WO2000018892A1

WO2000018892A1 - Histamine dehydrogenase, process for producing the same, method for quantitating histamine and quantification reagent

Info

Publication number: WO2000018892A1
Application number: PCT/JP1999/005131
Authority: WO
Inventors: Tsuneo Sato; Yasuhiko Imai; Tatsuo Horiuchi; Masaru Suzuki
Original assignee: Kikkoman Corporation
Priority date: 1998-09-25
Filing date: 1999-09-21
Publication date: 2000-04-06
Also published as: AU5654099A

Abstract

A novel histamine dehydrogenase which is usable in quantitating histamine performed over a broad scope including the fields of food industry, marine product industry, food sanitation, medical care and analytical instrument industry; a method for accurately quantitating histamine alone by a simple procedure with the use of the above histamine dehydrogenase neither carrying out any pretreatment for preliminarily eliminating impurities comprising biological amines (cadaverine, putrescine, etc.) nor adding stabilizers; and a quantification reagent therefor.

Description

Description Histamine dehydrogenase, its production method, histamine quantification method and quantification reagent Technical field

The present invention relates to a histamine dehydrogenase that can be suitably used for quantification of histamine performed in a wide field such as the food industry, the fishery industry, the food hygiene, the medical care, and the analytical instrument industry.

In addition, the present invention has high specificity for histamine, does not act (react) on biological amidin such as cadaverine or butrethine, acts well only on histamine, and has an optimum temperature. The present invention relates to histamine dehydrogenase having a temperature of 65 to 75 ° C.

The present invention also relates to histamine dehydrogenase which can be stably stored at room temperature for at least one week after dissolving the enzyme in an aqueous solution without adding a stabilizer.

The present invention also relates to a method for efficiently producing the histamine dehydrogenase.

Further, the present invention provides an open system using a simple apparatus in a short time without using a complicated pretreatment operation for removing interfering substances (impurities) from a sample by using the above histamine dehydrogenase. Also, the present invention relates to a method for measuring extremely small amounts (that is, with high sensitivity) of histamine without deteriorating the reliability of measured values. Further, the present invention can accurately quantify only histamine from a sample in which histamine and other amines are mixed, and can be operated easily, at low cost, in a short time and with high sensitivity. Further, the present invention relates to a histamine quantification method and a quantification reagent capable of quantifying histamine without impairing the reliability of the quantified value. Background art

Histamine is present in almost all tissues of animals and plants as well as bacteria-and histamine plays a role in the biological functions of these cells, such as proliferation, nucleic acid and protein synthesis, and regulation of enzyme activities. Clearly involved Have been.

Also, histamine has been shown to have various physiological effects in the human body, such as lowering blood pressure, smooth muscle contraction, and promoting glandular secretion.Histamine plays a role as a mediator in allergic reactions. Histamine has been regarded as important because it can be used as a clue for diagnosis and analysis of pathological conditions of allergic reactions.

Histamine is an amine having a molecular formula of C ₅ H ₉ N ₃ and a molecular number of 111, and having a chemical structure represented by the following formula (1).

Histamine is hardly present in fresh seafood and meat, but appears during its spoilage and is contaminated by microorganisms with strong histidine decarboxylase activity. It is produced from histidine by decarboxylation as shown in the following formula (1).

( 1 set

CH CHCOOH

_N u histidine decarboxylase

Histi

One CH ゥ CH ₂ NH ゥ

N v, ΝΗ + CO:

Histamine

Histamine is a chemical messenger of allergic reactions that occur in the body, so it is a toxic substance.

Symptoms include redness on the face within minutes to hours after eating, followed by itching, hives and eczema, and in severe cases, urinary measles spread throughout the body, causing bronchitis and a drop in blood pressure. Risk of rubbing.

Blue fish and beef, such as mackerel, bonito, tuna, sardine and horse mackerel, have high free histidine content and are said to be susceptible to histamine poisoning.Histamine poisoning from other protein foods Is reported to occur.

It has been pointed out that histamine poisoning accidents often occur in areas where large amounts of fish and shellfish are consumed, such as in Japan.

To prevent this poisoning accident, it is necessary to pay attention to the quality of seafood, especially freshness.However, even if no mere sensory findings show any abnormality, a significant amount of histamine of 100 to 50 Omg / l 00 g can be obtained. It can be included and is very difficult to manage.

Histamine in food is regulated in major countries of the world, with 5 to 6 mg ZL for beverages such as wine and liquid food, and 10 to 20 mg ZL OO g for seafood. .

For example, the US Food and Drug Administration (FDA), in 1982, adopted histamine as a freshness index and established an official analysis method based on fluorescence analysis.

In other words, if there is 10 to 2 Omg of histamine per 10 Og of canned tuna meat, it is considered that an unsuitable raw material is used, and if it is more than 50 mg per 100 g of canned tuna meat, it is healthy. It is considered harmful.

In addition, the FDA has revised the Compliance Policy / Guide (Comp 1 Alignment Guide 71.08.24) (Federa 1 Register Vol. 60, No. 149, 39754-5). (1 995)). In the United States, the HAC CP mandated a sanitary management system for fishery products in February 1997.

As a result, marine products exported to the United States are also covered, and in Japan, marine products exported to the United States have to take this control.

In FD A, histamine in fish and shellfish is an important control point: Critical Control Point (CCP), and its guidance standard is 50 ppm. Against this background, the need for histamine measurement is increasing in Japan as well.

—On the other hand, it is said that food poisoning caused by histamine occurs more frequently in Japan than in other countries, but there is currently no regulation on the amount of histamine in food.

However, for fishery products exported to the EU region, the regulated value of histamine l O S O mg / l O O g has been set as a chemical index of freshness.

As is clear from the above explanation, the importance of quantification of histamine has increased, and in response to this, measurement of histamine levels in food processing factories, food hygiene monitoring agencies, clinical laboratories, etc. has been simple and quick. There has been a strong demand for the development of a histamine quantification method that can be used.

As described above, FAD has established an official analysis method based on fluorescence measurement as a method for quantifying histamine. Fluorescence analysis is considered to be the most suitable method for the determination of histamine in fish and shellfish, and a similar method is used in Europe.

In the fluorescence analysis method, a fluorescent dye is produced by the condensation of histamine with 0-phthalaldehyde of a fluorescent reagent and histamine, and the intensity of the fluorescence is measured with a fluorescence spectrophotometer.

