JPS5937078B2 - Fermentation production method of androst-1,4-diene-3,17-dione - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the method for fermentation and production of androstol 1,4-diene-3°17-dione, and more specifically, the method for selectively producing androstol by using immobilized and proliferated viable bacterial cells. -1,4-diene-3゜17-dione (hereinafter "ADD")
”).

ADD is an important intermediate for female hormones such as follicular hormone and progesterone, and is a well-known compound useful as a raw material for drugs for treating gynecological diseases and oral contraceptives (pills), and has traditionally been used in the fermentation of sterols. Manufactured using production methods.

For example, Japanese Patent Publication No. 46-17951 and Japanese Patent Publication No. 17951
Publication No. 11998 discloses that microorganisms belonging to the genus Mycobacterium, Arthrobacterium, or Nocardia are cultured in a medium containing sterol in the coexistence of a chelating agent and an inhibitor such as nickel or cobalt. ADD
A method for manufacturing is disclosed, and also disclosed in Japanese Patent Application Laid-open No. 1983-
No. 10, JP-A-55-138394, U.S. Pat. No. 3,684,657, and U.S. Pat. No. 37,597
The specification of No. 91 discloses that ADD can be cured by culturing in a medium containing sterol using a mutant strain that lacks an enzyme that decomposes anthrostane compounds and whose parent strain is a microorganism belonging to the genus Mycobacterium. A method of manufacturing is described.

However, all of these conventionally proposed methods
Since microbial cells are grown and cultured in a state in which they are freely dispersed in a nutrient medium, the microbial cells and the desired product must be separated from the medium separately at the end of the culture, and the isolated bacteria must be separated from the medium. The cells must be discarded as they are, and for the next culture, new microbial cells must be precultured and then used for fermentation production, which is a complicated procedure.

Moreover, in this conventional fermentation production method, in addition to ADD, androst-4-ene-3,17-dione (hereinafter referred to as "4
-AD”) is produced as a by-product, so the target
There were serious drawbacks such as poor yield of DD and long culture time of 240 to 250 hours including preculture time.

The present inventors have conducted extensive research into an effective method for producing ADD without the above-mentioned drawbacks. As a result, the microorganisms to be used are entrapping immobilized in a natural or synthetic polymer gel in a state in which they can grow, and the sterol When the microbial cells entrappingly immobilized in the gel are grown and cultured using 4-AD as a substrate, 4-AD
ADD is selectively (or preferentially) produced with almost no by-products, ADD is obtained at a good yield, and the entrapping immobilized microbial cells can be repeated many times without pre-cultivation. It has been found that it can be used, and the present invention has been completed.

Therefore, according to the present invention, microbial cells having the ability to decompose the side chains of sterols entrappingly immobilized on a solid polymer carrier are cultured in a nutrient medium using sterols as a substrate,
The sterol is converted into androst-1,4-diene-3,1
Provided is a method for producing androst-1,4-diene-3,17-dione (ADD), which comprises selectively converting it to 7-dione (8DD).

The "sterols" used as substrates in the method of the present invention have the following formula.

In the formula, R represents a chain hydrocarbon group having 8 to 10 carbon atoms, and the B ring and/or the C ring may have a double bond. A general term for sterols represented by, for example, cholesterol, stigmasterol, α-1β- or γ-sitosterol, campesterol, ergosterol, brassicucasterol, fucosterol, lanosterol, agnosterol, etc. - Tertiary ester derivatives, such as cholesteryl oleate, cholesteryl palmitate, cholesteryl sulfate, cholesteryl acetate, cholesteryl hemisuccinate, cholesteryl benzoate, cholesteryl phosphate, etc.
C-3 ether derivatives, such as poIJ, t-xyethylene cholesteryl ether, polyoxyethylene lanolin alcohol ether, etc.; or oxidized derivatives thereof;
For example, cholic acid, lithocholic acid, deoxycholic acid, 4
-Cholestagene-3-one, ■, 4-Cholestagene-3
-one, 4.22-cholesfudien-3-one, 19-
Hydroxycholesterol and the like are included, and these can be used alone or in combination of two or more.

These sterols do not necessarily need to be used in pure isolated form; for example, wool fat, lanolin, hydrolysis products of lanolin (wool alcohol), alkaline cleaning oils from fish and squid oils, deodorized scums of vegetable oils, etc. Sterol-containing natural and processed products such as de-sludge, tall oil, etc. can also be used as substrates.

