Experiment No.

2: Qualitative Analysis on Salivary Amylase

Enzyme Activity
ABSTRACT: Enzymes are proteins that act as catalysts within living cells wherein catalysts
increase the rate at which chemical reactions occur without being consumed or
permanently altered themselves. Amylase, an example of an enzyme is a protein made by
pancreas and glands in and around the mouth and throat and has its substrate which is
starch that is a soft, white, tasteless powder polysaccharide comprising glucose monomers.
This laboratory experiment was aimed to determine the effects of various factors which
considers substrate concentration, enzyme denaturation, and pH of enzyme activity. The
effect of the substrate concentration, enzyme denaturation, pH, temperature, activators,
and enzyme concentration in the appearance of colors of the amylase upon mixing it with
the iodine solution is being identified in this study whereas the enzyme activity is measured
by the decrease and increase in the substrate concentration in a period of time.
Temperature, pH, substrate concentration and enzyme concentration affect the activity of
enzyme wherein the enzyme shows low activity at low temperatures and the rate of reaction
increases as the temperature rises. There is a certain temperature at which the enzyme is
maximally active and all these enzymes have optimum pH that affects whether the enzymes
become active or inactive.

Korrine G. Balais; Ma. Melanie Dalaine D. Carnaje; Elmy Joy A. Fernando; Jeri Anne O.
Fresnido; Marco C. Mamhot; Alexandra Therese Maramo, Angela P. Oris, Group 1, 1:30-
7:30pm, Monday, E610, BS BIO 2-1, Department of Biology, College of Science,
Polytechnic University of the Philippines. To be submitted on September 8, 2019.

Introduction

Enzymes are proteins that act as catalysts within living cells. Catalysts increase the
rate at which chemical reactions occur without being consumed or permanently altered
themselves. A chemical reaction is a process that converts one or more substances (known
as reagents, reactants, or substrates) to another type of substance (Christensen, 2018). As a
catalyst, an enzyme can facilitate the same chemical reaction over and over again. Like all
proteins, enzymes are composed of one or more long chains of interconnected amino acids.
Each enzyme possesses a unique sequence of amino acids that causes it to fold into a
characteristic shape. An enzyme's amino acid sequence is determined by a specific gene in
the cell's nucleus. This ensures that each copy of the enzyme is the same as all others.
Enzymes have different varieties and like other things it has its own composition,
functions, mechanisms, and reactions if it was subjected to other substances. One of its
types is the amylase. Amylase is a protein made by pancreas and glands in and around the
mouth and throat. It is a digestive enzyme that acts on starch in food, breaking it down into
smaller carbohydrate molecules (Marie, 2018). Amylase’s primary function is digestion,
but it may play a role in other facets of health as well, perhaps not directly, but as an
indicator.
Like any other enzymes, amylase has its substrate and it is known as starch. Starch
is a polysaccharide comprising glucose monomers joined in α 1, 4 linkages. It is a soft,
white, tasteless powder that is insoluble in cold water, alcohol, or other solvents. The basic
chemical formula of the starch molecule is (C6H10O5). In this experiment, a saliva–watery
liquid secreted into the mouth by glands, providing lubrication for chewing and
swallowing, and aiding digestion, will be subjected in different factors such as the substrate
concentration, enzyme denaturation, pH, temperature, activators, and enzyme
concentration. The students will find out what will be the effect of the aforementioned
factors to the appearance of colors of the amylase upon mixing the iodine solution into it.

Methodology

a. Equipment and Apparatus

The equipment used upon the accomplishment of this laboratory experiment
were hot plate and analytical balance, and other glassware.

b. Personal Protective Equipment

Since this experiment involved chemicals, personal protective equipment
were worn at all times. These included laboratory gown, disposable gloves, safety
goggles, closed shoes, and neat hair. Those PPEs asserted were utilized during the
experimental procedure.

c. Experimental Procedure

1. Effect of Enzyme Factors

1.1 Effect of Substrate Concentration

Three test tubes were labelled as A, B and C. In each tube, place 1.0 mL NaCl and
3 mL of saliva. Then, 3.5 mL of distilled water was added to tube A, 7 mL of distilled water
was added to tube B, and 9 mL of distilled water into tube C. Lastly, 7.5 mL, 4.0 mL and
2.0 mL starch solution was added to tubes A, B and C, respectively.

