KR100592465B1 - Method for preparing 4-androsten-3,17-dione and androstar-1,4-diene-3,17-dione using two-step fermentation process - Google Patents

Abstract

The present invention relates to 4-androsten-3,17-dione (Androst-4-ene-3,17-dione (AD)) and androsta-1,4-diene-3,17-dione (Androsta-1,4 -diene-3,17-dione (ADD)) is a method for preparing emulsified sterols or cyclodextrin-bound sterols, which are added to the medium, and are primarily used as microorganisms that are beneficial for the oxidation and side chain degradation of sterols. Culturing; And secondary culture of the oxidized and branched sterols into microorganisms advantageous for further branched decomposition. There is provided a method for producing AD / ADD.

The oxidation and branched chain decomposition process of the sterols used in the method of the present invention is not only performed in a short time, but also effective in producing high yield when AD / ADD is produced using the oxidized and branched sterols.

4-Androsten-3,17-dione, Androstar-1,4-diene-3,17-dione, sterol, cyclodextrin, emulsifier

Description

A method for Preparation of Androst-4-ene-3,17- using 4-androth-3,17-dione and androstar-1,4-diene-3,17-dione using two-step fermentation process dione and Androsta-1,4-diene-3,17-dione using Two-step Fermentation Process}

The present invention provides novel 4-androsten-3,17-dione (Androst-4-ene-3,17-dione; hereafter abbreviated as 'AD'), and androstar-1,4-diene-3,17 A method for preparing -dione (Androsta-1,4-diene-3,17-dione; hereinafter abbreviated as 'ADD'), more specifically emulsified sterol or cyclodextrin-sterol complex Is used as a microbial culture medium component to induce oxidative and branched hydrolysis of sterols by using strains that are excellent in oxidizing and branching of sterols first, and to prepare AD / ADD from secondary oxidized and branched sterols. A two stage fermentation process.

Steroid hormones are secreted by the human adrenal cortex, testes, ovaries, placenta, and corpus luteum, and are synthesized from cholesterol in the human body and androgens (androsterone, testosterone, etc.), which maintain the secondary sexual characteristics of men according to their physiological effects, Estrogens (estradiol, etc.), which are produced in the follicles, are secreted from the corpus luteum of the ovaries, and are used in the uterus to make pregnancy and maintain pregnancy (progesterone, etc.) and proteins. Glycocorticoids (such as cortisone and hydrocortisone), which are converted to glucose and hepatic glycogen, and inorganic corticoids (such as deoxycorticosterone and aldosterone), which play a very important role in balancing electrolytes and water in vivo. .

Due to the increase in stress and the effects of environmental hormones in the development of modern civilization, the balance of the hormone is broken in the human body, and many diseases are generated accordingly, and the aforementioned steroid hormone preparations are widely used as the therapeutic or adjuvant therapeutic agents of the disease. It is being used. In particular, the synthesized estrogens are essential for artificial insemination, procedure and treatment of infertile patients. Glucocorticoids consume protein to maintain glycogen storage in the body, which has an anesthetic effect that relieves the pain of several inflammations such as iris and arthritis. Adison disease, also known as fatal, can be treated by administering deoxycorticosterone and hydrocortisone.

As described above, various studies have been attempted for the synthesis of steroid hormones with increased demand, and a method of preparing and synthesizing AD / ADD, which is a precursor for steroid hormone synthesis, is common. The AD / ADD is prepared by metabolizing sterols by oxidative enzymes of microorganisms, wherein the substrate material may be cholesterol, cytosterol, camphorsterol, stigmasterol, ergosterol, and other sterol oxides. Because of this, there is a limit to increasing the substrate availability of microorganisms. Therefore, various methods for overcoming the above problems and increasing the yield of AD / ADD by increasing the microorganism availability of the sterols as a substrate have been studied.

