JPH0121760B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes deoxycholic acid and/or its salts to produce 12α-hydroxypregnar-1,
4-dien-3-one-20α-carboxylic acid and/
or a salt thereof, in detail, 12α using deoxycholic acid and/or a salt thereof as a substrate.
-Bacteria belonging to the genus Alcaligenes that produce hydroxypregnar-1,4-dien-3-one-20α-carboxylic acid and/or its salts are cultured in a medium containing deoxycholic acid and/or its salts.
12α-hydroxypregna-1,4-diene-3
12α characterized by producing -on-20α-carboxylic acid and/or its salt and collecting the same.
-Hydroxypregnane-1,4-dien-3-one-20α-carboxylic acid and/or a salt thereof. Conventionally, deoxycholic acid was treated with microorganisms.
12α-hydroxypregna-1,4-diene-3
Several methods are known for obtaining -on-20α-carboxylic acid. First, by PJ Barnes et al., Pseudomonas sp. NCIB10590
(Pseudomonas sp. NCIB10590) strain has been reported [J.Chem.Soc.Chem.
Commun, (1974), pp. 115-116 and
Tetrahedron, vol. 32, pp. 89-93 (1976)].
This method is used to grow Pseudomonas sp. in mineral salts medium 10 containing 0.1% deoxycholic acid.
NCIB10590 strain was cultured aerobically for 14 hours, and 10 g of deoxycholic acid was added to 12α-hydroxypregnathic acid-1,4-dien-3-one-20α-carboxylic acid.
657mg and 12β-hydroxyandroster 1,
960 mg of 4-diene-3,17-dione was obtained as the main product, and 12α-hydroxyandroster was obtained as the main product.
1,4-diene-3,17-dione, 12β-hydroxy-4-androstene-3,17-dione and
12ξ,17ξ-dihydroxy-4-androsten-3-one is obtained as a trace component. This method has a yield of 7% of 12α-hydroxypregnane-1,4-dien-3-one-20α-carboxylic acid.
The concentration of androstane-based by-products is low, and the concentration of the substrate deoxycholic acid is low.
It is not practical as it is as low as 0.1%. In addition, 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and 12β-hydroxyandroster 1,
Method for obtaining 4-diene-3,17-dione [see Biochem.Soc.Trans., Vol. 5, pp. 1758-1760 (1977)]; Bacteroides fragilis XF23 by RWOWen et al.
(Bacteroides fragilis XF23) strain or Bacteroides fragilis subsp. thetaiotaomicron E59 (Bacteroides fragilis subsp.
12α-Hydroxypregnane-1,4 from deoxycholic acid using thetaiotaomicron E59) strain.
-Dien-3-one-20α-carboxylic acid, 12β-
hydroxyandroster 1,4-diene-3,
17-dione, 12α-hydroxyandroster
Method for obtaining 1,4-diene-3,17-dione and 12β-hydroxy-4-androstene-3,17-dione [Biochem.Soc.Trans., vol. 5, 1711-
1713 (1977)], but all of these methods use anaerobic bacteria, require low concentrations of substrates, require long cultivation times, and are difficult to collect the target product. There are various problems in industrial fermentation production such as low yield. The present inventors used deoxycholic acid and/or its salt as a substrate to
4-dien-3-one-20α-carboxylic acid and/
As a result of long-term searches for microorganisms that can industrially advantageously produce 12α-hydroxypregnathic acid and its salts, certain bacteria belonging to the genus Alcaligenes have been found to produce 12α-hydroxypregnathic acid using deoxycholic acid and/or its salts as substrates.
We have discovered that 1,4-dien-3-one-20α-carboxylic acid and/or its salts can be produced with high selectivity and high yield, and have completed the present invention. The bacteria belonging to the genus Alcaligenes obtained by the present inventors include Alcaligenes fuecalis
D4020 (Alcaligenes faecalis D4020) strain (Feikoken Article No. 182) and Alcaligenes faecalis D4020-H405 (Alcaligenes faecalis D4020-
H405) strain (Feikoken Joyori No. 183). The Alcaligenes fuecalis D4020 strain is a wild strain obtained from soil, and the Alcaligenes fuecalis D4020-H405 strain is a mutant strain obtained by mutating the Alcaligenes fuecalis D4020 strain. The following table lists the mycological properties of these strains.

