Screening for ligninolytic enzymes in autochthonous fungal strains from Argentina isolated from different substrata

The production of different extracellular ligninolytic enzymes was studied in autochthonous fungal strains from Argentina isolated from litter derived from hydrocarbon-polluted sites and from basidiocarps frowing on wood in forests. The strains tested were cultivated in a carbon-limited medium with shaking. Laccase activity reached higher levels than aryl-alcohol oxidase and manganese-dependent peroxidase activities in liquid cultures from different fungi. No lignin peroxidase activity was found in any strain assayed. Some species are reported for the first time as producers of different ligninolytic enzymes.     

Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia

Summary This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer ‘ITS’ regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml^-1 within 15 days of incubation.     

α-mannosidase and mannanase of some wood-rotting fungi

Cultivation media from 11 wood-rotting fungi contained α-mannosidase and mannanase activity. α-Mannosidase was studied in more detail inPhellinus abietis and mannanase was studied more intimately in basidiomycetesPhellinus abietis, Trametes sanguinea andPholiota aurivella. Suitable cultivation conditions and optimum conditions for the production of α-mannosidase and mannanase were determined. Both enzymes are constitutive; mannanase is extracellular, α-mannosidase was found in both mycelium and cultivation medium.     

Screening of micro-organisms for decolorization of melanins produced by bluestain fungi

A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998     

Bioactivity of intra and extracellular substances from cyanobacteria and lactic acid bacteria on “wood blue stain” fungi

Cyanobacteria (photoautotrophic prokariota) have potential for the control of pathogenic bacteria and fungi. The effect of intra and extracellular products from cyanobacterial strains on the growth of fungi isolated from “wood blue stain,” was tested. Extracellular products were obtained by concentration and sterilization of the culture medium where cyanobacteria were grown. Cyanobacterial substances promoted or inhibited fungal growth according to the fungal and cyanobacterial strains tested. Extracellular products from Nostoc muscorum 79a and the methanolic extract from Microchaete tenera 84b biomass inhibited growth of Sphaeropsis sapinea 2157 (64.7 and 775.6%, respectively). Extracellular products of Nostoc piscinale 59 and biomass methanolic extract from N. muscorum 79a produced the highest growth promotion of Trichoderma boningii 452 (105.0%) and T. viride 993 (136.7%). Extracellular products of the heterotrophic lactic acid bacterium Streptococcus termophilus were also tested and strongly inhibited (64–92%) all the fungal strains. The tested fungi have different sensitivity to the bioactive substances present in the biomass and/or the culture medium of the studied cyanobacteria and lactic acid bacterium. N. muscorum 79a, M. tenera 84b, and S. termophilus have potential to control the wood blue stain fungi by a friendly environmental alternative.     

Biosorption of cadmium to mycelial pellets of wood-rotting fungi

Summary Fungal pellets of wood-rotting fungi (diameters ranged from 0.2 to 2 cm) were obtained by submerged cultivation on nutrient rich medium. Biosorption of cadmium (initial concentration, 10 mM) in batch system was followed by HPLC. Of 20 fungal strains tested, high abilities to accumulate cadmium were found in Fomitopsis pinicola (130.2 mg/g) and Trametes versicolor (109.5 mg/g). P. chrysosporium contained 84.5 mg Cd /g dry weight.     

Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five lignocellulose-degrading fungi by ferulic acid

At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurotus sajor-caju. At 0.5 g/l, howerver, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-caju) also secreted laccases. Laccase-secreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested.     

Intrinsic growth rate and cellobiohydrolase activity underlie the phylogenetic signal to fungal decomposer succession

During litter decay, different fungal decomposer genera reach their highest relative abundance at different times. We tested the long-standinghypothesis that this “peak decay stage” of fungi is related to the activity of their fungal extracellular enzymes that break down various plant biopolymers and related as well to the growth rate of fungi. Using 50 decomposer fungal species, spanning a range of peak decay stages, we measured (1) the activity of four polysaccharidases and two oxidases generated by each species, and (2) fungal species’ growth rates. We found that the activity of cellobiohydrolase and growth rate were negatively correlated with peak time point for filamentous fungi; fungi peaking early had greatest cellobiohydrolase activity and fastest growth. No relationships were found between peak decay stage and enzymes or growth for yeasts. These data suggest growth and resource use are important factors shaping succession during decay by the main fungal decomposers, but as-yetuninvestigated traits may explain the remainder of the variation in succession.     

