JP4581821B2 - Improved production of optically active 1,2-diols by microbial culture - Google Patents

Description

  The present invention relates to the production of optically active 1,2-diols containing optically active halogenohydrins that can be useful chiral building blocks in the synthesis of optically active compounds or their intermediates used in pharmaceuticals, agricultural chemicals, physiologically active substances and the like. Regarding the method. More specifically, the present invention relates to a method for producing optically active 1,2-diols by asymmetric assimilation resolution.

  Regarding a biological production method using a microorganism of optically active 1,2-diol containing optically active halogenohydrins, a method for producing optically active 1,2-propanediol using a resting cell of Pseudomonas microorganisms (Patent Document 1) ) And S-form 3-halogeno-1,2-propanediol (Patent Document 2) are known.

  Further, the present inventors cultivate a microorganism having the ability to assimilate and proliferate using an optically active substance as a carbon source in a medium containing the racemic body as a single carbon source, and recover the optically active substance remaining after the end of the culture. A method for producing an optically active form of 1,2-propanediol (Patent Document 3) or a method for producing an optically active form of 3-chloro-1,2-propanediol (method of asymmetric associative resolution) (Patent Document 3) Patent Literature 4, Patent Literature 5, and Patent Literature 6) are disclosed.

However, in the asymmetric assimilation method described in the above-mentioned document, it has been found that the ability to produce optically active substances by microorganisms varies greatly depending on the quality and lot of the medium used, especially the water quality used when preparing the medium.
Here, in general, calcium as a mineral component is one of important elements as a biological component, and Ca ²⁺ is known to be involved in the regulation of many life phenomena. So far, industrial microbial culture considering Ca ²⁺ concentration includes, for example, culture for production of unsaturated fatty acid (Patent Document 7), culture for decomposition of trichlorethylene (Patent Document 8), erythritol. It has been reported in the culture for the production of (Patent Document 9) and the like. However, in the production of optically active compound by asymmetric assimilation division method, the relationship between the Ca ²⁺ concentration and optically active form of a production capacity, namely Ca ²⁺ concentration and the reaction rate and three-dimensional discrimination (yield) and the relationship There is no report about.
JP-A-6-30790 JP-A-6-209781 JP 2002-253295 A Japanese Patent Laid-Open No. 3-191795 JP 2001-149090 A Japanese Patent Laid-Open No. 3-191794 No. 98/029558 JP 11-46758 A JP 11-2221092 A

  An object of the present invention is to provide a method for producing an optically active substance capable of producing optically active 1,2-diols having high optical purity in a short time and in a high yield.

As a result of repeated studies to solve the above problems, the present inventors have produced an optically active 1,2-diol by an asymmetric assimilation resolution method using microorganisms. The inventors have found that controlling the Ca ²⁺ concentration within a specific range significantly improves the speed of the optical resolution reaction and the yield of the optically active substance, thereby completing the present invention.

  The present invention has been completed based on the above findings, and provides the following method for producing an optically active substance.

    Item 1. The following formula (1)

(In the formula, R represents a methyl group, an ethyl group, a propyl group, a chloromethyl group, or a hydroxyethyl group.)
A first step of culturing a microorganism having the ability to assimilate the R-form or S-form of the compound represented by the above formula with a racemic isomer of the compound as a carbon source while controlling the Ca ²⁺ concentration in the medium at the start of the culture. When,
A method for producing an optically active substance, comprising: a second step of recovering the remaining optically active substance from the obtained culture.

Item 2. In the first step, the Ca ²⁺ concentration in the medium at the start of the culture was determined so that the optical purity of the optically active substance in which the compound represented by the above formula (1) remained was 98% e. e. The manufacturing method of claim | item 1 which controls so that time until it may become within 130 hours.

    Item 3. Item 3. The method according to Item 1 or 2, wherein the microorganism is a microorganism belonging to the genus Pseudomonas.

Item 4. Item 4. The production method according to any one of Items 1 to 3, wherein the Ca ²⁺ concentration in the medium at the start of culture is 3 to 40 ppm.

Item 5. The compound represented by the formula (1) is 1,2-propanediol, the Ca ²⁺ concentration in the medium at the start of the culture is 3 to 13 ppm in the first step, and the R-form 1,2-propanediol in the second step. The manufacturing method in any one of claim | item 1-4 which collect | recovers.