1.1 Effect of Enzyme Denaturation

Two test tubes were labelled as D and E. In each tube, place 1.0 mL NaCl, 3.5 mL
distilled water and 3 mL of saliva. In tube D, 3 mL of unboiled saliva was added while on
tube E, 3 mL was boiled for approximately 2 minutes before adding it to the tube.

1.2 Effect of pH

Label three test tubes F, G and H. In each tube, place 1.0 mL NaCl, 3 mL of saliva
and 7.5 mL starch solution. In tube F, add 2.5 mL distilled water and 0.5 mL HCL. In tube
G, add only 3 mL distilled water and in tube H, 2.5 mL distilled water and 0.5 mL NaOH.

2. Spot Test
 After preparing, a drop of each mixture was placed into a spot plate after mixing
the tube properly. Then, a drop of iodine solution was placed to the reaction mixture on the
spot plate and was observed for the development of a blue-violet coloration. This was
repeated for 15 times with a 1 minute interval for each drop.
If the reaction mixture fails to develop the color of iodostarch complex, Benedict’s
test was performed with 1 mL of the particular reaction mixture.

Results and Discussions

Enzyme activity is a measure of the catalytic ability and there are two
methods to measure enzyme activity: one is to measure the decrease in substrate
concentration in a period of time, and the other is to measure the increase in concentration
of a product after a period of time (Encyclopedia of Analytical Science, 2019).
Temperature, pH, substrate concentration and enzyme concentration affect the activity of
enzyme. Enzyme has low activity at low temperatures. The rate of reaction increases as the
temperature rises. There is a certain temperature at which the enzyme is maximally active-
this is called the optimum temperature (Brilliant Biology Student, n.d.).
In the experiment done, the factors that affected the enzyme activity were
substrate concentration (% or mL of starch), denaturation, and pH.

Table 1. Starch concentration. (✔) No starch. (x) Contains starch

RM Modifiers Minutes of Reaction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Benedict’s
Effect of Substrate Concentration (% or mL of Starch)
A x X ✔ ✔ x x ✔ ✔ x x ✔ X ✔ ✔ ✔
B X x x x x x x x x x X ✔ x x
C x X x x x x x x x x x X ✔ x x
Effect of Denaturation (Na-unboiled or native enzyme, De-boiled/denatured enzyme
D Na x x x x x x x x x x x X x x x
E De x x x x x x x x x x x X ✔ ✔ ✔
Effect of pH
F HCl x x x X x x x x x x x X x x x
G Water x x x X x x x x x x x X x x x
H NaOH x x x X x x x x x x x X x x ✔
.
 Table 2. Reaction A, B, and C on substrate concentration

Reaction
Reaction A

Reaction B

Reaction C

Increasing substrate concentration of an enzyme affects the enzyme activity by

increasing the rate of reaction to a certain point. Once the enzymes have bound, any
substrate increase will have no effect on the rate of reaction, as the available enzymes
will be saturated and working at their maximum rate (Khan Academy, 2019).
 Table 3. Reaction D and E on denaturation.

Reaction
Reaction D

Reaction E

Enzymes work consistently until they are denatured. When they are denatured,
enzymes are no longer active and cannot function. Some causes of enzymes to be denatured
are extreme temperatures and wrong levels of pH. When an enzyme is denatured, it loses
some of its original properties (Wiggins, n.d.).

Table 4. Reaction F, G, and H on pH.

Reaction
Reaction F
 Reaction G

Reaction H

All enzymes have optimum pH. That is where they work the fastest. As the pH
increases or drops from the optimum, the bonds that hold the enzyme in shape break which
makes the enzyme change shape. The active site also changes shape and the substrate no
longer fits. The enzyme is then denatured. The factor pH has a direct effect on when the
enzyme becomes active and inactive (Bernhardt, 2017).

