JPS6368099A - Analyzing method by using pyruvic acid oxydase and reagent thereof - Google Patents

Abstract

PURPOSE:To readily stably measure pyruvic acid, etc., by using a novel pyruvic acid oxydase having better heat stability than a conventional pyruvic acid oxydase, when pyruvic acid, ADP, etc., is measured by an enzymic method. CONSTITUTION:Lactobacillus.espi TE6103 strains (FERN-P No.8886) belonging to the genus Lactobacillus are cultured in a medium and a novel pyruvic acid oxydase thermally stable up to 45 deg.C when treated of 7pH for 10min is obtained by methods of extraction, purification, etc., from the culture medium. Then, the pyruvic acid oxydase is blended with FAD, thiamine pyrophosphate, phosphate, and at least one metal ion selected from magnesium ion, cobalt ion, manganese ion and calcium ion to prepare the aimed analytical reagent. The pyruvic acid, etc., can be precisely measured by using the analytical reagent.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides an analytical method using a novel thermostable pyruvate oxidase, that is, pyruvate oxidase that is thermostable up to 45°C at pH 7.0 and treated for 10 minutes. In particular, it relates to a method for measuring pyruvic acid and reagents therefor.

According to the present invention, it can be used not only for the measurement of pyruvate, but also for the measurement of substrates and enzymes that catalyze the pyruvate formation reaction. Typical examples of measurements are as follows.

■Glutamic acid-pyruvic acid transaminase or σ
- Ketoglutarate ■ Daltamic acid - Oxap acetate transaminase ■ Lactate dehydrogenase or lactate ■ Pyruvate kinase or ADP (prior art) Pyruvate oxidase is enzyme number E C1, 2, 3,
It is an enzyme that catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen. Location: Breakfast Bookstore), Lactobacillus plantarum (J
, Bactsriol, Vol. 160, pp. 273-278 (
1984)) and microorganisms of the genus Pediococcus, Sushileptococcus, and Aerococcus (Special Publication No. 58-40
It is known that various microorganisms such as Leuconostoc microorganisms (Japanese Unexamined Patent Application Publication No. 159777/1982) produce it.

However, the pyruvate oxidase produced by any of the strains does not have sufficient heat resistance, and there is a problem in the stability of the pyruvate measurement reagent using Koneru's enzyme.

(Problems to be Solved by the Invention) Based on the above background, the present inventor attempted to find a more practical reagent for measuring pyruvic acid with excellent stability.

(Means for Solving the Problems) The present inventors have discovered that TE-610, a microorganism belonging to the genus Lactobacillus isolated from soil in Tsuruga City, Fukui Prefecture,
It was discovered that three strains produce pyruvate oxidase with better thermostability than conventional pyruvate oxidase, and a special fraud application has already been filed.

Furthermore, it was discovered that pyruvate oxidase obtained from this strain can effectively measure pyruvate, and in addition, the enzyme activity of the enzyme reaction system that produces pyruvate can be measured.
The inventors have discovered that it is possible to quantify the enzyme in the enzymatic reaction system that produces pyruvate and the substrate of the enzymatic reaction system that produces pyruvate, and have arrived at the present invention. Furthermore, a reaction system consisting of the above pyruvate oxidase, FAD, thiamine pyrophosphate, phosphate, and at least one metal ion among magnesium ions, cobalt ions, manganese ions, and calcium ions is reacted with the pyruvate-containing sample system. It was discovered that pyruvic acid in a sample system can be measured satisfactorily by this reaction system, and that pyruvic acid can be measured simply and satisfactorily by adding a hydrogen peroxide detection system to this reaction system. Reached. That is, the present invention is an analytical method characterized by using pyruvate oxidase having excellent thermostability and having the following physicochemical properties.

■Real use: Catalyzes the reaction that produces acetyl phosphoric acid, carbon dioxide, and hydrogen peroxide from pyruvic acid, inorganic phosphoric acid, and oxygen.

