JP3007059B2 - Method for determination of formaldehyde oxidase and substance or enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel formaldehyde oxidase having a high substrate specificity which catalyzes a reaction of oxidizing formaldehyde in the presence of water and oxygen to form formic acid and hydrogen peroxide, a process for producing the same, The present invention relates to a method for quantifying formaldehyde using an enzyme or a substance or an enzyme involved in a reaction for producing formaldehyde by an enzymatic reaction, and a reagent for the quantification thereof.

[0002]

2. Description of the Related Art With respect to the presence of an enzyme that oxidizes formaldehyde, rabbit liver (Rabbit liver) is derived from animals.
[Journal of Chromatography (J. Chromat
ogr.), 475 , 363 (1989), Biotechnol.
Bioeng.), 27 , 447 (1985)], Guinea Pig liver [Archives of Biochemical and Biophysics (Arch. Bioche).
Biophys.), 242 , 213 (1985)], bovine liver [Biochemical Society of Transactions (Biochem. Soc. Tran).
s.), 1515 , 882 (1987)], and from microorganisms, Streptomyces [Arch. Microbiol.
131 , 351 (1982)], Pseudomonas ( Pseu
domonas ) (JP-A-58-17489) and the like.

[0003] However, these enzymes have low substrate specificity and, in addition to formaldehyde, are not limited to formaldehyde such as acetaldehyde, but also various other nitrogen-containing aromatic heterocyclic compounds and a wide range of compounds such as purines, hypoxanthines and quinines. Have been reported [Journal of Biological Chemistry (J. Biol. Chem.), 237 , 922 (1962);
239 , 2022 (1964), 239 , 2027
(1964)].

[0004] Therefore, when formaldehyde is quantified particularly in a biological sample due to low substrate specificity, acetaldehyde and various nitrogen-containing aromatic heterocyclic compounds coexisting in the sample affect the quantification. A method for quantifying formaldehyde using enzymes has not been realized. In the field of clinical examination, quantification of creatine concentration in biological samples such as serum and urine is extremely important as an index for neurological and muscular diseases, and quantification of creatinine concentration is extremely important as an index for kidney diseases and muscular diseases.

Conventionally, enzymatic quantification using creatininase, creatinase and sarcosine oxidase has been mainly used for quantifying creatine or creatinine concentration in a biological sample. However, there is no known method for converting creatine or creatinine to formaldehyde and then quantifying formaldehyde to determine creatine or creatinine.

[0006]

An object of the present invention is to provide formaldehyde oxidase having high substrate specificity and a method for producing the same. Another object of the present invention is to provide a method for quantifying formaldehyde or a substance or enzyme involved in a reaction for producing formaldehyde by an enzymatic reaction using the enzyme, and a reagent for quantifying the enzyme.

[0007]

SUMMARY OF THE INVENTION The present invention provides a method for acting on formaldehyde but not on acetaldehyde.
A novel formaldehyde oxidase that catalyzes the reaction of oxidizing formaldehyde in the presence of water and oxygen to produce formic acid and hydrogen peroxide, and a microorganism belonging to the genus Cylindrocarpon, which has the ability to produce the new formaldehyde oxidase, is cultured in a medium. And producing and accumulating formaldehyde oxidase in the culture, and collecting formaldehyde oxidase from the culture, which relates to a method for producing formaldehyde oxidase.

The present invention also relates to a novel formaldehyde oxidase and a reagent for quantifying formaldehyde comprising a reagent for quantifying hydrogen peroxide and a sample containing formaldehyde, wherein the novel formaldehyde oxidase is allowed to act on the reagent to remove oxygen or peroxidized by the oxidation reaction. The present invention relates to a method for determining formaldehyde, which comprises determining hydrogen oxide.

[0009] In addition, an enzyme involved in a reaction for converting a substance to be quantified into formaldehyde, a novel reagent for quantifying a substance to be quantified, comprising a novel formaldehyde oxidase and a reagent for quantifying hydrogen peroxide, and a sample containing the substance to be quantified, A method for quantifying a substance to be quantified, characterized by reacting an enzyme involved in a reaction for converting the substance to be quantified into formaldehyde and a novel formaldehyde oxidase, and quantifying oxygen or hydrogen peroxide generated by the oxidation reaction. About.

