EXPERIMENT No.
3
OBJECTIVE
To study the Activity of the enzyme lactase.
THEORY
Enzymes: An enzyme is a substance acting as a catalyst in the living organisms, which
regulates the rate at which chemical reactions takes place without itself taking part in the
process. Most chemical reactions that occur within all living organisms are regulated by
enzymes.
Factors affecting enzyme activity: Enzyme activity can be affected by a variety of factors,
such as temperature, pH, and concentration. Enzymes work best within specific conditions
such as temperature and pH ranges. A change in the sub-optimal conditions can cause an
enzyme to lose its ability to bind to a substrate.
Temperature: Raising temperature generally speeds up a reaction, and lowering temperature
slows down a reaction. However, extreme temperatures result in an enzyme to lose its shape
(denaturation) and stop working.
pH: Each enzyme works within an optimum pH range. A change in the pH results in enzyme
activity. Extreme pH values can cause enzymes to denature.
Enzyme and substrate concentration: Increasing the enzyme/substrate concentration will
speed up the reaction, as long as there is enzyme/substrate available to bind to. When all of
the enzyme/substrate is bound, the reaction no longer speeds up, as there will be nothing for
additional enzymes/substrate to bind to.
Mechanism of Enzyme Action
The basic mechanism by which the enzymes catalyze chemical reactions starts with the
binding of substrate to the active site of an enzyme. Active site is a specific region of an
enzyme that combines with the substrate. The substrate binding to the enzyme leads to change
in the distribution of electrons in the chemical bonds of the substrate and resulting in the
reactions that lead to the product formation. The active site has a unique shape that is
complementary to the shape of a substrate molecule. Enzymes specifically react with only one
or a very few similar compounds.
Lock and Key Theory: The Lock and Key model was first postulated in 1894 by Emil
Fischer. According to this, the lock is the enzyme and the key is the substrate. Only the
accurate substrate fits into the active site of the lock enzyme.
Induced Fit Theory: For many years, it was believed that enzyme-substrate binding took
place in “lock-and-key” manner. The induced fit model suggested that As the enzyme and
substrate come together, their interaction causes a slight change in the enzyme’s structure that
confirms an ideal binding arrangement between the enzyme and the substrate. This dynamic
binding maximizes the enzyme’s ability to catalyze its reaction.
Chemistry of enzyme Lactase:
Lactose, is a sugar found in milk is a disaccharide compound composed of glucose and
galactose. Ordinary table sugar sucrose is also a disaccharide composed of fructose and
glucose. The enzyme lactase breaks down lactose into glucose and galactose. Although lactose
is similar to sucrose, lactase will breakdown only lactose because of the shape of sugar. The
specificity of enzyme reaction of breaking down of sucrose and lactose is based on shape.
When hydrolysed by the enzyme lactase, lactose yields equi-molar quantities of D-glucose
and D-galactose.
Lactase
LACTOSE D- GLUCOSE + D-GALACTOSE
Lactase
SUCROSE GLUCOSE + FRUCTOSE (No reaction)
Lactose Intolerance: It is a digestive disorder where the body is unable to digest lactose sugar.
Symptoms of lactose intolerance include: flatulence, diarrhea, bloating, stomach cramps etc.
The body digests lactose using an enzyme called lactase. This breaks down lactose into glucose
and galactose, which can be easily absorbed into the bloodstream. People suffering from lactose
intolerance do not produce enough lactase, so the lactose stays in the digestive system, where it
starts getting fermented by gut bacteria. This results in the formation of gases, which cause the
symptoms associated with lactose intolerance. Depending on the underlying reason why the
body's not producing enough lactase, lactose intolerance may be temporary or permanent.
REQUIREMENTS
1. Lactase tablet
2. 15 ml of milk
3. Sucrose 5 gram
4. 10 and 100ml measuring cylinder
5. 500ml beaker
6. Test tubes with stand
7. Distilled water
8. Marker pen
9. Stopwatch
10. Hotplate/Burner
11. Glucose Test strips
12. Glass Rod
PROCEDURE
Preparation of Solutions: Keep following solutions ready before conducting the experiment:
1. Enzyme solution – Add and dissolve the lactase tablet to 200 ml distilled water.
2. Skim milk – Milk from which cream has been removed.
3. Sucrose solution – Add 5 gram of table sugar to 100 ml of distilled water. Stir to
dissolve the sugar.
4. Denatured Enzyme Solution –
i. Place 20 ml of enzyme solution into a test tube.
ii. Add 200 ml of water to a 500 ml beaker.
iii. Place the test - tube in the beaker.
iv. Place the beaker along with test – tube on a hot plate or burner.
v. Boil the water in the beaker for 30 min. Cool it down to room temperature.
After all solutions are ready follow the following steps:
5. Label the test – tube as –
a. A. Test – tube with skim milk + Enzyme solution.
VIII-
b. B. Test – tube with skim milk 1+ Water.
c. C. Test – tube with milk + Denatured enzyme solution.
d. D. Test – tube with sucrose solution + Enzyme solution.
6. In test – tube A, Add 2 ml of skim milk and 1 ml of enzyme solution.
7. Wait for 5 min. (or more) and test for glucose with the glucose test strip. Record
your data in the result table.
8. In test – tube B, Add 2 ml of skim milk and 1 ml of water and repeat step 7.
9. In test – tube C, Add 2 ml of skim milk and 1 ml of denatured enzyme solution and
repeat step 7.
10. In test – tube D, Add 2 ml of the sucrose solution and 1 ml of enzyme solution and
repeat step 7.
11. In test – tube E, Add 2 ml of the sucrose solution and 1 ml of water. Repeat step 7.
OBSERVATION
Find out presence and absence of glucose in the test – tube.
Type of solution Glucose result
Positive ‘+’ Negative ‘-‘
Test tube A: Skim milk + Enzyme
Test tube B: Skim milk + water
Test tube C: Skim milk + Denatured Enzyme
solution
Test tube D: Sucrose solution + Enzyme sol.
Test tube E: Sucrose solution + water
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