JPS62118898A - Microorganisms and methods for hydroxylating steroids at the 11β position using the microorganisms - Google Patents

Abstract

PURPOSE:To efficiently introduce hydroxyl group into a steroid, by inoculating Curvularia lunata having the ability to hydroxylate hydrogen atom of the steroid into a culture medium containing the steroid and cultivating the Curvularia lunata. CONSTITUTION:Curvularia lunata MCI1690 (FERM-P No.8515) is inoculated into a culture medium containing a steroid expressed by the formula and aerobically cultivated. Alternatively, oxidase produced by the above-mentioned microorganism is aerobically reacted therewith to introduce hydroxyl group into the 11beta-position of the above-mentioned steroid. The reaction is carried out under conditions of about 4-7 pH and about 20-30 deg.C for 12hr - 10 days and the accumulated oxidation product is fractionated and collected by methods, e.g. adsorption or extraction, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention provides hydroco-7zone (llβ
, lliα, 2/-trihydroxyderpregnone-3,-〇-dione) and tprednisolone (//β
The present invention relates to a novel method for producing ly(alpha,co/-)lihydroxy/,41-pregnagen-3,-0-dione) or substituted derivatives thereof.

(Prior Art) Conventional methods for producing hydrocortiscin and prednisolone, as well as their substituted derivatives, include 7-α,-nodihydro-4xedarpregnene-3,-〇-dione (hereinafter referred to as
Various methods are known, including a method in which rosJ (referred to as rosJ) or a substituted derivative thereof is hydroxylated at the 112-position using microorganisms, and a method in which desoxycorticosterone is hydroxylated and chemically converted to the 112-position.

Of these, the former method Occupy soft.

(J, A-a, s-, two, qg, t, tqzs),
Canning Hamelada, KoH, tqsr), Corticium Sasakie FO! Ko317 (Oorticium 5
asakii XFo! r2! Q ) (Bull, Ag
r, Ohem, Boa, Japan, Ko/, 390.
19! ), Streptomyces fradiae 313! Hiroono), Absidia elegans (Absidia, etc.) are known.

(Problems to be Solved by the Invention) In reactions using these strains, even the highest concentration reacted was only 0.0% w/v.

Moreover, the yield at this time is as low as that of ZOX.

(131 Nogi Research Annual Report No. 1 p, /1m/, , /
94λ) Also, according to Japanese Patent Application Laid-Open No. 2003-110003, it is stated that the yield can be improved (yield rt, b N ) by using α-acylooxysteroid, but the preparation The concentration is only 0-Ok'A.

As described above, these microorganisms have a low hydroxylation ability and require a large amount of cost if used for industrial production.

(Means for Solving the Problems) As a result of various studies on various microorganisms, the present inventors found that it was superior to previously known strains. separated. However, Kurbura Rialnata A-1007 strain, which is superior to conventional bacterial strains, produces a compound hydroxylated at two sites as a main by-product, although it has the ability to produce β-hydroxyl and hydroxyl.

Furthermore, the hydroxylation ability at position 112 was not high enough to be suitable for industrial production.

Therefore, in order to increase the yield and increase the hydroxylation ability at the 71β position, the present inventors
Mutation was carried out using 0ri as the parent strain using the conventional method (revised method) using ultraviolet light. As a result, the main by-product, a compound hydroxylated at position 20, was hardly produced, and the yield of yellowtail was significantly increased. In addition, the present invention was achieved by obtaining the microorganism Kurubular Rialnata MO Eng. 1690 strain, which had significantly improved hydroxylation ability at position 112.

That is, the gist of the present invention is the following general formula (+) (in the above formula,
R1 and R2 each independently represent a water atom, an alkyl group, a halogen atom, or a hydroxyl group, and R1 and R2 represent a hydrogen atom, a hydroxyl group, or an acyloxy group.

Further, the broken line in the Δ1 and 2 portions indicates a single bond or a -multiple bond. )
0urv&1artalunata, which has the ability to hydroxylate //β of steroids represented by
It resides in M O engineering xqo.

The present invention will be explained in detail below.