Further, the present invention relates to a reaction for producing formaldehyde comprising a substrate of an enzyme to be quantified involved in a reaction for producing formaldehyde, another enzyme involved in the reaction, a novel formaldehyde oxidase and a reagent for quantifying hydrogen peroxide. The reagent containing the enzyme to be quantified and the enzyme to be quantified involved in the reaction for producing formaldehyde are added to the sample containing the enzyme to be quantified and the substrate for the enzyme to be quantified to be involved in the reaction for producing formaldehyde. The present invention relates to a method for quantifying an enzyme which is involved in a reaction for producing formaldehyde, characterized by quantifying oxygen consumed by an oxidation reaction or hydrogen peroxide produced by reacting the enzyme with a novel formaldehyde oxidase.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the definition of the enzyme of the present invention, formaldehyde oxidase which acts on formaldehyde but does not act on acetaldehyde is defined as follows using formaldehyde and acetaldehyde as substrates using the same amount of enzyme. When the titer obtained by using formaldehyde as a substrate in the titer measuring method is set to 100, an enzyme whose relative titer obtained by using acetaldehyde as a substrate is 1.0 or less is shown.

In particular, when the same enzyme amount is used, various aldehydes are used as a substrate, and the titer obtained using formaldehyde as a substrate in the titration measuring method shown below is 100, an aldehyde other than formaldehyde is used. Enzymes having a relative titer of 1.0 or less, obtained using the same as a substrate, are preferred.

The physicochemical properties of the novel formaldehyde oxidase of the present invention are as follows. (1) Action It catalyzes the reaction of oxidizing formaldehyde in the presence of water and oxygen to produce formic acid and hydrogen peroxide. (2) Substrate specificity In a 50 mM phosphate buffer at pH 7.0, a substrate concentration of 15 m
Under the conditions of M, the activity of the enzyme on various substrates was measured,
Table 1 shows the results obtained by examining the relative activities with the activity against formaldehyde as 100.

[0014]

[Table 1]

From Table 1, it can be seen that the present enzyme acts specifically on formaldehyde and does not act on acetaldehyde. (3) Optimum pH 100 mM universal buffer (Johnson-lindsay buffer, basic biochemical experiment, pH 4.0 to 11.0)
The activity was determined by using the method described in Maki, Maruzen), and the optimum pH was examined. A pH-activity curve as shown in FIG. 1 was obtained. The optimum pH of this enzyme is 8.0 to 9.5. (4) pH stability 200 mM universal buffer having a pH of 4.0 to 11.0 (Johnson-Lindsay buffer, Basic Biochemistry Experiment, Vol. 6,
(Maruzen) for 15 minutes at 45 ° C., and the residual activity was measured. As a result, as shown in FIG.
It is stable in the range of 9.0. (5) Thermostability To examine the thermostability of this enzyme, 50 mM of pH 7.0 was used.
After being kept at each temperature for 15 minutes in a phosphate buffer, the residual activity was measured. As a result, it is stable up to around 55 ° C. as shown in FIG. (6) Optimum temperature The activity was determined using a 50 mM phosphate buffer at pH 7.0, and the optimum temperature was examined. As a result, a temperature-activity curve as shown in FIG. 4 was obtained. The optimum temperature of this enzyme is around 50 ° C. (7) Influence of Inhibitors The effects of various compounds on the activity using 50 mM phosphate buffer at pH 7.0 were examined using the compounds and concentrations shown in Table 2. The results are shown in Table 2.

[0016]

[Table 2]

This enzyme is inhibited by copper sulfate, NH ₂ OH, sodium azide and p-mericulibenzoic acid. (8) Molecular weight The molecular weight of the subunit of this enzyme measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is about 78,000, and the molecular weight measured by gel filtration is about 310 , 000. (9) Uniformity The enzyme produced a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. That is, about 60 in Tris-glycine buffer (pH 8.3).
After electrophoresis for 1 minute, a single protein band was observed by staining with Coomassie staining solution.

(10) Method for measuring the titer The titer of the enzyme is measured by the following method. 1) Reagent Substrate solution Formaldehyde is dissolved in 100 mM phosphate buffer (pH 7.0) to a concentration of 30 mM.

Coloring reagent Peroxidase (Toyobo, Grade III) was used at a final concentration of 1.
0 U / ml, 4-aminoantipyrine at a final concentration of 0.7
Dissolve 2 mM phenol in water to a final concentration of 12.6 mM. 2) Operation Add 1 ml of the coloring reagent to 1 ml of the substrate solution, and add 5 ml at 37 ° C.
Let react for minutes. Next, 0.02 ml of the enzyme solution was added, and 3
After shaking at 7 ° C. for 10 minutes to react, the absorbance at 500 nm is measured. As a control, water is used instead of the enzyme solution, and the difference in absorbance at 500 nm is determined. 3) Calculation method of titer One unit of formaldehyde oxidase is 1 unit at 37 ° C.
The amount of enzyme capable of decomposing 1 μmol of formaldehyde per minute is defined as one unit. Therefore, the titer (U / ml) per 1 ml of the enzyme solution can be obtained from the absorbance difference by the following formula (I).

