IE55533B1

IE55533B1 - Steroid bioconversion

Info

Publication number: IE55533B1
Application number: IE176783A
Authority: IE
Original assignee: Upjohn Co
Priority date: 1982-07-30
Filing date: 1983-07-27
Publication date: 1990-10-24
Also published as: NL8302596A; FR2531101B1; FR2531101A1; GB2123833A; DD213242A5; JPS5939299A; IE831767L; GB8317422D0; DE3322120C2; GB2123833B; CH657871A5; GB2131811B; JPH06225792A; GB2131811A; JPH0824596B2; DE3322120A1; GB8402640D0; JPH0614874B2

Description

3 3 The first therapeutic use of corticosteroids was demonstrated in the 1950's with the introduction of cortisone acetate treatment for rheumatoid arthritis. Further studies demonstrates that the insertion of unsaturation into the 1,2-position of hydrocortisone and cortisone gave the resultant 5 steroids, prednisolone and prednisone, enhanced potency and reduced drug-induced salt retention. Subsequently most other steroids used for the treatment of corticoid-responsive diseases have been synthesised so that they contain a double bond in the 1,2-position of the steroid molecule.

US-A-3284447 discloses the utility of pregna-l,4,9(ll)-trienes in the synthesis of diuretic corticosteroids substituted at carbon 16. US-A-4041055 discloses a process for the synthesis of corticosteroids from androsta-l,4-dienedione derivatives, demonstrating the utlility of androst-1-enes as important intermediates in the production of medically 15 useful steroids.

The bioconversion of 1,2-saturated steroids to the corresponding 1,2-dehydro steroids is disclosed in US-A-2837464, describing the 1-dehydrogenation of steroids in fermentation beers by Arthrobacter Corynebacterium) simplex; in US-A-3360439, describing the 1-dehydrogenation 20 of steroids by using A. simplex cells pre-treated with a lower alkanol or lower alkanone such as acetone before mixing with the substrate and a hydrogen carrier; and by Charney and Herzog in "Microbial Transformation of Steroids", Academic Press Inc., New York, pp 4-9, 236-261.

US-A-4035236 discloses a process for preparing 25 9a-hydroxyandrostenedione via fermentation of sitosterol, stigmasterol or cholesterol. US-A-4041055 discloses a general process for the synthesis of medically useful corticosteroids from this androstene. Intermediates involved in this chemistry can possess a 3-keto-4,9(ll)-diene configuration. 2.

Steroid bioconversions, and the taxonomy of micro-organisms which can cause 1-dehydrogenation, are described by Yamane et a^, Biotechnology and Bioengineering 21_ (1979) and by Sonomoto et al, Agric. Biol. Chem. 44 (1980) 1119-1126. Sonomoto et al. studied the 1-dehydrogenation of hydrocortisone by A. simplex. Two immiscible solvents, n-butanol and n-amyl alcohol, were tested but the bioconversion reaction did not proceed in the presence of these solvents. Water-miscible solvents such as 10¾ methanol-water and 10¾ propylene glycol-water were chosen as preferred solvents.

Yamane et studied the 1-dehydrogenation of 4-androstene-3,17-dione by Nocardia rhodocrous in a mixture comprising 50¾ benzene-heptane. The reported advantage of adding this solvent to bioconversion mixtures was to increase the reaction rate by a factor of 180. The preferred electron carrier for the Nocardia rhodocrous activity was phenazine methosulphate. The Nocardia rhodocrous enzyme was not active in the presence of menadione. The reaction was run at an organic solvent level only 20¾ saturated with steroid. It was demonstrated that increasing concentration of the steroid resulted in a decrease in the reaction rate. Oxygen was an essential reagent for the 1-dehydrogenation reaction. The solvent was 50¾ benzene-heptane. However, benzene is a known carcinogen. Phenazine methosulphate is too expensive and too unstable to render the reported microbial activity practical or useful in a production bioconversion process. The solvent is inflammable in the presence of oxygen. The method by which oxygen was introduced into the reactor, without producing an explosive environment, is not discussed.

