Environmental Science and Pollution Research

, Volume 25, Issue 20, pp 19989–20002 | Cite as

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

  • Diego Alberto Morales Urrea
  • Patricia Mónica Haure
  • Fernando Sebastián García Einschlag
  • Edgardo Martín ContrerasEmail author
Research Article


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 (q0) was studied. As a general rule, a noticeable increase in q0 was observed for pHs higher than 7. For a given pH, q0 increased as a function of the initial OII concentration. Besides, there was an inhibitory effect of high P concentrations on q0. 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.


Horseradish peroxidase Orange II Hydrogen peroxide Decolourization Kinetic model 





absorbance at the end of the a decolourization experiment


minimum absorbance obtained under the excess of hydrogen peroxide


dissociated form of the enzyme


resting state of the enzyme


compound I of the enzyme


compound II of the enzyme


non-dissociated form of the enzyme


horseradish peroxidase


Orange II


oxidation products of Orange II


hydrogen peroxide


critical hydrogen peroxide concentration


initial specific decolourization rate


reducing substrate


initial reducing substrate consumption rate


initial decolourization rate


reducing substrate consumption rate


decolourization rate


oxidation products obtained at basic conditions


oxidation products obtained at acid conditions


observable stoichiometric coefficient of decolourization



The authors gratefully acknowledge the financial support from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), by Universidad Nacional de la Plata (UNLP) and by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina.

Supplementary material

11356_2018_2134_MOESM1_ESM.docx (276 kb)
ESM 1 (DOCX 276 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Diego Alberto Morales Urrea
    • 1
  • Patricia Mónica Haure
    • 1
    • 2
  • Fernando Sebastián García Einschlag
    • 3
    • 4
  • Edgardo Martín Contreras
    • 1
    Email author
  1. 1.Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA)CCT - Mar del Plata CONICETMar del PlataArgentina
  2. 2.Facultad de IngenieríaUniversidad Nacional de Mar del Plata (UNMdP)Mar del PlataArgentina
  3. 3.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA)CCT - La Plata – CONICETLa PlataArgentina
  4. 4.Facultad de IngenieríaUniversidad Nacional de La Plata (UNLP)La PlataArgentina

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