JPH0919290A - Heat resistant and gamma-cyano-alpha-aminobutylic acid-synthetic enzyme and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new synthetic enzyme capable of producing γ-cyano-α- aminobutylic acid from O-acetyl-L-homoserine as a substrate. SOLUTION: This heat resistant and γ-cyano-α-aminobutylic acid-producing enzyme is produced by Bacillus stearothermophilus CN3 strain and has the following properties and actions; the enzyme produces γcyano-α-aminobutylic acid from O-acetyl-L-homoserine and a cyanide compound, the optimum pH: 7.5-8.5, the stable pH range: 6.0-10.5, the optimum temperature: 55-65 deg.C, temperature stability: stable until 60 deg.C when kept at pH 7.5 for 30 minutes, molecular weight: about 180kd by a gel filtration.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a heat-resistant γ-cyano-α.
-Aminobutyric acid synthase and a method for producing the same. More specifically, the present invention relates to O-acetyl-L-
A new γ that is capable of producing γ-cyano-α-aminobutyric acid using homoserine as a substrate and that has excellent thermal stability
TECHNICAL FIELD The present invention relates to cyano-α-aminobutyric acid synthase and a method for producing this synthase.

[0002]

2. Description of the Related Art γ-Cyano-α-
As an enzyme for producing aminobutyric acid, a bacterium belonging to the genus Chromobacterium ( C. violac
It is known that γ-cyano-α-aminobutyric acid synthase obtained by isolation from Eum ) (Biochemistry, 12 ,
p5369-5377, 1973).

Since this γ-cyano-α-aminobutyric acid synthase (hereinafter sometimes referred to as a conventional enzyme) has high reactivity with L-homocystine, L-homocystin is used as a substrate and cyanide is used. To produce γ-cyano-α-aminobutyric acid. However, when L-homocystine is used as a substrate, a by-product containing cyan is generated, which has a problem that the reaction efficiency is reduced and the purification of γ-cyano-α-aminobutyric acid is affected.

On the other hand, O-acetyl- is used as a substrate.
When L-homoserine is used, γ-cyano-α-aminobutyric acid can be produced without producing a by-product containing cyanide. However, the above-mentioned conventional enzyme reacts with O-acetyl-L-homoserine. Low reactivity (assuming that the reactivity to L-homocysteine is 100%, that to O-acetyl-L-homoserine is 5%), and that γ-
It was not possible to produce cyano-α-aminobutyric acid.

Further, in the case of a conventional enzyme, it is described that it is stable at 30 ° C. for 3 hours (the above-mentioned document), and the stability at high temperature was not preferable, so that γ-cyano-
There was also a problem that the reaction temperature for synthesizing α-aminobutyric acid had to be limited. The present invention has been made in view of the circumstances described above.
Γ-cyano-α using acetyl-L-homoserine as a substrate
-The object is to provide a new thermostable γ-cyano-α-aminobutyric acid synthase capable of producing aminobutyric acid and stable under high temperature conditions.

[0006]

In order to solve the above-mentioned problems, the present invention has the following properties (1) Action: γ-cyano-α-aminobutyric acid from O-acetyl-L-homoserine and cyanide (2) Optimum pH: 7.5 to 8.5 (3) Stable pH: 6.0 to 10.5 (4) Optimum temperature: 55 to 65 ° C (5) Temperature stability: pH 7. Stable up to 60 ° C. when kept at 5 ° C. for 30 minutes (6) A thermostable γ-cyano-α-aminobutyric acid synthase having a molecular weight of about 180 kd by gel filtration is provided.

Further, the present invention provides a bacterium which belongs to the genus Bacillus and has the ability to produce a thermostable γ-cyano-α-aminobutyric acid synthase having the above-mentioned properties (1) to (6). Thermostable γ-cyano-α-aminobutyric acid synthesis characterized by isolating γ-cyano-α-aminobutyric acid synthase from the bacterial cells of this bacterium by culturing in a cyano-α-aminobutyric acid synthase production medium A method for producing the enzyme is also provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The thermostable γ-cyano-α-aminobutyric acid synthase provided by the present invention has a high action on O-acetyl-L-homoserine (O-acetyl-L
-When the activity against homoserine is defined as 100%,
6.6% for L-homocystine), and when this O-acetyl-L-homoserine is used as a substrate for reaction with cyanide, γ-cyano-α-aminobutyric acid is efficiently produced without producing byproducts. can do. In addition, while the conventional enzyme is stable at 30 ° C. for 3 hours,
The synthetic enzyme of the present invention is stable up to 60 ° C., and is far superior to conventional enzymes in terms of stability.

