JPH06197778A - Production of phosphoenolpyruvic acid - Google Patents

Abstract

PURPOSE:To easily separate and purify a phosphoenolpyruvic acid under mild condition by forming the phosphoenolpyruvic acid from a carbon source using cultured product of yeast of the genus Saccharomyces, Hansenula, Pichia, Candida, Torulopsis, etc., and collecting the produced acid. CONSTITUTION:A yeast the belongs to the genus Saccharomyces, Hansenula, Pichia, Candida or Torulopsis (e.g. Torulopsis glabrata IFO 0622) is inoculated to a medium and cultured at 30 deg.C for 24 hr under shaking. The cultured liquid is subjected to centrifugal separation to collect the yeast cells. The yeast is made to react with a carbon source assimilable by the yeast (e.g. glucose) at 30 deg.C for 30hr and the produced and accumulated phosphoenolpyruvic acid is separated from phosphoric acid with a reverse osmosis membrane, etc. The phosphoenolpyruvic acid is incorporated with an alkali metal hydroxide and concentrated and crystallized in the form of an alkali metal salt to obtain the objective phosphoenolpyruvic acid under mild condition without using complicate operations.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for collecting and collecting phosphoenolpyruvate from a carbon source by using a carbon source assimilation enzyme system produced by yeast.

Phosphoenolpyruvate (hereinafter abbreviated as PEP) is a high energy phosphate compound and is an essential and important compound in the regeneration system of adenosine triphosphate (ATP).

[0003]

2. Description of the Related Art The chemical synthesis of PEP has long been known (Ch
em. Pev. , Vol. 61, 607 (1961))
As a method for producing PEP by biochemical means, Debaryomyces (Debaryomyces) is used.
s) Method using dried bacterial cells of the genus (J. Ferment.
Technol. , Vol. 65,225 (198
7)) is known.

[0004]

However, the yield of chemical synthesis is low and the yield is low, and the enzymatic method using dried cells is
There is a problem that the operation is complicated, such as adjustment of dried cells.

[0005]

Means for Solving the Problems The inventors of the present invention have earnestly studied the yield and the method for producing PEP with high yield and simple operation, and as a result, have reached the following invention.

That is, the present invention relates to Saccharomyces (S
genus accharomyces, Hansenula (Hans)
genus enula, Pichia genus, Candida genus or Torrlopsis (Tor)
A method for producing phosphoenolpyruvate characterized in that a phosphoenolpyruvate is produced and accumulated from a carbon source which can be assimilated by the yeast, using a culture of a yeast belonging to the genus is there.

Any yeast can be used in the present invention as long as it is a yeast belonging to the genera Saccharomyces, Hansenula, Pichia, Torlopsis and Candida. Among these, those having high ability to utilize various carbon sources can be preferably used. Specific examples of preferred yeast include, for example, Saccharomyces cerevisiae (I
FO 0213, 0538, 1950), Saccharomyces kluy.
veri (IFO 1982), Hansenula glucozyma Hansenula glucozyma (IF
O 1472), Pichia pastoris Pichia p
astoris (IFO 0948), Candida
Methanolica candida methanolica
(ATCC 26175), Candida lipolytica Candida lipolytica (IFO0
717), Torulopsis pinas Torulopsi
s pinus (IFO 0741), Torrlopsis
Grabrata Torulopsis grabrat
a (IFO 0622) and the like.

In the present invention, a yeast culture is used. The culture can be prepared by culturing the above yeast in a suitable nutrient medium. As a medium for culturing these yeasts, an ordinary natural or synthetic medium is used, but a natural medium containing an appropriate amino acid is preferably used.

The form of the yeast culture to be used in the present invention is arbitrary, and the yeast culture itself, cultured live cells, vacuum-dried cells, freeze-dried cells, dried cells by an organic solvent, etc. The dried bacterial cells, treated bacterial cells, etc. are included in the scope of the present invention. Of these, industrially, a culture itself obtained by culturing yeast in a nutrient medium is advantageously used.

