JPS5878593A - Preparation of d(-)-beta-hydroxyisobutyric acid - Google Patents

Abstract

PURPOSE:To prepare D(-)-beta-hydroxyisobutyric acid, by using a microorganism having the ability to convert isobutyl alcohol into the D(-)-beta-hydroxyisobutyric acid. CONSTITUTION:A microorganism, belonging to the genus Candida, Pichia, Torulopsis, Aspergillus, Choanephora, Wingea or Zygorhynchus, and having the ability to convert isobutyl alcohol into D(-)-beta-hydroxyisobutyric acid is cultivated by any one of the following methods: (A) The microorganism is cultivated in a culture medium containing the isobutyl alcohol under aerobic conditions. (B) The microorganism is cultivated in a culture medium under aerobic conditions, and the isobutyl alcohol is added to the resultant culture fluid and reacted therewith under aerobic conditions. (C) The microbial cell suspension separated from the culture fluid described above is reacted with the isobutyl alcohol under aerobic conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to optically active (-)-β- which is one of the raw materials useful for synthesizing various natural products having an optically active carbon skeleton or physiologically active substances such as pharmaceuticals. This invention relates to an industrially advantageous production method of hydroxyisobutyric acid using microorganisms.

Conventionally, as a method for preparing optically active (-)-β-hydroxyisobutyric acid, a method for optically resolving DL(±)-β-hydroxyisobutyric acid using a resolving agent such as optically active amines, and 2-methyl There are methods of asymmetric oxidation of =1.8-propanediol using acetic acid bacteria, but none of these methods can be carried out industrially from the viewpoint of yield and production cost.

Previously, the present inventors used isobutyric acid or methacrylic acid as raw materials to develop industrially advantageous (-)-β-
Established a method for producing hydroxyisobutyric acid (published in 1982)
6-8894, published in 1982-68895, etc.), but as a result of further research, D(-)-β was produced by microorganisms from isobutyl alcohol, which is cheaper than isobutyric acid and methacrylic acid.
- They discovered that hydroxyisobutyric acid can be produced and completed the present invention.

That is, the present invention uses inbutyl alcohol and D.
Dorsal Yandida, Pichia, Torulopsis, Aspergillus, Coanephora, which has the ability to convert into (-)-β-hydroxyisobutyric acid.

The present invention relates to a method for producing D(-) diβ-hydroxyisobutyric acid, which is characterized by allowing a microorganism belonging to the genus Wingea or the genus Chigorhynchus to act on the microorganism and collecting the produced D(-)-β-hydroxyisobutyric acid. .

Isobutyl alcohol used in the present invention D(-)-
Candida Rugosa is a microorganism that has the ability to convert into β-hydroxyisobutyric acid.
ugosa), candidaparapsio
dida parapsilosis), Pichjamembra aefaciens (Pichjamembra
naefacjens), Torulopsis candida, Aspergillus nig
er), Choanephora siluminans (Choaneph
oracircinans), fgo! Zygorhynchus moelleri
etc., and any nutrient source that can be assimilated by these bacteria can usually be used for these cultures. Carbohydrates such as glucose, sucrose, mannitol, etc. as carbon sources;
Alcohols including ethanol; hydrocarbons such as paraffin and olefins; organic acids such as acetic acid; soybean oil, etc. alone or in mixtures; nitrogen sources such as ammonium sulfate and ammonium phosphate; organic nutritional sources such as yeast extract and malt. It is possible to use a medium containing an appropriate mixture of nutrient sources used in normal culture, such as extracts, meat extracts, peptones, trace metal salts, vitamins, etc. As a method of culturing, the culture is carried out aerobically at a pH of 4.0 to 9.5 in a nutrient medium for 1 to 5 days at a temperature of 15 to 4'Oc.

A pH range of pH 6, Q to 9.5 is preferred for the conversion of isobutyl alcohol to D(-)-β-hydroxyisobutyric acid. In addition, as a method for causing microorganisms to act on inbutyl alcohol, a method that is carried out in parallel with the culture of microbial cells is, for example, an aerobic method in which isobutyl alcohol and the above carbon source coexist at a pH in the range of 4.0 to 9.5. There is a method of culturing the bacteria and accumulating D(-)-β-hydroxyisobutyric acid in the culture solution. A two-step method, for example, culturing bacterial cells aerobically in a nutrient medium at pH 4, Q to 9.5;
Isobutyl alcohol was added to the obtained culture solution to adjust the pH.
A method of maintaining 6.0 to 9.5 and reacting aerobically,
Alternatively, collect the bacterial cells from the obtained culture solution by centrifugation, etc., and use a solution with an appropriate composition of the bacterial cells, for example, M/15! , phosphate buffer (
pH 7.0), add isobutyl alcohol,
There is a method in which the reaction is carried out aerobically at pH 6 and in the range of θ to 9.5. In this case, bacterial cells that have been immobilized with sodium alginate or the like can also be used.

