JPH04218385A - Production of r(-)-mandelic acid - Google Patents

Abstract

PURPOSE:To efficiently obtain high-purity R(-)-mandelic acid by treating a specific bacterium with a mixture of R,S-mandelonitrile, etc., and prussic acid. CONSTITUTION:A bacterium [e.g. Pseudomonas sp. BC13-2 (FERM P-3,319)] (A) capable of stereo-selectively hydrolyzing nitrile group of R,S-mandelonitrile or a treated material (B) of the component A is obtained by selecting the bacterium from bacteria belonging to the genus Pseudomonas, Alcaligenes, etc. Then, a mixture (C) of 0.1-2.0wt.% mandelonitrile, 0.1-1.0wt.% benzaldehyde and 0.1-0.5wt.% prussic acid is reacted with 0.01-5.0wt.% based on mandelonitrile of dried cells of the component A or B at pH4-11.0 at 0-50 deg.C for 0.1-24 hours to give a reaction product (D). Then the component D is extracted and purified to directly produce a significant amount (50-100%) R(-)-mandelic acid.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for producing R(-)-mandelic acid. More specifically, using a microorganism that has the ability to asymmetrically hydrolyze mandelonitrile, R(-)
- A method for producing mandelic acid. R(-)-mandelic acid is industrially important as a raw material for the synthesis of cephem antibiotics and as a raw material for the synthesis of various pharmaceutical and agricultural products.

0002

[Prior art and its problems] R,S-mandelic acid (racemic form), which was chemically synthesized using a known method for producing R(-)-mandelic acid, was prepared by (1) racemic resolution by fractional crystallization [ See Japanese Patent Application Laid-Open No. 58-177933], (2
) Racemic resolution by chromatography [EP 98
707], (3) racemic ester and racemic resolution by enzymatic asymmetric hydrolysis [K. Mori
et al. , Tetrahedron 36,
91 (1980)],
(4) Chemical asymmetric synthesis method using chiral reagents [D.
A. Evans et al. , J. Am. C
hem. Soc. 107, 4346 (198
5) Reference] etc. In addition to the above-mentioned ester asymmetric hydrolysis method, biological methods include (5) Microbial asymmetric reduction of benzoylformic acid [JP-A-57-198
096], (6) Hydrolysis of R(-)-mandelonitrile asymmetrically synthesized by D-oxynitrilase [see JP-A-63-219388], (
7) Failure of racemic mandelonitrile or mandelamide by microorganisms of the genus Alcaligenes, Pseudomonas, Rhodopseudomonas, Corynebacterium, Acinetobacter, Bacillus, Mycobacterium, Rhodococcus or Candida. Method for producing R(-)-mandelic acid by simultaneous hydrolysis [JP-A-2-841
98] and the like are known.

However, any method based on racemic resolution complicates the process and reduces the yield at each step, and in the asymmetric synthesis method using a chiral reagent as a catalyst, the chiral reagent is expensive and However, there is a problem in that it is difficult to obtain products with high optical purity. In the biological method of asymmetric reduction of benzoylformic acid, the synthesis of the substrate and the NA
There are difficulties in maintaining the DH regeneration system, and sufficient research on industrialization of the D-oxynitrilase method has not yet been conducted. Regarding the production of R(-)-mandelic acid from racemic mandelonitrile or mandelamide, a predominant amount of the optically active form is not directly obtained from the racemic raw material;
The remaining unreacted nitrile or amide with the other optical activity is recovered and converted to the other optically active organic acid by hydrolysis with an acid, or made into a racemic form by alkali treatment, and used again as a raw material. and R(-)
-Used in the production of mandelic acid. Even in this method, the separation of the remaining optically active mandelonitrile or mandelamide and the process of racemization reaction by alkali treatment are complicated, resulting in a decrease in yield. As described above, the conventional methods include various problems, and it is difficult for any of them to be an industrially advantageous method for producing R(-)-mandelic acid.

0004

[Means for solving the problem] The present inventors have discovered that R(-
) - As a result of conducting studies aimed at developing an industrially advantageous production method for mandelic acid, we found that R,S-mandelonitrile or a mixed system of benzaldehyde and hydrocyanic acid was added to a near-neutral or basic aqueous medium. By treating the nitrile with a microorganism capable of hydrolyzing it, R can be quantitatively reduced.
, S-mandelonitrile, or benzaldehyde and hydrocyanic acid can be converted into R(-)-mandelic acid, and the present invention was completed.

