Bioactivity and Mycochemical Profile of Extracts from Mycelial Cultures of Ganoderma spp.

Fungal mycelium cultures are an alternative to natural sources in order to obtain valuable research materials. They also enable constant control and adaptation of the process, thereby leading to increased biomass growth and accumulation of bioactive metabolites. The present study aims to assess the biosynthetic potential of mycelial cultures of six Ganoderma species: G. adspersum, G. applanatum, G. carnosum, G. lucidum, G. pfeifferi, and G. resinaceum. The presence of phenolic acids, amino acids, indole compounds, sterols, and kojic acid in biomass extracts was determined by HPLC. The antioxidant and cytotoxic activities of the extracts and their effects on the inhibition of selected enzymes (tyrosinase and acetylcholinesterase) were also evaluated. The total content of phenolic acids in the extracts ranged from 5.8 (G. carnosum) to 114.07 mg/100 g dry weight (d.w.) (G. pfeifferi). The total content of indole compounds in the extracts ranged from 3.03 (G. carnosum) to 11.56 mg/100 g d.w. (G. lucidum) and that of ergosterol ranged from 28.15 (G. applanatum) to 74.78 mg/100 g d.w. (G. adspersum). Kojic acid was found in the extracts of G. applanatum and G. lucidum. The tested extracts showed significant antioxidant activity. The results suggest that the analyzed mycelial cultures are promising candidates for the development of new dietary supplements or pharmaceutical preparations.

Differences in Production, Composition, and Antioxidant Activities of Exopolymeric Substances (EPS) Obtained from Cultures of Endophytic Fusarium culmorum Strains with Different Effects on Cereals.

Exopolymeric substances (EPS) can determine plant-microorganism interactions and have great potential as bioactive compounds. The different amounts of EPS obtained from cultures of three endophytic Fusarium culmorum strains with different aggressiveness-growth promoting (PGPF), deleterious (DRMO), and pathogenic towards cereal plants-depended on growth conditions. The EPS concentrations (under optimized culture conditions) were the lowest (0.2 g/L) in the PGPF, about three times higher in the DRMO, and five times higher in the pathogen culture. The EPS of these strains differed in the content of proteins, phenolic components, total sugars, glycosidic linkages, and sugar composition (glucose, mannose, galactose, and smaller quantities of arabinose, galactosamine, and glucosamine). The pathogen EPS exhibited the highest total sugar and mannose concentration. FTIR analysis confirmed the ß configuration of the sugars. The EPS differed in the number and weight of polysaccharidic subfractions. The EPS of PGPF and DRMO had two subfractions and the pathogen EPS exhibited a subfraction with the lowest weight (5 kDa). The three EPS preparations (ethanol-precipitated EP, crude C, and proteolysed P) had antioxidant activity (particularly high for the EP-EPS soluble in high concentrations). The EP-EPS of the PGPF strain had the highest antioxidant activity, most likely associated with the highest content of phenolic compounds in this EPS.

Multilocus sequence analysis supports the taxonomic position of Astragalus glycyphyllos symbionts based on DNA-DNA hybridization.

In this study, the phylogenetic relationship and taxonomic status of six strains, representing different phenons and genomic groups of Astragalus glycyphyllos symbionts, originating from Poland, were established by comparative analysis of five concatenated housekeeping gene sequences (atpD, dnaK, glnA, recA and rpoB), DNA-DNA hybridization and total DNA G+C content. Maximum-likelihood phylogenetic analysis of combined atpD, dnaK, glnA, recA and rpoB sequence data placed the studied bacteria into the clade comprising the genus Mesorhizobium. In the core gene phylograms, four A. glycyphyllos nodule isolates (AG1, AG7, AG15 and AG27) formed a cluster common with Mesorhizobium ciceri, whereas the two other A. glycyphyllos symbionts (AG17 and AG22) were grouped together with Mesorhizobium amorphae and M. septentrionale. The species position of the studied bacteria was clarified by DNA-DNA hybridization. The DNA-DNA relatedness between isolates AG1, AG7, AG15 and AG27 and reference strain M. ciceri USDA 3383T was 76.4-84.2%, and all these A. glycyphyllos nodulators were defined as members of the genomospecies M. ciceri. DNA-DNA relatedness for isolates AG17 and AG22 and the reference strain M. amorphae ICMP 15022T was 77.5 and 80.1%, respectively. We propose that the nodule isolates AG17 and AG22 belong to the genomic species M. amorphae. Additionally, it was found that the total DNA G+C content of the six test A. glycyphyllos symbionts was 59.4-62.1 mol%, within the range for species of the genus Mesorhizobium.

