JP3694335B2 - Thermostable O-acetylserine sulfhydrylase and process for producing the same - Google Patents

Description

【００２１】
バチルス・ステアロサーモフィルスＣＮ３株の耐熱性Ｏ−アセチルセリンスルフヒドリラーゼの諸性質
酵素活性測定：１Ｍリン酸カリウム緩衝液（ｐＨ７．５）１０μｌ（終濃度５０ｍＭ）、水８０μｌ、０．８ｍＭピリドキサールリン酸２０μｌ（終濃度０．０８ｍＭ）、５０ｍＭ Ｏ−アセチル−Ｌ−セリン２０μｌ（終濃度５．０ｍＭ）、１００ｍＭシアン化カリウム２０μｌ（終濃度１０ｍＭ）および酵素液５０μｌからなる反応液（全量２００μｌ）を４５℃で１０分インキュベートした後、沸騰水浴中に２分間置いて反応停止し、ついで、１５０００ｒｐｍで１０分間遠心分離して上澄液をＨＰＬＣにかけ、酵素反応で生成したβ−シアノアラニンを定量することにより行った。かかる条件下で、１分間に１μｍｏｌのβ−シアノアラニンを生成する酵素活性を１ユニットと定義する。ＨＰＬＣの条件は、カラム：Ｉｎｅｒｔｓｉｌ ＯＤＳ−２（４．６Ｘ２５０ｍｍ）(ジーエルサイエンス株式会社製）、溶出液：１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．８）／ＣＨ₃ＣＮ（８５／１５）、流速：１ｍｌ／ｍｉｎ、検出：蛍光検出機（Ｅｘ：３４５ｎｍ、Ｅｍ４５０ｎｍ）、カラム温度：４０℃、サンプルボリューム：１０μｌであった。
【００２２】
（１）至適ｐＨ
ｐＨ６．０から１０．０の範囲の各ｐＨ下で、上記活性測定法により活性測定を行った（活性測定用反応液における緩衝液を以下のものに代えて反応を行った）。使用緩衝液は、２０ｍＭ ＭＥＳ（ｐＨ６．０〜６．５）、２０ｍＭ ＫＰＢ（ｐＨ６．０〜８．０）、２０ｍＭ ＭＯＰＳ（ｐＨ６．５〜７．５）、２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５〜９．０）および２０ｍＭ ＮＨ₄Ｃｌ−ＮＨ₄ＯＨ（ｐＨ８．５〜１０．０）であった。結果を図１に示す。図１より、本酵素の至適ｐＨは８．０であることがわかった。
【００２３】
（２）安定ｐＨ
また、本酵素を２０ｍＭ濃度の緩衝液に溶解し、６０℃、３０分保持した後、残存活性を測定した。使用緩衝液は、クエン酸／クエン酸ナトリウム（ｐＨ３．５〜５．５）、ＭＥＳ（ｐＨ６．０〜７．０）、ＫＨ₂ＰＯ₄−Ｋ₂ＨＰＯ₄（ｐＨ６．０〜８．０）Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５〜９．０）、ＮＨ₄Ｃｌ−ＮＨ₄ＯＨ（ｐＨ８．５〜１０．５）およびグリシン／ＫＣｌ−ＫＯＨ（ｐＨ１０．０〜１０．５）であった。結果を図２に示す。図２の結果より、本酵素の安定ｐＨは６．０〜１０．０であることがわっかった。
【００２４】
（３）至適温度
２０ｍＭリン酸カリウム緩衝液（ｐＨ７．５）に本酵素を溶解し、２０℃から８０℃までの範囲で上記酵素活性測定法により活性測定した。結果を図３に示す。図３より、本酵素の至適温度は４５℃であることがわかった。
【００２５】
（４）熱安定性
２０ｍＭリン酸カリウム緩衝液（ｐＨ７．５）に本酵素を溶解し、４０℃から９５℃までの各温度で３０分間酵素を処理し、残存活性を調べた。結果を図４に示す。図４より本酵素は熱安定性が非常に高く、７０℃まで安定であることがわかった。
【００２６】
（５）分子量およびサブユニット
液体クロマトグラフィーによるＴＳＫｇｅｌ Ｇ３０００ＳＷ（２０Ｘ３００ｍｍ、東ソー株式会社製）カラムを用いたゲル濾過により、分子量は７０，０００と算出された。また、ＳＤＳ−ＰＡＧＥによる分子量は３４，０００と算出され、本酵素は同一サブユニットの２量体酵素であると考えられた。
【００２７】
（６）基質特異性
前記活性測定用反応液において、Ｏ−アセチル−Ｌ−セリンに代えて種々の化合物を用いて活性を測定することにより、基質特異性を調べた。本酵素の基質特異性は高く、Ｏ−アセチル−Ｌ−セリン以外にはＬ−システインに対してわずかに作用した（Ｏ−アセチル−Ｌ−セリンを１００％とした場合の相対活性は４．８％）。なお、以下のアミノ酸は基質にならなかった。ＤＬ−ホモセリン、Ｌ−シスチン、Ｌ−ホモセリン、Ｌ−セリン、Ｌ−ホモセリン、Ｌ−メチオニン、Ｌ−アスパラギン、Ｌ−アスパラギン酸。
【００２８】
【発明の効果】
本発明によれば、熱安定性の非常に高いＯ−アセチルセリンスルフヒドリラーゼ、該酵素を産生する細菌株、該酵素の製造法が提供される。
【図面の簡単な説明】
【図１】 バチルス・ステアロサーモフィルスＣＮ３株のＯ−アセチルセリンスルフヒドリラーゼの至適ｐＨを示す図である。
【図２】 バチルス・ステアロサーモフィルスＣＮ３株のＯ−アセチルセリンスルフヒドリラーゼのｐＨ安定性を示す図である。
【図３】 バチルス・ステアロサーモフィルスＣＮ３株のＯ−アセチルセリンスルフヒドリラーゼの至適温度を示す図である。
