Mathematical analysis of two-phase mass transfer in a batch reactor for the chemical transformation of a steroid

A reactor is described for the conversion of the slightly water-soluble steroid testosterone (T) to 4-androstene-3, 17-dione (4-AD) by enzyme in the presence of excess cofactor. Since the enzyme is subject to substrate inhibition, reaction rates are strong functions of aqueous substrate concentration. High concentrations of the substrate, testosterone, per unit reactor volume are maintained within poly(dimethylsiloxane) beads that are suspended in the aqueous enzyme solution. Mass transfer (controlled by bead size, polymer to water volume ratio, enzyme loading) is used to control the degree and rate of conversion. The reactor dynamics are predicted over a wide range of reaction conditions. The product steroid is recovered in the polymeric beads from the enzyme solution.     

Structural and biochemical insights into 7β‐hydroxysteroid dehydrogenase stereoselectivity

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo‐ and regio‐selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β‐hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active‐site accessibility, the bases of the specificity for NADP⁺, and the general architecture of the steroid binding site. Comparison with 7α‐hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C‐terminal extension reshapes the substrate site of the β‐selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859–865. © 2016 Wiley Periodicals, Inc.     

Rat liver 3 alpha-hydroxysteroid dehydrogenase

3 alpha-HSD appears to be a multifunctional enzyme. In addition to its traditional role of catalyzing early steps in androgen metabolism, it will also oxidoreduce prostaglandins and detoxify trans-dihydrodiols (proximate carcinogens). Since these novel reactions have been quantified using homogeneous enzyme it is necessary to interpret the role of the enzyme in these processes in vivo with some caution. However, it is rare that such observations on a purified hydroxysteroid dehydrogenase have led to such important questions. Is the 3 alpha-HSD the only steroid dehydrogenase that transforms prostaglandins and trans-dihydrodiols? Are hydroxysteroid dehydrogenases and prostaglandin dehydrogenases the same enzymes in certain tissues? Does 3 alpha-HSD protect against chemical carcinogenesis in vivo? The inhibition of the purified dehydrogenase by therapeutically relevant concentrations of anti-inflammatory drugs also deserves comment. Is this hydroxysteroid dehydrogenase really an in vivo target for anti-inflammatory drug action? Could these drugs exert some of their pharmacological effect either by preventing glucocorticoid metabolism in some tissues or by preventing the transformation of PGF2 alpha (non-inflammatory prostanoid) to PGE2 (a pro-inflammatory prostanoid)? Could these drugs, by inhibiting trans-dihydrodiol oxidation, potentiate the initiation of chemical carcinogenesis? These and other important questions can be answered only by developing specific inhibitors for the dehydrogenase to decipher its function in vivo.     

Catalytic activity of noncovalent complexes of horse liver alcohol dehydrogenase,NAD+ and polymers,dissolved or suspended in organic solvents

Summary Noncovalent complexes were formed by lyophilization of aqueous solutions containing horse liver alcohol dehydrogenase, NAD⁺ and a polymer [ethyl cellulose or poly(vinyl butyral)]. The complexes expressed higher specific catalytic activity in organic solvents as compared to a corresponding amount of enzyme deposited on to Celite or lyophilized enzyme powder. The noncovalent complexes were soluble in toluene. In butyl acetate and methyl t-butyl ether, suspensions of fine particles were formed.     

Characterization of multiple forms of carbonyl reductase from chicken liver.

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.     

3(20)alpha-hydroxysteroid dehydrogenase activity of monkey liver indanol dehydrogenase

Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.     

Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.     

Histochemical and spectrophotometric demonstration of hydroxysteroid dehydrogenase activity in the presumed steroid producing cells of the brook lamprey (Lampetra planeri Bloch) during metamorphosis

Pronephric, opisthonephric, ovarian, and testicular tissue rich in presumed steroid producing cells taken from the brook lamprey during metamorphosis was investigated by histochemical and spectrophotometric methods. Histochemical results seemed to provide evidence for hydroxysteroid dehydrogenase activity using pregnenolone as substrate. Spectrophotometric evidence for hydroxysteroid dehydrogenase activity was obtained from presumed adrenocortical tissue, ovarian and testicular homogenates using pregnenolone, dehydroepiandrosterone, androsterone, and 3 beta, 17 beta-dihydroxy-5 alpha-androstane as substrates.     