[0020]

(Equation 1)

Next, a method for producing formaldehyde oxidase will be described. The enzyme belongs to the genus Cylindrocarpon, and the microorganism having the novel formaldehyde oxidase generating ability is cultured in a medium, formaldehyde oxidase is produced and accumulated in the culture, and formaldehyde oxidase is collected from the culture. Obtainable.

As the microorganism having the ability to produce formaldehyde oxidase of the present invention, any microorganism can be used as long as it belongs to the genus Cylindrocarpone and has the ability to produce formaldehyde oxidase of the present invention. Specific preferred microorganisms include Cylindrocarpon didymu
m ) FERM BP-5758.

[0023] Cylidro Kalpon Didim
The mycological properties of the species to which ndrocarpon didymum belong are described by Joseph C. Gilman, Manual of Soil Funjay.
Fungi) Second edition, pages 395-397 (1957),
The Iowa State College Pres
s, USA).

The above microorganisms were obtained from the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology, on November 28, 1996, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan, based on the Budapest Treaty. Deposited.

For the cultivation of the microorganism used in the present invention, a general mold culturing method is generally used. As the medium, any of a natural medium and a synthetic medium can be used as long as the medium contains a carbon source, a nitrogen source, an inorganic salt, and the like. Examples of the carbon source include carbohydrates, sugar alcohols,
Alcohols, organic acids and the like are used. Examples of the carbohydrate include glucose, sucrose, maltose, starch, molasses and the like. Examples of the sugar alcohol include glycerol, sorbitol, mannitol and the like. Examples of the alcohol include methanol, ethanol and the like. Examples of the organic acid include acetic acid, lactic acid, pyruvic acid, citric acid and the like.

As the nitrogen source, for example, inorganic or organic ammonium salts, nitrogen-containing organic substances and the like are used. Examples of the inorganic or organic ammonium salts include ammonia, ammonium chloride, ammonium carbonate, ammonium phosphate, ammonium acetate and the like. Examples of nitrogen-containing organic substances include urea, amino acids, peptone, NZ-amine, meat extract, corn steep liquor,
Casein hydrolyzate, yeast extract and the like are used.

As the inorganic salt, for example, potassium (I) phosphate, potassium (II) phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate and the like are used.
As a culture method, a liquid culture method, particularly a deep stirring culture method, is suitable. Culture is at pH 5.0-8.0, temperature 25-37.
C. for 1 to 7 days at room temperature or with aeration and stirring.

By culturing in this manner, formaldehyde oxidase is produced and accumulated mainly in the cells of the culture. The collection of formaldehyde oxidase from the culture is performed as follows. After completion of the culture, cells are collected from the culture by centrifugation or filtration. The cells are disrupted by an ultrasonic disrupter or the like to obtain a crude enzyme extract. This crude enzyme extract is usually used for enzyme purification, for example, salting out,
The treatment is performed by a method such as organic solvent precipitation, dialysis, ion exchange column chromatography, gel filtration, and freeze-drying. Thus, purified formaldehyde oxidase can be collected.

The reagent for quantifying formaldehyde of the present invention comprises the reagent for quantifying formaldehyde oxidase of the present invention and hydrogen peroxide. The reagent for quantifying a substance to be quantified according to the present invention comprises an enzyme involved in a reaction for converting the substance to be quantified into formaldehyde, a formaldehyde oxidase of the present invention, and a reagent for quantifying hydrogen peroxide.

Further, the reagent for quantifying an enzyme involved in the reaction for producing formaldehyde of the present invention is a substrate of the enzyme to be quantified which is involved in the reaction for producing formaldehyde, another enzyme involved in the reaction, It consists of formaldehyde oxidase and hydrogen peroxide. Each component can be supplied not only as a reagent coexisting in the same reagent, but also as a kit of a reagent in which specific components are combined as required.

As the formaldehyde oxidase of the present invention, an enzyme produced by the above-mentioned method is used, and an isolated and purified enzyme is preferred. The concentration of this enzyme in the reaction solution is
0.01-100 U / ml, preferably 0.1-10 U
/ Ml. Examples of the reagent for quantitatively determining hydrogen peroxide include a reagent comprising a peroxide active substance and a coloring reagent, a peroxide active substance and a fluorescent reagent, and a reagent comprising a peroxide active substance and a coloring reagent.

Examples of the peroxide active substance include peroxidase. The concentration of the peroxidation activating substance in the reaction solution is 0.01 to 100 U / ml, preferably 0.1 to 1 U / ml.
0 U / ml. As the color-forming reagent, any compound may be used as long as it is a compound which is oxidized in the presence of hydrogen peroxide and a peroxide activating substance to form a color.

