College of Allied Health Professions

Bachelor of Science in Medical Technology

Biochemistry
Name: Louana Angeli Josefin C. Ancheta SCORE
Section: BSN1-9
Department: CNAM
Date of Submission: 12/12/2024

TITLE
Laboratory Manual # 6
Enzymes

LEARNING OUTCOMES
By the end of this laboratory activity, students will be able to:
1. Explain how temperature and enzyme concentration affect the rate of enzymatic reactions.
2. Conduct experiments to observe changes in enzymatic activity under varying temperatures
and enzyme concentrations.
3. Analyze data collected from the experiment to identify trends and draw conclusions about
optimal conditions for enzyme activity.
4. Interpret graphs representing the relationship between temperature, concentration, and
enzymatic activity.
5. Demonstrate proper laboratory techniques for preparing and handling enzyme solutions and
substrates.
6. Relate experimental findings to real-world biological processes and industrial applications of
enzymes.

MATERIALS
Reagents:
● 1% starch solution

● Iodine solution
To be brought by students:
● Saliva

● Ice
Laboratory Equipment:
● Test tubes

● Beaker

● Electric water bath (37C)

● Group water bath (100C)

INSTRUCTIONS

REMINDERS

1. Put an aliquot portion of reagent in a vial by pouring the reagent from a reagent bottle using a funnel
and a graduated cylinder.

2. PIPETTES, DROPPERS, and SYRINGES cannot be used in transferring reagents.

3. DISPOSAL Dispose of used liquid materials into the waste bottle provided. Solid materials must be
returned to the counter or thrown into the solid waste container.

1. Effect of Temperature

a. Label 3 tubes as A, B, and C. Place 2.0 ml. of 1% starch solution into each of the 3 test tubes.
b. Label another 3 tubes with A-1, B-1, and C-1. Add 1.0 ml. saliva into each of the 3 tubes.
c. Place tube A-1 in an ice bath, B-1 in a 37 °C water bath, and C-1 in a boiling water bath for
about 5 minutes.
d. Transfer tube A into the ice bath and pour the contents of A-1 to tube A. Mix and place tube A
back into the ice bath. After 10 minutes, add 2 drops of the iodine solution. Note and record the
color of the solution.
e. Repeat the same procedure for B-1 to B and C-1 to C, using instead the 37 °C water bath for
tube B and the boiling water bath for tube C. Note and record the color of the solutions after the
10-minute incubation and addition of iodine solution.
f. Rank the tubes from 1 to 3 (with 1 for being colorless signifying greatest digestion, and 3 which
means least or no digestion). A dark blue solution indicates the presence of starch. Partial
digestion of starch results in a violet or red colored solution. A colorless solution indicates the
complete digestion of the polysaccharide.

2. Effect of Concentration

a. Prepare 4 clean and dry test tubes and label them from 1 to 4.
b. Place 2.0 ml of 1% starch solution into each tube. Add a drop of iodine solution into all the
tubes. Note and record the resulting color of the solution.
c. In a separate tube, warm 2.0 ml saliva in a 37 °C water bath for 5 minutes.
d. Tube 1 will serve as the control of the experiment.
e. Add 3 drops of warm saliva to tube 2. Mix quickly and note the time needed for complete
digestion to take place.
f. Do the same for tubes 3 and 4 but instead of 3 drops, add 6 drops of saliva to tube 3 and 10
drops of saliva to tube 4.
g. Rank the tubes from 1 to 3 (1-fastest digestion and 3-slowest digestion). Record all your
observations. A dark blue solution indicates the presence of starch. Partial digestion of starch
results in a violet or red colored solution. A colorless solution indicates the complete digestion of
the polysaccharide.
CRITICAL THINKING
FACTORS AFFECTING ENZYMATIC ACTIVITY

1. Effect of Temperature

a. What is optimum temperature? What is the optimum temperature of your enzyme? (5pts)

Generally, the optimum temperature is typically around 37°C. The temperature at which an
enzyme functions most effectively, achieving the maximum rate of reaction, is known as the
optimal temperature. The most effective catalysis occurs at this temperature because the
enzyme's active site is in the greatest possible condition to interact with its substrate.

b. In this procedure, identify which is the enzyme, the substrate, and the manipulated variable.
(6pts)

