Oxalic acid degradation in wood-rotting fungi. Searching for a new source of oxalate oxidase.

Oxalate oxidase (EC 1.2.3.4) is an oxalate-decomposing enzyme predominantly found in plants but also described in basidiomycete fungi. In this study, we investigated 23 fungi to determine their capability of oxalic acid degradation. After analyzing their secretomes for the products of the oxalic acid-degrading enzyme activity, three groups were distinguished among the fungi studied. The first group comprised nine fungi classified as oxalate oxidase producers, as their secretome pattern revealed an increase in the hydrogen peroxide concentration, no formic acid, and a reduction in the oxalic acid content. The second group of fungi comprised eight fungi described as oxalate decarboxylase producers characterized by an increase in the formic acid level associated with a decrease in the oxalate content in their secretomes. In the secretomes of the third group of six fungi, no increase in formic acid or hydrogen peroxide contents was observed but a decline in the oxalate level was found. The intracellular activity of OXO in the mycelia of Schizophyllum commune, Trametes hirsuta, Gloeophyllum trabeum, Abortiporus biennis, Cerrena unicolor, Ceriosporopsis mediosetigera, Trametes sanguinea, Ceriporiopsis subvermispora, and Laetiporus sulphureus was confirmed by a spectrophotometric assay.

Thromboelastometric Analysis of Anticancer Cerrena unicolor Subfractions Reveal Their Potential as Fibrin Glue Drug Carrier Enhancers.

In this study, the influence of two subfractions (with previously proven anti-cancer properties) isolated from wood rot fungus Cerrena unicolor on the formation of a fibrin clot was investigated in the context of potential use as fibrin glue and sealant enhancers and potential wound healing agents. With the use of ROTEM thromboelastometry, we demonstrated that, in the presence of fibrinogen and thrombin, the S6 fraction accelerated the formation of a fibrin clot, had a positive effect on its elasticity modulus, and enhanced the degree of fibrin cross-linking. The S5 fraction alone showed no influence on the fibrin coagulation process; however, in the presence of fibrin, it exhibited a decrease in anti-proliferative properties against the HT-29 line, while it increased the proliferation of cells in general at a concentration of 100 µg/mL. Both fractions retained their proapoptotic properties to a lesser degree. In combination with the S6 fraction in the ratio of 1:1 and 1:3, the fractions contributed to increased inhibition of the activity of matrix metalloproteinases (MMPs). This may suggest anti-metastatic activity of the combined fractions. In conclusion, the potential of the fractions isolated from the C. unicolor secretome to be used as a means of improving the wound healing process was presented. The potential for delivering agents with cytostatic properties introduced far from the site of action or exerting a pro-proliferative effect at the wound site with the aid of a fibrin sealant was demonstrated.

Lighting Conditions Influence the Dynamics of Protease Synthesis and Proteasomal Activity in the White Rot Fungus Cerrena unicolor.

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor. Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.

Oxalate oxidase from Abortiporus biennis - protein localisation and gene sequence analysis.

We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.

The wood decay fungus Cerrena unicolor adjusts its metabolism to grow on various types of wood and light conditions.

Cerrena unicolor is a wood-degrading basidiomycete with ecological and biotechnological importance. Comprehensive Biolog-based analysis was performed to assess the metabolic capabilities and sensitivity to chemicals of C. unicolor FCL139 growing in various sawdust substrates and light conditions. The metabolic preferences of the fungus towards utilization of specific substrates were shown to be correlated with the sawdust medium applied for fungus growth and the light conditions. The highest catabolic activity of C. unicolor was observed after fungus precultivation on birch and ash sawdust media. The fungus growing in the dark showed the highest metabolic activity which was indicated by capacity to utilize a broad spectrum of compounds and the decomposition of 74/95 of the carbon sources. In all the culture light conditions, p-hydroxyphenylacetic acid was the most readily metabolized compound. The greatest tolerance to chemicals was also observed during C. unicolor growth in darkness. The fungus was the most sensitive to nitrogen compounds and antibiotics, but more resistant to chelators. Comparative analysis of C. unicolor and selected wood-decay fungi from different taxonomic and ecological groups revealed average catabolic activity of the fungus. However, C. unicolor showed outstanding capabilities to catabolize salicin and arbutin. The obtained picture of C. unicolor metabolism showed that the fungus abilities to decompose woody plant material are influenced by various environmental factors.