JPS63133990A - Production of pyruvic acid - Google Patents

Abstract

PURPOSE:To efficiently produce pyruvic acid useful as a raw material for drugs and chemicals, by treating hydroxyacetone or methylglyoxal with an enzyme or a microorganism or an extract thereof. CONSTITUTION:An aqueous solution of hydroxyacetone or methylglyoxal containing a proper inorganic salt is treated with an enzyme such as alcohol oxidase, alcohol dehydrogenase, aldehyde oxidase, aldehyde hydrogenase, formaldehyde dehydrogenase, etc., a microorganism producing the enzyme or an extract thereof at pH4-9 at 4-40 deg.C by a method wherein the enzyme, etc., is directly dissolved or suspended or the enzyme, the microorganism or the extract is immobilized and the aqueous solution is directly brought into contact with the immobilized substance, the reaction is carried out and pyruvic acid is obtained from the reaction solution by organic solvent extraction method.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides hydroxyacetone (CH, COCH, OH)
Alternatively, the present invention relates to a method for producing pyruvic acid (CH, C0C00H) from methylglyoxal (CH, C0CHO).

Pyruvate is an intermediate metabolite produced in the glycolytic system, and plays an important role in sugar metabolism and amino acid metabolism in living cells, and is known to act as a metabolic promoter on cells.

Industrially, it has a wide range of uses as a raw material for the production of various amino acids such as alanine, tyrosine, and tryptophan, and as an intermediate raw material for the production of various pharmaceuticals. Therefore, if it can be produced at low cost, it will be extremely useful as a raw material for the synthesis of pillars and as a metabolic promoter.

(Prior art) Conventionally, methods for producing pyruvic acid include fermentation using various microorganisms, reacting sodium cyanide with acetyl chloride to synthesize acetyl cyanide, and hydrolyzing this, or using lactic acid or hydroxyacetone. A method of emulsifying the compound using a metal catalyst is known.

(Problems to be solved by the invention) However, the fermentation method and the method via acetyl cyanide have low yields and produce large amounts of by-products, and the method using a metal catalyst requires an expensive catalyst. Since regeneration is also complicated, it is not necessarily an advantageous method for producing pyruvic acid industrially and effectively. Therefore, wood inventors and others aimed to produce pyruvic acid for industrial purposes.
As a result of intensive research, we discovered that pyruvic acid can be efficiently produced by oxidizing inexpensive hydroquinacetone or its oxide, methylglyoxal, with enzymes, microorganisms, or its extracts.To complete the present invention. It's arrived.

(Means for Solving the Problems) The present invention is a method for producing pyruvic acid, which is characterized in that pyruvic acid is produced by reacting hydroquine acetone or methylglyoxal with an enzyme, a microorganism, or an extract thereof.

The enzyme used in the present invention is an enzyme that converts hydroquine acetone or methylglyoxal rubic acid, and examples thereof include alcohol okindase, alcohol dehydrogenase, aldehyde oxidase, aldehyde dehydrogenase, and formaldehyde dehydrogenase. Also, as a microorganism, meta/
Examples include bacteria, yeast, actinomycetes, and molds that have the ability to assimilate bacteria.

Enzymes that act on hydroquine acetone to produce pyruvate include (1) alcohol oxidase or θ0
Alcohol dehydrogenase, IIID(i) Alcohol oxidase GO A combination of alcohol dehydrogenase and aldehyde oxidase, aldehyde dehydrogenase or formal dehydrogenase is used.

The microorganism or extract thereof that produces pyruvate using the hydroxyacetone series may be any microorganism or extract thereof that produces the above-mentioned enzyme. Examples of microorganisms include bacteria such as Pseudomonas and Methylomonas, yeasts such as Satucharomyces, Pichia, Hannusella, Torulopsis, Candida, Chloechera, and Rhodotorula, actinomycetes such as Streptomyces, and molds such as Bryophragium and Paenromyces nato. .

As extracts from microorganisms, those obtained by applying various extraction methods to cultured cells or culture fluids of the above-mentioned microorganisms are used. Extraction methods include lysozyme, lytic enzymes such as Zymoliel, Dynomir, surfactants, and EDTA.
, using salts, acid treatment, alkali treatment, organic solvents, chaotropic ions, ultrasound, French press, and other physical crushing methods.

