JPH07289253A - Fructosylamino acid oxidase and its production - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme specifically acting on fructosyllysine, thus useful for diabetic diagnoses and food quality control and also useful for producing fructosyllysine and/or fructosyl N<alpha>-Z-lysine. CONSTITUTION:This fructosylamino acid oxidase is obtained by culturing e.g. Fusarium oxysporum S-1F4 (FERM-BP-5010) and has the following characteristics: (1) catalyzing such a reaction that an Amadori compound is oxidized in the presence of oxygen to form an alpha-ketoaldehyde, amine derivative and H2O2; (2) optimal pH is 8.0 and stable pH is 4.0-12.0; (3) optimal temperature is 45 deg.C and stable temperature is 20-55 deg.C; and (4) and molecular weight determined by gel filtration using a Cephacryl S-20 column is about 45000.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel fructosyl amino acid oxidase, and more specifically, a fructosyl amino acid oxidase derived from a bacterium of the genus Fusarium or Gibberella , a method for producing the enzyme, The present invention relates to an Amadori compound analysis method using an enzyme, and a reagent and a kit containing the enzyme. The present invention also provides fructosyl lysine and / or fructosyl as a substrate for the enzyme, which is useful for screening and / or culturing a fructosyl amino acid oxidase-producing bacterium.
The present invention provides a method for producing N ^α -Z-lysine, and a fructosyl lysine and / or fructosyl N ^α -Z-lysine-containing medium for screening and / or culturing fructosyl amino acid oxidase-producing bacteria.

[0002]

Amadori compounds are non-enzymatic and irreversible for amino groups and aldehyde groups when a substance having an amino group such as protein, peptide and amino acid and a reducing sugar such as aldose coexist. It is generated by binding and Amadori transition. The production rate of the Amadori compound is represented by a function such as the concentration of the reactive substance, the contact time, and the temperature. Therefore, it is considered that various information regarding substances containing these reactive substances can be obtained from the produced amount. The substance containing the Amadori compound includes foods such as soy sauce and body fluids such as blood. In the living body, a fructosylamine derivative, which is an Amadori compound in which glucose and an amino acid are bound, is produced. For example, a fructosylamine derivative obtained by glycating hemoglobin in blood is called glycohemoglobin, a derivative obtained by glycating albumin is called glycoalbumin, and a derivative obtained by glycating a protein in blood is called fructosamine. These blood levels reflect the average blood glucose level over a certain period of time in the past, and the measured value can be an important index for the diagnosis of diabetic condition and the management of symptoms. Above all, it is extremely useful. Further, by quantifying the Amadori compound in the food, it is possible to know the storage condition and the period after the production of the food, which is considered to be useful for quality control. Thus, quantitative analysis of Amadori compounds is useful in a wide range of fields including medicine and food.

Conventionally, as a method for quantifying Amadori compounds,
Method using high performance liquid chromatography [Chromatog
r.Sci.10: 659 (1979)], a method using a column packed with a boric acid-bonded solid [Clin.Chem.28: 2088 (1982)],
Electrophoresis [Clin. Chem. 26: 1598 (1980)], method utilizing antigen-antibody reaction [JJCLA 18: 620 (1993), equipment and reagents
16: 33-37 (1993)], Fructosamine assay [Clin.Ch
em.Acta 127: 87-95 (1982)], a method for colorimetric determination after oxidation using thiobarbituric acid [Clin.Chem.Acta112: 179-
204 (1981)] and the like are known, but they require expensive equipment and are not always accurate and quick methods.

In recent years, due to the characteristics of the enzyme (specificity of substrate, reaction, structure, position, etc.), the target substance can be selectively and rapidly analyzed accurately. Has become popular in the fields of clinical analysis and food analysis. An analytical method for quantifying an Amadori compound has already been proposed by allowing an oxidoreductase to act on an Amadori compound and measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated in the reaction (for example, Japanese Patent Publication No. -339
No. 97, Japanese Patent Publication No. 6-65300, Japanese Patent Laid-Open No. 2-
195900, JP-A-3-155780,
JP-A-4-4874, JP-A-5-192193, and JP-A-6-46846). Further, a method for quantifying glycated protein for the diagnosis of diabetes has also been disclosed (JP-A-2-195899, JP-A-2-19590).
No. 0, JP-A-5-192193, JP-A-6-4
6846).

The decomposition reaction of an Amadori compound with an oxidoreductase can be represented by the following general formula. _{^{R 1 -CO-CH 2 -NH-}} R 2 + O 2 + H 2 O → R 1 -CO-CHO + R 2 -NH + H 2 O 2 ( wherein, R ¹ is an aldose residue, R ² is As an enzyme that catalyzes the above reaction, for example, a fructosyl amino acid oxidase derived from a bacterium [a bacterium of the genus Corynebacterium (Japanese Patent Publication No. 6-65300, Japanese Patent Publication No. No. 33997), a bacterium of the genus Aspergillus (JP-A-3-155780)], and a ketoamine oxidase derived from the bacterium [Corynebacterium, Fusarium, Acremonium, or Debryomyces (JP-A-5-135). 192193 JP) Candida (Candida) spp fructosyl amine Daegu ligase from bacteria (JP-a 6-46846 Patent Publication), and Arukirurijina Ze [J.Biol.Chem.239 Volume, the 3790 over 3796
It can be prepared by the method described on page (1964)] and the like.

[0006]

However, the methods using these enzymes have the following problems. That is, the Amadori compound in blood, which is an index in the diagnosis of diabetes, is a glycated protein, which is usually produced by binding glucose to the ε position of a lysine residue in a protein molecule [J. Biol. Chem. 26: 13542-13545 (1986)]. Therefore, it was necessary to use an enzyme highly specific to fructosyl lysine for the measurement of glycated proteins. However, the existing Corynebacterium-derived enzyme does not act on fructosyl lysine, while the Aspergillus-derived enzyme acts on fructosyl lysine, but its activity on fructosyl lysine is higher than that on other Amadori compounds. Is low (see Table 5 below), and its action on glycated protein or its hydrolyzate has not been clarified. On the other hand, the ketoamine oxidase described in JP-A-5-192193 is an enzyme that acts specifically on the saccharification of the α-amine group of fructosyl valine, and is a glycated protein in which a saccharide is bound to a lysine residue. Cannot be measured accurately. Since fructosylamine deglycase has a high activity for difructosyl lysine, it is not possible to specifically measure the glycosylated product at the ε position of the lysine residue. Further, the method using alkyl lysinase has a problem that it acts on substances other than saccharides bound to lysine and has low specificity for glycated products, and accurate measurement could not be expected. Thus, conventional enzymes are not suitable for accurate quantification of glycosylated proteins, and the development of enzymes with high specificity for fructosyl lysine has been awaited. In general, in order for an enzyme-based analytical method to be accurate and useful, it is necessary to select an enzyme that is most suitable for the purpose of analysis. That is, reproducible and accurate analysis may not be possible unless an appropriate enzyme is used in consideration of various conditions such as the type of test substance that is a substrate of the enzyme, the state of the measurement sample, and the measurement conditions. There is. In order to select such an enzyme, activity, substrate specificity, temperature stability, pH stability and the like of various enzymes must be specified in advance. Therefore, it is desirable to produce more fructosyl amino acid oxidases and characterize them.

[0007]

Means for Solving the Problems The present inventors have conducted extensive studies for the purpose of providing a novel fructosyl amino acid oxidase that acts specifically on an Amadori compound, particularly a glycated protein, and as a result, the Fusarium genus ( Fusariu
m ) or Gibberella genus fungus was cultivated in the presence of fructosyl lysine and / or fructosyl N ^α- Z-lysine, and it was found that the enzyme of interest was produced, and the present invention was completed. . That is, the present invention uses a fungus of the genus Fusarium or Gibberella to produce fructosyl lysine and / or fructosyl N.
^The present invention provides a fructosyl amino acid oxidase produced by culturing in a medium containing ^α- Z-lysine. The fructosyl lysine and / or fructosyl N ^α -Z-lysine-containing medium used for culturing the fructosyl amino acid oxidase-producing bacterium of the present invention contains glucose and
Lysine and / or N ^α -Z-lysine at a temperature of 100 to 1
3-60 minutes at 50 ° C., fructosyl lysine obtained by autoclaving and / or fructosyl N ^alpha -Z- lysine containing (hereinafter, sometimes abbreviated as FZL). Therefore, the fructosyl amino acid oxidase of the present invention is an enzyme that acts specifically on fructosyl lysine and / or FZL. In the present specification, fructosyl lysine and / or FZ of the present invention are used.
L-specific fructosyl amino acid oxidase was labeled as fructosyl lysine oxida.
se) and have the same meaning, and hereinafter may be abbreviated as FLOD. The enzyme of the present invention can be produced by culturing a Fusarium or Gibberella genus capable of producing fructosyl amino acid oxidase in a medium containing fructosyl lysine and / or fructosyl N ^α -Z-lysine.

