EXPERIMENT 3

ENZYMES

B y G r o u p 2:
S i m o n e Apostol, K a t h y Aswit, Y v a Balanza,
C yr i n Butay, Marilen Serdenia, K i m A n g e l o
Tumaneng
 Enzymes play a crucial role in biochemical reactions by catalyzing
the conversion of substrates into products. One essential factor
influencing enzyme activity is pH, which can profoundly impact
their efficiency and effectiveness. For instance, amylase, an
enzyme responsible for breaking down starch into simpler sugars,
exhibits optimal activity within specific pH ranges. Salivary
amylase, found in human saliva, operates optimally at a pH of
around 6.7, while pancreatic amylase, produced in the pancreas,
functions best at a neutral to slightly alkaline pH of 7.0 to 7.4.
Deviations from these optimal pH ranges can result in reduced
enzymatic activity or even enzyme denaturation, underscoring the
3.21 critical role of pH in modulating enzymatic processes.

INFLENCE OF PH
1. To determine the influence of pH to enzymatic process of
Salivary amylase.
2. To determine the difference of each spot plate from each test
tube.

3.21
INFLUENCE OF PH
 T h e following observation pr e s e n ts o u r f i n d i n g s f r o m t h e experiment:

MINUTES T1 T2 T3 T4 T5 T6 T7 T8
pH=5.3 pH=5.7 pH=6.2 pH=6.7 pH=7.0 pH=7.4 pH=7.7 pH=8.0
2 Dark Dark Slight Light Light Light Very Light
black black black black black black light black
black
4 Black Black Black Light Light Lighter None Light
black black black black
6 Light Black Light None None None None Lighter
black black black
8 Almost Lighter None None None None None None
none black
10 None None None None None None None None
The table indicates that the originally dark color resulting
from adding a solution drop from each test tube to an iodine
solution is gradually transitioning to a lighter shade as the
pH of the buffer increases except at pH 8 which reaches the
alkaline range (salivary amylase did not act at this tube).
Moreover, with the passage of time, the black color produced
by the test tube as it remains immersed in a heated beaker is
progressively diminishing in intensity, thereby becoming
lighter.
 T h e following observation pr e s e n ts f i n d i n g s f r o m a n e x p e r i m e n t b y SHR SEM (2021):

shr sem (2021). Experiment 7- Salivary Amylase pH and Temperature- SCRIBD. C O M h t t p s : / / w w w . s c r i b d . c o m / d o c u m e n t / 5 4 2 5 6 3 3 3 0 / e x p e r i m e n t - 7 -

Salivary-amylase-pH-and-temperature

TIME (minutes) Test tube with Test tube with Test tube with
pH= 5 pH= 6.8 pH= 8
0 Blue color Blue color Blue color
2 Blue color Blue color Blue color

4 Blue color Blue color Blue color

- Blue color Blue color Blue color
x Blue color No change in color of Blue color
Iodine
Initially, the presence of starch causes the iodine solution to turn blue-black due to the
formation of a starch-iodine complex. When amylase is added, it speeds up the
breakdown of starch into smaller polysaccharides and eventually into maltose and other
products. As the starch is broken down, the iodine-starch complex is disrupted, leading to
a change in color from blue-black to a lighter color or even a complete disappearance of
the blue-black color, indicating the presence of the amylase enzyme and the breakdown of
starch. Moreover, higher temperatures can enhance the effectiveness of the amylase
enzyme in breaking down starch by increasing kinetic energy, leading to faster enzyme-
substrate collisions and accelerating the enzymatic reaction. However, excessive heat and
pH can denature the enzyme, so it's important to stay within the optimal temperature and
pH range for amylase function to maintain its effectiveness.
3.21
POST LAB QUESTIONS
1. If all the starch has not been used up, what
will happen when 1 drop of the reaction
mixture is added to the iodine solution? What
difference will be observed if all the starch
has been used up?

The iodine test is a method for determining the presence

or absence of starch in a solution. When all of the starch is
used up, the iodine solution becomes blue or purple when
combined with another iodine solution. In contrast, if all of
the starch has been used up and transformed into smaller
sugar molecules by amylase, the iodine solution will
remain yellow or brown, indicating full starch hydrolysis.
This test is very useful for monitoring starch hydrolysis
tests, including amylase.
 2. How is the time related to the enzymatic
activity of the amylase?

The response rate, an enzyme kinetics concept, describes

how time affects amylase's enzymatic activity. The
reaction rate is high at first because there are so many
starch molecules. The concentration of starch drops over
time, slowing the rate. This is referred to as the rate of
reaction decrease or rate decline. The rate may eventually
reach equilibrium, with the majority of the starch
transformed into sugar molecules. Experimental research
can test the reaction rate under controlled conditions to
obtain more exact results.
 3. What is the optimum pH for amylase
activity? Would you have predicte such value?

The optimum pH for amylase activity typically ranges

between pH 6.7 to 7.0, which is slightly acidic to neutral.
This range is because amylase enzymes work best in
environments where the pH is close to that of the human
body, where they are often utilized.
4. At a pH away from the optimum, what has
happened to the structure the active site to
cause inactivation?

At a pH away from the optimum, the structure of the

active site of amylase may undergo denaturation.
Denaturation is a process where the protein's secondary,
tertiary, or quaternary structure is disrupted, leading to a
loss of its biological activity.
 5. Enumerate the factors on which the pH
activity relationship of at enzyme depends.
Active site structure: The active site of an enzyme is sensitive
to changes in pH.
Ionization of amino acid residues: The pH of the environment
affects the ionization state of amino acid residues within the
enzyme's active site.
Optimal environment for catalysis: Each enzyme has an
optimal pH range in which it exhibits maximum catalytic
activity.
Hydrogen ion concentration: pH is a measure of hydrogen ion
concentration in a solution.
Stability of the enzyme structure: Extreme pH values can
denature enzymes by disrupting their secondary, tertiary, and
quaternary structures.
Buffering capacity: Buffering capacity of the solution can
influence the pH-activity relationship by helping maintain a
relatively constant pH despite changes in external pH
conditions.