JPS58179498A - Preparation of 11alpha-hydroxylated androstane compound by microorganism - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a synthetic raw material for adrenal cortical hormone, in high efficiency, by converting a sterol compound through an androstane compound to 11alpha-hdroxylated androstane compound using microorganisms. CONSTITUTION:A microbial strain belonging to Pseudonocardia genus and capable of converting a sterol compound (e.g. cholesterol) to an androstane compound and accumulating the product in the medium (e.g. Pseudonocardia methanolignica DIC-94125) is cultured in a nutrient medium containing a sterol compound, to accumulate the androstane compound in the medium. A microbial strain capable of hydroxylating the 11alpha-position of the androstane compound (e.g. Aspergillus ocharceus IFO-4345) is cultured successively in the above medium, and the 11alpha-hydroxylated androstane compound accumulated in the medium is separated therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION '4=The invention relates to a method for producing 11α-hydroxylated anthrostane compounds using microorganisms. Historically, Shade Nocardia (Ps@t+de+5ocardia) K11l was used as a styrene tJI material.
Anthrostane-based compounds are accumulated using a microorganism that is
The present invention relates to an extremely efficient production method for continuously producing 1α-hydroxylated anthrostane compounds in one tank.

Conventionally, Bacillus has been used as a microorganism that has the ability to convert various sterols to 11α-OH.
s) Genus, Pseudomonas (Ps@udotmomam
) Ilg, Streptomyces (8tr@pt@s
More than 300 types of microorganisms are known from 113 genera, including the genus Mycsi, the genus Mucor, and the genus Rhizopus. (Steroids and microorganisms, A
An example of an 11α-OH conversion reaction carried out by microorganisms on an industrial scale is the production of stigmasterol developed by US Company 771937. Corticoids (iill
During the manufacturing process (Iken Cortical Hormone), rhizogenes
Nigelika/su (Rh1xopusn+grieans)
, or Aspergillus ochraceus (Asp@r
One example is 11α-OH conversion of progeste a7 by S. gilrug oebraesum). In contrast, anthrostane compounds! 4-androstene-6,1
7-dione (hereinafter abbreviated as 4AD), 1 of 1,4-androstadiene-3°17-dione (hereinafter abbreviated as muDD)
Although 1α-hydroxy derivatives are very similar to the 11α-hydroxy derivatives of glogeste a7 fathom pregnane compounds as intermediates in the synthesis of corticoids, 11α-
As for the production of hydroxylated derivatives, a method for producing 11α-0H-ADDlF from Yui-ADD is a method using Aspergillus ochraceus (% open 111156-5).
No. 8497) is known. Moreover, this method is not based on a continuous fermentation method using two types of specific - as in the present invention, but is based on a one-stage fermentation method.

However, as a result of intensive research by the present inventors, microorganisms belonging to the genus Pseudonocardia that have the ability to convert and accumulate sterols into andromethane compounds such as ADD and 4-muD, and if kept, 4-mu DD, Efficient K11 (1-OH-ADD,
11 Discovered scales for producing 11α-hydroxylated anthrostane compounds such as Q-OH-4AD,
The present invention has now been completed.

The present invention relates to microorganisms belonging to the genus Pseudonocardia, such as Shootocardia, which have the ability to convert sterols into anthrostane compounds such as ADD and 4D, and the ability to accumulate these compounds in a culture medium.・Sterols are produced by culturing Methanoligna DIC-94125, DIC-94127, etc. to convert and accumulate sterols into anthrostane compounds, and further culturing organisms with 11α-hydroxylation ability. 11α-hydroxylated anthrostane compounds made from

It is to be controlled.

Since the present invention is a method of continuous fermentation in Japanese cypress, it is possible to directly produce 11α-hydroxylated anthrostane compounds without the need for separation of intermediate products, and the separation step involves separation of the compound σ). It only takes 1 Seki. On the other hand, the method of separating the anthrostane compound, which is an intermediate product, after production has the advantage of increasing the concentration of the intermediate product and removing unnecessary impurities; It is discontinuous and inefficient as it requires a lot of energy. However, the present invention is a continuous and efficient manufacturing method due to the advantages mentioned above.

The present invention will be explained in detail below.

