Enzyme function lab, lactase Fall 2020

Objectives:
1. Define the terms enzyme and substrate.
2. Explain the result of increasing the enzyme concentration in a reaction.
3. Explain the result of increasing the substrate concentration in a reaction.
4. Discuss the effect of pH on the activity of lactase.
5. Discuss the effect of temperature on the activity of lactase.

Introduction and Background:

Enzymes are protein molecules, which act to catalyze the chemical reactions in living things.
These chemical reactions make up the metabolism of the organism. There are two basic types of
reactions that occur in living organisms: catabolic reactions and anabolic reactions. Catabolic
reactions break larger molecules down into smaller molecules; anabolic reactions build larger
molecules from smaller molecules.
Lactase is a digestive enzyme found in the small intestine. This enzyme breaks down the
lactose sugar into its two component sugars, glucose and galactose.

Figure 1:

Lactose is the principle sugar found in milk and is found in all dairy products. People
who are lactose intolerant either have a primary or a secondary lactase deficiency. The primary
deficiency develops over time (mostly after the age of 2) whereby the body naturally produces
less lactase. Secondary deficiency is caused by some sort of damage to the small intestinal cells
that produce lactase (examples include Crohn’s disease, AIDS, surgical removal of the small
intestine, celiac disease and inflammatory bowel disease.) Lactose intolerance can run in
families as researchers have found that there is a genetic link.
Lactose intolerant people cannot digest lactose and suffer various types of intestinal
distress. People who are lactose intolerant commonly take a dietary supplement that contains
lactase. Lactase is sold under the commercial names of Lacteeze, Dairy Ease or Lactaid.

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Enzyme function lab, lactase Fall 2020

For this lab you will be doing simulation of the breakdown of lactose by the enzyme lactase.
The web site you will need is below.
https://sites.google.com/site/biologydarkow/lactase-enzyme-simulation

Start at “home”, read background, you can skip “explore model” then use the simulate tab to run
the experiments.
Initial questions:
1. The dependent variable for all of the experiments is the same. What is it and what are the
units of measurement?
Answer: Glucose concentration (mg/dL)
2. List the three independent variables you can test with this simulation.
Answer: Initial lactose, temperature, and pH
3. Write a hypothesis and a prediction (if/then format) for the experiment involving temperature.
Answer: if changing temperature change the rate of glucose production, then temperature is a
dependent variable of the glucose production.
Run the simulation as follows
Part 1: Temperature
Lactose 500 mg/dL
pH 7.0
Lactase 5mM
Do the following temperatures: 0, 25, 37, 42, 60 and 90 degrees Celsius.
After you change each temperature, hit run but do not hit reset, this will cause all the lines to
show on the same graph.

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Enzyme function lab, lactase Fall 2020

4. Which temperature(s) was/were the worst over the entire 20 minutes of the experiment?
0, 90
5. At the 5 minute time point which temperature(s) was/were best?
37, 42
6. Can you explain why three of the temperatures give the same data at 18 minutes? What has
happened in the experiment to get this result?
7. How does temperature affect enzyme activity?
The rate of an enzyme-catalyzed reaction increases as the temperature increases. However, at
high temperatures the rate decreases again because the enzyme becomes denatured and can no
longer function. As the temperature increases so does the rate of enzyme activity.
Part 2: substrate concentration
Temperature 25 degrees Celsius
pH 7.0
Lactase 5 mM
Do the following lactose concentrations, Lactose: 0, 50, 100, 200, 300. 400, 500 and 750 mg/dL
After you change the concentration, hit run but do not hit reset.

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Enzyme function lab, lactase Fall 2020

8. Each of the reactions forms a similar curve called a plateau. Why does this happen?
At low substrate concentration the reaction rate increases sharply with increasing substrate
concentration because their abundant free enzyme available (E) to bind added substrate. At high
substrate concentration, the reaction rate reaches a plateau as the enzyme active sites become
saturated with substrate (ES complex), and no free enzyme to bind the added substrate.
Part 3: pH
Lactose: 500 mg/dL
Temperature 25 degrees Celsius
Lactase 5 mM
Use the following pH values: 2, 4, 6, 8, 10, 12

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Enzyme function lab, lactase Fall 2020

After you change the pH, hit run but do not hit reset.

9. Which pH values resulted in the most enzyme activity as measured by glucose production?
8
10. Which ones resulted in the least. Why/How does pH affect enzyme activity?
2, 12
Extremely high or low pH values generally result in complete loss of activity for most enzymes.
Part 4: temperature with multiple trials for statistical analysis
Use the following link to get to the variable sample size experiment. The link is a little glitchy
so you can also find it under then enzymes tab on the web site you are currently on it is labeled
“enzymes with variable sample sizes”
https://sites.google.com/site/biologydarkow/lactase-with-variable-sample-sizes
Set Lactose to 100 mM
Lactase 5 mM
Sample size to 7.
Set pH to 7

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Enzyme function lab, lactase Fall 2020

You will do the experiment twice. For the first set of data, set the temperature to 25 degrees
Celsius. Select Run

After the 25 degree Celsius run, click on “tables of raw data” on the bottom right. Look at the 5
minute time point (5) and record the data for each sample in an excel file. You should have 7
data points for temperature 25.
Repeat the experiment keeping everything the same except the temperature, set it to 42 degree
Celsius. Select Run

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Enzyme function lab, lactase Fall 2020

After the 42 degree Celsius run, click on “tables of raw data” on the bottom right. Look at the 5
minute time point (5) and record the data for each sample in an excel file. You should have 7
data points for temperature 42.
Find the mean, standard deviation, standard error and 95% confidence intervals of the mean just
as you did in the statistical analysis lab for your data. Generate a table or a graph, using the
same format as in the statistical analysis lab. Discuss whether you support or reject your initial
hypothesis and what is the statistical basis for your decision.

Homework: the answers to all of the questions 1-10, the properly labeled graph or table
and your discussion of your results need to be turned in in a word for pdf format with your
last name and First initial in the file name. Make a new document, do not turn in the lab
itself.

Homework: the answers to all of the questions 1-10 embedded in the lab, the properly labeled
graph or table and your discussion of whether you supported or rejected your initial hypothesis
and what is the statistical basis for your decision need to be turned in in a word for pdf format
and uploaded to Canvas.. The instructions for making the graph or table were in your second
lab on statistical analysis and the excel file that you can use for doing the statistics is also posted
to Canvas. Make a new document, do not turn in the lab itself. I have posted the questions
below to make making a new document easier.

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Enzyme function lab, lactase Fall 2020

1. The dependent variable for all of the experiments is the same. What is it and what are the
units of measurement?
2. List the three independent variables you are testing with this simulation.
3. Write a hypothesis and a prediction (if/then format) for the experiment involving temperature.
4. Which temperature(s) was/were the worst over the entire 20 minutes of the experiment?
5. At the 5 minute time point which temperature(s) was/were best?
6. Can you explain why three of the temperatures give the same data at 18 minutes? What has
happened in the experiment to get this result?
7. How does temperature affect enzyme activity?
8. Each of the reactions forms a similar curve called a plateau. Why does this happen?
9. Which pH values resulted in the most enzyme activity as measured by glucose production?
10. Which ones resulted in the least. Why/How does pH affect enzyme activity?