JP2001314197A - Method for producing optically active hydroxyalkyl- alpha-aminocarboxylic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce an optically active hydroxyalkyl-α-aminocarboxylic acid from an α-ketolactone. SOLUTION: This method for producing the optically active hydroxyalkyl-α- aminocarboxylic acid and/or its salt, characterized by allowing the cells and/or treated product of a microorganism having an ability to stereoselectively convert the keto group of an α-ketocarboxylic acid into an amino group to act on the α-ketolactone.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing hydroxyalkyl-.alpha.-aminocarboxylic acid and / or a salt thereof from .alpha.-keto lactone represented by ketopantolactone by efficiently utilizing a specific microorganism. It relates to a method of manufacturing. L-pantonin or a lactone derivative thereof is useful as an important intermediate in the synthesis of medicinal and agricultural chemicals.

[0002]

2. Description of the Related Art
2-Amino-3,3-dimethyl-4-hydroxybutyric acid (pantonin) is produced by a chemical synthesis method and further treated under acidic conditions to give 3-amino-4,4-dimethyl-.
led to γ-butyrolactone (Nat. Prod. Lett. (199
4) 5,1, J. Biol. Chem. (1948) 175,867, Ber. Deutsch. C
hem. Ges. (1948) 81,323 and J. Am. Chem. Soc. (194
8) 70, 3088).

However, pantonin obtained by these methods is in a racemic form, and there has been no report on a method for producing an optically active pantonin in L-form or D-form. Using D-pantolactone as a starting material, an optically active lactone ((L) -3-amino-4,4,4) is obtained by trifluoromethanesulfonylation, azidation and reduction steps.
-Dimethyl-γ-butyrolactone) has been reported (Syn. Comm. 1994, 24, 557), and it is possible to lead to L-pantonin by further treating this compound under alkaline conditions.

[0004] However, this method is not practical because it has problems such as the use of expensive reagents and a decrease in the optical purity of the product. On the other hand, as a means for producing an L-form α-amino acid from α-ketocarboxylic acid, many methods using a biocatalyst such as a microbial enzyme are known (JP-A-7-250694, JP-A-7-250).
695, JP-A-63-146794, JP-B-63-38193, and J. of Biotechnology (199
7) 53, p29, etc.), and a method using α-ketolactone as a raw material is not known.

[0005] The present invention has been made in view of the above, and an object of the present invention is to provide a method for producing optically active hydroxyalkyl-α-aminocarboxylic acid and / or a salt thereof from α-ketolactone using a microorganism. And

[0006]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have made intensive studies to develop an efficient method for producing L-pantonin from ketopantolactone, and as a result, certain types of L-pantonin were produced. The present inventors have found that L-pantonin can be efficiently produced by allowing microbial cells to act on ketopantolactone, thereby completing the present invention.

That is, the gist of the present invention is to provide a microorganism having the ability to stereoselectively convert a keto group of α-ketocarboxylic acid to an amino group and / or a treated product thereof with α-ketocarboxylic acid.
The present invention relates to a method for producing an optically active hydroxyalkyl-α-aminocarboxylic acid and / or a salt thereof, characterized by acting on ketolactone.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The microorganism used in the production method of the present invention is not particularly limited as long as it is a microorganism having the ability to stereoselectively convert the keto group of α-ketocarboxylic acid to an amino group. (Ochrobactrum) genus,
Pseudomonas sp., Agrobacterium
(Agrobacterium) genus, Brevibacterium
ium) and microorganisms of the genus Sinorhizobium, among which more specifically Ochrobactrum anthropi, Ochrobactrum sp., Pseudomonas putida, Agro Agrobacterium tumefacien
s), Brevibacterium linens
nens) and Sinorhizob meliloti (Sinorhizob)
ium meliloti).

[0009] Specific strains of the above microorganisms include Ochrobactrum anthropi.
i) IFO12951 Ochrobactrum sp.IFO129
50 Pseudomonas putida IFO14796 Pseudomonas putida IFO12996 Agrobacterium tumefaciens
tumefaciens) IAM13129 Brevibacterium linen
s) IFO12142 Sinorhizobium melilot
i) Bacteria such as IAM12611 can be mentioned. Here, IFO stands for Fermentation Research Institute, and IAM stands for Institute for Molecular and Cell Biology, The University of Tokyo, and these strains are readily available.

