JPH05137571A - Thermostable adenosine-5'-phosphosulfate kinase and its production - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme, excellent in thermal stability, usable for a bioreactor and useful for producing 3'-phosphoadenosine 5-phosphosulfate, etc. CONSTITUTION:A bacterium of the genus Bacillus [e.g. Bacillus.stearothermophilus (NCA1503 strain)] is cultured in a culture medium at 60 deg.C for 3hr and the resultant product is then collected from the prepared culture to afford the objective thermostable adenosine-5'-phosphosulfate kinase, capable of acting on adenosine 5'-triphosphate and adenosine 5'-phosphosulfate and having the function to produce 3'-phosphoadenosine 5'-phosphosulfate, optimal pH of about 6.5-7.0 action temperature of about 50-70 deg.C, heat resistance in which the activity value of about 95% based on that before treatment is retained after the treatment in a buffer solution at about 60 deg.C for about 15min, about 50000 molecular weight measured by the gel filtration method and properties of usability as a bioreactor.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a thermostable adenosine-5'-phosphosulfate kinase (hereinafter referred to as AP) having a property that it can be effectively used in a bioreactor having excellent thermostability.
It is called S-kinase. ) And its manufacturing method.

[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. The relation between PAPS in the living body and some diseases represented by proteoglucan diseases has been reported [Brain Research, Vol. 20, p.341-360 (197).
0)]. As described above, the role of PAPS in the body is very important, and it is considered that it has a high utility value in the fields of medicine and the like. PAPS uses adenosine-5 'as described below.
It is a substance synthesized from -3 phosphate (hereinafter referred to as ATP) by the actions of two enzymes, adenosine-5'-3 phosphate sulfurylase (hereinafter referred to as ATP sulfurylase) and APS kinase.

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

Conventionally, APS kinase has a problem that the content per wet cell is very low and therefore PAPS cannot be synthesized in a large amount. Also, baker's yeast (Dry ba
ker's yeast, Sigma YSC-1) APS kinase at 30 ℃
When kept at room temperature, the residual activity was only about 8% in 1 hour, and APS kinase was extremely heat-labile.
S could not be synthesized efficiently.

For the above-mentioned reason, when PAPS is efficiently synthesized, or when APS kinase is used to measure the APS concentration in body fluids in order to investigate the relationship between APS and pathological conditions, it is industrially necessary. There was nothing that could be implemented, and there was a strong demand for commercialization.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention, from the above viewpoints, to provide a highly stable APS kinase and a method for producing the same.

[0007]

Means for Solving the Problems As a result of intensive studies aimed at efficiently obtaining a highly stable APS kinase, the present inventors have found that Bacillus acidocaldarius (Baci
llus acidocaldarius), Bacillus coagulans (B.coagulans), Bacillus licheniformis (B.li)
cheniformis), B. schlegeli
i), Bacillus stearothermophilus (B.stearothe)
Bacillus bacteria such as rmophilus) have been found to produce APS kinase having the above-mentioned properties, and have completed the present invention.

That is, the present invention provides a thermostable adenosine-5'-phosphosulfate kinase having the following physicochemical properties; and (a) Action: acts on ATP and APS to produce PAPS. (B) Optimum pH: about 6.5 to about 7.0. (C) Suitable temperature for action: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer solution at about 60 ° C. for about 15 minutes retains a value of about 95% of the activity before treatment. (E) Molecular weight: about 50,000 (gel filtration method). Bacillus bacterium (Bacillus sp.) Is cultivated, and a thermostable adenosine-5 'having the following physicochemical properties is obtained from the culture.
-A method for producing a thermostable adenosine-5'-phosphosulfate kinase, which comprises collecting phosphosulfate kinase. (A) Action: It acts on ATP and APS to generate PAPS. (B) Optimum pH: about 6.5 to about 7.0. (C) Suitable temperature for action: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer solution at about 60 ° C. for about 15 minutes retains a value of about 95% of the activity before treatment. (E) Molecular weight: about 50,000 (gel filtration method).

