JPS6250118B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of 9α-hydroxyandrost-4-ene-3,17-dione (hereinafter referred to as “9α
-OH-4AD). For details, see 9.
The present invention relates to a method for producing α-OH-4AD.

Because 9α-OH-4AD has a hydroxyl group (hereinafter sometimes abbreviated as “OH”) at the 9-position, it is easy to introduce a halogen atom at the 9α-position in an organic synthesis reaction, and there is also a double bond between C ₉ and C ₁₁ . After introduction, it became 11β
Easy to introduce OH. Most corticoids (adrenocortical hormones) have OH at the 11β position, and 9
Considering that having a halogen atom at the α position increases activity extremely, 9α-OH-4AD is an important precursor for corticoid production.

Several methods for producing 9α-OH-4AD using microorganisms are already known. Most of them introduce a hydroxyl group into the 9α position of androst-4-ene-3,17-dione (hereinafter abbreviated as 4AD); for example, U.S. Pat. Nos. 3,065,145 and 3,116,220. It is known from etc.

On the other hand, 9α-OH-4AD directly from sterols
Japanese Unexamined Patent Application Publication No. 52-54093 is known as a method for manufacturing the same. Here, a mutant strain of M. fortuitum belonging to the genus Mycobacterium (hereinafter abbreviated as "M.") is used.

Recently, the present inventors have succeeded in producing a larger amount of 9α-OH-4AD than sterols using a suddenly modified strain belonging to M. vaccae, and have achieved the present invention.

That is, the gist of the present invention is a method for producing 9α-OH-4AD, which comprises culturing M. vaccae MCI-1104 or M. vaccae MCI-1117 using sterols as a substrate. exists in

The present invention will be explained in detail below.

Conventionally known microorganisms belonging to M. batskae (hereinafter referred to as "wild strains") are 9α-OH-4AD.
9α-OH-4AD is not produced from sterols when cultured normally.

Therefore, the mutant strains M. batsukae MCI-1104 and MCI-1117 used in the present invention are
The 9α-OH-4AD enzyme possessed by the wild strain is deleted or has low activity, and such mutant strains do not produce 9α-OH-4AD under normal culture conditions.
It is clearly distinguished from the wild strain in that it produces a large amount of OH-4AD.

The former strain was obtained by treating M. batsukae ATCC No. 23034 as a parent strain with N-methyl-N'-nitro-N-nitrosoguanidine.

The latter strain was obtained using M. batsukae ATCC No. 23004 as the parent strain by treatment with ultraviolet light.

This strain has been deposited with the National Institute of Microbial Technology under the following number.

Microorganisms M. batsukae MCI-1104 No. 4741 M. batsukae MCI-1117 No. 4754 M. batsukae MCI-1104 and M. batsukae MCI-1117 are both M. Since it is a mutant strain obtained from the parent strain belonging to M. batsukae, it is naturally thought that it belongs to M. batsukae, and it is also a mutant strain that has the known 9α-OH-4AD production mutation described in JP-A No. 52-54093. It is clear that this strain is taxonomically different from the mutant strain M. Fortuitum NRRLB-8119.

The sterols used as substrates in the present invention are collectively referred to as various sterols, their C-3 esters, ether derivatives, or oxidized intermediates thereof. Various sterols are C-3 of the perhydrocyclopentanophenanthrene nucleus.
a hydroxyl group, usually a double bond at C-5,
It has a chain type side chain having 8 to 10 carbon atoms at C-17, and may have a double bond at C-7, C-8, C-9 (11), etc. in some cases. .

Examples of such various sterols include cholesterol, stigmasterol, campesterol, β-sitosterol, ergosterol, brassicasterol, fucosterol, lanosterol, agnosterol, dihydrolanosterol, dihydroagnosterol, α-sitosterol, and the like. It will be done. Preferred sterols are cholesterol, campesterol and β-sitosterol.

C-3 ester derivatives of 3β hydroxyl groups of various sterols and inorganic acids such as sulfuric acid or organic acids such as fatty acids can also be used as raw materials in the method of the present invention.

Examples of such C-3 ester derivatives include cholesteryl oleate, cholesteryl palmitate, cholesteryl sulfate, and the like.
Furthermore, C-3 ether derivatives obtained by, for example, a method of adding alkylene oxide to the 3β hydroxyl group of various sterols can also be used as raw materials in the method of the present invention.

