JPH0799993A - Production of optically active glycidol - Google Patents

Abstract

PURPOSE:To industrially obtain the subject compound of high optical purity by acting microbes belonging to e.g. Pseudomonas capable of optoselectively hydrolyzing glycidyl butyrate on glycidyl butyrate and hydrolyzing the resultant (R)-modification. CONSTITUTION:Microbes belonging to Pseudomonas, Rhodotorula or Rhizopus, capable of optoselectively hydrolyzing glycidyl butyrate are cultured and then made to act on racemic glycidyl butyrate. A hydrolase is then made to act on the optically active (R)-glycidyl butyrate to obtain the objective optically active (S)-glycidol useful as an intermediate for physiologically active substances such as medicines or agrochemicals or liquid crystal materials.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for producing an optically active glycidol compound, which comprises reacting a racemic glycidyl butyrate with a specific microorganism or enzyme.

[0002]

BACKGROUND OF THE INVENTION It is widely known that optical isomers exist in many of the compounds exhibiting biological activity. And, generally, the optical isomer exhibiting the desired biological activity of interest is limited to some isomers, and the other isomer does not exhibit the desired biological activity, or It is accompanied by undesirable physiological effects such as toxicity.

Therefore, in the case of a compound having an optical isomer, if there is no suitable means / method for obtaining a biologically active isomer, all the optical isomers in a mixed state are used as pesticides or pesticides. It is often used for pharmaceutical applications, and there have been problems that the original biological activity of the desired optical isomer is significantly reduced and that it is accompanied by undesirable physiological effects such as toxicity.

In order to overcome this problem, various methods for resolving optical isomers have been investigated. For example, an enzyme which prepares various ester derivatives of optical isomers to be resolved and optically selectively hydrolyzes the ester. Has been proposed, and a method for separating optical isomers from each other by utilizing the difference in solubility of the reaction system in a solvent, etc.,
In order to realize this method, research on microorganisms producing esterases having various optical selectivities is under way.

In such a technical background, optically active glycidol, which is one of the compounds exhibiting biological activity, has conventionally been regarded as important as a physiologically active compound for pharmaceutical or agrochemical use or a synthetic intermediate for liquid crystal materials. ing.

As a method for synthesizing optically active glycidol, a method for synthesizing optically active glycidol from D-mannitol (Fischer et al., J. Am. Chem. Soc., 64, p. 12).
91 (1942)) has been known for a long time, but this method was considered unsuitable for industrial production because it required many steps to prepare the final product. Recently, a method of asymmetric epoxidation of allyl alcohol (Shar
pless et al., Asymmetric Synthesis, Vol.5, Academic
Press, Inc., London, p. 247 (1985)) has been reported.However, although this method allows the preparation of the final product in a short step, it has a low optical purity during mass production. Is a point to be improved.

Further, in recent years, production of optically active glycidol utilizing an optically selective reaction of microorganisms and enzymes has been reported. For example, optically selective hydrolysis of racemic glycidyl butyrate by porcine pancreatic lipase (J. Am.Che
m.Soc., 106, p.7252 (1984), JP-A-1-285199),
Optical resolution of benzylglycidol precursor (J. Org. Chem., 5
5, p.4897 (1990)), asymmetric oxidation by alcohol dehydrogenases of the genera Acetobacter and Gluconobacter (European Patent Publication No. 0464905), etc.・ Although it is disclosed, these methods can obtain glycidol with high optical purity as compared with chemical synthetic methods, but the enzyme used is expensive and mass production is easy in terms of production technology. It included problems such as not being.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the problems of the above-mentioned prior art,
An object of the invention is to provide a method capable of industrially producing optically active glycidol having high optical purity.

That is, the gist of the present invention is that
Referring to FIG. 1, a microorganism that preferentially hydrolyzes an ester of glycidyl butyrate S-form, an enzyme produced by the microorganism, or an enzyme obtained from a culture of the microorganism is caused to act on glycidyl butyrate, and further the remaining The R-form glycidyl butyrate is treated with lipase or esterase to hydrolyze (R) -glycidyl butyrate.
Optical activity characterized by obtaining (S) -glycidol
This is a method for producing (S) -glycidol.

