Enzyme Catalysis with Applesauce and Pectinase
(adapted by A. Lyman-Buttler from Dr. J. Smith, Ball State Univ.)
Name:______________________________
Points
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Produce an outline of your practical biological investigation, including your purpose and hypothesis, and all information relevant
to the study design.
Identify your dependent and independent variables, their range, and how you will measure them.
Identify experimental constants (controlled variables) and their significance.
Demonstrate an ability to systematically record data.
Follow lab safety rules.
Experimentally determine the effects of pH and temperature on the rate of enzyme-catalyzed reactions.
Describe the benefits of graphing data and present different types of data appropriately in both graphs.
Evaluate any sources of error.
Present the findings of your investigation in a well organized scientific report.
Identify and explain the important features of a scientific report including
o discussion of the biological concepts involved (background)
o materials and methods
o data
o conclusions based on analysis of the data (results & discussion)
o evaluation of the investigation (discussion)
Peer review a lab report.
Background
In this lab the activity of the enzyme pectinase will be studied. Pectinase breaks down the carbohydrate pectin in applesauce.
Pectin is a common component of fruits; it helps the cells of the fruit stick to each other. When it is broken down, the applesauce turns
into apple juice. Purified pectinase is often used industrially in the manufacture of fruit juices. The effects of heat, acid, base, and
enzyme concentration will be determined by measuring the amount of juice produced as a function of time.
Enzymes are proteins that act as catalysts in living cells and carry out many of a cell’s metabolic processes. The chemical
that the enzyme acts upon is the substrate. In a typical enzyme-catalyzed reaction, the enzyme will bind briefly with the substrate,
allowing the substrate to form its products. After the products are formed, the enzyme molecule remains unchanged and continues to
react with more substrate molecules.
Because enzymes are proteins, they possess highly specific three-dimensional structures. As a result, most enzymes only
react with a specific substrate, and catalyze a single reaction, a property called specificity. An enzyme molecule has a region called
the active site which is the binding site for the substrate. This matching of active site to substrate is sometimes compared to a lock and
key, where the keyhole is the active site, and the key is the substrate. Due to the sensitivity of many proteins to changes in their
chemical and/or physical environment, the three-dimensional structure of enzymes is sometimes easily altered. When such a change
occurs, we say the enzyme has been denatured. Since denatured enzymes cease to undergo their normal chemical activities, rates of
reaction will be affected. Acids and bases (which change the pH) and excessive heat are common causes of enzyme denaturation.
Another factor that can change the activity of an enzyme would be the presence of chemicals called inhibitors that interfere
with the enzyme’s normal reactivity. Inhibition may be reversible or irreversible, depending on the nature of the inhibitor. If the
inhibitor acts by binding to the enzyme’s active site, it is called competitive inhibition.
Purpose: This lab activity actually involves four different experiments, with four different purpose questions:
1) How does an acid affect the rate of pectinase activity?
2) How does a base affect the rate of pectinase activity?
3) How does exposure to high temperatures affect the rate of pectinase activity?
4) How does enzyme concentration affect the rate of pectinase activity?
Page 1 of 4
Hypothesis & Prediction
You should be able to predict how each of the treatments will affect enzyme activity. You must give SPECIFIC reasons for your
predictions! (Hint: refer to Background & enzyme notes.) Write down your predictions below BEFORE beginning your experiment.
The hypothesis and prediction for the first treatment is done for you—use it as an example to complete the others.
1) If changes in pH cause enzymes to denature, then the reaction rate with an acid-treated enzyme will be
[ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative
control, because enzyme activity requires a close fit between the substrate and the enzyme’s active site, and denaturation of
the enzyme will alter the shape of the active site.