However, in this method, it is necessary to remove the interfering components before the condensation action, and to clean the sample, so that the time and means for performing cation or anion exchange resin column treatment cannot be avoided. It was something that took.

In general, when fish meat rots, biogenic amines such as cadaverine and butrethsin are produced almost simultaneously with histamine, so when quantifying histamine, biological amines other than histamine must be separated and removed. Have inconvenience.

There have been many studies on histamine quantification methods using chromatography.However, thin-phase chromatography and paper chromatography can measure many samples simultaneously using relatively inexpensive measuring equipment, but their quantitative performance is not sufficient. In gas chromatography, direct determination of non-volatile amines such as histamine is impossible, and it has been pointed out that the inconvenience of quantification after conversion to heptafluoroloptyril derivatives and the like has been pointed out. Recently, the use of histamine as an indicator of food freshness has been studied in Japan, and it is described in food analysis books as the best method for quantification by high performance liquid chromatography (HPLC).

For example, Yamanaka et al. Are conducting research on the determination of histamine in lean fish by the HPLC method (see the Japanese Journal of Food Science, 30, 377-400 (1989)).

In this study, a highly sensitive separation and quantification of seven simultaneously produced amines was an excellent scientific achievement.

However, this method requires complicated pretreatment operations, requires sophisticated equipment for measurement, and takes about 30 to 60 minutes for chromatographic operation.

In addition, as a method for quantifying histamine, an enzyme electrode coated with a monoamine oxidase-immobilized membrane is immersed in a meat extract containing histamine or other putrefactive amines, and changes in dissolved oxygen are measured to determine histamine. A method has been proposed (Ka rubeeta 1, Enzyme Micr ob. Tecn l. Vo l. 2, pp. 117-120, 1980).

However, this method has the disadvantage that it is not accurate (relative error is about 8%) because it measures the local dissolved oxygen concentration (DO) of the test solution.

As a method for quantifying histamine, histamine is quantified based on the amount of dissolved oxygen (DO) produced by adding a microbial enzyme reagent having histamine oxidase activity to a histamine-containing sample. A method is also known (see Patent 2717745, "Method for rapid quantification of histamine").

Compared to the conventional fluorescence analysis and HPLC method, this method does not require complicated troublesome substance removal operations or calibration operation using histamine standard solution, and has a response time of 1.5 minutes. As a simple and fast method, and also as an excellent method with high accuracy (0.98% relative error), it has already received good internal evaluation from FDA (Food Chemical News page 1 9, 1 ine 5-17, Sep. 26. 1994).

However, in this method, the output current of the oxygen electrode and the concentration of saturated dissolved oxygen are susceptible to temperature (temperature-dependent). All processes, including the air saturation operation, must be performed at a constant temperature (37 ° C). Therefore, a reactor equipped with a hot water jacket with thermostat to maintain the temperature of the working cell at a constant level is required. Had problems.

Also, the working cell equipped with the DO electrode can be difficult to measure DO accurately if any air bubbles (air) are mixed in it. In addition, the working cell had the inconvenience of being repeatedly used and having to be cleaned each time (that is, the disposable working cell cannot be used).

As described above, as a method for quantifying histamine, a method for quantifying histamine based on the decrease in dissolved oxygen (D 0) generated by adding an enzyme reagent having histamine oxidase activity derived from a microorganism to a histamine-containing sample is also known. Known (Patent 27177745) Powerful, but no method for quantifying histamine using histamine dehydrogenase is known.

On the other hand, enzymes that oxidatively decompose amines such as histamine include aminoxidase and amindehydrogenase.

Of the former enzymes, those that act on histamine are powerful, widely existing from microorganisms to higher animals, and many have broad specificities, and act on other amines to some extent.

The latter enzyme is a dehydratase that requires an electron acceptor other than oxygen to express the activity of aminolysis, but there are few reports of such enzymes.

The power known from the genus Pseudomonas (RRE ady eta 1.Biooch. J., 106, 245-255 1968) to date has been reported to date. In addition, this enzyme acts strongly on histamine and considerably affects other amines (see “Comparative Examples” in Table 1 “Substrates and Relative Activities” below).

Therefore, there is a difficulty in accurately quantifying only hissamine from a sample in which histamine and other amines are mixed.

In addition, microorganisms of No cardioidessimlex IF 0 1 20 67: (1) specifically degrade histamine, and other than histamine are vulnerable to agmatine It has no effect on other aromatic 'aliphatic monoamines, diamines and polyamines. (2) Both optimum pH and stable pH have 8.5, and (3) Km for histamine It is known to produce amine dehydrogenase, a dimer of the same subnet with a value of 0.095 mM, a molecular weight of 170,000 and a molecular weight of 84,000. (98, Japanese Society of Agricultural Chemistry, Abstracts of the meeting, p. 335). However, the optimum temperature and temperature stability range of this enzyme are not known. Also, enzymes are generally very unstable in aqueous solutions.

In addition, it is quickly deactivated at room temperature, and must be kept refrigerated or frozen. Therefore, each time the enzyme is used, the enzyme is used by dissolving the required amount in an aqueous liquid, and once prepared, it has a problem that it must be used within a short period of time.

Therefore, histamine dehydrogenase usually has a disadvantage that long-term stability cannot be ensured in an aqueous solution unless a stabilizer is added.

Therefore, the present invention is very useful for the enzymatic quantification method of histamine, and it is possible to measure histamine easily and simply with an unprecedented operation, and ③ it is possible to measure histamine with high accuracy. Histamine dehydrogenase that is stable for an extremely long time even after dissolving in a buffer solution and making it an aqueous solution without adding, ⑤ histamine dehydrogenase having high temperature stability, and ⑥ of the amines in the sample, A histamine that has high specificity for histamine, that is, it does not act on biological amides such as cadaverine and butrethsin, but acts only on histamine for the purpose of detection and can quantify it. It is an object of the present invention to obtain dehydrogenase and (1) to provide an efficient production method thereof.

Another object of the present invention is to provide a method for quantifying histamine using histamine dehydrogenase and a reagent for quantifying histamine, and the method for quantifying histamine comprises: It does not require fractionation or troublesome pretreatment to remove interfering substances (impurities). ⑩The time required for quantification is short, and quantification can be performed with extremely high accuracy. It can be measured using an instrument. (5) The working cell also has the advantage that it does not need to be operated under liquid-tight conditions (that is, it can be operated in an open system). Disclosure of the invention

The present inventors have conducted various studies in order to achieve the above object, and as a result, (1) the strain belonging to the genus Rhizobium does not act on cadaverine-putrescine and the like among the amides, but acts only on histamine. And it was found that histamine dehydrogenase having an optimum temperature of 65 to 75 ° C was produced. And (2) the enzyme is stable for an extremely long period of time even after dissolving in a buffer solution to form an aqueous solution without adding a stabilizing agent; (3) it has high temperature stability. It is extremely useful for enzymatic quantification of histamine, and histamine can be easily and accurately measured with simpler operation than ever before. When histamine or histamine salt was contained, it was found that the enzyme could be produced and accumulated in a considerable amount.