Microorganisms belonging to the genus Cardia that produce the anthrostane compounds used in the present invention are more suitable for producing anthrostane compounds than sterols, such as 4AD,
Microorganisms that have the ability to convert and accumulate MuDD singly and in large quantities in the culture medium can be used as natural mutants, artificial mutants,
Although any naturally isolated strain may be used, a mutant strain of Pseudonocardia methanolignica is preferable. This mutant strain has been deposited with the National Institute of Microbial Technology under the following number. That is, Pseudonocardia methanolignica (Pm@mdenocardia m@t) is a microorganism that has the ability to convert and accumulate sterols into 4AD.
hamoligml@a)DIC-94127(VI
Similarly, Pseudonocardia methanolignica DIC-94125 (ml) is a microorganism that has the ability to convert and accumulate ADD.
One of the representative strains is Engineering #1 Suizaki No. 6250), each of which has been deposited at the Microbial Technology Research Institute. The parent strain of 4 strains, 7 Eudonochaldeia methanolignica, was isolated from the natural world by the present inventors, and is a microorganism 1.
′! It has been deposited with the E-Technology Research Institute as microtechnical research donation No. 4747, and its ceramic properties book was published in the Special Publication No. 56-5335.
The content is the same as that described in Publication No. 2, so it will be omitted.

As a growth medium, any synthetic medium or natural medium can be used as long as it contains a mineral source, a nitrogen source, an inorganic salt, and vitamins.

The carbon source may be anything that can be assimilated (for example, glucose, sucrose, rhamnose, starch, malt S% molasses, methanol, ethanol,
Alcohols such as glycerin, hydrocarbons such as alkanes, α-olefins, and xylene, as well as organic mI such as acetic acid, citric acid, and lumyl! can be given.

As the i1 source, inorganic or organic substances such as ammonium sulfate, ammonium chloride, ammonium phosphorus, sodium nitrate, urea, peptone, and casein can be used.

As the inorganic salts, potassium, magnesium, zinc, iron, manga, steel, calcium, and other salts can be used.

In addition, yeast extract, meat extract, flavor liquid, malt extract, corn steep liquor, fish meal, vitamins, nucleic acids, amino acids, etc. may be added as necessary to increase the nutritional value of mold.

If an antifoaming agent is required, any commonly used antifoaming agent can be used.

Surfactants are particularly effective as sterol emulsifiers and are appropriately added to the culture medium. Typical surfactants include ionic emulsifiers, ie, fatty acid esters of ethylene oxide adduct polyglycol.

The sterols used in the present invention include cholesterol, tesmosterol, campesterol,
Halic [1] These include nastyrol, brassicasterol, ergosterol, β-sitosterol, stigmasterol, corbistyrol, clionasterol, polyferastyrol, fucosterol, etc. Among them, cholesterol, campesterol, and β-sitosterol are It is.

Ester derivatives of the above-mentioned sterols with organic or inorganic acids can also be used. These sterols can be purified, crude Mk, or a mixture, and it is also possible to use alkali-washed dark oils from sterol-containing natural products, such as wool fat, fish oil, and squid oil.

Furthermore, oxidized intermediates of various sterols or styrene ester derivatives are also used, such as 4LN, cholest-4-en-3-one, cholest-1,4-dien-3-one, and cholest-1,4-dien-3-one. -4,2
2-dien-3-one, 22.23-bisnorchola-5
Examples include -cholenic acid-3β-ol, 22.23-bisnorchola-1,4-dien-6-one-22-oic acid, pregnenochloride/progesterone, and the like.

When adding these sterols to the medium, they may be added to the growth medium in bulk, the whole amount may be added to the growth medium from the beginning, or the whole amount or divided portions may be added during the growth of the plant.

The degree of addition is usually 115 to 50 times the amount of medium.
g/l), preferably from 1 to 1011/l.

The sterols may be added as a powder after dry heat sterilization or autoclaving, but it is necessary to increase the contact between the sterols and the sterols, so it is more preferable. It is added after being made into a solution or suspension in acetone, methanol, ethanol, inglobanol, benzyl alcohol, ether, dimethylformamide, etc. in advance.

