Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli.

Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET-30a expression plasmid. The recombinant laccase was purified and visualized on SDS-PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg-1 , the optimum temperature and pH value were 60 °C and 4.0 respectively, measured by oxidation of 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Purified laccase activity under different metal ions and an inhibitor were tested, revealing that laccase activity increased by approximately 434.8% with Fe3+ , and 217.4% with Cu2+ at 10 mmol · L-1 concentrations, Mn2+ increased the laccase activity only at 5 mmol · L-1 , while Na+ increased activity at 1 mmol · L-1 but inhibited activity at 5 and 10 mmol · L-1 . SDS increased laccase activity at 1 mmol · L-1 , and inhibited activity at 5 and 10 mmol · L-1 .

A semi-rational approach to obtain an ionic liquid tolerant bacterial laccase through π-type interactions.

Laccases are particularly promising enzymes for biotechnology and bioremediation purposes. They are among the most effective enzymes capable of catalyzing the degradation of phenolic compounds with poor water solubility. The technological utility of lacasses can be enhanced greatly by their use in ionic liquids rather than in conventional organic solvents or in their natural aqueous reaction media. In the current study, a laccase from Bacillus HR03 has been engineered through a semi rational method. By screening a library of 450 clones, Glu188Tyr and Glu188Phe showed a distinct improvement in thermal stability and ionic liquid tolerance. In comparison with the wild type, selected mutants exhibited higher kcat/Km against ABTS in the imidazolium based ionic liquids, (1-ethyl-3-methyl imidazolium chloride [EMIm][Cl], butyl-3-methyl imidazolium chloride [BMIm][Cl] and hexyl-3-methyl imidazolium chloride [HMIm][Cl]). Glu188Tyr had a catalytic efficiency, two times greater when compared to the wild type in [HMIm][Cl]. Far-UV circular dichroism (CD) exhibited no significant changes in the secondary structure of the mutants and wild type. Glu188Tyr revealed a more compact structure using Near-UV CD and fluorescence spectroscopy that could account for its high thermal stability. According to bioinformatic analysis, π-π and anion-π interactions played the dominant role in stabilizing both variants.

Decolorization of Remazol Brilliant Blue R by a purified laccase of Polyporus brumalis.

A white rot basidiomycete Polyporus brumalis has been reported to induce two laccase genes under degradation conditions of dibutylphthalate. When this fungus was grown in a minimal medium, one laccase enzyme was detected by the native polyacrylamide gel electrophoresis. A laccase was purified through ammonium sulfate precipitation and ion exchange chromatography, and the estimated molecular weight was 70 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 °C, respectively. The K (m) value of the enzyme was 685.0 µM, and the V (max) was 0.147 ODmin(-1) unit(-1) for o-tolidine. Purified laccase showed effective decolorization of a dye, Remazol Brilliant Blue R (RBBR), without any laccase mediator. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was directly involved in the decolorization of RBBR.

Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg(-1) of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg(-1) specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0-6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and L: -cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.

Novel multienzyme oxidative biocatalyst for lignin bioprocessing.

A novel multienzyme biocatalyst, based on coimmobilization of the laccase and horseradish peroxidase by cross linking and layer-by-layer coating with polyelectrolyte, was designed, synthesized and applied at the development of an oxidative cascade process on lignin. The efficiency and specificity of the new LbL-multienzyme system, the occurrence of a synergy of the co-immobilized enzymes, the lignin oxidation pathway and the nature of the structural modifications occurred in treated lignins have been investigated in the present effort by means of GPC analysis and quantitative (31)P NMR techniques.

Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter.

Marasmius quercophilus is a white-rot fungus involved in carbon recycling in Mediterranean ecosystems because of its laccase production. Here we described the effect of metal ions and halide salts, on laccase activity in order to point out the action of such environmental pollutants on this enzyme of major importance. Furthermore we tested organic solvent effects on laccase reaction since reaction mixture including solvent can be used in the transformation of xenobiotics. In the case of metal ions, we found that chloride ions were responsible for inhibition while CuSO(4) and MnSO(4) enhanced laccase activity. When halides were tested, we showed the following degree of inhibition: F(-)>Cl(-)>Br(-). Furthermore we found that I(-) was oxidized by laccase with I(2) as the product of the reaction. With ABTS, 50% of the laccase activity remains for solvent concentration ranging from 40% to 60% depending on the solvent used while with syringaldazine solvent concentration ranged from 50% to 70%. The organic solvent effects observed were probably a result of enzyme denaturation and of both enhancement of oxidised product solubilisation and of substrate solubilisation (for syringaldazine). These results show that laccase from M. quercophilus is not rapidly inhibited by certain environmental pollutants which sustains its role in carbon turnover under pertubation. However the strong effect of chloride ion on laccase activity should be further investigated with in situ studies since this could drastically influence carbon recycling in litters from Mediterranean littoral locations.

Species-specific Cd-stress response in the white rot basidiomycetes Abortiporus biennis and Cerrena unicolor.

The effect of cadmium (Cd) on fungal growth, Cd bioaccumulation and biosorption, and on the formation of potential heavy metal response indicators such as thiols, oxalate, and laccase was investigated in the white rot fungi Cerrena unicolor andAbortiporus biennis. Only the highest Cd concentration employed (200 microM) inhibited growth of C. unicolor, whereas already lower Cd concentrations caused decreasing mycelia dry weights in A. biennis. Cd biosorption onto the mycelial surface was the predominant Cd sequestration mechanism in C. unicolor. Surface-bound and bioaccumulated Cd concentrations were essentially in the same range in A. biennis, leading to considerably higher intracellular Cd concentrations in A. biennis than in C. unicolor. Oxalate and laccase were produced by both of the fungal strains and their extracellular levels were elevated upon Cd exposure. Oxalate concentrations and laccase titres were considerably higher in C. unicolor than in A. biennis. Both fungi responded to increasing Cd concentrations by increasing intracellular amounts of thiol compounds (cysteine, gamma-glutamylcysteine, glutathione in both its reduced and oxidized form) but Cd application increased the amounts of thiols to a higher extend in A. biennis. Taken together, these species-specific responses towards Cd suggest that C. unicolor possesses a more efficient system than A. biennis to keep intracellular Cd concentrations low.

Uptake of cadmium ions in white-rot fungus Trametes versicolor: effect of cd (II) ions on the activity of laccase.

The effects of cadmium Cd (II) ions on the physiology and biological activity of Trametes versicolor, a strain belonging to white-rotting Basidiomycetes, were examined. Cd (II) ions were added to 10-day-old cultures grown on a liquid medium, or at the time of inoculation. Our experiments showed that T. versicolor is a good cadmium biosorbent from aqueous solution, this strain removing almost all the Cd (ll) ions over the first 2h of incubation by what appears to be a rapid, energy-independent surface binding phenomenon, at the rate of approximately 2mg Cd per g mycelial dry weight. An additional slower and energy-dependent transport mechanism was also present, taking in approximately 0.3mg Cd (II) perg dry weight. It is also shown that these Cd (II) ions significantly stimulate the activity of extracellular laccase when added to 10-day-old cultures.