Examples of such C-3 ether derivatives include polyoxyethylene cholesteryl ether.

It is obtained by reacting wool wax containing C-3 ester derivatives of the various sterols described above, lanolin, wool alcohol containing cholesterol obtained by hydrolysis of lanolin, and wool alcohol with ethylene oxide. It goes without saying that polyoxyethylene lanolin alcohol ether, which is a C-3 ether derivative, can also be used as a raw material in the method of the present invention.

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 in the process of the invention.

Additionally, oxidized intermediates of various sterols or their C-3 ester or ether derivatives can also be used as substrates in the process of the invention. Examples of such oxidation intermediates include various sterols, their C-3 esters, 4-en-3-one and 1,4-dien-3-one derivatives of C-3-ether derivatives, and specifically For example, Cholest-4-
en-3-one, cholestar-4・22-diene-3
-one, 3β-hydroxy-23,24-bisnorkol-5-ene-22-eutschacide, 3-oxo-23,24-bisnorkol-4-ene-22-eutschacide (hereinafter abbreviated as “4BN”), 3-oxo -Pregna-4・17(20) diene-20-carboxylic acid, 3-oxo-pregna-4・17(20) diene-20-carboxylic acid methyl, 3β-hydroxy-
androst-5-en-17-one, 4AD, and the like.

A carbon source, a nitrogen source, and an inorganic substance are appropriately added to the medium used in the method of the present invention.

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; Glycerides and fats such as soybean oil and cottonseed oil; Starch, Examples include sugars such as maltose, sucrose, glucose, and rhamnose. carbon source,
Natural nutritional sources, including nitrogen sources and other nutritional substances, include molasses, including high-test molasses, refined molasses and xylose molasses; bagasse, corn cob,
Alpha alpha, cornstarch liquor, day tailor's soluble, flavoring liquid, fishmeal, bran, meat extract, yeast, bittersweet extract, potato extract, malt extract, gluten, peptone, glutamate,
Asparagine, glycine, casein, casein decomposition products, skim milk, soybean meal, rapeseed meal, sesame meal,
Vegetable oil and fat meal such as linseed meal and cottonseed meal, and whole soybeans,
Oil seeds and oil fruits such as rapeseed, sesame, flaxseed, and cottonseed are included.

Inorganic substances include nitrogen sources such as ammonium sulfate and ammonium chloride; potassium and phosphorus sources such as dipotassium hydrogen phosphate;
Examples include salts of iron, copper, magnesium, manganese, cobalt, zinc, calcium, etc.; and ash of natural products such as molasses. Other vitamins can also be added if necessary.

The composition of the medium is selected depending on the type of bacteria used, but a carbon source, nitrogen source, potassium, phosphorus, and magnesium are essential medium components.

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 effective as emulsifiers for sterols, and are preferably added to the culture medium. As the surfactant, nonionic and anionic surfactants are preferred. Specific examples include polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, polyethylene glycol monostearate, and the like.

The culture temperature is usually 20 to 40℃;
The optimum temperature is around 25-35℃.

The pH of the medium is usually adjusted to 5-10, but 6-9
is preferred. Since the microorganism used in the present invention belongs to the genus M., it can withstand a pH of around 10, as is well known.

Although sterols, which are raw materials for the method of the present invention, are generally sterilized together with the medium, they 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 after being dissolved in a solvent such as dimethylformamide, or it is added as a suspension after being finely dispersed by ultrasonication. Further, it is preferable to add a surfactant at the same time because it promotes emulsification of substrate sterols and the like.

The culture time is not particularly limited, but 9α-OH-
4AD production increases rapidly. After that, the production amount of 9α-OH-4AD gradually increases with the culture time, but culturing for more than 20 days has little industrial significance.

After completion of the culture, the androstane compounds accumulated in the culture solution 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. 9α-OH-
4AD can be separated.

However, usually the culture is carried out by selecting appropriate culture conditions.
By selecting an appropriate extraction solvent, if 9α-OH-4AD predominates in the solvent extract of the culture solution, acetone, hexane, cyclohexane, etc. can be extracted from the solvent extract without chromatography after solvent distillation. 9α-OH-
You can obtain pure crystals of 4AD.

According to the present invention, 9α is more effective than cheap sterols.
It is possible to produce -OH-4AD with good yield.

