JPH0226957B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing valienamine and/or validamine. Of the present inventors, Kameda and Horii first treated validamycin A or validoxylamine A with the bacterial cells of Pseudomonas denitrificans. 4/2)-4-amino-6-hydroxymethyl-5-cyclohexene-1,2,3-triol] and validamine; 1L-(1,3,4/2,6)-4-
reported that it was possible to isolate amino-6-hydroxymethyl-1,2,3-cyclohexanetriol [J.
Chem.Soc.Chem.Commun, ) 1972, 746-747
page〕. However, in the production of valienamine and/or validamine by the above method, the strain does not grow using validamycins as the sole carbon source, and is obtained by culturing it in a medium containing other carbon sources, such as glucose. In addition, its ability to decompose validamycin is weak, and it is not suitable for mass production of valienamine and/or validamin.

As for the production method by chemical means, there is a method for producing validamine via hydrogenolysis reaction of validamycin A, validoxylamine, etc. [The Journal of Antibiotics, No. 24] Volume, pp. 59-63,
(1971)], but valienamine cannot be obtained by this method. Also, 2-O-
Methyl-L-chiroinositol (2-O-
Synthesis of valienamine from methyl-L-chiroinositol (H. Paulsen et al., Angew. Chem., Vol. 92, 930-
931 pages (1980)], but it requires many steps and is not suitable for mass production. In addition, a method for synthesizing DL-validamine by chemical synthesis means [Ogawa et al., Bulletin of the Chemical Society of Japan (Bull.Chem.Soc.Jpn.), Vol. 52,
pp. 1174-1176 (1979 and DL-valienamine [Ogawa et al., Chemistry Letters]
Letter), pp. 713-716 (1980)], but these methods yield the target compounds only as racemic compounds.

Furthermore, among the present inventors, Kameda and Horii discovered that a bacterial strain isolated from the soil of a paddy field in Kanazawa City, Ishikawa Prefecture was able to efficiently degrade validamycin into validamin and valienamine (Japanese Patent Application No. 55-128157
(Japanese Patent Application Laid-open No. 57-54593), Imai called this bacterium Flavobacterium saccharophilum IFO13984.
[1971, Japan Fermentation Engineering Society lecture abstracts, p. 242].

After that, Imai consulted with Kameda and Horii and searched for strains stored at the Fermentation Research Institute, and found that Flavobacterium heparinum (Flavobacterium heparinum)
Heparinum (species name), and was accepted into the Fermentation Research Institute in 1963 from ATCC via Nagoya University.
IFO12017 (ATCC13125) strain and Flavobacterium ceratolyteix (Flavobacterium
keratolyticus) from Kyushu University in 1980.
We found that strain IFO14087, which we received in 2007, degrades validamycin or validoxylamine.

However, in recent years, the genus Flavobacterium (Bergey's manual of determinative
bacteriology, 8th edition), it was reported that some strains should be transferred to the Cytophagaceae family [E. Callies et al., Antony Huang et al. Antonie
van Leeuwenhoek) Vol. 46, pp. 41-49 (1980)]
P. Christensen was also identified as Flavobacterium heparinum.
After examining the properties of ATCC13125 strain, we identified this strain as Cytophaga heparina.
Named [International Journal]
International Journal of Systematic Bacteriology
Bacteriology, Vol. 30, pp. 473-475 (1980)].

Imai is Flavobacterium ceratolyteix
It was found that strain IFO14087 contains menaquinone in its cells, and therefore is highly likely to be a species of Cytophagaceae. Original description of this strain [Manabu Kitamikado et al., Journal of the Agricultural University
of the Faculty of Agriculture, Kyushu
When comparing the description of Cytophaga heparina by P. Christensen (1979), Vol. 24, pp. 101-112 (1979)], differences were found between the two strains in terms of gelatin decomposition, starch decomposition, etc., and IFO14087
The strain is considered to be a different species from Cytophaga heparina.

The present invention was completed as a result of extensive studies based on these findings, and is directed to a microorganism that belongs to the genus Cytophaga and produces an enzyme capable of producing valienamine and/or validamine by acting on validamycin or validoxylamine. Alternatively, it is a method for producing valienamine and/or validamine, which is characterized in that the treated product is allowed to act on validamycin or validoxylamine.

Validamycin used in the method of the present invention is widely used as an agricultural antibiotic, and its structure consists of validoxylamine and D-glucose. Validoxylamine is currently A
and B are known, and validamycin is known to exist as A, B, C, D, E, and F due to the combination of validoxylamine A, B, and D-glucose [The Journal・
Of Antibiotics (J.Antibiotics)
Volume 25, pp. 48-53 (1972)].

In the method of the present invention, such individual validamycin, validoxylamine, or a mixture thereof can be used as raw materials, and for example, cultures of validamycin-producing bacteria or processed products thereof are advantageously used.

