JPH0134038B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a method for producing ursocholic acid as an intermediate raw material for producing ursodeoxycholic acid. [Prior art] 3α, 7β, 12α-trihydroxy-5β-cholane-
24-acid (3α, 7β, 12α-trihydroxy-5β-
Cholanic acid is a type of bile acid having a structure as shown in [ ] in Figure 4, and is called ursocholic acid. Also, Ursodeoxycholic acid
is a type of bile acid having the structural formula shown in brackets [ ] in Figure 5, is listed in the Japanese Pharmacopoeia, and has choleretic effects, gastric juice secretion promoting effects, and gallstone dissolving effects.
Conventionally, ursodeoxycholic acid has been produced by a chemical synthesis method using cholic acid as a starting material (for example,
“APPLIED AND ENVIR ONMENTAL
MICROBIOLOGY”, Vol.44, No.6, 1250
[1982]; JP-A-58-155098), a method for directly converting lithocholic acid to ursodeoxycholic acid using microorganisms (e.g., JP-A-58-155098, JP-A-59-27457) ), etc.
However, none of these uses ursocholic acid as a starting material or intermediate material. As a method for producing ursodeoxycholic acid using ursocholic acid as an intermediate raw material, there is a method by J. Derek Sutherland et al. (“Preparative Bioche mistry” Vol.12, No.
4, 307-321 [1982]). This method uses cholic acid as a starting material, converts the hydroxyl group at the 7α position to the 7β position, and then reduces the hydroxyl group at the 12α position to produce ursodeoxycholic acid. is as shown in the following equation. In this reaction, two types of enzymes are used to isomerize cholic acid to ursocholic acid, namely 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase.
Use NADP as a coenzyme. [Problems to be solved by the invention] In the enzymatic method of producing ursodeoxycholic acid from cholic acid via ursocholic acid, cholic acid, which naturally exists in large quantities and has high solubility in water, is used. Since it is not necessary to use organic solvents as raw materials, manufacturing costs are low and risks can be reduced. However, in the conventional method, two types of enzymes (7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase) and NADP are required for the conversion reaction from cholic acid to ursocholic acid, which greatly affects the production cost. In addition, it was necessary to pay attention to the stability of the enzyme. That is, the enzymes used were expensive and it was difficult to ensure quality stability. [Means for Solving the Problems] As a result of repeated research to solve the problems with the above-mentioned conventional methods, the present inventors have discovered that a certain genus of microorganisms has the ability to convert deoxycholic acid into ursocholic acid. By obtaining this knowledge and completing the method of the present invention based on this knowledge, the above-mentioned problems have been solved. That is, the method of the present invention
Pleurotus, Coriolus, Daedaleopsis, Panaeolus
Genus, Marasmius, Crinipellis, Pholiota
Ursocholic acid, characterized in that the deoxycholic acid is converted into ursocholic acid by contacting one or more types of microorganisms belonging to the genus Fusarium with deoxycholic acid, and the ursocholic acid is collected. This is the manufacturing method. Deoxycholic acid used in the method of the present invention is contained in large quantities in animal bile, second only to cholic acid, and there is no problem in supplying it. Furthermore, the microorganisms having the ability to convert ursocholic acid used in the method of the present invention are selected from the above-mentioned genera, and include, for example, Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus sagejakajiyu, and Pleurotus pulmonarius. (Pleurotus sajor
-caju), Pleurotus salmoneo-stramineus, Coriolus vesicolor,
Daedaleopsis styracina (Daedaleopsis)
styracina), Crinipellis stipitaria, Panaeolus sphinctrinus, Flammulia vertipes,
Fomes fuomentarius
fomentarius), Marasmius siccus, Pholiota nameko, Fusarium equiseti, and the like. Table 2 of Example 2 shows the amount of ursocholic acid produced by these representative microorganisms.
The strains shown in Table 2 are all publicly known deposited bacteria listed in the Fermentation Research Institute's catalog ("LIST OF CULTURES" 7th edition), and can be obtained from the Institute. Furthermore, mutant strains belonging to the above-mentioned genera and obtained by conventional methods can also be used as long as they have the ability to convert ursocholic acid. Methods for bringing the microorganism according to the present invention into contact with deoxycholic acid as described above include a method of culturing by adding deoxycholic acid to a nutrient medium suitable for the microorganism used, and a method of culturing by adding deoxycholic acid to a nutrient medium suitable for the microorganism used, and washing and suspending bacterial cells after culturing the microorganism used. A method such as dissolving deoxycholic acid in a liquid can be applied, but the contact method is not limited to this method. The method for culturing microorganisms according to the present invention can be applied to either liquid culture or solid culture, or it is also possible to isolate only the microbial cells obtained after culturing and use them as resting microbial cells. The medium contains xylose, glucose, galactose, fructose, mannose, and
Saccharides such as maltose, sutucarose, raffinose, starch, starch syrup, glycerol, sorbitol, organic acids and fatty acids such as acetic acid, citric acid, succinic acid, butyric acid, palmitic acid, alcohols such as ethyl alcohol, ethanol, amyl alcohol, hexane , hydrocarbons such as octene,
Alternatively, use natural nitrogen sources such as peptone, yeast extract, defatted soybeans, various amino acid mixtures, meat extract, corn steep liquor, and blackstrap molasses, as well as inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, sodium nitrate, and urea. can. In addition, various vitamins and various inorganic salts can be added as micronutrients, if necessary. These blending ratios are appropriately selected depending on the type of microorganism to be used so that it can grow most easily. The pH of the culture medium also varies depending on the type of microorganism used, but it is usually around PH2 to 10, and the culture temperature is around 15.
~35°C is preferred. In the case of liquid culture, the culture period is 2 to 14 days for basidiomycetes and 2 to 10 days for molds, and in the case of solid culture, it is 10 to 60 days for basidiomycetes and 5 to 5 days for molds.
