JP2001136997A - Production of (r)-1,2-diols - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a process for producing (R)-1,2-propane-diol that can carry out in an industrially large scale with excellent economy. SOLUTION: A microorganism is allowed to act on an enantiomer mixture of 1,2-diols and the resultant aqueous solution of (R)-1,2-diols is concentrated with a membrane to obtain the objective (R)-1,2-diols.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to (R) -1,2-
The present invention relates to a method for producing diols, particularly (R) -1,2-propanediol. The present compounds are useful as intermediates for various medical and agricultural chemicals, especially for synthetic antibacterial agents.

[0002]

2. Description of the Related Art
As a method for chemically producing (R) -1,2-propanediol, a method for asymmetric reduction of hydroxyacetone with an optically active ruthenium-BINAP catalyst (see JP-A-63-316744). A method for reducing lactide (see Japanese Patent Application Laid-Open No. 2-72129).
-44888, JP-A-64-75435)
Etc. are known. However, in the above-mentioned production method, the BINAP catalyst and hydroxyacetone are expensive, and the production method also requires an expensive reduction catalyst and the operation is complicated. In the production method, it is difficult to synthesize an optical resolving agent to be used, and application to production on an industrial level is not easy.

On the other hand, as a method for producing (R) -1,2-propanediol using an enzyme or a microorganism, a method for reducing hydroxyacetone using an enzyme (J. Org. Chem., 51, 25-36). , 1986) A method of causing Pseudomonas bacteria to act on a racemic 1,2-propanediol (see JP-A-6-30790) is known. However, in the production method of (1), the purified enzyme used is expensive. In the production method of (2), in addition to the fact that the target compound does not accumulate at a high concentration in the culture solution, (R)-
When isolating 1,2-propanediol from a culture solution, a large amount of water had to be distilled off. That is,
For the industrial production of (R) -1,2-propanediol, it was necessary to improve the price and operation.

[0004]

Means for Solving the Problems The present inventors have studied various methods for producing (R) -1,2-propanediol on an economically superior and industrial scale. That is, by solving the disadvantages of the conventional method that the target substance is accumulated at a high concentration in the culture medium and that a large amount of water can be easily removed, economically superior and satisfactory operability is satisfied. We thought that the manufacturing method could be established and conducted intensive studies. As a result, a method for accumulating a target substance at a high concentration in a medium can be solved by using a highly active microbial cell (or a processed product thereof) selected from the genus Brevibacterium and Corynebacterium. I found that it is possible. Furthermore, they have found that the target substance can be easily isolated from the culture solution by a method of removing water using a concentration membrane. The present invention has been completed based on these findings.

That is, the present invention relates to a method for producing (R) -form 1,2-diols, wherein a microorganism or a processed product thereof is allowed to act on an enantiomeric mixture of 1,2-diols.

Further, the present invention relates to each of the following. That is, the above-mentioned production method, wherein the microorganism is a microorganism having a feature of selectively assimilating the (S) form of a 1,2-diol; the microorganism significantly leaves the (R) form of a 1,2-diol. The above-described production method, which is a microorganism having characteristics; the above-mentioned production method, wherein the microorganism is one or more microorganisms selected from the genus Brevibacterium and Corynebacterium; the microorganism is Brevibacterium epidermidis, Brevibacterium linens, Brevibacterium ammonia
gene) and Corynebacterium glutamicum (Cory)
nebacterium glutamicum), which is one or more selected from the above-mentioned production methods;
2-propanediol, 1,2-butanediol, 1,
2-pentanediol, 1,2-hexanediol,
The above-mentioned production method which is 1,2-heptanediol or 1,2-octanediol; and the like.

The present invention also provides an aqueous solution in which (R) -1,2-diols remain by allowing a microorganism or a processed product thereof to act on an enantiomeric mixture of 1,2-diols. The present invention also relates to a process for producing (R) -1,2-diols, characterized in that the microorganisms are concentrated by using a microorganism; The above-mentioned production method, which is a microorganism having the above-mentioned method; The above-mentioned production method, which is a microorganism having a characteristic that the (R) form of a 1,2-diol is significantly left; The method as described above, wherein the microorganism is one or more microorganisms selected from the group consisting of Brevibacterium epidermidis.
rium epidermidis), Brevibacterium linens, Brevibacterium ammoniagene and Corynebacterium glutamicum
amicum), wherein the microorganism is Brevibacterium epidermidis; or the microorganism is Brevibacterium linens.
terium linens); the microorganism is Brevibacterium ammoniage
ne) is the above method; the microorganism is Brevibacterium
Glutamicum (Corynebacterium glutamicum), wherein the 1,2-diol is 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, 1,2. -The above-mentioned production method, which is heptanediol or 1,2-octanediol; the above-mentioned production method, wherein the film is a film capable of selectively removing water; and the above-mentioned production method, wherein the film is an RO film.

