JP2601356B2 - Fructosylamine oxidase, method for producing the same, method for quantifying amadori compounds using the enzyme, and reagents therefor - Google Patents

Description

The present invention relates to a novel enzyme fructosylamine oxidase, a method for producing the same, a method for quantifying an Amadori compound in a sample using the enzyme, and a reagent therefor. .

<Conventional technology and problems> Fructosylamine compounds are non-enzymatically produced as Amadori compounds when an aldose and a compound having an amino group come into contact with each other, and the concentration is proportional to the concentration of both and the contact time. It is known to increase. In other words, the concentration of the fructosylamine compound provides historical information about how much the sugar and the compound having an amino group have been in contact with the compound at what concentration or temperature, and therefore, the quantification of this compound is not possible. , Products containing large amounts of sugars and amino groups,
For example, it is useful for production and storage management of soy sauce, miso, infusion, and the like. In clinical tests, among these compounds in the blood, a fructosylamine compound bound to hemoglobin is converted to glycohemoglobin (Hb
A ₁₎ and referred also referred fructosyl amine compound mainly bound to albumin present in the serum and fructosamine, and actively measured as respectively reveal the pathology of diabetes.

However, the measurement method utilizing high performance liquid chromatography [Chromatogr.Sci. 10 659 (1979)], can be adsorbed solid bound with boric acid and packed in a column, a method for eluting the same [Clin. Chem. 28 2088 (1982)] and a method of performing electrophoresis using a gel as a bed [Clin. Chem. 26
1598 (1980)], but all have many drawbacks, such as operability and accuracy, or require expensive equipment. In addition, a method that has recently developed rapidly as a method for measuring fructosamine is a method of linking the reducing action of fructosylamine compound in alkalinity to the coloring of the dye [Cl
in. Chim. Acta. 127 87 (1983)], but there are problems in the specificity of the reaction and in the selection of a standard.

The present inventors have studied diligently to quantify these fructosylamine compounds using enzymes. as a result,
From bacteria belonging to the genus Corynebacterium in soil, the oxidase fructosylamino acid oxidase that acts on fructosylamino acids was discovered, and a patent application was filed earlier.
(JP-A-61-268178) However, although this enzyme acts well on Amadori compounds of α-amino acids, it is extremely weak or has no effect on fructose attached to the amino group of β or ε. Amino acids in the living body have an ε-amino group in addition to an α-amino group, and more of them are fructose compounds bound to the latter. Therefore, when aiming at the quantification of fructose bound to the ε-amino group of lysine, an enzyme of a bacterium belonging to the genus Corynebacterium cannot be used in view of its specificity.

The present inventors have further found that microorganisms in soil can search for enzymes having the activity of decomposing these compounds, and as a result, they can decompose not only α of amino acids but also fructose compounds attached to the amino group of ε. The present inventors have found a filamentous fungus belonging to the genus Aspergillus that produces a possible enzyme and completed the present invention.

<Means for Solving the Problems> The present invention will be specifically described below.

The physicochemical properties of the enzyme fructosylamine oxidase of the present invention are as follows.

(1) Action and substrate specificity In the presence of oxygen, the Amadori compound of α-amino acid, β-amino acid, ε-amino acid, D-amino acid is decomposed to form glycosone and hydrogen peroxide and the corresponding α-amino acid, β-amino acid.
It catalyzes reactions that produce amino acids, ε-amino acids, and D-amino acids.

In this formula, R ₁ is - [CH (OH)] is the _n -CH ₂ OH, R ₂
Is H or the side chain residue of an α-amino acid, R ₃ is — (CH ₂ ) _n H,
- (CH _{2) n} -COOH or _{- (CH 2) n -CH (} NH 2) -COOH
It is. _n is an integer of 0-4.

The enzyme has very little effect on Amadori compounds such as imino acid (for example, proline), N-methylamino acid (for example, sarcosine), which is a compound having only one H atom in the N atom, or morpholine. Is not shown. The amine moiety also has a small effect on fructosylamine, which is ammonia, but has no effect on reduced ketones in fructose residues, for example, glycitrylglycine. In addition, the reaction to sugar compounds other than fructose is much smaller than that of fructose.

