Fabrication of covalent immobilizers utilizing chitosan-magnesium chelating interactions

The chelating interactions amongst chitosan (CS) and Mg²⁺ were exploited to fabricate CS beads, with glutaraldehyde (GA) incorporated into the gelling solution to introduce the functionalities necessary for β-galactosidase (βGAL) covalent binding and also to provide additional cross-linking. The fabrication parameters of the GA/Mg-CS beads were systematically optimized by varying the concentrations of MgSO₄, GA, CS and acetic acid (AA). It was disclosed that the most efficient gelation solution comprised a mixture of 0.5% MgSO₄-1% GA, and that it was optimal to utilize a 2% CS solution in 0.5% AA. Structural, morphological and elemental characteristics of the beads were assessed using FTIR, SEM and EDX, respectively. The mechanical durability of the GA/Mg-CS beads was also corroborated. The GA/Mg-CS beads successfully immobilized βGAL, achieving immobilization efficiencies of up to 59.78%. Following immobilization, the βGAL optimal pH shifted from 5.3-4.9 to 4.2, whereas its optimal temperature was unchanged within the range of 56-60°C. The immobilized βGAL (iβGAL) demonstrated higher tolerance to solvents compared to its free homologue. Furthermore, the iβGAL preserved 75.25% of its initial enzymatic activity following 15 consecutive assay cycles. It also maintained 109.14% activity by the 50th storage day. Finally, the applicability of the iβGAL was demonstrated in 24 h whey-permeate lactose degradation experiments at 45 °C.