Fusarium oxysporum S-1F4 ( Fusarium oxyspor)
um S-1F4) (FERM BP-5010). In addition, as a bacterium of the genus Gibberella, Gibberella fujikuroi (IFO NO. 6356) ( Gibberella
fujikuroi ) or Gibberella fujikuroi (IFO NO.
6605) ( Gibberella fujikuroi ) and the like.

The present invention also provides a method for producing fructosyl lysine and / or fructosyl N ^α -Z-lysine, which is useful for screening and / or culturing a fructosyl amino acid oxidase-producing bacterium,
And fructosyl lysine and / or fructosyl N for culturing a fructosyl amino acid oxidase-producing bacterium
^A medium containing ^α- Z-lysine is provided. Conventionally,
Glycated proteins have been chemically synthesized by the method described in Japanese Patent Application Laid-Open No. 2-69664, but the synthesis required more than 8 days, and the operation was complicated and not practical. The present inventors have found that a glycated amino acid can be easily produced by allowing a monosaccharide and an amino acid having a free or protective group to coexist in a solution and autoclaving at 100 to 150 ° C. For example, glucose and lysine and / or N ^α -Z-lysine are autoclaved in a solution to give the desired fructosyl lysine and / or FZ.
L is easily obtained. For the purposes of the present invention, N ^α -Z-lysine is more preferred. Therefore, the present invention provides a method for producing a saccharified amino acid, which comprises coexisting a monosaccharide and an amino acid having a free or protective group in a solution and autoclaving at 100 to 150 ° C. . Further, the present invention is characterized in that 0.01 to 50% by weight of glucose and 0.01 to 20% by weight of N ^α -Z-lysine are autoclaved in a solution at 100 to 150 ° C. for 3 to 60 minutes. Fructosil N ^α -Z
-Provides a method for producing lysine.

As mentioned above, fructosyl lysine and / or
Alternatively, FZL is an aqueous solution containing 0.01 to 50% by weight glucose and 0.01 to 20% by weight lysine and / or N ^α -Z-lysine by autoclaving at 100 to 150 ° C. for 3 to 60 minutes, 0.01-0.5%
Fructosyl lysine and / or fructosyl N ^α −
It can be obtained as a Z-lysine aqueous solution (FIG. 1). Preferably, glucose 200 in a total volume of 1000 ml of solution.
g, N ^α- Z-lysine (10 g) was dissolved, and usually 120
Autoclave at 20 ° C for 20 minutes. Purified fructosyl N ^α -Z-lysine can be obtained by purifying the FZL aqueous solution obtained by the above-mentioned method of the present invention by reverse phase chromatography or ion exchange chromatography. The fructosyl lysine and / or FZL-containing medium can be prepared by adding fructosyl lysine and / or FZL obtained by the above-mentioned method of the present invention to an ordinary medium, but an appropriate mixture, that is, glucose 0 .
01 to 50% by weight, lysine and / or N ^α- Z-lysine 0.01 to 20% by weight, K ₂ HPO ₄ 0.1% by weight, Na
A mixture containing 0.1% by weight of H ₂ PO _4, 0.05% by weight of MgSO ₄ .7H ₂ O, 0.01% by weight of CaCl ₂ .2H ₂ O and 0.2% by weight of yeast extract (preferably pH 5.0). 6-
It can also be obtained by autoclaving 6.0) at 100 to 150 ° C. for 3 to 60 minutes. The medium is novel, is useful for screening and / or culturing FLOD-producing bacteria using fructosyl lysine and / or FZL as a substrate, and contributes to the development and research of FLOD. Medium of the present invention can be used to screen for growth possible all types of bacteria in the medium, as such bacteria, fungi, e.g., Fusarium (Fu
sarium ), Gibberella ( Gibberella ), Penicillium ( Penicillium ), Aspergillus ( Aspergillus )
, Bacteria such as Corynebactus
erium ), but not limited thereto. Therefore, the present invention also provides the above medium.

As the medium used for producing the FLOD of the present invention, a usual synthetic or natural medium containing a carbon source, a nitrogen source, an inorganic substance and other nutrient sources can be used. As the carbon source, for example, glucose, xylose, glycerin, etc. can be used, and as the nitrogen source, peptone, casein digest, yeast extract, etc. can be used. Furthermore, as inorganic substances, sodium, potassium, calcium,
What is contained in an ordinary medium such as manganese, magnesium, and cobalt can be used. FLOD of the present invention
Is best induced when cultured in fructosyl lysine and / or FZL containing medium. As an example of a preferable medium, fructosyl N ^α -Z-obtained by the method of the present invention
FZL medium using lysine (FZL) as a single nitrogen source and glucose as a carbon source (1.0% glucose, 0.03%).
5% FZL, 1.0% K ₂ HPO ₄ , 0.1% NaH ₂ P
_{O 4, 0.05% MgSO 4 ·} 7H 2 O, 0.01% CaCl 2
2H ₂ O and 0.01% vitamin mixture). A particularly preferred medium is 20 g glucose, 10 g FZL, 1.0 K ₂ HPO _{4 in} a total volume of 1,000 ml.
g, NaH ₂ PO ₄ 1.0 g, MgSO ₄ · 7H ₂ 0 0.5
g, CaCl ₂ .2H ₂ O 0.1 g and yeast extract 2.0 g
It is a medium containing (pH 5.6-6.0). The FZL-containing medium can be prepared by adding FZL to an ordinary medium or by autoclaving a medium containing glucose and N ^α -Z-lysine. Since the medium obtained by either method is brown due to the presence of fructosyl lysine and / or FZL, the FZL browning medium or GL (glycated lysine and / or glycated N ^α -Z-lysine) brown is used. It is called change medium.

Culturing is usually carried out at 25 to 37 ° C, preferably 28 ° C. The pH of the medium is in the range of 4.0 to 8.0, preferably 5.5 to 6.0. However,
These conditions are appropriately adjusted according to the state of each bacterium, and are not limited to the above. For example, Fusarium oxysporum S-1F4 is
When cultured for 20 to 40 hours, preferably 24 hours, FL
Although OD accumulates in the culture medium, 2
FLOD production was maximal when cultured for 4 hours
(See Figure 2). The thus-obtained culture product can be subjected to a conventional method to remove nucleic acids, cell wall fragments and the like to obtain an enzyme preparation. Usually, the enzyme activity is accumulated in the cells, so that the bacteria in the culture are ground and the enzyme is extracted. Grinding of cells can be done by autolysis, freezing, or by mechanical means or solvent.
Either ultrasonic treatment or pressurization may be used. Enzyme separation and purification methods are also known, such as salting out with ammonium sulfate, precipitation with an organic solvent such as ethanol, ion exchange chromatography, gel filtration, affinity chromatography, etc.
Perform in appropriate combination. For example, the culture is subjected to centrifugation or suction filtration to collect mycelium, washed, and then washed with 0.1 M Tris-H.
It is suspended in Cl buffer (pH 8.5) and the mycelium is disrupted by Dynomill. Then, the centrifuged supernatant is used as a cell-free extract and purified by treating with ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography.

However, for the purposes of the present invention, the FLO
D includes an enzyme-containing solution at any purification stage including a culture solution as long as it can catalyze the oxidation reaction of an Amadori compound, regardless of its degree of purification. Also,
Since the object of the present invention can be achieved even with only the site involved in the catalytic activity in the enzyme molecule, any fragment having Amadori compound oxidation activity is also included. The FLOD thus obtained is useful for the quantification of Amadori compounds, especially the glycated protein for the diagnosis of diabetes. Therefore, the present invention is characterized in that the fungus produces fructosyl amino acid oxidase by culturing the fungus in a medium containing a glycosylated amino acid and / or a glycosylated protein having a protecting group. The present invention provides a method for producing fructosyl amino acid oxidase. Furthermore, the present invention provides a strain belonging to the genus Fusarium or Gibberella and capable of producing fructosyl amino acid oxidase, fructosyl lysine and / or fructosyl N ^α -Z.
-Providing a method for producing fructosyl amino acid oxidase, which comprises culturing in a lysine-containing medium and recovering fructosyl amino acid oxidase from the culture. All FLODs produced by strains of these genera are useful for solving the technical problems to be solved by the present invention. However, their physicochemical properties are slightly different, and in the present specification, if necessary, fusarium.
FLOD-derived from Oxysporum S-1F4
S, FLOD derived from Gibberella fujikuroi is FLOD-
Call G. Hereinafter, the present invention will be described in detail.

