Tatyana Avramova

9α-Hydroxylation of 4-androstene-3,17-dione by resting Rhodococcus sp. cells

Abstract The effect of nitrogen and carbon nutrition, growth and biomass storage on the effectiveness of microbial 9α-hydroxylation of 4-androstene-3,17-dione was studied. The transformation was carried out by resting Rhodococcus sp. cells in nutrient deficient transformation medium at different pHs as well as in the presence of several solvents of the steroid compounds. A conversion ratio of ca 0·75 was observed with stationary phase cells grown on medium containing glucose and non-dehydrated casein hydrolysate. A strong influence of the nitrogen source on the effectiveness of the process was noticed. The presence of different alcohols in the reaction medium was found to increase the substrate conversion ratio to ca 0·85. A decrease in the transformation ability of stored cells was a logarithmic function of the storage time.

The inducibility of 9α-steroid hydroxylating activity in resting Rhodococcus sp. cells

Abstract The inducibility of the key enzyme systems controlling the microbial 9α-hydroxylation of 4-androstene-3,17-dione (AD) by resting Rhodococcus sp. IOC-77 cells was investigated. Consecutive induction of the 9α-steroid hydroxylating activity and the accompanying product degrading Δ 1 -steroid dehydrogenating activity of the organism was observed. The possibility of achieving a complete microbial conversion of AD into 9α-hydroxy-4-androstene-3,17-dione by blocking protein synthesis was demonstrated. The inducing properties of several androstane and pregnane compounds as well as the influence of some functional groups in their steroid structure were discussed.

The Importance of Rhamnolipid-Biosurfactant-Induced Changes in Bacterial Membrane Lipids of Bacillus subtilis for the Antimicrobial Activity of Thiosulfonates

The antimicrobial properties of methyl (MTS) and ethyl (ETS) esters of thiosulfonic acid alone and in combination with rhamnolipid-biosurfactant (RL) have been characterized for their ability to disrupt the normal physiological functions of living pathogens. Bactericidal and fungicidal activities of MTS and ETS and their combination with rhamnolipid were demonstrated on strains of Pseudomonas aeruginosa, Bacillus subtilis, Alcaligenes faecalis, and Rhizopus ngtricans. It was found that the combination of rhamnolipid and thiosulfonic esters has a synergistic effect leading to decreasing of bactericidal and fungicidal concentrations of MTS and ETS. More extensively was studied the effect of rhamnolipid on the lipid composition of B. subtilis bacterial membrane. To our knowledge, in this article is reported for the first time a remarkable increase of negatively charged phospholipid cardiolipin in the presence of rhamnolipid. The capacity of RL as a surface-active substance was confirmed by scanning electron microscopy (SEM). The occurrence of surface infolds and blebs on B. subtilis shown by SEM, was not accompanied by changes in membrane permeability tested by a live/dead viability staining for fluorescence microscopy. When RL was applied in combination with MTS, a dramatic permeability shift for propidium iodide was observed in vegetative cells.

Effect of Nitrogen Source in Cultivation Medium on the 9α-Hydroxylation of Pregnane Steroids by Resting Rhodococcus Sp. Cells

ABSTRACT The influence of the source of nitrogen in the cultivation media on 9α-hydroxylating activity of resting Rhodococcus sp. cells was investigated. Two pregnane steroids 3β-hydroxy-16α,17α-epoxy-5α-pregnane-20-one (5α-H) and 3β-hydroxy-16α,17α-epoxy-5- pregnen-20-one (Δ5) were used as substrates. Results obtained showed that the source of nitrogen in the cultivation medium significantly affected both the level of product accumulation and the dynamics of their formation. It is worth mentioning the difference in the hydroxylating activity of cell depot prepared on commercial dehydrated or freshly prepared nitrogen sources. Thus, the cultivation medium with Triptose (Difco) gave a Rhodococcus sp. cell depot actively accumulating hydroxylated derivatives from 5α-H while the most suitable for accumulation of such derivatives from Δ5 was media containing freshly prepared meat extract.

Bacterial Decolorization of Acid Orange 7 in the Presence of Ionic and Non-Ionic Surfactants

The effects of the non-ionic surfactant Triton® X-100, the cationic surfactant cetyltrimethylammonium bromide (CTAB) and the anionic surfactant sodium N-lauroyl sarcosinate (SLS) on the decolorization of the reaction medium containing the monoazo dye Acid Orange 7 (AO7) by Alcaligenes faecalis and Rhodococcus erythropolis were studied. It was found that the surfactants influenced in different ways the rate of decolorization. At all concentrations tested the non-ionic surfactant Triton X-100 decreased the decolorization rate of R. erythropolis. At concentrations above the critical micelle concentration (CMC) Triton X-100 upset the usually observed exponential decay of the dye with A. faecalis due probably to the existence of an outer membrane in this organism. In concentrations above the CMC the anionic surfactant SLS inhibited the decolorization and, at prolonged incubation, caused partial release of the bound dye. The cationic surfactant CTAB in concentrations above and below the CMC accelerated drastically the binding of AO7 to the cells causing a rapid staining of the biomass and complete decolorization of the reaction medium. An attempt was made for explanation of the observed differences by the negative electrostatic charge of the living bacterial cell.

Enhanced cell surface hydrophobicity favors the 9α-hydroxylation of androstenedione by resting Rhodococcus sp. cells

The achievement of an effective process of 9α‐hydroxylation of 4‐androstene‐3,17‐dione is of significant importance as it leads to the formation of the key intermediate 9α‐hydroxy‐4‐androstene‐3,17‐dione which is not possible by chemical means. In this study, the 9α‐hydroxylation of 4‐androstene‐3,17‐dione was carried out by resting Rhodococcus sp. cells. The ability of the naturally hydrophobic Rhodococcus to assimilate n‐alkanes was employed to obtain a cell depot with an intentionally increased cell surface hydrophobicity. The control Rhodococcus sp. cells were cultivated on medium containing glucose instead of n‐alkanes as a source of carbon and energy. Cells were harvested, washed from the cultivation media, and subjected to transformation of crystal androstenedione in buffer medium. The hydrophobicity of the n‐alkanes‐ and glucose‐grown cells, their total lipid content, and fatty acid composition were determined. The ultrastructure of the n‐alkanes‐ and glucose‐grown cells and their steroid hydroxylating activities were examined and compared. The results obtained in the present study showed that the intentionally achieved growth‐driven enhancement of the already hydrophobic Rhodococcus sp. cells made them even more compatible with the hydrophobic steroid substrate and enhanced its accessibility, which provided an increased steroid hydroxylating activity and lack of the accompanying product destruction.