The above-mentioned microorganisms are not only wild-type strains, but also mutant strains obtained by ordinary mutation treatment such as UV irradiation or NTG treatment, and recombinant strains induced by genetic techniques such as cell fusion or gene recombination. Or any strain. The host of the genetically modified strain may be the same genus as the parent strain or a different genus such as Escherichia coli as long as it is a transformable microorganism.

In the production method of the present invention, one or more of the above microorganisms are used in the form of cells and / or their processed products. Specifically, cells in a wet state obtained by culturing the microorganism and culturing the solution as it is or by centrifugation; those obtained by treating the cells with acetone, those obtained by freeze-drying, the cells A treated bacterial cell, such as one obtained by physically or enzymatically crushing, can be used. In addition, an enzyme fraction capable of stereoselectively converting the α-keto group of α-ketocarboxylic acid to an amino group from these cells or a processed product of the cells can be extracted and used as a crude product or a purified product. It is. Furthermore, the cells obtained in this manner, the treated cells, the enzyme fraction, and the like were immobilized on a carrier represented by a polyacrylamide gel, carrageenan gel, or the like, using a usual immobilization technique. It is also possible to use a thing etc. Therefore, in this specification, the term "microbial cells and / or their processed products" is used as a concept containing all of the above-mentioned microbial cells, processed microbial cells, enzyme fractions and their immobilized products.

The above-mentioned microorganism is usually used after being cultured, and this culture can be carried out according to a conventional method. The medium used for the culture contains a carbon source, a nitrogen source, inorganic ions, and the like that can be utilized by the microorganism. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and saccharose; glycerin, mannitol,
Polyalcohols such as xylitol and ribitol; organic acids such as citric acid and fumaric acid and the like are appropriately used. Nitrogen sources include inorganic nitrogen sources such as ammonium sulfate and sodium nitrate; NZ amine, tryptose, yeast extract,
Organic nitrogen sources such as polypeptone, urea, meat extract, corn steep liquor, soybean extract and the like can be used as appropriate. As inorganic ions, magnesium, potassium,
What contains metal ions, such as iron, manganese, and molybdenum, and phosphate ions may be used, but it is convenient to add inorganic salts that combine them.

In addition to these components, amino acids or vitamins can be added as a substance for promoting the reaction activity. Furthermore, it is also possible to add trace amounts of raw materials and products used in reactions such as DL-pantonin and ketopantolactone as enzyme inducers. In the culture, the pH of the medium is adjusted to a range of 3 to 10, preferably 5 to 9 under aerobic conditions, and the activity is maximized at a temperature of 10 to 45 ° C, preferably 20 to 35 ° C for 1 to 10 days. It is preferable to perform until it becomes.

In the production method of the present invention, α-keto lactone is used as a reaction raw material. Among them, the following general formula (1)

[0015]

Embedded image

Α-ketolactone represented by
The substituents R ^{1 to} R ^{3 in the} above general formula each independently represent a hydrogen atom; or a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec
And an alkyl group such as a -butyl group and a tert-butyl group. The alkyl group may be further substituted with a reaction-inactive group such as a hydroxyl group, an amino group, or a halogen atom. Among these substituents, a hydrogen atom or a group having 1 to 1 carbon atoms
4 straight-chain alkyl groups are more preferred. In the above general formula, n is an integer of 1 or more.
Is preferred.

The reaction raw material is preferably a compound in which R ¹ is an alkyl group and R ² and R ³ are hydrogen atoms. Specifically, ketopantolactone (R ¹ : methyl group, n =
1). In the present invention, the cells of the above-mentioned microorganisms and / or their treated products are allowed to act on α-keto lactone, which is the above-mentioned reaction raw material, in an aqueous medium to obtain optically active hydroxyalkyl-α-aminocarboxylic acid and / or Produce the salt.

