Precipitation and recovery of lysozyme denatured by urea and guanidine hydrochloride using anionic surfactant and acetone

Abstract

The surfactant precipitation and recovery method (SPR method) consist of a precipitation step, that is precipitation of protein-surfactant complex by the addition of aqueous surfactant solution to aqueous protein solution, and a recovery step, that is dissolution of precipitate in polar organic solvent and protein recovery by addition of trace amount of aqueous salt solution. When the denaturant was not included, the precipitation ratio of lysozyme increased as the AOT/lysozyme molar ratio increased, and after reaching 100%, the precipitation ratio decreased due to the redissolution of the precipitation. It was shown that lysozyme denatured by urea and GuHCl can be recovered by the SPR method. Lysozyme denatured with urea had a precipitation ratio and recovery ratio of almost 100% up to a urea concentration of 4 kmol/m3 at an AOT/lysozyme molar ratio of 28.6. As the urea concentration increased, the precipitation ratio and the recovery ratio decreased. Lysozyme denatured with GuHCl had a precipitation ratio and recovery ratio of about 75% at an AOT/lysozyme molar ratio of 71.5 and a GuHCl concentration of 0.6 kmol/m3. The specific activity and the secondary conformation of the recovered lysozyme were confirmed to be almost the same as those of the native lysozyme by measuring the enzyme activity and CD spectrum. It was shown that lysozyme denatured with urea and GuHCl can be recovered by the SPR method as lysozyme has native properties.

Keywords

• Protein recovery
• lysozyme
• anionic surfactant
• denaturant