JP3146641B2 - Method for producing optically active mandelic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing optically active mandelic acid.

[0002] optically active mandelic acid, a pharmaceutical raw material, such as penicillin or Sepharose porin antibiotics, yet is a useful compound as an optical resolving agent.

[0003]

2. Description of the Related Art As a method for producing optically active mandelic acid, an optical resolution method by fractional crystallization of a racemate, an optical resolution method by chromatography, an asymmetric organic chemical synthesis method and the like are known. As a method using microorganisms, a method of using a reductase (Kokoku 3 62393 JP), a method using a nitrilase (JP 3-2772
No. 92) is known. Also, a method utilizing the reducing power of microorganisms (Japanese Patent Publication No. 3-57752) has been proposed.

[0004]

However, methods such as the optical resolution method and the asymmetric synthesis method have drawbacks such as complicated operation and low optical purity, and the method using microorganisms has low yield and yield. It was not a satisfactory method in this respect.

[0005]

Means for Solving the Problems The present inventors have studied various methods for producing optically active mandelic acid and found that benzoylformic acid can be advantageously led to optically active mandelic acid by utilizing the reducing power of microorganisms. And found the present invention.

The present invention has the ability to convert benzoylformic acid to optically active mandelic acid, and has the ability to convert apiotricam.
chum), Candida, Torulopsis (T
orulopsis, Rhodotorula, Trichosporon, Rhodosporidium
soridium), Pichia, Kloec
genus kera), genus Hansenula, genus Saccharomyces, genus Klebsiella,
Rhodococcus sp., Microbacterium (M
icrobacterium, Corynebacteri
um), Brevibacterium, Arthrobacter, Enterobacter
genus, Micrococcus, Alcaligenes, Amycolatopsis
(Colatopsis) genus, Streptomyces (Streptomyces) genus and Nocardioides (Nocardioides) culture of at least one microorganism selected from microorganisms belonging to the genus,
A method for producing an optically active mandelic acid, which comprises reacting a cell or a treated product thereof with benzoylformic acid to generate and accumulate optically active mandelic acid, and isolating and collecting the optically active mandelic acid from a reaction solution. .

Hereinafter, the configuration of the present invention will be described in detail.

In the present invention, the compound has the ability to convert benzoylformic acid to optically active mandelic acid, and has the genus Apiotricum, Candida, Torulopsis, Rhodotorula,
Trichopolon, Rhodosporidium, Pichia, Cloeckella, Hansenula, Saccharomyces, Klebsiella, Rhodococcus, Micrococcus, Corynebacterium, Arthrobacter, Brevibacterium, Enterobacter, Miku loco Kkasu the genus,
At least one microorganism selected from microorganisms belonging to the genera Alcaligenes, Amycolatopsis, Streptomyces and Nocardioides is used.

Specific examples of such microorganisms include, for example, Apiotricum fumicola ATCC36992,
Candida Famata IFO0856, Candida Parapsilosis ATCC 7330, Torulopsis
Candida IFO0380, Rodorla Minuta AT
CC10658, Trichosporone ctaneum ATCC
36993, Rhodospolidium toluroides ATC
C10788, Pichia halofia ATCC 2424
0, Kloeckella magna IFO0868, Hansenula glucozaima ATCC 18938, Saccharomyces diastaticas IFO1439, Klebsiella
Oxytoka FERM P10097, Rhodococcus erythropolis ATCC 21035, Microbacterium ammoniaphyllum ATCC 15354, Brevibacterium lactofermentum ATCC 13869,
Corynebacterium glutamicum ATCC1303
2. Arthrobacter citreum ATCC 1162
4. Enterobacter aerogenes IFO1201
0, Micrococcus luteum IFO12708, Alcaligenes faecalis IAM1473, Amycolatopsis sulphurea ATCC 27624, Streptomyces binaseus IFO 13425, Nocardio albus ATCC 27980 and the like.

