Novel Affinity-Based Reversed Micellar System For Protein Extraction

Reversed micelles for the protein extraction have been extensively studied. Ionic surfactants were mostly employed to form the extractive reversed micellar systems, and problems remained for the denaturation of proteins and the low selectivity to proteins. The dissertation develops a novel affinity-based reversed micelles system formulated by nonionic surfactant, which is applied in protein extraction and purification.The noninonic surfactant of Span 85 is modified with CB as an affinity surfactant by a two-phase reaction. A novel reversed micelles formed by the mixture of Span 85 and CB-Span 85 conjugate are extensively characterized in the water content (W0), hydrodynamic radius (Rh) and aggregation number by water determination and laser scattering technology. The results show that the water content and hydrodynamic radius of the reversed micelles are significantly increased by the introduction of CB ligands (CB-Span 85 conjugate), and the reversed micelles with CB-Span 85 conjugate have wider aggregation number distribution than the Span 85 reversed micelles. Moreover, protein extraction yield by the affinity-based reversed micelles increase significantly with the coupled CB concentration, indicating that the extraction was based upon the affinity interactions between lysozyme molecules and the CB ligand. Protein recovery is carried out using a stripping solution of 1mol/L NaCl. The recovered proteins exhibite an activity equivalent to the native proteins and their secondary structure are also unchanged, which implies that the reversed micellar system is biocompatible.The dissertation investigates the effect of hexanol on the affinity-based reversed micelles due to the significant impact of the assistant in reversed micellar system. The addition of n-hexanol to the reversed micellar system resultes in significant increase in water content and hydrodynamic radius of the affinity reversed micelles. Moreover, the reversed micelles with hexanol reveal broader aggregation number distribution and larger average aggregation number than the reversed micelles without hexanol addition. Lysozyme extraction yield increases 16% with n-hexanol concentration increasing from 0 to 3 vol.%. It is considered due to the decreases in the micellar curvature and rigidity of themicellar interfacial layer, and the increase in the micellar interfacial fluidity, which is indicated that hexanol molecules contribute to the formation of all reversed micelles.Partitioning equilibria and kinetic analyses in the affinity-based reversed micelles have been studied. The partitioning isotherms of proteins in CB-Span 85 reversed micellar system can be approximated by the Langmuir equation. The extraction capacity for protein increases significantly with CB concentration and n-hexanol concentration. Kinetic analyses in forward extraction and in back extraction have gained clearer insight into the contributions of liquid-film mass transfers, steric hindrance effect, affinity associations and the interfacial resistance for protein accommodation and release.Furthermore, lysozyme is separated from the natural chicken ovalbumin solution and the purification factor reaches 23 through the first extraction by the affinity-based reversed micelles. The affinity-based reversed micellar system is recycled three times for lysozyme purification and the purification factor for lysozyme doesn't decrease greatly. The results indicate that the reversed micellar system would find potential application in protein separation.