Question

This is an assignment for my biochemistry class. We must determine the best column metric to accomplish the purification of a chicken egg white lysozyme.

Based on the properties of egg white lysozyme, choose the one best column matrix to accomplish the purification (Hint: Look up the molecular weight and pI value – go to the primary literature to fulfill the requirement in the underlined statement). Your column matrix options include either cation exchange or anion exchange.

Answer 1

Lysozyme is a ubiquitous enzyme present in secretions, body fluids and animal and human tissues , which catalyzes the hydrolysis of bacterial cell-wall sugars, specifically the β bonds between muramic acid and N-acetylglucosamine . Lysozyme is one of the most studied bacteriolytic enzymes. Because of its relative abundance in nature and its multiple functions and utilities, it is considered a model protein for different research studies. The hen egg white is still the starting material for lysozyme purification: considering its high concentration in such material – 3.5 % on dry basis – the explanation is evident if comparing this amount with that of other starting materials. Its low cost and high accessibility also contribute in the same way.From the point of view of the purification process design, lysozyme is a protein with singular physicochemical properties, especially for its high isoelectric point, which leads to believe that cation exchange chromatography is an ideal technique to purify it.Processes based on membrane filtration have also been used for lysozyme purification from egg white . However, membrane filtration technology is expensive and the processes for lysozyme purification are rather complex due to egg white high viscosity and molecular associations between the enzyme and other proteins - such as ovomucin - present in egg white. Despite the numerous strategies to purify lysozyme from egg white reported, very few have found industrial application. A classic production method at industrial level is crystallization from egg white in the presence of 5 % NaCl at pH 9.5.

This strategy, developed by Alderton and Fevold in 1964, has undergone several modifications. A yield of 60-80 % was achieved by using this technology, but the addition of a high concentration of salt and other additives precludes the further commercial utilization of egg white. A related industrial strategy is a two-step lysozyme isolation by ion exchange chromatography followed by crystallization: the egg white is mixed with an adequate amount of carboxymethyl cellulose and, after washing the matrix with water, lysozyme is eluted with 5 % NaCl and precipitated by pH adjustment to 9.5. The precipitate is then redissolved, desalted and concentrated.