JPS6247518B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides 9α-hydroxy-androst-4-ene-3,17-dione (hereinafter referred to as 9α-dione) produced by microorganisms.
-OH-4AD). Specifically, using a microorganism belonging to Corynebacterium equi (hereinafter abbreviated as C), 9α-OH-
The main purpose is to manufacture 4AD. Several methods have been known for a long time to convert 4AD to 9α-OH-4AD using microorganisms. For example, Nocardia sp.
[Dodson et al., Journal of
American Chemical Society, Volume 83;
4631 (1961) U.S. Reference], Cylindrocarpon radicicola
radicicola) [U.S. Pat. No. 3,065,146 and
3080298], Ascochytalinicola [US Patent No. 3116220]
This is a method using a method such as [see the specification of No. 1].
However, these techniques have not been put into practical use to date. The present inventors have recently discovered that by using C. equi, a genus that has not been described in conventional literature, it was possible to produce and accumulate 9α-OH-4AD from 4AD at a high concentration and yield that goes beyond conventional wisdom. We have discovered what can be done and arrived at the present invention. That is, the gist of the present invention is that androst-4
-9α- using a microorganism belonging to Corynebacterium equi using ene-3,17-dione as a substrate.
A method for producing a 9α-hydroxy steroid characterized by producing OH-4AD. The present invention will be explained in detail below. Bacteria belonging to C. equi used for the conversion of 4AD to 9α-OH-4AD include:
ATCC 6939, 7698, 7699, 10146, 21107,
21280, 21329, 21521 and 21690 each bacteria and
Bacterium IAM1038 is known. The conversion reaction may be carried out either in the state of growing cells or in the state of resting cells. In the case of resting bacterial cells, they are cultured in a medium that does not contain insoluble materials, collected by centrifugation, etc., and resuspended in a buffer containing 4AD, physiological saline, water, etc. to perform a conversion reaction. Stirring is required during the reaction, and in some cases, aeration is also required. Next, to explain in detail the case of using proliferated bacterial cells, a medium containing a carbon source, a nitrogen source, inorganic salts, and nutrients such as vitamins if necessary is sterilized, C. equi is inoculated into the medium, and cultured with shaking or aeration. Perform agitation culture. The medium used is as follows. Examples of carbon sources include n-paraffin,
Hydrocarbons such as α-olefin and xylene; Alcohols such as methanol, ethanol, glycerin, and higher alcohols; Organic acids and their salts such as succinic acid, acetic acid, and higher fatty acids; Starch, maltose, sucrose, glucose, rhamnose, etc. Sugars; oils and fats such as linseed oil, soybean oil, sesame oil, corn oil, rapeseed oil, palm oil, fish oil, beef tallow, and tallow oil. Natural nutritional sources including carbon sources, nitrogen sources and other nutritional substances include, for example, vegetable oil meal such as flaxseed oil, defatted soybean meal, rapeseed oil meal; molasses including high test molasses, refined molasses and xylose molasses; bagasse , Corn Cob, Alpha Alpha, Corn Staple Liquor, Day Tailor's Soluble, Flavor Liquid, Fish Meal, Bran, Meat Extract, Yeast, Yeast Extract, Potato Extract, Malt Extract, Gluten, Peptone, Glutamate, Asparagine, Glycine, Casein, Examples include casein decomposition products and skim milk. Examples of inorganic substances added to the medium include nitrogen sources such as ammonium sulfate and ammonium chloride; potassium and phosphorus sources such as dipotassium hydrogen phosphate; salts such as iron, copper, magnesium, manganese, cobalt, zinc, and calcium; and molasses. Examples include ashes of natural products. Other vitamins can also be added if necessary. The composition of the medium is selected depending on the type of fungi used, but a carbon source, nitrogen source, potassium, phosphorus, and magnesium are essential components of the medium. If an antifoaming agent is required, a known antifoaming agent may be added. Specific examples include polyoxyalkylene glycol, but it is not always necessary to add an antifoaming agent. Surfactants are often effective as emulsifiers for sterols. As the surfactant, nonionic and anionic surfactants are preferred. Specific examples include polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, polyethylene glycol monostearate, and the like. The reaction temperature is usually 20°C for both growing and resting bacterial cells.
