JPH0856666A - Heat-resistant o-acetylserinesulfhydrylase and production - Google Patents

Abstract

PURPOSE: To obtain the subject new enzyme excellent in heat stability, capable of producing β-cyanoalanine from O-acetyl-L-serine and a cyano compound and useful for clinical diagnosis, research, etc., by culturing a thermophilic bacteria belonging to the genus Bacillus having O- acetylserinesulfhydrylaseproducing activity. CONSTITUTION: A thermophilic bacteria belonging to the genus Bacillus having O-acetylserinesulfhydrylase-producing activity [e.g. Bacillus stearothermophilus CN3 (FERM-BP-4773)] is inoculated into an O-acetylserinesulfhydrylase-inducing culture medium and cultured at 60 deg.C for 24hr. The cultured liquid is subjected to centrifuge to collect bacterial cells. The bacterial cells are suspended in a buffer liquid and the cells are crushed by e.g. dinomill. The liquid is treated with centrifuge to collect the supernatant. This is purified through chromatography using e.g. an ion-exchange column or a hydrophobic column to obtain the objective new enzyme. The enzyme can produce β-cyanoalanine from O-acetyl-L-serine and a cyano compound. The enzyme has the following characteristics: an optimum pH, 7.0-9.0; a stable pH region, 6.0-10.0; an optimum temperature, 40-50 deg.C; and a molecular weight, 60000-80000.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to O-acetylserine sulfhydrylase, a method for producing the enzyme, a novel bacterial strain belonging to the genus Bacillus that produces the enzyme, and a method for producing the enzyme.

[0002]

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION O-
Acetylserine sulfhydrylase activity is known to be derived from some plants and microorganisms, and for example, derived from spinach (F. Ikegami et al., Phytochemistry, Vol. 27, 338).
5 to 3389 (1981), or derived from a bacterium belonging to the genus Chromobacterium (M. Anne and JK Christopher (JK).
Christopher), Biochimica et Biophysica Acta, No. 786.
Volume 123-132 (1984) and the like have been purified and examined for properties.

The enzyme reaction product β-cyanoalanine is
It is useful as a synthetic intermediate for various amino acid derivatives having biological activity, but all the O-acetylserine sulfhydrylases reported so far are unstable,
In particular, it had low thermal stability and was not suitable for the production of such useful substances. Therefore, there has been an urgent need to search for O-acetylserine sulfhydrylase having high thermostability.

[0004]

Under the above circumstances, the inventors of the present invention have conducted intensive studies and as a result, have found that the thermal stability of O is extremely high.
-Finding a bacterium belonging to the genus Bacillus that produces acetylserine sulfhydrylase and succeeding in isolating the thermostable O-acetylserine sulfhydrylase from the bacterium, the present invention has been completed. It was

That is, the present invention provides (1) a thermostable O-acetylserine sulfhydrylase having the following properties; Action: β-cyanoalanine is produced from O-acetyl-L-serine and cyanide. Optimum pH: 7.0-9.0 3. Stable pH: 6.0 to 10.0 4. Optimum temperature: 40 to 50 ° C 5. Thermal stability: Stable up to 70 ° C when kept at pH 7.5 for 30 minutes 6. Molecular weight: 60,000-80,000 by gel filtration
0, (2) The enzyme according to (1), which requires pyridorisal phosphate as a coenzyme, (3) O-acetyl-L-serine, L-cysteine
L-serine, O-methyl-L-serine, O-phosphoryl-L-serine, O-succinyl-L-serine or β-
An enzyme according to (1), which uses chloro-L-alanine as a substrate,
In particular, (4) the enzyme according to any one of (1) to (3), which is obtained from a thermophilic bacterium belonging to the genus Bacillus, (5) the bacterium is Bacillus stearothermophilus (B. stearothermophilus) (6) The enzyme is the Bacillus stearothermophilus CN3 strain deposited under the deposit number FERM BP-4773 at the Institute of Biotechnology, Institute of Industrial Science (4). )
It provides the described enzyme.

The present invention also provides (7) an O-acetylserine sulfhydrylase-inducing medium for a bacterium belonging to the genus Thermophilic Bacillus having the ability to produce O-acetylserine sulfhydrylase according to claim 1. And then isolating the O-acetylserine sulfhydrylase from the cells, particularly, (8) the O-acetylserine sulfhydrylase. The method according to (7), wherein the bacterium belonging to the genus Thermophilic Bacillus capable of producing sulfhydrylase is Bacillus stearothermophilus, (9) the O-acetylserine belonging to the genus Thermophilic Bacillus Bacteria capable of producing sulfhydrylase were deposited under the contract number FERMBP-
The method according to (7), which is the Bacillus stearothermophilus CN3 strain deposited under 4773.

