JP2761063B2 - Method for producing optically active 3-hydroxybutyric acid - Google Patents

Description

The present invention relates to a method for producing optically active 3-hydroxybutyric acid. More specifically, a method for producing optically active 3-hydroxybutyric acid, wherein a specific microorganism or a processed product thereof is allowed to act on an enantiomeric mixture of 1,3-butanediol, and the resulting optically active 3-hydroxybutyric acid is collected. About the method.

The optically active form of 3-hydroxybutyric acid is an important synthetic raw material for various medicines and agricultural chemicals.

[Problems to be solved by the prior art and the invention]

Conventionally, as a method for producing optically active 3-hydroxybutyric acid, a method of optically resolving a chemically synthesized racemate using a resolving agent (Helv. Chim. Acta., 64 , 1467 (1981)), a method of producing poly (organic) produced by microorganisms, -Hydroxybutyric acid obtained by hydrolysis (Helv. Chim. Acta., 65 , 459 (1982)), asymmetric reduction method of ethyl acetoacetate by microorganism (Helv. Chim.
Acta., 66 , 485 (1983)) or a method of producing from butyric acid using the β-oxidation system of yeast (Japanese Patent Application Laid-Open No. 58-158190), a method of producing a racemic 1,3-butanediol of the genus Rhodococcus. Method for producing by allowing microorganisms to act (JP-A-64-74999)
Is known.

However, looking at the characteristics of these production methods, there are respective advantages and disadvantages.The method using a resolving agent is expensive, and the method obtained by hydrolyzing poly-β-hydroxybutyric acid produces Things are expensive,
The asymmetric reduction method using microorganisms has various problems, such as low substrate concentration, and the method of producing from butyric acid using the β-oxidation system of yeast requires a long time for culturing or reaction of microorganisms. This is not necessarily an industrially superior method.

A method of producing a racemate of 1,3-butanediol by reacting a microorganism with the microorganism is considered to be an advantageous method from the viewpoint of the price of the raw material. However, in the method using the microorganism of the genus Rhodococcus, the substrate concentration is still low ( About 1%), which is not always an advantageous method. Although a method for producing the (R) form is known by this method, a method for producing the (S) form is not known at all.

[Means for solving the problem]

In view of these circumstances, the present inventors have made it possible for microorganisms to act on enantiomer mixtures of 1,3-butanediol, especially racemic 1,3-butanediol, which is industrially manufactured at low cost, by causing microorganisms to act on the mixture. The method of obtaining butyric acid is considered to be an excellent method, and screening is performed on a wider range of microorganisms in search of more excellent microbial strains, and strains producing (R) form and (S) form are produced. A strain was found and the present invention was completed.

The present inventors focused on a method for producing an optically active 3-hydroxybutyric acid by reacting a microorganism with an enantiomeric mixture of 1,3-butanediol as a method for obtaining optically active 3-hydroxybutyric acid, and searched for a microorganism suitable for this purpose. As a result, it belongs to the genus Candida, the genus Pichia, or the genus Schizoblastosporium, and has the ability to convert 1,3-butanediol into optically active (R) -3-hydroxybutyric acid. When a microorganism having the above or a processed product thereof is acted on an enantiomeric mixture of 1,3-butanediol, optically active (R) -3-hydroxybutyric acid is produced, and Candida (Candida)
Genus, Hansenula, Isatchenkia (I
ssatchenkia, Kluyveromyce
s), genus Pichia, saccharomycopsis (S
acharomycopsis, Bacillus, Corynebacterium, Citrobacter, or Enterobactor
acter), a microorganism having the ability to convert 1,3-butanediol to optically active (S) -3-hydroxybutyric acid, or a processed product thereof is allowed to act on an enantiomeric mixture of 1,3-butanediol. , Optically active (S)-
The inventors have found that 3-hydroxybutyric acid is produced, and completed the present invention.

Microorganisms used in the present invention include Candida (Ca
ndida), Pichia, Schizoblastosporium, or Bacillus (Bac)
microorganisms belonging to the genus illus that can produce (R) -form 3-hydroxybutyric acid from a mixture of enantiomers of 1,3-butanediol; and microorganisms belonging to the genus Candida, the genus Hansenula, and Isatchenkia
enkia), Kluyveromyces,
The genus Pichia, Saccharomycopsis (Saccharo)
genus mycopsis, genus Bacillus, genus Corynebacterium, Citrobactor
bacter) or Enterobacter
Any microorganisms belonging to the genus that can produce (S) -form 3-hydroxybutyric acid from a mixture of enantiomers of 1,3-butanediol can be used.

Specifically, as the producing strain of the (R) form, Candida utillis IFO 1086, IFO 063
9. Pichia heedii IFO 10020,
Pichia lindnerii ATCC 3265
8. Schizoblastosporium Kobayashi
porium kobayashi) IFO 1644 and the like.

