JPH0137112B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Pseudomonas strain UK113. 2-halo acid halide hydrolase is an enzyme used to produce optically active 2-hydroxycarboxylic acids from 2-halo acids. As a conventional method for producing this optically active 2-hydroxycarboxylic acid, a method for obtaining D-form and L-form 2-hydroxycarboxylic acid 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-hydroxycarboxylic acid can be easily obtained from 2-halo acid in high yield, and optically active 2-hydroxycarboxylic acid can be easily obtained from 2-halo acid. - This is a very advantageous method for industrially producing hydroxycarboxylic acids. However, conventionally known 2-halo acid halide hydrolases have been published in the Journal of Biological Chemistry (J.
Biol.Chem.) Volume 234, page 428 (1968) and European Journal of Biochemistry (Eur.J.Biochem.) Volume 21, page 99 (1971).
As stated in 2010), both
It acted only on the L-form of haloacid, producing D-form 2-hydroxycarboxylic acid, and did not act on the D-form 2-haloacid. Therefore, it has been impossible to produce L-form 2-hydroxycarboxylic acid, which is more important than D-form from a biochemical point of view, using conventional 2-halo acid halide hydrolases. In addition, since the raw material 2-halo acid exists as a racemate, which is a mixture of D-form and L-form, when conventional 2-halo acid halide hydrolase is used,
The D-form 2-haloacid remained as a raw material, which was economically disadvantageous. That is, 2-halo acid halide hydrolase obtained from conventional microorganisms
It has a major drawback in that it acts only on the L-form of 2-haloacids and not on the D-form. Therefore, from this perspective, the present inventors proposed 2-
As a result of conducting a wide range of natural screenings using the enrichment culture method in search of microorganisms capable of producing 2-halo acid halide hydrolase, which has the property of acting on the D-form of halo acids, Gokasho, Uji City, Kyoto Prefecture. It is thought that it belongs to the genus Pseudomonas from the soil of D.
A new bacterial strain has been discovered that has the property of assimilating 2-haloacids in the body, and this new bacterial strain has the ability to produce 2-haloacid halide hydrolase, which has the property of acting on the D-form of 2-haloacids. They discovered this and arrived at the present invention. That is, the present invention belongs to the genus Pseudomonas,
The subject matter is a Pseudomonas strain UK113 having the ability to produce 2-halogenocarboxylic acid halide hydrolase that acts on the D-form of 2-halogenocarboxylic acid. In the present invention, 2-haloacid has 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,
Derivatives of acetic acid such as monoiodoacetic acid, dichloroacetic acid, dibromoacetic acid or trichloroacetic acid, 2-
Chlorpropionic acid, 2-bromopropionic acid,
Derivatives of propionic acid such as 2,2-dichloropropionic acid 2-chlorobutyric acid, 2-bromobutyric acid,
Derivatives of butyric acid such as 2,2-dichlorobutyric acid, 2-
Examples include derivatives of valeric acid such as chlorovaleric acid, 2-promovaleric acid, and 2,2-dichlorovaleric acid.
It may also 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 five or fewer carbon atoms, or if the carboxylic acid is acetic acid, one It may be a compound in which ~3 is bonded. The mycological properties of the strain of the present invention are shown. The Manual of Microbiological Methods is used to examine this mycological property.
Microbiological Methods, Mc Grow-Hill
The methods and culture medium compositions described in Microbial Classification and Identification (edited by Takeji Hasegawa, University of Tokyo Press), and Specifications on Culture Media (written by Toshikazu Sakazaki, published by Naya Shoten) were used. (Morphological findings) 1 Cell shape and size; short rod-like. 0.5-1.0 micron x 1.5-3.0 micron. 2 Polymorphism; none. 3 Motility: Yes. 4 Flagella; present, polar flagella. 5 Spores: None. 6 Gram staining; negative. 7 Anti-acidity: None. (Growth status) 1 Juicy agar plate culture shape: circular. Peripheral; Entire edge. Protuberance; flat. Gloss: None. Surface: smooth. Color: Cream color. 2 Growth rate of meat juice agar slant culture: Good. Shape: filamentous. 3 Meat juice liquid culture surface growth: None. Turbidity; turbidity. Sediment: Yes. Coloring, bleaching; none. 4 Meat juice gelatin puncture culture Added 30% gelatin, gelatin does not liquefy. 5 Meat juice agar puncture culture; good surface growth. 6 Litmus milk Slightly alkaline, milk does not solidify. (Physiological properties) 1 Reduction of nitrate; none. 2 Denitrification reaction; none. 3 MR test; negative. 4 VP test; negative. 5 Indole formation; none. 6 Generation of hydrogen sulfide; none. 7 Hydrolysis of starch; none. 8 Use of citric acid; Yes. 9 Use of nitrates: Yes. 10 Use of ammonium salt; Yes. 11 Pigment formation; none. 12 Urease activity; none. 13 Oxidase reaction; positive. 14 Catalase activity; present. 15 OF test; oxidation tape. 16 Hydrolyzability of arginine; Yes. 17 Vitamin requirement: None. 18 Attitude towards oxygen; aerobic. 19 Accumulation of poly-β-hydroxybutyric acid; none. 20 Cleavage of aromatic ring; cleavage at ortho position. 21 Growth PH; PH5-8 22 Growth temperature; 20-35℃ [Generation of acid and gas from sugar] Sugar Acid Gas (1) L-arabinose + - (2) D-xylose + - (3) D-glucose + - (4) D-mannose + - (5) Franktose + - (6) D-galactose + - (7) Maltose - - (8) Cane sugar - - (9) Lactose + - (10) Trenhalose + - (11) D-sorbit + - (12) D-mannite + - (13) Inosit + - (14) Glycerin + - (15) Starch - - [Assimilation of carbon compounds] (1) Things that can be assimilated; D-2-chloropropionic acid. Glucose, 2-ketogluconic acid, inosyte, trehalose, lactose, benzylamine,
