JPH1075797A - Production of (r)-2-hydroxy-4-phenyl-3-butenoic acid by enzymatic method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for industrially and advantageously producing (R)-2-hydroxy-4-phenyl-3-butenoic acid useful as an intermediate for a medicine and an optically and pharmaceutically active material by acting a specific asymmetrically reducing enzyme on 2-oxo-4-phenyl-3-butenoic acid in the presence of NADPH. SOLUTION: The objective (R)-2-hydroxy-4-phenyl-3-butenoic acid is produced by acting an enzyme having following properties on 2-oxo-4-phenyl-3-butenoic acid in the method for producing (R)-2-hydroxy-4-phenyl-3-butenoic acid: the enzyme catalyses the production of (R)-2-hydroxy-4-phenyl-3-butylic acid by asymmetrically reducing 2-oxo-4-phenyl-3-butylic acid by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme; an optimum pH is about 6.5-7.0 (phosphate buffer); a stable pH range is 6-7; an optimum temperature is about 40 deg.C (pH7.0); thermal stability is at <=30 deg.C; the Km value of Michaelis constant for 2-oxo-4-phenyl-3-butylic acid is 0.87mM; a molecular weight (gel-filtration method) is about 50,000; and the enzyme is inhibited by mercury p-chlorobenzoate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid. (R) -2-hydroxy-4-phenyl-3-butenoic acid is a synthetic intermediate of various pharmaceuticals, optically active physiologically active compounds, and derivatives thereof.

[0002]

2. Description of the Related Art
As a method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid, a method of optically resolving a racemic form of the acid to form a bornylamine and a diastereomer and performing optical resolution
(Chem. Ber. 89, 671-67781956) and a method of producing by an asymmetric reduction method using a microorganism (JP-A-2-23876, JP-A-2-100688). It is difficult to say that the latter is excellent in terms of efficiency, and in the latter case, a substance having a low optical purity may be obtained by the action of many enzymes and the like coexisting in the cells.

[0003]

Means for Solving the Problems The inventors of the present invention have a simple method and a high optical purity of (R) -2-hydroxy-4-.
Focusing on the enzymatic method as a method for producing phenyl-3-butenoic acid, as a result of searching for enzymes suitable for this purpose, it was found that enzymes having the following physicochemical properties are suitable for that purpose, and the present invention completed.

That is, according to the present invention, an enzyme having the following physicochemical properties is allowed to act on 2-oxo-4-phenyl-3-butenoic acid to obtain (R) -2-hydroxy-4-phenyl having a high optical purity. This is a method for producing -3-butenoic acid.

The physicochemical properties of the enzyme are as follows: [1] Action and substrate specificity Reduced nicotinamide / adenine / dinucleotide / phosphoric acid (hereinafter referred to as NADPH) is used as a coenzyme and 2-oxo-4-phenyl Asymmetric reduction of butyric acid produces (R) -2-hydroxy-4-phenylbutyric acid.

[2] Optimum pH: around 6.5 to 7.0 (phosphate buffer) [3] Stable pH range: 6 to 7 [4] Optimum temperature: around 40 ° C. (pH 7.0) [ 5] Thermal stability; stable at 30 ° C. or less (pH 7.0, treated for 10 minutes) [6] Michaelis constant Km value for 2-oxo-4-phenylbutyric acid; 0.87 mM [7] Molecular weight: about 50,000 (gel Filtration method) [8] Inhibitor: pallachloromercuric benzoic acid.

The enzyme used in the present invention may be of any origin as long as it has the above-mentioned physicochemical properties. Preferred examples include lactic acid bacteria, more preferably Leuconostoc or lactobacillus. A microorganism strain belonging to the genus Bacillus can be used. Further, among them, preferred strains are Leuconostoc mesenteroides subspecies dextranicum (Leuconosto).
c mesenteroides subsp.dextranicum) IFO 3349, Leuconostoc mescentroides subspecies.
Dextranicum (Leuconostoc mesentoroides subsp.
dextranicum) ATCC 17072, Leuconostoc uenos ATCC 2731
0, Leuconosto Mesenteroides
c mesenteroides) AHU 1067, Lactobacillus
Lactbacillus fructosus IFO 351
6. Lactbacillus casei subsp. Casei IFO 120
04 and so on.

Any of these microorganisms can be suitably used, such as a wild-type strain, a mutant strain, or a recombinant induced by a genetic technique such as cell fusion or genetic engineering.