Cultivation is preferably carried out under aerobic conditions, and is generally carried out under aerated agitation and turbidity conditions. 9. The culture and reaction temperature is usually in the range of 25°C to 50°C, preferably around 35°C to 45°C. The pH of the culture medium can usually range from 4.5 to 90, but preferably from &b to Z5.

The anthrostane compounds obtained in this way are preferably 2 or 1,4-androstadiene-3,17-dione (referred to as ADD), 4-androstene-3,17-dione,
-dione (referred to as 4AD).

Next, the anthrostane compounds produced as described above, such as ADD and 4muD, are inoculated with microorganisms capable of converting them into 11α-OH and cultured to carry out a conversion reaction from 4muD and 4muD to 11α. -OH, 11α-0H-
DD is manufactured.

The microorganism used in the conversion reaction from an anthrostane compound to a 11α-0H-antrostane compound may be any microorganism that has 11α-OHI for the anthrostane compound, but is preferred. is selected from microorganisms belonging to the genera Aspergillus, Emericella, Neosartoria and Rhizogenes. For example 4AD
Representative examples of 11α-oH bacteria include Asbergus ochraceus IFO-4545, IFO-8559,
Aspergillus clavatas IFO-5857, Aspergillus nikah IFO-4407, Aspergillus
Rustus IFO-4113, Aspergillus venti IFO-4107, IFO-6578, IFO-887
9, Emerisella Nidorans IFO-8001, Neosartoria ficieri var ficieri IFO-5
866, IFO-8790% Rhizopus stolonifer
IFO-4781 is mentioned.

In addition, representative examples of 11α-OH conversion of mu DD include Aspergillus ochraceus IFO-4545, IFO-
8559゜Aspergillus venti IFO-410
7% IFO-6578, IFO-7126, IFO-8
879, Neosartoria ficieri perl ficieri IFO-8790.

The inoculation of these microorganisms capable of converting into 11α-OH is usually carried out after the amount of 4AD and ADD produced by the organisms used to produce anthrostane-based compounds, such as 4AD%ADD, reaches its maximum value, but in some cases, 4AD and ADD production may occur. It is also possible to inoculate during the melting process, and the inoculation method is usually to add a culture solution of a microorganism having 11α-OH conversion ability that has been separately cultured to a culture solution of a microorganism for 4μD1μDD raw brazing. Do it by doing this.

As for the culture medium composition of the microorganism capable of converting into 11α-OH, the above-mentioned growth medium for producing anthrostane compounds can be applied as is. It is desirable that the culture and reaction be carried out under aerobic conditions, and it is generally advantageous to carry out the culturing and reaction under conditions of aeration, stirring, and shaking. These culturing and reaction temperatures can normally range from 20°C to 45°C, but are preferably around 23°C to 35°C. The pH of the medium is usually 25~
It is possible within the range of 90, but preferably within the range of 4.5 to 15. Therefore, anthrostane compounds, that is, A
When starting the 11α-OH reaction of DD and 4AD, the temperature and p
Although it is desirable to change the reaction conditions for H, etc. to conditions suitable for the 11α-OH conversion reaction as described above, there is no problem even if the reaction is not carried out. Replacement biological culture tank for producing anthrostane compounds using a culture solution of a microorganism capable of converting into 11α-OH (the addition ratio can be in the range of 01% to 70%, but preferably in the range of 3% to 25%) be.

9. Depending on the size of the inoculum of the organism, a source of nutrients for microorganisms capable of converting to 11α-OH, a nitrogen source, and other nutrient sources may be added as necessary. . After the completion of the 11α-OH reaction, the target 11α-0H-antrostane compound is separated and collected from the culture solution using a conventional separation method. In other words, the culture solution after the reaction is mixed with ethyl acetate and chloroform. After extraction with a low polar organic solvent such as hequinane or cyclohexane, and concentrating the extract, it was placed in a column packed with an adsorbent such as silica gel or alumina, and then extracted with ether, benzene, chloroform, ethyl acetate, or methanol. , elute and fractionate using a solvent such as ethanol. After concentrating the eluate tank of 11α-0H-antrostane system-1 thus extracted and distilling off the solvent, ethyl acetate,
Benzene, chloroform, acetone, ether group
Obtained by crystallization from l solvent.