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.

Laccase production and metabolic diversity among Flammulina velutipes strains.

Twelve Flammulina velutipes strains originating from Poland were identified using internal transcribed spacer (ITS) region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was determined. All F. velutipes strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the F. velutipes strains up to five times. The highest catabolic activities were characteristic for only two strains with capabilities to decompose up to 22 carbon sources. The correlation between carbon repression and laccase production by F. velutipes was analyzed based on glucose assimilation by these strains. Moreover, the influence of metal ions (Cu(2+), Cd(2+)), veratric and ferulic acids, and temperature on laccase activities in the analyzed strains was determined. The results obtained proved that all the inducers influenced laccase expression in almost all the analyzed strains. However, the degree of induction depended not only on the strain used but also on the day of the induction.

Genetic diversity of the edible mushroom Pleurotus sp. by amplified fragment length polymorphism.

Pleurotus strains are the most important fungi used in the agricultural industry. The exact characterization and identification of Pleurotus species is fundamental for correct identification of the individuals and exploiting their full potential in food industry. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of 21 Pleurotus isolates of Asian and European origin. Using one PstI restriction endonuclease and four selective primers in an AFLP assay, 371 DNA fragments were generated, including 308 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished 21 Pleurotus sp. fungi. The coefficient of Jaccard's genome profile similarity between the analyzed strains ranged from 0.0 (Pleurotus sp. I vs. P. sajor-caju 237 and P. eryngii 238) to 0.750 (P. ostreatus 246 vs. P. ostreatus 248), and the average was 0.378. The AFLP-based dendrogram generated by the UPGMA method grouped all the Pleurotus fungi studied into two major clusters and one independent lineage located on the outskirt of the tree occupied by naturally growing Pleurotus species strain I. The results of the present study suggest the possible applicability of the AFLP-PstI method in effective identification and molecular characterization of Pleurotus sp. strains.

Feedback mode SECM study of laccase and bilirubin oxidase immobilised in a sol-gel processed silicate film.

Thin silicate films with immobilised enzymes catalysing dioxygen reduction, i.e. laccase and bilirubin oxidase (BOD), were deposited on glass and poly(methyl 2-methylpropenoate) (Plexiglas) surfaces in a sol-gel process by sol drop evaporation. Scanning electrochemical microscopy (SECM) images and approach curves were recorded using hexacyanoferrate(iii) as mediator in the feedback mode. Confocal laser scanning microscopy (CLSM) images in the reflection mode showed larger film thickness close to the edge of the film and laccase aggregates within the film. SECM images obtained using different dioxygen concentrations showed that the film edge and laccase aggregates exhibit higher enzymatic activity towards dioxygen reduction. SECM current-distance curves enabled the determination of kinetic information at the particular regions of the samples after numerical fitting of model parameters. The heterogeneous first order rate constant at the film border was estimated to be ca. 19 times higher than the value obtained when approaching to the centre of the film. The reason of higher laccase surface concentration at the film edge is carefully discussed. For comparison of laccase and BOD activities, silicate spots of 50 microm diameter were deposited on a single Plexiglas sample and examined using SECM. BOD exhibits much higher activity especially at neutral pH.

Purification of extracellular laccase from Cerrena unicolor.

Cerrena unicolor was found to produce large amounts of extracellular laccase when grown aerobically on the optimized Lindenberg and Holm medium in fermenter culture with an automatic pH control. The laccase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, affinity chromatography, and chromatofocusing. The enzymes isoforms were recovered with a 65- to 92-fold increase in specific activity and a yield for Ia1 = 6.7%; Ia2 = 27.5%; Ib = 9.7%; and IIa1 = 21%. The molecular mass of the purified enzymes proved to be 45, 47, 54, and 62 kD, respectively, as determined by size-exclusion high-performance liquid chromatography (HPLC). The isoelectric points were in the range of 4.7 to 4.2, and the carbohydrate content in the purified enzymes was between 1.6 and 3.5%.

NAD(P)-dependent glucose dehydrogenase: Applications for biosensors, bioelectrodes, and biofuel cells.

