Optimization of phenol removal with horseradish peroxidase encapsulated within tyramine-alginate micro-beads

Removal of phenolic compounds from water is of major interest over the years, since they are one of the most common pollutants in aqueous systems. Horseradish peroxidase (HRP) is the most investigated biocatalyst for this purpose. Inactivation of the enzyme is a major issue which can be successfully overcome by the enzyme immobilization on different polymers. In this study, tyramine-alginate micro-beads were used as carriers for the immobilization of horseradish peroxidase. The effect of the oxidation degree of tyramine-alginates on a specific activity of the enzyme was tested. An increase in the concentration of oxidized alginate from 2.5 to 20% resulted in a gradual increase in the specific activity from 0.05 to 0.67 U/mL. HRP immobilized within these micro-beads was tested for the phenol removal in a batch reactor. Reaction conditions were optimized to achieve a high removal efficiency and substantial reusability of the system. In this study, for the first time, an internal generation of hydrogen peroxide from glucose and glucose oxidase was employed in the phenol removal process with HRP immobilized on tyramine-alginate. Within 6 h of repeated use 96% of phenol was removed when the system for internal delivery of H2O2, composed of 0.187 U/mL of glucose oxidase and 4 mmol/L of glucose was employed. A common straightforward addition of hydrogen peroxide provided the removal efficiency of only 42%, under the same reaction conditions. The highest efficiency of the phenol removal (96%) was obtained with HRP immobilized within 20 mol% oxidized tyramine-alginate micro-beads. Fifteen mol% oxidized tyramine-alginate showed lower removal efficiency in the first cycle of use (73%) but more promising reusability, since the immobilized enzyme retained 61% of its initial activity even after four consecutive cycles of use.