Table 1 shows the activities of this enzyme and fructosyl amino acid oxidase (FAOX) described in JP-A-61-268178 with respect to each substrate.

The numerical values are ratios when the activity for D-fructosylglycine is set to 100.

*: Measured at 4 mg / ml in the reaction solution.

(2) Optimum pH: The optimum pH of this enzyme is 7.5 to 8.2 as shown in Fig. 1 when fructosylglycine is used as a substrate. The measurement was performed by measuring the absorption rate of the enzyme with an oxygen monitor. The buffers used in the figure are as follows.

○-○: 0.1 M potassium phosphate buffer ×-×: 0.1 M glycine-NaOH buffer (3) pH stability: 0.4 ml of various buffers containing 0.1 unit of the present enzyme was added at 37 ° C, 10
After heating for minutes, the remaining enzyme activity was examined. The result is as shown in FIG. The buffers used in the figure are as follows.

○-○: 0.1 M potassium phosphate buffer △-△: 0.1 M Tris-HCl buffer ×-×: 0.1 M glycine-NaOH buffer (4) Measuring method of titer: First method: Method for Colorimetric Determination of Hydrogen Oxide 2.8 ml of 0.1 M potassium phosphate buffer (pH 7.8) containing 0.005% 4-aminoantipyrine and 0.015% 2,4-dichlorophenolsulfonate is placed in a test tube, and 400 U / ml.
Add 10 μl of peroxidase solution. 37 temperature equilibrium
After reaching ℃, 0.1m enzyme solution with appropriate activity
l, 0.5 M fructosylglycine-0.1 ml was further added and reacted for 10 minutes, and the resulting dye was measured using a photoelectric colorimeter.
Measure absorbance at 510 nm. Separately, a graph is prepared using a standard solution of hydrogen peroxide in advance and examining the relationship with the amount of the formed dye. Using this graph, 37 ℃
Calculate the micromoles of hydrogen peroxide generated per minute and use this number as the activity unit in the enzyme solution used.

Method 2: Method for measuring the amount of oxygen absorbed by enzymatic reaction Transfer 2.9 ml of 0.1 M phosphate buffer (pH 7.8) to a measuring vessel of Oxygen Monitor manufactured by YSI, and add 0.5 M fructosylglycine-0.1 ml. And stirred at 37 ° C for 3 minutes to reach equilibrium with the dissolved oxygen and the temperature.Then, insert an enzyme electrode into the solution and seal it. Then, inject 30μ of the enzyme solution, and continuously record the enzyme absorption generated by a recorder connected to a monitor. And measure its initial speed. Similarly, a standard curve is prepared in advance between the oxygen concentration in the container and the recorded value, and the oxygen concentration is determined from the measured value using the standard curve. One unit is defined as the activity of an enzyme that causes absorption of 1 micromol of oxygen per minute at 37 ° C.

(5) Range of suitable temperature for action: Using fructosylglycine as a substrate, glycine produced by an enzymatic reaction at each temperature in a 0.1 M phosphate buffer (pH 7.8) was measured using an amino acid analyzer. The results are shown in FIG. 3, and the range of suitable temperature for the action of this enzyme is
30 ° C to 43 ° C.

(6) Thermal stability: 0.1 ml of an enzyme solution (0.1 M phosphate buffer, pH 7.8) containing 0.02 units of the purified enzyme was allowed to stand at each temperature for 10 minutes, and the remaining enzyme activity was examined. The results are as shown in FIG. 4. It is stable below 37 ° C., but 70% is inactivated at 50 ° C.

(7) Inhibition, activation and stabilization: Determined by measuring oxygen absorption in 0.1 M Tris-HCl buffer (pH 8.0). The effect of each substance at a concentration of 2 mM on this enzyme is as follows.

Hg ⁺⁺ , Cd ⁺⁺ , Ni ⁺⁺ , Zn ⁺⁺ , Co ⁺⁺ , Cu ⁺⁺ strongly inhibit,
Mg ⁺⁺ moderately inhibits. NaN ₃ and PCMB also strongly inhibit the enzymatic reaction. The activator and stabilizer for this enzyme are unknown.

(8) Purification method: This enzyme can be purified by the purification method described below.

