Metabolic coupling in the co-cultured fungal-yeast suite of Trametes ljubarskyi and Rhodotorula mucilaginosa leads to hypersecretion of laccase isozymes.

Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3-4), higher thermo-stability (60 °C), and broad enzyme kinetics (Km) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment.

An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization.

A novel extracellular laccase was purified from fermentation broth of the white rot fungus Trametes sp. F1635 by a three-step protocol including two consecutive ion-exchange chromatography steps on DEAE-Sepharose and SP-Sepharose, and a final gel-filtration on Superdex 75. The purified laccase (TsL) was a monomeric protein with the molecular mass of 64.8 kDa. It demonstrated high oxidation activity of 4.00 × 104 U/mg towards ABTS. Its N-terminal amino acid sequence was AIGPVADLTIINNAV which was unique and sharing high similarity of other fungal laccases. TsL was a yellow laccase based on absorption spectrum analysis. It demonstrated an acidic pH optimum of 2.6 and temperature optimum of 50 °C towards ABTS. The Km and Vmax values towards ABTS were estimated to 18.58 µM and 1.35 µmol/min, respectively. TsL manifested effective decolorization activity towards eriochrome black T (EBT), remazol brilliant blue R (RBBR), malachite green (MG), and eriochrome black T (EBT) (over 60%). Violuric acid (VA) and acetosyringone (AS) were the optimal mediators for the laccase in dye decolorization. Results suggest that TsL demonstrates great potential for dye decolorization and water treatment.

Quorum sensing molecule-farnesol increased the production and biological activities of extracellular polysaccharide from Trametes versicolor.

A novel strategy of exposing 2-day-old mycelia cultures to 0.8mM farnesol was developed to stimulate extracellular polysaccharide (EPS) production in Trametes versicolor submerged cultures. Farnesol, a quorum sensing molecule in fungi, could significantly increase EPS production by promoting polysaccharide biosynthesis and regulating mycelial morphology. EPS yield reached a maximum of 2.56g/L that was 2.7-fold greater than that of control cultures. Farnesol made T. versicolor develop into fluffy, loose and multi-hyphae morphology, which facilitated the excretion of intracellular polysaccharide into culture medium. Moreover, EPS from farnesol-induced cultures (EPS-F) with higher carbohydrate and uronic acid contents mainly contained high molecular weight polysaccharide (134kDa, 85%), and comprised glucose, mannose and galactose in a molar ratio of 34.2:2.1:1.0. These physicochemical properties led to stronger antioxidant and antitumor activities of EPS-F. This is the first report that farnesol can significantly improve the production of polysaccharide with higher biological activities. It provides a novel strategy to enhance the production and bioactivity of mushroom polysaccharide using microbial quorum sensing molecules.

Typification and Characterization of Trametes multicolor (Agaricomycetes), a Perspective Species of Medicinal Mushrooms.

Nomenclature revision and enlarged taxonomical descriptions are still needed for some well-known species whose interpretation is complicated by many nomenclature or taxonomical problems. The polyporoid fungus widely known as Trametes ochracea (= Coriolus zonatus) belongs to such a problematic group. At the same time, recent data show that this species, like its sister species T. versicolor, seems to be a perspective subject for fungal biotechnology and pharmacology. This article is devoted to stabilizing the nomenclature of the species in question via lectotypification and epitypification of Boletus multicolor. It will clarify the name T. multicolor as applied to this species is nomenclaturally correct and useful, free of further problems. An expanded species description and cultural characterization of epitype materials are presented.

[Preparation of protoplasts of the fungus Trametes hirsuta 072 and study of the effect of antioxidants on their formation and regeneration].

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 107/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES+ buffer during 4 h of incubation; in this case the titer reduction was 5­7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; ß-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of ß-carotene and quercetin by 18­24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of ß-carotene and quercetin; it was 0.1% in the presence of MES+. The rate of protoplast growth was two times higher in the presence of ß-carotene and quercetin.

Laccase produced by a thermotolerant strain of Trametes trogii LK13.

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%-50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g (-1) ) along with low cellulose (2 U g (-1) ) and xylanase (140 U g (-1) ) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes.