British Patent Specification No. 2,131,811 discloses a process for preparing a l,2-dehydro-3-keto-steroid which comprises exposing a 1.2- saturated-3-keto-steroid to Arthobacte simplex or Bacterium cvclooxydans, in the presence of an exogenous electron carrier and a water-immiscible solvent comprising an aromatic hydrocarbon.

According to the present invention, a process for preparing a 1.2- dehydro-Δ^-3-keto steroid comprises (i) air-drying or heat-drying cells of Arthobacter simplex or Bacterium cyclooxydans, in the absence of acetone, and (ii) exposing a 1,2-saturated- ^-3-keto-steroid to the 3. air-dried or heat-dried cells, in the absence of an aromatic hydrocarbon.

The reaction of step (ii) is preferably conducted in the presence of an exogenous electron carrier.

Advantages of the invention over the prior art are that the 5 bioconversion results in lower unconverted steroid substrate levels, that the process can be run at higher steroid concentrations, that fewer microbial cells (e.g. A. simplex), are required to convert a given quantity of steroid, and that the process is more tolerant of organic-soluble impurities present in the steroid, in the microbial cells, or produced from 10 the steroid by the cells. Further, A. simplex-catalysed reaction can be performed with more stable and less expensive electron carriers, such as menadione, than have been required for Nocardia rhodocrous. The enzyme in N. rhodocrous must be different from that produced by A. simplex.

The microbes used in the process of the present invention are known 15 as 1-dehydrogenators. Such microbes are listed by Charney and Herzog, supra.

Bacteria which 1-dehydrogenate steroids fall within two general groups established in Bergey's "Manual of Determinative Biology". 8th edition. The procedure described herein has been successfully demonstrated 20 for species from Arthobacter and Corynebacterium, genera that are included in "Part 17. Actinomycetes & Related Organisms".

Two micro-organisms which are publicly available, and are known as 1-dehydrogenation agents for steroids, are suitable for use in the invention. These are Bacterium cyclooxydans, ATCC 12673, and Arthrobacter 25 simplex, ATCC 6946. The former has been examined as a useful micro-organism, but the latter has been used much more extensively, for the 1-dehydroganation of steroids. These micro-organisms are respectively disclosed in US-A-3065146 and US-A-2837464.

The micro-organisms are grown in an aqueous nutrient medium 30 containing: (a) inorganic compounds such as nitrate, ammonium salts or organic nitrogenous compounds, e.g. yeast extract, peptone or cornsteep liquid, to provide nitrogen for growth; 4. (b) a carbon and energy source such as carbohydrates and sugar derivatives, oil, fatty acids and their methyl esters, alcohols, amino-acids or organic acids; and (c) ions and trace elements such as sodium, potassium, magnesium, phosphate, sulphate, manganese, copper, cobalt or molybdenum, in levels supplied by tap water or by the less refined medium ingredients (such as cornsteep liquor).

The organisms require oxygen present in the atmosphere for growth.

The temperature range for growth is 10-45 C for A. simplex. The optimum pH for growth is around neutrality.

The cells are induced for steroid-1-dehydrogenase activity by the addition to the medium of at least 0.005Ϊ w/v of a 1,2-saturated-i 4-3-keto-steroid compound such as androst-4-ene-3,17-dione or cortisone acetate. The inducer can be added at any point during the growth cycle. Cultures grown on nutrients such as lard oil will start synthesising the steroid-1-dehydrogenase rapidly, while cultures grown on glucose require substrate depletion before enzyme synthesis will occur. Incubation is continued for a period of at least 6 hours after the inducer is added.

The cells are then harvested, for drying.

