JPH08336397A - Assay using new nad synthase - Google Patents

Abstract

PURPOSE: To obtain a NAD synthase good in thermal stability and useful for determination of one component, either ATP, deamido-NAD, ammonia or ammo nium ions, in a liquid specimen. CONSTITUTION: This NAD synthase is obtained by culturing e.g. a Bacillus stearothermophilus H-804 strain (FERM BP-1408) and has the following properties: (1) capable of catalyzing enzymatic reaction of formula I, but incapable of catalyzing enzymatic reaction of formula II; (2) having substrate specificity to ammonia (including ammonium ions) ; (3) optimum pH value: 8.5-10.0 or so, and optimum temperature: 60 deg.C or so; (4) stable at 7.5-9.0 and at least at <=60 deg.C; (5) molecular weight: about 50000 (polyvinyl gel GPC); and (6) isoelectric point: pH5.3 or so (isoelectric point electrophoretic column). Using this synthase, NH3 (including NH4 <+> ) can be assayed esp. without being affected by the amino acid(s) in a liquid specimen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a measuring method using a novel NAD synthase. More specifically, the present invention provides the following method for measuring ATP, deamid-NAD and either ammonia or ammonium ion in a test solution in the presence of at least Mg ⁺⁺ ion or Mn ⁺⁺ ion. Catalyzes the enzymatic reaction of formula (a),
NAD synthase that does not catalyze the enzymatic reaction of the following formula (b) in the presence of g ⁺⁺ ions and uses ammonia and / or ammonium ions as substrates, and at least glutamine and asparagine as substrates (hereinafter, The present invention may be referred to as NAD synthase) may be applied to a test solution, and then the components consumed or produced in the reaction are measured.
The present invention relates to a method for measuring a component in a test liquid, which measures P, deamido-NAD, and any one component of ammonia and ammonium ions.

[0002]

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[0003]

2. Description of the Related Art Conventionally, NAD synthase (NAD ⁺ syn
theatase) is a rat liver [J. Biol. Ch
em. , 233, 493-500 (1958)], pig liver [J. Biol. Chem. , 236, 525-5
30 (1961)], yeast [J. Biol. Che
m. , 247, 4794-4802 (1972)],
E. FIG. coli [J. Biol. Chem. , 236, 1
494-1497 (1961); Biol. Che
m. , 242, 385-392 (1967)]. And in terms of enzyme classification,

[0004]

Embedded image

NAD synthase (E.
C. 6.3.1.5)

[0006]

[]

[0007]

[Chemical 4]

NAD synthase (E.
C. 6.8.5.1), both enzymes act as amino donors with ammonia (including ammonium ion) and L-glutamine as substrates, and NA
Regarding D synthase (EC 6.3.1.5),
[J. Biol. Chem. , 236, 1494-14
97 (1961); Biol. Chem. , 24
2, 385-392 (1967)], NH ₄ ⁺
Is reported to be 6.5 × 10 ⁻⁵ M and L-glutamine to have a Km value of 1.6 × 10 ⁻² M.

Further, NAD synthase (EC 6.3.
Regarding 5.1) [J. Biol. Chem. , 24
7, 4794-4802 (1972); Biol.
Chem. , 233, 493 to 500 (1958)], the Km value for NH ₄ ⁺ is 6.4 × 10 ³ M,
The Km value for L-glutamine is reported to be 5 × 10 ⁻³ M. Differences between the two enzymes are distinguished by whether or not they are inhibited by azaserine.

A conventionally known method for measuring the activity of NAD synthase is to reduce the NAD produced by the reaction with alcohol dehydrogenase (EC.1.1.1) and measure the reduced NAD produced at 340 nm. Or a method of measuring the generated NAD by a fluorescence method has been reported. In addition, the present inventors previously found that conventional NAD synthases (EC 6.3.1.5 and EC 6.3.5.
When measuring ATP, deamid-NAD and any one of NH ₃ , Gln or Asn amide donor in a test solution using 1), ATP, deamid-NAD, amide donor and The main reaction of NAD synthase acting in the presence of Mg ⁺⁺ is performed, and NAD generated by the main reaction is mixed with a redox reaction system using NAD as a coenzyme and a redox reaction system using reduced NAD as a coenzyme. In a test solution containing any one of ATP, amide-NAD, and an amide donor, which is characterized in that a coenzyme cycling reaction by combination is carried out and the components consumed or produced by the cycling reaction are quantified. A method for measuring the components has been reported (Japanese Patent Laid-Open No. 59-198995).

[0011]

As described above, the conventional N
The AD synthase utilizes L-glutamine as a substrate as an amino donor and catalyzes the enzymatic reaction of the following formula (a) in the presence of at least Mg ⁺⁺ ion or Mn ⁺⁺ ion to give Mg ^{+. In} the presence of ⁺ ion, the following formula (b)
N, which does not catalyze the enzymatic reaction of, uses ammonia and / or ammonium ions as substrates, and does not utilize at least glutamine and asparagine as substrates.
The AD synthase was unknown.

[0012]

Embedded image

Further, as described above, the conventional NAD synthase also has substrate specificity for L-glutamine, and therefore L-glutamine has a substrate specificity.
It was impossible to measure NH _{3 in} the presence of glutamine.

[0014]

Means for Solving the Problems In view of the above-mentioned problems, the inventors of the present invention have conducted extensive studies and found that a strain H-804 belonging to the genus Bacillus isolated from hot spring water in Tanoyu-cho, Beppu City, Oita Prefecture Catalyzing an enzymatic reaction of the following formula 9 (a) in the presence of a novel at least Mg ⁺⁺ ion or Mn ⁺⁺ ion, M
Produces a NAD synthase that does not catalyze the enzymatic reaction of the following formula 9 (b) in the presence of g ⁺⁺ ion and utilizes ammonia and / or ammonium ion as a substrate and does not utilize at least glutamine and asparagine as a substrate. I found that

[0015]

[Chemical 6]

That is, the present invention catalyzes the enzymatic reaction of the following formula (a) in the presence of at least Mg ⁺⁺ ions or Mn ⁺⁺ ions, and in the presence of Mg ⁺⁺ ions, the following formula (b): NAD synthase that does not catalyze the enzyme reaction described above, uses ammonia and / or ammonium ion as a substrate, and does not use at least glutamine and asparagine as a substrate (hereinafter sometimes referred to as NAD synthase of the present invention)

[0017]

[Chemical 7]

With respect to the above, ATP and deamido-N in the test solution
In measuring AD and any one of ammonia and ammonium ions, the NAD synthase of the present invention is allowed to act on a test liquid, and then the components consumed or produced in the reaction are measured.
This is a method for measuring a component in a test liquid, which measures any one component of ATP, deamido-NAD and ammonia or ammonium ion.