  Item 6. Item 6. The method according to Item 5, wherein the microorganism is Pseudomonas nitroreducens DS-S-RP8 strain (international deposit number: FERM BP-7793).

Item 7. The compound represented by the formula (1) is a medium using 3-chloro-1,2-propanediol as a carbon source, the Ca ²⁺ concentration in the medium at the start of the culture in the first step is 3 to 40 ppm, Item 5. The production method according to any one of Items 1 to 4, wherein S-form 3-chloro-1,2-propanediol is recovered in two steps.

  Item 8. Item 8. The production method according to Item 7, wherein the microorganism is Pseudomonas sp. DS-SI-5 strain (International Deposit Number: FERM BP-7080).

Item 9. The compound represented by the formula (1) is 3-chloro-1,2-propanediol, the Ca ²⁺ concentration in the medium at the start of the culture in Step 1 is 5 to 28 ppm, and in the second step, the R isomer Item 5. The method according to any one of Items 1 to 4, wherein 3-chloro-1,2-propanediol is recovered.

  Item 10. Item 10. The method according to Item 9, wherein the microorganism is a Pseudomonas sp. DS-K-2D1 strain (International Deposit Number: FERM BP-3096).

  By the method of the present invention, a stable and high production capacity for the production of optically active 1,2-diols containing optically active halogenohydrins was achieved. More specifically, in the production of optically active 1,2-diols containing optically active halogenohydrins by an asymmetric assimilation resolution reaction, 98% e. e. The optically active substance having the above high optical purity can be obtained. In addition, the yield of the optically active substance and the optical resolution reaction rate were small for each production batch, and a large production capacity was stably realized.

  Hereinafter, the present invention will be described in detail.

  The method for producing an optically active substance of the present invention comprises the following formula (1):

(In the formula, R represents a methyl group, an ethyl group, a propyl group, a chloromethyl group, or a hydroxyethyl group.)
A first step of culturing a microorganism having the ability to assimilate the R-form or S-form of the compound represented by the above in a medium containing a racemate of the compound as a carbon source, and the remaining optical activity from the obtained culture. And a second step of recovering the body. That is, the method of the present invention is a method for producing an S form or an R form of the compound represented by the formula (1) by an asymmetric assimilation resolution reaction using a microorganism.
In the method of the present invention, in the first step, the Ca ²⁺ concentration in the medium at the start of the culture is controlled. This Ca ²⁺ concentration is such that, for example, the optical purity of the optically active substance in which the compound represented by the above formula (1) remains is 98% e.e. e. It may be controlled so that the time until it becomes within 130 hours.

In the method of the present invention, batch culture in which the substrate is added all at once, fed-batch culture in which the substrate is added in multiple steps, or continuous culture in which the medium containing the substrate is continuously supplied and the product is continuously discharged. Any method may be adopted. 98% e. e. The “time” until the time indicates the culture time in batch culture and fed-batch culture, and the residence time in continuous culture. In continuous culture, the “Ca ²⁺ concentration in the medium at the start of culture” refers to the Ca ²⁺ concentration in the culture tank at the start of continuous culture. In addition, it is preferable to control the Ca ²⁺ concentration in the medium supplied during the culture. For example, the Ca ²⁺ concentration in the supplied medium is also within the allowable concentration range of Ca ^{2+ in} the medium at the start of the culture. 98% e.e. e. It may be controlled so that the residence time until it becomes becomes 130 hours.
The racemic isomer of the compound represented by the above formula (1) is used as a raw material compound for producing an optically active substance by the raw material asymmetric assimilation resolution method. Specifically, from the group consisting of 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, 3-chloro-1,2-propanediol, and 1,2,4-butanetriol Use the racemate of the compound of choice.

These compounds can be produced, for example, by hydrolysis from the corresponding epoxide under sulfuric acid acidity. Methods for producing diols from epoxides are described, for example, in Tetrahedr. Asymm. (1994) Vol. 5, No. 2, p239-246; and Bull. Soc. Chim. (1926) Vol. 39, p699-700. Yes.
Microorganism The microorganism used in the method of the present invention is a microorganism having the ability to selectively assimilate the R-form or S-form of the compound represented by the above formula (1). One microorganism may be used alone, or two or more microorganisms may be mixed and used as long as the microorganism can assimilate only the target optically active substance of the racemic raw material.

