in pH5 also read 0.0% within the first couple of trials,
however it appeared to increase exponentially shortly after and then actually decrease
constantly towards the end. With pH 7, the %transmittance began increasing right away
with 48.4% just after 5 minutes, but then continued to fluctuate around 84% for the
majority of the time. The readings with pH 9 were similar to those of pH 5, although its
%transmittance began increasing significantly from 10 to 15 minutes and it too
fluctuated during most of the experiment, at about 76%.
%Transmittance of Starch/Amylase Solution Over Time

Time
(minutes)

% Transmittance
pH 3

pH 5

pH 7

pH 9

0.0

0.0

0.0

0.0

0.0

0.0

48.4

0.0

10

0.0

0.6

81.4

9.8

15

0.0

26.5

86.5

59.5

20

0.0

52.1

81.0

70.7

25

0.0

62.1

87.0

79.0

30

0.0

75.5

84.5

75.7

35

0.0

74.4

85.2

79.9

40

0.0

73.7

87.0

80.3

45

0.0

72.8

85.1

77.7

Discussion:
According to the data collected, the hypothesis that the activity of enzymes would
increase as the pH approached pH 7, appeared to be correct. For example, pH 3 was
the lowest pH tested, deviating the most from pH 7. Its %transmittance never increased,

so this demonstrates that little to no starch was decomposed by the enzymes. Thus,
the enzymes did not function properly in pH 3. The second furthest pH was pH 5, which
actually had in increase in %transmittance up to a maximum of 75.5%, so it can safely
be assumed that the enzyme was functioning to some extent. However, it functioned to
a less extent than that of the pH 7, the optimal condition for this enzyme. This can also
be assumed because the highest %transmittance reached with pH 7 was 87.0%,
meaning that more of the starch was broken down and the enzyme was more active
overall. Increasing the pH to pH 9 did not make the activity of the enzyme increase, as
expected. The %transmittance of did not increase as much as that of the pH 7 mixture,
reaching only 80.3%, although the enzymes were actually more active than those in pH
5 despite being equidistant in magnitude from pH 7.
Like in every experiment, this one could not be said to be perfect. For example,
the beakers containing the amylase and starch solutions were left uncovered throughout
the entire lab. This could have led to some of the water being evaporated, increasing
the overall concentrations of starch and amylase in the solution. This would have
increased the reaction rate and therefore, the %transmittance, as there would be less
water interfering between the starch and amylase. This could have been prevented by
covering each beaker with a watchglass. Another source of error was that the cuvettes
were not perfectly clean. Although it may not have appeared to be happening,
transferring the cuvettes between lab partners most likely covered the cuvettes with oils
and other natural debris. This would have decreased the recorded %transmittance as
the debris would interfere with the amount of light allowed to pass through the cuvette.
Despite these errors, the experiment was successful. The initial hypothesis that
deviating from a pH of 7 would render an enzyme inactive was heavily supported by the
data collected. The mixture in pH7 appeared to be the optimal condition for the enzyme
as the %transmittance of the solution was significantly higher than in the other solutions.
This also allowed for the conclusion to be made that the reason the enzymes became
more inactive as the levels of pH increased or decreased from 7 was because the
proteins were being denatured. Knowing that structure is critical to the function of a
protein, this conclusion seems to be appropriate.

References:
"Enzymes." Chemistry for Biologists:. N.p., n.d. Web. 26 Nov. 2015.
"Introduction to Enzymes." Effects of PH (). N.p., n.d. Web. 26 Nov. 2015.
BBC News. BBC, n.d. Web. 26 Nov. 2015.