Further, the present inventors have determined that histamine can be quantified by allowing histamine dehydrogenase to act on a histamine-containing sample and measuring the product, and that histamine dehydrogenase can be added to the histamine-containing sample. Histamine can be accurately quantified by measuring reduced electron carrier, 4-imidazolyl acetoaldehyde or ammonia produced by the action of glycerol, and ⑨ A method for quantifying these histamine When using histamine dehydrogenase which specifically acts on histamine, it is necessary to accurately quantify only histamine from a sample containing both histamine and other amines. When enzymatically quantifying histamine in fish and shellfish and animal meat with reduced freshness, When using histamine dehydrogenase, which acts on min but not on cadaverine and putretsin, the cadaverine and the putrescine are not removed by fractionation (separation) of these fish and shellfish, We have obtained knowledge that can quantify histamine in livestock meat.

The present inventors have completed the present invention based on these findings.

That is, the first invention of the present invention is histamine dehydrogenase which specifically acts on histamine and has an optimum temperature of 65 to 75 ° C.

Further, histamine dehydrogenase having the following physicochemical properties is preferable. PT 99/5131 Action: One mole of histamine is produced by oxidative deamination in the presence of an electron acceptor to produce one mole of 4-imidazolyl acetate aldehyde and one mole of ammonia.

Substrate specificity: Acts specifically on histamine.

Optimal temperature: 65-75 ° C.

Conditions for deactivation by temperature: pH 8.0, treatment for 15 minutes, stable at 0 to 60 ° C.

Molecular weight: approx. 150, 000 (subunit approx. 71, 0000 X 2)

Further, the second invention is characterized by culturing a strain belonging to the genus Rhizobium and having the ability to produce histamine dehydrogenase in a medium, and collecting the histone dehydrogenase from the culture. A third invention is a method for producing a drogenase.The third invention is to cultivate the strain belonging to the genus Rhizobium and having the ability to produce histamine dehydrogenase in a medium containing histamine or histamine salt, and This is a method for producing histamine dehydrogenase, which is characterized in that the histamine dehydrogenase is collected from a product.

Further, a fourth invention is a method for quantifying histamine, which comprises causing a histamine-containing sample to act on histone dehydrogenase and measuring the product.

Further, the fifth invention is directed to making histamine-containing hydrogenase act on a histamine-containing sample, and measuring the resulting reduced electron carrier, 4-imidazolyl acetoaldehyde or ammonia. This is a special method for quantifying histamine.

Further, the fifth invention is characterized in that histamine dehydrogenase is added to a histamine-containing sample in the presence of an electron carrier and a reducing chromogenic reagent to carry out an enzymatic action, and to quantify the dye produced. This is a method for quantifying histamine.

A sixth invention is a reagent for quantifying histamine, which comprises (a) histamine dehydrogenase, (b) an electron carrier, and (c) a reduced-type electron carrier coloring agent. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the optimum pH of the present enzyme, Fig. 2 shows the stable pH range of the present enzyme, and Fig. 3 shows the optimum temperature range of the present enzyme. Figure 4 shows the yeast FIG. 5 is a diagram showing the thermostability of JP 5 nitrogen, and FIG. 5 is a diagram showing a calibration curve used for a histamine quantification method using the present enzyme. FIG. 6 is a graph showing a correlation between the amount of histamine contained in the canned mackerel meat boiled in mackerel by the quantitative method of the present invention and the conventional HPLC method. Best form.

First, as the histamine dehydrogenase used in the quantification method of the present invention, 1 mol of histamine was reacted with 1 mol of 4-imidazolylacetaldehyde and 1 mol of ammonia by oxidative deamination in the presence of an electron acceptor. Any enzyme having the activity of producing

By allowing histamine dehydrogenase to act on a histamine-containing sample and measuring the product, histamine can be quantified efficiently.

In addition, histamine can be quantified with high accuracy when histamine dehydrogenase is allowed to act on a histamine-containing sample and the resulting reduced electron carrier, 4-imidazolylacetoaldehyde or ammonia is measured.

In addition, histamine dehydrogenase is added to a histamine-containing sample in the presence of an electron carrier and a reducing chromogenic reagent to perform enzymatic action, and the resulting dye is quantified.The histamine is very easily and accurately quantified. it can.

In addition, in the method for quantifying histamine using histamine dehydrogenase, if histamine dehydrogenase which specifically acts on histamine is used, only histamine can be obtained from a sample in which histamine and other amines are mixed. Can be accurately determined.

In addition, when foods such as fish meat spoil (decrease in freshness), cadaverine and butrethsin are produced and accumulated at the same time as histamine. When using histamine dehydrogenase, which acts strongly on histamine but does not act on cadaverine and putrescine, in the quantification of histamine contained in such foods, cadaverine and putrescine are fractionated (separated). Histamine can be selectively quantified without removal.

The enzymes used here include, for example, the following developed by the present inventors. A novel histamine dehydrogenase having physicochemical properties (hereinafter sometimes referred to as “the present enzyme”) can be mentioned.

The details of the physicochemical properties of this enzyme are as follows.

(Action)

Oxidative deamination of 1 mole of histamine in the presence of an electron acceptor produces 1 mole of 4-imidazolyl acetoaldehyde and 1 mole of ammonia.

From this result, it was recognized that the catalyst represented by the following reaction formula was catalyzed.

Equation (2)

Hiss Evening Oxidation Type

Four

Huena Sinme

Sulfate

4-imidazolyl reduced type

Acetaldehyde phenome

—

(Substrate specificity)

Acts specifically on histamine. That is, it acts specifically on histamine, but has no or weak effect on other proteins.

