Covalent immobilization of phenylalanine dehydrogenase on cellulose membrane for biosensor construction

Bacillus badius phenylalanine dehydrogenase was covalent immobilized on amino-activated cellulose membrane by cross-linking with glutaraldehyde. The amount of immobilized enzyme was estimated as 440 μg/cm2, and the biocatalyst retained 82% of the initial PheDH activity. The functionalized membrane was further used as coating material for a glassy carbon electrode, which was employed for the construction of an amperometric biosensor toward l-phenylalanine. The enzyme electrode (poised at +700 mV versus Ag/AgCl) showed a linear amperometric response up to 9.1 mM l-phenylalanine with a detection limit of 25 μM. The biosensor reached 95% of steady-state current in about 25 s and its sensitivity was 177 μA/M cm2. The electrode retained full electrocatalytic activity after 16 days of storage at 4 °C in 50 mM sodium phosphate buffer pH 7.0.