General Biochemistry 2 Lab Manual 1
EXPERIMENT 2 – HYDROLYSIS OF STARCH BY
SALIVARY AMYLASE
OBJECTIVES AND BACKGROUND
Amylase, an enzyme present in saliva, catalyzes the hydrolysis of the glycosidic linkages
in starch. The effect of enzyme concentration, substrate concentration, temperature, pH, and
heavy metal cations on the activity of this enzyme will be investigated.
Metabolism
The chemical reactions that go on in living organisms, and which are essential for growth,
reproduction, movement, and all other vital functions, are individually and collectively referred
to as metabolism. Many thousands of such metabolic processes are occurring constantly in even
the simplest forms of life.
Enzymes are proteins that act as catalysts for metabolic reactions. They increase the rate of
the reaction, but do not influence the kind or amount of products formed. In general, each
metabolic reaction has to be catalyzed in the living organism by its own special enzyme.
The existence of enzymes in biological materials can be demonstrated by the effects they
bring about. In this experiment, digestive enzymes capable of breaking down such
polysaccharides as starch will be studied.
Food materials must be broken down by digestion before they can be absorbed and utilized
by the body. Consequently, if an organism has no enzyme capable of attacking a particular type
of carbohydrate, protein, or fat, that particular substance will have no food value for the
organism. Carbohydrates and fats can be broken down by strong acids or bases. However,
digestive enzymes can accomplish the same breakdown under conditions compatible with life,
that is, at moderate temperatures and physiological pH ranges.
Starch
The nutritional reservoir in plants is starch, which is actually a mixture of two
polysaccharides. Amylose, the unbranched type of starch, consists of glucose residues in α-l,4
linkage.
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Amylopectin, the branched form, has about one α-l,6 1inkage per thirty α-l,4 linkages.
Amylases
Hydrolysis of starch is the process of digestion. Enzymes called amylases catalyze only the
hydrolysis of α-1,4 glycosidic linkages in the amylose and amylopectin components of starch.
The hydrolysis does not proceed directly from polysaccharides to monomer units; rather, partial
hydrolysis products of intermediate size are obtained. These products are maltose and dextrin.
Maltose consists of two glucose units in α-1,4 linkage; dextrin is made up of several glucose
units joined by α-1,6 linkage in addition to α-1,4 linkages.
Amylase enzymes are present in saliva, so the digestion of carbohydrates begins in the
mouth. Digestion continues briefly in the stomach until the pH drops too low, and then is
completed in the intestines by the attack of another amylase.
The chemical conversion of starch to dextrins and maltose is a key part of the brewing step
in the production of beer. The amylase enzymes are added to the grain mixture as brewer's malt.
Approximately one third of the calories in regular beer are contributed by carbohydrate
remaining in the beer after fermentation. The carbohydrates remain because amylase is unable to
break down the α-l,6 glycosidic linkages in the amylopectin portion of the starch. To make "light
beer," less starch is used in the initial brewing step and a second enzyme, amyloglucosidase,
which can cleave the α-1,6 glycosidic linkages in amylopectin, is added in the fermentation step.
The light beer will have a lower carbohydrate composition because of the added
amyloglucosidase, and a lower alcohol content because of the reduced starch in the initial
fermentation mixture.
Study of Enzyme Activity
To follow the action of an enzyme, it is necessary to test for the appearance of a product or
the disappearance of a reactant over a measured period of time. In the reactions of amylase with
starch, you will test for the disappearance of starch by reacting samples of the reaction mixtures
General Biochemistry 2 Lab Manual 3
with iodine. Initially, an unhydrolyzed starch solution reacts with iodine to give a blue-black
color. The amylase catalyzes the hydrolysis of the α-1,4 glycosidic linkages, forming maltose
and dextrin. After the starch is hydrolyzed, the blue-black color produced with iodine is no
longer observed, and only the red or gold color of the iodine solution is seen. The faster the
amylase hydrolyzes the starch, the sooner the failure of the blue-black color to appear is
observed. If the blue-black color continues to appear each time it is tested, it can be concluded
that the enzyme is no longer active and that no hydrolysis of starch has occurred.
Enzyme activity depends on several factors, including enzyme concentration, substrate
concentration, pH, temperature, and presence of heavy metal cations. Your own saliva containing
the amylase enzyme will be used for this experiment, although the levels of amylase vary
considerably from one person to another. Each experiment must be timed. As you proceed with
each experiment, you will check enzyme activity by reacting a few drops of the reaction mixture
with iodine. The time at which the blue-black color of starch does not appear will be noted in
each experiment.
