JPH0424037B2 - - Google Patents

Description

[Detailed description of the invention]

<Industrial Application Field> The present invention relates to a method for producing pyruvic acid by a fermentation method. Pyruvate is an important intermediate in biological metabolism,
It is not only an effective raw material for the synthesis of various medicines and agricultural chemicals, but also a main raw material for the synthesis of amino acids such as L-tryptophan, L-cysteine, and L-tyrosine by enzymatic methods. Therefore, if it can be produced at low cost, it is useful as a raw material for various synthetics. <Prior Art> It is already known that pyruvic acid can be produced by a fermentation method using microorganisms of the genus Torulopsis (Journal of the Japanese Society of Agricultural Chemistry, Vol. 32, p. 573, Japanese Patent Application Laid-open No. 14789/1989). <Problems to be Solved by the Invention> However, in this conventional method, the amount of accumulated pyruvic acid and the yield are low, and it cannot be put into practical use industrially. In other words, Japanese Journal of Agricultural Chemistry No.
According to Volume 32, Page 573, the amount of pyruvate accumulated using Torulopsis candida is at most 0.5.
As low as g/. Furthermore, according to JP-A-62-14789, the amount of pyruvic acid accumulated using Torulopsis ETCH is as low as 5.1 g/at most. <Means and effects for solving the problems> Therefore, the present inventors have conducted intensive research on a method for producing pyruvic acid that can solve the above problems and has a high productivity. By imparting the property of simultaneously requesting isoleucine and valine to microorganisms that have the ability to produce pyruvate, the accumulated concentration of pyruvate,
It was discovered that the production yield was significantly improved, and the present invention was achieved. That is, the above object of the present invention is to produce and accumulate pyruvate in the medium by culturing a double isoleucine and valine auxotrophic strain of microorganisms that belong to the genus Torulopsis and have the ability to produce pyruvate, and to collect the same. This is achieved by doing so. That is, the present invention uses a mutant strain belonging to the genus Torulopsis that simultaneously requires isoleucine and valine for growth and has the ability to produce pyruvate to produce and accumulate pyruvate in a culture solution, and to produce and accumulate pyruvate from the culture solution. This is a method for producing pyruvic acid by a fermentation method, which is characterized by collecting pyruvic acid. Next, the present invention will be explained in detail. The mutant strain used in the present invention may be any microorganism belonging to the genus Torulopsis that has the ability to produce pyruvate and simultaneously requires isoleucine and valine for growth. Typical mutant strains used in the present invention include, for example, Torrolobsis glabrata x-
68 (FERM BP-1426) (nicotinic acid, thiamine,
pyridoxy, biotin, isoleucine, valine auxotrophy). This mutant strain is Torrolobsis glabrata TR-2026 (FERM BP-1425)
(requiring nicotinic acid, thiamine, pyridoxine, and biotin) as a parent strain and was induced by a conventional mutation treatment method. Such mutant strains can be produced by UV irradiation or N-methyl-N-
It can be easily obtained by performing usual mutation treatments such as nitro-N-nitrosoguanidine treatment and ethyl methanesulfonate (hereinafter abbreviated as EMS) treatment. The isoleucine and valine dual requirement here has a broad meaning and includes incomplete deletion type (so-called Leaky type). Furthermore, it also includes cases where the requirements are satisfied with biosynthetic precursors of isoleucine and valine. The medium used in the present invention may contain moderate amounts of carbon sources, nitrogen sources, inorganic salts, vitamins, etc. normally used in fermentation. , ethanol, methanol, etc. that can be used by the yeast used. Nitrogen sources include ammonium sulfate, ammonium nitrate,
Ammonium chloride, urea, peptone, meat extract, flavor liquid, and other organic and inorganic nitrogen compounds are used, and organic nitrogen compounds containing amino acids in a well-balanced manner are preferable. Inorganic salts include potassium phosphate,
Magnesium sulfate, iron, manganese, and other inorganic salts are used, and if necessary, thiamine,
Required vitamins such as niacin, pyridoxine, piothine, or yeast extract containing these,
A medium supplemented with corn steep liquor or other natural products may be used. During culture, as pyruvate is produced and accumulated, the pH decreases.
Therefore, it is effective for pyruvic acid production to adjust the pH to 3 to 7 with an alkali such as calcium carbonate, caustic soda, or caustic potash. A suitable temperature during culturing is 22°C to 32°C. After culturing,
Pyruvate accumulated in the system can be isolated and collected by conventional methods. For example, methods such as acidic ether extraction, phenylhydrazonation, and precipitation isolation may also be employed. <Examples> The present invention will be specifically explained below using Examples. Example 1 (Obtainment of L-isoleucine and valine double auxotrophic mutant) Torrolobus glabrata TR-2026
(FERM BP-1425) (nicotinic acid, thiamine,
After EMS treatment (1W/V%, 30℃ for 3 hours) of bacterial cells (requiring pyridoxine and biotin), the cells were inoculated onto a nutrient agar medium (GP agar medium, manufactured by Daigo Nutrient Chemical Co., Ltd.) for 30 minutes. Incubate for 2 days at °C.
Bacto Yeast Carbon Base (manufactured by DIFCO Co., Ltd., hereinafter referred to as
The samples were replicated on an agar medium (A) in which 5 g of ammonium sulfate was added to YCB medium (abbreviated as YCB medium) and an agar medium (B) in which 5 g of ammonium sulfate was added to YCB medium and 100 mg each of L-isoleucisine and valine were added. Next, the cells were cultured at 30°C for 3 days, and colonies that grew on the agar medium (B) but did not grow on the agar medium (A) were isolated. Next, agar medium
From (B), excluding colonies growing on each medium excluding L-isoleucine or L-valine, L-
Isoleucine and L-valine dual auxotrophic mutant Torlobussis glabrata X-68 (FERM BP
−1426) was obtained. Example 2 (Assay for isoleucine and valine auxotrophy) Each strain shown in Table 2 below was cultured on a GP agar slant medium for 24 hours, and a very small amount of the bacterial cells was scraped off.
Do not add L-isoleucine and L-valine
YCB agar medium and L-isoleucine only
A thin layer of YCB agar medium containing 0.01% L-valine, 0.01% L-isoleucine and 0.01% L-valine each was cultured at 30° C. for 4 days, and the presence or absence of growth was observed. No addition of L-isoleucine and L-valine, addition of only L-isoleucine,
A mutant strain that could not grow on an agar plate medium supplemented with only L-valine but grew on an agar plate medium supplemented with L-isoleucine and L-valine was designated as an L-isoleucine and valine double auxotrophic strain. The results are shown in Table 2, and it is clear that the L-isoleucine/valine dual auxotrophic strain Torlobussis glabrata It has acquired a dual requirement for L-isoleucine and L-valine.