Further, the present invention is characterized in that an aqueous solution containing (R) -1,2-diols obtained by the above-mentioned production method is concentrated using a membrane, and the aqueous solution of (R) -1,2-diols is concentrated. The present invention also provides the above method, wherein the film is a film capable of selectively removing water; and the above method, wherein the film is an RO film.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below.
In the present invention, a microorganism assimilating (S) -1,2-diols is allowed to act on an enantiomeric mixture of 1,2-diols to remove (S) -1,2-diols, )
It is characterized by obtaining -1,2-diols.

In the present invention, the microorganism used to obtain the (R) -1,2-diols is characterized by acting on an enantiomeric mixture to significantly leave the (R) -form of the 1,2-diols. There is no particular limitation as long as it is a microorganism having As such a microorganism, a microorganism selected from the genus Brevibacterium and the genus Corynebacterium is preferable. Further, these microorganisms may be strains such as wild strains, mutant strains, and recombinant strains induced by genetic manipulation or the like. The cells may be live cells, or may be cells that have been subjected to treatment such as crushed cells, acetone treatment, or lyophilization. Furthermore, an enzyme obtained from a culture solution of a microorganism or a microorganism cell can also be used. As used herein, the term "microorganism or processed product thereof" means this. The processed product obtained from a microorganism may be obtained from one kind of microorganism, but may be obtained from two or more kinds of microorganisms.

The culture 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, organic acids such as sugars, alcohols, citric acid, and acetic acid can be used. As the nitrogen source, organic acid ammonium salts such as ammonium chloride and ammonium sulfate, and inorganic organic nitrogen-containing compounds such as meat extract, yeast extract, and casein hydrolyzate can be used. In addition, factors that promote the growth of microorganisms, such as inorganic salts and vitamins, and substances that enhance the ability to produce the target compound of the present invention can be added as necessary. As a culture method,
The pH is in the range of 3.0-10.0, preferably 5.0-9.0, and the culture temperature is 10-60 ° C, preferably 25-40 ° C, anaerobically or aerobically at 12-72 under conditions suitable for the growth of the microorganism. It may be cultured for about an hour.

As a method for adding the enantiomeric mixture of 1,2-diols as a raw material, a method of adding an enantiomeric mixture of 1,2-diols to a culture solution after culturing a microorganism, or a method of centrifuging to remove cells. Separation, a method of adding the enantiomeric mixture of 1,2-diols to the separated cells, washing the cells as they are, or washing them with a buffer, etc., and then resuspending them in a buffer, water, etc., and culturing them as a medium for the growth of microorganisms And a method of adding an enantiomeric mixture of 1,2-diols from the beginning.

The enantiomeric mixture of 1,2-diols may be added directly as it is or may be added at once from the beginning of the reaction in a state of being dispersed in a surfactant. Good. The reaction may be carried out at pH 3.0-10.0, preferably 4.0-9.0, at a reaction temperature of 10-60 ° C, preferably 20-50 ° C, with gentle stirring for about 4 to 72 hours. The concentration of the enantiomeric mixture of 1,2-diols as a raw material is not particularly limited, but is preferably about 5 to 50%.

The desired (R) -1,2-diols remaining and accumulated by the action of microorganisms can be isolated as follows. First, bacterial cells are removed by filtration through a filter medium such as celite or diatomaceous earth or a membrane filter. Next, water is removed from the filtrate using a concentration membrane. The film to be used is not particularly limited as long as it can selectively remove water, but an RO film is preferable. The concentration of the (R) -1,2-diols in the concentrated solution can be set to an arbitrary concentration.
% Or more is preferable. Further, after the concentration is completed, the target substance can be isolated and purified from an aqueous solution containing the target substance (R) -1,2-diols at a high concentration by a method such as concentration under reduced pressure or solvent extraction. The (R)-obtained here
Although 1,2-diols can be used as such as intermediates for pharmaceuticals and the like, the method described in JP-A-3-20873 can be used to obtain 2,2-diols with very high purity.
-(R) -1-p-paranitrobenzoyloxy-2-
It is also possible to isolate alcohols.

