Nneoma Odoemena

Michael Lim
CRN:10956
3/1/16
Lab 4 Enzyme Physical Properties
Hypothesis:
With starch added in each test tube and amylase added to certain test tubes, the 3B
solution consisting of starch and Amylase at 37C with the Phosphate Buffer added will
hydrolyze starch producing a sugar called maltose. In the test tube it will present a yellow
color.
Introduction:
An Enzyme is a protein that reacts with a substrate and catalyzes biochemical
reactions. Enzyme activity depends on conditions that deal with the pH level,
temperature, substrate and substrate concentration. The general trend of how rapid an
enzyme reacts depends on increasing temperatures. The most optimal activity can be seen
from 30-40 C but for animal enzymes it denaturizes past 40 C. The pHs of the enzymes
differ amongst amylase. For pancreatic amylase the optimal pH is between 7.5-8, while
salivary amylase is 6.8. An acid phosphates optimal pH should have a low pH while an
alkaline phosphatase should have a high pH. In this lab an animal enzyme called alpha
amylase is used to react with starch in order to find the optimum activity of the enzyme at
different pH levels with Citrate Buffer (phH of 4.8) and Phosphate buffer (pH of 7) and
different temperatures (4C, 37C and 65C). The optimum pH for alpha amylase is 71
also showing activity between 30-40C. In this lab some test tubes will be exposed to
1

-amylase from porcine pancreas. (Product: A3176). Retrieved March 17, 2015, from www.sigmaaldrich.com

alpha amylase while others are exposed to distilled water to compare what happens in the
reaction.
Method:
In this procedure 0.1ml of starch is added to all test tubes because it is the substrate that
will be used to test the effect it has with or without Amylase. In all the Sets 0.9ml of
Citrate buffer was added in test tubes 1 and 2 which has a low pH of 4.8 and 0.9ml of
Phosphate Buffer was added to test tubes 3 and 4. These different buffers were added to
compare how the amylase and starch would be affected at different pH levels. Depending
on how high or low the pH was could affect the rate of the reaction. There area to look
for activity had to be between pH 5.5-8. All the test tubes were mixed and incubated for
15 minutes in different temperature conditions: 4C, 37C, and 65C also testing for the
optimum temperature that best allows activity for alpha amylase. In test tube 1 and 3 for
all the sets, 6.25 units/ml of alpha amylase was added then added. This could mean that
the solutions could be a negative or positive control based on whether the activity of the
enzyme is at its full potential. 0.1ml of distilled water was added to test tubes 2 and 4 for
all sets as a negative control because no amylase was presented. It was added to compare
the final reaction with the amylase. The test tubes were then mixed and a drop of iodine
reagent was added to all the test tubes. Iodine was added because it is an indicator to see
how starch is hydrolyzed by amylase. With iodine it can be seen which test tube formed
an iodine complex (blue color), the hydrolysis of starch (yellow color), or even an in
complete reaction with the starch (greenish color).

Results (table on separate sheet of paper)

For Set A in test tube 1 & 2 the color of the solution was a dark blue. For test tube 3 it
was a light blue and for test tube 4 it presented a darker blue. For Set B in test tube 1 & 2
the color of the solution was a deeper blue. For test tube 3 it was an orange colored
solution. For test tube 4 it was a green blue color. For Set C the test tube solution was a
blue color for 1 and 2. It was a green blue color for test tube solution 3 and 4.
Discussion:
In Set A all the colors were a dark shade of blue except test tube 3A which was a
lighter blue demonstrating an iodine complex for all. Since the temperature was at 4C , it
did not matter if the citrate or phosphate buffers were used with the addition of
amylase and distilled water because the optimal temperature in order for it to hydrolyze
was between 30-40C with a pH of 7. Since test tube 3A used the Phosphate Buffer its
reaction was slightly faster because of the pH. But in a general trend all the test tubes
solutions in set A reacted at a slower rate because of the low temperature. They all are
negative control because 1A and 3A did not react even with the amylase present and 2A
and 4A contained distilled water, so no reaction of the enzyme would be present as well
showing no full potential enzymatic activity.
For Set B, test tube 1B contained Amylase at a low pH of 4.8 (Citrate Buffer)
and was incubated at an optimal temperature of 37C, but due to the low pH level, the
final solution produced a deep blue color (iodine complex) which meant that the pH
changed the stability of amylase structure causing it to denaturize. Although it
contained amylase it tested negative for enzyme activity and was not in the 5.5-8 pH
range for Amylase. Due to the iodine complex the hydrolysis of starch was not possible.
For test tube 2B it contained the Citrate Buffer at 37C but only had distilled water

presenting a negative control and producing a deep blue color showing now activity of
the enzyme. For 3B it contained Amylase and presented both the optimal pH and
temperature with the phosphate buffer and temperature being at 37C. The mixture
produced a yellow colored solution called maltose. This demonstrated that the starch in
3B was hydrolyzed because it did not show blue color. No iodine complex was formed in
that test tube. 3B was the only positive control presented. For 4B it contained everything
3B had except distilled water was used instead of Amylase. It was a green blue color
and was a negative control. It was an incomplete reaction that appeared at a slow rate. For
Set C, the temperature was too high (65C) for the reaction so the Amylase in 1C and
3C denaturized with a blue and green blue color. 2C and 4C also appeared to have a blue
and green blue color with distilled water as the negative control. For 3C and 4C the color
green blue was presented exhibiting an incomplete reaction because it had the optimum
pH of 7 but not the optimum temperature.
Conclusion:
The hypothesis stated in the beginning is correct and supported by the data used
to record the activity of the experiment. In Set B the temperature was 37C which is in
the range (30-40 C) of optimum activity for animal enzymes such as Amylase from
porcine pancreas used in this lab. 3B had both the optimum temperature and pH of 7 used
by the Phosphate buffer allowing starch to hydrolyze and turn a yellow-orange color.
References
1.

-amylase from porcine pancreas. (Product: A3176). Retrieved March 17, 2015,
from www.sigmaaldrich.com