JPH03277292A - Production of optically active 2-hydroxycarboxylic acid - Google Patents

Abstract

PURPOSE:To enable obtaining of a high optical purity and rate of reaction at a low cost by reacting a racemic 2-hydroxynitrile with a microorganism of the genus Bacillus, etc., at the prescribed rate of reaction. CONSTITUTION:A racemic 2-hydroxynitrile (A) expressed by formula I [R is (un)substituted aryl or (un)substituted heterocyclic group] in an amount of 0.01-70wt.% is added to an aqueous medium to provide a raw material solution (B). On the other hand, a microorganism of the genus Alcaligenes, Acinetobacter or Bacillus is cultured in a culture medium to afford cultured microbial cells (C). The resultant microbial cells (C) in an amount of 0.05-20wt.% are added to the solution (B) and reacted at pH4-11 and 5-80 deg.C for 1-100hr until the rate of reaction attains >=50% to 100%. Thereby, a reaction completed solution (D) is obtained. The prepared ingredient (D) is then filtered to remove undissolved substances such as micrcbial cells. Impurities are subsequently extracted and removed with benzene, etc., at pH about 8.5 and the obtained solution is extracted with chloroform, etc., at pH about 2 to afford an extract (E), which is then purified by column chromatography to produce the objective optically active 2-hydroxycarboxylic acid expressed by formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing optically active 2-hydroxycarboxylic acids.

The optically active 2-hydroxycarboxylic acid obtained by the method of the present invention can be used as a raw material for pharmaceuticals such as antibiotics or sympathomimetic agents, as a raw material for agricultural chemicals, as a raw material for compounds having superdielectric properties,
Furthermore, it is a compound useful as an optical resolution agent.

(Prior art) Known methods for producing optically active 2-hydroxycarboxylic acids include optical resolution using fractional crystallization of a racemate, optical resolution using chromatography, and organic chemical asymmetric synthesis. However, these methods are complicated to operate and
It has disadvantages such as low yield and low purity of the product.

On the other hand, as for the method using microorganisms, a method using reductase (Japanese Patent Application Laid-Open No. 63-32492) is known;
It has the disadvantage of requiring expensive coenzymes. moreover,
Methods using the hydrolytic action of nitrile or amide are known (Japanese Patent Application Laid-open No. 88894/1988, Japanese Patent Publication No. 14668/1983, and Japanese Patent Application No. 160653/1999). these are,
This is a method that specifically acts on one isomer of a racemic nitrile and does not act on the other optical isomer. Therefore, in these methods, when the objective is to obtain a product with high optical purity, the reaction rate with respect to the racemic raw material is stopped at 50% or less.

(Purpose a) In view of the above-mentioned circumstances, an object of the present invention is to obtain an optically active 2-hydroxycarboxylic acid useful as an industrial raw material from a corresponding racemic nitrile by microorganisms or microorganisms. ait
The objective is to obtain the compound with high optical purity and high reaction rate through the action of compound ii.

(Means for Solving the Problems) In order to solve the above problems, the present inventors have conducted extensive studies on a method for producing optically active 2-hydroxycarboxylic acids using microorganisms, and have found that We have discovered a microorganism that converts racemic 2-hydroxynitrile into optically active 2-hydroxycarboxylic acid represented by formula (n). Further investigation revealed that because 2-hydroxynitrile is in an equilibrium state with the corresponding aldehyde and hydrocyanic acid in an aqueous medium, it is possible to produce products with high optical purity even if the reaction rate to the raw material is 50% or more. They discovered this and completed the present invention.

That is, in the present invention, racemic 2-hydroxynitrile represented by the following general formula (I) is treated with a microorganism belonging to the genus Alcaligenes, Acinetobacter, or Bacillus or a preparation thereof in an aqueous medium to produce the following general 2-hydroxynitrile. formula(
When obtaining the optically active 2-hydroxycarboxylic acid represented by ff), the reaction rate with respect to the racemic raw material was
% or more of optically active 2-hydroxycarboxylic acid.