The present invention will be explained in more detail in the following Examples, but the present invention is not limited thereto unless it goes beyond the gist thereof.

In the following examples, sterols, anthrostane compounds (4AD, ADD), and 11α-0H
- Qualitative and quantitative analysis of anthrostane compounds using thin layer chromatography, gas chromatography, and high performance liquid chromatography 1
It was confirmed by spin-off absorption spectra, magnetic resonance spectra and mass spectrometry.

Example 1 20g glucose, 10g coke steep liquor! i, Pegton 511. Phosphorus #21 Sodium 517. Camphor medium (pH 7,
0) Dispense 100 d into a 500 Bento Erlenmeyer-72 Scope, autoclave at 121°C for 15 minutes, and add 1 liter of Pseudonocardia Methanolig 2-strength IC-94127.
The seeds were inoculated using platinum loops and shaken at 40°C for 48 hours under ventilation to perform seed culture. This seed culture was inoculated into a 500 d Erlenmeyer flask containing 100 d of main culture medium having the same composition as the seed medium. The main culture was carried out in a rotary shaker at 40° C. and 20 Orpm, and 48 hours after the start of the culture, 5 ooq of cholesterol suspended in 517 Kml of methanol was added. After adding cholesterol,
A portion of the 128-hour nutrient solution was collected and the amount of anthrostane compounds produced was quantified using gas chromatography. As a result, 172 to 4AD was confirmed per flask. In addition, no other anthrostane compounds were produced. I haven't done it,
Cholest-4-ene, a cholesterol decomposition intermediate
Only 3-on produced 19 Da.

Next, glucose 20L cornstew liquor 5g, peptone 5g, 971!1 sodium 1g, phosphorus i12 potassium 3/, @@-r gune'/9A1y, ass1 iron 20
! Separate culture (48 hours of seed culture, 24 hours of main culture) was carried out in a 500 d capacity Erlen T-ear flask containing 100% of a medium (PH5, 6) consisting of 100% of Neosutruria fiscieri and 100% of water. IFO-586
4A at the 128th hour mentioned above.
D production culture 11 was inoculated. At this time, the pH was adjusted to 45. After inoculation, in a rotary shaker at 30℃, 220r
The reaction was carried out under the conditions of PI"l, the reaction was stopped at 96 hours, and all the culture solutions were combined and added with ethyl acetate jQ()ill.
[Extracted twice.

These extracts were combined to remove insoluble matter such as 4AD.
There was no residue of 11α-0H-4AD, and 76 μm of 11α-0H-4AD was produced.

Portrait: The 61-year-old benthos was hardly mentioned.

After concentrating this ethyl acetate extract, it was injected into a silica gel column, eluted with a benzene-ether system, and recrystallized from an acetone-chloroform system.
1α-0H-4AD5t5Jl was obtained. The obtained crystal was confirmed to be 11α-0H-4AD by melting point determination, quality 1 analysis, infrared absorption spectrum, and nuclear magnetic resonance spectrum.

Diarrhea case 2 In Example 1, Neosartoria ficieri var ficieri IFO-879 was used instead of Neosartoria ficieri var ficieri IFO-5866.
The same procedure as in Example 1 was carried out except that 0 was used. As a result of quantitative analysis using gas chromatography, 11α-〇H
-4AD was generating 40 Da. 6β- as a by-product
0R-4muD, 6β, and 11α-0H-4muD were produced at 7 and 34 days, respectively.

Example 3 Pseudonocardia methanolig'nica DIC-94
4mu D live by 127! ! was carried out in exactly the same manner as in Example 1. On the other hand, glycose 20I, ammonium nitrate 5
11 Phosphorus 11!2 Potassium 2J9. Separate culture (seed culture for 72 hours,
Aspergillus venti (musp@rgll1may@nti l) I which was subjected to main culture (48 hours)
1 lllL of culture solution 2011j of F 0-6578 was added to the above-mentioned 4muD production culture solution at the 144th hour. Portrait, 1
The amount of 4-muD produced after 44 hours was determined to be 181 da by gas chromatography.

The reaction was carried out in a rotary shaker at 30° C. and 22 rpm, and the reaction was stopped at 96 hours, and all the culture filtrate was combined and extracted twice with 100 d of ethyl acetate.