Helllink (Paul GM Hesselink et al., Enzyme Microbiology and Technology 1989, 11, 398-404) et al. Added cyclodextrin as a sterol-like collector to the medium to solubilize insoluble sterols in an aqueous medium. Steroid side chain degradation was promoted by Mycobacterium sp. As a result, the conversion of sterols to AD / ADD was more than doubled.

Cheong-Yi Lee et al., Applied microbiology and biotechnology, 1993, 38, 447-452, added 2% organic solvent (acetone) to increase the dispersibility of the sterols in the medium, In order to increase the conversion yield to AD / ADD, a two-step conversion process using two kinds of microorganisms was used. As a result, the yield of AD / ADD was increased than the case of using the organic solvent, but it is difficult to prepare a sterol solution having a concentration of 1% or more when 2% of acetone is added.

Lee, Kang-Min (Korean Journal of Biotechnology and Bioengineering, Vol. 7, No. 3, 161-165) used a method using microemulsion to increase the efficiency of sterols as substrates. Specifically, cytosterol, a type of sterol, was cross-linked with glutaldehyde, and then a microemulsion was prepared with a surfactant and added to the medium. As a result, it was confirmed that up to 120 mg of AD / ADD could be obtained per g of cells when 3 g of cytosterol was used.

Cedlaczek (L. Sedlaczek et al., Applied microbiology and biotechnology, 1999, 52, 563-571) and others have added glycine, a cell wall synthesis inhibitor, in the medium to reduce cross-links between cell wall peptides and peptidoglycans. We tried to increase the availability of sterols by breaking the (peptidoglycan) bond. As a result, the cell growth was reduced, but the production of AD / ADD increased by about 10%.

Ruth Goetschel et al., Enzyme Microbiology and Technology, 1992, Vol. 14, 390-395, et al. Cultured Rhodococcus erythropolis in lipozomal medium to enhance the oxidation of cholesterol.

In addition to the above studies, Linda Wang et al., The Journal of Biological Chemistry, 2000, Vol. 275, No. 10, 7224-7229, et al., Are mutations that do not biosynthesize mycolic acid, a major component of the mycobacterium cell wall. The experiments were carried out using sterols for hormonal intermediate conversion, and Amye (Aimee E. Belanger et al., Journal of bacteriology, 2000, 182, Issue 23, No. 6854-6856), reported that Mycobacterium smeg A mutant strain from which ddlA, a gene related to peptidoglycan biosynthesis of mycobacterium smegmatis, was removed was used.

The inventors have prepared a microbial Mycobacterium fortuitum EUG-119 which has a conversion rate of AD / ADD more than four times (2.1 g / L) higher than known microorganisms by using a chemical mutation process (Application No. 2001- 025702) Also, using a novel method of emulsifying sterols and a cyclodextrin-sterol complex extracted from milk as a microbial culture medium component, a development method of at least three times the conversion to AD / ADD has been developed and applied for. (Application No. 2001-025704).

However, while the foregoing methods have increased the conversion rate from sterols to AD / ADD, it is still estimated that the sterol availability of microorganisms is low. Therefore, there is still a need for a new method to further increase the yield of AD / ADD by increasing the sterol availability of microorganisms.

Therefore, the inventors have conducted extensive research. As a result, the addition of emulsified sterols or cyclodextrin-bound sterols into the medium, the primary cultivation of microorganisms beneficial for the oxidation and side chain degradation of sterols, It was confirmed that the secondary culture of the microorganisms producing AD / ADD in a high yield by the side chain decomposition action to increase the production yield and productivity significantly, the present invention was completed based on this.

An object of the present invention is to provide a novel AD / ADD production method that can increase the yield of AD / ADD by increasing the sterol availability of the microorganism.

AD / ADD production method of the present invention for achieving the above object by using the emulsified sterols and cyclodextrin-sterol complex as a substrate, the first to a microorganism excellent in the oxidation and side chain decomposition of sterols After oxidative and branched digestion, a two-step fermentation process is used to produce secondary AD / ADD with microorganisms that are highly capable of further branched degradation of the oxidized and branched sterols.