Enzyme pH Optimum
Lipase (pancreas) 8.0
Lipase (stomach) 4.0-5.0
Lipase ( castor oil) 4.7
Pepsin 1.5-1.6
Trypsin 7.8-8.7
Urease 7.0
Invertase 4.5
Mastase 6.1-6.8
Amylase (pancreas) 6.7-7.0
Amylase (malt) 4.6-5.2
Catalase 7.0
 Other factors that affect enzyme activity are temperature, inhibitors, and enzyme
concentration.
When temperature is increased, the collision between molecules increase because
increase in velocity and kinetic energy. There will be less time between collisions with
faster velocity. The optimum temperature for enzymes are 39 degree Fahrenheit and 4
degree Celsius. Temperature above 104 degrees Fahrenheit, 40 degrees Celsius, and the
enzymes will start to break down. The ends of the activity range of an enzyme is determined
by what temperature starts the activity and the temperature that breaks them down
(Santhosh, 2018). At cold temperatures, the enzyme molecules move slowly which reduces
frequency of enzyme-substrate collisions and therefore decreasing enzyme activity.
Crystalline structures are formed at point of freezing because molecular motions decrease
drastically and solid formation occurs (Graw, 2018).
Enzyme inhibitors are molecules that bind to enzymes which decreases enzyme
activity. An inhibitor can bind to an enzyme and stop a substrate from entering the
enzyme’s active site or prevent the enzyme from catalyzing a chemical reaction. Reversible
inhibitors bind to enzymes through weak non-covalent interactions such as ionic bonds,
hydrophobic interactions, and hydrogen bonds. These bonds can easily be remove. There
are three types of reversible inhibition, (1) competitive inhibitors are inhibitors that are
similar to the structure of the substrate that bind to the active site of the enzyme, (2)
uncompetitive inhibitors bind to the enzyme at the same time as the enzyme’s substrate but
in an allosteric site, (3) non-competitive inhibitors bind to allosteric site (not an active site)
which stops the enzyme’s activity by altering the shape of the active site (“Structural
Biochemistry/Enzyme/Reversible Inhibitors”, 2019).
When enzyme concentration is increased, it will speed up the reaction, as long as
there is an available substrate to bind to. Once all the substrate is bound, the reaction will
no longer speed up. (Khan Academy, 2019)

Summary

The effect of the temperature, inhibitors, pH and enzyme concentration in the

appearance of colors of the amylase upon mixing the iodine solution with it shows that the
enzyme has low activity at low temperature and the rate reaction gives rise as the
temperature increases. With this, it shows that there is certain temperature at which the
enzyme is maximally active and that they work faster on its maximum level. As the pH
increases or drops from the optimum, the bonds that hold the enzyme in shape break which
makes the enzyme change shape. Therefore, the factor pH has a direct effect on when the
enzyme becomes active and inactive. To sum it up, the observations in the activity show
that different factors do really have a part in rate of enzyme catalysis within a reaction.

Acknowledgements
We, the researchers, would like to acknowledge our parents for giving us the
opportunity to study at a university, such as PUP, that would pave the way for us to reach
our goals and aspirations. We would also like to extend our gratitude to the university that
allowed us to show and enhance our skills by providing us the things we need while also
teaching that life does not always hand everything, we need in a silver spoon. Appreciation
and thanks To Ms. Marie Dale T. Peralis, for guiding us and for the knowledge that she
shared with us throughout the experiment.\

References

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https://healthyeating.sfgate.com/functions-amylase-protease-lipase-digestive-enzymes-
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Gomes & Rocha-Santos. (2019). Encyclopedia of Analytical Science (Third Edition).

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biology 6049.html

(N. A.). Effect of Temperature on Enzyme Action. Retrieved from

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(N. A.). Structural Biochemistry/Enzyme/Reversible Inhibitors. Retrieved from

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