Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust.

Two laccase isoforms (lcc1 and lcc2) produced by Trametes versicolor, grown on oak sawdust under solid-state fermentation conditions, were purified and characterized. The two isoforms showed significant biochemical differences. Lcc1 and lcc2 had MWs of 60 and 100 kDa, respectively. Both isoforms had maximal activity at pH 3 with ABTS and 2,6-dimethyloxyphenol (DMP). Lcc1 was the most attractive isoform due to its greater affinity towards all the laccase substrates used. Lcc1 had Km values of 12, 10, 15 and 17 mM towards ABTS, DMP, guaiacol and syringaldazine, respectively. Lcc2 had equivalent values of 45, 47, 15 and 39 mM. The biochemical properties of lcc1 substantiate the potential of this enzyme for application in the treatment of contaminated water with low pH values and high phenolic content.

Induction of laccases in Trametes versicolor by aqueous wood extracts.

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 µl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml(-1)). The highest laccase activities detected were 1.92 ± 0.15 U ml(-1) (pine), 1.87 ± 0.26 U ml(-1) (cedar), and 1.56 ± 0.34 U ml(-1) (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85%), followed by pH 7 (50%) and pH 3 (15%). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.

Identification of volatile compounds produced by the bacterium Burkholderia tropica that inhibit the growth of fungal pathogens.

It has been documented that bacteria from the Burkholderia genera produce different kinds of compounds that inhibit plant pathogens, however in Burkholderia tropica, an endophytic diazotrophic and phosphate-solubilizing bacterium isolated from a wide diversity of plants, the capacity to produce antifungal compounds has not been evaluated. In order to expand our knowledge about Burkholderia tropica as a potential biological control agent, we analyzed 15 different strains of this bacterium to evaluate their capacities to inhibit the growth of four phytopathogenic fungi, Colletotrichum gloeosporioides, Fusarium culmorum, Fusarium oxysporum and Sclerotium rolffsi. Diverse analytical techniques, including plant root protection and dish plate growth assays and gas chromatography-mass spectroscopy showed that the fungal growth inhibition was intimately associated with the volatile compounds produced by B. tropica and, in particular, two bacterial strains (MTo293 and TTe203) exhibited the highest radial mycelial growth inhibition. Morphological changes associated with these compounds, such as disruption of fungal hyphae, were identified by using photomicrographic analysis. By using gas chromatography-mass spectroscopy technique, 18 volatile compounds involved in the growth inhibition mechanism were identified, including α-pinene and limonene. In addition, we found a high proportion of bacterial strains that produced siderophores during growth with different carbon sources, such as alanine and glutamic acid; however, their roles in the antagonism mechanism remain unclear.

Increasing Pleurotus ostreatus laccase production by culture medium optimization and copper/lignin synergistic induction.

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 26⁻² where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.

Halogenated pesticide transformation by a laccase-mediator system.

The transformation of organic halogenated pesticides by laccase-mediator system has been investigated. Twelve pesticides were assayed in the presence of nine different mediators. Acetosyringone and syringaldehyde showed to be the best mediators. The halogenated pesticides bromoxynil, niclosamide, bromofenoxim and dichlorophen were transformed by the laccase-syringaldehyde system showing catalytic activities of 48.8, 142.0, 166.2 and 1257.6nmolmin(-1)U(-1), respectively. The highest pesticide transformation rates were obtained with a mediator-substrate proportion of 5:1, one of the lowest reported so far for the laccase-mediator systems. The analysis of the main product from the dichlorophen transformation showed that an oxidative dehalogenation is involved in the catalytic mechanism. Adduct formation between the mediator syringaldehyde and the pesticides dichlorophen or bromoxynil was also found after enzymatic oxidation. The main goal of this work is to evaluate environmental-friendly mediators for the pesticide transformation, and the potential of laccase-mediator system to efficiently reduce the environmental impact of organic halogenated pesticides is discussed.

Chloroperoxidase-mediated transformation of highly halogenated monoaromatic compounds.

Peroxidase transformations of widely distributed pollutants, tetra- and penta-chlorinated phenols and anilines, were studied using different peroxidases. Chloroperoxidase from Caldariomyces fumago was able to transform tetra- and penta-chlorinated phenols and anilines, while horseradish peroxidase, lignin peroxidase from Phanerochaete chrysosporium and versatile peroxidase from Bjerkandera adusta were able only to transform the halogenated phenols. Chloroperoxidase showed a specific activity on pentachlorophenol two orders of magnitude higher than lignin peroxidase and horseradish peroxidase, and one order of magnitude higher than versatile peroxidase. The main product from peroxidase oxidation in all cases was a polymeric and insoluble material. The insolubilization of halogenated phenols and anilines permits their removal, reduces their bioavailability, and thus reduces their environmental impact. The other minor products from the enzymatic transformation of highly chlorinated compounds were determined by mass spectrometry. Tetrachloroquinone, dimers and trimers of halogenated compounds were also identified. Chloroperoxidase was able to halogenate tetrachloroaniline to form pentachloroaniline.

Effect of pollutants on the ergosterol content as indicator of fungal biomass.

Ergosterol content was determined in 20 white-rot fungi isolates and the values ranged from 2380 to 13060 microg g(-1) fungal biomass. Significant changes of ergosterol content according the physiological stage for Bjerkandera adusta 4312 and Coriolopsis gallica 8260 were found, showing the highest values during the stationary phase. However, in the case of Phanerochaete chrysosporium 3642, no changes were detected during growth. The effect of pollutants, such as heavy metals and fungicides, on the ergosterol content of C. gallica was determined. Heavy metals (Cu 80 ppm, Zn 50 ppm or Cd 10 ppm) and fungicides (thiram 3 ppm or pentachlorophenol 1.5 ppm) at concentrations that reduce the metabolic activity between 18% and 53% (pollutant-stressed cultures) did not affect the ergosterol content. Only the fungicide zineb (25 ppm) reduced significantly the ergosterol content in biomass basis. In soil experiments with Cu (80 ppm) or thiram (10 ppm) after 15 and 30 days of incubation, the ergosterol content in soil was linearly correlated to the fungal biomass C in both polluted and control soil cultures. The ergosterol content was independent of the presence or the absence of pollutants. Thus, these results indicate that ergosterol can be a useful indicator for fungal biomass in polluted soils, and can be applied for monitoring bioremediation processes.