For example, aniline or N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (HS
DA) or a phenol derivative such as phenol or N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (hereinafter abbreviated as EMSE) and a 4-aminoantipyrine ( Combination reagents of coupler compounds such as 4-AA) and 3-methyl-2-benzothiazolinohydrazine are exemplified.

Further, 3,3′-diaminobenzidine, o
-Dianisidine, 4-methoxy-1-naphthol, 2,
2'-azino-bis (3-ethylbenzothiazoline)-
6-sulfonic acid (ABTS), 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP), 10-N-carboxymethylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (CCAP) , N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) ) Methyl-4-dimethylaminophenyl] amine (BCMA), bis [3-bis (4-
Chlorophenyl) methyl-4-carboxyethylaminophenyl] amine and the like.

As the fluorescent reagent, homovanillic acid, p-
Examples include hydroxyphenylacetic acid, diacetylfluorescin derivatives (diacetylfluorescin, diacetyldichlorofluorescin, etc.). Luminescent reagents include luminol, isoluminol, pyrogallol, bis (2,4,6-trichlorophenyl) oxalate,
-Methoxy-1-naphthol, coumarin derivatives and the like.

The concentration of the coloring reagent, the fluorescent reagent and the luminescent reagent in the reaction solution is at least equimolar to the formaldehyde in the reaction solution, preferably 1 to 10,000 times, more preferably 10 to 10,000 times. It is a 000-fold molar amount. Normal,
0.01 to 100 mM is preferable, and 0.1 to 10 mM is more preferably used. The substance which can be quantified in the present invention is a substance which is converted into formaldehyde by an enzyme. The substance may be not only a substance that is converted to formaldehyde in a one-step reaction but also a substance that is converted to formaldehyde by a several-step enzymatic reaction. Examples of the substance include creatinine, creatine, sarcosine, methylamine and the like.

The enzyme involved in the reaction for converting the substance to be quantified into formaldehyde differs depending on the substance to be quantified. For example, when the substance to be quantified is creatine, creatininase, creatinase, sarcosine oxidase When the substance to be quantified is methylamine, amine oxidase can be mentioned. The enzyme to be quantified in the present invention is an enzyme involved in a reaction for producing formaldehyde. When the enzyme is creatininase, a substrate of the enzyme includes creatinine, and other enzymes involved in the reaction include creatinase and sarcosine oxidase. When the enzyme to be quantified is amine oxidase, the substrate of the enzyme includes methylamine.

The enzymes to be quantified involved in the reaction for producing formaldehyde include, for example, creatininase,
Creatinase, sarcosine oxidase, amine oxidase and the like can be mentioned. These quantitative reagents and kits thereof contain a buffer, an enzyme activator, a stabilizer, a surfactant, a preservative, and the like as necessary.

As the buffer, lactate buffer, citrate buffer, acetate buffer, succinate buffer, glycine buffer,
3,3-dimethylglutarate buffer, phthalate buffer,
Phosphate buffer, triethanolamine buffer, diethanolamine buffer, borate buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer,
Imidazole-acetate buffer, malate buffer, oxalate buffer, carbonate buffer, good buffer and the like.

Examples of the enzyme activator include dehydroacetic acid. Examples of the preservative include sodium azide, streptomycin sulfate and the like. Examples of the stabilizer include metal chelating agents such as ethylenediaminetetraacetic acid, and other polysaccharides such as soluble starch and derivatives thereof, which are commonly used as stabilizers for enzymes, albumin,
Proteins such as globulin and water-soluble polymer compounds such as polyethylene glycol can be used. Examples of the surfactant include Triton X-100.

A method for quantifying formaldehyde, characterized in that a novel formaldehyde oxidase is allowed to act on a sample containing formaldehyde, and oxygen consumed by the oxidation reaction or generated hydrogen peroxide is quantified. In the reaction of aldehyde oxidase, a sample containing formaldehyde is subjected to formaldehyde oxidase,
If necessary, a peroxide activating substance such as the above-mentioned peroxidase and a coloring reagent, a fluorescent reagent or a luminescent reagent, a quantitative reagent for hydrogen peroxide, and, if necessary, a buffer, an enzyme activator, a preservative, and stabilization. And an aqueous medium containing a surfactant and a surfactant at 20 to 50 ° C. for 1 to 15 minutes.

The amount of formaldehyde can be determined by quantifying the amount of oxygen lost by the reaction using, for example, an oxygen electrode method or a Warburg manometer. The generated hydrogen peroxide can be quantified, for example, with a hydrogen peroxide electrode. Preferably, a peroxide activating substance such as the above-described peroxidase and a reagent for determining hydrogen peroxide comprising a coloring reagent, a fluorescent reagent or a luminescent reagent are added during the formaldehyde oxidase reaction and then during or simultaneously with the start of the reaction. It can be determined by performing a hydrogen peroxide quantitative reaction. The dye, fluorescence and light amount generated by the reaction can be quantified by a conventional method.