【図４】 バチルス・ステアロサーモフィルスＣＮ３株のＯ−アセチルセリンスルフヒドリラーゼの熱安定性を示す図である。[0001]
[Industrial application fields]
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]
[Background Art and Problems to be Solved by the Invention]
O-acetylserine sulfhydrylase activity is known to be derived from some plants and microorganisms, for example, from spinach (F. Ikegami et al., Phytochemistry, Vol. 27). , 3385-3389 (1981), or from the genus Chromobacterium (M.Anne and JKChristopher, Biochimica et Biophysica) Acta), 786, 123-132 (1984), etc. have been refined and their properties have been investigated.
[0003]
The enzyme reaction product β-cyanoalanine is useful as an intermediate for the synthesis of various amino acid derivatives having biological activity, but all of the O-acetylserine sulfhydrylases reported so far are unstable. In particular, the thermal stability is low and it is 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]
[Means for Solving the Problems]
Under the above circumstances, as a result of intensive research, the present inventors have found a thermostable bacterium belonging to the genus Bacillus that produces O-acetylserine sulfhydrylase having a very high thermal stability. The present invention was completed by succeeding in isolating thermostable O-acetylserine sulfhydrylase.
[0005]
That is, the present invention
(1) a thermostable O-acetylserine sulfhydrylase having the following properties;
1. Action: produces β-cyanoalanine from O-acetyl-L-serine and cyanide Optimum pH: 7.0-9.0
3. Stable pH: 6.0 to 10.0
4). Optimal temperature: 40-50 ° C
5. Thermal stability: stable to 70 ° C. when held at pH 7.5 for 30 minutes Molecular weight: 60,000-80,000 by gel filtration
(2) The enzyme according to (1), which requires pyridosarlic acid 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-alanine The enzyme according to (1), 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 enzyme according to (4), wherein the bacterium is B. stearothermophilus,
(6) The bacterium is Bacillus stearothermophilus CN3 strain deposited under the accession number FERM BP-4773 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. .
