biochem EXP5.docx LATEST 2022
ION, PURIFICATION AND CHARACTERIZATION OF LYSOZYMES
FROM EGG WHITE
Introduction
The clear liquid of an egg is called egg white also known as albumin. It surrounds the yolk of the
egg providing protection and nutrition. It consists of 88% of water and only 11% of proteins and
remainder as carbohydrate, ash and 1% of lipids. There are a number of proteins that make up
the 11% of albumin. Ovalbumin 54%, has three fractions A1, A2 and A3 that differ from each
other based on their phosphorus content. It is a storage glycoprotein and calcium binder that
plays a vital role in storage of amino acids around the body (Abeyrathne, Lee and Ahn, 2013).
Ovomucoid 11%, is an allergen, has trypsin inhibitory activity and inhibits growth of
tumour cells. Ovomucin 3.5%, has an alpha subunit and a β‐subunit that are bound by disulfide
bonds. The β‐subunit has cytotoxic effect on cultured tumour cell, suppressing the cell.
Ovotransferrin 12%, has antibacterial and antiviral activities, involved in transport of iron to
target cells and aggregates by heating resulting in milky white gel. Lysozyme 3.5%, is highly
stable in acidic solution and heats at 100°C for 1-2- minutes. Their stability is due to 4 disulfide
bonds. It catalyzes the hydrolysis of 1,4-glycosidc linkage between N-acetylmuraminic acid and
N-acetylglucosamine in peptidoglycan layer of bacterial cells walls specifically the gram-
positive bacteria. This activity can be enhanced by EDTA, tripolyphosphate and butylparaben.
These are the main proteins. Ovomacroglobulin 0.5%, glycoprotein has ability to inhibit
hemagglutination. Cystatin 0.05%, is a sulphydryl proteinase activity inhibitor used as
antimicrobial, insecticidal and antiviral agent as well as control of cancer cell metastasis and
prevention of cerebral haemorrhage. Ovoflavoprotein 0.8%, flavoprotein binds to riboflavin in
1:1 ratio in egg white and transfers it from blood serum to albumen of egg while. Avidin 0.05%,
is a strongly basic glycoprotein which combines with biotin forming a stable complex that is not
absorbed by intestinal tracts of animals. Ovoinhibitor 1.5% inhibits proteolytic enzyme and
inhibits bacterial serine proteinase, chymotrypsin and fungal serine proteinase. Ovoglycoprotein
1.0% is a foaming agent consisting of G2 and G3 globulins. (Stevens, 1996). The egg white
proteins can be extracted through various approaches. Proteins can be salted out based on their
ionic strength or the salt concentration such as ammonium sulphate (NH4)2SO4 salt or other salts.
The (NH4)2SO4 is a highly soluble salt that stabilizes protein structure by preferential solvation
1
,and inhibits bacterial growth. Increasing the salt concentration decreases solubility of proteins
and at high ionic strength the protein will be precipitated out of solution. The dissociates of salt
NH4+ and SO42- are attracted to charges on compound being purified preventing water molecules
from binding to it and allowing precipitation (Coen, Blanch, and Prausnitz, 1995). Proteins can
also be separated by their Ip as they are less soluble in their zwitterion form of zero charge
allowing them to be precipitated out. Polar solvents like acetone and alcohol will remove the
bound water from protein and aid in separation. Another form of approach is sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that separates protein based on their
MW. The SDS is an anionic detergent that makes proteins negatively charged by attachment and
denaturing the proteins to allow separation solely based on size of the fragments. All fragments
move towards positive electrode with heavier fragments moving slower than the lighter
fragments (Al-Tubuly, 2000). The proteins are most commonly isolated through column
chromatography techniques such as ion-exchange chromatography in which different
immobilized resins can be used to isolate proteins based on their charge. Diethylaminoethyl
(DEAE) a positively charged resin can be used to elute out negatively charged bounded proteins
by increasing salt concentration and displacing those proteins. Cation exchanger
carboxymethylcellulose (CMC) is negatively charged and is used for elution of positively
charged proteins (Coskun, 2016). Biuret assay is used for testing presence of the peptide bonds
where peptides bonds react with the Cu2+ ions in alkaline solution forming a violet complex.
Nitrogen’s in peptides form coordination bond with the metal ion. The number of bonds formed
depends on the number of peptide bonds. The proteins is then measured at 540nm to form
calibration curve to find protein concentration. It does not depend on amino acid composition of
protein and requires at least 1mg of protein and it has low sensitivity (Gornall, Bardawill and
David, 1949).
Objectives
This experiment is aimed to isolate, purify and characterize the lysozyme protein from egg white
sample as well as analyzing their biological significance. This is done through different
techniques by dividing experiment into three weeks. In week one the isolation of different
fractions of lysozyme is done through ion-exchange chromatography with CMC resin, pH
alteration by buffer, centrifuge and dialysis to obtain 4 fractions of lysozyme protein; A, B, C and
2
, D. Dialysis used to isolate lysozyme D. In week 2 SDS-PAGE technique is used to separate
proteins based on their MW and to determine their MW. Another technique biuret assay is used
to determine the content of the lysozyme fractions with different dilutions, reading their
absorbance at 570nm through spectrophotometer and constructing standard curve to find the total
mass of protein and their purity concentration of fractions and the egg white. Week 3 is aimed to
determine the enzymatic activity of lysozyme fractions by their action on different dilutions of
Mycrococcus lysodeikticus microorganism and reading their absorbance at 500nm through
spectrophotometer.
Results
Week1
Table 1: The volume of the fractions of lysozyme and the egg white sample.
