This experiment is courtesy of

An Inquiry Approach to the Effects of Enzymes

Developers:

Jennifer Rinehimer
Wissahickon Middle School
Ambler, PA

Steven Pon
Radnor High School
Radnor, PA

Dr. Paul Reibach
Senior Plant Physiologist
Agricultural Products Development
Rohm and Haas Company

Dr. Rita Stevens
Agricultural Chemicals
New Product Process Manager
Rohm and Haas Company

Grade Level:

7 through 9

Discipline:

Biology, Biochemistry

Unit Goals:

Upon completion of this unit, the student will:

1. Recognize that enzymes are key components of chemical reactions in all living things.
2. Understand and apply the scientific method.
3. Practice safe laboratory procedure.

Unit Objectives:

Upon completion of this unit, the student will be able to:

1. Differentiate between protease and amylase enzymes in terms of function and target molecules.
2. Understand the role of enzymes during the germination of common seed plants.
3. Interpret quantitative and qualitative results.
4. Gather and analyze data from various enzymatic reactions and use this data to make conclusions.
5. Explain how temperature, concentration, and pH affect the activity of enzymes.

Background:

Living things utilize a simple sugar called glucose to provide energy for life processes. Organisms link excess glucose molecules together to form the molecule called starch for food storage. The organism cannot directly utilize starch molecules when it needs energy. It must first break down starch into glucose. This process requires the help of protein molecules called enzymes. Enzymes help break the bonds in a starch molecule so that the organism can use the smaller glucose units for energy.

Each type of enzyme controls specific types of reactions. Amylases are enzymes that target starch molecules. Human saliva contains an amylase. Another group of enzymes works primarily on protein molecules. These enzymes are known as proteases. Proteases degrade proteins into smaller pieces known as amino acids. Proteases can be found in common products such as meat tenderizer, pineapple, and papaya.

This series of activities is designed to explore the concept of an enzyme. The concept is developed through a series of investigations that manipulate specific variables. Students draw their own conclusions about enzyme activity by comparing and analyzing both quantitative and qualitative data.

This unit is designed using an inquiry-based approach to learning science. The students should be led and guided to ideas and concepts by the teacher. The unit is designed as a series of activities, not necessarily a day by day unit. In an inquiry-based environment, students are given time to explore answers to their own questions, further influencing their comprehension of the content. The students are asked to design their own experiments. A logbook or journal is recommended. A suggested way of performing the experiment is provided with each activity. Use this only as a guideline.

Advance Teacher Preparation:

You will need the following materials to complete 3 important tasks before the unit begins. The following materials are based on a class size of 30 students.

Plastic petri dishes (science catalog)		6/group (lids can be used)
Viable wheat seeds		30/group of three (approximately 15 grams)
Viable corn seeds		30/group of three (approximately 100 grams)
Viable barley seeds		30/group of three (approximately 15 grams)
Paper towels		3/group of three
Distilled water		8 liters
Agar (dry powder or tablets)		22 grams
Tincture of iodine (2% iodine)		30 mL
Knox Brand unflavored gelatin		5 boxes (28 grams each) (4 envelopes per box)
Corn Starch		1 box
Meat tenderizer (unspiced)		100 gram bottle
Papaya Enzyme (available in tablet form from stores such as GNC — check labels for papain)		approx. 90 tablets/bottle
Bromelain Enzyme (optional)		approx. 90 tablets/bottle
Amylase Enzymes (available in tablet form from stores such as GNC — check for amylase)		approx. 90 tablets/bottle
Mortar and pestle (small)		1
Hot plate		1
Stirring rods		2
400 mL beakers		2

Preparing the dishes:
The teacher should prepare a total of 12 dishes for each group of students. 6 dishes should be made with gelatin and 6 dishes should be made with agar. Suggested mix proportions:

Agar: 2 grams of powdered agar per 200 mL of water
Gelatin: 2 envelopes of gelatin per 200 mL of water

Before heating, add to the mixture 420-mg of starch per 200 mL water for the agar mixture only. Heat until boiling and pour into dishes. The dishes do not need to be covered — use the lids to double the quantity of available dishes. The agar will set quickly. The gelatin will need a little more time to completely set. These may be prepared 24-48 hours before they are needed. Dishes may be covered with wax paper after they cool.

Preparing the enzyme solutions:
Several enzyme solutions will need to be made. For the first activity, you will need two "unknown" solutions. For this, use one amylase and one protease. We suggest preparing the solutions as follows:

Amylase solution: a small amount of saliva with 5 mL of water.
Protease solution: 2 grams of meat tenderizer in 5 mL of water. Stir both until well mixed or dissolved.

