The strain of Mycobacterium sp. VKM Ac-1817D forms 9alpha-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M (r) approximately 58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.

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A nonlinear spectrometric method for determination of the stability constant (Ks) for cyclodextrin complex with steroid was developed. The method is based on calculation of the parameters of competitive cyclodextrin complexation by simultaneous fitting of two types of curves. Those of the first type are the dependencies of absorbance of methyl orange solution on the cyclodextrin concentration, the second type being the absorption curves of displacement of the dye, by steroid, from the cyclodextrin complex. With the method proposed, Ks values were calculated with standard deviation less than 10%. This method is validated by determination of Ks values using the phase-solubility technique. For neutral steroid molecules, the effect of pH on Ks was found to be insignificant. Ks values for the cyclodextrin-dye complex were determined for randomly methylated beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin and sulfobutylether-beta-cyclodextrin. More hydrophobic steroids were characterised by higher Ks values. Anionic beta-cyclodextrins showed high affinity for the steroids studied. Simple equipment and sufficient computing allowed recommendation of the method for express estimation of cyclodextrin's affinity for hydrophobic substrates.

Soybean extract residue (scum), a waste of soybean oil production, was examined as a raw material for C 17 -ketosteroid production. As a model process, its bioconversion to 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) by Mycobacterium sp VKM Ac-1817D was studied. The content of transformable sterols (sitosterol, stigmasterol and campesterol) in scum was estimated at ∼14%. The bioconversion of scum to 9-OH-AD was characterized by a long lag-period (300-350 h) followed by 9-OH-AD accumulation. The microbial or chemical elimination of fatty non-identified components resulted in sterol-enriched scum preparations. Effective conversion of these preparations by Mycobacterium sp was demonstrated: 9-OH-AD molar yield ∼65% was reached at 60 h from the scum preparation containing 10 g dm −3 transformable sterols. The process productivity was comparable with that for high quality-sitosterol of wood origin (tall-oil sitosterol).

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