Lead and Cadmium Co-exposure Mediated Toxic Insults on Hepatic Steroid Metabolism and Antioxidant System of Adult Male Rats

The redox status and steroid metabolism of liver of adult male rat exposed to lead (Pb) and cadmium (Cd) either alone or in co-exposure (0.025 mg/kg body weight intraperitoneally/15 days) was studied. Pb and Cd significantly accumulated in the liver. The activity of steroid metabolizing enzymes 17-βhydroxysteroid oxidoreductase and uridine diphosphate–glucuronyltransferase were decreased in experimental animals. 17-β-Hydroxysteroid dehydrogenase was reduced to 33%, 38%, and 24% on treatment of Pb, Cd, and co-exposure (Pb + Cd). Furthermore, the activity of uridine diphosphate–glucuronosyltransferase was significantly reduced to 27% (Pb exposure), 36% (Cd exposure), and 25% (co-exposure of Pb + Cd). Cd exposure exhibited more toxic effect than Pb, while co-exposure demonstrated the least. The activities of antioxidant enzymes, superoxide dismutase, catalase, glutathione reductase, and glucose-6-phosphate dehydrogenase decreased and glutathione peroxidase increased in mitochondrial and post-mitochondrial fractions. The level of lipid peroxidation increased, and cellular glutathione concentration decreased. Hepatic DNA was decreased, whereas RNA content and the activity of alanine transaminase remained unchanged. Histological studies revealed that only Cd-exposed groups exhibited cytotoxic effect. These results suggest that when Pb and Cd are present together in similar concentrations, they exhibited relatively decreased toxic effect when compared to lead and cadmium in isolation with regard to decreased steroid metabolizing and antioxidant enzyme activities. This seems that the toxic effect of these metals is antagonized by co-exposure due to possible competition amongst Pb and Cd for hepatic accumulation.     

Is There Any Steroid Hormone Formation in the Ovary of the Hagfish,Myxine glutinosa?

Localization of steroidogenic cells possibly existing in the hagfish was attempted using the following 3 methods: 1. Injection and autoradiographic localization of the labelled precursor cholesterol-4-¹⁴C, 2. Histochemical localization of 3β-ol dehydrogenase and other hydroxysteroid dehydrogenases and 3. Electron microscopic analysis of the gonad. It is concluded that if there is any steroid hormone formation in the ovary of the hagfish, it is extremely small and is not detectable by the methods used here.     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: II. Recovery and purification of pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

The novel approach of using aqueous two-phase systems for the elution of protein from ligand-coupled particles is investigated using pyruvate kinase and alcohol dehydrogenase from recombinant Saccharomyces cerevisiae and Cibacron blue F3G-A-coupled Sepharose CL6B (Blue-Sepharose) particles as a model system. The ligand-coupled particles distribute quantitatively to the polyethylene glycol-(PEG-) rich top phase and the recovered enzymes partition selectively to the dextran-(DEX-) rich bottom phase. An effective recovery and partial purification of pyruvate kinase and alcohol dehydrogenase from Blue-Sepharose particles using PEG8000-DEXT500 aqueous two-phase systems are demonstrated through a modest increase of salt concentration. The bioselective eluting agent, MgADP, which is useful in chromatographic operations, is not required for the process using aqueous two-phase systems. Recovery of pyruvate kinase, which is bound to ligand-coupled particles, in the DEX-rich bottom phase of aqueous two-phase systems can be up to 95% in one-step operations. The mixing time of ligand-coupled particles with aqueous two-phase systems is a major controlling variable. The salt concentration, the molecular weight of polymer, and the total volume of aqueous two-phase systems also influence the recovery of pyruvate kinase from ligand-coupled particles. The recovered enzymes in the DEX-rich bottom phase remain biologically stable over a long period of storage time. The concentration of product protein in a reduced volume and the easy separation from ligand-coupled particles are added advantages of the process using aqueous two-phase systems. Preliminary studies with goat polyclonal anti-pyruvate kinase-coupled Sepharose particles indicate that the process also may be applicable when a high-affinity ligand such as antibody is used. The experimental results and a theoretical derivation based on equilibrium models for binding/dissociation of ligands and proteins show that the process results in better recovery as compared to that of conventional bulk elution techniques.     

