NL8004741A - METHOD FOR PREPARING CHOLIC ACID DERIVATIVES BY MICROBIOLOGICAL WAY AND MICROORGANISMS FOR USE IN THE METHOD - Google Patents

Description

Br / Bl / lh / 4 m 4 Λ

Method for the preparation of cholic acid derivatives by microbiological means as well as micro-organisms for use in the method.

The invention relates to a process for the microbiological preparation of cholanic acid derivatives of the formula 1 in which X-sr-GH or the = 0 and R is hydrogen, an alkali metal or a 5 H

Alkaline earth metal as well as on microorganisms used in the process. More specifically, the cholic acid derivatives of the formula 1 of the invention can be produced in a high yield in a short period of time by culturing a specific microorganism in a culture medium containing bile acid or a salt thereof as a substrate.

The cholic acid of the formula 1 in which X 'ς ^ -ΟΗ

H

15, namely, 7, 7ef-dihydroxy-12-keto-5/5-cholic acid or a salt thereof is a suitable intermediate for the preparation of a solubilizing agent for gallstones, namely the chenodesoxygolic acid (CDGZ). Cholic acid derivative of the formula I, wherein X represents = 0, namely 70-hydroxy-3,1,12-diketo-5γS-cholic acid or a salt thereof is a suitable intermediate for the preparation of deoxygolic acid, which is a suitable starting material in the preparation of progesterone and adrenal corticosteroid derivatives.

It was already known that cholic acid derivatives of the formula I can be obtained by microbiological means using bile acid or a salt thereof as a substrate. For example, Hayakawa et al. Have described a method for preparing 30 Ct7 <* - dihydroxy-12-keto-5 ^> - cholic acid using Streptomyces gelaticus 1164 / The Journal of Biochemistry (Japan), Vol. 44, No. 2, pp. 109-113 (1957) and Proceedings of Japan Academy, "Vol. 32, pp. 519-522 (1956). Hasegawa et al. Have described a -2 method for preparing 2c (, 7i / -dihydroxy-12-keto-5fi-cholic acid using Aspergillus cinnamomeus strain HUT 2026 (Hiroshima Journal of Medical Science,

Full. 8, No. 3, pp. 277-283 (1959)). Likewise, 5 Kikuchi et al. Has described a method for preparing 30 '(, 7® <-dihydroxy-12-keto-5 | i-cholanoic acid using Staphylococcus epidermidis strain H1 (Journal of Biochemistry, Vol. 72, no. 1). pp. 165-172 (1972)) Furthermore, Hayakawa et al. has described a method for preparing!-hydroxy-3,12-diketo-5-3-cholanoic acid using Streptomyces gelaticus strain 1164 (Proceedings of Japan Academy, Vol. 32, 519-522 (1956)).

In these methods, however, the concentration of the bile acid used as substrate in the culture medium is low, for example, no more than 10 g / l. In experiments which finally led to the invention, it has been found that in the preparation of the cholanic acid derivatives according to the known methods it is necessary to use the bile acid in a low concentration by microbiological means, because the micro to be used therewith -organisms grow insufficiently or hardly when a bile acid concentration of 20 g / l or more is used in the culture medium. Moreover, with these known methods, too long a period of cultivation is necessary. It is therefore desirable to develop a process for the preparation of cholic acid derivatives, wherein they are obtained in high yield within a short period of time. According to the invention it has now been found that certain microorganisms belong to the genera. Arthrobacter, Brevibacterium and Corynebacterium can grow in a medium containing bile acid or a salt thereof as substrate in a concentration which can be within wide limits , and can produce the high yield cholic acid derivatives in a short period of time.

The object of the invention is now to provide a method for preparing a microbiological route of the cholanic acid derivatives of the formula 1, wherein they are obtained in a high yield within a short period of time. It is also an object of the invention to provide new microorganisms 80 04 * 1 * *> * -3- # capable of growing in a medium containing bile acid or a salt thereof as a substrate. This object, as well as other objects and advantages for the invention, will become apparent to those skilled in the art from the following detailed description.

According to the invention there is now provided a process for the microbiological preparation of a cholanic acid derivative of the formula. 1, which comprises a microorganism capable of growing in a medium which, as substrate, is bile acid or a salt thereof thereby producing the cholic acid derivative which microorganism belongs to one of the genera Arthrobacter, Brevibacterium and Corynebacterium is grown in a culture medium containing the substrate and the resulting derivative is collected.

The microorganisms to be used in the invention are the microorganisms isolated from the soil and mutants thereof, which are obtained by natural mutation or are formed, for example, by X-ray irradiation, ultraviolet rays, by means of a mutant agent, such as N-methyl-N'-nitro-N-nitroso guanidine, 4-nitroquinoline N-oxide, acriflavine or ethyl methanesulfonate or a combination thereof and the like.

Representatives deposited with the Fermentation Research Institute of the microorganisms capable of forming the cholic acid derivative of the formula 1 in a culture medium # containing bile acid or a derivative thereof, which can be used according to the invention, have been deposited with the Fermentation Research Institute , Agency of. Industrial Science and Technology # Japan (designated FERM below) and with the American Type Culture Collection 30 in the United States of America; (referred to below as ATCC). These are Arthrobacter strain CA-35 (FERM-P no. 5145? ATCC no. 31651), Arthrobacter strain CA-35-A589-29-32 (FERM-P no. 5522; ATCC no. 31652), Arthrobacter strain CA -35-A589-47 (FERM-P no. 5523? ATCC no. 31653), Arthrobacter strain CA-35-35 A849 (FERM-P no. 5524? ATCC no. 31654), Arthrobacter strain CA-35-A —1071-15 (FERM-P No. 5525; ATCC No. 31655), Arthrobacter strain CA-35-A-1448 (FERM-P No. 5526? ATCC No. 31656), Arthrobacter strain CA-35-A- 1475 (FERM-P no. 5527? ATCC no.

80 0 4 74 1 -4- 31657), Arthrobacter strain CA-35-A-1766-15 (FERM-P no. 5528? ATCC no. 31658), Arthrobacter strain CA-35-M-965-3 (FERM- P No. 5529, ATCC No. 31659), Arthrobacter strain CA-35-Y-37-12 (FERM-P No. 5530; ATCC No. 31660), Brevibacterium strain CA-6 (FERM-5 P No. 5144; ATCC No. 31661) and Corynebacterium strain CA-53 (FERM-P No. 5532; ATCC No. 31662). Arthrobacter strain CA-35, Brevibacterium strain CA-6 and Corynebacterium strain CA-53 are strains found in nature, while the other nine Arthrobacter strains are mutants of Arthrobacter strain CA-35. Arthrobacter strain. CA-35-Y-37-12 has been obtained by ultraviolet irradiation. The other eight mutations of Arthrobacter strain CA-35 were obtained by treatment of the original strain with N-methylN'-nitro-N-nitrosoguanidine.

The morphological, culture and physiological properties of these strains are set forth in Table A below. For comparison, the same properties of Arthrobacter simplex strain IAM 1660, which is related to the Arthrobacter 'strain CA-35, are also listed in Table A.

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Notes: 1) The symbols used with respect to the physiological properties have the following meanings: +: The trunk has the relative character or forms the relative product.

+: It is difficult to determine whether the strain has the relative character or whether it forms the relative product or not.

The strain does not have the relative character or 10 does not produce the relative product.

2) The symbols used in the production of acids and gases from carbohydrates have the following meanings: (i) growth, acids and gases:

The strain was grown in a Hugh and Leifson medium with the proviso that the carbon source was replaced with one of the carbohydrates 1-15 each, and growth and acid and gas production thereof were observed.

