JP2830378B2 - Production method of adenosine by fermentation method - Google Patents

Description

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

[Conventional technology]

Regarding adenosine production by fermentation, a method of producing adenosine by Bacillus subtilis having isoleucine requirement (Japanese Society of Agricultural Chemistry, 1964), a requirement for xanthine, histidine and threonine, and 8-
Adenosine production method using Bacillus subtilis having azaxanthin resistance (Agric. Biol. Chem., 35 , 1906 (197
1)), and a method using a mutant strain having guanine auxotrophy and having resistance to 8-azaadenine or 6-methylaminopurine (Japanese Patent Application No. 58-185313) was known.

However, all of these production methods have low adenosine accumulation concentration and have a problem in the stability of the strain.

[Problem to be Solved by the Invention] An object of the present invention is to provide a fermentation method of adenosine by a microorganism having a higher adenosine producing ability.

[Means for solving the problem]

The present inventor has conducted intensive studies to solve the above-described problems,
It belongs to the genus Bacillus and has been found that the above-mentioned object can be achieved by using a mutant having isoleucine auxotrophy, histidine auxotrophy and arginine auxotrophy, and a mutant having pyrimidine analog resistance, and completed the present invention. .

That is, the present invention is a microorganism belonging to the genus Bacillus, having isoleucine auxotrophy, histidine auxotrophy and arginine auxotrophy, and having adenosine producing ability, or belonging to the genus Bacillus, having pyrimidine analog resistance, and producing adenosine. The present invention relates to a method for producing adenosine by a fermentation method, which comprises culturing a microorganism having an ability to produce and accumulate adenosine in a culture solution and collecting the adenosine.

 Hereinafter, the present invention will be described in detail.

The strain that can be used in the present invention is not particularly limited as long as it is the above-mentioned microorganism, and the microorganism can be collected, for example, as follows. That is, a strain having the guanine requirement of Bacillus subtilis and having 8-azaadenine resistance and 6-methylaminopurine resistance is subjected to mutation treatment (N-methyl-N'-nitro-N-nitrosoguanidine and the like) by an ordinary method. ) And collect strains that require guanine and amino acids for growth of the strains, or various drug-resistant strains. From among these, screening can confirm that a strain that requires isoleucine, histidine, or arginine for growth or a strain that has primidine analog resistance accumulates a significant amount of adenosine in the culture solution. As a primidine analog, 4-
Aminopyrazolo [3,4-α] -pyrimidine, 4-amino-6-mercaptopyrazole [3,4-d] pyrimidine,
It is selected from 4-aminopyrazolo [3,4-d] pyrimidine 2'-deoxy-ribosite and the like.

The strains thus obtained are, for example, those requiring guanine, 8
Bacillus subtilis AJ12050 having resistance to azaadenine and resistance to 6-methylaminopurine (FERM P-7149)
Is a parent strain, and the following strains are obtained by mutating the parent strain.

(1) A strain having an isoleucine-, histidine-, or arginine-requiring requirement: Bacillus subtilis AJ12518 (FERM P-11473) (2) A strain having 4-aminopyrazolo [3,4-d] -pyrimidine resistance: Bacillus subtilis AJ 12519 (FERM P-11474) Experimental examples showing the requirements for amino acids and the degree of drug resistance of the strains exemplified in the present invention are shown below.

Experimental Example Amino acids were added to the following basic medium at the concentrations shown in Table 1.
In addition, a medium was prepared by adding each of the drugs to the concentrations shown in Table 1, 3 ml was dispensed into test tubes, sterilized, inoculated with each strain, and shake-cultured at 34 ° C for 20 hours. .

Basic medium Glucose 2 g / dl Ammonium chloride 0.5 第一 Potassium phosphate 0.1 マ グ ネ シ ウ ム Magnesium sulfate 0.04 ソ ー Sodium citrate 0.05 Ｌ L-glutamic acid 0.1 〃 Guanine 0.01 〃 Fe ⁺⁺ , Mn ⁺⁺ (each) 2 ppm pH (KOH 7.0) The growth status of each strain obtained by this culture is shown in Tables 1 and 2 as relative growth values.