The heat-resistant γ-cyano-α of the present invention
-Aminobutyric acid synthase requires pyridoxal phosphate as a coenzyme. In addition, the above-mentioned O-acetyl-L-
In addition to homoserine, L-homocystin can be used as a substrate. Specifically, this heat-resistant γ-cyano-α-
Aminobutyric acid synthase, Bacillus (Bacillus) bacterium belonging to the genus, and more specifically, may be a strain belonging to Bacillus stearothermophilus (B. stearothermophilus) can be isolated. Such strains belonging to Bacillus stearothermophilus include Bacillus stearothermophilus CN3 strain (accession number FERM BP) deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST.
-4773) can be illustrated as a suitable thing.

Next, the heat-resistant γ-cyano-α-of the present invention
A method for producing aminobutyric acid synthase will be described. This thermostable γ-cyano-α-aminobutyric acid synthase is obtained by culturing a bacterium belonging to the genus Bacillus in a γ-cyano-α-aminobutyric acid synthase production medium, Obtained by isolating -α-aminobutyric acid synthase. As bacteria belonging to the genus Bacillus,
A strain belonging to the above Bacillus stearothermophilus, preferably Bacillus stearothermophilus CN3
A strain can be used. The components of the γ-cyano-α-aminobutyric acid synthase production medium for culturing such bacteria include, for example, glucose, fructose, lactose, maltose, sucrose, starch and dextrin as carbon sources. Sugars, glutamic acid, amino acids such as serine, or organic acids such as fumaric acid, malic acid, succinic acid, or glycerol, as a nitrogen source, polypeptone, tryptone, yeast extract, malt extract, meat extract, corn steep liquor, soy flour Natural nitrogen sources such as hydrolysates, inorganic salts such as NaCl, dipotassium hydrogen phosphate, magnesium sulfate, etc., and trace metal components such as calcium chloride, boric acid, copper sulfate, potassium iodide, ferrous sulfate, Examples thereof include manganese sulfate and zinc sulfate.

Further, in order to isolate γ-cyano-α-aminobutyric acid synthase from the cells of the cultured bacteria, known purification methods can be used alone or in combination. For example, the culture solution is centrifuged to collect the bacterial cells, which are sonicated or Dyno-mill treated to disrupt the cells, and then centrifuged to obtain a cell-free extract. This is subjected to ammonium sulfate fractionation, desalting treatment by dialysis, etc., followed by ion exchange chromatography, hydrophobic chromatography,
The thermostable γ-cyano-α-aminobutyric acid synthase of the present invention can be isolated by performing hydroxyapatite chromatography, gel filtration chromatography and the like.

The heat resistance γ of the present invention will be described below with reference to examples.
The method for producing -cyano-α-aminobutyric acid synthase and its properties will be described in more detail, but the present invention is not limited to the following examples. The activity of γ-cyano-α-aminobutyric acid synthase in the following Examples was measured according to the method shown in the following Reference Example. Reference Example Enzyme activity was measured by 1M potassium phosphate buffer (pH 7.
5) 10 μl (final concentration 50 mM), 10 mM O-acetyl-L-homoserine 100 μl (final concentration 5 mM), 1
20 μl of 00 mM potassium cyanide (final concentration 10 m
M), 20 μl of 0.8 mM pyridoxal phosphate (final concentration 0.08 mM) and 50 μl of enzyme solution (total amount 200 μl) were incubated at 45 ° C. for 10 minutes and then placed in a boiling water bath for 2 minutes to stop the reaction. Let
Then, the supernatant obtained by centrifugation at 15,000 rpm for 5 minutes was subjected to high performance liquid chromatography (HPLC) to quantify γ-cyano-α-aminobutyric acid produced by the enzymatic reaction.

As the unit of enzyme activity, one unit was defined as the enzyme activity which produces 1 μmol of γ-cyano-α-aminobutyric acid per minute under the above-mentioned conditions. Also, H
The PLC conditions are: column: inert sils ODS-2 (inner diameter 4.6 × 250 mm: manufactured by GL Sciences), eluent: 20 mM sodium phosphate buffer (pH 6.8).
/ Acetonitrile (85:15).

