Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin

The electrochemical studies of laccase–mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases.     

New strains of basidiomycetes that produce bioethanol from lignocellulose biomass

Sixty six isolates were screened for ability of bioethanol production; dynamics of product accumulation and substrate utilization were investigated for two selected strains Trametes hirsuta MT-24.24 and Trametes versicolor IT-1. The strains’ efficiency was evaluated as bioethanol production by 1 g biomass. Strain T. versicolor IT-1 producing over 33 g/L of the ethanol for 9 d was selected. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and straw was shown with ethanol yields of 2.1, 1.6 and 1.7 g/L, respectively, for 9 d fermentation time.     

Printing of polymer microcapsules for enzyme immobilization on paper substrate

Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.     

Cloning of novel cellulases from cellulolytic fungi: Heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.     

Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five lignocellulose-degrading fungi by ferulic acid

At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurotus sajor-caju. At 0.5 g/l, howerver, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-caju) also secreted laccases. Laccase-secreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested.     

Laccase of the lignolytic fungus Trametes hirsuta: Purification and characterization of the enzyme, and cloning and primary structure of the gene

The main physicochemical characteristics of the major isoform of the laccase secreted by the fungus Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases (E _T1 ⁰ 790 ± 5), and it oxidizes with high efficiency various substrates of phenolic nature. The gene of this isoform was cloned, and its nucleotide sequence was determined. The length of the complete gene is 2134 bp. It comprises 11 exons and 10 introns. Analysis of the amino acid sequence of T. hirsuta 072 laccase demonstrated a high homology to the other laccases secreted by fungi of the genus Trametes.     

Improving the catalytic performance of fungal laccases in monoterpene-based reaction systems

Ternary systems consisting of monoterpenes (α-pinene or d-limonene), tert-butanol and water were used as reaction media to enhance the catalytic performance of laccases from various fungi sources (Trametes versicolor, T. hirsuta and Botrytis cinerea). The enzymes had improved catalytic efficiency (5- to 10-fold) in α-pinene-rich environment, while optimal reaction rates were in high-water content systems (15.5% v/v). The stability of laccases was significantly improved in monoterpene-based systems (up to 90% residual enzyme activity after 24 h at 30°C) in comparison with other non-conventional media. The results indicate that these ternary systems can increase the potential of laccases as catalysts for various oxidations.     

Synthesis of electroconductive polyaniline using immobilized laccase

A new method for synthesis of the conductive complex between polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanosulfonic acid) (PAMPS) was proposed; in this method, the immobilized laccase from the basidiomycete Trametes hirsuta is used as a biocatalyst for aniline oxidative polymerization. The conditions for laccase immobilization on CM cellulose by bifunctional Woodward’s reagent were optimized. The catalytic properties of immobilized and native laccases were compared. The immobilized laccase appeared an efficient catalyst for the oxidative radical polymerization of aniline on polysulfonic acid matrix at 4°C. It was demonstrated that the immobilized enzyme could be repeatedly used for enzymatic synthesis of this polymer. Several spectral characteristics of the PANI/PAMPS complexes synthesized at various pH values were studied. The conductance of PANI specimens produced using immobilized laccase as a catalyst was 13 mS/cm.     

Comparative Studies of Extracellular Fungal Laccases

Various basidiomycetes, ascomycetes, and deuteromycetes, grown in a sugar-rich liquid medium, were compared for laccase-producing ability and for the inducing effect of 2,5-xylidine on laccase production. Clear stimulation of the extracellular enzyme formation by xylidine was obtained in the cultures of Fomes annosus, Pholiota mutabilis, Pleurotus ostreatus, and Trametes versicolor, whereas Rhizoctonia praticola and Botrytis cinerea were not affected by the xylidine, and in the case of Podospora anserina a decrease in laccase activity was observed. The laccases were purified, and electrophoresis on polyacrylamide gels indicated a particular pattern for each laccase. The bands of the induced forms appeared only with basidiomycetes. The optimal pH of R. praticola laccase was in the neutral region, whereas the optima of all the other exolaccases were significantly lower (between pH 3.0 and 5.7). All laccases oxidized the methoxyphenolic acids under investigation, but there existed quantitative differences in oxidation efficiencies which depended on pH and on the nature (noninduced or induced) of the enzyme. The sensitivity of all enzymes to inhibitors did not differ considerably.     

A protein from Pleurotus eryngii var. tuoliensis C.J. Mou with strong removal activity against the natural steroid hormone,estriol: Purification,characterization, and identification as a laccase

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60 kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50 °C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.     

