JP3078067B2 - Thermostable adenosine-5'-3 phosphate sulfurylase and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a thermostable adenosine-5'-3 phosphate sulfurylase (hereinafter referred to as ATP sulfurylase) having properties useful for a bioreactor having excellent heat stability and a method for producing the same. It is about.

[0002]

2. Description of the Related Art 3'-phosphoadenosine-5'-phosphosulfate (hereinafter referred to as PAPS) is a sulfate group donor widely distributed from microorganisms to plants and higher animals. It has been reported that PAPS in the living body is associated with several diseases represented by proteoglycan diseases [Brain Research, Vol. 20, p. 341-360 (197)
0)]. Thus, the role of PAPS in vivo is very important, and it is considered that PAPS has high utility in fields such as medicine. PAPS is adenosine-5 ', as described below.
It is a substance synthesized from -3 phosphate (hereinafter referred to as ATP) by the action of two enzymes, ATP sulfurylase and adenosine-5'-phosphosulfate kinase (hereinafter referred to as APS kinase).

[0003] (In the formula, APS represents adenosine-5'-phosphosulfate.)

[0004] Conventionally, ATP sulfurylase, which is one of the enzymes important for PAPS synthesis, has a very low content per wet microbial cell, so that there is a problem that large amounts of PAPS cannot be synthesized. Also, baker's yeast (Dry baker '
s yeast, Sigma YSC-1)
When kept at ℃, the residual activity was only about 7% in 1 hour, and ATP sulfurylase was extremely unstable to heat.
PAPS could not be efficiently synthesized using this enzyme and APS kinase.

[0005] For the reasons described above, a method for efficiently synthesizing PAPS and a method for measuring the concentration of APS in body fluids by using the reverse reaction of the present enzyme to examine the relationship between APS and a disease state will be described. In such a case, there is nothing that can be implemented industrially, and there has been a strong demand for practical use.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a highly stable ATP sulfurylase and a method for producing the same from the above-mentioned viewpoints.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies with the aim of efficiently obtaining highly stable ATP sulfurylase, and as a result, have found that Bacillus acidocaldarius and Bacillus coagulans (B. coagulans), Bacillus licheniformis (B. licheniformis), Bacillus sheregeri (B. schl)
egelii), Bacillus stearothermophilus (B.stea)
It has been found that a bacterium belonging to the genus Bacillus such as Rothermophilus produces ATP sulfurylase having the above-mentioned properties, and the present invention has been completed.

That is, the present invention provides a thermostable adenosine-5'-3 phosphate sulfurylase having the following physicochemical properties; and (a) action: acts on ATP and SO ₄ ^2- to produce APS. (B) Optimum pH: about 7.5 to about 8.0. (C) Suitable temperature for operation: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer at about 60 ° C. for about 15 minutes maintains about 95% of the activity before treatment. (E) Molecular weight: about 76,000 (gel filtration method). A bacterium belonging to the genus Bacillus (Bacillus sp.) Is cultured, and a thermostable adenosine-5 'having the following physicochemical properties is obtained from the culture.
A method for producing a thermostable adenosine-5'-3-phosphate sulfurylase, comprising collecting -3-phosphate sulfurylase. (A) Action: Acts on ATP and SO ₄ ^2- to generate APS. (B) Optimum pH: about 7.5 to about 8.0. (C) Suitable temperature for operation: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer at about 60 ° C. for about 15 minutes maintains about 95% of the activity before treatment. (E) Molecular weight: about 76,000 (gel filtration method).

Hereinafter, the present invention will be described in detail. The thermostable ATP sulfurylase of the present invention has the following physicochemical properties. (A) Action: Acts on ATP and SO ₄ ^2- to generate APS. (B) Substrate specificity: In a normal reaction, sulfate and A
TP is the substrate. However, it has virtually no effect on AMP. In the reverse reaction, APS and pyrophosphate serve as substrates. (C) Optimum pH: The titer was measured by changing the pH of the buffer solution to be used according to the titer measuring method described later. As a result, the titer was about 7.5 to 8.0 as shown in FIG. (D) Stable pH: 4 ° C. in a buffer having a pH of 5.0 to 10.0.
After storage for 22 hours, the titer was measured according to the titer measurement method described later. The result is about 5.0 ~ as shown in FIG.
7.5. (E) Suitable temperature for action: The temperature was changed to 20 ° C to 80 ° C, and the titer was measured by the method described below. The optimal temperature for the operation is about 50 ° C to
0 ° C. (F) Heat resistance: After treatment in a buffer at about 60 ° C. for about 15 minutes, the activity was measured by the titer measurement method described later. As a result, the activity was 95% of the activity before the treatment. (G) Molecular weight: The molecular weight was measured by a gel filtration method. The molecular weight is about 76,000.

