JPH0147157B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes deoxycholic acid and/or its salt to produce 12α-hydroxypregnane-1,
4-dien-3-one-20α-carboxylic acid and/or
For details on the method for producing salts thereof, please refer to Pseudomonas albira D-235 (Pseudomonas albira D-235)
arvilla D-235) strain (Microtechnology Research Article No. 181)
were cultured in a medium containing deoxycholic acid and/or its salts to produce 12α-hydroxypregnane-1,4.
- 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and/or a salt thereof, characterized by producing and collecting the dien-3-one-20α-carboxylic acid and/or its salt; / or a method for producing its salt. Conventionally, deoxycholic acid was treated with microorganisms.
12α-hydroxypregna-1,4-diene-3
Several methods are known for obtaining -one-20α-carboxylic acid. First, PJBarnes et al. reported that Pseudomonas sp. NCIB 10590
(Pseudomonas sp. NCIB 10590) strain has been reported [J.Chem.Soc.Chem.
Commun, (1974), pp. 115-116 and
Tetrahedron, vol. 32, pp. 89-93 (1976)].
This method was developed for Pseudomonas sp. NCIB in mineral salts medium 10 containing 0.1% deoxycholic acid.
10590 strain was cultured aerobically for 14 hours, and 12α-hydroxypregna-1,
4-dien-3-one-20α-carboxylic acid 657mg
and 960 mg of 12β-hydroxyandrosta-1,4-diene-3,17-dione were obtained as the main product,
Furthermore, 12α-hydroxyandrosta-1,4-
diene-3,17-dione, 12β-hydroxy-4
-androstene-3,17-dione and 12ξ,
17ξ-dihydroxy-4-androstene-3-
It is obtained as a trace component. This method uses 12α-hydroxypregna-1,4-diene-
It is not practical because the yield of 3-one-20α-carboxylic acid is low at less than 7%, there are many androstane-based by-products, and the concentration of the substrate deoxycholic acid is as low as 0.1%. Regarding the Pseudomonas SP.NCIB 10590 strain used in this method, nothing has been reported other than that this strain is a bacterium belonging to the genus Pseudomonas collected from animal feces. In addition, 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and 12β-hydroxyandrosta-1,4
-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-
hydroxyandrosta-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 produce 12α-hydroxypregna-1,
4-dien-3-one-20α-carboxylic acid and/or
As a result of a long-term search for microorganisms that produce deoxycholic acid and/or its salts, we found that certain bacteria belonging to Pseudomonas albira produce 12α-hydroxypregnathic acid and/or its salts as substrates.
The present inventors have discovered that 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 Pseudomonas arvilla that the present inventors obtained include Pseudomonas arvilla D-235.
There is a strain (Feikoken Joyori No. 181). When this strain is cultured in a medium containing deoxycholic acid and/or its salts, it hardly produces 12β-hydroxyandrosta-1,4-diene-3,17-dione at any stage of the culture period. It is distinguished from the Pseudomonas sp. NCIB 10590 strain used in the method of Barnes et al. Pseudomonas albira D obtained by the present inventors
The mycological properties of the -235 strain are listed in the table below.