Hereinafter, the present invention will be described in more detail.

The novel AD / ADD production method of the present invention comprises the steps of adding the emulsified sterols or the sterols combined with the cyclodextrin to the medium, and primary culture with the microorganisms advantageous for the oxidation and side chain degradation of the sterols and the oxidation and side chains. Secondary culture with microorganisms advantageous for further sidechain degradation of the degraded sterols. At this time, in the first culture step and the second culture step of the microorganism, the first microorganism oxidation and side chain decomposition reaction and the second additional side chain decomposition reaction occur as in Scheme 1, respectively, to generate AD / ADD.

              First Oxidation / Side Cracking Secondary Side Branching

The emulsified sterols are used in an amount of 0.01 g to 10 g, preferably 0.1 g to 5 g, by weight of sterol per 100 ml of culture medium. When adding cyclodextrin-associated sterols to the medium, the cyclodextrin may be α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and the cyclodextrin-sterol complex is 0.07 per 100 ml of culture medium. g to 70 g, preferably 0.7 to 35 g.

As the primary and secondary microorganisms used in the present invention, all microorganisms using sterol as a carbon source can be used, and specifically, Asrobacter , Nocardia , Rhodococcus , Fusarium genus (Fusarium), Mycobacterium genus (Mycobacterium), micro tumefaciens in (Microbacterium), Pro tamino bakteo in (Protaminobacter), Brevibacterium genus (Brevibacterium), the genus Corynebacterium (Corynebacterium), Bacillus there are in (Bacillus), Serratia genus (Serratia), azo Sat bakteo in (Azptobacter), streptomycin MRS (Streptomyces), genus Alcaligenes (Alkaligenes), and the genus Pseudomonas (Pseudomonas) or the like. Representative strains belonging to the primary and secondary microorganisms include Arthrobacter simplex IAM 1660 , Norcardia erythropolis ATCC 4277 , Mycobacterium smegmatis IFO 3083 , Mycobacterium phlei IFO 3158 , and Mycobacterium fortuitum , Protaminobacter alboflavus ATCC 8458 , Brevibacterium liplyticum IAM 1398 , And Corynebacterium equi IAM 1038 , and the like, and mutant strains thereof may be used in the method of the present invention. Among the primary and secondary microorganisms, Asrobacter simplex, Brevibacterium lipoliticum, Rhodococcus erythropolis and Mycobacterium genus are preferred, and Asrobacter simplex and Brebibacte Leeum lipoliticum, Rhodococcus erythropolis, etc. are preferred, and as a secondary microorganism, Mycobacterium fortuitum EUG-119 produced by the present inventors by mutating Mycobacterium fortuitum (ATCC 29472) (Accession No .: KCCM-10259, Deposit Date: April 14, 2001) is more preferred because of its excellent ability to convert sterols and sterol oxides to AD / ADD.

The sterols used in the present invention are selected from the group consisting of cholesterol, cytosterol, camphorsterol, stigmasterol and ergosterol, with cholesterol, cytosterol, camphorsterol and stigmasterol being particularly preferred.

In the embodiment of the present invention was compared and analyzed the yield of the AD / ADD according to the method of the present invention and the conventional method, first, when using only the secondary microorganism 0.5 g / 100 ㎖ emulsified cholesterol of the present invention The yield of ADD was 130 mg / 100 ㎖, and cyclodextrin-cholesterol removed from milk was used to produce only 22.2 microorganisms of 92.2 mg / 100 ㎖ of AD / ADD per 0.5 grams of cholesterol added. In the above method, the primary microorganisms were cultured to induce oxidation and side chain degradation of sterols, and the secondary microorganisms were further cultured to induce further sterol side chain degradation, respectively, 259 mg / 100 ml and 189 mg. It was confirmed that / 100ml of AD / ADD is generated, which is about 2 to 3 times higher than the amount of AD / ADD prepared by a known method. Also, when 0.5 g / 100 ml of emulsified plant sterol is used as a substrate, primary microorganisms are cultured to induce oxidation and side chain decomposition of sterols, and secondary microorganisms are further cultured to induce further sterol side chain degradation. It was confirmed that 275 mg / 100 ml of AD / ADD was produced, which was able to produce AD / ADD in a nearly similar yield compared to using cholesterol as a substrate. In addition, in the case of using the sterols homogenized or emulsified by the conventional method, organic solvents and physical methods as a substrate, it was effective when the two-step fermentation process was applied.

