Amylase - Exploring digestion and evolution through a molecular machine
Grade level(s):High School (9-12), Grade 9, Grade 10, Grade 11, Grade 12
Topic:Enzymes, macromolecules, digestion, genetics, evolution
Amylase in our saliva breaks starch down into sugar. Human populations have variable copy numbers of the amylase gene due to differences in the amount of starch in their diet.
Before the lesson: Enzyme, digestion, carbohydrate, sugar, protein, amino acid, evolution, acid
After the lesson: Amylase, catalysis, positive selection
What you need:
- Magnetic printing paper (These 8mm ones from Custom Magnets work great, or use thinner version from Avery)
- Unsalted crackers, enough so that each student can have one (oyster crackers are small and easy to distribute to a large class, water crackers are also good)
- Purified amylase (available from SchoLAR chemistry for example at WARD'S)
- 0.125% (weight by volume) cornstarch in water stock solution: Make by dissolving 1.25g of cornstarch in 50 ml of room temp water and add to 950 ml of boiling water, as soon as the starch has been added to the boiling water remove from heat and stir. Let cool to room temperature before using
- 500mL PBS Buffer (pH 7-7.5). PBS buffer can be purchased for example at WARD'S
- 50mL of 20% (weight by volume) Dextrose in water. Dextrose can be purchased in health food stores or online, for example at Amazon.
- Iodine indicator solution: Add 5g of potassium iodide to 40ml of distilled water, then add 2.5 g of iodine crystals mix. Finish by adding water up to 50ml. Alternatively you can buy iodine solution as "Tincture of iodine" or " Lugol's solution" at a drugstore.
- 16% (volume by volume) glacial acetic acid in water. This will be referred to as "vinegar" however, vinegar is normally only 8% glacial acetic acid, so this solution is more acidic. Alternatively, regular household vinegar seems to work fine in the experiments as well.
- 1 x 200uL pipette and tips
- enough 1000uL pipettes for each student and pipette tips
- At least 3 x 600mL glass beakers (7 is best because then you don't have to reuse them during the lesson).
- 4 clear tubes that can hold at least 50mL of liquid (50mL falcon tubes are good).
- 2 clear tubes per student that can hold at least 3mL of liquid (5mL culture tubes are good).These are the spit dilution tubes.
- 3 clear tubes per student that can hold at least 15mL of liquid (15mL falcon tubes are good). These are the reaction tubes.
- 1 tube per 2 students that can hold 1.5ml of liquid (Eppendorf tubes are good). These are the vinegar tubes.
- 1 small weigh boat for each student. This will be the spit containers.
- Holders for all of these tubes - ideally enough so that each student pair has a holder for 5 tubes(broken down Styrofoam 15mL falcon tube holders are good)
- 1 500mL graduated cylinder
- 1 piece of gum per student, so that it is easier for them to make a lot of spit.
- Enough Latex gloves for the students
The first set of experiments are performed by the teachers as demonstrations with the aid of student volunteers.
The second set of experiments is performed by pairs of students.
Classroom, laboratory for student experiment part
90 minutes. Either a solid block or two separate 45 minute sessions. If splitting the lesson in two perform the intro and demos 1-4 on the first day and the student experiment on the second day.
The lesson is designed around two sets of experiments. The first set demonstrates that amylase is a digestive enzyme that degrades starch into sugar, can do so repeatedly and, like many enzymes, is sensitive to acid. The second set of experiments demonstrates the variability of amylase activity in different students' saliva.
Students should understand....
- that enzymes are proteins that are encoded by genes
- basic concepts of digestion: that you need to break down large building blocks (starch, proteins, fats) into smaller ones (sugar, amino acids, fatty acids)
- that evolution is a force that allows environmental pressures to affect our genes
The first goal is to reinforce the connection between a macroscopic biological process such as the digestion of starchy foods and the specific molecular actor that carries it out, in this case the enzyme amylase.
The second aim is to reinforce the concepts of positive selection and evolution. We will do this by comparing the starch degrading activity of individual’s saliva and tying the differences observed to current research showing that the number of amylase genes in human populations varies with the level of dietary starch.
Digestion: A person needs to break down the large building blocks that make up food (starch, proteins, fats) into smaller ones (sugar, amino acids, fatty acids). This is done so that we can use these small building blocks to make our own proteins and fats, as well as so that we can use sugar for energy.