For drying, cells are separated from the nutrient medium and concentrated by conventional means such as centrifugation or flocculation, filtration or ultrafiltration. The separated cells are preferably then dried under reduced pressure at 45 to 85 C, more preferably 45 to 75 C, and most preferably 55 to 75 C. Alternatively, they are air-dried, with heat. Air- and/or heat-drying may also be achieved by spray-drying or tumble drying.

Drying is preferably conducted to a moisture content of 1 to 10, preferably 5¾. Cells are stored at 5 C until used for bioconversion. Active dried cells can also be immobilised by standard techniques, such as entrapment within polyactrylamide gel and collagen or covalent coupling of the cells to a polyelectrolyte carrier as described in Methods in Enzymology, Vol. XLIV (1976), Academic Press, Inc., New York, pp 11-317.

. Bioconversion is accomplished by exposure of the prepared cells to the steroid substrate. Typically, the cells and steroid are suspended in a weakly buffered aqueous solution having a pH of 6 to 10, optimally 7.25 to 8,5. The amount of cells can range from 0.1 to 50 g/1. The steroid is 5 added at a weight ratio of 0.05 to 5.0 (steroidrcells). Cells levels of 8 to 10 g/1, with 5 to 10 g/1 steroid, are preferred when the bioconversion is run in an aqueous environment. An exogenous electron carrier is added in a catalytic amount, e.g. 0.5 mM menadione, to stimulate the reaction. Useful compounds include menadione (preferred), phenazine methosulphate, 10 dichlorophenol-indophenol, 1,4-naphthoquinone, menadione bisulphite, ubiquinones (Coenzyme Q) and vitamin K-type compounds. The mixture is incubated for 0 to 14 days at a temperature of 5 to 45 C. During incubation, the mixture has access to molecular oxygen and is preferably stirred. The rate of 1-dehydrogenation typically decreases with time. The 15 bioconversion can proceed to 98-100% completion in less than 24 hours, using 10 g/1 substrate.

Examples of the procedures which can be used are: (a) The steroid and menadione are suspended in a weakly buffered aqueous solution, followed by the addition of the dried cells. Surfactants 20 such as *Tween 80 can be added in low concentration, e.g. 0 to 5Ϊ, to aid in steroid suspension. (b) The cells are suspended in an aqueous buffer system, followed by the addition of the electron carrier dissolved in ethanol, methanol or acetone (not greater than 5¾ of final volume). The steroid substrate can 25 be added as a dry powder or dissolved (suspended) in a water-miscible organic solvent such as dimethylformamide, ethanol, methanol, acetone or dimethyl sulphoxide. (c) The dried cells are rehydrated in a small volume of buffer; more buffer or organic solvent is then added. Ethanol, acetone, butyl 30 acetate or methylene chloride, for example, is added to give a final organic solvent content of 0-95¾ (vol/vol). The steroid and electron carrier are added to initiate the reaction.

The bioconversion products and unconverted substrate can be recovered from the mixtures by conventional means. Steroids are typically recovered 35 by filtration, followed by extraction of the filter cake with an organic *Tween is Trade Mark 6. solvent such as acetone or methylene chloride. Alternatively, the whole bioconversion mixture can be extracted by mixing with a water-immiscible solvent such as butyl acetate or methylene chloride. The product is then isolated from the organic solvent.

