JP2003024048A - New microorganism and method for producing pyruvic acid by the microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new microbial strain having high pyruvic acid productivity and low assimilating potency of pyruvic acid and resistant to pyruvic acid of high concentration, and provide a method for producing pyruvic acid by using the microbial strain. SOLUTION: The microorganism belongs to the genus Candida and has high pyruvic acid productivity, especially Candida sp. MC-P0101 (FERM BP-7539).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism and a method for producing pyruvic acid by the microorganism. More specifically, the present invention relates to a novel microorganism belonging to the genus Candida and having pyruvic acid-producing ability, and a method for producing pyruvic acid in high yield in a short time using the microorganism.

[0002]

BACKGROUND OF THE INVENTION Pyruvate is an important intermediate of biometabolism in a glycolytic system that produces lactic acid (lactic acid fermentation) or ethanol (alcohol fermentation) from sugars, particularly glucose, and is highly reactive by itself. Since it is a ketocarboxylic acid, various α-amino acid production raw materials, metabolism promoting substances, cosmetic raw materials, including pharmaceutical synthetic raw materials and intermediates such as Parkinson's disease drug L-dopa and anti-influenza drugs,
It is a substance useful for various purposes such as medium components and raw materials for flavors.

In addition, pyruvate was found to be involved in the increased transport of glucose to myocytes, which
It is believed that ingestion of pyruvate can increase endurance during sports. In addition, it was found that pyruvate accelerates both body weight and body fat loss by two mechanisms: accelerated metabolism and accelerated fat utilization. For this reason, pyruvic acid alone or in combination with other compounds, diet preparations, pyruvic acid supplement preparations, transplant solutions and perfusion solutions during surgery, enteric preparations,
Oral composition, cosmetics, as well as intermediates of pharmaceutical raw materials, applied to medicines, foods, industrial chemicals, etc.,
Utilizing the acidity of pyruvic acid itself, it is increasingly used for applications such as acidulants and sterilization. Thus, in recent years, the demand for pyruvic acid has increased in various industries, and a large amount of pyruvic acid has been distributed.

[0004] A method for preparing pyruvic acid from a carbohydrate of a substrate by fermentation using a microorganism secreting pyruvic acid, which is one of these useful metabolites, has been studied and reported. ing. For example, Yarrowia lipolytica, which has a pyruvate-producing ability and has a reduced or deleted pyruvate-utilizing ability, is used.
The method of producing pyruvic acid by culturing the mutant strain of a) in a medium to which saccharides are added, allowing pyruvic acid to be produced and accumulated in the medium, and separating and collecting pyruvic acid from the medium (JP-A-9-252790). And a high-concentration pyruvic acid and / or halogenated pyruvic acid-resistant microorganism having a pyruvate-producing ability, particularly torulopsis glabrata (Torulopsis glabrata) is used to produce pyruvic acid. A method for producing pyruvic acid by separating and collecting (JP-A-2000-78996) and the like have been reported.

However, in the method disclosed in the above publication, pyruvic acid is a compound located at the center of biological metabolism, is used in many biosynthetic reactions, and has a high metabolic rate. It is difficult to accumulate in the microbial cells, and by-products such as ketoglutaric acid, glycerin, ethanol and lactic acid are produced in large amounts, and pyruvic acid itself is chemically highly reactive. There is a problem that it is very difficult to purify the pyruvic acid produced in the body. In addition to the above problems, many of the microorganisms used in the method disclosed in the above publication are deficient in the ability to synthesize amino acids and vitamins necessary for their growth, and therefore, these auxotrophic Specific nutrients required by microorganisms, that is, it is essential to add specific vitamins and amino acids to the medium, which is expensive depending on the type of vitamins and amino acids added,
Therefore, there is also a problem that pyruvic acid cannot be manufactured at low cost. Further, for the purpose of inexpensively producing pyruvic acid, it was also attempted to add a specific vitamin or amino acid to the medium with a small amount, but in such a method,
Along with the growth of microorganisms, these specific nutrients are deficient in the medium, which inhibits the growth of bacterial cells, resulting in a problem that the yield of the target metabolic intermediate pyruvic acid decreases.