Enzymes that act on methylglyoxal to produce pyruvate include (i) alcohol oxidase, (IO
Using alcohol dehydrogenase, Gii) aldehyde oxidase, Oo aldehyde dehydrogenase or (v) formaldehyde dehydrogenase.

The microorganism or extract thereof that acts on methylglyoxal to produce pyruvate may be any microorganism or extract thereof that produces the above-mentioned enzyme. Examples of microorganisms include those mentioned above. Examples of the extraction method include those mentioned above.

In the method of the present invention, pyruvic acid is produced by causing the enzyme, microorganism, or extract thereof to act on an aqueous solution of hydroxyacetone or methylglyoxal containing appropriate inorganic salts, and the inorganic salts that can be used in the present invention include: Metal salts such as potassium, sodium, manganese, magnesium, zinc, iron, copper, and titanium, and salts such as sulfuric acid, phosphoric acid, hydrochloric acid, nitric acid, and acetic acid can be used.

The pH of the reaction solution is 4 to 9, and the reaction temperature is 4 to 40°C. The reaction may be carried out by directly dissolving or suspending the enzyme, the microorganism, or the extract thereof, or by immobilizing the enzyme, the microorganism, or the extract, and bringing the reaction solution into contact with the immobilized product.

After the reaction is completed, the reaction solution is filtered or centrifuged to remove bacterial cells and other impurities, and the resulting in solution or centrifugation supernatant is added with, for example, hydrochloric acid to make a hydrochloric acid acidic solution with a pH of 2 or less. Afterwards, it is extracted with a hydrophobic organic solvent. The hydrophobic organic solvent used here is ethyl ether,
Methyl ethyl ketone, chloroform, petroleum ether, etc. are effective.

The organic solvent extract is concentrated under reduced pressure at 50° C. or lower, the concentrate is dissolved in cold water, and aqueous caustic soda solution is added to adjust the pH to around 8, followed by the addition of ethanol. By adding ethanol, pyruvic acid crystallizes as sodium pyruvate to form a precipitate, so the ethanol precipitate is collected by filtration or centrifugation, and dried under reduced pressure to be collected as a powder of sodium pyruvate.

In the present invention, as an intermediate raw material for manufacturing pharmaceuticals and industrial chemicals,
Alternatively, pyruvic acid, which is industrially very useful as a metabolic promoter, can be produced efficiently and at low cost.

(Example) Next, the present invention will be explained in more detail with reference to Examples.

Example 1 0.5%, Kt HP O
40.8%, FeCQ, -68,OO, 14% was dissolved in distilled water and adjusted to pH 7.0. The solution 10mff
100m! ! Dispense into a volume Erlenmeyer flask, add 100 U of alcohol okindase (manufactured by Toyobo), and add 30
The reaction was carried out by shaking at 13°C for an hour. After the reaction was completed, the amount of pyruvic acid in the reaction solution was determined and 0.047% of pyruvic acid was produced.

Example 2 Methylglyoxal 0.5%, K, HPO40.8
%, Fe CQ s ・6 H-00, 14%, NA
D5% was dissolved in distilled water and adjusted to pH 7.0. 10 m of the solution was dispensed into a Loomg Erlenmeyer flask, 50 U of formaldehyde dehydrogenase (manufactured by Toyobo) was added, and a shaking reaction was performed at 30'C for 110 hours. After the reaction was completed, the amount of pyruvic acid in the reaction solution was determined, and 0.36% of pyruvic acid was produced.

Example 3 Methanol 2.0%, polypeptone 0.05%, yeast extract 0.05%, corn steep force -0.5%, K
I HPO40.4%, ZnSO4*7Ht O0.0
02%, Mg5CL”7H,OO,075%, FeC
Dissolve 25”6Ht OO, 002% in tap water and adjust the pH
A culture medium adjusted to 5.0 and sterilized by autoclaving at 120°C for 20 minutes was obtained. 50 mff of this medium
Dispense g into Yosaka Lough flasks and pre-incubate 30 g with medium of the same composition.
Hansenula polymorpha I incubated at ℃ for 10 hours with shaking
A cultured cell fluid of the FO1476 strain was inoculated at 1.5 mff1 (equivalent to 3% per medium) and cultured with shaking at 30°C for 50 hours. After culturing, the mixture was centrifuged under cooling to obtain 1.1 g of viable bacterial cells.

