JP2837501B2 - Method for producing optically active diol - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing an optically active diol, which is useful as a raw material for synthesizing pharmaceuticals and agricultural chemicals, using a microorganism.

[Problems to be solved by conventional technology and invention]

Conventionally, as a method for producing an optically active diol using a microorganism, in the case of optically active 1,3-butanediol, an enantiomeric mixture of the compound has the ability to asymmetrically assimilate either enantiomeric mixture. A method of collecting a remaining optically active 1,3-butanediol by the action of a microorganism (Japanese Patent Application No. 1-107016, JP-A-1-320997) and the like are known. Also, in a similar manner, (R) -3-halogeno-1,2-propanediol (JP-A-62-122597, JP-A-62-158494, JP-A-63-158494)
Methods for producing (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, the microbial cells and the enantiomer mixture of the diol are contact-reacted under the optimal conditions, and after the optical purity of the target substance in the reaction solution reaches a predetermined value, until the used cells are completely separated from the reaction solution. During this time, a phenomenon was observed in which the optical purity of the target product suddenly decreased.

[Means for solving the problem]

In view of such circumstances, the present inventors have intensively studied measures to prevent a decrease in the optical purity of the product after the completion of the reaction. We have found that we can do this and completed the present invention.

That is, the present invention provides a method for producing an optically active diol in which a microorganism belonging to the genus Geotrichum or Candida is allowed to act on an enantiomeric mixture of diols and the remaining optically active diol is collected. An optically active diol characterized by preventing a decrease in the optical purity of the remaining optically active diol by subjecting the liquid to a treatment for preventing a decrease in optical purity selected from the following (1) to (5), alone or in combination. It is intended to provide a manufacturing method.

(1) The reaction completed solution is placed at least under aerobic conditions.

(2) Heat treatment of the reaction-terminated liquid.

(3) The reaction completed solution is cooled.

(4) Acidifying or basifying the reaction-terminated liquid.

(5) An organic solvent is added to the reaction completed solution.

As the diol that can be used in the present invention, any diol in the form of an enantiomeric mixture such as 1,2-diol and 1,3-diol can be used.Specifically, 1,3-butanediol, 1-diol and 1-diol can be used. , 2-propanediol, 1,2
-Butanediol, 3-halogeno-1,2-prohandiol, 1,2-pentanediol, 1,3-pentanediol, 3-phenyl-1,3-propanediol and the like.

As the microorganism that can be used in the present invention, the above-mentioned microorganisms that asymmetrically utilize one of the enantiomers of the enantiomeric mixture of diols and leave the optically active diol can be used. For example, in the case of 1,3-butanediol, microorganisms which asymmetrically assimilate one enantiomer of the enantiomeric mixture and leave the (R) form belong to the genus Candida and the genus Geotrichum. A microorganism belonging to the genus Candida can be mentioned as a microorganism that asymmetrically assimilates one of the enantiomers of the enantiomeric mixture and leaves the (S) form.

The mechanism by which these microorganisms asymmetrically assimilate one enantiomer of the enantiomeric mixture and leave the optically active diol is that the optically active substance remains by stereoselectively oxidizing the diol with the oxidoreductase system of the microorganism. It seems to be. 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, usually any medium that can 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, ethanol, 1,3-butanediol, alcohols such as glycerol or a mixture thereof, as a nitrogen source Can be used by appropriately mixing nutrients used in ordinary media such as ammonium sulfate, ammonium phosphate, urea, yeast extract, corn steep liquor, meat extract, peptone, etc., as well as inorganic salts and vitamins. If necessary, a factor that promotes the growth of the microorganism, a factor that enhances the ability to produce the target compound of the present invention, or a substance that is effective in maintaining the pH of the medium can be added.

As a culture method, the medium pH is 3.0 to 9.5, preferably 4 to
8. The cultivation temperature is 20-45 ° C, preferably 25-37 ° C, under anaerobic or aerobic conditions suitable for the growth of the microorganism.
The culture is performed for about 120 hours, preferably about 12 to 72 hours.

In the present invention, as a method of reacting the enantiomeric mixture of diols with the microbial cells, the enantiomeric mixture of diols is added in advance to the medium described above,
A method of simultaneously inoculating and inoculating a seed bacterium and culturing the cells, or a method of simultaneously performing cell growth and reaction, or a method of adding a mixture of diol enantiomers after a certain amount of cell growth is observed, and adding a mixture of diol enantiomers after culture is completed A method of separating cells after completion of the culture, and reacting this with an enantiomeric mixture of diols, or immobilizing the cells thus obtained by a known method,
A method of reacting with a mixture of diol enantiomers,
Various methods can be proposed.