Enzymes: Enzymes are proteins that can (a) carry out a specific reaction and (b) do so multiple times without getting used up. Amylase is an example of an enzyme. Amylase is found in our saliva and is responsible for starting to break down the starch that we eat. So amylase is an enzyme that carries out the specific reaction of breaking down starch into a simple sugar. Starch is made up of many molecules of glucose (the simple sugar) joined in a chain. When we say that amylase can catalyze the same reaction many times over we mean that it can break the bond between two glucose molecules in starch over and over without loosing its activity. This means that theoretically, if you gave a single amylase protein a lot of time and ideal conditions, the single amylase could break down all the starch that it comes into contact with. A useful analogy is that an enzyme is like a Philips head screwdriver. A Philips head screwdriver can catalyze the reaction of driving a screw into wood many times over without getting used up.
When we say that an enzyme catalyzes a specific reaction we mean that it is designed to do one thing very well, to the point where it can’t do other things. For example, although amylase can break down the sort of bond that joins glucose in starch chains it cannot break apart the bond that joins glucose in cellulose chains, despite the fact that both chains are made up of glucose joined together. Amylase is specialized to recognize and cut only the type of glucose-to-glucose bond found in starch, not the different kind of glucose-to-glucose bond found in cellulose. Another example is that amylase cannot break down proteins, which are chains of amino acids, while proteases, which break down proteins, cannot break down starch. If we go back to the Philips head screwdriver analogy we can say that while the Philips head screwdriver works very well with Philips head screws it doesn't work at all with flat-head screws, which need another type of “enzyme”, a flat-head screwdriver.
The enzyme magnet model kit included in the lesson is designed to help teachers make these points
Enzymes are sensitive to their environment. Examples of what we mean by environment are things such as the temperature an enzyme is in or the acidity of the liquid they are in. If an enzyme is heated too much it will misfold and change its shape. For example, when we cook an egg its white changes from a clear to opaque white. This is because the heat from cooking changes the shape of the proteins (enzymes are proteins) in the egg white which causes them to bunch together which in turn causes them to change color. Cooking meat is another example; raw meat tastes and looks different than cooked meat because the temperature from cooking changes the proteins in the meat. Usually when you change an enzyme’s shape it no longer works.
Acid also changes a protein’s shape. An example of this is what happens to milk when you add lime juice to it. The milk curdles because the acid in the lemon juice changes the shape of the proteins in milk causing them to bunch together. Another example is ceviche. Ceviche is a Spanish dish in which you cook thin slices of meat with acid, not with heat.
Because acid changes the shapes of proteins, which in turns damages the proteins, we use vinegar to stop the action of amylase in our experiment. Because acid changes the shape of proteins you also see that the solution of amylase with vinegar is cloudier than the solution of amylase without vinegar in it. Importantly vinegar does not change the color of the Iodine indicator used in our experiments.
How the indicator works: when the iodine atoms in the indicator are diluted in a water solution they are arranged in random order in the liquid. When they are randomly distributed they have a light yellow color. However, when starch -which is a long chain of glucoses- is added to the water solution it grabs onto iodine and organizes it in a non-random arrangement. Iodine in this non-random arrangement imposed by the starch chain is blue. When starch gets broken down into glucose it can no longer arrange iodine non-randomly, so the solution turns yellowish clear again.
Amylase and Evolution: The theory of evolution states that if an environmental pressure exists that favors one genetic outcome over another, over time long periods of time the favored genetic outcome will be selected for. In humans this has been very hard to show, and the paper included in this lesson is one of the only known examples in which evolution seems to have acted on a human trait.
The trait in question is the number of Amylase genes in a person’s DNA. In some cases the number of genes a person has in their DNA can be very different from person to person. Amylase is one of these genes. For example some people have as little as 2 copies of the amylase gene in their DNA while others have as many as 10 copies of the gene in their DNA.
The first thing that the scientists that wrote the paper show is that if a person has more genes that encode the amylase protein in their DNA that person will have more amylase in their spit. This means that a person who has only 2 copies of the amylase gene in their DNA is likely to have less amylase protein in their saliva than a person that has 4 copies of the amylase gene in their DNA.
