JPH0740946B2 - Fermentation method for producing pyruvic acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing pyruvic acid by a fermentation method.

Pyruvate is an important intermediate in biometabolism.
It is not only an effective synthetic raw material for agricultural chemicals, but also a main raw material for amino acid synthesis such as L-tryptophan, L-cysteine, and L-tyrosine by the enzymatic method. Therefore, if it can be manufactured at low cost, it is useful as various synthetic raw materials.

<Prior Art> It is already known that pyruvate can be produced by a fermentation method using a microorganism belonging to the genus Tollopsis (Journal of Agricultural Chemistry, Japan, Vol. 32, No. 573, JP 62-14789).

<Problems to be Solved by the Invention> However, in such a conventional method, the amount of pyruvic acid accumulated and the yield are low, and it cannot be industrially put into practical use. That is, according to the Journal of the Japanese Society of Agricultural Chemistry, Vol. 32, No. 573, the accumulated amount of privic acid using Torulopsis canandida is as low as 0.5 g /. In addition, JP-A-62-147
According to Japanese Patent Publication No. 89, the amount of accumulated pyruvic acid using Torulopsis ethiersi is as low as 5.1 g / at most. << Means and Actions for Solving Problems >> Accordingly, the inventors of the present invention have been able to solve the above problems and have further earnestly studied a method for producing pyruvic acid with high productivity. The present inventors have found that by imparting a property requiring arginine to a microorganism capable of producing pyruvic acid, the accumulated concentration of pyruvic acid and the production yield are remarkably improved, and the present invention has been accomplished.

That is, the above-mentioned object of the present invention is achieved by culturing an arginine-requiring strain of a microorganism belonging to the genus Tolulopsis and having a pyruvate-producing ability, thereby producing and accumulating pyruvic acid in the medium and collecting it. Is done.

That is, the present invention belongs to the genus Torulopsis (Torulopsis), which requires arginine for growth, and cultivates a mutant strain having pyruvic acid-producing ability to generate and accumulate pyruvic acid in the culture medium, A method for producing pyruvic acid by a fermentation method, which comprises collecting pyruvic acid.

Next, the present invention will be described in detail.

The mutant strain used in the present invention may be any microorganism as long as it is a microorganism belonging to the genus Torulopsis that has pyruvate-producing ability and requires arginine for growth.

Representative examples of the mutant strain used in the present invention include, for example, Torlovsis glabrata x-15 (FERMBP-14
23) (nicotinic acid, thiamine, pyridoxy, biotin,
Arginine requirement). This mutant strain was derived by a conventional mutation treatment method using Torulopsis glabrata IFO 0005 (nicotinic acid, thiamine, pyridoxine, biotin requirement) as a parent strain.

Such a mutant strain is obtained by irradiating a wild-type strain or a parent strain which does not require arginine for growth with ultraviolet rays or treatment with N-methyl-N-nitro-N-nitrosoguanidine to obtain ethyl methanesulfonate (hereinafter referred to as EMS). It can be easily obtained by subjecting to ordinary mutation treatment such as treatment.

Arginine requirement here has a broad meaning,
Incomplete deletion type (so-called Leaky type) is also included.
Furthermore, it also includes cases where the requirement for arginine as a biosynthetic precursor is satisfied.

The medium used in the present invention may be any medium which contains a carbon source, a nitrogen source, inorganic salts, vitamins and the like which are usually used for fermentation, and the carbon source includes sugars such as glucose and organic acids. , Ethanol, methanol, etc. that can be utilized by the yeast used are used. Ammonium sulfate, ammonium nitrate, ammonium chloride, urea, peptone, meat extract, taste liquid, and other organic and inorganic nitrogen compounds are used as the nitrogen source, and organic nitrogen compounds containing amino acids in a good balance are preferable. As inorganic salts, potassium phosphate, magnesium sulfate, iron, manganese, and other inorganic salts are used, and if necessary, required vitamins such as thiamine, niacin, pyridoxine, and biotin, or yeast extract containing these and corn steep A medium containing liquor and other natural products may be used.

