JPS63258586A - Production of pyruvic acid by fermentation - Google Patents

Abstract

PURPOSE:To produce pyruvic acid in high efficiency, by preparing a variant having lowered PDC activity from a microorganism capable of producing pyruvic acid and belonging to Torulopsis genus and culturing the variant in a medium. CONSTITUTION:Cell of Torulopsis glabrate IFO 0005 is treated with ethylmethane sulfonate to obtain a variant having PDC (pyruvate decarboxylase) activity lowered to <=70%, preferably <=50% of that of the mother strain, e.g. Torulopsis glabrate ACII33 (FERM BP-1424). The obtained variant strain is cultured in a medium containing carbon source, nitrogen source, inorganic salts vitamins, etc., 22-32 deg.C while controlling the pH of the medium to 3-7 with an alkali. Pyruvic acid can be separated from the cultured product.

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, and is used by various doctors and
It is not only an effective raw material for the synthesis of agricultural chemicals, but also a main raw material for the synthesis of amino acids such as L-tri-11-fan, L-cysteine, and tyrosine by the oxygen method. Therefore, if it can be produced at low cost, it is useful as a raw material for various synthetics.

<Prior art> Conventionally, as a method for producing pyruvic acid by fermentation, methods using yeast such as Satucharomyces and Candida, their mutant strains, basidiomycetes, or special bacteria have been known (Japanese Patent Publication No. 1983-1982). Publication No. 796, etc.).

<Problems to be Solved by the Invention> However, such conventional methods were not industrially advantageous because they produced a large amount of by-products such as ethanol, or the yield and yield were insufficient.

Means for Solving the Problems> Therefore, as a result of intensive research into a method for producing pyruvic acid that can solve the above problems and has higher productivity, the present inventors found that pyruvic acid, which belongs to the genus Torulopsis, The inventors have discovered that the accumulation degree and production yield of pyruvate can be significantly improved by reducing the pyruvate decarboxylase activity (hereinafter abbreviated as PDC activity) of a microorganism capable of producing it, and have arrived at the present invention.

That is, the above object of the present invention is achieved by culturing a strain of a microorganism with reduced PDC activity among the microorganisms belonging to the genus Torulopsis and having the ability to produce pyruvate, thereby producing and accumulating pyruvate in the medium, and collecting the pyruvate. It will be done.

That is, the present invention relates to Torulopsis (Torulopsis).
is), which has the ability to produce pyruvate and has lower pyruvate decarboxylase activity than its parent strain, is cultured to produce and accumulate pyruvate in the culture solution, and to produce 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 is succumbing to Torulopsis flexion and P
Any mutant strain may be used as long as it has a reduced DC activity and has the ability to produce pyruvate.

Typical mutant strains used in the present invention include, for example, Torulopsis glabrata ACII33 (FE
RMP-93/7) includes cotic acid, thiamine, pyridoxine, biotin requirement, and PDC activity reduction).

This mutant strain is Torulopsis glabrata IFO00
It is derived from cotinic acid, thiamine, pyridoxine, and biotin (requires 05).

Such mutant strains can be produced by treating the wild strain or the parent strain with UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine treatment, or ethyl methanesulfonate (hereinafter referred to as
1q can be obtained by selecting a strain that grows significantly better than the parent strain in a medium containing acetic acid from cells mutated by a conventional method such as EMS (abbreviated as EMS) treatment.

PDC activity in the present invention is determined by disrupting cells by ultrasonication, high pressure method using a French press, or other methods, and using a supernatant crude enzyme solution obtained by centrifugation, using pyruvate as a substrate, and supplementing thiamine pyrophosphate. It is measured by a method in which the acetaldehyde produced by reacting as an enzyme is reacted with 2,4-dinitrophenylhydrazine, and the 2,4-dinitrophenylhydrazone of the reacted acetaldehyde is quantitatively analyzed by high performance liquid chromatography.

Here, the PDC activity of the microorganism used in the present invention is expressed as a relative activity when the PDC activity of the parent strain is expressed as 100%.

The strain used in the present invention whose PDC activity is lower than that of the parent strain is preferably one whose PDC activity is reduced to 70% or less, preferably 50% or less, of the parent strain.

Even if the PDC activity is not reduced to 70% or less of the parent strain, as long as the PDC activity is reduced to 70% or less of the wild strain from which the strain was derived, the present invention The use of such strains is within the scope of the present invention, since similar effects are achieved.

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,
Other organic and inorganic nitrogen compounds may be used, preferably organic nitrogen compounds containing a balanced amount of amino acids. Potassium phosphate, magnesium sulfate, iron, manganese, and other inorganic salts are used as inorganic salts. A medium supplemented with cheap liquor, soybean, or other natural products may be used.

