Monika Osińska-Jaroszuk

New Bioactive Fungal Molecules with High Antioxidant and Antimicrobial Capacity Isolated from Cerrena unicolor Idiophasic Cultures

Three bioactive fractions, extracellular laccase (ex-LAC), crude endopolysaccharides (c-EPL), and a low molecular subfraction of secondary metabolites (ex-LMS), were isolated from the idiophasic cultures of the white rot fungus Cerrena unicolor. For the first time, we determined the antioxidant properties of these samples by chemiluminometric measurement (a) and assessment of the scavenging effect on ABTS (b) and the DPPH reduction rate (c). The highest reducing capability was found for the ex-LMS fraction: 39–90% for (a), 20–90% for (b), and 10–59% for (c) at the concentration of 6.25–800 µg/mL. The scavenging abilities of the C. unicolor c-EPL were between 36 and 70% for (a), 2 and 60% for (b), and 28 and 32% for (c) at the concentration of 6.25–800 µg/mL. A very high prooxidative potential was observed for the ex-LAC probes. The preliminary toxicity tests were done using the Microtox system and revealed the following percentage of the toxic effect against Vibrio fischeri: 85.37% for c-EPL, 50.67% for ex-LAC, and 99.8% for ex-LMS, respectively. The ex-LAC sample showed the antibacterial activity against Escherichia coli, c-EPL against Staphylococcus aureus, and ex-LMS against both bacterial strains, respectively, but the stronger inhibitory effect was exerted on S. aureus.

Exopolysaccharide from Ganoderma applanatum as a Promising Bioactive Compound with Cytostatic and Antibacterial Properties

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

Fungus Cerrena unicolor as an effective source of new antiviral, immunomodulatory, and anticancer compounds

In the report, three bioactive fractions from Cerrena unicolor: laccase (LAC), endopolysaccharides (c-EPL), and low molecular weight (ex-LMS) were tested for the first time towards their antiviral, immunostimulatory, cytotoxic and antiproliferative effect. The immunomodulatory activity was studied by means of THP-1-derived macrophages able to synthesize and secrete IL-6 and TNF-α. We used cervical carcinoma cell lines SiHa (ATCC, HTB-35) and CaSki (ATCC, CRL 1550) to determine antitumor activity and human skin fibroblasts (HSF) as a control. SiHa and L929 cell lines were used in the antiviral activity assay to propagate HHV-1 and EMCV, respectively. LAC was the most active against HSV at an early stage of viral replication, whereas the activity of laccase against EMCV was evident after incubation of the virus with LAC before and after the adsorption step. Moreover, the investigations showed that the fungal c-EPL fraction stimulated the production and secretion of TNF-α and IL-6 by THP-1-derived macrophages up to a level of 2000 pg/ml and 400 pg/ml, respectively. It was indicated for the first time that the LAC and ex-LMS fractions exhibited anticancer activity. This resulted from their cytotoxic or antiproliferative action against the investigated tumor cells at concentrations above 250 μg/ml and 10 μg/ml, respectively.

Amphotericin B-silver hybrid nanoparticles: synthesis, properties and antifungal activity

UNLABELLED High antifungal activity is reported, in comparison with commercially available products, of a novel hybrid system based on silver nanoparticles synthesized using a popular antifungal macrocyclic polyene amphotericin B (AmB) acting both as a reducing and stabilizing/capping agent. The synthesis reaction proceeds in an alkaline environment which prevents aggregation of AmB itself and promotes nanoparticle formation. The innovative approach produces monodisperse (PDI=0.05), AmB-coated silver nanoparticles (AmB-AgNPs) with the diameter ~7nm. The products were characterized using imaging (electron microscopy) and spectroscopic (UV-vis and infrared absorption, dynamic light scattering and Raman scattering) methods. The nanoparticles were tested against Candida albicans, Aspergillus niger and Fusarium culmorum species. For cytotoxicity studies CCD-841CoTr and THP-1 cell lines were used. Particularly high antifungal activity of AmB-AgNPs is interpreted as the result of synergy between the antifungal activity of amphotericin B and silver antimicrobial properties (Ag(+) ions release). FROM THE CLINICAL EDITOR Amphotericin B (AmB) is a common agent used for the treatment against severe fungal infections. In this article, the authors described a new approach in using a combination of AmB and silver nanoparticles, in which the silver nanoparticles were synthesized and stabilized by AmB. Experimental data confirmed synergistic antifungal effects between amphotericin B and silver. This novel synthesis process could potentially be important in future drug development and fabrication.

Characterization of cellobiose dehydrogenase and its FAD-domain from the ligninolytic basidiomycete Pycnoporus sanguineus

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in the culture supernatant of the selective delignifier Pycnoporus sanguineus maintained on a cellulose-based liquid medium. Cellobiose dehydrogenase was purified as two active fractions: CDH1-FAD (flavin domain) (40.4 fold) with recovery of 10.9% and CDH1 (flavo-heme enzyme) (54.7 fold) with recovery of 9.8%. As determined by SDS-PAGE, the molecular mass of the purified enzyme was found to be 113.4kDa and its isoelectric point was 4.2, whereas these values for the FAD-domain were 82.7kDa and pI=6.7. The carbohydrate content of the purified enzymes was 9.2%. In this work, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from fungus P. sanguineus were isolated, cloned, and characterized. The 2310bp full-length cDNA of cdh1 encoded a mature CDH protein containing 769 amino acids, which was preceded by a signal peptide of 19 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.

