JPS61280293A - Production of phosphoric acid ester of steroid compound - Google Patents

Abstract

PURPOSE:To obtain an easily water-soluble phosphoric acid ester of a steroid compound, by phosphatizing the alpha- or beta-type hydroxyl group of the steroid ring of a steroid compound with a mold belonging to Mortierella genus. CONSTITUTION:A steroid compound or its alkali metal salt is made to contact with the cell of a mold belonging to Mortierella genus and capable of converting a steroid compound to phosphoric acid ester (e.g. Mortierella ramanniana ramanniana Y2-1 strain) to obtain the phosphoric acid ester of the steroid compound or its alkali metal salt.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a microbiological method for producing phosphate esters of steroid compounds.

More specifically, a step of culturing a filamentous fungus belonging to the genus Mortierella that has the ability to phosphoric acid esterify a steroid compound, and a step of bringing the filamentous fungus belonging to the genus Mortierella into contact with a steroid compound or an alkali metal salt thereof. , a method for producing a phosphoric acid ester of a steroid compound or an alkali metal salt thereof by microbial conversion, which comprises a step of recovering a phosphoric acid ester of a steroid compound or an alkali metal salt thereof converted from the steroid compound or a salt thereof. It is.

For sulfuric acid esterification and glucuronic acid esterification of aromatic compounds by Jubei Nukiyoshi, hydroxybiphenyl Aspergillus toxiccarius (and
) by sulfuric acid esterification reaction (J, Bacterio
156, 49.1983) and the sulfuric acid and glucuronic acid esterification reaction of 1-naphthol and 4-hydroxybiphenyl by Cunninghamella elegans (Appl.

Environ, Microbio! ,,dan, 10
70.1982) has recently been reported, but at present, nothing is known about the phosphoric acid esterification reaction of steroid compounds and polycyclic hydrocarbons by microorganisms.

Furthermore, it has been revealed that steroid compounds have various physiological activities, and they have already been applied to several pharmaceutical products, and research is being conducted to expand their use. However, steroid compounds are generally poorly water soluble;
Easily water-soluble steroid compounds are manufactured by chemical synthesis through complicated steps. In addition, some of them are difficult to synthesize.

That is, the purpose of the present invention is to treat poorly water-soluble steroid compounds,
The object of the present invention is to produce readily water-soluble steroid compounds using microorganisms that have a strong ability to phosphorylate steroid compounds.

- As a result of intensive research on various bacterial strains using the phosphorylation of steroid compounds as an indicator, the present inventors discovered that
The filamentous fungus Mortierella lamagnana described in No. 795
Mortierella ramann
iana var, ramanniana) Y2-1
(Feikoken Jokyo No. 440) was found to exhibit strong phosphorylation activity against steroid compounds.

Specifically, we will screen for strains that have the ability to convert to phosphoric acid esters using tocholic acid, which is highly fat-soluble among steroid compounds and bile acids, or its amino acid foam combination as a substrate, from among the strains stored at the Yakult Honsha Central Research Institute. However, they discovered this activity in the filamentous fungus and completed the present invention.

That is, the structure of the present invention is to bring a filamentous fungus belonging to the genus Mortierella into contact with a steroid compound or an alkali metal salt thereof as a starting material to phosphorylate the α or β type hydroxyl group present in the steroid ring. This method produces phosphate esters of easily water-soluble steroid compounds.

More specifically, strains of filamentous fungi belonging to the genus Mortierella that have the ability to phosphorylate steroid compounds, typically Mortierella ramannia
By contacting the bacterial cells of strain Y2-1 (hereinafter simply referred to as strain Y2-1) with a steroid compound or its alkali metal salt, the steroid can be transformed using a microbial conversion method. A method for producing a phosphoric ester of a compound or an alkali metal salt thereof,
Specifically, the following methods are included.

(1) Cultivating the above-mentioned filamentous fungus in a culture medium containing a steroid compound or its alkali metal salt to accumulate the phosphate ester of the steroid compound or its alkali metal salt in the medium and collect it. Method.

(2) By bringing the cells obtained by culturing the filamentous fungus in a culture medium in which the filamentous fungus grows well into contact with a steroid compound or its alkali metal salt in a reaction solution,
A method for producing and recovering a phosphate ester of the steroid compound or an alkali metal salt thereof.

Next, each of the above methods will be explained in more detail. In the method (1) above, the steroid compound or its alkali metal salt may be added to the culture medium all at once at an appropriate time during the culture process, or may be added stepwise or continuously. The process may also be carried out batchwise, fed-batch or continuously.