In addition, as the strain producing the (S) body, Candida
Candida utilis IFO 0619, IFO 0988, Candida robusta AHU 3402, Candida rugosa IFO 1364, Candida krusei DSM 70073, Candida parapsilosis parapsilosis)
IFO 1396, Hansenula polymorpha DL1 (Hansenula p
olymorpha DL1) ATCC 26012, Hansenula Minuta (Han
senula minuta) IFO 0975, Hansenula Henricy (H
ansenula hericii) DSM 70272, Hansenula wickerhamii IFO 1706, Isatchenkia scuturata bar scuturata (Issa
tchenkia scutulata var.scutulata) IFO 10070, Kluyveromyces dr
asphilarum IFO 1012, Kluyveromyces lactis IFO 1903, Pichia farinosa IFO 1163, Pichia quercuum IFO 1276, Pichia pastoris DS Pichia heedii IFO 10019, Saccharomycopsis
Ripolitica (Saccharomycopsis lipolytica) IFO 155
0, Bacillus subtilis IFO 31
34, Bacillus pumilus ATCC 706
1. Corynebacterium mishiganense (Corynebacteri)
um michiganense) IFO 13762, Citrobacter freundii AHU 1534, or Enterobacter cloacae
e) ATCC 7256 and the like.

Any of these microorganisms can be suitably used, such as a wild-type strain, a mutant strain, or a recombinant strain derived by a genetic technique such as cell fusion or genetic engineering.

Microorganisms with IFO numbers are described in List of Cultures, 8th edition, vol.
8) and can be obtained from the IFO. Microorganisms with AHU numbers are described in the Catalog of Cultures, 4th edition (198
It is described in 7) and can be obtained from the Faculty of Agriculture, Hokkaido University. Microorganisms with ATCC numbers are American
Catalog of the Type Culture Collection (ATCC)
Bacteria Phages rDNA Vectors, 16th edition (1985), which can be obtained from the ATCC. Microorganisms with DSM numbers are Deutsch Sammlung von Mikroorga
It is described in Catalog of strains (1983) issued by nismen (DSM) and can be obtained from the DSM.

The medium for culturing the microorganism used in the present invention is not particularly limited as long as the microorganism can grow. For example, as the carbon source, any of the above microorganisms can be used.Specifically, glucose, fructose, sucrose, saccharides such as dextrin, sorbitol, ethanol, alcohols such as glycerol, fumaric acid, Organic acids and salts thereof such as citric acid, acetic acid and propionic acid, hydrocarbons such as paraffin and the like, and mixtures thereof can be used. As a nitrogen source,
For example, ammonium chloride, ammonium sulfate, ammonium salts of inorganic acids such as ammonium phosphate, ammonium fumarate, ammonium salts of organic acids such as ammonium citrate, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, urea, etc. An inorganic organic nitrogen-containing compound or a mixture thereof can be used. In addition, nutrients used for ordinary cultivation, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. Further, if necessary, a factor that promotes the growth of microorganisms, a factor that enhances the ability to produce the target compound of the present invention, for example, an enantiomeric mixture of 1,3-butanediol, or the pH of the medium
Substances effective for retention can also be added.

The enantiomer mixture of 1,3-butanediol referred to in the present invention means that the ratio of (R) form or (S) form of 1,3-butanediol may be any, but substantially, It goes without saying that a racemate having a ratio of 1: 1 is inexpensive and suitable.

As a culture method, the medium pH is 3.0 to 9.5, preferably 5 to 5.
-8, culturing at 20-45 ° C, preferably 25-37 ° C, under anaerobic or aerobic conditions suitable for the growth of the microorganism for 5-120 hours, preferably about 12-72 hours .

As a method for producing optically active 3-hydroxybutyric acid from a mixture of enantiomers of 1,3-butanediol, a method of adding an enantiomer mixture of 1,3-butanediol to a culture medium from the beginning or in the middle during culture of a microorganism. A method of adding an enantiomer mixture of 1,3-butanediol to a culture solution after completion of the culture, or a method of separating cells from the culture solution by centrifugation or the like, and then directly or after washing the cells, to a buffer solution, water, etc. 1,3 in resuspended
A method of adding an enantiomeric mixture of butanediol and reacting the mixture.

When the enantiomer mixture of 1,3-butanediol is added at the beginning or during the culturing, the addition concentration is not particularly limited, but is preferably about 0.1 to 10%.

When the reaction is carried out using the isolated cells, the reaction is carried out at pH 3 to 9, preferably pH 5 to 8, and at a temperature of 10 to 60 ° C, preferably
The reaction is performed at a temperature of 4040 ° C. for about 1 to 120 hours under stirring or standing. The use concentration of the enantiomer mixture of 1,3-butanediol is not particularly limited, but is preferably about 0.1 to 10%. At this time, the enantiomeric mixture of 1,3-butanediol is dissolved as it is or dissolved in water, or dissolved in an organic solvent that does not affect the reaction, or dispersed in a surfactant or the like, to cause the reaction. It may be added all at once or dividedly from the beginning.

