Biology 101 Lab: Enzymes
Introduction:
In this lab, you will examine two different enzymes. You will observe the action of enzymes, their
specificity, and effects of denaturing an enzyme. The enzymes you will investigate today are sucrase, and
lactase.
You will need to work with your group to perform the activities for each procedure. Complete the data
tables and questions for each test.
Reading:
Symbiosis lab manual pp. 83-86, Campbell pp. 151-160.
Caution:
You will be using heat and Benedict's reagent in this lab. Use caution and wear eye-protection!
Hydrolysis of Sucrose by the Enzyme Sucrase
Introduction:
The enzyme sucrase (invertase) catalyzes the hydrolysis of the disaccharide sucrose into its
monosaccharides glucose and fructose. Sucrase is utilized by yeast to breakdown sucrose in order to have
monosaccharides which can be used as its energy source.
Benedict's reagent (discussed in Lab #4) demonstrates a positive reaction with any reducing sugars,
which include monosaccharides and most disaccharides. Sucrose is NOT a reducing sugar since it does
not have a free carbonyl group and thus gives a negative reaction.
In this procedure, you will use sucrase to break down sucrose into its monosaccharide monomers. You
will also see the effects that result from denaturing sucrase.
Procedure:
l. Label 4 test tubes AI, A2, A3, and A4. Add the following to each test tube:
Into Tube AI, place 5 mL of 5% sucrose solution
Into Tube A2, place 5 mL of 5% sucrose solution and 2 mL of sucrase extract
Into Tube A3, place 5 mL of sucrose solution and 2 mL of boiled sucrase extract
Into Tube A4, place 5 mL of distilled water and 2 mL of sucrase extract.
2. Using the waterbath at the back of the room, heat to 30 - 35° C. Incubate the tubes in this warm water
bath for 35 minutes. (This is the optimal temperature range for sucrase activity).
3. Label 5 test tubes Bl, B2, B3, B4, B5. Add 2.5 mL of Benedict's reagent into each tube. Now transfer
to the B tubes:
Into Tube B 1, transfer the contents of Al
Into Tube B2, transfer the contents of A2
Into Tube B3, transfer the contents of A3
Into Tube B4, transfer the contents of A4
Into Tube B5, place 5 mL of 5% glucose solution
Tap each of the tubes to mix.
4. Fill a 250 mL beaker approximately 2/3 full with tap water and heat to just slightly boiling. Place all
the B tubes into a boiling water bath. DO NOT allow the water bath to get too hot. It should be heated
only high enough for bubbles to begin to rise from the bottom of the beaker.
After 3-4 minutes, remove the tubes and record the color.
Tube
# 1: sucrose
#2: sucrose + sucrase
Final Color
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#3: sucrose and boiled sucrase
#4: distilled water and sucrase
#5: glucose
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Interpretation
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1. Write out the chemical reaction that occurs when sucrase is added to sucrose in the presence of water.
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2. Which tube could be considered a positive control to show that the Benedict's reagent was working
properly? Explain.
3. For each of the 5 tubes, what was the result ofthe Benedict's test? Was this the expected result? Why
or why not?
Tube #1:
Benedict's test result:
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Expected result?
Explain:
Tube #2:
Benedict's test result:
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Expected result?
Explain:
Tube #3:
Benedict's test result:
Expected result?
Explain:
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Tube #4:
Benedict's test resultb>
Expected result?
Explain:
Tube #5:
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Benedict's test result:\:1}
Expected result?
Explain:
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Hydrolysis
of Lactose by Lactase
Introduction:
Lactose is the disaccharide found in milk. It is composed of two monosaccharides - glucose and
galactose. Sucrose (table sugar) is also a disaccharide and is composed of two monosaccharidesglucose and fructose.
Lactase is an enzyme that hydrolyzes lactose into its two monosaccharides. People who are lactose
intolerant have insufficient quantities of lactase and are unable to break down lactose. Lactase is
available for purchase in pill or liquid form for those who are lactose intolerant.
Lactase, like all enzymes, is specific for the type of substrates it is able to catalyze based on the shape of
the molecules. Lactase can only break down lactose.
In this portion of the lab, you will see lactase hydrolyze lactose into glucose and galactose. You will also
see the effects of having denatured lactase.
Procedure:
NOTE: Timing is critical in this test!
1. Label 5 test tubes 1,2,3,4,
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2. In Tube #1 add: 2 mL skim milk and 1 mL lactase enzyme solution.
Time for 2 minutes and test for glucose with the glucose test tape. Record your
result.
3. In Tube #2 add: 2 mL skim milk and 1 mL water.
Time for 2 minutes and test for glucose with the glucose test tape. Record your
result.
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4. In Tube #3 add: 2 mL skim milk and 1 mL heated lactase enzyme solution.
Time for 2 minutes and test for glucose t'h the glucose test tape. Record your
result.
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5. In Tube #4 add: 2 mL sucrose solution and 1 mL lactase enzyme solution.
Time for 2 minutes and test for glucose with the glucose test tape. Record your
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6. In Tube #5: 2 mL sucrose solution and 1 mL water.
Time for 2 minutes and test for glucose with the glucose test tape. Record your
result. I;
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Glucose Test Result (+ or-)
Tube#
# I: Milk and enzyme
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#2: milk and water
#3: milk and heated enzyme
#4: sucrose and enzyme
#5: sucrose and water
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1. Write out the lactose and lactase reaction.
2. Why did the enzyme react with lactose but not with sucrose?
3. What happened ~e
when it was boiled?
4. Enzymes can be denatured by reducing the pH. However, the commercially produced lactase is
formulated to work in the stomach. Would lowering the pH of the enzyme solution affect the working of
lactase? Why or why not?
5. Is the lactase reaction an example of a dehydration reaction or of hydrolysis?
6. For each of the 5 tubes, what was the result of the glucose test? Was this the expected result? Why or
why not?
Tube #1:
Glucose result:
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Expected?
Explain:
Tube #2:
Glucose result:
8
Expected?
Explain:
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Tube #3:
Glucose result:
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Expected?
Explain:
Tube #4:
Glucose result:
E.)
Expected?
Explain:
Tube #5:
Glucose result:8
Expected?
Explain:
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