JPS5931690A - Preparation of optically active lactic acid - Google Patents

Abstract

PURPOSE:To prepare an optically pure lactic acid having optical activity, economically, in an industrial scale, from an inexpensive raw material, with simple operation, by treating racemic 2-halogeno propionic acid successively with specific two kinds of a dehalogenases. CONSTITUTION:An L-2-halo-acid dehalogenase acting specifically to the L- isomer of 2-halogenopropionic acid (abbreviated as 2-halo-acid) is prepared by the extraction and purification of Pseudomonas putida. Separately, DL-2-halo- acid dehalogenase acting to both isomers of 2-halopropionic acid is separated and purified from a bacterial strain belonging to Pseudomanas genus. Racemic 2-halopropionic acid is treated with L-2-halo-acid dehalogenase to obtain a mixture of D-lactic acid and unreacted D-2-halopropionic acid. After separating the components, D-2-halopropionic acid is treated with DL-2-halo-acid dehalogenase to obrain L-lactic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing optically active lactic acid.

The method for producing optically active lactic acid is chemical synthesis.

Fermentation methods and enzyme methods are known. For example, lactic acid obtained by a chemical synthesis method using acetaldehyde and hydrocyanic acid as raw materials is a racemic acid that is a mixture of equal amounts of D and L forms. In order to obtain optically active lactic acid from this fusic lactic acid, it is necessary to separate it into D-lactic acid and r,-lactic acid using optically active amines tr or any optical resolving agent. In this method, the optical resolving agent used is expensive;
Furthermore, since the operation is extremely complicated, it is not an industrially advantageous method for obtaining optically active lactic acid. Also,
Optically active lactic acid can also be obtained by a fermentation method using microorganisms that accumulate D-lactic acid or L-lactic acid in a culture solution. However, this known method is industrially disadvantageous because not only the nutrient source for the microorganisms used is expensive, but also one more operation is required to extract and purify lactic acid from the culture solution.

On the other hand, methods for producing optically active lactic acid using an enzymatic method include 1, for example, Jarna M. Op American e-chemica.
Society magazines (J+Am, Chem, Soc,
) Vol. 102, pp. 7104-7105 (1980), using pyruvic acid as a raw material.

A method for producing D-lactic acid using D-lactate dehydrogenase is described. This method uses nicotinamide adenine dinucleotide (hereinafter N) as a coenzyme of D-lactate dehydrogenase.
It's called AD. ) into the reaction solution. Furthermore, since the coenzyme NAD is expensive, formic acid and formate dehydrogenase must also be added in order to reuse it.

The reaction system is complicated, and this method is not suitable for practical use.

Furthermore, the optical purity of D-lactic acid obtained by this method was 9
The optical purity was about 2%, which was not sufficient.

Therefore, the inventors of the present invention took into account these actual circumstances! ! fruit.

As a result of intensive research aimed at providing a method for producing optically active lactic acid with high optical purity using an inexpensive and easy operation,
By using the optical isomer of 2-halogenopropionic acid as a raw material and using nine reaction systems, especially two enzymes with different stereospecificities for the substrate, all of the objectives of the second generation were achieved. Heading 9 The present invention has been achieved.

Namely, the present invention is a method for producing optically active lactic acid, which is characterized in that, in producing optically active lactic acid, a racemic form of 2-halogenopropionic acid is used as a raw material and an enzyme is allowed to act on one reaction system. .

2-halogenopropionic acid (hereinafter referred to as 2) used in the present invention
-It is called halopropionic acid. ), the carbon t adjacent to the carboxy Vlt/group of propionic acid
Examples include compounds to which one halogen atom is bonded, and preferred examples include 2-chloropropionic acid, 2-bromopropionic acid, and 2-iodopropionic acid.

Examples of enzymes used in the present invention include dehalogenases that act specifically on one of the optical @l'1 forms of 2-halopropionic acid, and enzymes that act on both optical isomers of 2-halopropionic acids. Dehalogenating enzymes are preferred. One example of such an enzyme is 2-haloacid dehalogenase, especially VL-F (Paeu
L-2-haloate dehalogenase, which acts specifically on the L form of 2-haloacids, was extracted and purified from Pseudomonas spp.
- DL-2-haloacid dehalogenase which acts on both optical isomers of haloacid is preferred. Extraction, purification and enzymatic chemical properties of this T,-2-halo acid dehalogenase are described in Agrla, Blol, Chem.
) Mo1.46.837-838.

It has been described once in 1,000 years. Regarding the extraction, purification, physicochemical and enzymatic properties of DL-2-heroate dehalogenase, see Biochemistry Vol. 53, pages 828 and 1.
115, 1981.

