Trametes Versicolor Laccase Research Articles

Determination of phenolic acids using Trametes versicolor laccase

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1–1.0 μM caffeic acid, 0.05–0.2 μM ferulic acid, 2.0–14.0 μM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0–30.0 μM caffeic acid, 2.0–10.0 μM ferulic acid, 4.0–30.0 μM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.

Laccase-catalyzed mediated oxidation of benzyl alcohol: the role of TEMPO and formation of products including benzonitrile studied by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Substituted benzyl alcohol was oxidized enzymatically with a laccase-mediator system and the products were investigated as a function of time by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI-FTICRMS). With Trametes versicolor laccase (TVL), the mediator, 2,2',6,6'-tetramethylpiperidine-N-oxyl radical (TEMPO), undergoes oxidation and forms oxoammonium ion. Oxidized TEMPO oxidizes the alcohol and is simultaneously reduced to the N-OH form. The laccase then restores TEMPO back to the normal radical form and the oxidation cycle starts again. The role of TEMPO and the structures of its oxidized and reduced forms in the enzymatic oxidation process were clarified in collision-induced dissociation experiments and gas-phase hydrogen/deuterium (H/D) exchange reactions. The amounts of enzyme and mediator were significant for product formation: with greater amounts overoxidation products, the corresponding benzoic acid and benzonitrile were formed. Smaller amounts of laccase and mediator generated benzaldehyde in high yield. The reaction pathway for benzonitrile formation is discussed and it is suggested to start from benzaldehyde and the ammonia in the ammonium acetate buffer.

Targetting redox polymers as mediators for laccase oxygen reduction in a membrane-less biofuel cell

Electrodes modified with co-immobilized redox enzymes and redox polymers can be used to form membrane-less biofuel cells. In this communication, we report on our initial studies of a membrane-less biofuel cell concept using an osmium-based redox polymer for laccase-mediated reduction of oxygen coupled to glucose oxidase-mediated oxidation of glucose. We then present a thermodynamic examination of mediators of laccase oxygen reduction, and stemming from this, target two redox polymers of potential use, an osmium-based redox polymer ( E 0′+0.40 V vs. Ag/AgCl) and a ruthenium-based redox polymer ( E 0′+0.63 V vs. Ag/AgCl). The former shows promise for use in membrane-less biofuel cell cathodes, whilst the latter’s redox potential is too high to be an effective mediator of oxygen reduction by the Trametes versicolor laccase used in this study.

Crystal Structure of a Laccase from the FungusTrametes versicolor at 1.90-Å Resolution Containing a Full Complement of Coppers

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.

Dephenolisation of olive mill waste-waters by olive husk

Olive mill waste-waters (OMWW) are a by-product of olive-oil production and a major environmental problem in the Mediterranean area. The use of a polyphenol-oxidase from olive husk is proposed for the enzymatic removal of low-molecular phenolics in OMWW. For comparison purposes, a purified microbial polyphenol-oxidase ( Trametes versicolor laccase) was also employed. Though both enzymatic systems show relevant activity towards phenol polymerization, olive husk is much more appealing in view of possible applications because of its availability and extremely low cost, associated with excellent enzymatic activity and specificity.

Aromatic ring cleavage of a non-phenolic β- O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole

The novel cleavage products, 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)-1-formyloxypropane (II) and 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonate (III) were identified as products of a non-phenolic β- O-4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxylphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I), by a Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole (1-HBT). An isotopic experiment with a 13C-labeled lignin model dimer, 1,3-dihydroxy-2-(2,6-[U-ring- 13C]dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I- 13C) indicated that the formyl and carbonate carbons of products II and III were derived from the β-phenoxy group of β- O-4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase-1-HBT couple could catalyze the aromatic ring cleavage of non-phenolic β- O-4 lignin model dimer in addition to the β-ether cleavage, Cα-Cβ cleavage, and Cα-oxidation.

Oxidation of Polycyclic Aromatic Hydrocarbons (PAH) by Laccase of Trametes Versicolor

Laccase of Trametes versicolor was able to oxidize in vitro most of the 14 polycyclic aromatic hydrocarbons (PAH) tested. Acenaphthylene was removed by 37% followed by anthracene and benzo[a]pyrene which were oxidized by 18 and 19%, respectively. Lower but significant oxidation of about 10% was found for eight additional PAH: acenaphthene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, and perylene. Naphthalene, fluorene, and phenanthrene were recovered unchanged after incubation for 72 h with laccase. Addition of 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of PAH: acenaphthylene, acenaphthene, fluorene, anthracene, benzo[a]pyrene, and perylene were almost completely removed from the reaction mixture. Oxidation of pyrene and benzo[a]anthracene increased from 8 and 6% without a mediator to 48 and 53% in the presence of HBT. Other PAH were not significantly influenced by the addition of this mediator. PAH-quinones as oxidation products were formed from all PAH to different extents. A part of PAH was polymerized in the laccase/mediator system to products of weight-average molecular weight (MW) of approximately 1,500 Da. The correlation of the ionization potentials of PAH with the oxidation of these compounds is limited to the alternating PAH.

Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds.

Laccase of Trametes versicolor was generally able to oxidize anthracene in vitro. After 72 h incubation about 35% of the anthracene was transformed stoichiometrically to 9,10-anthraquinone. Transformation of anthracene increased rapidly in the presence of different mediators that readily generate stable radicals: 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1-hydroxybenzotriazole. For the reaction, the presence of both the laccase and the mediator was necessary. In the presence of 0.005 mM 1-hydroxybenzotriazole this conversion had removed 47% of the anthracene after 72 h; 75% of the substrate was oxidized during this period when ABTS (1 mM) was used as mediator. In contrast to reactions without or with only low concentrations of a mediator, there was a discrepancy between the disappearance of anthracene and the formation of 9,10-anthraquinone in mediator-forced reactions. Coupling-products of mediators with anthracene degradation products were found. Anthracene disappeared nearly completely after incubation for 72 h with laccase in a 0.1 mM solution of 1-hydroxybenzotriazole and was transformed to 9,10-anthraquinone in about 80% yield; 90% of the substrate was transformed in the presence of ABTS (2.0 mM) resulting again in 80% quinone. Phenothiazine was not effective in this system.

The effect of fungal laccase on fractionated lignosulphonates (peritan Na)

Two fractions obtained after chromatography of lignosulphonates on Sephadex G-50, varying in Mrs, were treated with extracellular Trametes versicolor laccase. After incubation of the low Mr fraction, polymerization was observed, while in the case of the high Mr fraction the reverse process occurred. As a result of depolymerization, five new lower Mr fractions appeared. The reaction reached peak level after 2 hr of incubation and then the quantities of the products diminished, possibly due to their repolymerization. These studies indicate that laccase possesses both polymerization and depolymerization activity though the former was predominant.