Decolorization of a chromophore molecule with immobilized horseradish peroxidase / Descoloração de uma molécula de cromóforo com peroxidase de rábano imobilizada

Priscila S. Corrêa, Suzana G. de Lima, Caio F. Pastusiak, Alfredo J. T. Bosco, Eliana M. Alhadeff


The enzymes can modify some effluent characteristics in order to increase the degradability, or the bioconversion of liquid  effluents. The oxireductases, laccases and peroxidases have been used due their high potentiality in many environmental treatments of natural and synthetic organic compounds as dyes, phenols and polyphenolics molecules. The performance of immobilized horseradish peroxidase on aminopropyl glass beads was investigated in this work in a decolorization reaction of methylene blue colorant. The experiments were conducted in batch conditions during 3 hours, with different aqueous solutions of peroxide hydrogen (H2O2) concentration solutions (2-10 mg/L), methylene blue (ME)  (5-20 mg/L) and the pH in the range from 4 to 8, according an experimental design proposed by the software STATISTICA®. After 3 hours of treatment the reduction of  the color was 60% when comparing to the original color and 50% when the immobilized enzymes were reused in five sequential batch treatment cycles for a 10 mg/L of H2O2 solution, 20 mg/L of ME  and pH 8.0.  Working in continuous process with two microreactors in series the system showed a good performance with 97% of decolorization in the first 15 minutes. After 1 hour of continuous treatment the percentage of the color removing was around 70%.



immobilized horseradish peroxidase; enzymatic decolorization; sequential enzymatic microreactors.

Full Text:



Alhadeff E.M., Salgado A.M., Pereira Jr. N., Valdman B., (2004). Development and Application of an Integrated System for Monitoring Ethanol Content of Fuels. Applied and Biochemistry Biotechnology, 113-116, 125-136.

Alhadeff E.M., Salgado A.M., Pereira Jr. N., Valdman B., (2005). A Sequential Enzymatic Microreactors System for Ethanol Detection of Gasohol Mixtures. Applied and Biochemistry Biotechnology,121, 1-12.

Axelsson J., Nilsson U., Terrazas E., Aliaga, T.A., Welander U., (2006). Decolorization of the Textile Dyes Reactive Red 2 and Reactive Blue 4 using Bjerkandera sp. Strain BOL 13 in a Continuous Rotating Biological Contactor Reactor, Enzyme and Microbiology Technology, 39, 32–37.

Bilal, M., Iqbal, H.M.N., Shah, S.Z.H., Hu, H., Wang, W., Zhang, X., (2016). Horseradish peroxidase-assisted approach to decolorize and detoxify dye pollutants in a packed bed bioreactor. Journal of Environmental Management, 183, 836-842.

Bilal, M., Iqbal, H.M.N., Hu, H., Wang, W., Zhang, X., (2017a). Enhanced bio-catalytic performance and dye degradation potential of chitosan-encapsulated horseradish peroxidase in a packed bed reactor system. Science of the Total Environmental, 575, 1352-1360.

Bilal, M., Rasheed, T., Iqbal, H.M.N., Hu, H., Wang, W., Zhang, X., (2017b). Novel characteristics of horseradish peroxidase immobilized onto the polyvinyl alcohol-alginate beads and its methyl orange degradation potential. International Journal of Biological Macromolecules, 105, 328-335.

Bilal M., Asgher M., Parra-Saldivar R., Hua H., Wang W. , Zhang X., Iqbal H. M.N., (2017c). Immobilized ligninolytic enzymes: An innovative and environmental responsive technology to tackle dye-based industrial pollutants – A review. Science of the Total Environment, 576, 646-659.

Bilal M., Rasheedb T., Iqbal H. M.N., Yand Y., (2018). Peroxidases-assisted removal of environmentally-related hazardous pollutants with reference to the reaction mechanisms of industrial dyes. Science of the Total Environment, 644, 1-13. .

Bradford M.M., (1976). A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding. Anal. Biochem. 72, 248–254.

Celebi M., Kaya M. A., Altikatoglu M., Yildirim H., (2013). Enzymatic Decolorization of Anthraquinone and Diazo Dyes Using Horseradish Peroxidase Enzyme Immobilized onto Various Polysulfone Supports. Appl Biochem Biotechnol. 171, 716–730.

Champagne P.P., Ramsay J.A., (2007). Reactive Blue 19 Decolouration by Laccase Immobilized on Silica Beads, Appl. Microbiol. Biotechnol. 77, 819–823.

Guaratini C.C.I., Zanoni M.V.B., (2000). Corantes Têxteis. Quím. Nova. 1, 71-1. Retrieved from

Jamal F., Signh S., Khatoon S., Mehrotra S., (2013). Application of Immobilized Pointed Gourd (Trichosanthes dioica) Peroxidase- Concanavalin A Complex on Calcium Alginate Pectin Gel in Decolorization of Synthetic Dyes Using Batch Processes and Continuous Two Reactor. System.J. Bioproces. Biotechniq. 3, 131-135. doi:10.4172/2155-9821.1000131.

Jamal F. and Goel T., (2014). Diethylaminoethyl Cellulose Immobilized Pointed Gourd (Trichosanthes dioica) Peroxidase in Decolorization of Synthetic Dyes. J Bioproces Biotech, 4:7, p.1-5, 187. doi: 10.4172/2155-9821.1000187.

Malania, R. S., Khannab, S., Moholkar, V. S., (2013). Sonoenzymatic decolourization of an azo dye employing immobilized horse radish peroxidase (HRP): A mechanistic study. Journal of Hazardous Materials, 256– 257, 90– 97.

Nicell J. and Wright H., (1997). A Model of Peroxidase Activity with Inhibition by Hydrogen Peroxide. Enzyme and Microb. Technol. 21, 302-310.

Pereira A. R.M., da Costa R. S.M., Yokoyama L., Alhadeff E.M., Teixeira L.A.C., (2014). Evaluation of Textile Dye Degradation Due to the Combined Action of Enzyme Horseradish Peroxidase and Hydrogen Peroxide. Appl. Biochem. Biotechnol. 174, 2741–2747.

Stolz A., (2001). Basic and Applied Aspects in the Microbial Degradation of Azo Dyes, Appl. Microbiol. Biotechnol., 56, 69-80.

Veitch N., (2004). Horseradish peroxidase: a Modern View of a Classic Enzyme. Phytochemistry, 65, 249-259. doi: 10.1016/j.phytochem.2003.10.022.

Yanto D.H.Y., Tachibana S, Itoh K., (2014). Biodecolorization of textile dyes by immobilized enzymes in a vertical bioreactor system, Procedia Environmental Sciences 20, 235 – 244.

Zaia D.A.M., Zaia C.T.V.V., Lichtig J., (1998). Determinação de Proteínas Totais Via Espectrofometria: Vantagens e Desvantagens dos Métodos Existentes. Química Nova, v. 21, n. 6, p. 787-793. Retrieved from