JP2936551B2 - Method for producing (R)-(+)-3-halolactic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to (R)-(+)-3-
The present invention relates to a method for producing halolactic acid. (R)-(+)-3-halolactic acid is a substance useful as a raw material for synthesizing optically active physiologically active substances such as pharmaceuticals.

[0002]

2. Description of the Related Art As for a biochemical preparation method of optically active (R)-(+)-3-chlorolactic acid, there is known a production method by asymmetric reduction of chloropyruvic acid using lactate dehydrogenase. (Journal of American
Chemical Society, 104, 44
52, 1982). In this method, since NADH is required as a coenzyme during the reaction,
It is necessary to supply H continuously, and a method of combining with the NADH regeneration system is used. However, this type of enzymatic reduction reaction combining the NADH regeneration system has many problems that are difficult to implement industrially. As a second method, (±) -3-halo-1,2
A method for producing optically active (R)-(+)-3-halolactic acid by asymmetric microbial oxidation of propanediol is known (Japanese Patent Publication No. 59-220193; Japanese Patent Publication No. 61-268197). No .: Journal of Fe
mentation Technology, 64
Vol. 251-254, 1986). Microorganisms of the genus Geotricum, Trichosporon, Endomyces, Hansenula, Candida, Sporidiobolus, and Roderomyces have been used. This method has the disadvantage that the yield is low. As a third method, (±) -3-
A method of obtaining (R)-(+)-3-halolactic acid by allowing a microorganism to act on halolactic acid (Japanese Patent Publication No. 61-268197) is known. Microorganisms include Candida, Cryptococcus, Endomyces, Hansenula, Roderomyces,
Pichia, Spolidibolus, Trichosporone, Alcaligenes, Bacillus, Corynebacterium, Aphanoacuscus, Aureobasidium, Baxella, Bacgia,
Microorganisms belonging to the genera Bisosilamis, Floridium, Cladosporium, Coniochetium, Eurotium and Gliocladium have been used. In this method, the optical purity is low when the yield is good, and the yield is low when the optical purity is high. As a fourth method,
A method is known in which a 2-haloacid dehalogenase is allowed to act on 2,3-dihalopropionic acid (Japanese Patent Application Laid-Open No. 2-113890), in which the cost of 2,3-dihalopropionic acid as a raw material is reduced. Has difficulties.

[0003]

As described above, since the prior art still has a difficulty in industrial implementation, a more efficient method for producing (R)-(+)-3-halolactic acid has been established. As a result of repeated studies to accomplish this, the present invention has been completed.

[0004]

According to the present invention SUMMARY OF], Pseudomonas (Pseudomonas) genus Arthrobacter (Arthrobacter) genus Esserihia (Escherichi a) the genus Saccharomyces (Sa
ccharomyces) belongs to the genus or Schwanniomyces (Schwanniomyces) genus, (±) -
By contacting a microorganism, a culture solution or a processed product thereof with a microorganism capable of producing (R)-(+)-3-halolactic acid from 3-halolactic acid with (±) -3-halolactic acid, By producing (R)-(+)-3-halolactic acid and collecting it, (R)-(+)-3-halolactic acid can be produced.

Hereinafter, the present invention will be described in detail. Microorganisms used in the present invention, Pseudomonas, Arthrobacter, Esserihia genus, belonging to the genus Saccharomyces or Gerhard <br/> Waniomisesu genus, acts on (±)-3-halo acid (-) - 3- Consumption conversion of halolactic acid (R)-(+)
-3-halolactic acid remains, and as a result, (R)-(+)
Any microorganism capable of producing -3-halolactic acid may be used. As a specific example, Pseudomonas aureofaciens ( Pseudomonas aureof)
aciens ) ATCC 13986, Pseudomonas dehagen
logenans) NCIB 9061, Arthrobacter citreus (Arthrobacter cit
reus ) ATCC 11624, Arthrobacter
Gloviformis ( Arthrobacter glob)
ifomis ) ATCC 8010, Esselicia
Escherichia coli ATCC
11303, Saccharomyces svalerieri ( Sacc)
haromyces chevalieri ) IFO
0222, Schwaniomyces oxydentalis ( Sc)
hwanniomyces occidentali
s) etc. ATCC 2607, 4 and the like.

