JPH02222677A - Purine nucleoside phosphorylase and production thereof - Google Patents

Abstract

NEW MATERIAL:Purine nucleoside phosphorylase having following physicochemical properties. Effect: purine nucleoside + Pi base + ribose-1-phosphoric acid; Substrate specificity: affect to purine nucleoside and base except adenine and adenosine; Optimum pH: 7-11.5. Does not inactivate even after treatment at 80 deg.C for 30 min at pH7-10; Optimum temperature: 80 deg.C. Stable even after treatment at 50-80 deg.C for 60min at pH7.0; Condition of inactivation: inactivates for 20min in treatment at 90 deg.C and pH7.0; Molecular weight: 68000 (by gel- permeation method with TSK gel G-3000SW), Molecular weight of sub-unit; 34000 (by SDS electrophoresis), etc. USE:Enzyme having excellent heat resistance and high reaction speed. PREPARATION:Microorganism belonging to genus Bacillus such as Bacillus stearothermophilus JTS 859 (FERM P-9666) is cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel purine nucleoside phosphorylase and a method for producing the same.

(Prior Art) To date, there are the following reports regarding purine nucleoside phosphorylases.

1, J, D, 5toeckler, C, Cambor
and R, E., Parks, Jr., [3ioc
hemistry-, 19, 102 (192, T, U,
Utagawa, H., Mar i sawa, S., Ya.
manaka, A, Yamazaki and 1
Y,Hfrose,,Agric.

Biol, Chem, 50.121 (1986) 3
, Y., Machida and T., Nakani.
shi, , Agric, Biol, Chem.

, 45, 1801. <1981> (Problems to be Solved by the Invention) The purine nucleoside phosphorylases described above had low thermostability.

The present inventors have conducted intensive research with the aim of providing a new enzyme that has better heat resistance and faster reaction rate than the previously known purine nucleoside phosphorylases.

(Means for solving the problems) The purine nucleoside bosphorylase of the present invention has the following properties.

■Action Nibrin nucleoside + Pi4\=-m- Base 2 Ribose-1-phosphate ■Substrate specificity: Acts on purine nucleosides and bases other than adenine and adenosine.

(1) Optimum 1) H and stable pH The optimal pH is pH 7 to 11.5. At pH 7 to 10, it is not inactivated even after 30 minutes of treatment at 80°C.

■Optimal temperature and range of action temperature Optimum temperature is 80℃ Stable at pH 7.0 and 50 to 80℃ even after 60 minutes of treatment Half-life at pH 7.0 and 80℃ is 16 hours (5): pH, Conditions for inactivation due to temperature, etc.: PH7.0
, deactivates in 20 minutes unless treated at 90°C.

■Inhibition, activation and stabilization No special activators or stabilizers are required.

Inhibited by mercuric chloride and parachloromercribenzoate.

■Molecular weight: 68000 (TSK gel G-3000S
(Gel filtration method using W) ■Molecular weight of subunit: 34,000 (SDS electrophoresis method) The purine nucleoside phosphorylase of the present invention has an optimal pH.
The properties are different from those known so far in terms of stable pH, optimum temperature, and effective temperature range.

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 JTS859, which was isolated from soil by the present inventors. 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 No. 9666 (
FERM P-9666).

The mycological properties of this strain were determined in Purge Aids Manual No. 2.
The results of the study based on the volume are shown below.

■、Form Bacillus: 5.4-6.5μm x 0.7-0.9μm Oval sporulation: 2.0-2.3 μm x 1.

1 to 1.2 μm, 1 per cell, located at terminal 2, culture properties NB medium: 62°C, 2 days culture on plate: white with some yellow, glossy, opaque, not raised, colonies Shape is on a circular wavy slant: white with slight yellow, glossy, opaque, no ridges, growth is moderate 3, biochemical properties 1, Gram stain positive 2, anaerobic culture, no growth, motile oxidase catalase Liquefaction of gelatin Lidomus milk OF test v-P test i Gas generation from glucose Production of acid from glucose Production of acid from arabinose Production of acid from mannitol Production of acid from xylose Growth in Sabouraud medium With slant liquid , Perihair positive, positive, liquefaction ability, coagulation, fermentation type, negative, no production, no production, no production, no production, no growth, growth, growth under 01001% lysozyme, no growth, under 0.02% azide. Growth No growth, growth under 7% NaCl No growth (grown up to 2%) Hydrolysis of casein Hydrolysis of starch with decomposition ability Generation of dihydroxyacetone with decomposition ability No formation, Elaguyoke test No growth, use of citric acid Negative Production of indole Negative urease activity Deamination of positive phenylalanine Negative arginine dehydrolase activity Degradation of positive tyrosine Negative 29 Production of levane Negative 30 Reduction of nitrate Positive 31 Denitrification ability of sodium ii acid Negative 32 Production of hydrogen sulfide Positive 33 Al nitrogen free Source Utilization Grows with NOl as the only nitrogen source Grows with NH as the only nitrogen source 34 GC content 47.3% 35 Growth temperature Optimum growth at 40-71°C 60-68°C 36 PH range pH 5,7- 8.5 optimum pH
Based on the above properties, this strain is identified as Bacillus stearothermophilus.