The time required for the hydrolysis of starch will be correlated to the relative enzyme
activity. When enzyme activity is high, the time for the starch to hydrolyze will be very short.
When the enzyme is operating poorly or not at all, the activity is low, and more time will be
required for the starch to hydrolyze. In some cases, the enzyme will be completely inactivated
and the blue-black color of the starch and iodine will persist throughout the entire experiment.
Graphs will be prepared showing the effects of concentration, pH, temperature, and heavy metal
cations on the relative enzyme activity.
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PROCEDURE
A. Effect of Enzyme Concentration
1. Collect approximately 5 mL of saliva in a medium test tube.
2. Place 2 droppers full of 1 % starch solution in each of 5 medium test tubes. Number the
test tubes 1-5.
3. Place the tubes in a 37-40 °C water bath. After 5 minutes, add the following amounts of
saliva to the test tubes as quickly as possible, mixing each solution thoroughly, and then
returning the tubes to the water bath. Do not overheat the bath or you will inactivate the
enzyme.
Test tube Number of drops of saliva
1 1
2 2
3 5
4 10
5 20
4. Prepare a spot plate for testing for the presence of starch in the samples by placing one
drop of iodine reagent in each of five depressions on the spot plate.
5. Two minutes after the addition of saliva, transfer one drop from each test tube (using a
different pipet for each tube) to a separate drop of iodine in the spot plate. Note the
color produced for each. Remember that the complex formed by starch and iodine is an
indigo blue. If the color of the iodine solution remains red or gold after adding the
starch solution, the starch has been completely hydrolyzed.
6. As soon as one of your starch solutions has hydrolyzed, use it to begin preparing the
solutions for Parts B and C below. Continue to test the remaining starch solutions that
have not yet hydrolyzed.
7. Clean the spot plate and then prepare it for the next testing by placing one drop of
iodine reagent in each of five depressions.
8. Repeat the testing at 5 minutes after the addition of saliva, and at 5 minute intervals
thereafter. Continue testing for 20 minutes, or until the blue-black color no longer
appears for each sample.
9. Record the time required for the hydrolysis of starch in each sample.
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B. Benedict's Test for Easily Oxidized Groups
The Benedict's reagent is a mild oxidizing agent consisting of the Cu+2 ion and the poly
atomic citrate ion (C6H6O7-2) in basic solution. The Cu+2 ion forms a complex with citrate; it is
wrapped by and protected within the citrate ion. This protection allows Cu+2 ion to remain
dissolved in the basic solution (otherwise, the extremely insoluble Cu(OH)2 would form). When
an easily oxidized compound is added to Benedict's reagent, and the solution is warmed, Cu+2
ion is reduced to Cu+1 ion. These ions cannot be protected from OH− by citrate, and precipitate as
copper(I) oxide (Cu2O). The Benedict's reagent has a bright blue color caused by the Cu+2 ion,
but Cu2O is a brick-red solid. Visible evidence of a positive Benedict's test is the disappearance
of the blue color of the solution, and the appearance of a brick-red precipitate. Monosaccharides
and disaccharides that contain aldehydes or α-hydroxy ketones will give a positive Benedict's
test, as these groups are easily oxidized to carboxylic acids.
Carbohydrates to be tested: hydrolyzed starch and fresh starch solutions.
1. In each of two medium test tubes place 2 droppers full of Benedict's reagent and 1 mL
of either fresh starch solution or the hydrolyzed starch from Part A.
2. Shake the contents of each tube well, and place both tubes at the same time in an
actively boiling water bath. Heat in the water bath for 3 minutes. On the report sheet,
record any changes in the colors or transparencies of the solutions, and in the formation
and color of precipitates.
C. Barfoed's Test for Monosaccharides
As is true of the Benedict's test, the Barfoed's test depends on the presence of easily
oxidized groups to reduce Cu+2 ion to Cu+1 ion to form the brick-red precipitate of Cu2O.
However, the Barfoed's reagent is slightly acidic rather than basic; this difference allows for
selectivity between the monosaccharides and disaccharides. Monosaccharides will test positive
within two to three minutes. Disaccharides with oxidizable groups require ten minutes or more
before they show the positive result.
