JPH0372279B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to 2-halogenocarboxylic acid (hereinafter referred to as 2-halogenocarboxylic acid)
It is called halo acid. The present invention relates to a method for producing an optically active 2-hydroxy acid using a 2-halo acid halide hydrolase having the property of acting on the D-isomer of the present invention. (Prior Art) 2-halo acid halide hydrolase is an enzyme used to produce optically active 2-hydroxy acids from 2-halo acids. As a conventional method for producing this optically active 2-hydroxy acid, a method for obtaining D-form and L-form 2-hydroxy acids using an optical resolution agent such as optically active amines is known.
However, this known method is not industrially advantageous because the optical resolving agent used is expensive and the operation is extremely complicated. Therefore, 2-halo acid halide hydrolase is used. Using this enzyme, optically pure 2-hydroxy acids can be easily obtained from 2-halo acids in high yields, and optically active 2-hydroxy acids can be easily obtained from 2-halo acids in high yields. This is a very advantageous method for industrially producing hydroxy acids. (Problems to be Solved by the Invention) However, conventionally known 2-halo acid halide hydrolases are
Volume, 428 pages (1968) and Journal of European Biochemistry (J.Eur.
Biochem.) Vol. 21, p. 99 (1971), etc., all of them act only on the L-form of 2-haloacids, producing D-form 2-hydroxy acids, and D
It had no effect on 2-haloacids in the body. Therefore, from a biochemical point of view, the production of L-form 2-hydroxy acids, which are more important than D-form, has not been possible using conventional 2-halo acid halide hydrolases. In addition, the raw material 2-haloacid exists as a racemate, which is a mixture of the D-form and the L-form, so when conventional 2-halo acid halide hydrolase is used, the D-form 2-haloacid is the raw material. It remained as it was, and was economically disadvantageous. (Means for solving the problem) Therefore, from this perspective, the present inventors
Optical activity 2 with high optical purity and low cost and easy operation
- As a result of intensive research aimed at providing a method for producing hydroxy acids, we found that using a racemic form of 2-halo acid as a raw material and using two enzymes with different stereospecificities for the substrate in the reaction system, the above-mentioned results were obtained. Find out that the purpose will be achieved. The invention has been completed. That is, the present invention uses a racemic form of a 2-halo acid as a raw material when producing an optically active 2-hydroxy acid using a 2-halo acid halide hydrolase that has the property of acting only on the L form of a 2-halo acid. The gist of the present invention is a method for producing optically active 2-hydroxy acids, which is characterized in that a 2-halo acid halide hydrolase having a property of acting on the D-form of 2-halo acids is used in the reaction system. In the present invention, 2-haloacid refers to the general formula R.
Compounds represented by CHX・COOH (X represents a halogen atom) or R・CX ₂・COOH, such as monochloroacetic acid, monobromoacetic acid, monoiodoacetic acid, dichloroacetic acid, dibromoacetic acid, or trichloroacetic acid. derivatives of acetic acid, such as
Propionic acid derivatives such as 2-chloropropionic acid, 2-bromopropionic acid, 2,2-dichloropropionic acid, 2-chlorobutyric acid, 2-bromobutyric acid,
Examples include derivatives of butyric acid such as 2,2-dichlorobutyric acid, and derivatives of valeric acid such as 2-chlorovaleric acid, 2-bromovaleric acid, and 2,2-dichlorovaleric acid. In addition, it may be a compound in which one or two halogen atoms such as chlorine or bromine are bonded to the second carbon atom of a carboxylic acid having 5 or less carbon atoms, or when the carboxylic acid is acetic acid, It may be a compound in which 1 to 3 are bonded. Next, the physicochemical properties of 2-halo acid halide hydrolase, which has the property of acting on the D form of 2-halo acid used in the present invention, will be shown. (1) Effect L-R・CHX・COOH+H ₂ O→D −R・CHOH・COOH+H ⁺ +X ^- D−R・CHX・COOH+H ₂ O→L −R・CHOH・COOH+H ⁺ +X ^- R・CX ₂・COOH+H ₂ O→R・CO・COOH +2H ⁺ +2X ^- Catalyzes the above reaction. In addition, R in the above general formula is H, H ₂ , HX,
Represents an atomic group such as X ₂ , CH ₃ , C ₂ H ₅ , C ₃ H ₇ , etc.