+: Stem growth occurs or an acid or a gas is formed.

+: It is difficult to observe whether the trunk is growing and whether an acid or a gas is being produced ... or not.

No growth of the strain occurs or no acid or gas is formed.

ii) Assimilation:

The culture medium, containing NH4NC> 3 (2 g / 1) KH2PO4 (2 g / 1), K2HPO4 (5 g / 1), MgS04.7H20 (0.2 g / 1), yeast extract (0.1 g / 1) and. one of the carbohydrates containing 1-15 (5 g / l) 30 was placed in a tube (21 mm diameter), the strain was grown therein with shaking and its assimilation (growth) was observed.

-: No growth of the trunk occurs.

35 +: There is little growth of the trunk.

+: Stem growth occurs.

++: Good growth of the trunk occurs.

+++: Very good growth of the trunk occurs.

80 04 74 1 -27-

Based on these morphological, culture and physiological properties, the classification of the strains has been undertaken according to Bergey's Mannual of Determinative Bacteriology 7th and 8th editions.

It is determined that Arthrobacter strain CA-35 has relationship with Arthrobacter simplex due to its microscopic observations, such as shape, Gram stain and the like as well as its physiological properties. However, Arthrobacter strain CA-35 differs from Arthrobacter 10 simplex strain IAM 1660 in that it produces pigment and assimilates carbohydrates as well as growth in a medium containing bile acid. Arthrobacter strain CA-35 can grow in a medium containing sodium cholate as the sole carbon source in a high concentration, such as about 20-500 g / l - 15, the main metabolic products of which are 7 <-hydroxy- 3,12-diketo-5 | 3-cholic acid, 3 or, 7 (i-dihydroxy-12-keto-5 | i-cholic acid, 7, 12 <-dihydroxy-3-keto-5-cholic acid and / or sodium salts thereof are formed, while Arthrobacter simplex strain IAM 1660 can hardly grow in a medium containing sodium cholate as the sole carbon source at a concentration of 10 g / l.

Arthrobacter strain CA-35 forms yellow to cream colored pigments. However, as a microorganism belonging to the genus Arthrobacter and showing the formation of a pigment, Arthrobacter oxydans, Arthrobacter aurescens and Arthrobacter ... ureafaciens are known. Arthrobacter strain CA-35, however, also differs from these microorganisms in that Arthrobacter oxydans and Arthrobacter aurescens are usually gram negative and starch hydrolysers and Arthrobacter ureafaciens are usually gram negative do not reduce a nitrate. It is therefore believed that Arthrobacter strain CA-35 is a new species belonging to the genus Arthrobacter because it differs from the strains of the standardized species belonging to the genus Arthro-35 bacter.

Although some of the mutants of Arthrobacter strain CA-35 are very different from the parent strain in the flagella, these mutants have been found to belong to Arthro- 80 0 4 74 1 -28 bacteria, because a mutant is generally of the same species if the parent strain is classified.

It was determined that Brevibacterium strain CA-6 belongs to the genus Brevibacterium based on its 5 microscopic observations, such as gram staining and the like, as well as its physiological properties. However, Brevibacterium strain CA-6 differs slightly from the other microorganisms belonging to the genus Brevibacterium because the other microorganisms have peritricheous flagella, while Brevibacterium strain CA-6 has a polar flagellum.

Furthermore, it was found that Coryriebacterium strain CA-53 has a close relationship with Corynebacterium egui.

The method according to the invention is carried out by a micro-organism capable of growing in a medium containing as substrate bile acid or a salt thereof, which micro-organism belongs to one of the genera Arthrobacter. Brevibacterium and Corynebacterium, as described above, in a culture medium containing bile acid or a salt thereof as a substrate.

In the invention, bile acid as such can be used as a substrate. An alkali metal salt of bile acid, such as sodium cholate, potassium cholate and the like, or an alkali metal salt of bile acid, such as calcium cholate, magnesium cholate and the like, preferably an alkali metal salt, may also be used. When a cholate is used, it is dissolved in water to form an aqueous solution. containing the cholate at a predetermined concentration. Likewise, a certain amount of an alkali metal compound or an alkaline earth metal compound, which may contain. bile acid a salt. are dissolved in water beforehand, after which bile acid is added to this solution to obtain an aqueous solution containing a cholate in a predetermined concentration. The concentration of the bile acid or salt thereof can vary within wide limits of about 1 - 500 g / l, calculated as bile acid. In connection with the yield and the desired cholanic acid ----------- derivatives of the formula 1, culture conditions and economic effectiveness such as practicality, workability and the like, it is recommended that the bile acid or salt thereof be used in a concentration of about 5-300 g / l, preferably about 10-200 g / l, calculated as bile acid.

Culturing can be carried out according to a known method, usually using a shaking or submerge culture using liquid medium.

As medium, a medium can be used which contains 10 nutritional components which can be assimilated by a microorganism to be used. The medium may contain bile acid or its salt as the sole carbon source, while an additional carbon source may also be present, such as a pentose (e.g. arabinose etc.) a hexose (e.g.

Glucose, mannose, fructose, galactose, etc.), a disaccharide (eg maltose, etc.), a starch decomposition product (eg dextrin, etc.), a sugar alcohol (eg sorbitol, etc.), a polyhydric alcohol (eg glycerol, etc. ), a mixture thereof and the like and / or other food ingredients, such as a polypeptone, a peptone, meat extract, malt extract, corn repellent, yeast extract, an amino acid, a mixture thereof and the like. Usually, the medium can have an additional carbon source and / or. other food ingredient is added, at a concentration of about 0.1-10 g / l. As a nitrogen source, for example, ammonium sulfate, ammonium chloride, arammonium phosphate, ammonium nitrate. sodium nitrate, potassium nitrate, a mixture thereof and the like are used. Usually, a nitrogen source can be added to the medium at a concentration of about 0.5-5 g / l.

Furthermore, as inorganic salts, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like can be added to the medium, usually at a concentration of about 0.1-10 g / l. Cultivation can be done in a shaking or submerge culture for 6 hours. up to 5 days 35 are carried out at a temperature of 25-35 ° C.

According to the process of the invention, the bile acid or salt thereof used as a substrate is converted into the cholanic acid derivatives of the formula 1 and a small amount of m 7i 1 -30-

multitude of lo (t12e <-dihydroxy-3-keto-5 | 1-cholanoic acid or salt thereof, if Arthrobacter strain CA-35 is used. It has been found that the cholanoic acid derivative of the formula 1, wherein X represents -OH, namely the 3θζ, 7c <cdihydroxy-12-5 H

keto-5 | 3-cholic acid. or salt thereof, mainly formed when the above-mentioned mutants of Arthrobacter strain CA-35 are used. Therefore, if the 2or, 7'-dihydroxy-12-keto-5 | S-cholanoic acid or salt thereof is desired, it is preferable to use these mutants, in particular Arthrobacter strain CAr35-A589-29- 32 (FERM-P no. 5522; ATCC no. 31652), Arthrobacter strain CA-35-A589-47 (EERM-P no. 5523; ATCC no. 31653) and Arthrobacter strain CA-35-A1766-15 (FERM -P no. 5528; ATCC no. 31658). In particular, Arthrobacter strain strain CA-35-A589-29-32 and Arthrobacter strain CA-35-A589-47 are preferred. When Brevibacterium strain CA-6 is used, the bile acid or salt thereof is converted into the cholic acid derivative of the formula 1 and a small amount of 7 ° -hydro-a4 xy-3,12-diketo-A-cholenic acid or salt thereof. When Coryne-20 bacterium strain CA-53 is used, the bile acid or salt thereof is converted into the cholanoic acid derivatives of the formula 1 and 7 ° -3-hydroxy-3,12-diketo-Z-1-cholic acid or salt thereof.