[0006]

Therefore, an object of the present invention is to provide a novel microorganism having a high pyruvate-producing ability but a low pyruvate-assimilating ability and a high concentration of pyruvate-resistant microorganism, and a pyruvic acid derived therefrom. It is to provide a production method of.

Another object of the present invention is to provide a novel microorganism having a low nutritional requirement for specific vitamins and amino acids and suppressing the production of by-products such as ethanol and lactic acid, and a method for producing pyruvic acid using the same. It is to be.

[0008] Still another object of the present invention is to provide a novel microorganism having a high assimilation rate of sugar and having ammonia resistance, and a method for producing pyruvic acid using the same.

[0009]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors succeeded in screening a microorganism satisfying the above-mentioned various characteristics. Further, as a result of further diligent studies on the method of culturing this microorganism, by culturing this microorganism in a medium in which the initial concentration of sugar in the medium was adjusted to a specific range, pyruvic acid was obtained in a short period at a high yield. I knew that I could produce it. The present invention has been completed based on the above findings.

That is, the above-mentioned various purposes are as follows (1)-
It is achieved by (5).

(1) It belongs to the genus Candida and has the following characteristics: (i) high pyruvate-producing ability but low pyruvate-assimilating ability; (ii) resistance to high-concentration pyruvate; iii) A microorganism having low auxotrophy to vitamins and amino acids; (iv) production of by-products is suppressed; (v) sugar assimilation rate is fast; and (vi) ammonia resistance.

(2) Candida SP MC-P0
101 strain (Candida sp. MC-P0101) FERM BP-75
39. The microorganism according to (1) above, which is 39.

(3) A method for producing pyruvic acid, which comprises using the microorganism according to (1) or (2) above.

(4) The method according to (3) above, wherein the microorganism according to (1) or (2) above is cultured in a culture solution having an initial sugar concentration adjusted to 50 to 200 g / liter to produce pyruvic acid. the method of.

(5) The microorganism according to (1) or (2) above is cultured to produce pyruvic acid in the culture solution.
The method according to (3) or (4) above.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The first aspect of the present invention is the genus Candida.
dida) and (i) high pyruvate production but low pyruvate utilization; (ii) resistant to high concentrations of pyruvate; (iii) low nutritional requirements for vitamins and amino acids; The present invention relates to a microorganism having the characteristics that (iv) production of by-products is suppressed; (v) sugar utilization rate is high; and (vi) ammonia resistance. A particularly preferred example of this microorganism includes Candida sp. MC-P0101 strain (Candida sp. MC-P0101), and this strain is obtained by the following screening method.
Candida utilis IFO 03
96 was obtained as a parent strain.

The second aspect of the present invention relates to a method for producing pyruvic acid, which comprises using the microorganism of the present invention. This method is especially suitable for the initial sugar concentration of 50 to 200 g.
By culturing this microorganism in a medium adjusted to a specific range of 1 / liter, pyruvic acid can be produced in a high yield in a short period of time.

The present invention will be described in detail below.

The present inventor has focused on the fact that it is necessary for pyruvate fermentation to control the metabolic decomposition of pyruvate by pyruvate dehydrogenase (PDC) using thiamine as a coenzyme. After the screening, secondary screening was carried out for pyruvate resistance in consideration of the fact that it is preferable to have resistance even in the presence of a high concentration of pyruvic acid. The screening method according to the invention will now be described in more detail below.

<Primary Screening> Candida utilis IFO 0396 strain cells were treated with N-methyl-N′-nitrosoguanidine (hereinafter abbreviated as “NTG”) by a conventional method. At this time, a commercially available YM agar medium (medium 1000
3 ml of Yeast Extract, De, per ml
xtrose 10g, Malt Extract 3
g, Agar 20 g, Peptone 5 g) to which sodium acetate was added so as to have a concentration of 1% by mass. NTG-treated cells were appropriately diluted and applied to this plate, and the cells were cultured at 24 ° C. for 48 hours, and the grown cells were collected.

