JPH01222787A - Reduction of beta-keto acids - Google Patents

Abstract

PURPOSE:To obtain high-purity beta-hydroxycarboxylic acids by reducing beta-keto acids in the presence of at least one kind of metallic ion selected from Fe, Al etc. using bacteria capable of reducing beta-keto group. CONSTITUTION:Firstly, beta-keto acid are introduced into a system in the presence of at least one kind of metallic ion selected from Fe, Al, Cr, Ce, and In. Thence, said beta-keto acid are reduced into the corresponding beta-hydroxycarboxylic acids using bacteria capable of reducing the beta-keto group introduced into the system. Said beta-keto acids are e.g., acetoacetic acid, acetoacetic alkyl esters, alpha- haloacetoacetic alkyl ester. Said bacteria capable of reducing the beta-keto group is e.g., Trichosporon sp., Rhodotorula sp.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing optically active β-hydroxycarboxylic acids by microbially reducing β-keto acids such as acetoacetate. be. Since these β-hydroxycarboxylic acids have an asymmetric carbon skeleton, they are highly useful substances that are used as modifiers for the synthesis of optically active substances such as chenamycin (antibiotics).

[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) As a method, a report using (R,R)-tartaric acid modified Raney nickel as a catalyst (Journal of the Chemical Society of Japan 10゜12
70 (1986), Japanese Unexamined Patent Publication No. 60-36442)
For the (b) method, Geotricum Candidum is used as a bacterial strain.
Report using Ill) (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] In order to solve the above-mentioned problems, the present inventors have conducted extensive research to reduce β-keto groups using a bacterium that has the ability to reduce β-keto groups. When producing the corresponding β-hydroxycarboxylic acids by reducing acids, if at least one metal ion selected from the group of iron, aluminum, chromium, cerium, and indium is present in the system, high-purity products can be produced with good yield. The inventors discovered that the desired object could be obtained and completed the present invention.

The β-keto acids targeted by the present invention include hydrogen, alkyl groups, halogenated alkyl groups, allyl groups,
aryl group, etc., R1: hydrogen, alkyl group, halogen, aryl group, allyl group, etc.; R3: hydrogen, alkyl group, allyl group, aryl group, etc.), typically acetoacetic acid, acetoacetic acid, etc. acetic acid alkyl ester,
α-Haloacetoacetic acid alkyl ester, γ-haloacetoacetic acid alkyl ester, acetoacetic acid allyl ester, α
-Haloacetoacetic acid allyl ester, γ-haloacetoacetic acid allyl ester, propionyl acetic acid, propionyl acetate, benzoyl acetate, benzoyl acetate,
Examples include β-ketopentanoic acid ester, and acetoacetic acid esters are practical.

Bacteria that reduce the β-keto group used in the present invention include Trichosporon, Rhodotorula, Debaryomyces, Cryptococcus, Torulopsis, Candida, Satucharomyces, Aureobasidium, Bryofradium, and Aspergillus. included.

Specific examples include trichosporon phthanium (IPo 119g),
Rhodotorula texensis ([FO0920), Rhodotorula rubra (IFollol), Debaryomyces hansenii (IFO0023).

Debaryomyces subglobosus (IFO0794)
, Cryptoboccus laurentii (IPo 06
09), Cryptobocchus neoformans (IP
o 0410), ) Lullopsis Candida (IF
OQ405), ) Lulopsis aeria (rFo
0g81), Candida utilis (IFO039)
6) , 'Candida lipolytica (IFO0717)
, Satucharomyces cerevisiae (IFO0635)
, Satucharomyces logos (IPo 027g),
Satucharomyces carlsbergensis (IFO046
1) , Rhodotorula barida (IPO0715), Rhodotorula minuta (IFOQ92Q), Debaryomyces subglobosus (IFO0794), Debaryomyces salmon (IFo 0060), Torulopsis coryculosa (IFO0381), Torulopsis candida (IFo 0380) ), Trichosporon cutaneum (IFO0173), Trichosporon fermentans (IFO1199), Aureobasidium pullulans (IFO4464),
Aureobasidium mansoni (IPO6421)
, Bryofradium virens (IFO6355), Bryofradium roseum (IPO5422), Aspergillus flavus (IPO4295), Aspergillus versicolor (IFO4105), and the like.

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. Also, Na, K.

Appropriate amounts of inorganic components such as Ca, Mg, P, and CI and vitamins are added.

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, culture medium, etc.), and adding sugars, etc. as an energy source. Then, β-keto acids were added to the system at a concentration of 0.01.
-50% by weight, preferably 0.05-10% by weight, and at 10-70°C, preferably 20-50°C,
It may be shaken, stirred, or allowed to stand at pH 3 to 8, preferably 4 to 7, for about 0.1 to 100 hours. or,
Any method can be used, such as separately immobilizing a bacterial strain and making it work, or using a reductase isolated from the strain.

Any method can be used as the reaction method, such as a batch method or a continuous method in which a tube or column is filled with the immobilized bacterial strain and β-keto acids are 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.

The β-keto acids are added to the system as they are or after being dissolved or dispersed in an organic solvent. The concentration range of the acids in the system is usually 0.01 to 50% by weight, and 0.01% by weight.
If it is ungrooved, there is no disadvantage in terms of reaction, but it is economically impractical.On the other hand, if it is larger than 50% by weight, problems such as a decrease in yield are likely to occur because the load is not applied to the bacterial cells. .