  Examples of microorganisms that can efficiently assimilate one optically active compound of the compound of formula (1) include microorganisms belonging to the genus Pseudomonas sp. Among them, Pseudomonas nitroreducens DS-S-RP8 strain (International deposit number: FERM BP-7793), Pseudomonas genus DS-SI-5 strain (international deposit number: FERM BP-7080), or Pseudomonas The genus DS-K-2D1 strain (international deposit number: FERM BP-3096) is a preferred strain.

  Pseudomonas nitroreducense DS-S-RP8 strain is a strain that can efficiently selectively utilize the S form of 1,2-propanediol. The Pseudomonas genus DS-SI-5 strain is a strain that can efficiently selectively assimilate the R form of 3-chloro-1,2-propanediol. Pseudomonas genus DS-K-2D1 strain is a strain that can efficiently selectively assimilate the S form of 3-chloro-1,2-propanediol.

In addition, 1,2-butanediol, 1,2-pentanediol, and 1,2,4-butanetriol also use microorganisms that can specifically assimilate one of the optically active substances in the medium at the start of culture. The production capacity of the optically active substance can be improved by controlling the Ca ²⁺ concentration.
The culture medium for culturing the microorganism as the seed culture is not particularly limited as long as the microorganism to be used can grow. For example, a known nutrient medium for culturing microorganisms containing peptone, yeast extract, glycerin and the like can be used. However, it is necessary to quantify in advance the Ca ²⁺ brought into the medium for performing the optical resolution reaction from the seed culture so that the Ca ²⁺ concentration in the medium in the asymmetric assimilation resolution reaction is within the appropriate range described above. There is.

The medium used for the asymmetric assimilation split reaction is not particularly limited as long as the Ca ²⁺ concentration at the start of the culture can be controlled within the above range, but if a minimal medium as shown below is used, Ca ²⁺ It is easy to adjust the concentration.

As the Ca ²⁺ source, a water-soluble salt that generates Ca ²⁺ can be used without limitation, but calcium chloride is preferably used. In addition, it is preferable to use ion-exchanged water for the preparation of the medium, so that it is not necessary to consider the amount of Ca ²⁺ contained in the water.

The optical purity of the remaining optically active compound of the formula (1) is 98% e.e. e. In order to make the time to reach within 130 hours, although it depends on the strain used, the raw material compound, the culture conditions, etc., the Ca ²⁺ concentration in the medium may be usually about 3 to 40 ppm.

Pseudomonas nitroredense DS-S-RP8 strain is used, for example, when a racemic form of 1,2-propanediol is asymmetrically resolved to obtain R-form 1,2-propanediol, The Ca ²⁺ concentration is preferably about 3 to 13 ppm, more preferably about 9 to 12 ppm.

Also, using Pseudomonas sp. DS-SI-5 strain, for example, racemic 3-chloro-1,2-propanediol is asymmetrically resolved to obtain S-form 3-chloro-1,2-propanediol. In such a case, the Ca ²⁺ concentration in the medium at the start of the culture is preferably about 3 to 40 ppm, more preferably about 7 to 37 ppm.

Also, using Pseudomonas sp. DS-K-2D1 strain, for example, racemic 3-chloro-1,2-propanediol is asymmetrically resolved to obtain R-isomer 3-chloro-1,2-propanediol. In this case, the Ca ²⁺ concentration in the medium at the start of culture is preferably about 5-28 ppm, more preferably about 9-27 ppm.

Within the above Ca ²⁺ concentration range, 98% e. e. The above target optically active substance is obtained.

In the present invention, the Ca ²⁺ concentration is a value measured by the ICP emission method.

  As the carbon source, a racemate of the compound represented by the formula (1) to be resolved (hereinafter sometimes referred to as “substrate”) is used. In order to perform optical resolution efficiently, it is preferable to use this substrate as a single carbon source.

