JPS6257312B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to optically active D(-)-, 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 β-hydroxyisobutyric acid using microorganisms. Conventionally, as a method for preparing optically active D(-)-β-hydroxyisobutyric acid, a method of optically resolving DL(±)-β-hydroxyisobutyric acid using a resolving agent such as optically active amines, 2- Methyl
There are methods for obtaining it from 1,3-propanediol by asymmetric oxidation using acetic acid bacteria, but none of these methods can be carried out industrially in terms of yield and manufacturing cost. The present inventors previously established an industrially advantageous method for producing D(-)-β-hydroxyisobutyric acid using microorganisms using isoenzymatic acid or methacrylic acid as raw materials (Japanese Patent Application Laid-Open No. 56-68394, (Sho 56-68395, etc.)
However, as a result of further research, it was discovered that D(-)-β-hydroxyisobutyric acid could be produced from isobutyraldehyde, isobutylamine, or isobutylamide through a microbial reaction.
The invention has been completed. That is, the present invention provides compounds of the genus Candeida, Pichia, Torulopsis, Aspergillus, Coanephora, which have the ability to convert isobutyraldehyde, isobutylamine, or isobutyramide into D(-)-β-hydroxyisobutyric acid.
D characterized in that the D(-)-β-hydroxyisobutyric acid produced is collected by allowing a microorganism belonging to the genus Wingea or the genus Chigorhynchus to act on it.
This is a method for producing (-)-β-hydroxyisobutyric acid. Isobutyraldehyde, isobutylamine or isobutylamide used in the present invention is
Microorganisms that have the ability to convert into (-)-β-hydroxyisobutyric acid include Candida rugosa, Candida parapsilosis, and Pichia membranefaciens.
membranaefaciens), Torulopsis candida, Aspergillus niger, Choanephora circinans, Zygorhynchus moelleri, etc. For these cultures, any nutrient source that can be assimilated by these bacteria can be used. For example, carbohydrates such as glucose, sucrose, and mannitol as carbon sources; alcohols such as ethanol and glycerol; hydrocarbons such as paraffins and olefins; organic acids such as acetic acid; soybean oil alone or a mixture thereof; ammonium sulfate as a nitrogen source; A medium can be used in which a nutrient source used in normal culture, such as ammonium phosphate, urea, yeast extract, malt extract, meat extract, peptone, etc. as an organic nutrient source, or trace metal salts, vitamins, etc., is appropriately mixed. The culture method is to adjust the pH of the nutrient medium to 4.0 to 9.5.
Cultivate aerobically at a temperature of 15 to 40°C for 1 to 5 days. A pH range of 6.0 to 9.5 is preferred for the conversion of isobutyraldehyde, isobutylamine or isobutyramide to D(-)-β-hydroxyisobutyric acid. In addition, as a method for making microorganisms act on isobutyraldehyde, isobutylamine, or isobutyramide, there is a method that is carried out in parallel with the culture of the microorganisms, such as a method in which isobutyraldehyde, isobutylamine, or isobutylamide is coexisted with the above nutrient source at PH4 There is a method of culturing aerobically in the range of .0 to 9.5 and accumulating D(-)-β-hydroxyisobutyric acid in the culture solution. From D(-)-
A method in which the conversion reaction to β-hydroxyisobutyric acid is carried out in two stages, for example, the production of bacterial cells is aerobically cultured in a nutrient medium at a pH range of 4.0 to 9.5, and the resulting culture solution is mixed with isobutyramide. Or add isobutylamine or isobutylamide to bring the pH to 6.0.
Collect the bacterial cells from the obtained culture solution by centrifugation, etc.
The bacterial cells are suspended in a solution with an appropriate composition, such as M/15 phosphate buffer (PH7.0), isobutyraldehyde, isobutylamine, or isobutylamide is added, and the reaction is carried out aerobically at a pH in the range of 6.0 to 9.5. There is a way to do this. In this case, bacterial cells that have been immobilized with sodium alginate or the like can also be used. Generally known extraction and purification methods can be used to collect D(-)-β-hydroxyisobutyric acid obtained through culture and reaction, but the D(-)-
Adjust the pH of the solution containing β-hydroxyisobutyric acid using sulfuric acid, etc.
Lower the temperature to around 2.0, and then add ammonium sulfate until it becomes saturated. Thereafter, extraction is carried out three times with equal volumes of ethyl acetate. When the solvent is removed from this under reduced pressure, a substance containing D(-)-β-hydroxyisobutyric acid is obtained in the form of a brown oil. Further, by dissolving this product in a small amount of benzene and performing silica gel column chromatography using a mixed solvent of benzene and acetone as elution, it can be easily separated from impurities. Furthermore, the amount of produced D(-)-β-hydroxyisobutyric acid can be easily determined by gas chromatography using a Shimadzu FAL-M10%/Simalite column [Hasegawa et al.; Journal of Fermentation Technology. , Vol. 59, p. 203 (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 Medium containing 2% glucose, 0.5% yeast extract, 0.3% peptone, and 0.3% meat extract (PH
7.0) Quillandida Rugoza IFO 0750 in 1,
Candida parapsilosis IFO 0708, Pichia membranefuaciens IAM4904, Torulopsis candida IFO 0380, Aspergillus niger IAM 2532, Coanephora cilcinanus HUT 1324, Wingea robertsii IFO
1277 and Tigorhynchus moellerii HUT 1305, and cultured in a 3-volume mini-jar fermenter at 30°C, aeration at 1 vvm, and stirring at 500 rpm for 24 hours. Then add 10% isobutyraldehyde to each culture.
g was added, and the reaction was carried out for 24 hours under the same conditions as culture while maintaining the pH at 7.0. Furthermore, 10 g of isobutylamine or isobutylamide was added to the culture solution of each bacteria that could be cultured in exactly the same manner, and the reaction was carried out for 24 hours under the same conditions as the culture while maintaining the pH at 7.0. D(-)-β- in the reaction solution thus obtained
Table 1 shows the results of gas chromatography analysis of the content of hydroxyisobutyric acid.