[0006] Mutants obtained by mutating these strains by an artificial mutation method, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagenic agent and the like can also be used.

[0007] In culturing the microorganism used in the present invention, an ordinary method for culturing a microorganism is generally used. As the medium, any of a synthetic medium and a natural medium can be used as long as the medium contains a carbon source, a nitrogen source, an inorganic substance, and substances necessary for growth of the microorganism that can be assimilated by the microorganism. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Further, depending on the assimilation ability of the microorganism, hydrocarbons, alcohols, organic acids and the like are also 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 powder, casamino acid and the like are used alone or in combination. others,
If necessary, add inorganic salts such as salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate, etc. be able to. Cultivation is carried out under aerobic conditions such as shaking culture or aeration-agitation culture at a temperature of 4 to 43 ° C, preferably 20 to 35 ° C.
C., pH 4 to 10, preferably 6.0 to 9.5, and usually completes in 1 to 10 days. The pH of the medium is preferably adjusted using sodium hydroxide or the like.

The contact of the microorganism used with (±) -3-halolactic acid can be carried out during the growth and culture of such a microorganism, or by adding (±) -3-halolactic acid to the grown culture. It can also be reacted. Further, after separating the cells from the culture, the cells or the processed cells were compared with (±) -3-
Halolactic acid can also be contacted in a suitable medium, for example, in a buffer. The treatment of the culture solution or the cells can be carried out without impairing the (-)-3-halolactic acid conversion activity in the cells.
A method in which the enzymatic reaction proceeds more easily is appropriately selected and performed. Specific examples of the processed product include a concentrate, a dried product, a processed product of a surfactant and / or an organic solvent, a cell obtained by centrifuging the culture, a crushed cell, and a dried cell. , Acetone-treated product, surfactant and / or organic solvent-treated product, lysed enzyme-treated product, immobilized cells or an enzyme sample extracted from the cells.

The (±) -3-halolactic acid used in the present invention is used in a free form or in a salt form such as a hydrochloride.
There is no particular limitation on the concentration of (±) -3-halolactic acid,
0.1 to 10% (weight to volume) is preferred. (+)-3
When carrying out the reaction for producing -halo lactic acid, the microorganism or a processed product thereof and (±) -3-halo lactic acid are subjected to 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 is usually completed 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, it is preferable to use a buffer solution as a reaction medium or adjust the pH during the reaction using sodium hydroxide or the like.

[0010] The reaction also occurs during the cultivation of the microorganism (±) -3.
-The method can also be carried out by adding halolactic acid to the culture solution. In this case, the amount of (±) -3-halolactic acid added may be such that the same concentration as in the reaction using the above-mentioned cells or the processed product thereof is obtained. Further, if necessary, the enzyme reaction can be promoted by adding an amount of the above-mentioned surfactant in the culture solution which does not inhibit the growth of the microorganism. After completion of the reaction, the (+)-3-halolactic acid generated (remaining) in the reaction solution is isolated by a known method, for example, means such as adsorption, desorption, concentration, and crystallization to an ion exchange resin.

[0011]

Examples of the present invention will be described below. (R)-(+)-by high performance liquid chromatography in Examples
The conditions for the fractional analysis of 3-chlorolactic acid and (S)-(-)-3-chlorolactic acid are as follows. Column: MCI GEL CRS 10W, 4.6 × 5
0 mm (manufactured by Mitsubishi Chemical), eluent: micromolar copper sulfate,
Flow rate: 1 ml per minute, sample volume: 10 microliters, detection: UV (254 nm). Example 1 Glucose 2%, diammonium phosphate 1%, phosphoric acid 1
10 m sterile medium with a composition of 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 shaking culture at 26 ° C. for 48 hours, cells were collected from the culture by centrifugation, washed with 50 mM phosphate buffer (pH 7.0), centrifuged again, and The cells are suspended in a 50 mM phosphate buffer (pH 7.0) containing ±) -3-chlorolactic acid to make 10 ml. The concentration of (±) -3-chlorolactic acid was final 0.2%. After the reaction mixture was shaken at 26 ° C. for 48 hours to react, 3-chlorolactic acid contained in the centrifuged supernatant was analyzed by high performance liquid chromatography. The results are as shown in Table 1. Although it depends on the strain, 50-80% of 3-chlorolactic acid remains, and the remaining (R)-(+) remains.
The optical isomer excess (ee) of -3-chlorolactic acid is 1
It was 00-38%. The yield was shown as a percentage with respect to the weight of the added (±) -3-chlorolactic acid.

