JP2727655B2 - Production of pyruvate by fermentation - Google Patents

Description

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

Pyruvic acid is an important intermediate in biological metabolism, and is an effective raw material for various medicines and agricultural chemicals, as well as a main raw material for the synthesis of amino acids such as L-tryptophan, L-cysteine, and L-tyrosine by the oxygen method. is there. Therefore, if it can be produced at low cost, it is useful as various synthetic raw materials.

<Prior Art> Conventionally, as a method for producing pyruvic acid by a fermentation method, a method using yeasts such as Saccharomyces and Candida and mutants thereof, basidiomycetes or special bacteria is known (Japanese Patent Publication No. -796 publication). Also,
As for pyruvate fermentation by a yeast belonging to the genus Torulopsis, a method using a wild strain (JP-A-62-275688, etc.) is known.

<Problems to be Solved by the Invention> However, such a conventional method is industrially advantageous because it has many by-products such as ethanol and α-ketoglutaric acid, or the yield and yield are not sufficient. Did not.

<Means and Actions for Solving the Problems> Accordingly, the present inventors have been able to solve the above-mentioned problems and have conducted intensive studies on a method for producing pyruvic acid with high productivity. The present inventors have found that by imparting a property having a high resistance to 2-deoxyglucose to a microorganism having an acid-producing ability, the accumulated concentration and production yield of pyruvic acid are remarkably improved.

That is, the above object of the present invention belongs to the genus Torulopsis, among microorganisms capable of producing pyruvate, by culturing a 2-deoxyglucose-resistant strain to produce and accumulate pyruvate in the medium, and collect this. That is achieved.

That is, the present invention is a microorganism belonging to the genus Torulopsis, which is resistant to 2-deoxyglucose and has pyruvate-producing ability, thereby producing and accumulating pyruvic acid in a culture solution. This is a method for producing pyruvic acid by a fermentation method, which comprises collecting an acid. Above all, no knowledge has been reported to date on imparting 2-deoxyglucose resistance to pyruvate fermentation producing bacteria belonging to the genus Torulopsis and improving pyruvate fermentation performance.

 Next, the present invention will be described in detail.

The mutant strain used in the present invention may be any microorganism that has pyruvic acid-producing ability and is resistant to 2-deoxyglucose and belongs to the genus Torulopsis.

Representative mutants used in the present invention include:
For example, 2-deoxyglucose-resistant mutant Torulopsis glabrata P95-21 (FERM P-10652) can be mentioned. This mutant strain is Torulopsis glabrata IFO 0005
Was used as a parent strain and was induced by a conventional mutation treatment method.

Such a mutant strain is obtained by irradiating the parent strain with ultraviolet light or N
-Methyl-N ^-- nitro-N-nitrosoguanidine treatment,
After a normal mutation treatment such as ethyl methanesulfonate (hereinafter abbreviated as EMS) treatment, it can grow significantly in a solid medium containing 2-deoxyglucose at a concentration such that the parent strain cannot grow sufficiently. What is necessary is just to obtain a suitable strain.

The 2-deoxyglucose-resistant strain in the present invention includes:
A strain having higher resistance than its parent strain, and preferably the final relative growth when cultured in a medium containing 2-deoxyglucose at a concentration such that the relative growth of the parent strain after 24 hours is 40% or less. Indicates 50% or more.

Here, the relative growth is measured by measuring the absorbance at 660 nm of the culture solution, and is expressed as the relative absorbance when the absorbance of the culture solution without addition of 2-deoxyglucose of each strain is expressed as 100%.

The medium used in the present invention may be any medium that contains moderately the carbon source, nitrogen source, inorganic salts, vitamins and the like usually used for fermentation. Examples of the carbon source include saccharides such as glucose and organic acids. , Ethanol, methanol and the like that can be used by the yeast used are used. As the nitrogen source, ammonium sulfate, ammonium nitrate, ammonium salt, urea, peptone, meat extract, taste liquor, and other organic and inorganic nitrogen compounds are used, and preferably organic nitrogen compounds containing amino acids in a well-balanced manner. As the 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 extracts and corn steep containing them A medium containing liquor and other natural products may be used.

