JPS58111695A - Preparation of 5'-inosinic acid by fermentation - Google Patents

Abstract

PURPOSE:To ferment and to prepare the titled substance efficiently, by cultivating a variant belonging to the genus Corynebacterium, having adenine demanding properties and glutamin analog resistance. CONSTITUTION:Corynebacterium equi AJ 11347, Corynebacterium equi AJ 11349, etc. as a parent strain is subjected to common variation induction procedure, to give a variant such as Corynebacterium equi AJ 11744, Corynabacterium equi AJ 11749, etc. having adenine demanding properties and glutamin analog resistance. The variant is cultivated in a liquid medium containing a carbon source, nitrogen source, adenine, etc. The cultivation is carried out under aerobic conditions at 24-40 deg.C at a pH of 5-9, and the collection of 5'-inosinic acid is carried out by removing a mold from the culture, treating the filtrate with both a decoloring resin and an anion exchange resin, to give an aqueous solution containing 5'-inosinic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 5"-inosinic acid by a fermentation method, and more particularly, it relates to a method for producing 5"-inosinic acid using a saccharide as a carbon source and directly producing 51-inosinic acid using a mutant strain of the genus Corynebacterium. Concerning efficient fermentation and production methods.

Conventionally, the method for producing 5°-inosinic acid, which has been widely used as a seasoning, involves converting hypoxanthine or inosine into 5'-inosinic acid by the action of microorganisms (Japanese Patent Publication No. 39-29858, Japanese Patent Publication No. 42-1186). , Special public school 4th year
5-37078) etc. were known.

In recent years, methods for converting inosine, etc. have been developed,
A method has been developed for directly fermenting and producing 5°-inosinic acid from sugars using mutant strains of the genus Bacillus or Corynebacterium (Japanese Patent Application Laid-open No. 55-150899, Japanese Patent Publication No. 56-32918).

The present inventors have conducted intensive research to develop a method for efficiently producing 51-inosinic acid from K using a direct fermentation method, and have found a mutant strain belonging to the genus Corynebacterium that has adenine auxotrophy and glutamine analog resistance. 5 of the amount stored in
The inventors discovered that a large number of mutant strains that produce 1-inosinic acid can be obtained, leading to the completion of the present invention. That is, the present invention involves culturing a mutant strain belonging to the genus Corynebacterium that has adenine auxotrophy and glutamine analog resistance and has the ability to produce 5°-inosinic acid in a medium to produce and accumulate 51-inosinic acid. The present invention relates to a method for producing inosinic acid, which is based on a fermentation method characterized in that the 5-inosinic acid is collected.

The microorganism used in the present invention is of the genus Srinebacterium genus t.
A mutant strain having adenine auxotrophy, glutamine analog resistance, and the ability to produce 51-inosinic acid is used. The following mutant strains are preferably used.

Corynebacterium ginsengii AJ117.4,000 “F
ERM-P 6256 (Ade”-, AZSr) Corynebacterium equii AJ 11749
FERM-P 6261 (Ade, DON
r) Corynebacterium equii AJ 11747
FERM P 6259 (Ade-, scr,
MSOr) Ad@”-: adenine auxotrophic AZ8r: azaserine resistant DONr: 6-shi7so5-t+7-L −/leleucine resistant SGr: sulfaguanidine resistant MSOr=methionine sulfoxide resistant The mutant strain of the present invention is , 51-inosine producing bacteria of the genus Mu, such as the adenine-auxotrophic Corynebacterium equii AJ11347 FERM-P4968, the adenine-auxotrophic and sulfaguanidine-resistant Corynebacterium equii AJ11349FERM-P4970
is used as a parent strain, and subjected to usual mutagenesis operations such as ultraviolet rays, X-ray irradiation, or NG (N-methyl-N
' -n1tro -N -n1tros.

guanidlne ) + DAPA ( Sodlu
m-P-dimsthylaminobenzene
diaso-5ulfonate), nitrous acid, etc., and then cultured on an agar plate containing an amount of glutamine analog that makes it impossible for the parent strain to grow, and select Comini to grow on the plate. This is what you get.

Examples of the above-mentioned parent strains include wild strains of the genus Corynebacterium, such as Corynebacterium equii AJ1723, Corynebacterium sp. It can also be obtained by using the parent strain as a parent strain and imparting choleniadenine auxotrophy and glutamine analog resistance.

The following experimental examples show the degree of resistance of the mutant strains of the present invention to glutamine analogs.

Experimental Example Liquid medium with the composition shown in Table 14. O and l were dispensed onto the test layer and sterilized by heating. A sterile-filtered glutamine analog aqueous solution was added to this to prepare a solution containing glutamine analogs at the concentrations shown in Table 2.

Guru w7su 10 g/z Mn5O*
・4H*O:, tomVz ammonium sulfate 2.
o Biotin 200μt/1MgSO4
・7H20G, 5 tt p-7mi/benzoic acid
1 mg/7K) I, Po,
1. Ott no vto'241 power tv
-nmu 1 〃FeSO4-nt, o
lie n Kazami, no acid 0.
5 t/lKW, PO41,0// 7 Denine so
mg/l*+Ll+1 @71j,',' ・-
Inoculate a certain amount of test bacteria cultured in a minimal medium without adding drugs to the above medium and shake at 31.5 U for 24 hours.
'Cultivated. The absorbance of each culture solution at 590 nm was measured to determine the growth rate. The results are shown in Table 2.

