JPH03103176A - Pyrimidine nucleoside phosphorylase and production thereof - Google Patents

Abstract

NEW MATERIAL:A substance having the following physical and chemical properties. Action: pyrimidine nucleoside + phosphoric acid base + ribose-1- phosphate. Substrate specificity: acting on pyrimidine nucleoside and bases except cytidine and cytosine. Optimum pH: 7-11. Stable pH: not being deactivated by treatment at 6.5-10.5 at 70 deg.C for 30 minutes. Optimum temperature: 70 deg.C. Proper temperature of action: stable even by treatment at pH7.0 at 50-70 deg.C for 60 minutes. Condition of deactivation: being deactivated by treatment at pH7.0-8.0 for 60 minutes. Molecular weight: 85,000 (gel filtration method by TSL gel G3,000 SW). Molecular weight of subunit: 54,000 (SDS electrophoresis), etc. USE:Useful for production of nucleic acid. PREPARATION:A bacterium such as Bacillus stearothermophilus JTS859 (FERM P-9666) belonging to the genus Bacillus is cultured.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a novel pyrimidine nucleoside phosphorylase and a method for producing the same. (Prior Art) Pyrimidine nucleoside phosphorylase is useful for producing nucleic acids, which are attracting attention as pharmaceuticals. It is an enzyme. To date, there are the following reports regarding pyrimidine nucleoside phosphorylase: 1.P.P.Sa
unders, B. A. Wilson and G.
．． F. Saunders. , J. Biol. Chem.
244.3691 (1967) 2. E. W. Yamada. , J. B i o l .
Chem. 243.1649 (1968) 3. J. J
．． Scocca. , J. Biol. Chem. 246.
6606 (1971) (Problem to be Solved by the Invention) However, the above-mentioned pyrimidine nucleoside phosphorylase had low thermostability. The purpose of the present inventors is to provide a new enzyme that has better heat resistance and faster reaction rate than the previously known pyrimidine nucleoside phosphorylases. With this goal in mind, we carried out intensive research. (Means for solving the problems) The pyrimidine nucleoside phosphorylase of the present invention has the following properties: ■Action: Pyrimidine nucleoside + phosphate ■Base
+ Ribose l-phosphate ■Substrate specificity: Acts on pyrimidine nucleosides and bases other than cytidine, cytosine, and their derivatives. ■Optimal pH and stable pH The optimum ρH is not inactivated even after 30 minutes of treatment at 70°C at pH 7-11 and pH 4.5-10.5. ■Optimum temperature and range of action temperatureOptimum temperature: 70℃ At pH 7.0, stable even after 60 minutes of treatment at 50-70℃pH 7.0, half-life at 70℃: 15.1 hours■+pH, temperature Conditions for inactivation: pH 7.0, 8
When treated at 0°C, it becomes inactive in 60 minutes. ■Inhibition, activation and stabilization No special activators or stabilizers are required. Inhibited by manganese chloride. ■Molecular weight: 85000 (TSK gel G-3000S
(Gel filtration method using W) ■Molecular weight of subunit: 54,000 (SDS electrophoresis method) The pyrimidine nucleoside phosphorylase of the present invention has a known range of optimal pH, stable pH, optimal temperature, and optimal temperature for action. It has different properties from things. Next, the method for producing the enzyme of the present invention will be explained. A preferred example of the enzyme-producing bacterium used in the present invention is Bacillus stearothermophilus/lty ci//l., which was isolated from soil by the present inventors. ! 5711
lθl7etz77p7i/〃s) J T S 8
There are 5 9. This strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its deposit number is FERM P-9666. The mycological properties of this strain were determined in Purge Aids Manual No. 2.
The results of the study according to the volume are shown below. 1. Morphology of rods = 5.4-6.5 μm x 0.7-0.9 μm Oval spore formation = 2.0-2.3 μm x 1.1-1.
