JPS61111699A - Production of optically active alpha-chloropropionic acid - Google Patents

Abstract

PURPOSE:To prepare the titled substance having high optical purity, easily, by hydrolyzing DL-alpha-chloropropionic acid ester in the presence of a cultured product of microbial strain belonging to Bacillus genus, Staphylococcus genus or Enterobacter genus. CONSTITUTION:A microbial strain such as Bacillus megaterium, Bacillus cereus, Staphylococcus warneri, Enterobacter cloacae, etc. is cultured, and a DL-alpha- chloropropionic acid ester is hydrolyzed in the presence of the cultured product, cultured microbial cells separated from the cultured product, or the extract of the cultured microbial cell. The reaction is carried out in an aqueous solution containing a buffering liquid, and the product is recovered by extraction or distillation.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing optically active α-chloropropionic acid.

[Conventional technology]

Optically active α-chloropropionic acid or its ester is used as a raw material for pharmaceuticals or agricultural intermediates.

This method for producing optically active α-chloropropionic acid and its esters has already been carried out by converting lactic acid into an excess amount of thionyl chloride lV.
A method is known in which this effect is applied. (Unexamined Japanese Patent Publication No. 56-7
(No. 745 Publication) [Problems to be Solved by the Invention] However, this method requires a large amount of thionyl chloride and generates troublesome hydrochloric acid gas and sulfur dioxide gas as reaction by-products. Appropriate methods of disposing of such by-products had to be taken care of. Furthermore, an effective reaction system requires the addition of a suitable base, such as pyridine or dimethylformamide, and methods for separating reaction products and treating bases can make the process more complicated.

[Means for solving problems]

The present inventor improved the drawbacks of the above-mentioned production method and conducted intensive studies on a more efficient production method of optically active α-chloropropionic acid 8 and its ester. As a result, Bacillus (
Bacillus genus, Nutaphylococcus (stap)
hylococcas) or Enterobacter (Kn
A hydrolysis reaction of D,L-α-chloropropionate is carried out in the presence of a culture of a strain belonging to the genus Terobacter, a culture of bacteria isolated from the culture, or an extract from the culture of bacteria. It was discovered that only mu-chloroglopionic acid ester μ was involved in the reaction to produce L-chloropropionic acid, and the present invention was completed based on this discovery.

Many W strains belonging to the genus Pacillus, Staphylococcus, and Enterobacter are used in the method of the present invention, but the strains used in the examples described below are Bacillus megaterlum,
Panrus cereus (B-a ereus), Staphylococcus warneri (staphylococca)
swarneriJ, and Enterobacter cloacani (DI'lt7σrO and αctor c oac
ae). These strains are available from the American Type Culture Collection.

Cultivation of this bacterial strain is carried out under aerobic conditions such as shaking culture or aerated agitation culture. Culture temperature is usually 20-35℃
It is. The medium is preferably pas, s~&5, and the culture period is usually 1 to 3 days.

Any type of nitrogen source and nitrogen source used in the culture medium may be used as long as they are available. That is, as carbon sources, glucone, fructose, sucrose,
Starch hydrolysates, various carbohydrates and organic acids such as molasses, acetic acid, and ethanol, 7-coles, and the like can be used. Nitrogen sources include ammonia, various inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium acetate, or meat extract, yeast extract, di-
Natural organic nitrogen sources can be used, such as steep liquor, casein hydrolyzate, and fishmee p1 soybean meal digest.

Many natural organic nitrogen sources can be both nitrogen and carbon sources.

Further, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, etc. may be used as necessary.

The enzyme source used in the present invention may be the culture of the present strain as it is, live bacterial cells collected from the culture solution by method E such as centrifugation, dried bacterial cells, or crushed or autologous bacterial cells. Processed bacterial cells obtained by treatments such as digestion and sonication, extracts from these bacterial cells, and crude enzymes obtained from the extracts can be used.

Of course, these immobilized enzymes or immobilized bacterial cells may also be used.

The hydrolysis reaction of α-chloropropionate is carried out in an aqueous solution containing a buffer solution. The reaction temperature is usually 20 to 45°C, preferably 25 to 55°C, and the pH of the reaction solution is usually 5 to 10, preferably s to Z5.

Alcohol of α-chloropropionate ester Methyl α-chloropropionate, whose alcohol moiety has 1 carbon number, has the fastest hydrolysis reaction rate, but the optical purity of the reaction product tends to decrease slightly. . Therefore, if it is necessary to maintain a high degree of optical purity, it is better to use ester μ having 2 or more carbon atoms.

As a method for recovering the product from the reaction solution, ordinary extraction separation and distillation separation methods can be applied, but the method is not necessarily limited to these methods. For example, α-ri cr a fa bio:/
L- produced by hydrolyzing acid ethyl μ
α-chloro7derobionic acid, ethanol, unreacted 1α-chloropropylene.

Ethyl pionate is obtained by extracting the reaction solution with n-hexane.
- By separating the hexane and aqueous layers, n-
Unreacted D-α-chloroglopionic acid ethyl μ in the hexane layer
Add ethanol to the aqueous solution layer, L-α-chloroglobion L
jZ, #. J: Hee! After separating inorganic salts such as monophosphate, diethyl ether was added to the aqueous solution layer, and ethanol and L-
After separating α-chloropropionate ethyl p into a diethyl ethanol layer and the other aqueous solutions, each solvent was evaporated and separated using an evaporator to obtain pure α.
-Chloropropion, D-α-ethyl chloropropionate can be obtained. Ethyl 200-α-chloroglopionate may be used as an intermediate as it is, but D-α-chloropropionic acid can be produced by an ordinary chemical hydrolysis reaction. n-hexane may be substituted with normal hydrophobic hydrocarbons, n-pentane,
N-hebutane, n-octane, benzene, toluene, etc. are preferably used. Can be substituted with diethyl ether.