【table】

【table】

[Table] Based on the mycological properties shown in the table above, Pseudomonas albira strain D-235 was identified. Pseudomonas albira D-235 strain is
Microscopic findings such as being a bacillus, having polar flagella, negative Gram staining, positive oxidase and catalase reactions, being aerobic, and O-F test. Because of its physiological properties, such as its oxidative nature, the
Of Determinative Bacteriology
Based on the 7th and 8th editions, it was identified as a bacterium belonging to the genus Pseudomonas. Furthermore, Pseudomonas albira D-235 strain has a large bacterial body size.
It has a size of 0.3 to 0.7 x 1.5 to 2.4μ, the color of the colonies on the agar plate is white and its surface is flat and exhibits moist light, it does not produce pigment, it does not liquefy gelatin, and it reduces nitrates. It was identified as a bacterium belonging to the Alvira species of the genus Pseudomonas, based on the fact that it does not produce acid and that it produces acid from glucose. 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. The method is carried out by culturing bacteria belonging to Pseudomonas albira 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 may normally be in the range of about 1 to 200 g/, but the
- Yield of hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and/or its salt,
Approximately 10~ from economical viewpoints such as culture conditions and operability
A range of 100g/ 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, bacteria belonging to Pseudomonas albira that produce 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and/or its salt using the above deoxycholic acid and/or its salt as a substrate are used. It is sufficient as long as it contains a nutrient source that can be assimilated and utilized. Deoxycholic acid and/or carbon sources are used as carbon sources.
or its salt may be used as a single carbon source, or deoxycholic acid and/or its salt may be combined with pentoses such as arabinose; glucose, mannose,
Hexoses such as fructose and galactose; Disaccharides such as sucrose and maltose;
Starch decomposition products; polyhydric alcohols such as sugar alcohols and glycerin; or polypeptone, peptone, meat extract, malt extract, cornstarch liquor, yeast extract, various amino acids, organic acids, etc. may be used in combination. As the nitrogen source, for example, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, or an organic nitrogen source such as polypeptone, peptone, meat extract, etc. is used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually carried out at 25 to 35°C for 10 hours to 7 days. 12α-Hydroxypregna-1,4-dien-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 extracts are collected and the solvent is distilled off to obtain the desired 12α-hydroxypregna-1,4-dien-3-one-20α.
- Carboxylic acids 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. When the culture filtrate or supernatant is made acidic in the above, 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid is obtained as a precipitate, so it can be directly filtered or centrifuged. However, in consideration of the operability of subsequent purification and other treatments, it is preferable to perform the above extraction operation. The extract or precipitate obtained by the above method contains, in addition to 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid, the remaining substrate deoxycol and/or its salts and secondary substances. 12α-hydroxypregna-1, which contains living organisms, can be extracted by adsorbing the extract or precipitate onto a silica gel column and eluting with a mixture of chloroform and ethanol.
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.
See Bar., vol. 88, p. 883 (1955)]. The present invention will be explained in more detail below using Examples. Example 1 Pseudomonas albira D-235 strain (Kaikoken Jokyo No. 181) was cultured by the following method. Deoxycholic acid 5.0g, ammonium nitrate 0.2g,
Potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate
Add tap water to 0.25g, magnesium sulfate heptahydrate 0.02g, yeast extract 0.01g and sodium hydroxide 0.5g to adjust the volume to 100ml (PH: 7.2),
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 inoculum grown in advance in the same medium as the above medium in a test tube shaker for 2 days was added to the above 500 ml Sakaguchi flask, and cultured with shaking at 30°C for 5 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 2 by adding hydrochloric acid to the obtained culture solution supernatant. This culture supernatant was mixed with 2/1 of chloroform methanol.
By extracting with 300 ml of the volumetric mixture and distilling off the chloroform/methanol mixture on a rotary evaporator, 2.9 g of a mixture of deoxycholic acid oxidation product and unconverted deoxycholic acid was obtained. Take a small portion of the above mixture, add methanol to it to make a 1% solution, and apply 20μ of this solution to a high-performance liquid chromatography system equipped with a Microbondapak C-18 column (manufactured by Waters, USA).
HLC-GPC-244 type). as mobile phase
A mixed solution of water/methanol in a 25/75 volume ratio adjusted to pH 4.0 was flowed at a flow rate of 1 ml/min, and detection was performed using a refractive index method, and the chromatogram shown in FIG. 1 was obtained. Peak A in this chromatogram,
Peak B, peak C, peak D, peak E and peak F were compared with the peaks of standard products and were determined to be 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid, 12α-hydroxy- 3-keto-4-cholenic acid, 12α-hydroxy-3-keto-5β-cholanic acid, 12α-hydroxy-
It was consistent with that of 3-keto-chola-1,4-dienoic acid, 12α-hydroxy-3-keto-5β-pregnane-20α-carboxylic acid and deoxycholic acid. Dissolve 2.8 g of the above mixture in a small amount of a 99/1 volume ratio mixture of chloroform/ethanol and
It was adsorbed onto a silica gel column. After washing this column with 500 ml of a mixture of chloroform/ethanol in a volume ratio of 99/1, 1 of the mixture of chloroform/ethanol in a volume ratio of 97/3 was applied to the column, and the solvent was removed from the resulting eluate using a rotary evaporator. By distillation, 310 mg of a mixture of deoxycholic acid oxidation products was obtained. Next, 500 ml of a mixture of chloroform/ethanol at a volume ratio of 95/5 was poured into the above column, the eluate was fractionated into a 10 ml fraction tube, and the fraction giving a single spot was collected by thin layer chromatography. The solvent was distilled off using a rotary evaporator to obtain 650 mg of a dry solid. By recrystallizing this with ethyl acetate, 12α-hydroxy pregnathyl
1,4-dien-3-one-20α-carboxylic acid
I got 450 mg. Furthermore, 200 ml of a mixture of chloroform/ethanol at a volume ratio of 90/10 was passed through the above column, and 1.2 g of unconverted deoxycholic acid was recovered from this eluate. 97/ of the previously obtained chloroform/ethanol
310 mg of a mixture of deoxycholic acid oxidation products from the elution fractions with the 3 volume ratio mixture was dissolved in a small amount of a 99/1 volume ratio mixture of chloroform/ethanol and adsorbed onto a 20 g silica gel column. 300 ml of a mixture of chloroform/ethanol with a volume ratio of 98/2 and 97/3 was poured into this column in this order, the eluate was fractionated into a 10 ml fraction tube, and a single sample was collected using thin-layer chromatography. The fractions giving spots were combined and the solvent was removed using a rotary evaporator. As a result, 80 mg of 12α-hydroxy-3-keto-5β-cholanic acid was obtained from the first half eluted fraction with a mixture of chloroform/ethanol at a volume ratio of 98/2, and 12α-hydroxy-3-keto-5β-cholanic acid was obtained from the second half eluted fraction. 10 mg of -4-cholenic acid was obtained. 5 mg of 12α-hydroxy-3-keto-5β-pregnane-20α-carboxylic acid was obtained from the first half of the elution fraction using a 97/3 volume ratio mixture of chloroform/ethanol, and 12α-hydroxy-3-carboxylic acid was obtained from the second half of the elution fraction. 10 mg of keto-chola-1,4-dienoic acid was obtained. Each of the obtained compounds was identified by the following method. 12α-hydroxypregna-1,4-diene-3
-one-20α-carboxylic acid mass spectrum m/z: 358 [M] ₊

Claims (1)

[Claims] 1 Pseudomonas alvira D-235
(Pseudomonas arvilla D-235) strain (Feikoken Article No. 181) was cultured in a medium containing deoxycholic acid and/or its salts to produce 12α-hydroxypregna-1,4-dien-3-one. -Production of 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid and/or its salt, which is characterized by producing and collecting 20α-carboxylic acid and/or its salt. Method.