The mycological properties of this novel NAD synthase-producing bacterium of the present invention are as follows. a. Morphological Features Using an ordinary agar slant medium, the cells were cultured at 50 ° C. for 1 to 3 days and microscopically observed. (1) Shape and sequence; rods with rounded ends, straight or slightly bent, form single or double chains. (2) Size: 0.5 to 1.0 × 1.6 to 5.5 μm (3) Motility: Exercise with peripheral hairs. (4) Spores; formed near the center or edge. The size is 0.8 to 1.0 × 1.0 × 1.5 μm, and the cells swell. b. Growth state in each medium (50 ° C.) (1) Ordinary agar plate medium It is grayish white to pale yellowish grayish white and semitransparent, grows linearly, grows slightly, and does not produce soluble pigment. (2) Ordinary agar slant medium It is translucent, grayish white to pale yellowish gray, and forms round and flat colonies. It does not produce soluble pigment. (3) Liquid medium It grows well and becomes cloudy uniformly, and it partially precipitates after 2-3 days. (4) BCP milk is unchanged. c. Physiological properties (+; Positive,-; Negative) Gram stain + KOH reaction-Acidic acid stain-Capsule formation-OF test (Hugh + Leifson medium) No change OF test (Modified medium) * 0 (Oxidation) Anaerobic growth -Catalase production + (weak) Oxidase production + (weak) Urease production (SSR medium) -〃 (Chris medium) -Lecithinase production Non-growing gelatin decomposition + starch decomposition + casein decomposition + esculin decomposition + arginine decomposition-cellulose decomposition-indole production -Hydrogen sulfide production + (lead acetate paper) Acetoin production-MR test-Reduction of nitrate-Growth in NaCl-free medium + Growth in 0.1% NaCl-containing medium + Growth in 0.25% NaCl- Growth at 65 ° C + Growth at 50 ° C + Growth at 37 ° C- Growth at H9.0-Growth at pH 8.0 + Growth at pH 5.6 + Growth at pH 4.8-Production of acid from sugar * (non-gas production) adonitol-L (+) arabinose + cellobiose + dulcitol -Meso-erythritol-D fructose-fucose-D galactose + D glucose + glycerin + inositol-inulin-lactose + maltose-D mannitol + D mannose + melezitose + rylmitol-sorbitol-sorbyl-sorbitose-D ribose + D-ribose. starch + Satsukarosu + trehalose + D-xylose ※ basal medium sugar pressurized ammonium medium _{(ASS) (NH 4) 2} HPO 4 1.0g KCl 0.2g MgSO 4 · 7H 2 O 0.2g yeast extract 1.0 g Agarose 3.0 g BTB 0.02 g Distilled water 1000 ml pH 7.0 ** Modified medium Medium supplemented with 10.0 g glucose in the above basal medium Usability test (Simons medium) Citrate-malonate- Gluconate-propionate-maleate-succinate-malate + availability test (Christenesen medium) Citrate-malonate-gluconate + propionate-maleate-succinate + Malate + From the above-mentioned mycological properties, this H-804 strain is a bacillus with rounded ends, straight or slightly curved, and is gram-positive,
It is a thermophilic bacterium that forms spores with a size of 0.5-1.0 × 1.6 × 5.5 μm, is weakly positive in the ability to produce catalase and oxidase, is non-motile, and oxidizes sugar (glucose). It is characterized by being a degrading bacterium. The taxonomic position of this bacterium having such various properties can be determined by Bergy's
Manual 8th Edition, 1974, Guide to Medical Bacterial Identification 2nd Edition, 1974 and Agricultural Han.
d book, 427, The genus Baci
When examined with reference to llus, the bacterium formed spores,
Since it can grow under aerobic conditions, it is determined to belong to the genus Bacillus.

Therefore, examples of highly (thermophilic) and thermotolerant strains belonging to the genus Bacillus include Bacillus subtilis, Bacillus coagillans, Bacillus licheniformis, Bacillus brevis and Bacillus stearothermophilus. Since the minimum growth temperature of this bacterium is 30 ° C. or higher, (A) Bacillus stearothermophilus and (B) Bacillus brevis are mentioned. A comparison and comparison of these properties is as follows. [+: Positive,-; negative, d: different depending on the strain].

This bacterium (A) (B) Size (μm) Width 0.5-1.0 0.5-0.1 0.6-0.9 Length 1.6-5.5 2 0.0-3.5 1.5-4.0 Gram stain + (easy decolorization) Indeterminate adventitious spore formation + ++ Swelling of cells by spores ++ motility − ++ Catalase production (+) ++ Under anaerobic condition Growth in --- Acetoin production --- Growth temperature Maximum (° C) 65 or higher 65-75 40-60 Minimum (° C) 37 or higher 30-45 10-35 Growth at pH 5.7 +-d 5% NaCl addition Growth in medium-d-Production from sugar Glucose + + + arabinose + d-xylose + d- Mannitol + production of gas from d d-sugar --- Use of starch decomposition + + d citrate --- d Nitrate Reduction-dd indole production --- Casein degradation + d + From the above comparative comparison, it was reported that this strain was in good agreement with Bacillus stearothermophilus, and that a non-motile strain was easily obtained in terms of motility. The results of comparative examination of the properties also agreed well with Bacillus stearothermophilus. Therefore, this H-80
4 strains of Bacillus stearothermophilus (Baci
lls stearothermophilus) H
It was identified and named -804. In addition, this strain was sent to the Institute of Microbiology and Biotechnology, Institute of Industrial Technology, Institute of Microbiology,
No. 9 (FERM P-8839), Microscopic Research Institute of Microbiology 140th
No. 8 (FERM BP-1408) ".