Germinating the seeds:
Begin this process at least five days prior to the beginning of the unit. Use C-fold paper towels if possible. Take approximately 30 seeds of the same type and place them in the fold of the paper towel. Roll the paper towel with the seeds inside and moisten with distilled water. Label the package as Day 1 / seed type (corn, barley, or wheat). Repeat this process with the other types of seeds. Enclose all three types from day one in aluminum foil and label the outside. There should be sets exactly like this one prepared for each group of three students.

Repeat this process each day for at least four more days in order to provide each group with five sets of seeds on the first day of the unit.

Safety Notes:

1. Iodine wash is mildly irritating to the skin and eyes. Safety glasses are recommended when using iodine solutions. Iodine solutions will permanently stain clothing, books, notebooks, and other porous materials. Iodine solutions will also stain skin but it will wash off within a few days.
   
   
2. Food coloring is not toxic. However, ingestion of anything in a science lab is not recommended. Food coloring will permanently stain clothing and other porous surfaces. Stains on skin will wash off within a few days.

Activity 1: What are Enzymes and How Do They Change Living Things?

• To use seeds in different stages of germination in developing the concept of an enzyme.
• To interpret experimental data in determining the presence or absence of starch in food sources and in different types of biological media.

• Three 45-minute class periods

• Sets of wheat/barley/corn seeds (prepared)
• logbooks
• agar and gelatin plates (one of each plate per group)
• unknown solutions with pipettes
• marking pen (for marking on plastic)
• iodine wash dropper bottles (about 5 drops iodine to a dropper bottle of water)
• small samples of different foods such as apple, potato, bread, ham, flour, sugar, and cheese for each group
• dry wheat, barley, and corn seeds for each group

1. Provide each group with five sets of seeds. Each set of seeds will contain seeds that began germination on a different day. In each set, there will be seeds from corn, barley, and wheat.
   
   
2. Ask the students to come up with a list of ways in which the seeds are different depending on which stage they are looking at.
   
   
3. Discuss the idea that some parts of the seed disappear over the course of 5 days. Come up with ideas on where these parts "go". Also discuss where the "new" parts come from. Lead them to the idea that the plant needs food to grow, just like people do. Where does the plants' food come from?
   
   
4. Provide each group of students with a gelatin plate and an agar-starch plate. Each group will also be given two unknown solutions (A & B). They should put a drop of each unknown on each of the plates and label them. It is recommended that the plates be allowed to sit for about 24 hours. Ensure that students label their plates. It is not necessary to cover plates.
   
   
5. To introduce the concept of starch, have several food items available for each group. Show the class what iodine looks like out of the bottle (brown-yellow in color). Have the students place a few drops of iodine solution on a small piece of bread. They will see that the iodine turns black. Have them do the same with other food items. A few recommendations are ham, cheeses, apple, potato, flour, and sugar.
   
   
6. Now, tie this concept into the plant seeds. Take a dry corn, wheat, and barley seed and put iodine on each of them. The students should begin to conclude that foods like bread and seeds are made up of the same thing because they react the same way to an iodine test. With teacher facilitation, they should realize that the substance that reacts with iodine is starch, a stored carbohydrate that is used as a food reserve in plants. The plant embryo inside of the seed uses this starch for energy to grow.
   
   
7. Distribute the dishes from step 4 (the A/B dishes) to the students (these dishes should have been sitting for 24 hours). Both of the dishes will be translucent but colorless in appearance. Allow the students to add an iodine wash solution to both plates. Swirl the solution around the plates and leave them sit for a few minutes. Pour off any additional iodine into the sink drains.
   
   
8. Ask the students the following leading questions and have them record their responses:
       a. Do you see any changes in the dish?
       b. What is the substance in the dish?
       c. What are the differences in the two dishes?
   
   
9. Allow the groups time to come to these conclusions. The students should conclude that there was starch in the agar plate and that the iodine turned the starch blue/black. They should also note what they see in the gelatin plate. They will explore that shortly.
   
   
10. What happened in the clear areas of the agar plate? Students should conclude that whatever was in the unknown solutions accounted for the difference. Explain to them that these "unknown" solutions contain proteins that break down substances into smaller particles. In this case, the starch was broken down, resulting in an area that did not stain.
    
    
11. Have the students work as a group to determine why the plates responded differently. They should come to a point where they conclude that the plates are made out of two different substances and that the unknown solutions acted differently depending on what the plates' material was.
    