30 days is appropriate. Deoxycholic acid can be added by adding deoxycholic acid to the medium in advance, or gradually adding deoxycholic acid to the microorganisms used after they have grown sufficiently in the medium. The latter method is preferred since deoxycholic acid has the property of inhibiting the growth of microorganisms. In the case of the former method, the amount of deoxycholic acid added is
The amount is preferably about 0.5 to 20 g/. In addition, in a method using dormant bacterial cells, the microorganism according to the present invention is cultured in advance, the bacterial cells are collected, washed with water, and then suspended in water or a buffer solution. Next, deoxycholic acid is added to this suspension, and the bacterial cells are brought into sufficient contact with the deoxycholic acid by aeration or shaking, thereby converting the deoxycholic acid into ursocholic acid. This method differs from the method described above in which deoxycholic acid is brought into contact with the microorganism according to the present invention in a medium, since the system in which ursocholic acid is converted and produced is mostly water or a simple inorganic salt solution. Purification of ursocholic acid is easy and economical.
Ursocholic acid produced by conversion from deoxycholic acid by the method described above is purified by a conventional method. For example, a culture solution or a suspension of resting bacterial cells is made acidic, an organic solvent (for example, diethyl ether) is added thereto, the ursocholic acid produced is extracted by shaking, and the organic solvent layer is then concentrated. Perform chromatography on a silica gel column to separate a fraction of ursocholic acid. This operation can be repeated several times to crystallize and obtain purified ursocholic acid. [Function] As mentioned above, the method of the present invention is applicable to the genus Pleurotus,
One of the microorganisms belonging to the genera Coriolus, Daedaleopsis, Panaeolus, Marasmius, Cliniperis, Fuoriota, and Fusarium
By bringing one or more species into contact with deoxycholic acid ([] in Figure 4), a hydroxyl group is added to the 7β position of deoxycholic acid, converting it to ursocholic acid []. The mechanism of action is shown in the equation shown in FIG. [Effects of the Invention] According to the method of the invention described above, ursocholic acid can be produced simply by bringing deoxycholic acid into contact with the microorganism according to the present invention as described above, and the reaction path is only one step. , extremely easy to process. Moreover, the microbial conversion reaction does not require the separate supply of enzymes and coenzymes [NAD(P)] as in conventional methods, making it more economical and requiring consideration of the stability of the enzymes and coenzymes. The need is completely eliminated. Further, since the raw material deoxycholic acid exists in large amounts naturally, there is no problem in its supply, and therefore ursocholic acid can be produced stably and at low cost. [Example] Example 1 PS medium (add 20 g of sucrose to the exudate of 200 g of potato, add water to make 1: "LIST
OF CULTURES, 7th ed.” (Published by Fermentation Research Institute)
Add 500 mg of deoxycholic acid to (p.
Dispense into books, sterilize at 120℃ for 30 minutes, and then add Pleurotus ostreatus.
This PS medium was inoculated from a medium in which IFO No. 30880 had been cultured on a slope in advance. Next, after shaking culture in a rotary shaker (120 rpm) at 27°C for 8 days, the culture solution was collected and diluted with 5N
The pH was adjusted to 2 to 3 with HCl, then twice the volume of diethyl ether was added, and the mixture was shaken and mixed for 5 minutes for extraction.Then, the centrifuged supernatant was collected and concentrated to dryness. This dried product was dissolved in ethyl alcohol to obtain a crude ursocholic acid solution, which was then purified by high performance liquid chromatography. The high-performance liquid chromatography device was LC-5A manufactured by Shimadzu Corporation, and the column was CLC-ODS (6 mmφ x 150 mm) manufactured by Shimadzu Corporation.
were used respectively. The developing solution is 10mM potassium phosphate buffer (PH
6.9): Using an acetonitrile mixture (7:3) at a flow rate of 0.6 ml/min, the sample charge amount for one time is 100 μ
And so. As a standard product of ursocholic acid, Alan F. Hofmann (Alan F. Hofmann:
Division of Gastroenterology, Department of
Medicine, School of Medicine, University of
California; 225 Dickinson street, San Diego;
Based on the fact that the peak retention time of this standard product in high-speed chromatography is 6.9 minutes, the retention time was
A fraction of 6.2 to 8.0 minutes was collected. This chromatography was performed 30 times. All the fractions obtained as described above were combined, acidified with hydrochloric acid (PH 2-3), extracted with shaking by adding twice the amount of ether, and concentrated. Next, the concentrated solution was subjected to silica gel chromatography (column: 10 mmφ x 300 mm, carrier: Merck Kieselgel Art.7734, 70-230).
mesh, developing solution; chloroform: methanol:
The ursocholic acid fraction was collected and crystallized from an aqueous ethanol system to obtain 20 mg of ursocholic acid. The following analysis was performed on the produced ursocholic acid. (1) Thin layer chromatography The thin layer chromatography plate used was Merck Kieselgel 60 (Art. 5721) and was developed with various solvents. Table 1 shows the composition of the developing solvent and the Rf value of the produced ursocholic acid. These Rf values matched those of the ursocholic acid standard product. (Display in Table 1 has been omitted.) (2) Gas chromatography The gas chromatography equipment used was GC-9A manufactured by Shimadzu Corporation, column 1m, "Gaschrome Q" as a carrier, and silicon in the liquid phase. DC−QF1(2
%) was used. The column temperature was 220°C, the injection temperature was 240°C, nitrogen was used as the carrier gas, and detection was performed using FID. As a result of the measurement, the retention time was 38.8 minutes, which matched the retention time of the standard product. (3) Melting point measurement The melting point of the produced ursocholic acid was 134°C, and that of the standard product was 133°C. (4) Elemental analysis The measured value of produced ursocholic acid is C: 67.8,
H: 9.72 (theoretical value C: 67.0, H: 10.0) (5) Molecular weight The measured value of the produced ursocholic acid is 409 (theoretical value
408).

