Chemical and Physical

Processes of Digestion
Lu, Mangaba, Nieveras, Ortiz, Ramos, Yatco
 Activity 1
Assessing Starch Digestion by Salivary Amylase
Objectives
1. Explain how enzyme activity can be assessed with enzyme assays
a. IKI assay
b. Benedict’s assay
2. Define enzyme, catalyst, hydrolase, substrate and control
3. Discuss specificity of amylase action
4. Discuss the possible effect of changes in temperature and pH on amylase
activity
Overview of Enzymes
● Enzymes have an optimal temperature and an optimal pH for maximum
activity.
● Human digestive enzymes have an optimal temperature equal to body
temperature (37°C) and most have an optimal pH near neutral.
● If the temperature or pH is too high, or if the pH is too low, enzymes will be
denatured
● Digestive enzyme are hydrolytic enzymes (hydrolases), which aids in the
breaking down of organic food molecules
○ By adding water to the molecular bonds, cleaving it between subunits
Introduction
● Focus of this activity is to investigate the hydrolysis of starch to maltose by salivary
amylase
● Starch digestion by amylase:
Amylase
Starch Water Maltose

● Salivary amylase: enzyme produced by salivary glands and is secreted in the mouth
● Enzyme assay: laboratory method of measuring enzymatic activity
○ IKI assay: test for the presence of starch
■ Blue or black: positive starch test
■ Diluted IKI color: negative starch test
○ Benedict’s assay test: for the presence of reducing sugars (glucose or maltose)
■ Green to reddish-brown: positive sugar test
■ No color changes: negative sugar test
Methodology
Methodology

Added IKI Reagent

Added Benedict’s Reagent

Results & Discussion
Experiment Data
Review Questions
1. List the substrate and the subunit product of amylase
- Substrate: Starch
- Product: Maltose
Amylase

Starch Water Maltose

Review Questions
2. What effect did boiling have on enzyme activity? Why? How well did the results
compare with your prediction?

- Increasing the temperature denatures the enzyme causing it to lose its

property to act on the substrate.
Review Questions
3. At what pH was the amylase activity the most active? Describe the significance
of this result.

- Enzymes are sensitive to pH the same way it is sensitive to temperature.

- Optimum pH of Amylase: pH 7.0
- Significance: Amylase can only work optimally in environments that have a pH
of 7.0 (Ex. Mouth and Duodenum)
Review Questions
4. Briefly the describe the need for controls and give an example used in this
activity.

- Controls are necessary to validate the results of the experiment.

4. Briefly the describe the need for controls and give an example used in this
activity.
Review Questions
5. Describe the significance of using a 37°C incubation temperature to test
salivary amylase activity.

- It mimics the internal temperature in the human body which is 37°C

- Significance: Increasing or decreasing the temperature reduces the enzymatic
ability of the salivary amylase as enzymes have a limited range of
temperature wherein it can work optimally.
Conclusion
● Amylase specifically catalyzes the hydrolysis of starch
○ Starch to maltose
● Temperature changes affects amylase activity
○ Enzymes denature when exposed to an increase in temperature
● pH changes affects activity of amylase
○ Amylase optimal activity is at pH 7.0
 Activity 2
Exploring Amylase Substrate Specificity
Objectives
1. Explain how enzyme activity can be assessed with enzyme assays
a. IKI assay
b. Benedict’s assay
2. Discuss the specificity that enzymes have for their substrate
3. Discuss the difference between the substrates starch and cellulose
4. Explain what would be the substrate specificity of peptidases
5. Explain how bacteria might aid in digestion
Overview of Enzyme Specificity
● Catalyze a specific reaction only
● Act on particular substrates only
○ Active site: enzyme pocket where substrate must fit into temporarily for catalysis to occur
○ Non-covalent bond (ionic bonds or hydrogen bonds)
Introduction
● Focus of this activity is on enzyme specificity for their substrates
● Starch will be hydrolyzed by salivary amylase:

