CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.
Title:

Test for the Effect of Temperature on Enzyme Activity.

Aim:

To Determine the Effects of Temperature on Enzyme Activity.

Introduction/Theory:

An enzyme may be defined as a protein that has catalytic

activity that is specific to a certain reaction(s). The enzyme
salivary amylase is produced by mammals and secreted in the
buccal cavity. This enzyme is capable of hydrolyzing starch.
Salivary amylase is classified as an endo-enzyme which means that
it hydrolyzes starch from the inside. Amylose hydrolyzes the 1
4 linkages of amylose which results in the formation of maltose
residues. Specific temperature and pH are required for enzymes to
function effectively and efficiently. A pH range of 5.6 - 6.9 is
optimum for salivary amylase. At 4C, salivary amylase has little
to no activity due to the insufficiency of energy / heat. As the
temperature increases from 4C, the activity of salivary amylase
increases steadily until it attains its optimal activity in its optimal
temperature range which is 32 C 37 C. After this temperature
range, the activity of salivary amylase decreases sharply. From 50
C the enzyme begins to denature, until 70 C at which the enzyme
is fully denatured and has zero activity. The structure of salivary
amylase is presented in figure 01.

Page 1 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.

Figure 1: Salivary Amylases Structure.

Starch is a polysaccharide that has a storage function in

plants. It is made up of glucose units linked by glycosidic bonds.
The chains made throughout the condensation reaction are either
linear or highly branched molecules. Linear molecules can be both
straight and helical, and these linear starch molecules are known as
Amylose. While the branched starch molecules are known as
Amylopectin. Natural starches, from plants, comprise of a
combination of amylose (10 - 25%) and amylopectin (75-90%). A
general structure of starch is shown in figure 02.

Page 2 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.

Figure 02: A general structure of starch.

Generally starch is considered to be insoluble. However, it
is soluble under certain conditions. In water, amylose is soluble
while amylopectin is insoluble. It is the opposite in organic
solvents. Starch can be found inside of the starch granules in plant
cells. These granules differ in size and shape according to species.
Iodine solution is used to test for the presence of starch.
The key to the iodine-starch reaction is the structure of the helical
amylose. A helix is either a coil or a spring. The presence of
promalt (which has -salivary amylase activity) would cause the
breakdown of the polysaccharide starch into simpler
carbohydrates, such as maltose/glucose (which are reducing
sugars.)
The red-brown iodine solution becomes blue-black in the
presence of starch. Therefore it is expected that the early parts of
the experiment there will be positive tests for starch .However, as
time processes and the starch is broken down into simple
carbohydrates (reducing sugars), the starch test would have
negative results.

Page 3 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.
The triiodide and pentaiodide ions formed in the iodine
solution are linear and fit inside the helix of the amylose. The
starch-iodide complex is formed as charge is transferred between
the starch and iodide ion. The transfer of charge between the starch
and the iodide ion alters the spacing between the energy levels.
This alternation results in the starch-iodide complex absorbing
light at a different wavelength, which results in an intense purple
colour know as blue-black. Foods which are high in amylose have
more intense blue-black colour when tested.
Materials Required:
1. Saliva
2. Distilled water
3. Iodine solution
4. 0.5% Starch solution
5. Timer
6. Camera
7. 10 test tubes with rack
8. Droppers
9. Measuring cylinders
10. Beakers
11. Ice water baths (0C, 10 C, 20 C and 30 C)
12. Water baths (40C, 50C, 60 C, 70 C, 80 C and 100 C)

Method:
1. In excess of 3 mL of saliva was provided from the buccal cavity of
one of the experimenters. The saliva was collected into a beaker.
3mL of the collected saliva was measured using a measuring
cylinder. This 3 mL of saliva was then diluted to 15 mL with
Page 4 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.
distilled water. The 15 mL of diluted saliva was used for the entire
experiment.
2. 10 test tubes were gathered and 3 mL of 0.5% starch solution was
added to each test tube.
3. Each of these 10 test tubes were then placed at a particular
temperature: 0, 10, 20, 30, 40, 50, 60, 70, 80 and 100 C by placing
it either in ice-bath or water bath.
4. After the tubes had reached their respective temperatures, 2-3
drops of the diluted saliva were added to the tubes and the contents
of the tube were mixed. The tube was then placed back into its ice
or water bath.
5. A few drops of the contents from the tubes were retrieved and
tested for starch by using iodine solution at one minute intervals.
6. The starch test was performed until there was no positive reaction
for starch.
Results:

The results that were gathered from this Laboratory Exercise are
tabulated in Table 1.

