A novel combination of prosthetic groups in a fungal laccase; PQQ and two copper atoms

Extracellular laccase (benzenediol: oxygen oxidoreductase EC 1.10.3.2) from the lignin-degrading fungus, Phlebia radiata, was shown to contain a novel combination of electron carriers as its prosthetic groups. In addition to two copper atoms per enzyme molecule, one molecule of PQQ was included as a cofactor. The EPR spectrum exhibits features of type 1 and type 2 copper atoms. In the enzymatic reaction 4 molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. During the reaction coniferyl alcohol is transformed to dilignols.     

Electron paramagnetic resonance studies of type 1 copper in type 2 depleted fungal laccase A

The electron paramagnetic resonance (EPR) spectra of type 1 copper(II) in 63Cu-enriched Coriolus versicolor laccase A (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) have been studied. The X-band EPR spectrum in type 2 copper-depleted [63Cu]laccase A exhibited well-resolved ligand superhyperfine structure in the g perpendicular region. This structure was assigned to an interaction with two nitrogens and two protons, an assignment which is consistent with a model in which the two nitrogens belong to two histidine ligands and the two protons are the methylene protons of a coordinating cysteine. It also requires the delocalization of a substantial amount of the type 1 copper(II) unpaired electron density onto the cysteine sulphur.     

包埋法固定化真菌漆酶及其应用研究

采用海藻酸钠包埋法固定真菌漆酶,海藻酸钠和CaCl2的最佳浓度分别为3%和4%,最佳给酶量为30U,最大回收率为48.0%.与游离漆酶相比,固定化漆酶的热稳定性有明显改善,最适反应pH向酸性方向漂移0.5,最适反应温度提高了5℃.使用固定化酶处理低浓度造纸废水,运行8批次后残留酶活为64%.     

Stable expression of a fungal laccase protein using transplastomic tobacco

Laccase catalyzes the oxidation of various phenolic compounds that can be used in a wide range of industrial applications such as waste detoxification and the textile industry. In the present study, we generated transplastomic tobacco plants to develop a reliable commercial source of laccase production. The stability of the laccase protein in the transgenic plants was increased by using the enhancer sequence from green fluorescent protein, resulting in three independent lines with high levels of laccase accumulation (up to 2?% of total protein); significant laccase activity, however, was not detected. Interestingly, the transplastomic lines showed slightly retarded vegetative growth, with a light green leaf color in comparison with the control, which may be attributable to copper deficiency induced by ligand chelation by abundantly produced laccase. These results suggest that the tobacco chloroplast is an efficient system for the mass production of laccase protein, but further studies are needed to obtain active enzyme.     

Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties

Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.     

A novel fungal laccase from Sordaria macrospora k-hell: expression,characterization, and application for lignin degradation

Bioprocess and Biosystems Engineering - The laccase has the ability to oxidize substituted phenols and the water is the sole byproduct, thus it has been employed to remove and/or modify the lignin...     

A single-step purification of an extracellular fungal laccase

Summary A single-step purification procedure forNeurospora crassa laccase is reported. It used Celite chromatography which permits the concentration of the extracellular enzyme from large culture volumes. This method is useful to isolate any fungal or plant laccase as well as other coppercontaining proteins. This work also takes advantage of the 100-fold induction of the enzyme by low concentrations of cycloheximide.

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Resumen Aquí se reporta un procedimiento de purificación en un solo paso para le enzima lacasa deNeurospora crassa. Consiste en el empleo de la cromatografía en Celite que permite la concentración de la enzima exocelular de grandes volúmenes de cultivo. Este método es útil para aislar cualquier lacasa de hongos o plantas así como otras proteínas que contienen sobre. En este trabajo, hemos aprovechado también la inducción de la enzima en más de 100 veces por bajas concentraciones de cicloheximida.

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Résumé Nous rapportons ici une procédure de purification en une étape de la laccase deNeurospora crassa. Elle consiste à employer la chromatographie sur Celite qui permet la concentration de l'enzyme exo-cellulaire à partir de grands volumes de milieu de culture. Cette méthode permet d'isoler n'importe quelle laccase fungique ou de plante ainsi que d'autres protéines à cuivre. Dans le présent travail, nous avons aussi tiré parti de l'induction au centuple de l'enzyme par de faibles concentrations en cycloheximide.

     

Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25 °C for enzyme reaction.     

X-ray structural studies of the fungal laccase from Cerrena maxima

Laccases are members of the blue multi-copper oxidase family. These enzymes oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and following the transfer of four electrons is reduced to two molecules of water. The X-ray structure of a laccase from Cerrena maxima has been elucidated at 1.9 A resolution using synchrotron data and the molecular replacement technique. The final refinement coefficients are Rcryst = 16.8% and Rfree = 23.0%, with root mean square deviations on bond lengths and bond angles of 0.015 A and 1.51 degrees , respectively. The type 1 copper centre has an isoleucine residue at the axial position and the "resting" state of the trinuclear centre comprises a single oxygen (OH) moiety asymmetrically disposed between the two type 3 copper ions and a water molecule attached to the type 2 ion. Several carbohydrate binding sites have been identified and the glycan chains appear to promote the formation of well-ordered crystals. Two tyrosine residues near the protein surface have been found in a nitrated state.     

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.     

Purification and properties of laccase from Ganoderma lucidum

Summary The enzyme laccase has been partially purified from the culture fluid of Ganoderma lucidum by acetone precipitation, ammonium sulphate fractionation and adsorption on alumina C gel. The enzyme has been shown to be specific for ortho and para hydroxyphenolic compounds, having Km values of 5.5×10^-5 M and 2.86×10^-5 M for catechol and hydroquinone respectively. The optimum pH for the oxidation of catechol and hydroquinone are 5.4 and 5.0 respectively. The enzyme is inactivated above 60°C and is inhibited by enzyme inhibitors and metal chelating agents like azide, cyanide etc.     

Production, purification, and properties of an extracellular laccase from Rigidoporus lignosus

Laccase from Rigidoporus lignosus, a white-rot basidiomycete, has been isolated from culture filtrates. The enzyme was purified to homogeneity and some of its structural and kinetic parameters have been determined. The effects of pH, temperature, and organic solvents on the activity and stability of the enzyme, under different conditions, were also assayed. The results we have obtained, including the rather broad substrate specificity of enzyme, combined with their relatively easy production and purification, suggest that laccase may be efficiently employed in a variety of biotechnology applications.     

A resonance raman investigation of perturbed states of tree and fungal laccase

Resonance Raman (RR) spectra of Rhus vernicifera laccase and Polyporus versicolor laccase in several perturbed states are reported. Coordination of fluoride to the type 2 copper of either laccase does not produce RR-detectable changes at the type 1 sites. Removal of the type 2 copper of Rhus laccase induces RR-detectable changes at the type 1 site that are most readily interpreted as arising from a heterogeneous sample. Freezing the Rhus laccase does not induce changes in its RR spectrum; further cooling to liquid nitrogen temperature induces a change in the type 1 site that is reflected in the RR spectrum. Electron paramagnetic resonance spectra confirm that the structure of the type 1 site is temperature dependent. Some variability in the Rhus laccase RR spectrum is observed in the absence of exogenous ligands or other perturbations; it is suggested that Rhus laccase preparations may be heterogeneous with respect to the structure of the type 1 copper site.