JP3107244B2 - Method for producing optically active diol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for producing an optically active diol, which is useful as a raw material for synthesizing pharmaceuticals and agricultural chemicals, using microorganisms.

[0002]

2. Description of the Related Art
As a method for producing an optically active diol using a microorganism, in the case of optically active 1,3-butanediol, a microorganism having an ability to asymmetrically assimilate either enantiomer is acted on an enantiomeric mixture of the compound. And collecting the remaining optically active 1,3-butanediol (Japanese Patent Application No. 1-107016,
-320997) and the like. In the same manner, (R) -3-halogeno-1,2-propanediol (JP-A-62-122597, JP-A-62-15)
8494, JP-A-63-251098),
Production methods such as (S) -1,2-diol (Abstracts of the 1989 Annual Meeting of the Japanese Society of Agricultural Chemistry) are known. These methods are extremely excellent methods for producing optically active diols, but have been found to have the following problems.

That is, a microbial cell and an enantiomeric mixture of a diol are brought into contact with each other under optimal conditions, and after the optical purity of the target substance in the reaction mixture reaches a predetermined value, the cells to be used are separated from the reaction mixture. Until the end, a phenomenon was observed in which the optical purity of the target product sharply decreased.

[0004]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies on measures to prevent the optical purity of the product after the reaction has been completed. As a result, it has been found that a decrease in the optical purity of the product can be prevented, and the present invention has been completed.

That is, the present invention relates to a method for producing an optically active diol in which a microorganism is allowed to act on an enantiomeric mixture of diols to collect the remaining optically active diol. Another object of the present invention is to provide a method for producing an optically active diol, which prevents a decrease in the optical purity of the remaining optically active diol.

In order to prevent a decrease in optical purity, a conventional method as described below can be used in combination with additives and the like which can be used in the present invention.

(1) The reaction-terminated liquid is placed at least under aerobic conditions.

(2) Heating the reaction-terminated liquid.

(3) The reaction-terminated liquid is cooled.

(4) The reaction-terminated liquid is subjected to acidification treatment or basification treatment.

(5) An organic catalyst is added to the reaction end solution.

The diols usable in the present invention include:
Any diol in the form of a mixture of enantiomers such as 1,2-diol and 1,3-diol can be used, and specific examples thereof include 1,3-butanediol and 1,2-diol.
Propanediol, 1,2-butanediol, 3-halogeno-1,2-propanediol, 1,2-pentanediol, 1,3-pentanediol, 3-phenyl-
1,3-propanediol and the like can be mentioned.

As the microorganism that can be used in the present invention, there can be used a microorganism that asymmetrically assimilates one of the enantiomers of the enantiomeric mixture of diols and remains the optically active diol.

For example, in the case of 1,3-butanediol, all the microorganisms described in Japanese Patent Application No. 1-107016 and JP-A-1-320997 relating to the present inventors can be used. .

[0015] Examples of these microorganisms include those which asymmetrically assimilate one enantiomer of the enantiomeric mixture and leave the (R) form, for example, genus Brevibacterium, genus Candida, Enterobacter
obacter, Geotrich
um) as a microorganism which asymmetrically assimilates one of the enantiomers of the enantiomeric mixture and leaves the (S) form, for example, Agrobacterium (Ag)
genus, Azotobacter (Azo)
bacterium, Bacillus
And microorganisms belonging to the genus, Candida genus, and the like.

The mechanism by which these microorganisms asymmetrically assimilate one of the enantiomers of the enantiomeric mixture and leave the optically active diol is based on the stereoselective oxidation of the diol by the oxidoreductase system of the microorganism. It is thought that the body remains. It is also presumed that the mechanism by which the optical purity of the reaction-terminated liquid decreases is that the optical purity decreases by reducing ketol, which is an oxidized form of the diol remaining in the reaction-terminated liquid.

