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Figure 1. The mechanism of Micrococcus lysodeikticus cell lysing with a lysozyme catalyst. The
process of lysozyme degradation where hydrolyzation in the peptidoglycan layer of bacterial cell
membranes breaks the beta-glycosidic linkage between N-acetylmuramic acid and N-acetyl glucosamine.
Experimental Enzyme Activity of Initial PurificationExperimental Enzyme Activity of Pure Lysozyme
Isolation and Purification of Lysozyme from Egg Whites: An Immune Response Ashlynn LaFlamme, Katie Carey, and Natalie Needy
Department of Biology and Environmental Science, Westminster College, Fulton, MO, 65251
Purification Method
Figure 3. The rates of absorbance (optical density at 450nm) per minute for
the egg white isolated lysozyme catalyzed assay reactions . Trial 1 assayed
0.1mL of isolated lysozyme, and trial 2 assayed 0.2mL of isolated lysozyme. The
two slopes represent the reaction rate of lysing Micrococcus lysodeikticus cells for
each reaction.
Assay
Lysozyme
Micrococcus lysodeikticus Cells (Intact)→ Micrococcus lysodeikticus Cells (Lysed)
Stock Solutions BlankActive Enzyme
Test Sample
Buffer
0.1M Potassium Phosphate buffer at pH
6.94
0.5mL 0.3mL
Substrate Suspension
9.0mg of Micrococcus lysodeikticus cells,
25mL buffer, and 5mL H2O
2.5mL 2.5mL
Lysozyme
0.002g of pure lysozyme per 200mL H2O or
0.1mL of egg extract lysozyme
0.0mL 0.2mL
Reaction Mechanism of Lysozyme
Add 100mL of egg white and
stir by hand for 5 minutes
Centrifuge with 4,000g
for 20 minutes as 4ºC
Pour off excess liquid
Prepare 1.5mL of 2% bentonite
suspension in 1% KCl pH 4.0
Wash clay with 0.5M
K3(PO4) pH 7.0
Wash clay with 30mL of 5%
pyridine pH 5. Repeat 3x.
Centrifuge with 4,000g
for 20 minutes as 4ºC
Add (NH₄)₂SO₄ to obtain a
concentration of 2.6M
Centrifuge with 4,000g
for 20 minutes as 4ºC
Collect supernatant
Follow assay procedure to
assess lysozyme activity
Assay Procedure
Preparation for procedure:
Table 1. The contents of the table organize and describe what materials the assay
consists of. It lists the stock solutions, concentrations, and volumes used in each
solution and test sample. The reaction which lysozyme facilitates during the assay
is also included, which lowers the activation energy to lyse Micrococcus
lysodeikticus cells.
Suspend Micrococcus
lysodeikticus
lyophilized cell in
25mL of K3(PO4) pH
7.0
Dilute to a final
volume of 30mL
with K3(PO4) pH 7.0
Stir solution and keep
in an ice bucket and
keep covered until
added to the assay
Figure 2. The rate of absorbance (optical density at 450nm) per minute for
the reaction of 100% pure lysozyme. This curve is the standard for our
experiment. It represents the rate at which pure lysozyme facilitates the substrate
Micrococcus lysodeikticus in lysing the cell walls of the bacterium in a buffer
solution.
y = -0.0143x + 0.0139
y = -0.0256x + 0.0497
-0.14
-0.12
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0 1 2 3 4 5 6 7
Ab
sorb
an
ce a
t 450n
m (
OD
)
Time (minute)
Initial Purification of Egg White Extract Assay
Trial 1 Trial 2
AbstractLysozyme is a natural antimicrobial enzyme found in a wide variety of organismal
immune responses. Its function is to catalyze the destruction of bacteria cell walls. This
study aims to isolate and purify lysozyme from hen egg whites while maintaining the
enzyme activity. The lysozyme was purified and isolated from egg white homogenate. Our
data were analyzed by Microsoft Excel to determine how much enzyme activity was found
in our isolated samples. It was concluded from our extraction and purification that we
achieved isolating an extract lysozyme sample which had a 27.6% slower reaction velocity
when compared to the purified lysozyme in assay. It is also noted that when the volume of
egg extract in assay was halved, the absorbance rate decreased by 56.6%. There was no
concluded significance in our findings, however. Further research, purification, and
experimentation will need to be conducted on enzyme activity and the characterization of
lysozyme to achieve full isolation.
