Monitoring the Enzymatic Oxidation of Xenobiotics by Hydrogen Peroxide through Oxidation–Reduction Potential Measurements

Abstract

In this work, three xenobiotics (orange II, phenol, and bisphenol A) were oxidized by hydrogen peroxide in the presence of a horseradish peroxidase (HRP) using a fed-batch system. During the experiments, the oxidation− reduction potential (ORP) of the reaction mixture was measured continuously. Results demonstrate that ORP values only increased when both substrates of the enzyme (hydrogen peroxide and the target compound) were present in the reaction mixture. For all of the tested pollutants, the continuous addition of hydrogen peroxide caused an increase in ORP values. When the reducing substrate was depleted, the addition of an excess of hydrogen peroxide caused a decrease of ORP values. The time at which ORP reached a maximum represented the end of the oxidation process. This maximum could be easily detected by means of the derivative of ORP as a function of time. To extend the application of the developed technique, the enzymatic oxidation of a binary mixture of BPA and OII was also followed using ORP measurements. Results were similar to those observed with only one reducing substrate. This work demonstrates that ORP measurements can be useful to maximize hydrogen peroxide efficiency through the controlled addition of the oxidant during the oxidation of OII, phenol, and BPA catalyzed by HRP. This approach allows a minimization of time and process costs since the reaction end-point can be easily detected on a real-time basis.

Immobilization of horseradish peroxidase onto electrospun polyurethane nanofiber matrices

“…While decolorization assays without P evaluate the adsorption of OII onto the different tested electrospun matrices, decolorization under the presence of P reflected both adsorption and oxidation processes. According to a previous work, 8 the optimum pH value to maximize the peroxidase activity of the tested HRP ranged between 9 and 10. Thus, adsorption tests were performed by immersing M, MA, or MAH membranes (1.0 ± 0.1 cm 2 ) in 4.4 ml of PB solution (100 mM, pH 9.0) containing OII (0.045 mM).…”

“…In a previous work, Morales Urrea et al 8 demonstrated that the oxidation of OII by hydrogen peroxide catalyzed by HRP produced an oxidation product (OP) that contributes to the absorbance of the reaction mixture. Because these OII oxidation products (OP) cannot be further oxidized by HRP under the tested conditions, the absorbance of the reaction mixture at a given time t ( A t ) is the contribution of OII ( A OII ), and OP ( A OP ) 8 : Taking into account that 1 mol of OII yields 1 mol of OP, 8 and assuming that the absorbance at the end of the experiment (A f ) is only due to the presence of OP (e.g., in the cases when the oxidation of OII is complete), the following expression can be obtained, where A 0 is the initial absorbance of the reaction mixture, is the ratio between the OII concentration at time t and the initial OII concentration, and is the ratio between final ( A f ) and initial ( A 0 ) absorbances. According to this definition, represents the maximum decolorization degree that can be achieved under the excess of oxidant when the oxidation of OII is complete.…”

“…However, in the cases when the OII oxidation was not complete (e.g., due to the inactivation of the enzyme or by the oxidant depletion, for example), the decolorization degree (R OII , %) was calculated as follows: where and are the initial and minimum absorbance values at 485 nm, respectively. Additionally, from the slope of the linear portion of the absorbance at 485 nm as a function of time, and considering that the molar attenuation coefficient of OII at 485 nm (ε OII ) is 19.5 ± 0.3 a.u./cm/mM, 8,9 the initial OII removal rate (V D0 , mM/h) was obtained. Then, the surface specific decolorization rate corresponding to the evaluated MAH (q 0 , μmolOII/h/cm 2 ) was calculated as follows: where V L (L) is the volume of the reaction mixture, and a (cm 2 ) is the area of the tested membrane.…”

“…To analyze OII decolorization profiles by hydrogen peroxide (P) catalyzed by free HRP and HRP‐containing matrices (MAH), in some experiments the HRP model developed by Morales Urrea et al 8 was used. This model represents consumption rates of OII (R S , mM/h), P (R P , mM/h), and the formation rate of oxidation products (R OP , mM/h): where [E], [S], and [P] represent enzyme, reducing substrate, and hydrogen peroxide concentrations, respectively.…”

“…Additionally, enzymatic processes are eco‐friendly cost‐competitive alternatives compared to physicochemical or microbiological methods 2,5,6 . In particular, horseradish peroxidase (HRP) has been successfully employed as a green catalyst to oxidize several xenobiotic compounds with hydrogen peroxide 7–9 …”