After cultivation, the products which have accumulated in the medium are separated from the medium and purified. First, the insoluble materials present in the medium, such as cells of the microorganisms and the like, are removed from the medium by a known method, such as filtration, centrifugation and the like. Then, the filtrate or supernatant obtained thereby is acidified by adding an acid, such as hydrochloric acid, to precipitate the products contained therein. At the same time, the bile acid or salt thereof used as a substrate, which remains without being converted, is also precipitated as bile acid. After separating the obtained precipitate, the filtrate or supernatant is further extracted with an inert organic solvent, such as ethyl acetate and the like and 80 0 4 74 1 -31-like and the solvent is distilled off from the extract to obtain a residue . The residual products and the substrate are almost completely recovered, respectively. recovered. The residue obtained is combined with the precipitate obtained as described above.

The mixture of products and bile acid thus obtained is then chromatographed to isolate the desired product. So. For example, the products obtained using -Arthrobacter strain CA-35 or a mutant thereof can be isolated in the following manner: the products and the bile acid are converted into the methyl esters thereof, after which the methyl esters obtained in this way are chromatographed on a column of silica gel by eluting sequentially. with chloroform, chloroform-ethanol (volume ratio of 99: 1) and. then chloroform-ethanoi (volume ratio of 97: 3). The desired Tctf-hydroxy-3,12-diketo-5H-cholanic acid methyl ester is eluted with chloroform. Elution with chloroform-ethanol (volume ratio of 99: 1) first makes one of these. the by-products, the 7th <, 12c <'- dihydroxy-3-keto-6-cholanoic acid methyl ester and then the desired 3a {f 7 <> (L-dihydroxy-12-keto-5] -3-cholanoic acid methyl ester. The methyl cholate is eluted eluted with chloroform-ethanol (volume ratio of 97: 3) These methyl-25 esters can be converted to the corresponding acids by hydrolysis by a conventional method If Brevibacterium strain CA-6 or Corynebacterium strain CA-53 is used, the products, on the other hand, are isolated, for example, in the following manner: the mixture of products with a bile acid is chromatographed directly on a silica gel by eluting successively with chloroform-ethanol (volume ratio of 98: 2) and then with chloroform-ethanol (volume ratio of 97: 3) The desired hydroxy-3,12-diketo-cholanic acid is eluted with chloroform-35 ethanol (volume ratio of 98: 2) ► By elution with chloroform-ethanol (volume ratio) of 97:3) first becomes a by-product, the T ^ hydroxy-S, 12-diketo-A-1-cholenic acid eluted, which is followed by the elution of the fl 7n 1 -32- desired 3 © (77o (-dihydroxy-12-keto-i-cholanoic acid).

The dihydroxy-12-keto-5-cholanic acid or salt thereof obtained by the process of the invention can be easily reduced by the Huang Minion reduction method to obtain: a useful solubilizing agent for gallstones, i.e. the chenodesoxy bile acid (CDGZ).

The 5 5 hydroxy hydroxy-3,12-diketo-5--cholanoic acid or salt thereof can be easily converted to deoxygolic acid, which is a useful starting material in the preparation of progesterone and adrenal corticosteroid derivatives by dehydration followed by hydrogenation by known methods.

The invention is explained in more detail below by means of the following examples and reference examples, which, however, are for illustrative purposes only and do not limit the invention.

Preparation of mutants.

An eye from an inoculation needle of Arthrobacter strain 20 CA-35 (FERM-E No. 5145; ATCC No. 31651) grown on a foam medium (Medium A: 0.5% NaOH, 5.0% Bile Acid, 0 .5% peptone, 0.5% yeast extract, 0.5% NaCl and 1.5% agar) was inoculated into 10 ml of a medium (medium B: 1% NaOH, 10% bile acid, 0.2% NH4NO3, 0.2% KH2P04, 0.5% K2HPC> 4, 0.02% MgS04.7H20 and 0.01% yeast extract) in a test tube (200 mm at a diameter of 21 mm) and for 24 hours at a temperature incubated at 30 ° C with shaking. Of the obtained culture, 0.3 ml at 10 ml of a medium (medium C: 0.05% NaOH, 0.5% bile acid, 0.5% glucose, 0.2% NH4NO3, 0.2% KH2PO4, 0 .5% K2HPO4, 0.02% MgSO4.7H20 and 0.01% yeast extract) were placed in a test tube (200 mm at a diameter of 21 mm) and shaken at a temperature of 30 ° C for 15-16 hours. incubated. The cells of the microorganism in the logarithmic phase were collected under aseptic conditions using a membrane filter (pore size of 0.45 µm), with 20 ml of a 0.1 M phosphate buffer (pH 7.0) washed and suspended in 25 ml of the same buffer.

A rautation treatment was performed by adding N-methyl-N1-nitro-N-nitrosoguanidine to a final concentration of 50 µg / ml to 4 ml of the above cell suspension in a test tube (200 µl). mm at a diameter of 21 mm) and incubating the mixture with shaking for 45 min at a temperature of 30 ° C. Under these conditions, lethal damage to Arthrobacter strain CA-35 was approximately 80%.

The cells treated in this way were collected by membrane filter (pore size of 0.45 µm) under 10 aseptic conditions, washed with 20 ml of a 0.1 M phosphate buffer (pH 7.0) and in 25 ml of the same buffer suspended. The resulting cell suspension was diluted with sterile normal saline and streaked on agar plates (medium D: 0.1% NaOH, 1.0% bile acid, 0.2% NH4NO3, 0.2% KH2K> 4, 0.5% K2HPO4 , 0.02% MgSO 4, 7H 2 O, 0.01% yeast extract and 1.5% agar), one. and other such that 300-500 colonies were formed per plate. The plates were incubated at a temperature of 30 ° C for 4 days. Each spotted colony formed in this way was isolated and kept at a temperature of 24 hours for 24 hours. Incubated at 30 ° C on a plate (medium E: 0.5% peptone, 0.5% yeast extract, 0.5% NaCl and 1.5% agar). Each colony formed on a medium E plate was again seeded on a medium D plate and incubated at 30 ° C for 20 hours.

Each colony of the above-mentioned plate of medium E, of which the re-expanded colony did not grow on the plate of medium D, was incubated for 24 hours at a temperature of 30 ° C on an oblique medium (medium F: 0). , 1% NaOH, 1.0% bile acid, 0.5% peptone, 0.5% yeast extract, 0.5% NaCl and 1.5% agar). An inoculation needle eye of this oblique culture was inoculated into 10 ml of a medium (medium G: 0.5% NaOH, 5.0% bile acid, 0.5% glucose, 0.2% NH 4 NO 3, 0.2 % KH2P04, 0.5% K2HP04, 0.02% MgS04.7H20 and 0.01% yeast extract) in a test tube (200 mm at a diameter of 21 mm) and incubated for 3 days at a temperature of 30 ° with shaking C.

When examining the products, they occur in any medium. Gathered, by thin layer chromatography, a microorganism was found which selectively produced 3c /, 7 xt-dihydroxy-80 04 74 1 -34-12-keto-5-cholanoic acid or salt thereof, which micro organism was designated Arthrobacter strain CA-35-M-965-3.

