Comparative Evaluation of Enzyme Immobilization Techniques with Impact on Activity and Stability

Enzyme immobilization can improve catalyst stability and reusability, although different immobilization strategies introduce trade-offs in catalytic performance and enzyme retention. This study compared free Escherichia coli β-galactosidase with preparations immobilized by physical adsorption on porous silica, glutaraldehyde-mediated covalent attachment to functionalized silica, and calcium-alginate entrapment. Catalytic activity, apparent Michaelis–Menten kinetics, protein loading, enzyme leaching, storage stability, operational reusability, and pH- and temperature-dependent activity were evaluated. Free β-galactosidase exhibited an apparent Vmax of 100 U and Km of 2.0 mM. Immobilization reduced apparent Vmax to 80, 60, and 90 U and increased Km to 2.5, 3.0, and 2.2 mM for adsorption, covalent attachment, and entrapment, respectively. After 21 days, residual activities were 50%, 60%, 85%, and 72% for free, adsorbed, covalently immobilized, and entrapped preparations, respectively. Covalent attachment demonstrated the greatest storage stability and resistance to enzyme release, whereas entrapment preserved comparatively high initial catalytic activity. All preparations exhibited maximum activity at pH 7.0, with temperature optima of 40–45 °C. These findings highlight method-dependent trade-offs between catalytic performance, enzyme retention, and operational stability, supporting application-specific selection of immobilization strategies.