Experiment #: 4

Determination of the Optimum pH and Temperature

of Salivary Amylase

Oa, Bill Clinton N.

Pasion, Mark Lorenz S.

*Pureza, Beatriz Soneth M.

Ragudo, Gabriel Phillip P.

Department of Biological Sciences

College of Science, University of Santo Tomas

Espaa, Manila, Philippines

ABSTRACT
Enzyme is a biological catalyst. An example of this is the salivary amylase found in saliva of animals. This salivary
amylase breaks down starch into maltose. It performs best at its optimum temperature and optimum pH, therefore,
changes in temperature an pH value affects the effectiveness of the enzyme. It was determined that the optimum
temperature of salivary amylase is within the range of 32-37C and the optimum pH is 6.7

Keywords: enzyme, pH, salivary amylase, temperature, optimum temperature, optimum pH, denatures

I. INTRODUCTION

According to Appling, an enzyme is a biological catalyst and is usually a protein

molecule but is sometimes a RNA molecule or ribonucleoproteins the proteins in enzymes are
usually globular. Catalysis is the acceleration of the chemical reaction by a substance which

undergoes no permanent chemical change. And so

enzyme as a catalyst speeds up the rate of

reaction in a living cell . These enzymes are the

most efficient catalyst known, according to

Campbell. It performs specific activities with the

aid of a substrate and it is very specific and may need a coenzyme for carrying out an activity. It

also has an active site where the substrate binds and forms a complex. The substances formed

after the reaction is called product. Figure 1 shows the enzyme-substrate complex.

Salivary amylase is an enzyme found in the saliva produce by the salivary gland of the

mouth and is responsible for breaking down starch, which is the first process of digestion. It is a

type of hydrolase enzyme which catalyzes hydrolysis reaction. Salivary amylase is -amylase

which cleaves internal is (1 4) linkages of both polymers. -Amylase degrades amylose to

maltose, a disaccharide, and a little glucose. (Appling et.al., 2016)

Enzyme activity can be affected by several factors. These factors include temperature,

pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or

activators but in this experiment, it will only focus on the effects of temperature and pH. As

mentioned above, proteins in enzymes are usually globular. The intra- and intermolecular bonds

that hold proteins in their secondary and tertiary structures are disrupted by changes in

temperature and pH. This affects shapes and so the catalytic activity of an enzyme is pH and

temperature sensitive. The objective of this experiment is to determine the optimum temperature

and the optimum pH of salivary amylase.

II. METHODOLOGY

Reagents:

Enzyme solution Acetate buffer

Buffered Starch Unbuffered Starch

1.1 M Iodine ( I 2 ) Solution

Materials:

Test Tube Spot plate

Dropper
Procedure:

In this experiment two effects on enzyme activity of salivary amylase were observed,

effect of temperature and effect of pH. Before the experiment proper, the class were divided into

two groups, team A and team B, and performed under similar situation. Each team had one

representative to prepare the enzyme solution with the use of their own saliva. 1 mL of saliva

was obtained and mixed with 9 mL distilled water and 30 mL 0.5% NaCl.

For the first activity, the effect of temperature was observed. It was done by putting 2 mL

of enzyme solution in a large test tube and labeled it as 37C (temperatures 4C, room

temperature (32C) , 50C, 60C and 70C were done by other groups). Next, in a separate test

tube, 2 mL of buffered starch was obtained. Both test tubes were incubated with the use of body

temperature for 10 minutes and were mixed immediately after incubation. 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; it has been recorded as 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; it has been one minute. The procedure

was repeated until light yellow colored form, it will be recorded as the last minute.

For the second activity, the effect of pH was observed. First 1 mL of acetate buffer with a

pH of 6.7 (pH 4.0, 5.0, 8.0 and 10.0 were done by other groups) and 1 mL of unbuffered solution

were mixed in a large test tube, then in a separate test tube, 2 mL of enzyme solution was

obtained. Both test tube were incubated for 10 minutes using the normal human body

temperature of 37C and were mixed immediately. Just like in the first activity 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; it has been recorded as 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; it has been one minute. The procedure

was repeated until light yellow colored solution was formed; it will be recorded as the last

minute. For both activities its optimum temperature and optimum pH were determined.

III. RESULTS AND DISCUSSION

It was mentioned earlier that salivary amylase is an enzyme obtained from the saliva. The

enzyme solution was prepared from the saliva of a student mixed with 9 mL water and 30 mL of

0.5 % sodium chloride (NaCl). The addition of water was to dilute the saliva into a more

appropriate concentration. Addition of NaCl is to disrupt the hydrogen ions that hold as a bridge

in the active site of the enzyme.