The above method is referred to below as "method M". According to the method M, a mutation treatment was performed using Arthrobacter strain CA-35-M-965-3 as the starting strain. As a result, three strains were found which selectively produced 3rd ^ 7th ^ dihydroxy-12-keto-5ji-cholic acid or salt thereof, which strains were referred to as Arthrobacter strain CA-35-A589, Arthrobacter strain CA-35 -A849, resp. Arthrobacter strain CA-35-A1071.

Two superior strains were identified from the colony of Arthrobacter strain CA-35-A589, which strains were referred to as Arthrobacter strain CA-35-A589-29-32, respectively.

Arthrobacter strain CA-35-A-589-47. Also, a superior strain was isolated from the colony of Arthrobacter strain CA-35-A1071, which was referred to as Arthrobacter strain CA-35-A-1071-15.

Furthermore, one superior strain was obtained using Arthrobacter strain CA-35-A849 as the starting strain. using method M, which superior strain was designated Arthrobacter strain CA-35-A-1448. Arthrobacter strain CA-35-A-1475 was obtained using Arthrobacter strain CA-35-Ari448 as a starting strain using the method M. Arthrobacter strain CA-: 35-A-1766 was obtained. Using Arthrobacter strain CA-35-A-1071. as the starting strain Using the method M, while Arthrobacter strain CA-35-A-1766-15 was isolated from the colony. Arthrobacter strain CA-35-A-1766 was obtained.

Arthrobacter strain CA-35-Y-37-12 was obtained using the following method: One eye of an inoculation needle of Arthrobacter strain CA-35 (FERM-P no »5145? ATCC no. 31651) placed on a oblique medium A was cultured, incubated in 10 ml medium B in a test tube (200 mm at a diameter of 21 mm) and incubated at 30 DEG C. for 20 hours with shaking. Cells of the microorganism were collected for 5 min at a. centrifuge temperature of 5 ° C under aseptic conditions at 10,000 revolutions / min. The cells were suspended in 10 ml of sterilized water. The suspension was placed in a Petri dish with a diameter of 7.5 cm, which was placed at a distance of 27 cm under an ultraviolet lamp (2537 &), of 15 W in a germ-free box and was irradiated with ultraviolet rays for 10 min. . Under these conditions, lethal damage to the Arthrobacter strain CA-35 was approximately 99.9%.

After the irradiation, the cells of the microorganism were collected using a membrane filter, washed with a 0.1 M phosphate buffer and suspended in the same buffer. The resulting suspension was diluted with normal saline and the culture steps described above using the media D-G. were repeated to obtain a superior strain, which selectively produced 3,7-dihydroxy-12-keto-5-cholanoic acid or salt thereof.

Example 1

Arthrobacter strain CA-35 (FEEM-P no. 5145; ATCC

No. 31651) was grown in the following manner;

Composition of the culture medium:

Bile acid 100 grams

Ammonium hydrate 2.0 grams. Potassium dihydrogen phosphate 2.0 grams

Dipotassium hydrogen phosphate 5.0 grams

Magnesium sulfate heptahydrate 0.2 grams

Yeast extract 0.1 gram

Sodium hydroxide 10 grams 30 Distilled water to 1.liter

The above ingredients were mixed together to give 1 liter of culture medium. 100 ml each of the culture medium was divided into 10 Sakaguchi bottles (500 ml volume) which were autoclaved at a temperature of 120 ° C for 15 min.

To the contents of each bottle was then added an aliquot of 10 ml of a seed culture obtained by previously cultivating the respective strain in the same medium with shaking fthn'47 & i -36- for a period of 2 days at a temperature of 30 ° C. Each bottle was then incubated on a shaker at 30 ° C for 2 days.

At the end of incubation, culture broth 5 was combined and centrifuged to remove the cells from the microorganism. The supernatant obtained was acidified by adding 600 ml of 1 n-aqueous hydrochloric acid to form a precipitate. The precipitate was separated while the residual solution was extracted with 1 liter of ethyl acetate. The ethyl acetate was distilled from the extract by rotary evaporator and the residue was combined with the above precipitate, whereby 85 g of a mixture of cholic acid derivatives and bile acid was obtained.

A small amount of the mixture was dissolved in methanol at a concentration of 1%. 10 µl of the solution was injected into a speed-acting liquid chromatograph equipped with a jiBondapak C-18 column (type HLC-GPC-244, manufactured by. Waters, United States of America) .

The column was eluted with water-methanol (30: 70 vol., PH 2.5) at a rate of 1 ml / min, while measuring the refractive index of the eluate.

The chromatogram obtained thereby is shown in the accompanying figure 1. The peaks A, B, C and D. in figure 1 correspond to the peaks of the reference standard of 'ib <s-hydroxy-3,12-diketo-5 | i-cholic acid, 3 or, 7th-dihydroxy-12-keto-5β-cholic acid, praise, 12-i-dihydroxy-3-keto-5-chloroic acid, respectively. bile acid.

If the compounds in the fractions correspond to the peaks A, B and C are isolated and their chemical composition is determined on the basis of the mass spectra, IR spectra and NMR spectra thereof, it appears further on the basis of these spectra that these compounds are the 7th-hydroxy-3,12-diketo-1-chloroic acid, 3s, C7th-dihydroxy-12-keto-5-chloroic acid. resp. To be {1,22-dihydroxy-3-keto-5 / i-cholic acid. In the accompanying Figures 2,3 and 4 the IR spectra (A, B and C) of the methyl esters of the compounds corresponding to the peaks A, B and C are shown in comparison. with the IR spectra of the reference standard (A *, B 'and C).

The melting points of the compounds and their methyl esters corresponding to peaks A, B and C are listed in Table B below.

TABLE B

Compounds Melting points of the Melting points of the methyl esters (° C) free acids (° C)

Found Literature Found Literature 10 Peak A. 150-152 152-154

Peak B 154-156 156-157 219-220 221-222

Peak C 168-169 169-172 185-186 186-188

The yield of the products and the amount of bile acid remaining without being converted were calculated based on the area ratio of the chromatogram of Figure 1. The results are shown in Table C below.

TABLE C

20 Connection Area- Conversion- Yield attitude number degree (%) or quantity.

TwHiydroxy-3,12-diketo-5/1-cholan- 28,54 28,54 24,3 acid (peak A) 25 3oC, 7 ^ -dihydroxy-12-keto-5 / i-cholan- 63,06 63 .06 53.6 acid (peak B) 7®ζ l2s »<- dihydroxy-3-keto-5 £ -cholic acid 1.54 1.54 1.30 (peak C) 30

Bile acid (peak D) 6.86 6.86 5.80

Example 2

The same procedure as described in Example 35 was repeated, however, 5.0 g / l of glucose was also added to the medium and 91.0 g of the mixture was obtained consisting of products and bile acid, without being converted was left behind. This 91.0 g of the mixture was dissolved in 270 ml of methanol, to which was added 9 ml of concentrated hydrochloric acid, and the resulting solution was refluxed for 20 min to dissolve the products and the bile acid. convert the methyl ester therefrom.

A column (1200 mm at a diameter of 70 mm) was filled with 1500 g of silica gel C-200, after which the above-mentioned methyl esters were absorbed. The column was eluted with chloroform to give 25.1 g of 1'-1-cycloxy-3,12-10 diketo-5J-cholic acid raethyl ester. The column was then eluted with chloroform-ethanol (99: 1 by volume), the first being 1.40 g of lc / T 12 © <- - dihydroxy-3-keto-phenol-chloranoic acid ethyl ester and then 56.4 g 3 =, Pf Pf Pf Pf Pf ^ddiliiliili--dro dro dro dro----l l l l l l l l l k k k k k k k k k 5 5 5 5 of methyl ester.

The methyl esters were hydrolysed to give 24.8 g of 7'-hydroxy-3,12-diketo-5'-3-chloroic acid, 1.38 g of 7th'-12''-dihydroxy-3-keto -'-cholanoic acid and 56.0 g 3'0. Dihydroxy-12-keto-5YS-cholic acid was obtained.

Example 3 The same procedure as described in Example 1 was repeated, except that different concentrations of bile acid were used and sodium hydroxide was added to the medium in an amount of 10% by weight, based on the bile acid used. The yield of products and the amount of bile acid remaining without conversion have been reported for each concentration of the substrate (bile acid) in Table D below.

TABLE D

(i 30 Compound -Yields_j, Substrate Concentration (g / l) i 50 200 300 ji 3 0Ct 7 # -dihydroxy- j 35 12-keto-5 /? -cholane 27.1 g 108.3 g 47.4 g, acid ij 7 <2-hydroxy- 3,12-diketo-5/3-cholane 12.3 g 49.0 g 21.4 g: acid 80 04 74 1 1 -39- 7 CL, 12 # -dihydroxy- 3-keto-5/3-cholane 0.7 g 2.65 g 1.20 g acid

Bile acid small amount 20.1 g 230 g

Example 4

The same procedure as described in Example 2 was repeated, however each bottle was incubated for 24 hours on a shaker at a temperature of 30 ° C to obtain 98.0 g of a mixture consisting of products and bile acid, without had been left behind. The mixture was subjected to rapid liquid chromatography under the same conditions as described in Example 1 to determine its composition. The results are shown in Table E below.

TABLE E

Compound Ratio-Number-0.37- * - dihydroxy-12-keto-5 / i-cholic acid 67.7 7o (-hydroxy-3.12-diketo-5 / * -cholic acid 8.3 lo (, 12D < -dihydroxy “3-keto-5 ^ -cholane-mr 4.0

Bile acid 20.0

Example 5

The same procedure as described in Example 20 was repeated, but an amount of 20 ml of seed culture obtained by cultivating at a temperature of 30 ° C for 24 hours was added to each bottle and 117.0 g of a mixture was obtained consisting of products and bile acid, which remained without being converted. The mixture was subjected to rapid chromatography under the same conditions as described in Example 1 to determine its composition. The results are shown in Table F below.

80 04 74 1 -40-

TABLE F

Compound Ratio Number 3 (7,7) - dihydroxy-12-keto-5-chloroic acid 63,3 7-hydroxy-3,12-diketo-5-chloroic acid 23,5 5 7 © o 12 © Dihydroxy-3-keto-5 | 4-cholic acid 2.0

Bile acid 11.2

Example 6

Arthrobacter strain CA-35-A589-29-32 (FERM-P no.

10 5521; ATCC No. 31652) was grown in the following manner:

Composition of the culture medium:

Bile acid 100 g

Ammonium nitrate 2> 0 g

Potassium dihydrogen phosphate 2, Q g 15 Dipotassium hydrogen phosphate 5.0 g

Magnesium sulfate heptahydrate 0.2 g

Yeast Extract 0.1 g

Sodium hydroxide 10 g

Glucose 5.0 g 20 Tap water up to 1 liter

The above ingredients except glucose were mixed with tap water and adjusted to a volume of 800 ml and then autoclaved at 120 ° C for 15 min. The glucose 25 was dissolved in 200 ml tap water and treated in the autoclave at a temperature of 110 ° C for 30 min.

After cooling, both solutions were combined to obtain 1 liter of culture medium. 100 ml aliquots of the culture medium were distributed under ten aseptic conditions between ten Sakaguchi bottles (volume of 500 ml). To the contents of each bottle were then added aliquots of 2 ml each of a seed culture obtained by cultivating the respective strain in the same medium for 14 hours with shaking at a temperature of 30 ° C. Each bottle was then incubated on a shaker at 30 ° C for 3 days.

8004 74 1 -41-

At the end of the culture, the culture broth was combined and centrifuged to remove the cells from the microorganisms. The supernatant obtained was acidified by adding 600 ml of 1 n aqueous hydrochloric acid to form a precipitate. The precipitate was separated and the remaining solution was extracted with 1 liter of ethyl acetate. The ethyl acetate was distilled from the extract using a rotary evaporator, and the residue was combined with the precipitate obtained as described above to obtain 99.3 g of a mixture of cholic acid derivatives and bile acid.

A small amount of the mixture was dissolved in methanol at a concentration of 2%. 10 µl of this solution was injected into a high speed liquid chromatography in the direction fitted with a juBondapak C-18 column (= type HLC-GPC-244).

The column was eluted with water-methanol (30:70 by volume, pH 4.0) at a rate of 1 ml / min, measuring the refractive index of the eluate.

The chromatogram obtained in this respect is shown in the accompanying figure 5. The peaks B and D in figure 5 correspond to the peaks of the reference standard of 30, 70 ^ dihydroxy-12-keto-5 | X-cholic acid, respectively. bile acid.

If the compound in the fraction corresponding to the peak · Ό is isolated and the chemical composition thereof is determined on the basis of the mass spectrum, the IR spectrum and the NMR spectrum, these spectra show that the compound is the 3D67c ^ dihydroxy-12-keto-^ -cholic acid.

The yield of the products and the amount of bile acid remaining without conversion were calculated based on the area ratio of the chromatogram of Figure 5. The results are shown in Table G below.

80 0 4 74 1

-42-TABLE G

Compounds Degree of conversion * Yield or (%) amount ___ (g) _ 3οζ 7 «C-dihydroxy-12-keto-5 | k-cholanoic acid (peak B) 98.85 98.16

Other derivatives of bile acid 0.59 0.59

Bile Acid (Peak D) 0.56 0.55 Φ: Turn conversion rate calculated based on area ratio.

Example 7

The same procedure as described in Example 5 6 was repeated, but instead of the 2 ml seed culture, a cell suspension of the microorganism prepared by centrifuging the cells of the microorganism was added to each bottle. separating 10 ml of the seed culture and suspending it in 10 ml of sterilized water to obtain 99.5 g of a mixture of products and residual bile acid. This 99.5 g mixture was dissolved in 270 ml of methanol, to which 9 ml of concentrated hydrochloric acid were added. The resulting solution was refluxed for 20 min to convert the products of the bile acid into its methyl esters.

A column (1200 mm at a diameter of 70 mm) was filled with 1500 g of silica gel C-200, after which the above-mentioned methyl esters were adsorbed. The column was eluted with chloroform-ethanol (99: 1 vol. Ratio) to give 102.0 g of 3 & 7 &quot; -dihydroxy-12-keto-5 &apos; -cholic acid methyl ester, which was hydrolyzed, yielding 98.5 g of 3c <, 7 ° <1-dihydroxy-12-keto-5yS-cholic acid. Example 8

The same procedure as described in Example 25 was repeated, but with the bile acid being used in different concentrations, the sodium hydroxide was added in a medium. amount of 10% by weight, based on the bile acid used, and 1N aqueous hydrochloric acid was added in an amount of 60 ml / g sodium hydroxide, which was used to form a precipitate.

80 04 74 1 -43-

The degree of conversion or yield of product and the amount of residual bile acid at each concentration of the substrate (bile acid) are listed in Table H below.