As a medium composition for culturing these microorganisms, any medium which can normally grow these microorganisms can be used. For example, as a carbon source, glucose, sucrose, sugars such as maltose, lactic acid, acetic acid,
Organic acids such as citric acid, alcohols such as ethanol, 1,3-butanediol, and glycerol or mixtures thereof, and nitrogen sources include ammonium sulfate, ammonium phosphate, urea, yeast extract, corn steep liquor, meat extract, and peptone. In addition, nutrients used in ordinary culture media such as inorganic salts and vitamins can be appropriately mixed and used.

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,
Alternatively, a substance effective for maintaining the pH of the medium can be added. As the medium method, the medium pH is 3.0 to 9.5, preferably 4 to 8, and the culture temperature is 20 to 45 ° C, preferably 25 to 45 ° C.
At 37 ° C, anaerobically or aerobically, under conditions suitable for growth of the microorganism, for 5 to 120 hours, preferably 12 to 72 hours.
Incubate for about an hour.

In the present invention, as a method for reacting the enantiomeric mixture of diols with the microbial cells, the enantiomeric mixture of diols is added in advance to the above-mentioned medium, and the inoculum is brought into contact therewith. A method of simultaneously performing cell growth and reaction by culturing, or a method of adding an enantiomeric mixture of diols after cell growth is observed to a certain extent, a method of adding an enantiomeric mixture of diols after culture, and Various methods can be proposed, such as a method of isolating the body and reacting the mixture with the diol enantiomer mixture, or a method of immobilizing the cells obtained in this manner by a known method and reacting with the diol enantiomer mixture. Various reaction conditions such as temperature, aeration and stirring conditions, substrate concentration, cell concentration, and reaction time when reacting the enantiomer mixture of the diol with the microbial cells by such various methods are not particularly limited, and It suffices if the most advantageous conditions for the production of the optical isomer are selected.

In this way, when the reaction between the enantiomer mixture of the diol and the microbial cells progresses and the optical purity of the desired optical isomer reaches a predetermined value, the amount of the reaction solution is several liters or less. If the amount is relatively small, microbial cells can be separated from the reaction solution in a short period of time, so there is not much problem. However, for example, the scale is increased to 10 liters or more, especially several m ³ or more as in industrial production. In such a case, it usually takes a considerable time for the cell separation step. In such a case, if the aeration and stirring were simply stopped after the reaction was completed and the mixture was allowed to stand, a phenomenon was observed in which the optical purity of the target optical isomer rapidly decreased as exemplified in Comparative Examples described later. If no fundamental countermeasures are taken against this phenomenon, it will have serious drawbacks as an industrial method for producing optically active diols based on this principle.

In view of such circumstances, the inventors of the present invention have conducted intensive studies on measures to prevent the decrease in optical purity, and as a result, it has been found that the above-mentioned drawbacks can be solved by adding an optical purity lowering inhibitor as described below. I found it. The optical purity lowering inhibitor that can be used in the present invention may be any as long as it can be added to the reaction termination solution to substantially prevent the reduction of the optical purity of the intended optical isomer,
Formic acid, 2-cyclohexane-1-one, cyclohexanone, methyl vinyl ketone, acetoin, isopropenyl acetate, acetol, ethyl formate, vinyl acetate, methyl isobutyl ketone, methyl ethyl ketone, allyl alcohol, formalin, 1-buten-3-ol , Etc., which can prevent a decrease in optical purity with a relatively small amount. The addition amount of the optical purity lowering inhibitor is preferably from 0.01 to 50% by weight, more preferably from 0.1 to 5% by weight, based on the reaction-terminated liquid.

After treating the reaction-terminated liquid in this manner, the cells are removed from the reaction liquid by a known method such as centrifugation or filtration. After appropriately concentrating and dehydrating the supernatant thus obtained, the target substance is extracted with an organic solvent such as ethyl acetate, and then dried with anhydrous sodium sulfate or the like, and the solvent is removed under reduced pressure. Is obtained. Further, it can be further purified by distillation.

The desired product can be obtained by directly dehydrating the supernatant under reduced pressure and then distilling it.
Activated carbon treatment or ion-exchange resin treatment, etc., and after pretreatment, dehydration and distillation can also yield the target product.

[0024]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to Examples.