[Table] Regarding the display of strain names, IAM is located at the Useful Bacterial Strain Storage Facility, Institute of Applied Microbiology, The University of Tokyo (1-1-1 Yayoi, Bunkyo-ku, Tokyo), and HUT is located at the Strain Storage Facility, Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University (Tokyo). These strains are stored in Shimami, Saijo-cho, Hiroshima City. After removing the bacterial cells from the resulting culture solution by centrifugation,
It was concentrated under reduced pressure to 200 ml, adjusted to pH 1.0 with sulfuric acid, and ammonium sulfate was added to make a saturated solution. Next, the mixture was extracted with twice the amount of ethyl acetate, and the solvent was removed under reduced pressure at below 50°C to obtain a brown oily substance. Add this oily substance to 5 times its weight of silica gel (Wako Gel Q).
-50) and applied it to a column prepared with benzene. Next, D(-)-β-hydroxyisobutyric acid was eluted with a benzene:acetone (3:1) solvent.
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 ^.
= -15.1 to 18.0° (C = 3, methanol), and it was confirmed that it was β-hydroxyisobutyric acid in the D(-) form. Example 2 After culturing Candeida Rugosa IFO 0750 for 24 hours in the same medium and conditions as in Example 1, the bacterial cells were collected by centrifugation and suspended in M/15 phosphate buffer (PH7.0) containing 1% glycose. , further added 10 g of isobutyraldehyde, and adjusted the pH with caustic soda.
The reaction was carried out for 24 hours under the same conditions as the culture while maintaining the temperature at 7.0. D in the reaction solution obtained in this way
As a result of gas chromatography analysis of the content of (-)-β-hydroxyisobutyric acid, it was found to be 3.1 mg/ml.
Accumulation of D(-)-β-hydroxyisobutyric acid was observed. Example 3 Using 1 g of isobutylamine instead of isobutyraldehyde, culturing and reaction were carried out in the same manner as in Example 2. As a result, 3.7 mg/ml of D(-)- was found in the reaction solution.
Accumulation of β-hydroxyisobutyric acid was observed. Example 4 Same as Example 2, the substrate was 10g of isobutyramide.
As a result of the reaction using
ml of D(-)-β-hydroxyisobutyric acid was observed to accumulate. Example 5 Medium containing 2% glucose, 0.5% yeast extract, 0.3% peptone, and 0.3% meat extract (PH
7.0) In addition to 1, isobutyraldehyde 0.5%,
Or add 0.5% isobutylamine or 0.5% isobutylamide and then
Inoculated with Lugoza IFO 0750. Stir this in a 3-volume jar fermenter at 30°C, with aeration of 1vvm.
The cells were cultured for 24 hours at 500 rom while adjusting the pH to 7.0. D(-) in the culture solution obtained in this way
-As a result of analyzing the accumulated amount of β-hydroxyisobutyric acid by gas chromatography, it was 1.2 mg/ml when using isobutyraldehyde as the raw material, 1.7 mg/ml when using isobutylamine as the raw material, and 1.7 mg/ml when using isobutylamide as the raw material. If 1.6mg/ml D
Accumulation of (-)-β-hydroxyisobutyric acid was observed.

Claims (1)

[Claims] 1. D(-)--
Candeida sp., Pichia sp., Torulopsis sp., Aspergillus sp., Coanephora sp., Wingea sp., which have the ability to convert into β-hydroxyisobutyric acid;
Alternatively, a method for producing D(-)-β-hydroxyisobutyric acid, which comprises treating a microorganism belonging to the genus Tigorhynchus and collecting the produced D(-)-β-hydroxyisobutyric acid. 2 The microorganisms are Candeida rugosa, Candeida parapsilosis, Pichia membranefuaciens, Torulopsis canandida, Aspergillus niger, Corenephora cilcinanus,
The method according to claim 1, wherein the plant is selected from the group consisting of Wingea robertii and Chigorhynchus moellerii. 3. The production method according to claim 1, which comprises culturing microorganisms in a nutrient medium and treating the obtained culture solution with a compound selected from the group consisting of isobutyraldehyde, isobutylamine, and isobutyramide. 4. A method according to claim 1 in which microorganisms are made to act on isobutyraldehyde, isobutylamine, or isobutylamide by culturing the microorganisms in a medium supplemented with a compound selected from the group consisting of isobutyraldehyde, isobutylamine, and isobutylamide. Manufacturing method. 5. Cultivating microorganisms in a nutrient medium, separating microbial cells from the obtained culture solution to prepare a cell suspension, and allowing it to act on a compound selected from the group consisting of isobutyraldehyde, isobutylamine, and isobutyramide. A manufacturing method according to claim 1.