FR2463809A1 - MICROBIAL PROCESS FOR PRODUCING CHOLANIC ACID DERIVATIVES AND THE MICROBES USED THEREIN - Google Patents

Abstract

A MICROBIAL PROCESS FOR PRODUCING A CHOLANIC ACID DERIVATIVE OF THE FORMULA: (CF DRAWING IN BOPI) OR X REPRESENTS --- OH OR O AND R

Description

The present invention relates to a microbial process for producing

  cholanic acid derivatives of formula:

0 1 0

(I)

X OH

  H o X represents ---- OH or = 0; and R represents a hydrogen atom II H or an alkali or alkaline earth metal and microbes used in this process. More particularly according to the invention, the cholanic acid derivatives of formula (1) can be produced rapidly with a high yield by culturing particular microbes in a culture medium containing cholanic acid or one of its

salts as substrate.

  The cholanic acid derivative of formula (I) where X

  H-dihydroxy-3a, 7a-keto-12-cholanic acid and its salts are useful as intermediates in the production of a solubilizing agent for gallstones, chenodeoxycholic acid. (CDCA). The cholanic acid derivative of formula (I) wherein X represents = 0, that is 7α-hydroxy-diketo-3,12-p-cholanic acid and its salts are useful intermediates for producing the acid. deoxycholic which is a starting material

  useful for producing progesterone and adrenocorticoid

  térotdes. It is known that the cholanic acid derivatives of formula (I) can be obtained by a microbial process which uses cholic acid or a salt thereof as a substrate. for example

  Hayakava et al. have described a process for producing dihydric acid

  5α-cholanic acid-3α, 7α-keto by the use of the strain Streptomyces gelaticus 1164 [The Journal of Biochemistry (Japan), vol. 44, No. 2, pp 109-113 (1975); and Proceedings of Japan Academy, vol. 32, pp. 519-522 (1956)]. Hasegawa et al. have described a process for producing dihydroxy-3a, 7a keto-12,55-cholanic acid by using the strain Aspergillus cinnamomeus HUT 2026 [Hiroshima

  Journal of Medical Science, Vol. 8, No. 3, pp. 277-283 (1959)].

  Kikuchi et al. also disclosed a method for producing 3-dihydroxy-7α-keto-5β-cholanic acid by use of the strain Staphylococcus epidermidis H-1 [Journal of Biochemistry,

  flight. 72, No. 1, pages 165 to 172 (1972)]. In addition, Hayakawa et al.

  have described a method for producing 7α-hydroxy-diketo-3,12-cholanic acid by use of the strain Streptomyces Relaticus 1164

  [Proceedings of Japan Academy, vol. 32, pp. 519-522 (1956)].

  However, in these processes, the concentration of the cholic acid used as substrate in a culture medium is low, that is to say does not exceed 10 g / l. Therefore,

  according to the applicant's experience, it is necessary to use

  a low concentration of cholic acid to produce cholanic acid derivatives according to known microbial processes because

  the microbes used develop little or badly when the concentration

  The amount of cholic acid in the culture medium is equal to or greater than 20 g / l. In addition, these known methods require too much culture time. It is therefore sought to develop a process for producing cholanic acid derivatives rapidly and in high yield. The Applicant has discovered that certain microbes belonging to the genera Arthrobacter, Brevibacterium and Corynebacterium can grow in a medium containing

  cholic acid or a salt thereof as a substrate for a concentration

  within a wide range and can be produced

  cholanic acid derivatives rapidly with a high yield.

  The subject of the invention is: a microbial process for producing the cholanic acid derivatives of formula (I) rapidly and in high yield; and new microbes that can grow in a medium containing cholanic acid or a salt thereof as a substrate. Other features and advantages of the invention

  will be better understood by reading the following description

  several embodiments with reference to the accompanying drawings in which: - Figure 1 is a chromatogram obtained by high speed liquid chromatography, where the relative refractive index (úln) is ordinate and the time (min) is in x, the product obtained in Example 1; - Figures 2, 3 and 4 respectively illustrate the

  infrared spectra. methyl esters of the corresponding compounds

  at peaks A, B and C (and reference standards A ', B' and C '), the wave numbers (cm) being on the abscissa; and FIGS. 5 to 15 are chromatograms similar to that of FIG.

Examples 6 and 9 to 18.

  The invention thus relates to a microbial process for producing a cholanic acid derivative of formula (I) which consists of

  to cultivate a microbe capable of developing in a con-

  holding cholic acid or a salt thereof as a substrate for producing the cholanic acid derivative, selected from the genera Arthrobacter, Brevibacterium and Corynebacterium, in a medium of

  culture containing the substrate and to collect the derivative product.

  The microbes used in the invention are those isolated from the soil and mutants obtained by natural mutation.

  products, for example by irradiation with X-rays, irradiation

  ultraviolet light, action a mutagenic agent such as

  N-methyl N-nitro N-nitroso guanidine, N-nitro-4-nitroquinone

  acid, acriflavine or ethyl methanesulfonate or any of their

combinations and the like.

  Applicant filed at the Research Institute

  on Fermentation, Ministry of Science and Industrial Technologies

  tribes of Japan (FERM) and the American Type Culture Collection of the United States of America (ATCC), the characteristic strains of microbes capable of producing cholanic acid derivatives of formula (I) in a culture medium containing cholic acid or one of its salts, which it obtained. These are: Arthrobacter CA-35 (FERM-P N 5145, ATCC N 31651), Arthrobacter CA-35-A589-29-32 (FERM-P N 5522, ATCC N 31652), Arthrobacter CA-35-A589- 47 (FERM-P N 5523, ATCC N 31653), Arthrobacter CA-35-A849 (FERM-P N 5524, ATCC N 31654), Arthrobacter CA-35-A-1071-15 (FERM-P N 5525, ATCC N 31655), Arthrobacter CA-35-A-1448 (FERM-P N 5526, ATCC N 31656), Arthrobacter CA-35-A-1475 (FERM-P N 5527, ATCC N 31657), Arthrobacter CA-35-A- 1766-15 (FERM-P N 5528, ATCC N 31658), Arthrobacter CA-35-M-965-3 (FERM-P N 5529, ATCC N 31659), Arthrobacter CA-35-Y-37-12 (FERM No. 5530, ATCC No. 31660), Brevibacterium CA-6 (FERM-β N 5144, ATCC No. 31661) and Corynebacterium CA-53 (FERM-β N 5532, ATCC No. 31662) Arthrobacter CA-35 strains, Brevibacterium CA -6 and Corynebacterium CA-53 are wild-type strains and the other nine strains of Arthrobacter are mutants of the Arthrobacter CA-35 strain. Arthrobacter strain CA-35-Y-37-12 was produced by ultraviolet irradiation. The eight other mutants of the Arthrobacter CA-35 strain were obtained by treatment of the

  parent strain by N-methyl N-nitro N-nitroso guanidine.

  Morphological, cultural and

  The physiological properties of these strains are shown in Table I below.

  For comparison, the characteristics of the strain Arthrobacter simplex IAM 1660 which is related to the strain Arthrobacter

  CA-35 are also included in Table I below.

  From these morphological characteristics, cul-

  physiological classification of strains according to Bergey's Manual of Determinative Bacteriology 7th and

8th edition.

  Arthrobacter strain CA-35 was determined to be

  in relation to Arthrobacter simplex because of its characteristics

  microscopic ticks, such as shape, Gram stain, and the like, as well as its physiological characteristics.However, the Arthrobacter CA-35 strain differs from the Arthrobacter simplex IAM 1660 strain by pigment production, carbohydrate uptake and culture in a medium containing cholic acid Arthrobacter strain CA-35 can grow in a medium containing sodium cholate as the only carbon source at a high concentration, about 20 to 500 g / cm 2. 1 to form as main metabolic product, 7α-hydroxy, 3-diketo-3,125-cholanic acid, 3α-dihydroxy-3α, 7α-keto-5β-cholanic acid, 7α-dihydroxy acid, 12a-cdto-3 5D-cholanic and / or their sodium salts while the strain Arthrobacter simplex IAM 1660 can hardly develop in a medium containing sodium cholate as the sole source of carbon at a concentration of 10 g / L. The strain Arthrobacter CA-35 produces color pigments yellow to cream. On the other hand, as Arthrobacter-producing and pigment-producing microbes, there are Arthrobacter oxydans, Arthrobacter aurescens and Arthrobacter ureafaciens. However, the Arthrobacter CA-35 strain differs from these microbes because Arthrobacter oxydans and Arthrobacter aurescens are generally gram negative and hydrolyze starch and Arthrobacter ureafaciens is generally gram negative and does not reduce a nitrate. Arthrobacter CA-35 appears to be a new species belonging to the genus Arthrobacter because it differs from strains of standard species

belonging to the genus Arthrobacter.

  Although some of the Arthrobacter CA-35 strain mutants differ completely from the parent strain with respect to flagella, it has been determined that these mutants belong to A. Arthrobacter because generally a mutant is classified in the same manner.

species as its parent strain.

