JPH04335886A - New enzyme, production thereof and production of optically active (r)-2-hydroxy-4-phenylbutyric acid - Google Patents

Abstract

PURPOSE:To efficiently produce the subject optically active (R)-2-hydroxy-4- phenylbutyric acid from 2-oxo-4-phenylbutyric acid. CONSTITUTION:An enzyme obtained by extraction and purification from a microorganism belonging to Leuconostic genus is allowed to act on 2-oxo-4- phenylbutyric acid on the presence of reduced type nicotinamde adenine dinucleotide phosphate NADPH as a coenzyme to asymmetrically reduce it. The above-mentioned enzyme has following physicochemical properties; Optimum pH: about 6.0, Stable pH region: 6-7, Optimum temperature: about 45 deg.C. Thermal stability: <=40 deg.C, Michaelis constant value Km for 2-oxo-4-phenylbutyric acid: 0.29mM, Molecular weight: 46000 and Inhibitor: mercury chloride and mercury para-chlorobenzoate.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a novel enzyme, a method for producing the same, and an optically active (R)-
The present invention relates to a method for producing 2-hydroxy-4-phenylbutyric acid.

0002

[Prior Art and Problems to be Solved by the Invention] In recent years, methods for obtaining optically active substances using enzymatic asymmetric reduction reactions have been studied. For example, Tokuko Sho 61-11591
Publication No. 1-27717, Japanese Unexamined Patent Publication No. 1986-2
No. 86 and Japanese Unexamined Patent Publication No. 63-32480,
Several types of dehydrogenases produced by lactic acid bacteria have been reported. These dehydrogenases asymmetrically reduce various 2-oxocarboxylic acids using reduced nicotinamide adenine dinucleotide (hereinafter referred to as NADH) as a coenzyme to produce the corresponding optically active 2-hydroxy Produces carboxylic acid.

Furthermore, Japanese Patent Application Laid-open No. 63-304979 discloses that reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH) is used as a coenzyme, and γ
-Substituted acetoacetate to (R)-γ-substituted-β-
As an enzyme that produces hydroxybutyric acid ester, a reductase extracted from a strain belonging to the genus Sporobolomyces has been disclosed.

On the other hand, optically active (R)-2-hydroxy-4-phenylbutyric acid is a compound useful as a synthetic intermediate for pharmaceuticals and the like. However, regarding the enzyme, 2-oxo-4-phenylbutyric acid is asymmetrically reduced and (R)
The activity of producing -2-hydroxy-4-phenylbutyric acid is not known.

[0005] Therefore, the object of the present invention is to obtain 2-oxo-4
- selectively asymmetrically reduces phenylbutyric acid to produce optically active (R
)-2-Hydroxy-4-phenylbutyric acid efficiently and a method for producing the same.

[0006] Another object of the present invention is to
Optically active (R)- from 2-oxo-4-phenylbutyric acid
An object of the present invention is to provide a production method that can efficiently obtain 2-hydroxy-4-phenylbutyric acid.

[0007]

[Structure of the Invention] As a result of searching for an enzyme capable of asymmetrically reducing 2-oxo-4-phenylbutyric acid to produce (R)-2-hydroxy-4-phenylbutyric acid, the present inventors discovered that leuconostoc The present invention was completed by discovering that microorganisms belonging to the genus Leuconostoc produce an enzyme capable of achieving the above object, and by clarifying the physicochemical properties of this enzyme.

That is, the present invention provides the following (1) to (8)
) A novel enzyme having the following physicochemical properties is provided.

(1) Action and substrate specificity: NADP
In the presence of H, 2-oxo-4-phenylbutyric acid is asymmetrically reduced to produce (R)-2-hydroxy-4-phenylbutyric acid (2) Optimum pH: around 6.0 (phosphate buffer )(
3) Stable pH range: 6-7 (phosphate buffer) (4)
Optimal temperature: Around 45°C (pH 6.0) (5) Thermal stability: Stable below 40°C (pH 7.2, treatment time 20 minutes) (6) Michaelis constant Km value for 2-oxo-4-phenylbutyric acid : 0.29mM (7) Molecular weight: 46,000 (gel filtration method) (8) Inhibitor: mercuric chloride and parachloromercuric benzoic acid The present invention also uses Leuconostoc.
The present invention provides a method for producing a novel enzyme, which involves culturing a microorganism belonging to the genus and collecting an enzyme having the above-mentioned physicochemical properties from the culture.