The concentration of steroid in culture is not particularly limited, but is preferably 0.1 to 100 g/J! is within the range of Before inoculating the microorganism, it may be grown in a suitable medium and then inoculated into the conversion medium. Additional nutrients or other sources of carbon, energy may be added as desired. Preferred carbon and energy sources are glucose, glycerol, blackstrap molasses, yeast extract, fatty acids, etc., and preferred nitrogen sources are ammonium salts, caustic liquor, soybean meal, cottonseed meal, fishmeal, beef extract, fish extract, etc. . Appropriate temperature is adopted for culturing,
The temperature is usually 10 to 45°C, preferably 25 to 30°C. The culture pH is usually 4 to 8, preferably 7 to 8.
It is 7.5. The preferred culture time is 48 hours or more, and optimally about 5 days.

The suitable time to harvest the target compound can be determined by monitoring one or more of the bacterial cell concentration, the carbon source concentration in the medium, the dissolved oxygen concentration, oxygen and carbon dioxide in the exhaust gas, or by various chromatographic analyzes of the culture filtrate. Determined by etc.

Next, in the method (2) above, all or part of the bacterial cells (spores) once separated from the culture medium may be administered into a reaction solution in which a steroid compound or an alkali metal salt thereof is suspended. Alternatively, all or part of the isolated bacterial cells may be immobilized using polyacrylamide, calcium alginate, etc., and then brought into contact with a steroid compound or its alkali metal salt. Furthermore, the reaction efficiency can be improved by adding an organic substance as an energy source, typically glucose, galactose, other carbohydrates, casein hydrolyzate, yeast extract, etc. to the reaction solution at a low concentration.

The converted product can be recovered in a variety of ways, including adsorption onto an ion exchange resin and subsequent elution, or extraction with a suitable water-miscible organic solvent followed by crystallization.

The growth form and physiological properties of the filamentous fungus of the genus Morteierella used in the present invention, typically the Y2-1 strain, were disclosed in Japanese Patent Application Laid-open No. 5
As described in No. 9-159795.

Examples of preferable compounds to be phosphoric acid esterified in the method of the present invention include the following, but are not limited to the compounds described herein, and include aromatic compounds, polycyclic hydrocarbon compounds, and the like. It is also possible to prepare each phosphoric acid ester by reacting with

cholesterol cholestane-3β-ol 17α-methyl-testosterone 5α-antrostane-3β, 17β-diol Δ5-
Androsten-3β,17β-diol Testosterone Androsterone Tehydroisoandrosterone to 4-Androsten-11β-ol-3,17-Siondeoxycorticosterone 17α-Hydroxy-pregnenolone Prednisolone Aldosterone Corticosterone Δ5-pI/Gne- 3β-ol-20-one 17α
-Hydroxy-progesterone hydrocortisone cortisone 17α-ethinylestradiol estrone estriol β-estradiolcholic acid chenodeoxycholic acid ursodeoxycholic acid deoxycholic acid lithocholic acid The present invention will now be described in more detail with reference to Examples. Not limited to these.

<Example 1> 50 g of glucose 1 5 polypeptone for 1 e of tap water
g, yeast extract 2 g 1KHzPO* 1g, K
JPO4,2g, Mg5Oa-71200,5
g, CaClzlomg, FeSO4□ 7Hz0
A liquid medium of 61 consisting of a mixture of 10 mg of thiamine hydrochloride and 1 g of taurolithocholic acid as a conversion substrate was introduced into a 1011 capacity fermenter, and the medium was incubated in a Sakaro flask at 27°C for 48 hours using a medium with the same composition. 5% of Y2-1 strain, which had been shake-cultured in a fermenter, was inoculated into a liquid medium in a fermenter, and the culture temperature was 27°C, stirring speed was 300 rpm, and aeration rate was 0.5 while adjusting the temperature to 17 to 7.5 p). The cells were cultured aerobically in VVM for 5 days. After culturing, the culture solution is cooled to 5° C. and centrifuged to obtain a clear supernatant liquid together with the bacterial cells and precipitated product. The supernatant is passed through a bed of a suitable polymeric nonionic adsorbent polystyrene-divinylbenzene copoly-r- (Amberlite XAD-2 is the preferred adsorbent) to adsorb the product. The bed is washed with deionized water to remove unwanted inorganics and then eluted with methanol or ethanol to recover the product.

This product is mixed with a product obtained by extracting the solid layer obtained by centrifugation with 70% hot water methanol or ethanol. This mixed extract was concentrated under reduced pressure to remove most of methanol or ethanol, and a concentrate was obtained.