2μm, 1 per 1m cell, located at the terminal 2. Culture properties: NB medium: 62°C, 2 days culture on plate: white with a slight yellow tinge. Shiny, opaque, and non-raised. Colony shape is circular wavy slant: white with a slight yellow tinge. Shiny, opaque, and not raised. Growth is medium 3. Biochemical properties 1 Positive Gram stain 2 Anaerobic culture 3 Motility 4 Oxidase 5 Catalase 6 Liquefaction of gelatin 7 Litmus milk 8 0F test 9 V-P test 10 Gas production from glucose 11 Acid production from glucose 12 From arabinose 13 Production of acid from mannitol 14 Production of acid from xylose 15 Growth in Sabouraud medium No slant growth, positive for pericilla, positive for liquefaction, coagulating fermentation type, negative for production, no production Liquid grown without producing soil Grows 16 Growth under 0.001% lysozyme No growth 17 Growth under 0.02% azide No growth 18 Growth under 7% NaCl No growth (2 19 Casein hydrolysis Degradation ability 20 Starch hydrolysis Degradation ability 21 Dihydroxyacetone production No production 22 Egg yoke test No growth 23 Utilization of citric acid Negative 24 Indole production Negative 25 Urease activity Positive 26 Deamination of phenylalanine Negative 27 Arginine dehydrolase activity Positive 1 28 Decomposition of tyrosine Negative 29 Levan production Negative 30 Reduction of w4 salts Positive 31 Denitrification ability of sodium nitrate Negative 32 Production of hydrogen sulfide Positive 33 Utilization of inorganic nitrogen sources N NH grown using Ox as the sole nitrogen source. 34 grown using as the sole nitrogen source OC content 47.3%35 Growth temperature Optimum growth at 40-71°C 60-68°C 36 ρH range pH 5.7-8.5 Optimum pH 6
．． 0 to 7.0 Based on the above properties, this strain is Bacillus stearothermophilus /liri//tts sh/7rρ//I
IIIρβIi/VS). The above strains may be cultured under the conditions normally used for Bacillus stearothermophilus. Preferably, the medium contains tryptone, veptone, or casamino acid as a nitrogen source, glucose or sucrose as a carbon source, and further contains yeast extract and sodium chloride.
Conditions such as 5°C, 180-22 Orpm 7' shaking culture or aerated agitation culture are best. In addition, the microorganism used in the present invention is not limited to Bacillus stearothermophilus JTS859, but any microorganism belonging to the genus Bacillus that produces pyrimidine nucleoside phosphorylase having the above-mentioned functions can be used. Pyrimidine nucleoside phosphorylase can be collected from culture according to standard methods.
As an example, after destroying the collected bacteria, heat treatment, acetone treatment, and pH treatment are sequentially applied, and then DEAE
(Diethylaminoethyl) cation resin ram, butyl group hydrophobic chromatography ram, and gel filtration methods can be shown. Using the pyrimidine nucleoside phosphorylase of the present invention, pyrimidine base and ribose-1-
Pyrimidine nucleosides can be efficiently produced from phosphoric acid. Hereinafter, examples of the present invention will be shown and explained in more detail. (Example 1) Bacillus stearothermophilus (Ea
cj//us. stearothermophjノ//
s) Culture of JTS859, 20g of bacterial collection, 10g of yeast extract, 3g of glucose, 3g of sodium chloride
A medium with a pH of 6.0 consisting of 11% and 11% water was used. Bacillus stearothermophilus JTS8 in this medium 2I
Add 3.2 x 10' of 59 spores and mix with K (diameter 6
Using a jar fermenter with 0m, 6 pieces each on the upper and lower parts, while rotating the stirring blades at 680 rpm,
．． 8 at 5vvm, culture temperature 65℃, pH 5.9-6.2
Cultured for hours. After culturing, the bacterial cells were centrifuged (10,000g, 4℃, 1
Bacteria were collected for 5 minutes. (Example 2) Separation and purification of enzymes 160 g of the obtained wet bacteria was suspended in 20 m M potassium phosphate (pH 7.0, hereinafter referred to as "buffer"), and Dynomil (manufactured by Willy A. Bachofen, Switzerland) was used. The bacterial cells were destroyed. [Add the equilibrated liquid, 1 4
After reducing the volume to 0.0 ml, centrifugation M (8300 g, 20 minutes) was performed to obtain 1300 ml of supernatant. The precipitate is 50
The suspension was suspended in 0 ml of buffer solution and treated with Dymomil for 5 minutes. After adding a buffer solution to bring the volume to 1030 ml, centrifugation (8300 g, 20 minutes) was performed to obtain 990 ml of supernatant. 990 ml of this supernatant was combined with 1300 ml of the previous supernatant and stirred gently at 63° C. for 1 hour. Then, centrifugation (8300g, 40 minutes) was performed. 2130 ml of supernatant was obtained. Add 200ml of acetone to 2130ml of heat-treated supernatant.