After combining these extracts and passing them through an e-filter, they were quantitatively determined using a Gask "Komatogelahui".
It was generating 1v. Also, almost no product was included.

Example 4 Mei J11fff 3 Aspergillus penthe 41FD
-65780ρ) Aspergillus venti IF
O-4107゜Same [FO-8879, Aspergillus
Ochraceus IFO-4345, -IFO-8559,
°γ Aspergillus sclavatus IFO-5867, Aspergillus niger IFO-4407, Aspergillus custus IFO-4113, Emericella nidra/s IFO-8001, and Rhizogeus stronipha-I
The same procedure as in Example 3 was carried out except that FO-4781 was used. The produced 11α-0H-4muD and by-products were determined by gas chromatography and are shown in Table 1.

Example 5 In implementation NI, β-
Sitosterol and campesterol 2: 111+
Compound, -X? Gumsteel, Cholest-4-en-3
The experiment was carried out in the same manner as in Example 1, except that -on was used. Table 2 shows the results of quantifying the produced 1'1α-0H-4AD by gas chromatography.

Table 2 Example 6 Seed medium (pH 7.0) 100 id consisting of 20 g of glycose, 10 p of corn methane liquor, 5 g of peptone, 3 g of monosodium phosphate, 7 y of potassium phosphorus #2, and 11 y of water
was dispensed into a 500 d Erle/Meyer flask, and 12
After autoclaving at 1°C for 15 minutes, one platinum loop of C. Pseudonocardia methanolignica DIC-94125 was inoculated, and the mixture was shaken under ventilation at 40°C for 48 hours to perform seed culture. A main culture medium consisting of the same composition as the 2 dl Ume medium for this seed culture, and a 500-volume Erle/Meyer flask containing 100-mL W! I planted a seed.

Main culture was carried out in a rotary shaker at 40℃, 2Q Orp.
48 hours after the start of culture, 500~ of S-filtered cholesterol in 5-methanol was added. 1f17 of the culture solution 144 hours after addition of cholesterol.
5 was collected and the produced anthrostane compound ant seed was placed in gas chromatography, and as a result, AD per flask.
D132Iv and 4AD15Iv were generated. Other anthrostane compounds were not produced at all, and cholest-4-en-3-one and cholesta-1,4-dien-5-one were each 91
I lived 1v, 25 da.

Next, glucose 20,9. Corn Methi Friker 5J,
Medium (pH 5,6) 10αm consisting of 5g peptone, 11 sodium phosphorus IJ, 5I potassium dipotassium phosphate, 1g magnesilla sulfate A, 20jv iron #11 sulfate, 11 water
Neosartoria fiscierival fiscieri IFO- which was separately cultured (48 hours of m culture, 24 hours of main culture) in a 500-volume Erle/Meyer flask containing tl-
When the culture solution 1511j of 8790 was added to the culture solution KIj for ADD children at the 144th hour mentioned above, the pH was adjusted to <
Adjusted to L5. After inoculation, use a rotary shaker for 3
The reaction was carried out at 0°C and 220 rpm, the reaction was stopped at 48 hours, and all the culture solutions were combined and diluted with 100% ethyl acetate.
A second sample was extracted.

After filtering the extraction liquid to remove impurities such as 1-unit, quantitative analysis was performed using gas chromatography. As a result, no residual ADD of 4tf<M was found. In addition, 6 units of 11α-0H-4AD were also produced, which is a byproduct of ADD fermentation, 4AD.
It is assumed that it was converted from

Next, after condensing this ethyl acetate extract, it was injected into a silica gel column, depolymerized in a chloroform-methanol system, and recrystallized from ether (as a result of repeated
Crystals of ~ were obtained. The obtained crystal was confirmed to be 11α-0H-ADD based on the melting point, mass spectrometry, infrared absorption spectrum, and magnetic resonance spectrum.

Example 7 Pseudonocardia methanoligni force DIC-941
The production of ADD in 25 was carried out in the same manner as in the experiment flJ6.

