JPH0151998B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention uses cholic acid (3α, 7α, 12α-trihydroxy-5β-cholanic acid) as a gallstone dissolving agent or as a choleretic agent ursodeoxycholic acid (UDC).
12-Keto-3α,7α- is an intermediate for the production of chenodeoxycholic acid (CDC), which is useful as a raw material for the synthesis of
The present invention relates to a method for efficiently producing dihydroxy-5β-cholanic acid (hereinafter abbreviated as 12-ketocholic acid) using microorganisms. [Prior Art] Conventionally, methods for producing 12-ketocholic acid from cholic acid using microorganisms include a method using microorganisms of the genus Arthrobacter (Japanese Patent Application Laid-Open No. 8796/1987, etc.);
Methods using microorganisms of the genus Previbacterium (Japanese Patent Application Laid-Open No. 56-29998, etc.) are known. The present inventors also discovered that specific microorganisms belonging to the genus Micrococcus and Corynebacterium produce 12-ketocholate from cholate added to the culture medium, and filed a patent application (Japanese Patent Application No. 57-227487 number, etc.). [Problems to be Solved by the Invention] However, in any of these methods, it is difficult to obtain high yield and high purity 12-ketocholic acid in a short time, and according to the findings of the present inventors, In the method of obtaining 12-ketocholate by directly adding cholate to the culture medium of microorganisms, 12-ketocholate is obtained.
- There are limits to the production rate of ketocholic acid, the amount of remaining cholic acid, the degree of coloring of the obtained 12-ketocholic acid, etc., and it cannot be judged as an efficient method. An object of the present invention is to obtain high-yield, high-purity 12-ketocholic acid in a short time from highly concentrated cholic acid. [Means for Solving the Problems] The present inventors have been working for many years on microorganisms that produce 12-ketocholic acid from cholic acid and using the microorganisms.
We have been conducting repeated research on methods for producing 12-ketocholic acid. As a result, a specific microorganism belonging to the genus Micrococcus was cultured, and the cells or a culture solution containing the cells were mixed with cholic acid and/or its salt in the coexistence of a boric acid buffer solution to cause a reaction. The present inventors have discovered that 12-ketocholic acid and/or its salts can be produced and collected in a manner that allows high-yield, highly pure 12-ketocholic acid to be obtained, and have completed the present invention. The microorganisms used in the present invention include, for example, Micrococcus SD-101 (Feikoken Bibori No. 6841), its mutant strains, genetically modified strains, etc., but are not particularly limited to these microorganisms. There is no particular limitation as long as the microorganism belongs to the genus Micrococcus and produces 12-ketocholic acid and/or its salt from cholic acid and/or its salt. The medium used in the present invention may be any medium as long as the microorganism can be grown by culturing,
For example, carbon sources include glucose, fructose, sucrose, acetic acid, ethyl alcohol, glycerin, etc.; nitrogen sources include organic nitrogen such as peptone, yeast extract, cornstarch liquor, and inorganic nitrogen such as ammonium sulfate and ammonium nitrate. . In addition, inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, manganese sulfate, and magnesium sulfate are added. The culture in the present invention can be carried out under aerobic conditions, for example, by an aeration stirring method or a reciprocating shaking method. The temperature may be anywhere from 20 to 38°C, but preferably 22 to 27°C. At temperatures lower than this range, the growth rate of microorganisms is slow, and at temperatures higher than this range, the conversion activity of microorganisms to produce 12-ketocholic acid and/or its salts from cholic acid and/or its salts is significantly impaired. PH during culture is 6.0~
9.0, but preferably at the early stage of culture.
The pH should be 7.0 to 8.0, and the pH should be adjusted when the culture has progressed to a certain extent.
A value of 5.8 to 6.2 is appropriate. At a pH lower than this range, the growth rate of the microorganism is slow, and at a pH higher than this range, the conversion activity of the microorganism to produce 12-ketocholic acid and/or its salt from cholic acid and/or its salt deteriorates. The culturing time is about 8 to 30 hours. The bacterial cells are extracted by processing the culture solution using methods such as centrifugation.
After collecting the bacteria, or after washing with physiological saline etc., it may be suspended in a buffer containing cholic acid and/or its salts, or the culture solution containing suspended bacteria and cholic acid and its salts may be suspended without collecting the bacteria. /or a buffer solution containing the salt thereof may be directly mixed. The resulting product is less colored if the bacteria are collected and then suspended and reacted, but from an industrial perspective, mixing without collecting the bacteria is easier. The concentration of cholic acid and/or its salt during the conversion reaction of the present invention may be 5 to 500 g/, and is preferably about 10 to 100 g/in consideration of conditions such as reaction time and operating method. The buffer is a boric acid buffer, specifically boric acid [orthoboric acid (H ₃ BO ₃ ), metaboric acid (HBO ₂ ), tetraboric acid (H ₂ B ₄ O ₇ ), etc.] and its water-soluble salts, For example, a mixture of sodium salt, potassium salt, ammonium salt, etc., or a mixture of boric acid and sodium hydroxide, potassium hydroxide, ammonium hydroxide, etc. is used. The concentration during the reaction is preferably 10 mmol/-500 mmol/. By adding boric acid, tetraboric acid or their salts, the formation of by-products can be suppressed to an extremely low rate. When using other buffers, such as phosphate salts or trishydroxymethylaminomethane salts, the yield of 12-ketocholic acid is higher than when no buffer is used, but a small amount of by-products are produced. No significant effect was observed as with the use of borates. The PH during exchange reaction may be 6.7 to 9.0, especially 6.7.