For cultivation of these microorganisms, a medium is used which is appropriately blended with an organic and inorganic carbon source, nitrogen source, vitamins, minerals, etc. which can be assimilated by these bacteria.
The pH of the medium varies slightly depending on the cells, and usually is p
H2 to 8 and pH 3 to 9 are usually preferable for bacteria. The temperature is usually 20 to 40 ° C., and the bacterium may be cultured aerobically or anaerobically for usually 4 to 20 days.

[0011] In the reaction of the present invention, cultures, cells or processed products of these microorganisms are used. Preferably, a cell suspension or a treated cell is used. The cell suspension referred to here is a cell obtained by centrifugation of the cell obtained by culturing, and the processed cell is a substance obtained by sonicating the cell obtained by culturing, For example, those immobilized on an acrylamide gel carrier by a known method may be mentioned.

An energy source is usually added to the reaction system of the present invention.

The energy source varies depending on the strain used, but generally, saccharides such as glucose, fructose, sucrose, glycerol and sorbitol are used.

The concentration of benzoylformic acid as a reaction substrate in a reaction solution can be usually about 0.1 to 5%. Regarding the method of addition, either batch or divided addition may be used.

[0015] The reaction temperature is usually from 20 to 40 ° C, preferably from 2 to 35 ° C. The pH of the reaction solution is usually 4.0 to 4.0.
8.5, preferably maintained between 6.0 and 8.0. The reaction time varies depending on the reaction temperature, but is usually 30 to 9 at 30 ° C.
0 hours.

As a reaction method, a substrate is added to the culture termination solution and shaken aerobically, or a saccharide is added as an energy source to a cell suspension or a processed cell, and then the substrate is added. There is a method of adding and shaking aerobically, and both can be adopted, but the latter gives better results.

Thus, by the reaction of the present invention, benzoylformic acid is asymmetrically reduced to produce optically active mandelic acid.

In order to isolate the thus produced optically active mandelic acid from the reaction solution, a general separation and purification method may be used. For example, after removing insoluble substances such as cells from the reaction solution by centrifugation, the pH of the reaction solution is adjusted to acidic, extracted with ethyl acetate, etc., dehydrated, and then dried to dryness or recrystallized under reduced pressure. Can be isolated and collected.

[0019]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

In the examples, the amount of mandelic acid produced and the ratio of R-form and S-form were determined by high performance liquid chromatography (H
PLC). The ODS column was used for the production amount analysis, and the ratio of the R-form and the S-form was determined by the Sumika Column SUMIPAK
A-3000 (Sumitomo Chemical Industries, Ltd.) was used.

In the examples, MA represents mandelic acid, the yield is represented by mol% of the produced optically active mandelic acid with respect to the reduced substrate, and R% represents the ratio of the (R) form in the produced mandelic acid. Express.

Example 1 Liquid consisting of sucrose 4%, polypeptone 2%, yeast extract 0.5%, potassium dihydrogen phosphate 0.5%
The culture land and pH7.0 with aqueous sodium hydroxide solution, poured into 18mmφ test tubes 5ml min at 120 ° C. in an autoclave,
Heat sterilization was performed for 20 minutes. Here, the various strains shown from a slant culture locations in Table 1, was inoculated one platinum loop and aerobically cultured for 48 hours on a shaker at 28 ° C.. Thereafter, the cells were separated by centrifugation and washed once with water to prepare the cells.
The cells were added to an 8 mmφ test tube, glucose was added so that the concentration became 5%, and benzoylformic acid was added so that the concentration became 1 wt%.
For 40 hours at pH 7.0.

The reaction solution thus obtained is subjected to HPLC
Table 1 shows the results of the analysis.

[0024]

[Table 1]

[0025] The same liquid culture ground as in Example 1, was dispensed 500ml into Sakaguchi flasks ten each 100ml min, the autoclave 120
Heat sterilized at ℃ for 20 minutes. Here in the Arthrobacter citreus ATCC11624 from a slant culture ground 1 platinum and ear inoculation, was incubated aerobically for 48 hours oice cormorant machine at 28 ℃. Then, cells of 1 liter of the culture solution were separated by centrifugation, washed once with water, adjusted, and added to a 5 liter Erlenmeyer flask, and glucose was further added to a concentration of 5%. Then, benzoylformic acid was added to a concentration of 0.5 wt%, and the mixture was aerobically shaken at a total volume of 500 ml for 80 hours at pH 7.0. Next, the cells were removed from the reaction solution by centrifugation, the reaction solution of 500 ml was concentrated to about 100 ml, the pH was adjusted to 2.0, and extraction was performed three times with 100 ml of ethyl acetate. After dehydration with, it is dried under reduced pressure and recrystallized with toluene.

