Short communication Exploitation of neglected horseradish peroxidase izoenzymes for dye decolorization Zoran Vuj  ci  c a, * , Barbara Janovi  c b , Nikola Lon  car a , Aleksandra Margeti  c b , Nata  sa Bo  zi  c b , Biljana Dojnov b , Miroslava Vuj  ci  c b a Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia b Institute of Chemistry, Technology and Metallurgy-Center of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia article info Article history: Received 25 September 2014 Received in revised form 8 October 2014 Accepted 8 October 2014 Available online Keywords: Horseradish peroxidase HRP Decolorization Reactive dyes Zymogram Izoenzymes abstract Horseradish peroxidase (HRP) is enzyme first described more than 200 years ago and yet there are still some aspects of this potent enzyme to be tackled. Researchers were focused on most abundant isoenzyme HRP C1A while remaining, particularly anionic isoenzymes were discarded inpurificationprocess. This work describes exploitation of those isoenzymes for removal of recalcitrant pollutants such as reactive dyes. Results demonstrated that not only these enzymes can decolorize dyes but also in some cases anionic forms are more efficient than commercially produced cationic HRP form. Enzyme concentration of 0.14 U ml 1 was found to provide maximum dye removal at optimized reaction conditions with dye concentration of 30 mg l 1 . Ma- jority of dyes tested were successfully decolorized at pH 5 or 7 while some dyes like Orange 2 and Reactive black 5 are decolorized most efficiently at pH 9. Anionic isoenzymes act by disrupting chromophore of Reactive black 5 while cationic HRP oxidize dye but leaves chromophore present. © 2014 Elsevier Ltd. All rights reserved. Introduction Horseradish peroxidase (HRP, EC 1.11.1.7) in the presence of hydrogen peroxide can oxidize variety of organic and inorganic compounds. Being seasonal plant horseradish is rather expensive source for enzyme purification yielding up to 100 mg of HRP isoenzyme preparation from 1 kg of roots and hence there are at- tempts to produce recombinant HRP (Krainer et al., 2014). Despite high cost and due to high activity of HRP it remains indispensible component of vast array of diagnostic kits and research tools. There are many different enzyme isoforms present in the roots of plants, but most of the large scale production uses cationic isoforms (Regalado et al., 2004; Veitch, 2006). Characterization of different isoenzymes is desirable in order to choose one that fits the most for specific application (Krainer et al., 2014). Since anionic isoforms are discarded in purification process of main isoform HRP C1A, they have no commercial values and this opens a new perspective in treatment of colored wastewater. HRP C1A is the only HRP isoform with solved structure, while much less is known about remaining isoforms. It is well known that HRP can polymerize phenolic and bisphenolic compounds, as well as aromatic amines such as dyes (Nicell et al., 1993; Bhunia et al., 2002; Mohan et al., 2005). Previous studies showed promising capability of HRP in decolorization of different textile and non-textile dyes (de Souza et al., 2007; da Silva et al., 2010), but not much data is available on efficacy comparison of anionic and cationic isoforms in dye removal. The objective of the present study was to compare anionic and cationic HRP isoforms in decolorization of different dyes depending on pH. Optimum conditions regarding concentration of hydrogen peroxide, dye and enzyme were tested in order to achieve best decolorization with minimal inputs. Material and methods Reagents All reagents and solvents were purchased from Merck (Darm- stadt, Germany) and SigmaeAldrich (St. Louis, MO, USA) unless otherwise stated. Fractionation of acidic and basic HRP isoforms Horseradish roots were purchased from local market in village Grabovac near Belgrade (Serbia) directly from producers. Homog- enized roots in distilled water were filtered through cheese cloth * Corresponding author. E-mail address: zvujcic@chem.bg.ac.rs (Z. Vuj ci c). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod http://dx.doi.org/10.1016/j.ibiod.2014.10.007 0964-8305/© 2014 Elsevier Ltd. All rights reserved. International Biodeterioration & Biodegradation 97 (2015) 124e127