H R-C-CN (I) In the above formulas (I) and (II), R represents a substituted or unsubstituted aryl group or a substituted or unsubstituted heterocyclic group. More specifically, examples of the aryl group include a phenyl group and a naphthyl group. The heterocyclic group includes at least one of nitrogen, oxygen, and sulfur as a heteroatom.
Those containing one or more seeds are preferred, and the number of carbon atoms is preferably 3 to 13, more preferably 3 to 6. Also,
Examples of the substituent include a lower alkyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms, an aryl group such as a phenyl group or a naphthyl group, a lower alkoxy group having 1 to 6 carbon atoms, a hydroxy group, a thiol group, a nitro group, Amino groups or halogen atoms such as fluorine, chlorine, iodine, and bromine are preferred.

The microorganisms used in the present invention include Alcaligenes (
AJcaligenes), Acinetobacter (Ac
inetobacter genus, Bacillus
It is a microorganism selected from among microorganisms belonging to the genus U.S.

Specifically, the following microorganisms can be used.

Alcaligenes faecalis
faecalis) ATCC8750, Acinetobacter sp.
A K 226 (FERM BP-2451), Bacillus subtilis
is) CN 5 (FE RMBP-2354)
.

Acinetobacter sp. AK 226 has already been isolated to produce unsaturated organic acids such as acrylic acid or methacrylic acid from the corresponding nitrile compounds (Japanese Patent Publication No. 632,596). This strain has been internationally deposited with the National Institute of Microbial Technology under the above number.

Bacillus subtilis CN 5 was isolated from soil as a nitrile-assimilating bacterium (Patent application No. 1-160
653), which has been internationally deposited with the National Institute of Microbial Technology under the above number.

The mycological properties of Acinetobacter sp. AK 226 and Bacillus subtilis CN 5 are as shown below.

The above mycological properties are summarized in Bergy's Bacteria Classification Book.
s Manual of Determin
amive BacLeriology 8th Edition (I
974)), and “Manual of Clinical Microbiology (Phylum anua 1 of C11)
nical Microbiology) 4th edition (I9
1985)].

Strain CN5 is primarily a Durham-positive bacillus and forms spores. Furthermore, it is clear that it belongs to the Bac i II Iaceae family because it has epiphytic flagella and is motile. Furthermore, the CN5 strain is aerobic and positive for catalase, so it belongs to the genus Bacillus. In addition, it does not produce gas than glucose, hydrolyzes starch, has a positive VP test, has nitrate reducing ability, grows at 50°C, and has 7% Na
This bacterium is Bacillus because it grows in CR-containing meat juice and can utilize citric acid in Coser citric acid medium.
It was identified as s 5ubtiliS.

The reaction method in the present invention is carried out by contacting a microorganism or a preparation thereof with a racemic nitrile represented by formula (I) in an aqueous medium.

Specifically, microorganisms or preparations thereof include cultures in which the microorganisms are cultured, microbial cells collected from the microorganisms, or processed microbial cells (for example, crushed microbial cells or enzymes isolated and extracted from the microbial cells), Furthermore, it refers to cells or treated cells that are immobilized on a carrier by an appropriate method.

The microorganisms used in the present invention can be cultured according to known methods. The medium used can be one known as a nutrient source for general microorganisms, including carbon sources such as glucose, glycerin, ethanol, sucrose, glutamic acid, acetic acid, and citric acid, nitrogen sources such as ammonium sulfate, ammonium chloride, and ammonia, and yeast. Organic nutrient sources such as extract, malt extract, peptone, and meat extract, and inorganic nutrient sources such as phosphoric acid, magnesium, potassium, iron, manganese, and lanthanum can be used in combination as appropriate. In addition, n-
A cyano compound such as butyronitrile or an amide compound such as caprolactam may be added. The pH of the medium is 5
You can choose between ~10 and the culture temperature is 18~50°C.
1 Preferably 25 to 40°C. The number of culture days is 0.5~
The culture may be carried out until the activity reaches its maximum within 10 days.