The L-2-haloate dehalogenase and D L-2-haloate dehalogenase used in the present invention 10 do not necessarily have to be pure, but can be prepared by using live bacterial bodies collected from a culture solution by a method such as centrifugation, or dried bacteria thereof. Grinding of the body or bacterial body,
It is also possible to use processed materials obtained from processing procedures such as autolysis and sonication, as well as extracts from these microbial cells and crude enzymes obtained from the extracts.

For example, the following method can be employed to produce optically active lactic acid in the present invention. That is, T4-2
- Halo acid dehalogenase catalyzes the dehalogenation reaction of L-2-halopropionic acid.

When 5-D-lactic acid is produced and L-2-helloate dehalogenase is applied to racemic 2-halopropionic acid, mixing of D-lactic acid and unreacted D=2-halopropionic acid is prevented. can get. By separating this D-lactic acid and D-2-halopropionic acid, D-lactic acid can be collected.

Next, DL-2-helloate dehalogenase acts on both optically compatible forms of 2-haloacids, producing 2-hydroxy acids with an inverted configuration from the substrate 2-haloacids. The resulting D-2-halopropionic acid is converted to L-lactic acid by the action of DL-2-heroate dehalogenase, and then
You can collect this. When performing the reaction using these two types of enzymes, the concentration (W/V%) of 2-halopropionic acid is preferably 0.1 to 80. More preferably 1 to 70. Optimally, the reaction may be carried out in the range of 5 to 5096. (2) When using -2-octaloyl dehalogenase, the pH of the reaction solution is preferably 7 to 12. More preferably 8 to 11. Optimally, it should be kept in the range of 9 to 10.5.

In addition, when using DL-2-heroate dehalogenase, the pH of the reaction solution is preferably 6 to 12. Further, preferably 7 to 11. It is preferable to keep it within the range of 8 to 10. As this enzymatic reaction progresses, the pH of the reaction solution decreases, so by adding an alkaline compound, the pH range shown in the table can be maintained. , potassium hydroxide, aqueous ammonia, etc., and any alkaline compound may be used as long as it does not inhibit the enzymatic reaction. Further, the temperature of the reaction solution when carrying out the enzyme reaction is preferably kept in the range of 10 to 60°C, more preferably 2 to 50°C, and preferably 60 to 45°C during one reaction. It is desirable to stir the liquid gently. The required amount of enzyme to be used can be determined by calculating the amount of 2-halopropionic acid used and the time required to complete the reaction. For example, 1
0ml of D-2-chloropropionic acid in 6 hours
The amount of Dr,-2-haloacid dehalogenase required for conversion to lactic acid is approximately 56 units. When adding this enzyme, the required amount may be added one time, or may be added in several portions. Furthermore, if the 9 reactions take a long time, there is a possibility that the 1 reaction liquid will be contaminated by microorganisms, so a preservative may be added to the 9 reaction liquid. Examples of preservatives to be used include:

Examples include sodium azide (NaN, ) + carbolic acid, but any preservative can be used as long as it does not inhibit the enzymatic reaction and does not directly react with lactic acid under the reaction conditions. Furthermore, as a method for separating D-lactic acid and D-2-halopropionic acid from each other, the difference in solubility in a solvent, silica gel carrier, Shiba column chromatography, or ion exchange chromatography can be applied. - Any means can be used as long as lactic acid and D-2-halopropionic acid can be separated from each other.

According to the present invention, optically active lactic acid can be produced in good yield from 2-halopropionic acid, which is an inexpensive raw material. Moreover, surprisingly, the optical purity of the lactic acid obtained by the present invention is close to 100% for both D-lactic acid. This is a lactic acid production method using Sen's enzyme 9, for example, Jhana I
Op American Chemical Society Magazine (J
, Am, Chem, Sec, ) Volume 102,
Since the optical purity of D-lactic acid obtained by the method described in 71114-7105 (1980) is 92%, the present invention is also excellent in that lactic acid with high optical purity can be obtained. ing. Furthermore, the 2-haloyl acid denogenase used in the present invention does not require coenzymes and exhibits only one activity.

For example, enzymes are very easy to operate in that they do not require special factors such as certain metal ions.

The present invention has created a method for obtaining optically pure lactic acid from inexpensive raw materials with easy operation.

The optically active lactic acid obtained according to the present invention 1 is useful as a raw material for the synthesis of physiologically active substances such as drugs and intermediates thereof, and can also be used as a biochemical reagent.

Next, the present invention will be specifically explained using examples.