[0015]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. “%” In the examples means “% by weight” unless otherwise specified. In the examples, 1,2-propanediol in the reaction solution was centrifuged to remove bacterial cells, and the supernatant was subjected to gas chromatography (column: Gaskuropack 54 60 /
80 mesh φ3 × 2m, temperature 190 ° C). The optical purity was determined by lyophilizing a part of an optically active 1,2-propanediol aqueous solution obtained by the reaction and reacting with p-toluenesulfonyl chloride in toluene in the presence of triethylamine. : CHIRALPAK AD / manufactured by Daicel Chemical Industries, Ltd., mobile phase: n-hexane / isopropyl alcohol = 85:15
, Wavelength: 254 nm, flow rate: 0.9 ml / min, temperature: 40
° C). (Retention time: (R) body 13.3 minutes
(S) body 14.8 minutes)

[Example 1] 5 m of medium for preparing bacterial cells having the following composition
is dispensed into a test tube of φ18 × 165mm and sterilized at 121 ℃ for 15 minutes
Brevibacterium epidermidis shown in Table 1 below
(Brevibacterium epidermidis) (IFO 14811)
Then, shaking culture was performed at 30 ° C. for 24 hours. << Bacteria preparation medium >> Meat extract 0.5%, Peptone 1.5
%, NaCl 0.5%, K_TwoHPO_Four0.5% (pH 7.0) Next, a reaction medium of the following composition and 1,2-propanediol
100 ml of a mixture of 1 g of an enantiomeric mixture of
(Propanediol concentration 1%) into a 500 ml Sakaguchi flask
Pour 5 ml of the above cell culture solution into
The cells were inoculated bacterially and reacted with shaking at 30 ° C for 24 hours. << Reaction medium >> Peptone 1.0%, Yeast extract 0.2%,
Na_TwoHPO_Four 0.25%, NaH _TwoPO_Four 0.25%, MgSO_Four・ 7H_TwoO 0.07
%, MnSO_Four・ 4H_TwoO 0.001% (pH 7.0) After completion of the reaction, the cells were removed by centrifugation and the resulting supernatant
After lyophilization of the solution, in toluene in the presence of triethylamine
Reaction with p-toluenesulfonyl chloride,
Optical purity was measured by high performance liquid chromatography. Ma
In addition, the supernatant was analyzed by gas chromatography for 1,2-
The quantification of the amount of propanediol was also performed. Got
Optical purity and yield of (R) -1,2-propanediol
The results are shown in Table 1.

Example 2 Brevibacterium linens (IFO121
42), Brevibacterium ammoniagenes (JCM1306), Coryne
bacterium glutamicum (IFO121168), Corynebacterium g
With respect to lutamicum (IFO121169), culturing was carried out using the same medium and method as in Example 1. The results are shown in Table 1.

[0018]

[Table 1] IFO: Institution for Fermentation Osaka JCM: Japan Collection of Microorganisms

Example 3 Brevibacterium epidermidis
For (IFO 14811), each solution obtained by mixing a reaction medium and 3 g, 5 g, and 10 g of racemic 1,2-propanediol 100
ml (1,2-propanediol concentration 3%, 5%, 10%)
Was dispensed into a 500 ml Sakaguchi flask, sterilized and cooled, and the solution 5 cultured in the medium for cell preparation as in Example 1 was added.
ml of the mixture was aseptically inoculated and incubated at 30 ° C. for 24 hours. After completion of the reaction, the optical purity and yield were determined by the same operation as in Example 1, and Table 2 was obtained.
It was shown to.