Substrate steroids for use in this invention belong to the 3-keto-androst-4-ene and 3-keto-pregn-4-ene series of steroids. Members of the androstene series include androst-4-ene-3,17-dione, androsta-4,901)-diene-3,17-dione and its 6"-fluoro, 6a-methyl or 16-methyl derivatives, and 1l0-hydroxyandro$t-4-ene-3,17-dione and its derivatives. Among steroids of the 3-keto-pregn-4-ene series which can be used are 17"-hydroxypregn-4-en- 20- yn-3~one and its 16-methyl derivatives; 118,21-dihydroxypregna-4,17(20)-dien-3-one and its 6a-methyl derivative; 20-chloropregna-4,9(11), 17(20)-trien-21-al-3-one; and various 3,20-diketo-pregn-4-enes, including 11,17,21-trihydroxy compounds such as hydrocortisone and its 6" -methyl derivative; 98,118-epoxy-17,21-dihydroxy compounds such as 98,118 -epoxy-17,21-di hydroxy-168-methylpregn-4-ene-3,20-dione; 3,20-diketo-4,9(ll)-pregnadienes such as 17",21-dihydroxypregna-4,9(11)-diene-3,20-dione and its 16a-methyl, 168-methyl and 16a-hydroxy derivatives or 17«-acetate ester; and 3,20-diketo-4,9(ll), 16-pregnatrienes such as 21- hydroxypregna-4,9(ll), 16-triene-3,20-dione and its 6a-fluoro derivative.

The 21-ester derivatives of those steroids containing a 21-hydroxyl group serve as substrates also. The preferred 21-esters are derived from lower fatty acids, e.g. acetic acid, or monocyclic carboxylic acids, e.g. benzoic acid.

The following Examples illustrate the invention. 7.

Example 1 Bacterium cyclooxydans (ATCC 12673) was inoculated into shake flasks containing a medium of cerelose, peptone and cornsteep liquor (6 g/1 of each), pH 7.0. The cultures were incubated on a rotary shaker at 28 C until glucose exhaustion occurred. Cortisone acetate (0.5 g/1) was added at that time and the flasks were incubated for an additional 16 hours. The cells were harvested by centrifugation, washed twice with water and then placed in a low vacuum oven at 45 C until dry. 0.5 g of the dried cells was rehydrated in 50 ml 50 mM phospahte buffer (pH 7.5) with stirring. Menadione was added to the cell suspension as an ethanolic solution (8.6 mg/ml ethanol) at a level of 0.025 ml/50 ml. The substrate was added as a dimethylformamide (DMF) Solution (100 mg 6 -methylhydrocortisone/ml DMF) to a final bioconversion concentration of 0.5 g/1. The mixture was incubated at 28 C with agitation. After 4 hours incubation, the steroid was 91¾ converted. 6 -Methyl prednisolone was recovered by conventional means.

Example 2 Arthrobacter sinplex (ATCC 6946) was grown in shake flasks in a medium containing 6 g/1 glucose, 6 g/1 cornsteep liquor and 6 g/1 of spray-dried lard water. The cultures were incubated at 28 C on a rotary shaker until glucose depletion occurred. At that time, cortisone acetate (0.15 g/1) was added to induce steroid-l-dehydrogenase synthesis. After overnight incubation, the cells were harvested by low speed centrifugation. The cell pellets were placed in a low vacuum oven at 55 C for 24 hours to dry. The dried material was transferred to an air-tight container and stored at 5 C.

The dried cells (10 g/1) were rehydrated in 50 mM phosphate buffer (pH 7.5) by stirring for 30 minutes. Menadione was then added to cell suspension as a dry powder, to a final concentration of 86 mg/1.

Micronised androsta-4,9(ll)-diene-3,17-dione was slurried in DMF and added to the bioconversion mixture at a level of 2.5 g steroid/1 and 2% (v/v) DMF. The mixture was incubated at 31 C with agitation in the presence of air for 24 hours. Androsta-l,4,9(ll)-triene-3,17-dione was recovered by conventional means. 8.