  Brevibacterium CA-6 strain was determined to belong to the genus Brevibacterium by microscopic examination as well as

  Gram stain and the like and its physiological characteristics

  cal. However, the Brevibacterium CA-6 strain differs somewhat from other microbes belonging to the genus Breyibacterium because other microbes have peritrichous flagella whereas the Brevibacterium CA-6 strain has a polar flagellum, etc. In addition, it was determined that the Corynebacterium strain

  CA-53 is in close relationship with Corynebacteriym equi.

  To practice the process of the invention, a microbe capable of growing in a medium containing cholic acid or a salt thereof is cultivated as a substrate, selected from the genera Arthrobacter, Brevibacterium and Coryobacterium, such as those above in a culture medium containing acid

  cholic or one of its salts is a substrate.

  In the invention, cholic acid itself can be used as a substrate. It is also possible to use an alkali metal salt of cholic acid, such as sodium cholate, potassium cholate or the like, or an alkaline earth metal salt of cholic acid, such as calcium cholate, cholate

  gnesium or the like and an alkali metal salt is preferred.

  When cholate is used, it is dissolved in water

  to prepare an aqueous solution containing cholate at a temperature

  predetermined centering. Alternatively, a certain amount of an alkali metal compound or an alkaline earth metal compound which forms a salt with cholic acid may be dissolved in water and cholic acid added to obtain a solution. aqueous solution containing a cholate at a predetermined concentration. The concentration of cholic acid or its salt may vary considerably

  significant in the range of about 1 to 500 g / l (in cholic acid).

  For issues of yield of the desired cholanic acid derivatives of formula (I), culture conditions and cost-effectiveness such as suitability and ease of processing and the like,

  it is recommended to use cholic acid or its salt at a

  centration from about 5 to 300 g / l and more preferably from about 10 to 200 g / l

in cholic acid.

  The culture can be carried out according to a known method

  and a culture with stirring or cultivation is generally carried out.

  submerged with a liquid medium.

  A medium containing nutrients that can be assimilated by the microbe used can be used. The medium may contain cholic acid or its salt as the only carbon source or may contain an additional carbon source, such as pentose (eg arabinose, etc.), hexose (e.g.

  glucose, mannose, fructose, galactose, etc.), a disaccharide

  wrinkle (eg maltose), a product of decomposition of starch

  (for example a dextrin, etc.), a polyhydric alcohol with a

  dique (for example sorbitol, etc.), a polyhydric alcohol (for example glycerol, etc.), a mixture thereof or the like and / or another nutritive substance, such as a polypeptone, a peptone, a meat extract , a malt extract, an infusion of mats, a

  yeast extract, an amino acid, a mixture thereof or the like.

  Generally, an additional carbon source and / or other nutrient may be added to a medium at a concentration of about 0.1 to 10 g / l. As a source of nitrogen, it is possible to use, for example, ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, one of their mixtures and the like. Generally, a source of nitrogen can be added to a medium at a concentration of about 0.5 to 5 g / l. In addition, as the inorganic salts, dipotassium phosphate or potassium dihydrogen phosphate may be added to a medium,

  magnesium sulphate, and the like, generally at a concentration

  from about 0.1 to 10 g / l. A shaking culture or submerged culture can be carried out at 25 ° to 35 ° C. for 6 hours. According to the process of the invention, the cholic acid or its salt which is used as substrate is converted into the cholanic acid derivatives of formula (I) and in a small amount of dihydroxy-7a, 12a keto acid. 35P-cholanic acid or a salt thereof when using the Arthrobacter CA-35 strain. The Applicant has discovered that when the above mutants of Arthrobacter CA-35 are used, the predominant cholanic acid derivative of

  formula (I) where X represents OH, that is to say dihydroxy acid;

  3ac, 7a keto-12 53-cholanic or a salt thereof. Thus, when 3-dihydroxy-3α, 7α-keto-5β-cholanic acid or a salt thereof is desired, it is preferable to use these mutants, in particular the Arthrobacter strain CA-35-A589-29-32 (FERM- No. 5522, ATCC No. 31652), Arthrobacter CA-35-A589-47 strain (FERM-P N 5523, ATCC No. 31653), and Arthrobacter CA-35-A-1766-15 strain (FERM-P N 5528). ATCC N 31658). In particular, the Arthrobacter CA-35-A589-29-32 strain and the Arthrobacter CA-35-A589-47 strain are especially preferred. When the Brevibacterium CA6 strain is used, the cholic acid or its salt is converted into the cholanic acid derivatives of formula (I) and a small amount of 7-hydroxy-3-diketo-3-amino acid. 4-cholenic or a salt thereof. When Corvnebaeterium strain CA-53 is used, the cholic acid or its salt is converted into the acid derivatives

  cholanic compound of formula (I) and 7-hydroxy-acid diketo-3,12-A-4-

cholenic acid or a salt thereof.

  After completion of the culture, the products which have accumulated there and are purified are separated from the medium. First of all,

  medium is removed by known methods such as filtering.

  centrifugation and the like, insoluble materials of the medium, such as microbial cells and the like. Then, the filtrate or the supernatant is acidified by adding an acid, such as hydrochloric acid, to precipitate the products. At the same time, the cholic acid or its salt, used as a substrate, which remains unconverted, is also precipitated as cholic acid. After separation of the precipitate obtained,

  The filtrate or supernatant is again extracted with an organic solvent.

  inert substance, such as ethyl acetate or the like, and

  the solvent of the extract by distillation, to obtain a residue.

  The remaining products are thus almost completely recovered and the

  substrate. This residue is combined with the precipitate previously obtained.

  The mixture of products and cholic acid is subjected

  thus obtained in a chromatography to isolate the desired product.

  For example, the following products can be isolated as follows:

  maintained by use of the strain Arthrobacter CA-35 or a mutant thereof: the products and cholic acid are converted into their methyl esters and the obtained methyl esters are subjected to chromatography on a column of silica gel with elutions chloroform, a chloroform-ethanol mixture (99/1, v / v) and then a chloroform-ethanol mixture (97/3, v / v). The desired methyl-7α-diketo-3,12 5β-cholanic acid methyl ester

  is eluted with chloroform. By elution with chlorinated

  form-ethanol (99/1, v / v) first one of the by-products, the methyl ester of 7-dihydroxy-7a, 12a-keto-5p-cholanic then the methyl ester of the acid. The desired 3-dihydroxy-3a, 7a-keto-5-cholanic acid is eluted, and the methyl cholate is eluted with chloroform-ethanol (97: 3, v / v), and these methyl esters can be converted to the corresponding acids by hydrolysis. If not, when using the Brevibacterium CA-6 strain or the Corvnebacterium CA-53 strain, the products can be isolated, for example, as follows: a mixture of the products and cholic acid is directly subjected to chromatography on silica gel with successive elutions with a chloroform-ethanol mixture (98/2, v / v) and then a chloroformethanol (97/3, v / v) mixture, the 7-hydroxy-acid diketo-3,12 5p- desired cholanic is eluted with the mixture

  chloroform-ethanol (98/2, v / v). By elution with chlorinated

  form-ethanol (97/3, v / v), is first eluted by-product 7a-hydroxy-diketo-3,12-C-4-cholenic acid and then dihydroxy-3a, 7a

keto-12 5p-cholanic desired.

  The 3α-dihydroxy-3α, 7α-keto-5β-cholanic acid or its salt obtained according to the invention can be easily reduced by the Huang Minlon reduction method to obtain a useful agent.

  As a solubilizer for gallstones, chenodeoxy-

chole (CDCA).

  The 7α-hydroxy-diketo-3,12-polycholanic acid or its salt can easily be converted into deoxycholic acid, which is a useful starting material for producing progesterone and adrenocorticosteroids, by dehydration and then hydrogenation.

according to a conventional method.

  The invention is illustrated by the examples and examples

  following non-limiting references.

  Preparation of mutants A loop of the strain Axthrobacter CA35 (FERM-P N 5145, ATCC N 31651) cultured on an inclined medium (medium A, NaOH 0.5%, cholic acid 5.0%, peptone 0 0.5%, yeast extract 0.5%, NaCl 0.5% and 1.5% agar) and 10 ml of medium (medium B, NaOH 1%, cholic acid%; NH4NO3: 0.2%, KH2PO4: 0.2%, K2HPO4: 0.5%, MgSO4.7H2O: 0.02% and yeast extract: 0.01%) in a test tube (200 mm x 21 mm) of diameter) and incubated with stirring at 30 C for 24 h. 0.3 ml of the culture obtained are added to 10 ml of a medium (medium C, NaOH 0.05%, cholic acid 0.5%, glucose 0.5%, N4N3 0.2%; KH 2 PO 4: 0.2 K 2 PO 4: 0.5%, MgSO 4, 7H 2 O: 0.02% and yeast extract: 0.01%) in a test tube (200 mm x 21 mm in diameter) and incubated in stirring at 30 C for 16 hours. The microbial cells were collected in the log phase with a filter membrane (pore size: 0.45 / um) under aseptic conditions, washed with 20 ml of 0.1 M phosphate tempon.

  (pH 7.0) and suspended in 25 ml of the same buffer.

  To perform mutagenic treatment, N-methyl N-nitro N-nitroso guanidine is added at the final concentration of 50 μg / ml to 4 ml of the above cell suspension in a test tube (200 mm × 21 mm in diameter) and the mixture is incubated with stirring at 30 ° C. for 45 minutes. In these circumstances, the rate

  lethality of the Arthrobacter CA-35 strain is approximately 80%.

