JPH0822222B2 - 2-Ketobutyric acid manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel microorganism and a method for producing 2-ketobutyric acid by the microorganism. 2-Ketobutyric acid is, for example, a raw material for isoleucine fermentation or a raw material for optically active hydroxybutyric acid, optically active aminobutyric acid, and antibiotic paulomycin A.

(Prior Art) Conventionally, as a method for industrially synthesizing 2-ketobutyric acid, a method is known in which diethyl oxalate and diethyl propionate are dehydrated and condensed, and then hydrolyzed with concentrated sulfuric acid. In addition, a method of obtaining acetaldehyde and cyanomethyl chloride as starting materials in multiple steps, a method of hydrolyzing 2-methanesulfonyloxycrotonic acid, a method of hydrolyzing etoxaryl propionic acid ethyl ester, and the like are known.

In addition to the above-mentioned chemical synthesis method, a method using a microorganism is described in Aerobaerae aerogenes (Aeroba
cte aerogenes), Micrococus lysodeiktricus, Torulopsis utilis, Escherichia
Dihydroxyacid dehydratas produced by Escherichia coli, Saccharomyces cerevisiae, etc.
e (4.2.1.9)) is known to have the ability to convert 2,3-dihydroxybutyric acid into a substrate and convert it into 2-ketobutyric acid (Wixom, RL et al., Biochim.Biophys.A).
cta. 53 , 433 (1961)).

(Problems to be Solved by the Invention) These conventional methods are not industrially advantageous methods because the chemical synthesis method requires multi-step synthesis or the starting materials are expensive. The method using a microorganism is not suitable for industrial production because the production yield is low. Therefore, it has been required to establish an inexpensive synthetic method. The present invention enables production of 2-ketobutyric acid at a lower cost than conventional methods.

(Means for Solving Problems) As a raw material for producing 2-ketobutyric acid, the present inventors have focused on 2,3-dihydroxybutyric acid that is easily organically synthesized as a threo body or an erythro body from inexpensive crotonic acid. did.
Then, in order to obtain a microorganism having an enzyme system that efficiently converts 2,3-dihydroxybutyric acid into 2-ketobutyric acid, a broad search was conducted from the natural world using a threo body as a substrate, and as a result, a microorganism belonging to Pseudomonas putida was found. , Treo-2,3
-The inventors have found that they have an enzyme system that efficiently converts dihydroxybutyric acid into 2-ketobutyric acid, and completed the invention.

That is, the present invention relates to Pseudomonas butida having the ability to convert 2,3-dihydroxybutyric acid into 2-ketobutyric acid.
In the presence of a microorganism or a treated product thereof, which belongs to the genus Pseudomonas and 5D3A strain (Microtechnology Research Institute No. 11126) and has the ability to convert 2,3-dihydroxybutyric acid into 2-ketobutyric acid, 2,3-dihydroxy This is a method for producing 2-ketobutyric acid, which comprises converting butyric acid into 2-ketobutyric acid to accumulate 2-ketobutyric acid, and collecting this.

Heretofore, it has not been known that Pseudomonas bacteria produce an enzyme system for converting 2,3-dihydroxybutyric acid into 2-ketobutyric acid. Therefore, the microorganism of the present application having an enzyme system for converting 2,3-dihydroxybutyric acid into 2-ketobutyric acid is novel.

The enzyme system used in the present invention utilizes the action of a bacterium belonging to the genus Pseudomonas to convert 2,3-dihydroxybutyric acid into 2-ketobutyric acid.

The present inventors searched from the natural world for a microorganism having an enzyme system that efficiently converts threo-2,3-dihydroxybutyric acid into 2-ketobutyric acid, and as a result, the 5D3A strain (Microtechnology Research Institute
No.) was isolated. The 5D3A strain has an activity of converting threo-2,3-dihydroxybutyric acid into 2-ketobutyric acid, but cannot utilize it. This property is advantageous for efficiently accumulating the converted 2-ketobutyric acid, and the 5D3A strain (Microtechnology Research Institute No. 11126) is preferable as the microorganism used in the present invention. Strain 5D3A (Microtech Lab. No. 11126)
The mycological properties of the are shown below.