(Equation 1) 1.77 g of (R) -mandelic acid having the formula:

[0026] Example 3 Glucose 5%, the liquid culture ground consisting of corn steep liquor 5% and pH6.0 with sodium hydroxide aqueous solution, 18Mmfai
Each 5 ml was dispensed into a test tube, and sterilized by heating at 120 ° C. for 20 minutes in an autoclave. Various strains shown here the slope culture locations in Table 1, was inoculated one platinum loop 63 hours at 28 ° C., and aerobically cultured on a shaker. Then, the cells were separated by centrifugation, washed once with water to adjust the cells,
Add the cells to an 18mmφ test tube, and then add 5%
And benzoylformic acid to a concentration of 1 wt%, to a total volume of 2 ml and 28 ° C.
For 17 hours at pH 7.0.

The reaction solution thus obtained was subjected to HPLC
Table 2 shows the results of the analysis.

[0028]

[Table 2]

Example 4 The same liquid medium as in Example 3 was added to 10 Sakaguchi flasks.
The mixture was dispensed in 00 ml portions and sterilized by heating at 120 ° C. for 20 minutes in an autoclave. Here in the candy parapucilosis (ATCC7330) from a slant culture areas 1 and platinum ear inoculation,
The cells were shaken at 28 ° C. for 63 hours and cultured aerobically. Then, cells of 1 liter of the culture solution were separated by centrifugation, washed once with water, adjusted, and added to a 5 liter Erlenmeyer flask, and glucose was further added to a concentration of 5%. Then, benzoylformic acid was added to a concentration of 1 wt%, and the total amount was 500 ml.
Shake aerobically at H7.0. Next, isolation was performed in the same manner as in Example 2, and the specific rotation was determined.

(Equation 2) 1.02 g of (R) -mandelic acid having the formula:

[0030]

According to the present invention, an optically active mandelic acid can be directly obtained from benzoylformic acid by an asymmetric reduction reaction using a microorganism, and can be industrially advantageously produced without complicated operations. Can be.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 7/42 C12R 1:84) (C12P 7/42 C12R 1:84) (C12P 7/42 C12R 1:78) (C12P 7/42 C12R 1:85) (C12P 7/42 C12R 1:01) (C12P 7/42 C12R 1:13) (C12P 7/42 C12R 1:15) (C12P 7/42 C12R 1:06) (C12P 7/42 C12R 1: 265) (C12P 7/42 C12R 1:05) (C12P 7/42 C12R 1: 465) (C12P 7/42 C12R 1: 365) (58) Field surveyed (Int. Cl. ⁷⁾ , DB name) C12P 7/42 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A method for converting benzoylformic acid to optically active mandelic acid, the method comprising the steps of:
Genus, Candida, Torulopsis
sis), Rhodotorula, Trichosporon
(Trichosporon), Rhodosporidium (Rhodosporidiu)
m) genus, Pichia genus, Kloeckera
Sp., Hansenula (Hansenula) spp., Saccharomyces (Sacc
haromyces, Klebsiella, Rhodococcus, Microbacterium
bacterium), Brevibacterium
Genus, Corynebacterium, Arthrobacter, Enterobacter
bacterium, Micrococcus, Alcaligenes, Amycola
topsis) genus, Streptomyces (Streptomyces) and Nocardioides (Nocardioides) at least one microorganism selected from microorganisms belonging to the genus, cultures or processed products thereof, benzoylformic acid, A method for producing optically active mandelic acid, comprising producing and accumulating active mandelic acid and isolating and collecting optically active mandelic acid from a reaction solution.