[0014]

【Example】

(1) Cultivation of Bacillus stearothermophilus CN3 strain Dried broth medium "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) for general bacteria was put into four test tubes (diameter 2.2 cm x length 19.5 cm). After dispensing, sterilizing at 120 ° C. for 20 minutes and cooling, Bacillus stearothermophilus CN3 strain was inoculated with one platinum loop each, and cultured at 58 ° C. for 18 hours with shaking to prepare a seed culture solution.

Soluble starch (1%), yeast extract (0.5%), and Mg were placed in four 2-liter culture flasks.
_{SO 4 · 7H 2 O (0.05} %), K 2 HPO 4 (0.
_{1%), FeSO 4 · 7H} 2 O (0.001%), L-
Medium consisting of glutamine (0.1%) (pH 7.2)
After 400 ml each was dispensed and sterilized at 120 ° C. for 20 minutes and cooled, 16 ml of the above seed culture solution (4 test tubes) was inoculated into each flask in an amount of 4 ml, and the temperature was adjusted to 58 ° C.
It was shake-cultured for 18 hours to prepare a preculture liquid.

Then, a defoaming agent "Adecanol LG126" (manufactured by Asahi Denka Co.) 0.0
200 liters of 160 liter medium supplemented with 1% (W / V)
Put in a jar fermenter of liter capacity, 120 ℃
After sterilizing for 20 minutes and cooling, the above preculture liquid 1.
6 liters were inoculated and cultured at 58 ° C. for 18 hours under the conditions of an aeration rate of 160 liters / minute and a stirring speed of 200 rpm. After the culture was completed, the bacterial cells were collected by Sharpless. (2) Isolation of thermostable γ-cyano-α-aminobutyric acid synthase from Bacillus stearothermophilus CN3 strain Isolation of thermostable γ-cyano-α-aminobutyric acid synthase by the following steps 1-8 did. 1. Preparation of cell-free extract: bacterial cell 66 obtained by the above culture
0 g of potassium phosphate buffer (20 mM, pH 7.5,
The mixture was suspended in 0.1 mM dithiothreitol (containing 0.1 mM dithiothreitol) so that the total amount became 2.5 liters, and then crushed with a grinding device "Dynomill" (manufactured by WAB). The disrupted liquid was centrifuged to remove the cell residue, and 2799 ml of a cell-free extract was obtained. 2. Heat treatment: leave the cell-free extract at 60 ° C. for 30 minutes,
The generated precipitate was removed by centrifugation to obtain a supernatant. 3. Ammonium sulfate 40-90% treatment: Ammonium sulfate was added to the supernatant liquid so as to be 40% saturated and left overnight, then
The deposited precipitate was removed by centrifugation, ammonium sulfate was again added to the obtained supernatant liquid so as to be 90% saturated, and the mixture was allowed to stand overnight, and the precipitate was obtained by centrifugation. 4. Purification by DEAE-Cellulofine A-500:
The precipitate was treated with 0.1 mM dithiothreitol and 0.0
It was dissolved in a 20 mM potassium phosphate buffer (pH 7.5) containing 1 mM pyridoxal phosphate and desalted with the same buffer using a dialysis membrane. This desalted solution was passed through a DEAE-Cellulofine A-500 column (8 cm in diameter x 22 cm in height) that had been equilibrated with the same buffer in advance to adsorb the enzyme, and 0.1 mM dithiothreitol and 0.01 mM were added.
After washing with 100 mM potassium phosphate buffer (pH 7.5) containing pyridoxal phosphate, 100 mM potassium phosphate buffer containing 0.1 mM dithiothreitol, 0.01 mM pyridoxal phosphate and 0.4 MKCl from the same buffer The enzyme was eluted by a gradient elution method to (pH 7.5) and the active fractions were collected. 5. Ammonium sulfate 60-75% treatment: Ammonium sulfate was added to the active fraction so as to be 60% saturated, and the mixture was allowed to stand overnight, the deposited precipitate was removed by centrifugation, and ammonium sulfate was added to the obtained supernatant again to 75%. The mixture was added so as to be% saturated and left overnight, and a precipitate was obtained by centrifugation. 6. Purification with phenyl-Toyopearl 650S: The precipitate was dissolved in 20 mM potassium phosphate buffer (pH 7.5) containing 30% saturated ammonium sulfate, 0.1 mM dithiothreitol and 0.01 mM pyridoxal phosphate, and the solution was previously dissolved in the same buffer. Equilibrated Phenyl-Toyopearl 6
After passing through a 50S column (diameter 2.5 × height 8.5 cm) to adsorb the enzyme and washing with the same buffer solution, 0.1 mM dithiothreitol and 0.01 mM were added from the same buffer solution.
The enzyme was eluted by a gradient elution method into 20 mM potassium phosphate buffer (pH 7.5) containing pyridoxal phosphate, and the active fractions were collected. 7. Purification by Sephacryl S-200HR: Ammonium sulfate was added to the active fraction to 80% saturation,
After standing overnight, a precipitate was obtained by centrifugation. This precipitate was added with 0.1 mM dithiothreitol and 0.2M.
50 mM sodium phosphate buffer containing NaCl (pH
Sephacryl S-200HR column (diameter 2.0 x height 106 cm) dissolved in 7.5) and equilibrated with the same buffer beforehand.
The enzyme was eluted with the same buffer and the active fractions were collected. 8. Purification by TSK gel-G3000SW: Ammonium sulfate was added to the active fraction to 80% saturation,
After standing overnight, a precipitate was obtained by centrifugation. This precipitate was dissolved in 100 mM sodium phosphate buffer (pH 7.0) containing 0.2 M NaCl, and TSK for HPLC was used.
Gel-G3000SW column (diameter 0.75 x height 6
0 cm) with the same buffer as the mobile phase at a flow rate of 0.7 ml /
It was injected under the condition of minute, and the active fraction was collected. The obtained enzyme is an electrophoretic uniform sample, and the specific activity is 147 U.
/ Mg. The total enzyme activity, total protein, specific activity, purification rate and yield in each of the above steps 1 to 8 were as shown in Table 1.

