Effects of Temperature and pH on the Enzymatic Activity of

Salivary Amylase

Gutierrez, Tracey Antaeus D., Ilagan, Ivylyn T., Jaen, Carmella Gloria D.*, Lim,

Leslie Cathleen T.

College of Science, University of Sto. Tomas, Espaa Blvd., Manila

Abstract

Salivary amylase or ptyalin was characterized in order to determine the effect of temperature and

pH on its activity to hydrolyze starch. Samples of the enzyme were subjected to different trials

involving changes in pH and temperature. The time wherein the hydrolysis occurred was noted

and its values were plotted in a graph. Results revealed the optimum pH as 6.7 and the optimum

temperature as 50C.

Introduction

Numerous reactions essential to life are characteristic of three-

dimensional proteins called enzymes. Enzymes are catalytic proteins produced

by living cells; they are able to accelerate chemical reactions up to about 10 3 to

107 more as compared to uncatalyzed reactions (Wellner, 2016; Moran, 1994). In

the absence of enzymes, these reactions would not take place at a significant

rate. In fact, most bodily chemical reactions would be become useless.

One important bodily activity that involves enzymes is the digestion of

food. The enzyme that aids in this process is called salivary amylase. Salivary

amylase, also known as -amylase or ptyalin, is an enzyme found in human

saliva, pancreatic juice, human breast milk, serum and certain tissues such as

the liver which is responsible for the breaking down of starch into maltose and

glucose by hydrolyzing the (1-4) linkages in starch (Enemchukwu, Ubaoji,

&Igwilo, 2013). Salivary alpha amylase functions as the first main step of the

process of digestion. The process of digestion begins with the chewing of food in

the presence of salivary alpha amylase in the mouth to convert the starch in food

into sugar into dextrins and maltose (Maureen, 2000). Starch is the the substrate

for ptyalin (-amylase). It is a carbohydrate whose major polymeric components

amylose and amylopectin (Wellner, 2016). Amylose is a linear polymer of

glucose, with all the residues linked together by (1 3 4) bonds while

Amylopectin is a branched chain polymer, with the branches starting at (1 3 6)

linkages along the chain of (1 3 4) linkages (Campbell, 2015).

The activity of enzymes is strongly affected by several factors, such as

temperature and pH (Horton, 2002). At certain levels of pH and temperature,

they take less time in producing products. The points at which the enzyme is at

its best state are known as the optimum pH and the optimum temperature.

Holum (1996) states that the functional activity of saliva occurs within, 5.8-7.1

range for pH and 35-37C for temperature. From that, a study done by

Enemchukwu, Ubaoji, and Igwilo (2013) concludes that the optimum pH for saliva

is pH 7 while the optimum temperature is 37C.

Hence, in this experiment (1) aims to examine the enzymatic activity and

specificity of salivary amylase depending on the different values of pH and

temperature and (2) determine the optimum pH and optimum temperature of

salivary amylase.

Methodology

Prior to the principal process in the experiment, an enzyme solution was

first prepared. This was done by first obtaining saliva samples from two members

of the class. One mL of this sample was taken and added to 9mL of distilled

water and 30mL of 0.5% NaCl. The resulting mixture of these solutions was

termed as the enzyme solution.

In order to determine the effect of temperature on the enzyme solution,

2mL of it was placed into a large test tube. After which, 2mL of buffered starch

solution (1% starch in phosphate buffer pH 6.7) was also placed in another large

test tube. Both tubes were placed in an ice bath (4C), consisting of a beaker

with ice, for 5 minutes. With both tubes remaining in the beaker, the buffered

starch solution was then poured into the other tube with the enzyme solution and

immediately mixed. Three drops of the mixture were then instantly put into a spot

plate, followed by the almost simultaneous addition of 0.001M Iodine solution.

The resulting mixture appeared purple in color. This was labeled as zero

minute. After one minute of the previous mixture remaining in the spot plate,

three drops of the incubated mixture and two drops of iodine solution were again

taken and placed into the adjacent spot plate well of the first mixture. This

solution was labeled one minute. Proceeding the following minute, the same

procedure was repeated until when the mixture in the spot plate well will appear
yellow. This was termed as the final minute was recorded as an amount of time

resulting from the 4C temperature.