Note that lactic acid was quantified and fixed by gas chromatography under the following conditions. The temperature at that time was 170°C for the column and 230°C for the injection section.
A glass column (length 2 m, inner diameter 5 ea) with 1 (manufactured by Gascro Kogyo Co., Ltd.) as a filler was used, a hydrogen flame detector (F'I
D). A calibration curve was prepared from the peak area obtained using a lactic acid solution with a known concentration in advance, and the amount of lactic acid in the sample was determined from this. In addition, lactic acid can be confirmed from the retention time in gas chromatography, but in addition to gas chromatography, for lactic acid isolated from one reaction solution, 'H-N MR spectrum measurement and processing in deuterium water are required. Lactic acid was identified by measuring its spectrum. The optical purity of lactic acid was determined by Methods Openzymatic Analysis.
fFh>zymatic Analyia) Vol. 5, pp. 1446 and 1492 (1974), ie.

It was determined by specifically separating and quantifying D- and L-lactic acid using an enzyme. For example, if the amounts of D- and L-lactic acid in the test solution measured by the above method are +A) and ('81 wg, respectively (A>B L), the optical purity (%) of lactic acid in the test solution is ( AB/A+B) It was calculated by calculating X100 and ll'.

Example 1 L-2-haloacid dehalogenase was prepared in advance using Agr.
ic, l'1io1. Chem, ) Volume 46,
837-838 (1982), and I) L-2-haloacid dehalogenase was prepared by the method described in Biochemical Journal, Vol. 56, pp. 828 and 1115 (1981). It was prepared using Method 1.

], 1 mol of D L -2-chloropropionic acid and 0
．． 059 sodium azide was dissolved in water, the pH was adjusted to 10.5 t using sodium hydroxide, and then water was added to adjust the pH to 100-. About 1 cup of this solution obtained above
Add 0 units of L-2-heroate dehalogenase.

The solution was kept at 50° C. for one hour while being gently stirred.

The pl+ of the solution was kept in the range 10-10.5 by adding 5N sodium hydroxide solution.

After about 12 and 24 hours, add 1 bottle to the above reaction solution.

Approximately 10 units of L-2-heroate dehalogenase were added in each case. Approximately 56 hours after the start of this reaction, concentrated hydrochloric acid was added to the reaction solution to adjust the pH to 2, and the mixture was dried under reduced pressure using a rotary evaporator, and ethanol was added thereto.
The ethanol-soluble fractions were collected and the ethanol was removed by evaporation. The resulting viscous liquid is dissolved in water and the formic acid type])
oweX 1 column (inner diameter 61. length 4 fl f
f)) After passing 1 liter of distilled water through the column, it was eluted with 1.5 liters of 11.2N formic acid solution, and further eluted with 2 liters of 1N formic acid solution. 0
．． The 2N formic acid elution fractions were collected, and water and formic acid were removed by evaporation using a rotary evaporator to obtain 4.059 D-lactic acid.

Next, water and formic acid were removed by evaporation from the 1N formic acid elution fraction in the same manner, and the obtained liquid and 0.059 sodium azide were dissolved in water, and the pH was adjusted using sodium hydroxide.
9.5 and water was added to make 100gj. Approximately 10 units of DL-2-heroate dehalogenase was added to this solution, and the solution was kept at a temperature of 5(]c with gentle stirring.

The pH of the solution was maintained in the range 9-9.5 by adding 5N lyllium hydroxide solution. After about 12 and 24 hours, about 10 units of DI, -2-haloacid dehalogenase were added. Approximately 36 hours after the start of the reaction, the pH of the reaction solution was adjusted to 2.0 using concentrated hydrochloric acid, and then
The mixture was dried under reduced pressure using a rotary evaporator, ethanol was added thereto, the ethanol-soluble fraction was collected, and -ethanol/I/ was removed by evaporation to obtain 3.6517 L-lactic acid.

Gas chromatography, 'U-NMR spectrum' and IR of D- and L-lactic acid obtained as in 1.2.
All of the analysis results such as spectra were consistent with the analysis results of commercially available genuine lactic acid, confirming that it was optically active lactic acid. Further, the optical purity of D- and L-lactic acid was 99% and 98%, respectively.

Patent applicant: Unitika Co., Ltd. 13-

Claims (2)

[Claims] (1) When producing optically active lactic acid, 2-
Using racemic halogenopropionic acid. A method for producing optically active lactic acid, characterized by allowing an enzyme to act in a reaction system. (2) Dehalogenation enzyme in which #l-element acts specifically on one optical isomer of 2-halogenopropionic acid and dehalogenation that acts on both optical isomers of 2-halogenopropionic acid. Claim 1 which is an enzyme
Manufacturing method described in section.