Marcin Grąz

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology.

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu2O, Al2O3, ZnO, CuFe2O4Zn, CdO, and MnO2) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu2O- and ZnO-amended plates was observed. ZnO, CdO and Cu2O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu2O, MnO2, ZnO and CuFe2O4Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu2O, ZnO and MnO2. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.

Nonlinear changes in the activity of the oxygen-dependent demethylase system in Rhodococcus erythropolis cells in the presence of low and very low doses of formaldehyde

The effect of exogenous, highly diluted formaldehyde on the rate of demethylation/re-methylation of veratric acid by the bacteria Rhodococcus erythropolis was studied using electrophoretic and microscopic techniques. The activity of 4-O-demethylase, responsible for accumulation of vanillic acid, and the levels of veratric and vanillic acids were determined using capillary electrophoresis. Formaldehyde was serially diluted at 1:100 ratios, and the total number of iterations was 20. After incubation of the successive dilutions of formaldehyde with the bacteria, demethylase activity oscillated in a sinusoidal manner. It was established using capillary electrophoresis that methylation of vanillic acid to veratric acid occurred at a double rate, as shown by the doubled fluctuation in the concentration of veratrate. There were also changes in the NADH oxidase activity, which is associated with methylation processes. Microscopic observations revealed the presence of numerous enlarged vacuoles in bacterial cells during the accumulation of large amounts of vanillic acid, and their disappearance together with a decrease in 4-O-demethylase activity. The presented results give evidence for the ability of living cells to detect the presence of submolecular concentrations of biological effectors in their environment and provide a basis for a scientific explanation of the law of hormesis and the therapeutic effect of homeopathic dilutions.

Oxalic acid, versatile peroxidase secretion and chelating ability of Bjerkandera fumosa in rich and limited culture conditions

Efficient ligninolytic systems of wood-degrading fungi include not only oxidizing enzymes, but also low-molecular-weight effectors. The ability of Bjerkandera fumosa to secrete oxalic acid and versatile peroxidase (VP) in nitrogen-rich and nitrogen-limited media was studied. Higher activity of VP was determined in the nitrogen-limited media but greater concentration of oxalic acid was observed in the cultures of B. fumosa without nitrogen limitation. Ferric ions chelating ability of Bjerkandera fumosa studied in ferric ions limited media was correlated with the increased level of oxalic acid. The presence of hydroxamate-type siderophores in B. fumosa media were also detected. Oxalate decarboxylase was found to be responsible for regulation of oxalic acid concentration in the tested B. fumosa cultures.

Laccase-mediated synthesis of a phenoxazine compound with antioxidative and dyeing properties – the optimisation process

This study demonstrates the optimisation of the main parameters of the laccase-mediated biosynthesis of high-intensity-coloured orange phenoxazine compound, 2-amino-3-oxo-3H-phenoxazine-8-sulfonic acid, and the antioxidative and dyeing properties. Among optimised parameters were the pH value, the activity of laccase, and the high concentration of the precursor as the necessary step in terms of dye synthesis scale-up. The high concentration of the precursor of ca. 10 g/L can be transformed totally by laccase at the activity of 30 U/g during 12 hours, in an optimised and standardised process in nearly 100% yield of synthesis. The obtained dye exhibited good dyeing properties determined according to the ISO standards. Antioxidative activities were detected for phenoxazinone dye using two independent methods, the chemiluminescence assay and the ABTS free radical-scavenging test, with the values of EC50 for the tested phenoxazine dye amounting 189.8 μg/mL and 1428 μg/mL, respectively. Despite the presence of the phenoxazine core in the structure of this dye, no antibacterial capacity was noted.

Decolourisation of anthraquinone-and anthracene-type dyes by versatile peroxidases from bjerkandera fumosa and pleurotus ostreatus D1

Abstract The catalytic properties of a versatile peroxidase from Pleurotus ostreatus D1 (Jacquin) P. Kummer were studied in comparison with that of a typical versatile peroxidase from Bjerkandera fumosa 137 (Per.:Fr) Karst. Decolourisation activities of both enzymes towards a wide range of dyes containing condensed aromatic rings (anthraquinone- and anthracene-type) were found. The anthraquinone dyes were decolourised rapidly by both tested peroxidases. The presence of polymerisation reaction products of Acid Blue 62, Basic Blue 22 and Reactive Blue 4 oxidation, and breakdown of aromatic rings of Alizarin Red were observed. The main catalytic constants (KM and Vmax) of the decolourisation reactions of anthraquinone dyes were calculated. In the case of Alizarin Red, inhibition of the activity of versatile peroxidase from P. ostreatus D1 by an excess of the substrate was observed. Independence from Mn2+ ions of the catalytic activity of versatile peroxidase from P. ostreatus D1 towards different substrates was revealed. Finally, differences in the catalytic activity towards anthracene-type dyes and monoaromatic substrates of both peroxidases were found.

Purification and electrophoretic characterization of bovine conglutinin.

