JPH04316496A - Production of (r)-(+)-halolactic acid - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject compound useful as a synthetic raw material, etc., for optically active physiologically active substance such as a medicine by reacting a (+ or -)-3 halolactic acid with a microorganism of the genus Pseudomonas, etc., and specifically metabolizing the (-)-3-halolactic acid. CONSTITUTION:A microorganism (e.g. Pseudomonas.aureofaciens ATCC13986), belonging to the genus Pseudomonas, Arthrobacter, Escherichia, Saccharomyces, Schwanniomyces or Torulopsis and having the ability to produce an (R)-(+)-3-halolactic acid from a (+ or -)-3-halolactic acid or its treated substance is catalytically reacted with a (+ or -)-3-halolactic acid to specifically metabolize the (-)-3-halolactic acid. The formed (R)-(+)-3-halolactic acid is then collected from a mixture of the (R)-3-halolactic acid with (S)-(-)-3-halolactic acid and the residual (R)-(+)-3-halolactic acid to afford the objective (R)-(+)-3-halolactic acid in high yield.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to (R)-(+)-3-
This invention relates to a method for producing halo-lactic acid. (R)-(+)-3-halolactic acid is a substance useful as a raw material for the synthesis of optically active physiologically active substances such as pharmaceuticals.

0002

[Prior Art] Regarding the biochemical preparation of optically active (R)-(+)-3-chlorolactic acid, there is a known method for producing optically active (R)-(+)-3-chlorolactic acid by asymmetric reduction of chloropyruvic acid using lactate dehydrogenase. Journal of America
a Chemical Society, 104
Vol. 4452, 1982). This method requires NADH as a coenzyme during the reaction, so it is necessary to continuously supply NADH by some means, and a method is used in which it is combined with an NADH regeneration system.
Enzymatic reduction reactions that combine regeneration systems have many problems that are difficult to implement industrially. The second method is known to produce optically active (R)-(+)-3-halolactic acid by a microbial asymmetric oxidation reaction of (±)-3-halo-1,2-propanediol. (Special Publication No. 59-220193: Publication No. 61-268197: Journal
of Fermentation Technology
ogy, vol. 64, pp. 251-254, 1986). Microorganisms of the genus Geotrichum, Trichosporon, Endomyces, Hansenula, Candida, Sporidiobolus, and Rhoderomyces are used as the microorganisms. This method has the disadvantage of low yield. As a third method,
(±)-3-Halolactic acid is treated with (R)-(
+) Method for obtaining -3-halolactic acid (Japanese Patent Publication No. 61-268
No. 197) is known. Microorganisms include Candida, Cryptococcus, Endomyces, Hansenula, Rhoderomyces, Pichia, Sporidiobolus, Trichosporon, Alcaligenes, Bacillus, Corynebacterium,
Microorganisms belonging to the genera Aphanoascus, Aureobasidium, Bacgela, Bacdia, Bysocilamis, Floridium, Cladosporium, Coniocetideum, Eurotium and Gliocladium have been used. This method has the disadvantage that when the yield is good, the optical purity is low, and when the optical purity is high, the yield is low. The fourth method is a method in which 2-halo acid dehalogenase is allowed to act on 2,3-dihalopropionic acid ((Japanese Patent Publication No. 2011-11)
3890) is known. This method has a drawback in terms of the cost of the raw material, 2,3-dihalopropionic acid.

0003

[Problems to be Solved by the Invention] As mentioned above, the conventional technology still has difficulties in industrial implementation, so a more efficient method for producing (R)-(+)-3-halolactic acid was established. As a result of repeated research, we have completed the present invention.

0004

[Means for Solving the Problems] According to the present invention, the genus Pseudomonas, the genus Arthrobacter, and the genus Esserichia (
Genus Escherichia, Saccharomyces
charomyces), Schwaniomyces (Sch
wanniomyces) or Torulopsis (
The cells, culture solution, or processed products of microorganisms belonging to the genus Torulopsis and having the ability to produce (R)-(+)-3-halolactic acid from (±)-3-halolactic acid (
±)-By contacting with 3-halolactic acid, (R)-
(R)-(+)-3-halolactic acid can be produced by producing (+)-3-halolactic acid and collecting it.