The reaction conditions in the present invention will be explained next. The aqueous medium used in the present invention is an aqueous medium such as water, a buffer solution or a culture solution. Furthermore, a homogeneous mixed medium consisting of an aqueous medium and a water-soluble organic solvent, or a two-phase system of an aqueous medium and an organic solvent can also be used, and these media are also within the scope of the aqueous medium in the present invention. Any organic solvent may be used as long as it has a concentration that does not significantly inhibit the reaction.

The racemate represented by formula (I) is added to the aqueous medium in powder or liquid form, or dissolved in the above-mentioned organic solvent. The concentration of the racemate represented by formula (I) is 0.
．． It is about 0.01 to 70% by weight, preferably 0.1 to 40% by weight, and does not need to be completely dissolved in the aqueous solvent. When using bacterial cells in the reaction, the concentration of bacterial cells is usually 0.05 to
It may be in the range of 20% by weight. The reaction temperature is 5 to 80°C, preferably 15 to 60°C, and the reaction pH is 4 to 11, preferably 6 to 10. The reaction time usually ranges from 1 to 100 hours. The racemate represented by formula (I) that is consumed may be replenished continuously or intermittently so that the concentration in the reaction solution is maintained within the above range. The reaction may be carried out sufficiently until the reaction rate is 50% or more, and the reaction rate is 60-60%.
It is carried out until it reaches 100%, preferably 80-100%.

Furthermore, the nitrile represented by formula (I) is in an equilibrium state of formula (III) below in the reaction medium.

H R-C-CN→R-CHO+ HCN (III)
Therefore, instead of the hydroxynitrile represented by formula N), the aldehyde represented by (III) and hydrocyanic acid (which may be a salt such as sodium cyanide or potassium cyanide) can be reacted under the above-mentioned reaction conditions.

Recovery of the target product in the present invention is carried out as follows. After removing insoluble matter such as bacterial cells from the reaction completed solution,
After adjusting the pH to a weak alkali, preferably around 8.5, impurities are extracted and removed using a solvent such as n-butanol, benzene, diethyl ether, or chloroform.
The desired product is recovered by making the pH acidic (around 2) and extracting with a solvent such as n-butanol, benzene, diethyl ether, or chloroform. Furthermore, the target product is purified by column chromatography using silica gel, eluting with a suitable solvent such as a mixture of hexane, diethyl ether, chloroform, and methanol, or by precipitating it as crystals.

The reaction mechanism of the present invention is that nitrilase, which is an enzyme that converts nitriles into carboxylic acids, selectively acts on one isomer of racemic nitriles, that is, the reaction rate of the enzyme is significantly different from that of the optical isomer. It is thought that this is based on the fact that there is a large difference between Furthermore, the other nitrile isomer remaining unacted has the formula (I[[)
Through an equilibrium reaction as shown in the following, it is naturally racemized to become a racemic nitrile shown by the formula (I), and the anti-2 further progresses. As a result, due to this natural racemizing power, the reaction rate for the racemic raw material is generally only 50% (when aiming for 100% optical purity), which is contrary to common sense, and the reaction rate exceeds 50%. It is thought that this can be done.

(Example) Next, the present invention will be explained in more detail with reference to Examples. However, these Examples do not limit the scope of the present invention.

Example 1 Production of R-(-)-mandelic acid Ammonium acetate 1%, yeast extract 0.5%, peptone 0.5%, monopotassium phosphate 0.12%, monopotassium phosphate 0.08%, chloride Cultured in 100 d of sterilized medium containing 0.1% sodium, 0.02% magnesium sulfate, 0.003% ferrous sulfate, and 0.1% n-butyronitrile and adjusted to pH 7.2. 4% of Alcaligenes faecalis ATCC8750 was inoculated.

