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Experiment 3
ACTIVITY OF SALIVARY AMYLASE
Carandang, Cruz, Pasumbal, Salem, Tolentino

ABSTRACT

The activity of salivary amylase enzyme was observed under different conditions. An amylase
solution was prepared containing 1% starch, 1% NaCl and phosphate buffer heated to 37°C to replicate
physiological conditions. The same setup was repeated and modified to simulate different enzyme and
substrate concentrations, temperature, and pH levels. The achromic point of each setup was used to
determine the effect of each factor on the enzyme’s activity. The experimental result for enzyme
concentration agreed with theoretical results, but the result for substrate concentration contradicts the
Michaelis-Menten and Lineweaver-Burk plots. Salivary amylase enzyme was also observed to have an
optimal range on both temperature and pH to reach maximum activity. The optimal pH range observed
was between 6.8 and 6.9, whereas the optimal temperature range for salivary amylase was observed at 37-
38℃.
INTRODUCTION described by the Michaelis-Menten equation:
[REF]
Enzymes are protein molecules in the cell that
function in catalyzing synthetic and metabolic
reactions. Enzymes are considered proteins
evidenced by the general inactivation in extreme The relationship described by the Michaelis-
temperatures and pH due to denaturation. Each Menten equation is a hyperbolic function. To
has a unique amino acid sequence and obtain a linear graph, the inverse of both initial
conformation, and most enzymes are specific to rate and substrate concentration are used called
a substrate or reactant. The specificity of the Lineweaver-Burk equation:[REF]
enzymes is shown by the presence of an active
site where only a specific substrate can fit.
[REF]
Aside from the concentrations of enzyme and
Salivary amylase, an enzyme found in saliva of substrate, temperature and pH are common
vertebrates, initiates the digestion of starch by factors that can affect the activity of an enzyme.
cleaving starch into disaccharides by hydrolysis. Enzymes, aside from having a substrate
Salivary amylase functions actively with the low specificity, have specific range of temperature
pH of saliva from 5.0-8.0 with an average of 6.5 and pH in catalysis reactions. Increasing
due to bicarbonates present in saliva.[REF] temperature leads to an increase in catalytic
activity, but increasing the temperature beyond
The activity of an enzyme can be measured the optimal range causes unfolding or
through enzyme kinetics or the rate of reaction. denaturation of proteins which destroys the
The rate depends on the concentrations of the conformation and activity. The activity of
substrate, product, and the enzyme. The affinity enzymes in acidic or alkaline solution varies
of the enzyme to its specific substrate is depending on the optimal pH range of the
correlated to the kinetics of the reaction as enzyme. For example, salivary amylase is active
at pH 6.5 found in saliva that has a pH range of
5.0-8.0. Not all enzymes have peak activity at

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neutral pH; pepsin found in the stomach is the spot plate was observed until the achromic
highly active at a very low pH of 1.5. (Randall, point is reached. The enzyme in the saliva may
et al, 2002). then be estimated in terms of the amylase units
by the equation:
In this experiment, the activity of salivary
amylase was observed and analyzed due to the
effect of different factors such as: (1) pH level,
(2) temperature, (3) substrate concentration, and The enzyme activity may also be obtained:
(4) enzyme concentration based on the achromic
point with iodine indicator to observe the
hydrolysis of starch by the activity of salivary
amylase. The rate of reaction of the hydrolysis
Where minutes corresponds to the achromic
by salivary amylase was further analyzed
point time in minutes, units of enzyme from the
through Michaelis-Menten and Lineweaver-
computed units of amylase in 1 mL saliva.
Burk plots.

The different effects of enzyme concentration,

MATERIALS AND METHODS
amount of substrate pH and temperature were
also observed similar to the prior enzyme
To observe the enzyme kinetics of salivary
activity. Different threshold values were
amylase, the digestion of starch as indicated by
employed so as to create a clear mark between
the color change of its products with iodine was
the varying variables within each factor.
monitored. The rate of hydrolysis may also be
obtained from the color changes that occurred
RESULTS
throughout the reaction of starch and the
intermediate product of dextrin with iodine.
Different parameters were subjected to variation
in order to observe their effect on enzyme
The salivary amylase was first prepared by
activity: enzyme concentration, substrate
letting a human sample chew a paraffin wax and
concentration, pH, and temperature.
letting the saliva flow into a beaker. The saliva
was then distilled in a ratio of 1:9 volume of
It can be inferred in Figure 1 that enzyme
saliva with distilled water respectively. The
activity is directly proportional to the
iodine-potassium iodide solution was prepared
concentration of the enzyme indicated by the
by dissolving 6 grams potassium iodide in 100
increasing trendline.
mL distilled water along with 4 grams of iodine
crystals. The starch solution of 1% concentration
The relationship of substrate concentration on
was prepared by having a ratio of 1:10 w/v
the enzyme activity is inversely proportional, as
1:100 dapat with water.
depicted in Figure 2 by its decreasing trendline.
The activity of amylase was estimated by
preparing a test solution containing 5 mL 1%
starch, 2 mL 1% NaCl and 2 mL phosphate
buffer maintained at 37 degrees celsius.
Addition of the enzyme amylase was done
afterwards and every minute, the color change in