This review discusses the physical and chemical properties of nicotinamide redox cofactor dependent glucose dehydrogenase (NAD(P) dependent GDH) and its extensive application in biosensors and bio-fuel cells. GDHs from different organisms show diverse biochemical properties (e.g., activity and stability) and preferences towards cofactors, such as nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). The (NAD(P)+) play important roles in biological electron transfer, however, there are some difficulties related to their application in devices that originate from their chemical properties and labile binding to the GDH enzyme. This review discusses the electrode modifications aimed at immobilising NAD+ or NADP+ cofactors and GDH at electrodes. Binding of the enzyme was achieved by appropriate protein engineering techniques, including polymerisation, hydrophobisation or hydrophilisation processes. Various enzyme-modified electrodes applied in biosensors, enzymatic fuel cells, and biobatteries are compared. Importantly, GDH can operate alone or as part of an enzymatic cascade, which often improves the functional parameters of the biofuel cell or simply allows use of cheaper fuels. Overall, this review explores how NAD(P)-dependent GDH has recently demonstrated high potential for use in various systems to generate electricity from biological sources for applications in implantable biomedical devices, wireless sensors, and portable electronic devices.

Thermoresponsive poly(N-isopropylacrylamide) gel for immobilization of laccase on indium tin oxide electrodes.

We report on the properties of hydrogel matrix for the immobilization of laccase on conductive supports. The poly(N-isopropylacrylamide) gel is attached firmly to the indium-tin oxide (ITO) electrode, following its silanization with dimethylethoxyvinylsilane. The enzyme entrapped in the gel structure remained active longer than in the solution, and its redox and catalytic properties could be investigated by voltammetric methods. The reduction signals of the active sites, T1 and T2, of the Cerrena unicolor laccase were determined to be 0.79 and 0.38 V, respectively. The laccase catalytic activity toward oxygen in poly(N-isopropylacrylamide) was found to depend strongly on temperature. Reversible swelling/shrinking of the matrix was studied at 30 and 35 degrees C. Shrinking of the gel at higher temperature considerably decreased the efficiency of the catalytic reaction, however, interestingly, did not lead to irreversible changes in the enzyme structure. At temperatures below that corresponding to volume phase transition, the catalytic properties of the film were fully restored. High catalytic efficiency of the gel immobilized enzyme made it possible to employ the gel covered electrode for monitoring oxygen in solutions.

Adsorption of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) on multiwalled carbon nanotubes-silicate film: application to bioelectrocatalytic dioxygen reduction.

Multiwalled carbon nanotubes were entrapped in sol-gel processed hydrophilic silicate thin film on tin-doped indium oxide support. Microscopic images show that the nanotubes form large agglomerates of largely separated nanotubes covered by silicate film. The measurements of capacitive current prove that approximately 10% of them remain electrochemically active. The surface confined cyclic voltammetry indicate adsorption of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) on this material. The oxidation charge estimated after the adsorption saturated shows that this compound is adsorbed on almost all the surface of the immobilised carbon nanotubes. After further modification of the electrode with extracellular laccase from Cerrena unicolor electrocatalytic dioxygen reduction is observed. The immobilised enzyme exhibits catalytic action whereas 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) adsorbed on carbon nanotubes serves as electron mediator between protein and electrode. Bioelectrocatalysis is also observed in the absence of adsorbed mediator but the efficiency of the process is approximately one order of magnitude smaller.

Analysis of the amino acid sequences of 18S RNA strains of Babesia canis isolated from dogs, including the analysis of serum proteome of protozoa infected dogs

The aim of this paper was to analyse the amino acid sequences of the 18S rRNA gene of Babesia canis strains and the proteomic analysis of the serum of dogs infected with three various genotypes: 18S rRNA B. canis. Material for the research was DNA B. canis obtained from dogs with babesiosis. In total, 60 DNA tested samples were divided into three groups (20 samples each). The groups were formed by DNA samples of the sequences marked as 18S RNA-A (group 1), 18S RNA-B (group 2), and 18S RNA-C (group 3). The basis for the classification of protozoa to a specific group was the location of relevant nucleotides (GA, AG, or TT) in position 150-151 of the tested nucleotide sequence 18S rRNA. Nucleotide sequences were transcribed into amino acid sequences and then analysed using DNASTAR software. From all 60 infected and ten healthy dogs (control group), the serum was taken to make proteomic tests using MALDI-TOF mass spectrometer. It was demonstrated that the mutations found in position 150 and 151 of the nucleotide sequence, result in a change of amino acid sequences. Moreover, it was also demonstrated that the disease course in dogs infected with different strains of protozoa is different. Each of the analysed strains of protozoa induced in the serum of infected animals the appearance of a protein fraction of mass 51 kDa, which may then be treated as a nonspecific disease marker used for the diagnosis of this disease but not to differentiate the protozoa strains.

ABTS-modified multiwalled carbon nanotubes as an effective mediating system for bioelectrocatalytic reduction of oxygen.