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 addition, in the following examples, 9α-OH-4AD
Quantification was performed by gas chromatography.

Also, in the examples below, percentages are by weight.

Example 1 Glucose 1.0%, meat extract 1.0%, peptone 1.0
% and 500 ml of seed medium (PH7.2) consisting of water
ml into a flask with a shoulder, steam sterilized at 120℃ for 15 minutes, inoculated with one platinum loop of M. batsukae MCI-1104, and shaken at 30℃ for 48 hours at 120 reciprocations/min with an amplitude of 7cm. Cultivate seeds. Add 2 ml of this seed culture to ground soybeans 5.0%, yeast 1.5%, K ₂ HPO ₄ 0.15
%, soybean oil 0.5%, MgSO ₄ 7H ₂ O 0.1%, silicone oil (Shin-Etsu Chemical Co., Ltd. trademark KM-70) 0.1%, cholesterol 2.0%, and water.
500 ml (PH7.0, steam sterilized at 120℃ for 20 minutes)
Inoculate 10 ml shoulder flasks. Main culture at 30℃
The culture was carried out under the conditions of 120 cycles/min and an amplitude of 7 cm, and the culture was stopped 200 hours after the start of culture, and all the culture solutions were combined and extracted twice with 1 part of ethyl acetate.

The extracts were combined to remove insoluble matter such as bacterial cells, and the steroid content was analyzed by gas chromatography. As a result, 0.83 g of 4α-OH-4AD was produced.

Example 2 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℃ for 15 minutes, inoculated with one platinum loop of M. batsukae MCI-1104, and incubated at 30℃ for 48 hours under conditions of reciprocating shaking at 120 cycles/min and an amplitude of 7 cm. Cultivate. 20ml of this seed culture solution was mixed with defatted soybean grinding 4.0%, yeast 2.0%, soybean oil 1.0%, rice bran 1.0%, NaNO ₃ 0.2%, K ₂ HPO ₄ 0.1%, MgSO ₄ .
Ten 500 ml shoulder flasks containing 50 ml of main culture medium (PH 7.0, steam sterilized at 120° C. for 20 minutes) consisting of 0.1% 7H ₂ O, 1.0% cholesterol and water are inoculated. The main culture was performed at 30°C with 100 reciprocations/min and 7 cm reciprocating shaking conditions.The culture was stopped 192 hours after the start of the culture, and the total culture solution was mixed with 1.5 ethyl acetate.
Extract with In this extract, 9α-OH-
0.52g of 4AD was present.

Example 3 In Example 1, instead of the substrate cholesterol, β-sitosterol (90%), a 2:1 mixture of β-sitosterol and campesterol, stigmasterol, cholest-4-en-3-one, cholesteryl oleate and Example 1 is repeated except using 4BN. Accumulated 9
The amount of α-OH-4AD is shown in Table 1.

Table 1 9α-OH-4AD (g) β-sitosterol 0.62 β-sitosterol + campesterol 0.58 stigmasterol 0.10 cholest-4-en-3-one 0.70 cholesteryl oleate 0.05 4BN 0.15 Example 4 Main culture medium in Example 1 In, cottonseed meal 4.0
%, soybean oil 1.0%, casein 2.0%, _{K2HPO4 0.2} %
Example 1 is repeated except that a medium consisting of 0.2% NaNO ₃ , 0.01% MgSO ₄ .7H ₂ O, 2.0% cholesterol and water is used.

The culture solution was extracted with ethyl acetate, and the solvent contained 9
0.88g of α-OH-4AD was present.

Example 5 Example 1 is repeated except that the microorganism used in Example 1 is M. batsukae MCI-1117 instead of M. batskae MCI-1104. The results showed that 1.15g of 9α-OH-4AD was produced.

Claims (1)

[Scope of Claims] 1. 9α-hydroxyandrost-4, which is characterized in that Mycobacterium batsukae MCI-1104 or Mycobacterium batskae MCI-1117 is cultured using sterols as a substrate. -ene-3,17-dione (hereinafter "9α-OH-4AD")
(abbreviated as ) manufacturing method. 2.9 caused by the microorganism described in claim 1
In the method for producing α-OH-4AD, the sterols are sterols, C-3 ester derivatives thereof,
A method characterized in that it is selected from the group consisting of C-3 ether derivatives or their oxidized intermediates.