The present invention will be described in detail below. The thermostable APS kinase of the present invention has the following physicochemical properties. (A) Action: It acts on ATP and APS to generate PAPS. (B) Substrate specificity: APS and A in the positive reaction
TP serves as a substrate. However, it has virtually no effect on AMP. In the reverse reaction, PAPS and ADP serve as substrates. However, 2'-phosphoadenosine-5'-phosphosulfate shows an activity of 0.005% or less of PAPS. (C) Optimum pH: As a result of measuring the titer by changing the pH of the buffer solution used according to the titer measuring method described later, it is about 6.5 to 7.0 as shown in FIG. (D) Stable pH: 4 ° C. in a buffer solution of pH 5.0 to 10.0
After storing for 20 hours, the titer was measured according to the titer measuring method described later. The result is about 5.0 ~ as shown in Fig.2.
It is 7.5. (E) Optimum temperature of action: The temperature was changed to 20 ° C to 80 ° C, and the titer was measured by the method described below. Optimal temperature is about 50 ° C to about 7
It is 0 ° C. (F) Heat resistance: After the treatment in a buffer solution at about 60 ° C. for about 15 minutes, the activity was measured by the titer measuring method described later, and as a result, the value was 95% of the activity before treatment. (G) Inhibition: Inhibition by phosphoric acid was observed when phosphoric acid was added to the assay method for titer described later and measurement was performed. Its inhibition constant KI is 90 mM. (H) Molecular weight: The molecular weight was measured by the gel filtration method. The molecular weight is about 50,000.

(I) Method for measuring titer: The measuring principle is ATP
When ATP sulfurylase (derived from brewer's yeast) is allowed to act with and MgSO ₄ as substrates, APS is produced. PAP produced from ATP and APS by acting an enzyme on this
S is quantified. [Reagent] Substrate: 100 mM ATP 1M Magnesium sulfate Buffer: 100 mM Tris-HCl buffer (pH 8.0) Enzyme: 50 u / ml ATP sulfurylase (from brewer's yeast) [Operation] 5 μl of the above substrate ATP and 5 μ magnesium sulfate
1, 385 μl of buffer solution and 5 μl of ATP sulfurylase are mixed by stirring, 100 μl of enzyme solution is added, and the mixture is reacted at 30 ° C. for 10 minutes. After completion of the reaction, the reaction is stopped by immersing it in boiling water for 1 minute. After centrifugation, the supernatant is analyzed by HPLC to quantify the PAPS produced. A blind test is carried out in the same manner for the case where no enzyme solution is added to confirm that PAPS is not produced. From the value thus obtained,
The PAPS concentration is quantified with respect to the value which has been previously subjected to HPLC analysis using an ATP solution having a known concentration. Regarding the display of enzyme activity, the amount of enzyme that produces 1 μmol of PAPS per minute is 1 U.

The thermostable APS kinase of the present invention is obtained from a bacterium of the genus Bacillus, and examples of the bacterium of the genus Bacillus include Bacillus acidocaldarius and Bacillus coagulans (B. coagulans), Bacillus licheniformis (B. licheniformis), Bacillus sheregeri (B.sc)
hlegelii), Bacillus stearothermophilus (B.st)
earothermophilus) and so on.

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

The means for obtaining a cell lysate according to the present invention includes:
For example, it can be obtained by collecting bacterial cells from a culture, and then crushing the cells by homogenizer, blender, mantongolin, dynomill, French press, ultrasonic treatment, freeze-thawing, lysozyme treatment and the like.

Next, a cationic polymer aggregating agent is added to the cell lysate (cell extract) to precipitate cell lysate and nucleic acid. As the cationic polymer flocculant used in the present invention, for example, polyaminoalkylmethacrylates, copolymers of polyaminoalkylmethacrylate and acrylamide, Mannich modified products of polyacrylamide, polydimethyldiallylammonium salts,
Examples thereof include polyvinyl imidazolines, polyacrylamides and amine polycondensates. The amount of the polymer flocculant added at that time varies depending on the type of the flocculant, but is 1 to 100 parts by weight of the dry weight of the crushed microorganisms.
40 parts by weight is preferred. This cationic polymer flocculant is previously dissolved in water, then added to the cell disruption solution and stirred for 10 minutes to 24 hours. Further, when pH adjustment is required, a buffer solution may be appropriately added so as to be 10 to 200 mM, and glucose is added in an amount of 1 to 100 parts by weight of the cell lysate for stabilizing the protein. You may add 50 weight part.

Next, the precipitated cell debris and nucleic acid are separated. For that purpose, for example, it may be left stationary, centrifuged, or filtered. A crude enzyme preparation can be obtained by these operations. In order to obtain a highly purified enzyme preparation, various chromatography such as gel filtration chromatography, hydrophobic chromatography, affinity chromatography, ion exchange chromatography and the like can be used. The APS kinase of the present invention has a higher purification rate, particularly when affinity chromatography is used, and is thus more preferable. A standard of the APS kinase of the present invention can be obtained by the method as described above.

[0016]

EXAMPLES The present invention will be specifically described below with reference to examples. In the examples, the protein amount of the enzyme preparation was determined by assuming that 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 bacterial cells. The wet bacterial cells obtained as described above were crushed by a freeze-thaw method, and then nucleic acid was removed using a polyacrylamide-based polymer flocculant. The resulting precipitate was removed by centrifugation to obtain a supernatant.