2) If ______________________________________________, then the reaction rate with a base-treated enzyme will be
[ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative
control, because ______________________________________________________________________________________
___________________________________________________________________________________________________
3) If ________________________________________________________, then the reaction rate with a heated enzyme will be
[ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative
control, because ______________________________________________________________________________________
___________________________________________________________________________________________________
4) If _________________________________________________, then the reaction rate with lower enzyme concentration will
be [ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative
control, because _______________________________________________________________________________________
___________________________________________________________________________________________________
Experimental Design
What is the dependent variable in this experiment? (Hint: look ahead at the directions) ______________________________________
List at least three experimental constants: __________________________________________________________________________
___________________________________________________________________________________________________________
What control(s) is (are) used? Be specific:__________________________________________________________________________
___________________________________________________________________________________________________________
Methods and Materials
Safety. WEAR GOGGLES. H2SO4 and KOH are corrosive substances that will cause serious chemical burns; if you get them on
yourself, put it immediately under running water, and tell Mr. LB. If you spill chemicals in the lab, notify Mr. LB (do NOT attempt to
clean it up yourself until given instructions). Any student seen working without goggles, or performing any other unsafe act, will be
removed from the lab and reported to the Student Management Coordinator.
Because different lab groups are using similar methods to collect the same kind of data, and are then sharing data, it is very important
that everyone pay careful attention to detail—don't screw up the procedure!
1. Which treatment will your group be doing? WRITE IT DOWN! ________
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2.
In a beaker, mix all your ingredients EXCEPT the applesauce using a glass stirring rod for 2 minutes. Then, add the
applesauce and stir for another 1 minute. The different treatments are:
Treatment
Name of treatment
1
positive control
1 container applesauce + 20 mL pectinase + 20 drops water
2
negative control
1 container applesauce + 20 mL water + 20 drops water
3
heated enzyme
1 container applesauce + 20 mL heated pectinase (60°C) + 20 drops water
4
enzyme w/ base
1 container applesauce + 20 drops KOH (strong base) + 20 mL pectinase
5
enzyme w/ acid
1 container applesauce + 20 drops H2SO4 (strong acid) + 20 mL pectinase
6
3.
4.
5.
6.
7.
Ingredients
lower enzyme concentration 1 container applesauce + 5 mL pectinase + 15 mL water + 20 drops water
Use a piece of pH paper to determine the pH of your mixture. Write down the pH of your solution here: _________
Set up your collection apparatus according to Mr. LB's demonstration. (You should have taken notes on this!)
Carefully pour the contents of the beaker into the funnel, being careful not to get any of it past the filter paper.
Start your stopwatch (or start watching the clock) and make your first measurement (should be 0.0 mL)
For all of the treatments, measure every 2 minutes for 30 minutes. Record in the table on the other side. Only record data for
the treatment YOUR group is doing; you will get data for the other treatments from the other lab groups.
Results
Record the volume of liquid produced at each of the times listed on the data table. You need to get some data from other groups, since
your group does only one of the treatments. All students are expected to submit a report with a completed data table.
Time
(min)
0:00
2:00
4:00
6:00
8:00
10
12
Volume (mL)
14
16
18
20
22
24
26
28
30
1
2
3
4
5
6
Using the data from your group AND from the other groups, draw one graph showing the results of all treatments. In your graph, plot
volume against time and use a separate color or symbol for each line. Include a key. In your key, do NOT use the treatment
number—use the treatment name! This is because somebody who reads your graph without reading the rest of your lab report
should still be able to understand your results. Make sure you include all the components of a good graph (you should already know
these) and pick the correct variable to plot on the x- and y-axis. Mr. LB has graph paper that you may use—ask if you need it. A
spreadsheet (like Excel) would be another good choice.
Discussion
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Analysis Questions
Answer the following questions. Many of the questions are based on the information in the “Background” section—read it!
1.
Treatments 1 and 2 are referred to as positive and negative controls. What does this mean? What was their purpose?
2.
What are experimental constants? Why is it important to identify them?
3.
Why was 15 ml water added to treatment 6? Why were 20 drops water added to treatments 1, 2, 3, and 6?
4.
Predict what may have happened in a treatment that was kept much colder than room temperature, e.g., at 5°C.
5.
What is the enzyme in this lab? What is the substrate?
6.
Describe the shape of the lines on your graph. What happens to the line near the end? Why do you think this happens?
7.
Suppose you were to repeat this experiment, but this time you add an inhibitor. Predict what would happen & why.
8.
Scientists have just discovered a new enzyme in the fruit fly Drosophila melanogaster. The function of the enzyme is to break
down fructose. What name would they probably give the enzyme and why? (Hint: look in your lecture notes.)
Discussion Paragraphs
Type a full 2-paragraph conclusion and attach it to this packet, or e-mail it to me. Write your own—no group papers!
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