On the other hand, the microbial cells used for the conversion of sterols have the C-17 side chain of sterols [the above formula (I
) has the ability to decompose the group R] in
As long as it has such side chain degrading ability, it can be selected from a wide range of microbial cells without being restricted by its taxonomic position.

Many microorganisms are known to have the ability to decompose sterol side chains [e.g. Agric, Biol.
, Chem-*42, 411 (1978), J.

Amer, Chem, Soc, 87 * 1387 (1
965), Agric.

Biol, Chem, 33.1644 (1969), etc.], even a bacterial cell whose ability is unknown can be easily assayed, for example, by the following screening method.

Sterol (
For example, add cholesterol) to a concentration of 0.1 to 0.5% and inoculate the test bacteria, and then
After culturing in a test tube at ℃, the culture solution was extracted with an equal volume of ethyl acetate, and the extract was developed by thin layer chromatography (developing solvent: chloroform), and the consumption of the substrate sterol and its associated production were determined by iodine coloring. By checking the physical spots, the test microorganisms are selected to have a rough side chain resolving power.

Furthermore, by carrying out mass culturing under the same conditions and confirming the product, the assay for microorganisms capable of decomposing side chains of sterols is completed.

Specific examples of microorganisms having the ability to decompose side chains of sterols include Arthrobacterium, Nocardia, Mycobacterium, Bacillus, Pseudomonas, Corynebacterium, Fusarium, and Brevibacterium. Examples include those belonging to the genus Protaminobacter, genus Microbacterium, genus Serratia, genus Azotobacter, and alkaline earth metals. In particular, those exemplified below can be mentioned as suitable examples.

All of the microbial cells exemplified below are stored in public preservation institutions and can be freely distributed to third parties.

In the examples below, the deposit number is in parentheses, and each abbreviation has the following meaning: ATCC: American Type Culture Contest.
Rection (USA) IFO: Fermentation Research Institute (Osaka) N■: Nagao Institute (Tokyo) IAM: University of Tokyo Applied Microbiology Research Institute (Tokyo) (1) Arthrobafter genus Arthrobafter simplex (ATCC6946)
) (2) Nocardia genus Nocardia coralina (IFO3338) Nocardia erythrophotus (NI9110) 1 (3) Mycobacterium genus Mycobacterium frey (IFO3158) Mycobacterium foleytrum (ATCC6842) Mycobacterium・Deiernhofferi (ATCC-
19340) (4) Bacillus sp.
5) Pseudomonas genus Pseudomonas putida (IFO3738) (6) Corynebacterium genus Corynebacterium equi (IAM1038) (7)
Fusarium genus Fusarium oxysporum (IFO5942) (8)
Brevibacterium genus Brevibacterium lipolyticum (IAM1398) (9) Protaminobacter genus Protaminobacter alboflabus (IFO3707
) (10) Serratia genus Serratia marsetuscens (IFO3046) aυ
Microbacterium lacticum (IAM1640) 02) Streptomyces genus Streptomyces coelicolor (IFO3176) 03) Azotobacter genus Azotobacter vinellandii DAM1078204)
Alkaline earth metal Alcaligenes faecalisis (IAM1015) The microorganisms used in the method of the present invention are not limited to the above-mentioned species, and these microorganisms can be used as parent strains and exposed to radiation, ultraviolet rays, or nitrosine. It is also possible to use mutant strains obtained by treatment with mutagenic agents such as guanidine derivatives.

According to the present invention, the microbial cells are entrapping immobilized on a polymeric solid carrier in a state in which they can grow and used.

Therefore, the solid carrier used for this entrapping immobilization allows microbial cells to proliferate on the carrier surface and/or within the carrier, and is substantially involved in a series of enzymatic reactions involved in the decomposition of the side chains of sterols. Suitable are natural or synthetic polymeric substances that have mechanical strength that does not adversely affect the ADD and preferably does not disintegrate during the manufacturing process of the ADD.

Such polymeric substances include, for example, alginic acid,
Examples include natural polymeric substances such as collagen, K-carrageenan, cellulose, agar, cellophane, and collodion, and synthetic polymeric substances such as polyacrylamide, polystyrene, polybutadiene, polyurethane, polyethylene glycol, and polypropylene glycol.

Among these, K-carrageenan, alginic acid, polyurethane, and polyethylene glycol are particularly suitable.