  Cells thus treated were collected with a filter membrane (pore size: 0.45 / um) under aseptic conditions, washed with 20 ml of 0.1 M phosphate buffer (pH 7.0) and suspended in 25 ml. ml of the same buffer. We dilute the

  cell suspension obtained with a sterile normal saline solution

  and plated on agar plates (D medium: NaOH: 0.1% cholic acid: 1.0% 7, NH 4 NO 3: 0.2%, KH 2 PO 4 0.2%, K 2 HPO 4: 0.5%, MgSO 4, 7H20: 0.02%, yeast extract: 0.01% and 1.5% agar) to obtain 300 to 500 colonies per bunch. The boots are incubated at 30 ° C. for 4 hours. Each punctiform colony formed is isolated and incubated on an agar plate (medium E, peptone: 0.5%, yeast extract: 0.5%, NaCl: 0.5% and 1.5% agar) at 30 ° C. for 24 hours. Each colony formed on the box of medium E is transplanted onto a bundle of medium D and incubated at 30 ° C. for h. Incubate on an inclined medium (medium F, NaOH 0.1%, cholic acid 1.0%, peptone 0.5%, yeast extract 0.5%, NaCl 0.5% and agar 1 , 5%) at 30 ° C. for 24 h each colony of the above box of medium E, the transplanted colony of which does not develop on the boot of medium D. One sows with a loop of the inclined culture 6 ml of medium (medium G, NaOH: 0.5, cholic acid: 5.0%, glucose: 0.5%, NH4NO3: 0.2%, KH2PO4: 0.2%, K2HPO4: 0.5%, MgSO4, 7H 2 O: 0.02% and yeast extract 0.1%) in a test tube (200 mm x 21 mm in diameter)

  and incubated with stirring at 30 C for 3 days.

  Thin layer chromatography examination of

  accumulated in each medium G enabled the claimant to

  discover a microbe that selectively produces dihy-

  keto-3a, 7a keto-12,55-cholanic acid or a salt thereof and which has been

  named Arthrobacter strain CA-35-M-965-3.

  The above procedure is hereinafter referred to as "mode

  According to the procedure M, a treatment was carried out

  mutagenic strain with the Arthrobacter CA-35-M-965-3 strain as a parent strain. The Applicant has thus discovered three strains that selectively produce 3α-dihydroxy-7α-keto-5β-cholanic acid or a salt thereof which have been designated respectively, strain Arthrobacter CA-35-A589, strain Arthrobacter CA-35-A849. and strain

Arthrobacter CA-35-A-1071.

  From a colony of strain Arthrobacter CA-35-A589 two superior strains were isolated and named respectively

  strain Arthrobacter CA-35-A589-29-32 and strain Arthrobacter CA-35-

  A589-47. An upper strain was also isolated from a colony of the Arthrobacter CA-35-A-1071 strain and named Arthrobacter strain

CA-35-A-1071-1015.

  In addition, an upper strain was produced using Arthrobacter strain CA-35-A849 as a parent strain.

  according to the procedure M and named Arthrobacter CA-35-A-1448 strain.

  Arthrobacter strain CA-35-A-1475 was produced using Arthrobacter strain CA-35-A-1448 as the parent strain according to procedure M. Arthrobacter strain CA-35-A-1766 was produced by use. Arthrobacter strain CA-35-A-1071 as a parent strain according to procedure M and Arthrobacter strain CA-35-A-1766-15.

  was isolated from a colony of this strain Arthrobacter CA-35-A-1766.

  Arthrobacter strain CA-35-Y-37-12 was produced

according to the following procedure.

  A loop of the Arthrobacter CA-35 strain (FERM-P N 5145, ATCC N 31651) cultured on an inclined medium A was taken and 10 ml of medium B were inoculated into a test tube (200 mm x 21 mm in diameter). ) and incubated with stirring at 30 C for 20 h. The microbial cells are collected by centrifugation (10,000 rpm) at 5 ° C. for 5 min under aseptic conditions. We

  put the cells in suspension in 10 ml of sterilized water. We are

  The suspension is triturated in a Petri dish (diameter 7.5 cm) placed at about 27 cm under a 15 W ultraviolet lamp (2537 A) in a sterile chamber and irradiated with ultraviolet light for min. In these conditions the case fatality rate of the strain

  Arthrobacter CA-35 is approximately 99.9%.

!

  After irradiation, the micro cells are

  well with a membrane filter, wash with phosphate buffer

  0.1 M and suspended in the same buffer. We dilute the

  The solution obtained with a physiological saline solution is repeated and the above culture is repeated with media D to G to obtain an upper strain which selectively produces dihydroxy-3a, 7a keto-12-cholanic acid or a salt thereof.

EXAMPLE 1

  The Arthrobacter CA-35 strain (FERM-P N 5145, ATCC N 31651) was cultured under the following conditions: Culture medium composition: Cholic acid 100 g Ammonium nitrate 2.0 g Monopotassium phosphate 2.0 g Dipotassium phosphate 5.0 g Magnesium sulphate heptahydrate 0.2 g Yeast extract 0.1 g Sodium hydroxide 10 g Distilled water qs 1 liter The above ingredients are mixed to obtain one liter of culture medium. 100 ml portions of the culture medium are divided into 10 500 ml Sakaguchi flasks and autoclaved at 120 ° C. for 15 minutes. 10 ml of seed culture obtained by pre-culturing the strain in the same medium with stirring at 30 ° C. for 2 days are added to each fraction. We incubate

  each flask on a shaker at 30 C for 2 days.

  When the culture is completed, the broths are combined

  cultured, and centrifuged to remove microbial cells.

  The supernatant obtained is acidified by addition of 600 ml of chloroacid

  aqueous hydride 1 To form a precipitate. Separate the precipitate

  and the remaining solution is extracted with 1 liter of ethyl acetate.

  The ethyl acetate is distilled off from the extract by rotary evaporation and the residue is combined with the above precipitate to obtain a mixture of cholanic acid and acid derivatives.

cholic (85 g).

  A small portion of the mixture is dissolved in methanol at a concentration of 1%. 10 μl of solution are injected into a high speed liquid chromatography apparatus equipped with a column / uBondapak C18 (HLC-GPC-244 type produced by Waters

in the United States of America).

  The column is eluted with a water-methanol mixture (30/70, v / v pH 2.5) at a flow rate of 1 ml / min and the index is measured.

of refraction of the eluate.

  The chromatogram obtained is illustrated in FIG.

  Peaks A, B, C and D of Figure 1 correspond to those of the standard

  Reference darts consisting of 7-hydroxyacid

  diketo-3,12 5P-cholanic acid, dihydroxy-3a, 7a keto-12 5P-cholo-

  of 7α-dihydroxy-7α, 12α-keto-3-cholanic acid and cholic acid. In addition, when isolating the compounds from the fractions

  15.Corresponding to peaks A, B and C and determining their constitu-

  from the mass spectrum, the infrared spectrum and the nuclear magnetic resonance spectrum, these spectra show that these compounds are, respectively, 7a-hydroxy-diketo-3,12g-cholanic acid, dihydroxy-3a acid, 7α-59-cholanic acid and 7α-dihydroxy-7α, 12α-keto-5β-cholanic acid. Figures 2, 3 and 4 illustrate the infrared spectra (A, B and C) of the methyl esters of the compounds corresponding to peaks A, B and C compared with

  those of the reference standards (A ', B' and C ').

  The melting points of the compounds corresponding to

  peaks A, B and C and their methyl esters are shown in table

II below.

  The yield of the products and the amount of cholic acid which remains untransformed from the surface ratios of the chromatogram of Figure 1 are calculated.

  are shown in Table III below.

EXAMPLE 2

  The procedure described in Example 1 is repeated except that 5.0 g / l of glucose is added to the mixture to obtain a mixture of products and choliqiu acid remaining unconverted (91). 0 g). The mixture (01.0 g) was dissolved in-270 ml of methanol and 9 ml of concentrated hydrochloric acid was added. The solution obtained is refluxed for 20 minutes to transform

  products and cholic acid in their methyl esters.

  We fill a column (1.200 mm x 70 mm diameter)

  1500 g of C-200 silica gel and the methyl esters are adsorbed therein.

  above. The column is eluted with chloroform to obtain

  1 g of methyl ester of 7-hydroxy acid diketo-3,12 5p-choline

  lanique. The column is then eluted with chloroform-ethanol

  (99/1, v / v) to obtain first 1.40 g of the dihydroxy-7a, 12a keto-5p-cholanic acid methyl ester and then 56.4 g of

  the dihydroxy-3α, 7α-keto-5β-cholanic acid methyl ester.

  These methyl esters are hydrolyzed to obtain 24.8 g

  of 7-12 hydroxy-diketo-3,12-p-cholanic acid, 1.38 g of dihydric acid,

  ka-7α, 12α-keto-5β-cholanic acid and 56.0 g of 3α-dihydroxy acid, 7a

keto-12 5p-cholanic.