The enzyme (amylase or saliva) in this experiment is responsible for breaking down the starch
which is the substrate into smaller sugars. As the test tubes are subjected to varying
temperatures (ice bath, warm water bath, and boiling water) to investigate the effects of
temperature on amylase activity, the manipulated variable is the temperature.The enzyme is the
saliva, the substrate is the starch, and the manipulated variable is the temperature (ice bath,
warm water, and boiling water).

c. How would you compare the degree of digestion in the different temperatures? (5 pts)

test tube A- there's a blue-black color when mixed then turned into light purple, meaning that the
starch wasn't digested.
test tube B- somehow clear, meaning the starch was digested by the saliva (enzyme).
test tube C- the color of the solution was somehow clear, meaning that the starch was partially
digested by the enzyme or saliva.

d. Provide an explanation for your observations in the different temperatures. (5 pts)

1. Ice Bath (Cold Temperature): Because molecules travel more slowly and have less
energy in cold temperatures, enzyme activity decreases. Iodine turns blue-black,
 indicating the presence of starch, as a result of amylase functioning significantly less
effectively and starch remaining largely intact.
2. Warm Water Bath (Optimal Temperature, around 37°C): Since this temperature is near
the enzyme's ideal temperature, amylase functions best at this temperature. Iodine
doesn't turn blue-black, instead, a clear with slight reddish-orange color has appeared, a
sign that starch has been broken down, because the enzyme's active site is in the best
possible form for doing so and the reaction occurs quickly.

3. Boiling Water (High Temperature): The amylase enzyme is probably denatured by the
high temperature when the test tube is subjected to boiling water, losing its structure and
becoming inactive. However, before the enzyme is completely inactivated, there may be
some partial starch breakdown, depending on how long the starch is exposed to boiling
water. If some of the starch has been broken down into smaller sugars, the solution may
become "lightly clear" and react with iodine differently than intact starch. Because some
of the starch has already been broken down into smaller, non-starch molecules that do
not react with iodine, the iodine may not turn completely blue-black. A lighter color or a
more translucent solution could be the outcome of this partial digestion.

e. Draw a graph showing the relationship between enzyme activity and temperature. (10 pts)
f. How can iodine solution determine the degree of digestion in the given tubes? Explain the
principle behind the use of this reagent. (10 pts)

Since iodine solution reacts specifically with starch to generate a distinctive blue-black hue, it is
used to measure the extent of starch digestion in the test tubes. This process happens when
iodine molecules form a blue-black combination with the helical shape of the starch molecule,
especially amylose. The idea behind iodine is that whereas smaller sugars, such as maltose,
which amylase breaks down into, do not react with iodine, starch will change color when iodine
is added. Thus, the amount of starch that is still present in the solution is indicated by the color
change—or lack thereof.

The presence of starch indicates that amylase has not sufficiently broken it down if the iodine
turns blue-black. This might happen if the enzyme activity was inhibited or retarded, as was the
case in cold or hot temperatures. The starch has either been fully or partially broken down into
smaller sugars that do not react with iodine, indicating effective digestion, if the solution is clear
or light yellow. The enzyme amylase is probably denatured in the boiling water test tube, and
while some starch may have been broken down prior to total inactivation, the degree of
digestion may cause iodine to change color only slightly or not at all. Thus, iodine acts as a
 visual indicator of starch digestion, and the intensity of the color helps determine the degree of
starch breakdown in the test tubes.

g. What is the health implication of this procedure? (10)

Amylase (saliva) is necessary for the breakdown of starches into simpler sugars that the body
uses as fuel, this process has health implications. Digestive issues like bloating, gas, or trouble
absorbing nutrients can result from disruptions in amylase activity, which can be caused by
temperature fluctuations, enzyme shortages, or digestive illnesses. For example, disorders such
as pancreatitis or amylase deficiency might affect the way starch is digested, resulting in
malabsorption of carbs and discomfort in the digestive tract. This demonstrates how crucial
amylase is to preserving healthy digestion and general metabolic function.