~40°C, but around 28-37°C is optimal. The pH of the reaction solution (buffer, culture medium, etc.) is usually 5 to 10.
It is preferably adjusted to 6 to 9. If the bacterial concentration is low, this conversion reaction will not proceed, so adjust or culture the C. genus bacteria concentration to 1 x 10 ⁹ /ml or more, preferably 1 x 10 ¹⁰ /ml or more. Things are terrible. 4AD, which is a raw material for the method of the present invention, may be sterilized together with the medium, or may be added to the medium after the start of culture. It is also possible to add in portions. In this case, it is added as it is after dry sterilization or moist heat sterilization, or it is added as a solution after being dissolved in a solvent such as dimethylformamide or methanol, or it is added as a finely dispersed suspension by ultrasonication. At that time, if a surfactant is added at the same time, 4AD
dispersion is often promoted. The time required for conversion cannot be determined uniformly depending on the bacterial concentration, temperature, pH, etc., but it is usually better to stop the reaction within 7 days, and in many cases it is better to stop the reaction within 1 to 2 days. Normally, the generated 9α-OH-4AD will undergo further decomposition and decrease as time passes, so quantify the amount generated over time, and if it starts to decrease, heat sterilization, acid/alkali addition, organic solvent addition, etc. Need to stop. The enzyme that converts 4AD to 9α-OH-4AD, that is, 9α-hydroxylase, is generally considered to be an inducible enzyme, so a small amount of C. equi seed culture is required.
It is better to add 4AD to induce the enzyme. After the conversion reaction, 9α- produced in the culture solution etc.
OH-4AD can be collected, separated and purified by known methods. For example, 9α-OH-4AD is extracted from a culture solution with several volumes of an organic solvent that is not easily miscible with water such as ethyl acetate, and the solvent is distilled off from the extract. uses porous resin, silica gel, alumina, etc. as an adsorbent, and petroleum ether,
benzene, chloroform, ether, acetone,
It can be separated from the decomposition product of 9α-OH-4AD, the substrate 4AD, other steroids, etc. by column chromatography using methanol, ethyl acetate, etc. as an eluent. However, if 9α-OH-4AD is the main component, pure crystals of 9α-OH-4AD can be obtained by dissolving it in acetone, hexane, cyclohexane, etc. and repeating recrystallization without using chromatography. 4AD used as a substrate in the method of the present invention is usually produced by microorganisms using sterols as a substrate. Although 4AD may be isolated as described above as a substrate, a reaction solution produced by a microorganism using sterols as a substrate may also be used as it is. Next, sterols are
9 by producing 4AD and inoculating it with C. equi.
A method for producing α-OH-4AD in a medium will be described in detail. First, the microorganism used to produce 4AD from sterols may be any known 4AD-producing microorganism. Such microorganisms include, for example, Mycobacterium parafitatum complexus MCI-0807 (see patent application "Method for producing androstol-4-ene-3,17-dione" dated April 17, 1972). , MCI-0801 bacteria, MCI-
Bacterium No. 0802 (see specification of patent application No. 52-124188), Bacterium Nocardia ariena MCI-0710, MCI-
Bacterium No. 0711 (see specification of patent application No. 133225/1971), Mycobacterium fortuitum NRRLB-8153
Mycobacterium sp. NRRL B-3805 (see US Pat. No. 3,759,791), Mycobacterium sp.
4AD-producing mutant strains such as Bacterium No. 3683 (see US Pat. No. 3,684,657) are known. It is not necessary to use mutant strains for 4AD production. For example, as shown in Japanese Patent Application Laid-Open No. 49-25192, dehydroepiandrosterone (hereinafter referred to as
Arthrobacter and DHA (abbreviated as DHA) as raw materials
4AD may be produced in a buffer solution or a medium using a non-mutated strain such as Simplex IAN No. 1660. Here, sterols refer to various sterols or their oxidized intermediates collectively. Various sterols have a hydroxyl group at the C-3 position of the perhydrocyclopentanophenanthrene nucleus, a double bond at the C-5 position, and a side chain with 8 to 10 carbon atoms at the C-17 position. In some cases, it may have a double bond at C-7, C-8, C-9 (11), etc. Examples of such various sterols include cholesterol, stigmasterol, campesterol, β-sitosterol, ergosterol, brassicasterol, fucosterol, lanosterol, agnosterol, dihydrolanosterol, dihydroagnosterol, α-sitosterol, etc. It will be done. Preferred sterols are cholesterol, campesterol, stigmasterol and sitosterol. Sterol-containing natural and processed products, such as alkaline-washed dark oils from fish and squid oils, as well as deodorized scums of vegetable oils, deodorized sludge, and tall oil, are likewise used as raw materials. Furthermore, oxidized intermediates of various sterols are also used as substrates. Such oxidation intermediates include 4-en-3-one or 1,4-one of various sterols.
