© 2005 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

Printed in U.S.A. Vol. 33, No. 2, pp. 112–116, 2005

Laboratory Exercises

Discovering an Accessible Enzyme: Salivary !-Amylase

PRIMA DIGESTIO FIT IN ORE: A DIDACTIC APPROACH FOR HIGH SCHOOL STUDENTS

Received for publication, August 16, 2004, and in revised form, November 28, 2004

Isabella Marini‡
From the Dipartimento di Fisiologia e Biochimica, Sezione di Biochimica, Università di Pisa, Via S. Zeno, 51,
56127 Pisa, Italy; Liceo Scientifico “Ulisse Dini,” via B. Croce, 36, 56125 Pisa, Italy; and Scuola di
Specializzazione per l’Insegnamento Secondario (SSIS), Toscana-Sede Università di Pisa,
via Possenti, 18, 56121 Pisa, Italy

Human salivary !-amylase is used in this experimental approach to introduce biology high school students
to the concept of enzyme activity in a dynamic way. Through a series of five easy, rapid, and inexpensive
laboratory experiments students learn what the activity of an enzyme consists of: first in a qualitative then
in a semi-quantitative way. They also learn how some environmental effectors can influence it. The choice
of a “human body” enzyme and not an anonymous commercial enzyme is a very attractive one for students.
This laboratory approach will be integrated with theoretical knowledge about !-amylase, starch, their
physiological meaning, and biotechnological applications.
Keywords: Salivary !-amylase, starch, enzyme teaching, biochemistry teaching, high school experiments.

In scientific education, it is important to help our stu-
dents have a better understanding of the fast-changing
world we live in. This is particularly true for biology. A
biology teacher today must integrate the classic concepts
with the latest developments. Students have a natural
curiosity for modern biology, and thus teachers may open
their pupils’ minds to the basic biological concepts. Bio-
chemistry is quite the ideal subject for this. In fact, this
“borderline” discipline can be considered as an “espe-
ranto” of life sciences. It is in fact simple to start from an
explanation in biochemical terms and then integrate that
with mutually beneficial fields and with theoretical knowl-
edge or vice versa. In this way, the goal of scientific edu-
cation—i.e. critical thinking through assimilating facts—is
achieved.
Starting with a laboratory approach is ideal for allowing
students to grasp meaningful concepts; because here the
students handle, observe, use instruments, learn tech-
niques, and acquire scientific concepts in a dynamic way
through questions, discussions, elaboration, charts, and
reports.
I began by analyzing the natural difficulties of students in
understanding the enzyme concept. In school texts, all the
descriptions are at a molecular level and often our stu-
dents are not up to the abstract thinking necessary to
understand them. In this way, knowledge is substantially
mnemonic and not rational, only because the concept is
not accessible. Because teaching results are generally
‡ To whom correspondence should be addressed: Diparti-
mento di Fisiologia e Biochimica, Sezione di Biochimica, Via S.
Zeno, 51, 56127 Pisa, Italy. Tel.: 39-050-2213180; Fax: 39-050-
2213170; E-mail: imarini@dfb.unipi.it.
112
successful when the hypothesized causal agent is observ-
able (consistently with Piaget’s theory of concrete opera-
tions preceding formal operations [1]), an essential first
approach would be to introduce the concept while working
at a phenomenological level on something that the student
can easily observe and experiment with.
I started from a Wohlgemuth Method protocol found in a
1971 text of clinical analysis [2] and I calibrated it to my
requirement; then I planned some other correlated exper-
iments to create an organic didactic unit about !-amylase,
with the Latin title: “Prima digestio fit in ore” (Lit. transla-
tion: the first digestion takes place in the mouth).
I chose a simple determination of salivary amylase ac-
tivity, because this enzyme can be extracted directly from
the students’ own bodies. It would be very different for
them if they used an “anonymous” commercial enzyme.
Moreover, this digestive enzyme is already in solution and
does not require homogenization or other treatments and
it is possible to study its activity without any instruments
except the eyes, using the iodine method.
SALIVARY !-AMYLASE
Salivary !-amylase (1,4-!-D-glucan glucano hydrolase;
EC 3.2.1.1) [3], a monomeric calcium-binding glycoprotein
(MW 56,000), is involved in preliminary carbohydrate di-
gestion. It catalyzes the hydrolysis of internal !,1– 4 glyco-
sidic bonds present, yielding a mixture of maltose, glu-
cose, oligosaccharides with varying lengths with an
!-configuration and !-limit dextrins, which constitute
branched oligosaccharides [4, 5]. The !-glicosidic bond is
very stable, having a spontaneous rate of hydrolysis of
!2 " 10#15 s#1 at room temperature. !-Amylase en-
hances this rate so enormously, i.e. 3 s#1, that it can be
This paper is available on line at http://www.bambed.org