Furthermore, if the temperature in the system is less than 0°C for a month, 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 decreases and killing increases, resulting in a decrease in yield.

There is no problem in coexisting sugars such as glucose and microbial substrates during the reaction. Such saccharides and microbial substrates can be added at any stage of the reaction, and can be carried out collectively, continuously, or in portions. Further, a practical reaction time is about 0.1 to 100 hours.

The feature of the present invention is that during the above reaction, iron, aluminum-lamb,
The point is that at least one metal ion selected from chromium, cerium, and indium is allowed to coexist in the system.

The ions are usually added to the system in salt form, such as nitrates, sulfates, halides, phosphates, acetates, and the like.

The amount added is 0.01 to 0.5% as salt to the reaction system.
0.005-0.1% of the reaction system as metal ions
The degree is effective.

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. get the object.

[Function] In the present invention, by coexisting the aforementioned metal ions, β-hydroxycarboxylic acids can be produced from β-keto acids in a high yield, and furthermore, compared with the conventional production method by asymmetric reduction, the optical It has the advantage of being excellent in purity, yield, and reproducibility.

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

Example 1 Aqueous solution I of urea 1%, ammonium sulfate 0.75%, phosphoric acid 0.5%
Q was placed in a 2Q jar fermenter, the pH was adjusted to 4.5 with IN sodium hydroxide, and the mixture was sterilized. Next is Satucharomyces cerevisiae (IFO0304) l
09 (dry weight of bacteria) was inoculated, and 800 ml of sterilized blackstrap molasses solution (containing 33% blackstrap molasses) was added dropwise at a rate of 50 ml/h to culture (30° C., PH 4, 5 constant). still,
Pure oxygen was supplied during the exponential growth phase so that dissolved oxygen was always 2-5 ppm.

After culturing for 24 hours, the bacterial cells were collected by centrifugation and washed with water once.
This was repeated to obtain 85 g (dry weight) of Satucharomyces cerepica. A reduction reaction was performed using the bacterial cells thus obtained.

That is, 39 g (dry weight) of bacterial cells was placed in a 1Q round bottom flask,
Add 7.5 g of glucose, 270 g of water, 0.4 g of ferric nitrate (nanahydrate), and add 19 g of ethyl acetoacetate as a substrate.
．． 29 (0,148 mol) was added, and the reaction was carried out at 30° C. with propeller stirring for 5 hours, followed by an extraction operation. That is, 200 cc of hexane was added, stirring was continued for 1 hour, and the emulsion was centrifuged (5000 rpm).
, 20 minutes), the obtained hexane layer was applied to a rotary evaporator, the hexane was distilled off, and 1
8.0 g of residue was obtained. According to gas chromatography, IR, and NMR analysis, the amount of ethyl β-hydroxybutyrate contained in this residue was +7.5 g (reduction yield 89.7%), and its specific optical density was [α]! It showed O+43.4 (C=1 chloroform), and the optical purity was 95% ee of the (S) form. The reaction was carried out in exactly the same manner except that the use of ferric nitrate was omitted in this reaction solution composition. As a result, the reduction yield was 60% and the (S)-isomer optical purity was 90% ee.

Examples 2 to 6 The results of experiments conducted in Example 1 by changing the type and amount of metal salt added are shown.

Examples 7 to 10 The results of experiments conducted in Example I by changing only the type of substrate are shown.

(The control example is the case without the addition of ferric nitrate.)NWA of the β-position substrate of the reduced product Yield of the reduced product Stereoconfiguration and optical purity Example 7 Octyl γ-chloroacetoacetate 90%
(R) 95%ee control example
85% (R) 90%ee Example 8 β-
Ethyl ketopentanoate 85% (S)94
%ee control example 5
0% (S) 89%ee Example 9 Methyl α-bromoacetoacetate 82% (S) 92%ee Control example
〃 53% (S)
90%ee Example 1 Oα Butyl methylacetoacetate
85% (S) 95%ee Examples II to 19 YM medium (yeast extract 49, malt extract 109, glucose 4 g, water IQ) was inoculated with the bacteria shown in the following table and incubated at 28°C.
After culturing for 40 hours, the bacteria were collected, washed once with distilled water, and subjected to the reaction.

That is, 100 ml of water was placed in a 500 ml Sakaro flask, and a predetermined amount of washed bacterial cells was added thereto to prepare a fungal suspension.

Next, after adding 0.2% of ferric chloride and 0.1% of dichromic nitrate (nonahydrate), a predetermined amount of acetoacetate shown in the table below was added, and the mixture was shaken at 28°C for 3 hours. After the reaction, 100 ml of ethyl acetate was added and stirred to perform an extraction operation. The ethyl acetate layer was put on a rotary evaporator and ethyl acetate was distilled off to obtain an extraction residue. The results of analysis of this extraction residue are shown (the control example is the case without the addition of metal salts).

[Effect] As described above, by adding a specific metal ion in the present invention, β-hydroxycarboxylic acids can be produced from β-keto acids, and have good optical purity and yield. The results were obtained.

Claims (1)

[Claims] 1. β-keto group reduction using a bacterium capable of reducing β-keto groups.
In producing the corresponding β-hydroxycarboxylic acids by reducing keto acids, β is characterized in that at least one metal ion selected from the group of iron, aluminum, chromium, cerium, and indium is allowed to coexist in the system.
-Method for reducing keto acids. 2. The reduction method according to claim 1, wherein the β-keto acids are acetoacetic esters.