This was cultured with shaking at 32°C for 18 hours. After culturing, collect the bacterial cells (990 μ dry cells) by centrifugation, and
After washing twice with M phosphate buffer (pH 6,5),
．． It was suspended in 1M phosphate buffer (pH 8°0) 160+d. 560 i of anzolonitrile to 100 i of this suspension
(2) was added, and the mixture was reacted at 32°C for 4 hours with stirring.

Bacterial cells were removed from the reaction solution by centrifugation. pH of supernatant
After adjusting to 8.5, diethyl ether 10011d
, and the organic layer was extracted and removed. pH of water layer is 1
．． 5, 100 parts of diethyl ether was added and the target product was extracted twice. After drying the extract under reduced pressure, 70℃ benzene approximately 30 + tl! When it was dissolved in water and left at room temperature, R-(-
)-mandelic acid was obtained.

[α)D −153° (C=1. H2O) Melting point: 130-132°C From the specific optical rotation, the R-isomer content was 100% e, e.

This sample was sent to the Journal of Chron+ato
When high performance liquid chromatography analysis was performed according to the method of Graphy, 216406 (I981), the R-isomer content was 99.99% e,e or more.

Example 2 Production of R-(-)-mandelic acid 42.1 mM of manzolonitrile was added to 10 suspensions of Alcaligenes faecalis ATCC8750 obtained in the same manner as in Example 1! The reaction progress was observed. Reaction 0. 5. 1. The amount of mandelic acid produced after 23 hours was 7°7, 14.6, 31.2, and 3, respectively.
It was 8.3mM.

After 3 hours, the bacterial cells were removed by centrifugation, and then R-(-)-mandelic acid 56μ (reaction rate 87
．． 5%).

[α) o 153° (C=L H2O) melting point;
From the specific optical rotation at 131-132°C, the R-isomer content was 100% e, e.

Example 3 Preparation of R-(-)-mandelic acid Benzaldehyde and KCN were added at 42.1 mM to a suspension of Alcaligenes faecalis ATCC8750 obtained in the same manner as in Example 1.
However, the pH was adjusted to 8.0) and the progress of the reaction was observed. The amounts of mandelic acid produced after 1, 2, and 4.6 hours of reaction were 8, 115, 9, 27, 4, and 38.2 mM, respectively. After 6 hours, the bacterial cells were removed by centrifugation, and 51 ml of R-(-)-mandelic acid (reaction rate 79.7%) was obtained in the same manner as in Example 1.

[α) o-153° (C=1. H2O) melting point; 1
From the specific optical rotation at 30 to 131°C, the R-isomer content was 100% e, e.

Example 4 Culture conditions for ATCC8750 strain Medium 100 WIi! was prepared from the medium shown in Example 1 with various inducers added instead of n-butyronitrile. Then, approximately 4% of Alcaligenes faecalis ATCC8750, which had been cultured in advance in the same manner as in Example 1, was added. This is 18
The culture was incubated with shaking at 32°C for an hour. The amount of grown bacterial cells after culturing and the production activity of R-(-)-mandelic acid from manzolonitrile were as shown in Table IC. The activity was vaguely determined as follows. To a solution of 7.8 micromoles of manzolonitrile dissolved in 1 d of IMF Lis-HCl buffer (pH 9,0), the cells collected by centrifugation after culturing were added at an appropriate concentration, and the mixture was incubated at 30°C for 30 After reacting for a minute, the produced R-(-)-mandelic acid was quantified. Quantification is
It was performed by high performance liquid column chromatography. Specifically, 0.1 M diammonium phosphate (pH 5,0
) and methanol at a ratio of 8:2 at a rate of 1-
This was performed by eluting each sample separately and detecting at 254 nm. The activity was expressed as R-(-)-mandelic acid (r+mole) produced per 1 dry cell per minute.