 As a result, the method of the present invention can be used very effectively for the purpose of obtaining AD / ADD in high yield.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited thereto.

Example 1 (Culturing of Microorganisms)

The primary culture of the primary microorganisms for the oxidation and side chain decomposition of sterols is to make a culture medium with the composition shown in Table 1, and then adjust the pH to 7.0 by adding sterol to 1 g / L, and 5 ml of the prepared medium. Was poured into a test tube, autoclaved at 121 ° C. for 15 minutes, and then cooled to inoculate Asrobacter simplex with platinum. After inoculation, seed culture was prepared by incubating at 30 ° C. and 200 rpm for 2 days.

Primary microbial seed culture medium composition ingredient weight% Ammonium Nitrate 0.1% Potassium phosphate (monobasic) 0.025% Magnesium sulfate 0.025% Yeast extract 0.5% Twin 80 0.05% water Add to 100 ml

In the case of the main culture of the primary microorganism, the pH of the main fermentation medium prepared in the composition of Table 1 was adjusted to 7.0, 50 ml of the prepared fermentation medium was put into a 500 ml stirred flask, and then autoclaved. Cholesterol was added to adjust the final concentration in the medium to 5g / ℓ after sterilization based on the second main culture volume, the final volume for the primary main culture was 85ml. After completion of the primary microbial culture, it was sterilized and secondary culture was performed by adding 10 ml of the secondary main fermentation medium and 5 ml of the secondary microbial seed culture solution.

Secondary microbial species culture for the side chain decomposition of more sterols was prepared in the culture medium with the composition shown in Table 2, and then adjusted to pH 7.0, 5 ml of the prepared medium was poured into a test tube and 15 minutes at 121 ° C. After autoclaving and cooling, Mycobacterium fortuitum ATCC 29472 and Mycobacterium Fortuitum EUG-119 (Accession No .: KCCM-10259) were inoculated with platinum. After inoculation, seed culture was prepared by incubating at 30 ° C. and 200 rpm for 4 days.

Secondary Microbial Seed Culture Composition ingredient weight% Yeast extract 0.1% nutrient 0.8% Glycerol 0.5% Twin 80 0.05% water Add to 100 ml

In the case of the main culture of the secondary microorganism, first, the pH of the main fermentation medium prepared with the composition of Table 3 excluding glucose was adjusted to 7.0, and the main fermentation medium was concentrated 10 times in a 500 ml stirred flask, followed by autoclaving. Was carried out. Glucose was prepared separately and sterilized at a 10-fold concentration and added so that the final volume of the main fermentation medium prepared above was 10 ml. The final volume of the main fermentation broth containing the seed culture was 100 ml.

 5 ml of the seed culture solution prepared above was inoculated into the prepared main fermentation medium, followed by incubation at 30 ° C. and 200 rpm under a reciprocating stirrer for 5 days.

Secondary microorganism main culture medium composition ingredient Weight (W%) glucose One% Yeast extract 0.5% Potassium phosphate (dibasic) 0.04% Potassium phosphate (monobasic) 0.08% Magnesium sulfate 0.02% Iron sulfate (II) 0.0005% Ammonium acetate 0.15% Zinc sulfate 0.0002% Manganese chloride 0.00005% Twin 80 0.05% water Add 1 liter

Comparative Example 1

When the cyclodextrin-cholesterol complex extracted from milk was used, the following method was performed to confirm the change in ADD production.