Preparation and characterization of bioactive products obtained via the solubilization of brown coal by white rot fungi

We investigated the solubilizing activity of the Basidiomycete fungi Trametes hirsuta and Trametes maxima, with respect to brown coal (lignite) during liquid phase cultivation. We found that the degrading capacity of the fungi is determined by the activity of the ligninolytic enzymes Mn peroxidase and lignin peroxidase. We assessed the growth-stimulating activity of biopreparations (BPs), based on the culture liquids (CL) of the studied fungal strains, which were grown on a rich or minimal medium. We found that the obtained BPs inhibited the growth of wheat shoots and roots at the germination stage, but they either had no effect at later stages of plant growth or showed a mild stimulation. When basidiomycetes were cultivated in the presence of brown coal, the obtained BPs stimulated root growth at the germination stage, and did not influence plant growth (Trametes hirsuta) or stimulated it (Trametes maxima) at later stages. Further, we report a pronounced detoxifying ability of the BPs in respect to the atrazine herbicide. We suggest that this effect is caused by the laccases action, that are present in the studied BPs.     

Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.     

Kinetic differences of purified laccases from six Pleurotus ostreatus strains

AIMS: Enzyme kinetics of purified laccases from six different Pleurotus ostreatus strains were determined in the oxidation of syringaldazine, guaiacol and ABTS. METHODS AND RESULTS: Significant differences in the kinetic constants were found. Catalytic activity (kcat) ranged from 19 to 941 U mg(-1) for syringaldazine, from 18 to 1565 U mg(-1) for ABTS, and from 4 to 44 U mg(-1) for guaiacol. The apparent affinity constants (KM) also showed significant differences between the different strains, from 12 to 52 micromol l(-1) for syringaldazine, from 8 to 79 micromol l(-1) for ABTS, and from 0.46 to 6.61 mmol l(-1) for guaiacol. No differences were found either on the effect of increasing concentrations of organic solvent (acetonitrile) or on the activity pH profile. The temperature profile was the same for all the P. ostreatus strains, except for the IE8 strain, which seems to be more sensitive to temperature. The kinetic and stability data from the six P. ostreatus strains were also compared with those obtained from other white rot fungi, Coriolopsis gallica and Trametes versicolor, showing clear differences. CONCLUSION: The different P. ostreatus isolates showed different kinetic constants. SIGNIFICANCE AND IMPACT OF THE STUDY: The different enzymatic properties of laccases from various P. ostreatus strains should be considered for a potential industrial or environmental application.     

Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus

Three new highly oxygenated and unsaturated metabolites named interfungins A (1), B (2), and C (3), which provide a diversity of hispidin class compounds in the fungi Inonotus and Phellinus, were isolated from the methanolic extract of the fruiting body of the fungus Inonotus xeranticus (Hymenochaetaceae). Their structures were established by spectroscopic methods. The existence of these functionalized metabolites implies that inoscavin A, davallialactone, and phelligridin F, which were previously isolated from the fungi Inonotus and Phellinus spp., are derived from 1. Compound 1 is derived from the condensation of hispidin and hispolon. Inoscavins B and C previously isolated from the fungus I. xeranticus are most probably derived from 2 which stemmed from the oxidative coupling of 3,4-dihydroxybenzalacetone and hispidin. This class of compounds exhibited significant free radical scavenging activity against the superoxide radical cation, ABTS radical anion, and DPPH radical.     

White-rot fungi capable of decolourising textile dyes under alkaline conditions

Twelve white-rot fungal strains belonging to seven different species were screened on plates under alkaline condition to study the decolourisation of the textile dyes Reactive Black 5 and Poly R-478. Three strains of Trametes versicolor (Micoteca da Universidade do Minho (MUM) 94.04, 04.100 and 04.101) and one strain of Phanerochaete chrysosporium (MUM 94.15) showed better decolourisation results. These four strains were used for decolourisation studies in liquid culture medium. All four selected strains presented more efficient decolourisation rates on Reactive Black 5 than on Poly R-478. For both dyes on solid and liquid culture media, the decolourisation capability exhibited by these strains depended on dye concentration and pH values of the media. Finally, the decolourisation of Reactive Black 5 by T. versicolor strains MUM 94.04 and 04.100 reached 100 %. In addition, the highest white-rot fungi ligninolytic enzyme activities were found for these two strains.     

Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the nonpregrown cultures. In both culture conditions, P. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/l BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost the same as that in the suspended culture. The estrogenic activity of 100 mg/l DMP decreased during biodegradation by P. ostreatus.     

Dibenzyl sulfide metabolism by white rot fungi

Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization. Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied. Trametes trogii UAMH 8156, Trametes hirsuta UAMH 8165, Phanerochaete chrysosporium ATCC 24725, Trametes versicolor IFO 30340 (formerly Coriolus sp.), and Tyromyces palustris IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone. The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation. Laccase activity (0.15 U/ml) was detected in the Trametes cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence. These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.     

Isolation and evaluation of terrestrial fungi with algicidal ability from Zijin Mountain,Nanjing, China

Approximately 60 fungal isolates from Zijin Mountain (Nanjing, China) were screened to determine their algicidal ability. The results show that 8 fungi belonging to Ascomycota and 5 belonging to Basidiomycota have algicidal ability. Of these fungi, Irpex lacteus T2b, Trametes hirsuta T24, Trametes versicolor F21a, and Bjerkandera adusta T1 showed strong algicidal ability. The order of fungal chlorophyll-a removal efficiency was as follows: T. versicolor F21a > I. lacteus T2b > B. adusta T1 > T. hirsuta T24. In particular, T. versicolor F21a completely removed algal cells within 30 h, showing the strongest algicidal ability. The results also show that all 4 fungal species degraded algal cells through direct attack. In addition, most of the tested fungi from the order Polyporales of Basidiomycota exhibited strong algicidal activity, suggesting that most fungi that belong to this order have algicidal ability. The findings of this work could direct the search for terrestrial fungi for bloom control.     

Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths.     

Role of autochthonous filamentous fungi in bioremediation of a soil historically contaminated with aromatic hydrocarbons

Nine fungal strains isolated from an aged and heavily contaminated soil were identified and screened to assess their degradative potential. Among them, Allescheriella sp. strain DABAC 1, Stachybotrys sp. strain DABAC 3, and Phlebia sp. strain DABAC 9 were selected for remediation trials on the basis of Poly R-478 decolorization associated with lignin-modifying enzyme (LME) production. These autochthonous fungi were tested for the abilities to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in the same contaminated soil. After 30 days, fungal colonization was clearly visible and was confirmed by ergosterol determination. In spite of subalkaline pH conditions and the presence of heavy metals, the autochthonous fungi produced laccase and Mn and lignin peroxidases. No LME activities were detected in control microcosms. All of the isolates led to a marked removal of naphthalene, dichloroaniline isomers, o-hydroxybiphenyl, and 1,1'-binaphthalene. Stachybotrys sp. strain DABAC 3 was the most effective isolate due to its ability to partially deplete the predominant contaminants 9,10-anthracenedione and 7H-benz[DE]anthracen-7-one. A release of chloride ions was observed in soil treated with either Allescheriella sp. strain DABAC 1 or Stachybotrys sp. strain DABAC 3, suggesting the occurrence of oxidative dehalogenation. The autochthonous fungi led to a significant decrease in soil toxicity, as assessed by both the Lepidium sativum L. germination test and the Collembola mortality test.     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.