Laccase multigene families in Agaricomycetes.

Here we present the results of the exploration of laccase multigene families (MGFs) in basidiomycetous fungi from different taxonomic groups using a next generation sequencing (NGS) technology. In our study, multiple laccase genes were identified in all of the investigated fungi (13 species) from Polyporaceae, Phanerochaetaceae, Meruliaceae, Pleurotaceae, Physalacriaceae, and Peniophoraceae families. It was shown that phylogenetic positioning of the newly identified sequences exhibit patterns of clusterization with respect to enzyme properties. This can be a potentially useful tool for selecting naturally existing laccases with different physicochemical characteristics relevant to different biotechnological applications. Moreover, the method developed in this study can be used in the screening of environmental samples and fast characterization of laccase MGFs in newly identified fungal species.

Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 µM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-µM and 10-µM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

Extracellular proteins of Trametes hirsuta st. 072 induced by copper ions and a lignocellulose substrate.

BACKGROUND: Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment. RESULTS: This study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attributed the observed extracellular LiP - like activity to the expressed versatile peroxidase (VP). An accessory enzyme, glyoxal oxidase, was found among the proteins secreted in the media during submerged cultivation of T. hirsuta both in the presence and in the absence of copper. However, aryl-alcohol oxidase (AAO) was not identified, despite the presence of AAO enzymatic activity secreted by the fungus. The spectra of the expressed enzymes dramatically changed depending on the growth conditions. Transfer from submerged cultivation to surface cultivation with the lignocellulose substrate switched off expression of exo-ß-1,3-glucanase and α-amylase and turned on secretion of endo-ß-1,3-glucanase and a range of glycosidases. In addition, an aspartic peptidase started being expressed instead of family S53 protease. For the first time, we report production of cerato-platanin proteins by Trametes species. The secretion of cerato-platanins was observed only in response to contact with lignocellulose, thus indicating a specific role of these proteins in degradation of the lignocellulose substrates. CONCLUSIONS: Our results suggest a sequential mechanism of natural substrate degradation by T. hirsuta, in which the fungus produces different sets of enzymes to digest all main components of the substrate during cultivation.

Draft Genome Sequence of the Fungus Trametes hirsuta 072.

A standard draft genome sequence of the white rot saprotrophic fungus Trametes hirsuta 072 (Basidiomycota, Polyporales) is presented. The genome sequence contains about 33.6 Mb assembled in 141 scaffolds with a G+C content of ~57.6%. The draft genome annotation predicts 14,598 putative protein-coding open reading frames (ORFs).

The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction.

Laccases, blue copper-containing oxidases, ≿ an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072, an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed.

Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes.

Diacylglyceryltrimethylhomoserines (DGTS) are betaine-type lipids that are phosphate-free analogs of phosphatidylcholines (PC). DGTS are abundant in some bacteria, algae, primitive vascular plants and fungi. In this study, we report inorganic phosphate (Pi) deficiency-induced DGTS synthesis in the basidial fungus Flammulina velutipes (Curt.: Fr.) Sing. We present results of an expression analysis of the BTA1 gene that codes for betaine lipid synthase and two genes of PC biosynthesis (CHO2 and CPT1) during phosphate starvation of F. velutipes culture. We demonstrate that FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite depletion in PC, both CHO2 and CPT1 were determined to have increased expression. We also describe the deduced amino acid sequence and genomic structure of the BTA1 gene in F. velutipes. Phylogenetic relationships between putative orthologs of BTA1 proteins of basidiomycete fungi are discussed.

Elucidation of the crystal structure of Coriolopsis caperata laccase: restoration of the structure and activity of the native enzyme from the T2-depleted form by copper ions.

Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. A new laccase was isolated from the basidiomycete Coriolopsis caperata strain 0677 and its amino-acid sequence was determined. According to its physicochemical properties and spectroscopic features, the laccase from C. caperata is a high redox-potential blue laccase. Attempts to crystallize the native enzyme were unsuccessful. The copper type 2-depleted (T2D) laccase was prepared and crystallized. The structure of T2D laccase from C. caperata was solved at 1.6 Šresolution, and attempts to reconstruct the T2 copper centre were performed using Cu(+) and Cu(2+) ions. The structure of T2D+Cu(+) laccase was solved at 1.89 Šresolution. It was shown that the T2D+Cu(+) laccase structure contained four copper ions in the active site. Reconstruction could not be achieved when the T2D laccase crystals were treated with CuSO4.

