Report: Lysozyme Extraction and Purification from Chicken Egg White

This report details the experimental process of isolating and purifying lysozyme from egg white, a mixture of proteins including the antibacterial enzyme lysozyme. The experiment utilizes ion-exchange chromatography with a CM cellulose column to separate lysozyme from other egg white proteins. The report includes the aims of the experiment, which are to purify lysozyme, monitor its purification through assays and SDS-PAGE, and assess the product quality. The report also mentions the use of UV absorbance and Lowry assay for protein concentration determination, and lysozyme activity assays. The discussion covers the importance of optimal conditions, such as pH and substrate concentration, for effective enzyme activity. Results from the lysozyme assay and Lowry assay are presented, along with data tables. The conclusion confirms the successful extraction and purification of lysozyme, highlighting the importance of column chromatography and the purity assessment using SDS-PAGE. The report also emphasizes the potential applications of purified lysozyme, such as understanding egg white allergies and its applications in biochemical research.

Discussion:
Young children are very much receptive to you are sensitive reactions caused by the
consumption of egg. The Cytoplasm of the unfertilized egg is known as egg white or albumin.
More than 20 types of proteins are present in egg white1. Among the proteins the allergens are,
1. Ovalbumin (Gal d 2)
2. Ovomucoid (Gal d 1)
3. Ovotransferrin (Gal d 3)
4. lysozyme (Gal d 4)
The lysosome is the catalytic enzyme which is known to digest the cell wall of the bacteria and
found abundantly in the albumin or egg white2. The lysosome is the important element present in
the egg white of hen and very much resistant to hydrolysis with Pepsin. The Ion exchange
chromatography is utilized to extract lysosome from egg white is the objective of the experiment
and verifying the purity of lysosome with the help of molecular and biochemical techniques is
the main objective of conducting this experiment.
The lysosome is first extracted from the egg white and it is purified with the help of size
exclusion chromatography. The column in the stationary phase consists of cross-linked gel
Matrix which is porous in nature. The size exclusion chromatography which is utilized for
separation and purifier in the lysosome is dependent on the size of the molecules3. The large
molecules elute at the initial mobile phase but the small molecules are trapped with the beads and
they elute at the end. The different types of protein present in albumin elute at different fractions
because of the size of the molecules to the purification process of the lysosome. Hypothetically it
is assumed that lysosome is small in comparison with the fractional range of the column which
1 Chen, C., Li, X., Yue, L., Jing, X., Yang, Y., Xu, Y., ... & Zhang, X. (2019). Purification and characterization of
lysozyme from Chinese Lueyang black-bone Silky fowl egg white. Preparative Biochemistry and Biotechnology, 1-
7.
2 Kouyoumdjian, A. J. M. (2019). The functionalisation and application of microporous micro-capillary films for the
chromatographic purification of biomolecules (Doctoral dissertation, University of Cambridge).
3 Onyenweaku, E. O., Oko, G. E., & Fila, W. A. (2018). Comparative Evaluation of Some Bioactive Compounds in
Raw and Boiled Egg Varieties: Eggs, Potential Nutraceuticals?. International Journal of Biochemistry Research &
Review, 1-7.