Table 1 shows the results of examining the relative activities of this enzyme for various substrates. Table 1 Substrate and relative activity (%) Substrate (10 mM) Comparative example of this enzyme (Note 1) Tyramine 0 0.4 Putretsucin 0 1 0 5.0 Serotonin 0

Davelin 0 6 0.3 Histamine 1 0 0. 0 1 0 0.0 Agmatine 9.9

Phenylethylamine 0

Spermidine 0 5 0.6 tryptamine 0 0 spermin 0 1 3.4

1,2-cyclohexanediamine 0

1, 3—propanediamine 1 2.9 2 8 1.3

1,4-cyclohexanediamine 0

1,6-hexanediamine 0 1 6 0.0

To 1, 7— "7" Tannona:; No 0

1, 8—Jamino octane 0

1, 9—Diaminononane 0

1, 10—Diaminodecane 0 Note 1) Comparative example: Pseudomonas spp. Amide dehydrogenase (RR E adyetal. Biochem. J., 106, 245-5, 255, 195, 688) Numerical values were obtained by calculation).

(Optimal pH)

The optimum pH was determined by measuring the activity of the enzyme at each ρΗ at 30 ° C using a Plitton-Robinson wide area buffer (ρΗ2 to Γ1.5) as a buffer.

Enzyme activity was measured using the following buffer: 2.4 mK 0.3 mM 1— Methoxyl 2 Mix 0.3 ml of PMS 0.1 m 1 mM WS T-8, pre-incubate at 30 ° C, then add 0.1 mM 1 of 10 mM histamine solution and 0.1 ml of this enzyme solution. The reaction was carried out at 30 ° C., and the reaction was carried out by measuring the amount of increase in reduced 11-Meth0Xy PMS generated in 60 minutes.

That is, the color development of WST-8 produced by the reaction with reduced 1-Methoxy PMS was measured by absorbance at 460 nm, and the increase in reduced 1-Methoxy PMS was measured.

FIG. 1 is a graph showing the optimum pH of the present enzyme. As shown in this figure, the optimum pH of the present enzyme is 9.0 to 11.5.

(Stable pH range)

The stable pH range was as follows: using a Plitton-Robinson wide area buffer (pH 2 to 11.5) as a buffer, treating at 30 ° C for 15 minutes at each pH, and remaining the activity of this enzyme at each pH. Was measured and determined.

FIG. 2 is a graph showing the stable pH range of the present enzyme. As shown in this figure, the stable PH range of the present enzyme is 7.0 to 11.5.

And it is almost completely deactivated at pH 4 or lower.

(Optimum temperature)

The enzyme activity of the present enzyme was measured at various temperatures (30 to 80 ° C) using the same substrate-enzyme mixture in the titer measurement method described later.

That is, 50 mM phosphate buffer (pH 8.0) 2.4 ml. 0.3 mM 1-Methoxy PMS aqueous solution 0.1 m 1 mM WST— 8 0.3 ml and lOmM histamine 0.1 ml of the enzyme solution, pre-incubation at a predetermined temperature, add 0.1 ml of the enzyme solution, react at a predetermined temperature, and form reduced 1-meth The activity of the enzyme was measured by measuring the increase in 0Xy PMS.

FIG. 3 is a graph showing the range of suitable working temperature of the present enzyme. As shown in this figure, the range of suitable working temperature of the present enzyme is 65 to 75 ° C.

(Deactivation condition by temperature)

Use the substrate / enzyme mixture in the titer measurement method described below at various temperatures. After treatment (pH 8.0) for 10 minutes, the residual activity of the enzyme was measured and determined.

Figure 4 is a graph showing the thermostability of this enzyme. As shown in this figure, this enzyme is stable up to around 60 ° C.

Above that, it is rapidly deactivated.

(Molecular weight)

The molecular weight of this enzyme was measured by high-performance liquid chromatography using a TSK-Ge1G30000 SW column (manufactured by Tosoh Corporation). The resulting molecular weight was estimated to be about 150,000. Also, the SDS polyacrylamide electrophoresis method was used to estimate the number of subunits of this enzyme to be about 71,000.

(Km value)

The Km value is 0.067 mM (pH 8.0) (relative to histamine) from the Linweber-Burk plot.

(Method of measuring titer)

The titer of the enzyme is measured by the following method, and the amount of the enzyme that produces 1 im01 of 41-imidazolylacetoaldehyde per minute is defined as 1 unit (1 U).

50 mM phosphate buffer (pH 8.0) 2.4 ml, 0.3 mM 1- Methoxy PMS aqueous solution 0.1 ml 1, 1 mM WST-8 aqueous solution 0.3 ml 1, 10 mM 0.1 ml of histamine solution and 0.1 ml of the present enzyme solution were added, and the mixture was reacted at 30 ° C. for 30 to 60 minutes.

The activity of the enzyme is the reduced form of the enzyme generated in the enzyme reaction.

The color development of WST_8 produced by reaction with PMS was measured at an absorbance of 460 nm. (Purification method)

Isolation and purification of this enzyme can be carried out according to a conventional method, for example, ammonium sulfate precipitation, organic solvent precipitation, adsorption treatment with an ion exchanger, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, and the like. Adsorption chromatography, affinity chromatography, electrophoresis, etc. can be used alone or in combination as appropriate.

Although the present enzyme used in the present invention has been described above, the present enzyme may have any of the above-mentioned functions and major physicochemical properties such as substrate specificity. Even those showing slight differences in quality are included as enzymes of the present invention.

This enzyme is extremely useful for the determination of histamine in fish to be measured and the determination of trace amounts of histamine in body fluids such as human serum and urine when measuring freshness of fish meat. is there.

By using this enzyme, it does not act on cadaverine-putrescine which does not need to be detected among the various amines contained in the fish meat or body fluid to be measured, and acts well only on the target histamine. This can be quantified accurately.

Next, a method for producing histamine dehydrogenase which specifically acts on histamine will be described.

The microorganism used here may be any microorganism as long as it is a strain having the present enzyme-producing ability, and may be a variant or mutant of this microorganism. As specific examples of the microorganism, any microorganism belonging to the genus Rhizobium can be mentioned.

For example, Rhizobium sp. (Rhizobbiumsp.) 419 (hereinafter sometimes referred to as "the present strain") can be mentioned, and a variant or mutant of the strain can also be used. This Rhizobium sp. 4-9 is a strain obtained by the present inventors by separating it from soil in Chiba Prefecture, and its bacteriological properties are as follows. Experiments for identification of mycological properties were mainly performed by Takeji Hasegawa, "Classification and Identification of Microorganisms", University of Tokyo Press (1975).