Examples include dien-3-one derivatives, and specific examples include cholest-4-en-3-one, cholest-1,4-dien-3-one, cholest-4,22-dien-3-one, and cholest-4,22-dien-3-one. on, 22,23-bisnorcholar-5-cholenic acid-3-β-ol,
22,23-bisnorchol-4-en-3-one-22
-Euctoxacid, pregnenolone, progesterone, DHA, etc. As for the culture method, it is preferable to culture under optimal conditions for 4AD-producing bacteria while producing 4AD, and after inoculating C. equi, to cultivate under optimal conditions for C. equi. Therefore, if the pH becomes extremely unstable during 4AD fermentation, it is possible to readjust the pH to around 7 by adding acid or alkali when inoculating with C. equi, and it is better to do so. There are many cases. It is generally better to inoculate C. equi when 4AD production is near its peak, rather than when 4AD production has just begun.
α-OH-4AD production will increase. When inoculating with C. equi, 4AD fermentation does not necessarily need to be stopped by heat treatment or the like. Even if C. equi is inoculated to a place where 4AD fermentation is still progressing, and both bacteria are mixed and cultured, 4AD to 9
α-OH-4AD is produced in large quantities. Although two-stage culture is possible in this way, the 4AD-producing fermentation is carried out in a medium that is optimal for the 4AD-producing bacteria, and usually there is no need to add new ingredients to it, and C. equi is inoculated and 9α- There is nothing wrong with generating OH-4AD. Just after 4AD fermentation
When it seems that nutrients are insufficient for adequate growth of C. equi, new ingredients such as meat extract, defatted soybeans, NH ₄ NO ₃ may be sterilized and added to the culture medium before or after inoculating C. equi. . The medium used for 4AD production fermentation varies depending on which 4AD producing bacteria are used, but as a general rule,
All of the medium components described above can be used as a medium for C. equi. The amount of inoculation of C. equi culture is usually 0.1 to 30%, preferably 1 to 30%, per culture of 4AD producing bacteria.
It is 15%. The product of the present invention, 9α-OH-4AD, is an important substance as an intermediate for the production of various steroids. Not only can various sex hormones be derived from this substance, but also because it has a hydroxyl group at the 9α position,
It has the great advantage of being able to easily introduce a hydroxyl group into the 11β position in high yield by an organic chemical reaction without relying on a microbial reaction. Therefore, this substance is an important substance as a raw material for the production of corticoids (corticoid hormones), which usually have a hydroxyl group at the 11β position. According to the present invention, 9α-OH-4AD can be produced in a medium at a high concentration of, for example, 0.5% or more. Also, 4AD from sterol, 9 from 4AD
By carrying out the two-step reaction of α-OH-4AD using fermentation, 9α-
This made it possible to produce OH-4AD at low cost. The present invention will be explained in more detail in the following examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof. In the Examples below, 9α-OH-4AD and other steroids were quantified by gas chromatography. Also, in the examples below, percentages are by weight. Example 1 Glucose 1.0%, meat extract 0.3%, peptone 1.0
%, salt 0.5% and water (PH7.0)
Dispense 100ml of into a 500ml kolben with a shoulder, and heat at 120℃.
