JP3146640B2 - Method for producing benzoylformic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing benzoylformic acid.

[0002] Benzoylformic acid is very useful as a raw material for optically active mandelic acid used in various pharmaceuticals.

[0003]

2. Description of the Related Art Conventionally, a method for producing a benzoyl formate by oxidizing a racemic mandelic ester by chemical means is known (JP-A-56-158733).
No.).

[0004]

However, a method for biochemically producing benzoylformic acid has not been found yet.
The present inventors have studied a method for efficiently producing benzoylformic acid using a microorganism under mild conditions.

[0005]

Means for Solving the Problems The present inventors have studied various methods for producing benzoylformic acid and found that mandelic acid, which can be synthesized at low cost by utilizing the oxidizing power of microorganisms, can be advantageously derived to benzoylformic acid. This led to the present invention. Accumulation of benzoylformic acid from mandelic acid using microorganisms has not heretofore been known and has not been carried out.

That is, the present invention has the ability to convert mandelic acid to benzoylformic acid and belongs to the genus Corynebacterium, Brevibacterium.
cterium), Amycolatopsis, Streptomyces, Rhodococcus (Rhococcus)
dococcus), Pseudomonas, Alcaligenes and Candida
da) A culture, a cell or a processed product of at least one microorganism selected from microorganisms belonging to the genus Gen. is allowed to act on mandelic acid to produce and accumulate benzoylformic acid, and benzoylformic acid is isolated and collected from the reaction solution A process for producing benzoylformic acid, characterized in that:

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

[0008] Mandelic acid used as a raw material in the present invention may be any of R-form, S-form and racemic-form. Usually, an industrially advantageous racemate is used.

[0009] In the present invention, it has the ability to convert mandelic acid to benzoylformic acid,
At least one microorganism selected from microorganisms belonging to the genus Brevibacterium, Amycolatopsis, Streptomyces, Rhodococcus, Pseudomonas, Alcaligenes and Candida is used.

[0010] Specific examples of such microorganisms include, for example, Corynebacterium glutamicum ATCC130.
32, Brevibacterium lactofermentum AT
CC13869, Amycolatopsis Sulfrear IFO
13270, Streptomyces binaseus IFO1
3425, Rhodococcus erythropolis IFO123
02, Pseudomonas fluorescens ATCC17
563, Alcaligenes faecalis IAM147
3, Candida parapsilosis ATCC 7330, and the like.

For cultivation of these microorganisms, a culture medium appropriately containing an organic and inorganic carbon source, nitrogen source, vitamins, minerals and the like which can be assimilated by these bacteria is usually used.
Usually, the pH of the medium is preferably pH 5 to 9. The temperature is usually 20 to 40 ° C., and the bacterium may be cultured aerobically for 1 to 7 days.

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 culturing cells obtained by centrifugation, and the processed cell is obtained by sonicating the cells obtained by culturing, for example, Examples include those immobilized on an acrylamide gel carrier by a known method.

The concentration of racemic mandelic acid, which is the reaction gas, in the reaction solution can usually be used in the range of about 0.1 to 5%. Regarding the method of addition, either batch or divided addition may be used.

[0014] The reaction temperature is usually 20 to 40 ° C, preferably 25 to 35 ° C. The pH of the reaction solution is usually 5-1.
1.0, preferably 7.5 to 9.0. 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 method in which a substrate is added to a culture termination solution and aerobically shaken, and a method in which a substrate is added to a cell suspension or a processed cell and aerobicly shaken. There is a law and both can be adopted, but the latter gives better results.

Thus, mandelic acid is oxidized by the reaction of the present invention to produce benzoylformic acid.

In order to isolate the benzoylformic acid thus produced from the reaction solution, a general separation and purification method may be used. For example, a target substance can be isolated and collected, such as a method of separating using a preparative liquid chromatography column or a method of isolating phenylhydrazone.

When the benzoylformic acid thus produced is reduced using a known benzoylformate-reducing bacterium or the like, optically active mandelic acid is efficiently produced.

[0019]

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

The amount of benzoylformic acid produced in the examples was analyzed by high performance liquid chromatography (HPLC) using an ODS column.

In the examples, BF represents benzoylformic acid, and the yield is represented by mol% of benzoylformic acid formed with respect to the reduced substrate.