First of all, Curvularia lunata in the present invention/
b 9 o (Urvularia 1unata
Ma engineering 1bqo) i13 engineering 9 [W#l” evening/ni issue (v
wnw p-1? The mycological properties are as follows.

A w Scientific properties Colonies grow well on potato textulose agar medium, exhibiting a velvety to comb-like appearance, gloss, and a grayish-black to dark brown to black color.

Sakae*zono thread is branched, has septa, pale to pale brown, and width up to i, s -48 m. Conidiophores are erect or supine on the hyphae, single, erect or curved, often zigzag upwards. Light brown, reddish-brown to deep reddish-brown, often pale at the top, smooth, L-number septa, length up to 5θμm, width! -9 μm 1 conidia are bolo-shaped conidia, round-shaped to spindle-shaped ellipsoids, 3-septate, lower 3rd cell is huge and curved, larger and darker than other cells, cells at both ends are Almost colorless to light brown, intermediate cells are brown to dark and smooth, -〇-30X9-1! The rμm umbilicus (htxum) does not protrude.

B. Taxonomic Consideration This species belongs to the genus Urvularia because it forms bolo-shaped conidia and has round to spindle-shaped conidia.

In the native country, the conidia have 3-septata and are directed downward. Therefore, this strain was identified as Ourvularia '1unata.

This Curvular Rialnata MOX (&90) has the ability to hydroxylate the 112th position of steroids.

The nutrient medium for this potato preferably contains a carbon source and a nitrogen source that can be assimilated by the fungus, as well as K and inorganic salts necessary for its growth. Carbon sources include glucose, lactose, sucrose, dextrin, starch, glycerin, etc., which are normally used for this purpose, and nitrogen sources include peptone, meat extract, casein, edamine, cornstarch liquor, etc. , yeast or yeast extract, nitrogen-containing organic substances such as soybean products, nitrogen-containing organic compounds such as nitrogen, amino acids, organic acid ammonium salts, and ammonium nitrate, ammonium phosphate, etc.
A desirable medium is prepared by appropriately blending inorganic nitrogen compounds such as ammonium sulfate and sodium nitrate, and inorganic salts necessary for bacterial growth such as potassium phosphate, enriched sodium, magnesium sulfate, and iron sulfate. can do.

An aqueous medium having a composition as described above has a pH of about
Adjusted to I-9.

As a method of cultivating bacteria, it is possible to use static culture, but since the bacteria themselves are aerobic, culture methods performed under aerobic conditions should be carried out using shaking culture or stirring. A deep culture method using aeration is advantageous in terms of implementation.

Hereinafter, a method for hydroxylating the /lβ position of steroids using the above-mentioned Kuruburarialnata MO Engineering/490 will be described.

The raw material steroids used in the present invention are represented by the general formula (J).

The amyloxy group is generally an aliphatic loxy group such as an acetoxy group or a propionyloxy group.

The timing of addition of the raw material steroid compound is selected at the start of culture or during continuous cultivation.

The raw material steroid compound can be used as it is in the form of a fine powder, or for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, ethyl acetate,
As a solution or suspension in a suitable solvent such as acetone, dioxane, dimethylformamide, etc., or as a solution or suspension in which a surfactant, dispersion i1, etc. are added, all at once or continuously over a certain period of time. or added intermittently. In particular, methanol and ethanol are used at 0% of the reaction solution. A good effect on the reaction can be brought about by adding an amount of ~1QS to the reaction solution.

The acidity of the medium, reaction temperature (cultivation temperature), reaction time (cultivation time), and other conditions in the reaction vary depending on the raw material steroid, the compound, the selected M1 group, the medium composition, etc. It is desirable to select and implement the most suitable conditions in each case. Therefore, the reaction is generally based on the starting pI! The process is carried out at a temperature of -0 to 0°C for about 10 days, but is not limited thereto.

In the reaction, the above-mentioned proliferating microbial cells may be used with other germ-static cells, immobilized microbial cells, and treated microbial cells.