Microorganisms with IFO numbers are listed in Listof Cultures, published by Fermentation Research Institute (IFO).
Tenth Edition, Volume 1 (1996), and can be obtained from the IFO. Microorganisms with AHU numbers are Catalogu issued by the Japan Federation of Microbial Strains (JFCC).
e of Cultures, 4th edition (1987), and can be obtained from the Faculty of Agriculture, Hokkaido University. Microorganisms with ATCC numbers are from the American Type Culture Collection.
(ATCC) Catalog of Bacteria PhagesrONA
Vectors, 18th Edition (1993).
Can be obtained from

[0010] The medium for culturing the microorganism for collecting the enzyme used in the present invention is not particularly limited as long as the microorganism can grow. For example, as the carbon source, any of the above microorganisms can be used, and specifically, glucose, fructose, sucrose, saccharides such as dextrin, sorbitol, ethanol, alcohols such as glycerol, fumaric acid, citric acid And organic acids such as acetic acid and propionic acid and salts thereof, hydrocarbons such as paraffin, and mixtures thereof. Examples of the nitrogen source include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, and ammonium phosphate;
Ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, meat extracts, yeast extracts, corn steep liquor, casein hydrolysates, inorganic or organic nitrogen-containing compounds such as urea, or mixtures thereof can be used. . In addition, nutrients used for ordinary culture, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. If necessary, a factor that promotes the growth of microorganisms, a factor that enhances the ability to produce the target compound of the present invention, or a substance that is effective in maintaining the pH of the medium can be added.

As a culturing method, the pH of the medium is from 3.0 to 9.0.
5, preferably 4 to 8, and a culture temperature of 20 to 45 ° C., preferably 25 to 37 ° C., under conditions suitable for anaerobic or aerobic growth of the microorganism, for 5 to 120 hours, preferably 12 to Incubate for about 72 hours.

The novel enzyme of the present invention can be purified from the microbial cells cultured in this manner by combining conventional enzyme purification methods.

For example, the culture is centrifuged to collect the cells, and a cell-free extract is obtained by sonication, etc., treated with streptomycin sulfate, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, gel filtration. And the like.

The activity of the enzyme of the present invention was measured as follows.

<Method of measuring activity> 2 ml of 200 mM phosphate buffer (pH 7.0), 0.5 ml of 12 mM potassium 2-oxo-4-phenylbutyrate solution, 0.1 ml of 4.5 mM NADPH solution
And an enzyme solution to form a total reaction system of 3 ml.
The decrease in absorbance at 340 nm of ADPH was followed by a spectrophotometer to determine the enzyme activity. The enzyme activity of 1 U was defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute under the above conditions.

The specific activity was defined as the U number of 1 mg of protein, and the protein was determined using a protein assay kit manufactured by Bio-Rad and bovine serum albumin as a standard substance.

Next, a method for producing optically active (R) -2-hydroxy-4-phenyl-3-butenoic acid from 2-oxo-4-phenyl-3-butenoic acid using this enzyme will be described. When (R) -2-hydroxy-4-phenyl-3-butenoic acid is produced from 2-oxo-4-phenyl-3-butenoic acid (substrate) using the enzyme of the present invention, a coenzyme together with the substrate is used. NADPH is required. The reaction is
Basically, a substrate, an enzyme, and NADPH may be mixed and reacted in an appropriate ratio under conditions that allow the activity of the enzyme to be stably expressed. The reaction is carried out at pH 5-9, preferably at pH 6.
The temperature is in the range of from 10 to 60 ° C, preferably from 20 to 4 ° C.
The reaction is carried out at 0 ° C. for about 1 to 120 hours under stirring or standing. The concentration of the substrate used is not particularly limited.
About 10% is preferable. Further, by incorporating a coenzyme recycling system by a known method, it is possible to produce more efficiently.

Further, the enzyme of the present invention can be used as an immobilized enzyme by immobilizing it on various immobilized carriers by a known method.

The optically active (R) -2-hydroxy-4-phenyl-3-butenoic acid produced by the above-mentioned reaction is collected by extracting the reaction solution with an organic solvent, and performing column chromatography, recrystallization and the like. It can be easily carried out by using a usual purification method.

[0020]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference examples and examples, but the present invention is not limited to these examples.