The ability of such a common redox mediator as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to undergo sorption on carbon surfaces is explored here to convert multiwalled carbon nanotubes (CNTs) into a stable colloidal solution of ABTS-modified carbon nanostructures, the diameters of which are approximately 10 nm (as determined by transmission electron microscopy). Subsequently, inks composed of fungal laccase (Cerrena unicolor) mixed with the dispersion of ABTS-modified CNTs and stabilized with Nafion, were deposited on glassy carbon and successfully employed to the reduction of oxygen in McIlvain buffer at pH 5.2. For comparison, the systems utilizing only ABTS-free CNTs and laccase as well as ABTS-modified CNTs did not show appreciable activity toward the oxygen reduction. The three-dimensionally distributed ABTS-modified CNTs are expected to improve the film's overall conductivity and to facilitate electrical connection between the electrode and the enzyme. The network film of ABTS-modified CNTs is rigid, and it is characterized by charge propagation capabilities comparable to the conventional redox polymers. The whole concept of utilization of CNTs modified with ultrathin films of redox mediators in the preparation of efficient bioelectrocatalytic films seems to be of general importance to electroanalytical chemistry and to the development of biosensors.

Scanning electrochemical microscopy study of laccase within a sol-gel processed silicate film.

The enzyme p-diphenol:dioxygen oxidoreductase (laccase, EC 1.10.3.2) was isolated from Cerrena unicolor fungus and embedded in a sol-gel film obtained by acidic condensation of TMOS. The gel was cast to thin films on glass. The laccase-containing silicate films were inspected by confocal laser scanning microscopy (CLSM), scanning force microscopy (SFM) and scanning electrochemical microscopy (SECM). CLSM images in the reflection mode showed aggregates within the silicate films. SECM images in the substrate-generation/tip-collection mode using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as electron donor for laccase showed that the position of aggregates coincides with increased enzymatic activity within the silicate film. The flux from individual aggregates was detected. SECM images in the redox competition mode confirmed the assignment and could exclude that topographic features observed by CLSM and SFM could be the reason for the image contrast. SFM images showed that the aggregates partially dissolve during prolonged exposure to aqueous buffer. The experimental setup allowed following one individual aggregate over time with all three microscopic techniques which enabled the collection of complementing information on morphology and catalytic activity as well as their development over time.

Laccase Enzyme Polymerization by Soft Plasma Jet for Durable Bioactive Coatings.

Conventional pin-to-point continuous wave Helium Corona plasma discharge was successfully used in Soft Plasma Polymerization (SPP) processes to immobilize into water and onto glass polymerized bioactive Cerrena unicolor laccase coatings. The coatings were tested for bioactivity and durability under water wash. The coatings showed up to 59% bioactivity relative to the native laccase in water deposition, undoubtedly due to damage to and fragmentation of monomer molecules by the active, energetic species in the plasma. However, plasma deposited laccase coatings on glass delivered 7 times the laccase activity of the same non-plasma deposition process in the coating after water wash. This latter result would seem to be due to the ability of the plasma to both crosslink monomer and more strongly bond it to the glass surface by a combination of surface cleaning and the creation of active, high energy sites in both glass and laccase molecules. FTIR analysis indicated that the core copper containing moieties at the centre of the molecule largely remain undamaged by this plasma type so that bonding and cross-linking reactions are likely to mainly involve species around the outer perimeter of the molecule. The chemical composition and structure of laccase biocoatings deposited by Corona SPP are described. The combination of the coating performance parameter values for retained activity and durability under water wash indicates that a relatively simple Corona plasma process for deposition of biocoatings, which directly polymerizes the monomer with no added matrix or encapsulant material, may offer enhanced solutions for biocatalyst, sensor or lab-on-a-chip applications.

Fungal polysaccharides as a water-adsorbing material in esters production with the use of lipase from Rhizomucor variabilis.

The extracellular crude Rhizomucor variabilis lipase was used for synthesis of flavor ester butyl caprylate and 1-butyl oleate often used as a diesel additive, a polyvinyl chloride plasticizer, a water-resisting agent, and an additive to hydraulic fluids. The influence of various reaction parameters such as the molar ratio, time, enzyme and substrate concentration, and effect of various fungal polysaccharides was estimated. The rate of catalyzed synthesis of esters largely depends on the solvent medium, and the maximum activity was found when n-hexane was used as a solvent. The maximum conversion yield of 58.2% and 59.3% was obtained for butyl caprylate and butyl oleate, respectively, under the following conditions: amount of free lipase 500 U; caprylic acid:butanol molar ratio 1:1; oleic acid:butanol molar ratio 2:1. The addition of naturally obtained fungal polysaccharides significantly enhanced the ester synthesis. The highest conversion rate of 95.2% was observed for butyl caprylate in the presence of AbEPS after 24 h with 500 U of free R. variabilis lipase. In the case of butyl oleate synthesis in the presence of LsPS, a maximum conversion yield of 91.2% was observed after the 24-h reaction.