EXAMPLE 3

  The procedure described in Example 1 is repeated, except that the concentration of the cholic acid is modified and sodium hydroxide is added to the medium at the rate of 10% by weight relative to to the cholic acid used. The yield

  products and the amount of cholic acid that remains without being

  formed for each concentration of the substrate (cholic acid)

in Table IV below.

EXAMPLE 4

  The procedure described in Example 2 is repeated, except that each flask is incubated on a stirrer at 30 ° C. for 24 hours to obtain a mixture of products and cholic acid which remains untransformed ( 98.0 g). This mixture is subjected to high speed liquid chromatography under the same conditions as in Example 1 to determine its composition. The results

  are shown in Table V below.

EXAMPLE 5

  The procedure described in Example 2 is repeated,

  except that 20 ml of culture culture are added to each bottle.

  The mixture was grown at 30 ° C for 24 hours to obtain a mixture of products and cholic acid which remained untransformed (117.0 g). The mixture is subjected to high chromatography

  under the same conditions as in Example 1 to determine

  undermine its composition. The results are shown in Table VI below.

after.

EXAMPLE 6

  The Arthrobacter strain CA-35-A589-29-32 (FERM-P N 5521, ATCC N 31652) was cultivated as follows: Culture medium composition: cholic acid 100 to ammonium nitrate 2.0 g Monopotassium phosphate 2.0 g Dipotassium phosphate 5.0 g Magnesium sulphate heptahydrate 0.2 g Yeast extract 0.1 g Sodium hydroxide 10 g Glucose 5.0 g Tap water qs 1 liter The above ingredients except glucose are mixed with tap water to a volume of 800 ml and autoclaved at 120 C for 15 min. The glucose is dissolved in 1 ml of tap water and autoclaved at 110 ° C. for 30 minutes. After cooling, the two solutions are combined to obtain one liter of culture medium. 100 ml portions of the culture medium are distributed in 10 500 ml Sakaguchi flasks under conditions of

  Aseptic. 200 ml of seed culture are added to each flask.

  cement obtained by prior culture of the strain in the same medium with stirring at 30 C for 14 h. Each bottle is incubated on a

stirrer at 30 C for 3 days.

  When the culture is complete, the culture broths are combined and centrifuged to remove microbial cells. The supernatant is acidified by adding 600 ml of 1N aqueous hydrochloric acid to form a precipitate. The precipitate is separated off and the residual solution is extracted with 1 liter of ethyl acetate. The ethyl acetate is removed from the extract by distillation with a rotary evaporator and the residue is combined with the above precipitate to obtain a mixture of derivatives.

  of cholanic acid and cholic acid (99.3 g).

  A small portion of the mixture is dissolved in methanol at a concentration of 2%. 10 μl of the solution are injected into a high speed liquid chromatograph equipped with

  of a column / uBondapak C-18 (type HLC-GPC-244).

  The column is eluted with a water-methanol mixture (30/70, v / v, pH 4.0) at a flow rate of 1 ml / min and the index is measured.

of refraction of the eluate.

  The chromatogram obtained is illstrated by FIG.

  The peaks B and D of FIG. 5 correspond to those of the reference standards consisting respectively of dihydroxy-3a, 7a

  keto-12 5j3-cholanic and cholic acid.

  When isolating the compound from the fraction corresponding to peak B, and determining its chemical constitution from the mass spectrum, the infrared spectrum and the nuclear magnetic resonance spectrum, these spectra show that this compound is

  3α-dihydroxy-3α, 7α-keto-5β-cholanic acid.

  The yield of the products and the amount of cholic acid which remains unconverted from the ratio of the surfaces of the chromatogram in Figure 5 are calculated.

in Table VII below.

EXAMPLE 7

  The procedure described in Example 6 is repeated, except that a suspension of microbial cells is prepared by centrifugally separating the microbial cells from the 10 ml of seed culture, which is suspended in 10 ml sterilized water and put in each bottle instead

  2 ml of seed culture to obtain a mixture of

  and remaining cholic acid (99.5 g). This mixture is dissolved

  (99.5 g) in 270 ml of methanol and 9 ml of hydrochloric acid are added.

  concentrated drique. The resulting solution is heated at reflux for 1 min to convert the products and cholic acid to their methyl esters. We fill a column (1.200 mm x 70 mm diameter)

  1500 g of C-200 silica gel and the methyl esters are adsorbed therein.

  above. The column is eluted with chloroform-

  ethanol (99/1, v / v) to obtain 102.0 g of dihydroxy-3a, 7a keto-12 5 P-cholanic acid methyl ester which is hydrolysed

  to obtain 98.5 g of 3α-dihydroxy-3α, 7α-keto-5β-cholanic acid.

EXAMPLE 8

  The procedure described in Example 6 is repeated, except that the concentration of the cholic acid is modified, and sodium hydroxide is added at a concentration of 10% by weight relative to to the cholic acid used and 1N aqueous hydrochloric acid was added at 60 ml per gram of sodium hydroxide used to form a precipitate. The conversion rate or yield of the product and the amount of cholic acid remaining for each concentration of the substrate (cholic acid) are

in Table VIII below.

* EXAMPLE 9

  The procedure described in Example 6 is repeated except that Arthrobacter strain CA-35-A589-47 (FERM-P N 5523, ATCC N 31659) is used instead of the Arthrobacter CA strain. 35-A589-29-32, the concentration of cholic acid in the medium is 50 g / l, the concentration of sodium hydroxide in the medium is 5 g / l and each bottle is incubated at 30 ° C. for 35 hours to obtain a mixture of products and cholic acid

remaining (49.31 g).

  The mixture is subjected to high speed liquid chromatography under the same conditions as in Example 6. The chromatogram obtained is illustrated in FIG. 6. Peaks A, B,

  C and D in Figure 6 correspond to those of the standard

  reference dyes consisting of 7α-hydroxy-diketo-3,12 P-cholanic acid, 3α-dihydroxy-3α, 7α-keto-5-cholanic acid, acid

  7α-dihydroxy-7α, 12α-keto-5β-cholanic acid and cholic acid.

  When fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction corresponding to peaks B, C and D contains a single component. The fraction corresponding to peak A consists of a mixture of 7-hydroxy-diketo-3,12 5 P-cholanic acid and a

other unidentified matter.

  The yield of the products and the amount of cholic acid which remains unchanged from the surface ratio of the chromatogram of Figure 6 are calculated.

in Table IX below.

EXAMPLE 10

  The procedure described in Example 9 is repeated except that Arthrobacter strain CA-35-A849 (FERM-P N 5524, ATCC 31654) is used instead of Arthrobacter strain CA-35-A589. -47 to obtain a mixture of products and cholic acid remaining

(49.34 g).

  The mixture is subjected to a liquid chromatography with

  high speed-under the same conditions as in Example 6.

  The chromatogram obtained is illustrated in FIG.

  Peaks A, B, C and D of Figure 7 correspond to those of the standard

  Reference dyes which are respectively 7α-hydroxy-diketo-3,12-p-cholanic acid, 3α-dihydroxy-3α, 7α-keto-5β-cholanic acid, 7α-dihydroxy-7α, 12α-keto-5β-cholanic acid. and

cholic acid.

  When fractions corresponding to peaks A, B, C and D are separated and subjected to layer chromatography

  thin, each fraction contains a single compound.

  The yield of the products and the amount of cholic acid which remains untransformed are calculated from the surface ratio of the chromatogram of Figure 7. The results

  are shown in Table X below.

EXAMPLE 11

  The procedure described in Example 9 is repeated, except that the Arthrobacter CA-35-A-1071-15 strain (FERM-P N 5525, ATCC N 31655) is used instead of the Arthrobacter strain. CA-35-A589-47 to obtain a mixture of products and cholic acid

remaining (49.31 g).

  The mixture is subjected to a liquid chromatography with

  high speed in the same conditions as in Example 6.

  The chromatogram obtained is illustrated in FIG. 8.

  Peaks A, B, C and D in Figure 8 correspond to those of the standard

  Reference darts consisting of 7-hydroxyacid

  diketo-3,12 5p-eholanic, dihydroxy-3a, 7a keto-12 5P-cholo-

  of 7-dihydroxy-7a, 12a-keto-5p-cholanic acid and cholic acid. When fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction corresponding to peaks B, C or D contains a single compound. The fraction corresponding to peak A is a mixture of 7α-hydroxy-diketo-3,12-polyolanic acid and another

unidentified.

  Product yield and quantity are calculated

  of cholic acid remaining from the ratio of chromatic surfaces

  Figure 8. The results are shown in Table XI below.

EXAMPLE 12

  The procedure described in Example 9 is repeated, except that strain Arthrobacter CA-35-A-1448 (FERM-P N 5526, ATCC N 31656) is used instead of the strain Arthrobacter CA. 35-A589-47 to obtain a mixture of products and cholic acid

remaining (49.30 g).

  The mixture is subjected to a liquid chromatography with

  high speed under the same conditions as in Example 6.

  The chromatogram obtained is illustrated in FIG. 9.

