Kinetics of Horseradish Peroxidase-Catalyzed Nitration of Phenol in a Biphasic System

The use of peroxidase in the nitration of phenols is gaining interest as compared with traditional chemical reactions. We investigated the kinetic characteristics of phenol nitration catalyzed by horseradish peroxidase (HRP) in an aqueous-organic biphasic system using n-butanol as the organic solvent and NO₂^- and H₂O₂ as substrates. The reaction rate was mainly controlled by the reaction kinetics in the aqueous phase when appropriate agitation was used to enhance mass transfer in the biphasic system. The initial velocity of the reaction increased with increasing HRP concentration. Additionally, an increase in the substrate concentrations of phenol (0-2 mM in organic phase) or H₂O₂ (0-0.1 mM in aqueous phase) enhanced the nitration efficiency catalyzed by HRP. In contrast, high concentrations of organic solvent decreased the kinetic parameter V_max/K_m. No inhibition of enzyme activity was observed when the concentrations of phenol and H₂O₂ were at or below 10 mM and 0.1 mM, respectively. On the basis of the peroxidase catalytic mechanism, a double-substrate ping-pong kinetic model was established. The kinetic parameters were K_m^H2O2= 1.09 mM, K_m^PhOH = 9.45 mM, and V_max = 0.196 mM/min. The proposed model was well fit to the data obtained from additional independent experiments under the suggested optimal synthesis conditions. The kinetic model developed in this paper lays a foundation for further comprehensive study of enzymatic nitration kinetics.