Example 9 The same procedure as described in Example 6 was repeated, except that Arthrobacter strain CA-35-A589-29-32 was used instead of Arthrobacter strain CA-35-A589t47 (FERM-P no. 5523; ATCC no. 31653), the bile acid and sodium hydroxide concentrations in the medium were adjusted to 50 g / l, respectively. 5 g / l and each bottle was incubated at 30 ° C for 35 hours to obtain 49.31 g of a mixture of products and residual bile acid.

The mixture was subjected to the same rapid liquid chromatography under the same conditions as described in Example 6.

The chromatogram obtained is shown in the accompanying figure 6. The peaks A, B, C and D in figure 6 correspond to the peaks of the reference standard of 20 Tc-hydroxy-S, 12-diketo-5-i-cholanoic acid, 3K, 7 ° f, dihydroxy-12-keto-5 / J-cholic acid, 70%, 12-dihydroxy-3-keto-5-cholic acid, or bile acid.

When the fractions corresponding to the peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction corresponding to the peak B, C or D appears to contain a single compound. The fraction corresponding to peak A consisted of a mixture of 70% (c, hydroxy-3,12-diketo-E-chloroic acid) and another unidentified substance.

The yield of the products and the amount of bile acid remaining in the unreacted state were calculated based on the area ratio of the chromatogram of Figure 6. The results are shown in Table 1 below.

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TABLE I

Connection Conversion rate Yield or, (%) how much fig) .... -'heid—— - '7' -hydroxy-3,12-diketo-5 # - cholanoic acid and unidentified 0.80 0.39 substance (peak A) '3a, 7öT-dihydroxy-12-keto- 5 / £ -cholcanic acid (peak B) 98.01 48.33 1a, 120f-dihydroxy-3-keto-5 /? -cholic acid (Peak C) 0.25 0.13 t fder \ 0.24 0.12 derivatives

Bile acid (peak D) 0.70 0.35

Example 10

The same procedure as described in Example 9 was repeated using Arthrobacter strain CA-35-A849 (FERM-P no. 5524; ATCCno. 31654) in place of Arthrobacter strain 5 CA-35-A589-47 and using 49.34 g of a mixture of products and residual bile acid was obtained.

The mixture was subjected to a rapid liquid chromatography under the same conditions as described in Example 6.

The chromatogram obtained in this respect is shown in the accompanying figure 7. The peaks A, B, C and D in figure 7 correspond to the peaks of the reference standard of praise-hydroxy-3,12-diketo-5 | & -cholanoic acid, 2PC 1.7% dihydroxy-12-keto-5-chloroic acid, 7% <12% dihydroxy-3-keto-3-cholic acid, 15. bile acid.

When the fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction is found to contain a single compound.

The yield of the products and the amount of bile acid remaining in the unreacted state were calculated from the area ratio of the chromatogram of Figure 7, 80 04 74 1 -46. The results are shown in Table J below.

TABLE J

i

Connection Conversion Yield or; degree (%) amount <?) 7 # -hydroxy-3,12-diketo-5-chloroic acid (peak A) 3.2 1.58 3 CL, 7Ci-dihydroxy-12-keto-.

5-Cholic acid (peak B) 83.5 41.20 I

Id, 12 <2-dihydroxy-3-keto-S-chloroic acid (peak C) 7.9 3.90% *. 2.8 1.38 derivatives

Bile acid (peak D) 2.6 1.28 I

Example 11 The same procedure as described in Example 9 was repeated, except that Arthrobacter strain CA-35-A-1071-15 (FERM-P no. 5525; ATCC no. 31655) instead of Arthrobacter strain CA-35-A589- 47 was used to obtain 49.31 g of a mixture of products and residual bile acid.

The mixture was subjected to rapid liquid chromatography under the same conditions as used in Example 6.

The obtained chromatogram is shown in the accompanying figure 8. The peaks A, B, C and D in figure 8 correspond to the peaks of the reference standard of 7c-hydroxy-3,12-diketo-5-chloroic acid, 7-dihydroxy-12-keto-5-chloroic acid, 1,22-dihydroxy-3-keto-chloroic acid, respectively. bile acid.

If the fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction corresponding to peak B, C or D appears to be a single compound. contain. The fraction corresponding to peak A consisted of a mixture of TP-hydroxy-3,12-diketo-5-S-cholic acid and another unidentified substance.

The yield of the products and the amount of residual bile acid were calculated based on the area ratio of the chromatogram of Figure 8. The results are summarized in Table K below.

10 TABLE K

_________________!

Conversion Yield or Connection s how many.

_______ grade (! S7 hero (g) 7CS-hydroxy-3,12-diketo-5/3-cholasic acid and nor 5 2 2.56 identified substance (peak A) 3o: .7 <2-dihydroxy-12-keto-i 5/5 cholic acid (peak B) 93.9 46.30

7CC, 12Ci-dihydroxy-3-keto-I

5 / & * chola * anic acid (peak C) 0.2 0.10 i

Other derivatives 0.2 0.10 '2j. Bile Acid ___ ° * 25_

Example 12

The same requirement as described in example.

9 was repeated, however using Arthrobacter strain CA-35-A-1448 (FERM-P no. 5526; ATCC no. 31656) instead of Arthro-30 bacterial strain CA-35-A589-47 and using 49.30 g of a mixture of products and residual bile acid was obtained.

The mixture was subjected to rapid liquid chromatography under the same conditions as described in Example 6.

The obtained chromatogram is shown in the accompanying figure 9. The peaks A, B, C and D in figure 9 correspond to the peaks of the reference standard of 7 ° -1 -1γγάΓθχγ-3, ΐ2- 80 04 741 -48-diketo- 5-Chloroic acid, 30S, 70-dihydroxy-12-keto-5 ^ r * cholic acid, 7 = 12,12-dihydroxy-3-keto-5 ^ -cholic acid, respectively. bile acid.

If the fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction corresponding to peak B, C or D appears to contain a single compound. The fraction corresponding to peak A consisted of a mixture of X 3 -hydroxy-3,12-diketo-S-chloroic acid and another unidentified substance.

The yields and products and the amount of bile acid remaining in the unreacted state were calculated based on the area ratio of the chromatogram of Figure 9. The results are summarized in Table L.

15 TABLE L

",. _. Conversion Yield or

Link . , How much- . .

grade (%) ^ 20 7a-Hydroxy-3,12-diketo-5/3-cholanoic acid and unidentified 6.5 3.20 indicated substance (peak A) 3 CL, 7Qf-dihydroxy-12-keto-5 / -chola.anic acid (peak B) 88.0 43.39 y 25 la, 12or-dihydroxy-3-keto-S / S'-cholanic acid (peak C) 0.3 0.15 y

Other derivatives 0.1 0.05

Bile acid. 5.1 2.51 i 30 _______; _!

Example 13

The same procedure as described in Example 9 was repeated, however, Arthrobacter strain CA-35-A-1475 (FEKM-P no. 5527; ATCC no. 31657) was replaced by Arthro-35 bacterial strain CA-35-A589-47 and obtained 49.35 g of a mixture of products and unreacted bile acid.

80 0 4 74 f% -49-

The mixture was subjected to rapid liquid chromatography under the same conditions as described in Example 6.

The chromatogram obtained is shown in the accompanying figure 10. The peaks A, B, C and D in figure 10 correspond to the peaks of reference standard of 7-f-hydroxy-3,12-diketo-5-chloroic acid, 3 = <tb> -dihydroxy-12-keto-^ -cholanoic acid, [(] -dihydroxy-S-keto -phl -cholic acid, or bile acid.