Based on the above mycological properties, Bergey's Manual
A search based on the classification criteria of the Determinative Bacteriology 8th Edition (1974) revealed that the 5D3A strain (11
Issue) Pseudomonas putid
a) was identified.

The present invention relates to a method for producing 2-ketobutyric acid using a microorganism belonging to the genus Pseudomonas.

Belonging to the genus Pseudomonas used in the present invention, 2,
Examples of the microorganism having the ability to convert 3-dihydroxybutyric acid into 2-ketobutyric acid include Pseudomonas putida 5D3A strain (also referred to as Pseudomonas putida TE3467) (Microtechnical Research Institute No. 11126). However, the strain is not limited to the strain. The processed product of the microorganism means an enzyme purified from a culture of the microorganism, an immobilized product of the enzyme, and the like.

Threo (or erythro), which is a raw material in the present invention
-2,3-Dihydroxybutyric acid is generally synthesized from crotonic acid. Regarding the synthetic method, a method of oxidizing crotonic acid with N-methylmorpholine-N-oxide in the presence of an osmium tetraoxide catalyst (Frank B. Armstrong, et al.
al., J. Chem. Soc. Perkin Trans.I, 1985,691 (1985))
In addition, a method of synthesizing crotonic acid in the presence of osmium tetroxide and hydrogen peroxide (FWBachelorand GAMiana, ca
nadian J. Chem., 47 , 4089 (1969)) and the like are known.

The medium composition for culturing the microorganism of the present invention,
Either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance and, if necessary, a small amount of a micronutrient. The carbon source used in the medium may be any of those that can be assimilated, such as glycerin, glucose and soluble starch. Although threo (or erythro) -2,3-dihydroxybutyric acid itself does not serve as a carbon source, it has the effect of inducing the enzyme system involved in this reaction system, and is preferably added to the medium. As the nitrogen source, ammonium salts, nitrates, etc. are used. For example, ammonium nitrate or ammonium sulfate. As the inorganic ions, phosphate ions, magnesium ions, potassium ions, etc. are appropriately used. Furthermore, as organic nutrients, yeast extract, peptone, meat extract, corn steep liquor and the like are appropriately used.

In the above medium, the microorganism of the present invention is used at a pH of 6 to 8 (preferably 7.2) and a temperature of 17 to 28 ° C (preferably 25 ° C).
Incubate for ~ 24 hours. The prepared culture of microorganisms contains threo (or erythro) -2,3-dihydroxybutyric acid 2-
It has an enzyme system that converts it to ketobutyric acid. Such a culture may be used as it is as an enzyme source in the reaction, or a live bacterial strain obtained by separating the culture by centrifugation or the like, dried bacterial cells, or ultrasonic treatment of the separated bacterial cells. It may be used as a treated product of cells obtained by treatment by a method such as self-digestion / grinding, or as a crude product or purified product of an extract obtained from these cells and an enzyme obtained from the extract. . Of course, it can also be used as these immobilized cells or immobilized enzyme.

In the method of the present invention, cells thus prepared, treated cells, immobilized cells, etc. are used as catalysts to convert threo (or erythro) -2,3-dihydroxybutyric acid in the reaction solution to give 2 -Generate and accumulate ketobutyric acid.

In order to increase the production rate of 2-ketobutyric acid in the reaction solution,
It is preferable to add a divalent cation in addition to the substrate threo (or erythro) -2,3-dihydroxybutyric acid. For example, the coexistence of 1 mM cobalt chloride in the reaction solution can significantly increase the production efficiency. . The amount of substrate used is not limited, but is preferably 100 mM. Enzymatic reaction is usually pH 8-10 (preferably 9.0), temperature is 10-40 ℃
(Preferably 25 ° C).

The reaction time is appropriately set depending on the purpose, but is usually 1 to 20 hours.

The 2-ketobutyric acid can be collected from the obtained reaction solution by, for example, a method such as fractionating the supernatant obtained by centrifuging the reaction solution by chromatography.

(Example) Hereinafter, although an example is given and demonstrated, these are illustrations and do not limit this invention.

In addition, threo-2,3-dihydroxybutyric acid was quantified by high performance liquid chromatography, and 2-ketobutyric acid was quantified by 2,4
-Spectrophotometrically with dinitrophenylhydrazine.