Also refer to “Bergey's Manu alof Determinative B acteriology”, 8th edition (1974), as a standard for classification identification. I made it. In addition, for phylogenetic analysis based on the base sequence of 16 S rDNA, the DNA database of the Japan DNA Data Bank was used.

Mycological properties of Rhizobium sp. (Rhizobbiumsp) 4-9 (A) Morphological properties

Microscopic observation [Bennet's medium (PH 8.0), culture at 30 ° C for 24 to 48 hours] a) Cell shape and size: 0.5 to 0.6 X.1.2 to 2.3 zm Oval bacilli o b) Cell polymorphism: None.

c) Mobility: Yes. Two to four flagella are observed.

d) Spore presence: None.

e) Gram staining: negative.

f) Acid resistance: negative.

(B) Growth status in each medium

a) Bennett agar culture: A round colony with a diameter of 1.5 to 2.5 mm is formed by standing culture at 30 ° C for 60 hours. The colonies have a whitish cream color and the surface is slightly raised in the center, shiny and produces a viscous substance. No pigment production is observed.

b) Bennett's liquid culture: In static culture at 30 ° C for 24 hours, it is slightly turbid and filamentous growth can be seen at the bottom.

In shaking culture, the entire medium becomes turbid.

c) Broth gelatin stab culture: grows along the puncture, but does not liquefy gelatin. _c d) Litmus milk culture: unchanged.

(C) Physiological properties

a) Nitrate reduction: Reduce.

b) Denitrification reaction: None.

c) MR test: negative

d) VP test: positive

e) Indole generation: Not generated.

f) Production of hydrogen sulfide: No production.

g) Starch hydrolysis: no hydrolysis.

h) Utilization of cunic acid: Use.

i) Use of inorganic nitrogen source: Uses nitrate, but does not use ammonium salt. j) Pigment formation: No formation.

k) Perase: positive

1) Oxidase: negative

m) Power cod: weak positive n) Range of growth: temperature 4-40 ° C, pH 5.5-9.0

o) Attitude towards oxygen: aerobic

p) 0—F test (Hugh—Leifson method): Oxidation

q) Decomposition of esculin: decomposed.

r) Deoxyribonuclease: negative

s) Tyrosine degradation: Does not degrade.

t) Casein decomposition: No decomposition.

u) Phenylalanine demineralization: negative

V) Tryptophan deaminase: negative

w) Generation of acids and gases from sugars: As shown in Table 2, acid formation from sugars other than D-merezitose is observed. No gas production is observed. Table 2 Generation of acid and gas from sugars Generation of acid Generation of gas

L-arabinose +

D—Xylose +

D—glucose +

D—Mannose +

D—Fructose +

D—galactose +

+

Sucrose +

Lactose +

Trenoperose +

D—sorbitol +

D—mannitol +

Inositol + Glyce mouth one hole +

+

D—Ribose +

Laugh Innos +

D—Selbiose +

Rhamnose +

Melibiose +

D-meresitrose This strain was determined to belong to the genus Rhizobium based on the above bacteriological properties.

In addition, phylogenetic analysis based on the base sequence of 16S rDNA was performed to estimate the taxonomic position of this bacterium, and it was confirmed that this strain was a bacterium belonging to the genus Rhizopium.

The strain is Rhiz ob ium 1 e gum i no sar um, R hiz ob i um et 1 i, Rh iz ob i um tropici, Rh iz ob i um hain an ensis, Rh iz ob i um mongo It was located in the same cluster as lense, Rhiz ob i um ga llicum. However, this strain forms a single branch in this cluster, and no related species could be estimated from the nucleotide sequence of 16 SrDNA.

In addition, when the results of the property tests of this strain were compared with those of the genus Rhizobium, the properties of this strain were different from those of any species.

For this reason, the strain was named Rhizobium 'SP (Rhizobbiumsp.) 419. This strain was submitted to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (1-1-1, Higashi, Tsukuba, Ibaraki Prefecture) on September 14, 1998 by FE RM P-16992 (FERM). BP-686 1). Next, a method for producing the present enzyme will be described.

The present enzyme is not limited by the type of microorganism, the culture method, and the conditions for producing the enzyme, as long as the purpose of the present invention is not particularly inhibited. In other words, any culturing method and any culturing conditions that provide an environment capable of growing a microorganism having the present enzyme-producing ability, for example, a microorganism belonging to the genus Rhizobium having the present enzyme-producing ability and producing the present enzyme can be employed.

As a culture method, a usual solid culture may be used, but a liquid culture method is preferable.

As the medium, any of a synthetic medium, a natural medium, and a semi-synthetic medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients appropriately.

The carbon source may be any assimilable carbon compound, for example, maltose, glucose, glycerin, fructose and the like.

As the nitrogen source, histamine or a histamine salt is desirable because the enzyme is induced and generated by histamine, but any nitrogen source that expresses the enzyme can be used. For example, yeast extract, polypeptone, meat extract, cornsprika, soy flour, amino acids, ammonium sulfate, ammonium nitrate and the like are used.

When histamine or histamine salt is added to the culture medium as a nitrogen source, the production and accumulation amount of the enzyme can be significantly increased. As the histamine salt, histamine hydrochloride and histamine phosphate are preferable.

Further, as the inorganic substance, various salts such as salt, potassium chloride, potassium phosphate monobasic, and potassium phosphate phosphate are preferred.

As other nutrients, various vitamins and the like can be used. Each of these nutrients can be used alone or in combination

In addition, you may add an antifoamer etc. as needed.

In order to produce the present enzyme using the liquid medium thus prepared, it is preferable to carry out aerobic culture by aeration and stirring submerged culture or shaking culture. At that time, the initial pH of the culture medium was adjusted to about 6.0 to 7.0, and at a temperature of about 25 to 37 ° C, preferably about 30 ° C, for 24 to 96 hours, preferably 4 Incubate for about 8 hours.

This culture produces and accumulates the present enzyme in the culture.

In order to collect the present enzyme from this culture, ordinary enzyme collecting means can be used. PT

Since the present enzyme is mainly present in the cells, it is preferable to separate the cells from the culture by, for example, filtration or centrifugation, and to collect the cells from the cells. In this case, the ability to use the cells as they are <<For example, a method of destroying the cells using various mechanical crushing means such as an ultrasonic crusher, French press, dynamill, etc. And a method of extracting an enzyme from cells using a surfactant such as Triton X-100 can be used alone or in combination.

Next, this is removed by filtration or centrifugation to obtain a crude enzyme solution of the present enzyme.