A range of ~7.2 is preferred. Even when a borate buffer is used, a small amount of by-products will be produced at a pH higher than this range. On the other hand, at a pH lower than this range, cholic acid precipitates and conversion activity is lost. The pH is adjusted using an aqueous solution of an alkali metal hydroxide such as sodium hydroxide and an acid such as hydrochloric acid or sulfuric acid. The temperature during the conversion reaction may be 20 to 45°C, but a range of 35 to 41°C is particularly preferred. At temperatures lower than this range, a large amount of cholic acid and/or its salt remains, and at temperatures higher than this range, the conversion activity is deactivated in a short period of time. The conversion reaction time varies depending on conditions such as cholic acid concentration, but 2
It will be carried out in about 72 hours. The product, ie, 12-ketocholic acid, can be recovered in good yield from the reaction solution by a known method. For example, the PH of the reaction solution may be lowered by adding an acid, and the resulting precipitate of 12-ketocholic acid may be filtered and then dried. After extracting the ketocholic acid, the solvent may be distilled off. [Operation] In the present invention, microorganisms are not cultured in a medium containing cholate, but are cultured in a medium not containing cholic acid and/or its salts, and the microorganisms are cultured in a medium containing cholic acid and/or its salts. By suspending or mixing in a buffer containing 12-ketocholic acid and/or its salts and reacting, it is possible to culture microorganisms under conditions that maximize the activity of converting cholic acid and/or its salts into 12-ketocholic acid and/or its salts. Moreover, since it is possible to accurately control conditions such as temperature and pH during the conversion reaction, high yield and high purity 12-ketocholic acid can be obtained in a short time. It is estimated that the synthesis of enzymes that produce 12-ketocholic acid and/or its salts from cholic acid and/or its salts within the bacterial cells is most active when the culture temperature is in the range of 22 to 27°C. Furthermore, it is estimated that the synthesis of the enzyme is most active when the pH during culture is 5.8 to 6.2. Regarding the conversion reaction temperature, it is assumed that the higher the temperature, the more the chemical equilibrium between cholic acid or its salt and 12-ketocholic acid or its salt exists on the side of 12-ketocholic acid or its salt. The optimal temperature of the enzyme that produces 12-ketocholic acid and/or its salt is around 40°C, and the enzyme that produces by-products from cholic acid and/or its salt and 12
- The optimal temperature for enzymes that produce by-products from ketocholic acid and/or its salts is thought to be lower than 37°C. Regarding the pH during the conversion reaction, the optimum pH for the enzyme that generates 12-ketocholic acid and/or its salt from cholic acid and/or its salt is around 6.8, and it is difficult to convert byproducts from cholic acid and/or its salt. The optimum pH of the enzyme that produces and the enzyme that produces by-products from 12-ketocholic acid and/or its salts is thought to be between 7.2 and 7.5. Additionally, boric acid (orthoboric acid, tetraboric acid, etc.)
and/or its salts strongly inhibit enzymes that produce byproducts from cholic acid and/or its salts and enzymes that produce byproducts from 12-ketocholic acid and/or its salts at a pH in the range of 5.8 to 6.2. It is estimated to be. [Effect] According to the present invention, specific microorganisms belonging to the genus Micrococcus are cultured, and the cells or a culture solution containing the cells are mixed with cholic acid and/or its salt in the presence of a boric acid buffer and reacted. By doing so, it is possible to obtain 12-ketocholic acid in higher yield and purity than with high concentration cholic acid and/or its salt.
Furthermore, this method of using microbial cells as a type of medium has the strong possibility of reducing costs by using the microbial cells repeatedly, or of immobilizing the microbial cells using known methods for other microorganisms. It is suggestive. Examples of the present invention will be shown below, but the present invention is not limited thereto. Example 1 A medium having the following composition was placed in a 5-jar fermenter and sterilized by heating in an autoclave at 120°C for 40 minutes. After cooling, adjust the pH to 7.2 with 2N sodium hydroxide solution and aseptically add glucose to 40%.
g was added. Add 500ml of the same medium to this medium in advance.
50 ml of a culture solution of Micrococcus SD-101 that had been precultured in an Erlenmeyer flask was added, and cultured at 25°C for 24 hours. As the bacteria grow, the pH decreases to 6.0
When the pH reached 14%, addition of 14% ammonia water was started to maintain the pH between 6.0 and 6.2. Next, this culture solution 1
and 200 mmol/containing 40 g of cholic acid.
of boric acid buffer (PH7.0) to adjust the pH to 7.0. This reaction solution was aerated and stirred at 37° C. for 26 hours, and then centrifuged. Adjust the pH of the supernatant to 2 with 1N hydrochloric acid.
The product was precipitated. After suction filtration, it was air-dried to obtain 12-ketocholic acid. The degree of progress of the conversion reaction was confirmed by analyzing the reaction solution at each time point after the start of the reaction using the method described below. The results are shown in Table 1. Medium composition Glucose 2% (sterilized and added separately) Ammonium sulfate 0.2% Monopotassium hydrogen phosphate 0.2% Dipotassium hydrogen phosphate 0.5% Yeast extract 0.2% Magnesium sulfate 0.05% Ferrous sulfate 4ppm Manganese sulfate 4ppm Tap water analysis method Liquid Chromatographic quantification. CDC the sample
Quantification was carried out using a modified internal standard method and a simple area percentage method using as an internal standard. Column: Shodex OPS pak F411 Pump: Model BIP-1 manufactured by JASCO Corporation Detector: Model Shodex-RI SE-31 Mobile phase: 75:25 methanol-water mixture, 0.02 mol of phosphoric acid/Column temperature: 30°C Liquid: 1ml/min Sample volume: 20μl

[Table] Example 2 The same procedure as in Example 1 was carried out except that the reaction temperature was changed to 41° C. from the 24th hour. The results were as shown in Table 2.

[Table] Example 3 Culture solution 1 cultured in the same manner as in Example 1 was taken and collected by centrifugation, containing 40 g of cholic acid.
The bacterial cells were suspended in 100 mmol/borate buffer 1 and the pH was adjusted to 7.0. Thereafter, the reaction was carried out in the same manner as in Example 2. The results were as shown in Table 3.

[Table] Example 4 The same procedure as in Example 1 was carried out except that the culture temperature was changed to 35°C. The results were as shown in Table 4.

[Table] Example 5 Everything was the same as in Example 1 except that the culture temperature was 27°C. The results were as shown in Table 5.

[Table] Example 6 Everything was the same as in Example 1 except that the pH during culture was maintained at 7.0. The results were as shown in Table 6.

[Table] Example 7 Except for using 200 mmol/tetraborate buffer (PH7.0) 1 containing 40 g of cholic acid in the reaction.
Everything was carried out in the same manner as in Example 1. The results were as shown in Table 7.

[Table] Example 8 The reaction was carried out in the same manner as in Example 1, except that 1 mol/mol boric acid buffer (PH7.0) containing 40 g of cholic acid was used in the reaction. The results were as shown in Table 8.

[Table] Example 9 The reaction was carried out in the same manner as in Example 1 except that the pH was maintained at 7.3 during the reaction. The results were as shown in Table 9.

[Table] Example 10 The same procedure as in Example 1 was carried out except that the reaction temperature was changed to 32°C. The results were as shown in Table 10.

[Table] Example 11 The same procedure as in Example 1 was carried out except that the reaction temperature was changed to 45°C. The results were as shown in Table 11.

[Table] Example 12 The reaction was carried out in the same manner as in Example 1, except that a 200 mmol boric acid buffer (PH7.0) containing 100 g of cholic acid was used in the reaction and the reaction time was extended to 48 hours. The results were as shown in Table 12.

[Table] Example 13 The reaction was carried out in the same manner as in Example 1, except that a 200 mmol boric acid buffer (PH7.0) containing 300 g of cholic acid was used in the reaction and the reaction time was extended to 72 hours. The results were as shown in Table 13.

[Table] Comparative Example 1 Cholic acid was added to the medium shown in Example 1 to a concentration of 20 mg/ml to adjust the pH to 7.0, and the same bacteria were precultured using the method shown in Example 1. amount was added and cultured at 35°C for 24 hours. Table 14 shows the concentrations of cholic acid and 12-ketocholic acid in the medium at each time of culture.

[Table] Comparative Example 2 The reaction was carried out in the same manner as in Example 1, except that 200 mmol/1 phosphate buffer (PH7.0) containing 40 g of cholic acid was used for the reaction. The results were as shown in Table 15.

[Table] Comparative Example 3 200 mmol/tris hydroxymethylaminomethane buffer (PH
7.0) All procedures were carried out in the same manner as in Example 1, except that 1 was used. The results were as shown in Table 16.

【table】

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Micrococcus and having the ability to produce 12-keto-3α,7α-dihydroxycholanic acid and/or its salt from cholic acid and/or its salt is cultivated in a nutrient medium. , the bacterial cells or a culture solution containing the bacterial cells are mixed with cholic acid and/or its salt in the presence of a borate buffer, and reacted, and 12-keto-3α,7α-dihydroxycholanic acid and 1. A method for producing 12-keto-3α,7α-dihydroxycholanic acid and/or a salt thereof, which comprises producing and collecting the salt. 2. The production method according to claim 1, wherein the microorganism used is Micrococcus SD-101 or a mutant strain thereof. 3 The culture temperature of microorganisms is 22 to 27℃, and the pH during culture is
5.8 to 6.2. The manufacturing method according to claim 1. 4 The reaction temperature of cholic acid and/or its salt is 35~
The manufacturing method according to claim 1, wherein the temperature is 41°C and the pH during the reaction is 6.7 to 7.2.