The Effects of Temperature and pH on the Enzyme Activity of Salivary

Amylase

Department of Biological Sciences, College of Science, University of Santo Tomas, Espaa

Blvd., Manila

Abstract: Salivary Amylase is an enzyme used to hydrolyze starch (polysaccharide) molecules, while its enzymatic
activity can be altered by changes in temperature and pH. The rates of enzymatic activity were measured by
introducing the enzymatic solution to different ranges of temperatures and pH. The Optimum Temperature for
Salivary Amylase is 37 C and the Optimum pH is 6.7.

Introduction:

Enzymes are biomolecules that act as catalysts in a chemical reaction, whereas most of

them are proteins. Catalysts are chemical agents that allow a chemical reaction to go at a faster

rate without undergoing any changes. In an enzymatic reaction, the molecules in the beginning of

the process are called substrates, and the enzyme then converts these substrates into different

products. The substances transformed after the reaction are often organic compounds. Enzymes

are known to be more efficient catalysts than any of the man-made catalysts yet devised by

giving a very high degree of specificity and selectivity. Like other catalysts, an enzyme does not

alter equilibrium, it only affects rates. Enzymes are the agents of metabolic function by which

they form metabolic pathways where nutrient molecules are degraded and energy is produced

and converted into useful metabolic forms.

Enzymes are remarkably modifiable biochemical catalysts that have three distinctive

features - regulation, catalytic power, and specificity. Regulation of enzyme activity is necessary

to the utilization and regulation of metabolism. It ensures that the rate of the metabolic reaction

is appropriate to the cells requirement by controlling the amount of enzyme protein produced by
the cell. It also regulates the interactions of the enzymes with some metabolic inhibitors and

activators, making it more rapid and reversible. Catalytic power refers to the ratio between the

enzyme-catalyzed rate to the uncatalyzed rate of the reaction. Enzymes exhibit extensive

catalytic power, hastening reaction rates as much as 1021 compared to uncatalyzed levels.

These results are far greater than with the use of other synthetic catalysts. Specificity on the other

hand refers to the selectivity of enzymes for their substrates. The substances upon which an

enzyme acts are called substrates. In the enzyme-catalyzed reaction, no waste products are

produced, for none of the substrates are diverted into nonproductive side reactions. The basis of

specificity is the exclusive interaction between an enzyme and a substrate during molecular

recognition based on structural complementarity. The site on the enzyme where the substrate

binds and catalysis occurs is known as the active site. Enzymes work by the lock and key

mechanism - a specific enzyme (key) is required to activate a substrate (lock). Enzymes can

sometimes be altered by changes in pH, temperature and concentration.

Enzymes are grouped into six major classes. Class 1, Oxidoreductases are the ones that

catalyze oxidation and reduction reactions. They include oxidases, reductases and catalases.

Kinases and transminases are included in Class 2, Transferases which transfer chemical groups

from one molecule to another molecule. Class 3, Hydrolases, catalyze the hydrolysis of chemical

bonds. Class 4, known as Lyases adds or removes groups from their substrates by forming

double bonds. Isomerases, Class 5, catalyzes the isomerization reaction. And lastly Class 6,

Synthetases or Ligases, forms bonds with ATP cleavages.

One of the most common enzymes known to man is Amylase. This enzyme is found in

human saliva. It has a shape that can recognize amylose (starch) a polysaccharide. It aids in the
hydrolyzation of -1,4-glycosidic linkages in amylose into disaccharides like maltose, and other

smaller sugar units. It performs best in the mouth where pH levels are neutral to slightly basic.

The objectives of the experiment are to determine the optimum pH and optimum

temperature to which amylase works best.

Methodology:

A. Effect of Temperature

An Enzyme solution was prepared by mixing 1mL of saliva with 9mL distilled water and

30mL of 0.5% NaCl. 2mL of the solution was then placed in a large test tube. In a separate

large test tube 2mL of Buffered starch solution was added. Both test tubes were incubated

for 5 minutes depending on the temperature of the water bath (room temperature). After 5

minutes, the contents of the test tube containing Buffered starch was transferred to the test

tube containing the enzymatic solution while it was still submerged in the water bath. Three

drops of the mixture was immediately mixed with 2 drops of Iodine solution onto the first

well of the spot plate. This was noted as the zero minute. After a one-minute interval, 3 drops

of the mixture was taken and mixed again with 2 drops of Iodine solution onto the second

well of the spot plate. This was noted as the first minute. The mixing of the iodine solution

and the mixture was repeated with a one-minute interval until a light yellow or light purple

color is observed. If no change in color was observed after 20 minutes, the time recorded was

automatically noted as . The time ellapsed was recorded and was then graphed to

determine the optimum temperature.