[0017]

[Table 1]

(3) Property test of thermostable γ-cyano-α-aminobutyric acid synthase isolated from Bacillus stearothermophilus CN3 strain The thermostable γ-cyano- of the present invention isolated by the above (2) The α-aminobutyric acid synthase was tested for optimum pH, stable pH, optimum temperature and temperature stability. Moreover, the absorption spectrum was measured. 1. Optimum pH: The buffer solution of the reaction solution for activity measurement in the enzyme activity measuring method described in the reference example is MES (pH 6.0 to
7.0), KPB (pH 6.0 to 8.0), MOPS
(PH 6.5 to 7.5), Tris-HCl (pH 7.
5 to 9.0), or _{NH 4 Cl-NH 4 OH (} pH
The enzyme activity was measured instead of 8.5 to 10.0). The results are shown in FIG. 1, and the optimum pH of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention is 7.5 to 7.5.
It was found to be 8.5. 2. Stable pH: The thermostable γ-cyano-α-aminobutyric acid synthase of the present invention was added to a buffer solution having a concentration of 20 mM, namely citric acid / sodium citrate (pH 3.5 to 5.5),
MES (pH 6.0 to 7.0), KPB (pH 6.0 to
8.0), Tris-HCl (pH 7.5 to 9.0),
Or _{NH 4 Cl-NH 4 OH (} pH8.5~10.
0) and glycine / KCl-KOH (pH 10.0-
10.5), and the residual activity was measured after holding at 60 ° C. for 30 minutes. The results are shown in FIG. 2, and it was revealed that the stable pH of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention is 6.0 to 10.5. 3. Optimum temperature: The thermostable γ-cyano-α-aminobutyric acid synthase of the present invention is added to 20 mM potassium phosphate buffer (pH
It was dissolved in 7.5) and was added to 30 by the enzyme activity measuring method of Reference Example.
The enzyme activity was measured in the range from ℃ to 70 ℃. The results are shown in FIG. 3, and the optimum temperature of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention is 55 to 65.
It was found to be ° C. 4. Temperature stability: The thermostable γ-cyano-α-aminobutyric acid synthase of the present invention was added to 20 mM potassium phosphate buffer (p
It was dissolved in H7.5) and kept at each temperature from 45 ° C. to 90 ° C., after which the residual activity was measured. The results are shown in FIG. 4, and it was revealed that the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention has extremely high thermal stability and is stable up to a temperature of 60 ° C. 5. Absorption spectrum The thermostable γ-cyano-α-aminobutyric acid synthase of the present invention is dissolved in a 20 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM dithiothreitol and 0.01 mM pyridoxal phosphate, and this solution is prepared. Targeting U-
The absorption spectrum was measured with a Model 3200 spectrophotometer (manufactured by Hitachi, Ltd.). The results are shown in FIG. 5 and Table 2, and the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention has a range of 410 to 440 nm, which is peculiar to an enzyme containing pyridoxal phosphate as a coenzyme. Absorption was seen.