(H) Method of measuring titer: The principle of measurement is ATP
When AMP and H ₂ O are used as substrates, AMP and pyrophosphate are produced. Pyrophosphatase is allowed to act on this, and the generated inorganic phosphate is quantified. [Reagent] Substrate: 100 mM ATP buffer: 100 mM Tris-HCl buffer (pH 8.0) Enzyme: 70 u / ml pyrophosphatase (Boehringer Mannheim Yamanouchi) Other salts: 100 mM sodium molybdate 1 M magnesium chloride Kit: Phospha C- Test Wako (manufactured by Wako Pure Chemical Industries) [Operation] 50 μl of the above-mentioned substrate ATP and 50 μl of buffer
Then, 50 μl of sodium molybdate, 5 μl of magnesium chloride, and 3 μl of pyrophosphatase are mixed with stirring, and then 100 μl of an enzyme solution is added and reacted at 30 ° C. for 10 minutes. After the reaction is completed, 50 μl of 3N sulfuric acid is added, and the mixture is stirred and mixed to stop the reaction. The generated inorganic phosphoric acid is quantified using an inorganic phosphoric acid measuring kit. As blind,
A solution treated in the same manner as that without addition of the enzyme solution is used. The enzymatic activity is expressed with the amount of enzyme that produces 2 μmol of phosphoric acid (1 μmol of pyrophosphate) per minute as 1 U.

[0011] The thermostable ATP sulfurylase of the present invention comprises:
It is obtained from a bacterium belonging to the genus Bacillus. Examples of such a bacterium belonging to the genus Bacillus include Bacillus acidocaldarius and Bacillus acidocaldarius.
Coagulans (B. coagulans), Bacillus licheniformis (B. licheniformis), Bacillus sheregeri (B. schlegelii), Bacillus stearothermophilus (B. stearothermophilus) and the like.

Next, a method for producing the thermostable ATP sulfurylase of the present invention will be described. The method for producing a thermostable ATP sulfurylase of the present invention comprises culturing a bacterium belonging to the genus Bacillus as described above, and collecting thermostable ATP sulfurylase from the culture. In the nutrient medium used for culturing Bacillus bacteria in the present invention, as a carbon source, for example, glucose, sucrose, fructose, starch hydrolyzate, molasses, sulphite pulp waste liquor, acetic acid, lactic acid and the like Organic acids, and furthermore, alcohols, fats, fatty acids, glycerin, and the like that can be used by bacteria to be used can be used, and as a nitrogen source, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, amino acid, peptone, meat extract, yeast Inorganic or organic substances such as extracts can be used. Further, as inorganic salts, for example, potassium, sodium, phosphoric acid,
Zinc, iron, magnesium, manganese, copper, calcium,
Salts such as cobalt, trace metal salts, corn steep liquor, vitamins, nucleic acids and the like may be used as necessary, and a general bacterial nutrient medium can be used. Bacteria belonging to the genus Bacillus may be aerobically cultured at 20 to 80 ° C, preferably 40 to 70 ° C, optimally 60 ° C for 2 to 6 hours using these media.

Means for obtaining a cell lysate in the present invention include:
For example, the cells can be obtained by collecting cells from a culture and then crushing the cells by a homogenizer, a blender, Manton-Gaulin, Dynomill, French press, sonication, freeze-thawing, lysozyme treatment, or the like.

Next, a cationic polymer flocculant is added to the cell lysate (cell extract) to precipitate cell lysates and nucleic acids. Examples of the cationic polymer flocculant used in the present invention include, for example, polyaminoalkyl methacrylates, copolymers of polyaminoalkyl methacrylate and acrylamide, Mannich-modified polyacrylamide, polydimethyldiallylammonium salts,
Examples thereof include polyvinyl imidazolines, polyacrylamides, and amine polycondensates. The amount of the polymer coagulant added at that time varies depending on the type of the coagulant, but is 1 to 100 parts by weight of the dry weight of the crushed microorganism.
40 parts by weight are preferred. After dissolving this cationic polymer flocculant in water in advance, it is added to the cell lysate and stirred for 10 minutes to 24 hours. When the pH needs to be adjusted, a buffer may be appropriately added so as to have a concentration of 10 to 200 mM, or glucose may be added to 100 parts by weight of cell lysate for stabilization of the protein. 50 parts by weight may be added.

Next, the precipitated cell debris and nucleic acid are separated. For this purpose, for example, it may be left standing, centrifuged, or filtered. By these operations, a crude enzyme preparation can be obtained. Furthermore in order to obtain a highly purified enzyme preparation can be used gel filtration chromatography, hydrophobic chromatography, affinity chromatography, the various types of chromatography ion exchange chromatography <br/> like. The ATP sulfurylase of the present invention has a high purification factor, particularly when affinity chromatography is used,
More preferred. According to the method as described above, the AT of the present invention
A sample of P sulfurylase is obtained.

