JPH01117792A - Production of 3(r)-hydroxybutyric acid ester - Google Patents

Abstract

PURPOSE:To obtain the title ester which is used for modification of optically active substances of optically high purity in high yield by allowing at least one of strains selected from Rhodotorula, Torulopsis, etc., to act on acetoacetic acid ester. CONSTITUTION:The reaction of an alcohol with ketene gives an acetoacetic acid ester (A) of the formula (R is alkyl) such as methyl acetoacetate. Then, at least one of strains selected from Rhodotorula, Debaryomyces, Torulopsis, Trichosporon, Aureobasidium, Glyocladium, and Aspergillus, such as Rhodotorula pallida is cultured in a medium containing saccharides to collect the resting cell bodies (B). The component (B), saccharides and the component (A) are dispersed in an aqueous medium in an amount of 0.01-50wt.% and they are stirred at 10-70 deg.C, pH3-8 for 0.1-100hr, extracted with hexane, then the extract is purified to give 3(R)-hydroxybutyric ester.

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention provides a method for reducing acetoacetate microbially.
The present invention relates to a method for producing optically active 3(R)-hydroxybutyric acid ester. Since this 3(R)-hydroxybutyric acid ester has an asymmetric carbon skeleton, it is used as a modifier for the synthesis of optically active substances such as chenamycin (antibiotic), and is a highly useful substance.

[Prior Art] Conventional methods for producing 3(R)-hydroxybutyrate by asymmetric reduction of acetoacetate include (a) a method using organic synthesis, and (b) a method using microorganisms.

(a) A report using (R,R)-tartrate-modified Raney nickel as a catalyst (Journal of the Chemical Society of Japan 10.12)
70 (19'86), Japanese Unexamined Patent Publication No. 60-36442), etc., and the (b) method uses Geotricum Candidum as a bacterial strain.
um) (He1v, Chim, Acta.

66.485 (1983)).

[Problems to be solved by the invention] With method (a), a product with high optical purity has not yet been obtained, and the catalyst is expensive, and with method (b), the yield is low. , there are problems such as poor reproducibility.

[Means for Solving the Problems] As a result of extensive research in order to solve the problems as described above, the present inventors found that Rhodotorula (R) was added to acetoacetic ester.
hodotorula), Debaryomyces (Deb
aryomyces), Torulopsis (Toru
1 ops is), genus Trichosporon (Tricho
sporon), Aureobasidium (Aureob
as idium), Gliocladium (Gli)
ocladium), Aspergillus (Aspergillus)
At least I PI of a strain selected from the genus Illus
The present inventors have discovered that 3(R)-hydroxybutyric acid ester with high optical purity can be obtained with good reproducibility and good yield when the compound is reacted with the following, and the present invention has been completed.

The starting material acetoacetate used in the present invention is, for example, the general formula (CH3COCH2C00R"J
This is a compound represented by R is an alkyl group, and methyl acetoacetate and ethyl acetoacetate are useful.

Examples of useful strains used in the present invention are as follows.

Rhodotorula palida (Rhodotorula pal)
(IFO0715), Rhodotorula m1nuta (IFO
0920), Debaryomyces subglobosus (D
ebary.

myces subglobosus) (IFO07
94), Debaryomyces salmon
yces 5ake) (IFO0060),
Torulopsis coryculosa (Torulopsis c)
oliculosa (IFo 0381), Torulopsis candida
da) (IFO0380), Trichosporoncutaneum
(IFO0173) , ) Lycosboro-77-
Mentans (Trichosporon fermen)
(IFO1199).

Aureobasidium pullulans
dium pullulans) (IFO4464)
, Aureobasidium mansoni (Aureob
asidium mansonii) (IFO64
-21), Gliocladium virens (Glioc
radium virens (IFO6355) Gliocladium roseum
roseum) (IFO5422), Aspergillus flavu
s) (IFO4295), Aspergillus versicolor
r) (IFO4105).

Various media can be used for culturing the strain used in the present invention, and carbon sources include various sugars, starches, etc., and nitrogen sources include yeast extract, meat extract, peptone, corn steep liquor, and inorganic salts. Besides, Na, Ni, Ca
, Mg, P, CI, and other inorganic components and vitamins are added in appropriate amounts.

The reaction method involves dispersing the culture solution of the strain, resting cells, or dried cells alone or in a mixture in an aqueous system (water, physiological saline, buffer solution, medium, etc.), adding sugars, etc. as an energy source, Next, add acetoacetate to a concentration of 0 in the system.
．． 01-50% by weight, preferably 0.05-10% by weight
10~70℃, preferably 20~5℃
0°C, pH 3-8, preferably 4-7, 0.1-10
It may be shaken, stirred, or allowed to stand for about 0 hours.