During the cultivation, the pH decreases as pyruvic acid is produced and accumulated. Therefore, adjusting the pH to 3 to 7 with an alkali such as calcium carbonate, caustic soda, or caustic potash is effective for pyruvic acid production. The temperature during the culturing is suitably from 22 ° C to 32 ° C. After completion of the culture, pyruvic acid accumulated in the system can be isolated and collected by a conventional method.

For example, acidic ether extraction, phenylhydrazonation and precipitation isolation can also be used.

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

Example 1 A. (Isolation of 2-Deoxyglucose-Resistant Strain) After treating cells of Torulopsis glabrata IFO 0005 with EMS (1 W / V%, 30 ° C. for 3 hours) by a conventional method, the cells were subjected to Bacto Yeast Carbon Base (manufactured by DIFCO Corporation)
(Hereinafter abbreviated as YCB medium), applied to an agar medium containing 5 g of ammonium sulfate and 40 mM of 2-deoxyglucose, cultured at 30 ° C. for 5 to 7 days, and obtained a highly resistant 2-deoxyglucose strain Torulopsis glabrata P95-21. I got

B. (Degree of Resistance of 2-Deoxyglucose-Resistant Strains) Each of the strains shown in Table 1 below was inoculated in a liquid medium obtained by adding 5 g / ammonium sulfate to a YCB medium, and cultured with shaking at 30 ° C. for 16 hours. The grown cells were inoculated into 5 ml of YCB medium containing 5 g / ammonium sulfate containing 2-deoxyglucose at concentrations of 0, 10 mM, 20 mM, 30 mM and 40 mM, respectively, and cultured at 30 ° C. for 24 hours. The degree of growth was examined. The results are shown in Table 1.

From the above, it is apparent that the 2-deoxyglucose-resistant strain Torulopsis glabrata P95-21 used in the method of the present invention has acquired higher resistance to 2-deoxyglucose than the parent strain.

Example 2 (Production of pyruvate) A fermentation medium containing 10% glucose, 0.3% ammonium sulfate, 0.5% polypeptone, 2mg / nicotinic acid, 400µg / pyridoxine / hydrochloride, 20µg / thiamine / hydrochloride, and 5µg / biotin / After dispensing 40 ml each into a Meyer flask, sterilizing and adding 4% of separately sterilized calcium carbonate, parent strain Torulopsis glabrata IFO 0005 and 2-deoxyglucose resistant strain Torulopsis glabrata P95-21.
Were inoculated and cultured at 30 ° C. for 60 hours. Pyruvic acid produced in the culture solution was quantified by high performance liquid chromatography.

 The results are shown in Table 2.

When the Torulopsis glabrata P95-21 of the present invention was used, both the accumulated concentration and the pyruvate absorption rate were significantly improved as compared with the parent strain.

Next, the culture solution 2 of Torulopsis glabrata P95-21 was used.
After sterilizing 00 ml, the supernatant was adjusted to pH 2.0 by adding hydrochloric acid, extracted with ethyl ether, then neutralized to pH 5.5 with sodium hydroxide, and concentrated under reduced pressure at 40 ° C. to about 50 ml. Ethanol was added dropwise to this concentrated liquid to obtain 6.59 g (purity: 97.5%) of sodium pyruvate.

<Effect of the Invention> According to the method of the present invention, the amount of pyruvic acid accumulated and the yield are improved, and it is possible to produce pyruvic acid at lower cost.

Claims (2)

(57) [Claims] (1) a member belonging to the genus Torulopsis,
A fermentation method comprising culturing a microorganism having tolerance to deoxyglucose and capable of producing pyruvate to produce and accumulate pyruvate in the culture solution, and collecting pyruvate from the culture solution; Method of producing acid. 2. The method according to claim 1, wherein the pyruvic acid-producing bacterium is Torulopsis.
2. A method according to claim 1 belonging to the genus glabrata spp.