[0019]

[Table 2]

[0020]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, .gamma.
A new thermostable γ-cyano-α-aminobutyric acid synthase capable of producing -cyano-α-aminobutyric acid is provided. This makes it possible to efficiently produce γ-cyano-α-aminobutyric acid.

[Brief description of the drawings]

FIG. 1 is a correlation diagram between relative activity and pH indicating the optimum pH of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention.

FIG. 2 is a correlation diagram of relative activity and pH showing stable pH of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention.

FIG. 3 is a correlation diagram between relative activity and temperature showing the optimum temperature of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention.

FIG. 4 is a correlation diagram between relative activity and temperature showing the temperature stability of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention.

FIG. 5 is a correlation diagram between wavelength and absorbance showing an absorption spectrum of the thermostable γ-cyano-α-aminobutyric acid synthase of the present invention.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technology display location (C12N 1/20 C12R 1:07) (72) Inventor Yuji Furuya Jusankenya, Kannabe Town, Fukaan District, Hiroshima Prefecture 20-9 (72) Inventor Tatsuhiko Kobayashi No. 307 Royal Center Building, 97 Shimobabacho, Jodo-ji, Sakyo-ku, Kyoto Prefecture (72) Inventor Masaru Shimizu 14 Nishinokyo-Hirakucho, Nakagyo-ku, Kyoto-shi, Kyoto Prefecture

Claims (8)

[Claims] 1. The following properties (1) Action: γ-cyano-α-aminobutyric acid is produced from O-acetyl-L-homoserine and cyanide (2) Optimum pH: 7.5-8.5 (3) Stable pH: 6.0 to 10.5 (4) Optimum temperature: 55 to 65 ° C (5) Temperature stability: Stable up to 60 ° C when kept at pH 7.5 for 30 minutes (6) Molecular weight: Thermostable γ-cyano-α-aminobutyric acid synthase having about 180 kd by gel filtration. 2. Pyridoxal phosphate as a coenzyme,
The thermostable γ-cyano-α-aminobutyric acid synthase according to claim 1, which produces γ-cyano-α-aminobutyric acid from O-acetyl-L-homoserine or L-homocystin and cyanide. 3. The thermostable γ-cyano-α-aminobutyric acid synthase according to claim 1, obtained by isolation from a bacterium belonging to the genus Bacillus . 4. A bacterium, Bacillus stearothermophilus (B. stearothermophilus) a thermostable γ- cyano -α- amino acid synthase according to claim 3. 5. The thermostable γ-cyano-according to claim 4, wherein the bacterium is Bacillus stearothermophilus CN3 strain (accession number FERM BP-4733) deposited at the Institute of Biotechnology, Institute of Biotechnology, Industrial Technology Institute. α-aminobutyric acid synthase. 6. The following properties, (1) action: producing γ-cyano-α-aminobutyric acid from O-acetyl-L-homoserine and cyanide (2) optimum pH: 7.5-8.5 (3) Stable pH: 6.0 to 10.5 (4) Optimum temperature: 55 to 65 ° C (5) Temperature stability: Stable up to 60 ° C when kept at pH 7.5 for 30 minutes (6) Molecular weight: Bacteria belonging to the genus Bacillus, which have a thermostable γ-cyano-α-aminobutyric acid synthase production capacity of about 180 kd by gel filtration, were cultured in a γ-cyano-α-aminobutyric acid synthase production medium. , A method for producing a thermostable γ-cyano-α-aminobutyric acid synthase, which comprises isolating the γ-cyano-α-aminobutyric acid synthase from the bacterial cells of this bacterium. 7. bacterium, Bacillus stearothermophilus (B. stearothermophilus) preparation of heat-resistant γ- cyano -α- amino acid synthase of claim 6 which is. 8. The thermostable γ-cyano-according to claim 6, wherein the bacterium is Bacillus stearothermophilus CN3 strain (accession number FERM BP-4733) deposited at the Institute of Biotechnology, Institute of Biotechnology, Industrial Technology Institute. A method for producing α-aminobutyric acid synthase.