represents a halogen atom such as chlorine or bromine. It acts on L-2-haloacid in the presence of water to hydrolyze the halogen atom to produce D-2-hydroxyacid. When the 2-haloacid is in the D form, the 2-haloacid in the L form is
Produces hydroxy acids. Furthermore, when the substrate is a 2-dihalo acid, a 2-keto acid is produced. (2) Substrate specificity The Michaelis constants (Km values) for D, L and DL-2-chloropropionic acids are 4.8 and 4.8, respectively.
1.1, 3.2mM. In addition, carbon number is 2~
Up to five 2-haloacids serve as substrates, and DL-2
-The reaction rates of other substrates when chloropropionic acid is taken as 100 are as shown in the following table.

[Table] (3) Optimal PH PH approx. 9.5 (measured at 30℃ using DL-2-chloropropionic acid as a substrate) (4) Stable PH range PH approx. 6 to 12 (4℃, 24 hours elapsed) (5) Suitable temperature for action and heat resistance The activity increases as the temperature increases from 20℃ to 45℃ at PH9.5, but when treated at 45℃ for 15 minutes, Activity decreases to less than 50%. (6) Potency measurement method: 0.1M Tris sulfate buffer (PH9.5) containing 50 micromoles of D, L-2-chloropropionic acid.
Add 1~100 μ of oxygen solution to 2 ml and incubate at 30℃ for 10 minutes.
After reacting for a minute, 0.1 ml of 3.6N sulfuric acid was added to stop the reaction. Chlorine ions in this reaction mixture were determined by the thiocyanate method. That is, 10% of mercury thiocyanate containing 0.1% per ml of the reaction mixture or its diluted solution.
1 ml of dioxane solution containing % ethanol, and 0.4 ml of 6N nitric acid solution containing 8% iron alum.
ml was added, the absorbance at 460 nm was measured, and the chloride ion concentration was determined from the calibration curve. One unit was defined as the amount of enzyme that produced 1 micromole of chloride ions per minute at 30°C. (7) Molecular weight The molecular weight was approximately 50,000 as determined by Sephadex G-150 gel chromatography. (8) Inhibition, Activation, and Stabilizer When various metal ions or additives were added and the enzyme activity was measured, as shown in the table below, it was inhibited by mercuric chloride, zinc, manganese, nickel, etc. Furthermore, no activator or stabilizer specific to this enzyme has been found so far.

【table】

[Table] Relative activity:%
In order to produce the above-mentioned 2-halo acid halide hydrolase, the following method can be adopted. That is, it can be obtained by culturing bacteria belonging to the genus Pseudomonas and collecting the above-mentioned 2-halo acid halide hydrolase from the culture. Preferred specific examples of bacteria belonging to the genus Pseudomonas include, for example, a new strain of Pseudomonas that the present inventors isolated from soil in Uji City, Kyoto Prefecture.
UK113 strain (Feikoken Bacterium No. 5666) is mentioned.
This strain has a particularly high ability to produce the above-mentioned 2-halo acid halide hydrolase, and is suitable for practical use. As a nutrient source for the medium used for culturing the bacteria belonging to the genus Pseudomonas, any nutrient source that can be assimilated by the bacteria can be used, and as a carbon source, for example, glucose,
Glycerin, alcohols, fats and oils, fatty acids, and 2-halo acids can be used, and nitrogen sources include, for example, ammonium sulfate, ammonia, ammonium chloride, ammonium phosphate, various amino acids,
Inorganic or organic substances such as peptone, meat extract, yeast extract, etc. can be used. Furthermore, as inorganic salts, for example, potassium, sodium, phosphoric acid, iron, zinc,
Various salts such as magnesium, manganese, copper, calcium, cobalt, molybdenum, etc., trace metal salts, corn staple liquor, vitamins, nucleic acids, etc. may be used as necessary, and a general nutrient medium for bacteria is used. can. Using these media, this strain is preferably grown.