[0016]

The present invention will be specifically described below with reference to examples. In the examples, the protein content of the enzyme preparation was determined on the assumption that the absorption 1 at 280 nm was 1 mg / ml of protein. Example 1 Glucose concentration 1%, yeast extract concentration 1%, phosphoric acid concentration
After sterilizing the medium containing 0.1% and some minerals,
The pH was adjusted to 6.5, and Bacillus stearothermophilus (NCA1503 strain) was inoculated and cultured. 60
After culturing at 3 ° C. for 3 hours, it was confirmed that glucose in the medium was consumed, and the cells were collected by centrifugation to obtain cells. After the wet cells obtained as described above were crushed by a freeze-thaw method, nucleic acids were removed using a polyacrylamide-based polymer flocculant. The resulting precipitate was removed by centrifugation to obtain a supernatant.

A 50 mM Tris-HCl buffer (p
When the supernatant was applied to a DEAE-Sepharose column equilibrated with H7.5), ATP sulfurylase was adsorbed. After sufficiently washing with the same buffer, the buffer was washed thoroughly with the same buffer and 0 to 500 mM sodium chloride. Elution was performed with a linear concentration gradient. The active fraction was collected and 1M
Ammonium sulfate was added to obtain. This was added to a 50 mM Tris-HCl buffer containing 1 M ammonium sulfate, pH7.
5 was applied to a phenyl sepharose column equilibrated. After sufficiently washing with the same buffer, 50 mM Tris-HCl buffer (pH 7.5) was sent. The obtained active fraction was collected, concentrated and dialyzed, and then 50 mM Tris-HCl buffer pH
The mixture was applied to a matrix gel blue A column equilibrated in 7.5. After sufficiently washing with the same buffer, the same buffer containing 1 M potassium chloride was sent. Collecting the active fraction,
A single band was obtained by polyacrylamide electrophoresis.

The specific activity of the enzyme preparation was 12.1 U / mg. When the obtained enzyme preparation was examined for various physicochemical properties, it had the properties described in (a) to (h) above.

Example 2 A medium containing 0.7% glucose, 0.8% yeast extract, 0.05% phosphoric acid and a small amount of minerals was sterilized, and the pH was adjusted to 7.0. Bacillus coagulans (ATCC 7050 strain) was inoculated and cultured. 62
After culturing at 4 ° C. for 4 hours, it was confirmed that glucose in the medium was consumed, and the cells were collected by centrifugation to obtain cells. After the wet cells obtained as described above were crushed by a French press, nucleic acid removal was performed using a polyacrylamide-based polymer flocculant. The resulting precipitate was removed by centrifugation and A
A crude extract containing TP sulfurylase was obtained.

A 50 mM Tris-HCl buffer (p
When the supernatant was applied to a DEAE-Sepharose column equilibrated with H7.5), ATP sulfurylase was adsorbed. After thoroughly washing with the same buffer, the buffer was washed with the same buffer at 0 to 0.4 M in concentration. Elution was performed with a linear concentration gradient of sodium. The active fraction was collected and concentrated. This was fractionated by Ultrogel ACA34 column chromatography using 50 mM Tris-HCl buffer (pH 7.5) as an eluent to obtain an active fraction. The active fraction was collected and applied to a hydroxylapatite column equilibrated with the same buffer. After washing well with the same buffer,
The same buffer containing 00 mM dipotassium phosphate was sent.
After concentration, purification was possible by polyacrylamide electrophoresis until a single band was given.

The specific activity of this enzyme preparation is 10.5 U / m
g. When the obtained enzyme preparation was examined for various physicochemical properties, it had the properties described in (a) to (h) above.

[0022]

The ATP sulfurylase of the present invention is far superior in thermostability to conventionally known ATP sulfurylase, and therefore has a great advantage to the industry.

[Brief description of the drawings]

FIG. 1 is a view showing an optimal pH curve of ATP sulfurylase of the present invention.

FIG. 2 is a view showing a pH stability curve of the ATP sulfurylase of the present invention.

FIG. 3 is a view showing the thermostability (thermostability) of the ATP sulfurylase of the present invention.

────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Hiroshi Nakajima 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Investigator, Central Research Laboratory, Inc. Mayumi Saito (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9 / 00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A thermostable adenosine-5'-3 phosphate sulfurylase having the following physicochemical properties. (A) Action: Acts on adenosine-5'-3 phosphate and sulfate ions to generate adenosine-5'-phosphosulfate. (B) Optimum pH: about 7.5 to about 8.0. (C) Suitable temperature for operation: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer at about 60 ° C. for about 15 minutes maintains about 95% of the activity before treatment. (E) Molecular weight: about 76,000 (gel filtration method). 2. A thermostable adenosine, comprising culturing a Bacillus bacterium (Bacillus sp.) And collecting a thermostable adenosine-5'-3-phosphate sulfurylase having the following physicochemical properties from the culture: A method for producing -5'-3 phosphate sulfurylase. (A) Action: Acts on adenosine-5'-3 phosphate and sulfate ions to generate adenosine-5'-phosphosulfate. (B) Optimum pH: about 7.5 to about 8.0. (C) Suitable temperature for operation: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer at about 60 ° C. for about 15 minutes maintains about 95% of the activity before treatment. (E) Molecular weight: about 76,000 (gel filtration method).