Abstract
Polyethyleneimine (PEI) glutaraldehyde-refined calcium pectinate (CaP)-agar beads were presented as improved covalent immobilization matrices. The CaP-agar beads exhibited incremented mechanical stability which facilitated their handling. The beads' concoction and activation processes were honed using the Box-Behnken design which recommended utilizing 5.4% agar, and a 2.95% PEI solution of pH 8.67. The honed CaP-agar beads established a more efficient ionic interaction with PEI which enabled the immobilization of more enzyme while utilizing less PEI than that required to activate the neat CaP beads. Furthermore, the activated CaP-agar beads granted superior operational stability to the immobilized enzyme, β-d-galactosidase (βgal), where it preserved 86.84 ± 0.37% of its precursive activity during its thirteenth reusability round. The CaP-agar immobilized βgal (iβgal) also showed incremented storage stability where it preserved 85.05 ± 3.32% of its precursive activity after 38 days of storage. The thermal stability of the iβgal was shown to be superior to that of the free enzyme as the iβgal exhibited incremented thermodynamic parameters, such as the t 1/2 values, the D values, the thermal denaturation activation energy, the enthalpies, and the Gibb's free energies. The βgal's immobilization onto the activated CaP-agar beads also shifted the enzyme's optimal pH from 4.6-5.1 to 3.3-4.9, whereas its optimal temperature was retained at 55 °C. The procured biocatalyst was exploited to efficiently hydrolyze the lactose in whey permeate.