LAB MANUAL MP 6 BIOPROCESS ENGINEERING 2

You are assigned to investigate about pepsin enzyme biological reaction activity. Find the
effect of pepsin enzyme concentration, temperature and pH towards its activity. What is the
substrate of the enzyme and are the products of the hydrolysis of the substrate.

INTRODUCTION

Enzymes are biological catalysts that are made of protein. Enzymes, like all catalysts,
speed up biochemical reactions without being used up, by lowering the activation energy of
the reactions. Since enzymes are proteins, they are eligible to denaturation in a variety of
ways, usually at extreme conditions (temperature, pH, etc). Enzymes work best at their
specific optimum conditions; most enzymes have optimum activity at a neutral pH and at
body temperature.

Enzymes are also very specific they only act on one substrate or one class of
related substrate molecules. The reason for this is that the active site of the enzyme is
complementary to the shape and polarity of the substrate. Typically, only one kind of
substrate will fit into the active site, like a key fitting in a lock.

In this experiment, we will work with enzyme pepsin. Pepsin is a protease that
begins digestion of proteins, breaking them into peptides and amino acids. Pepsinogen is
secreted by gastric glands of the stomach into the stomach. There, in the acid environment
of the stomach, pepsinogen is converted into pepsin.

Proteins are enormous molecules containing hundreds of amino acids that are linked
together into a chain like structure. Complete digestion breaks the links of the protein chain
freeing the individual amino acids one from another. The first stage of protein digestion
begins in the stomach where the enzyme pepsin reduces the large molecular chains into
many shorter chains. These smaller chains like fragments are known as polypeptides. Later
the polypeptides move out of the stomach and into the small intestine where their final
breakdown into amino acids takes place.

In this investigation we will observe the partial digestion of a protein called albumen
from an egg white. To speed the digestive process, the egg has been cooked and the egg
white finely ground. Grinding decreases particle size and increases the surface area
presented to the enzyme. The mixture of egg white and water has the appearance of milk
but, unlike milk the particles tend to settle out, for this is not a true solution. During
digestion the particles will disappear as they are broken down into soluble polypeptide
fragments. In the end only a clear solution will remain and all the particles of the egg white
protein will be gone.

Amount of albumin remaining Enzyme activity level

All None (0)
Most Low (1)
Some Moderate (2)
None High (3)
 The following reaction is investigated for this experiment:

Substrate Enzyme Product Test

Pepsin
Peptide Disappearance of
Protein
Amino acid egg white
H2O

Effect of Enzyme Concentration

During a catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an
enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favored by a
high concentration of enzyme and/or substrate. After the substrate is bound, the reaction
takes place, and then the product is released.

E + S <> ES > E + P

Effect of Temperature

All reactions are faster at a higher temperature. However, enzyme-catalyzed reactions

become slower or stop if the temperature becomes too high, because enzymes become
denatured at high temperatures. Therefore, enzymes have an optimum temperature that
corresponds to maximum activity. (At higher or lower temperatures, the activity of the
enzyme is lower.) The optimum temperature for pepsin is usually between 37C (the body
temperature) and 42C.

Effect of pH

Each enzyme has an optimum pH. Above or below an enzymes optimum pH, its activity is
lower. The optimum pH of a particular enzyme corresponds to the pH of its natural
environment. For many enzymes, this corresponds to pH values of around 7. For pepsin,
which is active in the stomach, the optimum pH is 2 (the pH of the stomach).

OBJECTIVE

To study the effect of pepsin enzyme concentration, temperature, and pH on its activity.

MATERIAL AND APPARATUS

Material Apparatus
Pepsin solution Test tubes
Egg white Dropper
Dilute HCl Water bath
Dilute NaOH Blender
PROCEDURE

Protein suspension preparation

1. Boil an egg to hardness, remove the shell, cut the egg in half and remove the yolk.
2. Place the hard egg white (albumin) in a blender with 100 mL of distilled water. Blend the
mixture at low speed and then high speed. Add another 100 mL of distilled water and
blend for several minutes.

* The blending should produce a suspension of very fine egg white particles and water.

3. Filter the suspension through a strain to remove any large particles of egg white.
4. Add distilled water to the filtrate to make a volume of 500 mL of suspension.

Effect of pepsin enzyme concentration on its activity

1. Label five test tubes 1-5. Place 5 mL of white egg solution in each of the first four test
tubes. Place 5 mL of pepsin solution in the fifth tube. Place all of the tubes in the 37C
water bath for 5 minutes.
2. Tube 1 will be the control and it will not contain any enzyme. Remove the tubes from
the water bath momentarily, and quickly add 3 drops of the warmed pepsin solution
from tube 5 to tube 2, add 6 drops of pepsin to tube 3, and add 9 drops of pepsin to
tube 4. Mix the tubes quickly by shaking them gently, and immediately put them back in
the 37C water bath. Record the time at which the enzyme is added.
3. Observe and record the enzyme activity level every 5 minutes for 25 minutes.

Effect of temperature on pepsin enzyme activity

1. Place 5 mL of egg white solution in each of three clean test tubes. Place 5 mL of pepsin
enzyme solution in 3 separate clean test tubes (so you will have a total of six test tubes:
3 containing albumin solution and 3 containing enzyme).
2. Place an egg white solution tube and a pepsin solution tube in the 37C water bath.
Place one tube of each in an ice-water bath, and one of each tube in a boiling water
bath. Keep the tubes in their baths for 10 minutes to allow them to reach the
temperature of their baths.
3. Read and record the temperature of the ice-water bath. Pour the pepsin solution into the
egg white solution, mix, and put the tubes back into the bath. Record the time.
4. Repeat step 4 for the 37C bath.
5. Repeat step 4 for the boiling water bath.
6. Observe and record the enzyme activity level every 5 minutes for 25 minutes.
Effect of pH on pepsin enzyme activity

1. Label four test tubes pH 2, 4, 7, and 10. In each tube, place 5 mL of the appropriate
buffer (with the corresponding pH). Add 5 mL of pepsin solution to each of the tubes.
Get 4 additional clean test tubes, and put 5 mL of egg white solution in each tube. Place
all 8 of these tubes in the 37C water bath for about 5 minutes to allow the temperature
to reach equilibrium.
2. Pour the contents of each egg white test tube into a different pepsin-buffer test tube.
Mix them and return the tubes to the 37C water bath.
3. Observe and record the enzyme activity level every 5 minutes for 25 minutes.