JPS5934891A - Preparation of azaserine by fermentation - Google Patents

Abstract

PURPOSE:To prepare azaserine useful as an antibacterial agent and antitumor agent, by culturing a microbial strain belonging to Microtetraspora genus. CONSTITUTION:Microorganism belonging to Microtetraspora genus and capable of producing azaserine, e.g. Microtetraspora glauca ATCC23057, is inoculated in a nutrient medium, and cultured at 25-40 deg.C, preferably 28-38 deg.C, at 4-10pH, preferably 6-8pH, for about 1-7 days in a liquid medium, and the objective azaserine is separated from the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing azaserine. More specifically, a fermentation method is characterized in that a microorganism belonging to the genus Microtetraspora and capable of producing azaserine is cultured in a nutrient medium, azaserine is accumulated in the culture, and the accumulated azaserine is collected from the culture. relates to a method for producing azaserine.

, azaserine is known as a substance having antibacterial and antitumor activities. Conventionally, azaserine has been produced using a strain belonging to Streptomyces fragilis, which has been reported along with the physicochemical properties of azaserine (Index of Antib
iotics from Actinomyotes,
167 pages, 1967, Handbook of An
Tibiotio Compounds Vow IV
part 145, 1980).

As a result of research on antibacterial substances produced by microorganisms of the genus Microtetraspora, the present inventor found that Microtetraspora glauca (Microtetraspora g'1auca)
) was found to produce azaserine, and the present invention was completed.

The present invention will be explained in detail below.

Any microorganism can be used as long as it belongs to the genus Microtetraspora and has the ability to produce azaserine.

Examples of suitable bacteria include Microtetraspora, Glauca ATC
C23057 is mentioned.

For culturing, a normal method for culturing actinomycetes is generally used. As the nutrient source, one containing a carbon source, a nitrogen source, a heat source, etc. is used.

As the return element source, glucose, starch, dextrin, mannose, fructose, sucrose, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, glycerin, organic acids, etc. may also be used. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate,
such as urea, or natural nitrogen-containing substances such as peptone,
Meat extract, yeast extract, dry yeast, corn steep
Soybean powder, soybean flour, casamino acids, etc. may be used alone or in combination. If necessary, add inorganic salts such as common salt, potassium chloride, calcium carbonate, phosphates such as potassium chloride, dipotassium phosphate, and alkali metal salts.
Organic or inorganic substances that promote the growth of the bacteria used and the production of azaserine can be appropriately added.

The most suitable culture method is a liquid culture method, especially a deep agitation culture method. The culture temperature is 25 to 40°C, especially 28 to 38°C, and the pH of the culture medium is adjusted to pH 4 to io, preferably 6 by adding aqueous ammonia or aqueous ammonia carbonate.
It will be held at ~8. When culture is carried out in liquid culture for usually 1 to 7 days, the target substance is produced and accumulated in the culture solution. When the production amount in the culture solution reaches the maximum, the culture is stopped, the bacterial cells are separated, and the target product is purified and isolated from the obtained culture solution.

The single MM 14 of azaserine from culture fluid utilizes separation and purification methods commonly used to isolate microbial metabolic products from their culture fluids.

Aspects of the present invention will be explained below using examples.

Example 1 Microtetraspora glauca ATCC23 as inoculum
057 is used. The strain was grown in a seed medium [20 liters of dextrin/1. Glucose 101/
l, peptone 10 f/l, :17-y ・Steep liquor 5 f/l, yeast extract 1 f/l, KH
2PO40,5f/t.

MySOa ・7Hz OO, medium pH 7.2 (before sterilization)) 300rJ with the composition of 28
Culture with shaking (220r, p, m, ) for 24 hours at ℃.

The obtained seed culture was inoculated at a rate of 5% (volume) into 15 tons of fermentation medium having the following composition in a jar fermenter with a capacity of 30℃, and was incubated at 30°C with aeration stirring (rotation speed 250 r, p
, m,; aeration rate 15 t/min).

Fermentation medium composition: glycerin 20 W/l, glucose 1
0f/1. , cottonseed meal 20 f/l, :I-7・steep liquor 5 f/l, 'dry yeast 10 f/t,
K2HPO40,5f/1. My 804・7H
z OO, 5f/l, pH 7.2 (before sterilization) with NaOH
Adjust with.

The pH of the medium was adjusted to pH 6.5-7. using ammonia water.
Adjust to 5. As a result of culturing for 72 hours, azaserine was used as a standard in the culture solution, and the value converted to this was 30μr/r.
Antibacterial substances were accumulated at the concentration of PLe.

Bacterial cells and precipitates were separated from the culture solution to obtain 13 tons of sap. This was packed with activated carbon (2t) and adsorbed into nine columns.
The active fraction was washed with water and eluted with 50% methanol, and after distilling off the methanol, it was freeze-dried to form a powder. This was dissolved in water-saturated butanol and developed with the same solvent in a silica gel column (500 resistance) packed with the same solvent.

The active fractions were collected and the solvent was distilled off, and then the solvent was distilled off.
0 and collect the active fraction by developing with the same solvent. By concentrating and drying these, 8 capes of diazaserine powder are recovered.

The physicochemical properties of the obtained azaserine are as follows, and are consistent with those of the standard product.

Elemental analysis value C: 34.80%, N: 24.409
g (actual value) H: 4.20'

Claims (1)

[Claims] Production of azaserine by a fermentation method characterized by culturing a microorganism belonging to the genus Microtetraspora and having azaserine-producing ability in a nutrient medium, accumulating azaserine in the culture, and collecting the accumulated azaserine from the culture. Law.