    
12. Introduce the concept of an enzyme. Explain that enzymes are very specific for the substances that they work on. Explain that agar is a vehicle for suspending starch. Amylases are enzymes that break down starch. On the other hand, gelatin is a protein. Proteases are enzymes that break down proteins. Some examples of substances containing enzymes are saliva, pineapple, papaya, LACTAID, and meat tenderizer.
    
    
13. Review the results from the two A/B dishes. Explain that where the amylase was working, there was a clear area when stained with iodine. Where a protease was working, the area appeared to be "watery" (in breaking down the protein, a molecule of water is formed when two amino acids of the protein are separated).
    
    

Activity 2: Dilution Process

• To learn how to prepare a serial dilution from a concentrated substance.

• One 45 minute period

• Food coloring
• six small (1 oz) condiment cups
• distilled water
• eye droppers or pipettes
• overhead projector
• transparency sheet (use a new sheet)

1. Frequently, the need for solutions of different concentrations arises. In order to set up solutions of identical chemical composition with varied concentrations, it is easiest to use a serial dilution process. A serial dilution process starts with a solution that is designated as a "concentrate". Using the concentrate, successively weaker solutions are mixed. This technique is handy for use in a case where the experimenter wishes to analyze the effect of varying solution concentrations.
   
   
2. Mark the condiment cups as follows:
   

   1:1

   1:10

   1:100

   1:50

   1:500

   1:1000

   
   
   
3. The cup for each dilution step will have a total of ten drops in it, just before proceeding to the next dilution step.
   
   
4. Place ten drops of food coloring into the cup marked 1:1. This means that there is 1 drop of concentrate for 1 drop of total liquid volume in the cup.
   
   
5. Use the pipette to place one drop from the 1:1 cup into the cup marked 1:10. Rinse the pipette with distilled water. Add nine drops of distilled water to the cup. There is now 1 drop of concentrate for 10 drops of total liquid in the cup, also known as a 1:10 dilution.
   
   
6. Use the pipette to place one drop from the 1:10 cup into the cup marked 1:100. Rinse the pipette with distilled water. Add nine drops of distilled water to the cup. There is now 1 drop of 1:10 for 10 drops of total liquid in the cup, also known as a 1:100 dilution.
   
   
7. Use the pipette to place one drop from the 1:100 cup into the cup marked 1:1000. Rinse the pipette with distilled water. Add nine drops of distilled water to the cup. There is now 1 drop of 1:100 for 10 drops of total liquid in the cup, also known as a 1:1000 dilution.
   
   
8. Use the pipette to place 2 drops from the 1:10 cup into the cup marked 1:50. Rinse the pipette with distilled water. Add 8 drops of distilled water to the cup. There are now 2 drops of 1:10 for 10 drops of total liquid in the cup, also known as a 2:100 dilution, which can be mathematically reduced to 1:50.
   
   
9. Use the pipette to place 2 drops from the 1:100 cup into the cup marked 1:500. Rinse the pipette with distilled water. Add 8 drops of distilled water to the cup. There are now 2 drops of 1:100 for 10 drops of total liquid in the cup, also known as a 2:1000 dilution, which can be mathematically reduced to 1:500.
   
   
10. As solutions become more diluted, it is successively more difficult to detect any color in the solutions. Label a new transparency sheet as follows:
    
    

    1:1

    1:10

    1:100

    1:50

    1:500

    1:1000

    H20

    
    
    Place the transparency sheet on the overhead projector and turn on the lamp. (Make sure it is level) Place one drop of each solution (use H₂0 for your control) on the transparency sheet near each corresponding dilution, and allow the heat from the lamp to evaporate the water from each drop. Even the drops that seem to have no color in them will show up as a small dot of coloring.
    
    Other dilution levels may be achieved by remembering to divide the drops of "concentrate" by the total volume of liquid.
    
    

Activity 3: Examine How Concentration Affects Enzyme Activity

• To determine if the concentration of enzyme in a solution affects its activity.

• Two or three 45 minute class periods

• meat tenderizer
• amylase tablets
• distilled water
• test tubes, test tube racks, and pipettes for dilution
• stirring rods
• mortar and pestle
• balance and weighing boats (an index card, folded to form a V-shape works)
• agar and gelatin plates
• marking pen
• iodine solution
• graph paper and ruler (optional)

1. Students will naturally think that if a little enzyme works, then more enzyme must work better! Give them the opportunity to test this hypothesis. They should be working with meat tenderizer (papain) for the protease and a commercial amylase tablet for their amylase.
   
