JPH0759593A - Production of optically active carboxylic acid, optically active isocarboxylic acid or optically active isoalkanol - Google Patents

Abstract

PURPOSE:To obtain an optically active carboxylic acid by a one-stage reaction using a simple separation means by biochemically oxidizing an isoalkyl compound with a specific microorganism. CONSTITUTION:An isoalkyl compound of formula I (R1 is an alkyl, an aromatic ring, a heterocyclic group or a sulfur-containing alkyl; (m) and (n) are independently 0, 1) is oxidized with a microorganism belonging to the genus Rhodococcus or Mycobacterium and capable of converting the compound of formula I to a carboxylic acid. The optically active carboxylic acid of formula II or formula III (C* is carbon atom imparted with optical activity) is easily separated from the reaction medium by extraction, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an optically active carboxylic acid having a methyl group or an ethyl group on the 2- or 3-position carbon atom, and an optical group having a methyl group on the 2-position carbon atom. The present invention relates to a method for producing an active isocarboxylic acid and a method for producing an optically active isoalkanol having a methyl group on the 2-position carbon atom. The optically active carboxylic acid, the optically active isocarboxylic acid, and the optically active isoalkanol produced by the method of the present invention are both
It is useful as a raw material used in the synthesis of various physiologically active substances having optical activity.

[0002]

As a method for producing an optically active carboxylic acid by a biochemical method, a racemic carboxylic acid is once produced and then converted into a racemic nitrile compound in which the carboxyl group of the compound is converted into a nitrile group. Then, a method of optically resolving the optically active carboxylic acid obtained by converting the nitrile group into a carboxyl group again by asymmetric hydration of a nitrilase enzyme or a microorganism having the enzyme [Appl.Env.Microbiol. 56, 10, 3125-3129 (1990)] has been proposed. However, this method has a problem in that a dangerous reagent such as cyan is required for the nitration step, the step is complicated, and the theoretical yield does not exceed 50% due to optical resolution. It was

Further, unlike the above-mentioned method, as a method for obtaining an optically active carboxylic acid without a nitration step, for example, a compound having an isopropyl group is oxidized using a filamentous fungus such as Cordiceps militaris. Then, a method for producing an optically active 2-arylpropionic acid (see Japanese Patent Laid-Open No. 62-63592) has been proposed. However, bacteria other than filamentous fungi oxidize the isopropyl group to give an optically active compound. There is no disclosure showing that the reaction can be converted to a different carboxylic acid.

In place of the above-mentioned method using filamentous fungi, there is desired a method for producing an optically active carboxylic acid by a novel biochemical method using a bacterium which can be easily cultured and without a nitration step. It is rare. That is, a novel method of producing an optically active carboxylic acid by a one-step biochemical process of oxidizing a raw material compound using a bacterium other than a filamentous fungus is desired. Alternatively, an optically active isoalkano-which can be converted into an optically active carboxylic acid by a conventional chemical oxidation method.
A new method for separating racemic isoalkanol by a biochemical method is desired.

[0005]

SUMMARY OF THE INVENTION The present invention is intended to solve the above problems, and an object of the present invention is to use a microorganism to
New method for producing an optically active carboxylic acid having a methyl group or an ethyl group on the carbon atom at the 3rd or 3rd position, a novel method for producing an optically active isocarboxylic acid, and further an optically active isoalkanol It is to provide a new method. In particular, by using a bacterium other than filamentous fungus as a microorganism and using a racemic isoalkyl compound or isoalkanol as a starting compound, an optically active compound having a molecular structure corresponding to the starting compound is obtained by a one-step biochemical process. Another object of the present invention is to provide a novel method for producing various isocarboxylic acids.

[0006]

Means for Solving the Problems The present inventor has conducted earnest research to solve the above problems, and is a microorganism collected from soil, which is an optically active carvone having a corresponding molecular structure from an isoalkyl compound. The presence of bacteria with the ability to convert to acids, and among the microorganisms,
It was found that there are bacteria having the ability to assimilate isoalkanol, and further that the product of these bacteria is an optically active isocarboxylic acid, the present invention has been completed.

That is, one method provided by the present invention is as follows.

[Chemical 7] (In the formula, R ₁ represents a substituent selected from the group consisting of an alkyl group, an aromatic ring, a heterocycle, and a sulfur-containing alkyl group,
m and n are 0 or 1) is oxidized by a microorganism belonging to the genus Rhodococcus or Mycobacterium and having the ability to convert the compound into a carboxylic acid.