(9) Molecular weight: The molecular weight of this enzyme was measured by a column gel filtration method using Sephadex G-200.
It was 80,000-83,000 in Tris-HCl buffer (pH 8.0).

(10) Isoelectric point As a result of measurement by disk focus electrophoresis, PI = 6.8
Met.

(11) Disk electrophoresis: Using Davis pH 9.4 gel, 4 mA / gel at 5 ° C, 70
Separation was performed, and the enzyme protein was stained with Coomassie Brilliant Blue G-250. As a result, when the acrylamide concentration of the gel was 7.5%, an almost single band having enzyme activity was observed at 2.5mm on the anode side (4.9cm for bromophenol blue) and at 5% at 11.5mm on the anode side. Was.

As described above, the present enzyme is a novel enzyme whose action and substrate specificity have never been known before.

Next, a method for producing fructosylamine oxidase according to the present invention will be described.

The microorganism used in the present invention belongs to the genus Aspergillus and may be any microorganism having an ability to produce fructosylamine oxidase. Specific examples thereof include Aspergillus sp. Can also be used. Aspergillus sp. 1005 is a strain newly isolated from the soil by the present inventors, and its bacteriological properties are as follows.

In addition, classification of bacteria is generally "The Genus Aspergillus (The Genus Aspergillus)" by The Reper (KBRaper) and Fennel (DIFennell) (The Weverky
Press).

(1) Growth condition in culture medium a) YpSs culture medium (growth at 25 ° C.) The growth was normal and spread to 37 mm incline on the fourth day, and thick white wool-like colonies were formed. The margins are slightly covered with colorless hyphae on the agar surface. Spores were formed at a diameter of 25 mm at the center, and the color was colorless at the periphery and light ocher (Colonial Buff) at the center. The backside was slightly pleated but no pigment formation was observed.

On the seventh day, the hyphae occupied 67 mm in diameter and 3 mm in the margin along the agar surface. Inside there was a white wool hypha with a width of 5 mm, and the inside was densely spore-grown.
The color was depressed yellow (Primrose Yellow) at the periphery, but was ocher (Olive Ocher) further inside, and dark brown (Brownish Olive) at the center. No pattern due to the color and shape of the colony surface was observed. On the back surface, an olive brown coloring was observed at a center diameter of 11 mm, but almost no folds were formed.

The mycelium is prominent, colorless and has partitions, and has a width of 2.
It is branched at 7-9.5 μm. The conidium-forming cells have cells with thickened hyphal cell membranes (Foot cells) and have no septum. Spore stalk with smooth surface, 10-12 μm in width, 1.5-2.4 in length
mm. Spherical conidiospores having a diameter of 31 to 37 μm are formed at the apical end, radial incisors are formed in two steps on the surface, and spores are formed at the tip. The spores are formed in a rosary, the spore head is radial, and is almost spherical with a diameter of 0.2 to 0.35 mm. Spores are approximately spherical with a diameter of 3-4 μm,
The surface is almost smooth and consists of one cell.

b) CIA medium (grown at Zapec-Yeast extract-Agar medium at 25 ° C) The growth is normal and colonies of 52 mm in diameter are formed on the fourth day,
Olivaceous Black (34 mm in diameter at the center)
The spores of the spores grow densely. However, dense white wool-like hyphae are observed on the outer periphery, and no pigment is generated. The apex extends above the aerial mycelium. The back has clear radial folds but no pigment formation.

On the 6th day, it spreads over the entire Petri dish with a diameter of 86 mm, and on the 7th day, the surrounding 2 mm is a place where slightly hyphae can be seen, and the entire surface is covered with black-brown spores.

The spores are very dense, but no surface pattern is seen. On the back, large radial folds are noticeable, but no pigment is produced.

c) Malt extract agar medium A colony having a diameter of 50 mm is formed on the fourth day, but the growth of aerial hyphae is small compared to other mediums and the whole is flat. Spore formation was observed at the center of 38 mm, and its color was dark ocher (Bu
ffy Olive), but the old part in the center is dark olive (Dark Olive). Spore stalks are short and thin compared to other media. Also, the density of spore formation is inferior to other media. No folds are seen on the back surface, and no pigment is formed.