That is, the present invention produces an ester-degrading enzyme having high optical selectivity to glycidyl butyrate and hydrolyzes racemic glycidyl butyrate to give optically active (R) -glycidyl butyrate. It is based on the knowledge of microorganisms belonging to the genus Rhodo-torula, Pseudomonas, or Rhizopus and the mutants of these microorganisms that are retained in the reaction solution. RIKEN Institute of Microbial Strains Preservation Facility (Japan Collecti, 2-1 Hirosawa, Wako)
on Microorganism, JCM), the following microorganisms: Rhodotorula rubr
a: JCM Catalog No. 3782), Rhodtorula Rubra (Rhod
otorula rubra: JCM Catalog No. 8117), Pseudomonas cepasia: JCM Catalog No.
2801), Rhizopus chinensis (JC)
M Catalog No. 5555), Rhizopu
s chinensis: JCM Catalog No. 5556), and Rhizopus chinensis: JCM Catalog No. 5
596) based on the findings.

For the culture of the above-mentioned microorganism, either a solid medium or a liquid medium which is usually used can be used, but the liquid medium is preferable in terms of the recovery efficiency of the produced enzyme. Further, as the carbon source used in the culture medium for the microorganism, a carbon source that can be assimilated by the microorganism can be used, and starch, sucrose, glucose, or the like is preferable because it can be industrially inexpensively used, and As the nitrogen source, natural nitrogen sources such as yeast extract, casein, corn steep liquor, peptone and meat extract, and inorganic nitrogen compounds such as ammonium sulfate, ammonium salt and urea can be used.

Considering good growth of microorganisms and enzyme production, the carbon source concentration is 1 to 20%, and the culture time is 16%.
The culture is carried out for about 48 hours, and any of stationary culture, aeration stirring, and shaking culture can be applied.

The racemic glycidyl butyrate which is the substrate for the enzymatic reaction in the method of the present invention is a known esterification method, for example, butyric anhydride or butyric acid halide is added to racemic glycidol, pyridine or triethylamine, tributylamine, It can be easily synthesized by reacting in the presence of an amine such as dimethylaminopyridine.

Furthermore, the enzymatic reaction in the method of the present invention comprises:
It is carried out by adding the above microorganisms, the enzyme produced by the above microorganisms, or the esterolytic enzyme collected from the culture of the above microorganisms to water or a buffer solution containing racemic glycidyl butyrate and stirring. In addition,
It is also possible to use the ester degrading enzyme as a crude enzyme in a treated product of microorganisms obtained by culturing.

Regarding the reaction conditions, in consideration of the reaction characteristics of the enzyme used, the reaction pH is 4-9, preferably 5-8, and the reaction temperature is 10 ° C-50 ° C, preferably 20 ° C-40 ° C. To adjust. After the reaction is completed, the generated glycidol and unreacted glycidyl butyrate are separated by a conventional method such as a solvent extraction method, a crystal precipitation method, or a column chromatography method. In the solvent extraction method, if n-hexane or heptane is used as a solvent, only unreacted optically active (R) -glycidyl butyrate can be recovered from the reaction solution with an efficiency of 90% or more, which will be described later. It became clear from the example.

Next, the separated (R) -glycidyl butyrate undergoes unfavorable side reactions such as ring opening and racemization of the epoxy ring in a conventional hydrolysis using an acid or a base. In the method of the present invention, the separated (R) -glycidyl butyrate is hydrolyzed in the neutral region using an ester hydrolase such as lipase or esterase to obtain (S) -glycidol. As the ester hydrolase that can be used in the method of the present invention, any of lipase and esterase having the ability to hydrolyze glycidyl butyrate can be used. Regarding the hydrolysis (enzyme) reaction, the reaction of glycidol Considering the characteristics, the reaction pH is adjusted to 6 to 8, preferably 6.5 to 7.5.