When culturing the above-mentioned strain, a medium containing tryptone, peptone, or casamino acid as a nitrogen source, glucose or sucrose as a carbon source, and further containing yeast extract and sodium chloride is used. Culture at 60-65℃
, shake culture at 180 to 22 rpm or aerated agitation culture.

As the microorganism used in the present invention, not only Bacillus stearothermophilus JTS859 but also any microorganism belonging to the genus Bacillus that produces purine nucleoside phosphorylase having the above-mentioned functions can be used.

Purine nucleoside phosphorylase is collected from the culture as follows: After destroying the collected cells, heat treatment, acetone treatment, and pH treatment are performed in sequence, and then a DEAE (diethylaminoethyl) cation resin column is used. , CM (carbomethoxy) anion resin column, butyl group hydrophobic chromatography column, phenyl group hydrophobic chromatography column, hydroxyapatite column, and gel filtration for isolation.

Purine nucleosides can be efficiently produced from purine bases and phosphoric acid using the purine nucleoside phosphorylase of the present invention. Examples of the present invention are shown below,
I will explain in more detail.

(Example) (Example 1) Bacillus stearothermophilus (Ba
ci l Ius, 5te) Culture of JTS859, 20g of bacterial collection peptone, 10g of yeast extract, 3g of glycose
A medium of p) 16.0 consisting of 11 g and 11 g of water was used.

Bacillus stearothermophilus JT in this medium 2I
Add 3°2 x 10' S859 spores, use a jar fermenter with stirring X (diameter 60 spores, 6 upper and lower each), rotate stirring blades at 680 rpm, aeration volume 1.5 vvm, and culture temperature. Culture was carried out at 65° C. and pH 5.9 to 6.2 for 8 hours.

After culturing, centrifuge the bacterial cells for 1M! (10000g.

Bacteria were collected at 4°C for 15 minutes.

(Example 2) Separation and Purification of Enzyme 160 g of the obtained wet bacteria was suspended in 500 mM potassium phosphate (pH 7.0, hereinafter referred to as M street solution J), and a 0M equilibrium solution in which the bacterial cells were destroyed with Dymomill was added. Add, 1400
ml, centrifugation (8300 g, 20 minutes) was performed to obtain 1300 ml of supernatant. The precipitate was suspended in 500 ml of buffer, 8M equilibrated solution treated with Dymomil for 5 minutes was added to bring the temperature to 10103O, and then centrifuged (8300g
, 20 minutes) to obtain 990 ml of supernatant. This supernatant 9
Combine 90 ml with 1300 ml' of the supernatant from above and heat at 63°C for 1 hour. ! Stir vigorously. Then, centrifugation (8
300 g, 40 minutes) to obtain 2130 ml of supernatant.

A 10° C. solution of 200 ml of acetone and 200 ml of buffer was added to 2130 ml of the heat-treated supernatant.

Furthermore, add 1.8N of acetone at -10℃, and
Stirred at ℃ for 15 minutes. Next, centrifugation (9000 g
, 5 minutes). 2.5 J of acetone at -10°C was added to the obtained supernatant, stirred for 15 minutes, and then centrifuged (9
000 g for 5 minutes) to obtain a precipitate. This precipitate was suspended in 800 ml of buffer to obtain 850 ml of acetone-treated enzyme solution.

1M phosphoric acid was added to 850ml of the acetone-treated I9 stock solution, the pH was adjusted to 4.75, and the mixture was kept at 0°C for 1111t. Centrifugation M (8900g, 1 hour) was performed to obtain a supernatant. IM potassium hydroxide was added to this supernatant to adjust the pH to 7.
The enzyme solution was adjusted to 0 and 840 ml of enzyme solution was obtained.

The obtained enzyme solution 840i was used as DEAE Toy.

It was added to a column (Φ45X 126m) packed with 200ml of Pearl 650M (manufactured by Tosoh).

Next, elution was performed with 20mM potassium phosphate JPN solution (p) 17°0) 2M, and then 20mM potassium phosphate and 0.05M, 0.1M, 0.15M, 0°2M, 0.
50 each of a solution of 5M sodium chloride at pH 7°0
Elution continued using 0 ml sequentially.