[0006]
The present invention also provides:
(7) A bacterium belonging to the genus thermophilic Bacillus having the ability to produce O-acetylserine sulfhydrylase according to claim 1 is cultured in an O-acetylserine sulfhydrylase-inducing medium; A method for producing O-acetylserine sulfhydrylase, characterized in that said O-acetylserine sulfhydrylase is isolated from
(8) The method according to (7), wherein the thermophilic bacterium belonging to the genus Bacillus having the ability to produce O-acetylserine sulfhydrylase is Bacillus stearothermophilus,
(9) Bacteria having the ability to produce the O-acetylserine sulfhydrylase belonging to the thermophilic Bacillus genus have been deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the accession number FERMBP-4773 The method according to (7), which is a Bacillus stearothermophilus CN3 strain.
[0007]
Furthermore, the present invention provides
(10) Provided with the Bacillus stearothermophilus CN3 strain deposited under the accession number FERM BP-4773 at the Institute of Biotechnology, AIST.
[0008]
Hereinafter, the present invention will be described in more detail.
The thermostable O-acetylserine sulfhydrylase of the present invention can be obtained by culturing a bacterium having the ability to produce O-acetylserine sulfhydrylase belonging to the thermostable Bacillus genus. Examples include Bacillus stearothermophilus CN3 strain and the like. The Bacillus stearothermophilus CN3 strain was isolated from nature by the present inventors and was assigned to the National Institute of Biotechnology, August 8, 1994 under the accession number FERM BP-4773. It has been deposited. The bacteriological properties of this bacterium are as follows.
[0009]
(A) Form 1. 1. Bacterial morphology: fine gonococci Mobility:-
3. Spore: + (oval)
4). Gram staining:-
[0010]
(B) Colony morphology: circular shape of 5 mm or less after 72 hours of culture, irregular, pale yellow translucent, smooth and flat surface
(C) Physiological properties Growth: Not grown at 37 ° C, grown at 45 ° C, 55 ° C and 67 ° C Intracellular microspheres:-
3. Litmus milk: Decomposition 4. Growth with 3% NaCl:-
Growth with 5.5% NaCl: −
Growth with 6.7% NaCl: −
7. Growth at pH 5.7:-
8). Acid production from glucose: +
9. Gas production from glucose:-
10. Egg yolk reaction: not growing under test conditions Casein degradation: +
12 Gelatin degradation:-
13. Tyrosine degradation: −
14 Starch hydrolysis: +
15. NO ₃ ^- from NO ₂ ^- Conversion to: +
16. Use of citric acid in Coser medium:-
17. Meller's arginine dihydrolase:-
18. ONPG: +
19. Acid xylose PWS: +
20. DNase:-
21. Catalase: +
22. Oxidase: +
[0012]
Based on the above bacteriological properties, classification was made according to Bargy's Manual of Systematic Bacteriology, and the CN3 strain was identified as Bacillus stearothermophilus.
[0013]
As the culture medium for the strain, it is not necessary to use a special culture medium, and a normal culture medium may be used. As carbon sources, sugars such as glucose, maltose, starch and dextrin, amino acids such as glutamic acid, organic acids such as fumaric acid and malic acid, or glycerol, nitrogen sources include polypeptone, yeast extract, soybean flour hydrolysis Natural nitrogen sources such as NaCl, inorganic salts such as NaCl, potassium phosphate, magnesium sulfate, and trace metal components such as calcium chloride, boric acid, copper sulfate, calcium iodide, ferrous sulfate, manganese sulfate, sulfuric acid Zinc or the like can be used.
[0014]
An example of a suitable medium composition for obtaining the thermostable O-acetylserine sulfhydrylase of the present invention is shown below. 1% polypeptone, 0.25% yeast extract, 0.1% glycerol, 0.1% (NH ₄ ) ₂ SO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.1% K ₂ HPO ₄ , and L-serine 0.1%.
[0015]
The culture pH is about 6.5 to about 7.5, preferably 7.2, the culture temperature is about 40 ° C. to about 70 ° C., preferably 50 to 60 ° C., and 12 to 48 hours, preferably 24 hours. Cultivate aerobically or with shaking. The thermostable O-acetylserine sulfhydrylase of the present invention can be obtained by isolating the enzyme from the thus cultured bacteria.