[0017]

EXAMPLES Preferred examples of the method of the present invention will be specifically described below, but the following examples are for illustrative purposes and should not be construed as limiting the present invention.

Example 1 Production of Bacteria In a 500 ml Erlenmeyer flask, 100 ml of a medium having the composition shown in Table 1 below was placed and pre-cultured in the same medium in advance. Rhodotorula rubra: JCM Catalog N
o.3782) inoculated with 1 ml of culture solution and rotary shaker for 16 hours at 30 ℃ (“MC-B-98R”: manufactured by Takasaki Scientific Instruments Co., Ltd.)
Shaking culture (160 rpm) was performed.

[0019]

[Table 1]

After completion of the culture, a cell culture solution having an absorbance (OD ₆₆₀ ) value of 32 was obtained.

Example 2: Production of bacterial cells In a 500 ml Erlenmeyer flask, 100 ml of a medium having the composition shown in Table 2 below was placed, and Pseudomonas cepasia (JCM Catalog No.) was pre-cultured in the same medium. .2801) culture solution (1 ml) was inoculated, and shake culture (160 rpm) was performed at 30 ° C. for 16 hours on a rotary shaker (“MC-B-98R”: manufactured by Takasaki Scientific Instruments Co., Ltd.).

[0022]

[Table 2]

After completion of the culture, a cell culture solution having an absorbance (OD ₆₆₀ ) value of 21 was obtained.

Example 3: Preparation of crude enzyme solution 100 ml of the medium used in Example 1 was added to a 500 ml Erlenmeyer flask.
l picked and grown on slant medium Rhizopus chinensis
(Rhizopus chinensis: JCM Catalog No.5555) inoculated with one platinum loop and rotary shaker (“MC-B
-98R ": Takasaki Scientific Instruments Co., Ltd.) shaking culture (160
rpm).

After culturing, the cells were homogenized (“Po
Lytron Homogenizer ": manufactured by Brinkman) was crushed for 1 minute to obtain a crude enzyme solution.

Example 4: Preparation of (S) -glycidol
1) To 7.5 ml of 50 mM phosphate buffer (pH 7.0), 2.0 ml of Rhodotorula rubra (JCM Catalog No. 3782) culture solution obtained in Example 1 and 0.1 g of glycidyl butyrate.
5 ml (3.7 mmol) was added, and the mixture was stirred at 30 ° C for 24 hours.

After completion of the reaction, 10 ml of n-hexane was added to the reaction solution to extract glycidyl butyrate. The extract was subjected to gas chromatography ("SIMADZU GC-4A
: Shimadzu Corporation), optical purity 98%
Of (R) -glycidyl butyrate was obtained with a yield of 21%.

50 mM phosphate buffer (pH 7.0) was added to this extract.
10 ml and 1 mg of lipase P (Pseudomonas sp., Manufactured by Nagase Seikagaku) were added, and the obtained (R) -glycidyl butyrate was added.
By hydrolyzing at 30 ℃, the optical purity of 99% or more
(S) -glycidol was obtained with a yield of 18%.

== Gas chromatographic analysis conditions == Column: Chiraldex G-TA, 30 m (manufactured by Astec) Temperature: Column initial temperature; 55 ° C (20 min), heating rate; 10
℃ / min, final temperature; 80 ℃ (37.5 min) injector temperature; 250 ℃, detector temperature; 250 ℃ Carrier gas: helium, 1 ml / min Detector: FID Example 5: Preparation of (S) -glycidol 2) Rhodotorula rubra obtained according to the method of Example 1
(Rhodotorula rubra: JCM Catalog No. 8117) culture solution
2.0 ml was added to 7.5 ml of 50 mM phosphate buffer (pH 7.0) together with 0.5 ml (3.7 mmol) of glycidyl butyrate, and the mixture was stirred at 30 ° C. for 24 hours.