Rapid multiple immunoenzyme assay of mycotoxins.

Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin-polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

Humic substances enhance growth and respiration in the basidiomycetes Trametes maxima under carbon limited conditions.

Humic substances (HS) represent the major reservoir of carbon (C) in ecosystems, and their turnover is crucial for understanding the global C cycle. Although basidiomycetes clearly have a role in HS degradation, much less is known about the effect of HS on fungal traits. We studied the alteration of physiological, biochemical, and morphological characteristics of Trametes maxima in the presence of HS. Both complete medium and minimal (C-limited) medium mimicking natural environmental conditions were used. Adding HS led to increased biomass yield, but under C-limited conditions the effect was more apparent. This result indicated that HS were used as an additional substrate and agreed with data showing a greater penetration of tritium-labeled HS into the cell interior under C-limited conditions. Humic substances induced ultra-structural changes in fungal cells, especially under C limitation, including reducing the thicknesses of the hyphal sheath and cell wall. In the minimal medium, cellular respiration increased nearly three-fold under HS application, while the corresponding effect in complete medium was lower. In addition, in the presence of inhibitors, HS stimulated either the cytochrome or the alternative pathway of respiration, depending on presence or absence of glucose in the medium. Our results suggest that, under conditions mimicking the natural environment, HS may play three major roles: as a surplus substrate for fungal growth, as a factor positively affecting cell morphology, and as an activator of physiological respiration.

Immunoassays of fungal laccases for screening of natural enzymes and control of recombinant enzyme production.

Because of the wide application of laccases in different biotechnological processes and intense studies of the enzymes from different sources, the development of efficient techniques for monitoring laccase level is a task of significant importance. Enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques were developed to control total content and isoform composition of laccases, including their recombinant preparations. Because glycosylated and nonglycosylated forms have different structures and sets of epitopes, two kinds of polyclonal antibodies were obtained and applied. The first antibody recognized the native (glycosylated) laccase purified from Trametes hirsuta and the second one reacted with recombinant (nonglycosylated) laccase expressed in Escherichia coli. Titers of the antibodies were analyzed by indirect ELISA with laccases isolated from several strains of basidiomycetes. The obtained cross-reactivity data for both antibodies demonstrated a correspondence with sequence homology of the laccases. The antibodies raised against recombinant (nonglycosylated) laccase had higher titers and thus were preferable for screening of recombinant laccase in cultural media. Thus, optimal antibody preparations were selected for screening of laccase-producing strains, and the control of recombinant enzymes and the efficiency of their use in immunochemical control of laccase levels were confirmed.

Estimation of uptake of humic substances from different sources by Escherichia coli cells under optimum and salt stress conditions by use of tritium-labeled humic materials.

The primary goal of this paper is to demonstrate potential strengths of the use of tritium-labeled humic substances (HS) to quantify their interaction with living cells under various conditions. A novel approach was taken to study the interaction between a model microorganism and the labeled humic material. The bacterium Escherichia coli was used as a model microorganism. Salt stress was used to study interactions of HS with living cells under nonoptimum conditions. Six tritium-labeled samples of HS originating from coal, peat, and soil were examined. To quantify their interaction with E. coli cells, bioconcentration factors (BCF) were calculated and the amount of HS that penetrated into the cell interior was determined, and the liquid scintillation counting technique was used as well. The BCF values under optimum conditions varied from 0.9 to 13.1 liters kg(-1) of cell biomass, whereas under salt stress conditions the range of corresponding values increased substantially and accounted for 0.2 to 130 liters kg(-1). The measured amounts of HS that penetrated into the cells were 23 to 167 mg and 25 to 465 mg HS per kg of cell biomass under optimum and salt stress conditions, respectively. This finding indicated increased penetration of HS into E. coli cells under salt stress.

Structure of native laccase from Trametes hirsuta at 1.8 A resolution.

This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.

