Name____________________________________________Date__________________Period___________
Protease Lab: Enzyme Hydrolysis
Standard:
1. Scientific progress is made by asking meaningful questions and conducting careful
investigations. As a basis for understanding this concept students will
1j: Recognize the issues of statistical variability and the need for controlled tests.
1k: Recognize the cumulative nature of scientific evidence.
1b. Students know enzymes are proteins that catalyze biochemical reactions without altering
reaction equilibrium and the activities of enzymes depend on the temperature, ionic
conditions, and the pH of the surroundings.
Objectives: In this lab students will:
1. Learn the process by which proteases work;
2. Analyze the results of enzyme hydrolysis;
3. Design an experiment which will test the enzymatic activity of proteases under different conditions; and
4. Contribute to class data for cumulative evidence and conclusion.
Introduction/ Background information:
In nature, there are enzymes called proteases that “digest”, hydrolyze or break down proteins. Many laundry
detergent manufacturers add them to the detergents in the hope that they will “digest” the protein stains off
our clothing! Most of these proteases are isolated from strains of Bacillus bacteria. These extracted bacterial
proteases are extremely stable at an alkaline (basic) pH, long-term storage and varying temperatures. These
bacteria are also genetically engineered in order to enhance the power of the proteases to withstand
degradation by bleach which is often added to brighten clothes. These detergents were one of the first
biotechnologically enhanced products available to the public. In this lab, we will be investigating how well
various detergents work and under which conditions they work the best!
In this lab students will:
Materials (per student group):
 8 test tubes and test tube racks
 Jell-O (regular, flavored, sugar free, etc.) and/or Knox gelatin
 Waxed pencil or marking pens
 Laundry detergents (if desired, allow students to bring detergent of their choice)
 Distilled water
Safety Precaution:
 Graduated cylinder
 Hot plate
 Cotton balls (or Para film)
detergents are
 Ruler
harmful if
 pH paper & scale
swallowed!!
 4 beakers
 stirring rod
Name____________________________________________Date__________________Period___________
Procedure:
Pre lab prep work (Teacher or student) Steps 1 -3 and 5 should be done a day ahead of the lab day.
1.
Bring 50mL of distilled water to a boil in a small beaker on the hot plate.
2.
Slowly add 18g of Jell-O to the boiling water while stirring with a glass rod. Stir and heat until
all materials are dissolved and the solution has briefly boiled.
3.
Using tongs, or “hot hands”, pour 3mL of the gelatin solution into the test tubes. Pour slowly and
try to minimize the number of air bubbles on the top of the solution. Cover each tube – either
with a cotton ball or Para film. Set the test tubes in the refrigerator to cool/solidify overnight.
4.
MEASURE and RECORD in your data chart the pH of your gelatin solution before discarding
any remaining solution.
5.
Make 10% solutions of the detergents provided. (Measure 10mL or 10g of the detergent. Place
in beaker. Measure 90mL of distilled water, add to beaker with detergent, and MIX well.) Label
the solutions carefully. RECORD in your data chart if any enzymes are listed in the ingredients.
6.
MEASURE and RECORD the pH of each 10% detergent solution.
7.
After the gelatin solution has solidified in the test tubes, mark the top level with a wax pencil, or
marking pen.
8.
For each detergent solution, use 2 test tubes : 1) add 10 drops of each detergent solution to the top
surface of each test tube; 2) add 10 drops of distilled water. Label your test tubes carefully. Set
test tubes aside for 24 hours.
Predict which test tubes will be “hydrolyzed”. RECORD predictions.
IF TEACHERS DESIRE TO ENCOURAGE FURTHER INQUIRY INVESTIGATIONS,
ALLOW STUDENTS TO USE THE REMAINING CLASS TIME FOR INDEPENDENT
WORK ON DESIGNING THE EXPERIMENT.
9.
10.
11.
After 24 hours, examine the test tubes. Notice that the gelatin has been liquefied in some tubes.
Use a ruler to measure (to the nearest mm) the depth of the liquefaction. Record data.
If time allows, set tubes aside for another 24 hours and repeat step #10.
Collect class data.
Extension Activity:
Design an experiment that would test one of the following variables:
o pH
o temperature (of detergent)
o concentration of the detergent (enzyme)
o time for hydrolysis
o effectiveness of the detergents in the presence of bleach
Be sure to write a detailed procedure and record all results.
Name____________________________________________Date__________________Period___________
What is the purpose of this lab?
Draw a labeled diagram of the experimental design.
Data:
pH of the gelatin solution _______
Detergent
Brand:
Detergent I:
Test tube 1
Test tube 2
Detergent II:
Test tube 1
Test tube 2
Detergent III:
Test tube 1
Test tube 2
Detergent IV:
Test tube 1
Test tube 2
pH
value
Enzymes listed
in ingredients
(list)
acidic?
basic?
neutral?
PREDICTIONS: Which
will hydrolyze the Jell-O?
Depth (mm) of Liquefaction of
Jell-O
24 hours
48 hours
Name____________________________________________Date__________________Period___________
Detergent I:
Detergent II:
Detergent III:
Detergent IV:
Test
tube1
Test
tube1
Test
tube1
Test
tube1
pH
Test tube
2
Test tube
2
Liquefaction (mm)
AVERAGE (mm)
Analysis:
1.
GRAPH the class average data for test tube 1 and test tube 2.
Test tube
2
Test
tube 2
Name____________________________________________Date__________________Period___________
2.
What was the purpose of the test tube containing only 10 drops of distilled water?
3.
Which detergents digested the most gelatin? The least?
4.
Did any detergent digest the gelatin solution that did not list an enzyme as an ingredient? Would
you guess that it has an enzyme? Could anything else explain the results?
5.
What other common household items might have proteases in them? Make a list of at least 3
items and discuss how the enzymes would “enhance” each item’s purpose/function.
Conclusion: Based on your as well as class results, summarize your learning, in general, about enzymes.