[0005] That is, the present invention is directed to Pseudomonas (P.
seudomonas), Alcaligenes (Al-
caligenes), Acinetobacter (Acin)
Etobacter genus or Caseobacter (Ca
-seobacter) and has the ability to stereoselectively hydrolyze the nitrile group of R,S-mandelonitrile, or the treated product is treated with R,S - by acting on a mixture of mandelonitrile or benzaldehyde and hydrocyanic acid,
This is a method for producing R(-)-mandelic acid, which is characterized in that a predominant amount of R(-)-mandelic acid is directly produced from raw materials R,S-mandelonitrile or benzaldehyde and hydrocyanic acid.

[0006] The present invention, which has the gist of the above, is based on R
, S-mandelonitrile is easily racemized by taking advantage of the property that mandelonitrile is easily racemized by dissociative equilibrium between benzaldehyde and hydrocyanic acid in a near-neutral or basic aqueous medium. By conjugating the reaction system with a microorganism having asymmetric hydrolysis activity for mandelonitrile, R,S-mandelonitrile can be directly converted into R
This is based on the finding by the present inventors that it can be converted to R(-)-mandelic acid in a predominant manner.

[0007] The microorganisms used in the present invention include Pseudomonas genus, Alcaligenes genus, Acinetobacter (
R(-)- belonging to the genus Acinetobacter or the genus Caseobacter
Mandelic acid producing bacteria, specifically Pseudomonas sp. BC13-2 (Feikokenjoyori No. 3319)
, Pseudomonas sp. BC15-2 (Feikoken Article No. 3320), Alcaligenes sp. BC12
-2 (Feikoken Bacteria No. 11263), Alcaligenes sp. BC20 (Feikoken Bacteria No. 11264), Alcaligenes sp. BC35-2 (Feikoken Article No. 3318), Alcaligenes sp. BC24
(Feikoken Bacteria Serial No. 12063), Acinetobacter
sp. BC9-2 (Feikokenjoyori No. 3317),
Caseobacter sp. BC4 (Feikokenjoyori No. 331
No. 6) and Caseobacter sp. The bacterial strain BC23 (Feikoken Bacteria No. 11261) can be mentioned. All of these microorganisms were discovered by the present inventor and have been deposited with the above numbers at the Institute of Microbial Technology, Agency of Industrial Science and Technology, and their mycological properties are as shown below. .

[Table 1]

[Table 2]

The above mycological properties are summarized in Bergey's Manual of Systematic Bacteriology.
matic Bacteriology, 1986)
Classified according to BC13-2 and BC15-2
The strains are Pseudomonas spp., BC12-2, BC20, BC35-2 and BC2.
4 strains are Alcaligenes
Genus, BC9-2 strain is Acinetobacter (Acinetobacter).
strains BC4 and BC23 were identified as bacteria belonging to the genus Caseobacter.

Next, embodiments of the present invention will be explained. The microorganisms used in the present invention are cultured using assimilable carbon sources (glycerol, glucose, sucrose, casamino acids, meat extract, yeast extract, malt extract, lactose,
This is done using a normal medium containing a nitrogen source (such as yeast extract), an inorganic salt essential for each microorganism (such as magnesium chloride, sodium sulfate, calcium chloride, manganese sulfate, iron chloride, zinc sulfate, etc.), etc. be exposed.

Nitriles (cinnamic acid nitrile, benzyl cyanide, isobutyronitrile, β-phenylpropionitrile, benzonitrile, 2-cyanopyridine, 3-cyanopyridine) at a concentration that does not significantly inhibit growth during the early or middle stage of culture. , 4-cyanopyridine, phenylsulfonylacetonitrile, ε-caprolactam, γ-butyronitrile, N(2-cyanoethyl)morpholine, and amides (isobutyramide, 4-pyridinecarboxylic acid amide, phenylacetamide, etc.) increase enzyme activity. The pH of the culture solution is preferably in the range of 4 to 10, and the culture is carried out aerobically at a temperature of 20 to 50° C. for about 1 to 7 days.