The present invention will be explained in detail below. The microorganisms used in the present invention belong to the genus Pseudomonas, Arthrobacter, Esselichia, Saccharomyces, Schwaniomyces, or Torulopsis, and are (±)-3-
It acts on halo-lactic acid and consumes and converts (-)-3-halolactic acid to leave (R)-(+)-3-halolactic acid, resulting in (R)-(+)-3-halolactic acid. Any microorganism can be used as long as it has the ability to produce it. A specific example is Pseudomonas aureofaciens.
ATCC 139
86, Pseudomonas dehalogenans (Pseudo
monas dehalogenans) NCIB
9061, Arthlobacter citreus (Arth
robacter citreus) ATCC 1
1624, Arthrobacter globiformis (Art
hrobacter globiformis) AT
CC 8010, Escheria coli (Escher
ichia coli) ATCC 11303, Saccharomyces chevalieri (Saccharamyc
es chevalieri) IFO 0222,
Schwann
iomyces occidentalis) ATC
C 26074, Torulopsis spherica (Tor
ulopsis sphaerica) ATCC
Examples include 8549.

[0006] Mutant strains obtained by mutating these strains by artificial mutation methods such as ultraviolet irradiation, X-ray irradiation, mutagenic agent treatment, etc. can also be used.

[0007] In culturing the microorganisms used in the present invention, conventional microorganism culturing methods are generally used. As the medium, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances that can be assimilated by microorganisms, and substances necessary for the growth of microorganisms. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of microorganisms, hydrocarbons, alcohols, organic acids, etc. may also be used. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean flour, casamino acid, etc. are used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate,
Inorganic salts such as calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate, etc. can be added. The culture is carried out under aerobic conditions such as shaking culture or aerated agitation culture at a temperature of 4 to 43°C, preferably 20 to 35°C.
, carried out at pH 4-10, preferably 6.0-9.5,
Usually completed in 1 to 10 days. The pH of the medium is preferably adjusted using sodium hydroxide or the like.

[0008] The microorganism used can be brought into contact with (±)-3-halolactic acid during the growth and culture of such microorganisms, or it can be carried out by adding (±)-3-halolactic acid to the grown culture. It can also be reacted. Furthermore, after separating the bacterial cells from the culture, the bacterial cells or the treated bacterial cells and (±)-3-
Halolactic acids can also be reacted in a suitable medium, for example in a buffer. The culture solution or bacterial cells can be treated without impairing the (-)-3-halolactic acid converting activity in the bacterial cells.
A method that facilitates the enzymatic reaction is appropriately selected and carried out. Specific processed products include culture concentrates, dried products, products treated with surfactants and/or organic solvents, as well as bacterial cells obtained by centrifuging the culture, crushed bacterial cells, and dried bacterial cells. Examples include acetone-treated products, surfactant and/or organic solvent-treated products, lytic enzyme-treated products, immobilized bacterial cells, and enzyme preparations extracted from bacterial cells.

The (±)-3-halolactic acid used in the present invention is used in the form of free or salt such as hydrochloride. There is no particular limit to the concentration of (±)-3-halolactic acid, but
0.1-10% (weight to volume) is preferred. (+)-3
- When carrying out the reaction to produce halo-lactic acid, microorganisms or their processed material and (±)-3-halo-lactic acid are mixed under suitable conditions at a temperature of 10 to 60°C, preferably 20 to 40°C,
The reaction is carried out at a pH of 4 to 10, preferably 7.0 to 9.5. The reaction usually completes in 1 to 10 days. The pH during the reaction is preferably maintained at a suitable pH (7.0 to 9.5). For this purpose, a buffer solution may be used as the reaction medium, or p
It is preferable to adjust H using sodium hydroxide or the like.

The reaction also occurs during the cultivation of microorganisms (±)-3
- It can also be carried out by adding halo-lactic acid to the culture solution. In this case, the amount of (±)-3-halolactic acid to be added may be the same concentration as in the reaction using the above-mentioned bacterial cells or a treated product thereof. Furthermore, if necessary, the enzymatic reaction can be promoted by making the above-mentioned surfactant present in the culture solution in an amount that does not inhibit the growth of microorganisms. After the reaction is completed, the (+)-3-halolactic acid produced (remaining) in the reaction solution is isolated by known methods such as adsorption to an ion exchange resin, desorption, concentration, and crystallization.

[0011]