β-alanine, L-valine, DL-arginine,
Betaine, chloroacetic acid, 2-chloropropionic acid, parahydroxybenzoic acid. (2) Things that cannot be assimilated; sucrose, geraniol, methanol, methylamine, oxalic acid. Based on the above mycological properties, Bevgey's Manual of Determinative Bacteriology
Bacteviology) 7th and 8th editions revealed that this strain is a Gram-negative bacillus, has polar flagella, is motile, aerobic, oxidase-positive, and oxidatively decomposes glucose. It is considered to be a bacterium included in the genus. Therefore, we searched for species included in the genus Pseudomonas and found that they are not auxotrophic and do not accumulate poly-β-hydroxybutyric acid.
It is thought to belong to Pseudomonas alcaligenes because it does not produce fluorescent dyes and the denitrification reaction is negative, but it does not grow at 41°C, does not hydrolyze gelatin, does not contain glucose, trehalose, 2-ketogluconic acid, They have different properties such as assimilating inosite and L-valine. Furthermore, Pseudomonas. Pseudomonas
putida), but it has different properties such as not producing a fluorescent dye and assimilating trehalose and inosites, and no known strain with the same mycological properties as this strain has been found. . In particular, there have been no reports of bacterial strains that have the property of assimilating D-form 2-haloacids. Based on the above, this strain is considered to be a new strain belonging to the genus Pseudomonas, so it was named Pseudomonas strain UK113 and deposited with the Institute of Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on July 30, 1981. Its microorganism accession number is FEIKEN Bibori No. 5666. As a nutrient source for the medium used for culturing the Pseudomonas strain UK113 of the present invention, any nutrient source that can be assimilated by this strain can be used. Examples of carbon sources include glycose, glycerin, alcohols, oils and fats, fatty acids,
Furthermore, 2-halo acids and the like can be used, and as the nitrogen source, inorganic or organic substances such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, various amino acids, peptone, meat extract, and yeast extract can be used. Furthermore, as inorganic salts, for example, potassium, sodium, phosphoric acid, zinc, iron, magnesium, manganese, copper, calcium, cobalt, molybdenum salts, trace metal salts, corn stapler, vitamins, etc. as necessary, Nucleic acids and the like may be used, and general nutrient media for bacteria can be used. Using these media, this strain is preferably grown.
10-36℃, more preferably 20-33℃, optimally
What is necessary is just to culture|cultivate aerobically at 26-30 degreeC for about 5-48 hours. The culture thus obtained can be used as it is as an enzyme source, but not only crude enzymes and purified enzymes, but also isolated live bacterial cells and processed products of isolated bacterial cells can also be used as enzyme sources. Next, the 2-haloyl acid halide hydrolase of the present invention is collected from the obtained culture, but it can be collected at any stage, such as culture, isolated live bacterial cells, processed isolated bacterial cells, crude enzyme, purified enzyme, etc. . As a purification method at that time, a usual enzyme purification method can be used. That is, after obtaining bacterial cells by centrifugation, the cells are disrupted by Manton-Gaulin, Dyno Mill, French press, ultrasonication, etc., and cell debris is removed by centrifugation to obtain a cell extract. Treated with streptomycin sulfate or protamine sulfate,
Furthermore, salting out treatment with ammonium sulfate or the like or organic solvent treatment with acetone or the like is performed, and for purification, ion exchange chromatography such as DEAE cellulose column, adsorption chromatography such as hydroxyapatite column, or sepadex chromatography is performed. It can be carried out in combination with gel chromatography using graphie or the like. In this way, the two
- Halo acid halide hydrolase can be isolated and purified. Next, the physicochemical properties of the 2-halo acid halide hydrolase thus obtained will be shown. 1 Effect LR・CHX・COOH+H ₂ O →D−R・CHOH・COOH+H ⁺ ＋X ^- DR・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 represents an atomic group such as H, H ₂ , HX, X ₂ , CH ₃ , C ₂ H ₅ or C ₃ H ₇ , and X represents a halogen atom such as chlorine or bromine. Hydrolyzes halogen atoms by acting on L-2-halo acid in the presence of water, and
-Produces 2-hydroxy acid. When the 2-haloacid is in the D form, an L form of 2-hydroxy acid is produced. In addition, when the substrate is 2-dihaloacid, 2
-Produces keto acids. 2 Substrate specificity The Michaelis constants (Km values) for D, L and DL-2-chloropropionic acid are respectively
They are 4.8, 1.1, and 3.2mM. In addition to this, the number of carbon atoms is 2
Up to ~5 2-haloacids serve as substrates, and DL-
The reaction rates of other substrates when 2-chloropropionic acid is taken as 100 are shown in the following table.