[0010]Furthermore, the present invention provides that in the presence of NADPH,
The optically active (R)-2-hydroxy- which causes the enzyme to act on 2-oxo-4-phenylbutyric acid to asymmetrically reduce it.
Also provided is a method for producing 4-phenylbutyric acid.

The origin of the novel enzyme of the present invention is not particularly limited, and any microorganism that can produce an enzyme having the above-mentioned physicochemical properties may be used. Preferred microorganisms are, for example, lactic acid bacteria, especially Leuconostoc.
c) It is a microorganism belonging to the genus. Among these microorganisms, the preferred strain is Leuconostoc dextranicum (
Leuconostocdextranicum)AT
It is CC27310.

[0012] The microorganisms assigned the ATCC numbers are listed in the American Type Culture Collection (
American Type Culture Col.
Catalog published by ATCC)
e of Bacteria Phages rDNA
Vectors, 16th Edition (1985), available from the ATCC.

Furthermore, these microorganisms may be wild strains, mutant strains, or recombinant strains induced by genetic methods such as cell fusion or genetic manipulation methods, as long as they produce enzymes having the above-mentioned physicochemical properties. Bacterial strains can also be suitably used.

[0014] The physicochemical properties of enzymes include unavoidable errors in measurement. Therefore, by way of example, the enzymes of the present invention also include enzymes having the following physicochemical properties.

(2) Optimum pH: about 5.5 to 6.5,
(3) Stable pH range: about 5.5 to 7.5, (4)
Optimal temperature: about 40 to 50°C, (6) 2-oxo-
Michaelis constant Km value for 4-phenylbutyric acid: 0.
Approximately 2 to 0.5mM (7) Molecular weight: approximately 40,000 to 50,000 The enzyme has the following physical and chemical properties as described in (1) to (8) above.
(9) In the presence of NADPH, acetaldehyde, n-
It is preferable to have the function of reducing aldehydes such as butyraldehyde, ketones such as 2-oxohexanoic acid and 2-oxooctanoic acid, and producing equimolar alcohols corresponding to these.

[0016] The enzyme selectively asymmetrically reduces 2-oxo-4-phenylbutyric acid in the presence of NADPH as a coenzyme rather than NADH.

The enzyme of the present invention can be obtained by culturing the above-mentioned microorganism in a medium and extracting and purifying the cultured cells.

[0018] The medium is not particularly limited as long as it allows microorganisms to grow. As a carbon source for the culture medium, any of the above-mentioned microorganisms can be used as long as they can be used; for example, sugars such as glucose, fructose, sucrose, dextrin, and starch; alcohols such as sorbitol, ethanol, and glycerol; fumaric acid, and citric acid. Organic acids such as acids, acetic acid and propionic acid and their salts; hydrocarbons such as paraffin; mixtures thereof, and the like can be used. Nitrogen sources include, for example, ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, ammonium phosphate; ammonium salts of organic acids such as ammonium fumarate, ammonium citrate; meat extract, yeast extract, malt extract, peptone, corn. steep liquor,
Inorganic or organic nitrogen-containing compounds such as casein hydrolyzate and urea; mixtures thereof can be used. In addition, the culture medium has
In addition to the above-mentioned components, nutrient sources used in normal culture, such as inorganic salts, trace metal salts, vitamins, etc., can be appropriately mixed and used. Furthermore, if necessary, factors that promote the growth of microorganisms, substances that are effective in maintaining the pH of the culture medium, and factors that increase the ability to produce the target compound of the present invention, such as 2-oxo-4-phenylbutyric acid, can also be added. .

[0019] The microorganisms are cultured under conditions suitable for growth, for example, the pH of the medium is 3.0 to 9.5, preferably 4 to 8;
The cultivation can be carried out at a culture temperature of 20 to 45°C, preferably 25 to 37°C. Cultivation of microorganisms can be carried out under anaerobic or aerobic conditions. The culture time is, for example, about 5 to 120 hours, preferably about 12 to 72 hours.

The enzyme of the present invention can be obtained by extraction and purification from cultured microbial cells. for example,
The culture is subjected to centrifugation to recover bacterial cells, and a cell-free extract is obtained by ultrasonic disruption or a combination of mechanical disruption and centrifugation. Next, the extract is, for example,
Streptomycin sulfate treatment, ammonium sulfate fractionation,
Enzymes can be obtained by purification using one or a combination of two or more conventional purification methods such as ion exchange chromatography, affinity chromatography, elution with an eluate, gel filtration, and ultrafiltration.