[0016]
In order to isolate the thermostable O-acetylserine sulfhydrylase of the present invention from the culture solution, known purification methods can be used alone or in combination. For example, the cells are collected by centrifuging the culture solution, disrupted by sonication or Dyno-mill treatment, and then cell-free extract is obtained by centrifugation. This was subjected to ammonium sulfate fractionation, desalting treatment such as dialysis, etc., and then subjected to ion exchange chromatography, hydrophobic chromatography, hydroxylapatite chromatography, gel filtration chromatography, etc., and the heat-resistant O-acetylserine of the present invention. Sulfhydrylase can be isolated. Further, since the enzyme of the present invention is a thermostable enzyme, purification efficiency can be increased by performing a heat treatment at 70 ° C. for 30 minutes, for example.
[0017]
Further, using the heat-resistant O-acetylserine sulfhydrylase of the present invention, cysteine, L-serine, O-acetyl-L-serine, O-methyl-L-serine, O-phosphoryl-L-serine, O- Clinical diagnosis by contacting succinyl-L-serine or β-chloro-L-alanine with cyanide labeled with ¹¹ C, ¹³ C, or ¹⁴ C in an aqueous medium such as potassium phosphate buffer. Alternatively, β-cyanoalanine or a derivative or salt thereof labeled with ¹¹ C, ¹³ C, or ¹⁴ C used for biochemical research can be synthesized.
[0018]
【Example】
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 cultured polypeptone 1%, yeast extract 0.25%, glycerol 0.1%, (NH ₄ ) ₂ SO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.05%, Dispense 8 ml each of K ₂ HPO ₄ 0.1% (pH 7.2) medium into two test tubes (2.5 × 20 cm), sterilize at 120 ° C. for 20 minutes, cool, and then Bacillus stearothermophilus The CN3 strain was inoculated one platinum ear at a time, and cultured with shaking at 60 ° C. for 24 hours to obtain a seed culture solution. 1.6 liters of a medium prepared by adding 0.01% (v / v) antifoaming agent (Asahi Denka Co., Ltd. Adecanol KG-126) to a medium having the same composition as described above was placed in a 2 liter jar fermenter, 120 ° C., After sterilization for 20 minutes and cooling, inoculate 16 ml of the above seed culture solution (for 2 test tubes) and culture under conditions of 60 ° C., 27 hours, aeration rate of 1.6 liters / minute and stirring speed of 300 rpm. And used as a preculture. Next, polypeptone 1%, yeast extract 0.25%, glycerol 0.1%, (NH ₄ ) ₂ SO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.05%, K ₂ HPO ₄ 0.1% , 160 liters of medium consisting of 0.1% L-serine (pH 7.2) was placed in a 200 liter jar fermenter, sterilized at 120 ° C. for 30 minutes, cooled, and inoculated with 1.6 liters of the above preculture Then, the cells were cultured at 60 ° C. for 24 hours under the conditions of an aeration rate of 120 liters / minute and a stirring speed of 200 rpm. After completion of the culture, the cells were collected by centrifugation with a sharp press (KS ultra-high speed centrifuge, manufactured by Kansai Centrifuge Co., Ltd.).
The obtained cells are divided into 8 equal parts (each 20 liters), suspended in appropriate amounts 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 subsequent enzyme production.
[0019]
Isolation of thermostable O-acetylserine sulfhydrylase from Bacillus stearothermophilus CN3 strain 60 liters of frozen cells were suspended in the above buffer solution to a total volume of about 2000 ml, and then sterilized by dynomill. The body was crushed. The disrupted solution was centrifuged to remove the cell residue, and 2100 ml of cell-free extract was obtained.