[Table] (6) Infrared absorption Spectrum of ursocholic acid produced by the method of the present invention [A] and spectrum of standard product [B]
is shown in Figure 1. (7) NMR The spectrum of ursocholic acid produced by the method of the present invention is shown in Figure 2, and the spectrum of the standard product is shown in Figure 3.
As shown in the figure. Example 2 PS medium 1 having the same composition as in Example 1 was prepared, and 500 mg of deoxycholic acid was added and dissolved therein to form the main culture medium. 100 ml of this medium was dispensed into ten 500 ml Sakaguchi flasks each, and after sterilization at 120°C for 30 minutes, the microorganisms shown in Table 2 were inoculated into this main culture medium from the medium previously cultured on a slant. Next, culturing was carried out under the same culture conditions as in Example 1, and the following
The conversion production reaction and purification treatment of ursocholic acid were carried out in the same manner as in Example 1. Each of the microorganisms shown in Table 2 was treated as described above, and the resulting amount of ursocholic acid produced was
Shown in the table. Example 3 Soybean flour-starch medium (20 g of whole soybean flour, 30 g of soluble starch, 1 g of KH ₂ PO ₄ , 2H ₂ O, MgSO ₄
7H ₂ O 0.5g, CaCl ₂・2H ₂ O 0.5g dissolved in 1 part water)
Dispense 100ml into a 500ml Sakaguchi flask and heat at 120℃20.
Sterilized for minutes. In this medium, Pleurotus sajor-caju IFO No.
30167 was inoculated and cultured at 27° C. for 4 days with shaking in a rotary shaker (120 rpm) to use as an inoculum.
Next, we added text culture medium (10 g of defatted corn meal, 20 g of dextrin, 17.4 g of K ₂ HPO ₄ , MgSO _{4.7H 2} O0.5
g, _CaCl2・_2H2O0.5g and deoxycholic acid
(0.5 g dissolved in 1 part water) 100 ml was dispensed into a 500 ml Sakaguchi flask and cultured for 8 days under the same conditions as the seed culture described above. After adjusting the pH of this culture solution to 2 to 3 with 5N hydrochloric acid, 2 times the amount of diethyl ether was added.
Extraction was carried out by shaking for a minute, and the centrifuged supernatant was collected and concentrated to dryness. Dissolve the obtained dry matter in 20 ml of ethanol,