Steam sterilize for 20 minutes. In this C. equi (equi)
One platinum loopful of ATCC No. 21329 was inoculated and cultured at 30°C for 52 hours using a reciprocating shaker with an amplitude of 7 cm and 120 reciprocations/min. 4ml of this seed culture solution, 4.0% cottonseed meal, 1.5% yeast
%, soybean oil 1.5%, NaNO ₃ 0.2%, K ₂ HPO ₄ 0.1%,
Main culture medium consisting of _MgSO4・_7H2O0.1g and water
Contains 50ml (PH7.0, steam sterilized at 120℃ for 20 minutes)
Inoculate one 500ml shoulder flask. Main culture is
Sterilized 4AD (powder) was carried out at 30°C under reciprocating shaking conditions of 120 reciprocations/min and an amplitude of 7 cm, and 30 hours after the start of culture.
Add 1.5 g aseptically. Thereafter, the culture is continued, and the culture is stopped 64 hours after the addition of 4AD, and the culture solution is extracted with 200 ml of ethyl acetate. As a result of gas chromatography analysis of 9α-OH-4AD in this extract, the presence of 509 mg of 9α-OH-4AD was confirmed. Since there was almost no raw material 4AD, there was no need to apply it to column chromatography.
As a result of recrystallization from acetone to heptane system, approx.
We were able to obtain 400 mg of pure 9α-OH-4AD crystals. On the other hand, it has the same composition as above, but only 1.5g from the beginning.
Prepare 50 ml of main culture medium containing 4AD in a 500 ml shoulder flask. After steam sterilizing this, the same amount of the aforementioned seed culture is simultaneously inoculated. After culturing this under the same conditions without adding 4AD midway, the culture is stopped after 94 hours. This culture solution was extracted with 200 ml of ethyl acetate, and the content of 9α-OH-4AD therein was measured, and it was confirmed that 423 mg of 9α-OH-4AD was present. Example 2 Glucose 1.0%, meat extract 0.3%, peptone 1.0
%, salt 0.5%, 4AD 0.02%, and water (PH 7.0), 100 ml of the seed medium (PH 7.0) is dispensed into a 500 ml shoulder bag and steam sterilized at 120°C for 20 minutes. One platinum loop of C. equi ATCC 21329 was inoculated into this.
48 on a reciprocating shaker with an amplitude of 7 cm and 120 reciprocations/min at 30℃
Incubate for hours. Combine all of these seed cultures and collect the bacteria by sedimenting them using refrigerated centrifugation. After washing twice with physiological saline, the cells are suspended in 25 ml of phosphate buffer (PH7.5, 1/20M) containing 1.0% 4AD. Put 25 ml of this suspension into three 500 ml shoulder flasks and shake under the same conditions as for seed culture. 7, 12, 27
After shaking for an hour, the mixture was extracted with 200 ml of ethyl acetate, and the 9α-OH-4AD content in the extract was measured. Table 1 shows the results. On the other hand, the results of seed culture without adding 4AD to the seed medium and then repeating the same procedure are shown in Table 1.

[Table] Example 3 C. equi was grown in the same manner as in Example 2.
Bacteria ATCC21329 was seeded and cultured. 2ml of this seed culture
, defatted soy flour 4.0%, molasses 4.0%, yeast 2.0%,
_{K2HPO4 0.25} %, _MgSO4・_7H2O0.1 %, starch 2.5
%, soybean oil 1.0%, 4AD 1.0%, and water in a 500 ml shoulder flask (PH 7.0,
Steam sterilization at 120℃ for 20 minutes). 30℃, 120
Culture is performed under the conditions of reciprocation/min and 7 cm amplitude, and the culture is stopped after 50 hours. After that, all the culture fluid was extracted with 200 ml of ethyl acetate, and 405 mg of 9α was found in the extract.
-OH- The existence of 4AD has been confirmed. On the other hand, 500 ml containing 50 ml of main culture medium of the same composition without the addition of 4AD.
Kolben with shoulders (PH7.0, 120℃, steam sterilization for 20 minutes)
Start culturing by inoculating 2 ml of the above-mentioned seed culture solution into two bottles, and at 30 hours, add 2 ml of dissolved 500 mg of 4AD.
of dimethylformamide was added to each colben. After that, the culture of one tube was stopped at 41.5 hours, and the culture solution was extracted with 200 ml of ethyl acetate, and the presence of 400 mg of 9α-OH-4AD was confirmed in the extract. The other one was cultured for 65.5 hours after adding 4AD, and extracted with 200ml of ethyl acetate.