Further, the present invention provides (10) a contract number F with the Institute of Biotechnology, Institute of Industrial Science and Technology.
It provides the Bacillus stearothermophilus CN3 strain deposited under ERM BP-4773.

The present invention will be described in more detail below. The thermostable O-acetylserine sulfhydrylase of the present invention can be obtained by culturing a bacterium having a thermostable O-acetylserine sulfhydrylase-producing ability belonging to the genus Bacillus. For example,
Bacillus stearot
hermophilus) CN3 strain and the like. The Bacillus stearothermophilus CN3 strain was isolated from the nature by the present inventors, and was deposited with the Accession No. FERM BP-4 on August 8, 1994 at the Institute of Biotechnology, Institute of Industrial Science and Technology.
Deposited as 773. The mycological properties of this bacterium are as follows.

(A) Form 1. Bacterial morphology: Fine bacilli 2. Motility: -3. Spores: + (elliptical) 4. Gram stainability:-

(B) Colony morphology: 5 m after 72 hours of culture
Circular less than m, irregular, light yellow translucent, smooth and flat surface

(C) Physiological properties 1. Growth: Does not grow at 37 ° C, but grows at 45 ° C, 55 ° C and 67 ° C. Intracellular microsphere: -3. Litmus milk: Degradation Growth at 4.3% NaCl:-Growth at 5.5% NaCl:-Growth at 6.7% NaCl:-7. Growth at pH 5.7: -8. Acid production from glucose: +9. Gas production from glucose: -10. Egg yolk reaction: Does not grow under the test conditions 11. Casein decomposition: +12. Gelatin decomposition: -13. Tyrosine degradation: -14. Starch hydrolysis: +15. Conversion of NO ₃ ⁻ to NO ₂ ⁻ : +16. Utilization of citric acid in Coser's medium: -17. Mellar arginine dihydrolase: -18. ONPG: + 19. Acid xylose PWS: +20. DNase: -21. Catalase: +22. Oxidase: +

[0012] Based on the above-mentioned mycological properties, Bargy's Manual of Systematic Bacteriol
, and the CN3 strain was identified as Bacillus stearothermophilus.

As the culture medium of the above-mentioned strain, it is not necessary to use a special medium, and an ordinary medium may be used. As the carbon source, glucose, maltose, starch, sugars such as dextrin, amino acids such as glutamic acid, or fumaric acid, organic acids such as malic acid, or glycerol, and as the nitrogen source, polypeptone, yeast extract, soybean powder hydrolysis As a natural nitrogen source and inorganic salt of things such as NaC
1, potassium phosphate, magnesium sulfate and the like, and as the trace metal component, calcium chloride, boric acid, copper sulfate, calcium iodide, ferrous sulfate, manganese sulfate, zinc sulfate and the like can be used.

An example of a suitable medium composition for obtaining the thermostable O-acetylserine sulfhydrylase of the present invention is shown below. Polypeptone 1%, yeast extract 0.25%, glycerol 0.1%, (NH ₄ ) ₂ SO ₄ 0.1%, MgSO _{4.
4 · 7H 2 O 0.05%,} K 2 HPO 4 0.1%, and L-0.1% serine.

The pH of the culture is 6.5 to about 7.5, preferably 7.2, and the culture temperature is about 40 to 70 ° C., preferably 50 to 60 ° C., for 12 to 48 hours, preferably. Culture for 24 hours aerobically or with shaking. The thermostable O-acetylserine sulfhydrylase of the present invention is
It can be obtained by isolating the enzyme from the bacteria thus cultured.

In order to isolate the thermostable O-acetylserine sulfhydrylase of the present invention from the above culture solution, 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, followed by centrifugation to obtain a cell-free extract. This is subjected to ammonium sulfate fractionation, subjected to desalting treatment by dialysis, etc., and then subjected to ion exchange chromatography, hydrophobic chromatography, hydroxylapatite chromatography, gel filtration chromatography, etc.
Acetylserine sulfhydrylase can be isolated. Further, since the enzyme of the present invention is a thermostable enzyme, purification efficiency can be improved by performing heat treatment at 70 ° C. for 30 minutes, for example.

