On-line Isolation Of Lysozyme From Egg White Using Multi-walled Carbon Nanotubes

Lysozyme is a hydrolytic enzyme widely applied in food industry, packaging industry, medicine and bioengineering. Quite a few isolation and purification approaches are currently available for lysozyme, including crystallization, precipitation, chromatography, ion-exchange, reverse micellar extraction, aqueous two-phase systems. Solid phase extraction (SPE) is a highly efficient simple sample pretreatment technique with advantages of high enrichment factor, low running cost and ease of automation. It is also widely used in the isolation of lysozyme from egg white.Lysozyme is the only alkali protein in egg white and is positively charged in aqueous solution. Carbon nanotubes's isoelectric point is 5.0 and after oxidation it is negatively charged. A novel method for the extraction of lysozyme from egg white using carbon nanotubes was developed based on the electrostatic interactions. Carbon nanotubes were packed into a SPE microcolumn after appropriate pretreatment, and the microcolumn was incorporated into a sequential injection system which facilitates online selective sorption of lysozyme in egg white. The retained protein was afterwards quantitatively eluted by using carbonate buffer. Various parameters affecting the adsorption and elution process were investigated to achieve optimized purification conditions. The results showed that lysozyme could be efficiently extracted by the microcolume packed with multi-walled carbon nanotubes. An enrichment factor of 12 and a sampling throughput of 10 h-1 were obtained.The purity of the lysozyme isolated from egg white was 100% and ca.0.4 g lysozyme could be extracted from 100 mL egg white. When considering that 13% of the egg-white is protein species, the isolated lysozyme counts to 3.6% of the total protein mass, which matched well the standard value.