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Chandler Kolb
Mrs. Donald
Honors Biology Green 2
6 November 2009
Lactase Enzyme Lab
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Problem: How does the enzyme, lactase, break down a specific substrate?
Research: An enzyme is a protein macromolecule found in our bodies that is needed to digest
many of the foods that we eat. These proteins act as catalysts because they accelerate the rate of
reaction by lowering the activation energy needed to proceed. Without enzymes, it would take
years to accumulate enough energy to just pick up your pencil, but with them, we have enough
energy to do it in a fraction of a second. As a function of an enzyme, a substrate (the substance
that is acted upon) combine with molecules to form a temporary enzyme-substrate complex, after
the products are formed the enzyme molecule releases them and is unchanged, making enzymes
reusable. An enzyme has a specific active site (where the substrate and the enzyme meet) and
only specific substrates can attach to these active sites. For example, like a lock and its key; a
science room key can open all of the science rooms in the building but cannot open the English
rooms, because the locks aren’t specific to that key. This is known as the Lock and Key
Hypothesis for enzymes. An enzyme can also be affected by temperature and pH levels. High
temperatures denature the enzyme meaning that the structure within the protein membrane
stretches out and disrupts the active site making it a very poor area to allow a substrate to
attach. An enzyme prefers a level of pH between 6 and 8 or a neutralized area, so an acidic
liquid alone or a basic liquid alone can affect enzymes functions differently.
Hypothesis: If the ability for an enzyme to breakdown a specific substrate in the presence of
different temperatures is related to the enzyme, lactase, then lactase will be able to break down
lactose only and there will be a presence of a positive glucose because of the specific shape of
the molecule and sucrose will not be broken down because the separation of glucose and
fructose isn’t possible since the molecule shape isn’t specific enough for this enzyme
particularly. Boiling the active enzyme solution will denature the enzymes active site and there
will be a negative presence of glucose.
If the ability for an enzyme to breakdown a specific substrate in the presence of different pH
levels is related to the enzyme, lactase, then lactase will break down lactose in the presence of a
low pH, or an acid, because we have hydrochloride acid in our bodies, therefore it would be
natural for it to break down in an acidic solution.
Materials:
1. 8 micro test tubes
2. Test tube rack
3. Milk
4. Active enzyme solution (lactaid)
5. Denatured enzyme solution
6. Sucrose solution
7. 1 M hydrochloric acid
8. Calcium carbonate (bleach)
9. 250 ml beaker
10. Water
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11. Stir rod
12. Pipettes
13. Glucose test sticks/strips (8)
14. Glucose test key
15. Petri dish
Procedure: Followed Lab Procedure
Data:
Part One: Temperature and Enzymatic Activity
Test Tubes
A (Milk and Enzyme
Solution )
B (Milk and Water)
C (Milk and
Denatured Enzyme
Solution)
D (Sucrose Solution
and Enzyme Solution)
E (Sucrose Solution
and Water)
The Glucose Stick’s
Color at 0 minutes
The Glucose Stick’s
Color after 30 seconds
Aqua
Sea Green
Aqua
Aqua
The Total Glucose
Present after the
Reaction (mg/dl)
+ 100 mg/dl Glucose
Present
Negative
Aqua
Aqua
Negative
Aqua
Aqua
Negative
Aqua
Aqua
Negative
Part Two: pH and Enzymatic Activity
Test Tubes
a. (1M Hydrochloric
Acid, Milk and Active
Enzyme Solution )
b. (Milk and Active
Enzyme Solution)
c. (Calcium carbonate
(Bleach), Milk, and
Active Enzyme
Solution)
Graph- See graph paper
The Glucose Stick’s
Color at 0 minutes
The Glucose Stick’s
Color after 30 seconds
The Total Glucose
Present after the
Reaction (mg/dl)
Aqua
Aqua
Negative
Aqua
Aqua
Negative
Aqua
Brown
+1000 mg/dl Glucose
Present
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Conclusion and Analysis:
The purpose of this lab was to observe how well the enzyme lactase could break down a
specific substrate in the presence of different temperatures and pH levels. Like predicted with the
presence of temperature, test tube A, with milk and lactaid, had a positive presence of glucose
after the reaction. This occurred because the enzyme, lactase, which was active in the lactaid
pill, was able to separate the molecule lactose, found in the milk, into glucose and galactose,
making a presence of 100 mg/dl of glucose after the reaction. However, the rest of the solutions
in that part of the experiment came out with a negative presence of glucose after their reactions.
Test tube B, with milk and water, was the control of these substrates because it is the normal
condition; no glucose was present after this reaction because there was no enzyme to break the
lactose apart. Test tube C, with milk and denatured enzymatic solution, is the exact opposite of
test tube A, but in this case when the lactaid pill was boiled, making the structure of the protein
stretch out within its membrane and then jumbled up the activation site where the lactase would
have separated lactose in glucose and galactose individually but the stretched out protein
content prevented this from happening. The enzyme solution in this test tube is called denatured
enzyme solution because when a high temperature makes an active site very poor it is known as
a denatured enzyme, which created a negative presence of glucose. Test tube D, with sucrose
solution and enzyme solution, created a negative presence of glucose because the enzyme lactase
is not able to break apart the molecule sucrose into glucose and fructose. Lastly, the test tube E,
with sucrose solution and water, didn’t have a positive presence of glucose because there wasn’t
even an enzyme to allow for the reaction to happen, and if there was lactase in this scenario, it
would have had the same outcome of test tube D. With the presence of different pH levels, test
tubes ‘a’ and ‘b’ had a negative presence of glucose after their reactions. But, test tube ‘c’,
containing bleach, milk and active enzyme solution had a very high presence of glucose after its
reaction. This is because the test tube ‘a’ had an acidic level to it and because where the lactase
is broken down in the body, in the Duodendum, there is a lot of hydrochloric acid from the
stomach coming in through the opening between this and the stomach, and therefore there must
be a basic liquid that is produced to neutralize the area. That is why the enzyme lactase prefers a
basic liquid over an acidic one. Test tube ‘b’, with milk and active enzyme, was the control for
this part of the experiment.
My first hypothesis was supported because the enzyme lactase was only able to break
down the molecule lactose into glucose and galactose. I also was supported when the denatured
enzymatic solution created a negative presence of glucose, because boiling the lactaid pill made
a poor active site on the enzyme. My second hypothesis however, was not supported, because I
thought it would be natural for the lactase to break down lactose in an acidic liquid because
that’s what is in our stomachs, but that is not the case. Lactase needs a basic solution because in
order for the Duodendum to be neutralized and the enzyme to work properly, there must be a
basic liquid and an acidic liquid to neutralize and make the Duodendum a good place for the
enzyme lactase to work properly and separate lactose into glucose and galactose.
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There were no systematic or random errors that affected my data. The practical
application for with this lab is directly related to individuals who may be Lactose Intolerant.
These individuals’ bodies are not able to digest the sugar, lactose, which is found in many milk
and dairy products because of an enzyme deficiency problem. Most diagnosed people use a pill
to put the active enzyme directly into their body. We used a pill known as lactaid in this lab to
create the active enzyme solution. If a person who is Lactose Intolerant eats milk or dairy
products, they may be miserable from problems like diarrhea, gas and sharp abdominal pain.
This lab shows when and in what circumstances the enzyme, lactase, can be broken down and
when it cannot be broken down.
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