Amylase
Starch Water Maltose Maltose Maltose

● Salivary amylase: enzyme produced by salivary glands and is secreted in the mouth
● Enzyme assay: laboratory method of measuring enzymatic activity
○ IKI assay: test for the presence of starch
■ Blue or black: positive starch test
■ Diluted IKI color: negative starch test
○ Benedict’s assay test: for the presence of reducing sugars (glucose or maltose)
■ Green to reddish-brown: positive sugar test
■ No color changes: negative sugar test
Starch & Cellulose
● Both are found in plants
○ Starch serves as energy storage
○ Cellulose provides rigidity to their cell walls
● Both are polymers of glucose
○ Starch: α-1,4-glycosidic bonds
○ Cellulose: β(1→4) glycosidic bonds
Methodology
Methodology

Added IKI Reagent

Added Benedict’s Reagent

Results & Discussion
Review Questions
1. Describe why the results in tube 1 and tube 2 are the same.

- The results in tube 1 and tube 2 are the same because amylase hydrolyzes
starch to maltose.
- Benedict’s test is a test used to detect the presence of reducing sugars
Review Questions
2. Describe the result in tube 3. How well did the results compare with your
prediction?

- As predicted, it will test positive in Iodine test and will test negative in
Benedict’s test.
- Amylase cannot hydrolyse cellulose
Review Questions
3. Describe the usual substrate for peptidase.

- The usual substrate for peptidase is protein (polypeptides) where it

hydrolyzes peptide bonds.
Review Questions
4. Explain how bacteria can aid in digestion.

- Bacteria possesses the enzyme cellulase which can digest cellulose.

Conclusion
● Amylase specifically catalyzes the hydrolysis of starch
○ Starch to maltose
● The substrate for peptidase is protein
● Bacteria aided in the digestion of cellulose
○ Enzyme cellulase
 Activity 3
Assessing Pepsin Digestion of Protein
Objectives
1. Explain how the enzyme activity of pepsin can be assessed with the BAPNA
assay.

2.Identify the substrate specificity of pepsin.

3.Discuss the effects of temperature and pH on pepsin activity.

4.Understand the pH specificity of enzyme activity and how it relates to human

physiology.
Introduction
In this activity we explore the digestion of proteins(peptides).

What is a peptide?

A peptide is 2 or more amino acids linked together by a peptide bond. It is

composed of a peptide chain with 10 to 100 amino acids and it is called
polypeptide.

The stomach contains chief cells which secretes a protein digesting enzyme
called pepsin. The pepsin hydrolyzes peptide bonds to form simpler and free
amino acids.
In this activity the students will use BAPNA as a substrate that asses pepsin
activity. BAPNA is a synthetic “peptide” that releases a yellow dye product when
hydrolyzed. It will turn yellow in the presence of an active peptidase such as
pepsin.
Methodology
1. We place 1 test tube in each slot(until 6) in the incubator.
2. We filled the test tubes with the ff:

Test tube #1: pepsin, BAPNA, pH 2.0 buffer

Test tube #2: pepsin, BAPNA, pH 2.0 buffer

Test tube #3: pepsin, deionized water, pH 2.0 buffer

Test tube #4: deionized water, BAPNA, pH 2.0

Test tube #5: pepsin, BAPNA, pH 7.0 buffer

Test tube #6: pepsin, BAPNA, pH 9.0 buffer

3. We lowered the test tube labeled #1 to be boiled in t he incubator

4. We then incubated all the 6 test tubes at 37°C for 60 mins.

5. We then put analyzed all the tubes in a spectrophotometer(an instrument that

measures the amount of light absorbed[called optical density] by a solution)

to measure how much yellow dye was released when BAPNA was “digested.”The
greater the optical density, the more BAPNA digestion by pepsin occurred.

6. We recorded the data from the experiment

Methodology
Methodology
Experiment Data
Results and discussion
Due to boiling it made the pepsin denatured and deactivated the action of the
enzymes. There was no activity in Tube #1 that had been boiled.

Tube # 2 turns yellow because of the presence of pepsin, BAPNA and a pH of 2.0
which has an acidic environment

Tube # 5 turns slightly yellow since pepsin and BAPNA is added but with the pH of
which is 7.0 is neutral
Review Questions
1. Describe the effect that boiling had on pepsin and how you could tell that it
had that effect.

