JPH05317076A - Production of adenosine by fermentation - Google Patents

Abstract

PURPOSE:To obtain adenosine in high efficiency at a low cost compared with conventional fermentation process by culturing a specific mutant belonging to the genus Bacillus in a liquid medium. CONSTITUTION:A mutant belonging to the genus Bacillus, requiring guanine for growth, resistant to thioadenyl sulfate and capable of producing adenosine [e.g. Bacillus subtilis AJ12708 (FERM P-12952)] is cultured in a liquid medium and the adenosine formed and accumulated in the culture liquid is separated therefrom. The cultivation is usually carried out at pH4-10 and 20-40 deg.C under aerobic condition for 2-5 days. The collection of adenosine from the culture liquid is carried out e.g. by separating bacterial cells from the liquid and treating the liquid with an ion-exchange resin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing adenosine by fermentation.

[0002]

2. Description of the Related Art Conventionally, with respect to adenosine production by fermentation, a method using Bacillus subtilis having an isoleucine requirement as a method for directly producing adenosine from a normal carbon source or a nitrogen source (FY 1937 Meeting), a method using Bacillus subtilis having xanthine, histidine and threonine requirement and having resistance to 8-azaxanthine (Agric. Biol. Che
m., 35 , 1906 (1971)), or belonging to the genus Bacillus,
There is known a method of using a mutant strain that requires guanine for growth and has resistance to 8-azaadenine and / or 6-methylaminopurine (JP-A-60-78593). However, all of these production methods have low adenosine productivity, and it has been desired to develop a production method of adenosine which is more advantageous in industrial production.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing adenosine by a fermentation method which is cheaper and more efficient than conventional methods.

[0004]

[Means for Solving the Problems] As a result of intensive studies to develop a method for producing adenosine by a fermentation method which is industrially advantageous over the conventional method, the present inventors have found that guanine auxotrophy belonging to the genus Bacillus is required. It has been found that the adenosine producing ability is dramatically improved by imparting thioadenyl sulfate resistance to an adenosine producing bacterium, and the present invention has been completed based on this finding.

That is, the present invention belongs to the genus Bacillus,
Fermentation method characterized by culturing a mutant strain that requires guanine for growth, has thioadenyl sulfate resistance and adenosine production ability in a liquid medium, adenosine is produced and accumulated in the culture medium, and is collected To provide a method for producing adenosine.

The mutant strain used in the present invention belongs to the genus Bacillus, requires guanine for growth, and has thioadenyl sulfate resistance and adenosine producing ability. Specific examples thereof are as follows. Is mentioned. Bacillus subtilis AJ12708 (F
ERM P-12952)

In addition, isoleucine auxotrophy for the above strains, 8
-Azaxanthine resistance, 8-azaadenine resistance and 6-
By imparting properties such as resistance to methylaminopurine, a strain having a higher adenosine-producing ability can be obtained, and such strains are included in the mutant strain of the present invention.

[0008] Such a mutant strain of the present invention uses a guanine-requiring adenosine-producing bacterium belonging to the genus Bacillus as a parent strain, and a normal mutation-inducing operation such as irradiation with ultraviolet rays or X-rays, or N-methyl- N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as NG), agar plate medium containing a concentration of thioadenyl sulfate treated with a chemical agent such as nitrous acid and mutated to prevent the parent strain from growing. It can be obtained by culturing in a plate and separating colonies that grow on the plate medium.

Known guanine-requiring adenosine-producing bacteria belonging to the genus Bacillus may be used, and the following strains are specifically used. Bacillus subtilis AJ12707 (FERM P-1295
1)

The method for obtaining the mutant strain used in the present invention and the degree of resistance to thioadenyl sulfate are shown in the following experimental examples.