  Peaks A, B, C and D in Figure 9 correspond to those of standard

  Reference darts consisting of 7-hydroxyacid

  diketo-3,12 5p-cholanic acid, dihydroxy-3a, 7a keto-12 5p-chola-

  7a-dihydroxy-7a, 12a-cdto-35P-cholanic acid and cholic acid. When fractions corresponding to peaks A, B, C and D are separated and subjected to thin layer chromatography, each fraction corresponding to peaks B, C or D contains a single compound. The fraction corresponding to peak A is an acid mixture

  hydroxy-7a diketo-3,12 5p-cholanic and other non-identical material

  fied. The yields of the products and the amount of cholic acid which remains unconverted from the ratio of the surfaces of the chromatogram in Figure 9 are calculated.

in Table XII below.

EXAMPLE 13

  The procedure described in Example 9 is repeated, except that the strain Arthrobacter CA-35-A1475 (FERM-P) is used.

  N 5529; ATCC N 31657) instead of the Arthrobacter strain CA-35-

  A589-47 to obtain a mixture of products and cholic acid which

  remains without being transformed (49.35 g).

  The mixture is subjected to liquid chromatography

  at high speed under the same conditions as in Example 6.

  The chromatogram obtained is illustrated in FIG.

  Peaks A, B, C and D of Figure 10 correspond to those of standard

  Reference darts consisting of 7-hydroxyacid

  diketo-3.12 5p-cholanic, dihydroxy-3a, 7a keto-12 53-chols-

  of 7α-dihydroxy-7α, 12α-keto-5β-cholanic acid and cholic acid. When fractions corresponding to peaks A, B, C and D are separated and subjected to layer chromatography

  thin, each fraction contains a single compound.

  The yield of the products and the amount of cholic acid are calculated from the ratio of the surfaces of the chrontogram

  Figure 10. The results are shown in Table XIII below.

after.

EXAMPLE 14

  The procedure described in Example 9 is repeated, except that the Arthrobacter CA-35-A-1766-15 strain (FERM-P N 5528, ATCC N 31658) is used instead of the Arthrobacter strain. CA-35-A589-47 to obtain a mixture of products and acid

cholic (49.27 g).

  The mixture is subjected to a liquid chromatography

  high speed under the same conditions as in Example 6.

  The chromatogram obtained is illustrated in FIG.

  Peaks A, B, C and D in Figure 11 correspond to those of the standard

  Reference darts consisting of 7-hydroxyacid

  diketo-3,12 5p-cholanic acid, dihydroxy-3a, 7a keto-12 5 $ -chola-

and cholic acid.

  When fractions corresponding to peaks A, B and D are separated, and subjected to thin layer chromatography,

  each fraction contains a single compound.

  Product yield and quantity are calculated

  of cholic acid remaining from the ratio of chromatic surfaces

  Figure 1. The results are shown in Table XIV.

below.

EXAMPLE 15

  The procedure described in Example 9 is repeated except that Arthrobacter strain CA-35-M-965-3 (FERM-P N 5529, ATCC No. 31659) is used instead of the Arthrobacter strain. CA-35-A589-47 to obtain a mixture of products and acid

cholic (49.36 g).

  The mixture is subjected to a liquid chromatography with

  high speed under the same conditions as in Example 6.

  The chromatogrammae obtained is illustrated in FIG. 12.

  The peaks A, B and D of FIG. 12 correspond to those of reference standards consisting respectively of 7α-hydroxy-diketo-3,12-cholanic acid, dihydroxy-3-acid, 7α-keto-5β-cholanic acid and

of cholic acid.

  When fractions corresponding to peaks A, B and D are separated and subjected to thin layer chromatography,

  each fraction corresponding to peaks B or D contains a single compound.

  The fraction corresponding to peak A is a mixture of 7-hydroxyacid

  diketo-3,12 5p-cholanic and other unidentified material.

  Product yield and quantity are calculated

  of cholic acid remaining from the ratio of chromatic surfaces

  Figure 12. The results are shown in Tables XV below.

EXAMPLE 16

  The procedure described in Example 9 is repeated, except that the Arthrobacter CA-35-Y-37-12 strain (FERM-P N 5530, ATCC N 31660) is used instead of the Arthrobacter strain. CA-35-A589-47 to obtain a mixture of products and acid

cholic (49.33 g).

  The mixture is subjected to a liquid chromatography with

  high speed under the same conditions as in Example 6.

  The chromatogram obtained is illustrated in FIG. 13.

  The peaks A, B, C and D of FIG. 13 correspond to those of reference standards consisting respectively of 7α-hydroxy-diketo-3,12-polycholanic acid, of 3α-dihydroxy-3α, 7α-keto-12-cholanic acid, 7α-dihydroxy-7α, 12α-keto-5β-cholanic acid and acid

cholic.

  When fractions corresponding to peaks A, B, C and D are separated, and subjected to layer chromatography

  thin, each fraction contains a single compound.

  Product yield and quantity are calculated

  of cholic acid remaining from the ratio of chromatic surfaces

  Figure 13. The results are shown in Tables XVI below.

EXAMPLE 17

  The procedure described in Example 1 is repeated except that the strain Brevibacterium CA-6 (FERM-P) is used.

  N 5144; ATCC No. 31661) instead of the Arthrobacter CA-35 strain.

  The mixture of products and remaining cholic acid obtained is subjected to high speed liquid chromatography.

in example 1.

  The chromatogram obtained is illustrated by FIG.

  When the compounds are isolated from the corresponding fractions

  at peaks E, A, B and D and their chemical constitution is determined from the mass spectrum, the infrared spectrum and the nuclear magnetic resonance spectrum, these spectra show that these compounds are, respectively, 7-hydroxyacrylic acid. diketo-3.12 ú4-cholenic acid, hydroxy-7a-diketo-3,12 5g-cholanic acid, acid

  3α-dihydroxy-3α, 7α-keto-5β-cholanic acid and cholic acid. The com

  set of fractions corresponding to peaks X and Y are unidentified.

  In the chromatogram of Fig. 14, the ratio of peak areas to peaks other than peaks X and Y (i.e.

  E / A / B / D areas) is 12.2 / 22.2 / 44.9 / 20.7.

EXAMPLE 18

  The procedure described in Example 1 is repeated, except that the Corynebacterium CA-53 strain (FERM-P N 5532, ATCC N 31662) is used instead of the Arthrobacter CA-35 strain.

  to obtain a mixture of products and cholic acid (82 g).

  The resulting mixture is subjected to chromatography

  at high speed under the same conditions as in the example

  ple 1, except that a mixture of water and metha-

  nol (30/70, v / v, pH 4.0) instead of a water-methanol mixture

(30/70, v / v, pH 2.5).

  The chromatogram obtained is illustrated in FIG.

  Peaks A, B, C and D in Figure 15 correspond to those of standard

  reference darts which are respectively 7-hydroxy-acid

  diketo-3.12 5P-cholanic acid, dihydroxy-3a, 7a keto-12 5e-chola-

  9-hydroxy-diketo-3,12-A4-cholenic acid and cholic acid.

  When the compounds are isolated from the corresponding fractions

  at peaks A, B and C, and quyon determines their chemical constitution.

  From the mass spectra, the infrared spectrum and the nuclear magnetic resonance spectrum, these spectra show that these compounds are, respectively, 7-hydroxy-3-diketo-3,12-P-cholanic acid, dihydroxy-3a, 7a acid. keto-12 5-cholanic and

  7-hydroxy-diketo-3,12 to 4-cholenic acid.

REFERENCE EXAMPLE 1

  10 g of the dihydroxy-3α, 7α-keto-12-cholanic acid thus obtained are introduced into a three-necked round-bottomed flask and 100 ml of ethylene glycol and an aqueous solution of potassium hydroxide (10 ml) are slowly added. g of potassium hydroxide + 20 ml of water). An additional 10 ml of 85% hydrazine hydrate is added and the mixture is refluxed at 100 ° C. for 2 hours. The temperature is gradually raised and the reflux is continued between 185

  and 190 C for 4 hours to distil off hydrate hydrate

  Zine. After cooling, the reaction mixture is diluted with an excess of water and the pH is adjusted to 3 to form a precipitate. The precipitate is collected by filtration, washed with water and

  it is air-dried to give 3-dihydroxy-3a, 7a-5-chloroacid

(CDCA); yield 8.5 g.

REFERENCE EXAMPLE 2

  In a three-necked round-bottom flask, 3 g of di-3,12 5 P-cholanic hydroxy7a acid obtained in the above examples is dissolved in 70 ml of pyridine and one mole of tosyl chloride is added. . The resulting mixture is allowed to react at 0 ° C. for 1 h and then refluxed for 5 h. After reflux, we add

  3N aqueous hydrochloric acid and the reaction mixture is extracted.

  with 100 ml of ether. The extract is distilled under reduced pressure to remove the ether and obtain 2.5 g of dicdto-3,12 & 6- and / or 47cholenic acid. The 2 g obtained are dissolved in 10 ml of 1N aqueous sodium hydroxide and 0.1 g of Raney nickel is added. After addition of 0.01 mole of hydrogen, the mixture is reacted overnight to give dihydroxy-3a, 12a 5p-cholanic acid (acid).

  deoxycholic) and dihydroxy-3a, 1203 5p-cholanic acid (makes

90%).

  Of course various modifications-may be

  provided by those skilled in the art to the devices or processes which have just been described solely by way of non-limiting examples without

depart from the scope of the invention.

TABLE I

  Characteristics Arthrobacter simplex Arthrobacter CA-35

IAM 1660

  Microscopic observation Shape Division Size (/ um) Flagella Spores Gram stain Acido-resistivity Cultural Observations Bunched broth Gelatin (in puncture) Milk sunflowers Bromcresol purple milk Physiological characteristics 1) Reduction of sticky nitrates (sometimes swollen or curved) 0.4 - 0.5 x 1 - 3 none none positivevariable none circular, slightly embossed, colorless, smooth, shiny, transparent liquefaction in reduced sunflower bag, clarified milk clarification, alkalization + sticks (sometimes broken or curved) rupture 0, 8 - 0.9 x 1.7 - 2.2 nil none positive circular voids, slightly raised, yellow to cream color, smooth, shiny, opaque no reduced sunflower liquefaction, milk unaltered alkaline, neither coagulated nor peptonized + N tg os o TABLEAUI (continued 1) Characteristics Arthrobacter simplex. Arthrobacter CA-35

IAM 1660

Denitrification - -

Methyl red test - -

Voges-Proskauer test - -

Indole production - -

H2S + + production

Hydrolysis of starch - -

  Use of citrates + + O Assimilation of sources + + mineral nitrogen Urease

Oxidase -

  Catalase + + Aerobic aerobic oxygen requirement Oxidation / fermentation test - oxidation oa Co% 0

BOARD

  Characteristics Arthrobacter simplex Arthorbacter CA-35

IAN 1660

  Production of acids and gases from carbohydrates 2) 1. Larabinose 2. D-xylose 3. D-glucose 4. D-mannose 5. D-fructose 6. D-galactose 7. Maltose 8. Sucrose 9. Lactose 10. Trehalose 11. Dsorbitol 12. D-mannitol 13. Inositol 14. Glycerol 15. Starch culture acids gas + _ _ + _ _ + _ _ + _ _ + _ _ + _ _ j _ _ _ + _ _ + _ _ + _ _ + _ _ + _ _ + _ _ + _ _ + _ _ z assimilation culture + + + + + + + + + + + + + + + + + + + acids gas assimilation + - - -it

+ _ + 4-F

+ - .. +

- +. +

- - I II

  + tN -J re> W to c CD 1% 0 I (continued 2)

BOARD

  Characteristics Arthrobacter CA-35-A589-29-32 Arthrobacter CA-35-A589-47 Microscopic Observation Division Form Size (/ unm) Flagella Spores Grim Acid Acid-Resistance Cultural Observations Broth agar in broth Broth agar in broth containing 10 g / l cholic acid Gelatin (in puncture) Milk sunflowers Bromcresol purple milk short sticks 0,8 - 1,0 x 1,3 - 2,0 polar flagella none positive nothingness circular, flat, yellow, smooth, shiny, opaque circular, flat, yellow, smooth, shiny, opaque liquefaction sunflower reduced, milk neither coagulated nor peptonized alkaline, neither coagulated nor peptonized short sticks 0.8 - 1.0 x 1.3 - 2.3 flagella none positive none circular, flat yellow, smooth, shiny, opaque circular, flat, white to pale cream, smooth, shiny, opaque liquefaction reduced sunflower, milk neither coagulated nor peptonized alkaline, nor coagulated or peptonized oo ro 0% os W CD - o - o I (continued 3) TABLE I (continued 4) Characteristics Arthrobacter CA-35-A589-29-32 Arthrobacter C & -35-A589-47 Physiological Characteristics 1) Nitrate Reduction + +

Denitrification -

Methyl red test -

Voges-Proskauer test -

Indole production -

H2S + + production

Hydrolysis of starch -

  Use of citrates + + Assimilation of mineral nitrogen sources ammonium + + nitrate + +

Urease -

  Catalase Oxidase + + Aerobic Aerobic Oxygen Requirement Oxidation / Fermentation Oxidation Test t> CD Bone or TAB LEAUI (Continued 5) Characteristics Arthrobacter CA-35-A589-29-32 Arthrobacter CA-35-A589-47 Production of acids and gases from carbohydrates 2) acids gas acids gas 1. L-arabinose '+ - + 2. D-xylose 3. D-glucose + 4. D-mannose

5. D-Fructose + - + -

6. D-galactose.

7. Maltose + - + -

8. Sucrose.

9. Lactose.

10. Trehalose.

u1. D-sorbitol.

12. D-.annitol -.

13. Inositol -

14. Glycerol -.

15. Aidon -

  oe D TABLE I (continued 6) Characteristics Arthrobacter CA-35-A849 Arthrobacter CA-35-A-1071-15 Microscopic Observation Form Division Size (μm) Flagella Spores Gram staining Acido-resistance Cultivation observations Bouillon agar broth Bouillon agar in a bunch containing 10 g / l of cholic acid Gelatin (in puncture) Sunflower milk Bromcresol purple milk short sticks 0.7 - 1.0 x 1.3 - 2.3 nil none positive circular, flat, yellow, none smooth, shiny, opaque round, flat, white to pale cream, shiny smooth, opaque liquefaction reduced sunflower, milk neither coagulated nor peptonized alkaline, nor coagulation or peptonization short sticks 0, 8 x 1.0 x 1.3 - 2, 5 none void positive void circular, flat, yellow, smooth, shiny, opaque circular, flat, white to pale cream, shiny smooth, opaque liquefaction sunflower reduced, milk neither coagulated nor peptonized alkaline, nor coagulated ptonisation en "N 0% os W oe - oC o TABLEAUI (cont'd 7) Characteristics Arthrobacter CA-35-A849 Arthrobacter CA-35-A-1071-15 Physiological Characteristics 1) Nitrate Reduction Denitrification Methyl Red Test Voges Test -Proskauer Production of indole Production of H2S Hydrolysis of starch Use of citrates Assimilation of nitrogen sources mineral ammonium nitrate Urease Oxidase Catalase Oxygen requirement Oxidation / fermentation test + + + + + + + + + + * + aerobic oxidation + aerobic oxidation TABLE I (continued 8) Characteristics Arthrobacter CA-35-A849 Arthrobacter CA-35-A-1071-15 Production of acids from hydrates 1. L-arabinose 2. D-xylose 3. D-glucose 4. D-mannose 5. Dfructose 6. D-galactose 7. Mucose 8. Sucrose 9. Lactose 10. Trehaloid 11. D-sorbitol 12. D-mannitol 13. Inositol 14. Glycerol 15. Starch and gas 2) carbon acids gas gas + + + + acids + + + + + + N o% 0 or TAB LEAUI (continued 9) Features Arthrobacter CA-35-A1448 Arthrobacter CA-35-A-1475 Microscopic Observation Shape Division Size (μm) Flagella Spores Gram staining Acido-resistance Cultural Observations Batch agar broth Bunch agar broth containing 10 g / 1 cholic acid Gelatin (in puncture) Sunflower Milk Bromcresol Purple Milk Short Sticks 0.7 - 1.0 x 1.2 - 2.0 Neon flagella positive None Circular, flat, yellow, smooth, shiny, opaque, circular, flat, white to pale cream, smooth, shiny, opaque liquefaction reduced sunflower, milk neither coagulated nor peptonized alkaline, neither coagulated nor peptonized short cocci sticks 1.0 - 1.3 x 1.2 - 1.8 flagella none positive circular, flat, yellow, smooth, shiny, opaque round, flat, white to pale cream, smooth, shiny, opaque liquefaction sunflower reduced, milk neither coagulated nor peptonized alkaline, nor coagulated or peptonized w> N o co o C QTABLEAUI (cont'd 10) Characteristics Arthrobacter CA-35-A-1448 Arthrobacter CA-35-A-1475 Physiological Characteristics 1) Nitrate Reduction + +

Denitrification -

Methyl red test -

  Voges-Proskauer test Indole production H2S + + production

Hydrolysis of starch -

  Use of citrates + + Assimilation of mineral nitrogen sources ammonium + + nitrate + +

Urease -

  Catalase Oxidase + Aerobic Aerobic Oxygen Test Oxidation / Fermentation Oxidation Test - N Co o. to TAB L WATER I (continued 11) Characteristics Arthrobacter CA-35-A-1448 Arthrobacter CA-35-A-1475 Production of acids and gases from carbohydrates 2 acids gas acids gas

1. L-arabinose + - - -

2. D-xylose - - -

3. D-glucose + - - -

4. D-mannose.

5. D-Fructose + - + -

6. D-galactose.

7. Maltose + - - -

8. Sucrose

9. Lactose.

10. Trehalose

11. D-sorbitol.

12. D-mannitol.

13, Inositol.