  Further, as described above, in the method of the present invention, batch culture may be performed in which the substrate is added all at once at the start of the culture, or fed-batch culture in which the substrate is added in divided portions may be performed. As a fed-batch culture method, for example, by determining the number of additions and addition amount of substrate from the acid, alkali titration, or substrate (carbon source) concentration in the reaction solution, which titrates the fluctuation of pH due to the progress of the reaction, There is a method of adding a substrate continuously or intermittently (Kasai et al., Journal of Biotechnology, 1997, 75, 255-275).

  In the method of the present invention, continuous culture can also be employed. As continuous culture, for example, as in fed-batch culture, the amount of substrate added is judged based on the acid or alkali titration titration of the change in pH due to the progress of the reaction or the substrate (carbon source) concentration in the reaction solution. There is a method of supplying a culture medium containing a substrate and obtaining a culture solution containing optically active substances that are continuously discharged (Japanese Patent Laid-Open No. 2004-041076).

  If the substrate (carbon source) concentration in the medium is too high, the growth of the microorganism may be hindered, that is, the microorganism has a growth inhibitory concentration with respect to the substrate. The asymmetric assimilation split reaction is a reaction accompanied by the growth of microorganisms, and it is necessary to keep the substrate concentration of the medium in culture below the concentration that inhibits the growth of microorganisms. Since the substrate is added in batches, it is possible to increase the total amount of substrate input compared to batch culture while keeping the substrate concentration of the culture medium during cultivation below the growth inhibition concentration.

  In the case of batch culture, the substrate concentration at the start of the culture is preferably about 0.1 to 15 w / v%, more preferably about 1 to 5 w / v%. If it is the said range, a substrate or an optically active substance can perform optical resolution efficiently, without preventing the growth of microorganisms.

  In addition, when fed-batch culture or continuous culture is performed, the total amount of substrate added is preferably about 0.1 to 15 w / v%, more preferably about 1 to 10 w / v% with respect to the medium. do it. Further, the substrate concentration of the medium during the culture is preferably about 1 to 7 w / v%, more preferably about 1 to 5 w / v%. If it is the said range, a substrate or an optically active substance can perform optical resolution efficiently, without preventing the growth of microorganisms. In the case of fed-batch culture, the substrate may be added in several portions, but the substrate concentration during the culture can be brought to the above range by adding appropriately as the reaction proceeds. The progress of the reaction can be measured from, for example, pH, substrate concentration, microbial turbidity, carbon dioxide emission and the like.

  Components other than the carbon source are not particularly limited, and components usually used for culturing microorganisms may be used. As the nitrogen source, for example, inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, and ammonium phosphate can be used. Moreover, in order to advance the asymmetric assimilation resolution reaction effectively, phosphates, magnesium salts, potassium salts, manganese salts, iron salts, zinc salts, copper salts and the like may be added as inorganic salts. Furthermore, various antifoaming agents can be added as necessary.

The pH of the medium during the culture may be, for example, about 4 to 10, preferably about 5 to 9, and may be about the optimum pH of the microorganism usually used within this range. When the pH of the medium gradually decreases or rises as the asymmetric associative separation reaction proceeds, add an appropriate alkali source or acid source to control the pH in the culture solution to be close to the optimum pH. do it. Since this addition amount matches the advancing rate of the asymmetric assimilation split reaction, the substrate can be added as an index when fed-batch culture is performed. For example, examples of the alkali source include alkali carbonate aqueous solutions such as sodium carbonate aqueous solution, potassium carbonate aqueous solution and ammonium carbonate aqueous solution; alkali hydroxide metal salt aqueous solutions such as sodium hydroxide aqueous solution and potassium hydroxide aqueous solution; or ammonia aqueous solution. As the acid source, a general acid such as hydrochloric acid or phosphoric acid may be used. It is preferable not to use a calcium salt in order to avoid fluctuations in the Ca ²⁺ concentration in the medium.

  The culture temperature is usually about 15 to 40 ° C., preferably about 20 to 37 ° C., and usually around the optimum temperature of the microorganism used within this range, for example, about 30 ° C. Moreover, although it changes with strains to be used, it may be usually aerobically cultured.