Experimental Example A guanine-requiring adenosine-producing bacterium AJ12707 derived from Bacillus subtilis ATCC6643 was cultured in a natural slant medium, and the grown cells were suspended in 100 mM phosphate buffer (pH 7.0) (10 per 1 ml). ⁹ to 1
( ¹⁰ cells were contained), NG was added to this so that the concentration was 250 μg / ml, and the mixture was kept at 34 ° C. for 30 minutes. After the NG-treated bacterial cells were thoroughly washed with the same buffer, they were applied to a minimal agar plate medium having the composition shown in Table 1 containing 500 μg / ml thioadenyl sulfate,
Culturing was carried out at 4 ° C for 4 days. A large one of the colonies grown on the plate was collected as a thioadenyl sulfate resistant strain. Among the resistant strains thus obtained, many were found to have more excellent adenosine-producing ability than the parent strain, and AJ12708 was obtained as one of them.

[0012]

[Table 1]

The resistance of the thus obtained mutant strain of the present invention and the parent strain thereof to thioadenyl sulfate was examined by the following method. Table 3 shows the minimum medium having the composition shown in Table 2.
Each concentration of thioadenyl sulphate was added to the test tube in an amount of 4 ml and sterilized by heating, and then inoculated with the cells of each strain thoroughly washed with the minimal medium shown in Table 2
Shaking culture was performed for 0 hours. After completion of the culture, 560 of the culture solution
Absorbance at nm was measured. Table 3 shows the growth rate in each medium as a relative value.

[0014]

[Table 2]

[0015]

[Table 3]

The liquid medium used in the present invention is a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, guanine and, if necessary, organic micronutrients such as amino acids, vitamins and nucleic acids. As the carbon source, it is sufficient that the mutant strain to be used can be used, sugars such as glucose, sucrose, fructose and maltose, starch saccharified solutions containing these sugars, sugar cane molasses, beet molasses, high test molasses, etc. Further, organic acids such as acetic acid and alcohols such as ethanol and glycerin are also used.
As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates and the like are used, as other organic nitrogen sources used auxiliary, oil meal, soybean hydrolyzate, corn steep liquor, yeast extract, Examples include peptone. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are appropriately used. As guanine as a nutrient, any of guanine, guanine mineral acid salt, guanosine, guanylic acid, liponucleic acid and the like can be used. In addition, the requirement for amino acids and the like may be imparted to the mutant strain of the present invention to further improve the yield of adenosine. In that case, it is necessary to supplement the required substance.

Cultivation of microorganisms is usually carried out under aerobic conditions at a pH of 4 to 10 and a temperature of 20 to 40 ° C.
The pH of the culture solution is adjusted with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas or the like. Thus, by culturing for 2 to 5 days, a significant amount of adenosine is produced and accumulated in the culture solution.

A method for collecting adenosine from the culture broth may be carried out according to a known method. For example, cells are separated and removed, and an ion exchange resin treatment method, a concentrated cooling crystallization method, a membrane separation method, or another method is combined. Performed by. In order to remove impurities, it may be purified by a conventional activated carbon adsorption method and reconstitution method.

[0019]

The present invention will be described in more detail with reference to the following examples.

Example 1 20 ml of a medium having the composition shown in Table 4 was dispensed into a 500 ml shake flask and heat-sterilized. Bacillus subtilis AJ1270 previously grown on a natural agar slant medium
7 and AJ12708 were inoculated in an amount of 1 platinum loop, and shake culture was performed at 34 ° C. for 72 hours. After the culture was completed, the amount of adenosine produced and accumulated in the culture medium was quantified by high performance liquid chromatography, and the results are shown in Table 5.

[0021]

[Table 4]

[0022]

[Table 5]

Claims (1)

[Claims] 1. A mutant strain belonging to the genus Bacillus, which requires guanine for growth, and has thioadenyl sulfate resistance and adenosine producing ability, is cultured in a liquid medium, and adenosine is produced and accumulated in the culture medium. A method for producing adenosine by a fermentation method, which comprises collecting