For example, a method for optically quantifying a dye produced by a color reaction is to use a commercially available spectrophotometer,
The absorbance is measured at a wavelength of ７750 nm using a reagent blank as a control. The wavelength used is preferably determined in consideration of the light absorption characteristics of the coloring reagent used and the light absorption characteristics of the reaction solution. Using a fixed amount of formaldehyde at a known concentration, the reaction is carried out in the same manner as described above, the increase in absorbance is measured, and a calibration curve is prepared. The unknown amount of formaldehyde is quantified using this calibration curve.

Further, an enzyme involved in the reaction for converting the substance to be quantified into formaldehyde and a novel formaldehyde oxidase are allowed to act on the sample containing the substance to be quantified, whereby oxygen consumed by the oxidation reaction or hydrogen peroxide generated is produced. The method of quantifying the substance to be quantified, which is characterized by quantification, is described. After the reaction to convert the substance to be quantified into formaldehyde enzymatically by a conventional method is completed, the above-mentioned quantitative reaction of formaldehyde is carried out during the reaction or simultaneously with the start of the reaction, and the amount can be quantified by the above-mentioned method.

Further, a sample containing the enzyme to be quantified involved in the reaction for producing formaldehyde is added to the sample containing the enzyme to be quantified for the reaction which produces formaldehyde,
Quantifying the enzyme involved in the reaction which produces formaldehyde to be quantified, characterized by reacting other enzymes involved in the reaction and novel formaldehyde oxidase to determine the amount of oxygen or hydrogen peroxide produced by the oxidation reaction Learn how.

The enzyme to be quantified can be quantified by the above-mentioned method by performing the above-mentioned quantitative reaction of formaldehyde during the reaction or simultaneously with the start of the reaction, after the reaction for enzymatically converting the substrate into formaldehyde by a conventional method. .

The case of creatine and creatinine as substances to be quantified will be described in more detail. Conventionally, for the determination of creatine and creatinine, a method is known in which creatininase, creatinase, and sarcosine oxidase are used to generate hydrogen peroxide and quantify the hydrogen peroxide by a conventional method.

The formaldehyde generated by this reaction is
By reacting the formaldehyde oxidase of the present invention to generate another molecule of hydrogen peroxide, hydrogen peroxide is generated twice as much as in the conventional quantitative system, and high sensitivity is realized. This reaction is shown below.

[0049]

Embedded image

That is, the present method is a quantitative system for quantifying creatine using creatinase and sarcosine oxidase,
Alternatively, a method of adding creatininase to this system and quantifying creatinine, adding formaldehyde oxidase to generate one molecule of creatine or two molecules of hydrogen peroxide from creatinine, and quantitate this.

When conventional formaldehyde oxidase is used, it also acts on aldehydes other than formaldehyde coexisting in the sample, so that even if the above-mentioned reaction is carried out, quantitative high sensitivity cannot be achieved. In carrying out the present invention, in the case of creatinine quantification, an appropriate buffer, creatininase, creatinase, sarcosine oxidase, and formaldehyde oxidase are added to a sample containing creatinine and reacted.

In the case of creatine determination, an appropriate buffer, creatinase, sarcosine oxidase, and formaldehyde oxidase are added to a sample containing creatine and reacted. The concentration of each substance in the reaction solution is 1 to 1000 U / ml for creatininase, preferably 35 to 1000 U / ml.
110 U / ml, creatinase 1-1000 U /
ml, preferably 35-110 U / ml, sarcosine oxidase is 1-1000 U / ml, preferably 7-U / ml.
25 U / ml, formaldehyde oxidase, 0.1
01-1000 U / ml, preferably 0.05-50 U
/ Ml, peroxidase is 0.1-1000 U / m
1, preferably 1 to 100 U / ml.

The case of creatine kinase as the enzyme to be quantified will be described in more detail. Creatine kinase is present in muscle tissue and the brain throughout the body, and in the area of clinical testing, measurement of creatine kinase activity is routinely measured in the diagnosis of muscle diseases, neurological diseases, brain diseases, heart diseases, etc. It is one of the important items.

Conventionally, creatine kinase has been quantified by various methods. However, by using the formaldehyde oxidase of the present invention, 2 mol of hydrogen peroxide is finally produced per 1 mol of the substrate by the following reaction. Quantification can be performed with high sensitivity.

