Available online at www.sciencedirect.com Enzyme and Microbial Technology 42 (2007) 70–75 Is the presence of dicarboxylic acids required in the MnP cycle? Study of Mn 3+ stability by cyclic voltammetry C. L ´ opez a,∗ , J.C. Garc´ ıa-Monteagudo b , M.T. Moreira a , G. Feijoo a , J.M. Lema a a Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain b Department of Physico-Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain Received 16 February 2007; received in revised form 7 August 2007; accepted 7 August 2007 Abstract The catalytic cycle of the enzyme manganese peroxidase (MnP) requires the presence of dicarboxylic acids to chelate and stabilize the oxidized and very unstable Mn 3+ , which is responsible for the final substrate oxidation. However, the enzymatic degradation of an azo dye, Orange II, was successfully performed in the absence of any carboxylic acid. To analyze this possible discrepancy, the effect of the presence of several organic acids (oxalic, malonic, tartaric and citric acids) was studied on the kinetics and the extension of the degradation of Orange II. The Mn 3+ chelating strength, an important factor that should influence the efficiency of the degradation, was determined for the different organic acids and the dye by cyclic voltammetry. Oxalic acid was determined to be the best chelator, followed by malonic, tartaric and finally, citric acid. Orange II was shown to act as a chelator, since the hydroxyl and sulfonic groups allow a stabilized complex to be formed, avoiding the use of any dicarboxylic acid. This distinctive property could be extended to other molecules with a potential binding capacity. © 2007 Elsevier Inc. All rights reserved. Keywords: Mn 3+ stabilization; Cyclic voltammetry; Manganese peroxidase; Carboxylic acids; Orange II; Chelation 1. Introduction The ability of ligninolytic fungi for the oxidative degradation of complex molecules is based on the production of extracellu- lar enzymes, such as peroxidases and oxidases. The study of the catalytic cycle gives the essential information about the con- ditions, cosubstrates and cofactors required for optimizing the degradation. In the case of the enzyme manganese peroxidase (MnP), the oxidation is performed by the Mn 3+ ion obtained in two sequential steps of the enzymatic cycle [1], which corre- spond to the reduction of the oxidized form MnP I to MnP II and the native form in the presence of Mn 2+ . Two Mn 3+ ions are generated in each cycle of enzyme. Mn 3+ is a strong oxidant (1.5 V) and its small size facilitates its ability to reach compounds that are not readily accessible to the enzyme which has a molecular weight of 40–50 kDa. How- ever, Mn 3+ is quite unstable in aqueous solution, and dismutation reactions occur to produce Mn 2+ and Mn 4+ , the latter precipi- tating as MnO 2 [2]. Ligninolytic fungi also produce significant ∗ Corresponding author. Tel.: +34 981 563100x16740; fax: +34 981 528050. E-mail address: clopez@usc.es (C. L ´ opez). concentrations of dicarboxylic acids which chelate the ion, thus greatly increasing its half-life and, therefore, the efficiency of the process. An ideal chelator should accomplish three main actions: (i) to assist in the dissociation between Mn 3+ and enzyme; (ii) to stabilize the ion Mn 3+ ; (iii) to not chelate the form Mn 2+ because Mn 2+ must be free to easily access the enzyme [3]. Chelation occurs due to the presence of at least two groups able to interact with the Mn 3+ ion, thus generating chains which lead to the stabilization of the ion. Di- and tricarboxylic acids, such as oxalic, malonic, tartaric or citric acid (Table 1), have the potential capability of chelation [4,5]. Also, some mono- carboxylic acids are able to form chelates provided that their structure presents another functional group that can interact with the ion. Lactic acid, with an alcoholic and a carboxylic group, is an example of this [3]. In previous works, the degradation of the azo dye, Orange II, by MnP was assessed in batch processes in the absence of chelating carboxylic acids [6]. MnP was purified and dialyzed to remove all organic acids from the fermentation broth. Assays were performed in the absence and presence of malonic acid (Fig. 1) and the results were very similar in the two cases, although in the presence of the organic acid the degradation was slightly faster. 0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.08.002