JPH035800B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing, accumulating, and collecting pyruvic acid from a carbon source using a carbon source assimilating enzyme system produced by yeast. Pyruvate is an important intermediate in biological metabolism,
It is not only a useful raw material for the synthesis of various medicines and agrochemicals, 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] Conventionally, as a method for producing pyruvic acid using yeast, a fermentation method using yeasts of the genus Satucharomyces, Candeida, etc. is known (Japanese Patent Publication No. 796/1983). [Problems to be solved by the invention] However, this method is carried out using a limited number of specific yeasts, especially yeasts that require thiamin, which have a weak carbon source utilization ability and a slow pyruvate production rate. There were some disadvantages such as low quality. In addition, other yeasts with high carbon source utilization ability had high pyruvate degrading activity and were unable to significantly accumulate pyruvate. [Means and effects for solving the problem] That is, the object of the present invention is to solve the problem by producing and accumulating pyruvate from a carbon source using a culture of yeast that has a strong ability to utilize various carbon sources. The purpose of the present invention is to suppress decomposition, and as a result of intensive research, the present inventors have arrived at the following invention. That is, the present invention uses a culture of yeast belonging to the genus Saccharomyces, the genus Hansenula, the genus Pichia, the genus Candida, or the genus Torulopsis to produce oxythiamine, pyrithiamine, or This is a method for producing pyruvic acid, which comprises producing and accumulating pyruvic acid from a carbon source that can be assimilated by the yeast in the presence of a salt thereof, and then isolating and collecting the pyruvic acid. As the yeast that can be used in the present invention, any yeast belonging to the genus Satucharomyces, genus Hansenula, genus Pichia, and genus Candeida can be used. Among these, those having high ability to utilize various carbon sources can be preferably used. Specific examples of preferred yeast include, for example, Saccharomyces cerevisie (IFO 0213,
0538, 1950), Satucharomyces kluyveri
Saccharomyces kluyveri (IFO 1892), Saccharomyces excigus
exiguus (IFO 0219) Hansenula capsulata (IFO 0974), Hansenula glucozyma (IFO
1472), Pichia pastoris
(IFO 0948), Candida cariosilignicola (IFO 1910), Candida methanolica
(ATCC26175), Candeida lipolyteica
Candida lipolytica (IF0 0717), Torulopsis
Torulopsis pinus (IFO 0741) Torulopsis glabrata
(IF00622) etc. In the present invention, a yeast culture is used. The culture can be prepared by culturing the yeast described above in an appropriate nutrient medium. As a medium for culturing these yeasts, ordinary natural or synthetic media can be used, but natural media containing an appropriate amount of amino acids are preferably used. The form of the yeast culture used in the present invention is arbitrary, and the yeast culture itself, cultured live cells, vacuum-dried cells, freeze-dried cells, dried cells such as dried cells using an organic solvent, etc. cells, treated bacterial cells, etc. are included within the scope of the present invention. Among these, the culture itself obtained by culturing yeast in a nutrient medium is advantageously used industrially. The carbon source which is the raw material for producing pyruvic acid may be any carbon source 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,
Sugar or sugar alcohol such as galactose, molasses, sorbitol, glycerin, acetic acid, citric acid,
Examples include organic acids such as lactic acid, alcohols such as methanol, ethanol, and propanol, and other hydrocarbons. More favorable results can be obtained by using sugar or sugar alcohol. The amount of yeast culture used in the present invention is preferably 1 to 50 g/, more preferably 6 to 15 g/in terms of dry cell concentration. Examples of oxythiamine or pyrithiamine or a salt thereof that can be used in the present invention include oxythiamine, pyrithiamine, oxythiamine hydrochloride, and pyrithiamine hydrochloride. Thiamine hydrochloride is preferred. The amount of oxythiamine or pyrithiamine or a salt thereof used in the production/accumulation system is usually 50 μmol/ to 10 mmol/, preferably 0.1 to 2.0 mmol/. In addition, many enzyme reactions in the production/accumulation system require magnesium ions, potassium ions, and phosphoric acid, and MgSO ₄ 7H ₂ O is 0.01 to 0.2%, preferably 0.02 to 0.1%, and KH ₂ PO ₄ is 0.05~2.0
%, preferably 0.1 to 0.5. During the production-accumulation reaction, the pH decreases as organic acids are produced, so for the production of pyruvic acid, the pH is usually adjusted to 3 to 8, preferably 4 to 6, with an alkali such as calcium carbonate, caustic soda, or caustic potash. It is effective for The temperature during the reaction is 20-32℃, preferably 24-30℃
is appropriate. In the present invention, in the production/accumulation system, pyruvic acid is normally produced and accumulated mainly through enzymatic reactions, but there is no problem even if pyruvate is produced and accumulated through fermentation, and conversely, pyruvate is generated and accumulated through fermentation. The production and accumulation of pyruvic acid may be the main activity. After the reaction is completed, the pyruvic acid produced and accumulated in the production accumulation system can be isolated and collected by a conventional method.
For example, it can be isolated by methods such as hydrochloric acid acidic ether extraction and ion exchange treatment. [Example] The present invention will be described below with reference to Examples. Confirmation and quantification of pyruvate produced in the Examples were performed by high performance liquid chromatography, lactate dehydrogenase method, etc. 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% glycose,
KH ₂ PO ₄ 0.2%, MgSO ₄ 7H ₂ O 0.05%, peptone
Medium 100 consisting of 1.0%, yeast extract 0.1%, PH6.0
ml was dispensed into a 500 ml shaking flask, sterilized, inoculated into one platinum loop, and cultured with shaking at 30°C for 24 hours. After culturing, collect the bacteria by centrifugation and collect the bacteria.
_{KH2PO4 0.2} %, _MgSO4・_7H2O0.05 %, _CaCO3
3.0%, oxythiamine hydrochloride 0.5mM or
60ml reaction solution containing 0mM, 5.0% glucose
(PH5.0) and subjected to shaking reaction at 30°C for 48 hours. Pyruvic acid produced in each reaction solution was as shown in Table 1.