To this cell-free extract, ammonium sulfate was added to 40% saturation, and after standing overnight, the deposited precipitate was removed by centrifugation, and the resulting supernatant was again saturated with ammonium sulfate to 90%. The mixture was added and allowed to stand for 5 hours, and a precipitate was obtained by centrifugation. This precipitate was dissolved in 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM dithiothreitol and desalted with the same buffer using a dialysis membrane. Ethanol that had been cooled to -80 ° C. in advance to a final concentration of 70% was added to 1065 ml of the desalted solution, 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) previously equilibrated with the same buffer, and washed with the same buffer. The active fractions were collected by elution of the enzyme from the same buffer by a gradient elution method into 100 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM dithiothreitol and 0.5 M NaCl.
Ammonium sulfate was added to this active fraction so as to be 80% saturated, and the mixture was left overnight and then centrifuged to obtain a precipitate. This precipitate was dissolved in 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM dithiothreitol and subjected to preparative electrophoresis (7.5% polyacrylamide gel). After the electrophoresis, the active portion in the gel was cut out, ground, and the enzyme was extracted using the same buffer.
Phenyl Sepharose CL- was added to this active fraction to 30% saturation and equilibrated in advance with 10 mM potassium phosphate buffer (pH 8.0) containing 30% saturated ammonium sulfate and 0.1 mM dithiothreitol. The enzyme was adsorbed through a 4B column (diameter 2.5 × height 12 cm), washed with this equilibration buffer, and 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM dithiothreitol from this buffer. The active fraction was collected by eluting the enzyme by the gradient elution method.
Octyl Sepharose CL- was added to this active fraction to 30% saturation, and equilibrated with 10 mM potassium phosphate buffer (pH 8.0) containing 30% saturated ammonium sulfate and 0.1 mM dithiothreitol in advance. The enzyme was adsorbed through a 4B column (diameter 1.5 × height 10 cm), washed with this equilibration buffer, and then washed with 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM dithiothreitol. The active fraction was collected by eluting the enzyme by the gradient elution method. It was confirmed that the enzyme preparation obtained here was electrophoretically single. The purification results are summarized in Table 1 below. In step 8, it was purified 218 times compared to the original cell-free extract.
[0020]
[Table 1]

[0021]
Various properties of thermostable O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain Enzyme activity measurement: 1 μL potassium phosphate buffer (pH 7.5) 10 μl (final concentration 50 mM), water 80 μl, 0.8 mM A reaction solution (total volume 200 μl) comprising 20 μl of pyridoxal phosphate (final concentration 0.08 mM), 20 μl of 50 mM O-acetyl-L-serine (final concentration 5.0 mM), 20 μl of 100 mM potassium cyanide (final concentration 10 mM) and 50 μl of enzyme solution. After incubating at 45 ° C. for 10 minutes, the reaction was stopped by placing in a boiling water bath for 2 minutes, and then centrifuged at 15000 rpm for 10 minutes, and the supernatant was subjected to HPLC to quantify β-cyanoalanine produced in the enzyme reaction. Was done. Under such conditions, the enzyme activity that produces 1 μmol of β-cyanoalanine per minute is defined as 1 unit. HPLC conditions are as follows: Column: Inertsil ODS-2 (4.6 × 250 mm) (manufactured by GL Sciences Inc.), eluent: 10 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.
[0022]
(1) Optimum pH
Activity was measured by the above activity measurement method under each pH in the range of pH 6.0 to 10.0 (reaction was performed by replacing the buffer in the reaction solution for activity measurement with the following). The buffer used was 20 mM MES (pH 6.0 to 6.5), 20 mM KPB (pH 6.0 to 8.0), 20 mM MOPS (pH 6.5 to 7.5), 20 mM Tris-HCl (pH 7.5 to 7.5). 9.0) and was _{20mM NH 4 Cl-NH 4 OH} (pH8.5~10.0). The results are shown in FIG. From FIG. 1, it was found that the optimum pH of the enzyme was 8.0.
[0023]
(2) Stable pH
Moreover, this enzyme was melt | dissolved in the buffer solution of a 20 mM density | concentration, The residual activity was measured after hold | maintaining for 30 minutes at 60 degreeC. The buffer used was citric acid / sodium citrate (pH 3.5 to 5.5), MES (pH 6.0 to 7.0), KH ₂ PO ₄ -K ₂ HPO ₄ (pH 6.0 to 8.0). Tris-HCl (pH 7.5 to 9.0), NH ₄ Cl—NH ₄ OH (pH 8.5 to 10.5) and glycine / KCl—KOH (pH 10.0 to 10.5). The results are shown in FIG. From the results of FIG. 2, it was found that the stable pH of this enzyme is 6.0 to 10.0.