Carbohydrates to be tested: hydrolyzed starch and fresh starch solutions.
1. In each of two medium test tubes place 1 dropper full of Barfoed’s reagent and 1 mL of
either fresh starch solution or the hydrolyzed starch from Part A.
2. Shake the contents of each tube well, and place both tubes at the same time into an
actively boiling water bath. Heat in the water bath for 5 minutes (no longer). During this
period, observe the tubes closely and record any change in color or clarity of the
solutions.
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D. Effect of pH
1. Place 2 droppers full of 1 % starch solution in each of 4 medium test tubes. Add the
following to the test tubes:
Test tube
1 5 mL of pH 3 buffer
2 5 mL of pH 5 buffer
3 5 mL of pH 6 buffer
4 5 mL of pH 9 buffer
5 5 mL of pH 11 buffer
2. Place the tubes in a 37-40 °C water bath. After 5 minutes, add 5 drops of saliva to each
of the test tubes as quickly as possible, mixing each solution thoroughly, and then returning
the tubes to the water bath. Do not overheat the bath or you will inactivate the enzyme.
3. Prepare a spot plate for testing for the presence of starch in the samples by placing one
drop of iodine reagent in each of five depressions on the spot plate.
4. Two minutes after the addition of saliva, transfer one drop from each test tube (using a
different pipet for each tube) to a separate drop of iodine in the spot plate. Note the color
produced for each.
5. Clean the spot plate and then prepare it for the next testing by placing one drop of iodine
reagent in each of five depressions.
6. Repeat the testing at 5 minutes after the addition of saliva, and at 5 minute intervals
thereafter. Continue testing for 20 minutes, or until the blue-black color no longer appears
for each sample.
7. Record the time required for the hydrolysis of starch.
8. Plot the relative enzyme activity (time for starch to hydrolyze) versus the pH of the
samples.
E. Inhibition of Enzyme Activity
1. Place 2 droppers full of 1 % starch solution in each of 4 medium test tubes. Add the
following to the test tubes:
General Biochemistry 2 Lab Manual 7
Test tube
1 1 mL of 0.1 M AgNO3 + 4 mL water
2 1 mL of 0.1 M NaCl + 4 mL water
3 5 mL of ethanol
4 5 mL of water
2. Place the tubes in a 37 -40 °C water bath. After 5 minutes, add 5 drops of saliva to each
of the test tubes as quickly as possible, mixing each solution thoroughly, and then returning
the tubes to the water bath. Do not overheat the bath or you will inactivate the enzyme.
3. Prepare a spot plate for testing for the presence of starch in the samples by placing one
drop of iodine reagent in each of four depressions on the spot plate.
4. Five minutes after the addition of saliva, transfer one drop from each test tube (using a
different pipet for each tube) to a separate drop of iodine in the spot plate. Note the color
produced for each.
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Data Sheet: Experiment 2
Name:___________________________________Student ID:_______________________
A. Effect of Enzyme Concentration
Test tube Drops of Saliva Starting time Time Blue Color Time (min.) for
Fails to Appear Starch Hydrolysis
1 1
2 2
3 5
4 10
5 20
Graph: Enzyme Activity (Time for Starch Hydrolysis) vs. Enzyme Concentration (Drops of
Saliva)
Questions:
1. How does the activity of the enzyme change as its concentration increases?
2. What are some reasons for this change in activity?
B. Benedict’s Test for Reducing Sugars
Observations Conclusions
Hydrolyzed
Starch
Fresh Starch
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C. Barfoed’s Test for Monosaccharides
Observations Conclusions
Hydrolyzed
Starch
Fresh Starch
Questions:
1. Are the results of the Benedict’s and Barfoed’s tests what you had expected? Explain your
answer by giving the structures and names of the products of the complete enzymatic hydrolysis
of starch.
D. Effect of pH
Time Blue Color Fails Time (min.) for Starch
pH Starting Time
to Appear Hydrolysis
3
5
7
9
11
Questions:
1. What are the effects of pH on the activity of the enzyme?
2. What are some reasons for effect of pH?
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E. Effect of Inhibitor
Added Reagent Observations after 5 Minutes Conclusions
AgNO3
NaCl
Ethanol
Water
Questions:
1. Which of the compounds added to the reaction tubes inhibited enzyme activity?
2. Explain how the compounds you mentioned above inhibit enzyme activity.