[Examples] Examples of the present invention are shown below. (R)-(+)- by high performance liquid chromatography in Examples
The conditions for the differential analysis of 3-chlorolactic acid and (S)-(-)-3-chlorolactic acid are as follows. Column: MCI GEL CRS 10W, 4.
6 x 50 mm (manufactured by Mitsubishi Kasei), eluent: micromolar copper sulfate, flow rate: 1 ml per minute, sample amount: 10 microliters, detection: UV (254 nm). Example 1 2% glucose, 1% diammonium phosphate, 1% phosphoric acid
10 m of sterile medium with the following composition: potassium 1%, meat extract 1%, peptone 1%, yeast extract 0.5%, sodium chloride 0.1%, (±)-3-chlorolactic acid 0.2%, pH 7.0
Inoculate the microorganisms shown in Table 1 into a large test tube containing
After culturing with shaking at 26°C for 48 hours, the bacterial cells were collected from the culture medium by centrifugation, and added to 50 mmol phosphate buffer (
After washing with pH 7.0), the bacterial cells were centrifuged again, and then
The bacterial cells are suspended in 50 mmol phosphate buffer (pH 7.0) containing (±)-3-chlorolactic acid to make 10 ml. The final concentration of (±)-3-chlorolactic acid was 0.2%. The reaction mixture was shaken and reacted at 26° C. for 48 hours, and then 3-chlorolactic acid contained in the centrifuged supernatant was analyzed by high performance liquid chromatography. The results are shown in Table 1, and although it varies depending on the strain, 50 to 80% of the 3-
Chlorolactic acid remains, and this residual (R)-(+)-3
-The optical isomer excess (ee) of chlorolactic acid is 100
It was ~38%. The yield was expressed as a percentage of the weight of (±)-3-chlorolactic acid added.

[Table 1]

Example 2 (±)-3-chlorolactic acid 3%, ammonium sulfate 0.2
%, dipotassium phosphate 0.2%, magnesium sulfate 0.05
%, meat extract 0.5%, peptone 1.0%, yeast extract 0.5%, glycerol 2%, manganese sulfate 0.001
%, zinc sulfate 0.001% and the pH was adjusted to 9.0 with sodium hydroxide in a large test tube containing Pseudomonas sp. ) was inoculated and cultured with shaking at 26°C for 48 hours, (+)-3-chlorolactic acid was produced in the culture supernatant at a concentration of 16 mg per ml, and the optical isomer excess rate (e.e. ) was 78%.

Example 3 Pseudomonas aureofaciens ATCC 1
3986 in the same manner as in Example 1, and the supernatant after removing the bacterial cells by centrifugation was
It was concentrated under reduced pressure to 1/0 of the liquid volume, and the pH of the concentrated liquid was adjusted to pH 2.5 with sulfuric acid, and extracted three times with twice the amount of ethyl acetate. The combined extracts were concentrated under reduced pressure and left in a cold room, and the precipitated crystals were filtered off to obtain 1.2 g of brown crystals. The crystals were dissolved in 5 ml of ethyl acetate, and silica gel (1.0 x 3
After adsorption through a column of 0 cm), the column was filled with n-
After washing with 50 ml of hexane, elution was carried out with a mixture of n-hexane and ethyl acetate (9:1 by volume), and (R)
The fractions of -(+)-3-chlorolactic acid were collected, concentrated, and dried to give 180 mg of white crystals. This crystal has a melting point (8
9℃), [α]25D+4.33° (C=10, water),
IR (Nujor) cm-1: 3450, 1722, elemental analysis value (C 28.84, H 3.85, Cl 2
8.36) agrees with the literature value, and (R)−(+
)-3-chlorolactic acid.

Example 4 In Example 1, (±)-3-chlorolactic acid was replaced with (±)-3-chlorolactic acid.
)-3-bromo lactic acid was used, and the results were shown in Table 2. (+)-3 with an optical isomer excess (ee) of 37 to 100% - Bromo lactic acid was produced.

[Table 2]

Example 5 In Example 3, (±)-3-chlorolactic acid was replaced with (±)-3-chlorolactic acid.
)-3-bromo lactic acid was used in the same manner as in Example 3 to obtain crystals of (R)-(+)-3-chlorolactic acid. Melting point 78°C, [α]25D+1.8° (C=10, water),
The optical isomer excess (ee) was 100%.

[0016]

Effects of the Invention According to the present invention, (R)-(+)-3-halolactic acid, which is useful as a raw material for the synthesis of optically active physiologically active substances such as pharmaceuticals, can be efficiently produced from (±)-3-halolactic acid. can do.

Claims (3)

[Claims] [Claim 1] Pseudomonas
as) genus, Arthrobacter
r) genus Escherichia,
Genus Saccharomyces, Genus Schwanniomyces,
Alternatively, a microorganism belonging to the genus Torulopsis and having the ability to produce (R)-(+)-3-halolactic acid from (±)-3-halolactic acid or a processed product thereof may be used to produce (±)-3-halolactic acid. to produce (R
A method for producing (R)-(+)-3-halolactic acid, which comprises collecting )-(+)-3-halolactic acid. 2. The method of claim 1, wherein the substrate is (±)-3-chlorolactic acid and the product is (R)-(+)-3-chlorolactic acid. 3. The method of claim 1, wherein the substrate is (±)-3-bromo lactic acid and the product is (R)-(+)-3-bromo lactic acid.