(I) Fusarium oxysporum S-1
FLODs produced by F4 The FLODs generally have the following physicochemical properties: 1) Catalyze the reaction of oxidizing Amadori compound in the presence of oxygen to produce α-ketoaldehyde, amine derivative and hydrogen peroxide; 2) Stable pH is about 4.0 to 12.0, optimum pH is 8.0); 3) stable temperature of about 20-55 ° C, optimum temperature of 45 ° C; 4) molecular weight of about 45,000 when measured by gel filtration method using Sephacryl S-200 column. (45kD
a).

Fusarium oxysporum S-1F4 ( Fusarium) which is a bacterium producing FLOD-S of the present invention
oxysporum S-1F4) (hereinafter referred to as S-1F4 strain) is a strain newly isolated by the present inventors from the soil, and its mycological characteristics are as follows. The classification of bacteria is
Mostly based on the description of "The Genus Fusarium " (CMI. 1971) by C. Booth. (1) Growth status in medium Growth in PDA medium, potato-sucrose agar medium, and oatmeal medium is very good in any medium. After culturing for 7 days in a thermostat at 25 ° C., it spreads like a felt over the entire Petri dish and exhibits white to pale purple. (2) Taxonomic properties The isolated bacteria were identified by culturing the microorganisms in oatmeal medium and observing the morphology of the conidia and conidiophor under a microscope. As a result, the color of the colony of S-1F4 strain was white to light purple, indicating that microconidia and macroconidia.
a) and a large number of chlamydospores are formed. Macroconidia is crescent-shaped and has 3 to 5 partition walls.
Since the shape of Microconidia is egg-oval and it forms small conidia in a pseudo-head shape, it was identified as Fusarium oxysporum . This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology under the deposit number FERM BP-5010 (deposit date: February 24, 1994).

The characteristics of FLOD-S produced according to the method of the present invention by the S-1F4 strain will be described in detail. 1. General inducing property FLOD-S is an inducible enzyme induced by fructosyl lysine and / or fructosyl N ^α- Z-lysine (FZL), which uses fructosyl lysine and / or FZL as a nitrogen source and glucose. It is produced by culturing Fusarium oxysporum S-1F4 in a medium containing fructosyl lysine as a carbon source and / or FZL.
FLOD-S is composed of glucose and lysine and / or N ^α −.
The enzyme is induced in a GL browning medium obtained by autoclaving Z-lysine together, but is not induced in a medium prepared by autoclaving glucose and lysine and / or N ^α -Z-lysine separately. Is a compound that acts specifically on the Amadori compound. Also, FLOD
-S was highly induced when lysine and arginine were used, and when glycine and lysine were compared, the induction effect was higher when lysine was used, so fructosyl lysine has a higher substrate activity.

2. Reaction Specificity and Substrate Specificity FLOD-S has the formula: R ¹ —CO—CH ₂ —NH—R ² + O ₂ + H ₂ O → R ¹ —CO—CHO + R ² —NH + H ₂ O ₂ (in the formula, R ¹ represents an aldose residue, R ² represents a protein residue) and has a catalytic activity in the reaction. In the above reaction formula, R ¹ is —OH, — (CH ₂ ) _n —, or — [CH (OH)] _n —CH ₂ OH (where n is an integer of 0-6), and R ² There _{^{-CHR 3 - [CONHR 3] m}} CO
OH (wherein R ³ is an α-amino acid side chain residue, m is 1-4
An Amadori compound represented by (expressing an integer of 80) is preferable as a substrate. Among them, compounds in which R ³ is a side chain residue of an amino acid selected from lysine, polylysine, valine, asparagine, etc., and n is 5 to 6 and m is 55 or less are preferable.

FLOD-S showed high specificity for FZL in both the partially purified enzyme and the purified enzyme. The partially purified enzyme was obtained by culturing the strain in a GL browning medium for 24 hours, collecting the mycelium by suction filtration, washing, and rinsing with 0.1M T.
The suspension was suspended in ris-HCl buffer (pH 8.5), the mycelium was crushed with Dynomill, and the supernatant obtained by centrifugation was subjected to ammonium sulfate fractionation, DE.
Prepared by purification by AE-Sephacel ion exchange chromatography. The purified enzyme was prepared according to the method described in Example 1 described later. The results are shown in Table 1 below.
And 2 are shown. Table 1 Substrate specificity of partially purified S-1F4-derived FLOD-S

[Table 1]

Table 2 Purified S-1F4-derived FLO
Substrate specificity of DS

[Table 2] From Tables 1 and 2, the FLOD-S of the present invention has higher activity against fructosyl N ^α -Z lysine (FZL) and also with fructosyl polylysine, and the Corynebacterium sp. Or Aspergillus sp. It can be seen that the substrate specificity is different from the known fructosyl amino acid oxidase derived from the genus.

3. pH and temperature conditions Measurement of pH conditions 0.1M acetic acid, potassium phosphate (K-P), Tris-HCl
And glycine (Gly) -NaOH buffer (pH 4.0-1)
After adding FLOD-S to 2.0) and incubating at 30 ° C for 10 minutes, the usual conditions (30 ° C, pH 8.0)
The activity was measured by. Measurement of temperature conditions 25 to 6 in 0.1M Tris-HCl buffer (pH 8.0)
After adding FLOD-S to the temperature condition of 0 ° C. and incubating for 10 minutes, the activity was measured under normal conditions. When measured by the above method, the stable pH range of FLD-S derived from S-1F4 is pH 4.0 to 12.0, preferably pH.
The optimum pH is 7.0 to 9.0, preferably 7.0 to 9.0, preferably 7.5 to 8.5, and most preferably 8.0 (Fig. 3).
reference). Further, the stable temperature region of S-1F4-derived FLOD-S is 20 to 55 ° C, which gradually deactivates at 30 ° C or higher, and completely deactivates at 60 ° C or higher. The enzymatic reaction proceeds efficiently at 30 to 50 ° C, preferably 40 to 50 ° C, more preferably 45 ° C (see Fig. 4).

4. Measurement of titer The titer of the enzyme was measured by the following method. (1) A method of measuring the produced hydrogen peroxide by a colorimetric method. A. Velocity method A 50 mM FZL solution was prepared by dissolving FZL prepared in advance in distilled water. 45 mM 4-
Aminoantipyrine, 60 units / ml peroxidase solution, and 60 mM phenol solution 100 each
μl, 0.1M Tris-HCl buffer (pH 8.0) 1m
l, and 50 μl of the enzyme solution are mixed, and the total amount is 2. with distilled water.
Make up to 9 ml. After equilibration at 30 ° C, 50 mM FZL
100 μl of the solution was added, and the absorbance at 505 nm was measured with time. From the molecular extinction coefficient (5.16 × 10 ³ M ^-1 cm ^-1 ) of the quinone dye produced, the micromoles of hydrogen peroxide produced per minute were calculated, and this number was used as the enzyme activity unit (unit: U). And

B. Terminal method The same treatment as in the above method A was performed, and after the addition of the substrate, 30 minutes
The enzyme activity is measured by measuring the absorbance at 505 nm after incubation at 0 ° C. and calculating the amount of hydrogen peroxide produced from a calibration curve prepared in advance using a standard hydrogen peroxide solution. (2) Method for measuring oxygen absorption by enzyme reaction Mix 1 ml of 0.1M Tris-HCl buffer (pH 8.0) and 50 μl of enzyme solution to make the total volume 2.9 ml with distilled water, and use rank Brothers oxygen electrode Put in the cell. After stirring at 30 ° C to equilibrate the temperature with dissolved oxygen, 50 mM F
Add 100 μl of ZL and measure oxygen uptake continuously with a recorder to get the initial velocity. The amount of oxygen absorbed in one minute was determined from the standard curve, and this was used as an enzyme unit.

5. Enzyme inhibition, activation and stabilization (1) Effect of metal Various metal ions with a final concentration of 1 mM were added for 5 minutes under the condition of 0.1M Tris-HCl buffer (pH 8.0). , 30 ° C
The activity was measured after preincubation with. The results are shown in Table 3 below. Table 3 Effect of metal ions on FLD-S activity derived from S-1F4

[Table 3] As is clear from Table 3, the activity of FLOD-S was slightly inhibited by divalent metal ions, and silver ions, mercury ions,
It is completely inhibited by copper and zinc ions.