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.

Purification and characterization of laccase from Sinorhizobium meliloti and analysis of the lacc gene.

The soil native bacterial strains were screened for laccase activity. Bacterial strain L3.8 with high laccase activity was identified as Sinorhizobium meliloti. The crude intracellular L3.8 enzyme extract was able to oxidize typical diagnostic substrates of plant and fungal laccases. Laccase L3.8 was purified 81-fold with a yield of 19.5%. The molecular mass of the purified bacterial laccase was found to be 70.0kDa and its pI was 4.77. UV-vis spectrum showed that L3.8 protein is a multicopper oxidase. The carbohydrate content of the purified enzyme was estimated at 3.2%. Moreover, the laccase active fraction was characterized in terms of kinetics, temperature, and pH optima as well as the effect of various chemical compounds on the laccase activity, and antioxidant properties, which indicated that the L3.8 laccase had unique properties that might be important in biotechnological applications. The lacc gene encoding S. meliloti laccase was cloned and characterized. The full-length sequence of 1950bp encoded a protein of 649 aa preceded by a signal peptide consisting of 26aa. Laccase L3.8 shared significant structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. Potential biotechnological importance of a newly identified laccase is discussed.

New alkaline lipase from Rhizomucor variabilis: Biochemical properties and stability in the presence of microbial EPS

A new strain of Rhizomucor variabilis producing an active extracellular lipase was identified and characterized in the present studies. The culture conditions were optimized and the highest lipase production amounting to 136 U/mL was achieved after 4 days of cultivation. The optimum pH (5.5) and temperature (28 °C) were determined as the best conditions for R. variabilis lipase production. The isolated enzyme preparation exhibited maximum activity at 40 °C and pH 8.0. Lipase from R. variabilis was stable up to 50 °C during 2 H retaining 80% of its initial activity. The enzyme was highly stable in the pH range of 7.0–9.0. Moreover, the addition of naturally obtained exopolysaccharides (EPS) significantly enhanced lipase activity. The presence of EPS derived from Ganoderma applanatum and Rhizobium leguminosarum enhanced the lipase activity, which was 22% and 31%, respectively, higher than that in the control experiments. Simultaneously, the pH activity profiles remained unchanged. The Michaelis–Menten constant and the turnover number of the enzyme for p‐nitrophenyl palmitate in the standard assay conditions were estimated at a level of 0.631 mM and 0.674 Sec−1. In conclusion, the results obtained in this work present a newly isolated lipase preparation stabilized with EPS or without modification as a very effective tool for industrial application.

The Influence of Biochemical Modification on the Properties of Adhesive Compounds

The main objective of this study was to determine the effect of biochemical modification of epoxy adhesive compounds on the mechanical properties of a cured adhesive exposed to various climatic factors. The epoxy adhesive was modified by lyophilized fungal metabolites and prepared by three methods. Additionally, the adhesive compound specimens were seasoned for two months at a temperature of 50 °C and 50% humidity in a climate test chamber, Espec SH 661. The tensile strength tests of the adhesive compounds were performed using a Zwick/Roell Z150 testing machine in compliance with the DIN EN ISO 527-1 standard. The examination of the adhesive specimens was performed using two microscopes: a LEO 912AB transmission electron microscope equipped with Quantax 200 for EDS X-ray spectroscopy and a Zeiss 510 META confocal microscope coupled to an AxioVert 200M. The experiments involved the use of a CT Skyscan 1172 tomograph. The results revealed that some mechanical properties of the modified adhesives were significantly affected by both the method of preparation of the adhesive compound and the content of the modifying agent. In addition, it was found that seasoning of the modified adhesives does not lead to a decrease in some of their mechanical properties.

Effect of exopolysaccharide from Ganoderma applanatum on the electrical properties of mouse fibroblast cells line L929 culture using an electric cell-substrate impedance sensing (ECIS) - Preliminary study.

UNLABELLED IIntroduction and objective. In recent years there has been intensified research on medicinal preparations of fungal origin. Some fungal polysaccharides may directly affect the inhibition of cancer cells proliferation which, stopping the cell cycle, leads to apoptosis. One of these substances (component of extract of Ganoderma spp) is extensively tested for its anti-cancer properties on the tumor cell lines. Electric cell-substrate impedance sensing (ECIS) is an in vitro impedance measuring system using alternating current (AC) to determinate the behaviour of the cells in physiological conditions. OBJECTIVE The aim of the study was to examine the electric properties (resistance, capacitance and impedance) of mouse fibroblasts cell line L929 after treatment by different concentration of crude exopolysaccharides from Ganoderma applanatum (GpEPS) in real time by ECIS technique. MATERIALS AND METHODS For the study, the L929 cell line culture was treated by different concentrations of GpEPS: C1=228.5 µg/mL; C2=22.85 µg/mL; C3=2.285 µg/mL; C4=0.2285 µg/mL; and C5=0.02285 µg/mL. Default optimal frequencies were used: Resistance (R) 4000Hz, Impedance (Z) 16000Hz, Capacitance (C) 64000Hz. RESULTS The study demonstrated that GpEPS had no significant effect on the resistance, capacitance and impedance cells cultures, which implies that there is no significant effect on the physiological processes of L929 fibroblasts. This indicates the possibility of using GpEPS preparation in anti-cancer therapy. CONCLUSIONS In the future, following further studies (comprising in preventive and therapeutic actions), GpEPS can be safely used in anti-cancer therapy which does not cause side-effects or damage to healthy cells.