JP2828729B2 - Method for producing optically active 1,3-butanediol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing optically active 1,3-butanediol, which is useful as a raw material for synthesizing pharmaceuticals such as agricultural chemicals, using microorganisms.

[Problems to be solved by conventional technology and invention]

Conventionally, as a method for producing optically active 1,3-butanediol using a microorganism, an enantiomer mixture of the compound is reacted with a microorganism having an ability to asymmetrically assimilate either enantiomer, and the remaining enantiomer mixture is reacted. To collect optically active 1,3-butanediol (Japanese Patent Application No. 1-1
No. 07016, JP-A-1-320997) and the like are known.

Although these methods are excellent methods for producing optically active 1,3-butanediol, there is a problem in that the concentration of the substrate charged during the reaction is low and the productivity of the target product is low. It has been desired to solve this problem, develop a highly productive production method that enables high-concentration preparation of the substrate.

[Means for solving the problem]

In view of such circumstances, the present inventors have conducted various studies to develop a production method with higher optical purity and a higher productivity in the production method of optically active 1,3-butanediol.
In this reaction, while controlling the concentration of the enantiomer opposite to the intended enantiomer in the reaction solution, 1,3
-It has been found that it is possible to improve the accumulation concentration of the target substance in the reaction solution by feeding and reacting a mixture of butanediol enantiomers, and it is possible to achieve a significant improvement in productivity. Completed the invention.

That is, the present invention relates to a method of allowing a microorganism to act on a mixture of enantiomers of 1,3-butanediol and remaining optically active 1,3-butanediol.
In the method for producing optically active 1,3-butanediol for collecting butanediol, the concentration of the enantiomer opposite to the enantiomer to be produced in the reaction solution is 0.1 to 0.1%.
The present invention relates to a method for producing an optically active 1,3-butanediol, characterized in that an enantiomeric mixture of 1,3-butanediol is fed and reacted while being controlled within a range of 3% by weight.

As a microorganism that can be used in the present invention, a microorganism having the ability to asymmetrically assimilate one enantiomer of an enantiomeric mixture of 1,3-butanediol and leave optically active 1,3-butanediol can be used. . All the microorganisms described in Japanese Patent Application No. 1-107016 and JP-A-1-320997 relating to the present inventors can be used. Examples of these microorganisms include a microorganism capable of asymmetrically assimilating an enantiomeric mixture of 1,3-butanediol and remaining the (R) form, for example, Brevibacterium (Brev
genus ibacterium, genus Candida, genus Enterobacter, Geotricum
microorganisms belonging to the genus Chum) or the like. Examples of microorganisms that asymmetrically assimilate an enantiomeric mixture of 1,3-butanediol and leave the (S) form include, for example, Agrobacterium genus and Azotobacter ( Azoto
and microorganisms belonging to the genus Bacillus, the genus Bacillus, and the like.

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 exci, 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 enantiomer mixture of 1,3-butanediol with the microbial cells, the enantiomer mixture of 1,3-butanediol is added in advance to the medium described above, A method of simultaneously initiating cell growth and reaction by inoculating and culturing a seed bacterium, or a method of adding an enantiomeric mixture of 1,3-butanediol after some degree of cell growth is observed, A method of adding an enantiomer mixture of 3-butanediol, a method of separating cells after completion of the culture, and reacting this with an enantiomer mixture of 1,3-butanediol,
Alternatively, various methods can be proposed, such as a method in which the cells obtained in this manner are immobilized by a known method and reacted with an enantiomeric mixture of 1,3-butanediol.

Various reaction conditions, such as temperature, aeration and stirring conditions, substrate concentration, cell concentration, reaction time, etc., when reacting the enantiomer mixture of 1,3-butanediol with microbial cells by such various methods are particularly limited. Instead, conditions that are most advantageous for the production of the desired enantiomer may be selected.

By the way, in performing the reaction of the enantiomer mixture of 1,3-butanediol with the microbial cells as described above, there is also a method in which the enantiomer mixture of 1,3-butanediol serving as a substrate is initially charged all at once. In this case, although it depends on the amount of cells to be used, in general, when the substrate concentration is several% or more, the substrate is inhibited, the reaction speed is reduced, and as a result, the reaction time is prolonged and the productivity is reduced. This is not necessarily the preferred method.

As a solution to such a problem, a so-called fed-batch reaction method is generally used, in which the substrate concentration is controlled within a range where substrate inhibition does not occur and the reaction is performed while maintaining the maximum reaction rate while continuously supplying the substrate. Many cases are taken.

As described in the following Comparative Examples and Examples also in the case of producing the target compound, the fed-batch reaction method has a shorter reaction time as compared with the case where the substrate concentration is set high from the beginning,
It was also found that the final accumulated concentration was increased and the productivity was improved.

Here, the substrate to be fed is a mixture of enantiomers of 1,3-butanediol, but it is the enantiomer opposite to the target substance that substantially becomes the substrate of the enzyme in the cells. It is important to control within a certain range. The concentration at this time is preferably 0.1 to
3% by weight, more preferably 0.5 to 1.5% by weight.

The feeding method may be intermittent or continuous. Furthermore,
Such a fed-batch reaction method is applicable to all of the above-mentioned reaction methods of microbial cells and an enantiomeric mixture of 1,3-butanediol.