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Figure 1. Effect of enzyme concentration on salivary
amylase enzyme activity.
Figure 2. Effect of starch concentration on salivary
amylase activity.
The effect of pH level on enzyme activity is
different from the former two factors in that
their relationship cannot be described as
proportional. It is shown in Figure 3 that enzyme
activity is highest at the optimum pH 6.8 and
any other pH would result to a low activity.
Figure 3. Activity of salivary amylase in different pH
levels.
Similar to pH, the enzyme activity also has an
optimum temperature at around 37℃ where it is
highest. As the temperature drifts away from the
optimal range so does the enzyme activity.
Figure 4. Activity of salivary amylase enzyme in
different temperatures.
The results gathered from the effect of substrate
concentration on enzyme activity contradicts the
theoretical literature. Therefore, a Michaelis-
Menten plot cannot be applied; the graph shown
in figure 2 is linear and has a negative slope
instead of a hyperbolic function. The same
situation was obtained when Lineweaver-Burk
plot is employed. The resulted graph has a
negative slope, in contrast with the expected
positive slope found in literature. The Michaelis-
Menten constant and maximum rate cannot be
properly calculated.
Subjective judgment on the achromic point or
systematic errors in obtaining solutions may be
the sources of error in the results.
Figure 5. Lineweaver-Burk plot of salivary amylase
activity.
*DISCUSS HOW SALIVARY AMYLASE
CLEAVES/HYDROLYZE STARCH ->
REACTION OF STARCH + I2 IN KI
DISCUSSION
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In the experiment, ptyalin or salivary amylase
initiated the digestion of starch into smaller
sugar molecules. (Randall et al, 2002). The
enzymatic activity can be visualized by plotting
the kinetic activity through the Michaelis-
Menten Plot. The rate of the reaction, vo and the
substrate concentration, [S] was obtained for the
graph of initial velocity vs substrate
concentration.
Estimation of Amylase Activity
Addition of the iodine solution to the enzyme
salivary amylase was added until no color
change was observed. This is known as the
achromic point or the point where the salivary
amylase on starch does not react anymore to
iodine indicating that the reaction has reached its
maximum capacity. The digestion mixture took
14 mins to reach the achromic point. The
minutes it took to reach the achromic point was
used to obtain the number of amylase in saliva
which was 0.0714 amylase units. The equation
used is shown in Figure 6. (See Appendix for
calculations)
Figure 6. Number of units of amylase in 1 mL saliva
equation
The estimated value of salivary amylase activity
was 0.051. It was calculated by multiplying the
amount of substrate, which is 10 mg/mL, with
the number of amylase units and dividing the
product with minutes it took to reach the
achromic point. The equation is shown in figure
7.
Figure 7. Enzyme Activity equation
Effect of Enzyme Concentration
Figure 1 shows that as the enzyme concentration
increases, the enzyme activity also increases
which depicts a directly proportional
relationship. This would imply that if the
enzyme concentration increased, the rate of
reaction would increase as well. This trend
follows the first-order kinetics found at lower
substrate concentrations. The rate of reactions
increases as the substrate concentration
increases. (Campbell & Farrell, 2013) This can
be visualized in the Figure 7 below.
Effect of the Amount of Substrate
Substrate concentrations increase as enzyme
concentrations increases which shows a directly
proportional relationship. However,
experimental results in Figure 2. showed an
inversely proportional relationship implying that
as the substrate concentration decreases, the
enzymatic activity increases. (Campbell &
Farrell, 2013)
Figure 8. Enzyme Substrate constants
Michaelis-Menten Equation is derived from
Figure 8 which shows that as the substrate
concentration [S] increases, the curve becomes
asymptotic to Vmax shown in Figure 9 and Figure
10.
Figure 9. Michaelis-Menten Equation
From Figure 9, Vo is the initial reaction rate,
Vmax is the maximal reaction rate, [S] is substrate
concentration and Km is the Michaelis-Menten
constant. The relationship of Vmax and [S] differs
depending on substrate concentrations. Vmax is
directly proportional to the substrate at low
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concentrations while Vmax becomes independent
of substrate concentrations at high
concentrations. When substrate concentration is
equal to Km , the Michaelis constant becomes
directly proportional to Vmax.