leads to trapping of lysosome molecules in the resin beads4. This will result in less amount of the
lysosome.
One of the most powerful media in the experiment is the enzymatic reaction which is utilized for
the detection of the presence of lysosome in egg white. The lysosome is composed of 128
residue of amino acids which are folded and form a globular structure5. When a substrate clamps
to the left of the structure tend to hydrolyze the peptidoglycan polysaccharide available with
bacterial cell walls and results in the osmotic lysis of the cell walls. It is a well-known fact that
the Gram-Positive Bacteria are more susceptible to the lysosome because of their exposure to the
extracellular environment6. The Gram-Negative bacteria have a thin layer of peptidoglycan
Therefore they are less susceptible to the lysosome. They have an outer membrane of
lipopolysaccharide which Shields them from being hydrolyzed.
The Gram-Positive Bacteria is taken as a sample to be used as a substrate to detect the activities
of lysosome as an enzyme. The presence of lysosome in comparison with the other major
proteins in albumin is detected with the help of lysosome assay. The lysosome is very important
for the research because it has the valuable capability of laying the Gram-Positive Bacteria. Like
other biomolecules, lysosome cannot be found in nature. They can only be extracted from
albumin and purified for further use. The method of extracting lysosome from albumin or egg
white is a simple process and does not require complicated apparatus and method of extraction.
The process of extraction is also not very time consuming, therefore, it is one of the most popular
ways to get lysosome for various use in biochemistry.
It is a known fact that lysosome is very much resistance to the hydrolysis with pepsin but with
the help of various experiments that can be stated that lysosome can be hydrolyzed with pepsin
when the pH is 1.2. Low pH is chosen to hydrolyze lysosome with pepsin in the simulated
gastric fluid. Optimal conditions are very effective and important to find the appropriate result.
The substrate concentration assays, as well as the pH essay, are used in this experiment to
4 Yang, Y., Park, J., You, S. G., & Hong, S. (2019). Immuno-stimulatory effects of sulfated polysaccharides isolated
from Codium fragile in olive flounder, Paralichthys olivaceus. Fish & shellfish immunology.
5 Fu, Q., Si, Y., Duan, C., Yan, Z., Liu, L., Yu, J., & Ding, B. (2019). Highly Carboxylated, Cellular Structured, and
Underwater Superelastic Nanofibrous Aerogels for Efficient Protein Separation. Advanced Functional Materials,
1808234.
6 Badan-Ara Marzdashti, R., Aghamaali, M. R., Varasteh, A., Nowruzfashkhami, M. R., & Sabkara, F. (2018).
Purification and characterization of lysozyme in Persian sturgeon, Acipenser persicus (Borodin, 1897) from the
Southwest Caspian Sea. Caspian Journal of Environmental Sciences, 16(4), 359-367.

understand the enzyme activity of lysosome as those activities vary with the change in the
concentration and buffer pH7. It is very important to have the appropriate concentration as the
higher concentration of the substrate.
There is a similarity on the binding properties of lysosome and the lysosome contaminated
proteins; therefore, it is very essential to determine the activity of lysosome using the
micrococcus suspension8. The amount of micrococcus which is appropriate for substrate
concentration of lysosome activity of 250 units is 0.4 mg per ml. It is very important to
determine the optimal pH where the bonding between the substrate and the enzyme is most
efficient it9. If an environment is too basic or too acidic it can cause obstacles for the bonding
between the enzyme and the substrate which results in the low activity of the lysosome. In the
lab, it is proved that pH 7 is the ideal pH for the activities of lysosome can be carried out very
effectively with the enzyme activity of 300 units.
Conclusions:
In the experiment, lysosome is successfully extracted from the album in out the egg white and it
is also purified using the column chromatography at the pH of 7. The concentration of the
lysosome which is very essential to determine in this experiment is successful to determine the
purity of lysosome with the help of lysosome assay. The purity of lysosome is confirmed with
the help of SDS page waste Assay. From the experiment, it can be successfully concluded that
column chromatography is very useful to extract lysosome from egg white because like other
enzymes lysosome is not available in nature and it should be extracted from the source10.
Purification of the lysosome is also very necessary procedure as the contaminated lysosome
7 Hazarika, C., Sarma, D., Puzari, P., Medhi, T., & Sharma, S. (2018). Use of Cytochrome P450 Enzyme Isolated
From Bacillus Stratosphericus sp. as Recognition Element in Designing Schottky-Based ISFET Biosensor for
Hydrocarbon Detection. IEEE Sensors Journal, 18(15), 6059-6069.
8 Morgenstern, J., Wang, G., Baumann, P., & Hubbuch, J. (2017). Model‐Based Investigation on the Mass Transfer
and Adsorption Mechanisms of Mono‐Pegylated Lysozyme in Ion‐Exchange Chromatography. Biotechnology
journal, 12(9), 1700255.
9 Kittelmann, J., Lang, K. M., Ottens, M., & Hubbuch, J. (2017). An orientation sensitive approach in biomolecule
interaction quantitative structure–activity relationship modeling and its application in ion-exchange chromatography.
Journal of Chromatography A, 1482, 48-56.
10 Brand, J., & Kulozik, U. (2017). Impact of the substrate viscosity, potentially interfering proteins and further
sample characteristics on the ion exchange efficiency of tangential flow membrane adsorbers. Food and bioproducts
processing, 102, 90-97.

cannot be utilized for various laboratory experiments11. The extraction and hydroxylation of both
commercial lysosome as well as the isolated lysosome are susceptible to the digestion of the
pepsin at lower pH. The pure lysosome is also very important to you understand the allergic
behavior of the egg whites and therefore they can be altered for the safe consumption of young
children12.
11 Angelo, J. M., Cvetkovic, A., Gantier, R., & Lenhoff, A. M. (2016). Characterization of cross-linked cellulosic
ion-exchange adsorbents: 2. Protein sorption and transport. Journal of Chromatography A, 1438, 100-112.
12 Mejía‐Manzano, L. A., Sandoval, G., Lienqueo, M. E., Moisset, P., Rito‐Palomares, M., & Asenjo, J. A. (2018).
Simulation of mono‐PEGylated lysozyme separation in heparin affinity chromatography using a general rate model.
Journal of Chemical Technology & Biotechnology, 93(7), 1980-1987.