In the present invention, the NAD synthase producing bacterium of the present invention belonging to the genus Bacillus includes Bacillus
Stearothermophilus H-804 is an example thereof, and not limited to this strain, all bacteria producing the NAD synthase of the present invention can be used in the present invention. Further, DNA which carries the genetic information of the NAD synthase of the present invention is isolated from the bacterium producing the NAD synthase of the present invention by genetic engineering, and the DNA is incorporated into the NAD synthase non-producing strain of the present invention,
A mutant strain mutated to express the NAD synthase-producing ability of the present invention can be obtained, and the NAD synthase of the present invention produced from the mutant strain and the measurement of components in a test solution using the enzyme The method is also included in the present invention.

The present invention is cultivated by a conventional method for producing the NAD synthase-producing bacterium of the present invention. The form of the culture may be liquid culture or solid culture, but it is industrially preferable to perform deep aeration stirring culture. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and examples thereof include glucose, sucrose, lactose, maltose, starch, dextrin, sugar concentrate, waste sugar concentrate, and glycerin. The nitrogen source may be any available nitrogen compound, and examples thereof include corn steep liquor, soybean flour, cottonseed flour, wheat gluten, peptone, meat extract, yeast extract, casein hydrolyzate, ammonium sulfate and the like. In addition, salts of phosphate, magnesium, calcium, potassium, sodium, zinc, iron, manganese, halogen and the like are used as necessary. In Bacillus stearothermophilus H-804, the culturing temperature may be appropriately changed within a range in which the NAD synthase-producing bacterium of the present invention grows and produces the present enzyme, but it is 48 to 70 ° C., particularly 55 to 55 ° C. 60 ° C is preferred. Although the culturing time varies depending on the culturing conditions, it suffices to terminate the culturing at an appropriate time in consideration of the time when the maximum titer of the present enzyme is reached, preferably 10 to 20 hours. When stirring by aeration, 200 to 400 r. p. m. Is sufficient under the conditions of.

The NAD synthase of the present invention is collected from the culture of the NAD synthase-producing bacterium thus obtained. Since the present enzyme is mainly contained in the microbial cells, the obtained culture The cells are harvested by means such as filtration or centrifugation, and the cells are disrupted by mechanical disruption treatment such as ultrasonic treatment, French press treatment or glass bead treatment, or enzymatic destruction such as lysozyme, and necessary. Depending on the above, a surfactant such as Triton X-100 (Triton X-100: trade name), Adecatol SO-120 (trade name), etc. may be added. The NAD synthase-containing solution thus obtained is concentrated or salted out without concentration with a soluble salt such as ammonium sulfate, or a hydrophilic organic solvent such as methanol, ethanol, acetone or isopropanol is used. The enzyme may be used to precipitate.

The precipitate is dissolved in water or a buffer solution,
If necessary, dialyze with a semipermeable membrane, and further chromatograph using an ion exchange resin such as DEAE-Sephadex, DEAE-Sepharose or DEAE-Cellulose, Carboxymethyl-Cellulose, Carboxymethyl-Sepharose, Carboxymethyl-Sephadex. And Sephadex G200, Sepharose CL-6B,
It is possible to obtain the purified enzyme by purification by chromatography using a gel filtration agent such as molecular sieve such as Cefacryl S-200, and then adding a stabilizer if necessary, and lyophilization. it can.

Next, the properties of the NAD synthase of the present invention obtained in the present invention will be described. (1) Molecular weight: about 50000 [Polyvinyl gel (trade name: GPC3000SW; manufactured by Toyo Soda Co., Ltd.) using a column (7.5 mm ID × 60 cm), and aldolase (rabbit muscle, N.W. 150,00) as a standard protein.
0), bovine serum albumin (MW 67,000), ovalbumin (chicken egg: MW 45,000),
By molecular sieve using cytochrome C (horse heart, MW 13,000). (2) Isoelectric point: around pH 5.3 [column for isoelectric focusing (manufactured by LKB), carrier ampoline pH 3.
5 to 10.0 (manufactured by LKB), 700 V, 48 hours after energization, 2 ml from the column (2.4 × 30 cm)
By fractionating each and measuring the pH and activity of each fraction. ] (3) Action;

[0027]

Embedded image

(4) Substrate specificity: It has substrate specificity for ammonia (including ammonium ion). Alternatively, 25 mM (NH ₄ ) ₂ S of the reaction solution I of the enzyme assay method described below is used.
Instead of O ₄ , 25 mM L-valine, L-homoserine, L-serine, L-alanine, L-methionine, L-
Tyrosine, L-threonine, L-leucine, L-isoleucine, L-arginine, L-phenylalanine, L-
Histidine, L- asparagine, adding L- glutamine, respectively, results of measuring the Supporting connexion activity as hereinafter described in the enzyme activity assay, the relative activity in the case of a 100 activity (NH _{4) 2} SO ₄ is, L- Valine, L-homoserine, L-serine, L-alanine, L-methionine, L-tyrosine,
L-threonine, L-leucine, L-isoleucine, L
-Arginine, L-phenylalanine, L-histidine, L-asparagine and L-glutamine are both 0.
0.