In the culture, the optical purity of the target optically active substance is usually 98% e.e. e. The reaction may be carried out until the above is reached, but the reaction may be stopped at a point of lower optical purity depending on the purpose of the optically active substance. Depending on the type, concentration, strain, culture conditions, etc. of the substrate, the optical purity is usually 98% e. e. The above optically active substance is obtained. Moreover, even if it is cultured for an excessively long time, the bacteria will not grow any longer, the asymmetric assimilation / division reaction will not proceed, and it is preferable to complete this time for economical production. It is preferable to determine the end point by monitoring the change in the pH of the medium accompanying the progress of the reaction, or measuring the production amount (residual amount) or optical purity of the target optically active substance by gas chromatography or the like.
Recovery of optically active substance The optically active substance remaining in the culture medium thus obtained may be recovered by a general method. For example, after removing cells from the culture solution by filter press, ultrafiltration, centrifugation, etc., the supernatant is concentrated by an evaporator and extracted with a solvent such as ethyl acetate or diethyl ether. Further, the extract is dehydrated with anhydrous magnesium sulfate and the like, and then the solvent is removed under reduced pressure, whereby an optically active syrup can be obtained. Further, it may be purified by conventional methods such as distillation and various chromatography.
EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. In the examples,% indicates w / v% unless otherwise specified.
<Used strain>
Pseudomonas nitroreducens DS-S-RP8 strain (international deposit number: FERM BP-7793), Pseudomonas sp. DS-SI-5 strain (international deposit) used in the following examples No .: FERM BP-7080) and Pseudomonas sp. DS-K-2D1 strain (International deposit number: FERM BP-3096) have already been deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. ing.
<Ca ²⁺ concentration measurement method>
The measurement was performed by the ICP emission method using an ICP emission analyzer (ICAP-575 Mark II manufactured by Japan Jarrell Ash). The apparatus conditions were an output of 1.2 kW and a torch +1.0 cm. The integration condition was one measurement with three integrations of a short wavelength of 3 seconds, a main wavelength of 3 seconds, and a long wavelength of 3 seconds.

Production of R-form 1,2-propanediol Autoclave sterilized at 121 ° C. for 15 minutes with a 500 ml baffle Erlenmeyer flask containing 100 ml of nutrient medium (pH 7.2) containing 2% yeast extract and 1% glycerin, A small amount of Pseudomonas nitroreducens DS-S-RP8 strain previously cultured in an agar nutrient medium having the same composition is inoculated with a platinum loop and cultured at 30 ° C. for 24 hours to obtain a seed culture solution. It was.

Next, 2.1 L of an inorganic component aqueous solution having the composition shown in Table 1 above was prepared with ion-exchanged water, and the Ca ²⁺ concentration was further adjusted using calcium chloride, and placed in a 5 L culture tank. , 2-propanediol was added in an amount of 100 g, and autoclaved at 121 ° C. for 15 minutes to prepare a minimal medium using racemic 1,2-propanediol as a single carbon source. In this way, four types of minimal media were prepared that were adjusted to have a Ca ²⁺ concentration of 3 ppm, 9 ppm, 12 ppm, and 15 ppm, respectively, at the start of culture. Of these Ca2 ⁺ concentrations in the minimal medium, the amount brought from seed culture is 1 ppm.

    100 ml of the above-mentioned minimal medium was added thereto, and the reaction (culture) was performed for 72 to 120 hours under the conditions of a temperature of 30 ° C., an aeration rate of 0.25 L / min, and a rotation speed of 500 rpm. The pH of the medium was measured and controlled using a pH controller, and controlled to a pH of 6.9 with a 25% (w / w) aqueous sodium hydroxide solution. Racemic 1,2-propanediol is added stepwise as the reaction proceeds so that the concentration of 1,2-propanediol in the culture does not exceed 5%, which is the growth inhibitory concentration of the strain. A total of 200 g of racemate was added. As a result of optical purity analysis by gas chromatography, the remaining R-form 1,2-propanediol was found to be 99.0% e.e. e. The reaction was terminated when the above was reached.