In addition, the thermostable O-acetylserine sulfhydrylase of the present invention is used to produce cysteine, L-serine,
O-acetyl-L-serine, O-methyl-L-serine,
O-phosphoryl-L-serine, O-succinyl-L-serine or β-chloro-L-alanine and ¹¹ C or ¹³ C
Alternatively, it is reacted with a ¹⁴ C-labeled cyanide in an aqueous medium such as a potassium phosphate buffer solution to react with it, and then used for clinical diagnosis or biochemical research. ¹¹ C or ¹³ C
Alternatively, β-cyanoalanine labeled with ¹⁴ C or its derivative or salt can be synthesized.

[0018]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Bacillus stearothermophilus CN3 strain culture polypeptone 1%, 0.25% yeast extract, glycerol _{0.1%, (NH 4) 2} SO 4 0.1%, MgSO 4 · 7
H ₂ O 0.05%, K ₂ HPO ₄ 0.1% (pH 7.
8 ml of the medium consisting of 2) in test tubes (2.5
(X20 cm), dispensed into 2 tubes, sterilized at 120 ° C. for 20 minutes, cooled, inoculated with one platinum loop of Bacillus stearothermophilus CN3 strain, and cultured with shaking at 60 ° C. for 24 hours to obtain a seed culture solution. . 1.6 liter of a medium obtained by adding 0.01% (v / v) of an antifoaming agent (Adekanol KG-126, Asahi Denka Co., Ltd.) to a medium having the same composition as described above was put in a 2 liter jar fermenter, 20 minutes sterilization, after cooling,
16 ml of the above seed culture solution (for 2 test tubes) was inoculated into this, and cultured at 60 ° C. for 27 hours under the conditions of an aeration rate of 1.6 l / min and a stirring speed of 300 rpm to obtain a preculture solution. . Next, polypeptone 1%, yeast extract 0.25%, glycerol 0.1%, (NH ₄ ) ₂ SO ₄ 0.1%, MgS.
_{O 4 · 7H 2 O 0.05%} , K 2 HPO 4 0.1%, L-
160 liters of a medium consisting of serine 0.1% (pH 7.2) was placed in a 200 liter jar fermenter, sterilized at 120 ° C. for 30 minutes, cooled, and then inoculated with 1.6 liters of the above preculture liquid, 60 Culturing was carried out at a temperature of 24 ° C. for 24 hours under the conditions of an aeration rate of 120 l / min and a stirring speed of 200 rpm. After the completion of the culture, the cells were recovered by centrifugation using a Sharpless (KS type ultra high speed centrifuge, manufactured by Kansai Centrifuge Manufacturing Co., Ltd.). The obtained cells were divided into 8 equal parts (each 20 liters), suspended in an appropriate amount of 10 mM potassium phosphate buffer (pH 8.0, containing 0.1 mM dithiothreitol), and frozen at -80 ° C. saved. This was thawed and used for the subsequent production of the enzyme.

Bacillus stearothermophilus CN3
Isolation of thermostable O-acetylserine sulfhydrylase from strain Strains of 60 liters of frozen cells were suspended in the above buffer so that the total amount was about 2000 ml, and the cells were crushed with Dynomill. The disrupted liquid was centrifuged to remove the cell residue, and 2100 ml of a cell-free extract was obtained. Ammonium sulphate was added to this cell-free extract at 40% saturation, left overnight and the precipitated precipitate was removed by centrifugation, and the resulting supernatant was again saturated with ammonium sulphate at 90%. The mixture was added and allowed to stand for 5 hours to obtain a precipitate by centrifugation. 0.1m of this precipitate
It was dissolved in 10 mM potassium phosphate buffer (pH 8.0) containing M dithiothreitol and desalted with the same buffer using a dialysis membrane. To 1065 ml of the desalted solution was added ethanol cooled to -80 ° C in advance so that the final concentration was 70%, and a precipitate was obtained by centrifugation. This was suspended in the same buffer and heat-treated at 70 ° C. for 30 minutes. After removing the precipitate by centrifugation, the supernatant was passed through a DEAE-Cellulofine A500 column (diameter 6.0 × height 18 cm) preliminarily equilibrated with the same buffer to adsorb the enzyme, and washed with the same buffer. The enzyme was eluted by a gradient elution method from the same buffer solution to a 100 mM potassium phosphate buffer solution (pH 8.0) containing 0.1 mM dithiothreitol and 0.5 M NaCl to collect active fractions. Ammonium sulfate was added to this active fraction to 80% saturation, and the mixture was allowed to stand overnight and then centrifuged to obtain a precipitate. This precipitate was added to 10 mM potassium phosphate buffer (pH: 0.1 mM dithiothreitol).
8.0) and subjected to preparative electrophoresis (7.5% polyacrylamide gel). After the migration, the active part in the gel was cut out, ground and the enzyme was extracted using the same buffer. Ammonium sulfate was added to this active fraction to 30% saturation, and phenyl sepharose CL was equilibrated beforehand with 10 mM potassium phosphate buffer (pH 8.0) containing 30% saturated ammonium sulfate and 0.1 mM dithiothreitol.
-4B column (diameter 2.5 x height 12 cm) to adsorb the enzyme, and after washing with this equilibration buffer, 10 mM potassium phosphate buffer (pH 8. 0 mM) containing 0.1 mM dithiothreitol from this buffer. The enzyme was eluted by the gradient elution method to 0) and the active fractions were collected. Ammonium sulfate was added to this active fraction at 30% saturation and
10% saturated ammonium sulfate and 0.1 mM dithiothreitol
Octyl Sepharose CL-4B column (diameter 1.5X) equilibrated with mM potassium phosphate buffer (pH 8.0)
The mixture is passed through a height of 10 cm) to adsorb the enzyme, washed with this equilibration buffer, and then 10 mM potassium phosphate buffer (pH 8.0.1 mM) containing 0.1 mM dithiothreitol.
The enzyme was eluted by the gradient elution method to 0) and the active fractions were collected. It was confirmed that the enzyme preparation obtained here was electrophoretically single. The purification results are summarized in Table 1 below. In step 8, the purification was 218 times that of the original cell-free extract.