considered as belonging to the most-efficient enzymes
known, increasing the rate 1015-fold.
Salivary !-amylase has a short-lived action. In fact, it is
swallowed with chewed food and subsequently inacti-
vated by extremely low gastric pH; amylase in fact has an
optimal pH around 7, and the pH of saliva is generally
between 6.4 and 7.0. !-Amylase is produced by salivary
glands and mainly from exocrine pancreas. In some path-
ological conditions, amylase increases in the blood when
the following occur: obstruction of pancreatic and salivary
ducts, severe renal insufficiency, and when there are acute
inflammation processes of parotid and pancreas. Meas-
uring !-amylase activity in serum, urine, and saliva is a
useful diagnostic tool in evaluating diseases of the pan-
creas and salivary glands.
!-Amylase is also found in a wide variety of microorgan-
isms belonging to Archaea as well as bacteria and has also
some secondary biological functions. It is important in the
oral microbial ecosystem. Perhaps it has a role in the
colonization and metabolism of bacteria there and in den-
tal plaque and caries formation [6].
Starch [7], the most abundant organic compound found in
nature after cellulose, has become a very important biopoly-
mer and is used in many industries as a feedstock material.
In several industrial processes, enzymes are used to trans-
form starch molecules into useful, added-value biochemi-
cals. !-Amylase is used in the sweetener food, textile, and
ethanol industries. Knowledge about !-amylase inactivation
in various conditions can optimize industrial processes and
has also an important economical significance [8, 9].
Plant biochemical mechanisms involving secondary
compounds protect seeds from attacks of herbivores such
as mammals, birds, insects, and microorganisms. One of
these—proteinaceous !-amylase inhibitor—is widely dis-
tributed in seeds of most cereal crops and some grain
legumes. It is assumed to be responsible for biochemical
defense, especially against insects, and has been compre-
hensively studied. Kidney beans, Phaseolus vulgaris, con-
tain an !-amylase inhibitor of insects and mammals, but
not of plants; its name is phaseolamin [10].
MATERIALS AND METHODS
• Phosphate buffer (50 mM) is prepared by dissolving the required
amount of dibasic phosphate in distilled water and titrating to pH
4 (buffer A), 5 (buffer B), 6 (buffer C), 6.5 (buffer D), and 7 (buffer
E) with monobasic solution before making up the final volume.
• Tris/HCl buffer (50 mM) is prepared by dissolving the required
amount of Tris in distilled water and titrating to pH 7 (buffer F),
7.5 (buffer G), 8 (buffer H), and 9 (buffer I) with HCl before making
up the final volume.
• Starch solution: 1 g of soluble starch diluted in 100 ml of 50 mM
phosphate buffer, pH 7.0.
• Iodine solution N/50: 20 g of KI and 12.7 g of iodine and distilled
water to 1 liter. This solution is diluted 1:5 with distilled water.
• Fehling solutions A and B.
• Iodine method: Iodine in aqueous solution with starch forms a
colored complex with high sensitivity and specificity. This iodine-
starch complex is blue-violet, while amylodestrine and maltose,
in the presence of iodine, become light pink or colorless. Starch
is used as indicator in redox reactions in which I2 appears or
disappears; e.g. in the reaction 2 S2O32– $ I2 7 2I# $ S4O62– for
detecting the peroxide number of an olive oil.
• Fehling’s test: An alkaline solution of cupric ions (Cu2$) is re-
duced to cuprous ions (Cu$) forming a yellow-red-colored pre-
113
cipitate of cuprous oxide (Cu2O) when heated in the presence of
reducing sugars.
• pH indicator strips.
• Saliva.
INITIAL STIMULUS: EATING THE SOFT PART OF BREAD
Describe sensations: Test with iodine before and after
chewing.
FIRST EXPERIMENT: THE COLORLESS POINT
Through a series of double dilutions we measure the
amount of saliva able to digest a fixed amount of starch.
When saliva is incubated with starch and the mixture
tested at intervals with iodine, the test color—initially
blue— changes successively to violet, then to pink, and
finally disappears as salivary !-amylase disrupts the
starch molecule and catalyzes hydrolysis of polysacchar-
ides to a mixture of oligosaccharides.
Procedure
The reagents used are starch and iodine solutions and
saliva.
In 11 test tubes we put 1 ml of buffer E and in the first we
add 1 ml of saliva. We do double dilutions passing 1 ml
from one test tube to the next. So we have a series of
progressive saliva dilutions from 1:2 to 1:1024. In the 11th
tube we put 1 ml of buffer E (blank). To all test tubes we
add 1 ml of substrate, stir, and incubate at 37 °C. After 15
min we add two drops of iodine solution to the mixtures
and wait (for a few seconds) for color development. We
have now to identify the tube with the maximum saliva
dilution, which is colorless. In such a tube the lowest
amount of amylase will be present that, in the adopted
assay conditions, is sufficient to completely hydrolyze the
substrate. Such a dilution value, which we call “colorless
number,” is proportional to the amount of amylase present
in the original saliva sample and is taken as the number of
arbitrary units of amylase activity. For example, if we have
the first seven test tubes without color, the colorless num-
ber (i.e. the dilution value of the saliva in the 7th tube, our
arbitrary amylasic units), is 128.
The colorless test tubes could be treated with Fehling’s
test to demonstrate the presence of reducing sugars, as
well as the disappearance of starch.
The comparison of the colorless number of each group
of students introduces the concept of individual differ-
ences and of normal-values-range.
The concept of arbitrary units and the relevant link of the
measurements with the arbitrary choice of the assay con-