The following temperature values to be tested were at room temperature,

which obtained 28C when measured, 37C, 50C, and 70C. This was achieved

by repeating the preceding procedure while only altering the temperature of the

bath as needed. After gathering all the final minutes from each temperature, the

values were plotted into temperature versus 1/time graph and connected in order

to form a bell shaped curve. The optimum pH will then be determined from the

graph.

The determination of the effect of changes in pH is similar to the

processes made for the effect of temperature. However, 1 mL of acetate buffer

(pH 4) and 1 mL of 2% unbuffered starch were mixed together in large test tube.

Similarly, 2 mL of the enzyme solution was also added in a separate large test

tube. Both test tubes were incubated for 5 minutes in a 37C water bath. After

this, the starch solution was poured into the tube containing the enzyme solution,

and immediately mixed, with the latter while remaining in the water bath. After

which, three drops of the resulting mixture were immediately taken and placed on

a well in the spot plate with two drops of iodine solution almost simultaneously

added. This resulting mixture appeared a dark purple. This well was labeled as

the zero minute. After one minute of the previous mixture remaining in the spot

plate, three drops of the incubated mixture and two drops of iodine solution were

again taken and placed into the adjacent spot plate well of the first mixture. This

solution was labeled one minute. This time at which the solution will appear
yellow was be termed as the final minute, and was recorded as an amount of

time resulting from pH 4.

Identical to the determination of the effect of temperature, everything was

done in a similar manner, although using different pH levels instead of different

temperatures by the appropriate buffered pH solution. The buffered solutions

used were the acetate buffer for pH 4 and pH 5, phosphate buffer for pH 6.7 and

pH 8, and bicarbonate buffer for pH 10. The temperature bath remained to be at

37C. After gathering the time values obtained at different pH levels, they were

plotted into a pH versus 1/time graph and connected in order to form a bell-

shaped curve. The optimum pH will then be determined from the graph.

Results and Discussion

Table 1. Effect of Temperature on Enzyme

Temperature (C) Time (min) 1/Time (min-1)

4 0

RT (28) 5 0.20

37 2 0.50

50 1 1.00

70 4 0.25
 1.2
optimum temperature: 50C
1
1/Time {min-1)
0.8
0.6
0.4
0.2
0
0 10 20 30 40 50 60 70 80
Temperature ( C)

Graph 1. Temperature vs. 1/Time Graph

Table 2. Effect of pH on Enzyme.

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

4 0

5 0

6.7 2 0.50

8 13 0.33

10 6 0.17

0.6
0.5 optimum pH:6.7
Time (1/min-1)

0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12
-0.1
pH

Graph 2. pH vs. 1/Time Graph

 Salivary amylase is an enzyme responsible for cleaving the the (14)

glucosidic bonds of amylose and amylopectins found in starch (Enemchukwu,

Ubaoji, Igwilo, 2013), and is the enzyme that is the focus of this experiment. In

order to obtain the enzyme, saliva samples were taken from two members in the

class. This is because according to Campbell (2015) saliva is contains the

enzyme to be studied, the -amylose. This pure saliva was diluted with distilled

water in order to obtain appropriate concentration. The addition of the sodium

chloride (NaCl) was done in order to provide the enzyme with a necessary

cofactor, the chloride ion, which further aids starch hydrolysis (Metzler, 2001).

Certain factors can the enzymatic activity of a protein such as salivary

amylase. Such factors are temperature and pH, which are independent variables

for this experiment. Futhermore, in both determinations of the effects of

tempearature and pH, the hydrolysis time will be the basis for results or the

dependent variable.

In determining the effect of temperature, a buffered starch solution

containing 1% starch in phosphate buffer was used. This starch is the substrate

to be acted upon by the amylase. The phosphate buffer was used because it has

a pH of 6.7, which is within the range wherein the salivary enzyme is functional

(Holum, 1996). The pH must remain constant because the only independent

variable in this experiment is the temperature no the pH. Changes in pH may

affect the results. Identical temperature was obtained in both solutions of the

buffered starch and enzyme solution by incubating both tubes in the same bath
and the same time. Once the states of the solutions are appropriate both were

mixed together. This mixing allows the amylase in the enzyme solution to start

the hydrolysis in the starch found in the starch solution. The mixture is kept on

the water bath to maintain its current state and conditions. The addition of three

drops of the incubated mixture with the two drops of Iodine solution, is done

immediately, if possible, almost simultaneously, in order to obtain a accurate

result regarding the moment when the amylase takes action on the starch. Iodine

here is used because it serves as a starch detector for the mixture on the spot

plate, appearing a dark purple color (Holum, 1996). This color indicates the

presence of starch in the solution. In the final minute wherein the iodine solution

does not produce anymore a purple solution shows that starch has been

hydrolyzed and it is now absent. This resulting solution appears yellow color after

adding iodine. It is at this state of time that the enzyme has hydrolyzed the

starch. This time, the final minute, was noted and plotted in a temperature versus