【table】

[Table] 6.1 mg of crude ursocholic acid was obtained by recrystallization. Example 4 Using the same culture medium as in Example 2 above, Fusarium
Fusarium equiseti IFO No.31095
Inoculum culture and main culture were performed. However, in this example, the amount of deoxycholic acid added to the main culture medium was 1.0 g/. Culture was carried out in the same manner as in Example 2, and the resulting culture solution was treated in the same manner as in Example 2 to obtain 460 mg of crude ursocholic acid. This crude ursocholic acid was subjected to high performance liquid chromatography and silica gel column chromatography under the same conditions as in Example 1, to obtain approximately 230 mg of substantially single ursocholic acid as determined by thin layer chromatography. Example 5 A PS medium having the same composition as in Example 1 was dispensed into three 500 ml Sakaguchi flasks at 100 ml each and sterilized under high pressure.
Next, Fusarium equiseti IFO No. 31095, which had been cultured on a slope in advance, was inoculated and cultured with shaking in a rotary shaker (120 rpm) at 28°C for 4 days. was cultured with aeration in an incubator (5 volumes) containing 2.5 volumes of a medium with the following composition. The culture conditions were 28℃, aeration rate of 1/min, and stirring rate of 120r.
It was set as pm. Medium composition Sucrose 40g / Peptone 10 〃 Yeast extract 1 〃 K ₂ HPO ₄ 17.4 〃 MgSO ₄・7H ₂ O 0.5 〃 CaCl ₂・2H ₂ O 0.5 〃 Deoxycholic acid 0.5 〃 Mineral (Note) 1 ml / (Note) Mineral FeSO ₄・7H ₂ O 1g/ MnSO ₄・6H ₂ O 1 〃 ZnSO ₄・4H ₂ O 1 〃 CuSO ₄・5H ₂ O 1 〃 NaMO ₆・2H ₂ O 1 〃 After the cultivation, filter the culture solution The mycelium produced during the culture was filtered from the spores, and further centrifuged (10,000 rpm, 15 min.) to collect the spores. The obtained spores were made into a suspension with sterile water at a spore count of 2 x 10 ¹⁰ /ml, and to 10ml of this suspension was added deoxycholic acid and phosphate buffer (PH7.5) at a concentration of 0.5g/0.1M, respectively. was added to make 100 ml. Then, add that 100ml to
It was dispensed into a 500 ml Sakaguchi flask, cultured at 28°C for 3 days with shaking in a rotary shaker (120 rpm), and 64 hours after inoculation, it was extracted and purified using the same method as in Example 1, and 0.25 g/Ursocol was extracted and purified using the same method as in Example 1. Obtained acid.

[Brief explanation of drawings]

FIG. 1 shows infrared absorption spectra of ursocholic acid [A] produced by the method of the present invention and standard ursocholic acid [B]. For ease of comparison, both are shown slightly shifted vertically. Figure 2 shows the ursocholic acid produced by the method of the present invention.
Figure 3 shows the NMR spectrum of a standard ursocholic acid. FIG. 4 is a formula showing the mechanism of action in the method of the present invention, where [ ] is deoxycholic acid and [ ] is ursocholic acid. FIG. 5 shows a reaction formula for producing ursodeoxycholic acid [] from ursocholic acid [].

Claims (1)

[Claims] 1. Pleurotus, Coriolus, Daedaleopsis, Panaeolus
Genus, Marasmius, Crinipellis, Pholiota
Production of ursocholic acid, which comprises converting deoxycholic acid into ursocholic acid by contacting one or more microorganisms belonging to the genus Fusarium with deoxycholic acid, and collecting the same. Law.