【table】

[Table] 3 Optimum PH PH approx. 9.5 (Measured at 30℃ using DL-2-chloropropionic acid as a substrate.) 4 Stable PH range PH approx. 6-12 (4℃, after 24 hours, original 5. Suitable temperature for action and heat resistance At PH9.5, activity increases as temperature rises from 20℃ to 45℃, but activity decreases to less than 50% after treatment at 45℃ for 15 minutes. do. 6. Method for measuring potency 0.1M Tris sulfate buffer (PH9.5) containing 50 micromoles of DL-2-chloropropionic acid2
1 to 100 µ of the enzyme solution was added to each ml, and after reacting at 30°C for 10 minutes, 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, to 1 ml of the reaction mixture or its diluted solution, 1 ml of a dioxane solution containing 10% ethanol containing 0.1% mercury thiocyanate and 0.4 ml of a 6N nitric acid solution containing 8% iron alum were added, and the absorbance at 460 mm was measured. 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 Cephadex G-150 gel chromatography. 8. Inhibiting, activating and stabilizing agents. 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] By using the 2-halo acid halide hydrolase produced by the Pseudomonas strain UK113 of the present invention, lactic acid can be produced in high yield from, for example, 2-chloropropionic acid. It becomes possible to produce L-lactic acid, which was impossible to produce with oxygen. Next, the present invention will be specifically explained using examples. Example 1 Ammonium sulfate 5g/, potassium phosphate 1g/, disodium phosphate 1g/, magnesium sulfate 0.05g/, ferrous sulfate 0.005g/,
Calcium hydroxide 0.005g/, manganese sulfate
0.0015g/sodium molybdate 0.0015g/
After heat sterilizing medium 248 adjusted to pH 7.0 at 120°C for 30 minutes, DL-2-chloropropionic acid
After adjusting the pH of 750g to 7 with sodium hydroxide, add sterilized 2 and remove Pseudomonas.
Inoculated with UK113 strain (Feikoken Bacteria No. 5666),
Culture was carried out with aeration and stirring at ℃ for 18 hours. After culturing, the cells were collected by centrifugation using a shear press to obtain 600 g of cells. The obtained bacterial cells were stored in a frozen state. Next, 600 frozen bacterial cells were suspended in 1.2 0.1M phosphate buffer (PH7.5), the cells were disrupted using Dynomil, and cell debris was removed by centrifugation. A crude extract was obtained. 2% protamine sulfate aqueous solution per 1 of this crude extract
After adding 200 ml and stirring thoroughly, the resulting precipitate was removed by centrifugation to obtain a Promitan supernatant.
Solid ammonium sulfate was gradually added to this supernatant.
It was set to 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°C). The generated precipitate was collected by centrifugation, dissolved again in 50mM phosphate buffer (PH7.5), and then diluted with 30 times the volume.
A crude enzyme solution was obtained by desalting by dialysis against 50 mM phosphate buffer (PH7.5). Prepare this crude enzyme solution in advance.
Equilibrated with 50mM phosphate buffer (PH7.5)
When the crude enzyme was passed through a DEAE cellulose column and eluted by gradually increasing the concentration of phosphate buffer, the desired 2-haloacid halide hydrolase was eluted at a phosphate concentration of approximately 100 mM. After collecting this fraction, concentrating and desalting, the eluate was further passed through a hydroxyapatite column equilibrated with 5mM phosphate buffer (PH7.5), and then
When elution was carried out using a linear gradient of 100 mM phosphate buffer, the desired 2-haloyl acid halide hydrolase was eluted at a concentration near 25 mM. After concentrating this elution fraction, purified 2-halo acid halide hydrolase was obtained by performing Sephadex G-150 column chromatography using 50mM phosphate buffer (PH7.5) as the eluent. . The thus obtained 2-halo acid halide hydrolase migrated to the anode side in 7.5% acrylamide disk electrophoresis at pH 9.4, giving a single band, and was detected by Sephadex G-150 gel chromatography.
The molecular weight was approximately 50,000. The yield was about 100 mg, the potassium value was about 35 units per mg of enzyme, and the degree of purification was about 60 when the crude extract was 1.

Claims (1)

[Claims] 1 Pseudomonas strain UK113 (Feikoken Bacterial Serial No. 5666) belonging to the genus Pseudomonas and having the ability to produce 2-halogenocarboxylic acid halide hydrolase that acts on the D-form of 2-halogenocarboxylic acid.