(2) Effects of various inhibitors Substances were tested in the same manner as the test for the effects of metal ions in (1) above. However, the final concentration of PCMB (mercuric parachlorobenzoate) was 0.1 mM, and the other concentrations were 1 mM. The results are shown in Table 4 below. In addition, regarding stabilization,
It was carried out by dialysis overnight against 50 mM Tris-HCl buffer (pH 8.5) to which 2 mM dithiothreitol (DTT) had been added, and then measuring the activity. The results are shown in Table 4 below. Table 4 Effect of various substances on S-1F4-derived FLOD-S activity

[Table 4] *: 0.1 mM As is clear from Table 4, FLOD-S activity is completely inhibited by PCMB (mercuric parachlorobenzoate).
It is also inhibited by phenylhydrazine and hydrazine. The influence of these inhibitors is that S
It is suggested that H group and carbonyl group are involved. On the other hand, the solvent that is stabilized by dithiothreitol and suitable for storage is dithiothreitol (DTT) 2 mM.
50 mM Tris-HCl buffer (pH 8.5).

6. Molecular Weight The molecular weight was measured by a column gel filtration method using Sephacryl S-200 and SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis). Column chromatography was 0.1M T containing 0.1M NaCl.
It was carried out using ris-HCl buffer (pH 8.5). The same procedure was performed for several proteins with known molecular weights, and the molecular weight of this enzyme was determined from the elution position. SDS-PAGE was carried out according to the method of Davis, using 10% gel, at 40 mA, and 3
After time migration, protein staining was performed with Coomassie Brilliant Blue G-250. Phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor as standard proteins were also electrophoresed in the same manner, and the molecular weight was determined from the calibration curve.
As a result of measurement, FLOD-S derived from S-1F4 has a molecular weight of about 45,000 (45 kDa) when measured by a column gel filtration method using Sephacryl S-200, and
Approximately 50,000 when measured by DS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
It was 0 (50 kDa) (see FIGS. 5 and 6).

7. Isoelectric point S-1 as a result of measurement by the disk focus electrophoresis method.
In the case of FLOD-S derived from F4, pI = 4.8.

8. Comparison with known enzyme The existing fructosyl amino acid oxidase derived from bacteria was compared with the S-1F4 derived FLOD-S of the present invention. Table 5 Comparison of fructosyl amino acid oxidases from various microorganisms

[Table 5] 1): T. Horiuchi et al. Agric. Biol. Ch
em., 53 (1), 103-110 (1989) 2): T. Horiuchi et al. Agric. Biol. Ch.
em., 55 (2), 333-338 (1991) 3): Specific activity to fructosyl-N ^α -Z-lysine 4): Specific activity to N-o-fructosyl-N ^α -formyllysine The following differences are observed between FLOD-S of E. coli and fructosyl amino acid oxidases derived from two other strains. (1) Difference in molecular weight: The FLOD-S of the present invention is produced as a monomer in the microbial cells, while the other two enzymes are produced as dimers in the microbial cells ( 2) Coenzyme: FLOD-S has covalently bound FAD as a coenzyme, while all other enzymes have non-covalently bound FAD. (3) Substrate specificity: Shows a difference in specificity for the substrate fructosyl lysine. That is, FLOD-S has high specificity for fructosyl lysine. On the other hand, although the enzyme derived from Corynebacterium does not act on fructosyl lysine, and the enzyme derived from Aspergillus acts on fructosyl lysine, its activity is lower than that of fructosyl valine. (4) Michaelis constant: This shows that the affinity of FLOD-S for the substrate FZL is higher than that of other enzymes. (5) Optimum pH, optimum temperature, isoelectric point, and inhibition by SH reagent: Differences between FLOD-S and the other two enzymes are shown.

(II) F produced by the genus Gibberella
LODs The FLOD-Gs generally have the following physicochemical properties. 1) It oxidizes an Amadori compound in the presence of oxygen to catalyze the reaction to produce α-ketoaldehyde, an amine derivative and hydrogen peroxide; 2) The optimum pH is 8.0; 3) The stable temperature is 20. ~ 50 ° C, optimum temperature is 35 ° C; 4) When measured by gel filtration method using 200 pg of Superdex, the molecular weight is about 47,000 (47 kDa).
Is.

The characteristics of the FLOD-G will be described in detail. 1. General inducing property FLOD-G is an inducible enzyme induced by fructosyl lysine and / or fructosyl N ^α- Z-lysine (FZL), which uses fructosyl lysine and / or FZL as a nitrogen source and glucose as a carbon source. It is produced by culturing a fructosyl amino acid oxidase-producing strain of Gibberella in a medium containing fructosyl lysine and / or FZL. FLOD-G is glucose and lysine and / or N ^alpha -Z- is lysine together be induced in a GL brown-colored medium obtained by autoclaving glucose and lysine and / or N ^alpha -Z- lysine separately autoclaved Since the enzyme is not induced in the medium prepared by treatment, the enzyme specifically acts on the Amadori compound.

2. FLOD-G of the reaction specificity and substrate specificity The present invention has the _{^{formula: R 1 -CO-CH 2 -NH}} -R 2 + O 2 + H 2 O → R 1 -CO-CHO + R 2 -NH + H ₂ O ₂ (in the formula, R ¹ represents an aldose residue and R ² represents a protein residue), and has a catalytic activity in the reaction. In the above reaction formula, R ¹ is —OH, — (CH ₂ ) _n —, or — [CH (OH)] _n —CH ₂ OH (where n is an integer of 0-6), and R ² There _{^{-CHR 3 - [CONHR 3] m}} CO
OH (wherein R ³ is an α-amino acid side chain residue, m is 1-4
An Amadori compound represented by (expressing an integer of 80) is preferable as a substrate. Among them, compounds in which R ³ is a side chain residue of an amino acid selected from lysine, polylysine, valine, asparagine, etc., and n is 5 to 6 and m is 55 or less are preferable.

The FLOD-G of the present invention has high specificity for fructosyl N ^α -Z-lysine as shown in Table 6 below. Table 6 Purified G. fujikuroi (IFO NO. 635
6) Substrate specificity of FLOD-G derived from

[Table 6] * 1: not detected * 2: fructosyl bovine serum albumin * 3: fructosyl human serum albumin From Table 6, FLOD-G of the present invention has an activity against fructosyl polylysine and also an activity against a protease digestion product of glycated protein. 3. pH and temperature conditions As a result of measuring the pH conditions in the same manner as in (I) 3, the optimum pH is 8.0 (Fig. 7). Also, 0.1M Tris-H
FLOD-G was added to a Cl buffer (pH 8.0) at a temperature of 20 to 60 ° C. and incubated for 10 minutes, and then the activity was measured under ordinary conditions. When measured by the above method,
The stable temperature range of the FLOD-G of the present invention is 20 to 50.
℃, preferably 20-40 ℃, more preferably 20 ℃, the enzymatic reaction is 20-50 ℃, preferably 20-4
It proceeds efficiently at 0 ° C., more preferably 35 ° C. (FIG. 8).

4. Measurement of titer Enzyme titer was measured by the following method. (1) A method of measuring the produced hydrogen peroxide by a colorimetric method. A. Kinetic method A 100 mM FZL solution was prepared by dissolving FZL obtained in advance with distilled water. 45 mM 4-
Aminoantipyrine, 60 units / ml peroxidase solution, and 60 mM phenol solution 100 each
μl and 0.1m Tris-HCl buffer (pH 8.0) 1m
l, and 50 μl of the enzyme solution are mixed, and the total amount is 2. with distilled water.
Make up to 95 ml. After equilibration at 30 ° C, 100 mM F
50 μl of ZL solution was added, and the absorbance at 505 nm was measured over time. From the molecular extinction coefficient (5.16 × 10 ³ M ^-1 cm ^-1 ) of the quinone dye produced, the micromoles of hydrogen peroxide produced per minute were calculated, and this number was used as the enzyme activity unit (unit: U). And

B. Terminal method The same treatment as in the above method A was performed, and after the addition of the substrate, 30 minutes at 30 ° C.
The enzyme activity is measured by measuring the absorbance at 505 nm after incubating in step 1 above and calculating the amount of hydrogen peroxide produced from a calibration curve prepared in advance using a standard hydrogen peroxide solution. (2) Method for measuring oxygen absorption by enzyme reaction Mix 1 ml of 0.1M Tris-HCl buffer (pH 8.0) with 50 μl of enzyme solution to make the total volume 3.0 ml with distilled water, and use rank Brothers oxygen electrode Put in the cell. After stirring at 30 ° C to equilibrate the temperature with dissolved oxygen, 50 mM F
Add 100 μl of ZL and measure oxygen uptake continuously with a recorder to get the initial velocity. The amount of oxygen absorbed in one minute was determined from the standard curve, and this was used as an enzyme unit.