After completion of the reaction, 10 ml of n-hexane was added to the reaction solution to extract glycidyl butyrate. The extract was analyzed by gas chromatography (“SIMADZU GC-4A”: manufactured by Shimadzu Corp.) according to the analysis conditions shown in Example 4 to find that (R) -glycidyl butyrate having an optical purity of 98% was obtained.
Obtained in 20%.

50 mM phosphate buffer (pH 7.0) was added to this extract.
10 ml and 1 mg of lipase P (Pseudomonas sp., Manufactured by Nagase Seikagaku) were added, and the obtained (R) -glycidyl butyrate was added.
By hydrolyzing at 30 ℃, the optical purity of 99% or more
(S) -glycidol was obtained with a yield of 17%.

Example 6: Preparation of (S) -glycidol
3) Pseudomonas cepasia (JCM) obtained in Example 2 was added to 7.5 ml of 50 mM phosphate buffer (pH 7.0).
2.0 ml of the culture solution of Catalog No. 2801) and 0.5 ml (3.7 mmol) of glycidyl butyrate were added, and the mixture was stirred at 30 ° C. for 24 hours.

After completion of the reaction, 10 ml of n-hexane was added to the reaction solution to extract glycidyl butyrate. When the extract was analyzed by gas chromatography under the analysis conditions shown in Example 4, (R) -glycidyl butyrate having an optical purity of 90% was obtained in a yield of 27%.

50 mM phosphate buffer (pH 7.0) 1
0 ml and 1 mg of lipase P (Pseudomonas sp., Manufactured by Nagase Seikagaku) were added, and the obtained (R) -glycidyl butyrate was added.
By hydrolysis at 30 ° C., (S) -glycidol with an optical purity of 93% was obtained with a yield of 24%.

Example 7: Preparation of (S) -glycidol
4) 2.0 ml of crude enzyme solution of Rhizopus chinensis (JCM catalog No. 5555) obtained in Example 3 and glycidyl butyrate were added to 7.5 ml of 50 mM phosphate buffer (pH 7.0).
0.5 ml (3.7 mmol) was added, and the mixture was stirred at 30 ° C for 24 hours.

After completion of the reaction, 10 ml of n-hexane was added to the reaction solution to extract glycidyl butyrate. When the extract was analyzed by gas chromatography according to the analysis conditions shown in Example 4, (R) -glycidyl butyrate having an optical purity of 99% was obtained in a yield of 26%.

50 mM phosphate buffer (pH 7.0) was added to this extract.
10 ml and 1 mg of lipase P (Pseudomonas sp., Manufactured by Nagase Seikagaku) were added, and the obtained (R) -glycidyl butyrate was added.
By hydrolysis at 30 ° C., (S) -glycidol with an optical purity of 99% was obtained with a yield of 22%.

Example 8: Preparation of (S) -glycidol
5) Rhizopus chinensis obtained according to the method of Example 3
(Rhizopus chinensis: JCM Catalog No. 5556) 2.0 ml of crude enzyme solution was added to 7.5 ml of 50 mM phosphate buffer (pH 7.0) together with 0.5 ml (3.7 mmol) of glycidyl butyrate and stirred at 30 ° C. for 24 hours. did.

After completion of the reaction, 10 ml of n-hexane was added to the reaction solution to extract glycidyl butyrate. When the extract was analyzed by gas chromatography under the analysis conditions shown in Example 4, (R) -glycidyl butyrate having an optical purity of 98% was obtained in a yield of 27%.

50 mM phosphate buffer (pH 7.0) was added to this extract.
10 ml and 1 mg of lipase P (Pseudomonas sp., Manufactured by Nagase Seikagaku) were added, and the obtained (R) -glycidyl butyrate was added.
By hydrolysis at 30 ° C., (S) -glycidol with an optical purity of 99% was obtained with a yield of 23%.

Example 9: Preparation of (S) -glycidol
6) Rhizopus chinensis obtained according to the method of Example 3
(Rhizopus chinensis: JCM Catalog No. 5596) 2.0 ml of crude enzyme solution was added to 7.5 ml of 50 mM phosphate buffer (pH 7.0) together with 0.5 ml (3.7 mmol) of glycidyl butyrate and stirred at 30 ° C. for 24 hours. did.