Any carbon source which is a raw material for producing pyruvic acid may be used as long as it can be assimilated by the yeast used in the present invention. Specific examples of preferred carbon sources include glucose, fructose, sucrose, mannose, mannitol, xylose, galactose, molasses, sorbitol, sugars or sugar alcohols such as glycerin, acetic acid, citric acid, organic acids such as lactic acid, methanol, ethanol. , Alcohols such as propanol, and hydrocarbons. By using sugar or sugar alcohol, more preferable results can be obtained.

The amount of the yeast culture used in the present invention is preferably 1 to 50 g / l, more preferably 5 to 20 g / l in terms of dry cell concentration.

The enzyme reaction in the product accumulation system is ATP,
Many require magnesium ions, potassium ions and phosphoric acid, and ATP is 0.1 mM to 15 mM,
Preferably 1mM~5mM, MgSO ₄ · 7H ₂
O is 0.01 to 0.2%, preferably 0.02 to 0.1
%, And KH ₂ PO ₄ is ₃ to 14%, preferably 6 to
It is usually used at a concentration of 13%.

During the production-accumulation reaction, p is generated as the organic acid is produced.
Since a decrease in H occurs, pH is usually 5 to 10, preferably 7 to 9 with an alkali such as calcium carbonate or caustic potash.
Is effective for phosphoenolpyruvate production.

The temperature during the reaction is suitably 20 to 37 ° C, preferably 25 to 32 ° C.

After completion of the reaction, the phosphoenolpyruvate produced and accumulated in the production and accumulation system is separated into phosphoenolpyruvate and phosphoric acid using a reverse osmosis membrane or the like, alkali hydroxide is added, and phosphoenolpyruvate alkali is added. It can be isolated by concentration and crystallization as an aqueous metal salt solution.

[0016]

EXAMPLES The present invention will be described below with reference to examples.

Confirmation and quantification of pyruvic acid produced in the examples were carried out by high performance liquid chromatography, an enzymatic method using pyruvate kinase and lactate dehydrogenase, and the like. The following analytical results were in good agreement with both of the above analytical methods and showed the same analytical values.

Example 1 Various yeasts shown in Table 1 were mixed with 0.5% glucose and KH _{2
PO 4 0.2%, MgSO 4 ·} 7H 2 O0.05%, 1.0% peptone, 0.1% yeast extract, after dispensing sterilized twice locations 100ml consisting pH6.0 to 500ml-volume shaking flask, 1 The cells were inoculated with platinum and cultured with shaking at 30 ° C. for 24 hours.

After the completion of the culture, the cells were collected by centrifugation to collect the cells, which were glucose 1%, KH ₂ PO ₄ 9% and MgSO ₄ .7H _2.
60 ml of reaction solution containing 0.05% O and 5 mM ATP
(PH adjusted to 7.5 with KOH aqueous solution), 30
Reacted at 30 ° C. for 30 hours.

The phosphoenolpyruvate produced in each reaction solution is shown in Table 1.

[Table 1]

Example 2 The carbon source of the reaction solution used in Example 1 was replaced with the carbon source shown in Table 2, and the results of culturing with the strains shown in Table 2 were shown in Table 2.
Shown in.

[0022]

[Table 2]

[0023]

INDUSTRIAL APPLICABILITY According to the present invention, phosphoenolpyruvate can be produced from a carbon source under mild conditions without complicated operations using a carbon source-utilizing enzyme system produced by yeast. .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12P 9/00 C12R 1:78) (C12P 9/00 C12R 1:84) (C12P 9/00 C12R 1:72) (C12P 9/00 C12R 1:73) (C12P 9/00 C12R 1:88)

Claims (1)

[Claims] 1. Saccharomyces
ces, Hansenula, Pichia, Candida
a) Phosphoenols characterized by producing and accumulating phosphoenolpyruvate from a carbon source that can be assimilated by the yeast using a culture of yeast belonging to the genus or the genus Torulopsis, and isolating and collecting the phosphoenolpyruvate. Method for producing pyruvic acid.