L. A. Golovleva

Characterization of an intradiol dioxygenase involved in the biodegradation of the chlorophenoxy herbicides 2,4-D and 2,4,5-T

Hydroxyquinol 1,2‐dioxygenase, an intradiol dioxygenase, which catalyzes the cleaving of the aromatic ring of hydroxyquinol, a key intermediate of 2,4‐D and 2,4,5‐T degradation, was purified from Nocardioides simplex 3E cells grown on 2,4‐D as the sole carbon source. This enzyme exhibits a highly restricted substrate specificity and is able to cleave hydroxyquinol (K m for hydroxyquinol as a substrate was 1.2 μM, V max 55 U/mg, K cat 57 s−1 and K cat/K m 47.5 μM s−1), 6‐chloro‐ and 5‐chlorohydroxyquinol. Different substituted catechols and hydroquinones are not substrates for this enzyme. This enzyme appears to be a dimer with two identical 37‐kDa subunits. Protein and iron analyses indicate an iron stoichiometry of 1 iron/65 kDa homodimer, α2 Fe. Both the electronic absorption spectrum which shows a broad absorption band with a maximum at 450 nm and the electron paramagnetic resonance spectra are consistent with a high‐spin iron(III) ion in a rhombic environment typical of the active site of intradiol cleaving enzymes.

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.

Aerobic bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) and its kinetic aspects

Aerobic bacterial degradation of PAHs is reviewed. Particular attention is paid to its kinetic aspects (rate and specificity). The general concepts of PAH biodegradation in nature and the role of aerobic bacteria in this process are described. The problem of PAH bioavailability and the mechanism of PAH penetration through bacterial cell wall are discussed. The key role of the reaction of PAH hydroxylation in controlling the rate and specificity of PAH biodegradation process is substantiated. The effects of competitive inhibition, intermediate inhibition, cross induction, and cometabolism are considered. The importance of microbial communities for PAH biodegradation in natural ecosystems is shown. The review contains the list of 138 references.

Degradation of Aniline by Delftia tsuruhatensis 14S in Batch and Continuous Processes

A Delftia tsuruhatensis strain capable of consuming aniline as the sole source of carbon, nitrogen, and energy at concentrations of up to 3200 mg/l was isolated from activated sludge of the sewage disposal plants of OAO Volzhskii Orgsintez. The strain grew on catechol and p-hydroxybenzoic acid but did not consume phenol, 2-aminophenol, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, 2-nitroaniline, 2-chlorophenol, or aminobenzoate. Aniline is degraded by cleavage of the catechol aromatic ring at the ortho position. Cells were immobilized on polycaproamide fiber. It was shown that the strain degraded aniline at 1000 mg/l in a continuous process over a long period of time.

Phenanthrene and anthracene degradation by microorganisms of the genus Rhodococcus

The cells of Rhodococcus opacus 412 and R. rhodnii 135 were adapted to phenanthrene and anthracene on a solid mineral medium. Preliminary adaptation of the strains accelerated the metabolism of polyaromatic hydrocarbons and provided for the ability of microorganisms to grow on pheanthrene as a sole carbon and energy source in a liquid mineral medium. It was shown that phenanthrene was mineralized by the strains through 7,8-benzocoumarin, 1-hydroxy-2-naphthoaldehyde, 1-hydroxy-2-naphthoic acid, salicylaldehyde, salicylate and catechol to the intermediates of tricarbonic acid cycle and partially transformed with the accumulation of the products of subsequent monooxygenation (3-hydroxyphenanthrene and phenanthrene dihydroxylated not in ortho-position). As a result of the adaptation of the strains to anthracene on a solid mineral medium, the obtained variant of strain R. opacus 412 was able to transform anthracene in a liquid mineral medium to anthraquinone and 6,7-benzocoumarin.

The Microbial Transformation of Phenanthrene and Anthracene

The transformation of phenanthrene and anthracene by Rhodococcus rhodnii 135, Pseudomonas fluorescens 26K, and Arthrobacter sp. K3 is studied. Twenty-one intermediates of phenanthrene and anthracene transformation are identified by HPLC, mass spectrometry, and NMR spectroscopy. P. fluorescens 26K and Arthrobacter sp. K3 are found to produce a wide range of intermediates, whereas R. rhodnii 135 oxidizes phenanthrene, resulting in the formation of a sole product, 3-hydroxyphenanthrene. Putative transformation pathways of phenanthrene and anthracene are proposed for the three bacterial strains studied. These strains can be used to obtain valuable compounds (such as hydroxylated polycyclic aromatic hydrocarbons) that are difficult to produce by chemical synthesis.