14. Glycerol +

15. Starch.

  0% o O TABLE I (continued 12) Characteristics Arthrobacter CA-35-A-1766-15 Arthrobacter CA-35-M-965-3 Microscopic Observation Shape Division Size (μm) Flagella Spores Gram staining Acido-resistance Cultivation observations Batch agar broth Bunch agar broth containing 10 g / l cholic acid Gelatin (in puncture) Sunflower milk Milk bromcresol purple short sticks 0.6 - 1.2 x 1.0 - 2.3 flagellum none positive none circular, flat, yellow, smooth, shiny, opaque circular, flat, yellow, smooth, shiny, opaque liquefaction reduced sunflower, milk neither coagulated nor peptonized alkaline, nor coagulated or peptonized short sticks cocci 1, O - 1,2 x 0 , 8 - 1.6 flagella none positive none circular, flat, yellow, smooth, shiny, opaque circular, flat, white to cream, smooth, shiny, opaque no reduced sunflower liquefaction, milk neither coagulated nor peptonized alkaline, nor coagulated neither peptonized w os Characteristics Arthrobacter CA-35-A-1766-15 Arthrobacter CA-35-M-965-3 Physiological Characteristics 1) Nitrate Reduction + +

Denitrification - -

Methyl red test - -

Voges-Proskauer test - -

Indole production -

Production of H2S + + Co

Hydrolysis of starch - -

  Use of citrates + + Assimilation of mineral nitrogen sources ammonium + + nitrate - + Urease Oxidase Catalase + + * Aerobic aerobic oxygen requirement Oxidation / fermentation test oxidation oxidation Ch o% o TAB LEAUI (continued 14) Characteristics Arthrobacter CA-35-A-1766-15 Arthrobacter CA-35-M-965-3 Production of acids and gases from carbohydrates 2) acids gas gases gas 1. L-arabinose + 2. Dxylose _ 3 D-glucose + + 4. D-mannose _ 5. D-fructose + + 6. D-galactose _ 7. Maltose + +

8. Sucrose.

  9. Lactose 10. Trehalose 11. D-sorbitol _

12. D-mannitol.

13. Inositol.

  14. Glycerol + _ 15. TAB LEAU I starch (continued 15) Characteristics Arthrobacter CA-35-Y-37-12 Brevibacterium CA-6 Microscopic observations Form Division Size (μm) Flagella Spores Gram stain Acido-resistance Observations Grow broth agar Broth agar broth containing 10 g / l cholic acid Gelatin (in puncture) Sunflower milk Milk bromcresol purple short sticks 0,8 - 1, O x 1,5 - 2,5 flagellum none positive nil / circular, flat, yellow, smooth, shiny, opaque circular, flat, - cream color, smooth, shiny, opaque liquefaction reduced sunflower, milk neither coagulated nor peptonized alkaline, neither coagulated nor peptonized sticks 1,0 x 2,5 - 4,0 polar flagellum none positive) none wavy, slightly raised, colorless, smooth, shiny, translucent peptonised liquefaction and becoming alkaline peptonized and becoming alkaline o ria 0% o% o TABLEAUI (continued 16) Characteristics Arthr CA-35-Y-37-12 Brevibacterium CA-6 Physiological Characteristics Nitrate Reduction + Denitrification + Methyl Red Test Voges-Proskauer Test Indole Production H2S + + Production Starch Hydrolysis + Citrate Use + + Assimilation of mineral nitrogen sources ammonium + nitrate + Urease Oxidase + Catalase + + Need aerobic aerobic oxygen

  Oxidation / fermentation oxidation test -

   o I (continued 17) Characteristics Arthrobacter Brevibacterium CA-6

CA-35-Y-37-12

* Production of acids and gases from carbohydrates 2) 1. Larabinose 2. D-xylose 3. D-glucose 4. D-mannose 5. D-fructose 6. Dgalactose 7. Maltose 8. Sucrose 9 Lactose 10. Trehalose 11. Dsorbitol 12. D-mannitol 13. Inositol 14. Glycerol 15. Starch acids gas + + + + + + culture + + + + + + + + + + + + + + + acids + + + + + + gas assimilation -I1 + +4+ + +44 +44 + | I; +1+ 4: N3 IN tu o o% 0

BOARD

  TABLEAU (cont'd 18) Characteristics Microscopic Observation Form Division Size (/ um) Flagella Spores Gram stain Acidoresistance Farming Observations Bunched Nutrient Agar Gelatin (in puncture) Sunflower Milk Btomocresol Purple Milk Physiological Bacteria 1) Nitrate Reduction Denitrification Red Test methyl test Voges-Proskauer Corynebacterium CA-53 sticks, in clubs break 1,3 - 2,4 x 1,0 - 1,2 nil none positive nothing 4w tw circular, flat, yellow, smooth, shiny, opaque no reduced sunflower liquefaction with alkaline brown sediment + o 0 0% TABLEAUI (continued 19) Characteristics Corynebacterium CA-53 Production of indole Production of H2S +

2 +

  Hydrolysis of starch Use of citrates + Assimilation of mineral nitrogen sources + Urease Oxidase + Catalase + Need for aerobic oxygen Oxidation / fermentation test Production of acids and gases from carbohydrates 2) acids gas 1. L-arabinose

2. D-xylose - -

3. D-glucose - -

4. D-mannose - -

5. D-fructose - -

6. D-galactose - -

  7. Maltose co o M) T A B L E A U I (cont. 20) Characteristics Corynebacterium CA-53 acids gas

8. Sucrose - -

9. Lactose -.

10. Trehalose -

11. D-sorbitol - -

12. D-mannitol - _

13. Inositol - -

14. Glycerol -

15. Starch -

  Notes: (1) The physiological symbols have the following meanings:

  +: The strain has the corresponding character or forms the corresponding product.

  + -: It is difficult to determine whether the strain has the corresponding character or form or not

the corresponding product.

  -: The strain does not have the corresponding character or does not form the corresponding product.

  0% oe o Remarks: 2) Symbols corresponding to the production of acids and gases from carbohydrates have the following meaning: a) Culture, acids and gases: The strain is cultivated in Hugh and Leifson's medium whose carbon source is one of the carbohydrates 1 to 15 and the culture and the production are observed

of acids and gases.

  +: The strain develops or produces an acid or a gas.

  + -: It is difficult to determine if the strain is growing or if an acid or a gas is o '

produced or not.

  -: The strain does not develop or there is no production of acid or gas.

  b) Assimilation: A culture medium consisting of Nt4NO3 (2 g / l), KH2PO4 (2 g / l), K2HPO4 (5 g / l), MgSO4, 7H20 (0) was placed in a tube (diameter 21 mm). 2 g / l), yeast extract (0.1 g / l) and one of the carbohydrates 1 to 15 (5 g / l), the broth is cultivated with stirring and observed

assimilation (culture).

-: The strain does not grow.

+: The strain grows weakly.

+: The strain grows.

++: The strain is growing well.

I + I: The strain grows very well.

o "tg co Co

BOARD

  Melting Point of Melting Point of Free Acid Methyl Esters Compounds (OC) (C) Found Literature Found Literature

Peak A 150-152 152-154 - -

  Peak B 154-156 156-157 219-220 221-222 Peak C 168-169 169-172 185-186 186188 El -J N a% os o - O0 II

T A B L E A U III

  Compounds Surface ratio Conversion rate Yield or amount (%) (g) 7-hydroxy-diketo-3,12-P-cholanic acid 28,54 28,54 24,3 (peak A) 3-dihydroxy-3a, 7a keto-12 P-cholanic 63.06 53.66 53.6 (peak B) 7α-dihydroxy-7α, 12α-p-cholanic acid 1.54 1.54 1.30 (peak C) cholic acid 6.86 6.86 5 , 80 (peak D) o0 of N o

TA B LEAU IV

  Yield Compounds Substrate Concentration (g / l)

200 300

  Dihydroxy-3a, 7a keto-12 27.1 g 108.3 g 47.4 g P-cholanic 7a-hydroxy-diketo-3.12 12.3 g 49.0 g 21.4 g g-cholanic Dihydroxy-7a acid , 12a keto-3 0.7 g 2.65 g 1.20 g g-cholanic Cholic acid small 20.1 g 230 g quantity N o O o o

TABLE V

  Compounds Ratio Dihydroxy-3a, 7a keto-12 5p-cholanic acid 67.7 7a-Hydroxy-diketo-3,12 5P-cholanic acid 8,3-Dihydroxy-7a, 12a-keto-3-polycholic acid 4.0 Cholic acid 20.0

T A B L E A VI

  Compounds Ratio Dihydroxy-3a, 7a keto-12 5A-cholanic acid 63.3 7a-hydroxy-diketo-3,12 5A-cholanic acid 23,5 Dihydroxy-7a, 12a-keto-5A-cholanic acid 2,0 Cholic acid 11 , 2 N ru 0% o no

TABLE VII

  Compounds Conversion rate * Yield or quantity Cop6s () (g) Dihydroxy-3a acid, 7a keto-12 98.85 98.16 P-cholanic (peak B) Other derivatives produced from 0.59 0.59 of the cholic acid Cholic acid (peak D) 0, 56 0.55

  * The conversion rate is determined from the surface ratio.