The amount of α-OH-4AD has already decreased, and 275 mg of 9α-
OH-4AD was confirmed. Example 4 Glucose 1.0%, meat extract 1.0%, peptone 1.0
% and 500 ml of seed medium (PH7.2) consisting of water
ml in a flask with a shoulder, steam sterilized at 120°C for 15 minutes, inoculated with one platinum loop of Mycobecterium paraphytitutum complex MCI-0807 (Feikokuken application form acceptance number 4476), and heated to 30°C. at 120
Seed culture is carried out for 48 hours under reciprocating shaking conditions of reciprocating motion/min and amplitude of 7 cm. Add 2 ml of this seed culture solution to 4.0 mL of crushed whole soybeans.
%, _NaNO3 0.2%, _{K2HPO4 0.2} %, _MgSO4・
Five 500 ml shoulder flasks (PH 7.0, steam sterilized at 120° C. for 20 minutes) containing 100 ml of main culture medium consisting of 0.1% 7H ₂ O, 1.0% cholesterol and water are inoculated. The main culture is performed at 30°C under conditions of reciprocating shaking at 120 reciprocations/min and an amplitude of 7 cm. PH at 200 hours after the start of culture
was aseptically adjusted to around 7, and further inoculated with 8 ml of C. equi ATCC No. 21329, which was seed cultured in the same manner as in Example 1. After inoculation, 30 minutes under the same shaking conditions.
Continue culturing for 48 hours at °C. After stopping the culture, all the culture fluids were combined and extracted with ethyl acetate in step 2. The steroid content in the extract was measured. As a result, 9α-OH-
4AD1.06g, cholesterol 0.35g, 4AD0.07
g, 20α-hydroxy-pregn-4-ene-3
- The presence of 0.18g of On was confirmed. Example 5 Glucose 1.0%, meat extract 1.0%, peptone 1.0
% and 500 ml of seed medium (PH7.2) consisting of water
Dispense into ml shoulder flasks, steam sterilize at 120°C for 15 minutes, and then remove Mycobacterium parafuthuitum.
Inoculate one loopful of Complexus MCI-0807.
Under conditions of reciprocating shaking at 30°C, 120 reciprocations/min, and an amplitude of 7 cm.
Incubate the seeds for 48 hours. Add 20ml of this seed culture solution to cottonseed meal.
4.0%, yeast 1.5%, soybean oil 1.0%, NaNO ₃ 0.2%,
_{800 ml of} main culture medium ( _PH
7.0, steam sterilized at 120°C for 20 minutes). The main culture was carried out at 30°C under the conditions of reciprocating shaking at 100 cycles/min and an amplitude of 7 cm, and at 240 hours after the start of culture, one plant was adjusted to pH around 7 without heat sterilization, and at the same time, the same procedure as in Example 2 was carried out. Inoculate 50 ml of C. equi ATCC No. 21329, which was seed-cultured. The other one was steam sterilized at 120℃ for 30 minutes.
After cooling, immediately adjust the pH to around 7, then use the same C.equi
Inoculate 50 ml of the seed culture of ATCC No. 21329. After inoculation, resume culturing in two flasks at the same time. After reopening
The culture was stopped at 69.5 hours, and the culture solution in each flask was extracted with 2.4 ml of ethyl acetate. As a result, C. equi
2.42g for those who were heat sterilized before inoculation, and 2.42g for those who were not heat sterilized.
2.88g of 9α-OH-4AD was produced. Example 6 Contains 100 ml of seed medium (adjusted to pH 7.2) consisting of 2.0% glycerol, 2.0% ground whole soybeans, 1.0% yeast extract, 0.2% NaNO ₃ , 0.1% K ₂ HPO ₄ and water.