In order to isolate and purify the present enzyme from the crude enzyme solution thus obtained, the above-mentioned purification method can be applied.

The isolation and purification of this enzyme can be carried out according to a conventional method, for example, ammonium sulfate precipitation, organic solvent precipitation, adsorption treatment using an ion exchanger, ion exchange chromatography, hydrophobic chromatography, gel filtration. Chromatography, adsorption chromatography, affinity chromatography, electrophoresis and the like are used alone or in an appropriate combination.

Next, the reagent for quantifying histamine of the present invention comprises (a) histamine dehydrogenase which specifically acts on histamine, (b) an electron carrier and (c) a reduced-type electron carrier coloring agent. It is a thing.

As an advantageous system for quantifying histamine, for example, as reaction reagents: (1) PH containing 0.3-0.3 U / m1 histamine dehydrogenase and 10-200 mM buffer 8 to 10 system, ② 0.003 to 3.0 mM O-electron carrier (for example, 10 M ethoxy PMS) and pH 8 to 10 containing 10 to 20 mM buffer Combinations of systems and pH 8-10 containing 10 to 20 mM buffer containing 0.1 to 10 mM WST-8 and ③.

Buffers used in these systems include, for example, phosphates such as potassium phosphate, tris-hydrochloride, acetate and the like.

To such a system, in addition to the above components, various conventional additives such as a solubilizing agent and a stabilizing agent may be added as necessary within a range not to impair the purpose of the present invention. Can also.

Specific examples include surfactants (Triton X-100, Bridge 35, Tween 80, cholic acid, etc.), reducing agents (mercaptoethanol, dithiothreitol, L-cysteine). ), Bovine serum albumin, sugars (glycerin, lactose, sucrose, etc.) can also be added.

These components may be added at an appropriate stage during the preparation of the above-mentioned system, and may be used alone or in combination of two or more.

The reagent of the present invention may be used in a dried or dissolved state, or may be impregnated in a thin-film carrier such as a sheet-impregnated paper.

The enzyme used may be immobilized by a conventional method and used repeatedly.

By using such a reagent of the present invention, histamine contained in various samples can be accurately quantified by a simple operation.

Next, the histamine quantification method of the present invention is, as described above, a histamine dehydrogenase which specifically acts on histamine in a histamine-containing sample but does not act on impurities such as cadaverine and putrescine. Is added and allowed to act, and the resulting product is measured. The histamine-containing sample may be any sample containing histamine (or a salt thereof), such as liquid and solid foods, biological substances such as urine and plasma, and biological tissues. And the like. Then, the sample is directly extracted or filtered with water, a buffer, or the like, and then concentrated to an appropriate concentration of histamine, or diluted with water, alcohol, a buffer, or the like, and quantified. May be served.

For quantification, the pH of these samples may be unadjusted, but the pH can be adjusted using an appropriate pH adjuster, for example, acid or alkali such as hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide, potassium hydroxide, etc. It is desirable to adjust to ~ 10.

The amount of the present enzyme to be applied to the histamine-containing sample is appropriately selected depending on the histamine content contained in the sample, the enzyme action conditions, and the like. 0 Add 3 to 3 U / m 1.

The enzyme can act on a histamine-containing sample, and any means can be used to measure the resulting product. A preferred method is to measure the reduced electron carrier, 4-imidazolyl acetoaldehyde, ammonia or the like generated by the action of the electron carrier. In this case, the temperature at which the present enzyme is allowed to act is 20 to 70 ° C, preferably 30 to 50 ° C. The action time at this time may be a time sufficient for decomposing the histamine of the histamine-containing sample, and is 1 to 60 minutes, preferably 2 to 20 minutes.

After completion of the operation, the means for quantifying the content of the reduced electron carrier, 41-imidazolylacetoaldehyde, ammonia and the like generated in the working solution is not particularly limited, and the measurement is performed using a known measuring means.

Then, quantification of histamine in the sample is performed using the histamine calibration curve prepared in advance by the same method.

As a method for quantifying reduced electron carriers, for example, histamine-containing samples can be used for tetrazolium-based electronic carriers such as phenazine meth- ol sulfate and meldable, and MTT, Nitro-TB, WST-8, etc. A method of quantifying the resulting dye by adding histamine dehydrogenase to the enzyme in the presence of a tetrazolium-based reducing chromogenic reagent is considered.

As a method for quantifying 4-imidazolyl acetoaldehyde, for example, 4-imidazolyl acetoaldehyde is allowed to react with aldehyde dehydrogenase, and a conjugation action at this time, that is, oxidized nicotinamide amide is used. Nucleotide (NAD +) → A method of measuring the amount of NADH produced by reduced nicotinamide adenine dinucleotide (NADH) by increasing the absorbance at 340 nm is considered. In addition, various known methods such as a ninhydrin action method, an indanol blue absorption spectrophotometric method, and an ion electrode method can be used for the method for determining ammonia. Next, a preferred example of the method for quantifying histamine of the present invention will be described.

First, to a sample containing histamine was added 0.003 to SUZml of histamine dehydrogenase, an electron carrier and a buffer of 10 to 200 mM, and a pH of 8 to 10 and a temperature of 3 to 10 mM. Enzymatic action at 0-50 ° C. The action time at this time may be a time sufficient to decompose histamine, and is 1 to 60 minutes, preferably 2 to 20 minutes. Next, the content of the generated reduced electron carrier, 41-imidazolyl acetoaldehyde, ammonia and the like was quantified by the above-mentioned method. Calculate the quantitative value of histamine in the sample using the calibration curve of histamine prepared in advance by the same method.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by these examples.

Example 1

(Method for producing histamine dehydrogenase)

Consists of glucose 1% (WZV), yeast extract 0.2% (WZV), histamine dihydrochloride 0.1% (W / V), K2HPO4 0.05% (W / V) and tap water 100 ml of a medium (pH 6.75) was placed in a Sakaguchi Kolben, and sterilized at 120 ° C for 15 minutes to prepare a medium. This was prepared for two tubes.

Inoculate one loopful of Rhizobium sp. (Riziz bibum sp.) 4-9 (F ERM BP—6861) into each, and incubate at 70 ° C for about 72 hours at 30 ° C. A seed culture was prepared by culturing with shaking at a shaking frequency of 140 rpm.

Then, the medium was sterilized in the same manner as described above, and 20 liters (L) of the prepared medium was placed in a 30-L jar arm mentor. ) Was aseptically inoculated and cultured under aeration and agitation for 48 hours under the conditions of 30 ° C., a rotation speed of 200 rpm, and an aeration of 10 LZ min.