At boiling temperature, the enzyme will denaturate and then it will not have any
activity against the BAPNA, as shown in in tube 1 with optical density of 0.00 and
no color change.
Review Questions
2. Was your prediction correct about the optimal pH for pepsin activity? Discuss
the physiologic correlation behind your results.

As seen in the test tube 2 the optimal pH for pepsin activity is 2.0, compared to
tube 5 and 6 the optical density was higher in tube 2.
Review Questions
3. What do you think would happen if you reduce the incubation time to 30
minutes for tube 5?

Basically it won't have enough time to break down as much BAPNA as it would
have with longer time.
Conclusion
An optical density greater than zero indicates that BAPNA digestion has occurred
and pepsin is active. It also indicates that more hydrolysis occurs.

The optimal pH of pepsin is at 2.0 which indicates that the environment is acidic.
This shows that pepsin is the most active in an acidic environment especially in
the stomach.

Heating process affects the activity of enzymes.

 Activity 4
Assessing Lipase Digestion of Fat
Objectives
● Explain how the activity of pancreatic lipase can be assessed by measuring
pH.
● Understand the pH specificity of lipase activity
● Understand the role of bile in fat digestion
● Discuss the challenges of using pH to measure digestion when comparing
lipase activity at various pH.
Introduction
● This activity explores fat digestion
● By far the most abundant fats of the diet are the neutral fats, also known as
triglycerides, a type of lipid that makes up fats and oils are nonpolar and
therefore very hydrophobic molecules. This insolubility presents a challenge
during digestion because these molecules tend to clump up together.
● Bile salts are secreted in the small intestine to emulsify fats during digestion.
Introduction
Introduction
● Lingual, gastric and pancreatic lipases are secreted during fat digestion.
● Lipase activity can be measured by monitoring the pH of the solution,
because some end products of fat digestion are acidic.
Methodology: Equipment
Methodology
Set-up:
Methodology
1. The following reagents were added to 6 test tubes:
Test tube 1: Lipase, Vegetable Oil, bile salts, pH 7.0 buffer
Test tube 2: Lipase, Vegetable Oil, deionized water, pH 7.0 buffer
Test tube 3: Lipase, deionized water, bile salts, pH 9.0 buffer
Test tube 4: Deionized water, Vegetable Oil, bile salts, pH 7.0 buffer
Test tube 5: Lipase, Vegetable Oil, bile salts, pH 2.0 buffer
Test tube 6: Lipase, Vegetable Oil, bile salts, pH 9.0 buffer
Methodology
2. Test tubes were incubated for 60 minutes at 37°C. The process of incubation
gently agitated the rack, evenly mixing the contents of each test tube.

3. Tubes were then placed in a pH meter to measure the final pH of the test
solutions.
Methodology
Experiment Data
Results & Discussion
Tube 1 investigated the action of bile on enzyme activity, and tube 2 examined
lipase activity without bile,

In test tube 5, even with the presence of bile salts and lipase, fat digestion does
not occur because the buffer (pH of 2.0) is already quite acidic.
Review Questions
1. Explain why lipase activity can’t be fully tested in tube 5.

Since the pH in test tube #5 is already very low, it is difficult to tell if fatty acids are
released.
Review Questions
2. Which tube has the highest lipase activity?

The correct prediction is tube #1, 7.0, which approximates the pH of the small
intestine
Review Questions
3. Explain why pancreatic lipase would be active in both the mouth and pancreas.

Since the activity of pancreatic lipase is highest at pH 7.0, the enzyme should be
active in the mouth and the pancreas.
Review Questions
4. Explain the process of bile emulsification of lipids and how it improves lipase
activity.

Bile serves to mechanically break up large globules of fat (through an

emulsification process) and produce small droplets that effectively increases the
surface area of the lipids.
Conclusion
● Lipase specifically catalyzes the hydrolysis of lipids.
● Temperature changes has an effect on the activity of lipase
● ·pH changes affects the activity of lipase

● A solution containing fatty acids liberated by activity of lipase has lower pH

than a solution without fatty acid production