ditions are emphasized to the students. For example, we
point out that the colorless number may change by using
different times of incubations or different amounts of
starch. The concept may then be expanded by pointing to
the relevance for the need of a conventional set of assay
conditions in order to compare the analysis of different
saliva samples and to the need for “less arbitrary units” to
compare the activity of different enzymes.
This experiment also allows students to make the ap-
propriate choice for a saliva dilution able to digest starch in
shorter times (few minutes), necessary for the following
experiments.
114
FIG. 1. pH influence on salivary !-amylase activity. For de-
tails, see the procedure of the second experiment.
SECOND EXPERIMENT: pH INFLUENCE
Enzymes require some conditions to work and one of
them is pH. Each enzyme has a pH value at which its rate
is an optimum and on each side of this optimum, its rate is
lower. The influence of pH may involve several different
types of effects because enzymes are proteins with many
ionic groups also present in the active site. Proteins are
stable only within a relatively limited pH range most often
near neutrality.
Procedure
The reagents used are starch and iodine solutions; saliva
that is diluted (generally) 1:10; buffers A, B, C, D, E, F, G,
H, and I; and pH indicator strips.
We test the saliva pH value.
We add to a test tube 300 "l of buffer A and 100 "l of the
starch solution. We test pH value (with pH indicator strips),
add 30 "l of the diluted saliva, and start with a
chronometer.
Then at intervals of 30 s, with a Pasteur pipette, we take
some drops of the mixture and assay it with the iodine
solution. We measure the time necessary for salivary
!-amylase to disrupt the starch molecule (when the color-
less point is reached). We repeat the same procedure with
buffers B, C, D, E, F, G, H, and I.
We transform the experimental data in enzymatic veloc-
ity (relative units: micrograms of hydrolyzed starch in 1
min) and plot as indicated in Fig. 1.
From the plot analysis students generally are able to
hypothesize the different amylase activity in the mouth and
in the stomach. If they have sufficient chemical knowledge
they can connect pH dependence of enzymatic activity to
ionization of amino acids and proteins. Obviously relative
units like micrograms of hydrolyzed starch in 1 min are
different from the “colorless number” arbitrary units of the
first experiment.
THIRD EXPERIMENT: TEMPERATURE EFFECT
The equilibrium constant for any chemical reaction as
well as the rate of the reaction depends strongly on
temperature; and enzyme-catalyzed reactions are no ex-
ception. For enzyme-catalyzed reactions, the rate in-
creases with temperature until a maximal rate is
achieved, but at a temperature above the maximum,
BAMBED, Vol. 33, No. 2, pp. 112–116, 2005
decreased rates are observed because of thermal dena-
turation of the enzyme.
Procedure
The reagents used are starch and iodine solutions; saliva
that is diluted (generally) 1:10; and buffer E.
We use a beaker with a hot plate or a container with ice
to have the desired temperature. We wait until the two test
tubes with enzymatic solution (solution 1) and starch so-
lution (solution 2) reach the desired temperature; at this
point we mix 500 "l of solution 1 with 100 "l of solution 2.
Every 30 s, with a Pasteur pipette, we take one drop of the
mixture and test with iodine solution; we repeat this pro-
cedure until the test solution is colorless and we make a
note of the time taken.
We repeat this procedure at 10, 20, 30, 37, 40, 50, 70,
and 90 °C.
At the end of the experience, we put the incubated
mixtures at 10 and 90 °C at room temperature for 5 or 6
min. Then we test with iodine solution.
We calculate reaction velocity in relative units (micro-
grams of starch transformed in 1 min) and plot experimen-
tal results.
This experiment could also be used to explain thermal
denaturation of proteins and food preserving methods.
FOURTH EXPERIMENT: SUBSTRATE CONCENTRATION EFFECT
For all enzymic processes, the rate of a reaction de-
pends upon the concentration of its substrate, other con-
ditions being constant. In this experiment, we study the
relationship between the initial velocity of the reaction and
the substrate concentration. Velocity increases hyperboli-
cally as the substrate concentration increases toward a
limiting maximal velocity. This indicates that the enzyme
must have a finite number of sites to combine with sub-
strates; when all sites are occupied, no further rate enhance-
ment occurs and the enzyme is saturated with substrate.
In the case of !-amylase, the substrate consists not only
of initial starch but also of its hydrolysis products; this is
why we wait until the colorless point is reached.
Procedure
The reagents used are starch and iodine solutions; saliva
that is diluted (generally) 1:10; and buffer E.
Here we use different starch concentrations with a fixed
amount of enzyme. We choose the lowest starch concen-
tration that is visible in the presence of Iodine.
We add to a test tube 800 "l of buffer E, 80 "l of the
starch solution, and then 30 "l of diluted saliva. We deter-
mine enzymatic activity as in the previous experiments
with the following final starch concentrations: 0.85, 1.25,
1.6, 1.9, 2.25, and 2.6 "g/ml. We calculate reaction veloc-
ity in relative units (micrograms of starch transformed in 1
min) and plot experimental results as in Fig. 2.
The curve obtained is a perfect means for introducing
the concepts of enzyme-substrate complex, saturation,
and the “Lock and Key” analogical model of Fischer [11].
With older students, the plot could be connected with the
kinetic constants, the Michaelis & Menten equation, the
Lineveawer-Burk plot, and their relative meanings.
 115