Steam sterilize a 500ml shoulder bag at 120℃ for 20 minutes. One platinum loop of Mycobacterium paraphytitutum complex MCI-0807 is inoculated into this. After inoculation, 120 reciprocations/min at 30℃, amplitude 7cm
Culture the seeds for 68 hours under reciprocating shaking conditions. Add 4 ml of this seed culture solution to 4.0% defatted ground soybean, 1.5% yeast, 1.0% soybean oil, 0.1% NH ₄ NO ₃ , 0.1% MgSO ₄ ,
Inoculate 50 ml of a main culture medium consisting of 0.1% K ₂ HPO ₄ 0.05% nonionic surfactant "Tween-60" manufactured by Kao Atlas, 2.0% of the sterols shown in Table 2, and water. After inoculation, culture at 30°C at 120 cycles/min and 7cm amplitude. After culturing for 240 hours, 4 ml of a seed culture of C. equi ATCC No. 21329 cultured in the same manner as in Example 2 was inoculated. At the same time NH ₄ NO ₃ 50
Add 5 ml of sterile water containing 200 mg of yeast extract. Then adjust the pH to around 7. After this, the culture was continued for another 48 hours, and the culture was stopped at the 48th hour, and the steroid was extracted from the culture solution with 200 ml of ethyl acetate. The amount of 9α-OH-4AD in this extract is listed in Table 2.

[Table] Example 7 Glucose 1.0%, Meat extract 1.0%, Peptone 1.0
% and 500 ml of seed medium (PH7.2) consisting of water
Dispense into two ml shoulder flasks, steam sterilize at 120°C for 15 minutes, and then remove N. ariena MCI-0710 (Feikoken Bacteria No. 4075) and MCI-0711 (Feikken Bacteria No. 4075). 4076) was inoculated with one platinum loopful of each strain at 30°C for 120 min.
Seed culture is carried out for 72 hours under reciprocating shaking conditions of reciprocating motion/min and amplitude of 7 cm. Add 2 ml of this seed culture solution to each crushed whole soybean.
6.0%, glycerin 4.0%, _{K2HPO4 0.15} %,
100 ml of main culture medium (A) consisting of NaNO ₃ 0.2%, MgSO ₄ 7H ₂ O 0.01%, cholesterol 1.0%, and water
(PH7.0, 120℃, steam sterilized for 20 minutes) into five 500ml shoulder flasks each. The main culture was carried out at 30°C under reciprocating shaking conditions of 120 cycles/min and a shaking width of 7 cm. 180 hours after the start of culture, the pH of the culture solution was adjusted to around 7, and C. equi was cultured in the same manner as in Example 2. (Equi) Inoculate 1 ml of the seed culture of ATCC No. 21329. After inoculation, continue culturing under the same shaking conditions for another 24 hours. Thereafter, 5 culture fluids were combined and extracted with ethyl acetate in step 2. MCI―
240 mg of 9α-OH-4AD in the extract of Bacterium No. 0710
It was being generated. On the other hand, 180 mg of 9α-OH-4AD was produced in the extract of MCI-0711. Example 8 Glucose 1.0%, meat extract 0.5%, peptone 0.5
Seed medium (PH7.2) consisting of % and NaCl 0.5% 100
Dispense ml into a 500ml flask with a shoulder and steam sterilize it at 120℃ for 15 minutes, then Mycobacterium sp.
NRRL B-3805 bacteria was inoculated with one platinum loop and heated to 120℃ at 30℃.
Seed culture is carried out for 48 hours on a reciprocating shaker with a vibration width of 7 cm and a reciprocating motion per minute. Add 2 ml of this seed culture to cholesterol 0.8
%, yeast extract 0.1%, meat extract 0.3%, peptone
0.5%, ground whole soybeans 4.0% and tallow fat 0.5%
Inoculate a 500 ml shouldered flask (PH 7.0, steam sterilized at 120 °C for 20 minutes) containing 50 ml of main culture medium consisting of: The main culture is cultivated under the same conditions as the seed culture. 150
1 ml of the seed culture of C. equi ATCC No. 21329, which was seed-cultured in the same manner as in Example 2, was inoculated at 1 ml. When extracted with 200ml of ethyl acetate 55 hours after inoculation, 9α-OH-4AD was found in the extract.
