KR820001188B1 - Process for producing steroidal alcohol - Google Patents

Abstract

Steroidal alcs. are produced by Mycobacterium species capable of producing 20α-hydroxymethylpregna-1, 4-dien-3-one (I) or 20α-hydroxymethylpregn-4-en- 3 -one (II) when provided with a sterol substrate. Thus, a starter culture was prepd. by inoculating a nutrient medium of glucose 1.0, meat ext. 1.0, and peptone 1.0% with M. parafortuitum and incubating at 30≰C for 72 h with shaking. The seed culture was then inoculated into a prodn. medium contg. soybean meal 4, K2HPO4 0.2, MgSO4.7H2O 0.1, fish oil residue 2, NaNO3 0.2, and cholesterol 0.8%, which was incubated at 30≰C for 160 h with shaking. Extn. of the resulting fermn. broth gave a cholesterol 0.05, I 2.65, and II 0.22g.

Description

How to prepare steroid alcohol

The present invention relates to a method for producing steroid alcohol, and more particularly to a method for producing steroid alcohol by a novel microorganism.

The microorganism belonging to the genus Mycobacterium (hereinafter abbreviated as "M") is 20α-hydroxymethylpregna-1,4-diene-3-silver (= 22-hydroxy-22,23-bisno Cola-1,4-dien-3-one) (hereinafter abbreviated as "HPD") and 20α-hydroxymethylpregan-4-en-3-one (= 22-hydroxy-22,23-ratio The production of snocola-4-en-3-one (hereinafter abbreviated as "4HP") itself is, for example, A) Applied Microbiology, Vol. 23, No. 1, pp. 72-77, 1972, USA B). US Pat. No. 3,684,657, and C) US Pat. No. 3,759,791, which is known.

However, the amount of HPD and 4HP (hereinafter, collectively referred to as HP) described in the three documents A to C described above is very small and has little industrial significance. That is, the amount of HPD produced was not about 20 µg / ml in Document A, 40 µg / ml in Document B, and 20 µg / ml in Document C, and only one of the examples in Document C was found for 4HP. Only 40 ㎍ / ml at maximum. Furthermore, in these documents the main product is endrostar-1,4-diene-3,17-dione (hereinafter abbreviated as "ADD") or endost-4-ene-3,17-dione (hereinafter, Abbreviated as "4AD", and HP's output is less than 1/10 of the main product.

However, HPD and 4HP are very useful intermediates that can be easily converted into steroids having the activities of corticosteroids (corticoids), progesterone, and protein anabolic hormones by conventional organic synthetic chemical reactions. The development of the manufacturing method was desired.

As a result of intensive studies in view of such circumstances, the inventors have found that any novel microorganism can be used to reach the present invention.

That is, the gist of the present invention is a method for producing a steroid alcohol, characterized by culturing a microorganism belonging to the genus M which can produce HPD or 4HP as a main product using sterols as a substrate.

Hereinafter, the present invention will be described in detail.

The microorganism used in the method of the present invention belongs to the genus M and is a microorganism capable of producing 4HP or HPD as a main product.

Of course, it goes without saying that such microorganisms may have HPD as the main product, 4HP as the main product, and HPD and 4HP as the main product.

Producing here with HPD or 4HP as the main product means that the molar percentage (%) of HPD + 4HP relative to the sterol material consumed exceeds 50%.

That is, the following expression is established.

Y = g ₁ ) G + g ₂ / G) × 100 ＞ 50

[Wherein Y is the yield (%), g ₁ is the production amount (mol) of HPD, g ₂ is the production amount (mole) of 4HP, and G is the consumption amount (mol) of sterols]

Microorganisms belonging to the genus M known in the art (hereinafter referred to as " wild wine ") do not produce HP as a main product even when cultured using sterols as a substrate as described above.

Therefore, the microorganism used in the present invention is clearly distinguished from wild wine in that HP is produced as a main product.

Such microorganisms include mutants that are depleted or attenuated by enzymes that degrade HPD and / or 4HP or enzymes that are activated later in metabolic pathways. In addition, such microorganisms may shift 4AD or ADD (e.g., 23,24-bisnocola-1,4-dinene from sterols by shifting the main metabolic pathway to HPD and / or 4HP to accumulate HPD and / or 4HP). Mutants lacking or weakening enzymes that are activated somewhere in the metabolic pathway to carboxylic acid).

As such a mutation strain, for example, M. parafortuitum complex MCL 0617 bacteria can be mentioned.

This strain was obtained by M. paraportumtum complex ATCC 25790 bacteria (= M. Neoaurum ATCC 25790 bacteria) as a parent strain and treatment with ultraviolet light.

