JP2750017B2 - Novel enzyme, method for producing the same, and method for producing optically active (R) -2-hydroxy-4-phenylbutyric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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]

2. Description of the Related Art In recent years, a method for obtaining an optically active substance by utilizing an enzymatic asymmetric reduction reaction has been studied. For example, Japanese Patent Publication No. 61-1159
No. 1, Japanese Patent Publication No. 27717/1995, Japanese Unexamined Patent Publication No.
No. 286 and JP-A-63-32480 report several types of dehydrogenases produced by lactic acid bacteria. These dehydrogenases asymmetrically reduce various 2-oxocarboxylic acids using reduced nicotinamide adenine dinucleotide (hereinafter, referred to as NADH) as a coenzyme, and form a corresponding optically active 2-hydroxycarboxylic acid. Produces carboxylic acids.

Japanese Patent Application Laid-Open No. Sho 63-304979 discloses that reduced nicotinamide / adenine / dinucleotide / phosphate (hereinafter referred to as NADPH) is used as a coenzyme and γ
-Substituted acetoacetic ester to (R) -γ-substituted-β-
As an enzyme for producing hydroxybutyrate, a reductase extracted from a strain belonging to the genus Sporobolomyces is 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, for the enzyme, the asymmetric reduction of 2-oxo-4-phenylbutyric acid
The activity of producing (R) -2-hydroxy-4-phenylbutyric acid is not known.

Accordingly, an object of the present invention is to provide 2-oxo-4
Another object of the present invention is to provide a novel enzyme that selectively asymmetrically reduces -phenylbutyric acid to efficiently produce optically active (R) -2-hydroxy-4-phenylbutyric acid, and a method for producing the same.

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

[0007]

The present inventors have searched for an enzyme capable of producing (R) -2-hydroxy-4-phenylbutyric acid by asymmetric reduction of 2-oxo-4-phenylbutyric acid. The present invention has been completed by finding that a microorganism belonging to the genus (Leuconostoc) produces an enzyme capable of achieving the above object, and clarifying the physicochemical properties of the enzyme.

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

(1) Action and substrate specificity: Asymmetric reduction of 2-oxo-4-phenylbutyric acid in the presence of NADPH,
(R) -2-Hydroxy-4-phenylbutyric acid is produced. (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: stable at 40 ° C or less (pH 7.2, treatment time 20 minutes) (6) Michaelis constant Km for 2-oxo-4-phenylbutyric acid Value: 0.29 mM (7) Molecular weight: 46000 (gel filtration method) (8) Inhibitor: mercury chloride and parachloromercuric benzoic acid The present invention also comprises culturing a microorganism belonging to the genus Leuconostoc, The present invention provides a method for producing a novel enzyme for collecting an enzyme having the above physicochemical properties from a culture.

[0010] Further, the present invention relates to a method for preparing NADPH,
Optically active (R) -2-hydroxy-, which is asymmetrically reduced by allowing the enzyme to act on 2-oxo-4-phenylbutyric acid.
A method for producing 4-phenylbutyric acid is also provided.

[0011] The origin of the novel enzyme of the present invention is not particularly limited, and may be any microorganism capable of producing the enzyme having the above-mentioned physicochemical properties. Preferred microorganisms are, for example, lactic acid bacteria, especially microorganisms belonging to the genus Leuconostoc. A preferred strain among these microorganisms is Leuconostoc dextranicum (Leuconostoc).
dextranicum) ATCC 27310.

[0012] The microorganisms with the ATCC number may be an American Type Culture Collection (American Type Culture Collection).
an Type Culture Collection, ATCC)
logue of Bacteria Phages rDNA Vectors, 16th edition (1
985) "and is available from the ATCC.

Furthermore, these microorganisms may be any of wild-type, mutant, or recombinant strains induced by genetic techniques such as cell fusion or genetic engineering, as long as they produce enzymes having the above-mentioned physicochemical properties. A strain can also be suitably used.