The strains thus obtained by the mutation treatment were collected due to the presence of acetic acid in the medium, and the pyruvic acid (hereinafter,
It can be expected to be a bacterium that can produce a large amount of “PA”. In other words, the obtained mutant strain has more P than the parent strain.
If the strain has a reduced DC activity and is capable of producing a large amount of PA, it is considered that the amount of PA accumulated in the fermentation broth is increased and the amount of ethanol accumulated is decreased.

In order to confirm the PA fermentation ability of the strain obtained in the above-mentioned primary screening, PA from the substrate glucose was confirmed.
The amount of accumulated ethanol was compared with that of the parent strain by flask culture. Table 1 shows the results of HPLC analysis of the culture broth after culturing at 30 ° C. for 40 hours in a flask. As shown in Table 1, PA was higher than that of the parent strain in any of the bacteria.
The accumulated amount was large and the accumulated amount of ethanol was small. In addition,
The medium composition was the same as the fermentation medium composition in the fermenter, and calcium carbonate was added to the medium at a rate of 40 g / L for the purpose of pH control.

[0023]

[Table 1]

<Secondary Screening> Among the strains selected in the above-mentioned primary screening, Candida utilis P-27 which had the highest PA accumulation was further added to commercially available YM agar medium at a ratio of 5% by mass to pyruvic acid. The cells were seeded on an agar plate to which sodium had been added, and the cells were cultured in the same manner as described above to collect bacterial cells considered to be resistant to high concentration of PA. It is considered that the strain thus collected is resistant to high concentration of PA and has a high PA production ability.

The strain obtained by the secondary screening was subjected to flask culture to confirm the accumulating ability in the same manner as the confirmation of the primary screening. 30 ℃ in a flask, 40
Table 2 shows the results of HPLC analysis of the culture broth after the time culturing. As shown in Table 2, among the selected strains, the strains having a significantly higher PA accumulation amount than the parent strain were selected as Candida sp. MC-P0101 (Candida sp. MC-P0).
I named it 101).

[0026]

[Table 2]

Next, Candida SP MC-P obtained from the above-mentioned primary and secondary screenings
When 0101 (Candida sp. MC-P0101) was analyzed for mycological properties, the results shown below were obtained.

(A) Cultural and morphological properties Candida cultivated in YM agar medium at 24 ° C. for 5 to 10 days
SP-MC-P0101 forms white to creamy, slightly glossy circular colonies.

Table 3 below shows the morphological characteristics when the cells were cultured on a YM agar medium at a growth temperature of 25 ° C.

[0030]

[Table 3]

(B) Physiological and chemical taxonomic properties Optimal growth conditions; pH: 5-6 Temperature: 25-35 ° C Range of growth; pH: 4-7 Temperature: 20-40 ° C Growth test: 0.01% cycloheximide;- Acetic acid production;- Sugar fermentation capacity:

[0032]

[Table 4]

Carbon compound utilization capacity:

[0034]

[Table 5]

Nitrogen compound utilization capacity:

[0036]

[Table 6]

The microorganisms screened in this manner are, as described above, Candida utilis (Candida).
utilis) IFO 0396 was obtained as a parent strain, and thus belongs to the genus Candida. The thus-screened microorganism has a high pyruvate-producing ability but a low pyruvate-assimilating ability; it has resistance to a high concentration of pyruvic acid; it has a low auxotrophy to specific vitamins and amino acids; ethanol. The production of by-products such as lactic acid and lactic acid is suppressed; the rate of assimilation of sugar is fast; and the property of having ammonia resistance and low oxygen requirement. Therefore, this microorganism was judged to be a novel microorganism, and Candida sp. MC-P0101 strain (Candida sp.
sp. MC-P0101) (hereinafter referred to as “MC-P0101 strain”). The MC-P0101 strain was deposited on April 10, 2001 at the Japan Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and the deposit number is FERM BP-7539.