A method for producing optically active (R)-2-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid will be explained below.

In the method of the present invention, the substrate 2-oxo-4-phenylbutyric acid is asymmetrically reduced by the enzyme having the above-mentioned physicochemical properties to form (R)-2-hydroxy-4-
In order to generate phenylbutyric acid, a substrate and a coenzyme NADPH are required.

[0023] The asymmetric reduction reaction may be carried out by mixing the substrate, enzyme, and NADPH in an appropriate ratio under conditions in which the activity of the enzyme is stably expressed. The pH of the reaction system is, for example,
It is about 5 to 9, preferably about 6 to 8. Further, the reaction temperature is, for example, about 10 to 60°C, preferably about 20 to 40°C. The reaction can be carried out for about 1 to 120 hours under stirring or standing still.

The concentration of the substrate is not particularly limited as long as the production amount and optical purity of the desired optically active substance are not reduced, but it is preferably about 0.1 to 10% by weight, for example. Further, the ratio of the substrate to the enzyme and the ratio of the coenzyme to the enzyme can be selected within a range that does not reduce the production amount and optical purity of the desired optically active substance.

Furthermore, by constructing a recycling system that recycles the coenzyme into the reaction system during the reaction, (R
)-2-hydroxy-4-phenylbutyric acid can be produced more efficiently. A coenzyme recycling system can be constructed by a conventional method that combines a separation means for separating the coenzyme from the reaction solution, a recycling line, and the like.

Furthermore, the enzyme of the present invention can be used as an immobilized enzyme by immobilizing it on various immobilization carriers by conventional methods such as adsorption, entrapment, covalent bonding, ionic bonding, cross-linking, etc. It is also possible to do so. The type of carrier is not particularly limited, and examples include polyacrylamide,
It may be cellulosic materials, styrenic polymers, DEAE-Sephadex, ion exchange resins, collagen, albumin, carrageenan, alginic acid, agar, etc.

The optically active (R)-2-hydroxy-4-phenylbutyric acid produced can be recovered by conventional separation and purification means. For example, the optically active substance can be easily obtained by subjecting the reaction solution to a conventional purification method such as membrane separation, extraction with an organic solvent, column chromatography, separation using an ion exchange resin, concentration under reduced pressure, or recrystallization. can. The optical purity of optically active (R)-2-hydroxy-4-phenylbutyric acid can be measured, for example, by high performance liquid chromatography (HPLC) using an optical isomer separation column.

[0028]

Effect of the invention: The novel enzyme of the present invention has 2-oxo-4
- selectively asymmetrically reduces phenylbutyric acid to produce optically active (R
)-2-hydroxy-4-phenylbutyric acid is efficiently produced.

According to the method for producing an enzyme of the present invention, an enzyme having the above-mentioned excellent properties can be obtained.

Furthermore, in the production method of the present invention, optically active (R)-2-hydroxy-4-phenylbutyric acid can be efficiently produced from 2-oxo-4-phenylbutyric acid by the action of the enzyme.

[0031]

EXAMPLES The present invention will be explained in more detail below based on Examples, but the present invention is not limited to these Examples.

[0032] The enzyme activity was measured by the following standard activity measurement method.

Total volume 1 containing 10 μM 2-oxo-4-phenylbutyric acid, 0.1 μM NADPH, 100 μM potassium phosphate buffer (pH 6.0) and an appropriate amount of enzyme solution.
A ml reaction system was prepared, and the enzyme activity was determined by monitoring the decrease in the absorbance of NADPH at 340 nm at 37° C. using a spectrophotometer. In addition, under the above conditions, 1μ per minute
The amount of enzyme that oxidized NADPH of M was defined as 1 unit (U) of enzyme activity.

Further, the specific activity was calculated as the number of units (U) per mg of protein. In addition, the protein amount was determined using bovine serum albumin as a standard substance.
) method.

Example 1 (purification of enzyme) Glucose 8%, yeast extract 1%, manganese sulfate 10p
pm, 18 L of a medium with a composition of 2% calcium carbonate was placed in a 30 L jar fermenter, heat sterilized, and then 500 ml of a culture of Leuconostoc dextranicum IFO27310 previously cultured in the same medium as above.
l was inoculated. The cells were cultured for 18 hours at 30°C, 200 rpm, and gas phase aeration.

