Co-immobilization of β-galactosidase and neutrase ® on biopolymer supports for integrated whey valorization

The co-valorization of lactose and proteins from dairy by-products offers both environmental and technological opportunities. In this study, bifunctional biocatalysts were developed by co-immobilizing β-galactosidase and Neutrase® onto glutaraldehyde-activated chitosan for the simultaneous hydrolysis of lactose and casein. Three co-immobilization strategies were examined, and the sequential configuration in which Neutrase® was immobilized prior to β-galactosidase (D3) exhibited the highest retained activities and the most pronounced thermal stabilization for both enzymes. The D3 derivative achieved immobilization yields of 76% for both enzymes and retained approximately 60–70% of its initial activity after five reuse cycles. Thermal inactivation analysis revealed a sixfold increase in Neutrase® stability (SF = 18.2) and a corresponding rise in activation free energy from 95.9 to 103.9 kJ.mol−1, indicating increased resistance to thermal denaturation. Kinetic modeling showed that β-galactosidase exhibited a fourfold increase in apparent reaction rate constant (k2 = 2.05 × 10−2 L.g−1.min−1), indicating that enzyme co-localization enhanced the apparent catalytic rate, while proteolytic activity was moderately affected by diffusional constraints (α = 0.40). The integrated kinetic and thermodynamic analysis indicated that co-immobilization can be associated with structural stabilization effects and interaction phenomena within the chitosan matrix. These findings support a sustainable and economically feasible approach for whey valorization.