JP3065754B2 - Novel xanthine dehydrogenase-T enzyme, measuring method using the same, and analytical composition containing the enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to adenosine, inosine, xanthosine, guanosine, adenine, guanine,
A novel xanthine dehydrogenase-T enzyme having no substrate specificity for thymidine and at least having substrate specificity for xanthine, and hypoxanthine or xanthine or hypoxanthine or xanthine present in a sample using the enzyme; The present invention relates to a method for quantifying the activity of a substrate or an enzyme involved in a reaction system for producing, and an analytical composition containing the same.

[0002]

2. Description of the Related Art Xanthine dehydrogenase is an enzyme that catalyzes the reaction of oxidizing hypoxanthine to xanthine and xanthine to uric acid [EC. It is known that NAD ⁺ is used as a coenzyme at the time of the oxidation reaction in 1.2.1.37]. That is, by measuring the amount of hypoxanthine and xanthine present in the sample, or NADH produced in an equivalent amount when oxidizing hypoxanthine and xanthine generated by a reaction system that generates hypoxanthine and xanthine, The presence or generated hypoxanthine and xanthine, or the substrate or enzyme activity involved in the reaction system for producing these substrates can be quantified (JP-A-62-32900).

[0003] Substrates involved in a reaction system for producing hypoxanthine and / or xanthine include adenosine, adenine, inosine, xanthosine, guanosine, and the like.
Guanine and the like include adenosine deaminase (EC. 3.5.4.4) and guanine deaminase (E
C. 3.5.4.3), purine nucleoside phosphorylase (EC.2.4.2.1), 5'-nucleotidase and the like are known.

[0004] These are useful as a screening for liver damage in clinical diagnosis. For example, in the case of guanine deaminase, xanthine produced using guanine as a substrate is oxidized using xanthine oxidase.
A method for measuring generated hydrogen peroxide is widely used.

In the case of adenosine deaminase,
The ammonia produced by the reaction is converted into α-ketoglutarate and reduced NADP using glutamate dehydrogenase.
A method for measuring the reduction of reduced NADP in the presence of is widely used. However, guanase activity measurement using xanthine oxidase is easily affected by reducing substances such as ascorbic acid in a sample, and adenosine deaminase activity measurement using glutamate dehydrogenase is
Each of them has a serious problem that the influence of ammonia present in the sample cannot be avoided.

Xanthine dehydrogenase is an enzyme derived from avian and rat liver [J. Biol. Chem. , 242
(18); 4097 (1967)] is known as a typical one, but other microorganisms include Streptomyces [Agr. Biol. Che
m. , 41; 1161 (1977); Bioche
m. 86, 45 (1979)], Micrococcus [J. Biol. Chem. , 242, 4108 (19
67)], Neurospora [J. Biol. Che
m. , 253, 2604 (1978)], Pseudomonas sp. [Biochim. Biophys. Acta, 4
10, 12 (1975)] and those of the genus Norcardeioides or Nocardia (Japanese Patent Laid-Open No. 61-170386).

However, these enzymes have a problem in substrate specificity. That is, the enzymes of the genus Streptomyces have an activity on guanine, and the enzymes of the genus Micrococcus and the genus Schudomonas have not only an activity on purines but also essentially an oxidase activity. Neurospora enzymes are
It is known that the activity is inhibited by low concentrations of hypoxanthine and xanthine, and that the enzymes of the genus Nocardioides or Nocardia have activity on inosine. Thus, each of the known xanthine dehydrogenases has problems and has not been put to practical use.

[0008]

The present invention has no xanthine oxidase activity and has no activity on at least adenosine, inosine, xanthosine, guanosine, adenine, guanine, purine, and thymidine. Thus, a novel xanthine dehydrogenase-T enzyme whose activity is not inhibited has been found, and the use of the enzyme of the present invention provides the quantification of xanthine present in a sample or the activity of a substrate or an enzyme involved in a reaction system for producing xanthine.

[0009]

In human blood, there are adenosine, inosine, xanthosine, guanosine, adenine, guanine, purine, etc., for example, about 0.1 mM for inosine and about 0.3 mM for adenosine. [Clin. Chem. , 36
(1), 81 (1990)]. For example, when trying to measure hypoxanthine or xanthine in blood using a conventional xanthine dehydrogenase, the presence of the above-mentioned substrate poses a problem as a correct error.

In particular, xanthine dehydrogenase of the genus Nocardeioides or Nocardia has a relative activity (to xanthine) of about 7% with respect to inosine. Has the drawback of giving a large measurement error, and cannot be used in a measurement system.

[0011] Further, when an attempt is made to measure guanine deaminase or adenosine aminase activity, if the conventional xanthine dehydrogenase has an activity on adenosine or guanine, it cannot be put to practical use.
In order to solve these problems, a wide screening was conducted from nature, and a strain producing a xanthine dehydrogenase-T enzyme having the intended performance was discovered.
The present invention has been completed.

That is, the present invention belongs to bacterial bacilli, and comprises xanthine dehydrogenase-T enzyme-producing bacteria and bacteria-N
o. 197 (10) [Bacteria No. 197
(10). Therefore, the present invention relates to bacterial bacilli, bacteria-No. 197 (10), wherein a microorganism capable of producing xanthine dehydrogenase-T enzyme is cultured in a culture medium, and xanthine dehydrogenase-T enzyme is contained in the culture.
A novel xanthine dehydrogenase-T enzyme obtained by producing and accumulating an enzyme and collecting a xanthine dehydrogenase-T enzyme that does not substantially act on adenosine, inosine, xanthosine, guanosine, adenine, and guanine, and using the same. The present invention relates to a method for determining the activity of a substrate or an enzyme, and an analytical composition containing the enzyme.

The adenosine, inosine,
The xanthine dehydrogenase-T enzyme, which does not substantially act on xanthosine, guanosine, adenine, and guanine, is preferably the strain belonging to the bacterial bacillus, the bacterial no.
197 (10) (Microtechnical Research Institute Co., Ltd., No. 3664), and may be obtained from any strain having the ability to produce the enzyme of the present invention. Bacteria belonging to bacterial bacilli isolated from the soil of Gunnamjima Town-No. 197 (10), and this strain is an example of a strain most effectively used in the present invention. The microbiological properties of this strain are as follows.

In the identification of this strain, the identification test was conducted using a guide for identifying medical bacteria (second edition), Microbio.
The method was carried out in accordance with the method of “Logical Methods” (1974), and the experimental results were obtained using Bergey's Manual.
f systematicBacteriological
al Vol. 1 (1984), eds., Vol. 2 (1
986), the same magazine, Vol. 3 (1989).

(1) Characteristics of growth Ordinary agar slant medium It grows well linearly. It is grayish white to pale ocher. No soluble pigment is produced. Ordinary agar plate medium A round, hill-shaped colony is formed. The surface is smooth, shiny and wet. It has a grayish white to pale ocher color, but does not produce soluble pigments. Liquid medium (peptone water) It grows well, is uniformly turbid, and forms a thin film. Litomos milk culture medium becomes alkaline.

(2) GCmol% of DNA 60 to 60.2 (3) Main isoprenoid quinone Q-10 (4) Morphological features Straight or slightly bent rod-shaped bacterium with rounded ends, single or double, occasionally short-chain I do. The size is 0.3-0.
Exercise with polar hairs, bipolar hairs or perihairs at 5 × 0.5-1.0 μm. No spore formation and no polymorphism. Sometimes arranged in a Y-shape.

(5) Physiological and biochemical properties [+;
(+); Weakly positive,-; negative, NT; not performed] Gram staining + KOH reaction-acid-fast staining-capsule formation-OF test (hugh-Leifson) NT

OF test (NH ₄ H ₂ PO ₄ ) as N source O (oxidation) Growth under aerobic conditions + Growth under anaerobic conditions-Growth temperature 42 ° C.-37 ° C.-30 ° C. + 20 ° C. + 10 ° C. NT Salt tolerance 0% + 1.5% + 3.0%-Growth pH 5.7-6.7 + 9.0 + 10.0-Gelatin degradation-Starch degradation-Casein degradation-

Esculin degradation + Cellulose degradation-Tyrosine degradation-Tween 80 degradation-Arginine degradation NT Catalase production + Oxidase production + Lecithinase production-Urease production (SSR)-

Urease production (Chris.) + Indole production-hydrogen sulfide production (lead acetate paper) + acetoin production (K ₂ HPO ₄ )-acetoin production (NaCl)-MR test-nitrate reduction test (gas production)-(NO ₂ ^- detection of) - (NO ₃ ^- detection of) +

Availability in Simmons medium (alkaline production) Citrate + malate + maleate-malonate-propionate-gluconate-succinate +

Availability in Christensen's medium (alkaline production) Citrate + malate + maleate-malonate-propionate-gluconate-succinate + gas production from glucose-

Production of acid from sugar Adonitol + L (+) arabinose + cellobiose + dulcitol + mesoerythritol-fructose + D-galactose + D-glucose + glycerin + inositol + inulin-

Lactose (+) maltose + mannitol + mannose + melezitose-melibiose + raffinose-L (+) rhamnose +

D-ribose + salicin-L-sorbose + sorbitol + starch-sucrose + trehalose + D-xylose +

As described above, this strain-No. 197 (1
The main property of 0) is a gram-positive rod-shaped bacterium that moves with periflagellate. It oxidatively degrades glucose to produce acids.
Catalase oxidase production, the main isoprenoid quine is ubiquinone-10 (Q-10). GC of DNA
mol is 60 to 60.2%.

This strain-No. Although 197 (10) is a Gram-positive bacterium, the quinone type is ubiquinone (Q-10). Determination of the type of isoprenoid quinone to be contained,
The types of isoprenoid quinones and the bacteria having the quinones are shown in Table 1 below, since they have been found to be useful for the classification of bacteria.

[0028]

[Table 1]

Some quinones are known to be reduced quinones. Table 1 shows that gram-positive bacteria contain menaquinone, but there is no report of a genus containing ubiquinone. Therefore, there is no description of the genus which coincides with the main properties of the present bacterium. 197 (1
No. 3) No. 3664 (FERM BP-36)
64).

The medium used for producing the enzyme using the novel xanthine dehydrogenase-T enzyme-producing bacterium of the present invention is appropriately selected from carbon sources, nitrogen sources, inorganic substances, and other nutrients in combination. However, xanthine or hypoxanthine is desirably added to the medium because the enzyme of the present invention is inductively produced by xanthine or hypoxanthine.

Examples of the carbon source include sucrose, glucose, waste sugar and the like. Examples of the nitrogen source include peptone, yeast extract, brain heart infusion (BHI), and the like. Metal salts such as potassium, magnesium, calcium, molybdenum and the like can be used.

For example, salting-out, dialysis, ion-exchange chromatography such as DEAE-Sepharose and Q-Sepharose (manufactured by Pharmacia), hydrophobic chromatography such as octyl-Sepharose, and Sephadex G-10
A highly purified enzyme preparation of the present invention can be obtained by appropriately combining gel chromatography methods such as 0, Cefacryl S-200 and the like.

According to the present invention, at least adenosine,
A sample containing hypoxanthine and / or xanthine, or a substrate or an enzyme involved in a reaction system that produces hypoxanthine and / or xanthine, which is not affected by inosine, xanthosine, guanosine, adenine, guanine, and thymidine. Converting the substrate into hypoxanthine and / or xanthine, or using the enzyme to generate hypoxanthine and / or xanthine, and using the existing or generated hypoxanthine and / or xanthine as a coenzyme,
In the presence of NADs or thio NADs, the enzyme of the present invention decomposes into uric acid, and the reduced N
By quantifying ADs or reduced thioNADs, the substrate or enzyme activity can be quantified.

The following are examples of the reaction system for producing hypoxanthine or xanthine. That is,

[0035]

Embedded image

In carrying out the reaction, xanthine dehydrogenase-T enzyme is added to the sample and reacted in an appropriate buffer, and the amount of reduced NAD or reduced thioNAD produced is quantified. Thus, the target substance can be quantified.

The xanthine dehydrogenase-T enzyme used in the present invention has substantially no action on adenosine, inosine, xanthosine, guanosine, adenine, guanine (and therefore has no substrate specificity),
Any enzyme that catalyzes the reaction of decomposing hypoxanthine and / or xanthine in the presence of ADs or thio NADs to produce stoichiometrically reduced NADs or reduced thio NADs Can be used.

The most preferred example of the enzyme is the enzyme xanthine dehydrogenase-T of the present invention, which is a xanthine dehydrogenase belonging to bacterial bacilli.
T enzyme producing bacterium, bacteria No. 197 (10).

As a buffer used in the reaction, a phosphate buffer, a Tris-HCl buffer, a glycine-sodium hydroxide buffer, or the like is used. The reaction is carried out at 20-60 ° C, preferably 20-40 ° C, pH 5-10, preferably pH 6.5-6.5.
Performed at 9.5 for 30 seconds to 60 minutes. When the subject to be measured is hypoxanthine or xanthine in the test liquid, the enzyme xanthine dehydrogenase-T enzyme of the present invention is added to the test liquid, and the enzyme is used to decompose hypoxanthine or xanthine, and the method described above. Can be determined.

When the object to be measured is a substance (substrate) related to hypoxanthine or a xanthine-forming reaction system, an enzyme, a substrate, and the like required for converting these substrates into hypoxanthine or xanthine are added. It may be converted into hypoxanthine or xanthine, decomposed by the action of xanthine dehydrogenase-T enzyme, and measured by the method described above.

When the object to be measured is hypoxanthine or an enzyme involved in a reaction system that produces xanthine, hypoxanthine or xanthine is produced by adding a substrate or the like of the enzyme whose activity is to be measured. It may be decomposed by the action of xanthine dehydrogenase-T enzyme and measured by the method described above. The above-described reaction may be performed sequentially one paragraph at a time for each reaction step,
It is also possible to carry out the reaction in one step by adding enzymes, substrates and the like required for each reaction step.

The measurement was performed using uric acid or reduced NADs (reduced thioNADs) produced by the above reaction [hereinafter referred to as NAD
H (also referred to as thioNADHs)] or the decrease in consumed NAD (thioNADs), etc., but in the present invention, the generated NADHs ( It is most convenient to quantify thioNADHs).

The measurement of NADHs (thio NADHs)
Each specific maximum absorption wavelength, for example, 340 nm for NADHs and 405 nm for thioNADHs
A method for measuring the increase in the amount of absorption of chromophore or a method for producing a dye by the action of an enzyme such as diaphorase in the presence of a chromogen and a hydrogen acceptor, and measuring the absorbance in the visible region of the reaction solution colored by the coloration of formazan. For example, in the case of xanthine, the color development of formazan may be measured by measuring the increase in absorption at 550 nm. As the hydrogen acceptor to be used, known acceptors such as methylene blue and nitro blue tetrazolium can be used, and they are commercially available.

As described above, in the present invention, NAD is added to hypoxanthine or a test solution containing xanthine.
Reacting the novel xanthine dehydrogenase-T enzyme of the present invention in the presence of thioNADs and / or thioNADs, and then quantifying the amount of measurable NADHs and / or thioNADHs produced by the reaction. The characteristic method of measuring hypoxanthine or xanthine includes not only the method of measuring hypoxanthine or xanthine present in the test solution but also the method of measuring the substrate or enzyme activity involved in the reaction system that produces hypoxanthine or xanthine. It must be noted that

The present invention further relates to a composition for analyzing a substrate or an enzyme activity, which is used for carrying out the above-mentioned assay method, which is a composition containing xanthine dehydrogenase-T enzyme. The following are schematically illustrated as examples of the composition for analysis. (1) When the subject to be quantified is hypoxanthine or xanthine present in the test solution, xanthine dehydrogenase-T enzyme, NADs and / or thioNADs, and reaction product quantification reagents and buffers.

(2) If the target to be quantified is hypoxanthine or a substrate or an enzymatic activity involved in a xanthine-forming reaction system, a reagent system or an activity necessary for introducing the substrate into hypoxanthine or xanthine The substrate of the enzyme to be measured and the reagent system required to guide this substrate to hypoxanthine. Xanthine dehydrogenase-
T enzymes and NADs and / or thio NADs, and reaction product quantification reagents and buffers.

Examples of the reagent for quantifying the reaction product in the above (1) include the above-mentioned reagents for coloring formazan for quantifying NADHs and other known reagents. The reagent system necessary for leading the substrate to be quantified to hypoxanthine or xanthine in the above (2) includes:
Enzymes required to direct the substrate to hypoxanthine or xanthine.

As this substrate, various substrates exemplified by the above-mentioned reaction system for producing hypoxanthine or xanthine, ie, adenosine, inosine, guanosine,
Guanine and the like, and examples of the enzyme include various enzymes exemplified in the same reaction system, that is, one kind of adenosine deaminase, purine nucleotide phosphorylase, guanase and the like, or a combination thereof. The substrate of the enzyme whose activity is to be measured and the reagent system necessary for introducing this substrate into hypoxanthine are the necessary components selected from the above-mentioned substrates and enzymes.

The xanthine dehydrogenase-T of the present invention
The properties of the enzyme are as follows. (1) Enzyme action As shown in the following formula, catalyzes a reaction for producing xanthine and NADH or thioNADH from hypoxanthine and NAD ⁺ or thioNAD ⁺ , and then catalyzes the reaction of generated xanthine and NAD ⁺ or thioNAD ^+. It catalyzes the reaction that produces uric acid and NADH or thioNADH.

[0050]

Embedded image

(2) Substrate specificity hypoxanthine 100% xanthine 75% uric acid 0 adenosine 0 inosine 0 xanthosine 0 guanosine 0 adenine 0 guanine 08-azaxanthine 08-azaguanine 0

(3) Molecular weight TSK gel G3000SW (0.75 × 6
(0 cm), and the molecular weight of the enzyme of the present invention measured by a gel filtration method was about 240,000.

(4) Isoelectric Point The isoelectric point of the enzyme of the present invention was 4.5 from focal electrophoresis using a carrier amphiplite having a pH of 3.5 to 10.0.

(5) Km value The Km value of the enzyme of the present invention with respect to hypoxanthine is 0.14.
mM, 0.24 mM for xanthine, 0.075 mM for NAD, 0.08 mM for thio-NAD.
mM.

(6) Thermal stability The enzyme solution of the present invention (0.1 u / ml) was prepared with 40 mM phosphate buffer (pH 7.5), and the remaining activity was measured after 15 minutes of heat treatment. As shown in FIG. 1,
Enzyme activity is stable at least up to 40 ° C, 60 ° C
Also retained 89% of the original activity.

(7) Optimum temperature Using 100 mM Tris-HCl buffer (pH 8),
The result of activity measurement at each temperature of 0, 45, 50, 55 and 60 ° C. is as shown in FIG. 2 and has the maximum activity around 55 ° C.

(8) pH stability The enzyme of the present invention (0.1 u / ml) was added to a 40 mM acetate buffer (pH 5.05 to 6.12-O-) and a phosphate buffer (pH
6.12 to 8.01-●-), Tris-HCl buffer (pH
7.84 to 8.58- △-) and glycine-sodium hydroxide buffer (pH 8.73 to 9.36- □-), and remaining activity after heating at 45 ° C for 15 minutes. As a result of the measurement, is shown in FIG. 3, and the enzyme activity maintains 90% or more in a wide pH range of pH 6.5 to 9.5.

(9) Optimum pH According to the enzyme activity measurement method described below, 100 m
100 mM phosphate buffer (pH 6.5-7.5- ●-), Tris-HCl buffer (pH 8.0-9.0- △-) and glycine-water in place of M Tris-HCl buffer (pH 8) A reaction solution was prepared using each buffer solution of sodium oxide buffer (pH 9.0 to 10.0- □-), and the activity was measured. The result is as shown in FIG. It shows maximum activity around.

(10) Xanthine dehydrogenase-T
Effect of metal ions and surfactant on enzyme activity Preparation of reaction solution 100 mM Tris-HCl buffer (pH 8.0) 1 mM NAD 2 mM xanthine 0.02 u / ml xanthine dehydrogenase-T
enzyme

In the above reaction solution, the metal salt is 10 or 1 mM.
The reaction was prepared at 37 ° C., and the increase in _{A340 nm} of NADH produced at that time was measured. The results are shown in Table 1. Further, a reaction solution was prepared so that the content of the surfactant was 0.5% in the reaction solution, the reaction was carried out at 37 ° C., and the increase in NADH _{340 nm} generated at that time was measured. The results are shown in Table 2. .

[0061]

[Table 2]

(11) Xanthine dehydrogenase-T
Method for measuring enzyme activity of enzyme (1) Composition of reaction solution 100 mM Tris-HCl buffer (p
H9.0) 2 mM NAD 2 mM Xanthine

(2) Enzyme activity measurement 1 ml of the reaction solution of (1) was dispensed into a 1 ml quartz cell having an optical path length of 1 cm, incubated at 37 ° C. for 5 minutes, and then 0.02 ml of an appropriately diluted enzyme solution was added. And the N produced by the reaction
The change in absorbance of ADH at 340 nm is measured over time. (3) Formula

[0064]

Embedded image 6.22: molecular extinction coefficient cm ² / umol 10: reaction time (min) Z: dilution factor

(12) Amount of Enzyme and Coenzyme Used in Reaction Solution When the amount of hypoxanthine and / or xanthine is to be measured, the amount of xanthine dehydrogenase-T enzyme to be used is appropriately adjusted according to the reaction conditions. Good, but 0.01 to 100 u / ml, usually 0.1 to 10 u / ml
Used in ml. The amount of NAD or thio NAD used as a coenzyme is 0.01 to 10 mM, usually 0.1 to 10 mM.
Used at ~ 2.0 mM.

For example, when measuring phosphate ions, the amount of purine nucleoside phosphorylase used is
It may be appropriately prepared depending on the reaction conditions, but 0.0
It is used at 1 to 100 u / ml, usually 0.1 to 20 u / ml.

[0067]

The bacterium belonging to the bacterial bacillus of the present invention -N
o. The xanthine dehydrogenase-T enzyme derived from 197 (10) has no enzymatic activity on adenosine, inosine, xanthosine, guanosine, adenine, guanine, and thymidine, and has much higher substrate specificity than existing xanthine dehydrogenase.

That is, using the enzyme of the present invention, N
The hypoxanthine and / or xanthine present or produced using the ADs and / or thioNADs is oxidized to uric acid, and the N produced at this time is oxidized.
When using a measurement reagent that measures ADHs or thioNADHs, it is possible to supply highly specific measurement reagents that are not affected by various substrates other than hypoxanthine or xanthine present in the sample. It is.

Further, since the enzyme of the present invention exhibits activity on xanthine produced by the reaction in the same manner as hypoxanthine, it is twice as sensitive when measuring the presence or produced hypoxanthine. Can be measured. When thio-NAD is used instead of NAD, the molecular extinction coefficient is about twice, so that twice the sensitivity can be obtained.

[0070]

EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 Bacterial No. Culture of 197 (10) Xanthin (Yamasa Shoyu Co., Ltd.) 1%, K ₂ HPO ₄ 0.
25%, MgSO ₄ 7H ₂ O 0.05%, Yeast extract (Kyoto Pharmaceutical) 0.1%, BHI (Difco)
100 ml of a liquid medium containing 1% pH 7.0 was dispensed into a 500 ml Erlenmeyer flask, sterilized by heating at 120 ° C. for 20 minutes, and then one platinum loop of bacteria-No. 197 (10) and inoculated at 28 ° C., 120 r. p. m. The culture was carried out in a shaking incubator for 60 hours to obtain 4.5 l of a culture solution of 50 of these (enzyme activity: 0.2 u / ml).

On the other hand, 0.05% of Disform CB442 (manufactured by NOF CORPORATION) was added as a defoamer to 20 l of the same medium composition as described above, and the mixture was charged into a 30 l jar arm mentor, sterilized by heating, and obtained as described above. 100 ml of the seeds were transplanted, the culture temperature was 28 ° C., the aeration rate was 20 l / min, and the internal pressure was 0.4.
kg / cm ² , stirring speed 200 r. p. m. For 40 hours to obtain 19 l of culture (enzyme activity: 0.3 u / ml).

Example 2 Separation and Purification of Enzyme 19 l of the culture obtained in Example 1 was collected by centrifugation and collected.
The suspension was suspended in 3 l of 0 mM Tris-HCl buffer (pH 8.0). This is a Dynomill (Willy A. Bachof)
en Maschinenfabrik TYPE
(KDL), the treated solution was centrifuged to remove the precipitate, and 2.9 l of the supernatant (enzyme activity 1.82)
u / ml).

This crude enzyme solution was passed through a column of 200 ml of DEAE-Sepharose buffered with 40 mM Tris-HCl buffer (pH 8.0), and then washed with 5 l of the same buffer. Then, 50, 150, 20 in the same buffer
400 ml of a buffer containing 0, 250, and 300 mM KCl was flowed. The xanthine dehydrogenase-T enzyme was eluted with a buffer containing 300 mM KCl to obtain 400 ml of an enzyme solution (enzyme activity: 9.0 u / ml).

The obtained enzyme solution was prepared using Amicon 24
CENTTRIFLO ULTRAFILTRATIO
The mixture was concentrated to 40 ml (enzyme activity: 85 u / ml) using N MEMBRANCE CONES CF25, and 0.3 MKC
l mM 100 mM Tris-HCl buffer (pH 8.0)
Sephaacryl S-200 (7.5 cm x 1) buffered with
(200 cm) using a column of molecular sieve, and collecting the active fractions in an amount of 200 ml (enzyme activity: 12 u / ml).
Was obtained. The resulting enzyme solution is 2
After dialysis overnight against 10 liters of 0 mM Tris-HCl buffer (pH 8.0), lyophilized to 445 mg (5.
(0 u / mg) of the enzyme preparation.

Example 3 Determination of hypoxanthine using NAD ⁺ as a coenzyme 40 mM Tris-HCl buffer (pH 8.5), 1 mM NA
Hypoxanthine was added to D ⁺ at 0.01 and 0.0, respectively.
A reaction solution was prepared at 2, 0.03, 0.04, and 0.05 mM, and the enzyme of the present invention was added at 1 u / ml.
The increase in _{A340 nm} of NADH produced before and after the reaction for 5 minutes was measured. The results are shown in FIG.

Example 4 Determination of Xanthine Using NAD as Coenzyme 40 mM Tris-HCl buffer (pH 8.5), 1 mM NA
X ⁺ is 0.01, 0.02,
A reaction solution was prepared at 0.03, 0.04, and 0.05 mM, and the enzyme of the present invention was added at 1 u / ml, and the A340 _nm increase of NADH generated before and after the reaction at 37 ° C. for 5 minutes was increased. The amount was measured. FIG. 6 shows the result.

Example 5 Determination of Hypoxanthine Using Thio-NAD as Coenzyme Hypoxanthine was added to 40 mM Tris-HCl buffer (pH 8.5) and 1 mM thio-NAD to 0.01 and 0.0, respectively.
A reaction solution was prepared at 2, 0.03, 0.04, and 0.05 mM, and the enzyme of the present invention was added at 1 u / ml.
A of thioNADH generated before and after the reaction for 5 minutes
The increase at _{405 nm} was measured. The result is shown in FIG.

Example 6 Determination of Xanthine Using Thio-NAD as Coenzyme Xanthine was added to 40 mM Tris-HCl buffer (pH 8.5), 1 mM thio-NAD, 0.01, 0.02,
A reaction solution was prepared at 0.03, 0.04, and 0.05 mM, and the enzyme of the present invention was added at 1 u / ml, and the A _{405 nm} of thioNADH generated before and after the reaction at 37 ° C. for 5 minutes was _measured . The increase was measured. The result is shown in FIG.

Example 7 Measurement of xanthine by coloring formazan 40 mM Tris-HCl buffer (pH 8.5), 1 mM NA
D, 0.2% Triton X-100, 5u / ml diaphorase (manufactured by Toyo Brewing Co., Ltd.), 0.025% nitro blue tetrazolium (manufactured by Wako Pure Chemical Industries, Ltd.), and the reaction solution was adjusted to 1u / ml of the enzyme of the present invention. Prepared, dispensed 1 ml into test tubes,
1, 2, 3, 4, 5 mM of the prepared xanthine was added to 0.0
The reaction was stopped at 37 ° C. for 5 minutes, 2 ml of 0.1N hydrochloric acid was added, and _{A550 nm} was measured. The result is shown in FIG.

Example 8 Inosine, purine nucleoside phosphorylase (PNP
L), Measurement of phosphate ion using the enzyme of the present invention and NAD 40 mM Tris-HCl buffer (pH 8.5), 1 mM
NAD, 20 mM inosine, 10 u / ml PNPL, 1 u
/ Ml of the reaction solution was prepared so as to be the enzyme of the present invention.
3, 4 and 5 mM of the prepared potassium phosphate was added in an amount of 0.01 ml each, and the N generated before and after the reaction at 37 ° C. for 5 minutes was added.
The amount of _{A340 nm} increase in ADH was measured. Figure 1 shows the results.
0. In addition, PNPL was prepared based on the following method. That is, the PNPL is a Bacillus cereus IFO.
13494 to J.I. Biol. Chem. , 246
(5), cultivation and purification by the method described in 1475-1480 (1971), and 21,000 U (enzyme activity 4
2u / mg) of the enzyme preparation was obtained.

Example 9 Measurement of Adenosine Deaminase Activity Using Xanthine Dehydrogenase-T Enzyme Adenosine deaminase (P from Ushihizo, manufactured by Sigma)
RODUCT NUMBER; A5773), the following experiment was performed.

Composition of reaction solution 100 mM Tris-HCl buffer (pH 8.5) 5 mM phosphate buffer (pH 8.5) 5 mM Adenosine (Sigma) 0.5% BSA (Sigma) 2 mM NAD (Oriental Yeast) ) 10 u / ml PNPL 1 u / ml xanthine dehydrogenase-T of the present invention
enzyme

Preparation of adenosine deaminase solution;
10 mM phosphate buffer containing 1% BSA (pH 6.
Using 5), the enzyme derived from Ushihizo was prepared at 1 u / ml.

Procedure 1 ml of the reaction solution was dispensed into a test tube, and the mixture was incubated at 37 ° C. for 3 minutes.
005, 0.01, 0.015, and 0.02 ml each were added, and the A340 _nm of NADH produced in exactly 5 minutes was measured. The results are shown in FIG.

Example 10 Measurement of Guanase Activity Using Xanthine Dehydrogenase-T Enzyme Rabbit liver-derived guanase manufactured by Sigma (PRODUCT)
The following experiment was performed using NUMBER; G5752).

Composition of reaction solution 100 mM Tris-HCl buffer (pH 8.5) 1 mM guanine (manufactured by Sigma) 0.5% BSA 2 mM NAD 1 u / ml Xanthine dehydrogenase-T of the present invention
enzyme

Preparation of guanase solution: Rabbit liver-derived enzyme was prepared at 0.5 u / ml using 10 mM Tris-HCl buffer (pH 8.0) containing 0.1% BSA.

Procedure 1 ml of the reaction solution was dispensed into a test tube, and incubated at 37 ° C. for 3 minutes.
01 and 0.02 ml each were added, and the A340 _nm of NADH produced in exactly 10 minutes was measured. The result is shown in FIG.
It was shown to.

Example 11 Measurement of Guanase Activity Using Xanthine Dehydrogenase-T Enzyme Rabbit liver-derived guanase manufactured by Sigma (PRODUCT)
The following experiment was performed using NUMBER; G5752).

Composition of reaction solution 100 mM Tris-HCl buffer (pH 8.5) 1 mM guanine (manufactured by Sigma) 0.5% BSA 2 mM thioNAD 1 u / ml Xanthine dehydrogenase-T of the present invention
enzyme

Preparation of guanase solution: Rabbit liver-derived enzyme was adjusted to 0.5 u / ml using 10 mM Tris-HCl buffer (pH 8.0) containing 0.1% BSA.

Procedure 1 ml of the reaction solution was dispensed into a test tube, and incubated at 37 ° C. for 3 minutes.
01, 0.015, and 0.02 ml were added, and the A _{415 nm} of the thioNADH produced in exactly 5 minutes was measured. FIG. 13 shows the result.

[Brief description of the drawings]

FIG. 1 is a diagram showing the thermostability of the enzyme of the present invention.

FIG. 2 is a diagram showing an optimum temperature of the enzyme of the present invention.

FIG. 3 is a diagram showing the pH stability of the enzyme of the present invention.

FIG. 4 is a graph showing the optimum pH of the enzyme of the present invention.

FIG. 5 is a diagram showing quantification of hypoxanthine using NAD.

FIG. 6 is a diagram showing quantification of xanthine using NAD.

FIG. 7 is a diagram showing quantification of hypoxanthine using thio-NAD.

FIG. 8 is a diagram showing the quantification of xanthine using thio-NAD.

FIG. 9 is a diagram showing quantification of xanthine by coloration of formazan.

FIG. 10 shows the results of measuring phosphate ions using inosine, PNPL, and NAD.

FIG. 11 shows the measurement of adenosine deaminase activity.

FIG. 12 shows the results of measuring the activity of guanase.

FIG. 13 shows the results of measuring the activity of guanase.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁷ Identification code FI (C12N 9/02 C12R 1:01)

Claims (6)

(57) [Claims] 1. It has the following physicochemical properties : ( a) molecular weight: about 240,000 ( b) isoelectric point: 4.5 ( c) thermal stability: stable up to 40 ° C., even at 60 ° C.
Retain the original 89% active (d) pH stability: pH6.5~9.5 (e) Optimum pH: pH 8.5 is around and also adenosine, inosine, xanthosine, guanosine, adenine, guanine, It does not show substrate specificity for thymidine, has at least substrate specificity for hypoxanthine and xanthine, and oxidizes to xanthine using hypoxanthine as a substrate using NADs and / or thioNADs as coenzymes. To produce reduced NADs and / or reduced thio NADs, or catalyze the reaction of oxidizing to uric acid using xanthine as a substrate to produce reduced NADs and / or reduced thio NADs. A novel xanthine dehydrogenase-T enzyme characterized. 2. A test solution containing hypoxanthine or xanthine has a substrate specificity for adenosine, inosine, xanthosine, guanosine, adenine, guanine, thymidine in the presence of NADs and / or thioNADs. Not shown, having at least substrate specificity for hypoxanthine and xanthine, utilizing NADs and / or thioNADs as coenzymes, and oxidizing to xanthine using hypoxanthine as a substrate to reduce NADs and / or 2. The xanthine dehydrogenase-T enzyme according to claim 1, which catalyzes a reaction for producing reduced thioNADs or oxidizing uric acid using xanthine as a substrate to produce reduced NADs and / or reduced thioNADs. And then the reduced NAD produced by the reaction
A method for measuring hypoxanthine or xanthine, characterized by quantifying the amount of thionadines and / or reduced thioNADs. 3. The test solution containing hypoxanthine or xanthine is a test solution present in the form of hypoxanthine or xanthine in the test solution, or produced from hypoxanthine or a precursor of xanthine. A test solution containing hypoxanthine or xanthine.
2. The measuring method according to 2 . 4. Hypoxanthine or xanthine produced from hypoxanthine or a precursor of xanthine,
4. The method according to claim 3 , wherein hypoxanthine produced from inosine and purine nucleoside phosphorylase, xanthine produced from this hypoxanthine, xanthine produced from guanine and guanase. 5. Adenosine, inosine, xanthosine,
It does not show substrate specificity for guanosine, adenine, guanine, and thymidine, has at least substrate specificity for hypoxanthine and xanthine, and uses NADs and / or thio NADs as coenzymes. Is oxidized to xanthine as a substrate to generate reduced NADs and / or thio NADs, or catalyzes a reaction to oxidize xanthine to uric acid as a substrate to generate reduced NADs and / or thio NADs Claim
1 novel Kisanchindehi de Rogenaze -T enzyme analysis composition containing as claimed. 6. The composition containing the xanthine dehydrogenase-T enzyme according to claim 5 , wherein the composition comprises NAD.
And / or analytical composition containing thioNADs.