Table Example 5 Process for producing R-(-)-mandelic acid using partially purified enzyme Example 1
Similarly, Alcaligenes faecalis ATCC87
50 was cultured for 500 days. The collected bacteria were treated with DTT (
dithiothreitol) 10IIIM in 0.03M potassium phosphate buffer (pH 6,5) 40-
Ultrasonication was performed at KHz for 7 minutes to disrupt the bacterial cells. The crushed bacterial cells were removed by centrifugation at 15,000×g for 20 minutes to obtain a cell-free extract. Add this to 1mM DTT
0.03M potassium phosphate buffer (pH 6,5
), the enzyme was passed through a DEAE-cellulose column, and trilase was added to the enzyme using a linear concentration gradient of 0.05M potassium phosphate buffer (pH 6.5) containing 0 to 0.5M sodium chloride and 1mM DTT. ) was eluted.

The active fraction was collected, ammonium sulfate was added to the mixture to give a concentration of 30%, and nitrilase was precipitated by centrifugation at 15,000×g for 20 minutes. The precipitate is In+
0.05M potassium phosphate buffer containing MDTT (pH
6,5) 2.511d, and dialyzed against the same buffer to partially purify the ammonium sulfate fraction. This purification process is shown in Table 2. The activity was measured in the same manner as in Example 4 except that the bacterial cells were replaced with enzymes. The unit of activity is expressed as the amount of enzyme that produces 1 μ5 ole of R-(-)-mandelic acid per minute.

Table ■, cell-free extract 31.8 24 0.0439 2.0 EAE-cellulose 20.3 76.1 0.267 5 units of enzyme solution obtained as above and manzolonitrile 5
0.1M potassium phosphate buffer (pH 8,0
)15〆 and reacted at 32°C for 6 hours. The product was purified in the same manner as in Example 1 to obtain 44 ml of R-(-)-mandelic acid. High performance liquid chromatography analysis revealed that the R-isomer content was 99.0%.

Example 6 Method for producing R-(-)-mandelic acid Similar to Example 1, Alcaligenes faecalis AT
CC8750 was cultured for 100m. The collected bacterial bodies are reduced to 0
．． After suspending in 10 d of 1M phosphate buffer (pH 8.0), a solution of 560 ml of manzolonitrile dissolved in 3 Mi of dimethyl sulfoxide was added, and the mixture was reacted at 32° C. for 4 hours with stirring. The product was purified in the same manner as in Example 1 to obtain 515 ml of R,-(-)-mandelic acid.

High performance liquid chromatography analysis revealed that the R-isomer content was 99.2%.

Example 7 Production of R-(-)-mandelic acid Ammonium acetate 1%, yeast extract 065%, peptone 0.5%, monopotassium phosphate 0.12%, dipotassium phosphate 0.08%, magnesium sulfate 0 .02%, ferrous sulfate 0.003%, sodium chloride O.1%, ε-caprolactam 0.1%, lanthanum chloride 0.15d,
Acinetobacter sp. AK was cultured in advance in sterile medium 100111 with a pH of 6.9.
226 was inoculated at 2% and cultured with shaking at 32°C for 18 hours.

After culturing, the bacterial cells were collected by centrifugation, washed twice with 0.0IM phosphate buffer (pH 6,5), and then diluted with 0.1M phosphate buffer (pH 6,5).
It was suspended in 20 d of phosphate buffer (pH 8,0).

Add 112 μm of manzolonitrile to this suspension and add 32 μm of manzolonitrile.
The reaction was allowed to proceed for 6 hours while stirring at °C. The product was purified in the same manner as in Example 1 to obtain 98 ml of R-(-)-mandelic acid. High performance liquid chromatography analysis revealed that the R content was 90%.