5 kg of beta cyclodextrin was added to 500 L of milk, and then homogenized at 500 rpm and 4 ° C. for 10 min using a homo-mixer. The resulting cyclodextrin-cholesterol complex was isolated by centrifuging the homogenized milk solution at 6000 rpm for 5 minutes at 4 ° C. The separated cyclodextrin-cholesterol complex was diluted with water so that the cholesterol concentration was 50 g / l, and once again homogenization was performed using a homo-mixer. 10 ml of the cyclodextrin-cholesterol-complex solution finally prepared by the above procedure (50 g / L of cholesterol) and 5 ml of the seed culture solution were added to the secondary main fermentation medium and cultured by the method of Example 1. AD / ADD production by a single process using a second microorganism Mycobacterium fortuitum ATCC 29472 was attempted.

The amount of AD / ADD produced was extracted twice with 1 ml of culture medium and 4 ml of 1: 1 mixed solvent of ethyl ether and petroleum ether, and then evaporated under reduced pressure, dissolved in 2-propanol, and analyzed by high pressure liquid chromatography.

As a result, it was confirmed that 92.2 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of cholesterol.

Example 2

Culture medium was prepared in the same manner as in Comparative Example 1 and cultured in the same manner as in Example 1. The primary microbial Asrobacter simplex was used to induce oxidation and side chain degradation of primary sterols and the production of AD / ADD using a secondary microbial Mycobacterium fortuitum ATCC 29472 .

As a result, it was confirmed that 189 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of cholesterol.

Comparative Example 2

The cholesterol was emulsified and then used.

Cholesterol and emulsifier are heated to the melting point, respectively, and dissolved completely. Then, the mixture is mixed at a ratio of 0.85 (w / w; cholesterol: emulsifier) and put into 80 g of water so that the concentration of cholesterol is 50 g / l. Was prepared.

10 ml of the emulsified cholesterol solution prepared in the above procedure (cholesterol 50 g / l) and 5 ml of the seed culture solution were added to the secondary main fermentation medium and incubated by the method of Example 1, and in the same manner as in Comparative Example 1, AD / ADD The amount of production was measured.

As a result, it was confirmed that 130 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of added cholesterol.

Example 3

Culture medium was prepared in the same manner as in Comparative Example 2 and cultured in the same manner as in Example 1. The primary microorganism was used to induce oxidation and side chain degradation of primary sterols using Asrobacter simplex and the production of AD / ADD using Mycobacterium fortuitum ATCC 29472 as secondary microorganisms. .

As a result, it was confirmed that 259 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of cholesterol.

Example 4

In Example 2, the primary microorganism was used to induce oxidation and side chain degradation of primary sterol using Asrobacter simplex, and the secondary microorganism was Mycobacterium portuitumum EUG-119 (Accession Number: KCCM-10259). The production of AD / ADD was attempted.

As a result, it was confirmed that 265 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of cholesterol.

Example 5

In Example 3, the primary microorganism was used to induce oxidation and side chain degradation of primary sterol using Asrobacter simplex, and the secondary microorganism was Mycobacterium portuitumum EUG-119 (Accession Number: KCCM-10259). The production of AD / ADD was attempted.

As a result, it was confirmed that 338 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of cholesterol.

Example 6

Emulsified plant sterols were prepared in the same manner as in Comparative Example 2 using plant sterols (sitosterol, camphorsterol, siigmasterol, and other sterol composition ratios 44: 24: 21: 11).

10 ml (50 g / l of plantsterol) of the emulsified plant sterol solution prepared in the above process was added as a substrate in the medium, and cultured in the same manner as in Example 3, that is, in the same manner as in Example 1, and the same as in Comparative Example 1. The amount of AD / ADD production was measured by the method. The strain used as the first microorganism was Rhodococos erythropolis, and after seeding, seed culture was prepared by incubating at 28 ° C. and 200 rpm for 2 days. In the case of the main culture of the primary microorganism was carried out as in Example 1, in the present example using plant sterol as a substrate was added 0.5% glucose to the culture medium of the primary microorganism. Secondary microorganisms were attempted to produce AD / ADD using Mycobacterium Fortuitum EUG-119 (Accession No .: KCCM-10259).