[0024]
(3) Optimum temperature The enzyme was dissolved in 20 mM potassium phosphate buffer (pH 7.5), and the activity was measured in the range from 20 ° C. to 80 ° C. by the above enzyme activity measurement method. The results are shown in FIG. From FIG. 3, it was found that the optimum temperature of this enzyme was 45 ° C.
[0025]
(4) Thermostability This enzyme was dissolved in 20 mM potassium phosphate buffer (pH 7.5), and the enzyme was treated at each temperature from 40 ° C. to 95 ° C. for 30 minutes, and the residual activity was examined. The results are shown in FIG. From FIG. 4, it was found that this enzyme has very high thermostability and is stable up to 70 ° C.
[0026]
(5) Molecular weight The molecular weight was calculated to be 70,000 by gel filtration using a TSKgel G3000SW (20 × 300 mm, manufactured by Tosoh Corporation) column by subunit liquid chromatography. Moreover, the molecular weight by SDS-PAGE was calculated to be 34,000, and this enzyme was considered to be a dimeric enzyme of the same subunit.
[0027]
(6) Substrate specificity In the reaction liquid for activity measurement, the substrate specificity was examined by measuring the activity using various compounds instead of O-acetyl-L-serine. The enzyme has a high substrate specificity and acted slightly on L-cysteine other than O-acetyl-L-serine (relative activity is 4.8 when O-acetyl-L-serine is taken as 100%). %). The following amino acids did not become substrates. DL-homoserine, L-cystine, L-homoserine, L-serine, L-homoserine, L-methionine, L-asparagine, L-aspartic acid.
[0028]
【The invention's effect】
According to the present invention, O-acetylserine sulfhydrylase having very high thermal stability, a bacterial strain producing the enzyme, and a method for producing the enzyme are provided.
[Brief description of the drawings]
FIG. 1 is a graph showing the optimum pH of O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain.
FIG. 2 is a graph showing the 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 the thermal stability of O-acetylserine sulfhydrylase of Bacillus stearothermophilus CN3 strain.

Claims (7)

Produced by Bacillus stearothermophilus (B. stearothermophilus) bacteria, heat-resistant O- acetyl sulfhydrylase having the following properties;
1. Action: produces β-cyanoalanine from O-acetyl-L-serine and cyanide Optimum pH: 7.0-9.0
3. Stable pH: 6.0 to 10.0
4). Optimal temperature: 40-50 ° C
5. Thermal stability: stable to 70 ° C. when held at pH 7.5 for 30 minutes Molecular weight: A dimer of subunits having a molecular weight of 34,000 by SDS-PAGE .   The enzyme according to claim 1, which requires pyridoxal phosphate as a coenzyme.   O-acetyl-L-serine, L-cysteine, L-serine, O-methyl-L-serine, O-phosphoryl-L-serine, O-succinyl-L-serine or β-chloro-L-alanine as a substrate The enzyme according to claim 1. The enzyme according to any one of claims 1 to 3, wherein the bacterium is Bacillus stearothermophilus CN3 strain deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the accession number FERMBP-4773 . Bacillus stearothermophilus bacterium having the ability to produce O-acetylserine sulfhydrylase is cultured in an O-acetylserine sulfhydrylase-inducing medium, and then the O-acetylserine sulfhydrylase is produced from the cells. A process for producing O-acetylserine sulfhydrylase, characterized in that 6. The method according to claim 5, wherein the bacterium is Bacillus stearothermophilus CN3 strain deposited under the accession number FERM BP-4773 at the Institute of Biotechnology, AIST . Bacillus stearothermophilus CN3 strain deposited under the accession number FERM BP-4773 at the Institute of Biotechnology, AIST .

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