Reference list:
 Angelo, J. M., Cvetkovic, A., Gantier, R., & Lenhoff, A. M. (2016). Characterization of
cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport.
Journal of Chromatography A, 1438, 100-112.
 Badan-Ara Marzdashti, R., Aghamaali, M. R., Varasteh, A., Nowruzfashkhami, M. R., &
Sabkara, F. (2018). Purification and characterization of lysozyme in Persian sturgeon,
Acipenser persicus (Borodin, 1897) from the Southwest Caspian Sea. Caspian Journal of
Environmental Sciences, 16(4), 359-367.
 Brand, J., & Kulozik, U. (2017). Impact of the substrate viscosity, potentially interfering
proteins and further sample characteristics on the ion exchange efficiency of tangential
flow membrane adsorbers. Food and bioproducts processing, 102, 90-97.
 Brand, J., Dachmann, E., Pichler, M., Lotz, S., & Kulozik, U. (2016). A novel approach
for lysozyme and ovotransferrin fractionation from egg white by radial flow membrane
adsorption chromatography: impact of product and process variables. Separation and
Purification Technology, 161, 44-52.
 Chen, C., Li, X., Yue, L., Jing, X., Yang, Y., Xu, Y., ... & Zhang, X. (2019). Purification
and characterization of lysozyme from Chinese Lueyang black-bone Silky fowl egg
white. Preparative Biochemistry and Biotechnology, 1-7.
 Fu, Q., Si, Y., Duan, C., Yan, Z., Liu, L., Yu, J., & Ding, B. (2019). Highly
Carboxylated, Cellular Structured, and Underwater Superelastic Nanofibrous Aerogels
for Efficient Protein Separation. Advanced Functional Materials, 1808234.
 Hazarika, C., Sarma, D., Puzari, P., Medhi, T., & Sharma, S. (2018). Use of Cytochrome
P450 Enzyme Isolated From Bacillus Stratosphericus sp. as Recognition Element in
Designing Schottky-Based ISFET Biosensor for Hydrocarbon Detection. IEEE Sensors
Journal, 18(15), 6059-6069.
 Kittelmann, J., Lang, K. M., Ottens, M., & Hubbuch, J. (2017). An orientation sensitive
approach in biomolecule interaction quantitative structure–activity relationship modeling
and its application in ion-exchange chromatography. Journal of Chromatography A,
1482, 48-56.

 Kouyoumdjian, A. J. M. (2019). The functionalisation and application of microporous
micro-capillary films for the chromatographic purification of biomolecules (Doctoral
dissertation, University of Cambridge).
 Mejía‐Manzano, L. A., Sandoval, G., Lienqueo, M. E., Moisset, P., Rito‐Palomares, M.,
& Asenjo, J. A. (2018). Simulation of mono‐PEGylated lysozyme separation in heparin
affinity chromatography using a general rate model. Journal of Chemical Technology &
Biotechnology, 93(7), 1980-1987.
 Morgenstern, J., Wang, G., Baumann, P., & Hubbuch, J. (2017). Model‐Based
Investigation on the Mass Transfer and Adsorption Mechanisms of Mono‐Pegylated
Lysozyme in Ion‐Exchange Chromatography. Biotechnology journal, 12(9), 1700255.
 Onyenweaku, E. O., Oko, G. E., & Fila, W. A. (2018). Comparative Evaluation of Some
Bioactive Compounds in Raw and Boiled Egg Varieties: Eggs, Potential Nutraceuticals?.
International Journal of Biochemistry Research & Review, 1-7.
 Yang, Y., Park, J., You, S. G., & Hong, S. (2019). Immuno-stimulatory effects of
sulfated polysaccharides isolated from Codium fragile in olive flounder, Paralichthys
olivaceus. Fish & shellfish immunology.