The following method is used to produce adenosine using these strains. The medium is an ordinary liquid medium containing a carbon source, a nitrogen source, inorganic salts, guanine and, if necessary, other micronutrients. As the carbon source, carbohydrates such as glucose, molasses and starch hydrolysate, organic acids such as acetic acid, propionic acid, pyruvic acid and citric acid, alcohols such as ethanol and propanol, and also hydrocarbons depending on bacteria can be used. . As the nitrogen source, inorganic nitrogen such as ammonium sulfate such as ammonium sulfate, ammonium nitrate, salt ammonium, and phosphorus phosphate, nitrate, urea, and an ammonium gas or casein hydrolyzate, and organic nitrogen such as amino acid can be used. Further, as guanine as an auxotroph, any of guanine, guanine mineral salt, guanosine, guanylic acid, ribonucleic acid and the like can be used. Also,
Addition of trace nutrients such as vitamins, amino acids and nucleobases as needed can often increase the amount of adenosine accumulated. In particular, when the strain requiring the amino acid of the present invention is used, the required substance must be added. The cultivation method has good aerobic conditions, and the cultivation temperature is 20 ° C to 40 ° C.
The range of ° C is good. In some cases, the fermentation temperature may be slightly changed during fermentation. PH at the start and during the culture
Is preferably adjusted to an optimal value in the range of 5.0 to 9.0. The pH can be adjusted using an inorganic acid, an organic acid or an alkali, as well as urea, calcium carbonate, aqueous ammonia, ammonium gas and the like. Thus 2-5
After culturing for days, a significant amount of adenosine accumulates in the medium. Further, by controlling the concentration of the residual carbon source to 0.5 to 2 g / dl and continuously feeding the carbon source, the accumulation of adenosine can be significantly increased.

In order to collect adenosine from the fermentation broth, for example, cells are separated and removed, and adenosine is isolated by treating with an anion exchange resin, a cation exchange resin or the like, or by using a combination of concentration and cooling crystallization. Alternatively, purification may be performed using a conventional activated carbon adsorption method and a recrystallization method.

 Hereinafter, the present invention will be further described with reference to examples.

〔Example〕

One loopful of the strains shown in Table 4 was inoculated into a 500 ml flask containing 500 ml of a seed medium having the composition shown in Table 3 at 34 ° C.
Cultured for hours.

1 ml of this culture solution was added to a 500 ml flask containing 20 ml of the main fermentation medium, and cultured at 34 ° C. for 72 hours. When adenosine in this culture solution was quantified by liquid chromatography, the amount of adenosine shown in Table 4 was produced and accumulated. Furthermore, by controlling the residual sugar to 0.5-2.0 g / dl in the culture of AJ-12519 and continuously feeding the carbon source, the accumulation of adenosine could be increased to 18.0 g / (culture 90 hours).

Culture solution 1 of strain AJ12519 obtained by the same method as above
After removing the cells from 0 by centrifugation, the supernatant was concentrated to 2 using an evaporator. The concentrate was left at 5 ° C. to generate adenosine crystals. The obtained adenosine crystals were melted in water, further adsorbed on activated carbon, eluted by a conventional method, and the adenosine fraction was collected, concentrated, cooled and crystallized to obtain 99 g of adenosine crystals.

[Effects of the Invention] By using the strain of the present invention, the accumulated amount of adenosine is improved, so that practical use at an industrial level is expected.

Claims (2)

(57) [Claims] (1) a member belonging to the genus Bacillus, which is required for isoleucine;
A method for producing adenosine by a fermentation method, comprising culturing a microorganism having histidine requirement and arginine requirement and having adenosine producing ability to produce and accumulate adenosine in a culture solution, and collecting the resulting adenosine. 2. A fermentation method comprising culturing a microorganism belonging to the genus Bacillus, having pyrimidine analog resistance and having adenosine producing ability to produce and accumulate adenosine in a culture solution, and collecting the resulting adenosine. Method for producing adenosine.