The entrapping immobilization of the microbial cells onto such a polymeric substance includes:
As long as the microbial cells after entrapping immobilization are maintained in a state where they can proliferate without being substantially killed, and there is no adverse effect on a series of enzymes involved in the side chain decomposition of sterols, it is generally carried out under mild conditions. , can be carried out by any method known per se.

For example, 4 of the above natural polymers (e.g., sodium alginate, K-carrageenan, agar, etc.) in which bacterial cells are suspended.
An aqueous solution at 0 to 50°C is gelled by leaving it at 0°C to room temperature or dropping it into a cooled aqueous solution containing metal ions such as calcium ions, potassium ions, aluminum ions, iron ions, etc. at appropriate concentrations. Method for immobilizing test microorganisms in [Biotechnol,
Bio ocug +19.387 (1977), Eur.
, J. Appl.

Microbiol 8, 241 (1979)],
A method of immobilizing bacterial cells by irradiating them with near ultraviolet rays, ultraviolet rays, or X-rays for a short period of time (1 to 5 minutes) to an aqueous solution of a synthetic polymer in which bacterial cells are suspended CFEBS Letters 6 No-9179 (
1976), Eur, J., Appl, Micr.
obiol.

Biotechnol 6, 207 (1979)] or a method of immobilizing bacterial cells in a polymer gel by simply mixing a bacterial cell suspension with a synthetic polymer having inocyanate groups at both ends CELI r, J. Appl. Mi
crobiol.

Biotechnol, 8, 143 (1979)
] etc., and according to these methods, a series of enzyme groups involved in the side chain decomposition reaction of sterols are not adversely affected in any way.

(The example described here is just one example.

) The amount of microbial cells immobilized on the carrier is not critical and can be varied widely depending on the type of polymer substance used and the type of microbial cells, but the amount of microbial cells immobilized on the carrier may be affected by leakage of microbial cells, enzyme activity, etc. Considering the mechanical strength of the carrier, etc., generally - the amount of microorganisms contained in 2 ml of 100 ml of preculture solution (cultivated for 20 to 50 hours) inoculated with microorganisms from a platinum loop (the amount is Although it varies depending on the bacterial cell, it is approximately 1 x 102 to I
10"), 250 m'if ~
20,! It is advantageous to use i', preferably 1 g to 3 g of polymeric material.

A solid carrier entrapping and immobilizing microbial cells is generally used after being molded into a shape such as a piece, tablet, cube, or film.

Its storage is usually immersed in physiological saline, potassium phosphate buffer (pH 6-8.1-50mM), or sterile water, and stored at 5°C to 30°C, or frozen.

The nutrient medium used for culturing the immobilized microbial cells prepared as described above can be prepared according to the microbial cells used, but in general, for actinomycetes, yeast extract (0%
4%), malt extract (1%), pH 7.0; for molds, malt extract (2%), peptone (0.1
%), pH 7.0, for yeast, malt extract (0.3%), yeast extract (0.3%), pH 7.0, and for bacteria, meat extract (1%), peptone. (Part 1), salt (Part 0, 5), and a pH of 7.0 are often used.

Many of the sterols used as substrates are generally insoluble or sparingly soluble in water, so the culture medium contains surfactants such as polyoxyethylene sorbicun triolate,
An appropriate amount of polyoxyethylene sorbicun monolaurate, polyoxyethylene monostearate, polyoxyethylene monopalmitate, sodium dodecyl ether sulfonate, cetyltrimethylammonium bromide, etc., usually at a concentration of 0.01 to 0.2% by weight. Alternatively, it is advantageous to add glyceride-containing substances, such as soybean lecithin, cottonseed oil, etc., preferably in a concentration of 0.2 to 5% by weight.

In particular, the addition of the latter glyceride-containing substance is effective and suitable for increasing the yield of ADD.

In addition, in order to increase the solubility of sterols etc. in the medium, water-miscible organic solvents such as dimethylformamide, dimethyl sulfoxide, acetone, methyl alcohol, ethyl Alcohol, n-furobyl alcohol, isopropyl alcohol, ethylene glycol, phlopylene glycol, glycerin, etc. may be used.

Furthermore, inhibitors of enzymes involved in the decomposition of steroid skeletons, such as salts of metals such as nickel and cobaltona, and chelating agents, such as α,α'-dipyridyl and 0-phenanthrone, are added to the medium as necessary. It's okay.

Such inhibitors and chelating agents are usually 0.1mM to 1mM.
It is preferable to use an amount that results in a concentration in the range of .