5. Inhibition, activation, and stabilization of enzymes (1) Effect of metal Add 0.1 mM final Tris-HCl buffer (pH 8.0) to each metal ion at a final concentration of 1 mM, and pre-heat at 30 ° C for 5 minutes. After incubation, activity was measured. The results are shown in Table 7 below. Table 7 Metal ion, G. fujikuroi (IFO NO.6
356) Effect on FLOD-G-derived activity

[Table 7] It is clear from Table 7 that cobalt ion, copper ion, zinc ion, mercury ion and iron (III) ion are inhibitory to the activity of FLOD-G of the present invention, and silver ion is completely inhibited.

(2) Effects of various inhibitors Substances were tested in the same manner as the test for the effects of metal ions in (1) above. The results are shown in Table 8. 5 studies for stabilization
It was performed by overnight dialysis against 0 mM Tris-HCl buffer (pH 8.5) to which 2 mM dithiothreitol (DTT) was added, and then measuring the activity. Table 8 Effect of various substances on FLOD-G activity

[Table 8] * 1: 0.1 mM As is clear from Table 8, FLOD-G activity was strongly inhibited by mercuric parachlorobenzoate, hydrazine, phenylhydrazine, hydroxylamine and aminoguanine, and SH group and carbonyl group were present in the enzymatic reaction. It is expected to play an important role. On the other hand, a solvent that is stabilized by dithiothreitol and suitable for storage is 50 mM Tris-HCl buffer (pH 8.5) supplemented with 2 mM dithiothreitol.

6. Molecular weight As a result of gel filtration method using 200 pg of Superdex, the molecular weight was about 47,000 (47 kDa) (FIG. 9). In addition, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is (I)
The molecular weight was found to be about 52,000 (52 kDa) by the same method as in No. 6 above, and it was shown that the molecular weight was about 52,000 (52 kDa) (FIG. 10). Therefore, it is clear that the FLOD-G of the present invention is a monomer.

7. Comparison with known enzyme The existing fructosyl amino acid oxidase derived from bacteria was compared with the FLOD-G of the present invention. Table 9 Comparison of fructosyl amino acid oxidases from various microorganisms

[Table 9] 1): T. Horiuchi et al. Agric. Biol. Che
m., 53 (1), 103-110 (1989) 2): T. Horiuchi et al. Agric. Biol. Che
m., 55 (2), 333-338 (1991) 3): Specific activity to fructosyl N ^α- Z-lysine 4): Specific activity to N-o-fructosyl N ^α -formyllysine The following differences are observed between FLOD-G and fructosyl amino acid oxidases from two other strains. (1) Difference in molecular weight: FLOD-G of the present invention is produced as a monomer, whereas the other two enzymes are produced as dimers. (2) Coenzyme: FLOD-G has covalently bound FAD as a coenzyme, while all other enzymes have non-covalently bound FAD. (3) Substrate specificity: FLOD-G does not act on fructosyl valine and is specific to fructosyl lysine, but the enzyme derived from Corynebacterium does not act on fructosyl lysine, and Aspergillus The genus-derived enzyme acts on fructosyl lysine, but its activity is lower than that on fructosyl valine. (4) Michaelis constant: Shows that the affinity of FLOD-G for the substrate fructosyl lysine is higher than that of other enzymes. (5) Differences such as optimum pH, optimum temperature, and inhibition by SH reagent indicate the difference between FLOD-G and the other two enzymes.

As described above, the FL produced by the method of the present invention by the bacteria derived from Fusarium and Gibberella.
OD is useful for quantifying Amadori compounds. Therefore,
The present invention also provides a method for analyzing an Amadori compound in a sample, which comprises contacting a sample containing the Amadori compound with the FLOD of the present invention and measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated. Is provided. The analysis method of the present invention is performed based on the measurement of the amount and / or the saccharification rate of glycated protein or the quantification of fructosamine in a biological component. The enzyme activity of FLOD is measured based on the following reaction. _{^{R 1 -CO-CH 2 -NH-}} R 2 + O 2 + H 2 O → R 1 -CO-CHO + R 2 -NH + H 2 O 2 ( wherein, R ¹ is an aldose residue, R ² is As the test liquid, any sample solution containing an Amadori compound can be used, and examples thereof include biological samples such as blood (whole blood, plasma or serum) and urine. Other foods such as soy sauce.

The FLOD of the present invention is allowed to act on the solution containing the Amadori compound in a suitable buffer. The pH and temperature of the reaction solution are set to conditions suitable for the enzyme used. That is, in the case of FLOD-S, pH 4.0 to 12.0,
The pH is preferably 8.0 and the temperature is 20 to 55 ° C, preferably 30 to 45 ° C, more preferably 45 ° C. Also, F
In the case of LOD-G, pH is 4.0 to 12.0, preferably 8.0, and temperature is 20 to 50 ° C, preferably 35 ° C. The amount of FLOD used is usually in the end point analysis method.
It is 0.1 unit / ml or more, preferably 1 to 100 unit / ml. Tris-HCl or the like is used as the buffer solution.

In the analysis method of the present invention, any one of the following methods for quantifying the Amadori compound is used. (1) Method Based on Amount of Hydrogen Peroxide Generated Regarding the amount of hydrogen peroxide and Amadori compound, which is measured by a quantitative method of hydrogen peroxide known in the art, for example, a coloring method, a method using a hydrogen peroxide electrode, etc. The Amadori compound in the sample is quantified by comparison with the standard curve prepared. Specifically, the titration method described in (I), 4 or (II), 4 above is applied. However, the FLOD amount is set to 1 unit / ml, an appropriately diluted sample is added, and the amount of hydrogen peroxide produced is measured. 4 for hydrogen peroxide coloring system
4-aminoantipyrine / N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, 4-aminoantipyrine / N, N-dimethylaniline, 4-amino instead of the aminoantipyrine / phenol system Antipyrine / N, N-diethylaniline, MBTH /
N, N-dimethylaniline, 4-aminoantipyrine /
A combination of 2,4-dichlorophenol and the like is possible. (2) Method based on oxygen consumption 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) was measured, and a standard curve prepared for the oxygen consumption and the amount of the Amadori compound was prepared. By comparison, the Amadori compound in the sample is quantified. In particular,
The measurement is carried out according to the measurement of the titer in (I), 4 or (II), 4 above. However, the amount of FLOD used is 1 unit / ml,
Add an appropriately diluted sample to determine the amount of oxygen absorbed.

The method of the present invention can be carried out using the sample solution as it is, but depending on the glycated protein of interest, it is preferable to carry out after releasing the lysine residue to which the sugar is bound in advance. For such purpose, there are cases where a proteolytic enzyme is used (enzymatic method) and cases where a chemical substance such as hydrochloric acid is used (chemical method), but the former is preferable.
Proteolytic enzymes that can be used in the method of the present invention are known to those skilled in the art, and examples thereof include trypsin, carboxypeptidase B, papain, aminopeptidase, chymotrypsin, thermolysin, dubucilisin, proteinase K, pronase and the like. Enzyme treatment methods are also known, and, for example, trypsin treatment can be performed by the method described in Examples below.

As described above, since the FLOD of the present invention has a high substrate specificity for fructosyl lysine contained in glycated proteins, it is possible to diagnose diabetes including the measurement of glycated proteins in blood samples. It is useful for As described above, when a blood sample (whole blood, plasma or serum) is used as the sample, the collected blood sample is used as it is or after being subjected to folding or the like. Further, the enzyme such as FLOD and peroxidase used in the method of the present invention may be used in a solution state, but may be immobilized on a suitable solid support. For example, by packing an enzyme immobilized on beads in a column and incorporating it in an automated device, daily analysis of a large number of samples such as clinical tests can be efficiently performed. Moreover, since the immobilized enzyme can be reused, it is preferable in terms of economic efficiency. Furthermore, by appropriately combining the enzyme and the color-developing dye, it is possible to obtain a kit for analysis of an Amadori compound which is useful not only for clinical analysis but also for food analysis.

Immobilization of the enzyme can be performed by a method known in the art. For example, it is carried out by a carrier binding method, a cross-linking method, an encapsulation method, a composite method or the like. Examples of the carrier include polymer gel, microcapsules, agarose, alginic acid, carrageenan and the like. The binding is carried out by a method known to those skilled in the art, utilizing a covalent bond, an ionic bond, a physical adsorption method, a biochemical affinity. When using immobilized enzyme, the analysis may be either flow or batch. As mentioned above,
Immobilized enzymes are particularly useful for routine analysis (clinical tests) of glycated proteins in blood samples. When clinical tests aim to diagnose diabetes, the diagnostic criteria are expressed as the glycated protein concentration, or the ratio of the glycated protein concentration to the total protein concentration in the sample (glycation rate) or fructosamine level. . The total protein concentration can be measured by a conventional method (absorbance at 280 nm, Lowry method, natural fluorescence of albumin, etc.).