[0020]

[Table 2]

Example 4 5 ml of a culture of Brevibacterium epidermidis (IFO 14811) cultured in a medium for preparing cells in the same manner as in Example 1 was dispensed into a 500 ml Sakaguchi flask, sterilized and cooled, and then cooled to 100 ml. Aseptically inoculate the culture medium for 3
Shaking culture was performed at 0 ° C for 24 hours. 1.2 L of a mixture of a reaction medium and 60 g of a 1,2-propanediol enantiomer mixture (1,2-propanediol concentration: 5%) was charged into a 2 L small culture apparatus (manufactured by Takasugi Seisakusho) and sterilized at 121 ° C. for 15 minutes. In addition, 60 ml of this bacterial cell culture solution was aseptically inoculated at 30 ° C.
The reaction was performed at 500 rpm and 1.0 vvm. Further, twice every 24 hours from the start of the reaction, an enantiomeric mixture of 1,2-propanediol was added in an amount of 60 g each (total amount of 180
g). 72 hours after the start of the reaction, the optical purity of (R) -1,2-propanediol determined by the same operation as in Example 1 was 97.2% ee, and the yield was 43%.

After 1.2 L of this culture was removed from the cells by centrifugation, it was filtered through 30 g of Celite. This aqueous solution is
Water was removed with a membrane Nanomax-95 (manufactured by Nippon Millipore Limited) to make 146 g. This aqueous solution was extracted with ethyl acetate, the ethyl acetate layer was concentrated, toluene was added, and the remaining water was azeotropically dehydrated under reduced pressure. (R) -1,2-propanediol 59.2 g, optical purity 97.1% ee.

Reference Example 1 (R) -1,2 obtained in Example 4
-Propanediol 50.0 g (97.1% ee 0.657 mol) and triethylamine 73.1 g (0.723 mol) in toluene 800 m
dissolved in p-nitrobenzoyl chloride 128.0 g
(0.690 mol) in 350 ml of toluene solution
It was added dropwise at 5 ° C. or less over 3 hours. Thereafter, the reaction was stirred at room temperature for 17 hours, 800 ml of water was added, and the mixture was stirred at 60 to 70 ° C. for 30 minutes to separate an aqueous layer. The same washing operation was performed twice, and the toluene layer was concentrated under reduced pressure.
Left for hours. The precipitated crystals are collected by filtration, and the crystals are collected under reduced pressure at 40 ° C.
After drying for 5 hours, 126.4 g of 2- (R) -1-p-nitrobenzoyloxy-2-propanol was obtained. This was subjected to high performance liquid chromatography (column: CHIRALPAK AS / manufactured by Daicel Chemical Industries, Ltd., mobile phase: n-hexane / isopropyl alcohol = 85: 15, wavelength: 254 nm, flow rate: 1.5 ml / m).
In temperature: 40 ° C), the optical purity was 10
It was 0.0% ee. (Retention time: (R) body 10.4 minutes
(S) body 7.9 minutes)

Claims (11)

[Claims] 1. A process for producing (R) -form 1,2-diols, wherein a microorganism or a processed product thereof is allowed to act on an enantiomeric mixture of 1,2-diols. 2. A microorganism or a treated product thereof is allowed to act on a mixture of enantiomers of 1,2-diols, and (R)-
(R) -1,2, wherein an aqueous solution in which 1,2-diols remain is obtained, and the aqueous solution is concentrated using a membrane.
-Preparation of diols 3. An aqueous solution containing (R) -1,2-diols is concentrated using a membrane.
Method for producing 2-diols 4. The method according to claim 1, wherein the microorganism has a characteristic of selectively assimilating the (S) form of 1,2-diols. 5. The method according to claim 1, wherein the microorganism is a microorganism having a characteristic of significantly remaining the (R) form of 1,2-diols. 6. The method according to claim 1, wherein the microorganism is one or more microorganisms selected from the genus Brevibacterium and the genus Corynebacterium. 7. The microorganism may be Brevibacterium epidermidis, Brevibacterium linens, Brevibacterium amoniagene.
iagene) and Corynebacterium glutamicum (Co
rynebacterium glutamicum)
7. The process according to claim 1,2,4,5 or 6 being more than one species. 8. The method of claim 1, wherein the 1,2-diol is 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol or 1,2. The process according to claim 1, 2, 4, 5, 6, or 7, which is -octanediol. 9. The method according to claim 1, wherein the 1,2-diol is 1,2-propanediol. 10. The method according to claim 2, wherein the film is a film capable of selectively removing water. 11. The method according to claim 2, wherein the film is an RO film.