   
2. They should begin this part of the process after completing the dilution exercise in Activity 2.
   
   
3. Allow the students to come up with a procedure to test how concentration of the enzyme affects the digestion of starch or protein. Their experimental design may differ from one group to the next. Encourage all feasible options. The best method for us was to take 5 g of Adolph's Meat Tenderizer and add it to 5 mL of distilled water, stirring until dissolved. This will give you a "concentrated" protease solution. This solution should be the starting point for their dilution series. To make the amylase solutions, use one tablet dissolved in 5 mL of distilled water to begin. Make it more concentrated by adding additional tablets to the same volume of water. Concentrations from 1-5 tablets should be sufficient. Allow the tablets some time to dissolve on their own. The students can then use either a stirring rod or mortar and pestle to help dissolve the tablets.
   
   
4. The students should label their dishes with the type of medium and the solutions they used on the bottom of the dish. Allow the dishes to sit for approximately 24 hours.
   
   
5. Examine the students' plates the next day for amylase activity (as evidenced by clear areas after an iodine stain) and protease activity (hydrolyzed area of the medium).
   
   
6. Students should conclude that a more concentrated enzyme shows a greater amount of activity, shown by a wider area. If their results appear to show a trend, you may wish to have them graph their data with concentration on the x-axis and size of area in mm on the y-axis.
   
   

Activity 4: How Does Length of Germination Affect Enzyme Activity?

• To examine seeds for an increase or decrease in enzyme activity over time.

• Two 45 minute class periods

• agar plates (3 per group)
• one corn seed from each day of germination plus a dry seed (6 total)
• one barley seed from each day of germination plus a dry seed (6 total)
• one wheat seed from each day of germination plus a dry seed (6 total)
• scalpel or exacto knife
• marking pen

1. Remind students that plant seeds contain starch as the primary food reserve. Some plant seeds store protein as the primary food reserve, and others store lipids (fats and oils) as the primary food reserve. Have students propose a hypothesis to answer this question: What happens to the stored starch in a germinating seed as days pass? Growing plants use amylase enzymes to break down starch so that glucose may be utilized for energy. Proteins are broken down by proteases, and lipids are broken down by lipases.
   
   
2. The students will test their hypothesis using the above materials in an experimental design that you help them develop. They should observe that the starch is stored inside of a seed coat. Therefore, if you really want to know how much starch is still present and how actively the enzyme is degrading the starch, the seed will have to be opened. The best way that we found to do this is by taking the seeds at each day of development and splitting them with a sharp knife. Supervise your students carefully while doing this or you may want to split the seeds for them.
   
   
3. We achieved the best results when we removed the embryo and took half of a seed, placing it cut-side down on the agar surface. We placed one half-seed for each day of germination on one dish. Keep each type of side on one dish.
   
   
4. You will have to allow time for this assay to work. When sufficient time has passed (24 hours), allow the students to stain the plates with iodine solution. The largest clear area should be around the "oldest" seeds. Have the students make conclusions as to why this happened and to compare the results to their original hypothesis. Also, it may be interesting to have them compare the appearance of the iodine stain in all three types of seeds.
   
   

Activity 5: Extracts from Germinating Seeds

• To analyze the enzyme activity level in liquid extracts from seeds at different stages of germination.

• Two - three 45 minute class periods

• 3 agar-starch petri dishes per group
• 12 small (1 oz) condiment cups
• Distilled water
• Eye dropper or pipette
• Iodine wash
• 1 Mortar/pestle per group
• 5 seeds of each (wheat, barley, and corn) germinating for 5 days
• 5 seeds of each (wheat, barley, and corn) germinating for 3 days
• 5 seeds of each (wheat, barley, and corn) germinating for 1 day
• 5 seeds of each (wheat, barley, and corn) which are still dry

1. Germinating seeds produce an amylase to digest food stored in the seed as a polysaccharide known as starch. The starch is broken down into the monosaccharide called glucose, which can be used directly by the growing plant embryo.
   Q. Is the level of enzyme the same or is it different for seeds in various stages of germination?
   Q. How can we design an experiment to answer this question?
   Q. What can serve as a control for the experiment?
   
   
2. One suggested experiment design: (if class time does not allow for all three types of seeds, use one or two types of seeds — crushing the seeds in the mortar will take about three to four minutes for each type and each germination stage)
   
   
3. Label petri dishes, one for each type of seed extract, and put five marker spots on the underside of each petri dish. Label the spots: H2O, dry, 1 day, 3 day, and 5 day.
   