The amount of 1,3-butanediol in the reaction solution was determined by gas chromatography (column: Thermon).
The optical purity was measured by 3,000, (2 m), and a temperature of 130 ° C.), after acetylating 1,3-butanediol of a sample with acetyl chloride by a conventional method, and then performing high-performance liquid chromatography using an optical resolution column (column: Daicel Chemical Industry Chiral Cell OB, Solvent: n-hexane / 2
-Propanol = 19: 1, wavelength 220 nm, flow rate 0.
5 ml / min).

Medium 10 having a composition of 2% glucose, 1% yeast extract and 0.01% silicone antifoam (pH 6)
0 liters into a 200 liter fermenter, 121
Heat sterilization at 15 ° C. for 15 minutes. Here, Geotricum candidum IFO pre-cultured in a medium having the same composition as above
Inoculate 1 liter of a 460 liter culture solution,
Then, stirring cultivation was performed for 24 hours under the conditions of stirring 190 rpm, aeration amount 0.4 vvm, and inner pressure of the fermenter 0.4 kg / cm ² .

Next, the culture was centrifuged to obtain 7 kg of freshly wet cells.

Using the whole amount of the raw wet cells, 5 kg of racemic 1,3-butanediol and 0.5 k of calcium carbonate were used.
g, silicone antifoaming composition 0.01 kg, total amount 100
Make up 1 liter of reaction solution and stir at 190 ° C. at 30 ° C.
m, aeration volume 0.4vvm, fermenter internal pressure 0.4kg / c
Under a condition of m ^2, the reaction was carried out by aeration and stirring. After the reaction starts, 5
Sampling was performed at 0 hour, and the concentration of the product and the optical purity were analyzed.
3 mg / ml, product is (R) -isomer and optical purity is 9
7.9% e. e. Met. The reaction was terminated here.

Next, 1 liter of the reaction-terminated liquid was taken, transferred to a 2.6-liter mini-jar fermenter, and stirred at 30 ° C. under aeration-free conditions at 50 rpm.
When kept for 4 hours, the optical purity was 79% e. e. Down to

Immediately after the completion of the reaction, 200 ml of the reaction-terminated liquid was placed in 13 1-liter Erlenmeyer flasks, and formic acid,
-Cyclohexane-1-one, cyclohexanone, methyl vinyl ketone, acetoin, isopropenyl acetate, acetol, ethyl formate, vinyl acetate, methyl isobutyl ketone, methyl ethyl ketone, allyl alcohol, formalin, 1-buten-3-ol for 24 hours, After gentle stirring at 30 ° C., the decrease in optical purity was measured.

The results obtained are shown in Table 1.

[0032]

[Table 1]

As can be seen from Table 1, the addition of an optical purity inhibitor to the reaction-terminated liquid can prevent a decrease in optical purity.

Reduction of optical purity = (optical purity after completion of reaction)-(30 ° C. · 2 after addition or non-addition of a reduction inhibitor)
Optical purity after holding for 4 hours)

[0035]

By using the method of the present invention, it has become possible to prevent a decrease in optical purity in a process for producing an optically active diol on an industrial scale.

Claims (2)

(57) [Claims] 1. An optic for collecting a remaining optically active substance by allowing a microorganism belonging to the genus Brevibacterium, Candida, Enterobacter, Geotricum, Agrobacterium, Azotobacter or Bacillus to act on the enantiomeric mixture. In the method for producing active 1,3-butanediol, the reaction-terminated liquid is formic acid, 2-cyclohexane-1-one, cyclohexanone, methyl vinyl ketone, acetoin, isopropenyl acetate, acetol, ethyl formate, vinyl acetate, By adding at least one optical purity lowering inhibitor selected from methyl isobutyl ketone, methyl ethyl ketone, allyl alcohol, formalin or 1-buten-3-ol, the optical purity of the remaining optically active substance is prevented from lowering. Optical activity 1,
A method for producing 3-butanediol. 2. The method for producing optically active 1,3-butanediol according to claim 1, wherein the microorganism is a microorganism belonging to the genus Geotricum.