[0014] The physicochemical properties of the enzyme 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) Optimum temperature: about 40 to 50 ° C., ( 6) Michaelis constant Km value for 2-oxo-4-phenylbutyric acid: about 0.2 to 0.5 mM (7) Molecular weight: about 40000 to 50,000 The enzyme has the physicochemical properties described in (1) to (8) above. in addition,
(9) An action of reducing aldehydes such as acetaldehyde and n-butyraldehyde and ketones such as 2-oxohexanoic acid and 2-oxooctanoic acid in the presence of NADPH to produce an equimolar alcohol corresponding thereto. It is preferred to have

The enzyme selectively asymmetrically reduces 2-oxo-4-phenylbutyric acid in the presence of NADPH as a coenzyme instead of NADH.

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

The medium is not particularly limited as long as the microorganism can grow. As the carbon source of the culture medium, any of the above microorganisms can be used, for example, sugars such as glucose, fructose, sucrose, dextrin, starch; alcohols such as sorbitol, ethanol, glycerol; fumaric acid, citric acid Organic acids such as acid, acetic acid and propionic acid and salts thereof; hydrocarbons such as paraffin; mixtures thereof and the like can be used. As the nitrogen source, for example, 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 extract, yeast extract, malt extract, peptone, corn Inorganic or organic nitrogen-containing compounds such as steep liquor, casein hydrolyzate, and urea; mixtures thereof can be used. In addition, the medium, in addition to the above components, nutrient sources used for normal culture,
For example, inorganic salts, trace metal salts, vitamins and the like as appropriate,
They can be used in combination. In addition, if necessary,
Factors that promote the growth of microorganisms, substances that are effective in maintaining the pH of the medium, and factors that enhance the ability to produce the target compound of the present invention, such as 2-oxo-4-phenylbutyric acid, can also be added.

Cultivation of microorganisms is carried out under conditions suitable for growth, for example, when the pH of the medium is 3.0 to 9.5, preferably 4 to 8,
The cultivation can be carried out at a temperature of 20 to 45 ° C, preferably 25 to 37 ° C. Cultivation of the microorganism can be performed under anaerobic or aerobic conditions. The culturing 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 extracting and purifying from the cultured microbial cells. For example,
The culture is subjected to centrifugation to collect the cells, and a cell-free extract is obtained by sonication or a method combining mechanical crushing and centrifugation. The extract is then treated with one or more ordinary purification methods such as, for example, streptomycin sulfate treatment, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, elution with an eluent, gel filtration, and ultrafiltration. By purifying in combination, an enzyme can be obtained.

Hereinafter, a method for producing optically active (R) -2-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid will be described.

In the method of the present invention, 2-oxo-4-phenylbutyric acid, which is a substrate, is asymmetrically reduced by the enzyme having the above-mentioned physicochemical properties to give (R) -2-hydroxy-4-
In order to produce phenylbutyric acid, coenzyme NADPH is required together with the substrate.

The asymmetric reduction reaction may be carried out by mixing a substrate, an enzyme and NADPH at an appropriate ratio under conditions where 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 performed under stirring or standing for about 1 to 120 hours.

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

Also, by constructing a recycling system for recycling the coenzyme to the reaction system during the reaction,
(R) -2-hydroxy-4-phenylbutyric acid can be produced more efficiently. The coenzyme recycling system can be constructed by a conventional method combining a separation means for separating the auxiliary enzyme 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 immobilized carriers by a conventional method such as adsorption, entrapment, covalent bonding, ionic bonding, cross-linking and the like. It is also possible. The type of the carrier is not particularly limited, for example, polyacrylamide,
Cellulose-based materials, styrene-based polymers, DEAE-Sephadex, ion exchange resins, collagen, albumin, carrageenan, alginic acid, agar, and the like may be used.