Various reaction conditions such as temperature, aeration and stirring conditions, substrate concentration, cell concentration, reaction time, and the like when reacting the enantiomer mixture of the diol with the microbial cells by such various methods are not particularly limited. It suffices if the most advantageous conditions for the production of the optical isomer are selected.

In this manner, 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 relatively small such as several liters or less. If it is, there is not much problem because microbial cells can be separated from the reaction solution within a short time, but for example, when the scale is large, 10 l or more, especially in the case of several m ³ or more like industrial production In general, it takes a considerable amount of time for the cell separation step. In such a case, when the aeration and stirring were simply stopped after the reaction was completed and 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 present inventors have conducted intensive research on this optical purity reduction prevention method, and as a result, the above-mentioned drawbacks have been solved by performing the optical purity reduction prevention treatment methods described below individually or in combination as described above. They found something that could be solved.

That is, the optical purity lowering prevention method is as follows.

(1) The reaction completed solution is placed at least under aerobic conditions.

(2) Heat treatment of the reaction-terminated liquid.

(3) The reaction completed solution is cooled.

(4) Acidifying or basifying the reaction-terminated liquid.

(5) An organic solvent is added to the reaction completed solution.

Here, each of the optical purity lowering prevention processing methods will be described in more detail.

(1) A method in which the reaction-terminated liquid is placed at least under aerobic conditions.

The aerobic condition referred to here may vary depending on the type of microorganism used and the like, but may be any condition as long as the optical purity of the target optical isomer does not substantially decrease. For example, the conditions are preferably such that the concentration of dissolved oxygen in the reaction solution can be maintained at 0.01 ppm or more.

(2) A method in which the reaction-terminated liquid is heat-treated.

The conditions of the heat treatment referred to herein may vary depending on the type of microorganism used, and the like, but may be any conditions as long as the optical purity of the target optical isomer does not substantially decrease. Preferably at 50-100 ° C for 10 minutes-5
The heating time is about 30 minutes to 1 hour at 60 to 80 ° C.

(3) A method of cooling the reaction-terminated liquid.

The condition for cooling and holding may vary depending on the type of microorganism used and the like, but may be any condition as long as the optical purity of the target optical isomer does not substantially decrease. It is preferably kept at 15 ° C or lower, more preferably at 10 ° C or lower.

(4) A method of subjecting the reaction termination solution to an acidification treatment or a basification treatment.

The conditions of the acidification treatment referred to herein may vary depending on the type of microorganism used and the like, but may be any conditions as long as the optical purity of the target optical isomer does not substantially decrease. It is preferably about pH 3 or less, more preferably about pH 2 or less. The conditions for the basification treatment may vary depending on the type of microorganism used and the like, but any conditions may be used as long as the optical purity of the target optical isomer does not substantially decrease. The pH is preferably about 8 or more, more preferably about 9 or more.

(5) A method of adding an organic solvent to the reaction-terminated liquid.

The organic solvent referred to here may be any solvent as long as it can substantially prevent the decrease in optical purity of the intended optical isomer, for example, acetone, ethanol, methanol, propanol, butanol, benzene, Examples include toluene and hexane. The amount of the organic solvent to be added is preferably 1 to 50% by weight, more preferably 5 to 20% by weight, based on the reaction-terminated liquid.

Although the various processing methods described above can achieve their purpose even when used alone, it is also possible to achieve the object by combining some methods.

After treating the reaction end solution in this manner, centrifugation,
Alternatively, the cells are removed from the reaction solution by a known method such as filtration. After appropriately concentrating and dehydrating the supernatant thus obtained, the desired product 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.
The desired optically active diol is obtained. Further, it can be further purified by distillation.

Also, the supernatant is directly dehydrated under reduced pressure, and then distilled to obtain the target product.After ultrafiltration membrane treatment, activated carbon treatment, or ion exchange resin treatment, and pretreatment,
The desired product can be obtained by dehydration and distillation.

〔Example〕

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

In addition, 1, in the reaction solution in these comparative examples and examples.
The amount of 3-butanediol was determined by gas chromatography (column: Thermon 3000, (2 m), temperature 130 ° C).
The optical purity was measured by 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: Chiral Cell OB manufactured by Daicel Chemical Industries, solvent: n-hexane / 2−
(Propanol = 19: 1, wavelength 220 nm, flow rate 0.5 ml. Min).