1/time graph. The time was reciprocated in order to show an inverse relationship

between time and the reaction rate. This reciprocation allows us to obtain the

speed of the reaction.

The optimum temperature is determined by looking at the highest point in

the graph. This is because, that point represents the temperature that allowed

the fastest hydrolysis. In this experiment the optimum temperature obtained was

50C (see Graph1). This means that at this temperature, salivary amylase works

most efficiently. However, this result must not occur because at high

tempeartures such as 50C and 70C the proteins are denatured. During
denaturation there is destruction of the non-covalent interactions between amino

acids in the enzyme, resulting to a change in its enzymes secondary or tertiary

structure and consequentially making it nonfunctional (Appling, Anthony-Cahill &

Mathews, 2016). The enzyme does not also become efficient at low

temperatures such as 4C because enzyme is in its very rigid or frozen inactive

form (Appling, Anthony-Cahill & Mathews, 2016). Thus, the perfect temperature

must be within the 35-37C as this temperature is just the right amount wherein

the protein is not inactive nor nonfunctional (Horum ,1996). The incorrect result,

concluding the optimum temperature to be 50C, may be attributed to human

error such as the unsimultaneouse addition of iodine to the mixture of the two

tubes or the failure to keep the tubes in the water bath during the incubation or

transfering by allowing the evaporation of water from the bath or by simply not

following instructions.

In determining the effects of pH, three different buffer solutions of different

pHs were used: acetate buffer for pHs 4 and 5, phosphate buffer for pHs 6.7 and

8, and bicarbonate buffer for pH 10. This was done in order to maintain the pH

level during examination, allowing the determination of effect of pH through the

measurement of the hydrolysis time. Both buffered starch solution and the

enzyme solution were put in a temperature bath at 37C in order to maintain

constant temperature for all. The temperature 37C was used because it is the

physiological temperature, at which most biomolecules function (Campbell,

2015). Mixing both the solutions allows the salivary amylase take action on the

starch, and start the hydrolysis reaction. The Iodine is immediately added in order
to obtain an accurate initial result for the data. Again, Iodine serves again as a

starch indicator, in which appears dark purple in the presence of starch. At the

final minute, when the mixture yields a yellow color, it indicates that the starch

has been completely hydrolyzed. Thus, the starch was been cleaved into pieces

by the enzyme is not present anymore in the solution. The hydrolysis time was

also recorded and its reciprocal was plotted in a graph.

Graph2 shows that optimum pH being 6.7. This is the level at which the

enzyme works most efficiently. This result matches with the statement Holum

(1996) indicating the optimum range to be within 5.8-7.1. The enzyme was not

efficient at a low pH, like pH 4, because the acidic environment causes

denaturation. At this state the hydrogen ion concentration increases, and

protonates most R groups present on the enzyme, destroying the noncovalent

interaction between the amino acids in the enzyme. This changes the enzymes

secondary or tertiary structure, finally destroying the whole protein (Appling,

Anthony-Cahill & Mathews, 2016). The high pH, like pH 8 and pH 10, also

denatured causes denaturation because the increased concentration of

hydroxide ions deprotonate the R groups in the enzyme to produce water. This

also causes the noncovalent interactions between the amino acids to be

destroyed, changing the shape and configuration of the protein, and finally

destroying the enzyme (Appling, Anthony-Cahill & Mathews, 2016).

Conclusion

The enzymatic activity of salivary amylase is dependent on certain

temperature and pH ranges. If the temperature is to high, the protein will be

denatured thus destroying the protein. If it is too low, the protein will be in a

frozen inactive state. At both extreme highs and lows of pH the protein is

denatured because of the high concentrations of hydrogen and hydroxide ions

respectively. Based on this experiment, its optimum temperature is 50.0C and

an optimum pH is 6.7. In this state, it produces the more products in a smaller

amount of time.

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