The dynamics of oxidase activity during cultivation of basidiomycetes from the genus Trametes Fr

Twenty strains of the wood-degrading fungi from the genus Trametes Fr., capable of synthesizing laccases, were screened according to the changes in the oxidase activity in a submerged culture. The range of maximal efficiency of various species with respect to extracellular oxidase activity was determined. The absence of correlation between the oxidase activity in a submerged culture and the size of colored zone on agar media (Bavendamm reaction) was demonstrated. The most efficient strains, T. hirsita 56 and T. ochracea 92-78, were used to produce laccases, homogeneous according to SDS electrophoresis data. A number of biochemical parameters characteristic of these enzymes were determined.     

Silver stained polyacrylamide gels and fluorescence-based automated capillary electrophoresis for detection of amplified fragment length polymorphism patterns obtained from white-rot fungi in the genus Trametes

Silver stained denaturing polyacrylamide gels (PAGEs) and fluorescent denaturing automated capillary electrophoresis (CE) were used to detect amplified fragment length polymorphism (AFLP) patterns obtained from white-rot fungi belonging to the genus Trametes. AFLP fingerprinting detected by the fluorescence-based method as well as by silver staining showed a high discriminatory power in differentiating nine strains of Trametes ochracea, nine strains of Trametes hirsuta and ten isolates of Trametes versicolor. UPGMA dendrograms derived from fluorescently labelled and silver stained AFLP patterns were similar, but a few differences were detected especially in the clustering of T. ochracea and T. hirsuta strains. Compared to silver-stained AFLP, detection of fluorescent AFLP was fast, reliable and easy to perform and it facilitated surveying with a computerized analysis system. Fluorescent CE seems to be well suited for studying similarity between Trametes species.     

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 fungu, Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases (E(T1) = 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 96.9%) to the other laccases secreted by fungi of the genus Trametes.     

Comparative study of the extracellular laccases from Cerrena unicolor 059 0784 and Pleurotus oastreatus 0432

The laccases of the basidiomycetes Cerrena unicolor 059, C. unicolor 0784, and Pleurotus oastreatus 0432 were assayed comparatively. The laccases were isolated as homogenous preparations with molecular weight 55, 56, and 57 kD, respectively. The three enzymes were found to be glycoproteins. The carbohydrate moiety of the glycoproteins included mannose, galactose, and N-acetylglucosamine. The carbohydrate moiety of the laccases from C. unicolor 059, C. unicolor 0784, and P. oastreatus 0432 accounted for 17, 23, and 24%, respectively. The pH optimum of the enzymes was at 4.0, 3.75, and 5.6, respectively. Thermal stability testing of laccases at 40 degrees C revealed that the C. unicolor 0784 enzyme was characterized by the highest thermal stability (after 172-h incubation the enzyme activity was maintained at a level of 25%). The Michaelis constant (Km) values of the reactions of oxidation of pyrocatechol, hydroquinone, and potassium ferrocyanide catalyzed by the basidiomycete laccases were determined.     

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.     

Laccase and other ligninolytic enzyme activities of selected strains ofTrametes spp. from different localities and substrates

Eighty-three strains belonging to three species of the genus Trametes FR. (T. versicolor, T. hirsuta and T. ochracea) collected in different localities and on different substrates were screened for laccase production. The production of other lignin-modifying enzymes--manganese peroxidase (MnP) and lignin peroxidase (LiP)--and the decolorization ability were also determined in 21 of them. Production variability was relatively high and no significant correlation was found between the origin of the strains (locality, substrate) and the enzyme production. Dikaryons of all 3 species (but not of all their strains) exhibited LiP activity, which was not detected in the respective monokaryons.     

