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Abstract
The enzymatic activity of the salivary amylase is being affected by several factors. These factors
include the temperature and pH. This experiment discusses how temperature and pH take role in
activating the enzymes. To explain how this happens, the rates of enzymatic activity of salivary
amylase in varying temperatures and pHs were measured and compared. A plot in the end shows
the results and comparison.
I. Introduction
Enzymes are natural biological catalysts that speed up the reactions occurring in living
organisms, both animals and plants. The use of a specific enzyme makes a typical metabolic
reaction a million times faster. Enzymes are being designed specifically for a certain chemical
reaction in the system. This is through the DNA, in the cell’s nucleus, which encodes instructions
for the cells to synthesized specific enzymes. An enzymatic activity consists of substrate, active
site & enzymes. Enzymes act on substrates. The specific site where enzymatic activity takes
place is the active site. The substance formed by the reaction is called product. The salivary
catalyze hydrolysis reactions where there is an addition of a water molecule to a bond resulting
in bond breakage. Amylase breaks down starch and turns it into maltose, a disaccharide. These
reactions have an important role in digestive process. The experiment has the main objective of
examining the enzymatic activity and specificity of salivary amylase depending on changes in
pH and temperature.
II. Materials
Spot plates
Test tubes
Medicine droppers
2% Unbuffered starch
III. Methodologies
In this experiment, two effects on the enzymatic activity of salivary amylase were
observed (effect of temperature & effect of pH). The first activity was the observation of the
effect of temperature. First, 2 mL of the enzyme solution was put in a large test tube and labeled
as 4 oC. Next, 2 mL of the buffered starch solution was put in a separate large test tube. Both test
tubes were incubated for 10 minutes in an ice bath at 4 oC. The two solutions were immediately
mixed. Three drops of the mixture were quickly taken and two drops of the iodine solution were
simultaneously added onto the first well of a spot plate. That has been the zero minute. After a
one-minute interval with continuous incubation, three drops again of the mixture were taken and
two drops of the iodine solution were added simultaneously onto the second well. That has been
the one minute. The step 5 was repeated until a light yellow-colored solution was observed. The
final time was noted. For the other temperatures (37 oC, 40 oC, 50 oC & 70 oC ), the steps 1 to 6
were repeated following the desired incubation temperature. Finally, the reciprocal of time (1/t,
min-1) in step 6 versus the temperature (T) was plotted and the optimum temperature of the
The last activity was the observation of the effect of pH. First, 1 mL of acetate buffer (pH
4) and 1 mL 2% unbuffered starch were mixed in a large test tube. 2 mL of the enzyme solution
was added in a separate large test tube. Both test tubes were incubated for 10 minutes in a 37 oC
water bath. The two solutions were immediately mixed. Three drops of the mixture were quickly
taken and two drops of the iodine solution were added simultaneously onto the first well of the
spot plate. That has been the zero minute. After a one-minute interval with continuous
incubation, three drops of the mixture were taken again and two drops of the iodine solution
were simultaneously added onto the second well. That has been the one minute. The step 5 was
repeated until a light-yellow colored solution was observed. The final time (t) was noted. The
steps 1 to 6 was repeated for the other pH (4, 5, 6.7, 8, 10) using the appropriate buffer. Finally,
the reciprocal of time (1/t, min-1) in step 6 versus the buffer pH was plotted and the optimum pH
A. Effect of Temperature
Temperature (T) Time (min) 1/t (min-1)
4oC ∞ mins 1/∞ / 0 mins-1
37 oC 5 mins 1/5 / 0.2 mins-1
40 oC 11 mins 1/11 / 0.09 mins-1
50 oC ∞ mins 1/∞ / 0 mins-1
70 oC ∞ mins 1/∞ / 0 mins-1
B. Effect of pH
Optimum pH (6.7,
0.07)
V. Discussion
The graph shows the reaction rate of the salivary amylase (reciprocal of time versus
temperature/pH). The rate of an enzyme catalyzed reaction as the temperature increases but this
is only up to a point. Enzymes work best at a certain temperature wherein the reaction rate will
be at a maximum. This is proved by the bell-shaped curve in the graph. This certain temperature
is called the optimum temperature. The results show that the peak of temperature-related reaction
rate is at 37oC. This is because the optimum temperature of this enzyme is at 37oC where the
enzymes and substrates are working at its best. This applies in the human body. At 4 oC, there is
little energy which makes the reaction slower. At 40oC, the enzymes and substrate are working a
lot quicker but due to the high temperature, some enzymes are denatured. At temperatures 50 oC
and above, all the enzymes are denatured due to destruction of secondary and tertiary structures.
At this temperature, the enzyme molecule vibrates too much to the extent that the enzymes lose
their three-dimensional structure. This destroys, as well, the active sites. Thus, the activity of the
pH also has a peak where the enzymatic reactions are at its greatest. An enzyme activity requires
a certain level of acidity and alkalinity to work at its best. The pHs higher or lower than the
optimum pH makes an enzyme activity slower and reduced. The optimum pH of the enzyme is
pH 6.7 which agrees to the rule of pH 6.7-7 as the optimum pH of salivary amylase. The pHs 4
and 5 are too acidic for a fast enzymatic reaction. At pH 8, the enzymes work fast but are slowly
being denatured due to much alkalinity. Enzyme activity at pH 10 will have all the enzymes
denatured.
In this activity, starch is the one being catalyzed by the enzyme solution. As mentioned
earlier, salivary amylase is a type of enzyme that breaks down starch and turns it into maltose, a
disaccharide. To test the presence of an enzyme activity between the two, iodine test was
performed. In this test, the rate of loss of reactant or increase of product is being observed.
Activators and inhibitors also affect the enzyme activity. Sodium chloride was added to the
enzyme solution to hasten the reaction and make it more possible. Chloride ions are important in
activating the salivary amylase. How does boiling affect the enzyme activity? Boiling increases
the temperature which destroys the proper shape of the enzymes. The enzymes become
the enzyme activity. Inability to maintain the optimum temperature and pH of a certain enzyme
can deter the assurance of accurate and reliable results. Enzymes are very important in the
systems of the living and a simple mistake can alter the process.
During the experiment, the required time involved in the experiment should be carefully
followed. The laboratory glasswares should be maintained clean to avoid mixing of unwanted
VII. References
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& Biological Chemistry, 2nd ed. Dubuque, Iowa: Wm-C Brown Publishers, 1997. Page
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Hein, M., Peisen, J.N., & Ritchey, J.M. Introduction to General, Organic, & Biochemistry in the
Laboratory, 8th ed. Hoboken, NJ: John Wiley & Sons, Inc., 2005. Page used:341-342
Henrickson, C.H., Byrd, L.C., & Hunter, N.W. A Laboratory for General, Organic, &
Biochemistry, 4th ed. New York: McGraw-Hill, 2005. Page used:355, 358-359
McKee, T. & McKee, J.R. Student Study Guide, Solutions Manual for use with Biochemistry:
The Molecular Basis of Life, 3rd ed. New York: McGraw Hill, 2003. Page used:59-60
Wilson, K. & Walker, J. Principles and Techniques of Biochemistry and Molecular Biology, 7th
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