JPS5921599B2 - Method for producing D(-)-β-hydroxyisobutyric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides various natural products having optically active carbon or
Optically active D(→-mono-β-hydroxyisobutyric acid), which is one of the reaction intermediates useful in the synthesis of physiologically active substances such as pharmaceuticals, can be industrially advantageously produced by utilizing the special ability of microorganisms. It's about how to do it.

Regarding the production of β-hydroxyisobutyric acid, several synthetic methods are known, including the reduction method of methylmalonic acid monomethyl ester, but all of them are optically inactive DL products. In addition to being difficult to use as a raw material for producing various optically active substances, it is also expensive to produce.

On the other hand, regarding the production method of optically active β-hydroxyisobutyric acid, Pseudomonas putida (Pseudomonas putida)
There is a method using isobutyric acid such as B. sputida (Biotechnology and Bioeng).
(ineering) 13, 203, 1971), these are all β-hydroxyisobutyric acids in the L(-l) form, and the DH form has not been obtained. Conventionally, D (→ body β
-There is no suitable industrially advantageous manufacturing method for hydroxyisobutyric acid, and the present inventors focused their attention on the development of the manufacturing method, and as a result of their research, they found that methacrylic acid, which was not found in the conventional knowledge of microbial metabolism, was - A microorganism with a metabolic pathway that converts it to β-hydroxyisobutyric acid is Candidia
a) We discovered the fact that D(→-β-hydroxyisobutyric acid) exists in the genus D(→-β-hydroxyisobutyric acid), which is more optically active than methacrylic acid, using microorganisms.

That is, the present invention provides a microorganism belonging to the genus Candeida that has the ability to convert methacrylic acid into D(tri)-β-hydroxyisobutyric acid, and a culture solution obtained by culturing the microorganism in a nutrient medium capable of assimilating it. The present invention also relates to a method for producing D(c)-β-hydroxyisobutyric acid, which is characterized in that it is treated with a suspension of bacterial cells of Paso. From methacrylic acid used in the present invention? Examples of microorganisms with a metabolic system that converts →-β-hydroxyisobutyric acid include Candida rugosa, Candida rugosa, and Candida rugosa.
Candidaparapsilosis
is), Candida utilis
In general, any nutrient source that can be assimilated by these bacteria can be used for this culture.

For example, carbohydrates such as glucose, sucrose, and mannitrate, alcohols such as ethanol, hydrocarbons such as paraffin and olefin, organic acids such as acetic acid and propionic acid, carbon sources such as soybean oil, or mixtures thereof, yeast scratches, peptone, and corn. A normal culture medium containing an appropriate amount of nitrogen-containing inorganic organic nutrients such as staple powder, ammonium sulfate, and ammonia, inorganic nutrients such as phosphate, magnesium, potassium, iron, and manganese, and vitamins such as biotin is used. It will be done. The culture method is to adjust the pH of the nutrient medium to 4.0-9.
5 and aerobically at 20 to 40°C for 1 to 5 days.

For the conversion of methacrylic acid to D(tri)-β-hydroxyisobutyric acid, a pH range of 6.0 to 9.0 is preferred. In addition, as a method for producing D(c)-β-hydroxyisobutyric acid, for example, methacrylic acid is used as the sole carbon source, or in coexistence with the above carbon sources, as a method carried out in parallel with the cultivation of bacterial cells. At PH4. There is a method of culturing aerobically in the range of O to 9.5 and accumulating D(c)-β-hydroxyisobutyric acid in the culture solution. A method in which the conversion reaction to β-hydroxyisobutyric acid is carried out in two separate stages, such as using methacrylic acid alone as a carbon source produced by bacterial cells, or using the above-mentioned carbon sources such as assimilable baraffin, or a nutrient medium containing a mixture of these. At PH4. The pH was adjusted to 6.0-9.5 by adding methacrylic acid to the resulting culture solution.
5 and react aerobically, or collect the bacterial cells from the obtained culture solution by centrifugation, etc., and transfer the bacterial cells to a solution of an appropriate composition, such as M/15 phosphate buffer (PH 8.5). methacrylic acid and a small amount of glucose and aerobically adjusted to pH 6. There is a method of carrying out the reaction in the range of 0 to 9.5.
In this case, the bacterial cells may be subjected to appropriate pretreatment such as toluene treatment in order to accelerate the reaction rate. As a method for collecting D(c)-β-hydroxyisobutyric acid obtained by culturing and reaction, conventional extraction and purification methods can be used, but the following methods can also be used.

For example, the pH of the resulting D(c)-β-hydroxyisobutyric acid-containing solution is lowered to around 1.0 with sulfuric acid or the like, and ammonium sulfate is added to achieve saturation. Thereafter, extraction is carried out three times with twice the volume of ethyl acetate. The solvent is removed at low temperature and under reduced pressure to obtain a substance containing D(iii)-β-hydroxyisobutyric acid in the form of a brown oil, which is then dissolved in a small amount of benzene and eluted with a benzene-acetone mixed solvent through a silica gel column. It can be easily separated from other impurities by chromatography. In addition, D(to) can be esterified by a known method of reacting with diazomethane etc.
-β-Hydroxyisobutyric acid methyl ester can also be separated by fractional distillation. D(to)-β-
Quantification of hydroxyisobutyric acid was performed by James, Rl Schaeffer.
etc. method (BiOtechnOlOgyandBlOe
mgimerimg, 13, 203, 1971).

