Abstract
ABSTRACT This study reports the immobilization of Aspergillus oryzae β‐galactosidase in a polyvinyl alcohol–sodium alginate (PVA–SA) hydrogel and its performance compared with the free enzyme. Through immobilization, the optimum temperature shifted from 40°C to 60°C, with the immobilized enzyme retaining high activity and exhibiting improved resistance to heat‐induced inactivation. Although maximum catalytic activity was observed at pH 5.0 for both free and immobilized forms, the immobilized enzyme sustained higher stability in near‐neutral and slightly alkaline environments. Kinetic analysis revealed that the K m value increased from 0.21 to 0.33 mM, indicating diffusion limitations, whereas the apparent V max rose from 0.40 to 1.71 U/mg protein. Storage experiments demonstrated improved stability at 4°C, with about 60% of the initial activity retained after 8 weeks, whereas freezing at −20°C accelerated inactivation. The immobilized enzyme retained more than 80% of its catalytic performance after three consecutive uses and still preserved approximately 65% following the fourth cycle. Lactose hydrolysis experiments confirmed efficient and sustained performance, reaching 78% conversion after 180 min.