Alexey Leontievsky

Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor

The toxicity of thirteen isomers of mono-, di-, tri- and pentachlorophenols was tested in potato-dextrose agar cultures of the white rot fungi Panus tigrinus and Coriolus versicolor. 2,4,6-Trichlorophenol (2,4,6-TCP) was chosen for further study of its toxicity and transformation in liquid cultures of these fungi. Two schemes of 2,4,6-TCP addition were tested to minimize its toxic effect to fungal cultures: stepwise addition from the moment of inoculation and single addition after five days of growth. In both cases the ligninolytic enzyme systems of both fungi were found to be responsible for 2,4,6-TCP transformation. 2,6-Dichloro-1,4-hydroquinol and 2,6-dichloro-1,4-benzoquinone were found as products of primary oxidation of 2,4,6-TCP by intact fungal cultures and purified ligninolytic enzymes, Mn-peroxidases and laccases of both fungi. However, primary attack of 2,4,6-TCP in P. tigrinus culture was conducted mainly by Mn-peroxidase, while in C. versicolor it was catalyzed predominantly by laccase, suggesting a different mode of regulation of these enzymes in the two fungi.

Transformation of high concentrations of chlorophenols by the white-rot basidiomycete Trametes versicolor immobilized on nylon mesh

Free-cell cultures of Trametes versicolor were compared with cultures immobilized on nylon mesh in a 2-litre bioreactor for transformation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP), added at intervals to the liquid culture medium over a period of 816 hrs. Increasing amounts of PCP from 200 ppm to 2000 ppm added batchwise to cultures permitted acclimatization of the fungus to these toxic pollutants. A total addition of 2000 ppm of 2,4-DCP and 3400 ppm PCP were removed from the immobilized cultures with 85% of 2,4-DCP and 70% of PCP transformed by enzymes (laccase and Mn-peroxidase), 5% 2,4-DCP and 28% PCP adsorbed by the biomass and 10% 2,4-DCP and 2% PCP retained in the medium at the termination of the fermentation after 1020 hrs. In contrast free-cell cultures in the same medium with the same addition regime of PCP and 2,4-DCP, transformed 20% 2,4-DCP and 12% PCP by enzyme action, adsorbed 58% 2,4-DCP and 80% PCP by the biomass, and retained 22% 2,4-DCP and 8% PCP in the medium. The use of nylon mesh as an immobilization matrix for removal of PCP and 2,4-DCP facilitates more efficient removal of chlorophenols and can be adapted to scale-up for application of large volumes of chlorophenol-containing aqueous effluents.

Microbial degradation of glyphosate herbicides (Review)

This review analyzes the issues associated with biodegradation of glyphosate (N-(phosphonomethyl)glycine), one of the most widespread herbicides. Glyphosate can accumulate in natural environments and can be toxic not only for plants but also for animals and bacteria. Microbial transformation and mineralization of glyphosate, as the only means of its rapid degradation, are discussed in detail. The different pathways of glyphosate catabolism employed by the known destructing bacteria representing different taxonomic groups are described. The potential existence of alternative glyphosate degradation pathways, apart from those mediated by C-P lyase and glyphosate oxidoreductase, is considered. Since the problem of purifying glyphosate-contaminated soils and water bodies is a topical issue, the possibilities of applying glyphosate-degrading bacteria for their bioremediation are discussed.

Two laccase isoforms of the basidiomycete Cerrena unicolor VKMF-3196. Induction, isolation and properties.

The laccase induction in submerged culture of basidiomycete Cerrena unicolor VKM F‐3196 was investigated. Cu2+ at concentration 0.1 mM was an optimum inducer of C. unicolor laccase. Two isoforms of laccase, namely LacC1 and LacC2, were isolated and characterized. The isoforms were shown to have different physical‐chemical and catalytic properties. On the basis of the MALDI TOF MS analysis of tryptic cleavage products of both the proteins and N‐terminal amino‐acid sequences analysis two isoforms of laccase (LacC1 and LacC2) were classified as products of two different genes. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Effect of ethanol on enzymatic activity of fungal laccases.