The method for culturing the microorganism of the present invention will be described below.

The medium used for culturing the strain of the present invention may be either a solid medium or a liquid medium, and a carbon source, a nitrogen source, an inorganic salt and other trace amounts which can be assimilated by the microorganism to be used. A medium containing components, which may be a synthetic medium or a natural medium.

The carbon source that can be used in the culture of the strain of the present invention is not particularly limited as long as it is a carbon source that can be assimilated by the strain. Specifically, in consideration of microbial assimilation, glucose, xylose, sucrose, maltose, α, α-trehalose, methyl α-D-glucoside, cellobiose, raffinose, melezitose, starch, starch hydrolyzate, molasses , Sugars such as molasses; alcohols such as glycerol; organic acids such as gluconic acid and lactic acid; hydrocarbons such as n-paraffin, n-hexadecane, and n-decane. These carbon sources may be used alone or in the form of a mixture of two or more kinds, and are appropriately selected depending on the kind of strain to be used, culture conditions and the like. Of these, glucose, starch, molasses of starch hydrolyzate, waste density and the like are preferably used, and glucose is particularly preferable. Further, the initial concentration of the carbon source in the medium is not particularly limited as long as the strain used grows well and can efficiently produce pyruvic acid, but the initial concentration of the carbon source in the medium is usually
50-200 g / liter, preferably 50-150 g
/ Liter. Particularly when culturing the MC-P0101 strain in a medium using glucose as a carbon source to produce pyruvic acid, the amount is preferably 50 to 200 g / liter, more preferably 75 to 120 g / liter.
It was found that by setting such a specific initial glucose concentration range, the assimilation rate of glucose can be significantly improved, fermentation can be completed in a short time, and thus a high yield of pyruvic acid can be produced in a short culture time. .

The nitrogen source that can be used in the culture of the strain of the present invention is not particularly limited and is appropriately selected depending on the type of the strain used and the culture conditions. Specifically, meat extract, peptone, polypeptone, yeast extract,
Soybean hydrolyzate, soybean powder, milk casein, casamino acid, various amino acids (valine, isoleucine, biotin,
Pantothenic acid, pyridoxine, thiamine, nicotinic acid,
L-lysine, etc.), corn steep liquor, cadaverine, ethylamine, other organic nitrogen compounds such as hydrolysates of animals, plants, microorganisms; organic ammonium salts such as ammonium acetate; ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium chloride, etc. Inorganic ammonium salts such as sodium nitrate; inorganic nitrates such as sodium nitrate; ammonia gas, ammonia water, and inorganic nitrogen compounds such as urea. These nitrogen sources may be used alone or in the form of a mixture of two or more kinds, and are appropriately selected depending on the kind of strain to be used, culture conditions and the like. Of these, yeast extract, peptone, corn steep liquor, aqueous ammonia, and ammonium sulfate are particularly preferably used. The initial nitrogen source concentration in the medium is not particularly limited as long as the strain used can grow well and produce pyruvic acid efficiently.

Inorganic salts that can be used in culturing the strain of the present invention include magnesium, manganese, calcium,
Chlorides such as sodium, potassium, copper, iron and zinc,
1 selected from phosphate, hydrochloride, sulfate and acetate
One kind or two or more kinds can be used. In addition, if necessary, thiamine, nicotinic acid, vitamins such as pyridoxine and biotin, antifoaming agents, vegetable oils, surfactants, and other trace substances such as natural products may be added to the medium to adjust the culture conditions. Stabilization may be attempted.

In the present invention, the culture of the strain is usually carried out under aerobic conditions by shaking, aeration (bubbling) or aeration and agitation culture, and the culture tanks used are batch type, repetitive batch type, continuous type. Regardless of the type or semi-batch type, the stirring tank type or the bubble column (air lift) type, it is appropriately selected depending on the type of the strain to be used, the fermentation capacity, and the like. When producing pyruvic acid using the MC-P0101 strain, MC-P0101 has a low oxygen requirement,
It can grow even with low aeration.

