Nina M. Myasoedova

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.

New efficient producers of fungal laccases

Two promising strains of laccase producers—Lentinus strigosus 1566 and Steccherinum ochraceum 1833—were found by screening of basidiomycetes. The cultivation conditions increasing the enzyme yield were selected. The maximal laccase activity was observed in the case of submerged cultivation of the mycelium immobilized on polycaproamide fibers in rich media in the presence of 2 mM CuSO4 in combination with the optimal inducer, namely, 2,6-dimethylphenol for L. strigosus and 2,4-dimethylphenol for S. ochraceum. Under these conditions, the activity of S. ochraceum laccase amounted to 33.1 U/ml and that of L. strigosus, to 186.5 U/ml. Anthracene was transformed with S. ochraceum laccase, and its oxidation to anthraquinone was demonstrated by mass spectrometry.

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.

Degradation of phenanthrene by the rhizobacterium Ensifer meliloti.

The biodegradation of the polycyclic aromatic hydrocarbon phenantherene by the rhizobacterial strain Ensifer meliloti P221, isolated from the root zone of plant grown in PAH-contaminated soil was studied. Bacterial growth and phenanthrene degradation under the influence of root-exuded organic acids were also investigated. Analysis of the metabolites produced by the strain by using thin-layer chromatography, gas chromatography, high-pressure liquid chromatography, and mass-spectrometry revealed that phenanthrene is bioconverted via two parallel pathways. The first, major pathway is through terminal aromatic ring cleavage (presumably at the C3–C4 bond) producing benzocoumarin and 1-hydroxy-2-naphthoic acid, whose further degradation with the formation of salicylic acid is difficult or is very slow. The second pathway is through the oxidation of the central aromatic ring at the C9–C10 bond, producing 9,10-dihydro-9,10-dihydroxyphenanthrene, 9,10-phenanthrenequinone, and 2,2′-diphenic acid. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites, has been reported for a bacterium of the Ensifer genus.

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.

Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinus.

The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296 K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.2 M CaCl2, 100 mM Tris-HCl pH 7.5. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, beta = 97.7 degrees , and contain 46% solvent. A complete native data set was collected to 1.4 A resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes.

Selective regulation of laccase isoform production by the Lentinus strigosus 1566 fungus

The effects of a number of culture medium components, such as peptone, yeast extract, mono- and disaccharides, copper ions, 2,6-dimethylphenol, and polycaproamide fiber, on the laccase activity dynamics in the culture liquid and laccase isoform production by the Lentinus strigosus 1566 fungus were studied. It was demonstrated that some saccharides selectively induced or inhibited the synthesis of different laccase isoforms. Similar action was exerted by copper ions, 2,6-dimethylphenol, and polycaproamide fiber, as well as by their combination. Selective in vivo regulation of the production of certain laccase isoforms by basidial fungi by means of altering the culturing medium composition can be utilised for various biotechnological purposes.

Myrothecium verrucaria F-3851, a producer of laccases transforming phenolic compounds at neutral and alkaline conditions

The conditions of submerged cultivation of the ascomycete Myrothecium verrucaria strain F-3851 were optimized in order to increase the yield of laccase in the culture liquid using the natural sources of carbon and energy (fresh rubbed potato tuber or floured grains of buckwheat, barley, oat, wheat, rye, rice, pea, or haricot). The pH-optima of oxidation of a number of laccase substrates (ABTS, 2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) by laccases of the culture liquid as well as substrate selectivity of laccases were investigated. The intermediates of transformation of phenylpropanoids (ferulic acid, p-coumaryl alcohol and coniferyl alcohol) by laccases of the culture liquid at neutral conditions were purified and identified. The ability of laccases of the culture liquid of M. verrucaria strain F-3851 to catalyze polymer compound formation during phenylpropanoid transformation was shown that offers the prospects of application of the laccases of M. verrucaria strain F-3851 for production of pharmacologically valuable polymers in a number of cellular biotechnologies carried out in neutral or alkaline environments.

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates.