JP3030306B2 - Method for producing 3-keto 7β-hydroxycholanic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

Industrial Applicability The present invention has a blood glucose lowering effect, a cholesterol lowering effect and an inhibitory effect on gallstone formation from ursodeoxycholic acid (Japanese Patent Publication No. 63-1957), and is useful as a therapeutic agent for liver disease (Showa 62-61920)
The present invention relates to a microorganism that produces 3-keto-7β-hydroxycholanoic acid, which is an intermediate for producing 3β, 7β-dihydroxycholanoic acid, and a method for producing 3-keto-7β-hydroxycholanoic acid using the microorganism.

[0002]

2. Description of the Related Art Heretofore, as a method for producing 3-keto-7β-hydroxycholanic acid, a method of selectively oxidizing the α-hydroxyl group at the 3-position of ursodeoxycholic acid has been known (Japanese Patent Publication No. Sho 63). No. 1957).

[0003]

However, this production method comprises four steps, and requires purification in each step, and has a problem in the reactivity and selectivity of each step. It was not satisfactory in terms of product purity.

[0004] In view of this situation, the present inventors have intensively studied a method for producing 3-keto-7β-droxycholanoic acid directly from ursodeoxycholic acid using a microorganism. As a result, the present inventors have discovered that a novel microorganism belonging to the genus Bacillus has 3-keto-7β-hydroxycholanic acid-producing ability, and completed the present invention.

That is, according to the present invention, a microorganism having the ability to produce 3-keto-7β-hydroxycholanic acid belonging to the genus Bacillus is cultured in a nutrient medium containing ursodeoxycholic acid, and 3-keto- 3-keto-7β consisting of producing 7β-hydroxycholanic acid and collecting it
-A method for producing hydroxycholanic acid is provided.

[0006] The present inventors have found that 3-keto-7β-amino acid in a medium containing ursodeoxycholate from soil in Ashikaga City, Tochigi Prefecture.
A bacterium capable of producing hydroxycholanic acid was isolated, and this strain was isolated from Bacillus sp. TTUR1-1 (B
acillus sp. TTUR1-1), and deposited on November 22, 1990 with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology (Microbial Laboratory No. 11860).

[0007] Isolation of bacteria from soil was carried out by the following method.

[0008] The soil is a sorghum medium I containing 5% sodium cholate [glucose 1%, yeast extract 0.1.
5%, potassium monohydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.02%, sodium carbonate 1%, pH1
0], and enrichment culture was performed at 30 ° C for 5 days. One loopful of the resulting culture was streaked on a Horikoshi medium I agar plate containing 5% sodium cholate, Was purely separated. These strains were grown at 30 ° C. in Horikoshi medium I, each containing 5% sodium ursodeoxycholate,
After culturing for 2 days, 3-keto-7β-hydroxycholanoic acid in the culture was quantified to obtain a strain having a high ability to convert 3-keto-7β-hydroxycholanoic acid.

The mycological properties of this fungus are as follows:
These tests and classification methods are described in the Vergees Manual of Systematic Bacteriology (BERG).
EY'S MANYUAL OF Systemati
c Bacteriology). Unless otherwise specified, a medium in which sodium carbonate was added and the pH was adjusted to 10 was used.

Mycological properties of TTUR1-1 bacteria (1) Form Form Bacillus Size 0.3-0.5 × 1.5-3.0 μ Flagella Yes Spores Yes (egg, formed at the end of cells. Spores) The sac swells.) 0.4-0.7 × 0.8-1.2μ Gram staining Change Acid resistance None

(2) Growth in each medium

(3) Physiological properties

[Table 1]

[0013] As a result of the above search, TTUR1-1 is an aerobic spore bacterium.
It is clear that the microorganism belongs to the genus (. However, it differs from general Bacillus microorganisms because the optimum pH for growth is on the alkaline side at around 10.

Further, Bacillus alcarophilus (Bacill), which is known as an alkalophilic Bacillus microorganism.
luchalophilus) and Bacillus alcalophilus subsp. halodurans (B
acillus alcalophilus subs
p. (Halodurans), the differences as shown in Table 2 were observed. In particular, the shape of the colony and the appearance around the colony were remarkably different.

On the other hand, most of the alkalophilic Bacillus microorganisms isolated from the natural world show a characteristic of a round shape and a whole shape in the shape of the colony and the surroundings, and TTUR
It does not match the properties shown by 1-1 bacteria.

Bacillus cereus 8-1 (FERM) is an alkaliphilic, irregular colony, and a fragmented periphery.
2885, JP-B-53-13708) and Bacillus
Alcalophilus 202-1 (Bacillus a)
lcalophylus 202-1, FERM267
4, Japanese Patent Publication No. 53-27786),
The differences shown in Table 2 were recognized with these bacteria.

[0017]

[Table 2]

Therefore, this bacterium is an aerobic spore bacterium, but as described above, it has various bacteriological properties, particularly, an optimum pH for growth is on the alkaline side near 10 and is different from known bacterial species. Therefore, it is appropriate to set the present bacterium as a new strain.

According to the present invention, 3-belonging to Bacillus genus
By culturing a microorganism capable of producing keto-7β-hydroxycholanoic acid in a nutrient medium containing ursodeoxycholic acid, 3-keto-7β-hydroxycholanic acid can be produced. The concentration of ursodeoxycholic acid in the nutrient medium is not particularly limited, but the target 3-keto-7β
-Yield of hydroxycholanic acid, 5 to 500 from culture conditions
g / l, preferably 40-300 g / l.

The medium that can be used in the present invention may be any medium as long as the microorganism can grow by culturing. Examples of the carbon source include glucose, fructose, maltose, sucrose, glycerin, Various carbohydrate raw materials such as starch, bran, molasses, etc., as a nitrogen source, organic nitrogen such as peptone, meat extract, yeast extract, corn steep liquor, soy flour, rapeseed oil cake,
Inorganic nitrogen such as ammonium nitrate, ammonium chloride and sodium nitrate can be used. In addition, it is preferable to add trace amounts of inorganic metal salts, vitamins, growth promoting factors and the like.

The culture in the method of the present invention can be carried out under aerobic conditions, for example, by aeration stirring or reciprocating shaking. The culturing conditions are not particularly limited, but generally speaking, the culturing is carried out at a temperature of 20 to 40 ° C., a pH of 7 to 11, and a period of about 1 to 6 days.

The desired 3-keto-7β-hydroxycholanic acid can be collected from the culture solution according to a conventional method. For example, there is a method of centrifuging a culture solution, acidifying a supernatant with diluted hydrochloric acid or diluted sulfuric acid, and recrystallizing a precipitated product.

[0023]

The present invention will be described below with reference to examples.
It goes without saying that the scope of the present invention is not limited to these examples.

The product was identified by high performance liquid chromatography under the following conditions. Column: Capsule Pack C18 Column (Type AG12
0, S-5μm, column size 4.6φ × 150mm,
Mobile phase; methanol / purified water / phosphoric acid (65/35 /
Flow rate; 1.7 ml / min detection; RI

Example 1 10 g of glucose, 5 g of peptone, 5 g of yeast extract, potassium monohydrogen phosphate 1
g, 0.2 g of magnesium sulfate heptahydrate in purified water 50
0 ml, and 50 g of ursodeoxycholic acid,
5 g of sodium hydroxide and 10 g of sodium carbonate were dissolved in 500 ml of purified water, and each was sterilized at 121 ° C. for 15 minutes. After cooling, they were mixed to form a medium (pH 10).

In a test tube (3φ × 19c)
m), 0.1 ml of a bacterial solution grown by shaking culture at 30 ° C. for 20 hours in a test tube containing 20 ml of the same composition except for ursodeoxycholic acid and sodium hydroxide from the above-mentioned medium beforehand is added. Inoculated aseptically. Thereafter, shaking culture was performed at 30 ° C. for 6 days.

After the culture, the cells were removed by centrifugation, and the resulting culture supernatant was acidified by adding dilute sulfuric acid to give 3-keto-7β-hydroxycholanoic acid and unconverted ursodeoxycholic acid. Precipitated. This precipitate was collected and dried to obtain 0.98 g of a white powder. A portion of this was taken and the production ratio of 3-keto-7β-hydroxycholanoic acid and ursodeoxycholic acid was determined by high performance liquid chromatography.
5% and ursodeoxycholic acid 20.5%. The mixture was recrystallized from methanol to give pure 3-keto-7.
β-hydroxycholanic acid was obtained.

Example 2 The operation was performed in the same manner as in Example 1 except that the shaking culture in Example 1 was changed to 3 days. 3-keto
The production ratio of 7β-hydroxycholanic acid and ursodeoxycholic acid is 3-keto-7β-hydroxycholanoic acid 5
6.6% and ursodeoxycholic acid 43.4%.

Example 3 The procedure of Example 1 was repeated, except that 150 g of ursodeoxycholic acid and 15 g of sodium hydroxide were used. The production ratio of 3-keto-7β-hydroxycholanoic acid and ursodeoxycholic acid was 52.6% of 3-keto-7β-hydroxycholanoic acid,
Ursodeoxycholic acid was 47.4%.

Example 4 10 g of glucose, 5 g of peptone, 5 g of yeast extract, and potassium monohydrogen phosphate 1
g, 0.2 g of magnesium sulfate heptahydrate in purified water 50
0 ml, and ursodeoxycholic acid 100
g, 10 g of sodium hydroxide and 10 g of sodium carbonate were dissolved in 500 ml of purified water, and sterilized at 121 ° C. for 15 minutes, respectively. After cooling, they were mixed to form a medium (pH 10).

100 ml of this medium was added to a Sakaguchi flask (50
After dispensing the above medium, urine deoxycholate and sodium hydroxide were previously removed from the above medium and placed in a test tube (3φ × 19 cm) containing 20 ml of the medium having the same composition.
0.1 ml of a bacterial solution grown by shaking culture at 20 ° C. for 20 hours was aseptically inoculated. Thereafter, shaking culture was performed at 30 ° C. for 4 days.

Thereafter, the same treatment as in Example was carried out to obtain crude 3-keto-7β-hydroxycholanic acid. 3-keto-7β
The formation ratio of -hydroxycholanic acid and ursodeoxycholic acid was 64.2 for 3-keto-7β-hydroxycholanoic acid.
% And ursodeoxycholic acid 35.8%.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:07)

Claims (2)

(57) [Claims] 1. Bacillus sp. T belonging to the genus Bacillus and capable of producing 3-keto-7β-hydroxycholanic acid
TUR1-1. 2. A microorganism belonging to the genus Bacillus and capable of producing 3-keto-7β-hydroxycholanoic acid is cultured in a nutrient medium containing ursodeoxycholic acid to produce 3-keto-7β-hydroxycholanoic acid in the culture. A method for producing 3-keto-7β-hydroxycholanoic acid, which comprises producing and collecting the resulting product.