When the reaction is carried out using the isolated cells, the cells may be used as they are, or may be those obtained by crushing the cells, treating with acetone, freeze-drying, or the like. Further, these cells or the treated cells are immobilized by a known method such as a polyacrylamide gel method, a sulfur-containing polysaccharide gel method (such as a carrageenan gel method), an alginic acid gel method, or an agar gel method. You can also.

The produced optically active 3-hydroxybutyric acid can be easily obtained by directly extracting from a culture solution or a reaction solution or after separating cells, extracting with an organic solvent, and using ordinary purification methods such as column chromatography and distillation. .

〔Example〕

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Racemic 1,3-butanediol 0.5%, yeast extract 0.1%
5%, peptone 1%, meat extract 0.5%, dipotassium phosphate
100 ml of a medium consisting of 0.1% and magnesium sulfate heptahydrate 0.05% (pH 7.0) was placed in a 500 ml Sakaguchi flask, sterilized, and each of the strains shown in Table 1 was inoculated, followed by reciprocal shaking culture at 30 ° C. for 48 hours. went.

After completion of the culture, 40 ml of the culture solution was taken, and the cells were separated by centrifugation and washed once with physiological saline to obtain viable cells.

After adding 4 ml of water to the viable cells to form a cell suspension, 4 ml of the suspension was collected in a test tube having a diameter of 18 mm. To this, 4 ml of a 4% solution of racemic 1,3-butanediol was added and reacted at 30 ° C. with shaking for 24 hours. After completion of the reaction, the bacteria were removed by centrifugation to obtain a supernatant.

5 μ of the supernatant was taken and the amount of 3-hydroxybutyric acid produced was quantified by high performance liquid chromatography (column: Shimadzu SCR-101H, solvent: 0.1% phosphoric acid, flow rate: 1 ml / min, detection: 210 nm).

The absolute configuration and optical purity of the thus produced 3-hydroxybutyric acid were determined as follows.

That is, the reaction supernatant was used as the ion exchange resin (Dowex1-x8, cl).
Through the column (2 ml) to adsorb 3-hydroxybutyric acid. Then, after washing with water, elution was performed with 4 ml of 1N hydrochloric acid. After removing hydrochloric acid under reduced pressure, methyl esterification was performed with methanol by a conventional method. Next, this sample was subjected to high performance liquid chromatography using an optical resolution column (column: Chiral Cell OD manufactured by Daicel Chemical Industries, 4.6 mm ID × 250 m
m, solvent: n-hexane / 2-propanol = 9: 1, flow rate: 0.5
ml / min, detection: 220 nm) to determine the absolute configuration and optical purity of the product.

 Table 1 shows the obtained results.

Example 2 Yeast extract 0.3%, malt extract 0.3%, peptone 0.5
%, Racemic 1,3-butanediol 2% medium (p
H6.0) 1 was placed in a 2.6-volume mini-jar fermenter, sterilized, and inoculated with the strains shown in Table 2 at 30 ° C., 1 vvm,
Culture was performed for 48 hours under the condition of 400 rpm.

After completion of the culture, the cells were removed by centrifugation to obtain a supernatant. Hydrochloric acid was added to the sample to adjust the pH to 2, then the solution was made into a saturated saline solution, and extracted three times with ethyl acetate 1. The extracts were combined, dried over anhydrous sodium sulfate, evaporated, and then distilled under reduced pressure to obtain a colorless oil. For these, the absolute configuration and optical purity of the product were measured in the same manner as in Example 1.

 Table 2 shows the obtained results.

[Effect of the Invention] The method for producing optically active 3-hydroxybutyric acid using the microorganism of the present invention makes it possible to produce optically active 3-hydroxybutyric acid by an industrially relatively simple method, It is very advantageous.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 41/00 BIOSIS (DIALOG) WPI (DIALOG) REGISTRY (STN) CA (STN)

Claims (2)

(57) [Claims] 1. An enantiomeric mixture of 1,3-butanediol is added to a mixture of Candida and Pichia.
a) Genus or Schizoblastosporium
(R) -3, which is produced by the action of a microorganism belonging to the genus porium and capable of converting 1,3-butanediol into optically active (R) -3-hydroxybutyric acid, or a processed product thereof. -A method for producing optically active 3-hydroxybutyric acid, comprising collecting hydroxybutyric acid. 2. The method according to claim 1, wherein the enantiomeric mixture of 1,3-butanediol is prepared by adding Candida genus, Hansenula
senula), Issatchenkia, Kluyveromyces, Pichia
a) Genus, Saccharomycopsis
Genus, Bacillus, Corynebacterium (Co
rynebacterium), Citrobacter
Or a microorganism belonging to the genus Enterobacter and having the ability to convert 1,3-butanediol into optically active (S) -3-hydroxybutyric acid, or an optically active substance produced by treating the microorganism. Na (S) -3-
Optical activity 3 characterized by collecting hydroxybutyric acid
-A method for producing a hydroxybutyric acid derivative.