On the 7th day, it spreads to a diameter of 75 mm, and spores are formed almost all over. The color of the spores is blackish brown (Olivaceous Bla
ck), the spore formation status differs concentrically from the center, and concentric patterns are observed as a whole. This pattern is also seen from the back, but no release of the dye to the agar is observed. No folds are observed.

d) Growth conditions pH 2.0 to 9.0 (approx. pH 4.5) Temperature 20 ° C to 45 ° C (optimal temperature 37 ° C) (2) Taxonomic position of this strain The nature and the aforementioned "The Genus
By comparing the mycological properties of the genus Aspergillus described in “The Genus Aspergillus”, the bacterium was determined to be a filamentous fungus belonging to the genus Aspergillus.

Spores are composed of one cell and are linked linearly but do not branch.

The conidiophore has a spherical head and is formed in a two-stage incisor.

The conidium stems from the Foot cell and has no septum.

The hypha has a partition and is colorless.

Furthermore, when the classification was performed based on the classification description of the genus Aspergillus from the growth state of the colonies grown on the Tzapek-agar medium, the colonies were classified into the Aspergillus niger group based on the following characteristics.

This results in a two-stage stroke.

The spore formation is radial, with the spore head forming a generally spherical shape as a whole, but the old spore head may divide slowly.

Apical sac is spherical (31-37 μm) and spore stalk is long enough.

The conidiospores have a dark color.

Furthermore, when classified according to the detailed description of the Aspergillus niger group, it was considered to be a fungus closely related to Aspergillus tulingensis from the following points.

This results in a two-stage stroke.

The color of the conidiospores is grayish brown (Olive
The conidiospores, which have a period of Brown), are initially colorless and transparent, but rapidly become colored and close to black.

Conidia are 3-4 μm in diameter and less than 5 μm.

No coloration is seen behind the colonies.

Conidiospores are often 2-3 mm and do not exceed 5 mm.

This strain has been deposited with the Ministry of International Trade and Industry at the National Institute of Microbial Industry and Technology, Microfabrication Research Institute, No. 2651 (FERM BP-2651).

Next, as a medium used in the present invention, any of a synthetic medium and a natural medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients. As a carbon source, for example, glucose, fructose, xylose,
Glycerin and the like can be used. As a nitrogen source, peptone, casein digest, or nitrogenous organic substances such as yeast extract can be suitably used. Salts such as sodium, potassium, calcium, manganese, magnesium, iron, and cobalt can be used as the inorganic substance.

In the present invention, fructosylamine oxidase can be obtained with the highest yield when cultured in a medium containing fructosyl amino acid. Preferable examples of the culture medium include, for example, fructosylglycine 1.0%, yeast extract 0.5%, polypeptone 0.5%, monopotassium hydrogen phosphate 0.2%.
%, Magnesium sulfate 0.05%, calcium chloride 0.01%,
(PH 6.5). The cultivation is usually carried out at a temperature in the range of 25 to 37 ° C, preferably around 30 ° C. Starting pH of culture
Is in the range of 6 to 8, but preferably around 6.5. Under such conditions, if cultured under shaking or deep stirring with shaking for 30 to 72 hours, fructosylamine oxidase is efficiently produced and accumulated in the culture.

Since this enzyme is usually present in cells, it is necessary to solubilize the enzyme by filtering the culture, collecting the cells, suspending the cells in an appropriate amount of buffer, and disrupting the cells. Fructosylamine oxidase can be obtained by removing nucleic acids, cell wall fragments and the like from the enzyme-containing solution thus obtained. If necessary, the present enzyme may be isolated and purified according to a conventional method, for example, (1) DEAE-cellulose column chromatography, (2) ammonium sulfate fractionation, (3)
Phenyl Sepharose column chromatography,
(4) A purified product can be obtained by using a method such as Sephadex G-200 column chromatography or a combination of other methods as necessary.
Specific examples of purification of the present enzyme are as follows.

After collecting the cells from the culture, the cells are suspended in a 0.02 M phosphate buffer (pH 8.0), and 1% Triton X-100 is added to dissolve the cells. After lightly grinding with a mixer, Dynomill [Shinmaru Enterprise (Sweden)]
The cells are disrupted using. Collect the supernatant by centrifugation,
DEAE-cellulose column (0.02M Tris-HCl buffer, p
(Equilibrated to H8.5) to adsorb the enzyme. 0.
After washing with 02M Tris-HCl buffer (pH 8.5), 0.5M
The enzyme is eluted at a salt concentration. The active fraction is collected and ammonium sulfate powder is added to this to a concentration of 6%. This is passed through a phenyl sepharose column equilibrated with a 0.02 M Tris-HCl buffer (pH 8.5) containing 6% ammonium sulfate to adsorb the enzyme. The column was equilibrated with the same buffer containing 2% ammonium sulfate, and then 0.02M Tris-containing 2% ammonium sulfate.
Elution is performed with a buffer having a concentration gradient formed by connecting hydrochloric acid buffer (pH 8.5) and 0.02 M Tris-hydrochloric acid buffer (pH 9.0) containing 10% ethanol. After concentration of the active moiety, it is applied to a DEAE-Sephadex A-50 column equilibrated in 0.02 M Tris-HCl buffer (pH 8.5). This is eluted with a 0 to 0.5 M saline gradient in the same buffer. The active portion is subjected to column chromatography on Sephadex G-200 equilibrated with a phosphate buffer containing 0.1 M salt, pH 7.5, to obtain a purified enzyme.

Next, a method for quantifying an Amadori compound according to the present invention will be described.

 The quantification method of the present invention is based on the following reaction.

In this formula, R ₁ is — [CH (OH)] _n —CH ₂ OH, _{where n} is 0 to
The integer 4 and R ₂ represent a side chain residue of an α-amino acid. Also R ₃
Is _{- (CH 2) n H,} - (CH 2) n -COOH or - (CH _{2) n} -C
H (NH ₂₎ is -COOH.

The test solution in the quantification method of the present invention may be any solution containing the Amadori compound shown in the above reaction formula, for example, containing a high concentration amino acid or sugar such as soy sauce or honey. Are preferably used. In the case of proteins and polypeptide chains derived from biological samples and the like, there is a problem to be solved by means of releasing them under mild conditions, but the effect of quantification is even greater.

When the above fructosylamine oxidase is allowed to act on the solution containing the Amadori compound, the pH is 6.5 to 10 and the temperature is 50 ° C or lower, preferably the pH is 7.5 to 8.5 and the temperature is 30 to 40 ° C.
The reaction is usually performed for about 10 to 20 minutes. For adjusting the pH, any buffer that can maintain the pH range of the reaction and does not inhibit the enzyme reaction is used, for example, potassium phosphate buffer, potassium phosphate-sodium carbonate buffer, citric acid -Sodium phosphate buffer and the like are preferred. The amount of fructosylamine oxidase used is usually 0.5 units / ml or more in the endpoint analysis.

In the present invention, the Amadori compound is quantified by any of the following measurement methods.

(1) Method for measuring hydrogen peroxide generated by enzymatic reaction: The amount of hydrogen peroxide generated by the reaction is quantified by a conventional method for quantitative determination of hydrogen peroxide, for example, a coloring method, a method using a hydrogen peroxide electrode, and the like. The Amadori compound is quantified from the prepared standard curve of the amount of hydrogen peroxide and the amount of the Amadori compound. When measuring hydrogen peroxide by a color development method, the operation is performed in the same manner as, for example, the measurement method described in the above-mentioned “Method for measuring titer”.

(2) Quantitative method based on oxygen consumption: In this quantitative method, a value obtained by subtracting the oxygen amount at the end of the reaction from the oxygen amount at the start of the reaction (oxygen consumption) is measured, and the oxygen consumption and Amadori prepared separately in advance are measured. The Amadori compound is quantified from a standard curve with the amount of the compound, and the measurement of the amount of oxygen is performed by a conventional method, for example, a Warburg pressure measurement method, an oxygen electrode method, or the like. When the oxygen consumption is measured by the oxygen electrode method, the operation is performed, for example, in the same manner as the measurement method described in the above-mentioned “Method for measuring titer”.

The reagent for quantifying an Amadori compound of the present invention has a pH range suitable for performing fructosylamine oxidase and enzymatic action, generally pH 6.5 to 10, preferably pH 7.5 to 8.5.
When measuring the reaction product further,
If necessary, a coloring agent and the like are appropriately combined.

As fructosylamine oxidase, liquid,
Any of powders may be used, and the amount of enzyme per sample is usually 0.5 units / ml or more for performing end-point analysis. Examples of the buffer include potassium phosphate buffer, potassium phosphate-
Sodium carbonate buffer, citrate-sodium phosphate buffer and the like are preferred.

As a coloring agent for measuring a reaction product, a substance that forms a color by reacting with the product is used.As a coloring agent for hydrogen peroxide, peroxidase and, for example, 4-aminoantipyrine / N, N-dimethyl Aniline, 4-aminoantipyrine / phenol, 4-aminoantipyrine / N, N-diethylaniline, ABTS, MBTH / N, N-diethylaniline, 4
-Aminoantipyrine / 2,4-dichlorophenolsulfonate and the like.

The reagent for quantifying Amadori compounds of the present invention is used in a cool and dark place,
It is preferable to store at a temperature of not more than ℃.

<Effects of the Invention> According to the present invention, it is easy to measure an Amadori compound, which has been difficult in the past, and to efficiently measure an Amadori compound that reflects the state during production and storage of foods such as soy sauce and infusions. Can be. In addition, those bound to protein or polypeptide chains derived from urine, blood, and living organisms can also be measured in the same manner after they have been released by the action of an appropriate peptidase, and are particularly used for measuring the pathological condition of diabetes. it can.

 Next, the present invention will be described with reference to examples.

<Example> Example 1 Aspergillus sp. 1005 (FERM BP-2651) was prepared by adding 1.0% of fructosylglycine, 0.5% of yeast extract, 0.5% of polypeptone, 0.2% of potassium phosphate, 0.2% of magnesium sulfate, and 0.01% of calcium chloride. The bacteria were inoculated into a Sakaguchi flask (capacity: 500 ml) containing 100 ml of the medium (pH 6.5) and cultured with shaking at 30 ° C. for 24 hours. This seed culture was inoculated in the same medium 2 in three mini-jar fermenters and subjected to aeration at a rate of 2 / min and a stirring speed of 400 rpm for 30 minutes.
Culturing was carried out at a temperature of 48 ° C. for 48 hours. The culture was filtered to collect the bacterial conditions. 0.02M Tris-
Hydrochloric acid buffer (containing 1% Triton X-100) pH 8.0,
250 ml was added to disperse the bacteria well and cooled to 4 ° C. with ice. This was lightly ground with a mixer, and then crushed by a dynomill (3000 rpm, 7 minutes). The crushing vessel was sufficiently cooled with ice-cold water. 12000 crushed liquid
The supernatant was collected by centrifugation at rpm for 15 minutes to obtain 275 ml of a liquid. This solution was equilibrated with 0.02 M Tris-HCl buffer pH 8.5, and the column was packed with DEAE-cellulose (diameter 9 c
m × 35 cm in length) to adsorb the enzyme. The active site was collected by elution with a 0 → 0.5M salt concentration gradient. Then, ammonium sulfate was dissolved in the enzyme solution at a rate of 6 g / 100 ml, and then a column (2.5 cm in diameter) packed with phenyl sepharose preliminarily equilibrated with a 0.02 M tris-hydrochloric acid buffer (pH 8.5) containing 6% ammonium sulfate. (Length 23 cm) to adsorb the enzyme. This was washed with the same buffer containing 2% ammonium sulfate to remove unnecessary proteins, and then contained 0.02M Tris-HCl buffer (pH 8.5) containing 2% ammonium sulfate and 10% ethanol.
Elution was carried out with a buffer having a concentration gradient created by connecting a 0.05 M Tris-HCl buffer (pH 9.0). After concentrating the active portion, a column packed with DEAE-Sephadex equilibrated in 0.02 M Tris-HCl buffer (pH 8.5) (diameter 2.
The enzyme was adsorbed over 5 cm x 35 cm in length). This was eluted with a salt gradient (0 to 0.5M) in the same buffer. The active part was converted to an Amicon ultrafiltration membrane device (fraction
000) and then [contains 0.1M salt in advance.
Equilibrated with 0.05 M Tris-HCl buffer (pH 7.5)] column packed with Sephadex G-200 (diameter 1.2 cm × length
Gel filtration over 100 cm). As a result of collecting active parts, 4.2
Unit / mg of protein was obtained. The yield was 27%.