Voltammetric determination of catalytic reaction parameters of laccase based on electrooxidation of hydroquinone and ABTS.

A convenient method for the measurement of the catalytic activity of laccase is proposed based on the voltammetric determination of catalytic reaction substrates: 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) and 1,4-benzenediol (hydroquinone). The measurement performed using microelectrodes working under spherical diffusion conditions is both accurate and simple, and allows to monitor parallely the consumption of substrate and formation of product of the catalytic reaction. The method proposed in this paper was compared with the two generally employed procedures based on oxygen measurement by Clark electrode and on spectrophotometry. The procedure described in the present paper was found to be simpler and more reproducible results were obtained than using Clark electrode. Compared to spectrophotometry a larger range of catalytic reaction substrates can be studied including colorless compounds.

Microbially mediated cadmium sorption/desorption processes in soil amended with sewage sludge.

A multi-compartment system was used to study the importance of microorganisms for Cd desorption from soil amended with sewage sludge and simultaneous resorption of the mobilized metal by soil constituents. Using this system made it possible to study the participation of microorganisms (Arthrobacter, Trichoderma), montmorillonite, humic acids, and iron oxides in resorption of the released Cd. A filter-sterilized water extract of root-free soil of pH 6.7 (RF) or RF supplemented with glucose (RFG) were used to mobilize Cd from soil at 14 degrees C in 48 h. Cadmium found in those extracts after 48-h incubation was recognized as bioavailable. Changes in pH values and enrichment of soil extracts with organic acids and siderophores resulted from microbial growth. RFG with lower pH and a higher content of ligands mobilized, on average, 40% of Cd introduced with sewage sludge amended soil, whereas RF mobilized only 20% of it. Sequential extractions of Cd at time 0 and Cd remaining in soil showed that RFG had mobilized Cd mostly from the fraction bound with Fe and Mn oxides. Microbial biomass accounted for only up to 3.4% (w/w) of the soil constituents used in the experiments but resorbed 25% of mobilized Cd. The chemical composition of mobilizing soil extracts and the solid-to-mobilizing-extracts volume ratio had a significant effect on the amount of bioavailable Cd. The results of the study suggest that microbial metabolites were involved in Cd mobilization, while the biomass of microorganisms was involved in Cd resorption as a biosorbent.

Purification and characterization of beta-mannosidases from white rot fungus Phlebia radiata.

A beta-mannosidase was purified from Phlebia radiata grown in a medium containing wheat bran or galactomannan as a carbon source. Maximal activity was observed at pH 5.5 and at 50 degrees C. Highly purified isoforms of beta-mannosidase (GM-1, GM-2) isolated from media containing galactomanan and (OT-1) media with wheat bran were obtained by means of column chromatography on Q-Sepharose, SP-Sepharose and chromatofocusing on Polybuffer Exchanger PBE-94.

Effects of culture conditions on production of extracellular laccase by Rhizoctonia praticola.

It was found that the soil-dwelling fungus Rhizoctonia praticola 93A was capable to produce laccase in submerged cultures. Effects of culture conditions on the enzyme biosynthesis in shaken flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably intensified by the addition of Cu2+ to a carbon-limited and nitrogen-sufficient culture medium (C/N = 0.98). When an optimized medium containing glucose (2 g/l) and L-asparagine (1.5 g/l) was used and enzyme synthesis was stimulated by addition of 5 microM Cu2+ before inoculation, maximal laccase activities obtained in a batch cultivation were, approximately, 1000 nkat/l. Under these conditions, addition to the medium of the aromatic inducer 2,5-xylidine (1 mM) led to a 10-fold increase in laccase activity. Laccase productivity in shaken flask cultures was also enhanced (to more than 4000 nkat/l on day 3) by using a medium with the initial pH of 7.5. Such a high value of the optimal medium pH for laccase production by R. praticola is exceptional among the ligninolytic fungi. In fermenter fungal cultures supplemented with cupric ions, the highest laccase activity (about 4000 nkat/l after 3 days' cultivation) was reached after 24-h incubation using a bioreactor with the aeration rate of 21/min, the agitation speed of 200 rpm, and a constant medium pH of 8.0.