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.