Asymmetric hydrolysis of mandelonitrile is carried out using bacterial cells isolated from the culture solution of the microorganisms cultured as described above or a treated bacterial cell product (crushed bacterial cells after multiplication, dried bacterial cells, or separated and purified microorganisms). Mandelonitrile asymmetric hydrolase, etc.) may be suspended in water or a buffer solution, and mandelonitrile or benzaldehyde and hydrocyanic acid may be allowed to coexist in this suspension. In the present invention, in order to racemize mandelonitrile as described above, it is essential to maintain the system near neutrality or basicity during the reaction, and the pH is kept at 4 to 11, preferably 6 to 10. adjust. In addition, although the reaction conditions in the present invention cannot be absolutely specified due to the sensitivity of the enzyme to benzaldehyde and hydrocyanic acid, the amount of mandelonitrile in the reaction solution is usually 0.1 to 2.0% by weight, preferably 0.5% by weight. ~1.0 wt%, benzaldehyde 0.
1 to 1.0% by weight, preferably 0.1 to 0.5% by weight, and prussic acid 0.1 to 0.5% by weight, preferably 0
．． 1 to 0.2% by weight, and the amount of microorganisms used relative to mandelonitrile is 0.01 to 5.0% as dry bacterial cells.
% by weight, reaction temperature is 0-50°C, preferably 10-3
The reaction may be carried out at 0°C for 0.1 to 24 hours.

R(-)-mandelic acid produced by the hydrolysis reaction is subjected to known methods such as centrifugation to remove microorganisms, and if necessary, ultrafiltration or the like to remove granular components, proteins, and polysaccharide components. If necessary, after treatment with activated carbon, high purity crystals can be obtained by concentrating under reduced pressure or extracting with an organic solvent under acidic conditions, and repeating recrystallization using ethyl acetate etc. .

[0013]

[Effect of the invention] According to the present invention, the optical purity is approximately 10.
At 0% ee, the selectivity to R(-)-mandelic acid is almost quantitative, and the dominant amount (50 to 100 %) of R(-)-mandelic acid, and it is also possible to stoichiometrically convert all raw materials to R(-)-mandelic acid, making it an extremely efficient R(-)-mandelic acid.
A method for producing (-)-mandelic acid can be provided.

[0014]

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the scope of the present invention is not limited to the Examples.

Example 1 (1) Culture The strains shown in Table 1 were cultured under the following conditions. i) Medium glycerol 2.0%
(W/V) Yeast extract
0.30% (W/V) monopotassium phosphate
0.68% (W/V) Disodium Phosphate
0.71% (W/V) Sodium Sulfate
0.28% (W/V) Magnesium chloride 0.04% (W/V) Calcium chloride 0.004% (W/V)
V) Manganese sulfate 4x10-4%
(W/V) Iron chloride 6
×10-5% (W/V) zinc sulfate
3 x 10-5% (W/V) agar
1.80% (W/V)
Benzyl cyanide 0.02% (W
/V) pH
7.5ii) Culture conditions Culture was carried out at 30° C. for 72 hours in the above plate medium.

(2) Asymmetric hydrolysis of mandelonitrile Bacterial cells were collected from the plate culture medium and diluted with 50mM phosphate buffer (pH
7.5) and suspended in 10 ml of the same phosphate buffer to prepare a resting cell reaction solution with a cell concentration such that OD630 = 1 to 50. Add mandelonitrile to this liquid
．． 2% (W/V) and 16-2% at 30°C.
The reaction was carried out for 4 hours. When the reaction completed liquid was analyzed by liquid chromatography (Shiseido ODS column), it was found that mandelic acid and ammonia were produced. In addition, a chiral cell for optical resolution (CHIRAL)
When the optical purity was analyzed using PAK WH column), it was found that R(-)-mandelic acid with high optical purity was produced. The results are shown in Table 1.

[Table 3]

Example 2 (1) Cultured Alkalinenes sp. The BC12-2 strain was cultivated under the following conditions. i) Medium (A medium) Glycerol 2.0%
(W/V) Yeast extract
0.30% (W/V) monopotassium phosphate
0.68% (W/V) Disodium Phosphate
0.71% (W/V) Sodium Sulfate
0.28% (W/V) Magnesium chloride 0.04% (W/V) Calcium chloride 0.004% (W/V)
V) Manganese sulfate 4x10-4%
(W/V) Iron chloride 6
×10-5% (W/V) zinc sulfate
3 x 10-5% (W/V) pH
7.5 (Medium B) Medium B is made by adding 0.02% (W/V) benzyl cyanide to medium A. ii) Culture conditions After culturing in medium A at 30°C for 72 hours, the obtained The cells were further cultured in medium B at 30°C for 48 hours.