The obtained culture solution was subjected to centrifugation to collect bacteria, and then washed with physiological saline. Obtained wet bacterial cells 200
10mM phosphate buffer (pH 7.2) (0.02%
(containing 2-mercaptoethanol) was added thereto, and the bacterial cells were disrupted by ultrasonication. 10000G of this crushing liquid,
Centrifugation was performed for 10 minutes to obtain 180 ml of supernatant.

[0037] This crude extract was equilibrated with the same buffer as above on a DEAE-cellulose column (10 x 20 cm).
was loaded. After washing this column with 1 L of the same buffer solution as above, the target enzyme was eluted using a stepwise method in which the KCl concentration was gradually increased, and it was eluted at a concentration of 0.2M.

After concentrating this active fraction by ultrafiltration,
In the same manner as above, it was loaded onto a DEAE-Toyopearl 650M column (4 x 30 cm) and eluted in the same manner, and the target enzyme was eluted at a KCl concentration of 0.15M.

After concentrating this active fraction in the same manner as above,
A Cellulofine GCL-2000 column (2 x 10
Gel filtration was performed using 0 cm). The active fraction was concentrated in the same manner as above, and the purity was examined by polyacrylamide gel electrophoresis according to a conventional method. A single band was detected, confirming the homogeneity of the enzyme preparation.

The purification process is summarized in Table 1.

[0041]

[Table 1] Example 2 (Substrate specificity) Using the enzyme preparation obtained in Example 1, reaction rates were investigated for the various substrates listed in Table 2 according to a standard activity measurement method. The activity against 2-oxo-4-phenylbutyric acid was 1
00, and the obtained results are shown in Table 2 as relative activity.

[0042] In this reaction, NADPH is
It cannot be replaced by ADH.

[0043]

[Table 2] Example 3 (Optimal pH) The optimal pH of the enzyme preparation obtained in Example 1 was investigated as follows. That is, the activity of the enzyme preparation was measured according to a standard activity measurement method while varying the type and pH of the buffer solution. In addition, when adjusting the pH, in the range of pH 5 to 8,
Using 00mM phosphate buffer, 2
00mM Tris-HCl buffer was used.

The activity measured in phosphate buffer (pH 6.0) was set as 100, and the results obtained are shown in FIG. 1 as relative activity.

From FIG. 1, the optimal pH of the enzyme preparation in the phosphate buffer is around 6.

Example 4 (pH Stability) The pH stability of the enzyme preparation obtained in Example 1 was investigated as follows. That is, 0.5 ml of the enzyme preparation was added to 2 ml of buffer solutions with different pH, and the mixture was treated at 20° C. for 20 minutes. In addition, at pH 5 to 7, 200mM phosphate buffer, pH
7 to 9, 200 mM Tris-HCl buffer was used. Next, 0.1 ml of the treated solution was sampled, 0.5 ml of 1M phosphate buffer (pH 7.0) was added, and residual activity was measured by a standard activity measurement method using 0.05 ml of the enzyme-containing solution.

The residual activity when treated with phosphate buffer (pH 6.0) was set as 100, and the results are shown in FIG. 2 as relative activity.

From FIG. 2, the enzyme preparation is stable at pH 6 to 7 in phosphate buffer.

Example 5 (Optimal Temperature) The optimal temperature of the enzyme preparation obtained in Example 1 was investigated as follows. That is, the reaction temperature was varied and the reaction rate was measured according to a standard activity measurement method. The reaction rate at a reaction temperature of 45° C. was set as 100, and the obtained results are shown in FIG. 3 as relative reaction rates.

From FIG. 3, the optimal temperature for the enzyme preparation is around 45°C.

Example 6 (Thermostability) The thermostability of the enzyme preparation obtained in Example 1 was investigated as follows. That is, after adding 200 mM phosphate buffer (pH 7.2) to the enzyme preparation and adjusting the pH to 7.2, it was treated for 20 minutes under different temperature conditions, and the residual activity of the treated solution was determined by standard activity measurement. It was measured by the method. And 20
The activity at °C is set as 100, and the results are shown in Figure 4 as residual activity (%).
Shown as

From FIG. 4, the enzyme preparation is stable at temperatures below 40°C.

Example 7 (Km value for 2-oxo-4-phenylbutyric acid) Using the enzyme preparation obtained in Example 1, 2-oxo-4-
When the concentration of phenylbutyric acid was changed and the activity was measured using a standard activity measurement method, the Km value was determined to be 0.29mM.
Met.