The following is a list of steroids which can be treated in accordance with the procedure of Example 2: androst-4-ene-3,17-dione 6a-fluoroandrosta-4,9(11)-diene-3,17-dione 6a-methylandrosta-4,9(11)-diene-3,17-d i one 16B-methylandrosta-4,9(11)-diene-3,17-dione 17a-hydroxypregn-4-en-20-yn-3-one 17a-hydroxypregn-4,9-(Π)-dien-20-yn-3-o ne 17a-hydroxy-lββ-methylpregna-4,9(11)-dien-20-yn-3-one Π β, 21-dihydroxypregna-4,7(20)-dien-3-one 21-acetoxy-11β-hydroxypregna-4,17(20)-diene-3-one 6a-methyl -Πβ, 21 -dihydroxypregna-4,17 (20 )-dien-3-one 20- chloropregna-4,9(11),17(20)-tri en-21-a1-3-one hydrocortisone 6a-methylhydrocorti sone 21 -acetoxy-Πβ, 17-dihydroxy-16B-methylpregn-4-ene-3,20-dione 21- acetoxy-9a-fluoro-llB,17-dihydroxy-16B-methylpregn-4-ene-3,20-dione 21-acetoxy-9B,lle-epoxy-17-hydroxy-16B-methylpregn-4-ene-3,20-dione 21-acetoxy-17-hydroxypregna-4,9(11)-diene-3,20-dione 21-acetoxy-16α,17-hydroxypregna-4,9(11)-diene-3,20-dione 21-acetoxy-17-hydroxy-16a-methylpregna-4,9(11)-diene-3,20-dione 21-benzoyloxy-17-hydroxy-163-methylpregna-4,9(11)-diene-3,20-dione 21-acetoxy-17-hydroxy-16B-methyhlpregna-4,9(11)-diene-,20-d ione 21-acetoxypregna-4,9(11),16-triene-3,20-dione 21-acetoxy-6a-fluoropregna-4,9(ll), 16(17)-triene-3,20-dione.

These substrates are converted to the following products, respectively: androst-l,4-diene-3,17-dione 6a-fluoroandrosta-l,4,9(ll)-triene-3,17-dione fix-methylandrosta-1,4,9(11)-tri ene-3,17-d i one 163 -methy landrosta-1,4,9(11)-triene-3,17-d ione 17a -hydroxypregna-1,4-dien-20-yn-3-one 17a-hydroxypregna-1,4,9{11)-trien-20-yn-3-one 17a -hydroxy-ΐδβ-methylpregna-1,4,9(11)-trien-20-yn-3-one 113.21- di hydroxypregna-1,4,17(20)-tr ien-3-one 113.21- dihydroxypregna-l,4,17(20)-trien-3-one and its 21-acetate 6 α-methyl-113,21-d i hydroxypregna-1,4,17(20)-tr ien-3-one 9. - chloropregna-l,4,9(11), 17(20)-tetraen-21-al-3-one prednisolone 6a-methylprednisolone 118.17.21- trihydroxy-160-methylpregna-1,4-diene-3,20-dione and its 21- acetate 9 «-fluoro-Π 0,17,21-trihydroxy-160-methylpregna-l,4-diene-3,20-dione and its 21-acetate 90,110-epoxy-17,21-dihydroxy-163-wethylpregna-1,4-diene-3,20-dione and its 21-acetate 17.21- dihydroxypregna-l,4,9{ll)-triene-3,20-dione and its 21-acetate 16«, 17,21-trihydroxypregna-1,4,9(11)-triene-3,20-dione and its 21-acetate 17.21- dihydroxy-16«-methylpregna-1,4,9(11)-triene-3,20-dione and its 21-acetate 21-benzoyloxy-17-hydroxy-16 0-methylpregna-1,4,9(11)-tri ene-3,20-di one 17.21- dihydroxy-163-methylpregna-1,4,9(11)-triene-3,20-dione and its 21-acetate 21-hydroxypregna-1,4,9(11),16-tetraene-3,20-dione and its 21-acetate 6«-fluoro-21-hydroxypregna-l,4,9(11),16-tetraene-3,20-dione and its 21-acetate.