In the present invention, the culturing conditions are appropriately selected depending on the composition of the medium, the culturing method, etc., and are not particularly limited as long as the strain can grow, and the kind of the strain used and other culturing conditions are used. It can be appropriately selected according to the above.
For example, the culture temperature is usually 20 to 40 ° C., preferably 25 to 35 ° C., although it depends on the type of strain.
The culturing time varies depending on the initial sugar concentration, the type of strain, etc., but is usually 30 to 70 hours. In addition, the pH of the medium suitable for culture is 4.0 to 7.0, preferably 5.0 to
It is 6.0. During the culturing period, the pH of the medium is lowered as pyruvic acid is produced and accumulated in the medium. Therefore, when such a decrease in pH occurs, it is preferable to adjust the pH within the above range with an alkali (including an aqueous solution form) such as ammonia, calcium carbonate, sodium hydroxide and potassium hydroxide. Alternatively, as described above, since the MC-P0101 strain has resistance to ammonia, when using this strain, aqueous ammonia may be added to the medium as both the nitrogen source and the neutralizing agent. The above-mentioned alkali may be used alone or in the form of a mixture of two or more kinds, and regardless of the state of solid or liquid, it is easy to handle and delicate pH adjustment is possible. Considering the points, it is more preferable to use the alkali in the form of an aqueous solution.

In the present invention, pyruvic acid is mainly released into the culture medium. The pyruvic acid thus produced may be used as it is without being isolated and purified, but the bacterial cells may be centrifuged or the like. After removal, it is preferably used after being isolated and purified by a conventional method. At this time, pyruvic acid
After completion of the culture, bacterial cells and other insoluble impurities mixed in the medium are removed by filtration or solid-liquid separation, and then can be isolated and recovered from the culture supernatant in the form of pyruvic acid or salt. The isolation / recovery method from the culture supernatant is not particularly limited, and known isolation / purification methods can be similarly used. Specifically, solvent extraction method, ion exchange chromatography method,
Examples thereof include a fractional precipitation method by insolubilization treatment, a fractional crystallization method by crystallization, a membrane separation method by a reverse osmosis membrane, and a concentrated crystallization method. More specifically, after culturing the strain of the present invention under predetermined conditions, the culture solution is filtered or centrifuged to remove bacterial cells and other insoluble impurities. Next, the obtained culture supernatant is acidified with hydrochloric acid or the like, extracted with an organic solvent such as ether, and the collected organic phase is added with an alkali such as an aqueous solution of sodium hydroxide or potassium hydroxide to adjust the pH.
After adjusting to about 6, the mixture is concentrated under reduced pressure at 50 ° C or lower. Furthermore, by adding ethanol to this concentrated liquid, sodium pyruvate or potassium pyruvate can be obtained as crystals. Alternatively, the pyruvic acid may be isolated by once converting the pyruvic acid into a phenylhydrazone and then performing precipitation isolation.

[0046]

EXAMPLES The present invention will now be described more specifically with reference to the examples of the present invention. In the following examples, "%" means "mass%" unless otherwise specified. Example 1 Seed medium containing 10 g / liter glucose, 5 g / liter
1 liter ammonium sulfate, 1 g / liter KH ₂
PO ₄ , 0.5 g / liter of MgSO ₄ .7H ₂ O,
It was prepared by dissolving 0.5 g / liter of yeast extract and 5 g / liter of polypeptone in distilled water, and further adding distilled water to adjust the total volume to 1000 ml.

50 ml of the seed medium thus prepared was placed in a 500 ml Erlenmeyer flask and the temperature was 121 ° C.
Autoclaved for 15 minutes. MC in this medium
-P0101 strain (FERM BP-7539) was inoculated with one platinum loop, and shake culture was performed at 30 ° C for 17 hours to prepare a preculture liquid.

Next, 2 liters of the fermentation medium prepared as described below was charged into a 3-liter bubble column type jar fermenter, and autoclaved at 121 ° C. for 15 minutes. This jar fermenter was inoculated with 20 ml of the preculture solution prepared above, and main culture was carried out at 30 ° C. for 45 hours while maintaining the pH of the medium at about 5.5 with a 10% aqueous solution of sodium hydroxide. went.