Decolorization of simulated textile dye baths by crude laccases from Trametes hirsuta and Cerrena unicolor

In this study crude laccases from the white‐rot fungi Cerrena unicolor and Trametes hirsuta were tested for their ability to decolorize simulated textile dye baths. The dyes used were Remazol Brilliant Blue R (RBBR) (100 mg/L), Congo Red (12.5 mg/L), Lanaset Grey (75 mg/L) and Poly R‐478 (50 mg/L). The effect of redox mediators on dye decolorization by laccases was also assessed. C. unicolor laccase was able to decolorize all the dyes tested. It was especially effective towards Congo Red and RBBR with 91 and 80% of color removal in 19.5 h despite the fact that simulated textile dye baths were used. Also Poly R‐478 and Lanaset Grey were partially decolorized (69 and 48%, respectively). C. unicolor laccase did not need any mediators for removing the dyes. However, T. hirsuta laccase was only able to decolorize simulated Congo Red and RBBR dye baths (91 and 45%, respectively) in 19.5 h without mediators. When using mediators the decolorization capability was enhanced substantially, e.g. Poly R‐478 was decolorized by 78% in 25.5 h. On the whole, both laccases showed potential to be used in industrial applications.     

Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent—A comparative study

Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:69–80, 2018     

Extracellular Laccases from Cerrena unicolor 059, Cerrena unicolor 0784, and Pleurotus oastreatus 0432: A Comparative Assay

Laccases of the basidiomycetes Cerrena unicolor 059, C. unicolor 0784, and Pleurotus oastreatus 0432 were subjected to a comparative study. The enzymes were isolated as homogeneous preparations with molecular weights of 55, 56, and 57 kD, respectively. The three enzymes were found to be glycoproteins. The carbohydrate moiety of the glycoproteins included mannose, galactose, and N-acetylglucosamine. The carbohydrate moieties of the laccases from C. unicolor 059, C. unicolor 0784, and P. oastreatus 0432 accounted for 17, 23, and 24%, respectively. The pH optima of the enzymes corresponded to 4.0, 3.75, and 5.6, respectively. Thermal stability tests (carried out at 40°C) demonstrated that the laccase of C. unicolor 0784 was characterized by the highest temperature resistance (the enzyme retained 25% activity after 172 h of incubation). The values of the Michaelis constant (K _M) were determined for the reactions of oxidation of pyrocatechol, hydroquinone, and potassium ferrocyanide catalyzed by the laccases of the basidiomycetes.     

Redox-mediated decolorization of synthetic dyes by fungal laccases

Laccases from the lignin-degrading basidiomycetes Trametes versicolor, Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents. Differences were attributed to the specific catalytic properties of the individual enzymes and to the structure of the dyes. Due to their higher oxidative capacities, the laccases from the two basidiomycetes decolorized dyes more efficiently than that of the ascomycete. The azo dye Direct Red 28, the indigoid Acid Blue 74 and anthraquinonic dyes were directly enzymatically decolorized within 16 h. The addition of 2 mM of the redox-mediator 1-hydroxybenzotriazole further improved and facilitated the decolorization of all nine dyes investigated. Laccases decolorized dyes both individually and in complex mixtures in the presence of bentonite or immobilized in alginate beads. Our data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries.     

Autoreduction and aggregation of fungal laccase in solution phase: possible correlation with a resting form of laccase

This paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. The issue that is reconsidered here is the previously proposed existence of "active" and "resting" forms of laccases. The presence of fungal laccases with partly reduced active sites is demonstrated. Of further interest is that an aggregated state in solution, not to our knowledge previously noted for laccase, has been found by using small-angle X-ray scattering as well as thorough analysis of the results of several biochemical experiments. Under some conditions, this aggregated state may correlate with the resting form of the laccases, although this resting form could have a broader significance. It was shown that Trametes ochracea laccase had some anomalous characteristics, which could be correlated with the high concentration of the "resting" enzyme. The mechanism of formation of resting laccase is suggested. Knowledge of the resting state is of importance for in vitro studies. Additionally, a suggestion about the possible regulatory role of this form in vivo is mentioned.     

Laccase from Melanocarpus albomyces binds effectively to cellulose

A laccase from the thermophilic fungus Melanocarpus albomyces was shown to bind to softwood and pure microcrystalline cellulose. The binding isotherm fitted well the Langmuir type one-site binding model. The adsorption parameters indicated that M. albomyces laccase binds with high affinity to cellulose with a relatively low maximum binding capacity, as compared to the values for various cellulases. The binding was shown to be reversible and not influenced by non-specific protein or 0.1-0.5 M Na2SO4. No binding was detected with laccases from Trametes hirsuta or Mauginiella sp., which suggests that binding to cellulose is typical for only some laccases.     

