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, Re < 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.