10-36℃, more preferably 20-33℃, optimally
The 2-halo acid halide hydrolase of the present invention is collected from the culture obtained by culturing aerobically at 20 to 30°C for about 5 to 48 hours. It can be collected at any stage, including processed products, crude enzymes, and purified enzymes. As a purification method, a normal enzyme purification method can be used. That is, after obtaining bacterial cells by centrifugation, etc., the bacterial cells are subjected to Manton-Gorlin, Dynomil,
After disrupting the bacteria using a French press, ultrasonication, etc., cell debris is removed by centrifugation to obtain a cell extract, which is then treated with streptomycin sulfate or protamine sulfate, followed by salting out with ammonium sulfate or the like, or with acetone, etc. For purification, ion exchange chromatography such as DEAE cellulose column,
This can be carried out in combination with adsorption chromatography using a hydroxyapatite column or the like, gel chromatography using a sepadex chromatography or the like. In this way, the above-mentioned 2-halo acid halide hydrolase can be isolated and purified. Further, the 2-halo acid halide hydrolase that has the property of acting only on the L-form of 2-halo acids used in the present invention is described, for example, in the above-mentioned Journal of Biological Chemistry (J.
Biol.Chem.) Vol. 243, p. 428 (1968) and Journal of European Biochemistry (J.Eur.Biochem.) Vol. 21, p. 99 (1971).
2 extracted and purified from Pseudomonas putida as described in
- Halo acid halide hydrolase. One of the 2-halo acid halide hydrolases used in the present invention acts on the D form of 2-halo acid, and therefore acts on L-2-halo acid to produce D-2-hydroxy acid, and further , acts on D-2-haloacid to produce L-2-hydroxy acid. Therefore,
Enables the production of L-2-hydroxy acids, which was impossible using conventional 2-halo acid halide hydrolases that act only on L-2-halo acids and do not act on D-2-halo acids. be able to. For example, in order to collect optically active lactic acid using DL-2-chloropropionic acid as the 2-halo acid, first, a conventionally known 2-halo acid halide hydrolase that acts only on the L-form is used. hand,
After converting L-2-chloropropionic acid to D-lactic acid, D-lactic acid and remaining D-2-chloropropionic acid are separated. As a separation method in this case, for example, a difference in solubility in a solvent or an ion exchange method can be applied. Next, the separated D-
2-chloropropionic acid may be converted to L-lactic acid using 2-halo acid halide hydrolase, which has the property of acting on the D form of 2-halo acid. In this way, when DL-2-chloropropionic acid is used as a raw material, optically active lactic acid can be collected in high yield, and L-lactic acid can be produced, which was impossible with conventional enzymes. becomes possible. Furthermore, conventional production of L-lactic acid is mainly carried out by a fermentation method using lactic acid bacteria, but in the present invention, L-lactic acid can be produced using a 2-halo acid halide hydrolase that has the property of acting on the D-form of 2-halo acid. When producing it, it is superior to conventional methods in terms of low raw material costs, non-pollution, and energy savings, which are the advantages of enzymatic methods, making it a very advantageous method for producing optically active lactic acid. be. Furthermore, optically active 2-hydroxy acids obtained using 2-halo acids as raw materials are useful as synthetic intermediates for pharmaceuticals or amino acids, and can also be used as biochemical reagents. (Example) Next, the present invention will be specifically explained using examples. Reference example 1 Ammonium sulfate 5g/, monopotassium phosphate 1g/, disodium phosphate 1g/, magnesium sulfate 0.05g/ferrous sulfate 0.005g/calcium hydroxide 0.005g/, manganese sulfate
0.0015g/, sodium molybdate 0.0015
g/, culture medium 248 adjusted to PH7.0 at 120℃ for 30
After heat sterilization for ℃ minutes, 750 g of DL-2-chloropropionic acid was adjusted to pH 7 with sodium hydroxide, and filtrated Pseudomonas strain 2 was added to incubate Pseudomonas strain UK113 (Feikoken Bacteria No. 5666). inoculate,
Culture with aeration at 30°C for 18 hours, then collect the bacterial cells by centrifugation using a shear press.
