Horseradish peroxidase-mediated decolourization of Orange II: modelling hydrogen peroxide utilization efficiency at different pH values

Abstract

Enzymatic decolourization of azo-dyes could be a cost-competitive alternative compared to physicochemical or microbiological methods. Stoichiometric and kinetic features of peroxidase-mediated decolourization of azo-dyes by hydrogen peroxide (P) are central for designing purposes. In this work, a modified version of the Dunford mechanism of peroxidases was developed. The proposed model takes into account the inhibition of peroxidases by high concentrations of P, the substrate-dependant catalatic activity of peroxidases (e.g. the decomposition of P to water and oxygen), the generation of oxidation products (OP) and the effect of pH on the decolourization kinetics of the azo-dye Orange II (OII). To obtain the parameters of the proposed model, two series of experiments were performed. In the first set, the effects of initial P concentration (0.01-0.12 mM) and pH (5-10) on the decolourization degree were studied at a constant initial OII concentration (0.045 mM). Obtained results showed that at pH 9-10 and low initial P concentrations, the consumption of P was mainly to oxidize OII. From the proposed model, an expression for the decolourization degree was obtained. In the second set of experiments, the effect of the initial concentrations of OII (0.023-0.090 mM), P (0.02-4.7 mM), HRP (34-136 mg/L) and pH (5-10) on the initial specific decolourization rate (q) was studied. As a general rule, a noticeable increase in q was observed for pHs higher than 7. For a given pH, q increased as a function of the initial OII concentration. Besides, there was an inhibitory effect of high P concentrations on q. To asses the possibility of reusing the enzyme, repeated additions of OII and P were performed. Results showed that the enzyme remained active after six reuse cycles. A satisfactory accordance between the change of the absorbance during these experiments and absorbances calculated using the proposed model was obtained. Considering that this set of data was not used during the fitting procedure of the model, the agreement between predicted and experimental absorbances provides a powerful validation of the model developed in the present work.

Immobilization of horseradish peroxidase onto electrospun polyurethane nanofiber matrices

“…In the cases when adsorption was the sole OII removal process (Figure 6(A,B)), UV/Vis spectra at the end of the assay had a lower intensity but a similar shape than the initial one. Conversely, UV/Vis spectra under the presence of P (Figure 6(C)) exhibited all characteristics corresponding to a typical OII oxidation assay catalyzed by free HRP 8,9 . As the oxidation proceeded, a decrease of OII characteristic absorbance bands at 485 and 430 nm was observed, and a new absorption band at 340 nm emerged due to the formation of OII oxidation products (OP).…”

“…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.…”

“…where α ¼ kd kc is the ratio between two constants. Equations ( 3), (9), and (10) were solved using the software GEPASI 3.30. 36 According to Morales Urrea et al, 8 at pH = 9, K P = 300 mM À1 , K I = 0.05 mM À1 , and α = 8.7 mM À1 .…”

“…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 …”

“…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][8][9] Due to the high cost associated with some enzymes, a key issue to develop an economically feasible enzyme-based process is the reutilization of the catalyst. In contrast with free enzymes, immobilized enzymes are easy to handle, and immobilization processes can often be designed to confer higher enzymatic stability, ensuring reusability, and achieving economically viable bioprocesses.…”