Since pH is lowered during the cultivation due to the production and accumulation of pyruvic acid, it is effective for pyruvic acid production to adjust the pH to 3 to 7 with alkali such as calcium carbonate, caustic soda and caustic potash. A temperature of 22 ° C to 32 ° C is suitable during the culture. After completion of the culture, the pyruvic acid accumulated in the system can be isolated and collected by a conventional method.

For example, methods such as extraction with acidic ether, phenylhydrazone conversion and precipitation isolation can also be adopted.

<Examples> Hereinafter, the present invention will be specifically described with reference to Examples.

Example 1 (Acquisition of L-arginine-requiring mutant strain) Torulopsis glabrata IFO 0005 (nicotinic acid,
Thiamine, pyridoxine, and biotin-requiring cells were subjected to EMS treatment (1 W / V%, 30 ° C for 3 hours) by a standard method, and then inoculated on nutrient agar medium (GP agar medium, Daigo Nutrition Chemical Co., Ltd.). Cultivated at 30 ℃ for 2 days, and then Bcto Yeast Carbon Base
(DIFCO Co., Ltd., hereinafter abbreviated as YCB medium), ammonium sulfate 5g /
Ammonium sulphate 5g /, L- on agar medium (A) and YCB medium
Each was rebursed on an agar medium (B) containing 100 mg / arginine. Then, the mixture was cultured at 30 ° C. for 3 days, colonies that grew on the agar medium (B) and did not grow on the agar medium (A) were isolated, and the L-arginine-requiring mutant strain Torulopsis glabrata X-15 (FERMBP- 1423) was acquired.

Example 2 (Test for arginine auxotrophy) Each strain shown in Table 2 below was cultivated on a GP agar slant medium for 24 hours, and a very small amount of the cells were scraped off, L-arginine was not added, and L-arginine was added at 0.01%. It was thinly applied to a YCB agar plate medium containing 5 g / mL of ammonium sulfate and cultured at 30 ° C. for 4 days, and the presence or absence of growth was observed. L-arginine-requiring mutant strains were those that could not grow on L-arginine-free agar plates and could grow on L-arginine-containing agar plates.

The results are shown in Table 2, and the L-arginine-requiring strain Torulopsis glabrata X-15 used in the method of the present invention was clearly compared with the parent strain of Torulopsis glabrata IFO 0005. Have acquired sex.

Example 3 (Production of pyruvic acid) A fermentation medium containing glucose 10%, ammonium sulfate 0.3%, polypeptone 0.5%, nicotinic acid 2 mg /, pyridoxine / hydrochloride 400 μg /, thiamine / hydrochloride 20 μg /, and biotin 5 μg / was prepared. Dispense 40 ml each into a Mayer flask, sterilize, and then add separately sterilized 4% calcium carbonate, and inoculate the parent strain Torulopsis glabrata IFO 0005 and the arginine-requiring mutant strain Torulopsis glabrata X-15 at 30 ° C. It was cultured for 60 hours. However, the mutant strain was cultured by adding 0.03% of L-arginine to the fermentation medium. The pyruvic acid produced in the culture solution was quantified by high performance liquid chromatography.

The results are shown in Table 1.

The yield was represented by the weight of pyruvic acid relative to the glucose consumed.

In the method using Torulopsis glabrata X-glabrata X-15 of the present invention, both the accumulated concentration and the yield of pyruvic acid are markedly improved over the parent strain.

Then, 200 ml of the X-15 culture solution was sterilized, hydrochloric acid was added to the supernatant to adjust the pH to 2.0, the mixture was extracted with ethyl ether, neutralized to pH 5.5 with caustic soda, and concentrated under reduced pressure at 40 ° C. , About 5 ml. Ethanol was added dropwise to this concentrated liquid to obtain 5.91 g (purity 97%) of sodium pyruvate.

<Effects of the Invention> According to the method of the present invention, the amount of pyruvic acid accumulated and the yield were improved, and it became possible to produce pyruvic acid at a lower cost.

Claims (1)

[Claims] 1. A mutant strain belonging to the genus Torulopsis, which requires arginine for growth and has a pyruvate-producing ability, is produced and accumulated in the culture medium to form and accumulate pyruvic acid. A method for producing pyruvic acid by a fermentation method, which comprises collecting more pyruvic acid.