Blue laccase from Coriolus versicolor and blue and yellow laccases from Panus tigrinus were isolated, purified and studied in acetate buffer solutions, with and without addition of various amounts of ethanol, using syringaldazine and 2,6-dimethoxyphenol as substrates. Effect of ethanol on blue laccases could be successfully described using the mixed inhibition model, over the range of 0–2.5 M ethanol concentrations. Yellow laccase from P. tigrinus behaves differently, which may be explained by the presence of some extra molecules in its structure, which possibly stabilize the enzyme and might be exchanged in ethanol solutions.

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C–P lyase incapable of degrading GP (C–P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C–P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C–P lyase II. O. anthropi GPK 3 also degraded MP via C–P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.

Production of ligninolytic enzymes of the white rot fungus Panus tigrinus

Abstract We developed optimal conditions for submerged cultivation of the white rot fungus Panus tigrinus , which increase the yield of extracellular ligninolytic enzymes. Adding (besides mineral salts) Tween-80, an increased concentration of manganese, and 3-methylbenzyl alcohol as inducer into the medium and using an optimal source of carbon, optimally immobilized mycelium and an optimal temperature shift, we succeeded in obtaining 99 nkat ml −1 Mn-peroxidase by NADH oxidation and 18 nkat ml −1 oxidase by syringaldazine in batch cultivation in flasks and a 3-1 laboratory bioreactor.

Ligninolytic enzymes of the fungus Panus tigrinus818: Biosynthesis, purification and properties

Abstract Extracellular Mn-dependent peroxidase and oxidase have been purified from the culture of white rot fungus P. tigrinus 8 18 grown on wheat straw. Some physico-chemical and ligninolytic properties of these enzymes have been investigated. Both enzymes have been shown to decompose a dimeric model compound of β-β′ type (and oxidase, also to demethoxylate substrate); to cleave Cα-Cβ in a nonphenolic model compound of β-1 type (Mn-peroxidase, also to split the Cβ-C4 bond); to be involved in lignin polymerization-depolymerization reactions. Based on the data presented and considering the absence of lignin peroxidase in the extracellular ligninolytic enzyme complex of P. tigrinus, Mn-peroxidase is suggested to be the key enzyme of the complex.

Oxidase of the white rot fungus Panus tigrinus 8/18

Extracellular oxidase of the white rot fungus Panus tigrinus earlier reported as laccase)contains copper but has no absorption spectrum typical of ‘blue’ oxidases. Thioglycolate and sodium azide inhibit the activity of this enzyme at concentrations 2.5–3 orders lower than those needed for fungal laccases. The oxidase of P. tigrinus oxidizes syringaldazine, coniferyl alcohol, ABTS, syringic acid, diaminobenzidine, guaiacol, catechol and vanillylacetone with different efficiencies. Oxygen consumption and no hydrogen peroxide formation were detected during substrate oxidation by P. tigrinus oxidase. It is proposed that P. tigrinus oxidase is a new ligninolytic enzyme.

Fungal Oxidoreductases and Humification in Forest Soils

Humification is aerobic, largely oxidative process of non-living organic matter biotransformation into recalcitrant humic substances (HS). HS comprise up to 90% of soil organic matter and represent a long-time sink for atmospheric CO2 with mean residence time of 102–103 years. Wood- and soil-colonizing fungi are the major driving force in humification, being involved in transformation of plant residues, synthesis, and degradation of HS. The chapter is focused on production of ligninolytic oxidoreductases by different groups of fungi and their role in humus synthesis and transformation in forest soils. White-rot fungi and litter-decomposing basidiomycetes producing acidic laccases and ligninolytic peroxidases are mainly involved in delignification and HS degradation, leading to release of small soluble fragments (fulvic acids, monomers) and CO2. Brown-rot fungi producing non-enzymatic oxidative agents and probably laccase are responsible for synthesis of high molecular weight humic acids from partially oxidized lignin. Ascomycetes produce non-ligninolytic peroxidases, neutral laccases, and tyrosinases and are mainly involved in synthesis of HS by partial lignin oxidation or extracellular polymerization of low molecular weight polyphenols. Laccases of ectomycorrhizae and lichens may participate in humus formation via polymerization of phenols, while tyrosinases may contribute to humic acid fraction via melanization.