On the other hand, sucrose 20J, corn steep liquor sy, *mother extract 211 peptone 31i, magnesium sulfate [15
A medium consisting of jr, #11 iron sulfate, 50 JlF, and water 11 (
Aspergillus ochraceus IFO-, which was separately cultured (48 hours of seed culture, 24 hours of single culture) in a 500 d Erlen 1-year flask containing 100 d of pH 5, 6)
8559 culture solution 20- was inoculated into the above-mentioned muDD production culture at 168 hours.

As a result of quantitative determination by gas chromatography, the DD yield at 168 hours was 158 da. or,
I was also generating 42 da.

Adjust the pH to 41:45Kll, and react with l'-79-C:X-4-J:I- under the conditions of 30°C and 220 rpn+, stop the reaction at 72 hours, and drain all the culture solution. A second extraction was performed with 100 m of ethyl acetate.

After filtering the extracts to remove insoluble substances such as 1-ADD, quantitative analysis was performed using gas chromatography. As a result, no unreacted ADD was found, and 86 Da of 11α-01 (-ADD) was produced. In addition, 11α-0H-4AD was found as a conversion product corresponding to 4muD, and 11α-0H-4AD was obtained from 9 sardines. , after 7
The remaining amount of ADD at 2nd hour is 1ls9Iv%11α-0R
-ADD generation-1 is 39Q and b-.

implementation? 118 Pseudonocardia methanolignica DIC-941
The production of ADD using 25V was carried out in exactly the same manner as in Example 6.

On the other hand, f le:2-s21. Ammonium nitrate5. ! i
', 'Dipotassium Jionate 2Ji1. Magnesium sulfate 0
Aspergillus spp. was cultured separately (seed culture for 72 hours, main culture for 48 hours) in a 100 d. Venti I
1120 wj of FO-6578 culture was withdrawn into the above-mentioned 168 hour muDD production culture. The amount of ADD produced at the 168th hour was determined to be 122 Iv by gas chromatography. Furthermore, 58 ■ of 4AD were also generated.

After inoculation, the pH was adjusted to 65 and the reaction was carried out in a rotary shaker at 50°C and 220 rpm.
The reaction was stopped at 96 hours, and all the culture solutions were combined and extracted twice with ethyl acetate 100W11. This extract was combined with insoluble materials such as bacterial cells to be quantified using PAD and gas chromatography.
Ig, and 54 das of 11α-0H-muDD were produced. In addition, as a conversion product corresponding to 4muD, 11α-0
H-4 MuD was also dazzled by 19 seconds.

4-10'i', rFO- In the same manner, 11α-OH conversion reaction was carried out using Aspergillus venti IF'σM6 and IF'0-8879. 11α-0H- at 96 hours after inoculation
The amount of ADD produced by gas chromatography is shown in Table 3. The same procedure as in Example 6 was carried out except that tigmasterol, cholest)-4-en-6-one, and cholesta-1,4-dien-6-one were used. The results of quantifying the generated 11α-0H-mu by DDtXl chromatography!
! 4.

Table 4 Continuation of page 1 0 shots: Tadayu Matsubayashi 4-3-18 Konakadai, Chiba City 0 shots Kazuyuki Miyayo 2-28-14 Gakuen Nishimachi, Kodaira City

Claims (1)

[Claims] t Conversion to a sterol a anthrostane compound,
By culturing a microorganism belonging to Pseudonocardia that has the ability to accumulate this compound in the medium, sterols are converted and accumulated into anthrostane compounds, and the anthrostane compounds are 11α-hydroxylated and added to the medium. 11α-hydroxylated anthrostane compounds made from sterols, characterized in that microorganisms having the ability to accumulate 11α-hydroxylated anthrostane compounds are subsequently cultured to accumulate 11α-hydroxylated anthrostane compounds in a medium. A method for producing a rostane compound. 2 Anthrostane-based compounds include 1,4-androstadiene-3,17-dione, 4-androstene-! 1
．． 17-Zion A special tfWf4-required range 1] J-Kyo method. 5 Microorganisms associated with Pseudonocardia h., Pseudonocardial odor DIC-94125,
-1 The method as described in Items 1 and 2 of the scope of special research, characterized in that it is DIC-94127. u %il characterized in that the microorganism having α-hydroxy flower salmon is a microorganism selected from the genus Aspergillus, the genus Emericella, the genus Neosartoria, and the genus Rhizopus.
'f range of determination The method described in the first, second, and third terms.