Example 8 (Inhibitors) The effects of various inhibitors on the enzyme preparation obtained in Example 1 were investigated as follows. That is, the inhibitors shown in Table 7 were added to a reaction system based on the standard activity method, and after incubating with the enzyme at 30° C. for 5 minutes, NADPH was added to start the reaction, and the activity was measured. The activity when no inhibitor was added was set as 100, and the obtained activities are shown in Table 3 as relative activities.

[0055]

[Table 3] From Table 3, the activity of the enzyme preparation is inhibited by mercuric chloride and parachloromercuric benzoic acid.

Example 9 (Molecular Weight) The molecular weight of the enzyme preparation obtained in Example 1 was measured by gel filtration using HPLC. That is, Asahipak GS-520 (0.75×60
cm) (manufactured by Asahi Kasei Co., Ltd.) as a column, the molecular weight was determined by HPLC method using a conventional method, and it was found to be 46,000.
Met.

Example 10 ((R)-2-hydroxy-4
-Production of phenylbutyric acid) 100mg of 2-oxo-4-phenylbutyric acid, 20ml
of water and adjusted to pH 6 with 1N NaOH. After adding 500 mg of NADPH to this solution and dissolving it,
20 ml of 0 mM phosphate buffer (pH 6.0) was added. Next, enzyme solution 5 obtained in the same manner as in Example 1
ml (200 U) was added and reacted at 30°C for 24 hours.

The reaction solution was adjusted to pH 2.5 with 1N sulfuric acid,
Sodium chloride was dissolved until saturated, and the reaction product was extracted twice with 50 ml of ethyl acetate. The organic layers were combined and the solvent was distilled off to obtain 80 mg of crude crystals. The crude crystals were recrystallized from toluene to produce 68 mg of crystals (yield: 68%).
I got it.

mp: 113.5°C [α]D = -8.5 (c = 1, ethanol) The obtained crystals were dissolved in a small amount of water and subjected to high performance liquid chromatography using an optical resolution column (column: Daicel Chemical industry (
Co., Ltd., Chiralcel OB, solvent: n-hexane/2-propanol = 19:1), and the absolute configuration and optical purity were measured, and it was found that the product and (R)-2-hydroxy-
The retention time completely matched that of 4-phenylbutyric acid, and the optical purity of the product was 100% e. e. Met.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the optimum pH of the enzyme preparation in Example 3.

FIG. 2 is a graph showing the stable pH range of the enzyme preparation in Example 4.

FIG. 3 is a graph showing the optimum temperature of the enzyme preparation in Example 5.

FIG. 4 is a graph showing the stable temperature range of the enzyme preparation in Example 6.

Claims (1)

[Scope of Claims] [Claim 1] A novel enzyme having the following physical and chemical properties (1) to (8). (1) Action and substrate specificity: In the presence of reduced nicotinamide adenine dinucleotide phosphate, 2-oxo-4-phenylbutyric acid is asymmetrically reduced to produce (R)-2-hydroxy-4-phenyl Generates butyric acid (2) Optimum pH: around 6.0 (phosphate buffer) (3)
Stable pH range: 6-7 (phosphate buffer) (4) Optimum temperature: around 45°C (pH 6.0) (5) Thermal stability: 4
Stable below 0°C (pH 7.2, treatment time 20 minutes) (6) Michaelis constant Km value for 2-oxo-4-phenylbutyric acid: 0.29mM (7) Molecular weight: 46000 (gel filtration method) (8) Inhibition Agents: mercuric chloride and parachloromercuric benzoic acid [Claim 2] In the presence of reduced nicotinamide, adenine, dinucleotide, and phosphoric acid, aldehydes and ketones are reduced to produce equimolar alcohols corresponding to these. The novel enzyme according to claim 1. [Claim 3] Leuconostoc
A method for producing a novel enzyme, which comprises culturing a microorganism belonging to the genus Toc and collecting the enzyme according to claim 1 from the culture. 4. An optically active compound (2-oxo-4-phenylbutyric acid) which causes the enzyme according to claim 1 to act on 2-oxo-4-phenylbutyric acid for asymmetric reduction in the presence of reduced nicotinamide adenine dinucleotide phosphate. R) Method for producing -2-hydroxy-4-phenylbutyric acid.