 As mentioned above, temperature has an effect on the enzyme activity of salivary

amylase. Temperature is the degree or intensity of heat present in a substance or object,

especially as expressed according to a

comparative scale and shown by a thermometer or

perceived by touch. Like in most chemicals, the

reaction rate slows down as the temperature

decreases and it increases as the temperature increases. As this chemical potential energy

increases it will be enough to weaken and break the bonds that determines the three dimensional

shape of the active proteins. This may lead to a thermal denaturation of the protein and thus

inactivate the protein. Thus too much heat can cause the rate of an enzyme catalyzed reaction to

decrease because the enzyme or substrate becomes denatured and inactive. Table 1 shows the

time a color reaction occurred when the mixture of enzyme solution with buffered starch solution

incubated in 37C is dropped with iodine solution.

Iodine test is used to determine the presence of starch in a solution. Starch will give a

dark blue color as an indication of its presence. But as the color turns to light yellow it denotes

the complete breakdown of starch in the solution. This light yellow color is achieved when it

reach its optimum temperature. It is when the enzyme shows its maximum activity at a specific

temperature. The rate of reaction above or below this point of temperature is slower. A tabular

form of the rate of reaction with different temperature is shown in table 2. To further explain it is

shown in figure 2, that at 4C the activity of enzyme is slowly due to lack of heat but as the

temperature raises the enzyme activity gets faster until it reach its optimum temperature. In this

experiment the optimum temperature of salivary amylase is said to be within the range of room

temperature which is 32C and human body temperature which is 37C. At this point, the
salivary amylase is at its highest activity; therefore its rate of reaction is faster compared to those

lower than the optimum temperature. It was also

shown that those temperature higher than the range,

which in this case is 50, 60, 70 C, the rate slows

down since the amylases active site is damaged and

its shape is changed. The substrate no longer fits into

the active site and enzymes are basically denatured. It is because at high temperature, the heat

vibrations inside the enzyme break the bond need to maintain the structure.

1.2
1
0.8
0.6
1 , ^(1)
0.4
0.2
0
4 roomtemp 37 50 60 70
Temperature (C)

Figure2. Effect of Temperature

It was also mentioned that pH also affects the

enzyme activity of salivary amylase. pH is a figure

expressing the acidity or alkalinity of a solution on a

logarithmic scale on which 7 is neutral, lower values are

more acid, and higher values more alkaline. Enzymes have active sites where the substrates

bind. These active sites are damaged or in other words their shape is changed by changing the

pH. Table 3 shows the time a color reaction occurred in the mixture of enzyme solution with

unbuffered starch and acetate buffer with pH 6.7.

 Just like the first activity, iodine test was also used to detect the presence of starch. Light

yellow color is achieved when it reached its most favorable pH value - the point where the

enzyme is most active known as the optimum pH.

Above and below this range, the reaction rate

reduces as enzymes get denatured. Extremely

high or low pH values generally result in

complete loss of activity for most enzymes. As

with activity, for each enzyme there is also a

region of pH optimal stability. A tabulated form of rate of reaction with chaing pH value is

shown in table 4.

As shown in figure 3, pH 4.0 the salivary amylase is in a too acidic environment. pH in a

too acidic environment causes amino acids to protonate which consequently denature enzyme.

As the pH level increase its rate of reaction is faster until it reaches its optimum pH 6.7. Here, the

amylase functions best. As the pH reaches higher than optimum pH, the rate of reaction begins to

slow down and denatured because of being in a too basic environment.

1.2
1
0.8
min^ (-1) 0.6
0.4
0.2
0
4 5 6.7 8 10
pH Value

Figure 3 Effect of pH
IV. CONCLUSION

Salivary amylase has its own optimum temperature and optimum pH. It is when the

salivary amylase works at its best that requires lesser amount of time and can produce more

product than those higher or lower than its optimum pH and temperature. The optimum

temperature of salivary amylase is within the range of room temperature, 32C and human body

temperature, 37C. While the optimum pH is said to be 6.7.

V. REFERENCES

Appling, D. R., Anthony-Cahill, S. J., & Mathews, C. K. (2016). Biochemistry: Concepts and

connections. England: Pearson Education Limited.

Campbell, M.K., & Farrell, S.O. (2013). Biochemistry. Canada: Cengage Learning.

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