A novel core-shell microsphere with hydrophobic and gel chromatography functions enhances protein purification

The spatial configuration and bonding conditions of polysaccharide microspheres make it difficult to distinguish molecules with similar hydrophobic properties in practical applications. Fortunately, medium modification offers more flexible chromatography conditions and higher selectivity, providing new solutions for complex purification environments. We synthesized hydrophobic composite core-shell agarose microspheres equipped with hydrophobic ligands and investigated the effects of hydrophobic ligand coupling on the morphology, particle size, pressure resistance, functional group change and protein adsorption capacity of microspheres. On this basis, the protein adsorption kinetics indicated that the best coupling temperature and time of phenyl and butyl were 40 °C, 8 h and 20 °C, 16 h, respectively. When the concentration of (NH 4 ) 2 SO 4 salt in the mobile phase is 2 M, and the pH is 7.0, the dynamic adsorption capacity of the medium for BSA reaches 12.6 mg/mL. The separation, purification efficiency, and resolution of these microspheres in a crude enzyme solution containing flavin monooxygenase improved significantly, demonstrating the practical implications and potential applications of this research. Overall, our findings reveal that this natural polysaccharide core-shell medium increases the modifiable sites of ligands, and can modify ligands in both the inner and outer layers, exhibiting its potential application in biomolecular separation.