The following is a further list of steroids which can be treated in accordance with the procedure of Example 1 or Example 2: 21 -acetoxy-9 α-fluoro-l10,16«,17-trihydroxypregn-4-ene-3,20-dione 21-acetoxy-6a,9«-difluoro-n 0,16a, 17-trihydroxypregn-4-ene -3,20-dione-16,17-acetonide 21-acetoxy-6«-fluoro-110-hydroxy-16«-methylpregn-4-ene-3,20-dione 21-acetoxy-6"-fluoro-110,17-dihydroxypregn-4-ene-3,20-dione 21-acetoxy-6a,9a-difluoro-110,17-dihydroxy-16«-methylpregn-4-ene-3,20-dione 21-acetoxy-9a-fluoro-110,16«,17-trihydroxypregn-4-ene-3,2O-dione- 16,17-acetonide 21-acetoxy-90,ll0-epoxy-6«-fluoro-16a,17-dihydroxypregn-4-ene-3,2O-dione-16,17-acetonide 21-acetoxy-9 0,110-epoxy-l6«-hydroxypregn-4-ene-3,20-di one 21-acetoxy-9 0,110-epoxy-16«,17-di hydroxypregn-4-ene-3,20-dione-16-l7- acetonide.

. These substrates are converted to the following products, respectively: 9a-fluoro-ll$, 16«,17,21-tetrahydroxypregna-l,4-diene-3,20-dione and its 21 -acetate 6a,9a-difluoro-llB,l(w,17,21-tetrahydroxypregna-l,4-diene-3,20-dione-16,17-acetonide and its 21-acetate 6a -fluoro-HS ,21 -dihydroxy-lfe -methylpregna-1,4-diene-3,20-dione and its 21-acetate 6a-fluoro-116,17,21-trihydroxypregna-l,4-diene-3,20-dione and its 21-acetate 6a,9a-difluoro-llB,17,21-trihydroxypregna-l,4-diene-3,20-dione and its 21-acetate 21-acetoxy-9a-fluoro-llP,16a,17-trihydroxypregna-l,4-diene-3,20-dione-16,17-acetonide 21-acetoxy-93,113-epoxy-6ct-fluoro-16a, 17-dihydroxypregna-l ,4-diene-3,20 -dione-16,17-acetonide 9B,116-epoxy-16a,21-dihydroxypregna-l,4-diene-3,20-dione and its 21-acetate 21-acetoxy-9B,11S-epoxy-16a,17-dihydroxypregna-l,4-diene-3,20-dione-16,17-acetonide.

Examples 3 and 4 Two different steroids were bioconverted with cells of A. simplex in fermentation beer (for comparison) and with dried A. simplex cells. After each bioconversion was judged to be finished (as indicated by no further decrease in residual substrate levels), the steroid in the mixture was extracted and isolated to provide yield data for each condition. The steroids were androsta-4,9(ll)-diene-3,17-dione (S^) and 21-acetoxypregna-4,9(11),16(17)-triene-3,20-dione (S2).

The following Table gives yields obtained by different bioconversions, either Ferm. (fermentation) or Dried (dried cells). "Useful steroid" is defined as either the Δ1 compound which can be used in further synthesis or the 1,2-dihydro substrate which can be recycled with another bioconversion to produce product. If any second crop is obtained, its yield is indicated in brackets. Π.

Substrate Amount (g/1) 10 10 8 8 Bioconversion Ferm. Dried Ferm. Dried Useful steroid (5) 77.2 (2.8) 82.6 (9.9) 18.1 79.6 Δ' Compound (¾) 2.6 (10.4) 100 (95) 59.1 97.8 Residue (¾) 87.4 (89.6) -(5) 40.9 2.1 Fermentation of exhibited poor 1-dehydrogenation. On fermentation of Sg, considerable amounts of other undesirable steroid molecules were also recovered.