[Table 1]

Example 2 (±) -3-chlorolactic acid 3%, ammonium sulfate 0.2
%, 2% potassium phosphate, 0.05% magnesium sulfate
%, Meat extract 0.5%, peptone 1.0%, yeast extract 0.5%, glycerol 2%, manganese sulfate 0.001
%, Zinc sulfate 0.001%, and a large test tube containing 10 ml of a sterilized medium adjusted to pH 9.0 with sodium hydroxide, in a large test tube, containing Pseudomonas sp. H1-1 (Microtechnical Laboratory No. 12128) ) Was inoculated and cultured with shaking at 26 ° C. for 48 hours, (+)-3-chlorolactic acid was produced at a concentration of 16 mg / ml in the culture supernatant, and the optical isomer excess (ee) was obtained. .) Was 78%.

Example 3 Pseudomonas aureofaciens ATCC 13
Reaction liquid 1 obtained in the same manner as in Example 1 using 986
The supernatant from which the cells were removed from 800 ml by centrifugation was 20
Concentrate the solution under reduced pressure to one-half the volume, and adjust the pH of the concentrated solution with sulfuric acid.
The mixture was extracted three times with twice the amount of ethyl acetate as H2.5. The extracts were combined, concentrated under reduced pressure, allowed to stand in a cold room, and the precipitated crystals were separated by filtration to obtain 1.2 g of brown crystals. This crystal is
Dissolve in ethyl acetate and silica gel (1.0 × 30
cm), the column was washed by flowing 50 ml of n-hexane and eluted with a mixture of n-hexane and ethyl acetate (9: 1 by volume) to give (R)-
The (+)-3-chlorolactic acid fraction was collected, concentrated and dried to give 180 mg of white crystals. This crystal has a melting point (89
° C), [α] ²⁵ _D + 4.33 ° (C = 10, water), I
R (Nujor) cm ^-1 : 3450, 1722, elemental analysis values (C 28.84, H 3.85, Cl 28.3)
From the data of 6), it is consistent with the literature value, and (R)-(+)-3
-Identified as chlorolactic acid.

Example 4 The results obtained in the same manner as in Example 1 except that (±) -3-bromolactic acid was used in place of (±) -3-chlorolactic acid are shown in Table 2. In this, (+)-3-bromolactic acid having an optical isomer excess (ee) of 37 to 100% was produced.

[Table 2]

Example 5 (R)-(+)-was prepared in the same manner as in Example 3 except that (±) -3-bromolactic acid was used instead of (±) -3-chlorolactic acid. Crystals of 3-chlorolactic acid were obtained. Melting point 78 ° C., [α] ²⁵ _D + 1.8 ° (C = 10,
Water) and the optical isomer excess (ee) was 100%.

[0016]

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

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 41/00 C12R 1:19) (C12P 41/00 C12R 1:85) (58) Investigated field (Int.Cl. ⁶ , DB name) C12P 41/00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. Pseudomonas (Pseudomon)
as) genus, Arthrobacter
r) genus, Escherichia genus,
A Saccharomyces (Saccharomyces) belonging to the genus
There is Schwanniomyces (Schwanniomyce
s) It belongs to the genus, and (R)-
A microorganism having the ability to produce (+)-3-halo lactic acid or a processed product thereof is contacted with (±) -3-halo lactic acid to collect the (R)-(+)-3-halo lactic acid produced. A process for producing (R)-(+)-3-halolactic acid, characterized in that: 2. The method according to claim 1, wherein the substrate is (±) -3-chlorolactic acid.
The method according to claim 1, wherein the product is (R)-(+)-3-chlorolactic acid. 3. The method according to claim 1, wherein the substrate is (±) -3-bromolactic acid,
The method according to claim 1, wherein the product is (R)-(+)-3-bromolactic acid.