After completion of the culture, 20 L of the culture solution was collected using a Microza (manufactured by Asahi Kasei Kogyo Co., Ltd., ultrafiltration membrane, registered trademark), and placed in 5 OmM phosphate buffer (pH 8.0). After washing the cells, the cells were suspended in about 1 L of the same buffer.

Next, the enzyme was purified by the following method.

Step 1: Preparation of crude enzyme solution

To the cell suspension were added 0.5%, 0.1% (W / V), and 2 O mM of Triton X—100, lysodium, and £ 0, respectively, and mixed. I left it.

Thereafter, the mixture was centrifuged (800 rpm, 60 min), and the supernatant was collected to prepare a crude enzyme solution.

Step 2: Ammonium sulfate fractionation

Ammonium sulfate was added to the crude enzyme solution, and the protein that precipitated at 40-60% saturation was collected by centrifugation (800 rpm, 60 min). The resulting precipitate was washed with 2% OmM phosphate buffer containing 13% (W / V) ammonium sulfate (P

H 7.0).

Step 3: Petilto Pearl 650 · Chromatography

The above enzyme solution was adsorbed onto a column of Putyltoyopearl 6.5 × 50 cm (2.5 × 30 cm), and then a 20 mM phosphate buffer solution containing 13% (W / V) ammonium sulfate ( pH 7.

0), and then 20 mM phosphate buffer (pH 7.0) containing 13% (W / V) ammonium sulfate and 20 mM phosphate buffer (pH 8 Using 5), the active fraction eluted with a phosphate buffer containing about 7 to 9% (W / V) ammonium sulfate was collected by the linear concentration gradient method.

Ammonium sulfate was added to the mixture to achieve a saturation of 60%, and the mixture was allowed to stand at low temperature for a long time. Then, the precipitate obtained by centrifugation (800 rpm, 60 min) was used to remove 20 mM tris-hydrochloric acid. The cells were dissolved in a buffer (pH 8.0). The enzyme solution was dialyzed against the buffer using a dialysis membrane.

Step 4: DEAE—Sephacel chromatography

The dialysate is adsorbed to the column (2.5 x 30 cm) packed with DEAE-Sephacel, and washed with 20 mM Tris-HCl buffer (pH 8.0). Then, elute with a phosphate buffer containing 0 M to 0 M sodium chloride by the linear concentration gradient method, and collect the active fraction eluted with a phosphate buffer containing about 0.4 M sodium chloride. Was.

(Enzyme purification sample)

The active fraction obtained by the above purification procedure was judged to be almost homogeneous by SDS-polyacrylamide gel electrophoresis, and was confirmed to be a purified sample.

This preparation had a total protein content of 1.06 mg, a total activity of 6.64 U, and a specific activity of 6.26 UZmg.

Example 2

(Stability test of solution histamine dehydrogenase)

The histamine dehydrogenase obtained in Example 1 above was added and dissolved in 50 mM Tris-HCl buffer at pH 9 to a concentration of 0.03 U / m1, and stored at 25. After 4 and 7 days, the activity was measured. Assuming that the activity of the first onset (day 0) was 100%, the residual activity after 4 days was 88% and that after 7 days was 80%, indicating that the activity was very stable.

Example 3

(Example of preparation of reagent for histamine determination)

The following three components were dissolved in purified water at the following concentrations or units, respectively, to prepare histamine quantification reagents comprising the three components. Table 3 Component composition of histamine determination reagent Component Concentration or unit Tris-HCl buffer (pH 9) 50 mM 2.4 mM l-Methoxy PMS (Note 1) (Electron carrier)

0.3 mM 0.1 ml

WST-8 (Note 2) (Reduced electron carrier—color former)

1 mM 0.3 ml Note 1: 1-Methoxy-5-methylphenazine sulfate

Note 2: 2 (2-Methoxy-14-nitrophenyl) 1-3- (4-Trophenyl) 1-5— (2,4-Disulfophenyl) 1-2H-Tetrazolium 1 sodium salt

Example 4

(Quantitative method for histamine, in which histamine dehydrogenase, which acts on histamine-containing samples as an enzyme, specifically acting on histamine but does not act on davaverine and putretsin), and measures the reduced electron carrier generated.

A histamine quantification reagent (reaction reagent solution) was prepared by mixing the three components shown in Table 3 immediately before measurement.

Using this histamine quantification reagent, histamine was quantified at a known concentration.

First, add 0.1 ml of each concentration of histamine standard solution to 2.8 ml of the reaction reagent solution. Incubated at 37 for 5 minutes. This is dispensed into a 96-well microplate at a rate of 200 μl at a time, and then histamine dehydrogenase (0.03 UZm 1) is added at 100 ° C., and the mixture is added at 37 ° C. for 3 hours. Acted for 0 minutes.

Then, after the action was started, the absorbance at 49 O nm was measured over time using a plate reader, and the value of the increase in absorbance (AOD) was determined.

A calibration curve was created from the relationship between this value (Y) and the histamine content (X).

Fig. 5 shows the calibration curve.

The equation of the calibration curve is as follows: y = 0.4755-0.00.016 (r = 0.9999).

This shows that there is a linear correlation between AOD and histamine content, which is effective as a calibration curve, and that the histamine concentration in the sample is 0.05 mM to 0.5 mM ( That is, it can be seen that a very small amount of histamine (about 5 to 50 ppm) can be quantified quickly and with high sensitivity.

Example 5

(Quantification of histamine contained in mackerel meat in canned mackerel)

1) Sample preparation

5 g of the canned mackerel meat boiled in boiled mackerel was quantified, 35 ml of 50 mM Tris-hydrochloric acid buffer (pH 8.5) was added, and the sample was made fine with a streamer. Heated. After standing to cool, the sample was made up to 50 ml with the same buffer solution, and this was filtered through a No. 2 filter paper and a 0.45 m disc filter and used as an analysis sample.

2) Preparation and quantification of histamine reagent

Preparation and quantification of histamine quantification reagents were performed in the same manner as in Examples 3 and 4.

The amount of histamine in the analysis sample was calculated from the equation of the calibration curve obtained in Example 4 using each A〇D obtained by analysis.

Comparative example

For comparison, histamine contained in the same analysis sample was quantified by the HPLC method (conventional method), which is said to have high reliability in the measured value in the conventional histamine quantification method. The correlation between the measured value of the method of the present invention (Example 5) and the measured value of the conventional method was examined. Figure 6 shows the results.