(2) Asymmetric hydrolysis of mandelonitrile Bacterial cells were separated from the resulting culture solution, washed with 50mM phosphate buffer (pH 7.5), and washed with 100mM of the same phosphate buffer.
1 to prepare a resting bacterial cell reaction solution (OD630
=50.48). Add 0.00 mandelonitrile to this solution.
2g was added and the reaction was carried out at 30°C. One hour after the start of the reaction, mandelonitrile completely disappeared,
R(-)-mandelic acid and ammonia were produced almost quantitatively. Further, 0.2 g of mandelonitrile was successively added every hour while the reaction continued for a total of 14 hours. Liquid chromatography (Shisei-do)
When analyzed using ODS column),
2.73% (W/V) (conversion yield; 88.93%) of R(-)-ammonium mandelate was accumulated 14 hours after the start of the reaction. After the reaction completed, the solution was sterilized by centrifugation, the pH was adjusted to 2 by acid treatment, and mandelic acid was extracted with ethyl acetate. The obtained mandelic acid solution was dried over anhydrous sodium sulfate, and then the organic solvent was distilled off to obtain crude crystals. The obtained crystals were recrystallized with ethyl acetate to obtain white powder crystals. The obtained crystals and standard mandelic acid were dissolved in 6N aqueous ammonia, and then dissolved at 1.0% (W
/V) was diluted with distilled water to obtain ammonium mandelate, and Table 2 shows the analysis results.

[Table 4]

Example 3 (1) Cultured Alcaligenes sp. Example 2 of BC35-2 strain
The cells were cultured under the same culture conditions. (2) Bacterial cells were obtained from the culture solution obtained by asymmetric hydrolysis of mandelonitrile in the same manner as in Example 1, and under the same reaction conditions as in Example 1 at a cell concentration of OD630 = 59.50. The reaction was started and an accumulation test was performed. After starting the reaction 1
By 4 hours, the substrate had completely disappeared, and after the start of the reaction, 42
The ammonium mandelate content in the reaction solution was 3.7 hours.
It was accumulated to 9% (W/V) (conversion yield; 89.09%). Thereafter, the same operations as in Example 2 were performed, and the results shown in Table 3 were obtained.

[Table 5]

Example 4 (1) Cultured Pseudomonas sp. Example 2 using BC12-2 strain
The cells were cultured under the same culture conditions. (2) Production of R(-)-mandelic acid from benzaldehyde and hydrocyanic acid From the obtained culture solution, bacterial cells were obtained in the same manner as in Example 1, and 100 m
1 to prepare a resting bacterial cell reaction solution (OD630
=50.5). Benzaldehyde and hydrocyanic acid were added to this solution to a final concentration of 15 mM each,
The reaction was started at 30°C. One hour after the start of the reaction, mandelonitrile and benzaldehyde produced by dissociation equilibrium completely disappeared, and R(-)-mandelic acid and ammonia were quantitatively produced. Further, when 15 mM each of benzaldehyde and hydrocyanic acid were added sequentially every hour, 2.6% (W/V) of R(-)-ammonium mandelate was accumulated 14 hours after the start of the reaction (conversion yield: 84.7 %). Further, when the same operation as in Example 2 was performed, the optical purity was 100%ee.

Claims (1)

[Claims] Belonging to the genus Pseudomonas, genus Alcaligenes, genus Acinetobacter or genus Caseobacter,
Microorganisms having the ability to stereoselectively hydrolyze the nitrile group of R,S-mandelonitrile or the treated product are mixed with R,S-mandelonitrile or benzaldehyde in a near neutral to basic aqueous medium. By acting on a mixture of hydrocyanic acid, a predominant amount of R(-) can be obtained directly from the raw material R,S-mandelonitrile or benzaldehyde and hydrocyanic acid.
-R(-) characterized by producing mandelic acid
- A method for producing mandelic acid.