The generated optically active (R) -2-hydroxy-4-phenylbutyric acid 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 with an ion exchange resin, concentration under reduced pressure, and recrystallization. it can. The optical purity of the 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]

The novel enzyme of the present invention is 2-oxo-4
-Phenylbutyric acid is selectively asymmetrically reduced to efficiently produce optically active (R) -2-hydroxy-4-phenylbutyric acid.

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

Further, 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 described below in more detail with reference to examples, but the present invention is not limited to these examples.

The enzyme activity was measured by the following standard activity measuring method.

A 1 ml reaction system 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 an enzyme solution was prepared. Then, the decrease in absorbance of NADPH at 340 nm was tracked with a spectrophotometer,
Enzyme activity was determined. Under the above conditions, 1 minute
The enzyme amount at which μM NADPH was oxidized was defined as 1 unit (U) of the enzyme activity.

The specific activity was calculated as the number of units (U) per mg of protein. The protein amount was measured by the Lowry method using bovine serum albumin as a standard substance.

Example 1 (Purification of enzyme) Glucose 8%, yeast extract 1%, manganese sulfate 10p
pm, 18 L of a medium having a composition of 2% calcium carbonate was placed in a 30 L jar fermenter, sterilized by heating, and then cultured in a medium similar to that described above in a culture medium of Leuconostoc dextranicum IFO27310, which had been preliminarily cultured in the same medium as described above.
1 was inoculated. The cells were cultured at 30 ° C. under the conditions of 200 rpm and gas phase aeration for 18 hours.

The obtained culture was subjected to centrifugation to collect the bacteria, and then washed with physiological saline. Obtained wet cells 200
g to 10 mM phosphate buffer (pH 7.2) (0.02%
200 ml of 2-mercaptoethanol) was added, and the cells were sonicated. This crushed liquid is 10,000
G, and centrifuged for 10 minutes to obtain 180 ml of a supernatant.

A column of DEAE-cellulose (10 × 20 cm) in which the crude extract was equilibrated with the same buffer as above.
Loaded. After the column was washed with 1 L of the same buffer as above, the target enzyme was eluted by the stepwise method of sequentially increasing the KCl concentration.

After the active fraction was concentrated by ultrafiltration,
In the same manner as described above, the column was loaded on a column (4 × 30 cm) of DEAE-Toyopearl 650M and eluted in the same manner. The target enzyme was eluted at a KCl concentration of 0.15M.

After concentrating the active fraction in the same manner as described above,
A column of Cellulofine GCL-2000 equilibrated with the same buffer as above containing 0.2 M KCl (2 × 10
(0 cm). The active fraction was concentrated in the same manner as described above, and the purity was examined by polyacrylamide gel electrophoresis according to a conventional method. As a result, it was detected as a single band, and the homogeneity of the enzyme preparation was confirmed.

The purification process is summarized in Table 1.

[0041]

[Table 1] Example 2 (Substrate Specificity) The reaction rates of various substrates shown in Table 2 were examined using the enzyme preparation obtained in Example 1 in accordance with the standard activity measurement method. The activity against 2-oxo-4-phenylbutyric acid is 1
The results are shown in Table 2 as relative activities.

In this reaction, NADPH is converted to N
ADH cannot be substituted.

[0043]

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

The activity measured with a phosphate buffer (pH 6.0) is defined as 100, and the obtained results are shown in FIG. 1 as relative activities.

As shown in FIG. 1, the optimum 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 examined as follows. That is, 0.5 ml of the enzyme preparation was added to 2 ml of buffers having different pH, and the mixture was treated at 20 ° C. for 20 minutes. At pH 5 to 7, 200 mM phosphate buffer, p
For H7-9, a 200 mM Tris-HCl buffer was used.
Next, 0.1 ml of the treatment solution was sampled, 0.5 ml of a 1 M phosphate buffer (pH 7.0) was added, and the residual activity was measured by a standard activity measurement method using 0.05 ml of a solution containing the enzyme.

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

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

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

FIG. 3 shows that the optimum temperature of the enzyme preparation is around 45 ° C.