The amount of sterols added as a substrate to the above medium is not critical and can be varied over a wide range, but it is usually 0.01 to 4% by weight, taking into account the solubility and conversion rate of sterols in the medium.
, preferably in an amount such that the concentration is 0.1 to 2% by weight.

The immobilized specimen can be cultured in a medium containing sterols as described above by a method known per se.

For example, the culture temperature is usually 20 to 50°C, preferably 20°C.
~30°C, and the pH of the medium is usually 5.
It can be adjusted to a range of 9 to 9, preferably 6 to 7.

As culture methods, shaking culture using a Sakaro flask and aerated agitation culture using a jar fermenter are easily carried out. The continuous culture method with aeration and agitation is the most practical.

Since ventilation is required, it is appropriate to use a seed type flow-type culture vessel.

Cultivation can be carried out for about 50 to 120 hours under the conditions described above.

As a result, ADD can be obtained with a yield of about 20 to 40%.

After completion of the culture, the produced ADD can be recovered and purified from the culture solution by methods known per se.

For example, when using a batch type incubator, first, the immobilized bacterial cell gel is separated from the culture solution by filtration, and the immobilized bacterial cell gel is incubated again in a new medium containing sterols and used repeatedly. becomes possible.

On the other hand, the culture solution was adjusted to pH 60, extracted with the same volume of dichloromethane, dried over anhydrous sulfuric acid, and the solvent was removed.

White crystals are obtained which are dissolved in methyl alcohol, ethyl alcohol or acetone.

The poorly soluble crystals are substrate sterols, which are filtered and subjected to the reaction again.

On the other hand, the mother liquor is removed, and ADD and 4-AD are separated by column chromatography using methylene chloride as a developing solvent.

Although the ADD and 4-AD thus obtained are highly pure, they can be further purified by recrystallization using n-hebutane.

When carrying out continuous culture, all that is required is to adjust the flowed out medium to pH 6.0 and then repeat the same procedure. According to the method of the present invention described above, unlike the conventional method, -1,4-diene-3°17-dione (8DD) is not only selectively obtained in good yield, but also the following advantages are obtained.

(b) Since the immobilized microbial cells used according to the method of the present invention are continuously incubated in a nutrient medium, the viable microbial cell layer on the carrier gel surface and/or inside the gel can be renewed, and therefore, the immobilized microbial cells can be renewed. It becomes possible to maintain the catalytic activity of the microorganism cells for a longer period of time.

(2) When preparing immobilized microbial cells, only a small amount of 0.1-20 wet microbial cells per 1 g of carrier may be used, eliminating the need for large-scale cultivation of viable microbial cells. be.

This means that androstole 1,4-diene-3,17
- Not only is it possible to reduce the cost of microbial cells required for the production of dione (ADD), but the time required for production is also significantly shortened (about 1/2 of the conventional fermentation method), which is economically and operationally advantageous. It is something.

(3) Although it varies depending on the microbial cells used, most of the immobilized and proliferated microbial cells are localized and clustered close to the surface of the carrier gel, so it is possible to prepare a product with high catalytic ability.

For example, highly hydrophobic sterols that are poorly soluble in water have a very low diffusion rate into the phase, but because viable bacterial cells are localized on the surface of the carrier, substrate sterols easily reach the surface of the phase. can come into contact with living bacterial cells, and the conversion reaction can proceed efficiently.

Furthermore, since the proliferating bacterial cells are densely packed on the surface, even in complex sequential reactions such as the lateral decomposition reaction of sterols, there are few by-products and the final product ADD can be converted very efficiently. converted.

(4) As mentioned above, the living bacteria involved in the conversion of sterols are localized on the surface of the carrier or, depending on the bacteria, inside the carrier, and therefore are not dispersed freely in the medium as in conventional fermentation methods.

Therefore, it is possible to separate the bacterial cells from the medium containing the product by simply passing through the phase at the end of the conversion reaction, especially in a medium containing a poorly water-soluble product such as ADD. Since it is not possible to remove the bacterial cells by centrifugation, the method of the present invention using immobilized and proliferated bacterial cells is very advantageous in terms of operation.

Furthermore, the isolated and recovered immobilized bacterial cells can be used repeatedly by incubating them in a new medium containing sterols, which is also economically advantageous.

The present invention will be further explained below with reference to Examples.