■Substrate specificity: oxaloacetate, DL-lactic acid, acetic acid, α-
It does not react with ketoglutaric acid and is specific for pyruvate.

■Thermal stability: 45℃ or less (pH 7, 0, 10 minute treatment) ■Optimal pH: Around 5.7 ■B value: Approximately 4X10-4M (pyruvic acid) ■Molecular weight:
Approximately 160.000 (Sephacryl S-30
(gel filtration method at 0) ■Isoelectric point: approx.
Cofactor: FAD and thiamine bitetraphosphate The present invention also uses pyruvate oxidase, FAD, thiamine pyrophosphate, phosphate, and magnesium ion, cobalt ion, manganese ion, and calcium ion. This is an analytical reagent characterized by containing at least one kind of metal ion.

The present invention measures components in a sample, particularly pyruvic acid, but the sample is not limited to human serum, food, and the like. In addition, in the present invention, by directly measuring pyruvate, substances that form pyruvate in the sample, such as α-ketoglutarate, lactic acid ADP, etc., and enzymes that catalyze the pyruvate formation reaction, such as GPT, GOT, lactic acid, etc. It also becomes possible to measure dehydrogenase, pyruvate kinase, etc.

The pyruvate oxidase with excellent thermostability used in the present invention is heat stable up to 45°C when incubated at pH 7.0 for 10 minutes. Any enzyme that catalyzes a reaction that produces carbon and hydrogen peroxide may be used, preferably pyruvate oxidase produced by Lactobacillus nis b. TE6103.

The mycological properties of the isolated and identified strain TE 6103 are shown below.

(a) Morphological size: 0.5 to 0.7 Form small round colonies at 30°C for 48 hours.

The surface is smooth and non-shiny. The color is pale yellow and no soluble pigment is formed.

(2) Meat juice agar slant culture weak growth (3) Meat juice liquid medium It grows by culturing at 30°C for 24 hours and becomes cloudy.

(4) Meat juice gelatin puncture culture It grows along the puncture line. Gelatin does not liquefy.

(5) No change in lid mass production c) Physiological properties (3) Formation of indole; negative (4) Formation of hydrogen sulfide: negative (5) Hydrolysis of starch; negative (6) Formation of pigment: none 7) Utilization of citric acid; Negative (8) Urease; Negative (9) Catalase; Negative Q oxidase; Negative (B) Growth range: Growth temperature 20°C to 45°C (Foundation) Attitude towards oxygen: Facultative anaerobic O-F test; production of acid from fermented sugars L-arabinose, D-xylose, D-glucose, D-mannose: + D-fructose: + D-galactose, malt sugar: + Sugar: +milk
Sugar Neat Releha Ro・Hirosu: - D-Sorbitney D-Mannitol: 10 Inosyl) Neelhamnose: 1 Melibiose: + (C) Other β-galactosidase Negative Arginine dihydrase: Negative Lysine decarboxylase: Negative Ornithine Carboxylase: Negative Tryptone 1-deaminase: Negative Experimental methods for identifying the above mycological properties are mainly described in "Classification and Identification of Microorganisms," edited by Takeharu Hase, Society Publishing Center (
(1975). In addition, as a standard for classification identification, Purging Aids Manual of Determinative Φ Bacteriology, 8th edition (1974) was referred to.

With the above mycological properties, the TK-6103 strain of this bacterium is a Gram-positive bacillus, negative for catalase and oxidase, and produces acid fermentatively from glucose, but does not produce gas from glucose. Determinative Bacteriology 8th Edition (
(1974), it is considered to belong to the genus Lactobacillus. Furthermore, Lactobacillus delbruecii (Llactobacillus delbruecii)
kji), but differences are observed in acid production from D-mannite and melibiose. Therefore, this bacterial strain was named Lactobacillus nis p strain TE6103. This bacterium has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under the microbial accession number 8886.