Extensive separation means are used to separate the oxidation products accumulated in the reaction medium. For example, there are adsorption methods in which the target substance is adsorbed on a suitable adsorbent such as alumina, Florisil (synthetic magnesium silicate), activated carbon, etc., and then eluted with a suitable polar solvent such as methanol or ethanol, or chloroform, methylene chloride, ethylene chloride, etc. Direct extraction using an organic solvent that can form two liquid phases with water, such as halogenated hydrocarbons or acetate esters, or a method that utilizes the difference in distribution ratio between co-liquid phases that are distributed countercurrently, or similar methods. The principle is chromatography using a suitable carrier such as sialumina, silica gel, cellulose/solde, etc.
Alternatively, there is a method that utilizes the difference in solubility, or an acylating agent using a hydrazide such as trimethylammonium acetate hydrazide or pyridinium acetate hydrazide or a lower fatty acid anhydride and a deoxidizing agent from a solution containing the target substance once extracted. Various separation means are selected and used as appropriate depending on the structure and functional group of the target substance to be separated, such as φ-throwing, in which the target substance is separated and collected as its functional conductor and then returned to its original form.

The product compound obtained by the method of the present invention is useful, for example, as an adrenocortical hormone active substance or a synthetic intermediate thereof.

(Examples) Although the following examples will be described in detail with reference to examples, the present invention is not limited to the following examples as long as they do not exceed the gist thereof.

Example 1 Ourvularia lunata (Ourvularia lunata)
Ata) MO/49Q was cultured in a rotary shaker for an hour at 27° C. using a Erlenmeyer flask with a 93θθ hex containing a 100% medium having the following composition.

Medium Pudov Sugar '01/10
0ml peptone cocorn staple liquor 〇, 5 (PHs, o) Next, this culture solution-1 was added to a jOO-containing Erlenmeyer flask containing 100- the same composition as the seed culture, and cultured in the same manner at ℃. .

After 7 days of culture, Oki/011 dissolved in methanol was added and cultured for 6 days, followed by reaction.

After the reaction, 1 Ioo of methyl alcohol was added, and m was removed by centrifugation to determine the amount of hydrocortiscin produced. The results are shown in Table 7.

Analysis was performed using high performance liquid chromatography.

Analysis conditions Eluent: Water/Methyl alcohol = Hiroshi/L Flow rate: /M
t/s* Detection: υVcosllnm Example 2. Culture and reaction were carried out in the same manner as in Example except that the raw material steroid was replaced with C8-/α-acetate and the number of days of culture was changed to day t.

SX potassium carbonate was then added IO- and the remaining ester was hydrolyzed in NJ atmosphere to produce hydrocortiscin. The results are shown in Table 7.

Example 3 Cultivation and reaction were carried out in the same manner as in Example 3, except that the raw material steroids Do1.08-/7α and col-diacetate were used.
produced prednisolone. The results are shown in Table 1.

(Effects of the Invention) According to the present invention, steroids can be efficiently hydroxylated at the /lβ position.

Claims (2)

[Claims] (1) The following general formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (In the above formula, R_1 and R_2 each independently represent a hydrogen atom, an alkyl group, a halogen atom, or a hydroxyl group,
R_3 and R_4 represent a hydrogen atom, a hydroxyl group or an acyloxy group. Also, the broken lines in parts △^1^ and ^2 indicate single bonds or 2
Indicates a double bond. ) has the ability to hydroxylate the 11β-position of steroids, and has been deposited as FIKEN Deposit No. 8515.
ia￥￥lunata￥)MCI1690. (2) The following general formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (In the above formula, R_1 and R_2 each independently represent a hydrogen atom, an alkyl group, a halogen atom, or a hydroxyl group,
R_3 and R_4 represent a hydrogen atom, a hydroxyl group or an acyloxy group. Also, the broken lines in parts △^1^ and ^2 indicate single bonds or 2
Indicates a double bond. ) in a medium containing steroids represented by Curvularia
￥ ￥lunata ￥) Introducing a hydroxyl group into the 11β position of the steroid by inoculating MCI1690 and culturing it aerobically, or by aerobically acting on the oxidase produced by the above bacteria. Characteristic method of hydroxylation at the 11β position of steroids.