The aqueous medium used here is water,
A buffer solution or a culture solution may be used. In this aqueous medium, a water-soluble organic solvent such as ethanol or a fat-soluble organic solvent such as ethyl acetate is added in an amount of about 1 to 5% in accordance with the degree of fat-solubility of the reaction raw material. It is also possible to appropriately contain them. α
The amount of ketolactone is 0.01% in the reaction solution;
Α-keto lactone is not necessarily completely dissolved in the aqueous medium.

When the enzyme involved in the reaction undergoes substrate inhibition by the reaction raw material, the reaction raw material α
-It is possible to further improve the accumulated amount of the product by continuously or intermittently adding ketolactone by the consumed amount. The amount of the microbial cells added to the reaction solution and / or the processed product thereof is added such that the concentration of the cells is about 0.01 to 20% by weight when the cells are added. When a treated product such as an enzyme is used, the specific activity of the enzyme is determined, and an amount is added so that the enzyme concentration corresponding to the above-mentioned cell concentration when added is added. This reaction may be performed under aerobic or anaerobic conditions, but is preferably performed under aerobic conditions.

Preferred reaction conditions for this reaction are a reaction temperature of freezing point to 70 ° C., preferably 10 to 50 ° C., a pH of 2 to 12, preferably 7 to 11, and a reaction time of about 1 to 100 hours. It is. In addition, this reaction is to convert a keto group into an amino group in a stereoselective manner. Examples of enzymes involved in this reaction include transaminase and dehydrogenase, and any of these may be used. These enzymes require an amino group donor during the reaction, and are amino acids such as L-aspartic acid, L-alanine and β-alanine; amines such as isopropylamine; and inorganic ammonium salts such as NH ₄ Cl. Is added to the reaction solution as appropriate, thereby dramatically increasing the reaction yield.

A coenzyme such as pyridoxal pentaphosphate; or glucose necessary for the regeneration of NAD (P) H;
The enzyme reaction can be promoted by adding an energy source such as formic acid, glucose dehydrogenase, or formate dehydrogenase as needed. By the above reaction, an optically active hydroxyalkyl-α-aminocarboxylic acid is obtained as a reaction product. Some of the above products are salts due to metal ions and the like coexisting in the reaction system.

These products are obtained by removing bacterial cells and / or processed products by centrifugation or the like, and then adding an inorganic acid such as hydrochloric acid or an organic acid such as p-toluenesulfonic acid to the reaction mixture. Hydroxyalkyl-α-aminocarboxylic acid is precipitated and separated by an electrofocusing method, or hydroxyalkyl-α-aminocarboxylic acid is isolated and purified from the reaction solution with an ion exchange resin. It can be purified.

Incidentally, (S) -2-amino-3,3-dimethyl-4-hydroxybutyric acid obtained by this reaction is a novel compound. In addition, the product is treated with an acid such as p-toluenesulfonic acid after the isolation / purification treatment to close the ring while maintaining the stereochemistry of the α-amino group,
Optically active aminolactone can be produced.

[0024]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Ochrobactrum sp. IFO129
50 cells were prepared by adding 0.5% of glucose and 1.0 of yeast extract.
%, Polypeptone 0.5%, sodium fumarate 0.5
%, L-aspartic acid 0.8%, KH ₂ PO ₄ 0.2
%, MgSO ₄ 0.8% and CaCl ₂ 0.1%, and inoculated into 500 ml of a pH 7.0 medium.
The cells were aerobically shake-cultured at 24 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation, washed with physiological saline, and then the cells were washed with 100 mg of ketopantolactone and 80 m of L-alanine.
g and 1 mg of pyridoxal pentaphosphate in 50 ml of phosphate buffer (0.1 M) and pH 9.0 with NaOH.
After adjusting to, a shaking reaction was carried out at 30 ° C. for 48 hours. HP using Daicel CHIRALPAK WH
When the concentration of L-pantonin in the reaction solution was measured by LC analysis, it was 1.4 mg / ml.

