Lab 4: Enzymes

ANSWER KEY
Pre-Lab Questions
1. How could you test to see if an enzyme was completely saturated during an
experiment? (2 pts)
To test this, youd simply see if the reaction was still taking place. If the enzyme was
completely depleted, the reaction could no longer occur.
2. List three conditions that would alter the activity of an enzyme. Be specific with your
explanation. (2 pts)
Temperature, salinity, and acidity are three conditions that affect enzyme activity.
Specific examples include:
Adding a salt packet to an enzyme would alter its activity--salt ions are often necessary
for optimal enzyme activity, so too much or too little salt will slow down the rate.
Enzyme activity increases with increasing temperature, until it becomes hot enough to
denature the protein (enzymes are proteins), then the activity would cease.
Enzymes have an optimum pH where they function the best. Higher or lower pH than
the optimum slows the rate of activity.
3. Take a look around your house and identify household products that work by means
of an enzyme. Name the products, and indicate how you know they work with an
enzyme. (2 pts)
Some examples include: contact lens cleaner (protease), dish soap (amylase), cheese
(rennin), meat (papain), and starch products (amylase).
Experiment 1: Enzymes in Food
Post-Lab Questions

(Table - 2 pts)
1. What were your controls for this experiment? What did they demonstrate? Why was
saliva included in this experiment? (2 pts)
This experiment is looking for the presence of the enzyme amylase. A positive control
would indicate the presence of amylase and so should be something that is KNOWN to
contain amylase. A negative control would indicate no amylase, so should be a
substance KNOWN to not contain amylase. As written, the positive control would be the
ginger root because it is known to contain amylase. The presence of amylase is
indirectly tested for by testing for the presence of starch with an IKI solution. Amylase
breaks down starch into its monomers of the simple sugar glucose, so if amylase is
present, there would be no starch because it would all be converted to sugars. If
amylase is present on the paper towel it would break down the starch. IKI is yellow, but
turns blue/black in the presence of starch. Once tested with IKI, a yellow color would
result indicating no starch.
The negative control will vary by student, but should be a substance known to NOT
contain amylase. One example of a negative control in this experiment is water
because there is no amylase in water. When you add IKI to water, it does not change
color.
Saliva is included in this experiment because saliva contains amylase. Therefore, the
amylase should break down the starch in the starch solution, causing the IKI to turn
yellow/brown upon exposure (indicating no starch left after the amylase has broken it
down to glucose.) This could also be considered a positive control because we know
amylase is present.
2. What is the function of amylase? What does amylase do to starch? (2 pts)
Amylase is an enzyme that breaks down starch into simple sugars. It is used for
digestive purposes in humans.
3. Which of the foods that you tested contained amylase? Which did not? What
experimental evidence supports your claim? (2 pts)
Answers will vary by the food products selected. If the IKI was yellow, that meant no
starch was present and so amylase has broken it down to sugars. If the IKI turned
blue/black, then starch is present, so amylase is not.
4. Saliva does not contain amylase until babies are two months old. How could this
affect an infants digestive requirements? (2 pts)
Amylase is required to break down starch in foods like cereal. Babies cant digest cereal
or other starch containing foods until their bodies start producing amylase.

5. There is another digestive enzyme (other than salivary amylase) that is secreted by
the salivary glands. Research to determine what this enzyme is called and identify it.
What substrate does it act on? Where in the body does it become activated, and why?
(2 pts)
The salivary glands offer many different digestive enzymes. One example is lysozyme.
Also known as muramidase, this enzyme provides a general antiseptic to kill some of
the bacteria which enters the mouth with food. Specifically, it has been found to provide
a defense against Gram-positive bacteria such as Bacillus and Streptococcus. It is also
found in other body fluids such as tears or milk, and is considered to be part of the
innate immune system. NOTE: the question asked for salivary enzymes.
Substance Resulting Color Presence of Starch?
6. Digestive enzymes in the gut include proteases, which digest proteins. Why dont
these enzymes digest the stomach and small intestine, which are partially composed of
protein? (2 pts)
The mucosal lining of the digestive tract contains protease inhibitors, preventing the
enzymes from digesting the stomach intestines. Furthermore, the coiled structure of the
proteins making up the organs hide the cleavage site. Only when proteins are
denatured (cooked or in high pH) do their cleavage sites reveal themselves so the
enzyme can take effect.
Experiment 2: Effect of Temperature on Enzyme Activity
Post-Lab Questions

(Table - 2 pts)
1. What reaction is being catalyzed in this experiment? (2 pts)
2 H2O2 --> 2 H2O + O2
2. What is the enzyme in this experiment? What is the substrate? (2 pts)

Catalase is the enzyme, found in the yeast. (Yeast is a living organism that produces the
enzyme catalase. Enzymes are proteins, so connecting with the lecture topic on gene
expression--a segment of DNA in the yeast contains the base pairs to make an mRNA
with the codons that would indicate the correct amino acids to be placed in the right
order to make the specific protein, catalase.)
The substrate it binds with is hydrogen peroxide.
3. What is the independent variable in this experiment? What is the dependent variable?
(2 pts)
The independent variable is the different temperature. The dependent variable is
enzyme activity which is measured indirectly by the balloon circumference which is
dictated by the amount of gas (oxygen) produced by the yeast.
4. How does the temperature affect enzyme function? Use evidence from your data to
support your answer. (2 pts)
The enzyme function increases as temperature increases. The yeast exposed to the
boiling water bath produced the most gas while the yeast exposed to the ice
bath/refrigerator produced the least amount of gas.
Tube Temperature (C) Balloon
5. Draw a graph of balloon diameter vs. temperature. What is the correlation? (2 pts)
The graph will show the correlation that as temperature increases, balloon
circumference increases. This reaction doesnt show it, but most enzymes have a
temperature-activity curve that looks like a parabola - activity increases up to a certain
temperature, but at higher temperatures the enzyme begins to break down (denature)
and activity plateaus and then decreases.

6. Is there a negative control in this experiment? If yes, identify the control. If no,
suggest how you could revise the experiment to include a negative control. (2 pts)

There is no negative control in this experiment. To include a negative control for this
experiment an additional test tube with hydrogen peroxide, but no yeast (enzyme) could
be included. If no yeast (enzyme) is present, then no reaction should occur and the
balloon should not inflate.
7. In general, how would an increase in substrate alter enzyme activity? Draw a graph
to illustrate this relationship. (2 pts)
Generally speaking, an increase in substrate alters enzyme activity similar to the way an
increase in temperature alters enzyme activity. An increase in substrate will increase
enzyme activity until a saturation point has been met; at which point, enzyme activity will
plateau and eventually decrease. The graph for this would look similar to the graph
provided in Question 5.
8. Design an experiment to determine the optimal temperature for enzyme function,
complete with controls. Where would you find the enzymes for this experiment? What
substrate would you use? (4 pts)
Answers will vary, but the experiment should include all the necessary steps to isolate
one variable and include a control(s). You should have indicated what the independent,
dependent, and controlled variables, and what you would use as a control.
Example: One way would be to use pineapples to produce the enzyme bromelain. The

substrate that could be used is the protein in meat that gets broken down by bromelain,
which causes the meat to be more tender. A negative control you could use would be a
piece of meat that was left untreated. All the meats would be identical cuts and source.