[Table] Also, Satsukaromyces cerevisiae IFO 0538
After sterilizing the reaction solution 1 in which the reaction was carried out in the presence of 0.5mM of oxythiamine (reaction solution containing 36.9 g/pyruvic acid), hydrochloric acid was added to the supernatant to adjust the pH to 2.0. extracted with ethyl ether,
Next, the pH was neutralized to 6.0 with caustic soda and concentrated under reduced pressure at 40°C to a volume of about 100 ml. Ethanol was added dropwise to this concentrated solution to obtain 25.8 g of sodium pyruvate (purity 97%). Example 2 Satucharomyces cerevisiae (IFO 0538) was cultured in the same manner as in Example 1. After the culture was completed, a reaction was carried out in the same manner as in Example 1 using a reaction solution in which 0.5 mM of oxythiamine was replaced with 2.0 mM of pyrithiamine, and 20.6 g/pyruvic acid was produced and accumulated after 60 hours. Example 3 Each yeast shown in Table 2 was cultured in the same manner as in Example 1. When the reaction was carried out in the same manner as in Example 1 by replacing glucose with the carbon source shown in Table 2, the amount of pyruvic acid produced in each reaction solution was as shown in Table 2.

〔Effect of the invention〕

According to the present invention, a significant amount of pyruvic acid can be accumulated even when using general yeast rather than yeast having a special mutation. Furthermore, when yeast with a strong carbon source assimilation ability is used, the decomposition of pyruvic acid can be suppressed and a significant amount of pyruvic acid can be accumulated. Therefore, the method of the present invention can be advantageously applied to industrial production.

Claims (1)

[Claims] 1 Saccharomyces spp., Hansenula spp., Pichia spp.
Using a culture of yeast belonging to the genus Candida or Torulopsis, pyruvic acid is produced and accumulated from a carbon source that can be assimilated by the yeast in the presence of oxythiamine or pyrithiamine or a salt thereof. 1. A method for producing pyruvic acid, which comprises separating and collecting the pyruvic acid.