IntroductionLysozyme plays two vital roles. The first includes protection to mammalian
and invertebrate bodies. In order to maintain proper health, lysozyme degradation of
the cell membrane of gram-positive bacteria must occur. This process occurs due to
hydrolyzation of the beta-glycosidic linkage between N-acetylmuramic acid and N-
acetyl glucosamine in the peptidoglycan layer of bacterial cell membranes (Figure 1),
which is the natural substrate for lysozyme. (Arnheim et al. 1972). Lysozyme can be
found in tears, blood, mucus, human milk, and egg whites and is a common immune
response. The second is the essential role in medical and biochemistry research. The
structure and characterization of lysozyme are consistent under a variety of
conditions (Strynadka and James 1991). Lysozyme can also be used commercially as
a food preservative because it inhibits the growth of bacteria which can prolong shelf
life. It is also researched for its use in pharmaceuticals and can be used as a
potentiating agent for antibiotics (Proctor and Cunningham, 1988). Due to the
stability of lysozyme, it is one of the most researched enzymes.
The characterization of lysozyme is well known. The average molecular
weight is 14,300 Daltons and it is located in the cell membrane (Alderton, 1944). The
structure is a singular polypeptide chain consisting of 129 amino acids with
additional no subunits (Berg, 2019). The thermal stability occurs at a pH of 5.0 and
the isoelectric pH range is between 10.5 pH and 11.0 pH (Alderton, 1944). The
active site has the architecture of a deep crevice with two domains, one for beta-sheet
structures and one for helical structures, linked by an alpha helix (Strynadka and
James, 1991).
This characterization information allows for a beginning knowledge of the
correct buffer pH, dialysis bag, and various equipment and methods needed to begin
isolating and purifying lysozyme from hen egg whites to achieve the maximum
activity of lysozyme.
References1. Alderton G, Ward WH, Fevold HL. 1944 Oct 22. Isolation of lysozyme from egg white.
The Western Regional Research Laboratory:44–58. [cited 2020 Feb
2]. https://www.jbc.org/content/157/1/43.full.pdf?sid=3a75259b-7e00-41f3-bf4b-
5ddbcbd4f402
2. Berg JM. Biochemistry. 8th ed. New York: Macmillan International Higher Education; 2019.
Dekina SS, Romanovska II, Ovsepyan AM, Bodyul MG, Toptikov VA,. 2015 Dec 1.
Directory of Open Access Journals. Biotechnologia Acta. [cited 2020 Feb
11]. https://doaj.org/article/d0ed5907a3ad4a3c80d3ae6842de43fc
3. Shugar D. 1952. The measurement of lysozyme activity and the ultra-violet inactivation
of lysozyme. Biochimica et Biophysica Acta 8:302–309.
4. Worthington V. 1993. Worthington enzyme manual: enzymes and related biochemicals.
New Jersey: Worthington Biochemical Corporation. p250-253.
5. Strynadka, N., and James, M.: Lysozyme Revisited - Crystallographic Evidence for
Distortion of an N-Acetylmuramic Acid Residue Bound in Site-D, J Mol Biol 220, 401, 1991
6. Arnheim N. et al., Chemical studies on the Enzymatic Specificity of Goose Egg White
Lysozyme, JBC Vol. 248, No 1 pp. 233-236 (1973)
7. Oliver WT, Wells JE. 2015 Aug 13. Lysozyme as an alternative to growth promoting
antibiotics in swine production. Journal of animal science and biotechnology. [cited 2020 Mar
10]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535397/
8. Proctor VA, Cunningham FE. 1988. The chemistry of lysozyme and its use as a food
preservative and a pharmaceutical. Critical reviews in food science and nutrition. [accessed
2020 Mar 8]. https://www.ncbi.nlm.nih.gov/pubmed/3280250
Results• The absorbance rate for trial 2 was fastest, with a reaction velocity of
-0.0256 OD/Min.
• Trial 1 had the slowest absorbance rate, with a reaction velocity of -0.0143
OD/Min (Figure 2).
• The pure lysozyme absorbance rate was -0.0338 OD/Min (Figure 1). This is
the experimental standard and represents 100% enzyme activity level.
• Trial 1 retained the least about of lysozyme activity, with only 42.31% of the
enzyme activity level (Figure 2).
• Trial 2 lysozyme activity was highest, with 75.74% of the total enzyme
activity (Figure 2).