Hydroxyacetone 5Va, KI HPO40, 35%
, K., HPo.

0.55% was dissolved in distilled water and adjusted to pH 6.0.

Dispense 10 mg of the solution into a 100 mQ Erlenmeyer flask,
Add 0.5 g of the obtained viable bacterial cells to this and hold at 10°C14
o Time tremor W reacted. After the reaction is completed, centrifuge to remove the bacterial cells, and remove the li! When liquid f was obtained and the amount of pyruvic acid in the supernatant liquid was determined, 0.079% pyruvic acid was produced.

Example 4 After dissolving 2 g of sodium alginate in 100 mf of distilled water and adjusting the pH to 7.0, alcohol okindase 2
000U was added and stirred. This was dropped into a 5% chloride solution and gelled into beads with a diameter of 0.5 to 1.0 mm.

The gel was packed into a glass column with a diameter of 'lOc and a length of 1 m, and 300 mQ of 20 mM phosphate buffer, pH 7.0, was passed through the column. Hydroxyacetone 5%, K, H
PO40,8%, FeCQs a6Hx OO,14
% was dissolved in distilled water and adjusted to I) H7,O, and 300 mQ of the solution was circulated through the column at a flow rate of 2.5 m91 min.

After the cyclic reaction at 30°C, the amount of pyruvic acid was determined, and 0.063% of pyruvic acid was produced.

Example 5 1.5 g of acrylamide monomer 1N, N'-methylenebisacrylamide somg, and 0.2 g of live yeast cells obtained in the same manner as in Example 3 were added to 20 mM phosphate buffer 1
) Dissolved in H7,0,8mg or! i! ! 5% of β-tumethylaminopropionitrile 1- Om Q as a polymerization accelerator and 1.0 m+ of potassium beroxo-2-sulfate as a polymerization initiator were added to the polymerization solution.
! was added and a polymerization reaction was carried out at 23°C for 10 minutes to obtain bead-shaped polyacrylamide gels having a diameter of 2 to 51 cm. Methylglyokyl 2.0%, K, HPO40.8%
, FeCts "68m OO, 14% was dissolved in distilled water and adjusted to 1) H6,O. 100mt of the solution was dispensed into a beaker and the prepared polyacrylamide gel 10
g was added thereto, and the mixture was reacted at 10° C. for 30 hours while stirring with a stirrer. After the reaction was completed, the amount of pyruvic acid in the reaction solution was measured, and 0.014% pyruvic acid was produced.

Example 6 In the same manner as in Example 1, 10
Prepare the same hydroquine acetone solution as in Example 1, dispense it, add alcohol okindase in the same manner as in Example 1, and shake it for 30 mm for 130 hours. After the reaction is completed. Take 100 mQ of the reaction solution, add hydrochloric acid to adjust the pH to below 2, and dilute with 100 mQ of ethyl ether.
The extracts were extracted twice and the extracts (notified with ether) were combined and concentrated under reduced pressure at below 50°C. After dissolving the precipitated pyruvic acid in 10 mQ of cold water, a caustic soda solution was added little by little to adjust the pH to 6.0, and ethanol was added dropwise to precipitate crystals of sodium pyruvic acid. Subsequently, sodium pyruvate was collected on a glass filter, washed with ethanol, and then dry-exploded under reduced pressure to obtain 0.14 g of powder. After accurately weighing 0.1 g of this powder and dissolving it in cold water, the amount of pyruvic acid was measured according to a conventional method using lactate dehydrogenase. As a result, 96.1% was sodium pyruvate.

(Effects of the Invention) According to the present invention, pyruvic acid can be obtained by reacting hydroquinoacetone or methylglyoxal with an enzyme, a microorganism, or an extract thereof. As shown in Examples 1 to 6, it has been revealed that the present invention enables the production of pyrupinic acid in a short time and is an efficient method for producing pyruvic acid.

Claims (1)

[Claims] 1. A method for producing pyruvic acid, which comprises reacting hydroxyacetone or methylglyoxal with an enzyme, a microorganism, or an extract thereof to produce pyruvic acid.