In order to avoid rapid decrease in PA in the reaction system as the hydrolysis reaction of α-chloropropionic acid ester progresses, it is desirable to use a mild solution with an appropriate α degree in the aqueous solution.

The present invention will now be explained with reference to Examples.

〔Example〕

Example 1 Bacillus megaterium (Bacillus megaterium)
eriumJ, Pasillus cereus (Bacillus supra)
8 cereus), Staphylococcus warneri (
Staphylococcus warneriJ, Enterobacter cloacani (Enterobacter, e.g.
r cloacae) in 100 m of a medium with the following composition.
A platinum loopful of inoculation was placed in an Erlenmeyer flask containing 300 ml of 100 ml of chlorine, and cultured at 30° C. for 24 hours.

Medium composition Meat extract 1.0% Peptone a. 5% yeast extract (L1% initial pq & 9) The culture solution was centrifuged, and the bacterial cells were collected and subjected to the next hydrolysis reaction.

D,L-α-chloropropionate ethyl varnish f)V
5. O gr , KHzPO4 5. The aqueous solution containing Q gr was adjusted to PI (&8) with a KOH aqueous solution, and the total volume was adjusted to 100 ml.
The reaction was carried out at °C for 24 hours with gentle shaking. After the reaction was completed, the reaction solution was extracted with n-hexane to separate unreacted entel, and the n-hexane layer and the aqueous reaction solution layer were measured separately using gas chromatography and a spectrophotometer. The measurement results are shown in Table-1.

Table 1 1. Pacifus megaterium 1.IZ Basilanus cereus 0.55 Staphylococcus 1.4 warneri 4. Enterobacter chloe CL2 Acae from the n-hexane extraction layer contains optically active unreacted α -Chloropropion was ethyl esterified.

Example 2 α-chloropropionyl ethyl used in Example 1 was converted to α-
The same experiment was carried out using methyl chloro10pionate instead. The results are shown in Table 2e.

Table 2 1. Pacillus megaterium 1.6 [Effects of the invention] By introducing a new process using highly selective microorganisms in the reaction process, optically active purity can be improved from D,L-α-chloropropionic acid ester. α-chloropropionic acid with a high content can be easily produced.

Patent Applicant Bundling Stock Company Procedures
Amendment 1. Description of the case 1982 Patent Application No. 230310 2 Title of the invention Process for producing optically active α-chlorobrobionoic acid 5 Amendment person Specification “Claims” and “Detailed Description of the Invention” σ) Patent The scope of claims is amended as shown in the attached sheet.

(2) The last line of the second page of the specification and the 10th line of the third vane
Line "Bacillus < Bacillus ) @ J"
terium) species, Bacillus cereus (Bacillus cereus)
lus cereus) species”7 amended.

(3) From line 11 on page 6 to line 3 on page 7, "The reaction is complete...you can do it." is separated using a separating funnel.The n-hexane layer contains unreacted ethyl-α-chloropropionate, and the aqueous layer contains lζethanol, L-α-chloropropionic acid,
and buffer IJ heptase, inorganic salts.

Next, diethyl ether is added to the aqueous solution layer for extraction, and ethanol and L-α-chloropropionic acid are extracted into the diethyl ether layer. After separating this diethyl ether layer, pure L-α-chloropropionic acid is obtained by evaporating diethyl ether and ethanol using an evaporator.

On the other hand, unreacted ethyl D-α-chloropropionate is n
- Can be separated and obtained from the hexane layer. ” he corrected.

(4) From the second line from the bottom of page 7 to the third line of page 8, "Bacillus megaterium proboscis... Φ" is changed to "Bacillus megaterium/proboscis..."
erium ) (ATCC89), Bacillus cereus (ATCC2
), Staphylococcus +7- Neri (3taphy
lococus warneri) (ATCC27
836), Enterobacter
oba+4er cloacae) (ATCC22
2) is corrected as follows.

(5) Same page 8'; 2nd line from F to 9th vane 2nd line "Unreacted ester φ...measured." is changed to "Unreacted ester was removed from the reaction solution. Reaction solution After extracting the aqueous layer with diethyl ether, ethanol and diethyl ether contained in the diethyl ether layer were removed by evaporation to obtain L-α-chloropropionic acid.

The amount of product was determined by analyzing the aqueous layer by gas chromatography, and the light intensity of the product was measured by a light meter. ” he corrected.

(6) The 8th line from the bottom of the 9th page = “2L5j” = “Produced α-chloropropionic acid [α]
° (The optical purity was 100%)".

Attachment Claims Staphylococcus (5taphY1ococcas)
genus, or Enoterobacter (EnterO4) aCt
D,L-α-chloropropionic acid ester is hydrolyzed in the presence of a culture of a strain belonging to the genus D, L-α-chloropropionate in the presence of a culture of a bacterial strain, a culture of bacteria isolated from the culture, or an extract from the culture of bacteria. A method for producing optically active α-chloropropionic acid.

Claims (1)

[Claims] In the presence of a culture of a strain belonging to the genus Bacillus, the genus Staphylococcus, or the genus Enterobacter, or a cultured bacterial cell isolated from a culture, or an extract from the cultured bacterial cell. A method for producing optically active α-chloropropionic acid, which comprises hydrolyzing D,L-α-chloropropionic acid ester.