The present invention also provides an analytical reagent or kit for an Amadori compound containing the FLOD of the present invention. The reagent for quantification of the Amadori compound of the present invention is
It consists of the FLOD of the invention, preferably pH 7.5 to 8.5, more preferably pH 8.0 buffer. When the FLOD is immobilized, the solid support is selected from polymer gels and the like, preferably alginic acid. F in reagent
The amount of LOD is usually 1 to 100 units / ml per sample when the end point analysis is performed, and the buffer solution is Tris-HCl (pH
8.0) is preferred. When quantifying the Amadori compound based on the production amount of hydrogen peroxide, as the coloring system, 4-aminoantipyrine / N-ethyl-N- (2-hydroxy) is used instead of 4-aminoantipyrine / phenol system as described above. -3-Sulfopropyl) -m-toluidine, 4
-Aminoantipyrine / N, N-dimethylaniline, 4
-Aminoantipyrine / N, N-diethylaniline, M
Combinations of BTH / N, N-dimethylaniline, 4-aminoantipyrine / 2,4-dichlorophenol and the like are possible. By combining the analytical reagent for the Amadori compound of the present invention with an appropriate color former and a color reference or standard substance for comparison, a kit useful for preliminary diagnosis and inspection can be obtained. The above-mentioned analytical reagents and kits measure the amount and / or glycation rate of glycated proteins in biological components,
Alternatively, it is used for quantifying fructosamine. Hereinafter, the present invention will be described in more detail with reference to examples.

[0045]

EXAMPLES Example 1 FLO from F. oxysporum S-1F4
Production and purification of DS F. Oxysporum S-1F4 ( Fusarium oxysporum S
-1F4: FERMBP-5010) to FZL 0.5%,
Inoculate 10 L of medium (pH 6.0) containing glucose 1.0%, dipotassium phosphate 0.1%, monosodium phosphate 0.1%, magnesium sulfate 0.05%, calcium chloride 0.01%, yeast extract 0.2%, and jar fermenter. Aeration rate of 2 L / min and stirring speed of 400 rpm.
The culture was carried out at 8 ° C. for 24 hours with stirring. The culture was collected by filtration. A part of the mycelium (200 g) containing 0.1 mM Tris-HCl hydrochloric acid buffer solution (pH 8.5) containing 2 mM DTT 1
It was suspended in L and the mycelium was crushed by Dynomill. The disrupted solution was centrifuged at 10,000 rpm for 15 minutes, and the resulting solution was used as a crude enzyme solution (cell-free extract). 40% in crude enzyme solution
Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added so as to be saturated, stirred, and centrifuged at 12,000 rpm for 10 minutes. Ammonium sulfate was added to the obtained supernatant so as to be 75% saturated, and the mixture was stirred and centrifuged at 12,000 rpm for 10 minutes. Precipitate was 50 mM Tris-H containing 2 mM DTT
It was dissolved in Cl hydrochloric acid buffer solution (pH 8.5) (hereinafter abbreviated as buffer solution A) and dialyzed against buffer solution A overnight. The dialysate was adsorbed on a DEAE-Sephacel column equilibrated with buffer A. After washing with the same buffer A, elution was performed with a linear concentration gradient of 0-0.5M potassium chloride. Collect the active fractions,
It was subjected to a 55% to 75% ammonium sulfate fraction and dialyzed against Buffer A overnight. Ammonium sulfate was added to the dialysate so as to be 25% saturated, and the dialysate was adsorbed on a phenyl-Toyopearl column equilibrated with buffer solution A containing 25% saturated ammonium sulfate. After washing with the same buffer, elution was carried out with a linear gradient of ammonium sulfate concentration 25-0% saturation.
The active fractions were collected, ammonium sulfate was added to 40% saturation, and the mixture was adsorbed on a butyl-Toyopearl column equilibrated with buffer solution A containing 40% saturated ammonium sulfate. After washing with the same buffer, elution was carried out with a linear concentration gradient of ammonium sulfate concentration of 40-0% saturation. The active fractions were collected, ammonium sulfate was added to 80% saturation, the mixture was stirred, then centrifuged at 12,000 rpm for 10 minutes,
The obtained precipitate was dissolved in 0.1M buffer A. The enzyme solution was subjected to Sephacryl S-200 gel filtration chromatography by equilibration with 0.1M buffer A containing 0.1M potassium chloride. The active fractions were collected and concentrated by ultrafiltration. Concentrate the Mon with Pharmacia FPLC system
Treatment with a Q column (elution with a 0-0.5M linear gradient of potassium chloride with buffer A) yielded 30-60 units of purified enzyme.

The UV absorption spectrum of the purified enzyme is shown in FIG. FIG. 11 suggests that this enzyme is a flavin enzyme. Further, the purified enzyme preparation was used to measure the molecular weight by a column gel filtration method using Sephacryl S-200 and sodium dodecyl sulfate / polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE). Column chromatography was performed using 0.1M Tris-HCl buffer (pH 8.5) containing 0.1M NaCl. The same procedure was performed for several proteins with known molecular weights, and the molecular weight of this enzyme was determined from the elution position. SDS-P
AGE uses phosphorylase B, bovine serum albumin (BSA), ovalbumin, carbonic anhydrase, soybean trypsin inhibitor as standard proteins,
The molecular weight was measured according to the method of Davis. That is, 10
% Gel, electrophoresis was performed at 40 mA for 3 hours, and protein staining was performed with Coomassie Brilliant Blue G-250. The molecular weight was calculated from the calibration curve. Measurement result, S-1
FL4-S derived from F4 has a molecular weight of about 4 when measured by a column gel filtration method using Sephacryl S-200.
It was 5,000 (45 kDa) and was about 50,000 (50 kDa) when measured by SDS-PAGE (see FIGS. 5 and 6). Furthermore, the FLO prepared in this example
With respect to the enzyme activity of D-S, pH and temperature stability, the influence of metals and inhibitors, etc., the values or properties described in each item of (I) above were shown.

Glucose is bound to a lysine residue
[J. Biol. Chem. 26: 13542-13545 (1986)] Glycated human serum albumin (Sigma)
Was used as a glycated protein substrate and quantified using the purified FLOD-S prepared in the above example. Example 2 Quantification of glycated albumin concentration 1) Trypsin treatment Reagent preparation A: 2% trypsin solution [dissolved in 0.1 M Tris-HCl hydrochloric acid buffer (pH 8.0)] B: 45 mM 4-aminoantipyrine aqueous solution C: 60 mM N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-toluidine D: 60 units / ml peroxidase solution E: 14 units / ml FLOD-S E 14 units / ml FLOD-S solution was prepared by the following method. That is, the F. oxysporum S obtained in Example 1
Purified FLOD-S derived from -1F4 was diluted with distilled water to 14 units / ml. FLOD reaction solution is 0.1
To 10 ml of M Tris-HCl hydrochloric acid buffer solution (pH 8.0), B ~
It was prepared by adding 1 ml of each solution E and adjusting the total amount to 30 ml with distilled water. The trypsin treatment was performed by mixing 30 μl of glycated human serum albumin (Sigma) with various concentrations (0-1.0%) and the same amount of the solution A, and incubating at 37 ° C. for 60 minutes.

2) Quantification Add 1 ml of FLOD reaction solution to the reaction solution treated with trypsin,
After incubating at 30 ° C. for 30 minutes, the absorbance at 555 nm was measured. Results are shown in FIG. In the figure, the vertical axis represents the absorbance at 555 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the glycated albumin concentration. The figure shows that there is a correlation between the glycated albumin concentration and the amount of hydrogen peroxide generated.

Example 3 Measurement of Saccharification Rate of Human Serum Albumin 150 mg of saccharified human serum albumin (Sigma) and 150 mg of human serum albumin (Sigma) were dissolved in 3 ml of 0.9% sodium chloride aqueous solution. When solutions having different saccharification rates were prepared by mixing these solutions and assayed using an automatic glycoalbumin measuring device (Kyoto Daiichi Kagaku), the saccharification rate was 24.1% to 5%.
It was 1.9%. A pretreatment mixture was prepared as follows using these solutions. Saccharified albumin solution 190 μl 6 mg / ml Proteinase K (Sigma) 20 μl 6 mg / ml Pronase E (Sigma) 20 μl 1.25% SDS 20 μl These mixtures were incubated at 55 ° C. for 30 minutes. Then, 1 part was taken out from each pretreatment sample and added to the reaction solution prepared as follows. 45 mM 4-aminoantipyrine solution 70 μl 60 mM phenol solution 70 μl 60 units / ml peroxidase solution 70 μl 0.1 M Tris-HCl hydrochloric acid buffer solution (pH 8.0) 700 μl 11.5 units / ml FLOD-S solution 30 μl The total amount was distilled water. It was 2 ml. 11.5 units / ml FL
The OD-S solution contained 0.1M Tris-HC so that the FLOD-S obtained by the method of Example 1 was adjusted to 11.5 units / ml.
It was prepared by diluting with 1 hydrochloric acid buffer solution (pH 8.0). This reaction solution was incubated at 30 ° C., and each treated sample was treated with 10
After adding 0 μl, the absorbance at 505 nm 30 minutes later was measured. FIG. 13 shows the relationship between the glycation rate of albumin and the absorbance obtained by this method. In the figure, the vertical axis represents the absorbance at 505 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the glycation ratio of albumin. The figure shows that there is a correlation between the saccharification rate of albumin and the amount of hydrogen peroxide generated.

