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A sequential esterification-deesterification strategy for agarose microspheres with expanded network and high ligand
Zizhou Chen1, Xiaoju Long1, Ziting Zhao1
1College of Ocean Food and Biological Engineering, Jimei University, Xiamen, 361021, China; National R&D Center for Red Alga Processing Technology, Xiamen, 361021, China; Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen, 361021, China; Xiamen Key Laboratory of Marine Functional Food, Xiamen, 361021, China.
Abstract:
Agarose microspheres form a dense gel network through strong intermolecular hydrogen bonding, which limits the accessibility of internal hydroxyl groups. This compact network can hinder the access of epichlorohydrin to internal hydroxyl groups and reduce activation efficiency, thereby constraining ligand loading and protein separation performance. To overcome this limitation, a sequential anhydride esterification-deesterification strategy was developed to construct agarose microspheres with an expanded network structure, high epoxy-group density and high-capacity cation-exchange functionality. Acetic anhydride (AA), succinic anhydride (SA), and octenyl succinic anhydride (OSA) were used to temporarily esterify agarose and regulate its network structure. Among the three anhydrides, OSA produced the most pronounced network expansion, despite its relatively low degree of substitution, which was attributed to the stronger structural perturbation associated with its bulky amphiphilic octenyl-succinyl substituent. Subsequent alkaline treatment removed the temporary ester groups and restored the previously esterified hydroxyl groups, while BDDE-mediated covalent crosslinking helped retain the expanded network structure. Under the optimized epoxidation conditions, OSAAM exhibited an epoxy-group density of 67.7 μmol/g, 49.8% higher than that of native AM treated under the same optimized conditions (45.2 μmol/g). After sulfonate functionalization, the OSAAM-SP medium exhibited an ion-exchange capacity (IEC) of 233.7 μmol/g, a static adsorption capacity of 377.57 mg/g (wet resin) in the absence of salt, a dynamic binding capacity of 206.7 mg/mL, and a recovery of 98.21%. The resulting medium was successfully applied to the purification of lysozyme from quail egg white. These results demonstrate that sequential regulation of the polysaccharide network and restoration of temporarily esterified sites can enhance subsequent functionalization and chromatographic performance.