Next, 500ml of the eluate fraction of 20mM potassium phosphate and 0.1M sodium chloride solution was pH-treated with 1M potassium hydroxide.
It was set at 8.0. This solution & DEAE Toy equilibrated with a solution of 2 Ont M potassium phosphate, 50 mM sodium chloride (pH 8,0).

pear + 650M 400a+l (Φ3
1×530III) column. Then 2
0mM potassium phosphate, 50mM sodium chloride solution (pH 8,0) 11 and 20mM potassium phosphate, 20
A linear gradient in which the sodium chloride concentration continuously increases was prepared using IJ using 0 mM sodium chloride solution (pH 8,0), and 20 ml portions were collected.

The 56th to 63rd fractions (166 ml) in which purine nucleoside phosphorylase activity was observed were collected and purified with 2M lactic acid.
It was set as H4.0.

This solution was equilibrated with 20mM potassium phosphate and 100mM sodium chloride solution (PH4.0).
yopearl 650M (manufactured by Tosoh) 20ml (Φ
26×38 pus) was added to the column. Then, 20mM potassium phosphate, 100mM sodium chloride solution (PH
4,0) 20 +nl, 20mM potassium phosphate, 4
00mM sodium chloride solution (pH 4,0) 100a+
Elution was continued sequentially with I, 20mM potassium phosphate, 500mM sodium chloride solution (pH 4°0>100+al).

50ml of active fraction was obtained from a 20mM potassium phosphate and 400mM sodium chloride solution. Add this solution to 2M KOH
The pH was adjusted to 7.0, and then 9.8 g of ammonium sulfate was added.
2MKOH'? "p)I was adjusted to 7.O. This solution was mixed with 20mM potassium phosphate, 1.5M
0) and then util Toyopearl
650M (manufactured by Tosoh) 50ml (Φl 6 x 25
0 m ) column.

20mM potassium phosphate, 1.5M ammonium sulfate solution (pH 7,
0) Eluted in 50ml. Then 2 including 1.5MK decline
Elution was performed by creating a linear gradient with decreasing ammonium sulfate concentration using 125 ml of 0 mM potassium phosphate and 125 ml of 20 mM potassium phosphate solution (pH 7,0).

The fraction in which purine nucleoside phosphorylase activity was observed was permeated with 75 mM potassium phosphate and concentrated to 5 ml by ultraf.

Next, using TSKgel DEAE 5PW (manufactured by Tosoh) (Φ7.5X75m), a potassium phosphate solution (pH 6,0> concentration was adjusted from 90mM to 150mM).
Increasing the concentration with a linear gradient over 5 minutes and elution at 150mM for 13 minutes resulted in an activity fraction of 17.8.
+al was obtained. The flow rate was 1 cl/min.

The resulting solution was ultrafiltered to a concentration of 4 mtk, 20 mM.
2 ml of ammonium sulfate saturated solution of potassium phosphate was added. Next, TSKgel DEAE5pw (Φ7.
5X75-), 20mM potassium phosphate 1.5M
[From low to 20mM potassium phosphate 7.5Mi low 60
1 at ammonium sulfate concentration of 0.75M with a linear gradient in minutes.
After 5 minutes of runoff, 26 ml of active compartment was obtained. Flow velocity is 1m
It was 1/min.

Next, 26m1 of this active section was treated with TSKgel HAl.
oo (Φ7.5X75, manufactured by Tosoh), the concentration of potassium phosphate solution (pH 6,8) was adjusted from 90mM to 100mM.
Increased with a linear gradient in 10 min to mM,
Further elution was performed for 10 minutes at 100mMt'' to obtain 1.7cm of the purine nucleoside phosphorylase of the present invention.The flow rate was la+l/min.

(Example 3) Physicochemical properties 0 Effect and substrate specificity Purine nucleoside phosporylase of the present invention 0°02M
200 uj of 20 mM nucleoside 2QmM potassium phosphate solution (pH 7,0) containing g was reacted at 60° C. for 10 minutes, and the generated base was determined. The results are shown in Table 1.

Moreover, the purine nucleoside phosphorylase of the present invention 0.0
Solution (p) 1 consisting of 2Mg, 20mM base, 20mM ribose-1-phosphate, 20mM potassium phosphate
7.0) 200 μm was reacted at 60° C. for 10 minutes, and the produced nucleoside was quantified. The results are shown in Table 2.