Substrate mass transport in two-phase partitioning bioreactors employing liquid and solid non-aqueous phases

The mass transfer of phenol and butyl acetate to/from water was studied in two-phase partitioning bioreactors using immiscible organic solvents and solid polymer beads as the partitioning phases in a 5-L stirred tank bioreactor. Virtually instantaneous mass transfer was observed with phenol in water/2-undecanone, and with butyl acetate in water/silicone oil systems. The mass transfer of butyl acetate to silicone oil was rapid irrespective of the viscosity of the partitioning phase. When Hytrel(?) polymer beads were employed as the partitioning phase, substrate transport to the polymer was found not to be externally mass transfer limited, but rather internally by substrate diffusion into the polymer. In contrast to gaseous, poorly soluble substrates studied in other works, mass transfer of soluble substrates such as phenol and butyl acetate to the polymer was unaffected by impeller speed but rather by polymer mass fraction.     

Potentiometric method for substrate analysis using immobilized NAD + -dependent oxidoreductase enzymes.

Two coenzyme-dependent oxidoreductases, glucose dehydrogenase and alcohol dehydrogenase, were immobilized in polyacrylamide gel over a platinum grid matrix and used as enzyme electrodes to measure their substrate concentrations in buffered aqueous solutions. The immobilized enzymes were used to oxidize their substrates in the presence of NAD +. Ferricyanide was used as the redox mediator and electroactive species. The determinations of glucose and ethanol were utilized to demonstrate and evaluate the performance of the system. The described methodology should be readily applicable to the analysis of numerous other substrates of coenzyme-dependent oxidoreductases.     

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.     

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.     

Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.

Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.     

Enzymatic properties of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are flavoproteins which catalyze the oxidative demethylation of dimethylglycine to sarcosine and sarcosine to glycine, respectively. During these reactions tightly bound tetrahydropteroylpentaglutamate (H4PteGlu5) is converted to 5,10-methylene tetrahydropteroylpentaglutamate (5,10-CH2-H4PteGlu5), although in the absence of H4PteGlu5, formaldehyde is produced. Single turnover studies using substrate levels of the enzyme (2.3 microM) showed pseudo-first-order kinetics, with apparent first-order rate constants of 0.084 and 0.14 s-1 at 23 and 48.3 microM dimethylglycine, respectively, for dimethylglycine dehydrogenase and 0.065 s-1 at 47.3 microM sarcosine for sarcosine dehydrogenase. The rates were identical in the absence or presence of bound tetrahydropteroylglutamate (H4PteGlu). Titration of the enzymes with substrate under anaerobic conditions did not disclose the presence of an intermediate semiquinone. The effect of dimethylglycine concentration upon the rate of the dimethylglycine dehydrogenase reaction under aerobic conditions showed nonsaturable kinetics suggesting a second low-affinity site for the substrate which increases the enzymatic rate. The Km for the high-affinity active site was 0.05 mM while direct binding for the low-affinity site could not be measured. Sarcosine and dimethylthetin are poor substrates for dimethylglycine dehydrogenase and methoxyacetic acid is a competitive inhibitor at low substrate concentrations. At high dimethylglycine concentrations, increasing the concentration of methoxyacetic acid produces an initial activation and then inhibition of dimethylglycine dehydrogenase activity. When these compounds were added in varying concentrations to the enzyme in the presence of dimethylglycine, their effects upon the rate of the reaction were consistent with the presence of a second low-affinity binding site on the enzyme which enhances the reaction rate. When sarcosine is used as the substrate for sarcosine dehydrogenase the kinetics are Michaelis-Menten with a Km of 0.5 mM for sarcosine. Also, methoxyacetic acid is a competitive inhibitor of sarcosine dehydrogenase with a Ki of 0.26 mM. In the absence of folate, substrate and product determinations indicated that 1 mol of formaldehyde and of sarcosine or glycine were produced for each mole of dimethylglycine or sarcosine consumed with the concomitant reduction of 1 mol of bound FAD.     