In this study a two-stage procedure for purification of conglutinin using affinity and ion-exchange chromatography was developed. To isolate conglutinin from bovine serum, its unique ability to bind to complement component iC3b was exploited. Incubation of bovine serum with chromatographic beads (TSK, Toyopearl HW-75 F) at 37 °C allows for iC3b deposition and subsequent binding of conglutinin. A single protein fraction eluted with ethylenediaminetetraacetic acid (EDTA) was then separated on an ion-exchange column in an NaCl gradient. The purification was evaluated by SDS-PAGE and western blotting. Conglutinin analyzed by SDS-PAGE under reducing conditions showed two main bands at 41 and 47 kDa and eight weaker bands. Nonreduced conglutinin appeared as a ladder pattern composed of many fractions ranging from 34 to 630 kDa. The bands at 34, 153, 174, 247, 338 and 387 kDa displayed the highest optical density. In the native conglutinin profile four fractions were observed, and the pI of this protein was below 8.5. The presence of sugar residues in the conglutinin molecule was detected using Schiffs reagent.

Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis

Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min(-1), respectively.

Intracellular distribution of cadmium during the growth of Abortiporus biennis on cadmium-amended media

Heavy metals are difficult to remediate and traditional remedial processes are expensive, so bioremediation technology using bacteria, fungi, or plants is of interest. Many studies have demonstrated that basidiomycetes fungi are able to growth under heavy metals stress. In this study the distribution of cadmium (Cd) in Abortiporus biennis cells was studied. Cd accumulated especially within cytoplasm and its presence caused changes in the cytoplasm appearance, which became denser in comparison to the cytoplasm of control cells. Vacuolization of cytoplasm and periplasmic region in A. biennis cells was also observed. The growth rate of A. biennis was inhibited up to 75% during the growth on medium amended with 1 mmol/L cadmium oxide. The presence of Cd in growing media inhibited oxalic acid secretion by A. biennis, but oxalate concentration increased together with elevated Cd concentration in growing medium. The influence of initial pH of growing media on the accumulation of Cd by A. biennis was also observed. The highest accumulation of Cd in mycelium was detected during A. biennis growth on media with a pH of 6. Studies addressing metals uptake by fungi and metal distribution in fungal cells may allow these organisms to be applied in bioremediation processes more effectively or to be used as bioindicators of contaminated environmental pollutions.

Determination of pamidronate in bisphosphonate-enriched bone cement by ion-pair hplc and capillary electrophoresis

Abstract The presence of pamidronate during local use of bisphosphonates (BP)-enriched bone cement was determined. The question was whether pamidronate implanted into the bone cement is eluted. The study was performed on 10 probes of BP-enriched bone cement located in 0.9% NaCl. The probes were incubated for 3 and 6 weeks. Ion-pair HPLC was used for the detection of pamidronate. Then, capillary electrophoresis was applied for quantitative analysis of pamidronate in the 3rd and 6th week after incubation. The presence of pamidronate, eluted from BP-enriched bone cement into 0.9% NaCl solution 3 and 6 weeks after incubation, was demonstrated. These results may explain the changes in the level of cytokine RANKL and bone turnover marker osteoprotegrin in rats’ serum treated with BP-enriched bone cement 3 and 6 weeks after surgery. The possibility of effective local use of BP-enriched bone cement in veterinary medicine was underlined. The results, and the former conducted research, point out that the clinical applications of BP-enriched bone cement in vivo may have some validity in the future.

Transcriptome-based analysis of the saprophytic fungus Abortiporus biennis – response to oxalic acid

In this study, the transcriptomic-based response of the white rot fungus Abortiporus biennis to oxalic acid induction was reported. The whole transcriptome of A. biennis was analysed using the RNA-based sequencing technology and Solid 5500 platform. De novo assembly of reads generated 37,719 contigs. A molecular function for 26,280 unique transcripts was assigned. The analysis of the A. biennis transcriptome predicted 635 hypothetical open reading frames encoding carbohydrate active enzymes distributed in 122 families. 82 genes were identified, whose expression level was significantly changed after oxalic acid addition. Among them, 18 genes were up-regulated and 64 genes were down-regulated. Genes coding for putative cellulose and hemicellulose degrading enzymes were predominantly up-regulated in the mycelium induced with oxalic acid; it was in the case of cellulases and xylanases (hemicellulases), in particular, β-glucosidase and endo-1,4-β-xylanases. On the contrary, several genes coding for lignolytic enzymes were down-regulated, with the significant repression level in the case of versatile peroxidase. Finally, we identified putative genes involved in oxalate metabolism. Among the transcripts detected in the A. biennis transcriptome, one was annotated as coding for putative oxalate decarboxylase (ODC) and nine transcripts were annotated as formate dehydrogenases (FDH). The addition of oxalic acid to the culture caused upregulation of the gene coding for ODC and three genes for FDH. Amongst the transcripts of putative FDH genes, one designated as NODE_36057, demonstrated the highest induction level recorded in this study after the oxalic acid addition.