JPH022589B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing valienamine.

The present inventors previously isolated the substance represented by the following formula from the natural world for the first time by allowing the bacterial cells of Pseudomonas denitrificans to act on validamycin A or validoxylamine A, and identified it as valienamine. named and reported. [Kameda et al., Journal of
The Chemical Society; Chemical Communication (J.Chem.Soc.Chem.Commun.)
1972, 746 pages] However, the production of valienamine by the above method requires that this strain does not directly assimilate validamycin A or validoxylamine A, and that cells obtained by culturing in a medium containing other carbon sources, such as glucose, are used. Moreover, its ability to decompose validamycin is weak, and it is not suitable for mass production of valienamine.

On the other hand, the present inventors searched for bacteria that can efficiently degrade validamycin, and collected Flavobacterium from the soil of rice fields in Kanazawa City, Ishikawa Prefecture.
A new strain of the genus Flavobacterium saccharo
philum n.sp., IFO 13984) was discovered and isolated in 1979.
It was reported as a new bacterial species by Imai in 1972 (Japan Society for Fermentation Engineering lecture abstracts, p. 242).
The present invention was completed based on the discovery that, including this bacterium, there are other bacteria in the genus Flavobacterium that can decompose validamycin or validoxylamine into valienamine at a good yield.

That is, the present invention is characterized in that a microorganism that belongs to the genus Flavobacterium and produces an enzyme that can act on validamycin or validoxylamine to produce valienamine, or a processed product thereof, is made to act on validamycin or validoxylamine. This is a method for producing valienamine.

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 it is known that validamycin exists as A, B, C, D, E, and F due to the combination of validoxylamines A and B and D-glucose.

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 Flavobacterium or its mutant strain, which has the ability to convert validamycin or validoxylamine into valienamine. (Flavobacterium saccharo-philum.
n.sp., IFO 13984, FERM-P No. 5707), etc. are used. Flavobacterium satucarophyllum is a Gram-negative, oxidase-positive, non-motile bacillus that grows well and oxidatively decomposes sugars using validamycin as the sole carbon source.
It uses many types of sugars as a single carbon source, but does not use Krebs cycle intermediates or sugar alcohols. Furthermore, sliding on the agar plane was not observed. (Japan Fermentation Engineering Society Conference, November 7, 1978)
(Japanese Oral Presentation, Lecture Proceedings, p. 242) The medium used for culturing the above-mentioned microorganisms in the method of the present invention may be in liquid or solid form as long as it contains a nutrient source that can be used by the strain; It is more suitable to use liquid culture when processing. 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, sugarcane, urea, ammonium salts (e.g., 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 to adjust the pH of the medium, or an appropriate amount of fats and oils, surface-enhancing agents, 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 for 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,
It refers to bacterial cells or crushed bacterial cells containing enzymes obtained by treatment with interfacial agents or organic solvents. 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 200 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 is a basic substance that is soluble in water and poorly soluble in general organic solvents, methods used for the isolation and purification of so-called water-soluble basic substances, such as ion exchange resin, activated carbon, Porous polymer, Cephadex,
Chromatography and adsorption/desorption methods using cephadex ion exchangers, cellulose, ion exchange cellulose, silica gel, alumina, etc. are advantageously used.

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

Example 1 Flavobacterium Satsukalofilm No.121
strain (IFO 13984) in 1% validamycin A, 1% ammonium sulfate, 0.7% potassium monohydrogen phosphate,
Potassium dihydrogen phosphate 0.3%, magnesium sulfate
inoculated into medium 2 containing 0.01% (PH7.1),
Shaking culture was performed at 27°C for 4 days. The resulting culture solution is centrifuged to remove bacterial cells and obtain a supernatant. This supernatant is passed through an Amberlite IRC-50 (ammonium type, 500 ml) column to adsorb valienamine, and after washing with water, it is eluted with 0.5N ammonia water. The eluate was concentrated under reduced pressure, and further
Pass through a x2 (OH ^- type, 500 ml) column and elute with water. Separate the ninhydrin positive section,
Concentrate to dryness under reduced pressure. Crystallize from 80% ethanol water to obtain 3.5 g of valienamine in the form of colorless needles.

Example 2 In a 2-Sakaguchi flask, 15 g of trypticase (manufactured by BBL) was dissolved in 500 ml of water, and after sterilization, Flavobacterium satsukalofilm strain No. 121 was inoculated and cultured with shaking at 28°C for 24 hours.

This culture solution was mixed with ammonium sulfate in a fermenter for 50 minutes.
Add 3 kg of validamycin A crude product (purified intermediate containing about 20% validamycin A, about 3% validamycin B, etc.) to an aqueous solution (30) of 300 g, potassium monohydrogen phosphate 210 g, potassium dihydrogen phosphate 90 g, and 3 g magnesium sulfate. Dissolve, add antifoam and add to sterile liquid.

The reaction solution was adjusted to pH 7.1 and stirred for 4 days under ventilation.

Centrifuge 20 minutes of the above reaction solution, and transfer the supernatant to an Amberlite IRC-50 (NH ⁺ ₄ type) column (10 minutes).
), wash the column with water, elute with 0.5N aqueous ammonia, and concentrate the eluted fraction under reduced pressure.
The residue was subjected to column chromatography using Dowex 1×2 (OH ^- type) and the column was eluted with water. The valienamine eluted fraction is concentrated under reduced pressure, and acetone is added to the concentrated solution for crystallization. Yield: 55.5g.

Example 3 In a Sakaguchi flask, add 15 g of trypticase to 500 g of water.
After dissolving in cc and sterilizing, Flavobacterium satsukalofilm No. 121 strain was inoculated and cultured with shaking at 27°C for 24 hours. Centrifuge the culture solution to collect bacterial cells,
Wash once with 0.1M phosphate buffer (PH7.0) to obtain about 2.5 g of wet bacterial cells. This bacterial cell was dissolved in a 0.1M phosphate buffer solution (PH7.0,
In addition to step 3), carry out the reaction at 27°C for 72 hours with shaking, and centrifuge the reaction solution to remove bacterial cells. The obtained supernatant liquid is treated in the same manner as in Example 1 to obtain 1.0 g of valienamine.

Example 4 Ammonium sulfate 1%, potassium monohydrogen phosphate
A mixture of validamycin E and F (approx. 3:2 mixture) in an aqueous solution (100 ml) containing 0.7% potassium dihydrogen phosphate, 0.3% potassium dihydrogen phosphate, and 0.01% magnesium sulfate.
Dissolve 1.0g (PH7.1), Flavobacterium
Satsukalofilm strain No. 121 was inoculated and cultured with shaking at 27°C for 4 days. The culture solution is centrifuged to remove bacterial cells, and the supernatant is treated in the same manner as in Example 1 to obtain 0.11 g of valienamine.

Claims (1)

[Claims] 1. A method for producing valienamine, which comprises causing a microorganism that belongs to the genus Flavobacterium and produces an enzyme that can act on validamycin or validoxylamine to produce valienamine, or a processed product thereof, to act on validamycin or validoxylamine. .