   
   
   
4. Crush five seeds with 20 drops of water using the mortar and pestle (follow the same procedure for both germinating seeds and dry seeds). Pour the resulting slurry into a small plastic condiment cup and label it. (e.g. Wheat — 5 day extract) Rinse the mortar and pestle with distilled water.
   
   
5. Repeat the procedure for each of the different number of days of germination and for each type of seed.
   
   
6. Place one drop of each wheat solution (try to draw only the liquid into the pipette) on the wheat petri dish. Repeat this procedure for the barley and the corn petri dishes. Place a drop of distilled water on the H₂O spot.
   
   
7. Extracts may be discarded or saved for advanced activity (See #9)
   
   
8. Set petri dishes in a safe place to sit overnight. Covers may be used, but they are not required.
   
   
9. After about twenty-four hours, visually examine petri dished for any change. Record observations. Why are some locations different in appearance?
   
   
10. Stain the petri dish with a weak iodine wash. After about two minutes, drain the iodine wash from the petri dishes and rinse gently with distilled water from a squeeze bottle. Examine the petri dishes after staining. Record observations and have group members discuss the possible reasons for any differences in appearance between each location on a given petri dish.
    
    
11. Advanced activity: This activity requires at least 12 glass culture tubes (5mm x 70mm is a good choice, but any very small test tubes will suffice)
    
    Q: Does heating or boiling an enzyme extract render it unable to digest starch?
    
    A suggested experiment design to test this: Place the liquid from each extract in a separate glass culture tube and immerse the bottom of each tube in a boiling water bath for about sixty seconds or until it just starts to boil (be careful not to boil the samples dry — some samples will have very little liquid to work with). Use a long, thin pipette to draw a drop of boiled extract. Place the drop on a fresh set of agar-starch petri dishes (as in step 6 above). Allow the petri dishes to stand for about 24 hours, then stain with iodine wash. Compare results with the results from the live enzyme extracts.
    
    

Activity 6: How Does pH Affect the Activity of an Enzyme?

• To change the pH of enzyme solutions with common products to determine how enzyme activity is affected

• Two 45-minute class periods

• Agar plates
• sodium bicarbonate (baking soda), dilute ammonia solution, or sodium carbonate (if available)
• vinegar
• pipettes
• weigh boats
• balance
• 2 50 mL beakers
• marking pen
• pH test strips for both acids and bases

1. The students should be starting to understand the specificity of enzymes. One factor that influences enzyme activity is pH. Depending on your students' background, you may have to introduce this concept first. pH level is a measure of the amount of particular types of ions produced by a compound when it is put in solution. A substance that is an acid releases H⁺ ions when placed in solution. Substances that are acidic have pH values below 7.0. Strong acids have a lower pH and release H⁺ ions almost completely in solution. Other substances release OH^- ions in solution. These solutions are called bases and have a pH above 7. Stronger bases have a higher pH and almost completely dissociate into OH^- ions. pH of 7.0 is neutral.
   
   
2. Tell the students that a common acid is vinegar (acetic acid) and a common base is baking soda (sodium bicarbonate). Have a volunteer from each group "donate" some saliva into two weighing boats.
   
   
3. Label two agar/starch petri dishes. One dish will be used to test the effect of acids on saliva (which they have already found has amylase in it) and the other will be used to test the effects of bases. Solicit ideas from the students on how they might do this. They may need some assistance if the concept of pH is new.
   
   
4. They can use the vinegar as is out of the container, since it is already a diluted form. Make a solution of baking soda by mixing approximately 700 mg of powder with about 10 mL of water.
   
   
5. Test the pH of saliva before starting by having the student volunteer put a pH test strip on their tongue. After they add the vinegar and baking soda to separate samples, take the pH of the saliva mixture and compare it to the original. Be sure to take the pH of the vinegar and baking soda without saliva also. The students should record all data from these test strips so that they can see how pH changes with the addition of an acid or base.
   
   
6. Now the students should test to see how this pH change affects the ability of amylase to digest starch. We suggest that the students have three test areas on each plate. One area will be saliva only. One area will be saliva mixed with either vinegar or baking soda, and the third area will be a drop of the vinegar or baking soda solution only.
   
   
7. Allow the dishes to sit about 24 hours. The students should then stain them with iodine solution to see where starch is still present. They should make observations about their results in their logbooks.