Laccase-catalyzed Reactions Research Articles

Carbon-oxygen bond formation by fungal laccases: cross-coupling of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide with the solvents water, methanol, and other alcohols

Laccase-catalyzed reactions lead to oxidation of the substrate via a cation radical, which has been described to undergo proton addition to form a quinonoid derivative or nucleophilic attack by itself producing homomolecular dimers. In this study, for the substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide, we show that, besides the quinonoid form of substrate, all products formed are nonhomomolecular ones. Indeed, without addition of a reaction partner, heteromolecular products are formed from the quinonoid form of the laccase-substrate and the solvents water or methanol present in the incubation assay. Consequently, in laccase catalyzed syntheses performed in aqueous solutions or in the presence of methanol or other alcohols, undesirable heteromolecular coupling reactions between the laccase substrate and solvents must be taken into account. Additionally, it could be shown at the example of methanol and other alcohols that C-O-bound cross-coupling of dihydroxylated aromatic substances with the hydroxyl group of aliphatic alcohols can be catalyzed by fungal laccases.

Nuclear amination catalyzed by fungal laccases: Comparison of laccase catalyzed amination with known chemical routes to aminoquinones

Laccases are able to initiate nuclear amination of p-hydroquinones with primary aromatic amines, resulting in the formation of the corresponding monoaminated and diaminated quinones. Two laccase catalyzed reactions are compared with established synthetic routes to aminoquinones, showing that formation of products from laccase catalyzed reaction is comparable with reaction using sodium iodate as oxidant. Advantages and disadvantages of laccase catalyzed amination are discussed. It is concluded that laccase catalysis is less suitable than sodium iodate oxidation for the amination of simple p-hydroquinones with simple amines.

Online kinetic studies on intermediates of laccase-catalyzed reaction in reversed micelle

Using water/AOT/ n-octane reversed micelle as the medium, the optical signal of the reactive intermediate of laccase-catalyzed oxidation of o-phenylenediamine, which was indetectable in aqueous solutions, was successfully captured. Thus online kinetic studies of the intermediate were accomplished. Two-way kinetic spectral data were acquired with stopped-flow technique. By resolving the data with global analysis software, both the kinetic curves and the absorption spectra of the components involved in the reaction process were simultaneously obtained. The whole reaction in the reversed micelle was proved to be composed of two successive steps, an enzymatic generation of the intermediate and a following nonenzymatic decay of the intermediate. A consecutive first-order kinetic model of the whole reaction was confirmed. The influences of microenvironmental factors of the medium (such as the pH value of the water pool and the water/AOT ratio) on the detection of the intermediate were also investigated.

Kinetics of laccase-catalyzed oxidative polymerization of catechol

Oxidative polymerization of catechol catalyzed by laccase enzyme from Trametes versicolor (ATCC 200801) in aqueous solution containing acetone was investigated in a batch system. The effects of initial catechol and dissolved oxygen concentrations on the initial reaction rate of oxidative polymerization were experimented. An interactive kinetic model as a function of catechol and dissolved oxygen concentrations was developed for enzymatic polymerization and corresponding biokinetic parameters have been evaluated for the first time. The parameters of the model, V max, K mm, K mO 2 and K i were found 0.247 g O 2 m −3 min −1, 70.75 g m −3, 10.4 g m −3 and 48.15 g m −3, respectively. The activation energy and reaction rate constant of oxidative catechol polymerization were calculated as 66.8 kJ mol −1 and 216.3 s −1, respectively. The estimated activation energy and rate constant of the reaction are of the order of magnitude with previously reported values for laccase-catalyzed reactions of different monomers.

Polymerization of guaiacol and a phenolic beta-O-4-substructure by Trametes hirsuta laccase in the presence of ABTS.

The reaction of the monomeric lignin model compound guaiacol and the beta-O-4-type dimer erol (1-(4-hydroxy-3-methoxyphenyl)-2(2-methoxyphenoxy)-propane-1,3-diol with laccase from Trametes hirsuta was studied in the presence of the mediator ABTS (2,2'-azino-di[3ethylbenzothiazoline-6-sulfonic acid]). The product mixtures were analyzed by means of aqueous-phase size exclusion chromatography (SEC) with 50 mM NaOH as eluent. Interestingly, in the laccase-catalyzed reaction with both substrates, the mediator not only functioned as an electron carrier but underwent coupling reactions with the substrate to give polymeric coupling products. The molecular weight of these copolymeric products was significantly higher than the molecular weight of products obtained without ABTS. After ultrafiltration, 33% and 21% of the initially applied ABTS could be found in the polymeric product fraction for the substrates guaiacol and erol, respectively, on the basis of nitrogen analysis. When ABTS was added to substrates after full laccase-catalyzed polymerization, the reaction proceeded toward higher molecular weights.

Degradation of trilinolein by laccase enzymes

Laccase enzymes were investigated for their potential to catalyze the oxidation of trilinolein and methyl linoleate. This study demonstrates that laccase enzymes can oxidize unsaturated fatty acid esters and their associated lipids. The reaction products resulting from laccase-catalyzed reactions with trilinolein were analyzed using combined reversed-phase high-performance liquid chromatography and mass spectrometry via an atmospheric pressure chemical ionization source. The dominant oxidation products detected were monohydroperoxides, bishydroperoxides, and epoxides. This paper presents the first detailed investigation into the interaction between laccase enzymes and lipids containing unsaturated fatty acids.