[Chemical 8] (Wherein R ₁ , m and n are the same as the compound represented by the general formula 7 above, C ^* represents a carbon atom to which optical activity is imparted), and the optically active carboxylic acid is separated. It is a method for producing an optically active carboxylic acid, which comprises obtaining the carboxylic acid.

Another method provided by the present invention is represented by the following general formula 9

[Chemical 9] (In the formula, R ₂ represents an alkyl group having 2 or more carbon atoms, an aromatic ring,
A heterocycle and a substituent selected from the group consisting of sulfur-containing alkyl groups, wherein p and q are 0 or 1), an isoalkanol represented by Rhodococcus or Mycobacterium, Oxidation with a microorganism capable of converting an isoalkanol to a carboxylic acid gives a compound of the general formula 1
0

[Chemical 10] (Wherein R ₂ and p and q are the same as the isoalkanol represented by the general formula 9 above, C ^* represents a carbon atom to which optical activity is imparted) Is a method for producing an optically active isocarboxylic acid.

Another aspect of the present invention is the following general formula 11

[Chemical 11] (In the formula, R ₃ represents a substituent selected from the group consisting of an alkyl group having 2 or more carbon atoms, an aromatic ring, a heterocycle, and a sulfur-containing alkyl group, and s and t are 0 or 1) The racemic isoalkanol is oxidized with a microorganism belonging to the genus Rhodococcus or the genus Mycobacterium and capable of converting the isoalkanol into a carboxylic acid.

[Chemical 12] (Wherein R ₃ and s and t are the same as the isoalkanol represented by the above general formula 11, C ^* represents a carbon atom to which optical activity is imparted), and the optically active isocarboxylic acid. Is produced and then the remaining optically active isoalkanol is separated and obtained, and the method is a method for producing an optically active isoalkanol.

In the compound represented by the above general formula 7 as a raw material, the group R _1- (CH ₂ ) _m-, which is composed of the substituent represented by R ₁ in the general formula 7, is present. Carbon is an asymmetric carbon, and there are two types of optical isomerism due to the carbon. In the present invention, in the general formula 7, when R ₁ is selected from an alkyl group, the alkyl group is preferably selected from the range of 3 to 20 carbon atoms. Further, it is more preferable that at least one hydrogen atom is present on each carbon contained in the constituted alkyl group R _1- (CH ₂ ) _m- . Examples of suitable alkyl groups for R ₁ include
n-propyl, isopropyl, n-butyl, isobutyl,
n-Pentyl, isopentyl and the like can be exemplified, and 2,5-dimethyl-
Examples thereof include 1-hexanol and 2,4-dimethyl-1-pentanol. When R ₁ is selected from an aromatic ring, the aromatic ring of choice includes an aromatic ring having an alkyl group or the like as a substituent. Examples of suitable aromatic rings for R ₁ include phenyl, alkylphenyl, naphthyl, alkylnaphthyl, etc., in which the number of condensed rings is selected to be 2 or less. Further, cumene can be named by using a specific compound name of aromatic hydrocarbon. , Diisopropylbenzene, cymene and the like can be exemplified as preferable compounds. When R ₁ is selected from a heterocycle, the heterocycle of choice includes a heterocycle in which an alkyl group or the like is present as a substituent. Examples of suitable heterocycles for R ₁ include furyl, thienyl, pyrrolyl, pyridyl, quinolyl, etc., in which the number of rings to be condensed is selected to be 2 or less. Pyridine and isopropylfuran can be exemplified as preferable compounds. When R ₁ is selected from sulfur-containing alkyl groups,
The sulfur-containing alkyl group is preferably selected from the range of 3 to 20 carbon atoms. Furthermore, it is more preferable that at least one hydrogen atom be present on each carbon contained in the sulfur-containing alkyl group. Examples of sulfur-containing alkyl groups suitable as R ₁ include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, and the like, and the specific compound name of thioether is used to indicate diisopropyl sulfide. And the like can be exemplified as preferable compounds.

