Immobilized polyphenol oxidase: Preparation, optimization and oxidation of phenolic compounds

Abstract

Polyphenol oxidase (PPO) was immobilized on chitosan/montmorillonite (CTS/MMT) and chitosan-gold nanoparticles/montmorillonite (CTS-AuNPs/MMT) composites, respectively. Taguchi method was applied to determine the optimal immobilization conditions for achieving the maximum enzyme activity. PPO immobilized on CTS/MMT (IPPO) and CTS-AuNPs/MMT (IPPO-Au) showed the highest enzyme activity at 15.61 × 10³ and 29.01 × 10³ U/g, respectively. IPPO-Au exhibited the higher stability and reusability than that of IPPO. The bio-catalytic performance of immobilized PPO was evaluated for the removal of phenol (PH), 4-chlorophenol (4-CP) and 2,4-dichlorophenol (2,4-DCP) in aqueous solution. The effects of pH, temperature, enzyme/substrate ratio, substrate concentration, reaction time on the phenolic compounds removal were investigated in detail. The results showed that, for both immobilized PPO, the optimal pH was 7, 6 and 5 for PH, 4-CP and 2,4-DCP, respectively, and the optimal temperature was 30 °C for all substrate. The optimal enzyme/substrate ratio and reaction time for IPPO-Au was lower than that of IPPO proved the higher catalytic efficiency. Chlorophenols showed the improved catalytic efficiency in comparison with PH for both immobilized PPO, while the effect of substrate chemical structure on the IPPO-Au properties was limited. The results suggested AuNPs presented in support played an important role on the enzyme activity and stability of immobilized PPO.

Keywords: Chitosan/montmorillonite composites; Gold nanoparticles; Immobilization; Optimization; Phenolic compounds; Polyphenol oxidase.