Comparative characterization of some fungal laccases

Laccase producing fungi were isolated from environmental samples. They were identified on the basis of ITS (Internal Transcribed Spacer) sequence analysis. The laccase production of the isolates were investigated and compared with those of other strains deriving from the Szeged Microbiological Collection and from mushroom producer’s spawn. The enzyme production were examined in various (e.g. basic, mineral and inducer containing) liquid media. The pH optimum determinations for laccase activities were carried out in cell free ferment broths, at pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 and 8, applying 25 mM succinate buffer. The investigations were based on ABTS [2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)] substrate measurement method. The results showed that under these conditions, among the ascomycetes strains investigated the best laccase producer was a Rhizoctonia solani isolate (the SzMC 6252J strain). Among the basidiomycetes tested, the best laccase producer was a Ganoderma isolate (HM3)

Isolation of bacteria antagonistic to pseudomonas tolaasii, the causative agent of brown blotch disease

The aim of this study was to isolate mushroom pathogenic Pseudomonas tolaasii antagonistic bacteria, and to investigate the inhibiting processes. P. tolaasii is a well known mushroom pathogen bacterium, which attacks oyster and button mushroom. The caused symptoms could be present as brown blotches on the caps, or browning and yellowing of the whole sporocarps. The toxin molecules, produced by the bacterium, disrupt the cellular membrane of the fungal cells via pore formation. The exclusion of the whole genus from the growing processes is difficult, because some species play key role in the healthy cap formation. Potential way of suppression is the artificially generated competition for the nutrient sources. In this case the production of enzymes and siderophores is important. Another opportunity to lowering the losses during the infection: the neutralization of the produced tolaasin toxin. This could prevent the browning and yellowing of the caps caused by P. tolaasii. WLIP (White Line Inducing Particle) produced by P. ’reactans’ is a well known tolaasin blocking molecule; it could form insoluble precipitate with the toxin. Some members of the Acinetobacter, Bacillus, Pedobacter and Sphingobacterium genera are well known toxin neutralizing species. The background of this phenomenon is still unknown. Examination of produced inhibitors depending on the molecule type (protein-analysis, thin layer and high pressure chromatography) could help us in the understanding of these processes. In this study, we isolated different bacteria having strong competition abilities. The best non-mushroom pathogenic strains were identified by partial 16S rDNA sequence analysis

Application of laccases, produced by ganoderma species, for the detoxification of some aniline and phenol derivatives

The objective of this work was to collect isolates of the white rot fungus Ganoderma from decaying woods and investigate their laccase producing and detoxification abilities against aniline and phenol compounds. Sporocarps were collected and the tissue culturing technique was used for fungal isolation. In these experiments specific medium, containing benomyl, dichloran and guaiacol was used. Laccase producing of the isolates were tested in liquid media containing different inducers. Crude enzyme extracts were prepared and characterized. The best laccase-producing strains were identified with partial ITS sequence analysis. Their secreted laccases were investigated with SDS-PAGE and the molecular weights of these enzymes were estimated. Degradation of 7 different aniline and phenol derivates (2,4-dichlorophenol, 2-methyl-4- chlorophenol, 3-chloroaniline, 4-chloroaniline, 2,6-dimethylaniline, 3,4-dichloroaniline and 3-chloro-4- methylaniline) were investigated, observing high degradation rates in most cases

Comparative characterization of some fungal laccases

Laccase producing fungi were isolated from environmental samples. They were identified on the basis of ITS (Internal Transcribed Spacer) sequence analysis. The laccase production of the isolates were investigated and compared with those of other strains deriving from the Szeged Microbiological Collection and from mushroom producer’s spawn. The enzyme production were examined in various (e.g. basic, mineral and inducer containing) liquid media. The pH optimum determinations for laccase activities were carried out in cell free ferment broths, at pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 and 8, applying 25 mM succinate buffer. The investigations were based on ABTS [2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)] substrate measurement method. The results showed that under these conditions, among the ascomycetes strains investigated the best laccase producer was a Rhizoctonia solani isolate (the SzMC 6252J strain). Among the basidiomycetes tested, the best laccase producer was a Ganoderma isolate (HM3)

Trichoderma isolates from vegetable rhizosphere samples : potential for the biological control of botrytis species

Members of the genus Trichoderma are wide-spread saprophytic fungi living in the soil and the rhizosphere of different plants. Due to their enzyme production abilities they are able to use complex biomolecules as carbon and nitrogen source. Many strains with very good biocontrol abilities against plant pathogenic fungi could be isolated from soils of agricultural areas. In this study we isolated Trichoderma strains from the rhizosphere of vegetables (pepper and lettuce) derived from gardens of different Hungarian cities (Szolnok, Kalocsa, Újszilvás, Kelebia). The isolates were identified by the sequence analysis of the ITS (internal transcribed spacer) region. The strains belonging to species that are not pathogenic to humans or cultivated mushrooms and possessing promising bicontrol potential were further investigated. The antagonistic abilities of the strains were studied against Botrytis species (B. cinerea, B. pseudocinerea) in in vitro confrontation tests and the extracellular enzyme systems of the strains were investigated in different liquid media. The knowledge of the correlation between the in vitro antagonistic abilities and enzyme production may contribute to our understanding of the biocontrol mechanism

Application of laccases, produced by ganoderma species, for the detoxification of some aniline and phenol derivatives

The objective of this work was to collect isolates of the white rot fungus Ganoderma from decaying woods and investigate their laccase producing and detoxification abilities against aniline and phenol compounds. Sporocarps were collected and the tissue culturing technique was used for fungal isolation. In these experiments specific medium, containing benomyl, dichloran and guaiacol was used. Laccase producing of the isolates were tested in liquid media containing different inducers. Crude enzyme extracts were prepared and characterized. The best laccase-producing strains were identified with partial ITS sequence analysis. Their secreted laccases were investigated with SDS-PAGE and the molecular weights of these enzymes were estimated. Degradation of 7 different aniline and phenol derivates (2,4-dichlorophenol, 2-methyl-4- chlorophenol, 3-chloroaniline, 4-chloroaniline, 2,6-dimethylaniline, 3,4-dichloroaniline and 3-chloro-4- methylaniline) were investigated, observing high degradation rates in most cases

Screening of trichoderma strains isolated from rhizosphere samples for laccase production

In this study we screened formerly isolated Trichoderma strains for laccase production on solid media supplemented with two different substrates, ABTS [2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)] or guaiacol. We detected outstandingly strong colour changes in the case of three Trichoderma strains in this experiment. The strains were identified based on internal transcribed spacer (ITS) sequence analysis as T. asperellum (SZMC 20786 and SZMC 20866) and T. atroviride (SZMC 20780). We also investigated the production of laccase enzymes in the case of these Trichoderma strains in two types of liquid media. The pH dependence of the secreted laccases was determined in cell free ferment broths at pH 3.5, 4, 5, 5.5, 6 and 6.5 adjusted with 25 mM succinate buffer. Laccase activities from liquid cultures were measured with ABTS as substrate. The results showed that the best laccase producer among the investigated Trichoderma strains was T. atroviride SZMC 20780 under these conditions. This strain shows the highest laccase enzyme activity on the second day of incubation in a rotary shaker at 25 °C