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The hydroxylase activities of new strains such as Curvularia lunata, C. geniculata, C. eragrostidis, C. prasadii, Ulocladium botrytis, Alternaria tenuis, and Fusarium oxysporum toward three steroid substrates, namely, androstenedione (AD), cortexolone (S), and dehydroepiandrosterone acetate (DAA), were characterized. The 9α-hydroxylase activity of C. lunata 1011 cells against S to form 9α-hydroxy-S was shown for the first time. It was found that C. geniculata 837 and F. oxysporum 11dn1 strains can hydroxylate substrates to form pharmacologically promising 7α-hydroxysteroids. C. geniculata 837 cells selectively hydroxylate AD, resulting in 7α-hydroxytestosterone, whereas F. oxysporum 11dn1 leads to the transformation of DAA to 7α-hydroxydehydroepiandrosterone.

The steroid-transforming activity of free and immobilized cells of Pimelobacter simplex VKPM As-1632 entrapped in an operationally stable macroporous polyvinyl alcohol cryogel was studied. It was shown that the macroporous matrix of the carrier did not create any diffusional limitations for steroid access to the cells or the removal of the transformation products from them. The optimal conditions for the hydrocortisone 1,2-dehydration into prednisolone by free and immobilized cells were elucidated. The immobilized biocatalyst was obtained in a granulated form and used in 32 successive cycles of steroid transformation. The average cycle duration was 45 min, and the prednisolone yield of during the first 20 cycles was 98%. It was established that the immobilized cells of the actinobacteria P. simplex retained high steroid-transforming activity over all of the transformation cycles. The physicochemical and diffusion characteristics of the polyvinyl alcohol gels and its granules were determined, and their high stability during repeated cycles of steroid transformation was shown. The results indicated that P. simplex immobilized cells represent an effective catalyst suitable for multiple use. Biomass consumption decreased upon its use, and product isolation, as well as culture storage, was much easier.

The main and side products of hydroxylation by the C. lunata VKPM F-981 mycelium of fourteen delta(4)-3-ketosteroids of the estrane, androstane, and pregnane series and six of their delta(5)-3beta-hydroxy analogues were identified by H1 PMR spectroscopy and comparison with standard samples. The obtained experimental data are considered in terms of the triangular model of the enzyme-substrate interaction. The dependence of the direction of hydroxylation of steroid molecules and the orientation of hydroxy groups on the structure of the initial substrate was revealed.

9alpha-Hydroxy derivatives were prepared from 11 steroids ofandrostane and pregnane series using Rhodococcus erythropolis VKPM Ac-1740 culture with 0.5-20 g/l substrate concentration in the reaction mixture. 9alpha-Monohydroxylation proceeded regardless of the substituent structure at C17. However, the structure of the steroid molecule influenced the time of complete conversion of the substrate and the yield of the transformation product. 9alpha-Hydroxy-androstenedione was obtained in 35 h in a yield of 85% when the maximum concentration of androstenedione (AD) was 10 g/l. 9alpha-Hydroxy-AD was also formed by the actinobacterium cells entrapped in poly(vinyl alcohol) cryogel beads. Nine successive transformation cycles were carried out using immobilized cells at 4.0 g/l concentration of AD in the medium. The yield of 9alpha-hydroxy-AD formed during six cycles (from two to eight with the duration of each cycle for 22-24 h) was 98%.

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.

Transformation of 16 delta5-3beta-hydroxy- and delta4-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-beta-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using specters of proton magnetic resonance and mass-specters, have been isolated. Hydroxylation of delta5-3beta-hydroxy-steroids occurred mostly in the C-7alpha position whereas hydroxylation of delta4-3-ketosteroids was in the C-11beta position. Only androst-4-en-3,17-dione, 9alpha-hydroxyl-androstenedione, and androsts-1,4-diene-3,17-dione were hydroxylated at C-14alpha position. Besides main 11beta-derivatives, the 6beta- and 7beta-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienon. The ratio of MCD to transforming steroid was 1 : 1 (mol/mol). Hydroxycortisone and 7alpha-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.

Hydroxylation activity of the mold fungi belonging to the orders Dothideales, Hypocreales, and Mucorales towards delta(5)-3beta-hydroxysteroids was studied. The fungi Bipolaris sorokiniana, Fusarium sp., and Rhizopus nigricans were able to introduce hydroxy group at position 7alpha; however, this ability was detected only at a low substrate load and with a low yield. A 7alpha-hydroxylase activity of the Curvularia lunata VKPM F-981 culture was shown for the first time. It was demonstrated that the studied strain was capable of stereo- and regioselective transformations of androstane 5-olefins at a load not less than 2 g/l. Conversion of pregnane steroids by this culture yielded both 7alpha and 11beta-hydroxy derivatives. The introduction of 7alpha-hydroxy group by this strain occurred concurrently with enzymatic hydrolysis of ester groups, which proceeded under mild conditions to give the corresponding alcohols in the cases of both 3-acetate of delta(5)-androstenes and mono- and triacetates of delta(5)-pregnenes.

Conditions of conversion of 17 alpha-methyltestosterone to methandrostenolone with the presence of modified beta-cyclodextrins (methylcyclodextrin, hydroxypropylcyclodextrin, and hydroxyethylcyclodextrin) in the steroid:cyclodextrin ratio 1:1 were studied. The experimental solutions of modified beta-cyclodextrins were prepared in deionized water with 5-7% methanol. Under the conditions found to be optimal, 1,2-dehydrogenation of 17 alpha-methyltestosterone was carried out with 2-4 g/l Pimelobacter simplex VKPM Ac-1632 biomass. At the substrate concentration 5-20 g/l, the reaction occurred for 1-15 h without any by-products. The maximum rate of methandrostenolone accumulation was observed with hydroxypropylcyclodextrin. The methylcyclodextrin solution can be reused for complete 17 alpha-methyltestosterone conversion at the concentration 5 g/l.