If the fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction is found to contain a single compound.

The yield of the products and the amount of residual bile acid were calculated based on the area ratio of the chromatogram of Figure 10. The results are shown in Table M below.

TABLE M

",. Conversion Yield or 2 o Compound Amount __ Degree (¾) (g) 7öT-Hydroxy-3,12-diketo-5 / S-Cholic Acid (Peak A) 3.26 1.61 i 3ar, 7 # -dihydroxy-12-keto - j 25 5/5-cholic acid (peak B) 92.0 4540 ii 7or, 120: -dihydroxy-3-keto- \ 5/2-cholic acid "(peak C) 0.20 0.10 y | an Other derivatives small amount

Bile acid (peak D) 4.54 2.24

Example 14

The same procedure as described in Example 35 was repeated, however, Arthrobacter strain CA-35-A-1766-15 (FERM-P no. 5528; ATCC no. 31658) was replaced by Arthrobacter strain CA-35-A589-47 and 49.27 g of a mixture of the products and bile acid were obtained, 80 04 74 1 -50-.

The mixture was subjected to rapid liquid chromatography under the same conditions as described in Example 6.

The obtained chromatogram is shown in the accompanying figure 11. The peaks A, B and D in figure 11 correspond to the peaks of the reference standard of T 1 -hydroxy-S, 12-diketo-E-1-cholanoic acid, Sef ' 7 or dihydroxy-12-keto-phenol-cholic acid, respectively. bile acid.

When the fractions corresponding to peaks A, B and D are separated and subjected to thin layer chromatography, each fraction appears to consist of a single compound.

The yield of the products as well as the amount of residual bile acid were calculated based on the surface reduction of the chromatogram of Figure 11. The results are shown in Table N below.

TABLE N

20 Compound Conversion Yield or raad9 degree Wheid66 Wj 7 <^ - hydroxy-3,12-diketo-5 /? - n m η π 'chola? Nzmir (peak A) u.zj υ, ιχ.

3 #, 7Qf-dihydroxy-12-keto-Q7 n7.

5/9 cholasanic acid (peak B)

Other derivatives - 0.75 0.37 j

. Bile acid (peak D) _ 2 ° ° ° ° I

* ° Example 15

The same procedure as described in Example 9 was repeated, however using Arthrobacter strain CA-35-M-965-3 (FERM-P no. 5529; ATCC no. 31659) in place of Arthrobacter strain CA-35-A589-47 and yielding 49.36 g of a mixture of products and bile acid.

The mixture was subjected to a rapid liquid chromatography under the same conditions as description regreg νρ <ψ} ψβ1ά 6.

-51-

The chromatogram obtained is shown in the accompanying figure 12. The peaks A, B and D in figure 12 correspond to the peaks of the reference standard of 7®-hydroxy-3, 12-diketo-5-chloroic acid, 33T, 7o Dihydroxy-12-keto-5 5-bcholanoic acid, respectively. bile acid.

When the fractions corresponding to peaks A, B and D are separated and subjected to thin layer chromatography, each fraction corresponding to the peak B or D appears to contain a single compound. The fraction corresponding to peak A consisted of a mixture of Tft-hydroxy-S, 12-diketo-5-chloroic acid and another unidentified substance.

The yield of the products and the amount of residual bile acid were calculated based on the area ratio of the chromatogram of Figure 12.

The results are shown in Table O below.

TABLE O

Connectincr 'Conversion Yield or 20 Veroxnazng how many,.

grade (%) hela (g) 7 or -hydroxy-3,12-diketo-5,6-cholanoic acid and unidentified substance (peak 'A) 3 #, 7a: -dihydroxy-12-keto- 25 5 / S * -cholic acid (peak B) 85.3 42.11

Other derivatives 2.0 0.99 'Bile acid (peak D) 4.1 2.02 i 30 Example 16

The same requirement as described in Example 9 was repeated, however using Arthrobacter strain CA-35-Y-37-12 (FERM-P no. 5530; ATCC no. 31660) in place of Arthrobacter strain CA-35-A589-47 and yielding 49.33 g of a mixture of products and bile acid.

The mixture was subjected to rapid liquid chromatography under the same conditions as described in Example 6.

80 04 74 1 -52-

The chromatogram obtained is shown in the accompanying figure 13. The peaks A, B, C and D in figure 13 correspond to the peaks of the reference standard of 7-hydroxy-xy-3,12-diketo-5-chloroic acid, 2P <,> f-dihydroxy-12-keto-5 | i-5-cholic acid, 7 ° C, 13> f-dihydroxy-3-keto-5-chloroic acid, resp. bile acid.

When the fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction is found to contain a single compound.

The yield of the products and the amount of bile acid remaining were calculated based on the area ratio of the chromatogram of Figure 13. The results are summarized in Table P below.

15 TABLE P

Compound Conversion Yield or Grade (%) Amount- (, _Z_heid_ 9n 7 <2-hydroxy-3,12-diketo-5 / 3- _ _ _ βη cholanoic acid (peak A) 3,7,7a-dihydroxy-12-keto - Qn n 44 4n 5/5-cholic acid (peak B), 90 0 44 * 40 7Qf, 12 <ni-dihydroxy-3-ketone -.

S / J cholic acid (peak C) * f 25

Bile acid (peak D) 2.0 0.98?

Example 17

The same procedure as described in Example 30 was repeated, but using Brevibacterium strain CA-6 (FERM-P No. 5144; ATCC No. 51661) in place of Arthrobacter strain CA-35.

The resulting mixture of products and residual bile acid was subjected to rapid liquid chromatography as described in Example 1.

The chromatogram obtained is shown in the accompanying figure 14.

If the compounds in the fractions corresponding to peaks E, A, B and D are isolated and their chemical composition determined on the basis of their mass spectra, IR spectra and NMR spectra, it appears from these spectra that these compounds have the 7th-hydroxy-3,12-5 diketo-α-cholenic acid, / bf-hydroxy-3,12-diketo-5 ^ 3-cholic acid, 3 or, 7-dihydroxy-12 -keto-5yi-cholic acid, resp. bile acid.

The compounds in the fractions corresponding to the peaks X and Y were unidentified substances.

In the chromatogram of Figure 14, the area to area ratio of the peaks excluding the peaks X and Y (i.e. the area ratio E: A: B: D) was 12.2: 22.2: 44.9: 20.7.

Example 18

The same procedure as described in Example 15 was repeated, however using Corynebacterium strain CA-53 (FERM-P no. 5532; ATCC no. 31662) in place of Arthrobacter strain CA-35 and using 82 g of a mixture of products and bile acid was obtained.

The resulting mixture was subjected to rapid liquid chromatography under the same conditions as described in Example 1, but using water-methanol (30:70 by volume, pH of 4.0) instead of water-methanol (by volume) of 30:70, pH of 2.5) was used as the eluent.

The chromatogram obtained is shown in the accompanying figure 15. The peaks A, B, C and D in figure 15 correspond to the peaks of the reference standard of X-hydroxy-3,12-diketo-5f & -cholic acid, Tctf-dihydroxy -12- 'Keto-54-cholic acid, T-hydroxy-3,12-diketo-A-cholenic acid, 30 respectively. bile acid. If the compounds in the fractions corresponding to peaks A, B and C were isolated and their chemical cells were strongly determined on the basis of the mass spectra, IR spectra and NMR spectra thereof, these spectra show that these compounds <RTI ID = 0.0> - </RTI> -hydroxy 3,12-35 diketo-S-cholanic acid, 3,4-dihydroxy-12-keto-5-1-cholanoic acid, respectively. the 75-hydroxy-3,12-diketo-A-cholenic acid.