As a result, it was confirmed that 275 mg / 100 ml of AD / ADD was generated per 0.5 g / 100 ml of added plant sterol.

In addition, when hormone biosynthesis was performed using Mycobacterium portuitumum EUG-119 in the above examples, the resultant AD / ADD has a specific ratio of ADD: AD of 9: 1 or more, that is, ADD is 90 Accounted for more than%. For reference, common strains have a ADD: AD ratio of 1: 1, but since ADD and AD are very similar structurally and chemically, they are difficult to purify, which increases the cost of purification and increases production cost. Considering that the unit price of ADD in circulation is 3 to 10 times higher than that of AD and is a more useful substance for producing physiologically high steroid hormones, this result is very useful industrially.

The results shown in the above Examples are shown in Table 4 below.

division Way Production amount (mg / ℓ) Conversion rate (%) Comparative Example 1 Cyclodextrin-Cholesterol Complex Single Process 922 25 Example 2 2-step process of Comparative Example 1 1,890 51 Comparative Example 2 Emulsified Cholesterol Monoprocess 1,300 35 Example 3 2-step process of Comparative Example 2 2,590 70 Example 4 Mycobacterium portuitum EUG-119 in Example 2 2,650 72 Example 5 Mycobacterium portuitum EUG-119 in Example 3 3,380 92 Example 6 Rhodococos erythropolis and plant sterols are used in Example 3. 2,750 89

As shown in Table 4, when the two-step fermentation process using the cyclodextrin-cholesterol complex and emulsified cholesterol was confirmed that the yield of AD / ADD is about 2 times higher than that of the other. In addition, when the two-step fermentation process using the emulsified plant sterol was confirmed that the yield is almost the same as the case using cholesterol.

The two-step fermentation process using the emulsified sterols and cyclodextrin-sterol complexes used in the method of the present invention is not only easy to implement, but as seen in the above examples, AD / ADD is used when using the method of the present invention. It is effective because it can be obtained in high yield.

Claims (7)

Selecting at least one of emulsified sterols or sterols bound to cyclodextrins and adding them to the medium, and primary culture with microorganisms which are beneficial for the oxidation and branched chain action of the sterols; And Secondary culturing with Mycobacterium fortuitum EUG-119 (Accession No .: KCCM-10259) as a microorganism favorable for further branched action of oxidized and branched sterols; AD (Androst-4-ene-3,17-dione) / ADD (Androsta-1,4-diene-3,17-dione) characterized in that it comprises a. The method of claim 1, wherein the sterols are selected from the group consisting of cholesterol, cytosterol, campestrol, stigmasterol, and ergosterol. The method according to claim 1, wherein the emulsified sterols are used in an amount of 0.01 g to 10 g by weight of sterol per 100 ml of culture medium. The method of claim 1, wherein when the sterols bound to the cyclodextrin are added to the medium, the cyclodextrin is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. The method according to claim 1 or 4, wherein the sterols bound to the cyclodextrin are used at 0.07 g to 70 g per 100 ml of culture medium. The method of claim 1, wherein the primary culture the microorganism Aspergillus bakteo in (Arthrobacter), no Arcadia in (Nocardia), Fusarium genus (Fusarium), also Cocos in (Rhodococcus) Mycobacterium genus (Micobacterium), the micro tumefaciens in (Microbacterium), Pro tamino bakteo in (Protaminobacter), Brevibacterium genus (Brevibacterium), the genus Corynebacterium (Corynebacterium), Bacillus (Bacillus), Serratia genus (Serratia), azo Sat bakteo in (Azptobacter ), characterized in that the Streptomyces is selected from the MRS (Streptomyces), Alcaligenes genus (Alkaligenes), Pseudomonas species (Pseudomonas), and the group consisting of the mutant strain. delete