[0055]

Embedded image

In the reaction formula, ADP indicates adenosine diphosphate and ATP indicates adenosine triphosphate. The concentration of each substance in the reaction solution was 0.1 to 100 for creatine phosphate.
0 mM, preferably 1-100 mM, ADP is 0.1
１０００1000 mM, preferably 1-100 mM, creatininase is 1-1000 U / ml, preferably 35-
110 U / ml, creatinase 1-1000 U /
ml, preferably 35-110 U / ml, sarcosine oxidase is 1-1000 U / ml, preferably 7-U / ml.
25 U / ml, formaldehyde oxidase, 0.1
01-1000 U / ml, preferably 0.05-50 U
/ Ml, peroxidase is 0.1-1000 U / m
1, preferably 1 to 100 U / ml.

The case where amine oxidase is used as the enzyme to be quantified will be described in more detail. Amine oxidase activity is significantly increased in organ fibrosis, especially in cirrhosis, and is useful for diagnosis of liver fibrosis. The activity of amine oxidase in serum is not always high, and a sensitive measuring method is required.

Conventionally, amine oxidase has been quantified by various methods. However, by using the formaldehyde oxidase of the present invention, 2 mol of hydrogen peroxide is finally produced per 1 mol of substrate by the following reaction. Quantification can be performed with high sensitivity.

[0059]

Embedded image

The concentration of each substance in the reaction solution is 0.1 to 1000 mM, preferably 1 to 100 mM, for methylamine.
M, formaldehyde oxidase, 0.01 to 10
00U / ml, preferably 0.05-50 U / ml, and peroxidase is 0.1-1000 U / ml, preferably 1-100 U / ml. Hereinafter, the present invention will be described specifically with reference to examples.

[0061]

【Example】

Example 1 Cylindrocarpon d dimum
idymum ) Production of formaldehyde oxidase by FERM BP-5758 Cylindrocarpon didim FERM BP-5
758 with glucose 1 g / dl, peptone 0.5 g / d
1, yeast extract 0.3 g / dl, malt extract 0.3 g /
Inoculate a 2 L Erlenmeyer flask containing 300 ml of a medium (pH 6.0) containing dl, and incubate at 30 ° C. for 48 hours.
Shaking culture was performed for a time. 600 ml of the obtained culture solution
Was inoculated into a 30 L jar fermenter containing 15 L of a medium having the same composition as the above-mentioned medium, and aerated and stirred at 30 ° C. for 3 days.

After completion of the culture, 15 L of the culture solution was centrifuged (1
(2,000 xg, 20 minutes)
The cells were suspended in 1,000 ml of M phosphate buffer (pH 7.0), crushed by Dynomill (manufactured by WABachofen), centrifuged (12,000 × g, 20 minutes), and the supernatant was collected. Ammonium sulfate was added to the supernatant to 80% saturation, and the precipitate was collected. A small amount of the resulting precipitate (about 2
(00 ml) in 60 mM phosphate buffer (pH 7.5), and the resulting solution was dialyzed against 5 L of the same buffer for 24 hours. An anion exchange resin DEAE-Cellulofine (DEAE-C) obtained by equilibrating the obtained dialysate (10 ml) with the same buffer.
ellulofine (manufactured by Chisso) column (1L, diameter 5c)
m). The active fractions that had passed through were combined, ammonium sulfate was added to this to 80% saturation, and the precipitate was collected by centrifugation (12,000 rpm, 20 minutes),
It was dissolved in 20 ml of a phosphate buffer (pH 9.0). The obtained solution was passed through a column (1 L, diameter 5 cm) of an anion exchange resin DEAE-Cellulofine (manufactured by Chisso Corporation) equilibrated with the same buffer (pH 9.0) to be adsorbed. After washing away the impure proteins with the same buffer, the protein was eluted with a concentration gradient of 0 to 1.0 M saline [10 mM phosphate buffer (pH 9.0)]. The active fractions eluted at a salt concentration of about 0.2-0.3M were combined, ammonium sulfate was added to the active fractions to 80% saturation, and the resulting precipitate was centrifuged (12,000).
rpm, 20 minutes) and dissolved in 10 ml of 10 mM phosphate buffer (pH 7.0). The resulting solution was dialyzed against 2 L of the same buffer for 24 hours to obtain a formaldehyde oxidase enzyme preparation.

The specific activity of the enzyme preparation was 50 U / mg.

Example 2 Reagents for Formaldehyde Quantification The following reagents were dissolved in distilled water so as to have the following concentrations to prepare formaldehyde quantification reagents. Phosphate buffer (pH 8.0) 50 mM 4-aminoantipyrine 0.36 mM EMSE 6.3 mM peroxidase 5.3 U / ml Formaldehyde oxidase 0.16 U / ml

Example 3 Determination of Formaldehyde To 2.9 ml of the reagent prepared in Example 2, 0.1 ml of various concentrations of formaldehyde solution was added to the final concentration of the reaction solution.
It was added to a concentration of 0.05 mM and reacted at 37 ° C. for 15 minutes, and the absorbance difference at 555 nm after 15 minutes was determined using a formaldehyde concentration of 0 mM as a control.