Example 6 (Thermal Stability) The thermal stability of the enzyme preparation obtained in Example 1 was examined as follows. That is, a 200 mM phosphate buffer (pH 7.2) was added to the enzyme preparation to adjust the pH to 7.2, followed by treatment under different temperature conditions for 20 minutes, and the residual activity of the treated solution was measured by standard activity measurement. It was measured by the method. And 20
The activity at 100 ° C. was defined as 100, and the results are shown in FIG.
As shown.

FIG. 4 shows that the enzyme preparation is stable at 40 ° C. or lower.

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 by a standard activity measuring method, and the Km value was determined,
M.

Example 8 (Inhibitor) The influence of various inhibitors on the enzyme preparation obtained in Example 1 was examined as follows. That is, the inhibitors shown in Table 7 were added to the reaction system according to the standard activity method, and the mixture was incubated with the enzyme at 30 ° C. for 5 minutes. Then, NADPH was added to start the reaction, and the activity was measured. The activity when no inhibitor was added was defined as 100, and the obtained activity is shown in Table 3 as a relative activity.

[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 a gel filtration method using an HPLC method. That is, Asahipak GS-520 (0.75 × 60 cm)
(Manufactured by Asahi Kasei Corporation) as a column,
The molecular weight was determined by HPLC to be 46,000.

Example 10 ((R) -2-hydroxy-4)
-Production of phenylbutyric acid) 100 mg of 2-oxo-4-phenylbutyric acid was added to 20 ml
And adjusted to pH 6 with 1N NaOH. After adding and dissolving 500 mg of NADPH to this solution, 10
20 ml of 0 mM phosphate buffer (pH 6.0) was added. Next, 5 ml (200 U) of the enzyme solution obtained in the same manner as in Example 1 was added, and the mixture was 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, the solvent was distilled off, and 80 mg of crude crystals were obtained. The crude crystals were recrystallized from toluene to give 68 mg of crystals (68% yield).
I got

Mp: 113.5 ° C. [α] _D = −8.5 (c = 1, ethanol) The obtained crystals are dissolved in a small amount of water, and high-performance liquid chromatography using an optical resolution column (column: Daicel) The product was subjected to a chiral cell OB (solvent: n-hexane / 2-propanol = 19: 1) manufactured by Chemical Industry Co., Ltd., and the absolute configuration and optical purity were measured. The product and (R) -2-hydroxy-4- The retention times with phenylbutyric acid are completely identical and the optical purity of the product is 100% e. e. Met.

[Brief description of the drawings]

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

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

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

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

Continuation of the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/04 C12P 7/52 CA (STN)

Claims (4)

(57) [Claims] 1. A novel enzyme having the following physicochemical properties (1) to (8). (1) Action and substrate specificity: Asymmetric reduction of 2-oxo-4-phenylbutyric acid in the presence of reduced nicotinamide / adenine / dinucleotide / phosphoric acid to give (R) -2-hydroxy-4-phenyl (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) 0) (5) Thermal stability: stable at 40 ° C. or less (pH 7.2, treatment time: 20 minutes) (6) Michaelis constant Km value for 2-oxo-4-phenylbutyric acid: 0.29 mM (7) Molecular weight: 46,000 (Gel filtration method) (8) Inhibitors: mercuric chloride and parachloromercuric benzoic acid 2. The novel enzyme according to claim 1, wherein aldehydes and ketones are reduced in the presence of reduced nicotinamide / adenine / dinucleotide / phosphate to produce an equimolar alcohol corresponding thereto. 3. A method for producing a novel enzyme, comprising culturing a microorganism belonging to the genus Leuconostoc and collecting the enzyme according to claim 1 from the culture. 4. The enzyme according to claim 1, which is in the presence of reduced nicotinamide / adenine / dinucleotide / phosphate.
A method for producing optically active (R) -2-hydroxy-4-phenylbutyric acid, which is asymmetrically reduced by acting on 2-oxo-4-phenylbutyric acid.