Example-1 A medium containing yeast extract (1% by weight), meat extract (1% by weight) and peptone (1% by weight) was adjusted to pH 7.0,
Sterilization was performed at 120°C for 20 minutes.

This medium contains Arthrobafter simplex (ATC).
C6946) was inoculated with a platinum loop, 140 times/at 30°C.
Cultured for 24 hours under shaking for 24 hours.

On the other hand, 4 g of K-carrageenan was dissolved in 100 ml of physiological saline by heating to 50°C, and 2 ml of the preculture solution containing viable cells prepared above was added to 25 ml of this solution and mixed thoroughly, followed by injection. The mixture was packed into a cylinder, dropped into a cooled divalent potassium chloride solution, and immobilized on bead-shaped (7) K-carrageenan gel.

After thoroughly washing this gel with sterile water, yeast extract (1% by weight)
), meat extract (1% by weight), peptone (1% by weight) and soybean lecithin (2.5% by weight) (pH 7,
0) Incubation was performed at 30° C. for 24 hours under shaking at 140 times/min at 100 d, and then 30079 (0.3% by weight) cholesterol was added as a substrate.

After incubation for 8 hours under the same conditions, α,α'-dipyridyl 15.6μ (0,016
weight) was added and incubated again under the same conditions for 72 hours.

After the reaction, the gel was filtered under aseptic conditions, the medium was extracted twice with dichloromethane (50 ml), and the reaction solution was subjected to TLC.
- As a result of quantitative analysis using a scanner, 1°4-androstadiene-3,17-dione (AD, D) was obtained in a yield of 36%.

Example-2 The immobilized bacterial cell gel prepared in Example-1 was added again to a new medium containing cholesterol, and the same operation as in Example-1 was performed, and the immobilized bacterial cell gel was repeatedly used. The ADD yield did not decrease, and no leakage of bacterial cells from the gel was observed during this period.

The results are shown in Table 1 below. Example-3 Pre-culture was carried out in the same manner as in Example-1, and 2 ml of this pre-culture solution was added to 50 rf of the sodium alginate aqueous solution of Part 2.
The mixture was added to Ll, mixed, and dropped into a cooled aqueous solution of 50 mM calcium chloride from a spray tube to prepare bead-shaped calcium alginate-immobilized bacterial cells.

After thoroughly washing this gel with sterile water, this gel was incubated in the same manner as in Example 1.
DD was obtained with a yield of 25%.

Example-4 2 moles of toluene diisocyanate with an average molecular weight of about 20
00-3000 and reacted at 80°C for about 3 hours to produce a polyurethane having an average molecular weight of about 2500-3500, which has one incyanate group at both ends and polyethylene oxide in the main chain. adjusted.

On the other hand, Arthrobafter simplex (ATCC6946) was pre-cultured in the same manner as in Example 1, and 2 ml of this pre-culture solution was mixed with polyurethane I prepared as described above.
After adding g and mixing thoroughly to immobilize the bacterial cells in the gel,
The gel was left to stand at 4° C. for about 1 hour to fully harden, and the obtained immobilized product was cut into cubes of 3×3×3 mm.

When the immobilized gel cells thus prepared were incubated in a medium containing cholesterol in exactly the same manner as in Example 1, ADD was obtained at a yield of 30%.

Example 5 Arthrobafter simplex (ATCC6946) was precultured in the same manner as in Example 1, and 1 g of acrylamide and 50 μm of NN' to methylenebisacrylamide were dissolved in 2 ml of this preculture solution.

Next, 1 ml of a 5 wt% aqueous solution of NNN'N'-tetramethylenediamine and 1 rILl of a 2.5 wt.
The mixture was left at room temperature for 5 minutes and then at room temperature for 1 hour to obtain immobilized bacterial cells.

When this immobilized product was cut into cubes of 2 x 2 x 2 mm and incubated in a medium containing cholesterol in the same manner as in Example 1, ADD was obtained with a yield of 20%. Ta.

Examples 6 to 17 A carrageenan-immobilized bacterial cell gel was prepared by the method shown in Example 1 using 2 ml of the preculture solution cultured as in Example 1 in a medium suitable for culturing various microbial cells. I got it.

This was incubated in a medium having the medium composition shown in Table 2 below to attempt conversion to cholesterol-free ADD.

The results are shown in Table 3 below.

However, the concentration of soybean lecithin in each region is 2.5% by weight, and the concentration of αα′α diirisyl is 0.016% by weight.
It is.

Further, the substrate cholesterol concentration was 0.3%.

