Ludmila Alexeevna Golovleva

Ability of actinobacteria Rhodococcus opacus 1CP to survive under unfavorable conditions and retain its biodegradation activity was assessed. The morphological and ultrastructural features of R. opacus 1CP cells degrading benzoate in the presence of oxidants and stress-protecting agents were investigated. The cells of R. opacus 1CP were resistant to oxidative stress caused by up to 100 mM H₂O₂ or up to 25 μM juglone (5-oxy-1,4-naphthoquinone). After 2 h of stress impact, changes in the fatty acid composition, increased activity of antioxidant enzymes, and changes in cell morphology and ultrastructure were observed. The strain retained its ability to degrade benzoate. Quercetin had a protective effect on benzoate-degrading cells of R. opacus 1CP. The strategy for cells survival under unfavorable conditions was formulated, which included decreased cell size/volume and formation of densely-packed cell conglomerates, in which the cells are embedded into a common matrix. Formation of conglomerates may probably be considered as a means for protecting the cells against aggressive environmental factors. The multicellular conglomerate structure and the matrix material impede the penetration of toxic substances into the conglomerates, promoting survival of the cells located inside.

Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding α- and β-subunits of protocatechuate 3,4-dioxygenase and to two genes of the R. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.

The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L⁻¹. The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200–250 mg L⁻¹) and high (4 g L⁻¹) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate.

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.

The review summarizes the authors’ own data and reports by other researchers concerning the degradation of stable organic compounds by actinobacteria. Properties of these microorganisms are characterized. They include the ability to survive and to maintain metabolic activity during prolonged exposure to adverse environmental conditions, as well as the presence of enzymes with a broad substrate specificity, which enables these bacteria to decompose natural and synthetic substances. The transformation pathways of key intermediates and the ability of actinobacteria to develop novel pathways are discussed. Approaches to increasing the destructive activity of bacterial cultures are presented.

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...

Bacterial strain Bacillus sp. VT-8 was shown to use trinitrotoluene (TNT) as the sole source of carbon, nitrogen, and energy, as well as to carry out its co-oxidation. Resistance of Bacillus sp. VT-8 to high TNT concentrations was shown. Efficient detoxification of TNT-contaminated soil and water samples was demonstrated. This strain may be recommended for TNT biodegradation at concentrations of up to 140 mg/L due to its high degradation activity and the absence of toxic effect of TNT on Bacillus sp. VT-8.