【table】

【table】

[Table] Based on the mycological properties shown in the table above, the Alcaligenes faecalis D4020 strain and the Alcaligenes faecalis D4020-H405 strain were identified. Alcaligenes fuecalis strain D4020 is a bacillus, has periflagella,
Microscopic findings such as negative Gram staining, positive oxidase and catalase reactions, aerobic status, O-F
Based on the physiological properties such as oxidative test results, it was identified as a bacterium belonging to the genus Alcaligenes based on the Verge's Manual of Determinative Bacteriology, 7th and 8th editions. . Furthermore, Alcaligenes fuecalis strain D4020 was identified as a bacterium belonging to the Alcaligenes fuecalis species, because it does not liquefy gelatin, does not change the milk other than to become alkaline, and does not have a denitrification reaction. Additionally, mutant strains are generally considered to belong to the same species as their parent strains, and the Alcaligenes fuecalis strain D4020-H405 was determined to be a bacterium belonging to the Alcaligenes fuecalis species. The production of 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and/or its salt by the method of the present invention involves the production of 12α-hydroxypregnathic acid and/or its salt using deoxycholic acid and/or its salt as a substrate. It is carried out by culturing bacteria belonging to the genus Alcaligenes that produce -1,4-dien-3-one-20α-carboxylic acid and/or its salts in a medium containing deoxycholic acid and/or its salts. Specifically, the salt of deoxycholic acid is a salt of an alkali metal such as sodium or potassium, or a salt of an alkaline earth metal such as calcium or magnesium. The concentration of deoxycholic acid and/or its salt is usually about 1 to 200
12α-hydroxypregnane-1,4-dien-3-one-20α produced.
- Approximately 10 to 100 g/carboxylic acid and/or its salt from economical viewpoints such as yield, culture conditions, and operability.
A range of is preferred. The culture method is basically the same as that used for aerobic culture of general microorganisms, but usually a shaking culture method using a liquid medium or an aerated agitation culture method is used. As a medium, use the above deoxycholic acid and/or its salt as a substrate and 12α
-Hydroxypregnane-1,4-dien-3-one-20α-carboxylic acid and/or its salts, as long as it contains a nutrient source that can be utilized by bacteria belonging to the genus Alcaligenes. As the carbon source, deoxycholic acid and/or its salt may be used as the sole carbon source, or glucose, glycerin, peptone, meat extract, yeast extract, etc. may be used in combination with deoxycholic acid and/or its salt. Examples of nitrogen sources include inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, and potassium nitrate, or polypeptone, peptone,
Organic nitrogen sources such as meat extracts are used. Also,
In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. Culture conditions are not specific, but usually at 25-35℃ for 10
Shake culture or aerated agitation culture is performed for 7 days. 12α-Hydroxypregnane-1,4-diene-3-one- accumulated in the culture medium in this way
20α-carboxylic acid and/or its salt can be separated and collected by known methods. For example, the culture filtrate or supernatant obtained by separating and removing bacterial cells and other insoluble components in the culture solution by filtration or centrifugation is acidified by adding an acid such as hydrochloric acid or sulfuric acid, and then organic solvents that dissolve and phase separate from water, such as ethyl acetate, chloroform,
Extract using a mixture of chloroform and methanol. The obtained extract is collected and the solvent is distilled off to obtain the desired 12α-hydroxypregnane-1,4-dien-3-one.
20α-carboxylic acid can be recovered. This extraction operation using an organic solvent can be carried out not only on the culture filtrate or supernatant, but also on the culture solution itself, and can also be carried out without adding an acid to the culture solution, culture filtrate, or supernatant in advance. In the above, when the culture filtrate or supernatant is acidified, 12α-hydroxypregnane-1,4-
Since dien-3-one-20α-carboxylic acid is obtained as a precipitate, it can also be recovered as it is by filtration or centrifugation. The extract or precipitate obtained by the above method contains 12α-hydroxypregnane-1,4-dien-3-one-
In addition to 20α-carboxylic acid, it contains almost no residual substrate deoxycol and/or its salt and by-products, and can easily be purified by recrystallization from water/methanol, for example. 4-dien-3-one-20α-carboxylic acid can be obtained. 12α-Hydroxypregna-1,4-dien-3-one-20α-carboxylic acid obtained by the method of the present invention can be used for hormonal steroids such as prednisone by eliminating the carboxylic acid at position 20. It can be derived into 12α-hydroxypregna-1,4-diene-3,20-dione, which is a raw material for synthesis [H.Ruschig et al., Chem.Ber.,
88, p. 883 (1955)]. The present invention will be explained in more detail below using Examples. Reference example Method for obtaining mutant strains Medium 1 (composition: deoxycholic acid 0.5%, sodium hydroxide 0.05%, peptone 0.5%, yeast extract