The question is: Has this genetic trait (the number of amylase genes in your DNA) been selected for? In this case the environmental pressure that selects a genetic outcome would be how much starch a human population normally eats. For example, if a population eats a lot of starch it would be beneficial for the people in that population to have a lot of copies of the amylase gene in their DNA. This way these people would have a lot of amylase protein in their spit with which to break down the starch in their food. On the other hand, people with very little starch in their diet would not need to have a lot of copies of amylase genes in their DNA because since they don’t eat a lot of starch they don’t need a lot of amylase protein in their spit to degrade it.
To answer this question the scientists who wrote the article included in this lesson compared the number of amylase genes in human populations that are historically “high” starch consumers and populations are that historically “low” starch consumers. The high starch populations were two agricultural populations (one European, one Japanese) as well as Hadza hunter-gatherers from Tanzania who rely extensively on starch-rich roots and tubers. The low-starch populations included Biaka and Mbuti rainforest hunter-gatherers from the Central African Republic and Congo, as well as Datog pastoralists from Tanzania and the Yakut, a pastoralist, fishing society from Siberia.
The scientists found that people that had traditionally high starch diets had more copies of the amylase gene in their DNA than people with traditionally low starch diets. They suggest that this is evidence of a selective pressure (diet) leading to a genetic outcome (number of amylase genes in DNA).
Before the lesson:
- Print magnetic enzyme and substrate pieces (see attachment) on magnetic printing paper and cut out the individual pieces. Laminate the pieces to make them last longer.
- Print out the attached starch consumption map in color. Have enough for each pair of students.
- For demo #2: Pour 250mL of water into a 600mL glass beaker. Label as water. Make 250mL of 2% dextrose solution (25mL of 20% dextrose solution + 225mL of water) and pour into another 600mL glass beaker. Label as sugar. Make 250mL of 0.0125% Starch solution (25mL of 0.125% Starch stock solution + 225mL of water) and pour into another 600mL glass beaker. Label as starch.
- For Demos # 3 and #4: Make 1L of 0.0125% of starch solution in water (1:10 dilution of 0.125% starch stock solution). Add 200uL of iodine indicator, and stir. Split this solution equally into 4 x 600mL glass beakers, so each beaker gets 250mL. These are the solutions to which you will add amylase or PBS in demo #3, and vinegar and Amylase or PBS in Demo #4.
- Add 1g of amylase powder per tube to two 50mL falcon tubes and label as amylase. Add 50mL per tube of PBS to two 50mL falcon tubes and label as PBS. Add 50mL per tube of Vinegar to two other 50mL falcon tubes and label as Vinegar.
- For Demo #5 (student experiment): Make 500mL of 0.0125% of starch solution in water (1:10 dilution of 0.125% starch stock solution). Add 200uL of iodine indicator, and stir. Aliquot 5mL of this solution into 3 reaction tubes per student pair (I.e. if you have 10 student pairs you will need 30 tubes. We recommend that you make extra tubes in case students make a mistake during the lesson). One of the three tubes will be the control. Add 500uL of PBS and 500uL of vinegar to the control tubes on top of the 5mL of blue starch solution. The other 2 tubes are the reaction tubes for the students. Each student pair should have a control tube, and two reaction tubes. Aliquot 1.5mL of vinegar into tubes. Each pair gets one of these.
Lesson Implementation / Outline
Introduce digestion, which starts in our mouth.
DEMO #1: Each student takes a small cracker, and places it in their mouth, chews it 20 times and lets the mush sit on their tongue.
THINK-PAIR-SHARE: After the students perform the demo pair the students and ask one half of the pairs to think about one question and the other half about the other question. Questions:
-What happened to the cracker and why?
-Did the cracker become sweeter as time went on, why or why not?
Once some of the pairs have given their answers use them as jump off points to introduce the following concepts:
-Enzymes break down food
-Crackers are made out of starch, which we cannot use for energy. When we put them in our mouths enzymes in saliva break down the starch into sugar, which we can use for energy.
CALL OUT: What is meat made out of? (proteins) What is it broken down to? (amino acids) What do we use amino acids for? (To make our own proteins). (If there is enough time repeat questions but with fats. Made out of fatty acids, glycerol backbone and fatty acids, broken down for energy and to make our own, different fats).
First part of the lesson: Amylase is an enzyme that breaks starch down into sugar. Explain what an enzyme is and how it works. Introduce the tools we will use in the experiments.