Table 2 Relative growth rate AJ11549. 100 6 3 5A
Jl1744100 74 3 6AJl1
749 100 5 92 5AJ1174
7 100 5 2 86 The glutamine analog used in the present invention refers to one whose inhibition of the growth of microorganisms of the genus Corynebacterium is partially or completely relieved by the coexistence of glutamine, and specifically, glutamine Examples include 5-oxo-L-norleucine (hereinafter abbreviated as DoN) and methionine sulfoxide (hereinafter abbreviated as 1.MSO).

The medium for culturing the mutant strain of the present invention is a conventional liquid medium containing a carbon source, a nitrogen source, inorganic salts, adenine and, if necessary, other micronutrients. As the carbon source, carbohydrates such as glucose, molasses, and starch hydrolyzate, organic acids such as acetic acid and gluconic acid, and alcohols such as ethanol can be used. As a nitrogen source, ammonium sulfate,
Ammonium nitrate, ammonium chloride, ammonium phosphorus, ammonia gas, etc. can be used.

Further, as the adenine as a nutritional substance, any of adenine, adenine mineral salt, adenosine, adenylic acid, ribonucleic acid, etc. can be used. Furthermore, if micronutrients such as vitamins, amino acids, and nucleobases other than adenine are added as necessary, the amount of 51-inosinic acid accumulated can often be increased.

The cultivation method is preferably carried out under aerobic conditions, and the cultivation temperature is preferably in the range of 24 to 40C. Depending on the situation, the fermentation temperature may be slightly changed during fermentation.

It is desirable to adjust the pH of the culture solution to 5.0 to 9, Or-, at the start of the culture and during the culture. For pH adjustment, inorganic acids, organic acids or alkalis, as well as urea, calcium carbonate, aqueous ammonia, ammonia gas, etc. can be used. Thus, a significant amount of 5'-inosinic acid accumulates in the medium after 2 to 7 days of culture.

In order to collect 51-inosinic acid from the fermentation liquid, for example, the bacterial cells are separated and removed, and the filtrate is treated using a combination of a decolorizing resin and an anion exchange resin, or a combination of an anion exchange resin and a cation exchange resin. In this case, a relatively pure aqueous solution containing 5-inosinic acid can be obtained.

Examples will be described below.

Example 1 A medium having the composition shown in Table 3 was dispensed into 20w1 500 d shoulder flasks and sterilized by heating at 120C for 10 minutes.

Table 3 (pH 6,5) 1 bottle of this medium, 2 ounces of the bacterial strains shown in Table 4/1 course
/As yeast extract 1 t/l, peptone lot/l, salt 5 t/l, agar 2 ot/l (pH 7,2) slant culture medium for 18 hours at 31.5 U. Three platinum loops were inoculated and cultured with shaking at 34C for 5 days. The amount of 5I-inosinic acid accumulated in the culture medium was
As shown in the table.

Table 14 Accumulated amount p of 51-inosinic acid: J 11 s 49 6.3 AJ
11744 11.4A
J11749 15.9A
J11747. 14.7 Example 2 The liquid medium asomg in Table 3 was mixed with 1. It was placed in an Ot capacity jarp amentor and sterilized by heating at 120C for 10 minutes. This was inoculated with seed bodies (3 slants) of Corynebacterium equii AJ 11744 prepared in the same manner as in Example 1, and 34t? ventilation for 51 minutes (t/
s V, V, M, ), stirring (so.

rpm) culture was performed. In addition, the p of the culture solution during the culture period
H was adjusted to 7.01 using ammonia gas.

After the culture is completed, 5°-inosinic acid is present in the culture solution at 11.1%.
I had accumulated 9/. 1 ton of this culture solution was centrifuged to remove bacterial cells, and the clarified solution was collected at pi+3. o and adsorbed onto activated carbon. After washing with water, it was eluted with 50% aqueous methanol solution ioz and concentrated to a volume of 100 mA'. Next
After adjusting the pH to 7.5 with NaOH and adding the same amount of methanol, 5'-IMPφ2Na・7.5H,0 was converted to crystal 8.
．． 8f was obtained.

Patent Applicant Ajinomoto Co., Ltd. Procedural Amendment - (Method) 1. Indication of the case Patent Application No. 208753 of 1982 2. Process for manufacturing 5-inosinic acid by method of naming the invention #1 3. Person filing the amendment Relationship to the incident Patent applicant address: 5B Kyobashi-chome, Chuo-ku, Tokyo (Date of shipment)
(April 21, 1975) 5. Number of inventions increased by amendment None 1. Contents of amendment (1) Title of invention on page 1 of specification
``Method for producing inosinic acid'' is corrected to ``Method for producing 5'-inosinic acid by fermentation method''.

(2) Line 9 from the bottom of the first specification, “Claims”
should be corrected to read "detailed description of the invention."

-5:

Claims (1)

[Claims] A mutant strain of the genus Corynebacterium that has adenine auxotrophy and higlutamine analog resistance and has the ability to produce 5'-inosinic acid is cultured in a medium to produce and accumulate 5°-inosinic acid, A method for producing 5°-inosinic acid by a fermentation method, which comprises collecting 5°-inosinic acid.