Laccase produced by a thermotolerant strain of Trametes trogii LK13

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g−1) along with low cellulose (2 U g−1) and xylanase (140 U g−1) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes.

Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

Reduced graphene oxide hydrogels and xerogels provide efficient platforms for immobilization and laccase production by Trametes pubescens.

Fungal immobilization is an interesting topic in enzyme production and bioprocess development. The properties of graphene (i.e. large surface area, hydrophobicity), together with the possibility of producing it at low cost and with tailor-made properties, make this popular material worthy of investigation as a support for fungal immobilization. In the present paper, 3D-organized structures of reduced graphene oxide (rGO) in hydrogels and their dried derivatives (xerogels) were synthesized, characterized and investigated as potential supports for the immobilization of the white-rot fungus Trametes pubescens. It was found that the morphology of the hydrogels and xerogels was not influenced by the synthesis conditions; however the 3D structure was preserved after drying and formation of xerogels. Both, hydrogels and xerogels have been shown to be suitable supports for the immobilization of T. pubescens. Additionally, xerogels promoted increased laccase activities and maximum activity values of about 20 ± 1 U/mL were attained. These activities were much higher than those obtained with other well-known inert supports. Nevertheless, no relationship between support morphology and productivity was found. The encouraging results obtained have paved the way for the development of novel graphene-based supports for microorganism immobilization.

Growth inhibition and antioxidative response of wood decay fungi exposed to plant extracts of Casearia species.

UNLABELLED: Ligninolytic fungi take part in critical processes in ecosystems such as nutrient recycling; however, some fungal species can be pathogenic to forest and urban trees and deteriorate wood products. The tropical flora is an important source of antimicrobial compounds environmentally safer than traditional wood preservatives. Therefore, this study aimed to evaluate the inhibitory activity of ethanol plant extracts of Casearia sylvestris and Casearia decandra on the white-rot wood decay basidiomycetes Trametes villosa and Pycnoporus sanguineus. In addition, the effect of the extracts on the fungal antioxidative metabolism was studied. Among the different substances present in the extracts, the phytochemical analyses identified a clerodane diterpenoid (C. sylvestris) and cinnamic acid, hydroquinone and ß-sitosterol (C. decandra). The extracts inhibited the fungi up to 70% and caused hyphal morphology changes. The extracts triggered oxidative stress process as indicated by the increased levels of the antioxidant enzymes catalase and glutathione reductase. Therefore, the Casearia extracts are a potential source of natural biocides to control wood decay fungi, and one of the mechanisms of action is the oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The Casearia plant extracts exhibited important antifungal activity on wood decay fungi and triggered oxidative stress process, an inhibitory mechanism rarely studied in filamentous fungi exposed to plant extracts. Therefore, a starting point was provided for the development of natural compounds-based products as an alternative to chemical fungicides. In addition, subsidies were given to further studies in order to elucidate in more detail how compounds present in extracts of native tropical plants affect the physiology of fungi.

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor affect differently the proliferation rate of HepG2 cells.

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 µg mL(-1) on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 µg mL(-1) of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.

Ultrasound-intensified laccase production from Trametes versicolor.

An efficient intermittent ultrasonic treatment strategy was developed to improve laccase production from Trametes versicolor mycelia cultures. The optimized strategy consisted of exposing 2-day-old mycelia cultures to 5-min ultrasonic treatments for two times with a 12-h interval at the fixed ultrasonic power and frequency (120 W, 40 kHz). After 5 days of culture, this strategy produced the highest extracellular laccase activity of 588.9 U/L among all treatments tested which was 1.8-fold greater than the control without ultrasound treatment. The ultrasonic treatment resulted in a higher pellet porosity that facilitated the mass transfer of nutrients and metabolites from the pellets to the surrounding liquid. Furthermore, the ultrasonic treatment induced the expression of the laccase gene (lcc), which correlated with a sharp increase in both extracellular and intracellular laccase activity. This is the first study to find positive effects of ultrasound on gene expression in fungal cells. These results provide a basis for understanding the stimulation of metabolite production and process intensification by ultrasonic treatment in filamentous fungal culture.