The present enzyme utilizes ammonia (including ammonium ion), and at least L-valine, L-homoserine, L-serine, L-alanine, L-methionine, L-tyrosine, L-threonine, L- Leucine, L
-Isoleucine, L-arginine, L-phenylalanine, L-histidine, L-asparagine, L-glutamine are not used. (5) Optimum pH; dimethyl glutaric acid-sodium hydroxide buffer solution (p
H5.0-7.0), Tris-HCl buffer (pH 6.5-
9.0), glucine-sodium hydroxide buffer (pH
8.5 to 10.0) and 10 times after the enzyme reaction.
The reaction was stopped by heating at 0 ° C for 10 minutes, and the N
The result of measuring the amount of AD is as shown in FIG. pH
It has an optimum pH in the vicinity of 8.5 to 10.0. (6) pH stability: This enzyme was treated with 50 mM dimethyl glutaric acid-sodium hydroxide buffer solution (pH 5.0 to 7.
0), Tris-HCl buffer (pH 6.5-9.0), glycine-sodium hydroxide buffer (pH 8.5-10.
0) and treated at 60 ° C. for 15 minutes, and the residual activity was measured according to the enzyme activity measuring method described below.
It is as shown in FIG. It is stable in the pH range of 7.5 to 9.0. (7) Thermal stability; 50 mM Tris-HCl buffer (pH 8.
The present enzyme of 0) was dissolved, heat-treated at each temperature for 15 minutes, and the residual activity was measured according to the enzyme activity measuring method described below. The results are shown in FIG. It is stable at least at 60 ° C or lower. (8) Optimum temperature; 50 ° C, 55 ° C, 60 ° C, 65 ° C, 7
Reaction solution I of the enzyme activity measuring method described below at each temperature of 0 ° C.
The reaction was performed for 10 minutes, then immediately cooled, and the reaction solution II was added at 37 ° C., and the results of measurement according to the enzyme activity measuring method described later are shown in FIG. It has an optimum temperature around 60 ° C.

Regarding the optimum temperature, 50 ° C., 55 ° C., 60 ° C., 65 ° C., 70 ° C. were obtained using a solution of the enzyme in 50 mM Tris-HCl buffer (pH 8.0).
At each temperature of ° C and 75 ° C, the enzyme activity measurement method described below (note that in the case described below, 90% purity deamide-NAD manufactured by Sigma was used, but in this measurement, 99% purity deamid manufactured by Oriental Yeast was used. Reaction solution I (using NAD)
After 10 minutes of reaction, the mixture was immediately cooled, and at 37 ° C., a reaction solution II (50 mM of EDTA was used; diaphorase and alcohol dehydrogenase were manufactured by the same company but used in different lots) was added. As a result of measuring the activity according to the enzyme activity measurement method, the relative activity when the activity at 70 ° C was defined as 100% was 43% at 50 ° C, 62% at 55 ° C, and 7% at 60 ° C.
8%, 94% at 65 ° C and 81 at 75 ° C
%, And the optimum temperature was judged to be around 70 ° C. From the above, the method for measuring the activity of the present enzyme is affected by the purity of the deamide-NAD used and the lot difference of diaphorase and alcohol dehydrogenase,
Furthermore, it is presumed that it was influenced by the concentration of EDTA, and it was not recognized as a difference in the properties of the enzyme itself. (9) Activation and activity inhibitor: 5 mM in the case of the metal ions shown in Table 1 in the reaction solution I of the enzyme activity measuring method described later,
20 mM for EDTA, 0.1 for surfactant
It was added so that it became%, and it measured according to the enzyme activity measuring method. The results are shown in Table 1. As Ni ⁺⁺ ion, strongly inhibited by NiCl ₂ (5 mM), 20 mM
No activity appears with EDTA.

Further, among the reagents used in the enzyme activity measuring method described below, MnCl ₂ (3 mM) was added under the condition that MgCl ₂ (5 mM) was not included as Mg ⁺⁺ ions, and the method was followed in accordance with the enzyme activity measuring method described below. As a result of activity
Compared to the case where the reaction solution containing gCl ₂ (5 mM) was used, when the reaction solution containing MnCl ₂ (3 mM) was used,
It shows a relative activity of 150%.

[0032]

[Table 1]

* Without additive: MgCl 2 as Mg ⁺⁺ ion
₂ (5 mM) is included. The following is each metal ion and a surfactant added thereto. (10) Enzyme activity measurement method Activity measurement method Reaction solution I 50 mM Tris-HCl buffer solution pH 8.0 10 mM KCl 5 mM MgCl ₂ 0.05% Bovine serum albumin 2 mM ATP 0.5 mM Deamid-NAD 25 mM (NH ₄ ) ₂ SO ₄ Reaction solution II 50 mM Tris-HCl buffer solution pH 8.0 10U diaphorase / ml (manufactured by Asahi Kasei, Bacillus-producing bacteria) 3% ethanol 10U alcohol dehydrogenase / ml (manufactured by Toyobo, yeast-derived) 0.025% NTB (nitro) Tetrazolium blue) 0.1% Triton X-100 (trade name) 10 mM EDTA reaction solution I 0.3 ml was placed in a small test tube, heated to 37 ° C, 50 μl of enzyme solution was added, and reacted exactly at 37 ° C for 10 minutes. And add 0.7 ml of reaction solution II to stop the reaction solution. It was the start of the cycling reaction with. The cycling reaction was performed at 37 ° C for exactly 5 minutes and 0.1N HC
After stopping the cycling reaction by adding 1 ml of 2.0 ml, the absorbance at 550 nm was measured and the enzyme activity was determined from the value at that time. The activity was calculated according to the following formula.

[0034]

[Equation 1]

ΔA550: Absorbance of sample ΔS550: Absorbance of standard solution (0.1 mM NAD) 0.005: Sample amount (ml) 10: Reaction time f: Dilution ratio The reaction system of the present invention is summarized as follows. .

[0036]

[Chemical 9]

The test solution of the present invention may be any solution containing at least one test component of ATP, deamid-NAD and ammonia (including ammonium ion) in the above reaction system. Examples include a test liquid containing one of the test components in advance, and a test liquid containing the test component produced or consumed by another enzyme reaction system.

A preferred example of the above-mentioned enzyme reaction system is A
An enzyme reaction system that consumes or produces TP, deamid-NAD, NH ₃ (including ammonium ion) and does not involve coenzymes such as NAD and reduced NAD, for example, the following enzyme reaction system, It does not limit the subject matter of the present invention. (1) Enzyme reaction system creatine kinase (EC 2.7.3.2) that produces ATP Reducing agents; β-mercaptoethanol, reduced glutathione, cysteine, N-acetylcysteine, dithiothreitol and the like.

