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.

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.

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.

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.

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.

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.

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.

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.

The phenotypic and genomic diversity of Aspergillus strains producing glucose dehydrogenase.

Twelve Aspergillus sp. strains producing glucose dehydrogenase were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was investigated. Moreover, partial gdh gene sequences were determined and aligned. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of twelve Aspergillus isolates. Using one PstI restriction endonuclease and five selective primers in an AFLP assay, 556 DNA fragments were generated, including 532 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished twelve Aspergilli fungi. The AFLP-based dendrogram generated by the UPGMA method grouped all the Aspergillus fungi studied into two major clusters. All the Aspergillus 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 analyzed strains up to three folds. All of the studied strains mainly decomposed carbohydrates.

Determination of biodiversity of Coprinus comatus using genotyping and metabolic profiling tools.

Coprinus comatus strains (CCMs) originating from Poland were identified using ITS region sequencing. Based on the sequences obtained, the genetic relationship between the CCM strains was determined and a clear separation of all strains into two main clusters was obtained. The Coprinus strains were also genetically characterized for the first time by the AFLP technique. The analysis showed that the CCMs separated into four main clusters and a high complication of a UPGMA-based dendrogram was achieved. C. comatus strains included in the analysis displayed an AFLP profile similarity level in the range from 44 to 66%. The highest similarity coefficient, 0.490, was found between CCM12 and CCM13, and the lowest (0.202) between the CCM2 and CCM5 isolates. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mycelial growth. The analysis allowed comparison of the functional diversity of the CCM strains and revealed a broad variability within the analyzed Coprinus species based on substrate utilization profiles. Significant differences (2-48) have been shown in the substrate richness values. The Biolog experiments proved to be a good profiling technology for studying the diversity in shaggy manes due to metabolic differences and demonstrated that all the strains might be considered individually. It is evident that the strain metabolic grouping does not correlate with the grouping based on the ITS sequences and AFLP profiles, however, some similarities may be observed.

Characterization of Cellobiose Dehydrogenase from a Biotechnologically Important Cerrena unicolor Strain.

Cellobiose dehydrogenase (CDH), a secreted flavocytochrome produced by a number of wood-degrading fungi, was detected in the culture supernatant of a biotechnologically important strain of Cerrena unicolor grown in a modified cellulose-based liquid medium. The enzyme was purified as two active fractions: CuCDH-FAD (flavin domain) (1.51-fold) with recovery of 8.35 % and CuCDH (flavo-heme enzyme) (21.21-fold) with recovery of 73.41 %. As CDH from other wood-rotting fungi, the intact form of cellobiose dehydrogenase of C. unicolor is a monomeric protein containing one flavin and one heme b with molecular mass 97 kDa and pI = 4.55. The enzyme is glycosylated (8.2 %) mainly with mannose and glucosamine residues. Moreover, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from the fungus C. unicolor were isolated, cloned, and characterized. The 2316-bp full-length cDNA of cdh1 encoded a mature CDH protein containing 771 amino acids preceded by a signal peptide consisting of 18 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.

Genetic and metabolic intraspecific biodiversity of Ganoderma lucidum.

Fourteen Ganoderma lucidum strains from different geographic regions were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship between the analyzed strains was determined. All G. lucidum strains were also genetically characterized using the AFLP technique. G. lucidum strains included in the analysis displayed an AFLP profile similarity level in the range from 9.6 to 33.9%. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mitochondrial activity. The analysis allowed comparison of functional diversity of the fungal strains. The substrate utilization profiles for the isolates tested revealed a broad variability within the analyzed G. lucidum species and proved to be a good profiling technology for studying the diversity in fungi. Significant differences have been demonstrated in substrate richness values. Interestingly, the analysis of growth and biomass production also differentiated the strains based on the growth rate on the agar and sawdust substrate. In general, the mycelial growth on the sawdust substrate was more balanced and the fastest fungal growth was observed for GRE3 and FCL192.

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.

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.

Purification and properties of an α-(1 → 3)-glucanase (EC 3.2.1.84) from Trichoderma harzianum and its use for reduction of artificial dental plaque accumulation.

Extracellular α-(1 → 3)-glucanase (mutanase, EC 3.2.1.84) produced by Trichoderma harzianum CCM F-340 was purified to homogeneity by ultrafiltration followed by ion exchange and hydrophobic interaction chromatography, and final chromatofocusing. The enzyme was recovered with an 18.4-fold increase in specific activity and a yield of 4.3%. Some properties of the α-(1 → 3)-glucanase were investigated. The molecular mass of the enzyme is 67 kDa, as estimated by SDS/PAGE, its isoelectric point 7.1, and the carbohydrate content 3%. The pH and temperature optima are 5.5 and 45°C, respectively. The enzyme is stable over a pH range of 4.5-6.0 and up to 45°C for 1 h. The Km and Vmax under standard assay conditions are 0.73 mg/ml and 11.39 x 10(-2) µmol/min/mg protein, respectively. The enzyme activity is stimulated by addition of Mg(2+) and Na(+), and significantly inhibited by Hg(2+). The α-(1 → 3)-glucanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-(1 → 3)-glucans. The 20-residue N-terminal sequence of the enzyme is identical with those of other α-(1 → 3)-glucanases from the genus Trichoderma, and highly similar to those from other fungi. The purified α-(1 → 3)-glucanase was effective in preventing artificial dental plaque formation. The easy purification from fermentation broth and high stability, and the effective inhibition of oral biofilm accumulation make this α-(1 → 3)-glucanase highly useful for industrial and medical application.

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.

Hybrid biobattery based on arylated carbon nanotubes and laccase.

Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm(2) and the open circuit potential was 1.5 V.

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%.

Poly-o-aminophenol as a laccase mediator and influence of the enzyme on the polymer electrodeposition.

Applicability of poly-o-aminophenol (POAP) as a redox mediator for fungal laccase is investigated. Laccase has been entrapped by means of electrochemical polymerization. The obtained layers have been characterized by cyclic voltammetry, as well as by spectroscopic methods. The enzyme activity has been verified by the standard test using syringaldazine. Laccase immobilized in the POAP matrix catalyses oxygen reduction without any additional mediators. POAP is able to mediate the electron transfer between the enzyme active site and the electrode surface similarly to poly-o-phenylenediamine which has been studied previously, but its redox potential is shifted significantly towards positive values. The role of laccase in electrodeposition of POAP has been studied. It has been found that the presence of the enzyme influences the structure of electrodeposited films. Furthermore, laccase facilitates the electrodeposition. The monomer-o-aminophenol (OAP) belongs to typical laccase substrates. The polymer can be precipitated from the solution containing the monomer and laccase. The morphology of POAP formed by laccase differs from typical polymer samples synthesized chemically or electrochemically. It contains round microstructures composed of nano-needles. Laccase is therefore a promising polymerization initiator for synthesis of nanostructured conducting polymers.