ml and 200 ml of buffer solution (-10°C) was added. Furthermore, add 1.81 liters of acetone at -10°C,
Stir for 15 minutes at ~10°C. Next, centrifugation (90
00g for 5 minutes). After adding 2.5 liters of acetone at -10°C to the obtained supernatant and stirring for 15 minutes, centrifugation (9000 g, 5 minutes) was performed to obtain a precipitate. This precipitate was suspended in 800 ml of buffer. 850 ml of acetone-treated enzyme solution was obtained. IM phosphoric acid was added to 850 ml of acetone-treated enzyme solution, ρH was set to 4.75, and the mixture was kept at 0°C for 1 hour. Centrifugation (8900 g, 1 hour) was performed. A supernatant was obtained. IM potassium hydroxide was added to this supernatant and the pH was adjusted to 7.0 to obtain 840 ml of enzyme solution. 840 ml of the obtained enzyme solution was added to DEAE Toy.
It was added to a column (Φ45 x 126 m) packed with 200 ml of opearl 650M (manufactured by Tosoh). Elution was then carried out with 2M 20mM potassium phosphate buffer (pH 7.0), followed by 20mM potassium phosphate buffer (pH 7.0). O'
Elution was performed using 11 solutions each of pH 7.0 consisting of 5M, 0.1M, 0.15M, 0.2M, 0, and 5M sodium chloride in sequence. Then 20mM potassium phosphate, O.
The enzyme activity was found in fraction 11 eluted from IM sodium chloride solution. The same amount of 20 mM potassium phosphate solution (pH 7.0) was added to the obtained elution fraction, and the pH was adjusted to 8.0 with IM potassium hydroxide solution. Add this solution to DEAE
Toyopearl 650M (manufactured by Tosoh>450
It was added to a column (Φ31 x 53 mm) packed with ml. Furthermore, the sodium chloride concentration increased with a solution (pH 8.0) of 20mM potassium phosphate and 50mM sodium chloride (pH 8.0) and a solution (pH 8.0) of 20mM potassium phosphate and 200mM sodium chloride (IJ). A gradient was prepared and eluted to obtain 176 ml of active fraction. After adding 27.9 g of ammonium sulfate to both portions, the pH was adjusted to 7.0 with potassium hydroxide. This solution was pre-equilibrated with a solution (pH 7.0) consisting of 1.2 Mil EE ammonium and 20 mM potassium phosphate.
It was added to a 100 ml (Φ16 x 300 m) column of Toyopearl 650S (manufactured by Tosoh), and eluted with 100 ml of a solution (pH 7.0) consisting of 1.2 MI ammonium and 20 mM potassium phosphate. Furthermore, 250 ml of a solution consisting of 1.2 M ammonium sulfate and 20 mM potassium phosphate and a 20 mM potassium phosphate solution (p
H7. 0 ) 2 5 0 a+1 to create a gradient of decreasing ammonium sulfate concentration and elute. 75 ml of the active fraction was collected, dialyzed against 20 mM potassium phosphate solution, and concentrated to 14.2 a+l by ultrafiltration. 14.2 ml of the obtained active fraction was dialyzed against 0.2 M potassium phosphate solution (pH 7.0) and concentrated to 7 ml by ultrafiltration. The resulting solution was diluted with 20mM potassium phosphate solution (pH 7.
DEAE-5PW (manufactured by Tosoh, Φ7) equilibrated with
．． 5x75m). Then a solution consisting of 20mM potassium and 50mM sodium chloride (pH 8.
0) and a solution consisting of 20mM potassium phosphate and 225mM sodium chloride (ρH 8.0), the flow rate was 1 m with a gradient of increasing sodium chloride concentration.
It eluted at 1/min. The obtained solution was diluted with 0.2M potassium phosphate solution (pH 6.
8) was added to TSKgel-G3ooosw (manufactured by Tosoh, Φ7.5X750 shoulder). Then 0.