Pyruvate kinase (EC 2.7.
1.40) Acetate kinase (EC 2.7.2.1) Carbamate kinase (EC 2.7.2.2) Aspartate kinase (EC 2.7.2.4) Phosphoglycerate kinase (EC 2.7.2.
3) Arginine kinase (EC 2.7.3.3)

[0040] (2) as an enzyme reaction system a) a water-soluble ammonium salts utilizing water-soluble ammonium salts or NH ₃ can emit NH ₃ are ammonium chloride,
Ammonia water, ammonium sulfate, ammonium nitrate,
Examples are inorganic or organic ammonium salts capable of releasing ammonium ions such as ammonium acetate and ammonium citrate. b) Nicotine amidase (EC 3.5.1.19) Nicotinamide + H ₂ O → Nicotinic acid + NH ₃ + H ⁺ c) Glutamyl-peptide-glutaminase (EC
3.5.1.44) L- glutaminyl - peptide + H ₂ O → L- glutaminyl - peptide + NH ₃ d) arginine deaminase (E.C.3.5.3.6) L- arginine + H ₂ O → Shitosarin + NH ₃ + H ⁺ e) guanine deaminase (EC 3.5.4.3) guanine + H ₂ O → xanthine + NH ₃ + H ⁺ f) adenosine deaminase (EC 3.5.4.4) adenosine + H ₂ O → Inosine + NH ₃ + H ⁺ g) Creatinine deaminase (EC 3.5.4.2)
1) Creatinine + H ₂ O → N-methylhydantoin + NH
₃ + H ⁺ h) threonine dehydrase (EC 4.2.1.1)
6) L-threonine + H ₂ O → 2-oxobutyric acid + CO ₂ + N
H ₃ ⁺ H + i) aspartic acid ammonia - lyase (E.C.4.
3.1.1) L-aspartic acid → fumaric acid + NH ₃ + H ⁺ j) L-methionine γ-lyase (EC 4.4.1.
11) L-methionine + H ₂ O → 2-oxobutyric acid + methanethiol + NH ₃ + H ⁺ k) methylaminoglutamate methyl tranferase (EC.2.1.1.21)

[0041]

[Chemical 10]

(3) The measurement of deamino-NAD and NH ₃ (including ammonium ion) in a test solution, which is characterized by the measurement of AMP produced by the present enzymatic reaction, is exemplified by the following enzymatic reaction system. Adenylate kinase (EC 2.7.4.3)

[0043]

[Chemical 11]

Pyruvate kinase (EC 2.7.
1.40)

[0045]

[Chemical 12]

Pyruvate oxidase (EC 1.
2.3.3)

[0047]

[Chemical 13]

Measurement of NH ₃ (including ammonium ion) deamino-NAD in the test liquid by measuring the produced H ₂ O ₂ in the presence of peroxidase Lactate dehydrogenase (EC.1.1.1.1. 27) Pyruvate + NADH + H ⁺ → L-lactic acid + NAD NAD in the presence of excess lactate dehydrogenase and NADH
In the present invention, the reduction of A ₃₄₀ due to the oxidation of H is not limited to the reaction system containing ATP and NH ₃ consumed or produced in the above-mentioned enzyme reaction system, and the enzyme activity used in the enzyme reaction system A reaction solution for measuring, quantifying the consumed substrate or the produced product may also be used as a test solution.

ATP and NH in these enzyme reaction systems
The quantitative purpose of ₃ is to measure the activity of the enzyme in the enzyme reaction system and to measure any one of the other components used. In this enzyme reaction system, a certain amount of a reagent other than the component to be measured may be used. In this case, the amounts of the test solution and the reagent to be measured may be appropriately set and changed according to the purpose to be measured and the conditions to be selected, and are not particularly limited.

Further, as an oxidation-reduction reaction system using NAD synthase of the present invention as a coenzyme for NAD, for example, dehydrogenase (E ₁ ) which forms a reaction of consuming NAD to produce reduced NAD and the reaction system or by the substrate (S _1), can be used a reaction system by dehydrogenases that can be both a coenzyme NAD and NADP (E ₁₎ and its substrate (S _1). The above-mentioned dehydrogenase is not particularly limited as long as it consumes at least NAD as a coenzyme, and any dehydrogenase may be used as long as it acts on a specific substrate used in excess to produce reduced NAD. It may be the enzyme of origin.

Examples of these enzymes and their substrates are described in "Enzyme Handbook". For example, lactate dehydrogenase (EC 1.1.1.12) and L-lactate, glycerol dehydrogenase (EC 1.1.1.6) and glycerol, glycerol-3-phosphate dehydrogenase (E.
C. 1.1.1.8) and glycerol-3-phosphate, glucose dehydrogenase (EC.1.
1.47) and glucose, malate dehydrogenase (EC 1.1.1.13) and L-malate,
Glutamate dehydrogenase (EC 1.4.1.
2) and L-glutamate, 3-α-hydroxysteroid dehydrogenase (EC 1.1.1.1.50)
And 3-α-hydroxy steroids.

The amount of enzyme used in these redox reactions differs depending on the enzyme titer, the type of substrate and the coenzyme cycling rate. The base mass is one mole ratio of substrate consumed per cycle, and a much larger molar amount than the coenzyme to be cycled is used. It may be decided based on time. Further, the concentration may be at least the concentration at which the reaction rate of the oxidoreductase shows the maximum. It can usually be present in a concentration range of 0.1 mM to 100 mM.

Examples of the reaction system using reduced NAD as a coenzyme include a reaction system of an agent (E ₂ ) that consumes at least reduced NAD and produces NAD and its substrate (S ₂ ). Examples of the reaction system of a substance include a reaction system of an oxidoreductase that consumes at least reduced NAD to produce NAD and its substrate, and a reaction system of an electron transfer substance and a tetrazolium salt.

Coenzyme Cycling Reaction System (a) Redox Reaction System Using NAD as Coenzyme; (B) a transfer reaction system using NADH as a coenzyme; NH _3: To Fugan positive monovalent ammonium ions of ammonia.