From the results shown in FIG. 6, there is a linear correlation between the method of the present invention (Example 5) and the conventional method, and the equation showing the correlation is y = 1.0045X-4.665. 6 (r = 0.998), and it was found that the quantification method of the present invention showed a very good correlation with the conventional HPLC method.

This indicates that the method for quantifying histamine of the present invention has high reliability in measured values.

In addition, the measurement is performed using a simple device that can perform quantification with extremely high sensitivity, requiring no complicated pretreatment operation for removing interfering substances (impurities) from the sample, requiring a short time for quantification. It can be seen that the working cell does not need to operate under liquid tight conditions (ie it can be operated in an open system).

Example 6

(Quantification of histamine in a sample in which histamine and other amines (cadaverine and putrescine) are mixed)

In the method for quantifying histamine in Example 4 above, instead of the `` histamine standard solution at each concentration '', `` cadaverine and butretsucin at the same concentration as the histamine were mixed in the histamine standard solution at each concentration ''. Histamine was quantified in exactly the same manner except that a “sample solution to be used” was used.

For comparison, histamine was quantified by the HPLC method (conventional method) in the same sample solution as described above.

Examination of the correlation between these measurements gave the same results as in Figure 6. In other words, when `` sample solutions in which cadaverine and putrescine are mixed with the same concentration of histamine in the histamine standard solution of each concentration '' were used, the reaction solution was compared with the histamine standard solution. No difference was observed in the amount of coloring.

Therefore, it can be seen that the method for quantifying histamine according to the present invention can efficiently quantify only histamine without being affected by other amines. In particular, cadaverine and putrescine are produced at almost the same time as histamine when fish meat rots. In contrast to the method, it was necessary to fractionate (separate) and remove them by some method. In the present invention, however, an enzyme having a substrate specificity that acts strongly on histamine but does not act on cadaverine or butretzin is used. It can be seen that histamine can be quantified without using any cumbersome and time-consuming separation operation. Industrial applicability

The present invention provides a novel histamine derivative which acts specifically on histamine, does not act (reacts) on biological amines such as cadaverine and butrethsin, and has an optimum temperature of 65 to 75 ° C. Provided are drogenase and a method for producing the same.

The new histamine dehydrogenase can be stably stored at room temperature for at least one week after dissolving in an aqueous solution without adding a stabilizer.

This new histamine dehydrogenase can be used to determine the amount of histamine in the fish meat to be measured and the trace amount of histamine contained in body fluids such as human serum and urine when measuring the freshness of fish meat. This enzyme is extremely useful for the determination of cadaverin and putrescine, which is required to be detected among the various amides contained in the fish meat or body fluid to be measured. However, it works well only for the target histamine, and it can be quantified with high accuracy.

In addition, cadaverine and butretsucin are amides that are produced at the same time as histamine when fish meat rots, and these must be separated and removed by some conventional method for enzymatic determination of histamine. On the other hand, the histamine quantification method using the present enzyme has the advantage that it has excellent characteristics, so that a cumbersome and time-consuming separation operation is not required at all.

The present invention provides a new method for enzymatic quantification of histamine in histamine-containing samples using a novel histamine dehydrogenase.

In addition, since histamine-containing dehydrogenase is allowed to act on a histamine-containing sample, the amount of reduced electron carrier, 4-imidazolylacetoaldehyde or ammonia produced is measured, so that histamine can be accurately quantified.

In addition, in the method for quantifying histamine using this novel histamine dehydrogenase, a complicated pretreatment operation for removing interfering substances (impurities) in the sample is performed. It is possible to measure very small amounts of histamine (that is, with good sensitivity) in a short time, using a simple device, in an open system, and without impairing the reliability of the measured values. .

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

The scope of the claims

1. Histamine dehydrogenase which specifically acts on histamine and has an optimum temperature of 65 to 75 ° C.

2. A method for producing histamine dehydrogenase, which comprises culturing a strain belonging to the genus Rhizobim and having the ability to produce histamine dehydrogenase according to claim 1 in a medium, and collecting the histamine dehydrogenase from the culture.

3. A strain belonging to the genus Rhizobium and having the ability to produce histamine dehydrogenase according to claim 1 is cultured in a medium containing histamine or a histamine salt, and the histamine dehydrogenase is cultured from the culture. A method for producing histamine dehydrogenase, comprising:

4. A method for quantifying histamine, characterized by allowing histamine dehydrogenase to act on a sample containing histamine and measuring the product.

5. A method for quantifying histamine, which comprises reacting histamine dehydrogenase on a sample containing histamine and measuring the reduced electronic carrier, 4-imidazolylacetoaldehyde or ammonia produced.

6. Histamine, characterized in that histamine dehydrogenase is added to a histamine-containing sample in the presence of an electron carrier and a reduced-type electron carrier coloring agent to perform an enzymatic action, and to quantitate the produced dye. Quantitation method.

7. The method for quantifying histamine according to claim 6, wherein the electronic carrier and the reduced electronic carrier color former are tetrazolium-based compounds.

8. The method for quantifying histamine according to any one of claims 4 to 7, wherein the histamine-containing sample is a food or drink or a body fluid.

9. The histamine according to any one of claims 4 to 8, characterized in that histamine dehydrogenase is a histamine dehydrogenase which acts strongly on histamine but does not act on cadaverine and putrescine. Quantification method.

10. The method for quantifying seven stamin according to any one of claims 4 to 9, wherein the histamine dehydrogenase according to claim 1 is used as histamine dehydrogenase.

1 1. Immobilization of histamine dehydrogenase as histamine dehydrogenase The method for quantifying histamine according to any one of claims 4 to 10, which utilizes an immobilizing enzyme c.

1 2. A reagent for quantifying histamine containing (a) histamine dehydrogenase, (b) an electron carrier and (c) a reduced-type electronic carrier color former.

13. The histamine determination reagent according to claim 12, wherein a tetrazolium-based compound is used as an electron carrier, and a reduced tetrazolium-based compound is used as a reduced-electron-carrier color former.

14. A histamine dehydrogenase, which is a histamine dehydrogenase, which is a histamine dehydrogenase which has a strong effect on histamine but does not act on cadaverine and putrescine. 13 For quantification of histamine according to any of 3 s¾ medicine o

1 5. The reagent for quantifying histamine according to any one of claims 12 to 14, wherein the histamine dehydrogenase according to claim 1 is used as histamine dehydrogenase. .