The microorganism used in the method of the present invention may be any microorganism belonging to the genus Cytophaga or its mutant strain, which has the ability to convert validamycin or validoxylamine into valienamine and/or validamine, such as Cytophaga heparina ( Cytophaga heparina,
IFO12017, ATCC13125), and IFO14087 strains are used.

The medium used for culturing the above-mentioned microorganisms in the method of the present invention may be either liquid or solid as long as it contains a nutrient source that can be used by the strain, but it is more appropriate to use a liquid medium when processing a large amount. be. The culture medium contains a carbon source that can be assimilated by the above microorganisms,
Digestible nitrogen sources, inorganic substances, micronutrients, etc. may be added as appropriate. Carbon sources include, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, etc., oils and fats (eg, soybean oil, lard oil, chicken oil, etc.), and nitrogen sources include, for example. Meat extract, yeast extract, dried yeast, soybean flour, corn steep liquor, peptone, cottonseed flour, blackstrap molasses, urea, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.), and others are used. Furthermore, salts containing sodium, potassium, calcium, magnesium, etc., metal salts such as iron, manganese, zinc, cobalt, nickel, etc., salts such as phosphoric acid, boric acid, etc., and salts of organic acids such as acetic acid, propionic acid, etc. are used as appropriate. . Other amino acids (e.g., glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (e.g., dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nicotinic acid, B ₁₂ ,C,E
etc.), nucleic acids (eg, purines, pyrimidines, derivatives thereof, etc.), and the like. Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added for the purpose of defoaming.

The culturing method may be static culture, shaking culture, or aeration/agitation culture. It goes without saying that for large-scale treatment, it is desirable to use so-called deep aeration agitation culture. It goes without saying that culture conditions vary depending on the condition and composition of the medium, the type of strain, the culture method, etc.;
It is best to select the initial pH to be around neutral at a temperature of 20°C to 45°C. In particular, the temperature during the middle stage of cultivation is between 24°C and 37°C.
℃, and the initial pH is preferably 6.5 to 8.5. Cultivation time may be about 6 to 100 hours, but especially 16 to 100 hours.
Good after 60 hours.

The "culture" used in the present invention refers to what is obtained by the above-mentioned culture.

In the present invention, the microbial cells obtained in this way or a processed product thereof can be used, and the term "processed product" here refers to the culture obtained above subjected to physicochemical treatments such as filtration, centrifugation, ultraviolet separation, etc. Sonication, French press treatment, alumina grinding, lytic enzyme treatment,
This refers to bacterial cells obtained by treatment with surfactants or organic solvents, or crushed bacterial cells containing enzymes. Enzymes obtained by purification by known methods, or bacterial cells or enzymes immobilized by known methods can also be used.

The method of the present invention is carried out by bringing the raw material compound into contact with the above-mentioned microorganism or its treated product. The concentration of the raw material compound in the reaction solution is suitably 1 to 5%.
A reaction temperature of 20 to 45°C and a pH of 5 to 8 are suitable, but a temperature of 24 to 30°C and an initial pH of 6.5 to 7.5 are particularly good. The reaction time varies depending on the growth state and amount of microorganisms added to the decomposition reaction solution, but is suitably 24 to 300 hours, more preferably 48 to 100 hours. In addition, the reaction may be performed under static conditions or under conditions of shaking, aeration, or stirring;
Aeration or stirring is better. In the reaction solution, reaction accelerators, enzyme stabilizers,
Preservatives (penicillin antibiotics, aminoglycoside antibiotics, etc.) may be added.

To collect the target product from the reaction solution, the means normally used for collecting microbial metabolites are used alone, in combination in any order, or repeatedly. That is, for example, filtration, centrifugation, concentration, drying, freeze-drying, adsorption, desorption, methods that utilize differences in solubility in various solvents (for example,
(precipitation, crystallization, recrystallization, etc.), chromatography, etc. In addition, utilizing the fact that valienamine and validamine are basic substances that are soluble in water and poorly soluble in general organic solvents, methods used for the isolation and purification of so-called water-soluble water-based substances, such as ion exchange resin, Chromatography and adsorption/desorption methods using activated carbon, high porous polymers, Cephadex, Cephadex ion exchangers, cellulose, ion-exchanged cellulose, silica gel, alumina, etc. are advantageously used.

Next, the present invention will be explained with reference to Examples.

Example 1 (a) In a two-Sakaguchi flask, 15 g of Trypticase (manufactured by BBL) was dissolved in 500 ml of water, and after sterilization, Cytophaga heparina (IFO12017, ATCC13125) was inoculated, and the mixture was incubated at 28°C.
Incubate with shaking for 24 hours. This culture solution was dissolved in 30 g of polypeptone, 210 g of yeast extract, and 90 g of sodium chloride in 30 g of water in a 50 fermenter, and 15 g of an antifoaming agent (Actocol, manufactured by Takeda Pharmaceutical Co., Ltd.) was added to adjust the pH to 7.1. After that, add to the sterilized preculture medium and incubate at 28 °C for 24 hours with aeration and agitation.
Incubate for hours. 5 of this culture solution, 200
Ammonium sulfate 1.0Kg, potassium phosphate-hydrogen 0.7Kg, potassium dihydrogen phosphate in fermenter
0.3Kg, magnesium sulfate 0.01Kg, and 10% of the crude solution of validamycin A (validamycin A content: approximately 20%) were dissolved in 100% of water, an antifoaming agent (0.05Kg) was added, the pH was adjusted to 7.1, and sterilized. Transfer to main fermentation medium. The reaction is carried out by culturing at 28°C for 96 hours with aeration and stirring.

