Download Document

Amylase Enzyme LAB
a) Enzymes
Enzymes, which are a type of protein, are biological catalysts found in all living
things. What do enzymes do? An enzyme’s job is to change molecules. An enzyme may
add atoms to a molecule, remove atoms from a molecule, split a large molecule into two
smaller molecules, or join together smaller molecules to form a larger molecule. But the
important point is that enzymes always change molecules. The term “chemical reaction”
means a change in a molecule, so the best way to define enzymes is to say enzymes are
proteins that carry out chemical reactions.
Enzymes act as catalysts to increase the rates of chemical reactions. Without the
enzymes in our digestive tract for example, it would take us about 50 years to digest a
single meal!
Enzymes work in this way: They begin by binding to the molecule that they are going
to change (called the substrate molecule). Each enzyme has a special crevice, called its
active site, where it binds its substrate molecule. Once the substrate is in the active site,
the enzyme changes it and then releases it. The changed substrate is called the product.
b) Optima of enzymes
The speed at which an enzyme converts substrate to product is called the rate of the
enzyme. For example, if an enzyme converts 40 micrograms of substrate into 40
micrograms of product in one second, we say that the enzyme’s rate is 40 micrograms
product per second.
Biologists have found that the temperature and pH of the enzyme’s environment can
change its rate. For example, the graph below shows the different rates at different pH’s
for a certain enzyme.
Enzyme
Rate
(product made per second)
Name:
Date:
Due Date:
Notice that this enzyme’s fastest rate occurs at pH 8. This is called the “optimum pH” of
the enzyme. Not all enzymes have a pH optimum at 8, but usually an enzyme’s optima
are the same as its natural environment. For example, enzymes that are found in the
stomach (an acidic environment) have optima at acidic pH’s (usually pH 2 or 3).
Why does the enzyme’s rate decrease if it is not at its pH optimum? The answer is
that the enzyme becomes denatured (unfolded). Denaturing an enzyme disrupts its active
site, and therefore the enzyme loses the ability to bind (and change) its substrate.
Enzyme
Denatured enzyme (not functional)
The pH is not the only environmental factor that can change an enzyme’s rate. The
temperature also has an effect. The effect of temperature on a certain enzyme is shown on
the graph below. Notice that there is a “temperature optimum” (a temperature where the
enzyme has its highest rate).
Usually this is the
temperature of the enzyme’s
Enzyme
natural environment. For
rate
example, most enzymes in
(Product
the human body have a
made
temperature optimum at
per second)
37 ºC because that is body
temperature.
Why does the enzyme rate
decrease when its
temperature is above its
optimum? For the same
reason explained for pH: The
enzyme is denatured. In other
words, high temperatures
unfold enzymes.
Why does the enzyme rate decrease when its temperature is below its temperature
optimum? You might think at first that the enzyme is denatured, but that is not correct.
The reason for the rate decrease is that molecules move slower at colder temperatures.
You can think of the enzyme molecule and its substrate molecules going into “slow
motion” at colder temperatures. They are not denatured, they are simply slowed.
2
Name:
Date:
Due Date:
Lab Purpose:
In this laboratory exercise you will observe the action of the enzyme amylase on
starch. Amylase is present in our saliva and in secretions from the pancreas, and begins to
act on the starch in our food while still in the mouth.
Starch is a polymer made from many glucose
molecules connected together (figure at right). It is
used by many organisms as a way to store glucose for
later use.
For organisms to be able to use the energy in
starch the polymer must be broken down into its
simpler glucose units. In animals, this hydrolysis
reaction is catalyzed by the enzyme amylase.
Benedict's solution is a test reagent that reacts positively with simple reducing
sugars like maltose, but will not react with starch. A positive test is observed as the
formation of a brownish-red cuprous oxide precipitate. A weaker positive test will be
yellow to orange.
Materials
Cornstarch solution
Amylase enzyme solution
Vinegar
Benedict’s (in dropper bottle)
Water dispensing bottle
3 transfer pipettes
100 ml beaker
3 test tubes
Test tube rack
Masking tape to label tubes
Hot plate
Timer/stopwatch
Procedures
1. Fill the 100-mL beaker about 3/4 full of water and place on the hot plate
for a boiling water bath. Keep the water JUST AT BOILING.
2. Label 3 test tubes A, B and C. Add 2 mL of amylase enzyme solution into
each test tube.
3. Into tube A, add 2 mL of vinegar. Into tubes B and C, add 2 mL of water.
Stir the tubes to mix.
4. Place tube B into the boiling water bath for 5 minutes. After the five
minutes, remove from the bath, and place back into the test tube rack.
3
Name:
Date:
Due Date:
5. Add 1 mL of the starch solution to each tube and mix. Allow the tubes to
sit for 10 minutes, occasionally mixing.
6. Add 5 drops of Benedict's solution to each tube and stir to mix. Place the
tubes in the hot water bath. The reaction takes several minutes to begin.
OBSERVATIONS:
Tube A: Starch + amylase enzyme solution treated with vinegar (acid)
Was the Benedict’s test positive or negative?
_______________________
What does this
indicate?_____________________________________________
____________________________________________________
____________________________________________________
_____________________________________
Tube B: Starch + amylase enzyme solution and water, treated in a boiling
water bath
Was the test positive or negative? _______________________
What does this
indicate?_____________________________________________
____________________________________________________
____________________________________________________
_____________________________________
Tube C: Starch + amylase enzyme solution
Was the test positive or negative? _______________________
What does this
indicate?_____________________________________________
____________________________________________________
____________________________________________________
_____________________________________
When done, wash all instruments and put them back where you obtained
them. The plastic transfer pipettes can go into the regular trash.
4
Name:
Date:
Due Date:
ANALYSIS QUESTIONS:
1. In this lab, what weak acid denatured the protein?
2. What was the purpose of placing one test tube in a hot water bath?
3. If there were some experimental errors that could affect your test,
describe them here.
4. What can you conclude about the best working conditions for
amylase?
5