Using 2M potassium phosphate solution (pH 6.8), flow rate 0
．． Elution was performed at 5 ml/min to obtain 12.1 mg of the pyrimidine nucleoside phosphorylase of the present invention. (Example 3) Physical and chemical properties ■Action and substrate specificity Pyrimidine nucleoside phosphorylase of the present invention 0.1
250 μl of a 20 mM potassium phosphate solution (pH 7.0) containing 4 μg of nucleoside was reacted at 60° C. for 20 minutes, and the generated 1= group was quantified. The results are shown in Table 1. Furthermore, the pyrimidine nucleoside phosphorylase 0 of the present invention
．． 14μg, 20mM base, 20mM ribose-1-phosphate and 20mM potassium phosphate solution (pH
7.0) 250 μl was reacted at 60°C for 20 minutes, and the produced nucleoside was quantified. The results are shown in Table 2. Table 1 Table 2 (blank below) ■Optimal pH and stable pH Pyrimidine nucleoside phosphorylase of the present invention 0.5
60 lmjl of 20mM potassium phosphate solution (pH 3.0-13) containing 5μg of 20mMS-methyluridine
'C for 20 minutes, and the produced hyboxanthin was measured. The results are shown in Figure 1. High enzyme activity was observed between pH 7 and 11.5. To examine stable p}I, 0.55 μg of the pyrimidine nucleoside phosphorylase of the present invention was added to 90 μl of 20 mM
Citrate buffer (containing 20mM potassium phosphate), 2
0mM glycine buffer (contains 20mM potassium phosphate). After aging at 70°C for 30 minutes in 20mM Lis-HCl solution (containing 20mM potassium phosphate) and 20mM potassium phosphate buffer, the remaining enzyme activity was quantified. The results are shown in Figure 2. Enzyme activity was not inactivated at pH 6.5 to 10.5. ■Optimum temperature and range of temperature for action Pyrimidine nucleoside phosphorylase of the present invention 0.5
1 ml of 20mMS-methyluridine, 20mM potassium phosphate solution (pH 7.0) containing 5 μg was added to 40-80%
The reaction was carried out at ℃, and the released hypoxanthin was quantified. The results are shown in Figure 3. At temperatures below 70°C, the higher the temperature, the faster the production rate, and at 80°C, the enzyme was deactivated within 30 minutes. In addition, the half-life of the enzyme in 2'OmM potassium phosphate solution (pH 7.0) is 15 at 70°C as shown in Figure 4.
．． It was one hour. ■Inhibition, activation and stabilization of the pyrimidine nucleoside phosphorylase of the present invention 0.5
20mM MS-methyluridine containing 5μg, 20mM potassium phosphate solution and 1mM various metal ion solutions (pH
7.0>lml was reacted at 60°C for 20 minutes, and the produced hyboxanthin was quantified. M g S O a ・7 H t O, H g C
Iz. C u S O. -5H. O, ZnSO4
・7H2 0, CHx ICOOH. No inhibition was observed with EDTA and parachloromercribenzoate, but strong inhibition was observed with Mn(12.4H20).
■Gel P with molecular weight TSK gel G-3000SW (Tosoh M)
Under the law, it was 85,000. The molecular weight of the subunit by SDS electrophoresis was 54,000. ■Amino acid composition Analyzed using an amino acid analyzer (Hitachi H835). The results are shown in Table 3. Table 3 (Effects of the Invention) Nucleic acids can be easily obtained from bases using the pyrimidine nucleoside phosphorylase of the present invention.

[Brief explanation of drawings]

FIG. 1 is a drawing showing the pH dependence of the pyrimidine nucleoside phosphorylase of the present invention. FIG. 2 is a diagram showing the pH stability of the pyrimidine nucleoside phosphorylase of the present invention. Figure 3 is a diagram showing the range of suitable temperature for action. Figure 4 is a diagram showing the half-life of enzyme activity. Origin: Japan Tobacco Inc. 58 remaining surnames (γ.)

Claims (1)

[Claims] 1. Pyrimidine nucleoside phosphorylase (1) having the following physical and chemical properties: pyrimidine nucleoside + phosphate ⇔ base +
Ribose-1-phosphate (2) Substrate specificity: Acts on pyrimidine nucleosides and bases other than cytidine, cytosine, and their derivatives. (3) Optimum pH and Stable pH The optimal pH is pH 7 to 11. At pH 6.5 to 10.5, it is not inactivated even after 30 minutes of treatment at 70°C. (4) Optimum temperature and range of action temperature The optimal temperature is 70°C, and at pH 7.0, it is stable even after 60 minutes of treatment at 50 to 70°C (5): Conditions for inactivation depending on pH, temperature, etc.: pH 7 .0
, it is deactivated in 60 minutes when treated at 80°C. (6) Inhibition, activation and stabilization No special activators or stabilizers are required. Inhibited by manganese chloride. (7) Molecular weight: 85000 (TSK gel G-3000S
(Gel filtration method using W) (8) Molecular weight of subunit: 54,000 (SDS electrophoresis method) 2. A microorganism belonging to the genus Bacillus having the ability to produce the enzyme according to claim 1 is cultured, and the enzyme is extracted from the culture. A method for producing pyrimidine nucleoside phosphorylase, which comprises collecting pyrimidine nucleoside phosphorylase.