(b) The reaction solution obtained in (a) was centrifuged, and the supernatant was passed through and adsorbed on an Amberlite IRC-50 (NH ⁺ ₄ type, manufactured by Rohm and Haas) column (30). After washing the column with water (90), 0.5N
Elute with aqueous ammonia. Collect the elution fractions (fractions No. 5 to 10; each fraction 10),
Concentrate under reduced pressure to approximately 3.8%.

1/10 volume (380 ml) of the above concentrated solution was subjected to column chromatography (1.8) using Dowex 1×2 (OH ^- type, manufactured by Dow Chemical Company), and the column was eluted with water. Each elution fraction was analyzed by thin layer chromatography [silica gel 60F ₂₅₄ (manufactured by Merck & Co., Ltd.); developing solvent, n-propyl alcohol/acetic acid/water (4:1:1); color reagent, ninhydrin; valienamine Rf = 0.42, Validamine
Check with Rf=0.35]. The eluted fractions of validamine (1.4 to 2.0) are collected, concentrated under reduced pressure, and then lyophilized to obtain 3.4 g of white powder of validamine.
Crystallization is carried out in methanol-ethanol. The eluted fractions of valienamine (2.25-3.8) are collected and concentrated under reduced pressure, and acetone is added to the resulting syrupy substance to obtain crystals of valienamine (12.7 g).

Example 2 Validamycin A 1%, ammonium sulfate 1%,
Adjust the aqueous solution (2) of potassium monohydrogen phosphate 0.7%, potassium dihydrogen phosphate 0.3%, and magnesium sulfate 0.01% to pH 7.1, and add Cytophaga heparina (IFO12017,
ATCC13125) and culture with shaking at 27°C for 4 days. The culture solution was centrifuged to remove bacterial cells, and the supernatant was adsorbed onto a column (500 ml) of Amberlite IRC-50 (NH ⁺ ₄ type, manufactured by Rohm and Haas).
After washing with water, elute with 0.5N ammonia water. Concentrate the eluate under reduced pressure, and transfer the concentrated solution to a dowex 1×2
(OH ^- type, manufactured by Dow Chemical Company) (500 ml) and elute with water. Each eluted fraction is examined by thin layer chromatography in the same manner as in Example 1-(b). The eluted fractions of Validamine were collected first, concentrated under reduced pressure, and then lyophilized to obtain a white powder of Validamine (0.76 g).Then, the eluted fractions of Valienamine were collected, concentrated under reduced pressure, and evaporated to dryness. %
Crystallization from ethanol water yields valienamine crystals (1.62 g).

Example 3 In a 2-Sakaguchi flask, add 15 g of trypticase to water.
Dissolve in 500ml, inoculate with Cytophaga heparina (IFO12017, ATCC13125) after sterilization, and incubate at 27℃.
Incubate with shaking for 24 hours. The culture solution is centrifuged to collect bacterial cells, which are washed once with 0.1M phosphate buffer (PH7.0) to obtain about 2.1 g of wet bacterial cells. The cells were added to a solution of validoxylamine A (3.0 g) in 0.1 M phosphate buffer (PH 7.0, 2), and the reaction was carried out at 27° C. for 72 hours with shaking. The reaction solution was centrifuged to remove bacterial cells, and the supernatant was transferred to Amberlite IRC-50 (NH ⁺ ₄ type,
Validamine (100 mg) and valienamine (285 mg) are obtained by adsorbing the sample into 100 ml) and treating in the same manner as in Example 2.

Example 4 Ammonium sulfate 1%, potassium monohydrogen phosphate
A mixture of validamycin E and F (approximately 3:2 mixture) in an aqueous solution (50 ml) containing 0.7% potassium dihydrogen phosphate, 0.3% potassium dihydrogen phosphate, and 0.01% magnesium sulfate.
Dissolve 0.50g, adjust the pH to 7, and after sterilize, Cytophaga heparina (IFO12017, ATCC13125)
and culture with shaking at 27°C for 4 days. The culture solution is treated in the same manner as in Example 1 to obtain validamine (34.5 mg) and valienamine (14 mg).

Claims (1)

[Claims] 1. Valienamine, which is characterized in that a microorganism belonging to the genus Cytophaga that produces an enzyme capable of producing valienamine and/or validamine by acting on validamycin or validamycin or validamycin, or a processed product thereof, is activated to act on validamycin or validoxylamine. and/or a method of manufacturing Validamine.