This strain has been deposited as Accession No. 4258 (FERM-P 4258) by the Japanese Institute of Microbiological Technology.

Regarding the parent strain of the bacterium, the parent strain ATCC 25790 belongs to the M. paraportuitum complex as a conclusion of a joint study by the International Working Groupon Mycobacterial Taxonomy of 13 experts. See Saito Hajime, Tabamura Michio, International Journal of Cystematic Bacteriology, Vol. 27, pp. 75-85 (1977, USA).

The bacterium ATCC 25790 was included in the new M. aurum Tabamura prepared as Tangcho Tabamura 309, and it was prepared using Tabamura # 358 (ATCC 23366) as a reference strain. Tachimura Tabio, Medicine and Biology, Vol. 72, pp. 270 to 273, 1966].

Subsequently, Mr. Tabamura established M. neoaurum Tabamura as a different strain of M. aurum as a reference strain of Tabamura # 3503 (ATCC 25795), and ATCC 25790 was M. au. It was said to belong to rum [Taramura Michio, Medicine and Biology, Vol. 85, pp. 229–233, 1972].

However, according to the recent literature (transfer) of Saito, Tabamura et al., ATCC 25790 bacteria are not members of M. aurum, but are members of M. neoaurum [Saito, Tabamura et al., Transfer reference 77 See FIG. 1]. In addition, in the same literature, M. paraporturitum Tabamura, M. aurum Tabamura, M. neoaurum Tabamura, M. dieronhoferi Benikek & Kohasz ) And "Kanazawa Strains" are extremely close to each other and are indistinguishable at this stage, so these five species are not distributed and should be treated collectively as M. Paraportitum complex. It is concluded.

Tabamura et al. Have reached almost the same conclusions in other literature, saying that M. aurum and M. neoaurum are treated as members of the M. paraportitum complex or as subspecies of M. paraportutumum [Journal of General Micmo]. See Journal of General Microbiology, Vol. 98, pp. 511-517 (UK, 1977).

For the comparison of the general properties of the M. paraportuitum complex and the related species, Saito Hajime (TB, 50, 402, 1975 Japan) Saito et al. (Ex. 75, 80-81, Japan, 1977) And Tabamura et al. (Exchange, p. 515, 1977, Japan).

As mentioned above, the taxonomic position of the parent strain ATCC 25790 Tsukamura # 309 belongs to the M. paraportitum complex according to the conclusion of the chief authority in this area.

M. paraportuitum complex is a group of gram-positive, anti-acidic, non-motile, non-spore-forming, rapidly developing, rapidly growing scotochromogenic mycobactreia, or Runyon. Group IV)

On the other hand, the mutant strain MCI-0617 isolated from the parent strain ATCC 25790 had no microbial difference except that the former formed rough colony on the agar plate compared to the parent strain. As such, the mutant strain MCI 0617 belongs to the M. paraportuitum complex because it belongs to the M. paraportuitum complex.

Thus, although not surprisingly, these mutant strains have been introduced as U.S. Patent Nos. 3,759,791 and 3,684,657 as 4AD and ADD production mutant strains, M. SP. NRRL B-3683 and MRRL B-3805. [This strain is identified as M. baccae in Japanese Patent Application Laid-Open No. 52-105289].

For a comparison of the taxa, see Bursey Handbook, 8th edition ["Bergey's Manual of Determinative Bacteriologyed. K", pp. 695-696, Williams & Wilkins Co., 1974, USA). See also Hajime (Transfer, p. 402, 1975), Saito et al. (Ex., P. 80-81, 1977), and Tabamura et al. (Ex., P. 515, 1977).

In addition, MCI-0617 bacterium differs from NRRL B-3805 and B-3683 bacterium in terms of degradability of ADD, 4AD and 9α-OH-4AD, and the former grows slightly as a substrate, but the latter does not grow. There is a difference.

As such, the mutant strain MCI-0617 is considered to belong to the M. paraportuitum complex and it is obvious that the strain is distinct from the known ADD and 4AD production mutants.

The sterols used as the substrate in the present invention are collectively called sterols by combining various sterols, their C-3 esters, ether derivatives or their oxidizing intermediates. The various sterols include a hydroxyl group at C-3, a double bond at C-5, and a C-17 side chain having 8 to 10 carbon atoms in C-17 of the perhydroxycyclopentanophenanthrene nucleus, and in some cases C- 7, C-8, C-9 (11) or the like may have a double bond.