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

Example 1 Glucose 2%, yeast extract 0.5%, peptone 0
．． Quandida Rugosa I in a medium (PH6.O) containing 3%, 0.3% meat scratches, and 0.1% methacrylic acid (PH6.O).
FOO75O (CandidarugOsa), 1F
00591, Candida parapsilosis IFOO7
O8 (CandidapanapsilOsis) was inoculated and cultured for 20 hours at 30°C in a 31-volume mini jar fermenter with aeration of 1 VVM and 1 stirring at 500 rpm.

Thereafter, methacrylic acid 20f7 was added to each culture solution, the pH was adjusted to 8.5 with caustic soda, and the reaction was further cultured for 48 hours under the same conditions. Each of the obtained reaction solutions was diluted with sulfuric acid to PHL. O, and ammonium sulfate was added to make a saturated solution. Next, the mixture was extracted three times with equal amounts of ethyl acetate, the extract was dehydrated over anhydrous sodium sulfate, and the solvent was removed under reduced pressure below 40°C to obtain a yellowish brown oil. This oily substance was applied to a column using 5 times the weight of silica gel (Wako Gel Q5O) adjusted with benzene.

Benzeneliacetone (9:1) at 4 times the initial column volume
It was washed with a solvent to elute and remove unreacted methacrylic acid, and then eluted with benzenelyacetone (3:1) solvent to elute -β-hydroxyisobutyric acid. The obtained D(c)-β-hydroxyisobutyric acid fractions were collected and the solvent was removed under reduced pressure to obtain a syrup-like substance. What was obtained in this way was gas chromatography, silica gel thin layer chromatography, etc. Analysis and other analysis confirmed that it was highly pure β-hydroxyisobutyric acid.

This is a digital automatic polarimeter PMl manufactured by Union Giken.
As shown in Table 1, it was confirmed that it was DH-β-hydroxyisobutyric acid. Example 2 Normal paraffin 3%, diammonium phosphate 1.3
%, potassium phosphate 0.7%, sodium chloride 0.0
1%, zinc sulfate 0,006%, ferric sulfate 0.009%
, medium containing 0.5% yeast extract (PH6.5) 11
inoculated with Quyandida Rugoza IFOO75O, 3
1 volume mini jar fermenter at 30℃, ventilation 1.5
The cells were cultured for 72 hours using VVM and stirring at 700 rpm, and the resulting culture solution was centrifuged to collect cells, which were further washed twice with 0.9% saline to obtain cells.

This was suspended in M/15 phosphate buffer (PH 8.5), 307 ml of methacrylic acid and 27 ml of glucose were added, and the PH
The temperature was adjusted to 8.5 and reacted for 48 hours under the same conditions as culture. After that, extraction and purification was performed in the same manner as in Example 1.
(c)-β-hydroxyisobutyric acid 13.5y1[α]M
-13.45 kg (c=12.5 ethanol) was obtained. Example 3 Glucose 1.5%, yeast extract 0.5%, peptone 0.3%, meat scratches 0.3%, methacrylic acid 0.5%
, medium containing calcium carbonate 0.5 (PH6.O) 1
1 was inoculated with Candeida parapsilosis IFOO7O8 and cultured in a mini jar at 30°C with aeration of RwMl at 500 rpm for 24 hours, and then the pH was adjusted to 8.
D(c)-β was extracted and purified in the same manner as in Example 1 from the culture solution maintained at 0.0 with caustic soda and further cultured for 24 hours.
-Hydroxyisobutyric acid 1.3y1 [α] sweet = -13.4
4 do (c=12.

5 ethanol) was obtained.

Example 4 Using Candeida utilis IFOO396, 8.8 r of D(c)-β-hydroxyisobutyric acid was obtained by culturing, extraction and purification in the same manner as in Example 1.

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Candeida that has the ability to convert methacrylic acid into D(-)-β-hydroxyisobutyric acid, obtained by culturing the microorganism in a nutrient medium capable of assimilating it. A method for producing D-(-)-β-hydroxyisobutyric acid, which comprises reacting with a culture solution or a cell suspension thereof. 2 Microorganisms such as Candeida Rugosa and Candeida Rugosa
The method of manufacturing according to claim 1, wherein the plant is Parapsilosis or Candida utilis. 3. Claims 1 or 2 in which methacrylic acid alone or in combination with one or more carbohydrates, alcohols, organic acids, or hydrocarbons that can be assimilated is used as a carbon source for microorganisms. Manufacturing method described in section. 4. The manufacturing method according to claim 3, wherein the microorganism is cultured at a pH in the range of 4.0 to 9.5. 5. Claim No. 5, which involves culturing one or more types of carbohydrates, alcohols, organic acids, or hydrocarbons that can assimilate microorganisms as a carbon source, and allowing the resulting culture solution or bacterial cell suspension to act on methacrylic acid. The manufacturing method according to item 1 or 2. 6 Cultivate microorganisms at a pH range of 4.0 to 9.5, and react the culture solution or bacterial cell suspension with methacrylic acid at pH 4.0 to 9.5.
The manufacturing method according to claim 5, which is carried out within the range of H6.0 to 9.5.