This was adsorbed onto a column packed with 350 g of Sephadex LH20, and the fat-soluble fraction was eluted with a mixed solvent of chloroform:methanol (1:1) containing 0.01M NaCl, and then the water-soluble fraction was collected with methanol. . This was concentrated to dryness under reduced pressure to obtain 3.5 g of solid.

Next, the above solid matter was added to DBAE Sephadex A-2560g.
The phosphate ester fraction of taurolithocholic acid was collected by elution under a 0 to 1M salt concentration gradient using a column packed with .

This target fraction was adsorbed onto the XAD2 resin, washed with deionized water, desalted, and eluted with methanol to obtain 2.1 g of crystals of sodium salt of taurolithocholic acid-3-phosphate.

<Example 2> The concentration of taurolithocholic acid in the medium of Example 1 was reduced to 0.1 g.
/12 medium, pH 7, aeration rate 0.5vvms
The Y2-1 strain was cultured for 48 hours at a stirring speed of 300 rpm. The bacterial cells were collected with gauze, washed with pH 7 phosphate buffer, and then divided into 2 livers, 041 g, glucose 10
g1 yeast extract 065g and taurolithocholic acid 0.
2 g of distilled water and 11 g of distilled water (<pH 7.5) to a bacterial cell concentration of 20 g/12.
The denaturation reaction was carried out at ℃ for 60 hours. Thereafter, the same operations as in Example 1 were performed to obtain 0.1 g of sodium salt crystals of taurolithocholic acid-3-phosphate per 11 reaction liquids.

The crystals recovered in Example (11, (2)) of the present invention were chemically identified using the analysis method listed below.

(1) Secondary ionization mass spectrometry (SIMS) Since the molecular peak (M++) (+) is observed at 630, the molecular weight is 629.

(2) Infrared absorption spectrum The results measured by the KBr method are shown in FIG.

C-O stretching vibration of po-φ at 120Qcm-', 10
Absorption indicating P,0 stretching vibration is observed at 2102O'.

(3) Melting point Decomposed at 280°C.

(41' H-N M R Sample measurements were performed in heavy water.

■ 4.58ppm H-C-OH (m) 3rd place 4.
06ppm LC(t) Taurine side chain methylene 3.56
ppm HzC(t) Taurine side chain methylene 1.42p
pm H:+C(d) 21st place], 41ppm 83
C(S) 19th place 11-16pp H3C(S) 18
Position ■ 1H at position 3 is ' at position 3 of the substrate taurolithocholic acid.
The fact that the magnetic field is shifted down from H (4.10 ppm) indicates that the phosphate group is ester bonded to the 3α hydroxyl group of taurolitocholic acid.

(5) Release of phosphate ester by phosphatase One crystal obtained in Example 1 was dissolved in 50 ml of glycine buffer at pH 10.4, and 100 units of alkaline phosphatase (Bovine International
Mucosa-derived Sigma 1VkLP-5521,) was added and dephosphorylation was performed at 37°C for 7 hours. After the reaction was completed, the amount of phosphorus liberated in the reaction solution was quantified by colorimetric assay (Deniges Method Agricultural Chemistry Experiment Book Vol. 1, P103, Akebono, Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University), and 48.9 μg was detected. This is the theoretical content of 49.2
It almost matches μg. In addition, taurolithocholic acid liberated by alkaline phosphatase treatment was measured using high-performance liquid chromatography exclusively for bile acids (Pile Pack and Enzyme Back System manufactured by JASCO Corporation).
1.60 μmole of taurolithocholic acid was detected. This means that the theoretical content of taurolithocholic acid contained in the sodium salt of phosphoric ester of taurolithocholic acid is 1.59 μmol1. It almost matches e. Based on the fact that the molecular weight is 629 from the mass spectrometry analysis, the presence of phosphate groups in the infrared absorption spectrum, and the fact that the substrate is taurolithocholic acid, it was determined that phosphoric acid esterification had occurred, and furthermore, 'H- It was confirmed from the NMR spectrum that phosphoric acid was ester bonded to the hydroxyl group of the substrate.

Furthermore, since phosphoric acid was liberated by phosphatase treatment, this crystal was identified as the sodium salt of taurolithocholic acid-3-phosphate.