As a method for collecting D(-)-β-hydroxyinobutyric acid obtained by culture and reaction, a conventional extraction and purification method can be used. Lower the pH of the solution to around 2.0 using sulfuric acid or the like, and then add ammonium sulfate until it becomes saturated. After that,
Extraction is carried out 8 times with equal volumes of ethyl acetate. When this is removed under reduced pressure and the agent is removed, a substance containing D(-)-β-hydroxyisobutyric acid is obtained in the form of a brown oil. Furthermore, this product can be easily separated from impurities by dissolving it in a small amount of penzene and performing silica gel column chromatography with elution with a benzene-acetone mixed solvent.

In addition, the determination of the produced (-)-β-hydroxyisobutyric acid is as follows:
This can be easily carried out by gas chromatography using a ShiXzu PAL-MIO%/Simalite column (Hase et al., Journal of Fermentation Technology 59° 208 (1981)).

Next, the present invention will be explained with reference to Examples, but the present invention is not limited only to the Examples.

Example 1 2 glucose, 0.5% yeast extract, 0 peptone
．． 8%, meat extract 0.8t, isobutyl alcohol 0.
Candida Φ rugosa IFO 0750 ＼ Kiya / Deida balapsilosis IFO 0708, Pichia fistula aefaciens IAM 4904, Torulopsis candida IF in a medium (pH 7,0) containing 5.
0 0880, Aspergillus niger IAM2582
, Koane Robertzi IFO1277, Chigorincas
Moelleri HUT 1805, and 8 were cultured in a mini jar fermenter at 80° C., aeration at 1 vvm, and stirring at 500 rpm for 20 hours.

Thereafter, the pH was maintained at 7.0 and culture was continued for an additional 24 hours.

Table 1 shows the results of gas chromatography analysis of the content of D(-)-β-hydroxyisobutyric acid in the culture fluid thus obtained.

After removing the bacterial cells from the obtained culture solution by centrifugation, 200*/
The mixture was concentrated under reduced pressure, adjusted to pH 1, Q with sulfuric acid, and made into a saturated solution with ammonium sulfate. Next, the mixture was extracted with twice the amount of ethyl acetate, and the solvent was removed under reduced pressure at 50° C. or lower to obtain a yellow oily substance.

This oily substance was applied to a column prepared with benzene using 5 times the weight of silica gel (Wako Gel Q-50).

Then, D(-) was prepared using benzene:acetone (3:1) solvent.
-β-hydroxyinobutyric acid was eluted.

The obtained D'(-)-β-hydroxyisobutyric acid fractions were collected, the solvent was removed under reduced pressure, and the optical rotations were measured after dissolving in methanol. 18
．． Obtain the value of 0° (C=8, methanol) and D(-
) was confirmed to be β-hydroxyisobutyric acid.

Example 2 Glucose 2%, yeast extract 0.5%, peptone 0
．． 8%, meat extract 0.8% (pH 7.0)
) if Candeida Rugoza IPO0750'i
The cells were inoculated and cultured for 20 hours at 30° C., aeration at 1 vvm, and stirring at 500 rpm in an 8-inch mini-jar fermenter. Thereafter, the bacterial cells were collected by centrifugation, and a solution of M/15 phosphate buffer (p
H7.0), and the reaction was carried out for 24 hours under the same conditions as the culture while maintaining the pH at 7.0 with caustic soda. Thereafter, 10F isobutyl alcohol was further added and the reaction was carried out for 24 hours. As a result of gas chromatography analysis of the content of D(-)-β-hydroxyisobutyric acid in the culture solution obtained in this way, the content of D(-)-β-hydroxyisobutyric acid was 9.2 μ/d.
-)-β-Hydroxyisobutyric acid accumulation was observed. Furthermore, D(-)-β-hydroxyisobutyric acid was purified in the same manner as in Example 1, and the optical rotation was measured, and the result was [α]25--17.
7° (C = 8° methanol), and the β-
It was confirmed to be hydroxyisobutyric acid.

In this example, almost the same amount of D(-)-β-hydroxyisobutyric acid was accumulated even when inbutyl alcohol was directly added to the culture solution without separating the bacterial cells from the culture solution. was recognized.

Patent applicant Kanebuchi Chemical Industry Co., Ltd. Agent: Patent Attorney Makoto Asano -

Claims (5)

[Claims] (1) Add this to isobutyl alcohol and D(-)-
D(-)- produced by reacting microorganisms belonging to the genus Candida, Pichia, Torulopsis, Aspergillus, Coanephora, Wingea, or Tigorhynchus that have the ability to convert into β-human'roxyisobutyric acid.
D characterized by collecting β-hydroxyisobutyric acid
A method for producing (-)-β-hydroxyisobutyric acid. (2) Claim 1, in which the microorganism is Candida rugosa, Candida parapsilosis, Pichia menfranaefaciens, Torulopsis canteita, Aspergillus nica, Coanephora cirsinans, Wingea robertsii, or Chigorhynchus moellerii. Manufacturing method described. (3) The production method according to claim 1, in which the microorganisms are cultured in a medium to which isobutyl alcohol is added, thereby allowing the microorganisms to act on isobutyl alcohol. (4) The production according to claim 1, in which microbial cells are separated from a culture solution obtained by culturing microorganisms in a nutrient medium to prepare a cell suspension, and the suspension is reacted with isobutyl alcohol. Law. (5) The manufacturing method according to claim 1, in which isobutyl alcohol is added to a culture solution obtained by culturing microorganisms in a nutrient medium and acts thereon.