Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths

Amperometric detection of mono- and diphenols at Cerrena unicolor laccase-modified graphite electrode: correlation between sensitivity and substrate structure

Graphite electrode modified with laccase from Cerrena unicolor served as a biosensor for detection of 30 phenolic compounds with different structures. Some correlations of the sensor response to the structures of substrates are discussed. This biosensor responded to: (i) nanomolar concentrations of some of the selected phenolic compounds, e.g., 2,6-dimethoxyphenol,coniferyl alcohol, caffeic acid, DOPAC and hydroquinone, (ii) micromolar concentrations, e.g., ferulic acid, syringic acid, dopamine, 3,4-dihydroxybenzoic acid and DL-noradrenaline, and (iii) millimolar concentrations in the case of phenol and 4-hydroxybenzaldehyde. Among the ortho- or para-substituted phenols, the sensitivity of the C unicolor laccase-modified electrode increased in the following order -H, -CH3, -OH, -OCH3 and -NH3+ but in the case of para-substituted phenols, the K-m(app) values were lower. The sensitivity of the laccase electrode increased with an additional -OH group in para-substituted phenols. In the case of the selected compounds, kinetic data from electrochemical flow injection system were compared with those obtained from experiments in solution. (c) 2005 Elsevier B.V. All rights reserved

Use of laccase-modified electrode for amperometric detection of plant flavonoids

In this study, laccase from Cerrena unicolor as a highly active enzyme was adsorptively immobilized on the surface of graphite electrode. The electrode inserted into flow-injection system served as a biosensor for detection of polyphenols, including flavonoids. We used catechin hydrate, epicatechin, epicatechin gallate, prodelphinidin, and caffeic acid as a target compounds in determination. The performed investigations demonstrate that laccase from white rot fungus C unicolor can be applied for the construction of biosensors useful for determination of flavonoids and related compounds in flow-injection system. Electrodes modified with laccase yield responses for both simple compounds (caffeic acid) and compounds with three or more phenolic and non-phenolic rings, although with different sensitivity. (C) 2004 Elsevier Inc. All rights reserved

Characterization of graphite electrodes modified with laccases from Trametes hirsuta and Cerrena unicolor and their use for flow injection amperometric determination of some phenolic compounds

Fungal laccases from Trametes hirsuta and Cerrena unicolor were immobilized on spectroscopic graphite electrodes using physical absorption. The laccase-modified graphite electrodes were mounted as the working electrodes into a wall jet flow through an electrochemical cell and then used for flow injection amperometric determination of different phenolic compounds of relevance both for environmental and clinical analysis. At optimum conditions for determination of catechol serving as a model compound, the output response signals of the laccase-modified electrodes were recorded as the result of injections of 50 mu L of solutions with different phenolic Substrates into the carrier citrate buffer solution with a working potential of -50mV vs. Ag vertical bar AgCl. Statistical aspects were applied and the Michaelis-Menten constants evaluated were correlated to the chemical structure or the investigated phenolic compounds

Novel textile dye obtained through transformation of 2-amino-3-methoxybenzoic acid by free and immobilised laccase from a Pleurotus ostreatus strain

Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting lac- case-mediated transformation efficiency included the selection of type and pH value of buffer, reaction tem- perature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40 mM sodium-tartrate buffer pH 5.5 containing 3 mM of the substrate, efficiently transformed by 2 U of free laccase per 1 mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C18) incubated in optimum conditions of 40 mM phosphate buffer pH 7.0 containing 1 mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40 °C and 60 °C in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U

Development of a laccase-modified electrode for amperometric detection of mono- and diphenols. The influence of enzyme storage method

Results are reported for biosensors based on two different preparations of the same enzyme, laccase from Cerrena unicolor, one lyophilized and one stored frozen at - 18degreesC, for monitoring phenolic compounds. The enzyme was adsorbed on graphite electrodes and these were used in a flow through wall jet cell connected to a flow injection set-up. The electrodes were used at - 50 mV vs. AgAgCl. The effect of PH, flow rate of the carrier buffer was investigated as well as the operational and storage stability. Biosensors based on the frozen enzyme preparation were shown to be superior for biosensor construction

Direct electron transfer reactions of laccases from different origins on carbon electrodes

Electrochemical studies of laccases from basidiomycetes, i.e., Trametes hirsuta, Trametes ochracea, Coriolopsis fulvocinerea, Cerrena maxima, and Cerrena unicolor, have been performed. Direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. For all mentioned high potential laccases direct electron transfer (DET) has been registered at spectrographic graphite and highly ordered pyrolytic graphite electrodes. The characteristics of DET reactions of the enzymes were analysed under aerobic and anaerobic conditions. It is shown that the T I site of the laccase is the primary electron acceptor, both in solution (homogenous case) and at surface of the graphite electrode (heterogeneous case). A mechanism of ET for the process of the electroreduction of oxygen at the laccase-modified graphite electrodes is proposed and the similarity of this heterogeneous process to the laccase catalysed oxygen reduction homogeneous reaction is concluded. (c) 2005 Elsevier B.V. All rights reserved