Example 1 Glycerin 0.5%, threo-2,3-dihydroxybutyric acid 0.
3%, yeast extract 0.15%, ammonium sulfate 0.1%, monopotassium phosphate 0.15%, dipotassium phosphate 0.15%, salt 0.
Add 50 ml of medium (pH 7.2) containing 1%, magnesium sulfate heptahydrate 0.02% and other trace elements to a flask with a shoulder at 120 ° C.
Sterilized for 20 minutes. This was inoculated with Pseudomonas and Putida 5D3A strain (Microtechnology Research Institute No. 11126), and shake-cultured at 25 ° C for 12 hours. Cells were collected from this culture by centrifugation and then containing 100 mM threo-2,3-dihydroxybutyric acid.
100mM Tris-HCl buffer (pH 9.0), cell concentration 40mg
Suspended to be / ml. At the same time, during suspension, cobalt chloride was added to a concentration of 1 mM (total volume 10 m
l). This reaction solution was kept at 25 ° C. for 0 to 24 hours while shaking to measure the amount of 2-ketobutyric acid produced. The results are shown in Table 1.

Also, the same operation was performed when the cell concentration was 10, 20, 80 mg / ml. The results are shown in FIG. 1 including the case where the bacterial cell concentration is 40 mg / ml.

Fig. 1 shows the cells collected by centrifugation with 100 mM trio-2,3-dihydroxybutyric acid-containing 100 mM Tris-HCl buffer (pH 9.0) at cell concentrations of 10, 20, 40, 8 respectively.
The reaction solution was suspended so that the concentration became 0 mg / ml, and at the same time, cobalt chloride was added at a concentration of 1 mM during suspension (total volume 1
0 ml) shows the amount of 2-ketobutyric acid produced when kept at 25 ° C. for 0 to 24 hours while shaking.

The symbol ○-○ in the figure indicates the case where the bacterial cell concentration is 10 mg / ml, and the following △-△ is 20 mg / ml, □-□ is 40 mg / ml, and ●-● is 80 mg / ml.
, Respectively.

Example 2 Glycerin 0.5%, yeast extract 0.15%, ammonium sulfate 0.1%, monopotassium phosphate 0.15%, dipotassium phosphate 0.15%, salt 0.1%, magnesium sulfate heptahydrate 0.02%
0 to 2.0 mM in the medium (pH7.2) containing other trace elements
Add threo-2,3-dihydroxybutyric acid in the range of
50 ml was put into a shoulder flask and sterilized at 120 ° C. for 20 minutes.
Each of them was inoculated with Pseudomonas putida 5D3A strain (Microtechnology Research Institute, No. 11126), and after shaking culture at 25 ° C for 12 hours, the cells were collected by centrifugation.

The collected cells were suspended in 100 mM trio-2,3-dihydroxybutyric acid-containing 100 mM Tris-hydrochloric acid buffer (pH 9.0) at a cell concentration of 20 mg / ml. At the time of suspension, cobalt chloride was added at the same time to a concentration of 1 mM (total volume 10 ml). Shake 25 of these reactions.
The temperature was kept at 24 ° C for 24 hours, and the amount of 2-ketobutyric acid produced was measured. The results are shown in Table 2.

(Effects of the Invention) The present invention relates to novel microorganisms and
It is intended to provide a method for producing ketobutyric acid, and it is clear that 2-ketobutyric acid can be efficiently produced, as shown in the examples.

[Brief description of drawings]

 FIG. 1 shows the amount of 2-ketobutyric acid produced.

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Claims (2)

[Claims] 1. A Pseudomonas putida 5D3A strain having an ability to convert 2,3-dihydroxybutyric acid into 2-ketobutyric acid (Microtechnical Research Institute No. 11126). 2. A method for converting 2,3-dihydroxybutyric acid into 2-ketobutyric acid in the presence of a microorganism belonging to the genus Pseudomonas and having an ability to convert 2,3-dihydroxybutyric acid into 2-ketobutyric acid or a treated product thereof. Then, 2-ketobutyric acid is allowed to accumulate, and this is collected, which is a method for producing 2-ketobutyric acid.