Full Paper Bioelectrocatalysis of Plant Peroxidases Immobilized on Graphite in Aqueous and Mixed Solvent Media Zuzana Brusova, a Elena E. Ferapontova,* b, c Ivan Yu. Sakharov , d Edmond Magner, a Lo Gorton c a Materials and Surface Science Institute and Department of Chemical and Environmental Science, University of Limerick, Plassey, Co. Limerick, Ireland b Group of Bioinformatics, Weblab, IT Centre, Voskhod 26a, Novosibirsk 630102, Russia c Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden *e-mail: elena.ferapontova@analykem.lu.se; ferapontova@nsk.fio.ru d Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119899, Russia Received: July 15, 2004 Final version: September 3, 2004 Abstract The bioelectrocatalytic function of two plant peroxidases, horseradish peroxidase (HRP) and a newly purified royal palm tree-leaf peroxidase (RPTP), was studied on graphite electrodes in aqueous buffer solutions, over the pH range 7.0 – 3.0, and in aqueous buffer solutions, at pH 6.0, containing different amounts of a polar organic cosolvent, i.e., ethanol and acetonitrile. The kinetics of direct and of mediated (using catechol as mediator) reduction of H 2 O 2 at the HRP- and RPTP-modified graphite electrodes were analyzed amperometrically at  50 mV(vs. Ag j AgCl j 0.1 M KCl) in a flow-through wall-jet electrochemical system. Values of the apparent rate constant of the heterogeneous electron transfer between the enzyme and graphite, k s , the rate constant for enzymatic reduction of H 2 O 2 , k 1 , and the rate constant of mediated reduction of H 2 O 2 , k 3, were determined using the modified Koutecky-Levich approach. Analysis of the variation of the rate constants and the response of the peroxidase-modified electrodes to H 2 O 2 with pH and content of the organic cosolvent demonstrated that maximal peroxidase bioelectrocatalytic activity occurred at pH 5.0 – 6.0 and at concentrations of ethanol of 10 – 20% v/v. At a lower pH, higher concentrations of ethanol, and in aqueous solutions of acetonitrile, the bioelectrocatalytic activity of the peroxidase-modified electrodes drastically decreased. However, contrary to the data for homogeneous catalysis, both peroxidases were bioelectrocatalytically active even in 95% organic co-solvents, thus demonstrating a stabilizing effect of the enzyme immobilization on the bioelectrocatalytic performance of the peroxidases. RPTP immobilized on graphite demonstrated lower overall activity but a higher pH- and organic cosolvent-stability than HRP. Keywords: Horseradish peroxidase, Royal palm tree peroxidase, Bioelectrocatalysis, Acetonitrile, Ethanol 1. Introduction Biocatalysis in organic/water-restricted organic media pro- vides a variety of advantages both for fundamental studies of enzyme catalysis and in biotechnology, e.g., in organic synthesis, biosensor development, and environmental chemistry [1 – 4]. As indicated by Klibanov [1, 5, 6], these advantages include: 1) increased solubility of reactants, especially in the case of hydrophobic substrates, 2) enhanced stability of enzymes in appropriate organic media, 3) control of enzymatic selectivity and specificity, 4) elimination of nonenzymatic and side reactions favorable in aqueous media, 5) modulation of “molecular memory” of the enzyme, 6) control of enzyme complex formation with ligands which are insoluble in organic solvents. Organic solvents can induce extensive changes in enzyme activity and specificity, since the molecular structure of enzymes and, consequently, their activity depend on numerous noncovalent interactions, i.e., ionic and hydrophobic/hydro- philic interactions, hydrogen bonding etc. [7]. The main disadvantage of biocatalysis in a nonaqueous environment is the drastically reduced enzymatic activity compared to that in aqueous media [8], which can arise from factors such as dehydration, structural effects, including enzyme denatura- tion and solvent-active site interactions, and substrate solvation effects. Immobilization of enzymes can protect the catalytically active conformation against structural perturbations and inactivation, and in some instances enhance the specific activity of enzymes in organic media [9 – 12]. To gain deeper insight into the mechanisms that define the catalytic function of redox enzymes in such media, we have examined the effects of two polar organic solvents, ethanol and acetonitrile, on the bioelectrocatalytic performance of heme-containing plant peroxidases (hydrogen peroxide oxidoreductase EC 1.11.1.7), specifically, horseradish per- oxidase (HRP) and the newly isolated royal palm tree peroxidase (RPTP), both immobilized onto graphite elec- trodes. HRP (isoenzyme C, pI 8.8) has been extensively used in the construction of biosensors [13], wastewater bioreme- diation and biotransformation of various drugs and chem- icals [14], synthesis in organic media, e.g., for phenolic polymerization [15], and as an indicator in HRP-based 460 Electroanalysis 2005, 17, No. 5 – 6  2005 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim DOI: 10.1002/elan.200403182