BIOLOGY 101

PRACTICAL 3: THE EFFECTS OF pH AND TEMPERATURE ON

ENZYME ACTIVITY

NAME: OARABILE FLORAH PULENG

PROGRAM: BSc EARTH AND ENVIRONMENTAL SCIENCES
STUDENT ID: 22000968
INTRODUCTION
Protein catalysts in biological systems are known as enzymes, according to research by Hertz,
McMillan, and Russell. According to Hertz et al., an enzyme's active site, where it attaches to
a protein or other substance during a reaction, is where a substrate—the substance an enzyme
acts on—binds. The substrate is then transformed into products by the enzyme. After the
products have left the area, the enzyme is ready to join with a different substrate and carry out
the process once more.
The induced-fit model, which is in line with Sadava et al. (2014), describes how the active
site alters when it interacts with the substrate. According to Eggebrecht and Zedalis (2015),
an enzyme-substrate complex is created when an enzyme binds to its substrate. This complex,
among other things, decreases the reaction's activation energy and speeds up its advancement.
As mentioned by Hertz et al. (2015), enzymes speed up reactions by reducing their activation
energy. According to Sadava et al. (2017), enzymes don't affect how much the free energy
changes or the equilibrium point of a process.
Certain non-protein molecules must be linked to certain enzymes in order for them to operate.
As stated by Savada et al., these are known as cofactors (2014). As an illustration, carbonic
anhydrase cofactor zinc ion. Coenzymes are what are referred to as other, say Eggebrecht and
Zedalis (2018). They increase an enzyme's activity. According to Sadava et al. (2017), certain
chemical inhibitors have the ability to bind to enzymes and slow the rates of the reactions
they catalyse. For instance, numerous medicinal medications like toxins and aspirin.
Enzymes are specialized, according to Hertz et al. Pepsin and trypsin are a couple examples
of specialized enzymes. Only certain circumstances allow enzymes to work. pH,
concentration of the enzyme, substrate, and product are those requirements. Temperature is
another environmental component that may impact enzyme function, according to
Wallenstein et al. (2012). There is no catalysis from the enzyme when the temperature is too
high because the active site is denatured. The enzymes won't have enough energy to work
when these conditions are below optimal levels.
The only factors considered in this experiment were the impact of temperature and pH on
enzyme activity. Amylase was the enzyme that processed starch and a combination of starch
and other substances. The experiment was put to the test to see if the hypothesis was
supported by it.
AIM
The goal of the experiment is to determine how the amount of heat and cold, as well as how
basic or acidic a material is, affects how quickly enzymes function in the body. Additionally,
ascertain the pH and temperature at which amylase performs best.

HYPOTHESIS
I believe that enzymatic activity will increase with a really high pH and temperature and vice
versa. Due to an increase in kinetic energy that causes molecules to move, heat typically has
the tendency to speed up reactions.

METHOD
This experiment was divided into two parts. Part A and Part B.
Part A of the experiment examined how temperature affects the activity of enzymes. One
through eight test tubes were assigned numbers. 1% starch solution was used in test tubes one
through four, whereas 1% amylase solution was placed in test tubes five through eight. The
first and fifth tubes were stored in ice coolers, the second and sixth tubes in 50°C water baths,
the third and seventh tubes in 70°C water baths, and the fourth and eighth tubes in 100°C
water baths. After waiting about five minutes, tubes were put into tubes 1, 2, 3, and 4,
respectively. The mixing time was noted (0 minutes). Tubes 1, 2, 3, and 4's positions were
adjusted. The contents from each test tube were sucked out onto a white hollow tile for 60
minutes, divided into 10-minute intervals, and a drop of iodine solution was added to check
for starch. Results were recorded for 10 minutes.
The experiment in Part B examined how pH affects the kinetics of enzymes. The labels on
the test tubes read "1 to 6." One millilitre of a 1 M sodium hydrogen carbonate solution, one-
half millilitre of a 1 M sodium hydrogen carbonate solution, one millilitre of water, one
millilitre of a 1 M acetic acid, and one millilitre of a 1 M acetic acid were added to test tubes
one through six, respectively. The pH of each mixture was then examined after 2mm of 1%
amylase solution had been added to each test tube. The pH paper's colour was then compared
to a pH-coloured chart. A water bath set to 50°C was then used to place the test tube. For an
hour, the pH and iodine solution were tested every 10 minutes recording results.
RESULTS
Section A
Table 1 Title; EFFECTS OF TEMPERATURE ON ENZYME ACTION

