JPS6188894A - Preparation of optically active alpha-hydroxy acid by microorganism - Google Patents

Abstract

PURPOSE:To produce an optically active alpha-hydroxy acid useful as pharmaceuticals, etc., economically in an industrial scale, by separating a racemic alpha-hydroxy acid amide compound in a culture medium using a microorganism capable of producing exclusively D-alpha-hydroxy acid compound or L-alpha-hydroxy acid compound. CONSTITUTION:A racemic alpha-hydroxy acid amide compound (e.g., D,L-pantamide) is used as a raw material, and a microbial strain capable of decomposing alpha- hydroxy acid amide compound and producing exclusively D-alpha-hydroxy acid compound or L-alpha-hydroxy acid compound [e.g. Aeromonas hydrophila (FERM-P No.7360)] is cultured in a medium containing a phsophate, calcium chloride, glucose, etc., without stirring, to produce the objective optically active alpha-hydroxy acid compound in the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an optically active α-oxy' acid compound using a racemic α-oxyacid amide compound as a raw material. More specifically, the present invention uses a racemic α-oxyacid amide compound as a raw material, decomposes the α-oxyacid amide compound, and produces only a D-α-oxyacid compound or an L-α-oxyacid compound. Provided is a method for producing an optically active α-oxyacid compound, the method comprising producing an optically active α-oxyacid compound in a culture medium using a microorganism capable of producing . It is.

Optically active α-oxyacid compounds are useful substances as starting materials for related optically active compounds, some as drugs themselves, or as raw materials for medical products. Developing an efficient production method for is extremely important.

Conventionally, generally, optically active α-oxy acid compounds are
The racemic oxy/acid compound obtained by chemical synthesis is
It is produced by optical resolution or direct production by fermentation. Optical resolution methods include the diastereomer method using an appropriate base and the direct crystallization method, but they have disadvantages such as expensive reagents and low fractionation rates, and fermentation methods are , most often use sugar as a substrate, and have the disadvantage that their range of application is extremely narrow.

As a result of intensive research into industrial methods for producing optically active α-oxyacid compounds, the present inventors have discovered a method for advantageously obtaining optically active α-oxyacid compounds by utilizing the asymmetric hydrolysis ability of microorganisms. I found out. The present invention is based on this knowledge.

The present inventors have discovered Aeromonas hydrophylla (Feikoken Bibori No. 7360), Moraxella phenylpyruvica (
asymmetrically hydrolyzing a D,L-α-oxyacid amide compound as a raw material using
Optionally D-, , :tjQl or α with L-activity
- Succeeded in producing oxyacid compounds.

A method for searching for microorganisms capable of producing germs using only the D-α-oxy/fusion compound or only the L-α-oxy acid compound used in the present invention can be carried out, for example, by the following method.

That is, microorganisms are separated by supplying soil, sewage, dust, air, etc., and using a liquid medium or solid medium in which microorganisms can grow.

The isolated bacterial strain is tested for asymmetric hydrolysis ability. For example, dipotassium phosphate 0.7%, potassium phosphate 06%, magnesium sulfate 0.05%, calcium chloride o,oos%, ferric chloride 0.0005%, glucose 0.5%, D, L- ) The isolated bacteria are spread in a liquid medium consisting of 02% ξantamide and cultured aerobically at 27° C. for 6 to 5 days. Collect the grown bacterial cells, D, L
- Add α-oxinamide and allow to react for 1 to 3 hours.

After the reaction, the optical purity of the produced α-oxyacid is measured using chromatography, optical rotation, etc.

By such a method, the present inventors have identified more than 200 types of microorganisms that have the ability to produce optically active α-oxyacid compounds from L-α-oxyacid amide compounds, obtained from approximately 80 persimmon soils. An intensive search was made for isolated IY4. As a result, it was confirmed that the two types of bacteria mentioned above had excellent performance.

A search for the taxonomic position of the selected fungi revealed that they were allegorized to Aeromonas hydrophila and Moraxella phenylpyruvica, respectively. The determination of species and genus is based on the Vernorth Manual of Determinative Bacteriology (8th edition 197).
4) It was carried out according to the classification book.

The raw material used in the method of the present invention has the formula % [in the formula, r is C1-12 branched or straight-chain alkyl or alkenyl, substituted lower alkyl (the substituent is /% , hydroxy, lower alkoxy 7, amine,
alkylamino, meaning carboxy), allyl, substituted allyl (where the substituents are lower alkyl, lower alkoxy/, haloken), aralkyl, aralkenyl, heterocyclic aralkyl, heterocyclic aralkenyl] It is a racemic form of α-oxyacid amide compound.

In addition, L-α-oxyacid amide compounds (or L-α-oxyacids) can be easily produced from L-α-oxyacids. A good yield can be obtained by reacting overnight in a medium-sized room L-.

Other adhesives and L-α-oxyacid amide compounds are 0!゛S.

The corresponding compound can be easily obtained in good yield from L-α-oxyacid according to the method described in J.D. 5ynthesis 2dan 62 (1940).

The α-oxy Table 1 compound having the desired optical activity is, for example, a compound represented by the formula R-CH-COOH B [wherein R has the same meaning as above].

In carrying out the present invention, the aforementioned microorganisms isolated from soil, such as Aeromonas hydrophylla (Feikoken Bibori No. 7360) or Moraxella phenylpyruvica (
For example, the microorganism microorganisms microorganisms were cultured with shaking in a suitable medium containing L-bantamide, and after culturing,
The bacterial cells are separated by centrifugation, filtration, etc., and an L-α-oxyacid amide compound is added to the bacterial cells to suspend them and allowed to react at an appropriate temperature. After the reaction, bacterial cells are removed from the reaction solution, and then purification operations such as fractional extraction and chromatography are performed. In this way, the desired optically active α-oxy acid compound can be obtained. Conditions for separation, extraction, purification, etc. are appropriately selected depending on the physical properties of the starting material and target material.