Thus, various sterols include cholesterol, stigmasterol, camphorsterol, cytosterol, ergosterol, brassicasterol, fucosterol, lanosterol, agnosterol, dihydrolanosterol, dehydroagnosterol, and the like. Preferred sterols are cholesterol, camphorsterol and cytosterol.

In addition, C-3 ester derivatives of 3β hydroxyl groups of various sterols and inorganic acids such as sulfuric acid or organic acids such as fatty acids are also used as raw materials of the method of the present invention.

Cholesteryl oleate, cholesteryl palmitate, cholesteryl sulfate, etc. as such a C-3 ester derivative are mentioned. In addition, C-3 ether derivatives obtained by, for example, a method of adding alkylene oxide to 3β-OH of various sterols are also used as raw materials of the method of the present invention.

Polyoxyethylene cholesteryl ether etc. are mentioned as such C-3 ether derivatives.

C obtained by reacting ethylene oxide with depressed arool and melancholy alcohol containing cholesterol obtained from wool paper (wool wax), lanolin and hydrolysis of lanolin containing C-3 ester derivatives of the various sterols described above Of course, polyoxyethylene lanolin alcohol ether, which is a -3 ether derivative, is also used as a raw material of the method of the present invention.

Alkali-washed dike oil from fish oil or squid oil, as well as sterol-containing natural products and processed products, such as deodorized scum, deodorized sludge and earth oil of vegetable oil, are also used as raw materials of the method of the present invention.

In addition, oxidation intermediates of various sterols or their C-3 ester or ether derivatives are also used as substrates of the process of the invention. Such oxidizing intermediates include various sterols, their C-3 esters, and 4-en-3-one or 1,4-dien-3-one derivatives of C-3 ether derivatives, specifically, for example, Kore Str-4-en-3-one, Coresta-1,4-diene-3-one, Coresta-1,4-diene-3-one, Coresta-4,22-diene-3-one, and the like. .

A carbon source, a nitrogen source, and an inorganic substance are suitably added to the medium used by the method of the present invention.

As a carbon source, For example, Hydrocarbon; Alcohols such as methanol, ethanol, organic acids such as succinic acid and acetic acid, and salts thereof, glycerides such as soybean oil and cottonseed oil, and sugars such as fats and oils, starch, maltose, sucrose, glucose and tamnose. Natural sources of nutrients containing nitrogen, nitrogen sources and other nutrients include molasses, vargas, cooncove, arpalfa, coonstack liquor, distirazolsor, undiluted liquor, which contains high test molasses and xylose molasses. Fish meal, bran, meat extract, yeast, yeast extract, potato extract, malt extract, gurten, peptone, glutamate, asparagine, glycine, casein, casein degradant, skim milk, soybean meal, rapeseed meal, horsetail, flax seed Vegetable oils, such as gourd and cottonseed gourd, and oilseeds, such as rapeseed, rapeseed, horse, linseed, and cottonseed, and fruit trees.

Examples of the inorganic substance include yuan, salt and the like, such as a nitrogen source and dipotassium phosphate; Salts such as iron, copper, magnesium, manganese, cobalt, zinc and calcium; molasses; and compounds of natural products. Vitamins can also be added as needed.

The composition of the medium is selected according to the type of fungal species used, and carbon source, nitrogen source, potassium, phosphorus and magnesium are indispensable as the medium component.

What is necessary is just to add a well-known antifoamer. Specific examples thereof include polyoxyalkyrene glycol, and the like, but it is not necessary to add an antifoaming agent.

Surfactant is effective as an emulsifier of sterols, and it is preferable to add in surfactant. As surfactant, a nonionic and anionic thing is preferable. Specifically, polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, polyethyleneglycol monostearate, etc. are mentioned, for example.

The incubation temperature is usually 20 to 40 ℃, the optimum is around 30 to 35 ℃.

The pH of the medium is usually adjusted to 5 to 10, with 6 to 9 being preferred. Since the microorganism used in the present invention belongs to the genus M, it can withstand a pH of around 10 as is well known.

Sterols which are raw materials of the method of the present invention are generally sterilized together with the medium, but may be added to the medium after the start of the culture. In addition, divided addition is also possible. In this case, it is added as it is after sterilization or wet heat sterilization, or dissolved in dimethylformamide or the like as a solution, or finely dispersed and suspended by sonication to be added as a suspension. The addition of a surfactant at the same time is preferable because emulsification of substrate sterols and the like is promoted.

Incubation time is not particularly limited. Usually, about 3 days after addition of raw material sterols, the yield of HP increases rapidly. After that, the production of HP gradually increases with the incubation time, but culturing for 20 days or more is of little industrial significance.