In the isoalkanol represented by the above general formula 9 as a raw material, the carbon at the 2-position where the methyl group exists is an asymmetric carbon, and two kinds of optical isomers due to the carbon exist. . In the present invention, in general formalized 9, when selecting the R ₂ from alkyl groups having more than 2 carbon atoms, the alkyl group R ₂ alkyl group composed of R ₂ - the total number of carbon atoms of - (CH _{2) p} It is preferable to select in the range of 3 to 20. Furthermore, it is more preferable that at least one hydrogen atom is present on each carbon contained in the alkyl group R _2- (CH ₂ ) _p- . Examples of suitable alkyl groups for R ₂ include n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl and the like, and the specific compound name of isoalkanol is 2,5- Dimethyl-1-
Hexanol and 2,4-dimethyl-1-pentanol can be exemplified as preferable compounds. When R ₂ is selected from an aromatic ring, the aromatic ring of choice includes an aromatic ring having an alkyl group or the like as a substituent. Examples of suitable aromatic rings for R ₂ include phenyl, alkylphenyl, naphthyl, and alkylnaphthyl, which select the number of condensed rings to be 2 or less. Further, using specific compound names in which aromatic ring names are described, 2-phenyl-1-propanol, 2-phenyl-
Examples of preferable compounds include 1-butanol and 2-naphthyl-1-propanol. When R ₂ is selected from a heterocycle, the heterocycle of choice includes a heterocycle in which an alkyl group or the like is present as a substituent. As an example of a suitable heterocycle for R ₂ , a furyl for selecting the number of condensed rings to be 2 or less,
Thienyl, pyrrolyl, pyridyl, quinolyl and the like can be exemplified, and pyridyl isopropanol, furyl isopropanol and the like can be exemplified as preferable compounds by using specific compound names of heterocyclic compounds. When R ₂ is selected from a sulfur-containing alkyl group, the sulfur-containing alkyl group has 3 carbon atoms.
It is preferable to select in the range of -20. Furthermore, it is more preferable that at least one hydrogen atom be present on each carbon contained in the sulfur-containing alkyl group. Examples of the sulfur-containing alkyl group suitable as R ₂ include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, and the like, and using a specific compound name having a thioether bond, 2-Isopropylthio-1-propanol etc. can be illustrated as a preferable compound.

The isoalkanol represented by the above general formula 11 as the raw material is the same as the isoalkanol represented by the above general formula 9, and in the present invention, the general formula 11 R ₃ in the formula is R _{2 in} the general formula 9 above.
It can be selected by the same means and, and preferred substituents as R ₂ is a suitable displaceable group as R _3.

The microorganism used in the present invention belongs to the genus Rhodococcus or the genus Mycobacterium and has the ability to convert the compound represented by the general formula 7 into a carboxylic acid. Specifically, it is isolated from soil. Rhodococcus sp. 11B (Deposit No. FERM P-13654, Institute of Biotechnology, Agency of Industrial Science and Technology) and Mycobacterium sp. D84-
1 (same as FERM P-13657), Mycobacterium sp. D59-1
(The same FERM P-13656), Mycobacterium sp. D28-1 (the same)
FERM P-13655). These strains have been deposited with the above numbers at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry. The mycological properties of these strains are shown in Table 1.

[0014]

[Table 1]

Further, in the method for producing an optically active isocarboxylic acid using the isoalkanol of the present invention as a raw material, and the method for obtaining an optically active isoalkanol from a racemic isoalkanol, Rhodococcus or Mycobacteria is used. A strain belonging to the genus Umm and having the ability to convert the isoalkanol represented by the general formula 9 or the general formula 11 into a carboxylic acid can be used. In addition, as an example of a preferable specific strain, the above-mentioned Rhodoco
ccus sp. 11B (Deposit No. FE, Institute of Biotechnology, AIST)
RMP-13654), and generally, a suitable strain can be easily selected from the group consisting of strains having the ability to convert the compound represented by the general formula 7 into a carboxylic acid.

In the present invention, a strain of the above-mentioned microorganism or a culture thereof or a bacterial body in which the strain is preliminarily grown,
In addition to the reaction medium to which the compound as the raw material is added, in order to bring the bacteria and the reaction medium components into sufficient contact, for example, a technique such as stirring, air blowing with a nozzle, or shaking is applied, and the aerobic condition is applied. Cause the bacteria to oxidize. During the reaction, the temperature of the reaction medium may be maintained at 35 ° C. or lower, preferably 25 ° C. or higher, and the pH of the reaction medium may be adjusted to the range of 3 or more and 9 or less, preferably 4 or more and 8 or less. The reaction time is appropriately selected depending on the density of the bacterium in the reaction medium and the amount of the raw material compound to be added, but in general, the reaction time may be selected in the range of 5 hours to 100 hours. .

As the medium used for culturing the microorganism used in the present invention, a medium usually used for culturing a bacterium belonging to the genus Rhodococcus or Mycobacterium can be used. As a medium used for culturing, for example, as a carbon source, glucose, sucrose, maltose, glycerol or the like, as a nitrogen source, ammonium nitrate or ammonium phosphate which is an inorganic nitrogen compound, and or peptone which is an organic nitrogen compound, Corn steep liquor, amino acids, and the like, inorganic salts such as sodium phosphate, calcium phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate, or manganese sulfate are added, and yeast extract or the like is added as another nutrient source. Used. Table 2 shows an example of a medium composition used when preparing a culture of the above-mentioned microbial strain or when preparing a microbial cell in which the strain is preliminarily grown. Table 3 shows an example of the composition of the above reaction medium.

[0018]

[Table 2]

[0019]

[Table 3]

The optically active carboxylic acid produced in the reaction medium by oxidizing the starting material compound in the reaction medium by the bacterium is extracted from the reaction medium after completion of the reaction, Separation can be carried out using a conventional method such as solid-liquid separation, neutralization precipitation, fractional distillation and the like. Further refined,
Only the optically active carboxylic acid of interest can be collected. Also, when the target optically active carboxylic acid is an optically active isocarboxylic acid, it can be separated by the same method as above, and further purified to obtain only the target optically active isocarboxylic acid. can do.

On the other hand, when a racemic isoalkanol is used as a raw material compound, the above reaction does not convert it into an optically active isocarboxylic acid, and the remaining isoalkanol becomes optically active. After separating the optically active isocarboxylic acid from the reaction medium in advance, the remaining optically active isoalkanol in the reaction medium should also be separated from the reaction medium by a conventional method such as extraction, solid-liquid separation, or fractional distillation. You can Alternatively, it is separated from the reaction medium directly or simultaneously with the optically active isocarboxylic acid by a conventional method such as extraction or fractional distillation, and then by a conventional method such as solid-liquid separation, neutralization precipitation or fractional distillation. By carrying out separation and purification, only the desired optically active isoalkanol can be collected.

[0022]

INDUSTRIAL APPLICABILITY According to the present invention, a compound represented by the general formula 7 is used as a raw material compound in a one-step reaction, and by a simple separating means, a methyl group or An optically active carboxylic acid having an ethyl group can be obtained. In addition, an optically active isocarboxylic acid having a methyl group or an ethyl group at the 2-position carbon can be obtained by a one-step reaction using an isoalkanol represented by the general formula 9 as a raw material compound and by a simple separation means. The acid can be obtained. Further, an optically active isoalkanol can be obtained by a one-step reaction by using an isoalkanol represented by the general formula 11 as a raw material compound.

[0023]

【Example】

(Example 1) As a growth medium, (NH ₄ ) ₂ HPO ₄ 4.0 g, Na _{2
HPO 4 · 12H 2 O 2.5g,} KH 2 PO 4 2.0g, MgSO 4 · 7H 2 O 30m
g, CaCl ₂ .2H ₂ O 60 mg, yeast extract 200 mg, and glucose 10 g were dissolved in 1 l of deionized water, and the pH was adjusted to 7.2 to prepare a medium. 50 ml of the growth medium was dispensed into a 500 ml Sakaguchi flask and sterilized in an autoclave at 121 ° C. for 20 minutes. Rhodococ
After inoculating 3 platinum loops of cus sp. 11B (Deposit No. FERM P-13654 of the Institute of Biotechnology, Institute of Industrial Science and Technology) into the growth medium prepared and sterilized as described above, the mixture was shake-cultured at 30 ° C. for 24 hours, and What culture | cultivated the bacterium was used as an inoculum liquid. Next, 2% of the inoculum solution was added to the growth medium prepared and sterilized as described above.
(V / v) 50 inoculated plants were prepared. Each was shake-cultured at 30 ° C. for 48 hours. In addition, 2 after the start of culture
After 4 hours, 1% (v) of n-heptane was added to the culture solution.
/ V) was added and the flask was sealed with a rubber stopper to continue the culture. The cells produced by the culture 100mM phosphate buffer (pH 7.2) and reaction medium _{(K 2 HPO 4 1.74g, MgSO} 4 · 7
H ₂ O (1.50 g) and FeSO ₄ · 7H ₂ O (50 mg) were dissolved in pure water (1 l), and the pH was adjusted to 8.0. The suspension was turbid to prepare a bacterial suspension.

Each 50 ml of the bacterial suspension was dispensed into a 500 ml Sakaguchi flask and 500 μl of 2,5-dimethylhexane.
Was added to each flask. Then, the flask was tightly stoppered, and cultured with shaking at 30 ° C. for 24 hours. The cells were removed from the culture solution obtained above by centrifugation. The obtained residual liquid was acidified with hydrochloric acid, and solvent extraction was performed with an equal amount of diethyl ether. Then, the extracted diethyl ether layer was separated, dehydrated with sodium sulfate, and then the solvent diethyl ether was distilled off to obtain 20 mg of a transparent syrup. When the syrup was analyzed by gas chromatography and mass spectrometry, the product contained in the syrup was identified as 2,5-dimethylhexanoic acid. Furthermore, the purity of the product was calculated to be 96.5% based on the area ratio of the product peak measured by gas chromatography analysis under the following conditions. The product, 2,5-dimethylhexanoic acid, was confirmed to be optically active, the measured optical rotation showed a negative value, and its optical purity was calculated to be 74% ee.

Example 2 (NH ₄ ) ₂ HPO ₄ 4.0 g, Na ₂ HPO ₄
12H ₂ O 2.5 g, KH ₂ PO ₄ 2.0 g, MgSO ₄ 7H ₂ O 30 mg, C
A medium in which 60 mg of aCl ₂ · 2H ₂ O and 200 mg of yeast extract are dissolved in 1 liter of deionized water, and the pH is adjusted to 7.2 (the same medium as in Example 1 except that glucose is removed). Add 20 ml to a 500 ml Sakaguchi flask and
Sterilization was carried out in an autoclave at 21 ° C for 15 minutes. Then, NAG (Nutrient Aga manufactured by Oxoid Co., Ltd. was previously added to this medium.
1% glucose added to r) medium at 30 ° C, 7
Inoculate 3 platinum loops of cells of Mycobacterium sp. D84-1 (Deposit No. FERM P-13657, Institute of Biotechnology, Institute of Biotechnology, Industrial Technology Institute) plated for 2 hours, and inoculate 1% of 2,5-dimethylhexane
After the addition, shaking culture was performed at 30 ° C. for 5 days. The obtained cultured bacterial cells were washed with 100 mM phosphate buffer (pH 7.2) and the same reaction medium as in Example 1, and then suspended again in 5 ml of reaction medium to prepare a bacterial suspension.

50 μl of 2,5-dimethylhexane was added to 5 ml of the bacterial suspension, which was sealed and shake-cultured at 30 ° C. for 24 hours. From the culture broth obtained as described above, a solvent was extracted by the same operation as in Example 1, and the solvent diethyl ether was further distilled off to obtain a transparent syrup. From the gas chromatographic analysis and the mass spectrometry spectrum of the syrup, the obtained product was the same product as in Example 1 and was identified as 2,5-dimethylhexanoic acid. After methyl esterifying the product, gas chromatography analysis using a column for optical resolution (Chiraldex GT-A manufactured by Tokyo Kasei) was performed, and two peaks were recognized as the product. The mass spectrometric spectra were the same and were identified as methyl 2,5-dimethylhexanoate. The product peak area ratio is 92.5: 7.5,
From this, the optical purity was calculated to be 85% ee.

(Example 3) Using the bacterial suspension of Rhodococcus sp. 11B strain prepared in the same manner as in Example 1, cumene was added as a substrate and the same culture as in Example 2 was performed. The cells were removed from the culture solution obtained above by centrifugation. The obtained residual liquid was acidified with hydrochloric acid, and solvent extraction was performed with an equal amount of diethyl ether. Then, the diethyl ether layer after extraction was separated, dehydrated with sodium sulfate, and then the solvent diethyl ether was distilled off. Gas chromatographic analysis of the obtained residue revealed that α
Two peaks were observed which were consistent with the standards for -phenylpropanol and α-phenylpropionic acid. Also,
It was confirmed that the GC-MS spectrum of this peak also matched the spectrum of the standard product. Gas chromatographic analysis by the internal standard method showed that α-phenylpropanol (2-phenyl-1-propanol) was 407 mg / l and α-phenylpropionic acid (2-phenylpropionic acid) was 677 mg / l in the culture solution. It was measured that they were produced at the respective concentrations. Α-Phenylpropionic acid contained in this residue was methyl esterified by a conventional method, and then gas chromatographic analysis was performed using a column for optical resolution.
The ratio of R-form and S-form of methyl phenylpropionate is 12:88, from which the optical purity is 76% ee.
Was calculated. In addition, the separated α-phenylpropanol also has optical activity, and the ratio of its R-form and S-form is 7
It was analyzed as 3:27, from which the optical purity was calculated to be 46% ee.

(Example 4) Using the bacterial suspension of Rhodococcus sp. 11B strain prepared in the same manner as in Example 1, diisopropyl sulfide was added as a substrate and the same culture as in Example 1 was carried out. The cells were removed from the culture solution obtained above by centrifugation. The obtained residual liquid was acidified with hydrochloric acid, and solvent extraction was performed with an equal amount of diethyl ether. Then, the extracted diethyl ether layer was separated, dehydrated with sodium sulfate, and then the solvent diethyl ether was distilled off to obtain 270 mg of a transparent syrup. The purity of the product contained in this syrup was 98.5% by gas chromatography analysis. Further, the product contained in this syrup was identified as isopropylthioisopropionic acid from the GC-MS spectrum and the IR spectrum. When this syrup was made into a diastereomer using (S)-(-) phenylethylamine and the ratio was analyzed by gas chromatography, the ratio of diastereomers was 28.5: 71.5.

(Example 5) By the same method as in Example 2, Mycobacterium sp. D59-1 strain and Mycobacterium s
Each of p. D28-1 strain bacterial suspensions was prepared. Using the bacterial suspension, 2,5-dimethylhexane was added as a substrate and the same culture and post-treatment as in Example 2 were performed. D59-1
The mass spectrometric spectra of the respective products obtained using the bacterial suspension of the strain and the bacterial suspension of the D28-1 strain were both the same as those of 2,5-dimethylhexanoic acid. Further, after subjecting these to methyl esterification, gas chromatographic analysis was carried out using an optical resolution column in the same manner as in Example 2. As a result, the area ratio of optical isomer peaks was D59-1 strain. In this case, the optical purity was 90.1: 9.9, and the optical purity was calculated to be 80.2% ee. When the D28-1 strain was used, the optical purity was 91.3: 8.7, and the optical purity was 8
It was calculated to be 2.6% ee.

Example 6 As a growth medium, (NH ₄ ) ₂ HPO _{4 was used.
4.0g, Na 2 HPO 4 · 12H} 2 O2.5g, KH 2 PO 4 2.0g, MgSO 4 · 7
H ₂ O 30 mg, CaCl ₂ · 2H ₂ O 60 mg, and yeast extract 200 m
It has a composition in which g is dissolved in 1 liter of deionized water, and the pH is 7.
A medium adjusted to 2 was prepared. 5 ml of the growth medium with an inner diameter of 2
Dispense into a 4 mm test tube and place in an autoclave at 121 ° C.
Then, it was sterilized for 20 minutes. 1 of Rhodococcus sp. 11B (Deposit No. FERM P-13654, Institute of Biotechnology, Institute of Biotechnology, AIST)
The amount of platinum loop was inoculated into the growth medium prepared and sterilized as described above, 500 μl of 2,5-dimethylhexane was added, the container was sealed with a rubber stopper, and the strain was cultured at 30 ° C. for 3 days with shaking to culture the bacterium. Was used as the inoculum solution. Next, 50 ml of the above-mentioned growth medium was dispensed into a 500 ml Sakaguchi flask, 0.4% (v / v) of the inoculum solution was inoculated to the sterilized product, and 500 μl of 2,5-dimethylhexane was further added. After the addition, the flask was tightly closed with a rubber stopper, and cultured at 30 ° C. for 7 days with shaking. The cells produced by the culture 100mM phosphate buffer (pH 7.2) and reaction medium _{(K 2 HPO 4 1.74g, MgSO} 4 · 7H
₂ O 1.50 g, FeSO ₄ .7H ₂ O 50 mg dissolved in 1 l of pure water, and further washed with a medium having a pH adjusted to 8.0) and then resuspended in 5 ml of reaction medium, A bacterial suspension was prepared.

5 ml of the bacterial suspension was placed in a test tube having an inner diameter of 24 mm, and 25 μl of racemic α-phenylpropanol (2-phenyl-1-propanol) was added as a substrate.
Then, the flask was stoppered tightly and cultured with shaking at 30 ° C. for 20 hours. The cells were removed from the culture solution obtained above by centrifugation. The resulting residual liquid is acidified with hydrochloric acid,
The solvent was extracted twice with an equal amount of diethyl ether.
Then, the extracted diethyl ether layer was separated, dehydrated with sodium sulfate, and then the solvent diethyl ether was distilled off to obtain a transparent syrup. When the syrup was analyzed by a gas chromatography method, two peaks corresponding to the standard products of α-phenylpropanol and α-phenylpropionic acid were observed. It was also confirmed that the GC-MS spectrum of this peak also matched the spectrum of the standard product. From the gas chromatography analysis by the internal standard method, α-phenylpropanol (2-phenyl-1-propanol) remained at a concentration of 3.56 g / l in the culture broth, and α-phenylpropionic acid ( 2-phenylpropionic acid) is 0.77 g / l
It was measured to be produced at a concentration of.

After α-phenylpropionic acid contained in this syrup is methyl-esterified by a conventional method,
Optical resolution column (Astec, Chiraldex GT
Gas chromatographic analysis according to A) revealed that the optical purity (absolute configuration) of α-phenylpropionic acid was found to be 50.4% ee from the area ratio of the R and S peaks of α-phenylpropionic acid methyl ester. It was calculated as (S body). On the other hand, when gas chromatographic analysis was performed using a column for optical resolution, α
-The optical purity (absolute configuration) of phenylpropanol is 1
It was calculated to be 9.5% ee (R form). From the transparent syrup in which α-phenylpropionic acid and α-phenylpropanol are mixed, a weakly alkaline (pH about 10) alkaline aqueous solution is added to the syrup to dissolve α-phenylpropionic acid in the aqueous solution. Then, extract only α-phenylpropanol with diethyl ether as a solvent, then make the remaining aqueous solution acidic (about pH 2), and then extract only α-phenylpropanol with diethyl ether as a solvent. Can be separated.

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Claims (3)

[Claims] 1. The following general formula 1 (In the formula, R ₁ represents a substituent selected from the group consisting of an alkyl group, an aromatic ring, a heterocycle, and a sulfur-containing alkyl group,
m and n are 0 or 1) is oxidized by a microorganism belonging to the genus Rhodococcus or Mycobacterium and capable of converting the compound into a carboxylic acid to give a compound represented by the general formula 2 ] (Wherein R ₁ , m, and n are the same as the compound represented by the above general formula 1, C ^* represents a carbon atom to which optical activity is imparted), and the optically active carboxylic acid is separated. A method for producing an optically active carboxylic acid, characterized by being obtained. 2. The following general formula 3 (In the formula, R ₂ represents a substituent selected from the group consisting of an alkyl group having 2 or more carbon atoms, an aromatic ring, a heterocycle, and a sulfur-containing alkyl group, and p and q are 0 or 1) The resulting isoalkanol is oxidized by a microorganism belonging to the genus Rhodococcus or the genus Mycobacterium and capable of converting the isoalkanol into a carboxylic acid. (Wherein R ₂ and p and q are the same as the isoalkanol represented by the above general formula 3, C ^* represents a carbon atom to which optical activity is imparted), and the optically active isocarboxylic acid. A method for producing an optically active isocarboxylic acid, which comprises: 3. The following general formula 5: (In the formula, R ₃ represents a substituent selected from the group consisting of an alkyl group having 2 or more carbon atoms, an aromatic ring, a heterocycle, and a sulfur-containing alkyl group, and s and t are 0 or 1) The racemic isoalkanol is oxidized with a microorganism belonging to the genus Rhodococcus or the genus Mycobacterium and having the ability to convert the isoalkanol into a carboxylic acid. (Wherein R ₃ and s and t are the same as the isoalkanol represented by the above general formula 5, C ^* represents a carbon atom to which optical activity is imparted), and the optically active isocarboxylic acid. And then isolating and obtaining the remaining optically active isoalkanol, the method for producing an optically active isoalkanol.