Lab Report Biology Foundation in Science

Name: Fatimatuzzahra’ binti Hardiyono

No. Matric: FIS09210001
Lecture’s Name: Madam Roskiyani binti Mistamiruddin
Lab Experiment: Factors Affecting Enzyme Activity
Lab Date: 22 June 2022 Sem: Semester 3 Intake: September 2021

Introduction:

An enzyme is a protein molecule that serves as a biological catalyst or a substance that can increase
the rate of a reaction without changing its own structure. Enzymes catalyze reactions that occur
inside living things (i.e. metabolic reactions). They work by binding to a substrate (reactant) at
their active site to form an enzyme-substrate complex and converting the substrate into a product(s)
of the chemical reaction. Once the enzyme converts the substrate it releases the product(s) and
leaves the reaction in the same exact form it was introduced. Because enzymes are not altered
during the reactions they can be used over and over again. Enzyme + Substrate? (Enzyme-substrate
complex). Enzyme + Product Enzymes usually catalyze only specific reactions. For example, the
enzyme amylase only catalyzes the conversion of starch to maltose. For instance, most enzymes
found in the human body function most efficiently at 37 ºC (98.6 ºF). When an enzyme is outside
its optimal temperature and pH range it works more slowly or sometimes does not work at all. Like
all proteins, enzymes are made up of a chain of covalently bonded amino acids (primary structure)
that bend, turn and fold to form secondary, tertiary, and quaternary structures, which make up its
3D shape. The shapes of enzymes are the result of weak bonds (i.e. hydrogen bonds) that occur
between non-adjacent amino acids. This 3D shape allows them to properly catalyze reactions.
Enzyme denaturation occurs when it is subjected to excessive heat or extremely high or low pH
(denaturing conditions). When an enzyme is denatured it loses its quaternary, tertiary, and
secondary structure and becomes a chain of amino acids linked by peptide bonds (or covalent
bonds that occur between adjacent amino acids). This primary structure usually stays the same,
because the peptide bonds are too strong to break. However, enzymes that have lost their 3D shape
no longer function.
Objective:

1. To define catalyst, enzyme, substrate, product, optimal conditions, denaturation, peptide

bond, and hydrogen bond.
2. To distinguish when an enzyme is functional and when it is denatured (non-functional).
3. To identify the effects of environmental factors, such as heat and acidity, on enzyme
activity.
4. To test for amylase activity on starch molecules using the iodine test.
5. To understand the difference between the primary and tertiary structure of proteins

Material and Apparatus:

4 test tubes.
Solution A: A solution of water (no amylase) and starch.
Solution B: A solution of untreated amylase and starch.
Solution C: A solution of amylase treated with HCl (a very strong acid) and starch.
Solution D: A solution of amylase that has been boiled for 5 minutes and starch.
Iodine (to test for starch).
Wax pencil to mark the tubes.
Tube rack.
A beaker of boiling water.
A 37 ºC water bath.

Procedure:

Part A
Four solutions will be set up. This experiment will contain 2 controls for comparison and 2
experimental tubes. One of the experimental tubes will test the effects of a low pH on the enzyme
(tube 3) and one of the experimental tubes will test the effects of extreme heat on the enzyme (tube
4). The solutions were made immediately before the lab to cut down on time.
 1. 4 test tubes labelled as A, B, C, and D.
2. 1 full dropper of distilled water and starch was added into test tube A.
3. 1 full dropper of untreated amylase and starch was added into test tube B.
4. 1 full dropper of amylase treated with HCl and starch was added into test tube C.
5. 1 full dropper of amylase that has been boiled for 5 minutes and starch was added into test
tube D.
6. 4 tubes were in the 37 ºC water bath.
7. The tubes were incubated for 15 minutes.

Part B
Now each tube will be tested for the presence of starch by using the iodine test. If starch is still
present in the tube then the amylase was not functional (i.e. it did not change the starch into
maltose).

1. Two drops of iodine were added to each test tube.

2. The test tubes were swirled until obtained changes in colour.
3. The observation was recorded in the table below.

Data Recording:

Test tube Colour Changes of Iodine

A

Turns black
B

Colourless
C

Yellow
D

Almost colourless (have a small spot of yellow colour)

Discussion and Conclusion:

From the data recording above, in test tube A, the changes of colour occurred because there was
no presence of an amylase enzyme (because test tube A only has distilled water) that can simplify
starch into simple sugar (maltose), which makes the iodine colour (yellow) turns black (because
iodine reacts with the starch; which mean there still have the presence of starch in that solution).
For test tube B, the iodine turns colourless which shows that the enzyme amylase hydrolyses starch
completely. For test tube C, the iodine colour remains the same (yellow). It is because the enzyme
amylase was denatured due to decreasing pH. Enzyme amylase is most active at pH 6.8, the
reaction rate will decrease as hydrochloric acid (HCL) provides a much acidic environment (pH
for HCL between 1.5 to 3.5) for amylase to function at its maximum strength. Lastly, for test tube
D, the amylase enzyme was exposed to high temperatures. When exposed to extreme temperatures,
enzymes denature. Upon boiling enzymes, since they are heat-sensitive, they easily deactivate.
Almost all enzymes get deactivated over 47°C temperature. Boiling enzymes break the ionic and
hydrogen bonds which are held in place. Its structure of it is disintegrated and hence they will be
unable to form a complex with the substrate. But according to the observation above, the solution
turns almost colourless with a small spot of yellow colour appearing which shows that even at high
temperatures, the amylase enzyme can still break starch down slowly but not completely. To
conclude, the effectiveness of an enzyme will be affected if one of many contributing factors was
present.

Questions:

1. What is the function of acid hydrochloric (HCL)?

The function of acid hydrochloric in this experiment is as an enzyme inhibitor. An enzyme

inhibitor is a molecule that binds to an enzyme and blocks its activity.

2. Why do we need the 37°C water bath?

 37°C is human body temperature. At this temperature, most enzyme functions are
performed because the enzymes are able to retain their structure at that temperature,
allowing them to break down complex molecules efficiently.

3. List down and explain factors that contribute to the enzyme activity.

Factors Explanation
Substrate The activity of an enzyme also increases with the increase in substrate
concentration concentration. If the substrate concentration increases, then the
availability of the active site would decrease. This will affect the activity
of an enzyme and limit the reaction rate.
pH Each enzyme has its optimal pH in which they work. For example,
pepsin and trypsin work on acidic pH. The enzymes are globular
proteinaceous structures, formed by the interaction of the hydrogen bond
between the side chains of the protein. Any change in the causes
deionization of the side-chain which results in the denaturation of the
enzyme.
Temperature Each enzyme works at its optimal temperature. Any alteration in
temperature affects the activity of an enzyme, and it also leads to the
denaturation of an enzyme.
Enzyme Each enzyme requires cofactors (inorganic ion or protein organic
cofactor and molecules) for its work. The non-availability of these cofactors
coenzyme decreases the activity of an enzyme.
Enzyme The inhibitors of an enzyme bind to the active site which affects the
inhibitors activity of an enzyme.
References:

1. Toppr. (2020). Explain the factors affecting enzyme activity.

https://www.toppr.com/ask/en-my/question/explain-the-factors-affecting-enzyme-
activity/

2. Wikipedia. (2022). Enzyme Inhibitor. https://en.wikipedia.org/wiki/Enzyme_inhibitor