TABLE VIII

  Conversion rate or yield Compounds Substrate concentration (g / l)

1 5 10 20 50 100 200

  Dihydroxy-3a, 7a keto-12 P-cholanic acid 96.7% * 98.0% 7 9.3 g 19.5 g 49.6 g 98.2 g 129.2 g Cholic acid 0.4% 0 7% * 0.1 g 0.2 g 0.1 g 0.6 g 70.0 g * Conversion rate u W ooc "C

TABLE IX

  Compounds. Conversion rate Yield or amount Compounds (%) (g) C.) (g) 7α-hydroxy-diketo-3.12 5p-0.80 0.39 cholanic acid and unidentified material (peak A) Dihydroxy-3a acid, 7a keto-12 5p-98.01 48.33 cholanic (peak B) 7α-dihydroxy-7a, 12a-keto-5p-0.25 0.13 cholanic (peak C) Other derivatives 0.24 0.12 Cholic acid ( peak D) 0.70 0.35 Vn w w w w w w w w w w w w

PAINTINGS

  Compounds Conversion rate Yield or quantity M% (g) Hydroxy7a acid diketo-3, 12 3.2 1.58 P-cholanic (peak A) Dihydroxy-3a acid, 7a cepto 83.5 41.20 P-cholanic (peak B) dihydroxy-7a, 12a keto-3 7.9 3.90 e-cholanic acid (peak C) Other drifts 28 1.38 Cholic acid (peak D) 2.6 1, 28 v, w oe o oD

BOARD

  N w on o CD% oCompositesCalculation Yield or Quantity CompoundsM) 7a-Hydroxy-diketo-3.12 5s2 2.56 f-cholanic acid and unidentified material (peak A) Dihydroxy-3a acid, 7a keto-12 93.9 46.30 P-cholanic (peak B) dihydroxy-7a, 12a keto-3 0.2 0.10 b-cholanic (peak C) Other derivatives 0.2 0.10 Cholic acid (peak D) 0, 0, 25 XI

TABLE XII

  Compounds Conversion rate Yield or amount Compounds (%) (g) 7-hydroxy-diketo-3,12 6,5 -3,20 P-cholanic acid and unidentified material (peak A) Dihydroxy-3a, 7a keto-12 acid 88 43,39 P-cholanic (peak B) 7α-dihydroxy-7a, 12α-keto-3 0.305 P-cholanic acid (peak C) Other derivatives O, 1 0.05 Cholic acid (peak D) 5 , 1 2.51 a Ln & N 0% o% C

T A B L E A XIII

  Compounds Conversion rate Yield or amount (%) (g) Hydroxy7a acid diketo-3,12 3,26 1,61 59-cholanic (peak A) Dihydroxy-3a acid, 7a keto 92,0 45,40 P-cholanic ( peak B) dihydroxy-7a, 12a keto-3 0.20 0, 10 e-cholanic acid (peak C)

Other derivatives small quantity -

  Cholic acid (peak D) 4.54 2.24 n 0% Ms o% o ho c0

TA B LEAU XIV

  Conversion rate Yield or amount Compounds (%) (g) Hydroxy7a diketo-3.12 0.23 0.11 P-cholanic acid (peak A) dihydroxy-3a, 7a keto acid 97.02 47.80 59-cholanic ( peak B) Other derivatives O, 75 0.37 Cholic acid (peak D) 2.0 0.99 tn y M on o o do

TABLE XV

  Compound Conversion rate Yield or quantity omos.s. M%) (g) 7-Hydroxy-diketo-3,12-8,64-P-cholanic acid and unidentified material (peak A) 8α-dihydroxy-3α, 7α-keto-12 85.3 42,11 P-cholanic acid (peak B) Other derivatives 2.0 0.99 Cholic acid (peak D) 4.1 2.02 o0 w on Co CD "O

TABLE XVI

  Con ditions Conversion rate Yield or quantity (%) (g) Hydroxy7a acid diketo-3.12 7.7 3.80 P-cholanic (peak A) Dihydroxy-3a acid, 7a keto 12 90.0 44.40 P-cholanic (peak B) dihydroxy-7a, 12a keto-3 0.3 0.15 P-cholanic acid (peak C) cholic acid (peak D) 2.0 0.98 In O o0 ho Co

Claims (13)

R E V E N D I C A T ATIONS   A microbial process for producing a cholanic acid derivative of the formula: COOR H X OH   X represents OH or = O and R represents a hydrogen atom, an alkali metal or an alkaline earth metal, characterized in that it consists in cultivating a microbe capable of developing in a medium containing cholic acid or a salt thereof as a substrate for producing the cholanic acid derivative, selected from the genera Arthrobacter, Brevibacterium and Corvnebacterium, in a culture medium containing the substrate and recovering the derivative product.   2. The microbial method according to claim 1, wherein   in that the microbe was selected from the group consisting of: Arthrobacter CA-35 (FERM-P # 5145; ATCC # 31651), Arthrobacter CA-35-A589-29-32 (FERM-P # 5522; ATCC No. 31652),   Arthrobacter CA-35-A589-47 (FERM-P No. 5523, ATCC No. 31653), Arthroplasty   CA-35-A849 bacterium (FERM-P No. 5524; ATCC No. 31654), Arthrobacter   CA-35-A-1071-15 (FERM-P No. 5525; ATCC No. 31655), Arthrobacter CA-35-   A-1448 (FERM-P No. 5526, ATCC No. 31656), Arthrobacter CA-35-A-1475 (FERM-P No. 5527, ATCC No. 31657), Arthrobacter CA-35-A-1766-15 (FERM-P 5288, ATCC No. 31658), Arthrobacter CA-35 M-965-3 (FERM-P No. 5529, ATCC No. 31659), Arthrobacter CA-35-Y-37-12 (FERM-P No. 5530, ATCC No. 31660) , Brevibacterium CA-6 (FERM-P No. 5144, ATCC No. 31661),   and Corvnebacterium CA-53 (FERM-P No. 5532; ATCC No. 31662).   3. Microbial process according to claim 1, characterized   in that the cholanic acid derivative produced is the acid   3α-dihydroxy-7α-keto-5β-cholanic acid.   4. A microbial process according to claim 3, characterized   in which the microbe was selected from the group consisting of: Arthrobacter CA-35-A589-29-32 (FERM-P # 5522; ATCC # 31652), Arthrobacter CA-35-A589-47 (FERM- No. 5523, ATCC No. 31653), Arthrobacter CA-35-A849 (FERM-P No. 5524, ATCC No. 31654), Atthrobacter CA-35-A-107115 (FERM-P No. 5525, ATCC No. 31655), Arthrobacter   CA-35-A-1448 (FERM-P No. 5526; ATCC No. 31656), Arthrobacter CA-35-   A-1475 (FERM-P No. 5527, ATCC No. 31657), Arthrobacter CA-35-A-1766-15 (FERM-P No. 5528, ATCC No. 31658), Arthrobacter CA-35-M-965-3 (FERM- No. 5529, ATCC No. 31659), and Arthrobacter CA-35-Y-37-12 (FERM-P No. 5530; ATCC 31660).   5. Microbial process according to claim 1, characterized   in that the substrate is used at a concentration of 1 to 500 g / l in cholic acid.   6. Microbial process according to claim 5, characterized   terized in that the substrate is used at a concentration of 5 to 300 g / l in cholic acid.   7. Microbial process according to claim 6, characterized   achieved by using the substrate at a concentration of 10 to 200 g / l in cholic acid.   8. Arthrobacter sp. characterized in that it is capable of growing in a medium containing cholic acid or a salt thereof as a substrate for producing cholanic acid derivatives of formula: 0   COOR x OH OH represents OH or 30 and R represents a hydrogen atom, H   an alkali metal or an alkaline earth metal.   9. Arthrobacter sp. according to claim 8, characterized   In that it consists of Arthrobacter CA-35 strain (FERM-P No. 5145, ATCC No. 31651, Arthrobacter CA-35-A589-29-32 (FERM-P No. 5522, ATCC No. 31652), Arthrobacter CA. 35-A 589-47 (FERM-P No. 5523, ATCC No. 31653), Arthrobacter CA-35-A 849 (PERM-P No. 5524, ATCC No. 31654), Arthrobacter CA35-A-1071-15 (FERM-P 5525, ATCC No. 31655), Arthrobacter CA-35-A-1448 (FERM-P No. 5526, ATCC No. 31656),   Arthrobacter CA-35-A-1475 (FERM-P No. 5527; ATCC No. 31657), Arthroplasty   CA-35A-1766-15 (FERM-P No. 5528; ATCC No. 31658), Arthrobacter   CA-35-M-965-3 (FERM-P No. 5529; ATCC No. 31659), or Arthrobacter CA-35-   Y-37-12 (FERM-P No. 5530, ATCC No. 31660).   10. Brevibacterium sp. characterized in that it is   able to grow in a medium containing chelate acid   or a salt thereof as a substrate for producing cholanic acid derivatives of the formula: COOR o X represents --OH or = O and R represents a hydrogen atom, H   an alkali metal or an alkaline earth metal.   11. Brevibacterium sp. according to claim 10, characterized in that it consists of the strain Brevibacterium CA-6 (FERM-P No. 5144, ATCC No. 31661).   12. Corynebacterium sp. characterized in that it is -   able to grow in a medium containing chelate acid   or a salt thereof as substrates for producing cholanic acid derivatives of the formula: where X represents OH or = O and R represents a hyrogen atom, H   an alkali metal or an alkaline earth metal.   13. Corynebacterium sp. according to claim 12, characterized in that it consists of the strain Corynebacterium CA-53 (FERM-P No. 5332, ATCC No. 31662).