80 04 74 1 -54-

Reference example 1

10 g of the 3 ((7, 7-dihydroxy-12-keto-5-choline) acid obtained in the manner described above was placed in a three-neck (round bottom) flask, after which 5 were slowly added 100 ml of ethylene glycol and an aqueous potassium hydroxide solution (10 g of potassium hydroxide plus 20 ml of water) was added, further 10 ml of 85% hydrazine hydrate was added and the resulting mixture was refluxed for 2 hours at a temperature of 100 ° C. The temperature was gradually increased, then reflux was continued for another 4 hours at a temperature of 185-190 ° C to distill off the hydrazine hydrate. After cooling, the reaction mixture was diluted with excess water and pH of 3 was set, "15 being a precipitate. The precipitate was collected by filtration, washed with water and air dried to obtain 8.5 g of SeOT-2-dihydroxy-S / 1-cholanzaur (CDGZ).

Reference Example 2 In a three-necked (round bottom) flask, 3 g of the 7 <-Hydroxy-3,12-diketo-5'-cholanoic acid obtained according to the above examples were dissolved in 70 ml of pyridine, after which 1 mol of tosyl chloride was added. The resulting mixture was brought to conversion at a temperature of 0 ° C for one hour and then refluxed for an additional 5 hours. After refluxing, 3N aqueous hydrochloric acid was added and the reaction mixture was extracted with 100ml ether. The ether was distilled off from the extract under reduced pressure to obtain 2.5 g of Λ-η ** 30 and / or fa-3,12-diketo-cholic acid. Then, 2 g of the obtained acid in 10 ml of a 1 n. sodium hydroxide solution dissolved in water, adding 0.1 g of Raney nickel. After adding 0.01 mole of hydrogen, the mixture was allowed to react overnight, whereby 30% 12-r-dihydroxy-5-chloroic acid (deoxygalic acid) and 3%, 12o-dihydroxy- -cholic acid (yield; 90%) was obtained.

8004 74 1

Claims (25)

A method for the microbiological preparation of a cholanic acid derivative of the formula 1 of the formula sheet, wherein X or = 0 and R is hydrogen, alkali 5 H represent metal or alkaline earth metal, characterized in that a microorganism capable of growing in a medium containing as substrate bile acid or a salt thereof and thereby producing the cholic acid derivative 10, which microorganism belongs to one of the genera Arthrobacter, Brevibacterium and Corynebacterium, are grown in a culture medium containing the substrate and the derivative obtained is collected. Method according to claim 1, characterized in that the microorganism used is Arthrobacter strain CA-35 (FERM-P no. 5145; ATCC no. 31651), Arthrobacter strain CA-35-A589-29-32 (FERM -P No. 5522; ATCC No. 31652), Arthrobacter strain CA-35-A589-47 (FERM-P No. 5523; ATCC No. 31653), Arthrobacter strain CA-35-A849 (FERM-P No. 5524; ATCC No. 31654), Arthrobacter 20 strain CA-35-A-1071-15 (FERM-P No. 5525; ATCC No. 31655), Arthrobacter strain CA-35-A-1448 (FERM-P No. 5526; ATCC No. 31656), Arthrobacter strain CA-35-A-1475 (FERM-P No. 5527; ATCC No. 41657), Arthrobacter strain CA-35-A-1766-15 (FERM-P No. 5288; ATCC No. 31658), Arthrobacter strain CA-35-M-965-3 (FERM-25 P no. 5529; ATCC no. 31659), Arthrobacter strain CA-35-Y-37-12 (FERM-P no. 5530; ATCC no. 31660), Brevibacterium strain CA-6 (FERM-P No. 5144; ATCC No. 31661) or Corynebacterium strain CA-53 (FERM-P No. 5532; ATCC No. 31662). 3. A method according to claim 1, characterized in that the produced cholic acid derivative is 3, 7 <* - dihydroxy-12-keto-5 | & -cholic acid. Method according to claim 3, characterized in that the microorganism Arthrobacter strain CA-35-A589-29-32 (FERM-P no. 5522; ATCC no. 31652), Arthrobacter strain CA-35- 35 A589-47 (FERM-P no. 5523; ATCC no. 31653), Arthrobacter strain CA-35-A849 (FERM-P no. 5524; ATCC no. 31654), Arthrobacter strain CA-35-A-1071-15 (FERM-P no. 5525; ATCC no. 31655), Arthrobacter strain CA-35-A-1448 (FERM-P no. 5526; ATCC no. 80 0 4 74 t -56- 31656), Arthrobacter strain CA- 35-A-1475 (FERM-P no. 5527; ATCC. No. 31657), Arthrobacter strain CA-35-A-1766-15 (FERM-P no. 5528; ATCC no. 31658), Arthrobacter strain CA-35 -M-965-3 (FERM-P No. 5529; ATCC No. 31659) or Arthrobacter strain CA-35-Y-37-5 12 (FERM-P No. 5530; ATCC No. 31660). Process according to one or more of the preceding claims, characterized in that the substrate is used in a concentration of 1-500 g / l, calculated as bile acid. 6. Process according to one or more of the preceding claims, characterized in that the substrate is used in a concentration of 5-300 g / l, calculated as bile acid. Process according to one or more of the preceding claims, characterized in that the substrate is used in a concentration of 10-200 g / l, calculated as bile acid. Arthrobacter sp., Which is capable of growing in a medium containing bile acid or a salt thereof as substrate, to form cholanoic acid derivatives of the formula 1 of the formula sheet wherein X is S OH or = O and R is hydrogen , H 20 represent alkali metal or alkaline earth metal. Arthrobacter strain CA-35 (FERM-P no. 5145; ATCC no. 31651). Arthrobacter strain CA-35-A589-29-32 (FERM-P No. 5522; ATCC No. 31652). 11. Arthrobacter strain CA-35-A589-47 (FERM-P No. 5523; ATCC No. 31653). Arthrobacter strain CA-35-A849 (FERM-P No. 5524; ATCC No. 31654). Arthrobacter strain CA-35-A-1071-15 (FERM-P No. 30 5525; ATCC No. 31655). Arthrobacter strain CA-35-A-1448 (FERM-P No. 5526; ATCC No. 31656). Arthrobacter strain CA-35-A-1475 (FERM-P No. 5527; ATCC No. 31657). Arthrobacter strain CA-35-A-1766-15 (FERM-P No. 5528; ATCC No. 31658). Arthrobacter strain CA-35-M-965-3 (FERM-P No. 5529; ATCC No. 31659). 8004 74 t -57- 18. Arthrobacter strain CA-35-Y-37-12 (FERM-P No. 5530; ATCC No. 31660). 19. Brevibacterium sp. which is capable of growing in a medium containing bile acid or a salt thereof as substrate, to form cholanic acid derivatives of the formula 1 of the formula sheet, wherein X «ς ^ -ΟΗ or = 0 and R Η hydrogen, alkali metal or alkaline earth metal. 20. Brevibacterium strain CA-6 (FERM-P No. 5144; 10 ATCC No. 31661). 21. Corynebacterium sp. who is able to. " growing in a medium containing bile acid or a salt thereof as a substrate, to form cholanic acid derivatives of the formula 1, wherein X <ςτ-ΟΗ or = 0 and R is hydrogen, alkali metal or 15 H propose alkaline earth metal. 22. Corynebacterium strain CA-53 (FERM-P No. 5532; ATCC No. 31662). 8004 74 1