Detoxification of high concentrations of trinitrotoluene by bacteria

The ability of the strains-destructors of various aromatic compounds to utilize trinitrotoluene (TNT) up to concentration of 70 mg/l was shown. An increase in the TNT concentration from 100 to 150 mg/l did not inhibit its conversion rate by the Kocuria palustris RS32 strain. The Acinetobacter sp. VT11 strain utilized TNT as a sole substrate for growth; 3,5-dinitro-4-methyl anilide acetate and 2,6-dinitro-4-aminotoluene were identified as intermediates of TNT degradation by active strains of Pseudomonas sp. VT-7W and Kocuria rosea RS51. At the same time, 4-methyl-3,5-dinitroformamide was discovered for the first time upon the TNT destruction by the bacteria strains of Rhdococcus opacus 1G and Rhdococcus sp. VT-7. The active bacterial strains achieved an 82-90% destruction of TNT when they were introduced into the soil.

Phenol degradation by Rhodococcus opacus strain 1G

During cultivation in a liquid medium, the bacterium Rhodococcus opacus 1G was capable of growing on phenol at a concentration of up to 0.75 g/l. Immobilization of Rhodococcus opacus 1G had a positive effect on cell growth in the presence of phenol at high concentrations. The substrate at concentrations of 1.0 and 1.5 g/l was completely utilized over 24 and 48 h, respectively. The key enzymes of phenol degradation (two catechol 1,2-dioxygenases and muconate cycloisomerase) were isolated. One of the dioxygenases was very unstable. By substrate specificity, another enzyme belonged to catechol 1,2-dioxygenases of the classical ortho-pathway. Chlorocatechols and chlorophenols served as competitive inhibitors of catechol 1,2-dioxygenases. The inhibitory effect of other aromatic compounds was less significant. Our results suggest that this strain holds promise for bioremediation of phenol wastewater.

New Approaches to Increasing the Yield of Laccase from Panus tigrinus

We optimized the conditions for laccase production by the lignolytic fungus Panus tigrinus 8/18. 2,4-Dimethylphenol was used as an aromatic inducer. Introduction of 2,4-dimethylphenol and 2 mM CuSO4 into a rich medium was followed by a tenfold increase in the yield of this enzyme. Additional treatment of the medium with perftoran (an oxygen-transporting agent) and immobilization of the fungus on polycaproamide fibers significantly increased the activity of laccase in the medium. Thus, optimum conditions for cultivation of P. tigrinus were found, which allowed an increase in laccase activity in the medium 25-fold as compared to that achieved using any other method described previously.

Crystallization and preliminary crystallographic analysis of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E: a novel dioxygenase involved in the biodegradation of polychlorinated aromatic compounds.

Hydroxyquinol 1,2-dioxygenase (HQ1,2O) from Nocardioides simplex 3E, an enzyme involved in the aerobic biodegradation of a large class of chloroaromatic compounds such as 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T), has been crystallized. HQ1,2O, which specifically catalyzes the intradiol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), an intermediate in the degradation of a variety of aromatic pollutants, to maleylacetate, has been recently purified to homogeneity. The enzyme is an homodimer composed of two identical subunits in a alpha 2-type quaternary structure, has a molecular weight of about 65 kDa and contains a catalytically essential Fe(III) ion. Crystals of HQ1,2O obtained using 2% PEG 400 and 2 M ammonium sulfate at pH 7.5 as precipitants belong to the orthorhombic space group P212121, with unit-cell parameters a = 81.15 (6), b = 86.79 (7), c = 114.93 (8). Assuming one dimer per asymmetric unit, the Vm value is 2.51 A3 Da-1. A complete native data set to 1.8 A resolution has been collected on a laboratory source. This is the first intradiol dioxygenase which specifically catalyzes the cleavage of hydroxyquinol to give diffraction-quality crystals.