Example 8 Production method of R-(-)-mandelic acid Glucose 1%, yeast extract 0.5%, peptone 0.5
%, monopotassium phosphate 0.12%, dipotassium phosphate 0
．． 08%, magnesium sulfate 0゜02%, ferrous sulfate 0
．． Sterilizing medium 1 containing 0.003%, sodium chloride 0.1%, imbutyronitrile 0.15% and pH 7.2
00d! 4% of Bacillus subtilis CN 5, which had been previously cultured in the same medium, was inoculated and cultured with shaking at 32°C for 20 hours. After culturing, collect the bacterial cells by centrifugation,
After washing twice with 0.01M phosphate buffer (pH 7.0), 0.1M phosphate buffer (pH 8.0) 20-
suspended in. 112 μm of manzolonitrile was added to this suspension, and the mixture was reacted at 32° C. with stirring for 6 hours. The product was purified in the same manner as in Example 1 to obtain 106 ■ of R~(-)-mandelic acid. High performance liquid chromatography analysis revealed that the R content was 92%.

Example 9 Production of R-(-)-4-hydroxy-3-methoxymandelic acid Vanillin and KCN were added to 20 mM of a suspension of Alcaligenes faecalis ATCC8750 obtained in the same manner as in Example 1. (however, the pH was adjusted to 8.0), and the mixture was reacted at 30° C. for 5 hours with stirring. Bacterial cells were removed from the reaction solution by centrifugation. After adjusting the pH of the supernatant to 8.5, 10 ml of diethyl ether was added and the organic layer was extracted and removed. After adjusting the pH of the aqueous layer to 1.5, 10 ml of diethyl ether was added.
The target product was extracted twice. After drying the extract under reduced pressure, it was dissolved in approximately 2-2-benzene at 70°C and left at room temperature, resulting in 34.8■ (reaction yield 88%) of R-(-)-4-.
Hydroxy-3-methoxymandelic acid was obtained.

[α]D −130° (C=1.820) Melting point; 15
The specific optical rotation at 2° C. and the R-isomer content were 99.2% e,e.

Example 10 Production of (-)-2-thienylglycolic acid Alcaligenes faecalis ATCC8 obtained in the same manner as in Example 1
2-thiophene glycolonitrile (α-hydroxy-2-thiophene acetonitrile) was added to a 10-ml suspension of 750 ml and reacted at 30° C. for 15 hours with stirring. Bacterial cells were removed from the reaction solution by centrifugation. After adjusting the pH of the supernatant to 8.5, diethyl ether 10111 was added and the organic layer was extracted and removed. After adjusting the pH of the aqueous layer to 1.5, diethyl ether 10W11 was added to extract the target product twice. After drying the extract under reduced pressure, dissolve it in about 2 d of benzene and add 1
When left at 0°C, the reaction yield was 23.7cm (reaction yield 75%).
) of (-)2-thienylglycolic acid was obtained.

Cα) o 96.5° (C=I, H, O) melting point;
Based on the specific optical rotation at 83°C, the (-) body volume was 98.2% e, e.

(Effects of the Invention) By utilizing the present invention, an optically active 2-hydroxycarboxylic acid can be produced using a microorganism using an optically inactive substance as a raw material under reaction conditions at room temperature and normal pressure. Furthermore, according to the present invention, the reaction rate for raw materials can be reduced by 50%.
With the above, an optically active substance with high optical purity, for example, 90% e,e or more, preferably 98% e,e or more can be obtained. Furthermore, since the reaction rate is actually close to 100%, there is no need to recover and reuse raw materials. The above points are extremely advantageous economically.

Although the present invention has been described in detail and particularly with reference to embodiments thereof, various changes and modifications may be made thereto without departing from the spirit and scope of the invention. , should be obvious to those skilled in this technical field.

(Has power)

Claims (1)

[Scope of Claims] A racemic 2-hydroxynitrile represented by the following general formula (I) is reacted with a microorganism belonging to the genus Alcaligenes, Acinetobacter or Bacillus or a preparation thereof in an aqueous medium to form a racemic 2-hydroxynitrile represented by the following general formula (I). A method for producing an optically active 2-hydroxycarboxylic acid, which is characterized in that when obtaining the optically active 2-hydroxycarboxylic acid represented by (II), the reaction rate with respect to a racemic raw material is 50% or more. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R represents a substituted or unsubstituted aryl group or a substituted or unsubstituted heterocyclic group.)