Effect of Enzyme Concentration

a. In this procedure, identify the enzymes, the substrate, and the manipulated variable.(6 pts)

Enzyme: The enzyme involved in this process is amylase, found in saliva, which facilitates the
conversion of starch into simpler sugars.
Substrate: The substrate utilized is starch, a complex carbohydrate that amylase breaks down into
smaller sugars such as maltose.
Manipulated Variable: The manipulated variable is the quantity of saliva (specifically, the number of
drops) introduced to the starch solution. This variable is changed to examine its impact on the rate of
starch digestion.

b. Account for the result obtained after adding in the tubes containing the starch solution.(5
pts)

When iodine is introduced to the starch solution in the test tubes, it interacts with the starch, producing
a blue-black hue. The presence of iodine signifies that the starch remains unbroken. The varying
results across the tubes illustrate the action of amylase in saliva on starch:

Tube 1 (Control): With no saliva present, the starch stays unaltered, and the iodine gives the solution a
blue-black color, indicating no digestion has occurred.
Tube 2 (3 drops of saliva): Amylase begins to decompose some of the starch, leading to partial
digestion. The iodine may display a violet or red color, reflecting some breakdown, though digestion is
not yet complete.
Tube 3 (6 drops of saliva): An increase in saliva leads to a faster breakdown of starch. The resulting
solution may exhibit a reduced color (violet or red), suggesting incomplete digestion.
Tube 4 (10 drops of saliva): The highest concentration of amylase breaks down nearly all or all of the
starch, probably resulting in the solution becoming colorless, signifying the complete digestion of starch
into simpler sugars.

c. How would you compare the degree of digestion in the different amounts of saliva (8 pts)

Tube 1 (Control): There is no digestion present since no saliva is added, which means the starch
remains unchanged, and the solution remains blue-black.
Tube 2 (3 drops of saliva): Some digestion takes place, as the solution may appear violet or red,
signifying that a portion of the starch has been broken down, but it is not yet complete.
Tube 3 (6 drops of saliva): Digestive activity is more rapid compared to Tube 2, with the solution likely
becoming lighter in hue (less intense violet/red), indicating greater starch breakdown.
Tube 4 (10 drops of saliva): The solution might turn colorless, showing that the starch has been entirely
digested, representing the highest level of digestion.

d. Provide an explanation for your observations in the different amounts of saliva.(10 pts)
Tube 1 (Control): In the absence of saliva, amylase is not present, so starch remains intact and does
not undergo breakdown. The solution remains blue-black as iodine interacts with the unbroken starch.
Tube 2 (3 drops of saliva): With a small quantity of saliva, a limited amount of amylase is available,
resulting in only a portion of the starch being digested, which produces a violet or red color.
Tube 3 (6 drops of saliva): An increase in the amount of saliva adds more amylase, accelerating the
starch digestion process. This results in a greater breakdown of starch, leading to a lighter hue that
indicates partial digestion.
Tube 4 (10 drops of saliva): With the highest volume of saliva, the concentration of amylase becomes
sufficient to almost completely digest the starch, resulting in a colorless solution, which signifies
complete starch digestion.

e. Explain the principle behind the use of this reagent. (10 pts)
Iodine solution, the reagent used in this process, reacts specifically with starch. Iodine's ability to
combine with the helical structure of starch, particularly the amylose component, forms the basis for its
application. Iodine attaches itself to starch and gives a solution of starch its distinctive blue-black hue.
Only in the presence of starch does this reaction take place. Iodine won't form the blue-black complex if
amylase has broken down the starch into smaller sugars (like maltose). Instead, the solution will turn
violet, red, or colorless, signifying partial or full digestion.

f. What is the health implication of this procedure? (10 pts)

Energy Production: The body uses carbohydrates as its primary energy source. Amylase aids in the
conversion of starches into sugars that can be absorbed and converted into energy.
Digestive Health: Bloating, indigestion, and nutrient malabsorption are among the digestive issues that
can result from impaired amylase activity.
Amylase Deficiency: Some people may not produce enough amylase, which can make it difficult for
them to digest starchy foods and result in discomfort, gas, and diarrhea.
Temperature Sensitivity: The experiment demonstrates that high temperatures have the ability to
denature enzymes, which may have an impact on digestion. Amylase's capacity to break down starch
can be impacted by cold or fever, which could lead to digestive issues.
Pancreatic Health: Disorders such as pancreatitis can impair the pancreas's ability to produce amylase,
which further impedes the breakdown of starches and results in inadequate nutrient absorption.