Voltammetric monitoring of laccase-catalysed mediated reactions

Six different compounds capable of mediating laccase-catalysed reactions have been tested by cyclic voltammetry. They exhibited quasi-reversible electrodic behaviour with formal redox potentials ranging from 150 to 800 mV ( E 0′ vs. SCE). The immersion of a laccase-coated glassy carbon electrode (GCE) in mediator solutions generated large cathodic catalytic currents easily recorded by cyclic voltammetry at low-potential scan rates. This current showed two well-defined pH profiles, which correlated with the variation of the mediator redox potentials at the pH range tested. The relevant effect of temperature on the activity of laccase has been assessed here. Likewise, it was shown that the current record varied with the substrate concentration. This trend fitted Michaelis–Menten kinetics, which allowed us to give an estimation of the affinity of the fungal laccase for the different mediators.

Reaction kinetics for laccase-catalyzed polymerization of 1-naphthol

Laccase-catalyzed oxidative polymerization of 1-naphthol was carried out in a closed system containing acetone and sodium acetate buffer. The effects of initial 1-naphthol and dissolved oxygen concentrations on the initial reaction rate were investigated. A multiplicative mathematical model, using a function of 1-naphthol and dissolved oxygen concentrations, was developed for enzymatic polymerization and the corresponding biokinetic parameters have been evaluated for the first time. The activation energy and reaction rate constant of the laccase-catalyzed 1-naphthol polymerization were calculated as 57 kJ/mol and 311 l/s, respectively. The activation energy calculated was in the typical range of 30–60 kJ/mol and rate constant was of the order of magnitude of previously reported values for laccase-catalyzed reactions with different monomers.

The application of ESR-spin Stable Technique in Laccase Chemical System

A method for the effective generation fo high steady state concentration of semiqumone radicals was established in laccase/O2 system by using ESR-spm stable technique in static condition.The result of ESR demonstrated that the laccase-catalyzed reaction of these substrates underwent the stage of semiqumones.

Micelle Enhanced Spectrofluorimetric Determination of L-ascorbic Acid Based on Laccase-linked Coupling Reaction Using Flow-injection Stopped-flow Technique.

A novel spectrofluorimetric method for determination of L-ascorbic acid is described. It is based on the inhibition of L-ascorbic acid on the formation of 2, 3-diaminophenazine, which is an oxidation product of o-phenylenediamine catalyzed by laccase, in the presence of Brij-35, which shows strong enhancement on the fluorescence(at λem/λex=530nm/430nm) of the product, while no effect on the laccase-catalyzed reaction. The mechanism of o-phenylenediamine oxidation reaction catalyzed by laccase in the presence of L-ascorbic acid is discussed. L-ascorbic acid is determined in the range of O.02-2.Oμg/ml with a detection limit of 0.014μg/ml. The method is applied to the determination of L-ascorbic acid in pharmaceuticals, the results agree well with the nominal values and those obtained by the reference method.

Laccase/glucose oxidase electrode for determination of glucose

The electrode involves a layer of co-immobilized glucose oxidase and laccase in a gelatin membrane placed over a modified oxygen electrode. Hexacyanoferrate(III) is added to the samples to oxidize reductive interferents such as ascorbic acid, and the hexacyanoferrate(II) formed is re-oxidized by a laccase-catalyzed reaction. Ascorbic acid is completely eliminated up to a concentration of 20 mM in the sample.

The mechanism of electron transfer in laccase-catalysed reactions

1. 1. The reaction of the electron acceptors in Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) have been studied with stopped-flow and rapid-freeze EPR techniques. The studies have been directed mainly towards elucidation of the role of the type 2 Cu 2+ as a possible pH-sensitive regulator of electron transfer. 2. 2. Anaerobic reduction experiments with Rhus laccase indicate that the type 1 and 2 sites contribute one electron each to the reduction of the two-electron-accepting type 3 site. There is also evidence that the reduction of the type 1 Cu 2+ triggers the reduction of the type 2 Cu 2+. 3. 3. Only at pH values at which the reduction of the two-electron acceptor is limited by a slow intramolecular reaction can an OH − be displaced from the type 2 Cu 2+ by the inhibitor F −. 4. 4. A model describing the role of the electron-accepting sites in catalysis is formulated.

Oxidative polymerization of lignins by laccase in water-acetone mixture.

The enzymatic oxidative polymerization of five technical lignins with different molecular properties, i.e. Soda Grass/Wheat straw Lignin, Organosolv Hardwood Lignin, Soda Wheat straw Lignin, Alkali pretreated Wheat straw Lignin, and Kraft Softwood was studied. All lignins were previously fractionated by acetone/water 50:50 (v/v) and the laccase-catalyzed polymerization of the low molecular weight fractions (Mw < 4000 g/mol) was carried out in the same solvent system. Reactivity of lignin substrates in laccase-catalyzed reactions was determined by monitoring the oxygen consumption. The oxidation reactions in 50% acetone in water mixture proceed with high rate for all tested lignins. Polymerization products were analyzed by size exclusion chromatography, FT-IR, and (31)P-NMR and evidence of important lignin modifications after incubation with laccase. Lignin polymers with higher molecular weight (Mw up to 17500 g/mol) were obtained. The obtained polymers have potential for applications in bioplastics, adhesives and as polymeric dispersants.