Further, after completion of the reaction, the reaction supernatant from which the cells were removed by centrifugation was washed with a cation exchange resin SK1B (H +
After passing through a column (type), the column was washed with water and eluted with dilute aqueous ammonia. The fractions of L-pantonin were collected and concentrated to dryness to obtain 55 mg of L-pantonin. Example 2 Ochrobactrum sp. IFO129
50 cells, 0.5% glucose, 0.4 yeast extract
%, Polypeptone 1.0%, NaCl 0.5% and K
1 of a medium containing 0.2% ₂ HPO ₄ and having a pH of 7.0
The cells were inoculated into 00 ml and aerobically shake cultured at 30 ° C. for 24 hours. After the completion of the culture, the cells were collected by centrifugation, washed with a physiological saline, and the cells were washed with ketopantolactone 20m.
g, glucose 60mg and ammonium formate 60mg
Was suspended in 10 ml of a phosphate buffer solution (0.1 M) containing, and adjusted to pH 9.0 with NaOH, followed by a shaking reaction at 30 ° C. for 48 hours. When the concentration of L-pantonin in the reaction solution was measured, it was 1.2 mg / ml.

Example 3 Using the cells shown in Table 1, the same culturing operation and reaction operation as in Example 1 were performed. Table 1 shows the obtained results.

[0027]

Table 1 Microorganisms L-Pantonin production [mg / mL] Ochrobactrum anthropi IFO12951 0.8 Pseudomonas putida IFO14796 1.0 Pseudomonas putida IFO12996 0.4 Agrobacterium tumefaciens IAM13129 0.6 Brevibacterium lineens IFO12142 0.4 Sinorizobiium IA From the results of 0.6 or more, according to the production method of the present invention, L-
It can be seen that pantonin and the like can be efficiently produced.

[0028]

According to the production method of the present invention, it is possible to efficiently produce optically active hydroxyalkyl-α-aminocarboxylic acid from α-keto lactone using a microorganism.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:13) C12R 1:13) (C12P 13/04 (C12P 13/04 C12R 1:40) C12R 1 : 72) Inventor Makoto Ueda 1000 Kamoshita-cho, Aoba-ku, Yokohama-shi, Kanagawa Prefecture F-term in Yokohama Research Laboratory, Mitsubishi Chemical Corporation 4B064 AE03 CA02 CC03 CD08 DA01 DA11 4H006 AA01 AB84 BN10

Claims (7)

[Claims] The present invention relates to an optical system characterized in that microbial cells having a capability of stereoselectively converting a keto group of an α-ketocarboxylic acid to an amino group and / or a treated product thereof are allowed to act on α-ketolactone. Active hydroxyalkyl-
A method for producing α-aminocarboxylic acid and / or a salt thereof. 2. The method according to claim 1, wherein the hydroxyalkyl-α-aminocarboxylic acid and / or a salt thereof is in the (S) -form. 3. An α-keto lactone represented by the following general formula (1): (Wherein R ^{1 to} R ³ are each independently a hydrogen atom or an alkyl group, and n is an integer of 1 or more). Manufacturing method. 4. The method according to claim 1, wherein the α-ketolactone is ketopantolactone. 5. A microorganism capable of stereoselectively converting a keto group of α-ketocarboxylic acid to an amino group is a microorganism of the genus Ochrobactrum or Pseudodom.
ona), Agrobacterium, Brevibacterium and Sinorhizobium are microorganisms arbitrarily selected from the group consisting of the genus Agrobacterium, Agrobacterium, Brevibacterium and Sinorhizobium. The production method described in 1. 6. A microorganism capable of stereoselectively converting a keto group of an α-ketocarboxylic acid to an amino group includes Ochrobactrum anthropi, Ochrobactrum sp., And Pseudomonas putida. putida), Agrobacterium tumefacien
s), Brevibacterium lens
inens) and Sinorhizobium meliloti (Sinorhizo)
The method according to any one of claims 1 to 4, which is a microorganism arbitrarily selected from the group consisting of Bium meliloti). 7. An (S) -2-amino- represented by the following formula:
3,3-dimethyl-4-hydroxybutyric acid. Embedded image