The fermentation medium was 100 g / liter glucose, 2.5 g / liter ammonium sulfate,
After dissolving 0.5 g / liter KH ₂ PO ₄ , 0.25 g / liter MgSO ₄ .7H ₂ O, 0.5 g / liter yeast extract, and 5 g / liter polypeptone in distilled water, further distilled water And the total volume is 1000m
It was prepared by adjusting to 1.

After culturing for a predetermined time, the culture supernatant excluding the bacterial cells was analyzed by liquid chromatography. Pyruvate was quantified by high performance liquid chromatography and NADH absorbance measurement method using lactate dehydrogenase, and the results of both analysis methods were in good agreement. The results are shown in Table 7 below.

Comparative Example 1 In Example 1, the parent strain Candida utilis IF was used instead of the MC-P0101 strain.
Culture was performed in the same manner as in Example 1 except that O 0396 was used and the culture time was 30 hours, and the culture supernatant was analyzed in the same manner as in Example 1. The results are shown in Table 7 below.

Example 2 In the same manner as in Example 1, 10% aqueous ammonia was used as a pH adjusting agent in place of the 10% aqueous sodium hydroxide solution, and a medium without ammonium sulfate was used as the fermentation medium. The cells were cultured as described above, and the culture supernatant was analyzed in the same manner as in Example 1. The results are shown in Table 7 below.

[0053]

[Table 7]

From Table 7, it is shown that the MC-P0101 strain of the present invention produces a high yield of pyruvic acid in the culture broth, and particularly significantly suppresses the by-product of ethanol.

Example 3 In a 500 ml Erlenmeyer flask, 50 ml of a fermentation medium prepared in the same manner as in Example 1 except that the glucose concentration was changed as shown in Table 8 below was placed, and 121 ° C., 15
Pressurized steam sterilization was performed for a minute. MC-P01 was added to this medium.
One platinum loop was inoculated with 01 strain and 4% calcium carbonate was added to the medium.
At a temperature of 30 ° C., as shown in Table 2 below,
A culture solution was prepared by shaking culture.

The amount of pyruvic acid contained in the culture supernatant of each of the thus prepared culture broths was analyzed by the same liquid chromatography as in Example 1. The results are shown in Table 8 below.

[0057]

[Table 8]

Example 4 In a 500 ml Erlenmeyer flask, 50 ml of the seed medium prepared in the same manner as in Example 1 was placed, and the temperature was 121 ° C. and the temperature was 15 ° C.
Pressurized steam sterilization was performed for a minute. MC-P01 was added to this medium.
One platinum loop of the strain 01 was inoculated and shake-cultured at 30 ° C. for 17 hours to prepare a preculture liquid.

Next, 2 liters of the fermentation medium prepared in the same manner as in Example 1 was charged into a 3 liter airlift type jar fermenter, and autoclaved at 121 ° C. for 15 minutes. This jar fermenter was inoculated with 20 ml of the preculture solution prepared above, and main culture was carried out at 30 ° C. for 45 hours while keeping the pH of the medium at 5.5 with a 10% aqueous solution of sodium hydroxide. It was

The amount of pyruvic acid contained in the culture supernatant of the thus-prepared culture broth was analyzed by liquid chromatography in the same manner as in Example 1. The results are shown in Table 9 below.

Comparative Example 2 In Example 3, the parent strain Candida utilis IF was used instead of the MC-P0101 strain.
Culturing was performed in the same manner as in Example 1 except that O 0396 was used, and the amount of pyruvic acid contained in the culture supernatant was analyzed by the same liquid chromatography as in Example 1.
The results are shown in Table 9 below.