E ₁ : Dehydrogenase which consumes NAD and S ₁ as substrates and catalyzes a reaction to produce reduced NAD and P ₁ E ₂ : Consumes reduced NAD and S ₂ to produce NAD and P ₂ . Agents that catalyze the reaction that occurs S ₁ : reduced substrate of E ₁ S ₂ : oxidized substrate of E ₂ P ₁ : oxidized product of S ₁ P ₂ : reduced product of S _{2 When} using NH ₃ The reaction system of is represented by the formula as follows.

[0056]

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As an oxidoreductase that consumes reduced NAD and produces NAD, at least reduced NAD is used as a coenzyme, and N acts by acting on an excessive amount of a specific substrate (S ₂ ).
Dehydrogenase that produces a reduced product (P ₂ ) of AD and S ₂ , or at least a reduced NAD as a coenzyme, and a reduced NAD that has as an acceptor a cytochrome, a disulfide compound, quinone, or an analog thereof: a receptor Any oxidoreductase may be used, and its origin is not limited. Examples of these enzymes and their substrates or receptors are described in "Enzyme Handbook".

Examples of dehydrogenase and its substrate include lactate dehydrogenase (EC.1.1.
1.27) and pyruvate, alcohol dehydrogenase (EC.1.1.1.1) and acetaldehyde, glycerol dehydrogenase (EC.1.1.1.1)
And dihydroxyacetone. Also NAD
Examples of H: receptor oxidoreductase include cytochrome b ₅ reductase (EC.1.6.2.2), diaphorase (EC.1.6.4.3) and the like. As receptors, methylene blue, flavins, quinones,
2,6-dichlorophenol indophenol and the like can be mentioned.

The combination of reduced NAD: receptor oxidoreductase and receptor is not particularly limited as long as it is an enzyme that uses reduced NAD as a coenzyme and an electron acceptor. A preferred combination is diaphorase (E
C. 1.6.4.3) and the tetrazolium salt, and methylene blue, NAD dehydrogenase (EC.
1.6.99.3) and cytochrome C and the like, and the concentration used may usually be in the range of 0.05 to 100 U / ml. The concentration of tetrazolium salt used is rather limited by the water solubility of both the tetrazolium salt and the ultimately formed formazan, but can usually be present in the temperature range of 1 μg to 100 μg per ml of reagent.

As the electron transfer substance, reduced NAD
Examples include substances that have the ability to oxidize to AD and that do not adversely affect the coenzyme cycling reaction, such as phenacin metal sulphate, meldora blue, and pyrocyanine. The concentration to be used may be set according to the cycling rate, but is usually 5 per 1 ml of reaction solution I.
It may be present in the concentration range of μg to 0.5 ml.

The above-mentioned cycling reaction is usually not performed at room temperature.
At a temperature of around 37 ° C, preferably 30-37 ° C
Done. The reaction time is not particularly limited,
It may be carried out for 1 minute or longer, preferably 5 minutes or longer. Scheduled
To stop the reaction quickly at the end of the
It is carried out by adding hydrochloric acid, phosphoric acid or the like. Rhino
After carrying out the cling reaction, the smell of this cycling reaction
The amount of energy consumed or produced is quantified.
E is the part to be formed₁Reduced substrate (S ₁) Oxidized raw
Product (P₁) Or E₂Oxidized substrate (S₂) Reduction student
Product (P₂) Should be targeted, and
Is E₁Reduced substrate (S₁) Or E₂Oxidized substrate of
(S₂), The P₁, P₂, S
₁, S₂The amount of any one of the components may be quantified.
Briefly, it is colorless in the substrate state and in the product state
When changing the absorption wavelength that causes color change or fluorescence
It is to use the quantitative method of the product. For example tetrazoli
Umium salt as substrate (S₂), Return the generated formazan
Original product (P₂) At most, colorimetrically correct this formalin
It is a measure. Further flavins and quinones
Substrate (S₂), The substrate (S
₂) Consumption is measured based on its unique absorption wavelength
Then, it may be quantified.

In the above reaction, it is preferable to add a surfactant in order to help prevent the precipitation of formazan formed from the tetrazolium salt. Examples of the surfactant include nonionic surfactants such as Triton X-100 and ADECATOL SO-145. The concentration used can be in the range of 0.01 to 3% with respect to the reagent. By adding this surfactant, the measured value can be increased and the formazan dye can be stabilized.

The colorimetric quantification of the resulting formazan dye may be carried out by measuring the absorbance (OD) at a specific absorption wavelength of formazan. For example, the absorbance is measured at a wavelength of 500 to 550 nm to quantify the substance to be measured. It can be carried out. According to the present invention, not only the end point method but also a late method and a dry chemical method (film method, immobilization method) are possible.

[0064]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto.

[0065]

Reference Example 1 Production Method Peptone 1%, glucose 0.
5%, K ₂ HPO ₄ 0.05%, NaCl 0.05
%, And 40 liters of liquid medium (pH 7.6) containing 0.05% MgSO ₄ .7H ₂ O at 120 ° C., 20
Bacillus stearothermophilus (Bacillus S) pre-cultured in a medium having the same composition as above after sterilization for a minute
200 ml of inoculum of T. thermophilus) H-804 was inoculated, and the aeration rate was 40 at 10 ° C. for 10 hours.
Liter / min, stirring speed 150 r. p. m. Aeration culture was performed. After culturing, collect the cells by centrifugation and collect the cells 0.1%.
10 mM Tris-HCl buffer containing lysozyme (pH
8.0) Dispersed in 500 ml, reacted at 37 ° C. for 30 minutes, and lysed.

This solution was treated with 5000 r.p.m. p. m. After centrifugation for 10 minutes, 450 ml of supernatant was obtained. Ammonium sulfate was added to the supernatant to fractionate it with ammonium sulfate (0.5 to 0.71 saturation), and the precipitate was dissolved in 50 ml of 10 mM Tris-HCl buffer (2 /
U) and dialyzed against 5 liters of this buffer. The insoluble matter generated in this dialysate was centrifuged (15000 r.p.m.).
p. m. It was removed in 10 minutes). Supernatant (20U)
Buffered with 10 mM Tris buffer (pH 8.0)
EAE-Sepharose CL-6B column (25 x 5c
m), and eluted with a concentration gradient method of 0-0.5M NaCl. Fractions eluted with 0.25 to 0.3 M NaCl were collected (80 ml, 16.5 U) and concentrated using Amicon Centriflow Membrane Corn CF25, and then Sephadex G-100 (3.6 × 80 c).
Purification was carried out in m) and the active fractions were collected and used as a purified standard (50 ml, 14 U).

[0067]

Example 1 Measurement of ATP in Specimen Reaction solution I 50 mM Tris-HCl buffer pH 8.0 10 mM KCl 5 mM MgCl ₂ 0.05% Bovine serum albumin 1 mM Deamino-NAD 50 mM (NH ₄ ) ₂ SO ₄ 100 mU Present invention NAD Synthetic enzyme / ml Reaction solution II 50 mM Tris-hydrochloric acid buffer pH 8.0 10U diaphorase (manufactured by Asahi Kasei Corp., derived from Bacillus) 3% ethanol 10U alcohol dehydrogenase (manufactured by Toyobo Co., Ltd.,
Derived from yeast) 0.025% nitrotetrazolium blue 0.1% Triton X-100 15 mM EDTA Reaction solution I 0.3 ml was placed in a small test tube and heated to 37 ° C., and then 0, 10, 20, 30, 40 μM ATP solution. 5 μl of each was added, and after reacting at 37 ° C. for 10 minutes, 0.7 ml of reaction solution II was added, reacted exactly at 37 ° C. for 5 minutes, and then 2.0 ml of 0.1N HCl was added to stop the reaction. Absorbance was measured at 550 nm. The result is
Good linearity was obtained as shown in FIG.

[0068]

Example 2 Measurement of ammonium ion 50 mM (NH in the reaction system I shown in Example 1_Four)₂S
O_FourTo the reaction solution containing 5 mM ATP instead of
5, 10, 15, 20 μM (NH_Four)₂SO _FourSolution
5 μl was added and the same operation as in Example 2 was performed. That conclusion
As for the fruit, good linearity was obtained as shown in FIG.

[0069]

Example 3 Measurement of creatinine Reaction solution I 50 mM Tris-HCl buffer pH 8.0 10 mM KCl 5 mM MgCl ₂ 1 mM ATP 0.05% bovine serum albumin 1 mM deamino-NAD 20 U / ml creatinine deiminase (Kodak) 100 mU / ml The NAD synthase reaction solution I of the present invention I (0.3 ml) was placed in a small test tube, heated to 37 ° C., and 1, 2, 3, 4 mg / dl creatinine solution was added to 10 ml.
μl was added, and the same operation as in Example 2 was performed. The result was as shown in FIG. 7, and good linearity was obtained.

[0070]

Example 4 Method for measuring ammonium ion 50 mM Tris-HCl buffer pH 8.0 10 mM KCl 5 mM MgCl ₂ 1 mM ATP 0.05% bovine serum albumin 1 mM deamino-NAD 100 mU / ml The NAD synthase of the present invention 1 ml of the above reaction solution is small. In a test tube, after heating to 37 ° C., 10 μl of 0, 25, 50, and 100 mM (NH ₄ ) ₂ SO ₄ solutions were added, reacted at 37 ° C. for 20 minutes, and then the absorbance was measured at 340 nm. As a result, good linearity was obtained as shown in FIG.

[0071]

Example 5 Measurement of generated AMP Reaction solution I 50 mM Tris-HCl buffer pH 8.0 10 mM KCl 5 mM MgCl ₂ 1 mM ATP 0.05% Bovine serum albumin 0.05% Triton X-100 1 mM Deamid-NAD 10 U / ml Myokinase (from Bacillus Sigma) 10 U / ml Pyruvate Kinase (Sigma)
Bacillus-derived) 10 mM Phosphoenolpyruvate 100 mU / ml NAD synthase reaction solution II of the present invention II 50 mM phosphate buffer pH 8.0 100 mU / ml Pyruvate oxidase (Sigma)
100 mU / ml peroxidase (manufactured by Sigma) 0.2% phenol 0.3% 4-aminoantipyrine The reaction solution I (1 ml) was placed in a small test tube and heated to 37 ° C. and then 0. 5, 1.0, 1.5, 20 mM (NH ₄ )
Add 10 μl of each ₂ SO ₄ solution, and add 30
After reacting for 0.1 minutes, 0.1 ml of reaction solution II was added,
After reacting at 30 ° C. for 30 minutes, the absorbance was measured at 500 nm. As a result, good linearity was obtained as shown in FIG.

[0072]

Example 6 Deamid-NAD Assay Method Reaction solution 50 mM Tris-HCl buffer pH 8.0 10 mM KCl 5 mM MgCl ₂ 0.05% Bovine serum albumin 50 mM (NH ₄ ) ₂ SO ₄ 1 mM ATP 100 mU / ml Inventive NAD synthesis Enzyme 3% Ethanol 10U Alcohol dehydrogenase 0.1% Triton X-100 1 ml of reaction solution was placed in a small test tube and heated to 37 ° C. and then 0,
After adding 10 μl each of 2.5, 5, and 10 mM deamid-NAD solutions and reacting at 37 ° C. for 30 minutes,
Absorbance was measured at 0 nm. As a result, good linearity was obtained as shown in FIG.

[0073]

Example 7 Optimum concentration of metal ion Reaction solution I 50 mM Tris-HCl buffer pH 8.0 10 mM KCl 0.05% Bovine serum albumin 2 mM ATP 0.5 mM Deamid-NAD 25 mM (NH ₄ ) ₂ SO ₄ reaction solution II 50 mM Tris-HCl buffer pH 8.0 10U diaphorase 1 ml (from Asahi Kasei's Bacillus production bacteria) 3% ethanol 10U alcohol dehydrogenase / ml (from Toyobo Co., Ltd. yeast) 0.025% NTB (nitrotetrazolium blue) 0.1 % Triton X-100 (trade name) 10 mM EDTA Reaction solution I contained MgCl ₂ 0.25 mM and 0.50, respectively.
mM, 0.75 mM, 1 mM, 2 mM, 3 mM, 4 m
M, MnCl ₂ 0.25 mM, 0.50 mM, 0.75
mM, 1 mM, 2 mM, 3 mM, and 4 mM were added, and the activity was measured according to the enzyme activity measuring method. The relative activities are shown in FIG. 11 for the MgCl ₂ addition group and in FIG. 12 for the MnCl ₂ addition group. As a result, the optimum concentration was 3 mM in the MnCl ₂ addition group.

[0074]

INDUSTRIAL APPLICABILITY The present invention can be used for quantifying ATP, deamido-NAD and any one component of ammonia or ammonium ion in a test liquid by using the NAD synthase of the present invention, and particularly, the test liquid This makes it possible to measure ammonia (including ammonium ion) without being affected by the amino acids contained therein. In addition, it is also useful in the above-mentioned various analyzes from the viewpoint that it is a thermostable enzyme that is stable up to 60 ° C. in terms of thermostability of the NAD synthase of the present invention as a preferred embodiment.

[Brief description of drawings]

FIG. 1 is an optimum pH curve of NAD synthase of the present invention.

FIG. 2 is a pH stability curve of NAD synthase of the present invention.

FIG. 3 is a thermostability curve of NAD synthase of the present invention.

FIG. 4 is an optimum temperature curve of NAD synthase of the present invention.

FIG. 5 is a quantitative curve of ATP in the present invention.

FIG. 6 is a quantitative curve of ammonia measured by absorbance at 550 nm.

FIG. 7 is a quantitative curve of creatinine.

FIG. 8 is a quantitative curve of ammonia measured by absorbance at 340 nm.

FIG. 9 is a quantitative curve of ammonia by measuring AMP produced.

FIG. 10 is a quantification curve of deamide-NAD.

FIG. 11 is an optimum addition curve with respect to the MgCl ₂ addition concentration.

FIG. 12 is an optimum addition curve with respect to MnCl ₂ addition concentration.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // (C12N 9/00 C12R 1:07)

Claims (17)

[Claims] 1. ATP and deamide-NAD in a test solution
And either ammonia or ammonium ions
In measuring one, at least Mg⁺⁺Ion again
Is Mn⁺⁺In the presence of ions, the enzymatic reaction of the following formula (a)
Catalyst and Mg ⁺⁺The enzyme of formula (b) below in the presence of ions
Ammonia and / or ammonium without catalyzing the reaction
Use mu ions as a substrate, and at least glutamic
NAD compounds that do not use arsenic and asparagine as substrates
The enzyme is allowed to act on the test solution, and then it is consumed in the reaction.
Specialize in measuring the costed or generated components
ATP, deamido-NAD and ammonia
Or one of the ammonium ions is measured
A method for measuring the components in the test liquid. [Chemical 1] 2. The measuring method according to claim 1, wherein the produced NAD is measured in measuring the produced component. 3. The NAD produced is measured by reacting it in the presence of an oxidoreductase having NAD as a coenzyme and its reduced substrate, and measuring the resulting reduced NAD.
The measurement method described in. 4. A redox reaction system using NAD as a coenzyme and a reduced NA in measuring the produced NAD.
The coenzyme cycling reaction is performed in combination with a redox reaction system using D as a coenzyme, and the components consumed or produced by the cycling reaction are quantified.
The measurement method described in. 5. The assay method according to claim 4, wherein the redox reaction system using NAD as a coenzyme is an enzyme reaction system using dehydrogenase that consumes NAD to produce reduced NAD and its substrate. 6. Dehydrogenase and its substrate are lactate dehydrogenase and L-lactate, alcohol dehydrogenase and ethanol, glycerol dehydrogenase and glycerol, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate, glucose dehydrogenase and glucose, malate dehydrogenase. And L-
The assay method according to claim 5, which is one of malate, glutamate dehydrogenase and L-glutamate, 3-α-hydroxysteroid dehydrogenase and 3-α-hydroxysteroid. 7. The measuring method according to claim 4, wherein the redox reaction system using reduced NAD as a coenzyme is an enzyme reaction system using an oxidoreductase that consumes reduced NAD to produce NAD and a substrate thereof. 8. A redox enzyme and its substrate are lactate dehydrogenase and pyruvate, alcohol dehydrogenase and acetaldehyde, glycerol dehydrogenase and dihydroxyacetone, glycerol-3-phosphate dehydrogenase and dihydroxyacetone phosphate, malate dehydrogenase and ogizaroacetate, The measuring method according to claim 7, which is either a 3-α-hydroxysteroid dehydrogenase or a 3-ketosteroid. 9. The assay method according to claim 7, wherein the oxidoreductase and its substrate are reduced NAD: receptor oxidoreductase and its receptor. 10. Reduced NAD: Receptor oxidoreductase and its receptor are reduced NAD dehydrogenase and flavins, quinones, 2,6-dichlorophenol indophenol, ferricyanide salt, tetrazolium salt, cytochrome C or oxygen. The measuring method according to claim 9, wherein 11. The assay method according to claim 7, wherein the oxidoreductase and its substrate are diaphorase and a tetrazolium salt or resazurin. 12. The measuring method according to claim 4, wherein the redox reaction system using reduced NAD as a coenzyme is a reaction system using an electron carrier and a tetrazolium salt. 13. The method according to claim 12, wherein the electron transfer substance is phenazine-metasulfate, Meldola blue or pyrocyanine. 14. The measuring method according to claim 1, which comprises measuring the generated AMP in measuring the generated component. 15. The measuring method according to claim 14, wherein the AMP is measured by measuring the produced pyruvic acid in the presence of ATP, in the presence of adenylate kinase, phosphoenolpyruvate, and pyruvate kinase. 16. The measuring method according to claim 15, wherein pyruvic acid is measured by allowing pyruvate oxidase to act and measuring the produced H ₂ O ₂ . 17. A method for measuring pyruvate, which causes NA to decrease lactate dehydrogenase in the presence of NADH.
The measuring method according to claim 15, which comprises measuring the amount of DH.