Lysozyme Isolation and Purification

Lysozyme, also known as mucopeptide N-acetyl-muramoylhydrolase, is widely distributed in many tissues in plants and animals. It was discovered in 1922 by Alexander Flemming while he was suffering from a cold and let some of his nasal secretions fall on a plate of bacteria and later noticed that the bacteria in the vicinity of the mucus from his nose had dissolved. He was able to establish that the anti-bacterial activity in the mucus was due to an enzyme, the enzyme he called lysozyme because it breaks down the cell walls of bacteria and causes them to lyse.

Lysozyme was the first enzyme whose three-dimensional structure at the atomic level was determined. Its shape resembles that of a three-dimensional Pac Man; it's an egg-shaped molecule with a deep cleft. The cleft, which resembles Pac Man's mouth, is the active site of the enzyme -- the place where long polymers (polysaccharides) found in the cell walls of bacteria and fungi are cut into small subunits. The breakdown of these polymers results in the formation of holes in the cell walls and cell lysis, where cells burst and cellular contents escape. (This is why the name, "lysozyme", coined by Flemming, is apt.)

When the polysaccharide substrate binds to the lysozyme cleft, part of the cleft closes a bit (reminding us again of Pac Man) and distorts polysaccharide subunits; at the same time two acidic amino acids of the lysozyme active site add an [H+] and [OH] across the glycosidic bond between two monomers, thus hydrolyzing the polymer. So, the name "hydrolase" is also apt.

Knowledge of the structure and activity of lysozyme has given scientists insight toward understanding the structure and behavior of proteins in general and has served as a model ever since its structure was elucidated over 30 years ago. Experiments with lysozyme also provide valuable insight and the conceptual basis for many of the peptide therapies currently being developed by biotechnology companies.

Lysozyme offers other advantages, it is in plentiful supply and the starting material (hen eggs) is inexpensive. The procedures we will use mimic procedures used to purify recombinant proteins from bacterial cultures; in fact we could use these same procedures to purify recombinant lysozyme from recombinant cells engineered by the protein engineers doing the experiments mentioned in the paragraph above. Beautiful color pictures of three-dimensional