FIG. 5 shows the results.

Example 4 Reagents for Determination of Creatine The reagents shown below were dissolved in distilled water so as to have the concentrations shown below to prepare a reagent solution for creatine determination. Phosphate buffer (pH 8.0) 50 mM 4-aminoantipyrine 0.36 mM EMSE 6.3 mM peroxidase 5.3 U / ml formaldehyde oxidase 0.16 U / ml sarcosine oxidase 0.5 U / ml creatinase 5.0 U / ml

Example 5 Determination of creatine 0.1 ml of creatine solutions of various concentrations were added to 2.9 ml of the reagent prepared in Example 4 at a final concentration of 0 to 0.05 m.
M, and reacted at 37 ° C. for 15 minutes. The absorbance difference at 555 nm after 15 minutes was determined using a creatine concentration of 0 mM as a control.

As a comparative experiment, the same test was carried out using the reagent shown in Example 4 except that formaldehyde oxidase was omitted. FIG. 6 shows the results.

Example 6 Reagents for Quantifying Creatinine The following reagents were dissolved in distilled water so as to have the following concentrations to prepare creatinine quantitative reagent solutions. Phosphate buffer (pH 8.0) 50 mM 4-aminoantipyrine 0.36 mM EMSE 6.3 mM peroxidase 5.3 U / ml formaldehyde oxidase 0.16 U / ml sarcosine oxidase 0.5 U / ml creatinase 5.0 U / ml creatinine Nase 64.5U / ml

Example 7 Determination of Creatinine Creatinine was added to the reagent solution prepared in Example 6 so that the final concentration was 0 to 0.05 mM, and the solution was added at 37 ° C. for 15 minutes.
The absorbance at 550 nm after the reaction for one minute was measured using a reagent blank as a control. As a comparative experiment, the same test was performed using the reagent shown in Example 6 except that formaldehyde oxidase was removed. FIG. 7 shows the results.

Example 8 In the same manner as in Example 7, a sample prepared to a concentration of creatinine of 0.01 mM was measured. As a result, 550 nm
Was 145 mABS when no formaldehyde oxidase was added, whereas it was 292 mAB when added.
It was almost twice that of BS and could be measured with good sensitivity. The coefficient of variation when the same reagent was measured five times was 3.5 for the former.
% And the latter 0.7%, the reproducibility was significantly improved.

[0073]

By using the formaldehyde oxidase of the present invention, substances in a biological sample can be quantified with high sensitivity and high reproducibility.

[Brief description of the drawings]

[Fig. 1] Cylind
1 shows the pH-activity curve of formaldehyde oxidase from C. rocarpon didymum FERM BP- 5758 .

[Figure 2] Cylind Carpon Didim
2 shows the pH-stability curve of formaldehyde oxidase from C. rocarpon didymum FERM BP- 5758 .

[Figure 3] Cylind
FIG . 2 shows the thermostability curve of formaldehyde oxidase derived from FERM BP- 5758 .

Fig. 4 Cylind Carpon Didim
1 shows a temperature-activity curve of formaldehyde oxidase derived from FERM BP- 5758 .

[Fig. 5] Cylind Carpon Didim
rocarpon didymum ) is a calibration curve of formaldehyde using formaldehyde oxidase derived from FERM BP- 5758 , the vertical axis represents absorbance after 15 minutes, and the horizontal axis represents formaldehyde concentration (mM).

[Fig. 6] Cylind
rocarpon didymum ) Calibration curves of creatine with and without the addition of formaldehyde oxidase derived from FERM BP- 5758 . The vertical axis represents absorbance after 15 minutes, and the horizontal axis represents creatine concentration (mM).

[Explanation of symbols]

 − ● − When using formaldehyde oxidase − ○ − When not using formaldehyde oxidase

[Fig. 7] Cylind Carpon Didim
rocarpon didymum ) Calibration curves of creatinine with and without the addition of formaldehyde oxidase derived from FERM BP- 5758 . The vertical axis represents absorbance after 15 minutes, and the horizontal axis represents creatinine concentration (mM).