Go back to Cracker and introduce the idea that the enzyme that broke starch down into sugar is called amylase.
WHITEBOARD DEMO: What do digestive enzymes do? They break down larger molecules like starch, which we cannot use, into smaller molecules like sugar, which we can't use.
Use Enzyme substrate magnets to make three points:
-An enzyme breaks down larger molecules into smaller molecules like sugars.
-An enzyme is specific for its substrate: the enzyme that breaks down starch won't be able to break proteins down.
-An enzyme is reusable, it doesn't get used up in the reaction, so it can perform it over and over again.
DEMO #2: Introduce the iodine indicator.
Show that if a student stirs 100uL of Iodine stock solution into 250mL of water the solution is clear to very light yellow.
Show that if a student stirs 100uL of Iodine stock solution into 250mL of sugar solution the solution is to very light yellow.
When a student stirs 100uL of Iodine stock solution into 250uL of 0.0125% Starch solution the solution turns blue.
DEMO # 3: Introduce amylase reaction and show that the enzyme can be used more than once.
Ask a student to add 50mL of PBS to a 250mL starch iodine solutions. This solution should remain purple.
Now ask the student to stir in 50mL of amylase solution (1g amylase in 50mL PBS. Add the PBS right before the demo, so that the amylase is as fresh as possible) into one of the 250mL starch-iodine solutions. This solution should become clear in about 1min.
To reinforce how enzymes work and clear up commen misconceptions, ask students....
a.) What would you expect to see if more iodine solution was added? (no change, because the starch needed to react with the iodine has already been broken down by the amylase)
b) What would you expect to see if addtional starch solution was added? (blue color should return since the amylase has not yet broken it down into sugars. That blue color should eventually disappear again as the amylase breaks down the newly added starch)
c) What would have happened in the original reaction, if we had a most more concentrated starch solution? (the solution would have taken longer to clear as the amylase has more substrate to break down)
DEMO # 4: Show that adding vinegar to the reaction does not allow it to happen.
Intro this part of the demo by using the magnet set to show that a protein whose shape has been changed can no longer catalyse its reaction. Useful examples of proteins being changed are fried eggs (the heat makes the whites turn white) or ceviche, where thin slices of meat are cooked by acid.
Ask a student volunteer to stir in 50mL of vinegar into each one of the remaining 250mL starch-iodine solutions (there shoud be two). Both solutions should remain blue.
Ask the student to add 50mL of amylase solution (1g amylase in 50mL PBS, add the PBS right before the demo, so that the amylase is as fresh as possible) to one of the beakers with vinegar and 50mL of PBS to the other. The beaker with the amylase will remain blue, but cloudy (due to the vinegar changing the shape of the amylase protein), while the beaker with the PBS will also be blue, but less cloudy. Compare these beakers to the ones from demo #3 in which amylase cleared the blue solution.
Explain that acid changes the enzyme's shape which prevents it from breaking starch down. Use magnetic pieces to explain this concept.
Second part of the lesson: Introduces the idea that our environment can affect our genetic makeup over evolutionary time and that differences in the amount of starch in different populations' diets has led to variability in how much amylase different human populations have.
STUDENT INVESTIGATION: Variation in student's amylase activity.
Students will work in pairs. Each pair of students will be given:
- The protocol sheet and experiment key
- 1 marker to label reaction and saliva dilution tubes
- 3 x 15 ml falcon tubes filled with 5 ml of starch/iodine solution (0.0125% cornstarch, 0.02% potassium iodide, 0.01% iodine).
- 2 x 5 ml test tubes filled with 1 ml of PBS. One of the 15mL tubes will be marked 'Control' and will have had 0.5mL of vinegar, and 0.5mL of PBS added to it beforehand. Each student will be instructed to mark one of each type of the other two tubes with their name. These tubes will be used to carry out the amylase reactions and saliva dilutions, respectively.
- 2 weigh boats, one for each student to collect saliva in for their amylase reaction
- 1 laboratory timer or stopwatch to time the amylase reactions. The reactions will be carried out for 1 minute.
- 1 x 1.5ml tube filled with 1.5 mL of 16% Acetic Acid (it will be labelled 'Vinegar'). Students will use the vinegar to stop their reactions after 1 minute.
The investigation should begin with a demonstration on how to use a mirco-pippette if students are not familiar using them.
