JPS594996B2 - Production method of inosine by fermentation method - Google Patents

Description

[Detailed description of the invention] This invention relates to a method for producing inosine by fermentation.

Conventionally, inosine has been produced using a mutant strain of the genus Bacillus.As a result of our study to further improve the conventional production method, we have developed a mutant strain of the genus Bacillus that has the ability to produce inosine. It was found that a mutant strain endowed with stronger 51-nucleotidase (3135) activity than the parent strain produces inosine at a higher yield.

That is, the present invention involves culturing a mutant strain belonging to the genus Bacillus that requires adenine, has stronger 5'-nucleotidase activity than the parent strain, and has the ability to produce inosine, and inosine produced and accumulated in the medium. This is a method for producing inosine by a fermentation method, which is characterized by collecting inosine.

The microorganisms used in the method of the present invention have the above-mentioned properties, and such microorganisms can be mutated from their parent strains by conventional methods.

Schoonifugu mutants with strong 51-nucleotidase activity can be easily produced by selecting strains that release phosphate at a high rate using 5'-nucleotides as substrates.

Furthermore, if the mutant strain of the present invention is further endowed with properties that are conventionally known to increase inosine production, such as amino acid requirement, purine analog resistance, and 51-guanylate reductase deficiency, the production capacity will be increased. Highly mutant strains can be obtained.

Parent strains used to induce the mutant strains of the present invention include Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, and the like, which are conventionally known to produce inosine.

Next, a specific example of mutagenic induction of such a microorganism will be shown.

Experimental example Bacillus subtilis AJ11045 (F'ERM-P
3589 was treated with N-methyl-N'-nitro-N-nitronganidine (1000[gamma]/-) at 0[deg.]C for 40 minutes and then irradiated with ultraviolet light for 1 minute.

This was applied to the following medium, and the resulting colonies were sprayed with 8 mM p-nitrophenyl phosphate solution, and the strains that produced large yellow halos were collected.

From among the strains with strong alkaline phosphatase activity obtained in this way, the rate of release of inorganic phosphoric acid using 5'-IMP as a substrate was measured.
Nucleotidase activity was measured.

Thus, AJ11043 (F'
BRM-P3587) was obtained.

A mutant strain AJ11044 (FERM-P35) with stronger 57-nucleotidase than AJ11046 (FBRM-P3590) was obtained using the same method as above.
88) was obtained.

Medium Nidris(hydroxymethyl)aminomethane 3FJ/13
Nail 3g/1
Mg504・7H200,259/1 0a012・2H200,013fj/l glycose 2g/II peptone
29/l adenine
0.1 g/1 (nH8,o) The specific activity of the 57-nucleotidase of the mutant strain of the present invention relative to the parent strain is as shown in Figure 1. This is a conventional medium containing sources, inorganic ions, adenine and other required substances, and other appropriate organic micronutrients as required.

As the carbon source, carbohydrates such as glucose and sucrose, alcohols such as ethanol, glycerin and sorbitol, and organic acids such as acetic acid can be used as appropriate.

As the nitrogen source, ammonium salt, ammonia water, ammonia gas, etc. are used.

The best culture conditions are aerobic conditions, and the fermentation temperature is 2.
The culture time is usually 2 to 5 days at 5°C to 40°C.

In some cases, it may be a good idea to change the culture temperature slightly during the culture.

The pH at the start of culture and during culture should be between 5.0 and 9.0 (
To adjust the pH, an inorganic acid, an organic acid, an alkaline substance, calcium carbonate, ammonia gas, aqueous ammonia, urea, etc. can be used.

To collect inosine from the culture solution, the bacterial cells are separated and removed, the supernatant is treated with an ion exchange resin, the inosine fraction is collected and concentrated, and an organic solvent is added.

Inosine precipitated by this operation is removed from water or an organic solvent (80%
EtoH).

The thus obtained purified crystals were found to be consistent with pure guanosine based on elemental analysis, ribose coloration, ultraviolet absorption curve, and infrared absorption curve.

Example 1 Seed medium glucose 6 #/dllNH4
C10,3y7di KH2PO40,059/d1 MgS04・7H200°04fJ/dl Dog bean protein acid hydrolyzate 4 7727! /dll ribonucleic acid
0.5 g/dl xanthine
5 ■/dJI? Mn50 ・4H201
~/d1 Fe50 ・7H201m9/d1 pH 7,4 (KOH) Main fermentation medium glucose 8 fJ/dllN
H4C11,5g/dll' KH2PO40,05,! iZ/dA MgSO,・7 H2O0,049/dllFeSO4
・7H201■/d1 MnS04・4H201"9/dA? KC!1 2.1 g/dll
Ribonucleic acid 0.1 g/di! Xanthine 5 ■/d7DL-methionine 0.03g/d7 soybean protein hydrolyzate
3.2 d/dllCaC03 (separate sterilization)
2 El/di! pH 7.0 (KOH) Fill a 500ml shaking flask with 50rnl of the above seed medium, inoculate it with the microorganisms shown in Table 2, and incubate at 34°C for 16 hours.
Cultured with shaking for hours.

On the other hand, the main fermentation medium 20- is 500m71! Place in a shaking flask, inoculate with 1d of the above seed culture solution, and incubate at 34°C for 7 days.
The culture was incubated with shaking for 2 hours.

Inosine in the culture solution was separated by paper chromatography, extracted with 0.1 NHO1, and the absorbance at 250 mμ was measured. As a result, 2.0 g/dl of inosine was produced and accumulated.

A culture solution 11 of AJ11044 was prepared in the same manner as above, the bacterial cells were removed, and the filtrate was treated with an ion exchange resin in a conventional manner, and then the inosine fraction was collected and concentrated.

Crude inosine crystals were obtained, recrystallized, and purified to obtain 12 g of crystals.

Claims (1)

[Claims] 1. Cultivate a mutant strain that belongs to the genus Bacillus, requires adenine, has stronger 5'-nucleotidase (3135: activity) and inosine-producing ability than the parent strain, and collects inosine produced and accumulated in the medium. A method for producing inosine by a fermentation method, characterized by: