JPH0272873A - Nucleosideoxidase and analyzing method utilizing the same - Google Patents

Abstract

NEW MATERIAL:Nucleosideoxidase YT-1 capable of reacting with various kind of nucleosides such as inosine without reacting with a base such as hypoxanthine, nucleside such as inosinic acid, ribose, etc. and having 5.0-6.0 optimum pH, leaving >=95wt.% of original activity at pH 6.0 and 60 deg.C for 15min and inactivating at 70 deg.C for 15min, having about 130,000 molecular weight and isoelectric point of pH 5.3 and inhibited by potassium cyanide or sodium azide, having no maximum absorption at >=450n m in a neutral buffer such as phos phoric acid buffer and containing iron. USE:For analysis of nucleoside amount of liquid to be tested. PREPARATION:A microorganism having a nucleosideoxidase YT-1-producing ability such as Pseudomonas maltophilia LB-86 (FERM P-9813) is cultured to provide the nucleosideoxidase YT-1.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to a new enzyme that catalyzes the oxidation reaction of nucleosides, specifically, to oxidize nucleosides through the reaction of nucleosides with molecular oxygen to form nucleosides-5'-aldehydes.
The present invention relates to a novel nucleoside oxidase YT-1 that produces 5'-carboxylic acid but does not produce hydrogen peroxide as a by-product, and a novel analytical method using this enzyme.

BACKGROUND OF THE INVENTION As enzymes that act on nucleosides, an enzyme (nucleoside oxidase) that catalyzes the oxidation reaction shown by the following formula is known, as well as hydrolase and deaminase.

The above-mentioned nucleoside oxidase has conventionally been typically used, for example, in Pseudomonas putida (Pseudomonas putida).
It is isolated and purified from microorganisms such as P. sputida, and is used for the production of nucleoside-5'-carboxylic acids, the expression of nucleosides, and the activity measurement system of enzymes that generate or decrease nucleosides through reactions. It is used in various ways.

However, the use of the oxidase inevitably generates H2O2 in the oxidation reaction system, and for example, in the production of the nucleoside-5'-carboxylic acids,
It is necessary to add catalase etc. to the reaction system to decompose the above H2O2, and the H2O2 generated in the above reaction system
The method of measuring and quantifying nucleosides by measuring 2ffi has the disadvantage of requiring the use of peroxidase.

Moreover, the above measurement method has disadvantages such as a complicated measurement system (for example, Japanese Patent Application Laid-Open No. 57-58883;
7-68794, JP-A-57-94300, etc.).

Problems to be Solved by the Invention The present inventors have been conducting extensive research on nucleoside oxidases originating from various microorganisms, and in the process, they discovered a strain belonging to the genus Pseudomonas that was newly isolated from soil. discovered that it has the ability to produce an enzyme that catalyzes an oxidation reaction different from that of conventional nucleoside oxidases, and also succeeded in isolating and purifying the enzyme and elucidating its properties. They also succeeded in developing a measurement technique, which led to the completion of the present invention.

Means for solving the problem, that is, the present invention provides an enzyme that catalyzes the oxidation reaction of nucleosides.
A novel nucleoside oxidase, which produces nucleoside-5'-carboxylic acid via nucleoside-5'-aldehyde through the reaction of the nucleoside with molecular oxygen, but does not produce hydrogen peroxide as a by-product. Y
T-1 and a system containing or producing nucleosides are used as test solutions, and the nucleoside oxidase YT-1 is applied to a mixture of the above test solution and a coloring reagent that develops color by oxidation, and the test solution is The present invention relates to a method for analyzing a test liquid, characterized in that the degree of color development of the color reagent described above is measured in proportion to the change in the nucleoside base.

The nucleoside oxidation reaction catalyzed by the enzyme of the present invention is:
It is shown by the following formulas (1) and (2), and is clearly distinguished from the oxidation reactions of nucleoside oxidases known so far. Of course, there have been no known nucleoside oxidases that catalyze the reactions represented by formulas (1) and (2).

Furthermore, the nucleoside oxidase YT-1 of the present invention is
As will be explained in detail later, the enzyme has the unique property of exhibiting laccase-like activity depending on the amount of the nucleoside at the same time as the oxidation reaction of the nucleoside represented by the above formula; It is completely different from nucleoside oxidase. The analytical method of the present invention utilizes the laccase-like activity specific to the enzyme to determine the degree of color development of a color reagent, for example, by determining the absorbance.
Such an analysis cannot be performed using conventional nucleoside oxidases.

Hereinafter, the method for producing the enzyme of the present invention, its properties, and the analytical method of the present invention using the same will be sequentially explained.

The enzyme of the present invention can be produced using the enzyme-producing bacterium belonging to the genus Pseudomonas. Examples of the enzyme-producing bacteria used here include a strain newly isolated by the present inventors and having the following properties.

■, Mycological properties (A) Morphological properties ■ The cells are bacilli, and the size is 0. 5X1. 5~
It is 1.7 μm.

■ It is motile and the flagella are extremely flagella.

■ Spores are not formed.

■ Negative in Durham staining.

(B) Culture findings■ Growth on broth agar plate is moderate, colonies are circular, and the surface is smooth and yellow.

■ Meat juice agar slant culture The growth is moderate and the surface is smooth and yellow.

■ Meat, juice liquid culture It grows on the surface and its growth is moderate.

■ Meat juice gelatin puncture culture grows on the surface and liquefies gelatin (Strati
form).

(C) Physiological properties ■ Nitrate reduction Negative ■ Denitrification reaction Negative ■ Indole formation Negative ■ Hydrogen sulfide formation Negative ■ Starch hydrolysis Negative ■ Nitrate utilization Negative ■ Ammonium salt utilization Positive ■ Pigment production Fluorescent dye , biocyanin, and xanthomonadin are not produced. Broth agar may produce a brown, diffusible pigment.

■ Urease negative [strain] Oxidase negative to four-sided ■ Catalase positive ■ Growth range: Does not grow at pH 4 or 5. Does not grow at 4°C and 41°C. The appropriate growth pH is between 5 and 8, and the temperature is 35.
°C is optimal.

0 Attitude towards oxygen Aerobic ■ 0-F test (Hugh Leifson method) Oxidative ■ Production of acid from sugars - glucose Positive D-fructose Positive D-maltose Positive D-galactose Positive D-xylose Positive D-mannitol Negative sucrose Negative Lactose Positive @ Aesculin degradation Positive 0 A, Luginine dihydrase Negative @ Lysine decarboxylase Positive 0 Ornithine decarboxylase
Negative [phase] Phenylalanine deaminase negative■
Egg yolk reaction negative ■ Tween 20
Decomposition of positive ■ Decomposition of Tween 80
Positive [phase] Accumulation of polybator hydroxybutyrate Negative [phase] Requires auxotrophic methionine or cystine.

Based on the above properties, we searched for this strain by purging (Bcr
gcy's Manual of Systemati
According to C Bacteriology (1984)), this strain is: ■ Gram-negative rod and does not form spores; ■ It is catalase positive; ■ It moves by ultraflagella; and ■ It does not oxidize glucose. The Pseudomonas genus (P.
It was recognized that it belongs to the genus seudomonas). Additionally, ■ requires cystine or methionine for growth, ■
Negative for arginine dihydrolase; ■Does not accumulate polybata hydroxybutyrate; ■Colonies are yellow; ■Does not produce xanthomonadin;
■ Pseudomonas maltophilia (Pseudomonas maltophilia)
tophilia).

Based on the above results, the present inventors identified this strain as a strain of Pseudomonas maltophilia, and it is known as Pseudomonas maltophilia LB-86 (Pseudomonas maltophilia).
As maltophlla LB-88).

The strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with the above indication, and was approved by the Ministry of International Trade and Industry as a certified microorganism strain No. 9813 (FERM P).
-9813).

The nucleoside oxidase YT-1 of the present invention comprises the above-mentioned L
It can be produced by culturing microorganisms that belong to the genus Pseudomonas, including the B-86 strain, its mutants, etc., and have the ability to produce the above-mentioned nucleoside oxidase YT-1.

Cultivation of the above microorganisms can be carried out in a suitable nutrient-containing medium. As this medium, any synthetic medium, semi-synthetic medium, or natural medium containing various carbon sources, nitrogen sources, inorganic substances, etc. that are commonly used for culturing microorganisms belonging to the genus Pseudomonas can be used. Specifically, the above-mentioned carbon sources include various carbon compounds that can be assimilated by microorganisms, such as higher fatty acids, starch decomposition products (dextrin), maltose, lactose, glucose, molasses, and fructose, used alone or in combination of two or more. It will be done. Examples of the nitrogen source include peptone, meat extract, yeast extract, soybean flour, cottonseed flour, and corn steep liquor.

As the inorganic substance, for example, in addition to common salt, inorganic salts such as phosphates and sulfates of inorganic metals such as potassium, sodium, magnesium, calcium, zinc, and iron may be used as appropriate.

The above-mentioned microorganisms can be cultured by liquid culture or solid culture, and is preferably carried out aerobically under aeration and agitation conditions. Culture conditions such as pH of the medium, culture temperature, culture time, etc. are preferably selected from conditions that are suitable for the growth of the microorganism to be cultured and that produce as much of the desired enzyme as possible.

For example, the pH of the medium is in the range of about 6 to 8, and the culture temperature is about 20.
Preferably, the temperature is selected from the range of ~35°C. The culture time may be selected so that the production and accumulation of the desired enzyme reaches its maximum, and is usually selected from a range of about 12 to 48 hours. Of course, each culture condition, culture method, etc. described above can be changed as appropriate depending on the type of microorganism used, external conditions for culture, etc.

The culture thus obtained usually contains, in its cell division,
The target enzyme is accumulated. The collection and purification of this enzyme can be carried out in accordance with various conventional methods conventionally employed for producing enzyme 2 and the like using microorganisms. Examples of the above-mentioned purification means include means that utilize solubility in a solvent, means that utilizes a difference in ionic bonding force, means that utilizes a difference in molecular world, means that utilizes a difference in isoelectric point,
Means utilizing differences in hydrophobicity, etc. can be employed alone or in appropriate combinations.

In detail, the collection of the target enzyme from the above bacterial cell divisions is as follows.
This involves suspending wet bacterial cells obtained by collecting them using a conventional method such as centrifugation in a phosphate buffer, Tris buffer, etc.
This can be carried out by appropriately combining various bacterial cell treatment methods such as ultrasonication, French press, and lysozyme treatment, and then a crude enzyme-containing liquid can be obtained.

The above-mentioned crude enzyme-containing solution can be further purified by a conventional method to obtain a purified enzyme preparation.

As a means for this purification, for example, a precipitate is first collected from the crude enzyme-containing solution by a fractional precipitation method using an organic solvent such as ethanol or acetone, or a salting-out method using ammonium sulfate, aluminum sulfate, etc., and then the precipitate is This can be carried out by subjecting it to chromatography using an ion exchanger such as diethylaminoethyl dextran or a gel slipping agent such as polyacrylamide gel, and the purified enzyme preparation thus obtained can be further purified into a purified powder preparation by freeze-drying or other operations. It is also possible to do this.

The enzyme of the present invention obtained as described above has the above formula (
In addition to being characterized by having an enzymatic action that catalyzes the oxidation reactions shown in 1) and (2), it is also characterized by having the following physicochemical properties.

(1) Substrate specificity: Acts on various nucleosides such as inosine. It does not act on bases such as hypoxanthine, nucleotides such as inosinic acid, ribose, etc.

(2) Temperature and pH stability: pH 6, 0, 95% or more remained in 15 minutes at 60°C, 70%
It is inactivated in 15 minutes at ℃.

Further, when treated at 37° C. for 60 minutes, 95% or more remains at pH 5.0 to 6.0.

(3) Optimum pH: pH 5.0 to 6.0.

(4) Molecular weight: approximately 130,000.

(by gel filtration method) (5) Isoelectric point: pH 5.3.

(6) Effect of inhibitors Inhibited by potassium dianate and sodium azide.

(7) Visible absorption There is no absorption maximum at 450 nm or above in a neutral buffer such as diphosphate buffer. Oxidized enzymes that have not been reduced by the substrate, dinitzion (sodium hydrosulfide), etc., do not have an absorption maximum at 450 nm or more.

(8) Metal content: Contains iron.

The above-mentioned physicochemical properties and other properties will be described in detail in Examples below.

The enzyme nucleoside oxidase YT-1 of the present invention having the above properties can be used for quantitative determination of nucleosides in the same manner as conventional nucleoside oxidases. That is,
Since the enzyme of the present invention oxidizes nucleosides using molecular oxygen as an electron acceptor, it can be used to quantify nucleosides by measuring oxygen consumption. According to this method, by using the enzyme of the present invention, nucleosides can be measured in a reaction system that uses nucleosides as a substrate or a reaction system that produces nucleosides,
This makes it possible to measure the activity of various enzymes, such as enzymes that catalyze reactions that produce nucleosides, such as 5'-nucleotidase. Furthermore, for example, by measuring the amount of nucleosides in an extract of seafood, the freshness of the seafood can be determined, and it is also useful, for example, in the production of nucleoside-5'-carboxylic acid.

In particular, the enzyme of the present invention has a unique property of exhibiting a laccase reaction depending on the amount of nucleoside in the presence of the nucleoside simultaneously with the oxidation reaction of the nucleoside. Moreover, this laccase-like activity is completely proportional to the amount of nucleoside, and therefore, by utilizing this property, it is possible to oxidize typical laccase substrates such as hydroquinone, as well as Polyporus versicolor.
Like the original laccase, it can generate pigments such as quinoneimine by oxidatively coupling 4-aminoantipyrine with phenol or aniline-based substances, and the amount of nucleosides can be determined by measuring the amount of this pigment produced. be able to. Furthermore, by using the enzyme of the present invention, the initial rate of the enzymatic reaction that liberates nucleosides can be accurately and simply determined spectrophotometrically.

The present invention also provides a new method for assaying a test liquid that utilizes the laccase activity specific to the enzyme of the present invention.

In detail, this assay method uses a system containing or producing nucleosides as a test solution,
It is carried out by applying the nucleoside oxidase YT-1 of the present invention to a mixture of a coloring reagent that develops color through oxidation and the above test liquid, and measuring the degree of color development of the above coloring reagent that is proportional to the change in the amount of nucleosides in the test liquid. Ru.

The test solution used in the above may be any system as long as it contains nucleosides or is capable of releasing or producing nucleosides (nucleoside production, free enzyme reaction system). Representative examples of the test liquid include the following canned liquids.

(1) Sample solution for measuring enzyme activity in which 5'-nucleotides such as 5'-IMP and 5'-AMP are exposed to 5'-nucleotidase to release nucleosides such as inosine and adenosine (2) 3'-IMP Sample solution for measuring enzyme activity (3) in which 3'-nucleotidase acts on 3'-nucleotides such as 3'-AMP to release nucleosides such as inosine and adenosine (3) Contains nucleosides produced by decomposition of biological components For example, a sample solution for measuring the freshness of fresh fish, meat, etc. (4) Activity measurement of ribonuclease or deoxyribonuclease in which ribonuclease or deoxyribonuclease is applied to RNA5DNA, oligonucleotides, etc., and alkaline phosphatase or the like is further applied to the resulting nucleotides to release nucleosides. (5) Reagent sample solution of nucleosides such as adenosine, deoxyuridine, xanthosine, thymidine, uridine, guanosine, cytidine, deoxyinosine, etc. (6) For measuring the activity of enzymes such as inosinase that use nucleosides as substrates and hydrolyze them. Sample liquid.

Among the above, the sample solution for enzyme activity measurement described in (1), that is, the nucleoside release enzyme reaction system, can be specifically exemplified by a system containing human serum and nucleotides.
When the assay method of the present invention is applied to this system, the 5'-nucleotidase activity in the human serum is measured, which is very useful as a liver function testing method. Conventionally, methods for measuring the activity of 5'-nucleotidase in human serum include methods that measure inorganic phosphorus released using 5'-AMP as a substrate, and enzymatic methods that utilize adenosine deaminase and glutamate dehydrogenase. However, the above methods for measuring inorganic phosphorus have drawbacks such as long reaction times, complicated operations, and the need to correct coexisting alkaline phosphatase activity.Also, even the enzymatic method described above has disadvantages in that the measurement system is However, the assay method of the present invention is very simple to operate, has higher sensitivity than conventional methods, and usually has a reaction time of about 20 minutes. The 5'-nucleotidase activity in target human serum can be measured in a very short time of ~5 minutes.

In addition, the coloring reagents that develop color through oxidation used in the assay method of the present invention include reagents that are oxidized with a single compound and exhibit absorption in the visible region, and reagents that are oxidized with a combination of two or more compounds and condensed to produce an absorption in the visible region. Included are reagents that exhibit absorption. Examples of the above single compounds include various laccase substrates, such as 0-tolidine, o-toluidine, 0-dianisidine, 1O-N-methylcarbamoyl-3,7-dimethylamino-1O-H-phenothiazine, bis[3-bis( Examples include aniline-based substances such as 4-chlorophenyl)-methyl-4-dimethylaminophenyl]amine.

In addition, as a combination of two or more of the above-mentioned compounds, a combination of a so-called coupler and a Trinder reagent (hydrogen donor), as typified by 4-aminoantipyrine and phenol, which have been widely used as clinical diagnostic agents, may be used. can be mentioned. Specific examples of the above couplers include 4-
In addition to aminoantipyrine (4-AA), 2゜6-dibromoaminophenol, 3-methyl-benzothiazolinone hydrazone, etc., and as specific examples of Trinder reagent (hydrogen donor), in addition to phenol, β- Chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, N,N-dimethylaniline (DMA), N
-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-aningine (ADO3), N-ethyl-N
-(2-hydroxy-3-sulfopropyl)-aniline (ALO8), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOO8)
), N-ethyl-N-sulfopropyl)-m-anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline・Sodium salt (DAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAO3), N-sulfoprobyl-3,5-dimethoxyaniline ( HDAPS),
N-(2-hydroxy-3-sulfonropyl)-3,
5-dimethoxyaniline (HDAO8), N-ethyl-N-(2-hydroxy-3-sulfoprovir)-3
, 5-dimethylaniline (MAO3), N-ethyl-
N-sulfopropyl-3,5-dimethylaniline (M
APS), N-ethyl-N-sulfoprobyl-m-
Toluidine (TOPS), N2-ethyl-N2- (
Examples include 3-methylphenyl)-N'-acetylethylenediamine (EMAE).

According to the assay method of the present invention, first, the enzyme of the present invention is allowed to act on a mixture of the above-mentioned test liquid and a coloring reagent. This is usually done by adding a coloring reagent to a test solution and then adding the enzyme of the present invention, or adding the test solution to a solution in which the enzyme of the present invention is added to a coloring reagent.
This is then carried out by leaving the mixture at a predetermined temperature for a predetermined period of time, and color development of the coloring reagent is observed due to the enzymatic reaction. In the above, the test liquid can be appropriately diluted and used. Examples of the diluent used here include various buffer solutions such as phosphate buffer and Tris-HCl buffer, water, and the like. Further, the amount of each reagent used in the enzyme reaction system is arbitrarily determined and is not particularly limited, but the concentration of coloring reagents in the reaction system is usually about 0.1 to 1.
The amount of the enzyme of the present invention is approximately 0.05 to 10 units per reaction solution, and the nucleoside concentration of the test solution is approximately 0 to 0.2 mM in the reaction solution. It is preferable to use each in an amount within the following concentration range. In addition, the reaction conditions of the enzyme reaction system are not particularly limited, but are usually about 20 to 40°C.
The reaction time can be appropriately determined depending on the amount of the enzyme of the present invention used, the reaction temperature, pH, etc., and is generally very short, and the reaction is completed within about 20 minutes. Furthermore, if the pH of the enzyme reaction system is within the range of approximately 3 to 10, the reaction will proceed satisfactorily;
A range of approximately 4 to 8 is preferred.

In the method of the present invention, the change in the amount of nucleosides in the test solution can then be measured by measuring the degree of color development of the enzyme reaction mixture. The degree of color development can be measured by spectroscopically measuring the absorption in the visible region of a substance that exhibits absorption in the visible region, such as a dye produced by an enzyme reaction, according to a conventional method. For example, in a system that produces a quinoneimine dye from 4-aminoantipyrine and phenol, the degree of color development can be measured by measuring the absorbance at 500 nm using a spectrophotometer. Representative examples of other systems and actual examples of absorbance measurement in each system will be described in detail in Examples below.

Furthermore, in the above analysis method of the present invention, when an enzyme reaction system that liberates nucleosides, such as a solution containing a nucleotidase, is used as the test solution, the reaction using the enzyme of the present invention is carried out in a cell of a spectrophotometer, Enzyme activity such as the above-mentioned nucleotidase can also be measured by determining the increase in absorbance per unit time.

As described above, the present invention provides a new analytical method that utilizes the enzyme of the present invention and is extremely effective for quantifying nucleosides and measuring the activity of various enzymes in systems that generate and release nucleosides.

EXAMPLES Examples will be given below to explain the present invention in more detail.

In addition, nucleoside oxidase activity was measured by the following method.

<Nucleoside oxidase activity measurement method> 1ml of 100mM phosphate buffer (pH 6.0) containing 7mM inosine
2 into the cell of the enzyme electrode, add 5μQ of the enzyme preparation,
The initial rate of enzyme consumption is determined at 25°C. That is, 2
The decrease in the amount of dissolved oxygen from 250 μM in terms of saturated dissolved oxygen at 5°C is measured over time using an oxygen electrode, and the consumption of 1 μmol of oxygen per minute is 1 unit (U).
).

Example 1 Pseudomonas maltophilia LB-86 (Pseu
domonas maltophila LB-86
, Kaikoken Bacteria Serial No. 9813) was inoculated into a 500-capacity Sakalo flask containing 100 drops of bouillon medium (1% meat extract, 1% peptone, and 5% NaCQO, the same hereinafter). A seed culture is obtained by culturing with shaking (110 rpm) at ℃ for 12 hours.

The obtained seed culture was inoculated (inoculated) into a 6Q bouillon medium that had been previously sterilized in a jar fermenter with a capacity of 10 f2 and 0.07% of the antifoam agent "Adekanol" (manufactured by Asahi Denka Kogyo Co., Ltd.) was added. n: 2%). Culture temperature 25
°C, stirring 25 rpm.

Cultured for 12 hours under aeration conditions of m6Q/min, and after completion of culture,
The bacteria were collected, suspended in IQ phosphate buffer (pH 6,0), and subjected to ultrasonication at 20 KHz and 200 W for 15 minutes. In this way, a bacterial cell extract is obtained.

The nucleoside oxidase activity of this product is 1.2U
/ It was a precept.

Example 2 Pseudomonas maltophilia LB-86 (Feikoken Bacteria No. 9813) was inoculated into a Sakalo flask containing 100 or 500 volumes of broth medium, and incubated at 25°C for 12 hours.
A seed culture is obtained by shaking culture (110 rpm) for hours.

The obtained seed culture was placed in a pre-sterilized 100% yeast extract/glucose medium (yeast extract 2.5%, glucose 3%, KCQo,
1%, KH2PO40, 1%, MgSO4・7H200
, 05%, pH 7.2) (inoculation amount: 2%).

Culture was carried out for 24 hours at a culture temperature of 25°C and stirring at 110 rpm. After the culture was completed, the bacteria were collected, and the cells were suspended in a phosphate buffer solution (pH 7,0) at a concentration of 30 per flask. Ultrasonication is carried out under the same conditions as in Example 1. In this way, a bacterial cell extract is obtained.

The nucleoside oxidase activity of this product is 7, OU
/ It was a precept.

Example 3 272Ttl of bacterial cell extract obtained in the same manner as in Example 2
After adding 20% streptomycin sulfate 60% to Q and removing the resulting precipitate by centrifugation, ammonium sulfate is added to give 35% saturation. After removing the formed precipitate, ammonium sulfate is further added to 60% saturation. After leaving in a cold place overnight to form a precipitate, the precipitate is collected and dissolved in 20 mM phosphate buffer (pH 6,0). This was placed in a dialysis tube and dialyzed for - days, then preliminarily prepared with 20mM phosphate buffer (
DEAE-Toyovar 650 equilibrated at pH 6,0)
The column was applied to an M column (manufactured by Toso Corporation), washed thoroughly with the same buffer, and then eluted with a linear gradient of 0 to 0.5M sodium chloride.

The active fraction was concentrated using an Amicon membrane (manufactured by Amicon) with a fractionation molecule ff120000, and was further equilibrated with 20 mM phosphate buffer (pH 6,0) using Sephacryl S-20.
0 column (manufactured by Pharmacia) and perform gel filtration. The eluted active fractions are collected, concentrated again using an Amicon membrane with fractionation molecules ff120000, and gel filtration using Sephacryl S-200 is repeated.

The active fractions eluted above are collected and freeze-dried to obtain purified nucleondooxidase YT-1.

This purified product was disk electrophoretically single.

The results of the above 1 and purification are summarized in Table 1 below.

Below in Table 1, a test example of the properties of the purified nucleoside oxidase YT-1 sample of the present invention obtained in Example 3 will be described in detail.

(1) Enzyme action test I ■ Detection of oxidation reaction products 10 U of the standard enzyme was dissolved in an appropriate amount of water, placed in a dialysis tube, and placed in 100 mQ of 7 mM inosine solution for reaction.

A portion of the reaction solution was collected at appropriate times between 0 minutes and 800 minutes of reaction, and subjected to high performance liquid chromatography (HPLC).
) was analyzed. The HPLC is fine SIL018 (
Using a F1neSILcm8 (manufactured by JASCO Corporation) column,
50mM phosphate buffer (pH 3,0)/methanol=9515 was used as the solvent, and the flow rate was 1. Absorbance detection was performed at 260 nm at 5 mQ/min.

The results at a given time are shown in FIG.

In the figure, the X axis shows retention time (minutes), the y axis shows reaction time (incubation time, minutes), and the Z axis shows absorbance at 260 nm.

The following can be seen from Figure 1. That is, inosine decreases over time, resulting in two peaks (P-1 and P-2).
) appeared, and p-1 increased at the beginning of the reaction, then gradually decreased and disappeared, and finally only P-2 remained.

When the above P-1 was separated by HPLC and reacted with a nucleoside oxidase enzyme preparation, it was confirmed that it changed to P-2 with the consumption of oxygen. On the other hand, P-2 did not change even when subjected to the reaction with the above enzyme preparation.

From this result, the enzyme preparation of the present invention oxidizes inosine and P-
It was found that it has the effect of producing P-1 and further oxidizing P-1 to produce P-2.

■Identification of reaction products■-I Preparation of P-1 and P-2 Add 100U of this enzyme preparation in a dialysis tube to an inosine aqueous solution (2mg/mQ) 100N12, and heat at 37°C without aeration and stirring. The reaction was allowed to proceed overnight to produce p-2. After removing the dialysis tube, hydrochloric acid was added to the reaction solution to adjust the pH to 3.0, and the resulting p-2 crystals were collected by centrifugation. This was washed with a hydrochloric acid solution of pH 3.0 and dried under reduced pressure to obtain crystalline 190B of P-2.

In addition, put 10 U of this enzyme preparation into a dialysis tube, and
In the same manner as in the preparation of 2, it was added to an aqueous inosine solution and reacted. The reaction product was collected over time and analyzed by HPLC,
At the time when the amount of P-1 accumulated increased (37°C, about 30 minutes),
The dialysis tube containing the enzyme was removed to stop the reaction. After concentrating this reaction solution under reduced pressure, the p-1 fraction was separated by HPLC and purified by freeze-drying to obtain P-1 powder.

■-2 Analysis of base components and sugars of P-1 and P-2 P-1
, P-2 and inosine in IN hydrochloric acid at 100°C.
, hydrolyzed for 1 hour. The bases and sugars contained in each decomposition solution were analyzed.

As a result, all base components exhibited the same behavior as the standard hypoxanthine product in thin layer chromatography and HPLC. From this, the base components of P-1 and P-2 were both identified as hypoxanthine, which is the same as inosine.

In addition, in the sugar analysis by thin layer chromatography mentioned above, D-ribose was detected from the inosine decomposition solution, but a spot was detected at a different position from D-ribose from the P-1 and P-2 decomposition solutions. It was done. From this, P-
It became clear that the sugar moiety of inosine in 1 and P-2 was oxidized.

(1)-3Identification of P-1 P-1 was reduced with 1.2 equivalents of sodium borohydride. When this reduced product was analyzed by thin layer chromatography and HPLC, its behavior was consistent with that of a standard product of inosine. Therefore, the reduction product of p-1 with sodium borohydride was identified as inosine. Therefore, inosine-5'-aldehyde is the only structure that can be reduced by sodium borohydride and returned to the substrate inosine. This was also supported by the nuclear magnetic resonance and infrared spectra of P-1. From the above, P-1 was identified as inosine-5'-aldehyde.

■-4Identification of P-2 C1C13-N spectrum and Hl of crystal specimen of P-2
-NMR spectra were measured.

C1C13-N is completely coupled and decoupled, Hl-NMR is dimethyl sulfoxide (DM
This was done by measuring the spectrum in SO-ds) and the spectrum after adding heavy water (D20).

As a result, all signals could be assigned as shown in Table 2 below based on their chemical shifts, binding constants, etc., and p-2 could be identified as inosine-5'-carboxylic acid.

Table 2 H HOH However, in the table, S indicates a singlet and d indicates a doublet.

(2) Reaction stoichiometry IU of this enzyme preparation was added to 100 mM phosphate buffer containing 50 nmoles of inosine, and the amount of oxygen consumed, amount of P-2 produced, and amount of hydrogen peroxide were measured.

As a result, the oxygen consumption for 50 nmoles of inosine was 5
47 nmoles of P-2 were produced in 1 nmole.

Furthermore, no hydrogen peroxide was detected.

Next, IU of this enzyme preparation was added to 100 mM phosphate buffer containing 100 nmoles of Pl, and oxygen consumption ffl,
P-2 production 0 and hydrogen peroxide amount were measured.

As a result, 97 nmoles of P-2 were produced at an oxygen consumption of 48 nmoles in 100 nmoles P-1. In this case too, no hydrogen peroxide was detected.

Further, in the above reaction, no difference was observed in the amount of oxygen consumed even when IU of catalase was added, and from this point as well, it was revealed that hydrogen peroxide was not generated in this reaction.

From the above results, the enzyme of the present invention shows that 1 mole of inosine is 1 mole of inosine.
Oxidize 72 moles of oxygen as an electron acceptor to produce 1 mole of inosine-5'-aldehyde and 1 mole of water;
Furthermore, 1 mole of inosine-5' aldehyde is oxidized using 1/2 mole of oxygen as an electron acceptor to form 1 mole of inosine-5'aldehyde.
It was revealed that 5'-carboxylic acid was produced.

(2) Enzyme action test ■ Identification of reaction products when 1,4-hydroquinone is used as a substrate and stoichiometry of 100 mM phosphoric acid containing 0.5 μmol of inosine and 0.2 μmol of 1°4-hydroquinone Buffer solution (pH 7,
IU of this enzyme preparation was added to 0)1 reaction, reacted for 10 minutes at room temperature, and the reaction product was analyzed by HPLC.

As a result, the elution position coincided with that of the p-quinone standard product.

Furthermore, their ultraviolet spectra also matched.

Therefore, the reaction product was identified as p-quinone.

In addition, IU of this enzyme preparation was added to 100 mM phosphate buffer (pH 7.0) 1+1112 containing 0.06 μmol of inosine and 0.12 μmol of 1,4-hydroquinone, and reacted for 10 minutes at room temperature. The oxygen consumption was measured using an oxygen electrode.

As a result, the oxygen consumption was 0.115 μmol, of which 0.055 μmol was obtained by subtracting 0.06 μmol of oxygen used for the oxidation of inosine, which was 0.12 μmol of 1,
This means that it was used for the oxidation of 4-hydroquinone.

Therefore, this enzyme oxidizes 1 mole of 1,4-hydroquinone with 1/2 mole of oxygen to produce 1 mole of p-quinone and 1 mole of p-quinone.
It was found to produce mol of water. This means that the conventionally known laccase (Laccase EC1, l)
This conforms to the reaction of OJ, 2).

(2) Relationship between nucleoside oxidation and laccase action No reaction occurs when this enzyme is mixed with a laccase substrate such as 1,4-hydroquinone. However, when nucleosides such as inosine and adenosine are coexisting in the above reaction system, a laccase reaction occurs. That is, the laccase activity of this enzyme requires the presence of nucleosides, and this fact was confirmed by the following test.

0.06 μmol inosine and 0.02.0.06.0
．． 100 mM phosphate buffer (pH 7,0) containing either 12 or 0.6 μmol of 1,4-hydroquinone
+nQ was treated with IU of this enzyme. The results of examining the oxygen consumption and p-quinone production at this time are shown in Table 3 below.

Table 3 From Table 3 above, this enzyme converts 1 mole of nucleoside into 1
It is confirmed that when a mole of oxygen is oxidized as an electron acceptor, simultaneously and separately from the oxidation of the nucleoside, a laccase reaction is carried out using 1 mole of oxygen as an electron acceptor.

■ Effect on phenol and 4-aminoantipyrine This enzyme causes an oxidative coupling reaction between phenol and 4-aminoantipyrine in the presence of nucleosides, similar to conventionally known laccases (derived from Polyporus persicolor, etc.). to produce a red quinoneimine dye. The red pigment produced at this time is different from the quinone imine pigment produced by the conventional oxidation reaction of phenol-4-aminoantipyrine with peroxidase in the presence of hydrogen peroxide in its ultraviolet and visible absorption spectra and elution position by HPLC. It is confirmed that they are the same.

The relationship between the production of quinoneimine dye (degree of color development) by the enzyme of the present invention and the amount of nucleoside was investigated as follows. That is, inosine was used as the nucleoside, and 10 m
100 containing M phenol and 4-aminoantipyrine
Add a prescribed concentration of mM phosphate buffer (pH 7,0) to BrrIQ, add IU of this enzyme to the solution, and react.
Color development was performed. The results are shown in FIG.

In the figure, the horizontal axis shows the inosine concentration (nanomole), and the vertical axis shows the absorbance at 500 nm.

From FIG. 2, a linear relationship is established between nucleoside synthesis and color development, and it is clear from this that the present enzyme can be used in colorimetric dials of nucleosides by utilizing the above reaction.

(3) Substrate specificity test Using the various compounds shown in Table 4 below as substrates, the relative activity of this enzyme against these (the activity against inosine is 1
00) was measured.

The results are also listed in Table 4.

Table 4 The absorption spectra of the sample liquid before and after were determined.

The results are shown in Table 5 below.

From Table 5, it is clear that it does not act on nucleobases or sugars alone, nor does it act on nucleotides.

It is also understood that the sugar moiety of the nucleoside on which this enzyme acts may be either ribose or deoxyribose, and the base moiety may also be of various types.

(4) Specificity test for laccase substrate 7m of this enzyme
The substrate specificity of laccase-like activity in the presence of M-inosine was investigated as follows. That is, 1 unit of this enzyme was added to a sample solution containing 7 mM of inosine and 1 mM of various laccase substrates shown in Table 5 below, and left for 5 minutes. It is found to act on various laccase substrates.

In addition to inosine, the expression of the above-mentioned laccase-like activity also requires
It has been confirmed that any nucleoside can be used as long as it is a substrate for various nucleoside oxidases as shown in Table 4.

(5) Temperature and pH test ■ Temperature stability: This enzyme was dissolved in phosphate buffer (pH 6,0) for 100 min.
After being treated for 5 minutes, the residual activity was measured.

The results are shown in Figure 3 (horizontal axis = temperature, vertical axis = residual activity), and it is clear that this enzyme is stable at 60°C for 15 minutes.

■ pH stability: This enzyme was treated at 37°C for 60 minutes at each pH,
Thereafter, residual activity was measured at pH 6.0.

The results are shown in FIG. 4 (horizontal axis=pH, vertical axis=residual activity), and it can be seen that this enzyme is stable at pH 5 to 6.

■ Optimal pH: 11 as shown in Figure 5 (horizontal axis = pH, vertical axis = relative activity)
The optimum pH of this enzyme was 5-6. In addition, in Figure 5 (1
) indicates acetate buffer (μ=0.05), and (2) indicates phosphate buffer (μ=0.05).

■Optimal temperature: The accuracy of the oxygen electrode used to measure the activity of this enzyme decreases at temperatures above 40°C, making accurate measurements impossible.

In the temperature range up to 40°C, the highest activity was shown at 40°C.

(6) Molecular weight measurement test Sephacryl S-200 (manufactured by Pharmacia) column (
Diameter 2. The molecular weight of this enzyme was measured by the gel clearing method using 5c+nX length 90 cm).

As a result, the molecular weight of this enzyme was determined to be 130,000.

(7) Isoelectric point test Using carrier amphorite (pH 3.5-10.5), cooling (4°
C) Electrophoresis was performed for 48 hours to determine the isoelectric point of the enzyme.

The isoelectric point of the obtained enzyme was pH 5.3.

(8) Inhibitor effect test This enzyme solution (0.02U) was tested in the presence of various inhibitors.
H6, 0, 25℃, after 10 minutes of inosinini action,
Activity was measured.

The results are shown in Table 6 below.

From Table 6 above, it can be seen that this enzyme is inhibited by potassium cyanide, sodium azide, N-bromosuccinimide, and the like.

(9) Ultraviolet and visible absorption spectral analysis A spectral analysis of the enzyme in 100 mM phosphate buffer (pH 6,0) was performed.

The results are shown in FIG.

(10) Metal content As a result of measurement by atomic absorption spectrometry, it was confirmed that this enzyme contained iron, but molybdenum, copper, manganese, and zinc were not detected.

(11) Effect of metal ions on activity This enzyme solution (0
,02U) in the presence of various metal ions at pH 6.0,
After acting on inosine at 25° C. for 10 minutes, the activity was measured to examine the effects of various metal ions on the enzyme activity.

The results are shown in Table 7 below.

Table 7 (12) Km value As a result of determining the apparent Km value for inosine, the Km
The value was 4.4×10 −5 M.

(13) Amino acid composition This enzyme was prepared under reduced pressure using 6N hydrochloric acid at 24.48 or 72
After hydrolysis for a period of time and sufficient removal of hydrochloric acid, 0.09M
It was dissolved in a citrate buffer (pH 2.2), and the amino acids in this solution were analyzed using JLC200, Type A automatic amino acid analyzer manufactured by JEOL Ltd.

The content of tryptophan (Trp) is 4.2N NaOH
After hydrolysis at 110° C. for 24 hours, measurements were made in the same manner. Cystine (Cys) was quantified by oxidizing the enzyme with performic acid and then decomposing it with hydrochloric acid [S, Moore
, J. Biol.Chem., 238. 235
(1963)).

The results are shown in Table 8 below.

Table 8 (14) Disk electrophoresis using 7% polyacrylamide gel (pH 9,5) using the method of Davis, J. B., Davis, Ann, N.
,Y.

Acad, Sci., 121, 404 (198
4) Two disk electrophoresis cycles of this enzyme were performed according to (4)). One part was subjected to protein staining, and the other part was cut into sections of 1/1 m to measure the activity, and the electrophoretic position of each was investigated.

As a result, the enzyme protein showed a single band, and its migration position coincided with the active migration position.

Examples of the analytical method of the present invention using the enzyme of the present invention will be shown below. In each of the following cases, the enzyme used in Example 3 is
The purified nucleoside oxidase YT-1 sample obtained in .

Example 4 4-aminoantipyrine 23.4 mg 5% phenol 2mf25QmM
HEPES buffer 98 Decomposition (pH 7,5) Total 100 Peng Coloring solution with the above composition 2.0 Peng and nucleoside oxidase 10
μΩ (0.35 U) was added, and then 0.5 μΩ of test solution containing adenosine at various concentrations was added. After adding 31TII2 and reacting the mixture at 30° C. for 20 minutes, the absorbance at 500 nm was measured.

The results are shown in FIG. In the figure, the vertical axis represents the absorbance, and the horizontal axis represents the adenosine concentration of the test solution (mmol).
shows.

From FIG. 7, it can be seen that adenosine in the test liquid can be easily and favorably quantified by absorbance measurement.

Example 5 4-aminoantipyrine 2B,4DN-ethyl-N=(2-hydroxy-3-sulfopropyl)-3°5-dimethylaniline 178.7mg50m
M phosphate buffer - (pH 6,0) 100 mQ meter 100 Coloring solution with the above composition 2. 10 μQ (0.35 U) of nucleoside oxidase was added to the omc, and then 0.0 μQ (0.35 U) of nucleoside oxidase was added to the test solution containing various concentrations of inosine, guanosine, and deoxyadenosine. After adding 3mf2 and reacting the mixture at 30°C for 20 minutes, the absorbance at 630r+m was measured.

The results are shown in the same manner as in Fig. 7, and Fig. 8 (inosine measurement results), Fig. 9 (guanosine measurement results), and Fig. 10 (
Deoxyadenosine measurement results).

It can be seen from the 2nd grade figures that the amount of each nucleoside in the test liquid can be easily and favorably quantified by absorbance measurement.

Example 6 4-aminoantipyrine 23.4 mg N-ethyl-N-(2-hydroxy-3-sulfonropyl) -m-toluidine 171.3 mg φ2 sodium glycerol-2-phosphate 1.76 g MnS
O4-4~6H2019, 4mg50mM HEPE
S buffer (pH 7,5) 100 or total
100 precepts Add 10 μQ of nucleoside oxidase to coloring solution 2.0 with the above composition.
(0.35 U), and then a test solution containing various concentrations of 5'-nucleotidase (manufactured by Sigma, 5'-ND
Control E) Add 0.1 ml, then substrate 11
．． 5mM 5'-adenylic acid (5'-AMP) solution 0.2
5550I per unit time using a spectrophotometer.
The increase in absorbance of 11 was measured for about 5 minutes.

