V. E. Nikitina

Reduction of Organic and Inorganic Selenium Compounds by the Edible Medicinal Basidiomycete Lentinula edodes and the Accumulation of Elemental Selenium Nanoparticles in Its Mycelium

We report for the first time that the medicinal basidiomycete Lentinula edodes can reduce selenium from inorganic sodium selenite (SeIV) and the organoselenium compound 1,5-diphenyl-3-selenopentanedione-1,5 (DAPS-25) to the elemental state, forming spherical nanoparticles. Submerged cultivation of the fungus with sodium selenite or with DAPS-25 produced an intense red coloration of L. edodes mycelial hyphae, indicating accumulation of elemental selenium (Se0) in a red modification. Several methods, including transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), and X-ray fluorescence, were used to show that red Se0 accumulated intracellularly in the fungal hyphae as electron-dense nanoparticles with a diameter of 180.51±16.82 nm. Under designated cultivation conditions, shiitake did not reduce selenium from sodium selenate (SeVI).

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.

Phenol oxidase activity in bacteria of the genus Azospirillum

Phenol oxidase activity was detected for the first time in a number of strains belonging to various Azospirillum species. Both extracellular and intracellular activities of laccase, Mn-peroxidase, lignin peroxidase, and tyrosinase were observed. Extracellular enzymes were found to have higher activity. Significant differences in phenol oxidase activities were observed between species and strains.

Bioremediation of oil-polluted soil with an association including the fungus Pleurotus ostreatus and soil microflora

The possibility of application of the Pleurotus ostreatus D1-soil microflora to bioremediation of oil-polluted soils was studied. The fungus degraded mainly the aromatic fractions, whereas soil microflora intensely degraded paraffin and naphthene oil fractions. Introduction of the fungus Pleurotus ostreatus D1 to soil induces degradation of a wider range of oil hydrocarbons. It is reasonable to further investigate the discovered phenomenon in order to improve procedures of remediation of oil-polluted soils.

Enzymatic formation of gold nanoparticles by submerged culture of the basidiomycete Lentinus edodes

We report for the first time that the medicinal basidiomycete Lentinus edodes can reduce Au(III) from chloroauric acid (HAuCl4) to elemental Au [Au(0)], forming nanoparticles. Several methods, including transmission electron microscopy, electron energy loss spectroscopy, X-ray fluorescence, and dynamic light scattering, were used to show that when the fungus was grown submerged, colloidal gold accumulated on the surface of and inside the mycelial hyphae as electron-dense particles mostly spherical in shape, with sizes ranging from 5 to 50nm. Homogeneous proteins (the fungal enzymes laccase, tyrosinase, and Mn-peroxidase) were found for the first time to be involved in the reduction of Au(III) to Au(0) from HAuCl4. A possible mechanism forming Au nanoparticles is discussed.

Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. The lectin activity in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C:N ratio, (9.5–12):1) on day 15–18 of culturing at pH 8.0–9.0.

Hemagglutinating activity and motility of the bacterium Azospirillum brasilense in the presence of various nitrogen sources

Hemagglutinating activity of the Azospirillum brasilense strain Sp245 grown in liquid media and the swarming motility of those bacteria grown in semisolid media vary significantly depending on the nitrogen source. In media with nitrate or nitrite, an increase in the hemagglutinating activity and a decrease in the swarming circles’ diameter of Sp245 were observed, compared to bacteria grown in the presence of ammonium or N2. A ∼67-kDa hemagglutinin exhibiting affinity to the O-specific polysaccharide, an acidic D-rhamnan (OPS-I), was isolated from the surface of Sp245 cells. Introduction of the hemagglutinin into the media resulted in a decrease in the Sp245 cell motility while not affecting its mutants lacking the acidic D-rhamnan or the Sp245.5 mutant with a different OPS structure. Cells of strain Sp245.5 demonstrated hemagglutinating activity two times higher than that of the parent Sp245 strain and formed “diffuse” colonies, rather than distinct swarming circles Sp245 formed when grown in a semisolid medium. The data obtained demonstrate that intercellular contacts mediated by the interaction between the surface hemagglutinin and OPS-I, which is sensitive to environmental factors, affect the collective motility of cells.

Isolation and characterization of Lentinus edodes (Berk.) singer extracellular lectins

Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 ± 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 ± 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.

Hemagglutinating activity of the fungus Lentinus edodes (Berk.) sing [Lentinus edodes (Berk.) Pegler]

The hemagglutinating (HA) activity of the submerged mycelium and the culture liquid (CL) of four strains ofLentinus edodes was studied. The HA activity of the CLs proved to be much higher than that of mycelia. The carbohydrate specificity of fungal agglutinating factors was determined. HA activity was investigated as a function of the inoculum size, cultivation temperature, and culture age. The agglutinating activity of different morphogenetic structures ofL. edodes F-249, including mycelium, brown mycelial mat (MM), primordia, and fruiting bodies, was studied. MM was found to possess the maximum HA activity, which can be explained by the possible involvement of agglutinins in the formation of MM, which is composed of glued hyphae.

Biosynthesis of gold nanoparticles by Azospirillum brasilense

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparticles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au+3 (HAuCl4) to Au0. Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.