50 mM Tris-HCl buffer (p
When the supernatant was applied to a DEAE-Sepharose column equilibrated with H8.0), APS kinase was adsorbed. Therefore, after thoroughly washing with the same buffer, 0 to 500 mM sodium chloride was added using the same buffer. Elution was performed with a linear concentration gradient. The active fraction was collected and ammonium sulfate was added to it so that it would be 70% saturated. After centrifugation,
The precipitate was dissolved in the same buffer and dialyzed. After applying this to a Matrex gel blue A column equilibrated with the same buffer, it was thoroughly washed with the same buffer, and a phosphate buffer pH 7.2 containing 1.5 M potassium chloride was sent. Ammonium sulfate was added to the obtained active fraction to a concentration of 800 mM, and the mixture was applied to a phenylcellulofine column equilibrated with a 50 mM Tris-HCl buffer solution containing 8.0 mM ammonium sulfate (pH 8.0). After thoroughly washing with the same buffer, 50 mM Tris-HCl buffer pH 8.0 was sent.
The active fraction was collected, concentrated, and subjected to polyacrylamide gel electrophoresis to obtain a single band.

The specific activity of the enzyme preparation was 1.1 U / mg. When the physicochemical properties of the obtained enzyme preparation were examined, it had the properties described in (a) to (h) above.

Example 2 After sterilizing a medium containing glucose concentration of 0.7%, yeast extract concentration of 0.8%, phosphoric acid concentration of 0.05% and some minerals, the pH was adjusted to 7.0, Bacillus coagulans (ATCC7050 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 bacterial cells. The wet bacterial cells obtained as described above were crushed with a French press, and then nucleic acid was removed using a polyacrylamide-based polymer flocculant. The resulting precipitate was removed by centrifugation to remove A
A crude extract containing PS kinase was obtained.

50 mM Tris-HCl buffer (p
When the supernatant was applied to a DEAE-Sepharose column equilibrated with (H8.0), APS kinase was adsorbed. Therefore, after thoroughly washing with the same buffer, 0 to 0.8 M salinization was performed using the same buffer. Elution was performed with a linear concentration gradient of sodium. The active fraction was collected, concentrated and dialyzed. This was applied to a hydroxylapatite column equilibrated with the same buffer. After thoroughly washing with the same buffer,
The same buffer solution containing 100 mM dipotassium phosphate was fed. After concentrating the eluted active fraction, Sephadex G using 50 mM Tris-HCl buffer (pH 8.0) as the eluent
Fractionation was performed by -100 column chromatography to obtain an active fraction. After concentration, the product could be purified by polyacrylamide electrophoresis until a single band was obtained.

The specific activity of this enzyme preparation is 1.9 U / mg
Met. When the physicochemical properties of the obtained enzyme preparation were examined, it had the properties described in (a) to (h) above.

[0022]

The APS kinase of the present invention is far more thermostable than the conventionally known APS kinases, and therefore has a great advantage for the industrial world.

[Brief description of drawings]

FIG. 1 is a diagram showing an optimum pH curve of APS kinase of the present invention.

FIG. 2 is a diagram showing a pH stability curve of the APS kinase of the present invention.

FIG. 3: Thermostability (heat resistance) of the APS kinase of the present invention
FIG.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Hiroshi Nakajima 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Ltd. Central Research Laboratory

Claims (2)

[Claims] 1. A thermostable adenosine-5′-phosphosulfate kinase having the following physicochemical properties. (A) Action: adenosine-5'-3 phosphate and adenosine-
Acts on 5'-phosphosulfate to produce 3'-phosphoadenosine-5'-phosphosulfate. (B) Optimum pH: about 6.5 to about 7.0. (C) Suitable temperature for action: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer solution at about 60 ° C. for about 15 minutes retains a value of about 95% of the activity before treatment. (E) Molecular weight: about 50,000 (gel filtration method). 2. A thermostable adenosine comprising culturing a bacterium of the genus Bacillus (Bacillus sp.) And collecting a thermostable adenosine-5′-phosphosulfate kinase having the following physicochemical properties from the culture. A method for producing -5'-phosphosulfate kinase. (A) Action: adenosine-5'-3 phosphate and adenosine-
Acts on 5'-phosphosulfate to produce 3'-phosphoadenosine-5'-phosphosulfate. (B) Optimum pH: about 6.5 to about 7.0. (C) Suitable temperature for action: about 50 ° C to about 70 ° C. (D) Heat resistance: The activity after treatment in a buffer solution at about 60 ° C. for about 15 minutes retains a value of about 95% of the activity before treatment. (E) Molecular weight: about 50,000 (gel filtration method).