Enzymatic cross-linking of gelatine with laccase and tyrosinase

Conventional cross-linking of proteins involves the use of toxic chemicals. Here, cross-linking of gelatine and gelatine hydrolysates with tyrosinases from Botryosphaeria obtusa (BoT1 and BoT2), Agaricus bisporus (AbT) and from Verrucomicrobium spinosum (VsT) and with laccases from Trametes hirsuta (ThL) and T. versicolor (TvL) was demonstrated. Enzymatic oxidation of tyrosine residues was indicated by UV/VIS and fluorescence spectroscopy and further confirmed by oxygen consumption measurements. Using a model substrate (Tyr-Ala) dimerization was demonstrated by using RP-HPLC and LC-MS. Enzymatic cross-linking significantly increased the molecular weight of the soluble material up to the point of precipitation as demonstrated by both SDS-PAGE and size exclusion chromatography. The effect of cross-linking was further enhanced in the presence of phenolic molecules such as catechin.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Preparation and characterization of bioactive products obtained via the solubilization of brown coal by white rot fungi

We investigated the solubilizing activity of the Basidiomycete fungi Trametes hirsuta and Trametes maxima, with respect to brown coal (lignite) during liquid phase cultivation. We found that the degrading capacity of the fungi is determined by the activity of the ligninolytic enzymes Mn peroxidase and lignin peroxidase. We assessed the growth-stimulating activity of biopreparations (BPs), based on the culture liquids (CL) of the studied fungal strains, which were grown on a rich or minimal medium. We found that the obtained BPs inhibited the growth of wheat shoots and roots at the germination stage, but they either had no effect at later stages of plant growth or showed a mild stimulation. When basidiomycetes were cultivated in the presence of brown coal, the obtained BPs stimulated root growth at the germination stage, and did not influence plant growth (Trametes hirsuta) or stimulated it (Trametes maxima) at later stages. Further, we report a pronounced detoxifying ability of the BPs in respect to the atrazine herbicide. We suggest that this effect is caused by the laccases action, that are present in the studied BPs.     

Laccase-mediated functionalization of chitosan by caffeic and gallic acids for modulating antioxidant and antimicrobial properties

A new strategy for the functionalization of chitosan with caffeic acid (CA) or gallic acid (GA) using laccase from Trametes versicolor is presented for the first time, yielding a product with modulated antioxidant and antimicrobial properties. UV-vis spectroscopy coupled to HPLC-SEC analysis and cyclic voltammetry kinetic studies showed that laccase catalyzes the oxidation of phenolic acids to electrophilic o-quinones, which undergo new oligomer/polymer-forming structures originated by C-C coupling between the benzene rings and C-O-C coupling involving phenolic side-chains. Furthermore, pH tunable reactions/interactions of the laccases oxidized o-quinones with nucleophilic amino groups of chitosan were determined with FTIR and ¹H NMR spectroscopy's. The highest antioxidant activity was found to be for chitosan modified with phenolic acids at pH 4.5, exhibit also an increased activity against Escherichia coli and Listeria monocytogenes compared to untreated chitosan.     

Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM

A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respectively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase production. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after optimization of the hydrolysis parameters. The results of a confirmation experiment under the optimum conditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass.     

Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases

Cross-linked enzyme aggregates (CLEA^®) were prepared from laccases from three different sources: Trametes versicolor, Trametes villosa and Agaricus bisporus. The effect of the various parameters – nature of the precipitant, pH, temperature, glutaraldehyde concentration and cross-linking time – on the activity recovery and storage and operational stability of the resulting CLEAs was different. The laccase CLEAs exhibited the expected increased stability compared to the free enzyme but there was no direct correlation with the number of surface lysine residues in the latter. It is clearly not the only parameter influencing the properties of the CLEA. Co-aggregation with albumin did not improve the stability. The laccase CLEAs, in combination with the stable N-oxy radical, TEMPO, were shown to be active and stable catalysts for the aerobic oxidation of linear C₅–C₁₀ aliphatic alcohols, to the corresponding aldehydes, in aqueous buffer (pH 4). Rates were an order of magnitude higher than those observed with the corresponding free enzyme and the CLEAs could be recycled several times without appreciable loss of activity. The addition of water immiscible or water miscible solvents showed no further improvement in rate compared with reactions in aqueous buffer alone.     

A novel ethanol-tolerant laccase,Tvlac, from <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Objectives

To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source.

Results

Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 U ml^?1 within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60 °C, respectively. Its activity was stimulated by ethanol at 10 % (v/v) which increased the V ₀.

Conclusions

The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.

     

Tyrosinase Activity Discovered in Trametes spp

A total of 54 strains of white rot fungi belonging to 12 closely related species of genera Trametes,Coriolopsis, Cerrena and Lenzites were tested for tyrosinase activity. In the majority of the strains preliminary spot test using p-cresol gave mostly negative results, the activity being detected only in four strains of three different Trametes species (Trametes cervina, T. hirsuta, T. versicolor). The tyrosinase activity of these strains (one strain of T. cervina, T. hirsuta H3, T. hirsuta H7 and T. versicolor V14) was then confirmed spectrophotometrically. Tyrosinase activity has not yet been described in any Trametes species.     

Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Abstract

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing.

Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket.

Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.     

Dibenzyl Sulfide Metabolism by White Rot Fungi

Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization. Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied. Trametes trogii UAMH 8156, Trametes hirsuta UAMH 8165, Phanerochaete chrysosporium ATCC 24725, Trametes versicolor IFO 30340 (formerly Coriolus sp.), and Tyromyces palustris IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone. The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation. Laccase activity (0.15 U/ml) was detected in the Trametes cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence. These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.