Example 4 Determination of Glycated Protein in Blood Sample Glycated protein in blood was quantified by the method of Example 2 using the serum of diabetic patient as a substrate and FLOD-S purified in Example 1. Conventionally, fructosamine in serum and hemoglobin A1 in whole blood have been used as a blood glucose control index for diabetic patients.
The c (HbA1c) value is used. In this embodiment,
The FLOD of the present invention using an outpatient diabetic blood sample.
The degree of correlation between the glycated protein (serum albumin) measurement by the enzyme method using -S and the conventionally used index was examined. The results are shown in FIGS. The vertical axis in the figure shows the absorbance at 555 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis shows the fructosamine value in the patient serum used as the sample (FIG. 14) and the hemoglobin Alc value in the whole blood (FIG. 15). Each figure shows that there is a correlation between the measurement result of glycated protein using FLOD-S and the conventional blood glucose control index of diabetic patients. That is, the usefulness of the method of the present invention as an index was confirmed. In addition, patients with high HbA1c and fructosamine levels should have high glycated protein (serum albumin) levels. Since fructosamine is a glycosylated protein in serum, it correlates well with the in vivo lifespan of albumin, which is its main component. But H
Since bA1c has different lifespans of hemoglobin and albumin in vivo, and its glycation site is a valine residue, its correlation with glycated proteins is expected to be inferior to the correlation between fructosamine and glycated proteins. 14 and 1
This can be read from 5.

Example 5 G. Fujikuroi (IFO N
O. Production and purification of FLOD-G derived from G. Fujikuroi (IFO NO. 6356; Gibberella f
ujikuroi ) FZL 0.5%, glucose 1.0%, dipotassium phosphate 0.1%, monosodium phosphate 0.1%,
Magnesium sulfate 0.05%, calcium chloride 0.01
%, Yeast extract 0.2% containing medium (pH 6.
0) Inoculate 10 L, and use a jar fermenter at 28 ° C. under the conditions of aeration rate of 2 L / min and stirring speed of 400 rpm.
The culture was carried out with stirring for 24 hours. The culture was filtered and collected. 170 g (wet weight) of mycelium containing 2 mM DTT,
Suspend in 1 L of 1M Tris-HCl buffer (pH 8.5),
The mycelium was crushed with a dyno mill. Disintegrate with 10,0
The solution obtained by centrifugation at 00 rpm for 15 minutes was used as a crude enzyme solution (cell-free extract). Ammonium sulfate was added to the crude enzyme solution to 40% saturation, and the mixture was stirred and centrifuged at 12,000 rpm for 10 minutes. Ammonium sulfate was added to the obtained supernatant so as to be 75% saturated, and the mixture was stirred and centrifuged at 12,000 rpm for 10 minutes. The precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.5) (buffer A) containing 2 mM DTT, and dialyzed against buffer A overnight. The dialysate was adsorbed on a DEAE-Sephacel column equilibrated with buffer A. Buffer A
After washing with water, elution was carried out with a linear concentration gradient of 0-0.5M potassium chloride. Active fractions were collected, subjected to 55% to 75% ammonium sulfate fraction, and dialyzed against buffer A overnight. Ammonium sulfate was added to the dialysate so as to be 25% saturated, and the dialysate was adsorbed on a phenyl-Toyopearl column equilibrated with buffer solution A containing 25% saturated ammonium sulfate. After washing with the same buffer, ammonium sulfate concentration 25-
Elution with a linear gradient of 0% saturation. Collect the active fractions, 40
Ammonium sulphate was added so that it would be% saturated, and it was adsorbed on a butyl-Toyopearl column equilibrated with buffer A containing 40% saturated ammonium sulphate. After washing with the same buffer, ammonium sulfate concentration 40-0%
Elution was performed with a saturated linear concentration gradient. Collect the active fractions, 8
Ammonium sulfate was added to 0% saturation, and after stirring,
Centrifuged at 00 rpm for 10 minutes, and the resulting precipitate was 0.1M.
It was dissolved in buffer solution A. The enzyme solution was subjected to Sephacryl S-200 gel filtration chromatography containing 0.1M potassium chloride and equilibrated with 0.1M buffer A. The active fractions were collected and concentrated by ultrafiltration. Treating the concentrate with a Pharmacia FPLC system using a Mono Q column (0-potassium chloride with buffer A).
Elution with 0.5 M linear concentration gradient), 30-60
A unit of purified enzyme was obtained.

The purified enzyme preparation thus obtained was subjected to SDS-PAGE.
In, the phosphorylase B, bovine serum albumin (BSA), ovalbumin, carbonic anhydrase and soybean trypsin inhibitor were used as standard proteins, and the molecular weight was measured according to the method of Davis. That is,
Using 10% gel, electrophoresis was performed at 40 mA for 3 hours, and protein staining was performed with Coomassie Brilliant Blue G-250. As a result of obtaining the molecular weight from the calibration curve, it was shown that the molecular weight of the subunit was about 52,000 (52 kDa) (FIG. 10). In addition, in the molecular weight measurement by gel filtration with Superdex 200 pg, it was about 47,000 (47 kDa), as is apparent from the calibration curve in FIG. The enzymatic activity of FLOD-G prepared in this example, p
Regarding the H and temperature stability, the influence of metals and inhibitors, etc., the values or properties described in each item of the above (II) are shown.

Example 6 Quantification of fructosyl polylysine A 0.1% fructosyl polylysine solution was added to BMY.NB.
When assayed using the T assay, it was found to contain 750 μmol / l fructosamine. A series of samples varying from 0 to 750 μmol / l was prepared by diluting this solution with distilled water. For each dilution, mix with an equal volume of 0.05% trypsin and mix at 37 ° C for 1 hour.
Incubated for hours. Then, a part of each pretreatment sample was taken out and added to the reaction solution prepared as follows. 45 mM 4-aminoantipyrine solution 50 μl 60 mM phenol solution 50 μl 60 units / ml peroxidase solution 50 μl 0.1 M Tris-HCl buffer solution (pH 8.0) 500 μl 7.6 units / ml FLOD-G solution 30 μl Total amount 1300 μl with distilled water And 7.6 units / ml
The FLOD-G solution was adjusted so that the FLOD-G derived from Gibberella obtained by the method of Example 5 was adjusted to 7.6 units / ml.
It was prepared by diluting with 1 M Tris-HCl hydrochloric acid buffer (pH 8.0). Incubate this reaction at 30 ° C,
200 μl of each treated sample was added, and the absorbance at 505 nm after 30 minutes was measured. The relationship between fructosamine obtained by this method and the absorbance is shown in FIG. In the figure, the vertical axis is 5
The absorbance at 05 nm (corresponding to the amount of hydrogen peroxide), the horizontal axis represents the fructosamine value. The figure shows that there is a correlation between the fructosamine level and the hydrogen peroxide generation rate.

Example 7 Measurement of glycation rate of human serum albumin 150 mg of glycated human serum albumin (Sigma) and 150 mg of human serum albumin (Sigma) were dissolved in 3 ml of 0.9% sodium chloride aqueous solution. When solutions having different saccharification rates were prepared by mixing these solutions and assayed using an automatic glycoalbumin measuring device (Kyoto Daiichi Kagaku), the saccharification rate was 24.1% to 5%.
It was 1.9%. A pretreatment mixture was prepared as follows using these solutions. Saccharified albumin solution 190 μl 6 mg / ml Proteinase K (Sigma) 20 μl 6 mg / ml Pronase E (Sigma) 20 μl 1.25% SDS 20 μl These mixtures were incubated at 55 ° C. for 30 minutes. Then, a part of each pretreatment sample was taken out and added to the reaction solution prepared as follows. 45 mM 4-aminoantipyrine solution 70 μl 60 mM phenol solution 70 μl 60 units / ml peroxidase solution 70 μl 0.1 M Tris-HCl hydrochloric acid buffer (pH 8.0) 700 μl 7.6 units / ml FLOD-G solution 30 μl It was 2 ml. This reaction solution was incubated at 30 ° C., 100 μl of each treated sample was added, and the absorbance at 505 nm after 30 minutes was measured. FIG. 17 shows the relationship between the glycation rate of albumin and the absorbance obtained by this method. In the figure, the vertical axis represents the absorbance at 505 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the glycation ratio of albumin. The figure shows that there is a correlation between the saccharification rate of albumin and the amount of hydrogen peroxide generated.

