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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.

Pectins are a group of heterologous polysaccharides capable of forming hydrogels and applicable in many industrial processes. A new type of modified pectin was synthesized by periodate oxidation and reductive amination with dopamine and sodium cyanoborohydride. The success of modification was confirmed by UV–Vis,FTIR, and 1H NMR spectroscopy. The obtained dopamine-pectin could form hydrogels by ionic crosslinking of carboxyl groups with calcium or by crosslinking phenol groups with laccase. For enzymatic crosslinking with laccase from Streptomyces cyaneus expressed in E. coli, isolation and purification of the enzyme was done. Using emulsion-based enzymatic crosslinking polymerization, dopamine-pectin microbeads with immobilized laccase were made. The immobilized laccase showed improved thermal and pH stability in comparison to the free enzyme. The immobilized biocatalyst effectively decolorized various dyes: Amido Black 10B, Reactive Black 5, and Evans Blue. After ten cycles of repeated use, the microbead immobilized laccase could still decolorize 60% and 36% of Amido Black 10B and Reactive Black 5, respectively.