Fabrication and Evaluation of Polyvinyl Alcohol–Chitosan Hydrogels for the Physical Entrapment of β‐Galactosidase

ABSTRACT

In this study, commercially sourced β‐galactosidase was immobilized by physical entrapment within a polyvinyl alcohol–chitosan (PVA–CS) hydrogel matrix, and its catalytic behavior and stability were evaluated in comparison with the free enzyme. The depletion‐based activity immobilization yield was 72.79%, while the protein‐based immobilization yield was 69.3% ± 2.4%. Immobilization shifted the optimum temperature from 40°C for the free enzyme to 50°C for the immobilized preparation and the optimum pH from 5.0 to 6.0. Direct nonlinear Michaelis–Menten analysis of matched Bradford‐protein‐normalized fixed‐30 min assay velocities yielded apparent K _m values of 2.97 and 1.32 mM and apparent V _max values of 16.90 and 22.98 U/mg protein for the free and immobilized preparations, respectively. These kinetic parameters were interpreted as assay‐dependent apparent values rather than direct measures of intrinsic substrate affinity or catalytic turnover. The immobilized enzyme retained more than 60% of its initial activity after six reuse cycles, but activity declined to approximately 8% by the 12th cycle, indicating limited long‐term operational durability. Overall, these findings show that physical entrapment within a PVA–CS hydrogel modifies the apparent catalytic and stability characteristics of β‐galactosidase and provides a basis for further optimization of hydrogel‐based immobilization systems.