FIG. 2. Substrate concentration ef-

fect on salivary !-amylase activity.
For details, see the procedure of the
fourth experiment.

FIFTH EXPERIMENT: INHIBITION OF !-AMYLASE

BY PHASEOLAMIN
The rate of an enzyme-catalyzed reaction can be de-
creased by specific inhibitors; compounds that combine
with the enzyme and prevent normal enzyme-substrate
interactions, thereby diminishing the rate of the reaction.
Many drugs are inhibitors of metabolic reactions, and mo-
lecular pharmacology is largely dependent on knowledge
of inhibition of enzymes. Fundamental information about
enzymes themselves is also revealed by enzyme inhibition
studies.
A source of a specific inhibitor of !-amylase is the FIG. 3. Inhibitory effect of phaseolamin on salivary !-amy-
kidney bean, which contains phaseolamin, a proteina- lase. For details, see the procedure of the fifth experiment.
ceous inhibitor of the enzyme.
Obviously in handling human saliva students must wear
Procedure gloves.

The reagents used are starch and iodine solutions; saliva
that is diluted (generally) 1:10; and buffer E.
Dry bean seeds are suspended in tap water for 5– 6 h;
then they are homogenized by being ground in a mortar
and pestle with buffer E (about 500 mg seeds/ml buffer)
and standing for 3 h at 4 °C. This extract is then centri-
fuged at 15,000 " g for 15 min; supernatant is used as a
phaseolamin sample.
Phaseolamin action is tested for !-amylase inhibition by
adding 100 "l of supernatant to 300 "l of buffer E and 30
"l of diluted saliva. After 15 min of incubation, 100 "l of the
starch solution is added to the mixture, which is then
tested with iodine. As a control, the phaseolamin sample is
added to the enzyme in the presence of starch, which
prevents inhibition. The experimental results are reported
in Fig. 3.
By means of this experiment the concepts of enzyme
inhibition and inactivation, and natural defense mecha-
nisms can be successfully introduced.
GENERAL NOTES
These experiments do not use or generate any hazard-
ous reagent; however the weighing of iodine needs atten-
tion and wearing a safety mask is necessary because
iodine is easily sublimated. Starch solubilization must be
carried out with warm water and this operation obviously
needs attention; so it is preferable to supply the students
with the iodine and starch solutions previously prepared.
STUDENT PITFALL
Experiments are very simple to do, but some mistakes
can occur in the result analysis. My experience shows that
care needs to be taken in accurate pipetting and calculat-
ing the right dilution. Students commonly need help, at the
beginning, in thinking in terms of micro quantities, calcu-
lating reaction velocity, converting experimental results to
relative enzyme units, and plotting data. Sometimes I must
remind my students that “colorless number” arbitrary units
of the first experiment are different from relative units such
as micrograms of hydrolyzed starch in 1 min.
TIMING OF LABORATORY
My 3 years of experience with second year Liceo stu-
dents show that 2 h are sufficient for each experimental
block; laboratory work is then followed by 1 h of discus-
sion on obtained results and on formulation and testing of
hypothesis. At the end of the five experiments, another
hour is dedicated to a comprehensive discussion.
STUDY QUESTIONS
1. Why don’t you digest cellulose?
2. How could you quantitate reaction velocity?
3. What is the amylase activity in your saliva?
4. How could you explain enzyme behavior at different pH levels?
5. What happens to salivary amylase at low gastric pH?
6. How could you describe and explain the shape of the pH
curve?