After the end of the culture, HP accumulated in the culture liquid can be collected and separated economically by a known method.

For example, crude HP is obtained by extracting HP from the culture solution with an organic solvent which is difficult to be mixed with water such as several times the amount of ethyl acetate and distilling the solvent from the extract solution. If it is necessary to separate HPD, 4HP, substrate sterols or by-products, porous resin, silica gel, alumina, etc. are used as adsorbent and petroleum ether, benzene, chloroform, ether, acetone, methanol, ethyl acetate, etc. The product can be separated by column chromatography used as.

To purify the isolated HPD or 4HP, respectively, recrystallization may be repeated with a suitable solvent such as 10% ethanol or hexane.

According to the method of the present invention, HP can be obtained in a yield of at least 50 mol%, usually at least 70 mol% per sorbosterols. In addition, according to the method of the present invention, if the culture is carried out at a high substrate concentration, the industrial advantage is also great in that the amount of HP increases in proportion.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples unless they exceed the gist of the present invention.

In the following examples, the qualitative and quantitative determination of sterols and derivatives thereof, HP, remaining sterols and by-product steroids was carried out by gas chromatography.

In the following examples,% is based on weight.

Example 1

100 ml of medium (pH 7.2) consisting of 1.0% glucose, 1.0% meat extract, 1.0% peptone and water was dispensed into a 500 ml shoulder flask, steam sterilized at 12 ° C. for 15 minutes, and then M. paraportumtum complex Inoculate MC 10617 with platinum and incubate for 72 hours at 30 ° C. under reciprocating shaking conditions of 120 round trips / minute and amplitude of 7 cm. 2 ml of this seed culture solution was degreased soybean meal 4.0%, fish paste 2.0%, K ₂ HP ₄ 0.2%, NaNO ₃ 0.2%, NgSO ₄ 7H ₂ O 0.1%, soybean oil 1.0% (pH 7.0, sterilized at 120 ° C for 20 minutes) 10 ml 500 ml shoulder flasks containing 0.8 ml of cholesterol and 50 ml of this culture medium (pH 7.0, steam sterilization at 120 ° C. for 20 minutes) are inoculated.

The culture was performed at 30 ° C. under reciprocating shaking conditions of 120 reciprocation / min and amplitude of 7 cm. The culture was stopped at 160 hours after the start of the culture, and the cultures were combined and extracted twice with 2 L of ethyl acetate.

The extracts were combined and the insolubles, such as cells, were filtered, and the amount of steroid was analyzed. The result was 0.05 g of residue cholesterol, 2.65 g of HPD, and 0.22 g of 4HP. This is the cell in which HP was produced in about 85% yield per large consumption of cholesterol. 0.03 g of ADD was present in the byproducts.

When the extract was subjected to silica gel column chromatography to extract 20% ethyl acetate-n-hexane as a solvent, HPD and 4HP were separated from each other and further separated from substrate cholesterol and by-products. Fractions of each of the eluted HPD and 4HP were collected and the solvent was distilled off and recrystallized twice from 10% ethanol heptane to obtain 2.45 g of HPD and 0.17 g of 4HP. Melting point, mass spectrometry, NMR, and IR were in agreement with the literature data.

Example 2

In Example 1, Example 1 was repeated except having used the various steroids shown in following Table 1 instead of cholesterol as a raw material steroid.

TABLE 1

Example 3

Except for using the culture medium in Example 1, a cultured soybean meal containing 6.0%, soybean 1.0%, K ₂ HPO ₄ 0.25%, MgSO ₄ · 7H ₂ O 0.1% and various concentrations of cholesterol in the culture medium Example 1 was repeated. The results are shown in Table 2.

TABLE 2

Example 4

In Example 1, a 5-liter shoulder flask containing 1.0% glucose, 1.0% meat extract, 1.0% peptone, 1.0% surfactant "Tween-80" from Kao Atlas, and 800 ml of medium containing water was prepared. Example 1 was repeated except that the culture was inoculated with 20 ml of the seed, and then the main culture was carried out for 30 hours at 30 ° C., 100 round trips / minute, and shaking of 7 cm in amplitude. As a result of analyzing the ethyl acetate extract, it was confirmed that 0.90% of HPD and 0.76 g of 4HPO were present. Cholesterol remained 4.8 g.

Claims (1)

20α-hydroxymethylpregna-1,4-dien-3-one, 20α-hydroxymethylpregine- characterized by culturing Mycobacterium paraportumtum complex MCI 0617 strain with sterols as a substrate. Process for preparing 4-en-3-one or mixtures thereof.