Alcaligenes fuecalis grown on slants with 0.5% sodium chloride, 0.5% sodium chloride and 1.5% agar).
Culture medium 2 (composition: 2% deoxycholic acid, 0.2% sodium hydroxide, 0.2% ammonium nitrate, 0.1% monopotassium phosphate, 0.6% dipotassium phosphate, sulfuric acid) was prepared using a loopful of D4020 strain in a test tube. Magnesium heptahydrate 0.02% and yeast extract
0.02%) and cultured with shaking at 30°C for 8 to 10 hours. Medium 3 (composition: deoxycholic acid 0.5%, sodium hydroxide 0.05%, glucose 0.1%, ammonium nitrate 0.2%, monopotassium phosphate 0.1%, dipotassium phosphate 0.6 %, magnesium sulfate heptahydrate 0.02%, and yeast extract 0.02%) and cultured at 30°C for 10 to 15 hours. Next, the cells in the logarithmic growth phase were collected by aseptic filtration using a 0.45μ membrane filter, washed with 20ml of 0.1M phosphate buffer (PH: 7.0), and suspended in 25ml of the same buffer. Ta. Mutation treatment was carried out by adding N-methyl-N'-nitro-N-nitrosoguanidine to the mixture at a final concentration of 20 μg/ml and shaking at 30° C. for 10 to 15 minutes. Filter and collect the mutant-treated bacterial cells using a 0.4μ membrane filter, and add 20ml of 0.1M phosphate buffer (PH: 7.0).
After washing with water, the cells were suspended in 20 ml of the same buffer. The obtained bacterial suspension was diluted with sterile physiological saline and mixed with medium 4 (composition: deoxycholic acid 0.5%, sodium hydroxide 0.05%, ammonium nitrate 0.2%, phosphoric acid 1
Potassium 0.1%, dipotassium phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02%
The mixture was plated on an agar plate containing 1.5% agar and 1.5% agar so that 500 to 1000 colonies appeared, and then cultured at 30°C for 3 to 4 days. After isolating a microscopic colony among the colonies that appeared on a slant of medium 1, a loopful of it was prepared in a test tube in advance in medium 5 (composition: deoxycholic acid 0.2%, sodium hydroxide).
0.02%, glucose 0.1%, ammonium nitrate 0.2
%, monopotassium phosphate 0.1%, dipotassium phosphate
0.6%, magnesium sulfate heptahydrate 0.02%, and yeast extract 0.02%) and cultured with shaking at 30°C for 24 hours. The products in each culture solution obtained were assayed by thin layer chromatography,
Target 12α-hydroxypregna-1,4-
We found a strain that selectively accumulates diene-3-one-20α-carboxylic acid and named it Alcaligenes faecalis strain D4020-H405. Example 1 Alcaligenes fuecalis D4020 strain (Kaikoken Joyori No. 182) was cultured by the following method. Deoxycholic acid 2.0g, ammonium nitrate 0.2g,
Potassium dihydrogen phosphate 0.12g, dipotassium hydrogen phosphate
Add tap water to 0.61g, magnesium sulfate heptahydrate 0.02g, yeast extract 0.02g and sodium hydroxide 0.2g to adjust the volume to 100ml (PH: 8.4),
This was used as a culture medium. This medium was placed in a 500 ml Sakaguchi flask and steam sterilized at 120°C for 15 minutes. 10 ml of the inoculum grown in advance in the same medium as the above one in a test tube shaker for one day was added to the above 500 ml Sakaguchi flask and cultured with shaking at 30°C for 3 days. After culturing, the culture solution was collected and the bacterial cells were removed using a centrifuge, and the pH of the culture solution supernatant was adjusted to 3 by adding hydrochloric acid to the obtained culture solution supernatant. The resulting precipitate was filtered, thoroughly washed with water, and then dried to obtain 950 mg of 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid. The obtained 12α-hydroxypregna-1,4-
Take a portion of diene-3-one-20α-carboxylic acid, add methanol to it to make a 1% solution, and apply 10μ of this solution to a high-performance liquid chromatography system equipped with a Microbondapak C-18 column (Waters Inc., USA). manufactured by HLC-GPC-244). As a mobile phase, a mixed solution of water/methanol in a volume ratio of 25/75 adjusted to pH 4.0 was flowed at a flow rate of 1 ml/min, and detection was performed using a refractive index method. The area ratio of each peak in the obtained liquid chromatogram was measured using an integrator (Shimadzu Chromato Pack C-
R1A), and from this area ratio, the above 12α-hydroxypregnane-1,4-dien-3-one
The purity of 20α-carboxylic acid was determined to be 93%. In addition, the substance corresponding to the main peak of liquid chromatography was separated by thin layer chromatography, and 12α-hydroxy pregnate was extracted using the following method.
It was identified as 1,4-dien-3-one-20α-carboxylic acid. Mass spectrum m/z: 358 [M] ⁺

Claims (1)

[Claims] 1. Bacteria belonging to the genus Alcaligenes that produce 12α-hydroxypregnane-1,4-dien-3-one-20α-carboxylic acid and/or its salts using deoxycholic acid and/or its salts as substrates were treated with deoxycholic acid. and/or a salt thereof to produce 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and/or a salt thereof, and collecting the same. 12α-Hydroxypregna-1,4-diene-
A method for producing 3-one-20α-carboxylic acid and/or a salt thereof.