7. How does the heat treatment affect enzyme activity?
116
8. How could you describe and explain the shape of the tem-
perature curve?
9. How does substrate concentration affect enzyme activity?
10. How could you describe and explain the shape of the sub-
strate concentration curve?
11. What is an enzyme inhibitor?
12. What is the biological meaning of phaseolamin?
DISCUSSION OF RESULTS
I carried out these five experiments with 14- to 15-year-
old students with no particular difficulties. Analysis, dis-
cussion, and elaboration of experimental results demon-
strate that all students learn the enzyme concept at a
qualitative level and almost all of them at a semi-quantita-
tive level. A critical component of scientific knowledge,
and also of experimental sciences teaching, is the stu-
dent’s formulation and testing of hypothesis as attempts at
explanations for some observations. Once a hypothesis
has been formulated, it is tested by construction of an
if/and/then argument, and the use of evidence in a hypo-
thetic-predictive fashion. In this case the second, third,
and fifth experiments have been very useful to the stu-
dents right data interpretation. I found that well-designed
instructions brought about significant and general
improvements.
Prima digestio fit in ore, with its pedagogical and didac-
tic meanings, was also carried out with graduate students
of the Specialization School for Teaching in Secondary
Schools (SSIS) of the Natural Sciences Section. They ap-
preciated the structure and organization of the project, and
the didactic feed-back as well as the integrated theoretical
themes such as: ionic equilibrium, protein structure, pro-
BAMBED, Vol. 33, No. 2, pp. 112–116, 2005
tein folding, characteristics of enzymatic reactions, and
biotechnology.
Acknowledgments—I thank Professor Umberto Mura, Depart-
ment of Physiology and Biochemistry, University of Pisa, for his
constant encouragement and his precious suggestions. I am
also grateful to Prof. Piero Luigi Ipata, Department of Physiol-
ogy and Biochemistry, University of Pisa, who introduced me to
biochemistry didactic. Special thanks are due to Prof. Alessan-
dra Bianchi and Dr. G. Phillips for the English revision of the
manuscript.
REFERENCES
[1] B. Inhelder, J. Piaget (1958) The Growth of Logical Thinking from
Childhood to Adolescence, Basic Books, New York.
[2] F. Pasquinelli (1971) Manuale per tecnici di laboratorio, Ed. Rosini,
Firenze, pp. 714 –717.
[3] D. Voet, J. G. Voet (2004) Biochemistry, 3rd Ed., J. Wiley & Sons Inc.,
Hoboken, NJ, pp. 367– 459.
[4] L. Kandra, G. Gyemant (2000) Examination of the active sites of
human salivary !-amylase (HSA), Carbohydr. Res. 329, 579 –585.
[5] E. A. MacGregor, S. Janecek, B. Svensson (2001) Relationship of
sequence and structure to specificity in the !-amylase family of
enzymes, Biochim. Biophys. Acta 1546, 1–20.
[6] F. A. Scannapieco, G. Torres, M. J. Levine (1993) Salivary !-amylase:
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[7] D. Voet, J. G. Voet (2004) Biochemistry, 3rd Ed., J. Wiley & Sons Inc.,
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[8] M. J. van der Maarel, B. van der Veen, J. C. Uitdehaag, H. Leemhuis,
L. Dijkhuizen (2002) Properties and applications of starch-converting
enzymes of the !-amylase family, J. Biotechnol. 94, 137–155.
[9] W. D. Crabb, J. K. Shetty (1999) Commodity scale production of
sugars from starches, Curr. Opin. Microbiol. 2, 252–256.
[10] J. J. Marshall, C. M. Lauda. (1975) Purification and properties of
phaseolamin, an inhibitor of !-amylase, from the kidney bean,
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