The unreacted α-oxyacid amide having optical activity can be racemized in the presence of an alkali, subjected to appropriate treatment, and used repeatedly.

The present invention will be specifically described below with reference to Examples.

Example 1 Preparation of Aeromonas hydrophila inoculum solution Dipotassium phosphate 702, Potassium phosphate 60? Dissolved in purified water (pH 7.2), sterilized, added 0.5 ml of magnesium sulfate, 0.5 ml of calcium chloride,
1%' ferric chloride 0.25-, glucose 50? ,D,L
- Dissolve 2.02 ml of bantamide in 50 ml of purified water, sterilize it by filtration, and mix. Dispense 5 d each into sterilized test tubes, and inoculate one platinum loop of Aeromonas hydrophila (Feikoken Bibori No. 7560) isolated from soil onto an agar medium with the above composition (agar 1.5%). An inoculum solution of Aeromonas no idrophylla was prepared by statically culturing the mixture aerobically at 27°C for 3 days.

Example 2 Preparation of Moraxella phenylpyruvica inoculum liquid Moraxella phenylpyruvica isolated from soil was placed in a medium 5- having the same composition as in Example 1.
No. 9) was inoculated with one platinum loop and cultured stationary aerobically at 27° C. for 6 days to obtain an inoculum solution of Moraxella phenylpyruvica.

Example 6 Synthesis of D-bento acid 1 me of Aeromonas hydrophila seed solution obtained in Example 1 was added to 500 ml of a medium having the same composition as in Example 1.
and culture with shaking at 27°C for 5 days.

5. Wet bacteria are collected from culture medium a by centrifugation. Obtained Of.

2.2r of bacterial cells were added to 3.0 mZ of o, 4ba D, L-t'r nitamide' phosphate buffer solution (pH 7,2) and reacted at 67°C for 1 hour. After removing the bacterial cells by centrifugation, chromatography and ethyl acetate extraction yielded D-.nitric acid 58+119 (yield 62, optical purity 94%). Optical purity is determined by lactonizing pantoic acid, Ar+alythcal Blochemistr.
y 1129-16 (1981) Example 4 Synthesis of D-Manoderic Acid Aeromonas hydrophylla hygrophila 2.0? I got it. Bacterial body 1.5? 0,1M
D, L-mandelamide phosphate buffer solution (pH
7,2) Add to 10- and react at 27°C for 1 hour.

After removing the microbial cells by centrifugation, acidification with hydrochloric acid and extraction with ether produced mandelic acid 52 #+9 (specific optical rotation [α': l, -116° (C = 0.5, H2
O) ) was further crystallized with ethyl acetate-hexane (mixing ratio 1:1) to obtain 45 mg of D-mandelic acid (specific optical rotation [α] 6° = -145° (c = i, o ethanol)
m, p, 131-152°) were obtained.

Example 5 Synthesis of L-band acid 300 mj of medium having the same composition as in Example 1! Moraxella phenylpyruvica inoculum solution (1 go) obtained in Example 2
Culture with shaking at 27°C for 5 days. Wet bacteria 2.01 was obtained from the culture solution by centrifugation.

Add 1.22 bacterial cells to 0.2 MD, L-puffed amide phosphoric acid mild fI solution (pH 7.2) 2.5-
React at 37°C for 4.5 hours. After removing bacterial cells by centrifugation, L was removed by chromatography and ethyl acetate extraction.
-15 tnq of pantoic acid (yield 62%, optical purity 90%)
I got it.

Example 6 Synthesis of L-mandelic acid Moraxella humidus 157 was obtained in the same manner as in Example 5. Add 0.11vlD of bacterial cells 1.17 and 10rnl of L-mandelamide phosphate buffer solution (pH 7,2),
The reaction was carried out at ℃ for 2 hours. After removing the bacterial cells by centrifugation,
The reaction product mandelic acid 64 m9 (specific optical rotation [α] 9 = +113° (C
=0.5, we get I(2o:l). It was crystallized from a mixed solution of ethyl acetate and hexane (1:1), and L-mandelic acid 56 m9 (specific optical rotation [α] t0-+146° (C=
1.0° ethanol) m, p, 131-162°)
I got it.

α-oxy acid compounds having optical activity, such as D-α-
Examples of oxyacids include D-/ξnodonic acid and D-tartaric acid, and examples of L-α-oxyacids include L-lactic acid and L-IJmalgamic acid. It is widely used as a raw material for various substances, and is often found to contain optically active α-oxyacids as constituents of recently discovered useful microbial metabolites, such as bestatin. The method of the present invention is an extremely useful method for obtaining the 0-form or L-form of these α-oxyacids.

Patent applicant: Fuji Pharmaceutical Co., Ltd. Patent attorney: Takao Minami
'l -゛-゛1

Claims (1)

[Claims] 1) Using a racemic α-oxyacid amide compound as a raw material, the α-oxyacid amide compound is decomposed to produce only a D-α-oxyacid compound or only an L-α-oxyacid compound. 1. A method for producing an optically active α-oxyacid compound, which comprises producing an optically active α-oxyacid compound in a culture medium using a microorganism capable of producing the optically active α-oxyacid compound. 2) The optically active α-oxy acid according to claim 1, wherein the microorganism having the ability to produce only the D-α-oxy acid compound is Aeromonas hydrophylla (Feikokenbokuyoku No. 7360). Method of manufacturing the compound. 3) The optically active α-oxy acid according to claim 1, wherein the microorganism having the ability to produce only the L-α-oxy acid compound is Moraxella phenylpyruvica (Feikokenbokuyori No. 7359). Method of manufacturing the compound.