Table 01: Results that were gathered from this Laboratory Exercise.
1

10

Temperature / C

10

20

30

40

50

60

70

80

100

Ti

Test Tube Number

Page 5 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda

me / minutes

O Neil.
1

10

Key:
: Starch Present
: Starch not Present

Discussion:

At 0 C, the starch was not hydrolysed after four minutes. As stated

in the theory this was due to salivary amylase having little to no activity
due to the insufficiency of energy / heat at this temperature.
At 10 C and 20 C, the starch was not hydrolysed after six minutes
and ten minutes respectively. Because these temperatures are not the
optimum temperature at which salivary amylase works (32 C - 37 C), it
is not surprising that the starch was not hydrolysed.

Page 6 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.
At 30 C, which is very close to the optimal temperature range for
salivary amylase (32 C - 37 C), the starch was hydrolysed by the third
minute. This was expected to happen as the activity of salivary amylase
would be high at this temperature.
At 40 C, the starch was not hydrolysed after ten minutes. Because
this temperature is beyond the optimum temperature at which salivary
amylase works (32 C - 37 C), it is not surprising that the starch was not
hydrolysed.
At 50 C and 60 C, the starch was not hydrolysed after ten
minutes in each case. Because these temperatures are not the optimum
temperature at which salivary amylase works (32 C - 37 C) along with
the fact that at these temperatures, the enzyme salivary amylase gets
denatured, it is not surprising that the starch was not hydrolysed.
At 70 C and 60 C, the starch seemed to have hydrolysed after
five minutes. Because this temperature is not the optimum temperature at
which salivary amylase works (32 C - 37 C) along with the fact that at
this temperature, the enzyme salivary amylase gets denatured, it is
surprising that the starch was hydrolysed. The experimenter suspects that
this was an experimental error, perhaps the colour change was
misinterpreted or salivary amylase of a temperature close to the optimal
range for salivary amylase like 30 C was added at the fifth minute instead
of the salivary amylase at 70 C.
At 80 C and 100 C, the starch was not hydrolysed after five and
six minutes respectively. Because these temperatures are not the optimum
temperature at which salivary amylase works (32 C - 37 C) along with
the fact that at these temperatures, the enzyme salivary amylase was
denatured, it is not surprising that the starch was not hydrolysed
Page 7 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.
Conclusion:

According to this Laboratory Exercise, it can be concluded that;

The temperature at which the enzyme salivary amylase exists

affects it activity
Salivary amylase has high activity at 30 C
The Activity of Salivary amylase is so low at temperatures
other than 30 C, that the activity cannot be detected.

Recommendations:

It is recommended that;

That the pH of the systems being studied be maintained at the

optimal pH for salivary amylase, which is, 5.6 - 6.9. If this
parameter is not controlled then the true effect of temperature
on the activity of salivary amylase cannot be known as the pH

affects the activity of salivary amylase as well.

The source of amylase should be from recognized supplier
such as Sigma-Aldrich and not the experimenters tongue. This
should be so because the amylase in the saliva of an
experiment would not be standard and introduce more variables

into the experiment.

Sophisticated equipment should be used to provide water or ice
baths at accurate temperatures since having to manually adjust
the temperatures of the baths is tedious and this can lead to
affect the accuracy of the experiment.

Page 8 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.

References
1. Wang, N.S. (n.d.). Experiment No. 5 Starch Hydrolysis by Amylase. Retrieved on
September 29, 2015, from http://www.eng.umd.edu/~nsw/ench485/lab5.htm
2. Iodine Test for Starch. (n.d.). Retrieved September 2, 2015, from
http://brilliantbiologystudent.weebly.com/iodine-test-for-starch.html
3. Brewing Aids |. (n.d.). Retrieved September 28, 2015, from
http://gilbertsonandpage.com/products/brewing-aids/
4. The Effects of Temperature and pH. (2012, December 3). Retrieved September 30, 2015.

Page 9 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.

Appendix 1
A few images of the Laboratory Exercise being carried out are as follows; image one shows the
preparation of one of the water baths while image two and three show the enzyme being
subjected to various temperatures.

Page 10 of 11

CHM 4103 Lab Report 2.

GOKHUL, Raja.

September 28, 2015.

Registration Number: 14/0705/0763.

Lecturer: Professor Brij B. Tewari

USI: 1015485.

Group 1; Group Partners: Leanna Simon and Alicia Sukhdeo, Xiomara Griffith and Amanda
O Neil.
1

Page 11 of 11