Further, any method can be adopted, such as separately immobilizing a bacterial strain and making it work, or using a reductase isolated from the strain.

Any method can be used for the reaction, such as a bulk method or a continuous method in which the immobilized bacterial strain is packed into a tube or tower and the acetoacetic ester is allowed to flow down.

As a medium for such a reaction, not only water but also an organic solvent compatible with water, such as a mixed water/organic solvent system such as alcohol or acetone, can be used. It is of course necessary to select an organic solvent that is not harmful to microorganisms.

Acetoacetate is added to the system as it is or after being dissolved or dispersed in an organic solvent. The concentration range of the ester in the system is usually 0.01 to 50% by weight, and if it is less than 1% by weight, there is no disadvantage in terms of reaction, but it is economically impractical; If the size is larger, problems such as less load on the bacterial cells and a decrease in yield are likely to occur.

Furthermore, if the temperature in the system is less than 10°C, the activity of the bacteria will decrease, while if it exceeds 70°C, the death of the bacteria will increase, and in both cases the yield will decrease.

If the pH in the system is less than 3 or greater than 8, the activity of the bacteria will decrease and the death rate will increase, resulting in a decrease in yield.

There is no problem in coexisting sugars such as glucose and microbial substrates during the reaction. The addition of such saccharides and microbial substrates can be carried out at any stage of the reaction, and can be carried out either batchwise, continuously or dividedly. Further, the reaction time is approximately 0.1 to 100 hours.

After the reaction is complete, the reaction solution is extracted with an organic solvent such as ethyl acetate or hexane, and the solvent is distilled off, or the bacterial strain is separated using a conventional method such as centrifugation and recovered by direct distillation. 3(R)-hydroxybutyric acid ester is obtained.

[Function] In the present invention, by using the above-mentioned strain, 3(R)-hydroxybutyric acid ester can be produced from acetoacetic ester, and further, optical purity and purity are improved compared to the conventional production method using asymmetric reduction. It has the advantage of being excellent in both yield and reproducibility.

[Example] Hereinafter, the present invention will be explained in more detail by giving examples.

Example I YM medium (yeast extract 4g, malt extract 109, glucose 4g, water 1 (2, PH4, 5) was inoculated with Rhodotorula parulida (IFo 0715), cultured with shaking at 28°C for 40 hours, and bacteria were collected. Afterwards, the cells were washed with ion-exchanged water to obtain viable bacterial cells.

100 ml of ion-exchanged water was placed in a 500 ml Sakaro flask, and the above-mentioned live bacterial cells (7.0 g as dried fluorophores) were added to prepare a suspension.

Next, 3.5 g of methyl acetoacetate was added, and the mixture was reacted with reciprocating shaking at 28° C. for 3 hours. After the reaction is complete, add 10 ethyl acetate
The ethyl acetate layer was dried over anhydrous magnesium sulfate, and the ethyl acetate was distilled off. When the extracted residual aroma was confirmed by gas chromatography and IRlNMR, it was found to be methyl 3(R)-hydroxybutyrate.

The yield was determined by high performance liquid chromatography and was 8.
1%, and the produced ester showed a value of [α), -38 (chloroform solvent, concentration 5.0%).

The above results are shown in Table 1.

Examples 2 to 14 Using the bacterial cells shown in Table 1, experiments were carried out according to Example 1 to obtain the corresponding 3(R)-hydroxybutyric acid esters. The results are also shown in Table 1.

(Left below) [Effects] As described above, by using the above-mentioned strain in the present invention, 3(R)-hydroxybutyrate can be produced from acetoacetate, and the optical purity, yield and Good results were obtained in terms of reproducibility.

Patent Applicant Nippon Gosei Kagaku Kogyo Co., Ltd. Procedural Amendment Document January 1986/L1 Day 3, Person making the amendment Relationship to the case Patent Applicant Address 9-6 Nozaki-cho, Kita-ku, Osaka (zip code 530) )
Name (410) Nippon Gosei Kagaku Kogyo Co., Ltd. 5. Correct "Pallida" to "Valida" on page 7, line 9 of the amended statement of contents.

Claims (1)

[Claims] Rhodotoru to acetoacetic acid ester
genus Debaryomyces (la), Debaryomyces
s), the genus Torulopsis, the genus Trichosporon, the genus Aureobasidium, the genus Gliocladium, and the genus Aspergillus. Features 3 (
R)-Production method of hydroxybutyric acid ester.