During culture, pI decreases as pyruvic acid is produced and accumulated, so it is effective for pyruvic acid production to adjust it to 1) H3 to 7 with an alkali such as calcium carbonate, caustic soda, caustic potash, or ammonia. A suitable temperature during culturing is 22°C to 32°C. After completion of the culture, pyruvate accumulated in the system can be isolated and collected by a conventional method.

For example, methods such as acidic ether extraction and phenylhydrazonation followed by precipitation isolation can also be employed.

Effect> In the present invention, reducing the PDC activity level is
It is estimated that it helps reduce the metabolism of pyruvate to acetaldehyde and increases the conversion rate of glucose to pyruvate, thereby increasing pyruvate accumulation.

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

Example 1 A, (PDC activity decrease number obtained) Torulopsis glabrata IFO0005 (requiring cotic acid, thiamine, pyridoxine, and biotin) was subjected to EMS treatment (1 W/V%, 3 hours at 30°C) using a conventional method. Afterwards, it was inoculated onto a nutrient agar medium (GP agar medium, manufactured by Daigo Nutrient Chemical Co., Ltd.), and cultured at 30'C for 2 days. Each hydrate was replicated onto a basic agar medium (B) containing 59/j2.

Next, we collected colonies that grew significantly better on the main agar medium (B) than on the basic agar medium (A) after culturing at 30'C for 3 days, and cultured them in a fermentation medium consisting of the components shown in Table 1. Pyruvate fermentation was carried out to determine the accumulation of pyruvate and the sugar yield, strain ACn33 (FERMP-9317), which showed improved yield.
) was obtained. Next, regarding the strain ACn33 and its parent strain,
PDC activity was measured as follows.

B. (Measurement of PDC activity) Add the basal medium shown in Table 1 to a 500 ml Sakaro flask.
The test strains IFO0005 and ACII33 were each inoculated into the culture medium, and cultured with shaking at 30°C for 30 hours. The bacterial cells were separated from the culture solution, washed twice with 0.2 M potassium-phosphate buffer (pH 7.2), suspended in 10 m of the above buffer, sonicated (20 minutes), and centrifuged. Insoluble matter was removed, and the resulting supernatant was used as an enzyme solution.

Next, prepare the enzyme reaction solution shown in Table 2, and
Enzyme reaction was carried out for 1 minute, 0.5mM 2,4-dinitrophenylhydrazine dissolved in 2N hydrochloric acid was added to stop the reaction, and the aldehyde was left standing for 15 minutes to hydrazonate, and 2ml of methanol was added to convert the hydrazone compound. was dissolved, and 2,4-dinitrophenylhydrazone of acetaldehyde was subjected to constant ω analysis using high performance liquid chromatography.

Note that a reaction solution without pyruvic acid was used as a control solution.

Table 2 As shown in Table 3, the PDC activity of strain ACII33 was reduced to about 1/2 of that of the parent strain.

Table 3 Example 2 (Production of pyruvic acid) Sodium acetate and
A medium supplemented with 5 g/l of trihydrate was dispensed into 11 Meyer flasks in 40 rrIi portions and sterilized at 115° C. for 15 minutes. Next, strains IF0 0005 and ACII3
3 was inoculated in platinum loops and cultured in rotation at 30°C for 60 hours. The amount of pyruvic acid produced in the culture solution was determined by high performance liquid chromatography. The results are shown in Table 4.

Note that the sugar yield was expressed as 1ffi of pyruvic acid relative to consumed glucose.

As is clear from the results in Table 4, the method using Torulopsis glabrata ACn33 of the present invention example significantly improves both the accumulated concentration and production yield of pyruvic acid compared to the parent strain. The reason for the improvement is presumed to be that the metabolism of generated pyruvate to acetaldehyde was suppressed due to a decrease in PDC activity, and the conversion rate of glucose to pyruvate increased.

Next, after sterilizing 200 d of the culture solution of ACn33, hydrochloric acid was added to the supernatant to bring the pH to 112.0, extracted with ethyl ether, and then the pH was neutralized to 5.5 with caustic soda and then heated at 40°C.
It was concentrated under reduced pressure to a volume of about 5 ml. Ethanol was added dropwise to this concentrated solution, and sodium pyruvic acid 6.629 (purity 98%) was added.
) is 1q.

<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] A mutant strain belonging to the genus Torulopsis that has the ability to produce pyruvate and has lower pyruvate decarboxylase activity than its parent strain is cultured to produce and accumulate pyruvate in the culture solution. A method for producing virupic acid by a fermentation method characterized by collecting pyruvic acid.