 B. Effect of pH

An Enzyme solution was prepared by mixing 1mL of saliva with 9mL distilled water and

30mL of 0.5% NaCl. 2mL of the solution was then placed in a large test tube. In a separate

large test tube a mixture of 1mL of Unbuffered starch solution and 1mL of the assigned pH

(pH=5) was added. Both test tubes were incubated for 5 minutes in a 37 C water bath. After

5 minutes, the contents of the test tube containing unbuffered starch was transferred to the

test tube containing the enzymatic solution while it was still submerged in the water bath.

Three drops of the mixture was immediately mixed with 2 drops of Iodine solution onto the

first well of the spot plate. This was noted as the zero minute. After a one-minute interval, 3

drops of the mixture was taken and mixed again with 2 drops of Iodine solution onto the

second well of the spot plate. This was noted as the first minute. The mixing of the iodine

solution and the mixture was repeated with a one-minute interval until a light yellow or light

purple color is observed. If no change in color was observed after 20 minutes, the time

recorded was automatically noted as . The time Dlapsed was recorded and was then

graphed to determine the optimum pH.

Results and Discussion:

The effects of Temperature and pH in the enzyme activity of Salivary Amylase was

determined by introducing the enzyme solution to different temperatures and pH and by

recording and measuring its rate. Upon the preparation of the enzyme solution, 0.5% NaCl was

added to activate the salivary amylase present to hydrolyze starch. The positive result or starch
 breakdown is indicated by a change in color of the solution from a dark blue color to a light

yellow or light purple solution.

A. Effect of Temperature

(Table 1. Results for the Effect of Temperature on Salivary Amylase)

Time (mins) Reciprocal of Time (

min1
Temperature (T)
4 C 0
28 C 4 0.25
37 C 2 0.50
50 C 3 0.33
70 C 0
Optimum Temperature: 37 C

At extreme temperatures the rate of enzyme activity increases because at high

temperature, the substrate molecules move faster so an enzyme is likely to come in contact with

its designated substrate more quickly. However enzymes are active within 38 C to 40 C. Below

38 C enzymes are less active and weak while above the range of 38 C to 40 C , the enzymes

are denaturated causing it to lose its biological enzymatic activity as shown in Table 1 .An

enzyme has its own optimum temperature where going above or beyond this temperature results

to a lost in the enzymes functionality.

0.6
0.5
0.4
0.3
1/ time ((
0.2
0.1
0
0 10 20 30 40 50 60 70 80

Temperature C
 (Figu

re 1. Graph of the reciprocal of time VS the temperature)

Figure 1 shows that enzymatic activity when plotted against temperature results as a bell-

shaped curve. The highest peak indicates the optimum temperature which is 37 C. At 4 C the

enzymatic reaction of salivary amylase does not or slowly occurs. But as the temperature

increases to 28 C (room temperature) and 37 C, the enzymatic reaction of salivary amylase

occurs since amylase is capable to hydrolize in such temperatures. But as the temperature

increases to 50 C and 70 C the enzymatic activity decreased in rate. The decline occurs because

temperature affects enzyme conformation. Thus the enzyme and the substrate no longer fits

together properly. The salivary amylase was denatured already.

B. Effect of pH

(Table 2. Results for the Effect of pH on Salivary Amylase)

Time (mins) Reciprocal of Time (

min1
pH
4 0
5 0

6.7 2 0.50
8 0
10 5 0.20
Optimum pH: 6.7

Enzymes act only on specific pH levels where they are most catalytically active. Most

enzymes have an optimum pH range of 7-8. Changes in pH also affect the enzymatic activity of
 salivary amylase. Like with temperature, pH can alter the structure and arrangement of enzymes.

Increase and decrease in pH causes the denaturation of enzymes resulting to a lost in the

enzymes functionality.

0.6
0.5
0.4
0.3
1/time ((
0.2
0.1
0
3 4 5 6 7 8 9 10 11

p
H

(Figure 2. Graph of the reciprocal of time VS the pH)

Figure 2. shows that when plotted against the pH, the enzymatic activity results as a bell-

shaped curved as well. The optimum pH is indicated by the highest peak which is 6.7. At a pH of

4 and 5, the solution is too acidic for the salivary amylase to function. The known optimum pH

for the action of salivary amylase ranges from 5.6 to 6.9 where it was observed in Figure 2. But

as the pH increases to 8 the Salivary Amylase was denatured. However as the pH increases to 10,

the Salivary amylase was functioning again. This error may be obtained by inaccurate timing and

measurement during the experiment. The pH effects enzyme reactions because it affects the

specific structure of the active site of the enzyme. The Amino acids which make up proteins are

affected by pH as they readily react with acids and bases. As the shape or polarity of the active

site changes, the effectiveness of the catalyst changes as well.

Conclusion:

Through the experiment, the optimum temperature of Salivary Amylase was determined

to be 37C and its optimum pH is 6.7.

References:

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Online:
Investigating the effect of pH on amylase activity. Retrieved September 20, 2016, from

http://www.nuffieldfoundation.org/practical-biology/investigating-effect-ph-amylase-activity