Comparative Example 1 Glucose 2%, Yeast extract 1%, Silicone defoamer 0.
100 l of a medium having a composition of 01% (pH 6) was placed in a 200 l fermenter and sterilized by heating at 121 ° C. for 15 minutes. Here, Geotricum candidam pre-cultured in a medium with the same composition as above
Inoculate culture 1 of IFO 4601, agitate at 30 ° C, 190 rpm,
Under aeration conditions of 0.4 vvm and an inner pressure of the fermenter of 0.4 kg / cm ² , aeration and agitation culture was performed for 24 hours.

Next, this culture solution was centrifuged to obtain 7 kg of fresh wet cells.

Using the whole amount of the raw wet cells, racemic 1,3-butanediol (5 kg), calcium carbonate (0.5 kg), silicon defoamer (0.
A reaction solution having a composition of 01 kg and a total volume of 100 l was formed, and the reaction was carried out at 30 ° C. under agitation at 190 rpm, aeration of 0.4 vvm, and an internal pressure of the fermenter of 0.4 kg / cm ² under aeration. Sampling was performed 50 hours after the start of the reaction, and the concentration and optical purity of the product were analyzed. The 1,3-butanediol concentration was 21.3 mg / ml, the product was the (R) -isomer, and the optical purity was 97.9%. was ee. The reaction was terminated here.

Next, one of the reaction-terminated liquids was taken, transferred to a 2.6-liter mini-jar fermenter, and kept at 30 ° C. while stirring at 50 rpm under aeration-free conditions.

This reaction end solution is sampled with time,
The change in the optical purity of the (R) -form 1,3-butanediol was tracked.

 Table 1 shows the obtained results.

As can be seen from this, when the reaction-terminated liquid is left under anaerobic conditions, the optical purity of the product rapidly decreases.

Example 1 The reaction-terminated liquid obtained in Comparative Example 1 was mixed with three 2.6 l
Taken by mini fermenters, stirring 100 rp each
Under the conditions of m and 30 ° C., the aeration amount was changed and maintained in the range of 0.1 vvm to 0.5 vvm.

For each of the cases set in this way, sampling was conducted over time, and changes in the optical purity of the (R) -form 1,3-butanediol were tracked. The dissolved oxygen concentration at the time of sampling was measured.

 Table 2 shows the obtained results.

As can be seen here, a decrease in optical purity could be prevented by keeping the reaction-terminated liquid under microaerobic conditions.

Example 2 200 ml of the reaction-terminated liquid obtained in Comparative Example 1 was placed in three Erlenmeyer flasks, each of which was placed in an incubator set at 5, 10, and 15 ° C., and held under gentle stirring. Starting from when the temperature of the reaction-terminated liquid reached a predetermined value, the samples were sequentially sampled with time and the optical purity was measured. Table 3 shows the obtained results.

As can be seen, cooling the reaction-terminated liquid and keeping it at 15 ° C. or lower can prevent a decrease in optical purity.

Example 3 In the same manner as in Example 2, 200 ml of the reaction-terminated liquid was placed in three Erlenmeyer flasks, each placed in an incubator set at 50, 60, and 70 ° C., and after reaching a predetermined temperature under gentle stirring. Heat treatment was performed for 1 hour. After the completion of the heat treatment, the system was cooled with gentle stirring and reached a temperature of 30 ° C., and thereafter, the sample was sequentially sampled over time while maintaining the temperature at 30 ° C. to measure the optical purity. Table 4 shows the obtained results.

As can be seen here, a reduction in optical purity can be prevented by heating the reaction-terminated liquid at 50 ° C. or higher for 1 hour.

Example 4 In the same manner as in Example 3, 200 ml of the reaction-terminated liquid was placed in each of six Erlenmeyer flasks, and 0.1 N hydrochloric acid or 0.1 N sodium hydroxide solution was added to adjust the pH to 1, 2, 3, and 8, 9, 10 respectively. did. Thereafter, the sample was placed in an incubator set at 30 ° C., sampled sequentially with time while maintaining the temperature at 30 ° under gentle stirring, and the optical purity was measured. Table 5 shows the obtained results.

As can be seen here, the reaction termination solution is pH 3 or less or pH 8
By maintaining the above, a decrease in optical purity can be prevented.

Example 5 In the same manner as in Example 3, 200 ml of the reaction-terminated liquid was placed in three Erlenmeyer flasks, and acetone, ethanol, and methanol were each added to a concentration of 10%. After that, put it in an incubator set at 30 ° C, sample it with time while keeping it at 30 ° C with gentle stirring,
Optical purity was measured. Table 6 shows the obtained results.

As can be seen here, the addition of an organic solvent to the reaction-terminated liquid can prevent a decrease in optical purity.