[0055]

INDUSTRIAL APPLICABILITY The FLOD of the present invention differs from the conventional homologous enzyme fructosyl amino acid oxidase in substrate specificity and specifically acts on fructosyl lysine.
Therefore, it is useful for the development of new clinical analysis and food analysis methods, and contributes greatly to the diagnosis of diabetes and the quality control of food. In particular, it is considered that the amount and / or glycation rate of glycated protein in blood is used as an index to help diagnose the pathological condition of diabetes. Further, the glycated protein can be accurately quantified by the Amadori compound analysis reagent and analysis method using the FLOD of the present invention, which can contribute to the diagnosis of diabetes and the management of symptoms. Further, fructosyl lysine and / or FZL useful for screening and / or culturing of fructosyl amino acid oxidase-producing bacteria can be easily obtained by the method for producing fructosyl lysine and / or FZL of the present invention. Further, the GL browning medium containing fructosyl lysine and / or FZL is FL
It is useful for efficiently culturing OD-producing bacteria.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the amount of fructosyl N ^α -Z-lysine produced by autoclaving 1% N ^α -Z-lysine and the glucose concentration.

FIG. 2 is a graph showing the relationship between the amount of FLOD produced in a culture medium and the culture time.

FIG. 3 is a graph showing the relationship between the activity of S-1F4-derived FLOD-S in a solvent and pH.

FIG. 4 is a graph showing the relationship between the activity of S-1F4-derived FLOD-S in a solvent and temperature.

FIG. 5 is a graph showing the results of molecular weight measurement of S-1F4-derived FLOD-S by gel filtration using Sephacryl S-200.

FIG. 6: SDS-P of purified FLOD-S derived from S-1F4
FIG. 3 is a copy of a photograph showing a migration pattern by electrophoresis obtained by AGE.

FIG. 7 is a graph showing the relationship between activity of Giberella-derived FLOD-G in a solvent and pH.

FIG. 8 is a graph showing the relationship between the activity of FLOD-G derived from Gibberella in a solvent and temperature.

FIG. 9 is a graph showing the results of measuring the molecular weight of purified FLOD-G derived from Gibberella by gel filtration using Superdex 200 pg.

FIG. 10: SDS- of purified FLOD-G derived from Gibberella
FIG. 6 is a copy of a photograph showing a migration pattern by electrophoresis obtained by PAGE.

FIG. 11: Absorption spectrum of purified S-1F4-derived FLOD-S.

FIG. 12 is a graph showing the relationship between the concentration of glycated human serum albumin used as a substrate and the amount of hydrogen peroxide produced by the FLOD action.

FIG. 13 is a graph showing the relationship between the saccharification rate of albumin and the amount of hydrogen peroxide produced by the FLOD action.

FIG. 14: Serum fructosamine level and F in diabetic patients
The graph which shows the relationship with the glycated protein concentration measurement value by LOD-S.

FIG. 15 is a graph showing the relationship between the hemoglobin Alc value in whole blood of diabetic patients and the glycated protein concentration measurement value by FLOD-S.

FIG. 16 is a graph showing the relationship between the fructosamine value and the amount of hydrogen peroxide produced by the FLOD action.

FIG. 17 is a graph showing the relationship between the saccharification rate of albumin and the amount of hydrogen peroxide produced by the FLOD action.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1: 645) (C12N 9/06 C12R 1:77) (C12N 1/14 C12R 1:77) (C12N 1/14 C12R 1: 645) (72) Inventor Masayuki Yagi 1 Nishitokugoku Santandacho, Ukyo-ku, Kyoto City, Kyoto Prefecture 4 Nishi-Kyogoku housing complex, Room 302 (72) Fumiyo Funatsu, 40, Niseko, Hirakata-shi, Osaka Prefecture No. 2

Claims (16)

[Claims] 1. A fructosyl amino acid oxidase produced by culturing a bacterium of the genus Fusarium or Gibberella having the ability to produce fructosyl amino acid oxidase in a fructosyl lysine-containing medium. 2. The fructosyl lysine-containing medium contains glucose and lysine and / or N ^α -Z-lysine at a temperature of 1.
The fructosyl amino acid oxidase according to claim 1, which contains fructosyl lysine obtained by autoclaving at 0 to 150 ° C for 3 to 60 minutes. 3. A bacterium belonging to the genus Fusarium is Fusarium oxysporum S-1F4.
4) (FERM BP-5010), the fructosyl amino acid oxidase according to claim 1. 4. A fructosyl amino acid oxidase according to claim 1, which has the following physicochemical properties: 1) Oxidation of an Amadori compound in the presence of oxygen to form an α-ketoaldehyde or amine derivative. And catalyze the reaction to generate hydrogen peroxide; 2) stable pH is 4.0 to 12.0, optimum pH is 8.0; 3) stable temperature is 20 to 55 ° C, optimum temperature is 45. 4) The molecular weight is about 45,000 (45 kD) when measured by gel filtration using a Sephacryl S-200 column.
a). 5. A bacterium belonging to the genus Gibberella is Gibberella fujikuroi (IFO NO. 6356) ( Gibberella fujikuroi ).
Or Gibberella fujikuroi (IFO NO.6605)
( Gibberella fujikuroi ) The fructosyl amino acid oxidase according to claim 1. 6. A fructosyl amino acid oxidase according to claim 1, 2 or 5, which has the following physicochemical properties: 1) An Amadori compound is oxidized in the presence of oxygen to form an α-ketoaldehyde or amine derivative. And catalyze the reaction to generate hydrogen peroxide; 2) optimum pH is 8.0; 3) stable temperature is 20-50 ° C, optimum temperature is 35 ° C; 4) Superdex 200 pg is used Molecular weight is about 47,000 (47 kDa) when measured by gel filtration method
Is. 7. A Fusarium oxysporum S-1F4
( Fusarium oxysporum S-1F4) (FERM BP-
5010). 8. A method for producing a saccharified amino acid, which comprises coexisting a monosaccharide and an amino acid having a free or protective group in a solution and subjecting the mixture to a high-temperature pressure treatment. 9. An autoclave of 0.01 to 50% by weight glucose and 0.01 to 20% by weight lysine and / or N ^α -Z-lysine in a solution at 100 to 150 ° C. for 3 to 60 minutes. The method according to claim 8, wherein fructosyl lysine and / or fructosyl N ^α- Z-lysine is produced. 10. Glucose 0.01 to 50% by weight, lysine and / or N ^α -Z-lysine 0.01 to 20% by weight, K ₂ HPO ₄ 0.1% by weight, NaH ₂ PO ₄ 0.1% by weight. _{%, MgSO 4 · 7H 2 O} 0.05 wt%, CaCl ₂ ·
Fructosyl lysine and / or fructosyl obtained by autoclaving a mixture containing 0.01% by weight of 2H ₂ O and 0.2% by weight of yeast extract at 100 to 150 ° C. for 3 to 60 minutes.
^N α -Z- lysine-containing medium. 11. A method of culturing a fungus in a medium containing a glycosylated amino acid and / or a glycosylated protein having a free or protective group, thereby causing the fungus to produce fructosyl amino acid oxidase. A method for producing a fructosyl amino acid oxidase. 12. A genus of Fusarium or Gibberella,
Fructosyl lysine and / or fructosyl N ^α-
The method according to claim 11, which comprises culturing in a medium containing Z-lysine and recovering fructosyl amino acid oxidase from the culture. 13. A sample of Amadori in a sample, characterized in that the sample containing the Amadori compound is contacted with the fructosyl amino acid oxidase according to claim 1 and the consumption of oxygen or the generation of hydrogen peroxide is measured. Analytical method of compounds. 14. The sample is a biological component, and the Amadori compound is analyzed by measuring the amount and / or saccharification rate of glycated protein in the biological component, or by quantifying fructosamine. The method described. 15. A reagent or kit for the analysis of an Amadori compound containing the fructosyl amino acid oxidase according to claim 1. 16. Amount and / or amount of glycated protein in biological components
16. The analytical reagent or kit according to claim 15, which is used for measuring a saccharification rate or quantifying fructosamine.