[0020]

[Table 1]

Bacillus stearothermophilus CN3
Properties of thermostable O-acetylserine sulfhydrylase of the strain Enzyme activity measurement: 1M potassium phosphate buffer (pH 7.
5) 10 μl (final concentration 50 mM), water 80 μl, 0.8
20 μl of mM pyridoxal phosphate (final concentration 0.08 m
M), 50 mM O-acetyl-L-serine 20 μl
(Final concentration 5.0 mM), 100 mM potassium cyanide 2
After incubating a reaction solution (total volume 200 μl) consisting of 0 μl (final concentration 10 mM) and enzyme solution 50 μl at 45 ° C. for 10 minutes, the reaction was stopped by placing it in a boiling water bath for 2 minutes,
Then, centrifugation was performed at 15000 rpm for 10 minutes, the supernatant was subjected to HPLC, and β-cyanoalanine produced by the enzymatic reaction was quantified. Under such conditions,
The enzyme activity that produces 1 μmol of β-cyanoalanine in 1 minute is defined as 1 unit. The condition of HPLC is column: Inertsil ODS-2 (4.6X250).
mm) (manufactured by GL Sciences Inc.), eluent: 1
0 mM sodium phosphate buffer (pH 6.8) / CH ₃
CN (85/15), flow rate: 1 ml / min, detection: fluorescence detector (Ex: 345 nm, Em450 nm), column temperature: 40 ° C., sample volume: 10 μl.

(1) Optimum pH The activity was measured by the above activity measuring method under each pH in the range of pH 6.0 to 10.0 (the buffer solution in the activity measuring reaction solution was changed to the following one). The reaction was performed). The buffer used is 20 mM MES (pH 6.0 to 6.5),
20 mM KPB (pH 6.0 to 8.0), 20 mM M
OPS (pH 6.5 to 7.5), 20 mM Tris-
HCl (pH 7.5-9.0) and 20 mM NH ₄ C
was _{l-NH 4 OH (pH8.5~10.0)} . The results are shown in Fig. 1. From FIG. 1, the optimum pH of this enzyme is 8.0.
It turned out to be.

(2) Stable pH Further, the present enzyme was dissolved in a buffer solution having a concentration of 20 mM to prepare 60
After holding at 30 ° C. for 30 minutes, the residual activity was measured. The buffer used is citric acid / sodium citrate (pH 3.5-
5.5), MES (pH 6.0 to 7.0), KH ₂ PO _{4
-K 2 HPO 4 (pH6.0~8.0) Tris} -HCl
_{(PH7.5~9.0), NH 4 Cl-} NH 4 OH (pH
8.5-10.5) and glycine / KCl-KOH
It was (pH 10.0-10.5). The results are shown in Figure 2. From the result of FIG. 2, the stable pH of this enzyme is 6.0 to 1.
I knew it was 0.0.