The results are shown in FIG. In the figure, the vertical axis shows the increase in absorbance per minute, and the horizontal axis shows the 5'-nucleotidase concentration (U/Q).

From FIG. 11, it can be seen that the amount of nucleotidase in the test liquid can be easily and favorably quantified by absorbance measurement.

Example 7 4-aminoantipyrine 23.4 mg 5%,
Phenol 2 or MnSO4・
4-6H2019, 4mg 50mM HEPES buffer (pH 7,5) 98 11IQ meter 100 Coloring solution with the above composition 2. 10 μQ (0.35 U) of nucleoside oxidase was added to o+nc, and then a test solution containing various concentrations of 3'-nucleotidase (manufactured by Sigma) was added.
．． Add 1 mO, then 11.5mM 3' substrate
-Adenylic acid (3' = AMP) solution 0.211Q was added, and the increase in absorbance at 505 nm per unit time was measured for about 5 minutes using a spectrophotometer.

The results are shown in FIG. 12, similar to FIG. 11.

The figure shows that the amount of nucleotidase in the test solution can be easily, quickly, and satisfactorily quantified by absorbance measurement.

Example 8 To a coloring solution having the same composition as the coloring solution shown in Example 6, 10 μQ (0.35 U) of nucleoside oxidase and 0.0 μl of human serum were added. Add 1 mQ, then 5'-AMP(11,5
mM) 0.2TnQ was added and 555n was added using a spectrophotometer.
57 in human serum by measuring the increase in absorbance of m.
- Nucleotidase activity was measured.

The above measurement was repeated 15 times using the same serum sample, and the results are shown in Table 9 below.

From Table 9, Table 10, and the above table, it is clear that according to the present measurement method, 5'-nucleotidase activity in human serum can be measured with high reproducibility.

Example 9 In the same manner as in Example 8, 5′ −
Nucleotidase activity was measured.

For comparison, a conventional enzymatic method (C, L, M, Arkesteijn, J.,
Cl1n.

Chem, Cl1n, B1och+;m,, Vol,
14. p155-158(197B):] was performed to measure the enzyme activity.

The obtained results (U/Q) are shown in Table 10 below.

According to the table above, this measurement method can be used to replace the enzyme method, which has drawbacks such as the conventional measurement system being complicated and requiring a preliminary reaction of about 30 minutes before starting the reaction. It is clear that the 5'-nucleotidase activity in the protein can be measured with high sensitivity and in a short time using a very simple operation.

Example 10 100mM phosphate buffer containing various concentrations of inosine (
pH 6,0)2mf2, 1mMo-)luidine solution 1
Add mQ, then 10 μQ of nucleoside oxidase
(0.35 U) was added and reacted at 37° C. for 15 minutes, and the absorbance of the reaction solution at 480 nm was measured.

The results obtained are shown in FIG. In the figure, the vertical axis is 48
The absorbance at 0 nm is plotted on the horizontal axis, and the amount of inosine in the reaction solution (μ
mole).

From the figure, it is clear that the amount of inosine in the test liquid can be satisfactorily measured by utilizing the proportional relationship between the inosine content of the test liquid and the absorbance.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of high performance liquid chromatography of a reaction product obtained by carrying out an oxidation reaction with the present enzyme using inosine as a substrate according to the enzyme action test I of the present invention. Figure 2 shows the relationship between the degree of color development of a quinone imine dye obtained by carrying out an oxidation reaction with this enzyme using inosine as a substrate in the presence of phenol and 4-aminoantipyrine and the above substrate concentration according to enzyme action test H of the present invention. It is a diagram. FIG. 3 is a diagram showing the temperature stability of this enzyme. FIG. 4 is a diagram showing the pH stability of this enzyme. FIG. 5 is a diagram showing the optimum pH of this enzyme. FIG. 6 is a diagram showing the results of spectrum analysis of this enzyme in 100 mM phosphate buffer (pH 6.0). FIGS. 7 to 10 are graphs showing the relationship between the amount of nucleosides in various test liquids and the measured value of absorbance according to Examples 4 and 5. FIGS. 11 and 12 are graphs showing the relationship between the amount of nucleotidase in various test liquids and the measured absorbance value according to Examples 6 and 7. FIG. 13 is a graph showing the measurement results (relationship between the amount of inosine and the measured absorbance value) according to Example 10. (That's all) Diagram Notion Time (Threat) Diagram Inosine 11 degrees (nanomole) Diagram H Diagram Diagram Inosine 'J Farm Road (millimol) Diagram U Hironucleotidase degree (I + I) 3'-nucleotidase 511 vine (, lJg) Figure O10 Guanosine: /R < mmol) Figure

Claims (4)

[Claims] (1) An enzyme that catalyzes the oxidation reaction of nucleosides, which produces nucleoside-5'-carboxylic acid via nucleoside-5'-aldehyde through the reaction of the nucleoside with molecular oxygen, but hydrogen peroxide is a secondary enzyme. A novel nucleoside oxidase YT-1 characterized in that it does not produce nucleoside oxidase. (2) A system containing or producing nucleosides is used as a test solution, and the nucleoside oxidase YT-1 according to claim 1 is allowed to act on a mixture of the test solution and a coloring reagent that develops color by oxidation; A method for analyzing a test liquid, characterized by measuring the degree of color development of the above-mentioned coloring reagent, which is proportional to a change in the amount of nucleosides in the test liquid. (3) The analytical method according to claim (2), wherein the test liquid is selected from a sample containing nucleosides, an enzyme reaction system that liberates nucleosides, and an enzyme reaction system that degrades nucleosides. (4) The test solution is a nucleoside-releasing enzyme reaction system containing human serum and nucleotides, and the 5'-
The analytical method according to claim (2), wherein nucleotidase activity is measured.