<Example 3> 50g of glucose and 5% of polypeptone for 11 parts of tap water
g, yeast extract 2g, KIhPOt 2g-K
zHPOa 3g 5Mg5O4-7Hz00.5g,
Fe5Oa '71hO10mg, CaC1z
10mg of thiamine hydrochloride, 10ml of thiamine hydrochloride, and 0.5g of each of the 26 types of steroid compounds shown in Table 1 as substrates for phosphoric acid esterification.
Dispense 100ml into 1ml Sakalo flask and heat at 121°C.
Y was sterilized for 10 minutes, cooled to room temperature, and cultured with shaking at 27°C for 2 days in the above medium without steroids.
A 5% culture solution of strain 2-1 was inoculated. Each Sakaro flask was cultured at 27°C with shaking at 120 strokes per minute for 3 days. After culturing, the culture solution was adjusted to pH 6 with 5N hydrochloric acid and 5N sodium hydroxide for 8 cycles, and 20ml of the solution was diluted with 40ml of
The extracts were each extracted with 1 ml of n-phenol and concentrated to dryness under reduced pressure. -Next, 10ml of glycine buffer at pH 10.4 was injected into half of each concentrate, and after ultrasonication to make a homogeneous solution, 12.5 units of alkaline phosphatase (Sigma P-5521) derived from small and medium intestine mucosa was added. was added, and the dephosphorylation reaction was carried out at 37°C for 7 hours.

Next, this reaction solution was extracted with 20ml of n-butanol,
It was concentrated to dryness under reduced pressure. A certain amount of the n-butanol-extracted sample of the culture solution prepared in this way and the alkaline phosphatase-treated sample was transferred to silica gel using a developing solvent system of n-butanol:acetic acid:water (10:5:3). (Merck Kieselgel G-60, F254
．． A thin layer chromatography analysis of 0.2511) was carried out, and the presence and amount of spots that disappeared upon phosphatase treatment was quantified using a TLC scanner (Shimadzu Dual Wave System, TLC Scanner -).

Table 1 shows each substrate and its phosphorylation efficiency by the y2-1 strain.

Table I Efficiency of phosphorylation of each steroid by Y2-1 strain Steroid Phosphorus ester (%) Cholesterol 30 Cholestane-3
β-ol 4017α-methyl-testosterone 505α-antrostane-3β,
6517β-diol Δ5-androstene-3β, 6017β-
Dioltestosterone 55androsterone 25tehythroisoandrosterone 15Δ4-androstene-11β-
10 All-3,17-cyonedeoxycorticosterone 517α-hydroxy-pregnenolone 30 Prednisolone
15 aldosterone
5 corticosterone 2
0△5-Pregnen-3β-ol-4520-one 17α-hydroxy-progesterone 10 Hydrocortisone 10 Cortisone
1517α-ethinylestradiol 5-estrone
75 Estriol 10β
-estradiol 80 cholic acid
45chenodeoxycholic acid
35 Ursodeoxycholic acid
40 Deoxycholic acid 45 Lithochol 50

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing the basic structural formula of the conversion reaction substrate according to the present invention, and FIG. 2 is an infrared absorption spectrum of the sodium salt of taurolithocholic acid-3-phosphate produced in Example 1. be.

Claims (4)

[Claims] (1) A step of culturing a filamentous fungus belonging to the genus Mortierella that has the ability to phosphoric acid esterify a steroid compound, and a step of bringing the filamentous fungus belonging to the genus Mortierella into contact with a steroid compound or an alkali metal salt thereof; A method for producing a phosphoric acid ester of a steroid compound or an alkali metal salt thereof by microbial conversion, the method comprising the step of recovering a phosphoric acid ester of a steroid compound or an alkali metal salt thereof converted from a steroid compound or a salt thereof. (2) The step of contacting a filamentous fungus belonging to the genus Mortierella that has the ability to phosphoric acid esterify a steroid compound with a steroid compound or an alkali metal salt thereof is carried out in a culture medium for culturing the filamentous fungus. A method for producing a phosphoric acid ester of a steroid compound or an alkali metal salt thereof by microbial conversion according to claim 1, wherein the step is a step of: (3) further comprising the steps of collecting a filamentous fungus belonging to the genus Mortierella that has the ability to phosphorylate a steroid compound from a culture medium, and preparing a reaction solution in which the filamentous fungus is suspended; The method of converting a phosphoric ester of a steroid compound or an alkali metal salt thereof by microbial conversion according to claim 1, wherein the step of bringing the bacteria into contact with the steroid compound or an alkali metal salt thereof is a step carried out in the reaction solution. Production method. (4) The filamentous fungus belonging to the genus Mortierella is Mortierella lamanyana lamanyana (￥Mortiere
lla￥￥ramanniana￥￥var￥. ¥ra
A method for producing a phosphoric acid ester of a steroid compound or an alkali metal salt thereof by microbial conversion according to claims 1 to 3, wherein the phosphoric acid ester of a steroid compound or an alkali metal salt thereof is obtained by converting the phosphoric acid ester of a steroid compound or an alkali metal salt thereof.