Development of printable bioactive paper containing laccase

Compatibility of Trametes versicolor and Trametes hirsuta laccases was studied with polymers used for flexographic inks. The aim was to produce bioactive paper with ability to change color. Optimum pH for the stability of Trametes versicolor and Trametes hirsuta laccases was determined during storage at room temperature for 60 days. The optimum pH for the stability of both laccases was 8–9. The stabilization effect of flexo printing inks on the enzymes was tested in liquid form and when coated on paper. Sulfo polyester resin HZ1100D stabilized the two laccases both in solution and on paper. For example, Trametes versicolor laccase remained stable for at least 8 weeks when coated with HZ1100D polymer. Furthermore, the adsorption of the flexo inks to cellulose was studied with quartz crystal microbalance with dissipation monitoring (QCM-D). It was observed that HZ1100D also adsorbs well on cellulose over a wide pH range. The results suggested that laccases are well suited to bioactive paper applications. Large scale manufacturing of bioactive paper products by flexo printing would be possible because of the compatibility of laccases with flexo inks.     

Isolation and Purification of Enzymes from Ligninolytic Complex of the Basidial Fungus <Emphasis Type="Italic">Trametes pubescens</Emphasis> (Schumach.) Pilat and Study of Their Properties

A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.     

Regio- and stereoselective reductions of dehydrocholic acid

Dehydrocholic acid (DHCA), an unnatural bile acid, is manufactured by oxidation of cholic acid. Its biotransformation by two basidiomycetes (Trametes hirsuta and Collybia velutipes) is reported. These mycelia showed different affinities for the substrate and selectivities of attack: T. hirsuta in particular regio- and stereoselectively reduced the 3-keto group to yield 3 alpha-hydroxy-7,12-diketo-5 beta-cholan-24-oic acid (7,12-diketolithocolic acid) as the main product. A number of different chemical reductions were carried out on DHCA; among them hydrogenation with Raney Nickel in water under high-intensity ultrasound proved highly regio- and stereoselective, yielding 7,12-diketolithocolic acid exclusively. (1)H and (13)C resonances were assigned in details thanks to a series of 1D and 2D NMR runs including DEPT, NOESY, H-H COSY, gHSQC and gHMBC.     

Increase of the detoxification potential of basidiomycetes by induction of laccase biosynthesis

The effect of oxidoreductase inducers guaiacol and syringaldazine on the ability of Coriolus hirsutus, Coriolopsis fulvocinerea, Cerrena maxima, and cocultivated Coriolus hirsutus/Cerrena maxima to degrade atrazine in submerged cultures was studied. All the basidiomycetes reduced atrazine concentration with and without syringaldazine or guaiacol. The degree of atrazine degradation was higher in induced cultures (77-98% vs. 68-94% without induction). Of the four cultures, the highest detoxifying potential was observed in Coriolopsis fulvocinerea with and without an inducer (98% with guaiacol), and the lowest was in Cerrena maxima. Inducers decreased the residual atrazine concentration differently in the different cultures. A long-term increase of laccase production was observed in both induced and uninduced cultures, whereas the activity of Mn-peroxidase decreased. The results indicate that laccase plays a larger role in atrazine biodegradation than Mn-peroxidase.     

Fungal decomposition of oat straw during liquid and solid state fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

The dynamics of oxidase activity during cultivation of basidiomycetes from the genus Trametes Fr.

Twenty strains of the wood-degrading fungi from the genus Trametes Fr., capable of synthesizing laccases, were screened according to the changes in the oxidase activity in a submerged culture. The range of maximal efficiency of various species with respect to extracellular oxidase activity was determined. The absence of correlation between the oxidase activity in a submerged culture and the size of colored zone on agar media (Bavendamm reaction) was demonstrated. The most efficient strains, T. hirsita 56 and T. ochracea 92-78, were used to produce laccases, homogeneous according to SDS electrophoresis data. A number of biochemical parameters characteristic of these enzymes were determined.     

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.     

Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.