Kinetic and theoretical comprehension of diverse rate laws and reactivity gaps in Coriolus hirsutus laccase-catalyzed oxidation of acido and cyclometalated Ru(II) complexes.

The reactivity of the acido Ru(II) complexes cis-[RuCl(2)(LL)(2)], [RuCO(3)(LL)(2)], cis-[RuCO(3)-(bquin)(2)] (LL = 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen); bquin = 2,2'-biquinoline) and cyclometalated Ru(II) derivatives of 2-phenylpyridine and 4-(2-tolyl)pyridine [Ru(o-C(6)H(4)-2-py)(phen)(2)]PF(6) (1), [Ru(o-C(6)H(3)-p-R-2-py)(bpy)(MeCN)(2)]PF(6) (2), and [Ru(o-C(6)H(3)-p-R-2-py)(phen)(MeCN)(2)]PF(6) (3) (R = H (a), Me (b)) toward laccase from Coriolus hirsutus has been investigated by conventional UV-vis spectroscopy at pH 3-7 and 25 degrees C. The acido and cyclometalated complexes are readily oxidized into the corresponding Ru(III) species, but the two types of complexes differ substantially in reactivity and obey different rate laws. The acido complexes are oxidized more slowly and the second-order kinetics, first-order in laccase and Ru(II), holds with the rate constants around 5 x 10(4) M(-1) s(-1) at pH 4.5 and 25 degrees C. The cyclometalated complexes 1-3 react much faster and the hyperbolic Michaelis-Menten kinetics holds. However, it is not due to formation of an enzyme-substrate complex but rather because of the ping-pong mechanism of catalysis, viz. E(ox) + Ru(II) --> E(red) + Ru(III) (k(1)); E(red) + 1/4O(2) --> E(ox) (k(2)), with the rate constants k(1) in the range (2-9) x 10(7) M(-1) s(-1) under the same conditions. The huge values of k(1) move the enzymatic oxidation toward a kinetic regime when the dioxygen half-reaction becomes the rate-limiting step. Cyclometalated compounds 1-3 can therefore be used for routine estimation of k(2), that is, the rate constant for reoxidation for laccases by dioxygen. The mechanism proposed was confirmed by the direct stopped-flow measurements of the k(2) rate constant (8.1 x 10(5) M(-1) s(-1) at 26 degrees C) and supported by the theoretical modeling of interaction between the bpy analogue of 1 and Coriolus hirsutes laccase using Monte Carlo simulations.

Crystallization and preliminary X-ray analysis of a four-copper laccase from Coriolus hirsutus.

Laccase from the fungus Coriolus hirsutus has been purified. Crystals of the enzyme suitable for X-ray structure analysis have been obtained under optimized crystallization conditions using polyethylene glycol as a precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.65, b = 74.01, c = 124.83 A, and contain 40% solvent and a single molecule of laccase in the asymmetric unit. X-ray data were collected to 1.85 A at the copper edge and the four copper sites have been located from the anomalous signal. The obtained SAD phases with subsequent density modification produced a promising initial electron-density map.

Laccase and Mn-peroxidase production by Coriolus hirsutus strain 075 in a jar fermentor.

The white-rot fungus Coriolus hirsutus strain 075 excretes considerable amounts of laccase and Mn-peroxidase into culture broth over a brief production time. The effects of agitation speed, temperature, aeration and inoculum amount on laccase production using a 10-l fermentor were studied. The optimum fermentation conditions were a 15% inoculum, an aeration rate of 0.88 vvm, an agitation speed of 160 rpm, and a temperature of 28 degrees C. By optimizing the fermentation conditions, the laccase activity reached 80+/-3 U/ml in 3 d and the purified enzyme output was 30 mg/l. The laccase and Mn-peroxidase were purified by means of isoelectrofocusing and ion-exchange chromatography. The pIs of the laccase isoenzymes were 4.2 and 4.5. Mn-peroxidase had only one isoenzyme with a pI of 3.2. The optimum pH was 4.5 for laccase with syringaldazine as the substrate and 5.0-5.3 for Mn-peroxidase with Mn(+2) and H2O2 as the substrates. The laccase and Mn-peroxidase retained 50% of their activities at 50 degrees C after 55 h and 12 h of incubation time, respectively.