TIME Test tube 1 Tem Test tube Temp Test tube Tem Test tube 4 Temp
(mins) p 2 (℃) 3 p (℃)
(℃) (℃)
0 Brown-blue 2-4 Brown- 50 Brown- 70 Brown- 90
black blue black blue black blue black
10 Brown- 2-4 Brown- 50 Remains 70 Remains 90
blue black blue black brown brown
20 Brown- 2-4 Brown- 50 Remains 70 Remains 90
blue black blue black brown brown
30 Brown- 2-4 Brown- 50 Remains 70 Remains 90
blue black blue black brown brown
40 Brown- 2-4 Brown- 50 Remains 70 Remains 90
blue black blue black brown brown
50 Brown- 2-4 Remains 50 Remains 70 Remains 90
blue black brown brown brown
60 Brown- 2-4 Remains 50 Remains 70 90
blue black brown brown

Section B
Table 2 title; EFFECTS OF pH ON ENZYME ACTION
TIME Test p Test pH Test pH Test p Test pH Test pH
(Mins tube1 H tube 2 tube 3 tube 4 H tube 5 tube
) 6
0 Brow 8 Brown 9 Blue- 6 Brow 7 Brow 4 Blue 4
n black n n black
10 Blue- 10 Blue- 9 Blue 7 Blue- 6 Blue- 4 Blue 4
black black black black black -
black
20 Blue- 9 Black 9 yello 5 yello 4 Blue- 4 Blue 3
black w w black -
black
30 Blue- 10 Black 10 Blue- 3 Blue 2
black black -
black
40 Blue- 9 Black 10 Blue- 4 Blue 3
black black -
black
50 black 9 Black 10 Blue- 4 Blue 3
black -
black
60 black 9 Black 10 Blue- 3 Blue 4
 black -
black
KEY
Brown-blue black means that starch is present
Remains brown means that is starch displayed

DISCUSSION
The pattern of how amylase responds to changes in temperature and pH was examined in this
study using statistical techniques and observational skills. According to Gomez Villages et al.
(2014), amylases are a family of enzymes that break down starch into sugars.
The experiment disproves my theory. The experiment's findings for part A contradict the idea
that an extremely high temperature causes a speedier reaction. We had to wait 20 minutes for
amylase to start breaking down starch in test tube one. This demonstrates that a low
temperature does not supply the enzyme with sufficient kinetic energy to function, supporting
the hypothesis put out by Eggebrecht and Zedalis (2018) that the sample in the 50°C water
bath may have been tampered with in order to skew the results. It was too hot for amylase to
function, according to the results of samples taken from water baths at 70°C and 100°C.
Eggebrecht and Zedalis (2018) interpret this as a sign that the enzyme's active site had
denatured and was no longer capable of breaking down starch. This conclusion further
suggests that amylase is substrate-specific, as Hertz et al. (2015) had claimed.
Part B's findings support the first half of the hypothesis, which asserts that high pH causes
faster reactions. According to the findings from test tubes one and two, the starch was totally
dissolved. This suggests that amylase can function in simple circumstances. Test tube results
three and four reveal a very faint presence of starch. Amylase can therefore function in
neutral to slightly acidic environments. According to the results for test tubes five and six,
amylase is incapable of functioning in highly acidic environments. Since the test tube
samples show a colour change from black to blue, no starch was broken down. Starch must
therefore be present. Amylase did not function.
According to Trincone (2018), amylase has applications in the chemical, pharmaceutical, and
textile sectors as well as in the treatment of cellulosic materials, leather, and detergents, to
mention a few. Sidhu et al. (2018) claim that microbial enzymes like lipase and protease aid
in the creation of cheddar cheese and milk coagulation. Mehta and Satyanarayana claim that
the demand for consumer products and biofuels are the main drivers of the ongoing growth in
the worldwide market for microbial enzymes (2016). Among the most common industrial
enzymes used in the baking, alcohol, detergent, and textile industries are Amylase, according
to Yan and Wu (2016), in the biotechnological industries, amylase is improved by altering its
structural makeup as well as by cloning and producing specific amylase in various species.

CONCLUSION
This experiment's objective has been accomplished. While the second half of the theory was
shown to be incorrect, the first half was demonstrated to be accurate. As a result, the
experiment was a success because it revealed new data and helped determine the hypothesis'
actual findings. The findings suggest that temperature and pH are ideal for enzymes.
According to the findings, amylase functions best at a pH of 4 and a temperature of 50°C.

REFERENCES
Eggebrecht, J& Zedalis, J, 2018, Biology for AP Courses. USA; OpenStax
Sadava, D, Hillis, D.M, Heller, C.H& Berenbaum, M.R. 2014, LIFE; The Science of
Biology,10th ed. USA; Sinauer Associates and Freeman & Co
Hertz, P.E, McMillan, B & Rusell, P J, 2015, Biology; The Dynamic Sciences. 4 th ed. USA;
Cengage Learning