The relationship between the Ca ²⁺ concentration, the reaction time, and the yield is shown in Table 2 below. The yield indicates the ratio of the optically active substance remaining with respect to the initial substrate (carbon source) concentration. As is apparent from Table 2, when the Ca ²⁺ concentration is 3 to 12 ppm, the optical purity of the remaining R-form 1,2-propanediol is 99% e.e. e. The reaction time was fast especially at 9 ppm. In the case of 3 ppm, the asymmetric assimilation resolution reaction was delayed, and the yield, that is, the stereoselectivity, was slightly reduced, but was in a practical range. In the case of 15 ppm, the asymmetric assimilation resolution reaction is inhibited and the optical purity is 98% e.e. e. I did not reach the above.

  After completion of the culture, the cells were removed by centrifugation, and the supernatant was concentrated with an evaporator and extracted with diethyl ether. Next, the extract was dehydrated with anhydrous magnesium sulfate, and then the solvent was removed under reduced pressure to obtain 1,2-propanediol syrup.

This 1,2-propanediol was trifluorolated with trifluoroacetic anhydride in dichloromethane, and then a capillary column G-TA (0.25 mm (ID) × 30 m (length)) manufactured by Astec Corporation was used. The optical purity was measured by gas chromatography. The analysis conditions are as follows.
Column temperature: 60 ° C
Detector temperature: 200 ° C
Carrier gas: Nitrogen flow rate: 0.5 ml / min
Detector: FID
Split ratio: 200/1
Retention time of 1,2-propanediol: R-form is 11.4 minutes,
S body is 17.6 minutes

The S-form 3-chloro-1,2-propanediol-producing microorganism was Pseudomonas sp. DS-SI-5. The seed culture solution was prepared in the same manner as in Example 1 except that a medium containing 1% each of peptone, yeast extract, and glycerin, or a medium containing 2% yeast extract and 1% glycerin was used.

Next, 63 g of racemic 3-chloro-1,2-propanediol was added, and the Ca ²⁺ concentration of the medium at the start of culture (after inoculating the seed culture) was 7 ppm, 20 ppm, 25 ppm, 30 ppm, and 37 ppm, respectively. 6 minimal media were prepared in the same manner as in Example 1 except that the concentration was adjusted to 43 ppm. Of the Ca ²⁺ concentration in the medium at the start of the culture, the amount brought from the seed culture is 3 ppm or 7 ppm.

  100 ml of the aforementioned seed culture solution was added thereto, and a reaction (culture) was performed for 70 hours under the conditions of a temperature of 30 ° C., an aeration rate of 0.25 L / min, and a rotation speed of 510 rpm. The pH was measured and controlled using a pH controller, and the decrease in pH due to the dechlorination reaction was titrated with a 25% (w / w) aqueous sodium hydroxide solution to control the pH to 6.9.

Racemic 3-chloro-1,2-propanediol was continuously added using a pH controller at a ratio of 1.8 times the number of moles of sodium hydroxide added by titration, and a total of 200 g of racemic body was added. Then, the addition was stopped (by Kasai et al., Biotechnology Society, 1997, 75, 255-275) (however, when the Ca ²⁺ concentration was 43 ppm, the total amount of racemate added was 148 g). The concentration of racemic 3-chloro-1,2-propanediol in the culture did not exceed 5%, which is the growth inhibitory concentration of the strain used. As a result of optical purity analysis by gas chromatography, the remaining S form 3-chloro-1,2-propanediol was found to be 99.0% e.e. e. The reaction was terminated when the time reached above.

The relationship between the Ca ²⁺ concentration, the reaction time, and the yield is shown in Table 3 below. As is clear from Table 3, when the Ca ²⁺ concentration is 7 to 37 ppm, the optical purity of the remaining S-form 3-chloro-1,2-propanediol is 99% e.e. e. And especially at 37 ppm, the reaction time was fast. In addition, at 43 ppm, since the activity of the bacterium decreased, only 148 g of the racemic substrate (carbon source) could be fed, and the bacterium was inactivated during the culture, so that the optical purity was 98% e.e. e. It did not become more.

  After completion of the culture, the cells were removed by centrifugation, and the supernatant was concentrated with an evaporator and extracted with diethyl ether. Next, the extract was dehydrated with anhydrous magnesium sulfate, and then the solvent was removed under reduced pressure to obtain 3-chloro-1,2-propanediol syrup.