Example 6 Glucose 2%, Yeast extract 1%, Silicone defoamer 0.
100 l of a medium having a composition of 01% (pH 6) was placed in a 200 l fermenter and sterilized by heating at 121 ° C. for 15 minutes. Here, a culture solution 1 of Candida parapsilosis IFO 1396 precultured in a medium having the same composition as described above was inoculated, and the mixture was stirred at 30 ° C. and stirred at 190 rp.
m, aeration rate 0.4Vvm, under conditions of internal pressure 0.4 kg / cm ² in the fermentor, 12
Aeration and agitation culture was performed for hours. Thereafter, 3 kg of a heat-sterilized racemic 1,3-butanediol was added to this culture solution, and the culture was further continued under the same conditions. Sampling over time and measuring the optical purity of the product
Optical purity 96.5% e. 50 hours after adding 1,3-butanediol.
(R) form of e. Here, the reaction is terminated,
The temperature was reduced to 10 ° C., the stirring was set to 50 rpm, and the amount of air flow was set to 0.1 vvm, and the bacteria were sequentially removed by a continuous centrifuge.

When the amount of (R) -1,3-butanediol produced and the optical purity of 93.7 l of the obtained supernatant were measured, 97.0% e.
e. produced 1.25 kg.

This supernatant was treated with an ultrafiltration membrane (fraction molecular weight: 30,000, membrane area: 5 m ² : manufactured by Daicel Chemical Industries), and then concentrated and dehydrated under reduced pressure. Thereafter, the mixture was distilled under reduced pressure (30 to 40 Torr) at 120 to 130 ° C. to obtain 1.05 kg of a purified product (purity 98.5
%, Optical purity 96.8% ee).

Example 7 2% glucose, 1% yeast extract, silicone defoamer 0.
100 l of a medium having a composition of 01% (pH 6) was placed in a 200 l fermenter and sterilized by heating at 121 ° C. for 15 minutes. Here, Candida utilus IF pre-cultured in a medium with the same composition as above
The culture solution 1 of O 1086 was inoculated, and cultured under aeration at 30 ° C. for 24 hours under agitation at 190 rpm, aeration of 0.4 vvm, and an internal pressure of the fermenter of 0.4 kg / cm ² . Thereafter, 7.5 kg of fresh wet cells were obtained by centrifugation. Using the whole amount of the raw wet cells, a reaction solution of a total amount of 100 l was composed of a racemic 1,3-butanediol (5 kg), calcium carbonate (0.5 kg), and a silicon defoamer (0.01 kg), and stirred at 30 ° C. 190rpm, aeration 0.4vvm, internal pressure of fermenter
Under a condition of 0.4 kg / cm ^2, the reaction was carried out by aeration and stirring.

Sampling was performed 50 hours after the start of the reaction, and the concentration and optical purity of the product were analyzed. The 1,3-butanediol concentration was 20.3 mg / ml, the product was (S) form, and the optical purity was 9%.
It was 8.9% ee. The reaction was terminated here. Thereafter, the reaction solution was heated at 50 ° C. for 1 hour,
Next, after cooling, the bacteria were removed by a continuous centrifuge to obtain a supernatant.

The supernatant contains 98.7% ee (S) -form 1,3-
A total of 2.04 kg of butanediol was contained.

〔The invention's effect〕

By using the method of the present invention, a decrease in optical purity can be prevented in the production of an optically active diol on an industrial scale.

Claims (7)

(57) [Claims] 1. The method according to claim 1, wherein said enantiomeric mixture of diols is of the genus Geotrichum or Candid.
a) A method for producing an optically active diol in which a microorganism belonging to the genus is acted to collect the remaining optically active diol,
An optical activity characterized by preventing the decrease of the optical purity of the remaining optically active diol by performing the treatment for preventing optical purity decrease selected from the following (1) to (5) individually or in combination with the reaction completed solution. A method for producing a diol. (1) The reaction completed solution is placed at least under aerobic conditions. (2) Heat treatment of the reaction-terminated liquid. (3) The reaction completed solution is cooled. (4) Acidifying or basifying the reaction-terminated liquid. (5) An organic solvent is added to the reaction completed solution. 2. The method according to claim 1, wherein the diol is 1,3-butanediol. 3. The method according to claim 1, wherein the aerobic condition is a dissolved oxygen concentration of 0.01 ppm or more. 4. The method according to claim 1, wherein the heat treatment comprises heating the reaction-terminated liquid to 50 ° C. or higher. 5. The method according to claim 1, wherein the cooling treatment is to keep the reaction-terminated liquid at 15 ° C. or lower. 6. An acidification treatment of pH 3 or less, and a basification treatment of pH 8 or less.
The method according to claim 1, wherein the reaction-terminated liquid is held as described above. 7. The method according to claim 1, wherein the organic solvent is acetone, methanol or ethanol.