JP3484917B2 - Method for producing hypoxanthine - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing hypoxanthine. More specifically, it relates to a method for producing hypoxanthine using a microorganism.

[0002]

BACKGROUND OF THE INVENTION Hypoxanthine is an intermediate metabolite of nucleic acid widely existing in animals, plants, microorganisms and the like, and is a precursor of inosine and inosine acid production. Hypoxanthine also exists as a base of inosinic acid, which is used as a precursor of DNA and RNA components or as an umami component of foods, and is an important substance that forms the core of many purine-based nucleic acid metabolisms. Hypoxanthine is produced by deaminase reaction of adenine (adenine deaminase reaction) or deamidation reaction of adenine by nitrous acid, or by phosphorolysis of inosine by nucleoside phosphorylase, and when it is produced in a large amount,
The method using a deaminase requires a large amount of adenine deaminase, and the method for deamidating adenine with nitrous acid has a problem that a substance after the reaction needs to be treated.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for efficiently producing hypoxanthine using a microorganism.

[0004]

The object of the present invention is as follows.
A microorganism belonging to the genus Pseudomonas having the ability to produce hypoxanthine is cultured in an adenine-added complete medium, preferably an adenine-added complete medium whose pH is adjusted to 7.5 to 10.5 to allow hypoxanthine to be produced and accumulated, and this is collected. To be achieved.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION As a microorganism belonging to the genus Pseudomonas having an ability to produce hypoxanthine,
For example, P. solanacearum No. 206-04 (FERM P-15545) is used. This microorganism was cultivated and separated from hot spring water in Otari Village, Nagano Prefecture by the following method.

That is, L-broth as a complete medium (tryptone 1%, yeast extract 0.5%, NaCl 0.5%, pH 7.0 before sterilization) about 0.005% or more, generally 0.05% adenine 3 ml medium Was placed in a test tube, 1 ml of the hot spring water collected was added thereto, and the mixture was incubated at 40 ° C. for 72 hours with shaking, and then separated.

This P. solanacearum No. 206-04 has the following mycological properties. A. Morphology (1) Cell shape and size: bacillus, 0.5 μm × 1 μm (2) Motility: Yes (3) Spores: No (4) Gram stainability: Negative B. Growth state broth agar plate culture in medium: yellowish brown, smooth C.I. Physiological properties (1) Reduction of nitrate: Negative (2) VP test: Positive (3) Indole formation: Negative (4) Hydrogen sulfide formation: Negative (5) Citric acid utilization: Negative (6) Dye formation : Negative (7) Urease: Negative (8) Oxidase: Positive (9) Lysine degradation: Negative (10) Ornithine degradation: Negative (11) Arginine degradation: Negative (12) Growth range: pH 5-8, temperature 30-50 ℃ (13) Attitude toward oxygen: Facultative anaerobic (14) Acid production medium from sugars: Peptone 2g, NaCl 5g, K ₂ HPO ₄ 0.3g, carbohydrate 10
g, bromthymol blue 0.08g, agar 15g, distilled water 1000m
l (pH7.1) Concentration of addition: 1% D-glucose + D-mannitol + Adnit-L-arabinose + inosit-D-sorbit-maltose-sucrose +

Based on the above mycological properties,
As a result of searching by y's Mannual of Determinative Bacteriology 9th edition, it was confirmed to be a bacterium belonging to the genus Pseudomonas solanacearum .

When carrying out the method of the present invention, adjusting the pH of the adenine-added complete medium to 7.5 to 10.5, preferably 8.5 to 9.5, the hypoxanthine production rate is significantly improved. If the pH is higher than this, adenine in the medium will precipitate. The pH of the complete medium containing adenine is adjusted with an aqueous sodium hydroxide solution.

[0010]

【Example】

Example 1 P. solanacearum No. 206-04 (FERM P-15545) was added in an amount of 50 ml.
After preculturing by shaking culture in L-broth liquid medium overnight, in a culture medium 500 ml containing 0.05% adenine in L-broth liquid medium, using a rotary culture tank with a baffle plate. The main culture was carried out by culturing for 72 hours under the conditions of stirring rpm of 4000 rpm and 40 ° C.

After carrying out these series of operations, the strain was extracted from the culture medium using a centrifuge, and the amount of hypoxanthine produced was quantified by HPLC. ODS-80 as a column
TM was used and 0.5M ammonium formate was used as a developing solvent, and the column was developed at a column temperature of 35 ° C. And the detection wavelength 254n
Hypoxanthine production was quantified using m-
It was confirmed that hypoxanthine was produced at a production amount of about 800 to 900 μg per 1 ml of a culture medium containing 0.05% adenine in th liquid medium.

When a complete medium containing no adenine was used, hypoxanthine was not produced.

Example 2 3 ml of a culture medium prepared by adding 0.05% adenine to L-broth liquid medium (pH 7.0) was placed in a test tube, and P. solanac was added to the medium .
earum No.206-04 the (FERM P-15545) was added, to obtain a pre-culture broth is performed for 24 hours with shaking cultured under conditions of 37 ° C., then, pH of adenine added complete medium with sodium hydroxide solution Was adjusted within the range of 7.5 to 10.0, 1.5 ml of the preculture liquid was added to 8.5 ml of this medium, and shaking culture was again carried out at a culture temperature of 37 ° C. for 100 hours.

The relationship between the pH of the complete medium containing adenine and the production rate of hypoxanthine is shown in the following table. The adenine assimilation rate in this case is 0.75 to 1.66 μg / ml ・ hr, and p
The value was higher than 0.65 μg / ml · hr at H7.0. pH Hypoxanthine production rate (μg / ml ・ hr) 7.0 0.65 7.5 0.75 8.0 0.91 8.5 1.31 9.0 1.66 9.5 1.54 10.0 1.16

[0015]

INDUSTRIAL APPLICABILITY A microorganism belonging to the genus Pseudomonas having the ability to produce hypoxanthine can efficiently and inexpensively produce hypoxanthine, which is a precursor of inosine and inosine acid used as umami components.

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Claims (2)

(57) [Claims] 1. A method for producing hypoxanthine, characterized in that a microorganism belonging to the genus Pseudomonas having the ability to produce hypoxanthine is cultured in a complete medium containing adenine to produce and accumulate hypoxanthine, which is then collected. . 2. The method for producing hypoxanthine according to claim 1, wherein an adenine-added complete medium having a pH adjusted to 7.5 to 10.5 is used.