Controlled Delivery of H₂O₂: A Three-Enzyme Cascade Flow Reactor for Peroxidase-Catalyzed Reactions

Peroxidases are promising catalysts for oxidation reactions, yet their practical utility has been hindered by the fact that they require hydrogen peroxide (H₂O₂), which at high concentrations can cause deactivation of enzymes. Practical processes involving the use of peroxidases require the frequent addition of low concentrations of H₂O₂. In situ generation of H₂O₂ can be achieved using oxidase-type enzymes. In this study, a three-enzyme cascade system comprised of a H₂O₂ generator (glucose oxidase (GOx)), H₂O₂-dependent enzymes (chloroperoxidase (CPO) or horseradish peroxidase (HRP)), and a H₂O₂ scavenger (catalase (CAT)) was deployed in a flow reactor. Immobilization of the enzymes on a graphite rod was achieved through electrochemically driven physical adsorption, followed by cross-linking with glutaraldehyde. Modeling studies indicated that the flow in the reactor was laminar (Reynolds number, R < 2000) and was nearly fully developed at the midplane of the annular reactor. Immobilized CAT and GOx displayed good stability, retaining 79% and 84% of their initial activity, respectively, after three cycles of operation. Conversely, immobilized CPO exhibited a considerable reduction in activity after one use, retaining only 30% of its initial activity. The GOx-CAT-GRE system enabled controlled delivery of H₂O₂ in a more stable manner with a 4-fold enhancement in the oxidation of indole compared to the direct addition of H₂O₂. Using CPO in solution coupled with GOx-CAT-GRE yields of 90% for the oxidation of indole to 2-oxyindole and of 93% and 91% for the chlorination of thymol and carvacrol, respectively.