Example 5 Arthobacter simplex was grown in a 5-litre "Microferm" fermentor, induced for steroid-1-dehydrogenase synthesis, and harvested by centrifugation. Portions of the recovered cell paste were subjected to two different preparation methods. The first was the procedure disclosed 15 herein which consisted of drying the cells under reduced pressure at 55 C. The second method was the procedure recommended in US-A-3360439 for the preparation of acetone-dried cells; cells were mixed with acetone, harvested and dried at 5 C under reduced pressure. 9 11 The dried cell preparations were then used to bioconvert Δ ’ -20 androstenedione in shake flasks at 10 g/1 cells and 10 g/1 steroid.

Results are given below. Bioconversion activity is calculated, in each case, as a percentage of the total weight of product formed and of substrate added, with respect to the weight of product formed, per hour per cell weight.

% Residual Bioconversion How Dried Time (h) Substrate Activity Acetone 1 98.7 0.013 4 95.5 0.0Π 24 84.1* 0.007 Heat 1 85.1 0.149 4 23.2 0.192 24 7.2* 0.039 (*) Residual levels did not decrease with further incubation. 12.

Example 6 1 g dried A. simplex cells, prepared as described in Example 2, was resuspended in 100 ml 50 mM phosphate buffer (pH 7.5). Menadione, dissolved in 3A ethanol, was added to a final concentration of 86 mg/1. 0.5 g 11P-hydroxyandrostenedione was added to the flask. The mixture was incubated on a rotary shaker at 31 C for one day. When sampled, two products were observed, by thin layer chromatography of a methylene chloride extract. Approximately 95% of the steroid was present as 11 -hydroxyandrosta-l,4-diene-3,l7-dione. The remainder was unconverted substrate.

The following is a list of steroids which can be treated in accordance with the procedure of Example 6: 11P-hydroxy-16e-methylandrost-4-ene-3,17-dione llP-hydroxy-16a-methylandrost-4-ene-3,17-dione 6a-f1uoro-11p-hydroxyandrost-4-ene-3,17-dione 6a-methyl-116-hydroxyandrost-4-ene-3,17-d io ne 1la-hydroxyandrost-4-ene-3,17-dione androst-4-ene-3,11,17-trione.

These substrates are converted to the following products, respectively: 1 IP-hydroxy-16$ -methylandrosta-1,4-diene-3,17-dione IIP-hydroxy-lfo-methylandrosta-l,4-diene-3,17-dione 6a-fluoro-llP-hydroxyandrosta-l,4-diene-3,17-dione 6"-methyl-11P-hydroxyandrosta-1,4-diene~3,17-dione lla-hydroxyandrosta-l,4-diene-3,17-dione androsta-1,4-diene-3,11,17-trione. 13.

Claims

1. A process for preparing a l,2-dehydro-4 -3-keto-steroid, which comprises (i) air-drying or heat-drying cells of Arthobacter simplex or Bacterium cyclooxydans, in the absence of acetone, and 5 (iil exposing a l,2-saturated-a^-3-keto-steroid to the air-dried or heat-dried cells, in the absence of an aromatic hydrocarbon.

2. A process according to claim 1, wherein step (ii) is conducted in the presence of an exogenous electron carrier.

3. A process according to claim 2, wherein the electron carrier is 10 menadione, phenazine methosulphate, dichlorophenolindophenol, 1,4-naphthoquinone, menadione bisulphite, an ubiquinone (Coenzyme Q) or a vitamin K-type compound.

4. A process according to claim 2, wherein the electron carrier is menadione. 15 5. A process according to any preceding claim, wherein, in step (i), the cells are dried to a moisture content of 1 to 10¾. 6. A process according to any preceding claim, wherein, in step (i), the cells are dried under reduced temperature at a temperature of 45 to 85 C. 20 7. A process according to claim 5, wherein the temperature is 55 to 75 C. 8. A process according to claim 1, substantially as described in any of the Examples. Dated this 27th day of July 1983, TOMKINS & CO. ^ppr^cants' Agents, (signed) 4 C L A I M s

5. Dartmouth Road,m DUBLIN 6 14.