Examples of microorganisms used in the present invention include the above-mentioned Lactobacillus nis b. TE6103; however, the microorganisms are not limited to this one, and belong to the Lactobacillus genus, and produce pyruvate oxidase with excellent thermostability. can all be used in the present invention. Pyruvate oxidase-producing bacteria with excellent thermostability can use either synthetic or natural media as long as they contain appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients that are used in the usual enzyme production methods. . For example, glucose as a carbon source,
Sucrose, dextrin, pyruvate, molasses, etc. are used. Examples of nitrogen sources include M-containing natural products such as peptone, meat extract, yeast extract, and casein hydrolyzate, and inorganic or organic amino acids such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium citrate, and glutamic acid. Nitrogen compounds are used.

Culture is usually carried out by shaking culture or aerated agitation culture. The culture temperature is preferably controlled in the range of 20 to 40°C, preferably around 30°C, and the culture pH is controlled in the range of 5 to 8, preferably p) 16 to 7. It can be carried out under conditions other than these if the strain used grows.

The culture period is usually 1 to 4 days, and pyruvate oxidase is produced and accumulated within the bacterial cells.

The present enzyme may be purified by any commonly used purification method. For example, the extraction method may include ultrasonic crushing, mechanical crushing using glass beads, a French press, a surfactant, and the like.

Furthermore, for the extract, there are known salting-out methods such as ammonium sulfate and Glauber's salt, metal aggregation methods such as magnesium chloride and calcium chloride, aggregation methods such as protamine and polyethyleneimine, and DEAE (diethylaminoethyl) cephalase.
It can be purified by ion exchange chromatography such as CM (carboxymethyl) Cef7'O-s. The method for measuring the activity of pyruvate oxidase is as follows.

0.15M K-phosphate buffer pH 7,010d0.
3% 4-aminoantipyrine 1go 0.6%
Phenol 17!6mM Thiamine pyrophosphate 1-0.3mM F A D-
NA2 1-150U/-peroxidase 1-30mM EDTA ・Na2
1 dO, 3MMgSO41ml distilled water
3- After preparing the above reaction mixture, take 2.5 tnt into a test tube and add 0.5-tnt of 0.3M potassium pyruvate to a test tube.
Additionally, prewarm at 37°C for 5 minutes. Enzyme solution 0.05
+I! 7! Add and mix gently, then add 3 to the water as a control.
The change in absorbance at 500 nm is recorded with a spectrophotometer controlled at 7°C, and the change in absorbance per minute is determined from the initial linear portion (ΔODtegt).

Blind testing was performed using 50 mMK-! instead of the enzyme solution. Add 0.05 m of J acid buffer pH 7.0 and perform the same operation as above to determine the change in absorbance per minute (△0Dblank).
k).

The display of pyruvate oxidase activity is 1 under the above conditions.
1 unit (U) of activity that produces micromoles of hydrogen peroxide
And so.

Pyruvate oxidase used in the present invention was isolated by the following method.

1. Cultured meat extract 0.2%, polypeptone 1%, yeast extract 0.
4% Tween 800.1% (v/v), K2HPO
4-3H200, 25%, Na-7 cetate 3H2
00.5%, diammonium citrate 2%, MgSO4
-7H200,02%, MnSO4Φ7H200,02
% (pH 7.0) was transferred to a Soomg volume flask and autoclaved at 121°C for 15 minutes. After cooling, a 5% aqueous solution of sodium pyruvate, which had been sterilized by filtration, was added aseptically for 10 minutes. A loopful of Lactobacillus nis bee strain TE6103 was inoculated into the same medium as a seed culture, and cultured with shaking at 30°C for 24 hours to obtain a seed culture solution. Next, 5.4 t of the same medium was transferred to a 10 t jar fermentor, and autoclaved at 121°C for 15 minutes.
After cooling, 600 td of 5% aqueous sodium pyruvate solution, which had been sterilized by filtration, was added aseptically. Add this to 100 seeds of culture solution.
Transfer d, 300 rpm % ventilation f42t/aclimate, 30
℃21. It was cultured for 4 hours. pH is 1 to 6.5 or higher
Controlled with 0 N NaOH. Pyruvate oxidase activity during culture was 23 mU 7 ml-broth.