(3) Optimum temperature This enzyme was dissolved in a 20 mM potassium phosphate buffer (pH 7.5), and the activity was measured in the range of 20 ° C. to 80 ° C. by the above enzyme activity measuring method. The results are shown in Fig. 3. From FIG. 3, it was found that the optimum temperature of this enzyme was 45 ° C.

(4) Thermostability The present enzyme was dissolved in 20 mM potassium phosphate buffer (pH 7.5), treated with the enzyme for 30 minutes at each temperature from 40 ° C to 95 ° C, and the residual activity was examined. FIG. 4 shows the results. From FIG. 4, it was found that this enzyme has very high thermostability and is stable up to 70 ° C.

(5) Molecular weight and subunits TSKgel G300 by liquid chromatography
The molecular weight was calculated to be 70,000 by gel filtration using a 0SW (20 × 300 mm, manufactured by Tosoh Corporation) column. The molecular weight by SDS-PAGE was 34,
It was calculated to be 000, and this enzyme was considered to be a dimeric enzyme having the same subunit.

(6) Substrate specificity The substrate specificity was investigated by measuring the activity of various compounds in the reaction solution for activity measurement, instead of O-acetyl-L-serine. The substrate specificity of this enzyme is high, and it slightly acts on L-cysteine other than O-acetyl-L-serine (relative activity is 4.8 when O-acetyl-L-serine is 100%). %). In addition,
The following amino acids did not serve as substrates. DL-homoserine, L-cystine, L-homoserine, L-serine, L-
Homoserine, L-methionine, L-asparagine, L-
Aspartic acid.

[0028]

The present invention provides O-acetylserine sulfhydrylase having extremely high thermostability, a bacterial strain producing the enzyme, and a method for producing the enzyme.

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH of O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain.

FIG. 2 is a diagram showing pH stability of O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain.

FIG. 3 is a graph showing the optimum temperature of O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain.

FIG. 4 is a diagram showing thermostability of O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1:07) (72) Inventor Gunner Antoni S-75185 Uppsala, Sweden (No address is displayed. ) Uppsala Academiska Shukhugh Set, Uppsala University Pty Center (72) Inventor Bengt Longström, S-75185, Sweden Uppsala (No address) Uppsala Academiska Shukhugh Set, Uppsala University University Pee Center

Claims (10)

[Claims] 1. A thermostable O-acetylserine sulfhydrylase having the following properties; Action: β-cyanoalanine is produced from O-acetyl-L-serine and cyanide. Optimum pH: 7.0-9.0 3. Stable pH: 6.0 to 10.0 4. Optimum temperature: 40 to 50 ° C 5. Thermal stability: Stable up to 70 ° C when kept at pH 7.5 for 30 minutes 6. Molecular weight: 60,000-80,000 by gel filtration
0. 2. The enzyme according to claim 1, which requires pyridoxal phosphate as a coenzyme. 3. O-acetyl-L-serine, L-cysteine, L-serine, O-methyl-L-serine, O-phosphoryl-L-serine, O-succinyl-L-serine or β-chloro-L. -The enzyme according to claim 1, which uses alanine as a substrate. 4. The enzyme according to any one of claims 1 to 3, which is obtained from a thermophilic bacterium belonging to the genus Bacillus. 5. The enzyme according to claim 4, wherein the bacterium is Bacillus stearothermophilus. 6. The enzyme according to claim 4, wherein the bacterium is Bacillus stearothermophilus CN3 strain deposited under the deposit number FERMBP-4733 at the Institute of Biotechnology, Institute of Industrial Science and Technology. 7. A thermophilic bacterium belonging to the genus Bacillus having the ability to produce O-acetylserine sulfhydrylase according to claim 1, is cultured in an O-acetylserine sulfhydrylase-inducing medium, and then the O which is characterized in that the O-acetylserine sulfhydrylase is isolated from bacterial cells.
-A method for producing acetylserine sulfhydrylase. 8. The method according to claim 7, wherein the bacterium belonging to the thermophilic Bacillus genus capable of producing O-acetylserine sulfhydrylase is Bacillus stearothermophilus. 9. A bacterium having the thermophilic Bacillus genus capable of producing the O-acetylserine sulfhydrylase, has a contract number FERM at the Institute of Biotechnology, Institute of Industrial Science and Technology.
The method according to claim 7, which is Bacillus stearothermophilus CN3 strain deposited under BP-4773. 10. Bacillus stearothermophilus CN3 strain deposited under the deposit number FERM BP-4773 at the Institute of Biotechnology, Institute of Industrial Science and Technology.