Lysozyme fractions Volume (ml)
A 79
B 50
C 34
D 30
Egg white (source of lysozyme 15.8
fractions)
Week 2
3
ION, PURIFICATION AND CHARACTERIZATION OF LYSOZYMES
FROM EGG WHITE
Introduction
The clear liquid of an egg is called egg white also known as albumin. It surrounds the yolk of the
egg providing protection and nutrition. It consists of 88% of water and only 11% of proteins and
remainder as carbohydrate, ash and 1% of lipids. There are a number of proteins that make up
the 11% of albumin. Ovalbumin 54%, has three fractions A1, A2 and A3 that differ from each
other based on their phosphorus content. It is a storage glycoprotein and calcium binder that
plays a vital role in storage of amino acids around the body (Abeyrathne, Lee and Ahn, 2013).
Ovomucoid 11%, is an allergen, has trypsin inhibitory activity and inhibits growth of
tumour cells. Ovomucin 3.5%, has an alpha subunit and a β‐subunit that are bound by disulfide
bonds. The β‐subunit has cytotoxic effect on cultured tumour cell, suppressing the cell.
Ovotransferrin 12%, has antibacterial and antiviral activities, involved in transport of iron to
target cells and aggregates by heating resulting in milky white gel. Lysozyme 3.5%, is highly
stable in acidic solution and heats at 100°C for 1-2- minutes. Their stability is due to 4 disulfide
bonds. It catalyzes the hydrolysis of 1,4-glycosidc linkage between N-acetylmuraminic acid and
N-acetylglucosamine in peptidoglycan layer of bacterial cells walls specifically the gram-
positive bacteria. This activity can be enhanced by EDTA, tripolyphosphate and butylparaben.
These are the main proteins. Ovomacroglobulin 0.5%, glycoprotein has ability to inhibit
hemagglutination. Cystatin 0.05%, is a sulphydryl proteinase activity inhibitor used as
antimicrobial, insecticidal and antiviral agent as well as control of cancer cell metastasis and
prevention of cerebral haemorrhage. Ovoflavoprotein 0.8%, flavoprotein binds to riboflavin in
1:1 ratio in egg white and transfers it from blood serum to albumen of egg while. Avidin 0.05%,
is a strongly basic glycoprotein which combines with biotin forming a stable complex that is not
absorbed by intestinal tracts of animals. Ovoinhibitor 1.5% inhibits proteolytic enzyme and
inhibits bacterial serine proteinase, chymotrypsin and fungal serine proteinase. Ovoglycoprotein
1.0% is a foaming agent consisting of G2 and G3 globulins. (Stevens, 1996). The egg white
proteins can be extracted through various approaches. Proteins can be salted out based on their
ionic strength or the salt concentration such as ammonium sulphate (NH4)2SO4 salt or other salts.
The (NH4)2SO4 is a highly soluble salt that stabilizes protein structure by preferential solvation
1
,and inhibits bacterial growth. Increasing the salt concentration decreases solubility of proteins
and at high ionic strength the protein will be precipitated out of solution. The dissociates of salt
NH4+ and SO42- are attracted to charges on compound being purified preventing water molecules
from binding to it and allowing precipitation (Coen, Blanch, and Prausnitz, 1995). Proteins can
also be separated by their Ip as they are less soluble in their zwitterion form of zero charge
allowing them to be precipitated out. Polar solvents like acetone and alcohol will remove the
bound water from protein and aid in separation. Another form of approach is sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that separates protein based on their
MW. The SDS is an anionic detergent that makes proteins negatively charged by attachment and
denaturing the proteins to allow separation solely based on size of the fragments. All fragments
move towards positive electrode with heavier fragments moving slower than the lighter
fragments (Al-Tubuly, 2000). The proteins are most commonly isolated through column
chromatography techniques such as ion-exchange chromatography in which different
immobilized resins can be used to isolate proteins based on their charge. Diethylaminoethyl
(DEAE) a positively charged resin can be used to elute out negatively charged bounded proteins
by increasing salt concentration and displacing those proteins. Cation exchanger
carboxymethylcellulose (CMC) is negatively charged and is used for elution of positively
charged proteins (Coskun, 2016). Biuret assay is used for testing presence of the peptide bonds
where peptides bonds react with the Cu2+ ions in alkaline solution forming a violet complex.
Nitrogen’s in peptides form coordination bond with the metal ion. The number of bonds formed
depends on the number of peptide bonds. The proteins is then measured at 540nm to form
calibration curve to find protein concentration. It does not depend on amino acid composition of
protein and requires at least 1mg of protein and it has low sensitivity (Gornall, Bardawill and
David, 1949).
Objectives
This experiment is aimed to isolate, purify and characterize the lysozyme protein from egg white
sample as well as analyzing their biological significance. This is done through different
techniques by dividing experiment into three weeks. In week one the isolation of different
fractions of lysozyme is done through ion-exchange chromatography with CMC resin, pH
alteration by buffer, centrifuge and dialysis to obtain 4 fractions of lysozyme protein; A, B, C and
2
, D. Dialysis used to isolate lysozyme D. In week 2 SDS-PAGE technique is used to separate
proteins based on their MW and to determine their MW. Another technique biuret assay is used
to determine the content of the lysozyme fractions with different dilutions, reading their
absorbance at 570nm through spectrophotometer and constructing standard curve to find the total
mass of protein and their purity concentration of fractions and the egg white. Week 3 is aimed to
determine the enzymatic activity of lysozyme fractions by their action on different dilutions of
Mycrococcus lysodeikticus microorganism and reading their absorbance at 500nm through
spectrophotometer.
Results
Week1
Table 1: The volume of the fractions of lysozyme and the egg white sample.
Lysozyme fractions Volume (ml)
A 79
B 50
C 34
D 30
Egg white (source of lysozyme 15.8
fractions)
Week 2
3
