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.
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