This 3-chloro-1,2-propanediol was dissolved in 2-propanol, converted to glycidol with a 12.5N aqueous sodium hydroxide solution, and then capillary column A-PH (0.25 mm (ID) × 30 m (Astech) The optical purity was measured by gas chromatography using length)). The measurement conditions are as follows.
Column temperature: 45 ° C
Detector temperature: 200 ° C
Carrier gas: Helium flow rate: 0.5 ml / min
Detector: FID
Split ratio: 100/1
Glicidol retention time:
R form is 33.8 minutes, S form is 35.2 minutes

Pseudomonas (Pseudomonas sp.) DS-K-2D1 strain was used as an R-form 3-chloro-1,2-propanediol-producing microorganism. The seed culture solution was prepared in the same manner as in Example 1 except that a medium containing 1% each of peptone, yeast extract, and glycerin was used.

Next, except that 63 g of racemic 3-chloro-1,2-propanediol was added and that the Ca ²⁺ concentration of the medium at the start of culture was adjusted to 3 ppm, 9 ppm, 15 ppm, and 27 ppm, respectively. In the same manner as in Example 1, four types of minimal media were prepared. Of the Ca ²⁺ concentration of the culture medium at the start of the culture, the amount brought in from the seed culture is 3 ppm.

  100 ml of the aforementioned seed culture solution was added thereto, and a reaction (culture) was performed for 70 hours under the conditions of a temperature of 30 ° C., an aeration rate of 0.25 L / min, and a rotation speed of 450 rpm. The pH was measured and controlled using a pH controller, and the decrease in pH due to the dechlorination reaction was titrated with a 25% (w / w) aqueous sodium hydroxide solution to control the pH to 6.9.

In the same manner as in Example 2, racemic 3-chloro-1,2-propanediol was continuously added at a ratio of 1.35 times the number of moles of sodium hydroxide titrated, and a total of 160 g of racemic body was obtained. When it was added, the addition was stopped (however, 139 g in a medium having a Ca ²⁺ concentration of 3 ppm). The concentration of racemic 3-chloro-1,2-propanediol in the culture did not exceed 5%, which is the growth inhibitory concentration of the strain used. As a result of optical purity analysis by gas chromatography, the remaining R-form 3-chloro-1,2-propanediol was found to be 99.0% e.e. e. The reaction was terminated when the above was reached.

The relationship between the Ca ²⁺ concentration, the reaction time, and the yield is shown in Table 4 below.

As is apparent from Table 4, when the Ca ²⁺ concentration is 9 to 27 ppm, the optical purity of the remaining R-form 3-chloro-1,2-propanediol is 99% e.e. e. In particular, in the case of 9 ppm, the reaction time was fast. In the case of 3 ppm, after 45 hours from the start of the culture, the molar ratio of chloro ions generated as a result of decomposition of the substrate and sodium hydroxide required for neutralization fluctuates in a constant manner, and an effective fed-batch reaction occurs. could not. Therefore, the optical purity of the remaining substrate during the reaction is 99% e.e. e. The carbon source was insufficient, the predetermined racemic substrate was fed and added, and the reaction was stopped without optical resolution. As a result, only 139 g (87% of the initially planned 160 g) could be divided. When the molar ratio of the fed substrate to sodium hydroxide is increased, the reaction byproduct glycidol (T. Suzuki et al, Appl. Microbiol. Biotechnol., 1993, 40, 273-278) The concentration increased, and the growth of bacterial cells and the asymmetric assimilation resolution reaction were inhibited. Table 4 shows the Ca ²⁺ concentration and the reaction results.

  The optical purity of R-form 3-chloro-1,2-propanediol in the culture was measured in the same manner as in the R-form of Example 2.

Continuous culture at 121 ° C. for 15 minutes, in a 500 ml baffled Erlenmeyer flask containing a liquid medium containing 10 g / l of glycerin autoclaved, 10 g / l of yeast extract, and 10 g / l of peptone (pH 7.2) in advance. Pseudomonas sp. Grown on the nutrient medium plate. A cell of DS-S-RP8 was inoculated into one platinum loop and cultured at 30 ° C. for 24 hours to obtain a seed culture solution.

