JPS5818076B2 - Method for producing ribonucleic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing Nyororibonucleic acid by fermentation.

Ribonucleic acid is a substance for which an inexpensive industrial production method is required as a raw material in the production of sodium 5'-inosinate and sodium 5'-guanylate, both of which have taste effects.

As a result of various studies on the production method of ribonucleic acid, the present inventors discovered that they belong to the genus Bacillus and were able to collect a large number of mutant strains that produce large amounts of ribonucleic acid from mutant strains that are sensitive to surfactants. It was successful and the present invention was completed.

The related strain used in the present invention is a mutant strain that belongs to the genus Bacillus and is sensitive to surfactants.

Such mutant strains are Bacillus subtilis (Ba-cilis
Using a Bacillus microorganism such as I Fo 3214 as a parent strain, it is subjected to ultraviolet irradiation treatment, X-ray irradiation treatment, or N-methyl-N-nitro-
It can be obtained by performing usual mutagenesis treatment such as N-nitrosoguanidine treatment.

The surfactant in the present invention includes nonionic, cationic, and anionic surfactants, but in particular, higher alcohol sulfate having 10 to 18 carbon atoms, and higher alcohol sulfate having 12 to 18 carbon atoms. Desirable examples include polyoxyethylene sorbicun fatty acid ester, fatty acid amide sulfonate having 12 to 18 carbon atoms, and alkylamine salt having 8 to 23 carbon atoms.

Among these, the surfactant considered to be the most effective is sodium dodecyl sulfonic acid.

Mutant strains of the present invention are often sensitive to more than one of these surfactants.

"Mutant strain sensitive to surfactants" in the present invention
What is meant by suspending bacterial cells in the logarithmic growth phase in a solution containing a surfactant (turbidity IFO3214:0.310°N-32:0.
315), refers to a mutant strain that is easier to lyse than the parent strain when the degree of bacteriolysis is measured by measuring the decrease in turbidity (absorbance).

Examples of the mutant strains of the present invention include the following.

Bacillus subtilis N-32FERN-32FER:
Sensitive to sodium dodecyl sulfonic acid.

Examples showing the sensitivity of the mutant strains used in the present invention to surfactants are shown below.

[For each experimental example 1e, put 4 ml of medium A containing 10 g of glucose, 10 g of meat extract, 10 g of peptone, and 5 g of salt and adjust the pH to 7.0 into a 20 ml lfj test tube, sterilize it, and inoculate the test bacteria into it. Then, the mixture was cultured with shaking at 30°C for 18 hours to obtain a seed culture solution.

This was inoculated at 2% into a sterilized medium by adding 20 ml of medium A to a 5'00 ml cylinder.

After culturing at 37°C for 6 hours, the bacterial cells were separated to obtain bacterial cells in the early stage of logarithmic growth. 11 containing a certain amount of 0.02% sodium dodecyl sulfonic acid
The suspension was suspended in 50 mol Tris-HCl buffer (pH 7.2) and the absorbance at 560 mμ was determined over time at 37°C to measure the percentage decrease in turbidity.

The results are shown in Table 1 as the degree of bacteriolysis (ratio to the degree of bacteriolysis when no surfactant is included).

As a medium for culturing such a different strain, a conventional medium containing a carbon source, a nitrogen source, an inorganic salt, and, if necessary, organic micronutrients can be used.

As the carbon source, glucose, fructose, sucrose, maltose, mannose, mannitol, inositol, sorbitol, starch and starch saccharification liquid, molasses, etc. can be used.

Suitable nitrogen sources include ammonium salts, aqueous ammonia, ammonia gas, and urea.

If necessary, inorganic ions such as potassium salts, phosphates, magnesium salts, etc. are added to the medium.

Amino acids, vitamins, etc. are added to the medium as necessary as organic micronutrients.

The culture method requires good aerobic conditions, and the culture temperature is 25-40℃.
Good range of °C.

Depending on the case, the fermentation temperature may be changed during the culture.

It is desirable to adjust the pH of the culture solution to 5.0 to 9.0 at the beginning and during the culture.

To adjust the pH, inorganic acids, organic acids, alkalis, and even urea, calcium carbonate, aqueous ammonia, ammonia gas, etc. can be used.

Thus, after 2 to 7 days of culture, a significant amount of ribonucleic acid accumulates in the culture medium.

Ribonucleic acid in the culture solution was quantified by creating a standard curve using yeast ribonucleic acid using the Orsin method.

To isolate and collect the ribonucleic acid produced and accumulated in the culture solution, remove the bacterial cells from the culture solution, extract the ribonucleic acid with phenol, and add 2% sodium acetate and twice the volume of cold ethanol. This can be carried out by a conventional method, such as adding .

Example 1 20 ml of the culture medium having the composition shown in Table 2 was dispensed into 500 shaker flasks and sterilized by heating at 115° C. for 110 minutes.

The bacterial strains shown in Table 3 were added to this medium with 2% glucose, 1% peptone, 1% meat extract, 0.5% salt, and 2% agar (pH
A loopful of bacterial cells cultured for 20 hours on a slant containing 7,0) was inoculated and cultured with shaking at 34°C for 72 hours (11
0 times/min 7 Confucius @) was performed.

After completion of the culture, the amount of ribonucleic acid shown in Table 2 was accumulated in the culture solution.

Culture solution 21 of N-32 was prepared in the same manner as above, and bacterial cells were removed by centrifugation.

The pH of the supernatant was adjusted to 7.0, and ribonucleic acid was extracted with phenol.

Phenol <2% Sodium Acetate Add twice the volume of cold ethanol to precipitate 7'J Bonukyuan, collect this and divide into 0.5M chloride.0.01M1-! J Soot-acid buffer (pH 7
, 3) and filtered through a Millipore filter (HAWP 0
．． 45 mμ).

2% sodium acetate and twice the amount of cold ethanol were added to this filtrate to precipitate ribonucleic acid, which was further washed with cold ethanol and dried to obtain ribonucleic acid 6.47.

Example 2 N-32 was cultured in the same manner as in Example 1 using glucose, mannose, fructose, sucrose, mannitol, inositol, sorbitol, or soluble starch as the carbon source instead of maltose among the medium compositions shown in Table 2. did.

Then, ribonucleic acid in the culture solution was quantified using the Orsin method.

The results are shown in Table 3.

Claims (1)

[Claims] 1. A mutant strain belonging to the genus Bacillus that is sensitive to surfactants and has the ability to produce and accumulate ribonucleic acid in a medium is cultured in a liquid medium, and the ribonucleic acid produced in the medium is collected. A method for producing ribonucleic acid.