Biological Chemistry

Enzymatic Activity of Salivary Amylase

Ellicia Vern Mendoza, John Michael Joseph Napa, Nicanor Olanka, *Maria Christina Paine
Group 6, 3Bio-3
Department of Biological Sciences, College of Science,
University of Santo Tomas, España, Manila 1055

Date Submitted: December 11, 2010

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

amylase, produced by salivary glands, is a type of a hydrolase enzyme (amylase). Hydrolases

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

Enzyme solution (1 mL saliva + 9 mL distilled H2O + 30 mL 0.5% NaCl)

Buffered starch (1% starch in phosphate buffer pH 6.7)

0.001 M Iodine Solution

Spot plates

Test tubes

Medicine droppers

Constant temperature bath (4oC, 37 oC, 40 oC, 50 oC, 70 oC)

2% Unbuffered starch

Water bath set at 37 oC

Acetate buffer solutions (pH 4&5)

Phosphate buffer solutions (pH 6.7 & 8)

Bicarbonate buffer (pH 10)

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

amylase was determined.

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

of the amylase was determined.

IV. Data & Results

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

pH Time (min) 1/t (min-1)

4 ∞ mins 1/∞ / 0 mins-1
5 19 mins 1/19 / 0.05 mins-1
6.7 14 mins 1/14 / 0.07 mins-1
8 29 mins 1/29 / 0.03 mins-1
10 ∞ mins 1/∞ / 0 mins-1

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

enzyme is reduced to zero.

If there is an optimum temperature, there is also an optimum pH. Similar to temperature,

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

denatured hindering the activity to be performed.

VI. Conclusion & Recommendation

 In conclusion, the experiment highlights the importance of knowing the factors that affect

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

chemical that may affect the results.

VII. References

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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:

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Wilson, K. & Walker, J. Principles and Techniques of Biochemistry and Molecular Biology, 7th

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