122mg was present. Example 9 Glucose 1.0%, malt extract 0.3%, yeast extract 0.3%, peptone 0.5%, K ₂ HPO ₄ 0.2%,
Seed medium consisting of 0.2% Na ₂ HPO ₄ and water in two 50 ml shoulder flasks (PH 7.0, sterilized at 120°C for 20 minutes) each containing Arthrobaeter simplex IAM 1660 and Brevibacterium lipolyticum. (Brevibacterium lipolyticum) IAM No. 1398 is inoculated and cultured for 24 hours at 30° C., 120 cycles/min, and shaking at 7 cm amplitude. 5 ml of this culture solution,
A 500 ml shoulder flask containing 500 mg of DHA and 50 ml of 1/30 M phosphate buffer (PH 7.0) (110°C, 5
The cells were inoculated into a tube (steam sterilized for 1 minute) and shaken at 30° C. for 116 hours at 120 strokes/minute and an amplitude of 7 cm. then 120
℃ and sterilized for 20 minutes. At this stage, from the results of a small amount of sampling, the total volume of the buffer contained 170 mg of DHA for Brevibacterium, 301 mg of 4AD, 17 mg of androstar 1,4-diene-3,17-dione (ADD), and 194 mg of DHA for Arthrobacter.
It was confirmed that 273mg of 4AD and 13mg of ADD were present. After sterilization, 4 ml of C. equi ATCC No. 21329, which was seed-cultured in the same manner as in Example 2, was inoculated into the sterilized solution. Conversion to 9α-OH-4AD is performed under the same shaking conditions for 38 hours at 30°C after inoculation. After 38 hours, extract the steroids with 200 ml of ethyl acetate. As a result, 240 mg was found in the Brevibacterium extract and 220 mg in the Arthrobacter extract.
9α-OH-4AD was present. Example 10 In Example 1, C. equi subsp. mucilaginosus (C. equi) was used instead of C. equi ATCC No. 21329 as a microorganism.
Example 1 is repeated, except that Bacterium subsp. mucilaginosus) ATCC 21521 is used. As a result, when 4AD was added midway, 470 mg of 9α-OH-4AD was present in the culture extract (200 ml of ethyl acetate), whereas when 4AD was added at the time of medium preparation, 505 mg of 9α-OH-4AD was present in the culture extract (200 ml of ethyl acetate). It was confirmed that

Claims (1)

[Claims] 1. Using a microorganism belonging to Corynebacterium equi with androst-4-ene-3,17-dione (hereinafter abbreviated as 4AD) as a substrate, 9α-hydroxy-androst-4-ene- A method for producing a 9α-hydroxysteroid, which comprises producing a 3,17-dione. 2. In the method described in claim 1,
The microorganism is Corynebacterium equi
A method characterized by using ATCC No. 21329 bacteria. 3. The method according to claim 1 or 2, wherein the 4AD is produced by a microorganism using sterols as a substrate. 4. In the method described in claim 3,
A method characterized in that the 4AD-producing microorganism is a 4AD-producing mutant strain belonging to the genus Mycobacterium. 5. In the method described in claim 4,
A method characterized in that the 4AD-producing microorganism is a 4AD-producing mutant strain belonging to Mycobacterium parafortuitum complexus. 6. In the method described in claim 5,
A method characterized in that the microorganism that produces 4AD is Mycobacterium paraforthitutum complexus MCI-0807. 7. In the method recited in claim 3,
The microorganism that produces 4AD belongs to the genus Nocardia.
A method characterized by using a 4AD-producing mutant strain. 8. In the method described in claim 7,
A method characterized in that the microorganism that produces 4AD is a microorganism belonging to Nocardia ariaena. 9. In the method recited in claim 8,
The microorganism that produces 4AD is Nocardia ariaena.
A method characterized by using MCI-0710 bacteria. 10. The method according to claim 8, wherein the 4AD-producing microorganism is Nocardia ariaena MCI-0711. 11 In the method according to any one of claims 3 to 10, the sterols are sterols, C-3 ester derivatives thereof, C-3
A method characterized in that the ether derivative is selected from the group consisting of ether derivatives or oxidized intermediates thereof. 12 In the method according to any one of claims 1 to 11, the microorganisms belonging to the genus Corynebacterium to be used are pre-contained with 4AD.
A method characterized in that the method is used after culturing in a containing medium and inducing enzymes.