Extraction and partial purification of the lectin Concanavalin A from Canavalia ensiformis using NADES and affinity chromatography on macroporous cryogels functionalized with D-glucosamine

The development of selective and sustainable strategies for lectin recovery is important for improving purification efficiency while preserving the biological activity of these proteins. In this context, the combination of green extraction systems with carbohydrate-functionalized macroporous matrices represents a promising approach for Concanavalin A (ConA) recovery. This study investigated the extraction and partial purification of ConA from jack bean seeds ( Canavalia ensiformis ) using natural deep eutectic solvents (NADES), followed by macroporous cryogels functionalized with D-glucosamine. The extraction and purification processes were monitored by protein quantification, hemagglutinating activity, and SDS-PAGE, while the cryogels were physicochemically characterized. Among the evaluated extraction systems, the citric acid–glucose NADES (CA–Glu, 1:1) showed the highest total protein recovery (20.19%), compared with 14.72% for aqueous extraction. The synthesized cryogels exhibited high total porosity (∼90%), which was preserved after D-glucosamine functionalization. Notably, the functionalized cryogel showed an adsorption capacity of 29.665 ± 1.332 mg/g for standard ConA, compared with 9.770 ± 1.052 mg/g for the non-functionalized matrix, representing an approximately threefold increase. During purification of the NADES extracts, the total protein recovered in the eluates remained within a narrow range (0.234–0.250 mg), despite differences in the initial protein content of the extracts, supporting the selective contribution of the immobilized affinity ligand. Hemagglutinating activity was preserved after adsorption and elution, with increased specific activity in most systems, indicating enrichment of the biologically active lectin fraction. SDS-PAGE confirmed the presence of ConA at approximately 25 kDa under denaturing conditions and showed a reduction in secondary protein bands after affinity purification. Overall, the integration of NADES-assisted extraction with D-glucosamine-functionalized macroporous cryogels provides a promising strategy for the partial purification of ConA, combining enhanced adsorption capacity, preservation of the macroporous structure, and maintenance of lectin biological activity.