An amperometric biosensor based on laccase immobilized in polymer matrices for determining phenolic compounds

An amperometric enzyme electrode based on laccase for determining phenolic compounds is proposed. The following three types of polymer materials were used for enzyme immobilization on the surface of a glassy-carbon electrode: positively charged cetyl ethyl poly (ethyleneimine) (CEPEI) and negatively charged commercial Nafion and Eastman AQ 29D polymers. The advantages and disadvantages of each of the above polymers for enzyme immobilization are discussed. The detection limits of the model phenolic compounds hydroquinone and pyrocatechol in a buffer solution on laccase immobilization in a Nation membrane were 3.5 x 10(-8) and 5.0 x 10(-8) M, respectively, at a signal-to-noise ratio of 3. Electrodes with laccase immobilized in Nation and Eastman AQ 29D membranes exhibited the shortest response time. The operating stability and the stability in storage can be significantly improved by the additional incorporation of gelatin in the polymer matrices. Gelatin prevents enzyme inactivation as a result of enzyme modification by the free-radical oxidation products of phenolic compounds

Electrochemistry and kinetics of fungal laccase mediators

The screening of potential redox mediators for laccase was performed using homogeneous Trametes hirsuta laccase. Heterogeneous (electrochemical) and homogeneous (oxidation by laccase) reactions of the different types of the enhancers (mediators) of the enzyme were investigated. It was discovered that derivatives of phenyl-methyl-pyrazolones and benzoic acid, as well as N-hydroxynaphthalimide were efficient substrates for the laccase. The characterization of several representatives from each class was carried out using electrochemical and enzyme kinetics methods. The kinetic parameters for the oxidation of phenyl-methyl-pyrazolones and 3-(6-hylroxy)-aminobenzoic acid were comparable to those for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by the laccase, whereas the rate of enzymatic oxidation of N-hydroxynaphthalimide was sufficiently lower. Electrochemical experiments demonstrated that only oxidation of phenyl-methyl-pyrazolones and N-hydroxynaphthalimide yielded several high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, whereas derivatives of benzoic acid showed low-potential intermediate, which was not able to oxidized lignin model compound. Phenyl-methyl-pyrazolones was about 50% as effective in degrading veratryl alcohol compared to ABTS as judged from HPLC kinetic studies, whereas N-hydroxynaphthalimide showed the same efficiency as ABTS. Phenyl-methyl-pyrazolones and hydroxynaphthalimides may be of commercial interest for oxidoreductase-catalyzed biodegradation of different xenobiotics. (c) 2005 Elsevier B.V. All rights reserved

Direct electron transfer between copper-containing proteins and electrodes

The electrochemistry of some copper-containing proteins and enzymes, viz. azurin, galactose oxidase, tyrosinase (catechol oxidase), and the "blue" multicopper oxidases (ascorbate oxidase, bilirubin oxidase, ceruloplasmin, laccase) is reviewed and discussed in conjunction with their basic biochemical and structural characteristics. It is shown that long-range electron transfer between these enzymes and electrodes can be established, and the mechanistic schemes of the DET processes are proposed. (c) 2004 Elsevier B. V. All rights reserved

Laccases with Variable Properties from Different Strains of <i>Steccherinum ochraceum</i>: Does Glycosylation Matter?

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and characterized seven laccases from different strains of Steccherinum ochraceum obtained from regions of central Russia. Although all seven laccases had the same primary sequences, there was a little variation in their molecular weights and thermostabilities. Moreover, statistically significant differences in laccases&#8217; catalytic parameters of oxidation of phenolic substrates and ABTS were observed. After the deglycosylation of four selected laccases by Endo H and PNGase F, their affinities to pyrocatechol and ABTS became the same, suggesting a substantial role of N-linked glycosylation in moderation of enzymatic properties of laccases