• The pure lysozyme assay sample was calculated to contain an concentration
of 4.662x10-5 mol/L.
y = -0.0338x - 0.056
-0.21
-0.16
-0.11
-0.06
-0.01
0.04
0 0.5 1 1.5 2 2.5 3 3.5 4
Ab
sorb
an
ce a
t 450n
m (
OD
)
Time (minute)
Pure Lysozyme Assay
DiscussionIn order to determine the activity of our experimental egg white lysozyme
isolation, we compared our results to the pure lysozyme absorbance velocity,
which was our standard concentration assay (Figure 2). Only the concentration of
pure lysozyme in the assay was determined, and had a 6.993x10-4 M. The sample
which had the fastest absorbance rate was trial 2, which used 0.2mL of egg white
isolated lysozyme (Figure 3). The sample in trial 1 had the slowest absorbance rate
(Figure 3). When compared to the pure lysozyme, trial 2 was 27.6% slower and
trial 1 was 81.0% slower (Figure 4). Trial 1 used half as much egg extract
lysozyme as trial 2 and the reaction velocity of trial 1 was reduced by 56.6%
compared to trial 2.
Since the substrate concentration was kept constant throughout at 0.30g/L of
suspended Micrococcus lysodeikticus in buffer and water, we can assume that the
quantity of enzyme is directly proportional to the velocity of the reaction. We can
also assume that the percent activity is directly related to the amount of lysozyme
in the samples since this was the only variable altered. This allows us to correlate
the decrease in enzyme activity with the decrease of lysozyme concentration
added to the assay. This explains why trial 1 was much slower and expressed less
enzyme activity than trial 2. Lysozyme only has one subunit, therefore it cannot
bind to more than one substrate at a time. The increase of egg white extract
lysozyme was the only variable altered, indicating that a higher concentration of
lysozyme in trial 2, under the conditions of our assay, was more effective at
catalyzing the reaction than trial 1. The increase of enzyme activity compared to
the increase of lysozyme concentration can be seen in figure 3 and appears to be
directly related. I would suggest further testing of various isolated enzyme
concentrations in order to determine which yields the greatest amount of activity
and calculating the significance of these findings.
In order to determine if there is a significant comparison between
our isolated lysozyme and the purified lysozyme, further purification methods
are needed. These methods would include dialysis, gel chromatography,
and lyophilization. Further purification would promote higher activity levels of
the enzyme. In addition to purification, a Bradford test is needed to determine
the exact protein concentration of lysozyme. This concentration of lysozyme
can be compared to the purified lysozyme. Throughout the isolation
and purification process, it is important to note that lysozyme can be inhibited
by surface-active reagents, such as dodecyl sulfate, alcohols and fatty
acids. It is also essential to store the enzyme in its optimal conditions, such as
maintaining the pH and temperature. Keeping the enzyme in its optimal conditions
will prevent the enzyme from degradation, thus inhibiting activity.
0
0.2
0.4
0.6
0.8
1
0.00E+00 5.00E-06 1.00E-05 1.50E-05 2.00E-05 2.50E-05 3.00E-05 3.50E-05 4.00E-05 4.50E-05 5.00E-05
% A
cti
vit
y (
OD
/Min
ute
)
Lysozyme Concentration (M)
Lysozyme Activity vs. Lysozyme Concentration
Enzyme Activity Results
Figure 4. The percent of lysozyme activity graphed over the concentration of
lysozyme using reaction velocity for all three assay samples. Each point represents
the activity and concentration of lysozyme in the reaction, representing pure lysozyme,
trial 2, and trial 1 values. The amount of enzyme present positively correlates directly
with the percent activity of the reaction.
AcknowledgementsWe would like to thank the Westminster College Biology and
Chemistry departments, and more specifically, Dr. Johanna Morrow for her
invaluable help and knowledge during this arduous process. We would also like
to thank Alana Funk for managing lab supplies and materials, as well as assisting
us in our waste processing and disposal.
Trial 1
Trial 2
Pure Lysozyme
Assay Procedure:
1. Set spectrophotometer-20 to 450 nm and 25ºC.
2. Pipette 2.5 ml of Micrococcus lysodeikticus lyophilized cell suspension into
a cuvette and incubate for 4-5 minutes to achieve temperature equilibrium.
3. Prepare the blank sample by pipetting 0.5 ml of buffer solution to the cuvette
to give a total volume of 3.0 ml.
4. Zero spectrophotmoeter-20 using the prepared buffer-substrate blank.
5. Once ready to record enzyme activity, add 0.1 mL of diluted enzyme to the
cuvette and immediately begin recording the absorbance.
6. Record the absorbance value in 15 second intervals for the first 120 seconds,
then in 30 second intervals until the reaction has reached 5 minutes.