Example 2 4-aminoantipyrine 0.05% 0.3 ml 2,4-dichlorophenolsulfonate 0.15% 0.3 ml phosphate buffer 0.2 M, pH 7.5 1.5 ml peroxidase 400 units / ml 10 μ fructosyl valine 1.5 mM 0 The total volume was adjusted to 2.80 ml by adding 100 μ distilled water.

Add 200 μl of fructosylamine oxidase (containing 165 units / ml) to the test tube containing the reaction solution, and add
After reacting at 7 ° C. for 10 minutes, the coloring dye was colorimetrically determined at 510 nm. As a result, a proportional relationship was observed between the added fructosyl valine and the absorbance.

Example 3 1.5 ml of 0.2 M potassium phosphate buffer (pH 7.5) was placed in an oxygen measuring container of an oxygen monitor (manufactured by YSI, USA), catalase (5000 units / ml) 20 μ, fructosylamine oxidase (165 units) / μl) 200μ and distilled water
1.2 ml was added and stirring was continued at 37 ° C. for 3 minutes to reach equilibrium. After the oxygen electrode was inserted and sealed, 10 µ of a soy sauce solution (adjusted to pH 7.0 and then diluted twice with water) was added to react. The progress of the reaction was all recorded by a recorder connected to a monitor, and the reaction result was measured 10 minutes later. As a result, the change in the oxygen concentration on the 74 scale was read from before the reaction.
Similarly, when the procedure was performed using water instead of fructosylamine oxidase, a change of 14 scales was read. The difference was 60 scale. This value was calculated to be 0.25 μmol from a calibration curve obtained by performing the same operation using a fructosylglycine standard solution instead of the soy sauce solution.

Example 4 Reagent for Measurement Preparation of Reagent A: 5 mg / 75 ml 4-aminoantipyrine [0.15 M potassium phosphate buffer (pH 7.0)] B: 15 mg / 15 ml N, N-dimethylaniline, C: 132 units fructosylamine -Oxidase + 660 units peroxidase / 10 ml When using, mix 3 types and use 2.5 ml of reagent solution for 0.5 ml of sample solution.

[Brief description of the drawings]

FIG. 1 is a graph showing the optimum pH of the present enzyme, FIG. 2 is a graph showing the stable pH of the present enzyme, and FIG. 3 is a graph showing the optimum temperature range for the action of the present enzyme. FIG. 4 is a graph showing thermal stability. The buffers used in FIGS. 1 and 2 are as follows. ○-○: 0.1 M potassium phosphate buffer △-△: 0.1 M Tris-HCl buffer ×-×: 0.1 M glycine-NaOH buffer

Claims (4)

(57) [Claims] 1. A fructosylamine oxidase having the following physicochemical properties: (A) Action and Substrate Specificity The Amadori compound is oxidized in the presence of oxygen to catalyze a reaction to produce α-ketoaldehyde, an amine derivative, and hydrogen peroxide. The Amadori compound works well on derivatives of α-amino acids, β-amino acids, ε-amino acids, and D-amino acids. (B) Optimum pH and stable PH range: The optimal pH range is pH 7.5 to 8.5 (phosphate buffer) when fructosylglycine is used as a substrate, and the stable PH range is 7.5 to 11.0. (C) Suitable temperature range for operation: 30 ° C to 43 ° C. (D) Thermal stability: stable against heating at 37 ° C. for 10 minutes, but deactivated by 70% or more by heating at 50 ° C. for 10 minutes. (E) Molecular weight: The value measured by a gel filtration method using a Sephadex G-200 column is about 80,000 to 83,000. 2. A method for culturing a strain belonging to the genus Aspergillus and having the ability to produce fructosylamine oxidase according to claim 1 in a medium, and collecting the fructosylamine oxidase from the culture. The method for producing fructosylamine oxidase according to claim 1, characterized in that: 3. A sample containing an Amadori compound is treated with the fructosylamine oxidase according to claim 1 to measure the amount of oxygen consumed by the oxidation reaction, or to measure hydrogen peroxide generated by the reaction. A method for quantifying an Amadori compound. 4. A reagent for measuring an Amadori compound comprising the fructosylamine oxidase according to claim 1.