After completion of the reaction, 10 ml of n-hexane was added to the reaction solution to extract glycidyl butyrate. When the extract was analyzed by gas chromatography under the analysis conditions shown in Example 4, (R) -glycidyl butyrate having an optical purity of 99% was obtained in a yield of 25%.

50 mM phosphate buffer (pH 7.0) was added to this extract.
10 ml and 1 mg of lipase P (Pseudomonas sp., Manufactured by Nagase Seikagaku) were added, and the obtained (R) -glycidyl butyrate was added.
By hydrolysis at 30 ° C., (S) -glycidol with an optical purity of 99% was obtained with a yield of 21%.

[0044]

EFFECTS OF THE INVENTION According to the method of the present invention, optically active (S) -glycidol, which is expected to be applied in various fields, can be easily produced with high optical purity as compared with the conventional chemical synthesis method. That is, an excellent method suitable for industrial mass production is provided.

[Brief description of drawings]

FIG. 1 is a schematic view showing a manufacturing process of the present invention.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technology display location (C12P 41/00 C12R 1: 845) (72) Inventor Tomoko Umesato 2-3-2 Muroya, Nishi-ku, Kobe City, Hyogo Prefecture Nagase & Co., Ltd. Research and Development Center

Claims (5)

[Claims] 1. A method for producing an optically active glycidol compound, which comprises the following steps: (1) Rhodotorula genus, Pseudomonas (Ps)
eudomonas) or Rhizopus genus,
And culturing a microorganism having the ability to optically and selectively hydrolyze glycidyl butyrate, (2) act the microorganism on racemic glycidyl butyrate, optical activity (R)-
Glycidyl butyrate is produced, and (3) the optically active (R)-
Optical activity characterized by including a step of hydrolyzing glycidyl butyrate by causing a hydrolase to act, and (4) recovering the produced optically active (S) -glycidol.
(S) -Method for producing glycidol. 2. A method for producing an optically active glycidol compound, which comprises the following steps: (1) genus Rhodotorula, Pseudomonas (Ps)
eudomonas) or Rhizopus genus,
And culturing a microorganism having the ability to optically and selectively hydrolyze glycidyl butyrate, (2) the enzyme produced by the microorganism to act on racemic glycidyl butyrate, optically active (R) -glycidyl butyrate Produces (3)
The optically active (R) -glycidyl butyrate is hydrolyzed by the action of a hydrolase, and (4) the produced optically active (S) -glycidol is recovered, which is characterized in that (S) -Method for producing glycidol. 3. A method for producing an optically active glycidol compound, which comprises the following steps: (1) genus Rhodotorula, Pseudomonas (Ps)
eudomonas) or Rhizopus genus,
And culturing a microorganism having the ability to optically and selectively hydrolyze glycidyl butyrate, (2) act the enzyme obtained from the culture of the microorganism on racemic glycidyl butyrate, optical activity (R) -Producing glycidyl butyrate, (3) hydrolyzing the optically active (R) -glycidyl butyrate by acting a hydrolase, and (4) recovering the produced optically active (S) -glycidol. And a step of producing optically active (S) -glycidol. 4. The method for producing an optically active (S) -glycidol according to claim 1, wherein the hydrolase is a lipase or an esterase. 5. The microorganism is Rhodotorula rubra (Rho
dotorula rubra: JCM Catalog No. 3782), Rhodotorula rubra: JCM Catalog No. 811
7), Pseudomonas cepasia
: JCM Catalog No. 2801), Rhizopus chinensis (R
hizopus chinensis: JCM Catalog No.5555), Rhizopus chinensis: JCM Catalog N
5556), Rhizopus chinensi
s: one or more microorganisms selected from the group consisting of JCM Catalog No. 5596), The method for producing an optically active (S) -glycidol according to any one of claims 1 to 4.