[Table] (Note) +: Growth -: No growth
Ile: Isoleucine Val: Valine Example 3 (Production of pyruvic acid) Glucose 10%, Ammonium sulfate 0.3%, Potassium phosphate 0.1%, Magnesium sulfate heptahydrate 0.05%,
Polypebutone 0.5%, nicotinic acid 2mg/, pyridoxine hydrochloride 400g/, thiamine hydrochloride
Fermentation medium containing 20g/, biotin 5g/1
After sterilization, add 4% of separately sterilized calcium carbonate and inoculate the parent strain Torulopsis glabrata TR-2026 and the isoleucine and valine double requesting mutant strain Torulopsis glabrata X-68. , 60 at 30℃
Cultured for hours. However, the mutant strain contains L-
Culture was performed with the addition of 0.01% each of isoleucine and L-valine. Pyruvate produced in the culture solution was quantified using high performance liquid chromatography. The results are shown in Table 1.

[Table] The yield was expressed as the weight of pyruvic acid relative to consumed glucose. The method using Torrolobsis glabrata Next, after sterilizing 200 ml of the X-68 culture solution, add hydrochloric acid to the supernatant to adjust the pH to 2.0, extract with ethyl ether, and then neutralize the pH to 5.5 with caustic soda.
It was concentrated under reduced pressure at °C to a volume of approximately 5 ml. Add ethanol dropwise to this concentrate and add 5.91g of sodium pyruvic acid.
(purity 97%) was obtained. <Effects of the Invention> According to the method of the present invention, the accumulated amount and yield of pyruvic acid have been improved, and it has become possible to produce pyruvic acid at a lower cost.

Claims (1)

[Claims] 1. Cultivating a mutant strain belonging to the genus Torulopsis that simultaneously requires isoleucine and valine for growth and having the ability to produce pyruvate, producing and accumulating pyruvate in the culture solution,
A method for producing pyruvic acid by a fermentation method, which comprises collecting pyruvic acid from a culture solution.