1 L (pH 6.9) of an inorganic component aqueous solution shown in Table 1 of Example 1 was prepared with ion-exchanged water, Ca ²⁺ concentration was further adjusted to 10 ppm using calcium chloride, and racemic 1, A 2 L culture tank containing a minimal medium supplemented with 40 g of 2-propanediol was autoclaved at 121 ° C. for 15 minutes. To this, 40 ml of seed culture solution prepared in the same manner as in Example 1 was added, and batch culture was performed for 15 hours under the conditions of a temperature of 30 ° C., an aeration rate of 0.1 L / min, and a rotation speed of 500 rpm. At that time, the cell turbidity was 8.6 OD (turbidity at 660 nm), and the 1,2-propanediol concentration at that time was 2.2%. The pH was measured and controlled using a linked pH controller, and the pH was controlled to 6.9 with a 3N aqueous sodium hydroxide solution.

Next, the culture was transferred to continuous culture. A 2 L culture tank containing 1 L (pH 6.9) of an inorganic component aqueous solution containing 10 ppm of Ca ²⁺ as described above was sterilized by autoclaving at 121 ° C. for 15 minutes. Connected. In the first and second tanks, the conditions such as temperature, aeration volume, pH, etc. were the same as in batch culture. Furthermore, 100 g / L of racemic 1,2-propanediol as a single carbon source was added to the same aqueous solution as the inorganic component aqueous solution containing 10 ppm of Ca ²⁺ as described above to obtain a supply medium for continuous culture. Continuous culture was performed by adjusting the medium supply flow rate so that the microbial turbidity in the first tank was maintained at 11 OD (turbidity at 660 nm). As a result, a steady state was obtained at a medium supply rate of 0.0159 L / hour. The residence time at this time was 126 hours. In the first tank, the 1,2-propanediol concentration was 6.8%, the specific growth rate μ was 0.0159 (hour- ¹ ), and the specific substrate consumption rate ν was 0.048 (g / OD · time · L). Maintained. At this time, the cell turbidity in the second tank was 14 OD (turbidity at 660 nm), and the 1,2-propanediol concentration was 3.8%. When the optical purity of 1,2-propanediol in the culture broth flowing out from the second tank was measured in the same manner as in Example 1, it was found to be 98% e.e. e. The above R-isomer 1,2-propanediol.

Claims (6)

A microorganism belonging to the genus Pseudomonas sp. Having the ability to assimilate the S form of 1,2-propanediol was cultured at the start of culture in a medium containing a racemic form of 1,2-propanediol as a single carbon source. A first step of culturing at a Ca ²⁺ concentration of 3 to 13 ppm in the medium ;
A second step of recovering the remaining R-form 1,2-propanediol from the obtained culture.
A process for producing an optically active form of R-form 1,2-propanediol . The method according to claim 1, wherein the microorganism is Pseudomonas nitroreducens DS-S-RP8 strain (International deposit number: FERM BP-7793). A microorganism belonging to the genus Pseudomonas having the ability to assimilate the R form of 3-chloro-1,2-propanediol is obtained by using a racemic form of 3-chloro-1,2-propanediol as a single carbon source. A first step of culturing at a Ca ²⁺ concentration of 3 to 40 ppm in the culture medium at the start of the culture in a medium containing
A second step of recovering the remaining S-form 3-chloro-1,2-propanediol from the resulting culture.
A method for producing an optically active substance of S-form 3-chloro-1,2-propanediol . The production method according to claim 3, wherein the microorganism is a Pseudomonas sp. DS-SI-5 strain (International Deposit Number: FERM BP-7080). A microorganism belonging to the genus Pseudomonas having the ability to assimilate the S form of 3-chloro-1,2-propanediol is obtained by using a racemic form of 3-chloro-1,2-propanediol as a single carbon source. A first step of culturing at a Ca ²⁺ concentration of 5 to 28 ppm in the culture medium at the start of the culture in a medium containing
A second step of recovering the remaining R-form 3-chloro-1,2-propanediol from the resulting culture.
Process for producing optically active form of R-form 3-chloro-1,2-propanediol . The production method according to claim 5, wherein the microorganism is a Pseudomonas sp. DS-K-2D1 strain (international deposit number: FERM BP-3096).