600g of bacterial cells were obtained. The obtained bacterial cells were stored in a frozen state. Next, 600 g of frozen bacterial cells were suspended in 1.2 0.1M phosphate buffer (PH7.5), the cells were disrupted using Dynomill, and cell debris was removed by centrifugation.
- A crude extract containing haloacid halide hydrolase was obtained. After adding 200 ml of a 2% aqueous protamine sulfate solution per 1 portion of this crude extract and stirring thoroughly, the resulting precipitate was removed by centrifugation to obtain a protamine supernatant. Solid ammonium sulfate was gradually added to this supernatant to achieve 40% saturation (4°C). The generated precipitate was removed by centrifugation, and solid ammonium sulfate was gradually added to the supernatant to achieve 70% saturation (4
℃). The generated precipitate was collected by centrifugation and dissolved again in 50mM phosphate buffer (PH7.5).
Next, add 30 times the volume of 50mM phosphate buffer (PH7.5)
A crude enzyme solution was obtained by dialysis and desalting. This crude enzyme solution was passed through a DEAE cellulose column equilibrated in advance with 50mM phosphate buffer (PH7.5) and eluted by gradually increasing the phosphate buffer concentration. - Halo acid halide hydrolase was eluted. This fraction was collected, concentrated, desalted, and further added to 5mM phosphate buffer (PH
Pass the eluate through a hydroxyapatite column equilibrated with 7.5) and remove from 5mM phosphate buffer.
When elution was carried out using a linear gradient of 100 mM phosphate buffer, the target 2-haloyl acid halide hydrolase was eluted at a concentration near 25 mM. After concentrating this elution fraction, purified 2-haloyl acid halide hydrolase can be obtained by performing Sephadex G-150 column chromatography using 50mM phosphate buffer (PH7.5) as the eluent. Ta. The 2-halo acid halide hydrolase thus obtained migrated to the anode side in 7.5% acrylamide disc electrophoresis at pH 9.4, giving a single band.
The molecular weight was approximately 50,000 as determined by Sephadex G-150 gel chromatography. Its yield is approx.
At 100 mg, it shows a titer of about 35 units per mg of enzyme,
The degree of purification was approximately 60, compared to 1 for the crude extract. Example 1 2 ml of 0.1M Tris sulfate buffer (PH9.5) containing 50 micromoles of D and L-2-chloropropionic acid
2- obtained by the method described in Journal of European Biochemistry (J.Eur.Biochem.), Vol. 21, p. 99 (1971).
Adding about 1.5 units of 2-halo acid halide hydrolase, which has the property of acting only on the L-form of halo acids,
After reacting at 30°C for 2 hours, D and L-lactic acid in the reaction solution was quantified. As a result, production of D-lactic acid (47 micromoles) was observed, but no L-lactic acid was detected. Next, 1.5 units of the purified enzyme collected in Reference Example 1 was added, and the reaction was carried out at 30°C for 2 hours. When L-lactic acid in the reaction solution was quantified, it was found that 52 micromoles of L-lactic acid. was being generated. These results are summarized in Table 1. To quantify lactic acid, add 30mM to 0.5ml of 0.5M glycine buffer (PH9.0) containing 0.4M hydrazine.
NAD (nicotinamide adenine dinucleotide)
Add 50μ each of test solution and D or L-
2.5 units of lactate dehydrogenase was added, and the increase in absorbance per unit time at 340 nm at 25°C was measured, and the lactic acid in the test solution was quantified from a calibration curve prepared in advance using a calcium lactate solution of known concentration.

[Table] As is clear from Table 1, optically active 2-hydroxy acids can be produced by the method of the present invention. (Effects of the Invention) According to the present invention, an optically pure 2-hydroxy acid can be produced from inexpensive raw materials with easy operation.

Claims (1)

[Claims] 1. When producing an optically active 2-hydroxy acid using 2-halo acid halide hydrolase, which has the property of acting only on the L-form of 2-halogenocarboxylic acid, a racemic form of 2-halogenocarboxylic acid is used as a raw material, and the reaction is carried out. D of 2-halogenocarboxylic acid in the system
1. A method for producing an optically active 2-hydroxy acid, which comprises using a 2-halo acid halide hydrolase that has a property of acting on the human body.