When the reaction is performed as described above and the optical purity of the target enantiomer reaches a desired value, the cells are removed from the reaction solution by a known method, and the process proceeds to a purification step. After appropriately concentrating and dehydrating the obtained supernatant, the desired product is extracted with an organic solvent such as ethyl acetate, and then dehydrated with anhydrous sodium sulfate or the like.
When the solvent is removed under reduced pressure, the desired optically active 1,3-butanediol is obtained. Further, it can be further purified by distillation. The target product can be obtained by directly dehydrating the supernatant under reduced pressure and then distilling, and after pretreatment by ultrafiltration membrane treatment, activated carbon treatment, or ion exchange resin treatment, etc., dehydration and distillation The desired product can be obtained.

〔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 Glycol 2%, Yeast Extract 1%, Silicone Defoamer 0.
Medium 15 having a composition of 01% (pH 6) was placed in a 30-volume jar fermenter and sterilized by heating at 121 ° C. for 15 minutes. Here, one preculture of Geotricum candidum IFO 4601 precultured in a medium having the same composition as described above was inoculated at 30 ° C.
With stirring of 400 rpm, aeration of 0.5 vvm, internal pressure of fermentation tank of 0.4 kg / cm ²
Under the conditions described above, aeration and stirring culture was performed for 20 hours.

Next, 1 was taken from this culture solution and centrifuged to obtain 70 g of fresh wet cells.

Using the whole amount of the viable cells, 50 g of racemic 1,3-butanediol, 0.1 g of silicon defoamer, and water were added to make up a total amount of 1 reaction solution, which was placed in a 2.6-volume mini jar fermenter. , At 30 ° C, stirring 400rpm, aeration 0.5vvm, pH3
(Adjusted with sulfuric acid or caustic soda solution) under aeration and agitation to react. The reaction was terminated when the optical purity reached 97% ee. The reaction time was 38 hours. 17 g of (R) -1,3-butanediol was produced in the reaction completed solution.

Comparative Example 2 The culture solution 1 obtained in Comparative Example 1 was placed in a 2.6-vol minijar, and the pH was controlled at 3 with 0.1N sulfuric acid or 0.1N sodium hydroxide solution at 30 ° C. and stirring at 400 rpm.
Under the condition of aeration of 0.5 vvm, 50 g of racemic 1,3-butanediol was added and reacted. The reaction was terminated when the optical purity reached 97% ee. The reaction time was 35 hours. 18 g of (R) -1,3-butanediol was produced in the reaction completed solution.

Example 1 The culture solution 3 obtained in Comparative Example 1 was centrifuged, and 210 g of the obtained fresh wet cells were divided into three parts. 0.1 g of the silicon antifoaming agent was added to 70 g of the fresh wet cells, and water was added to the rest. A reaction solution of a total amount of 1 was composed of 3 liquids, put in a 2.6-volume mini jar as in Comparative Example 1, and stirred at 30 ° C. under 400 rpm, aeration of 0.5 vvm, and pH 3 (adjusted with sulfuric acid or caustic soda solution). No. 1 jar was 0.1-0.5%, No. 2 jar was 0.5-1.5%, and No. 3 jar was 1.5% -2.5%. %, And finally, a total of 70 g as a racemate was continuously supplied while reacting. The reaction was terminated when the optical purity reached 97% ee.

Reaction time of each jar, (R)
Table 1 shows the concentrations of -1,3-butanediol.

Example 2 The culture solution 1 obtained in Comparative Example 1 was placed in a 2.6-vol minijar, and the pH was controlled at 3 with 0.1N sulfuric acid or 0.1N caustic soda solution. While controlling the racemic form of 1,3-butanediol so that the concentration of the (S) form falls within the range of 0.5 to 1.5%, finally, a total of 80 g of the racemic form is continuously supplied while reacting and reacted. Was. Optical purity 97%
The point at which ee was reached was regarded as the end of the reaction. The reaction time was 58 hours, and the concentration of (R) -1,3-butanediol in the reaction-terminated liquid was 34 g /.

The reaction-terminated liquid was centrifuged to obtain 900 g of a supernatant. The supernatant is passed through an ultrafiltration membrane (molecular weight cut off 30,000, effective membrane area 0.25
m ² : manufactured by Daicel Chemical Industries, Ltd.), and then concentrated and dehydrated with a rotary evaporator under reduced pressure, and distilled at 120 to 130 ° C. under reduced pressure (30 to 40 torr) to obtain (R) -1,3
-25 g of a purified butanediol were obtained.

〔The invention's effect〕

By using the method of the present invention, in the production of optically active 1,3-butanediol on an industrial scale, a high concentration of the substrate can be charged, and the productivity can be greatly improved.

Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12P 41/00 BIOSIS (DERWENT) WPI (DERWENT)

Claims (2)

(57) [Claims] 1. A method for producing an optically active 1,3-butanediol, wherein a microorganism is acted on an enantiomeric mixture of 1,3-butanediol to collect the remaining optically active 1,3-butanediol. The concentration of the enantiomer opposite to the enantiomer in the reaction mixture is 0.1 to 3% by weight.
A process for adding an enantiomeric mixture of 1,3-butanediol while allowing the mixture to react within the range described above, and reacting the mixture. 2. The process according to claim 1, wherein the concentration of the enantiomer opposite to the enantiomer to be produced in the reaction mixture is controlled within the range of 0.5 to 1.5% by weight.