Students follow the procedure on the student hand-out:
- Each student gets a piece of gum and puts on gloves.
- After students label their tubes properly, each student will be ask to spit a few times into a weigh boat until they produce 0.5 ml of saliva (for the weigh boats we're using, this means about 1/3-1/2 full).
- Students will pipette 0.5 ml of their saliva slowly and carefully into the 5 ml PBS test tubes that are labelled with their name. Students should mix their saliva in with the buffer by pipetting up and down a few times.
- Then, students should pipette 0.5 ml of their diluted saliva into the 15 ml starch iodine tube marked with their name to start the amylase reaction. Only one member of each pair will be able to do the amylase reaction at a time.
- The other member of the pair should act as the timer. The timer's job is to start the 1 minute count down at the same time as their partner starts the amylase reaction. They should also give their partner a 30 second warning, which allows their partner to prepare for stopping the amylase reaction.
- To prepare for stopping the reaction, students should unscrew the reaction tube top, being careful not to spill any of the reaction, then take 0.5 ml of vinegar into their pipette and be ready to put the 0.5 ml of vinegar into the reaction tube when the timer stays stop.
- After one student in each pair finishes their reaction, partners switch roles and the second member of each pair carries out their reaction while the first acts as the timer.
- After the pair has carried out their 2 reactions, one person should add 0.5 ml of vinegar into the control tube.
- When all students have finished the exercise we will ask them to compare the color of their amylase reactions with the control and compare their individual reactions.
Ask students to talk about their comparisons in pairs and then ask students to report out what they concluded. Then, combine tubes from multiple groups on one rack. Ask students to draw conclusions about the relative amount of amylase in each tube, and if there is time, order the tubes from highest to lowest amount of amylase.
SHOUT OUT: Ask students where we get most of the carbs in our diets.
Point out that most of these are derived either from grains or from fruits and that grains have mostly starch in them while fruits have mostly sugars in them. Point out that there are still regional differences in where we get carbs: In the southern US people eat more grits, in South America they eat more Quinoa.
Ask students what people did 100,000-10,000 years ago to get their carbs, when there were no supermarkets.
They either farmed grains, hunted, or gathered fruits. All of which imply very different amounts of starch.
MAP ACTIVITY : Pass out map of world grain consumption in 1995. Ask students about what the map shows; what are some countries that have a high/low starch comsumption? Why might this be? Explain that some differences are due to modern industrial practices but that some of them reflect historical tendencies (for example, historically there was more hunting and gathering in central africa, while in eurasia, there has been more farming. This is due to soil conditions, climate, etc.)
CALL OUT: Ask students two questions:
-If there is a lot of starch in your diet is it beneficial to have a lot of amylase? (Yes/No) Why?
-If there is not a lot of starch in your diet is it beneficial to have a lot of amylase? (Yes/No) Why?
Use this as a start off point to introduce positive selection:
SLIDESHOW: Use Slides showing how a person living tens of thousands of years ago who produces more amylase in a high starch environment will more likely be healthy and produce more offspring than somebody who has less amylase. Whereas there is no such benefit in a low starch environment.
This part of the lesson introduces the concept of positive selection of beneficial genetic mutations.
Introduce the Scientific paper attached and show that scientists performed several experiments to confirm that people with different levels of starch in their diet have different copy numbers of amylase in their DNA.
Discuss the following questions with your students:
- What does the amount of amylase in your saliva tell you?
- Can you change the amount of amylase in your saliva through your diet?
- Do students with a high amount of amylase in the class have an evolutinary advantage over other students with a lower amount?
Once the conclusions are reached, and if there is time, you can ask students what are the possible reasons why some people's saliva reacted more than other peoples saliva other than the different copy number of amylase? It might be useful to have students list the things we are controlling for in our experiment. Then ask students what are somethings we are not controlling in this experiment? Have students think-pair and share out their ideas. The ideas will be listed on the board. We will explain to students that this sort of brainstorming is what scientists do when they are designing experiments. In order to understand the effect that many types of variation have on an experiment, scientists design controls to see the effect of individual of variables. Next, we will ask the students how they propose to test some of the potential variables in this experiment.
Extensions and Reflections
Note: It is highly advised that teachers test the student experiment themselves before having students carry it out. If you are having trouble getting the experiment to work as expected, please see the Troubleshooting Student Experiment attachment for advice.