[Explanation of symbols]

 − ● − When using formaldehyde oxidase − ○ − When not using formaldehyde oxidase

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 645)

Claims (17)

(57) [Claims] 1. A microorganism belonging to the genus Cylindrocarpon, which catalyzes a reaction of oxidizing formaldehyde in the presence of water and oxygen to form formic acid and hydrogen peroxide, which acts on formaldehyde but not on acetaldehyde. Derived from
New formaldehyde oxidase. 2. A compound which acts on formaldehyde but has
Does not act on aldehydes, forms in the presence of water and oxygen
Reaction of oxidation of aldehyde to form formic acid and hydrogen peroxide
The catalyze new Formaldehyde having the following physical and chemical properties:
Hydoxidase. (A) Optimum pH: 8.0-9.5 (shown in FIG. 1) . (B) Stable pH: 7.0-9.0 (shown in FIG. 2) . (C) Thermal stability: stable up to 55 ° C. (shown in FIG. 3) . (D) Suitable temperature for operation: around 50 ° C. (shown in FIG. 4) . (E) Influence of inhibitors: Inhibited by copper sulfate, NH ₂ OH, sodium azide, p-mericuribenzoic acid. (F) Molecular weight: about 78,000 (SDS-PAGE method)
Approximately 310,000 with the following subunits (gel filtration) . 3. The method according to claim 1, which belongs to the genus Cylindrocarpon.
Or a method of producing formaldehyde oxidase, which comprises culturing the microorganism capable of producing formaldehyde oxidase according to 2 in a medium, producing and accumulating formaldehyde oxidase in the culture, and collecting formaldehyde oxidase from the culture. 4. A reagent for quantifying formaldehyde, comprising the reagent for quantifying formaldehyde oxidase according to claim 1 or 2 and hydrogen peroxide. 5. A method for quantifying formaldehyde, which comprises causing a formaldehyde oxidase according to claim 1 or 2 to act on a sample containing formaldehyde, and quantifying oxygen disappeared by the oxidation reaction or hydrogen peroxide produced. 6. A reagent for quantifying a substance to be quantified, comprising an enzyme involved in a reaction for converting a substance to be quantified into formaldehyde, a reagent for quantifying formaldehyde oxidase according to claim 1 and hydrogen peroxide. 7. The reagent according to claim 6, wherein the substance to be quantified is creatine, and the enzymes involved in the reaction for converting the substance to be quantified into formaldehyde are creatinase and sarcosine oxidase. 8. The substance to be quantified is creatinine,
7. The reagent according to claim 6, wherein the enzymes involved in the reaction for converting the substance to be quantified into formaldehyde are creatininase, creatinase and sarcosine oxidase. 9. An enzyme involved in a reaction for converting a substance to be quantified into formaldehyde and a formaldehyde oxidase according to claim 1 or 2 on a sample containing the substance to be quantified, and the amount of oxygen eliminated by an oxidation reaction. Alternatively, a method for quantifying a substance to be quantified, characterized by quantifying generated hydrogen peroxide. 10. The substance to be quantified is creatine,
The method according to claim 9, wherein the enzymes involved in the reaction for converting the substance to be quantified into formaldehyde are creatinase and sarcosine oxidase. 11. The method according to claim 9, wherein the substance to be quantified is creatinine, and the enzymes involved in the reaction for converting the substance to be quantified into formaldehyde are creatininase, creatinase and sarcosine oxidase. 12. Formaldehyde comprising a substrate of an enzyme to be quantified involved in a reaction for producing formaldehyde, another enzyme involved in the reaction, formaldehyde oxidase according to claim 1 or 2 and a reagent for quantifying hydrogen peroxide. Reagent for the enzyme involved in the reaction to be performed. 13. The enzyme to be quantified is creatine kinase, the substrates of the enzyme to be quantified are creatine phosphate and adenosine diphosphate, and the other enzymes involved in the reaction are creatinase and sarcosine oxidase. Item 13. The quantitative reagent according to Item 12. 14. The reagent according to claim 12, wherein the enzyme to be quantified is amine oxidase, and the substrate of the enzyme to be quantified is methylamine. 15. A sample containing an enzyme to be quantified which is involved in a reaction for producing formaldehyde, a substrate of an enzyme to be quantified which is involved in a reaction for producing formaldehyde,
The enzyme involved in the reaction is reacted with other enzymes involved in the reaction and the formaldehyde oxidase according to claim 1 or 2 to determine the amount of oxygen disappeared by the oxidation reaction or the amount of hydrogen peroxide produced. The method of quantifying the enzyme to be quantified. 16. The enzyme to be quantified is creatine kinase, the substrates of the enzyme to be quantified are creatine phosphate and adenosine diphosphate, and the other enzymes involved in the reaction are creatinase and sarcosine oxidase. Item 16. The method according to Item 15. 17. The method according to claim 15, wherein the enzyme to be quantified is amine oxidase, and the substrate of the enzyme to be quantified is methylamine.