2. Collect 6 tons of isolation culture using a centrifuge, and add 50mMK-I.
J acid buffer pH 7,01007! Suspended at . The mixture was treated with an ultrasonic crusher (marine electric electrode, 19 KHz) for 15 minutes, and centrifuged to obtain a supernatant. The supernatant was adjusted to 50 mM
DEA equilibrated with K-phosphate buffer pH 7.0
It was subjected to E-Sepharose GL-6B (manufactured by Pharmacia) column chromatography, and pyruvate oxidase activity was determined in the 0 to 0.5M NaCL elution fraction. After concentrating the eluate using an ultrafilter, 50mM K-phosphate buffer p
Gel filtration was performed using Sephadex G-200 (manufactured by Pharmacia) equilibrated with H7.0. The result is 541
Pyruvate oxidase of J was obtained. The pH activity of the obtained enzyme is shown in FIG. 1, and the thermostability is shown in FIG. pH
The activity was measured using 50 mM acetate buffer in the pH range of 5 to 6.5 and 50 mM K-phosphate buffer in the pH range of 6 to 8. Thermal stability was measured at pH 7,
The residual activity was expressed after treatment for 0 and 10 minutes.

The physicochemical properties of the enzyme used in the present invention are shown below.

Action: Catalyzes the reaction that produces acetyl phosphoric acid, carbon dioxide, and hydrogen peroxide from pyruvic acid, inorganic phosphoric acid, and oxygen.

CHsCOC00H+HOPOa2 +02→CHaC
OOPO32+C0r) H202 Optimum pH: around 5.7 Thermal stability: 45°C or less (pH 7, 0, 10 minute treatment) h
Value: Approximately 4X10-4M (pyruvic acid) molecules*: Approximately 16
0,000 (gel filtration method using 5ephacryl S-300) Isoelectric point: approx. 4.4 (Carrier ampholy
Focused electrophoresis method using TC) Substrate specificity: Does not react with oxaloacetate, DL-lactic acid, acetic acid, or α-ketoglutaric acid, but is specific to pyruvic acid.

Foot-after: FAD and thiamine biruphosphate In the present invention, a reagent containing the above-mentioned pyruvate oxidase and inorganic phosphoric acid is added to a sample, and the amount of hydrogen peroxide, carbon dioxide, or acetyl phosphate produced is measured. Alternatively, the amount of pyruvic acid in the sample can be determined by measuring the amount of oxygen or inorganic phosphoric acid consumed. In particular, in the reaction system containing the thermostable pyruvate oxidase of the present invention, in order to perform the enzymatic reaction with pyruvate favorably, F
AD, thiamine biphosphate, phosphate, and at least one metal ion selected from magnesium ions, cobalt ions, manganese ions, and calcium ions may be used, and a detection system for hydrogen peroxide, which is a product of this reaction, may be used. When used, the chromogen may be appropriately selected and used as necessary. As a hydrogen peroxide measurement system,
For example, the following system can be considered.

(1) Peroxidase and color former - peroxidase, thiaminoantipyrine and phenol O peroxidase, thiaminoantipyrine and N,
N-dimethylaniline O peroxidase, methylbenzothiacylinyl hydrazone (IFBTH) and N-ethyl-N-sulfobyl-m-anisidine (ESPAS) 10 peroxidase, thiaminoantipyrine and N-
Ethyl-N-(2-hydroxy-3-sulfopropyl)
-m-) luidine (EMSPT) (2) Hydrogen peroxide electrode method In the present invention, the amount of enzyme used may be any amount that allows the enzymatic reaction to proceed sufficiently, and the content of pyruvic acid in the sample system, The reagent of the present invention may be changed as appropriate depending on the temperature, time, etc. of the enzymatic reaction, but for example, the reagent of the present invention contains pyruvate oxidase with excellent thermal stability, about 0.1 to 20 u/-, phosphoric acid 5
~50mM, FAD 1~20μMW degrees, thiamine pyro-7 osphate around 100~500μM, metal ions around 1~50mM, buffer pH 6~7.5, and 4-aminoantipyrine o, o as a hydrogen peroxide detection system.
o s~0°05%, phenol 0.01~0.1
% and about 1 to 10 u/- of peroxidase. To measure a pyruvate-forming substance or an enzyme that catalyzes the pyruvate-forming reaction, the reagent of the present invention is used in combination with a conventionally known reagent that induces pyruvate.