(Left below) Nucleoside inosine adenosine anosine xanthine cytidine uridine 5-/methyluridine 2--deoxyinosine 2--deoxyadenosine 2'-deoxyguanosine thymidine 2--deoxyuridine 2'-deoxycytidine Amount of base produced 0.22mM 0. 18 0.10 0.46 0.33 Table 2 Base Generated nucleoside hyboxanthin C169mM adenine
O Xanthine 0,52 Cytosine 0 Uracil 0 Thymine 0 ■Optimal pH and stable pH Purine nucleoside phosphorylase of the present invention 0°68μ
5 ml of a 20 mM inosine, 20 mM potassium phosphate solution (pH 3.5 to 13) containing g was reacted at 60° C. for 15 minutes. The produced hyboxanthin is shown in Figure 1, pH 7
High enzyme activity was observed between 11.5 and 11.5.

To check the stable pH, 0.17 μg of the purine nucleoside phosphorylase of the present invention was added to 50 μM of 20 mM citric acid [1 equilibration (containing 20 mM potassium phosphate) = 20 m
8 each in Mglycine Mffr solution (containing 20mM potassium phosphate), 20mM) Lis-HCl buffer (containing 20mM potassium phosphate), 20mM potassium phosphate M solution
The reaction was carried out at 0°C for 300 minutes, and the residual enzyme activity was quantified. The results are shown in Figure 2.

Enzyme activity was not inactivated even at pH 7-10.

■Optimum temperature and range of action temperature Purine nucleoside phosphorylase of the present invention 0.085
1 ml of 20mM inosine and 20mM potassium phosphate solution (pH 7,0) containing ug was reacted at 40 to 90°C,
Hyboxanthin produced was quantified. The results are shown in Figure 3.

Below 80°C, the higher the temperature, the faster the production rate is at 290°C.
The enzyme was deactivated within 20 minutes.

In addition, 20mM potassium phosphate and ZmM inosine solution (
The half-life of the enzyme at pH 7,0) is 1 as shown in Figure 4.
It was 6 hours.

■Inhibition, activation and stabilization of the purine nucleoside phosphorylase of the present invention 0°09μ
20mM inosine containing g, 20mM potassium phosphate solution and 1ml of various metal ion solutions (pH 7°0) at 60°C.
The mixture was reacted for 20 minutes, and the produced hyboxanthin was quantified.

Mg5Oa-7H20, MnCjl x' 4H,
01CuSOa' 5Hx 01ZnSOa ・7H
No inhibition was observed with z 01CH* I C0OH and EDTA, but strong inhibition was observed with Hg Cj x and parachloromercribenzoate.

■Gel r using molecular weight TSK Gel G-3000SW (manufactured by Takun)
Under the law, it was 68,000.

The molecular weight of the subunit by SDS electrophoresis is 340
It's 00.

(2) Amino acid composition Analyzed using an amino acid analyzer (Hitachi IJ8835). The results are shown in Table 3.

Table 3 Constituent amino acids aspartic acid Threonine Serin glutamic acid glycine alanine Valine methionine isoleucine Leucine Tyrosine Phenylalanine lysine histidine arginine proline Mol% 8,04 4, 30 3,08 12.64 10.07 10.80 8, 59 3,68 8, 55 7, 26 3, 26 2, 32 4, 07 5,96 5, 49

[Brief explanation of the drawing]

FIG. 1 is a drawing showing the pH dependence of the purine nucleoside phosphorylase of the present invention. FIG. 2 is a diagram showing the pH stability of the purine nucleoside phosphorylase of the present invention. FIG. 3 is a diagram showing the range of suitable temperature for action. FIG. 4 is a diagram showing the half-life of enzyme activity. Number of minutes / Time (minutes)

Claims (1)

[Claims] 1. Purine nucleoside phosphorylase having the following physical and chemical properties (1) Action: Purine nucleoside + Pi base + ribose-1-phosphate (2) Substrate specificity: Purine nucleosides other than adenine and adenosine and acts on bases. (3) Optimum pH and Stable pH The optimal pH is pH 7 to 11.5. At pH 7 to 10, it is not inactivated even after 30 minutes of treatment at 80°C. (4) Range of optimal temperature and action temperature The optimal temperature is 80°C and pH 7.0 at 50°C. Stable even after 60 minutes of treatment at 80°C to 80°C. (5) Conditions for inactivation depending on pH, temperature, etc.: pH7.0
, it is deactivated in 20 minutes when treated at 90°C. (6) Inhibition, activation and stabilization No special activators or stabilizers are required. Inhibited by mercuric chloride and parachloromercribenzoate. (7) Molecular weight: 68000 (TSK gel G-3000S
(Gel filtration method using W) (8) Molecular weight of subunit: 34,000 (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. 1. A method for producing purine nucleoside phosphorylase, which comprises collecting .