Example 1 A liquid medium consisting of sucrose 2%, polypeptone 4%, yeast extract 0.5% and potassium dihydrogen phosphate 0.5% was adjusted to pH 7.0 with an aqueous sodium hydroxide solution, and 5 ml each was placed in an 18 mmφ test tube. Dispense and place in an autoclave at 120 ° C for 2 hours.
Heat sterilized for 0 minutes. One platinum loop was inoculated with each of the various bacteria shown in Table 1 from the slant culture medium and cultured aerobically on a shaker at 28 ° C. for 63 hours. Thereafter, the cells were separated by centrifugation and washed once with water to prepare the cells, and the obtained cells were added to an 18 mmφ test tube containing 5 ml of a 10 g / l racemic mandelic acid aqueous solution. PH at 28 ° C for 40 hours
The mixture was shaken at 7.0 to react. The reaction solution thus obtained was centrifuged, and the supernatant was analyzed by HPLC. The results are shown in Table 1.

[0023]

[Table 1]

Example 2 Candida parapsilosis ATC using a liquid medium consisting of 4% glucose and 5% corn steep liquor
Except for using C7330, the same procedures as in Example 1 were carried out. As a result, 2.3 g / l of BF was accumulated, and the yield was 67.4.
%Met.

Example 3 100 ml of the same liquid medium as in Example 1 was placed in a 500 ml Sakaguchi flask, and sterilized by heating at 120 ° C. for 20 minutes in an autoclave. Here, Amycolatopsis
Inoculate one platinum loop of Sulfur IFO 13270, 28 ° C
For 63 hours on a shaker. Thereafter, the cells were separated by centrifugation, washed once with water to prepare the cells, and the obtained cells were added to 100 ml of a 20 g / l racemic mandelic acid aqueous solution, and the pH was adjusted to 7 at 30 ° C. for 25 hours. .
The sample was shaken at 5 to react. This reaction solution was centrifuged, and the supernatant from which the cells had been removed was subjected to HPLC (column: SKge / ODS1).
20A (55 mm x 60 cm) to obtain 680 mg of the desired benzoylformic acid (isolation yield: 72.1%).

Reference Example (combination with known reducing bacteria) Pseudomonas fluorescens ATCC in Example 1
Using 17563, benzoylformic acid was produced in the same manner except that S-mandelic acid was used as a substrate, and the supernatant was used as a substrate for the next reduction reaction.

As a known reducing bacterium, Rhodotorula minuta A
Using TCC10658, a liquid medium consisting of 4% of glucose and 5% of corn steep liquor was adjusted to pH 6.0 with an aqueous solution of caustic soda, placed in an 18-mmφ test tube in a volume of 5 ml, and sterilized by heating at 120 ° C. for 20 minutes in an autoclave. One platinum loop was inoculated and shake-cultured at 28 ° C. for 63 hours.
Then, cells were separated by centrifugation, washed once with water,
The cells were prepared.

Next, the reduced bacteria thus prepared were added to 3 ml of the supernatant containing benzoylformic acid obtained by oxidizing the above S-mandelic acid, and glucose was further added to give a concentration of 3%. The reaction was carried out by aerobically shaking at 45 ° C for 45 hours.

The reaction solution obtained in this manner is converted to HPL
As a result of analysis in C, 8.5 g / l of mandelic acid was accumulated,
The reaction yield was 91%, and the optical purity of R-mandelic acid was 9
It was 5.3%.

[0030]

According to the present invention, benzoylformic acid can be efficiently produced from mandelic acid under mild conditions by an oxidation reaction using a microorganism.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI (C12P 7/40 C12R 1: 465) (C12P 7/40 C12R 1: 465) (C12P 7/40 C12R 1:39) (C12P 7/40 C12R 1:05) (C12P 7/40 C12R 1:72)

Claims (1)

(57) [Claims] The present invention has the ability to convert mandelic acid to benzoylformic acid and has the ability to convert corynebacterium (Corynebacteri).
um), Brevibacterium, Amycolatopsis, Streptomyces
(Streptomyces), Rhodococcus, Pseudomonas, Alcaligenes (Alcal
genes) and at least one microorganism selected from microorganisms belonging to the genus Candida and the genus of the genus Candida. A method for producing benzoylformic acid, comprising isolating and collecting benzoylformic acid from benzene.