(Examples) The present invention will be explained below with reference to Examples, but the present invention is not limited by these in any way.

Example I P I FES buffer pH 7,050mMF A D
Na2 10 ttM'thiamine pyrophosphate 0.2mM Mg5O<
10mMKH2PO45mM 4-aminoantipyrine 0.01% phenol 0.02% peroxidase 5u/-pyruvate oxidase 2 LVfRt Reaction solution with the above composition: (Separate d and preheat at 37°C for about 5 minutes, Pyruvine mjJ+) rum aqueous solution (3-15mM) 2
After adding 0 μt, the mixture was reacted at 37° C. for 10 minutes, and the absorbance at 500 nm was measured after the reaction. The result is the third
As shown in the figure, good linearity was obtained.

Example 2 PIFBS buffer, pH 7,050mMFAD/Na2
10μM thiamine bilophosphate) 0.2mM Mg5O<
10mMKHzPO<
5mM Phosphoenolpyruvate II
O, 5mM pyruvate kinase
10u/l117!4-aminoantipyrine
0.01% phenol
0.02% peroxidase s
I)/at pyruvate oxidase 2
u/sg The reaction solution 3- consisting of the above composition was fractionated, and 37
Preheat at ℃ for about 5 minutes, add ADP aqueous solution (3 to 1
After adding 20 pL of 5mM), the mixture was reacted at 37°C for 1°, and the absorbance at 500 nm was measured after the reaction. As a result, good linearity was obtained as shown in FIG.

(Effects of the Invention) In the present invention, by using pyruvate oxidase which has better thermal stability than conventional pyruvate oxidase, an analytical reagent having excellent solution stability, for example, a reagent for measuring pyruvate can be produced. be able to.

Further, by using immobilized oxygen, for example, as electrode membranes, beads, etc., a wide range of measurement systems becomes possible.

[Brief explanation of the drawing]

FIG. 1 shows the optimum pH of pyruvate oxidase of the present invention. The second vl shows the thermostability of the pyruvate oxidase of the invention. FIG. 3 shows a calibration curve for pyruvate using the pyruvate oxidase of the present invention. Figure 4 shows AD using pyruvate oxidase of the present invention.
A calibration curve for P is shown.

Claims (1)

[Scope of Claims] 1. An analytical method characterized by using pyruvate oxidase having excellent thermostability and having the following physicochemical properties. (1) Action: Catalyzes the reaction that produces acetyl phosphoric acid, carbon dioxide, and hydrogen peroxide from pyruvic acid, inorganic phosphoric acid, and oxygen. (2) Substrate specificity: oxaloacetate, DL-lactic acid, acetic acid,
It does not react with α-ketoglutaric acid and is specific for pyruvic acid. (3) Thermal stability: 45°C or less (pH 7.0, 10 minutes treatment) (4) Optimum pH: Around 5.7 (5) Km value: Approximately 4 x 10^-^4M (pyruvic acid) (
6) Molecular weight: approximately 160,000 (Sephacryl
Gel filtration method using S-300) (7) Isoelectric point: approximately 4.4 (Carrier ampho
(8) Cofactor: FAD and thiamine pyrophosphate 2, pyruvate oxidase described in claim 1, FAD, thiamine pyrophosphate, phosphate and magnesium ion, An analytical reagent containing at least one metal ion selected from cobalt ions, manganese ions, and calcium ions.