Steroid metabolism and profile of steroidogenic gene expression in Episkin: high similarity with human epidermis

The skin is a well-recognized site of steroid formation and metabolism. Episkin is a cultured human epidermis. In this report, we investigate whether Episkin possesses a steroidogenic machinery able to metabolize adrenal steroid precursors into active steroids. Episkin was incubated with [14C]-dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) and their metabolites were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS). The results show that the major product of DHEA metabolism in Episkin is DHEA sulfate (DHEAS) (88% of the metabolites) while the other metabolites are 7alpha-OH-DHEA (8.2%), 4-dione (1.3%), 5-androstenediol (1.3%), dihydrotestosterone (DHT) (1.4%) and androsterone (ADT) (2.3%). When 4-dione is used as substrate, much higher levels of C19-steroids are produced with ADT representing 77% of the metabolites. These data indicate that 5alpha-reductase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 3alpha-hydroxysteroid dehdyrogenase (3alpha-HSD) activities are present at moderate levels in Episkin, while 3beta-HSD activity is low and represents a rate-limiting step in the conversion of DHEA into C19-steroids. Using realtime PCR, we have measured the level of mRNAs encoding the steroidogenic enzymes in Episkin. A good agreement is found between the mRNAs expression in Episkin and the metabolic profile. High expression levels of steroid sulfotransferase SULT2B1B and type 3 3alpha-HSD (AKR1C2) correspond to the high levels of DHEA sulfate (DHEAS) and ADT formed from DHEA and 4-dione, respectively. 3beta-HSD is almost undetectable while the other enzymes such as type 1 5alpha-reductase, types 2, 4, 5, 7, 8, and 10 17beta-HSD and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) (AKR1C1) are highly expressed. Except for UGT-glucuronosyl transferase, similar mRNA expression profiles between Episkin and human epidermis are observed.     

A quick method for the determination of inhibition constants.

The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants.     

Production of 13beta-alkyl-3-methoxy-8,14-seco-1,3,5(10),9(11)-gonatetraen-14beta-ol-17-ones and 13beta-alkyl-3-methoxy-8,14-seco-1,3,5(10),9(11)-gonatetraen-17alpha-ol-14-ones by microbial enzymes.

For the purpose of producing hydroxy-keto-seco-steroids in which hydroxyl group is attached to a carbon atom having the R-configuration, numerous biochemically active microorganisms were tested without any success. The hydroxysteroid oxidoreductase enzymes of the investigated bacterial, yeast and fungal strains were suitable only for the production of 17beta-ol-14-one and 14alpha-ol-17-one derivatives. The required compounds were prepared by combinations of enzymatic reactions with chemical reduction. (i) By hydroxysteroid oxidoreductase of Saccharomyces uvarum and Saccharomyces drosophilarum, 17beta-ol-14-one and 14alpha-ol-17-one derivatives of 14,17-dione, respectively, were obtained. (ii) The above compounds were acetylated then reduced by sodium borohydride. (iii) 14beta,17beta-diol-17-acetate and 14alpha,17alpha-diol-14-acetate were dehydrogenated by dehydroxysteroid oxidoreductase of Nocardia sp. and Mycobacterum sp., respectively, in the presence of steroid esterase. The reaction mixture contained either 14beta-ol-17-one or 17alpha-ol-14-one derivatives, since oxidation by hydroxysteroid oxidoreductase was limited to the hydroxyl group attached to a carbon atom having the S-configuration.