Comparative Example 3 Torulopsis glabrata I, which is known to produce pyruvic acid in high yield
FO 0005 strain was added to Bacto Yeast Carbon Base medium (Difc
One platinum loop was inoculated into 5 ml (manufactured by O Co.) and shake-cultured at 30 ° C. for 24 hours to prepare a preculture liquid.
Next, a medium (composition: 115 g / liter glucose, 5 g / liter ammonium sulfate, 0.5 g / liter polypeptone, 1 mg /
Liter nicotinic acid, 0.02 mg / liter thiamine hydrochloride, 40 g / liter calcium carbonate) 50
Inoculate a 500 ml Erlenmeyer flask containing 100 ml, 180r
The cells were cultured for 70 hours under the conditions of pm and an amplitude of 30 cm.

The amount of pyruvic acid contained in the culture supernatant of the thus-prepared culture broth was analyzed by the same liquid chromatography as in Example 1. The results are shown in Table 9 below.

Comparative Example 4 Yarrowia lipolytica ATCC, which is known to produce pyruvic acid in high yield
20363 as a pre-culture medium (composition: 10% glucose, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ · 7H ₂
O, 4% polypeptone, 4% CaCO ₃ , pH 5.5)
Inoculate one platinum loop in 200 ml, at 30 ℃ for 48 hours,
The preculture liquid was prepared by shaking culture. Next, basal medium (composition: 10% glucose, 0.1% KH
2.5 liters of ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O, 4% polypeptone, pH 5.5) were inoculated with 200 ml of the preculture solution prepared above and cultured at 30 ° C. for 96 hours. .. The culture was carried out with a sodium hydroxide aqueous solution in a jar fermenter while maintaining the pH at 5.5.

The amount of pyruvic acid contained in the culture supernatant of the thus-prepared culture broth was analyzed by the same liquid chromatography as in Example 1. The results are shown in Table 9 below.

[0066]

[Table 9]

From the results shown in Table 9, MC-of the present invention
It is shown that the P0101 strain can produce a large amount of pyruvic acid in a short time as compared with the parent strain and other strains known to produce pyruvic acid in high yield.

[0068]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a novel microorganism belonging to the genus Candida and capable of producing pyruvic acid, and particularly Candida sp. MC-P0101 of the present invention. Strain (Candida sp.MC-P010
1) FERM BP-7539 has a high pyruvate-producing ability but a low pyruvate-assimilating ability; it has resistance to high-concentration pyruvic acid; low auxotrophy to specific vitamins and amino acids; It has the characteristics that the production of by-products is suppressed; the rate of assimilation of sugar is fast; and that it has ammonia resistance.

The present invention also provides a method for producing pyruvic acid, which comprises using the novel microorganism of the present invention. In particular, the initial glucose concentration in the culture medium was 7
By adjusting to a specific range of 5 to 120 g / liter, the assimilation rate of sugar by microorganisms can be significantly improved and fermentation can be completed in a short time. Therefore, a high yield of pyruvic acid can be obtained in a short culture time. Can be produced.

Further, Candida sp. M of the present invention
C-P0101 strain (Candida sp. MC-P0101) FERM B
Since P-7539 produces pyruvic acid in the culture medium, continuous culture is possible, and therefore P-7539 is suitable for mass production.

Claims (5)

[Claims] 1. A genus belonging to the genus Candida and having the following characteristics: (i) high pyruvate-producing ability but low pyruvate-utilizing ability; (ii) resistance to high-concentration pyruvate; (iii) ) Microorganisms having low nutritional requirements for vitamins and amino acids; (iv) production of by-products is suppressed; (v) sugar utilization rate is high; and (vi) ammonia resistance. 2. Candida SP MC-P0101
The microorganism according to claim 1, which is a strain (Candida sp. MC-P0101) FERM BP-7539. 3. A method for producing pyruvic acid, which comprises using the microorganism according to claim 1 or 2. 4. The method according to claim 3, wherein the microorganism according to claim 1 is cultivated in a culture solution in which the initial sugar concentration is adjusted to 50 to 200 g / liter to produce pyruvic acid. 5. The method according to claim 3 or 4, wherein the microorganism according to claim 1 or 2 is cultured to produce pyruvic acid in the culture solution.