The pH in both cases is 7.0.

Examples 18-23 The same procedure as in Example 1 was carried out except that the sterols shown in Table 4 below were used instead of cholesterol as the substrate, and the K-carrageenan-immobilized growing fungus Arthrophta simplex (ATCC6946) was used. Incubation was performed using

The results are shown in Table 4 below. Example 24 Maleated polybutadiene 10 with an average molecular weight of about 2000
A monoester derivative of maleated polybutadiene was obtained by reacting 0 g with 30 g of 2-hydroxyethyl methacrylate in toluene.

To this was added 1.5 g of benzoin ethyl ether to obtain liquid maleated polybutadiene (hereinafter abbreviated as PBM-2000).

Arthrobacter simplex (ATCC6946) was precultured in the same manner as in Example 1, and this preculture solution 2
rni! was added to a 3M benzene solution containing 1g of the PBM-2000 resin synthesized as described above, mixed thoroughly in the coexistence of Tween-85 (0.2%) as a surfactant, and then mixed into a 4×4 solution. After pouring into a framed transparent polyester film and covering the top and bottom with the same film, it was irradiated with near ultraviolet light of 300-360 nm for 3 minutes.

In this way, a film-like gel of Earthbacter simplex immobilized on a resin called PBM-2000 was obtained.

This was cut into 5 x 5 pieces, washed with sterile water, added to a medium containing cholesterol and incubated in the same manner as in Example 1, and the ADD was 5 with a yield of 15%.
4-AD was obtained with a yield of %.

Example 25 200 ml of agar was added to 1077 Zl of distilled water, dissolved by boiling, and then cooled to 50°C.

When the temperature of the solution reached 50°C, Arthrobutter Simplex (ATCC6) was added in the same manner as in Example 1.
After adding culture solution 1a pre-cultured with 946) and mixing thoroughly, the mixture was left to stand at room temperature for 20 hours while maintaining sterile conditions.

The gel thus obtained was cut into 3X3X3 cubes, which were soaked in glutaraldehyde (1% aqueous solution) for 10 minutes and then in hexamethylene diamine (1% aqueous solution) for 5 minutes.
After soaking for a minute, it was washed with sterile water.

When the thus obtained gel was incubated in a medium containing cholesterol in the same manner as in Example 1, ADD was obtained with a yield of 30%, and 4-AD with a yield of 4%.
was gotten.

Comparative Example A medium containing yeast extract (1% by weight), meat extract (1% by weight) and peptone (1% by weight) was adjusted to pH 7.0,
Sterilization was performed at 20°C for 20 minutes.

This medium contains Arthrobafter simplex (ATC).
C6946) was inoculated with a platinum loop, 140 times/at 30°C.
Cultured for 24 hours under shaking for 24 hours.

This culture solution 2a was mixed into 100 ml of a medium (pH 7.0) consisting of yeast extract (1% by weight), meat extract (1% by weight), peptone (1% by weight) and soybean lecithin (2.5% by weight).
ILl plus 2 ml under 140 shaking/min at 30°C.
Incubation was carried out for 4 hours, after which 3007Q (0.3% by weight) cholesterol was added as a substrate.

After incubation under the same conditions for 8 hours, αα'α dilysyl 15.6% (0,016% by weight) was added as an inhibitor, and the mixture was incubated again under the same conditions for 72 hours.

After the reaction was completed, the medium was diluted and extracted twice with methane (50 m7), and the reaction solution was quantified using a TCL-scanner.
The yield of ADD) was 29%.

Claims (1)

[Claims] 1. Using sterols as a substrate, microorganisms having the ability to decompose the side chains of sterols entrappingly immobilized on a polymeric solid carrier are cultured in a nutrient medium, and the sterols are converted into androst-1. , 4-diene-3°17-dione. , 4-diene-3°17-dione. 2. The microbial cells are of the genus Arthrobacter, Nocardia, Mycobacterium, Bacillus, Pseudomonas, Corynebacterium, Fusarium, Brevibacterium, Protaminobacter, Microbacterium, Serratia The method according to claim 1, which is a bacterial species belonging to the genus Azotobacter or alkaline earth metals. 3. The method according to claim 1, wherein the sterol is a sterol, a C-3 ester derivative thereof, a C-3 ester derivative thereof, or an oxidized derivative thereof. 4. The method according to claim 1, wherein the solid carrier is a natural or synthetic polymeric substance.