EXERCISE 5: EFFECT OF

PHYSIOCHEMICAL FACTORS
ON FUNCTIONALITY OF
CELLULAR MOLECULES
The Effect of Chemical and Physical Factors on the
Functioning of Lysozyme

Merapelo J Thekiso
201700141
The Effect of Chemical and Physical Factors on the Functioning of Lysozyme
Aim
The aim of this laboratory experiment is to investigate the effect of temperature on the
defensive role of lysozyme, the effect of lysozyme on the bacterial cell wall and the light
scattering properties of M. luteus.
Introduction
Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrolase is an
antimicrobial enzyme produced by animals that forms part of the innate immune system.
Lysozyme, enzyme found in the secretions (tears) of the lacrimal glands of animals and in
nasal mucus, gastric secretions, and egg white. Discovered in 1921 by Sir Alexander Fleming,
lysozyme catalyzes the breakdown of certain carbohydrates found in the cell walls of certain
bacteria. Lysozyme can break the chemical bonds in the outer cell wall of the bacteria.
Bacterial cell walls contain a layer of peptidoglycan, which is the specific site that lysozyme
targets. If lysozyme is present, the bacterial cell wall gets damaged and the absorbance of
bacterial suspension decreases, and the decrease can be taken as a reasonable measure of
the enzyme’s activity. Gram-positive cells are quite susceptible to this lysis as their cell walls
have a high proportion of peptidoglycan. Gram-negative bacteria are less susceptible due to
the presence of an outer membrane and a lower proportion of peptidoglycan. Lysozyme
functions best at a pH range of 6.00-9.00 and a temperature range of 30-90oC with its
maximal activity being at pH 6.6 and temperature of 40oC.
Methods
Refer to the University of Botswana, Faculty of Science, Department of Biological Sciences,
Cell Biology, Bio 211, Laboratory Manual, Academic Year 2018/2019, pages 17-18, Part A,
Part B and Part D.
Results

Absorbance @ 600nm (ABS)

0.5

1.5

2.5
0

2
0
Fig. 1: The Relationship Between Amount of

0.02
Bacteria and Absorbance

0.04
0.06
Volume (ml)
0.08
0.1
0.12
0.14
0.16

Trend: As the amount of bacteria (ml) increases, the absorbance at 600nm (ABS) increases.
 Absorbance @ 600nm (ABS)

1.62

1.64

1.66

1.68

1.72

1.74

1.76
1.7
0
Fig. 2: The Rate of Lysozyme Activity

20
40
60
Time (s)
80
100
120
140

Trend: As the time increases, the absorbance (ABS) of bacterial suspension at 600nm
decreases.
 Trend: As the time increases, the absorbance (ABS) of bacterial suspension decreases
gradually and slowly or becomes constant for different temperatures of lysozyme.
2.2
Absorbance @ 600nm (ABS)

2
1.8
1.6
1.4
1.2
0 20 40 60 80 100 120 140
Time (s)
0 Degrees Celsius 30 Degrees Celsius
37 Degrees Celsius 65 Degrees Celsius
85 Degrees Celsius
Fig. 3: The Effect of Temperature on the
Defensive Role of Lysozyme
Discussion
The amount of M. luteus is directly proportional to the absorbance because as the amount
of bacteria is increased absorbance is also increased as seen in Fig.1 above. In Fig.3, the rate
of absorbance decrease was higher for temperature 37oC because it is the closest to the
40oC optimal temperature for lysozyme activity. Both Gram-positive and Gram-negative
bacteria contain peptidoglycan in the cell wall. The peptidoglycan layer of Gram-positive
bacteria is generally much thicker than that of Gram-negative organisms. In Gram-positive
cells the peptidoglycan may thus constitute ~50% of the dry weight of the cell wall. Even
though the peptidoglycan layer is thinner in Gram-negatives, their peptidoglycan is less
accessible as it is sandwiched between the cytoplasmic membrane and an outer membrane
composed of lipopolysaccharides, phospholipids and protein. The presence in Gram-
negative cells of this outer membrane offers Gram-negative bacteria an effective barrier to
lysozyme action. In turn, this barrier renders Gram-negative bacteria intrinsically more
resistant than Gram-positive organisms to lysozyme attack and bacteriolysis. In other words,
the difference in the cell wall layer structures explains why lysozyme is especially active
against Gram-positive organisms. Perturbation of the outer membrane of Gram-negative
bacteria by chemical or physical means may render Gram-negative organisms more
susceptible to peptidoglycan degrading enzymes (Zeuthen and Bogh-Sorensen, 2003). In
Fig.2, as the time increases the absorbance of bacterial suspension decreases. The lysozyme
activity was carried out over in a period of two minutes and therefore it was not possible to
conclude how long it would take for the lysozyme in the tears to destroy the amount of M.
luteus in the test tube but from a study on the Preparation of Lysozyme Eye Drops and its
Effect on Rabbit Model of Bacterial Keratitis conducted by Dave (2010) it took about six
minutes for lysozyme to destroy bacteria under the very same conditions as those outlined
in the procedure. A reasonable amount of time is needed for lysozyme to work effectively
and if more lysozyme is added to an assay mixture it would decrease the reaction time
required for it to destroy M. luteus. Lysozyme, one of the most powerful natural
antibacterial and antiviral compounds known to man, has been used in foods and
pharmaceuticals for over three decades as it naturally inhibits the growth of many spoilage
organisms, increases a healthy shelf life and ensures food safety. It also boosts the immunity
system. due to its lytic activity on the cell wall of gram-positive micro-organisms. These
organisms are responsible for many infections of the human body as well as the spoilage of
various foods.
Conclusion
In conclusion, the results obtained afford us the ability to conclude that there was lysozyme
activity as the absorbance of bacterial suspension decreased signaling the destroying of M.
luteus. It was also concluded that the optimal temperature for lysozyme activity is 37 oC and
a pH of 6.4 due to the use of a phosphate buffer of the same pH throughout the whole
experimental procedure.
References
Dave, J. (2010) Role of Lysozyme in Body Defense. Biochem, 35-36.
Zeythen, P. and Bogh-Sorensen, L. (2003) Food Preservation Techniques. Elsevier.