Stabilisation and kinetic characterisation of penicillinase immobilised on carbodiimide activated carbon paste electrode

This study describes the covalent immobilisation of penicillinase from Bacillus licheniformis onto a carbon paste electrode (CPE) functionalised with N-cyclohexyl-N′-(2-morpholinoethyl) carbodiimide (CMC). The carbodiimide reagent served to activate carboxyl groups on the electrode surface, facilitating amide bond formation between the enzyme and the support matrix. The CMC-modified CPE created a microenvironment conducive to preserving enzymatic function and enhancing stability. Several immobilisation parameters were systematically investigated, including enzyme loading, coupling duration, and the influence of pH and temperature. Optimal activity was achieved at an enzyme loading of approximately 20–22 µg/cm2, with binding equilibrium attained after roughly 24 h. The immobilised enzyme exhibited a pH optimum of 6.5, comparable to that of the free enzyme, yet retained greater activity across both acidic and alkaline conditions. Post-immobilisation analysis showed the temperature for maximum enzyme activity increased from 37 °C to 42 °C. Kinetic characterisation revealed an increase in apparent Km from 0.0429 to 0.0951 mM and a decrease in Vmax from 1.024 to 0.272 µmol·min−1·mg−1, suggesting the presence of diffusional limitations and steric hindrance. Operational stability assessments showed that the immobilised enzyme retained approximately 82% of its initial activity after 30 days (10 reuse cycles) and around 47% after 90 days (30 cycles). These findings underscore the promise of CMC-modified CPE as a durable and effective platform for penicillinase immobilisation, with potential applications in biosensor development and biocatalysis.