Figure 10. Michaelis constant equation
From the equation in Figure 10., Km is the
substrate concentration which the reaction
reaches the half of the maximum rate. This is the
substrate concentration at which significant
catalysis can occur.
Figure 11. The rate of a catalysis reaction by an
enzyme is dependent on substrate concentration.
(adapted from Campbell & Farrell, 2013).
By obtaining the reciprocal of Figure 9., the
Michaelis-Menten equation, the Lineweaver-
Burk equation can be obtained which is a plot of
the reciprocal of the substrate against the
reciprocal of the velocity. This equation is
important in determining the types of inhibition
if competitive, noncompetitive, or uncompetitive
because of its linear nature as opposed to the
Michaelis-Menten plot which has has a
hyperbolic line when plotted.
Figure 12. Lineweaver-Burk Equation.
Figure 13. A Lineweaver-Burk plot illustrating the
derivation of Km and Vmax from x and y-intercepts
respectively. (adapted from Randall et al, 2002).
Effect of pH
Most proteins have net charges in physiological
conditions which lead to increased attraction to a
certain substrate. This would lead to an increase
in the rate of reaction. Because enzymes have
amino acid side chains of different charges, the
side chains may affect the conformation of the
enzymes. Some enzymes work better when
certain amino acids are protonated or
deprotonated which causes the protein to fold in
its native conformation where the enzyme is
most active. (Randall et al, 2002)
As seen from Figure 3, there is a peak in the
graph which depicts the optimum pH, or the the
pH range at which the activity of enzymes is
maximized which in the case of the salivary
amylase, it is at optimum at 6.7-7.0. (Randall et
al, 2002)
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During the experiment, the enzymatic activity
and the effect of pH coincides with the
theoretical results. There is a peak at 6.8-6.9
which is close to the theoretical value.
Effect of temperature
Reaction rates increases as temperature
increases with respect to the activity of
molecules when heated. Higher temperatures
allow for an increase in molecular activity
therefore increasing the formation of enzyme-
substrate complexes. However, an increase in
temperature increases the probability of protein
denaturation, which disrupts the conformation of
polypeptide chains. Hence, the reaction rate
reaches a maximum when denaturation is
balanced by enzyme-substrate reactivity and this
would show a peak in the graph, which is is the
optimal temperature. The rate of enzyme
destruction by heat is balanced by the increase in
enzyme-substrate reactivity, and the two effects
of elevated temperature cancel. At that
temperature the reaction rate is maximal. Upon
reaching a certain temperature denaturation is no
longer balanced and reaction rates decrease.
At higher temperatures, enzymes are destroyed
and the rate of reaction rapidly increases which
contributes to lethal effects of excessive
temperatures. (Randall et al., 2002).
CONCLUSION
Salivary amylase was subjected to different
factors in order to determine their effect on its
enzymatic activity. It was observed
experimentally that the relationship between the
enzyme activity and the enzyme concentration is
directly proportional with each other which
follows first-order kinetics in low substrate
concentrations.
The effect of substrate concentration was
observed to be inversely proportional with the
enzyme activity of amylase through the graph
produced, but the theoretical explanation states
otherwise. The activity of salivary amylase
should be directly proportional to starch
concentration as stated by the Michaelis-Menten
equation as well in the Lineweaver-Burk
equation. Subjective judgment on the achromic
point or systematic errors in obtaining solutions
may be the sources of error in the results.
The effect of pH and temperature on salivary
amylase activity both illustrate that enzymes
have optimal range in which the catalytic
activity is at maximum. The optimal pH range
observed was between 6.8 and 6.9 where some
side chains are protonated as well as
deprotonated in order to achieve the enzyme’s
maximum activity. Salivary amylase activity
also increases with increasing temperature, but
increasing the temperature beyond 40℃ showed
decreased activity due to the denaturation of
salivary amylase.The optimal temperature range
for salivary amylase was observed at 37-38℃.
REFERENCES
Campbell, M.K., & Farrell, S.O. (2013).
Biochemistry (8th ed.). Stamford, CT: Cengage
Learning.
Randall, D. J., Burggren, W. W., French, K., &
Eckert, R. (2002). Eckert animal physiology:
Mechanisms and adaptations. New York: W.H.
Freeman and Co.