Fig. 4. Michaelis–Menten kinetic studies: comparative Michaelis–Menten kinetics studies for aqueous solutions of nALP, cALP, ALP-PS, and ALP-PS in the biocatalytic silica NPs-PEI sponge. Reaction mixture comprised of 1.5 mL glycine buffer (100 mM, pH 8.8 containing 1 mM MgCl₂·6H₂O); 1.5 mL substrate solution (paranitrophenyl phosphate, pNPP) with concentration range 30 to 450 μM and 100 μL, 0.1 mg mL⁻¹ diluted ALP solution for nALP, cALP, ALP-PS. Small cylindrical sponges were used for assaying ALP-PS activity (0.7 mL volume containing 0.25 mg total protein), these sponges were equilibrated to ambient temperature prior to assay. Snapshots bottom right inset, show cylindrical sponges before and after reaction suspended in the cuvette with the help of a thread. Enzyme assay was carried out at 37 °C for 15 min with constant stirring, monitored continuously at 410 nm using UV-visible spectrophotometer.