Learning Outcomes: The Course Learning Outcomes assessed by this assignment are: Apply the fundamental principles of biochemistry and molecular biology concepts with a focus on analytical techniques,...

Lab Activity - Purification and analysis of Lysozyme
Aim
The aim of this lab activity was to demonstrate how we can isolate a single protein of interest from the complex mixture of biomolecules. To understand this, in the present activity the Lysozyme enzyme was isolated and purifies from the egg’s white and its purity was analysed by different analytical techniques like protein assay, enzyme activity assay and gel electrophoresis.
Introduction
Lysozyme, also known as LZ, muramidase or N-acetylmuramylhydrolase, is an important component of non-specific innate immune response of animal’s body. It is a naturally occu
ing enzyme mainly found in neutrophils, macrophages, serum, saliva, milk, honey and hen’s egg white. Lysozyme enzyme has antimicrobial activity and protects us from various bacterial infections mostly the infections caused by gram positive bacteria. It attacks the shielding cell walls of bacteria by cleaving its peptidoglycan component which is made up of long ca
ohydrate chains cross-linked by short peptide strands. It is a glycoside hydrolase enzyme which specifically catalyse the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues of peptidoglycan and disrupts the integrity of bacterial cell wall. Damage to the peptidoglycan cell wall ultimately causes the death of bacterial cell as due to the loss of their protective cell wall their delicate plasma-mem
ane gets exposed to the harsh su
ounding conditions of high osmotic pressure, high temperature etc. and gets damaged. Due to its antibacterial nature it is used in therapeutic drugs as well and therefore needs to be extracted from its rich sources like milk, saliva, hen’s egg white etc. Since egg’s white contains large amount of lysozyme as lysozyme comprises about 3.5% of the total protein by weight present in the egg’s white. Lysozyme is the most basis single subunit protein with molecular weight of 14.6 KDa and isoelectric pH of 11. So, in this lab activity we uses egg’s white for the extraction of lysozyme and purified it. The level of purity was evaluated by different analytical techniques like protein assay, activity assay and b and electrophoresis. The procedure of the different methods used and the results obtained are discussed below in the methodology and results section.
Methods used for the isolation and purification process
· Steps used for the isolation of Lysozyme-
1. In the first step egg’s white is separated from the fresh hen’s egg and collected into 100 ml measuring cylinder. The collected volume should be around 25 ml.
2. Then the collected egg’s white was transfe
ed into a 250ml beaker, placed on ice, about 100 mL (4 volumes) of Buffer 1: 0.025M NaHCO3 having pH 9.0 was added and stir gently with a plastic rod.
3. Now the diluted egg white was filtered through a Mira cloth in a funnel, mixed gently and marked as fraction A.
4. After this, 2.0 mL of Fraction A was taken in a 10 mL measuring cylinder, added with 200 mg of ca
oxymethyl cellulose (CMC) resin and mixed gently.
5. This mixed is then transfe
ed into the centrifuge tubes and centrifuge at 2500 rpm for 5 min.
6. The supernatant was then decanted and transfer to another tube as fraction B.
7. The lysozyme gets bounded to the CMC pellets which were then washed with buffer and stored as fraction C.
8. Finally in5 ml of fraction C, 5 ml of buffer is added, mixed gently and dialysed.
9. Later the resultant liquid is stored as fraction D
10. These fractions were then used for lysozyme activity estimation by double-beam spectrophotometry, quantitative assay of protein by Lowry estimation and molecular weight estimation by SDS PAGE.
· Steps used for the lysozyme assay –
1. To perform the lysozyme...