Reference Example Purification of enzyme 8% glucose, 1% yeast extract, 10 pp manganese sulfate
18 l of a medium having a composition of m.sup.2 into a 30-liter fermenter and heat-sterilized, followed by Leuconostoc oen.
os) ATCC 27310 was inoculated. While maintaining the pH at 6.5 with 1N aqueous sodium hydroxide, 10
The cells were cultured at 0 rpm with aeration of 0.1 vvm for 24 hours. The culture is centrifuged (5 ° C., 10,000 rp) with a cooling centrifuge.
m, 10 minutes), and the precipitated cells were collected. After washing 145 g of the wet cells twice with 300 ml of physiological saline, 200 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 3 mM 2-mercaptoethanol was added, and the cells were crushed with a French press. The cooling centrifuge (5
C., 10,000 rpm, 10 minutes) to obtain 200 ml of a supernatant (crude enzyme solution).

While the crude enzyme solution was cooled (5 ° C.), salting out was performed under the condition of ammonium sulfate 0.8 saturation. The precipitate generated by salting out is cooled in a centrifuge (5 ° C., 10,000 rpm).
, 10 minutes) to collect the precipitate.
Dialysis was performed against a 20 mM potassium phosphate buffer (pH 8.0) containing mercaptoethanol. Then 3mM2-
The column was loaded on a column (DEAE-Toyopearl 650M, 2.5 × 30 cm) equilibrated with a 20 mM potassium phosphate buffer (pH 8.0) containing mercaptoethanol. After loading, it was washed with 500 ml of 20 mM potassium phosphate buffer (pH 8.0) containing 3 mM 2-mercaptoethanol, and the target enzyme was eluted by a gradient elution method in which the concentration of sodium chloride was linearly increased from 0 to 1 M. After concentrating those active fractions by ultrafiltration, 50 mM potassium phosphate buffer containing 3 mM 2-mercaptoethanol (pH
7.5) Equilibrated column (Toyopearl HW-55)
F, 2.5 × 100 cm). The active fractions were collected and used as a partially purified enzyme preparation. The table shows the purification steps.

[0023]

[Table 1] [Example] 2-oxo-4-phenyl-3-butenoic acid 5
0 mg was dissolved in 50 ml of distilled water, and adjusted to pH 6.5 by adding a 1N aqueous solution of potassium hydroxide. Add NADPH 2 to this solution
After adding and dissolving 50 mg, 50 ml of 200 mM potassium phosphate buffer (pH 6.5) was added. Then, in the same manner as in Reference Example, Leuconostocoeno
s) 100 mg of a partially purified enzyme preparation purified from ATCC 27310 was added and incubated at 30 ° C. for 10 hours. 2-oxo-4-phenyl-3 in the reaction solution
-After confirming that all the butenoic acid has been converted, 1
N sulfuric acid was added to bring the pH to 1. Then 100 ml of ethyl acetate
And extracted twice. After separating the ethyl acetate layer, ethyl acetate was distilled off using a rotary evaporator to remove (R)-
45 mg of crude crystals of 2-hydroxy-4-phenyl-3-butenoic acid were obtained. The crude crystals were reconstituted with ethanol to give (R) -2-hydroxy-4-phenyl-
4 mg of 3-butenoic acid crystals were obtained. The crystals are dissolved in a small amount of distilled water and subjected to high performance liquid chromatography using an optical resolution column (mobile phase; 0.5 mM copper sulfate: CH3CN).
(4: 1), column; CHIRALPAKWH φ4.
6 mm × 250 mm, column temperature; 50 ° C., detection; A25
4, the absolute configuration and optical purity were measured at a flow rate of 1.7 ml / min). e. Met.

[0024]

According to the method of the present invention, (R) -2-hydroxy-4-phenyl-3-butenoic acid can be advantageously produced.

Claims (2)

[Claims] (1) An enzyme having the following physicochemical properties is allowed to act on 2-oxo-4-phenyl-3-butenoic acid: (R) -2-hydroxy-4-phenyl-3- A method for producing butenoic acid. Physicochemical properties of enzyme [1] Action and substrate specificity As a coenzyme, reduced nicotinamide / adenine / dinucleotide / phosphoric acid (hereinafter referred to as NADPH), asymmetric reduction of 2-oxo-4-phenylbutyric acid, Produces (R) -2-hydroxy-4-phenylbutyric acid. [2] Optimum pH; around 6.5 to 7.0 (phosphate buffer) [3] Stable pH range; 6 to 7 [4] Optimum temperature; around 40 ° C (pH 7.0) [5] Heat Stability: stable at 30 ° C or less (pH 7.0, 10 minutes treatment) [6] Michaelis constant Km value for 2-oxo-4-phenylbutyric acid; 0.87 mM [7] Molecular weight; about 50,000 (gel filtration method) [8] inhibitors; balachloromercury benzoic acid 2. The method according to claim 1, wherein the enzyme is produced by the genus Leuconostoc or Lactobacillus.