JPH08154672A - Fructosylaminoacid oxidase and its production - Google Patents

Abstract

PURPOSE: To obtain the subject oxidase useful for e.g. analysis of Amadori compounds, by culturing in a fructosyllysine-containing medium Fusarium sp. bacteria capable of producing the fructosylaminoacid oxidase. CONSTITUTION: This fructosylaminoacid oxidase having activity to fructosyllysine and fructosylvaline is obtained by culturing in a fructosyllysine- containing medium Fusarium sp. bacteria capable of producing the fructosylaminoacid oxidase, e.g. Fusarium oxysporum f. sp. lini(IF-No.5880), etc. This oxidase can catalyze reaction of oxidizing the Amadori compounds in the presence of oxygen to form an α-keto-aldehyde, an amine derivative and H2 O2 .

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 , a method for producing the enzyme, and an Amadori compound using the enzyme. And the reagents and kits containing the enzyme.

[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 in which hemoglobin in blood is glycated is called glycohemoglobin, a derivative in which albumin is glycated is called glycoalbumin, and a derivative in which protein in blood is glycated is called fructosamine. These blood levels reflect the average blood glucose level in a certain period in the past, and the measured value can be an important index for the diagnosis and management of the symptoms of diabetes. 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 [Chromato
gr.Sci.10: 659 (1979)], a method using a column packed with a boric acid-bonded solid [Clin. Chem. 28: 2088-2094 (198).
2)], electrophoresis [Clin. Chem. 26: 1598-1602 (1980)], method utilizing antigen-antibody reaction [JJCLA 18: 620 (199)
3), Instruments / Reagents 16: 33-37 (1993)], Fructosamine assay [Clin.Chim.Acta 127: 87-95 (1982)], Colorimetric determination after oxidation using thiobarbituric acid. [Clin.Chi
m.Acta 112: 197-204 (1981)] is known,
It required expensive equipment and was not always an accurate and fast method.

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). Furthermore, a method for quantifying glycated protein for the diagnosis of diabetes has also been disclosed (JP-A-2-195899, JP-A-2-1959).
No. 00, Japanese Unexamined Patent Publication No. 5-192193, No. 6
-46846).

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 ₂ + H ₂ O ₂ (wherein R ¹ represents an aldose residue, R ² represents an amino acid, protein or peptide residue) As an enzyme that catalyzes the above reaction, Are known. 1. Fructosyl amino acid oxidase: genus Corynebacterium (Japanese Patent Publication No. 5-33997)
Japanese Patent Publication No. 6-65300) and Aspergillus (JP-A-3-155780). 2. Fructosylamine deglycase: Candida ( Ca
ndida ) (JP-A-6-46846). 3. Fructosyl amino acid degrading enzymes: Penicillium (Penicillium) (JP-A-4-4874). 4. Ketoamine oxidase: Corynebacterium,
4. Fusarium genus, Acremonium genus or Debryomyces genus (Japanese Patent Laid-Open No. 5-192193) 5. Alkyl lysinase: J. Biol. Chem. 239, 379
Prepared by the method described on page 0-3796 (1964).

[0006]

However, the methods using these enzymes have the following problems. That is, glycated proteins in blood, which are indicators in the diagnosis of diabetes, are glycated albumin, glycated hemoglobin, and fructosamine. Glycated albumin is the ε of lysine residue in protein molecules.
Glucose is bound to the position [J. Biol. Ch
em. 261: 13542-13545 (1986)]. Glycated hemoglobin is described in [J. Biol. Chem. 254: 3892-3898 (1979)], glucose is also bound to the N-terminal valine of the hemoglobin β chain. Therefore, it was necessary to use an enzyme having high specificity for fructosyl lysine and fructosyl valine for the measurement of glycated protein which is an index of diabetes. However, existing enzymes derived from the genus Corynebacterium do not act on fructosyl lysine, and the enzymes derived from the genus Aspergillus have not been clarified about the action on glycated proteins or hydrolysates thereof. On the other hand, JP-A-5-192193
The ketoamine oxidase described in the publication can decompose fructosyl valine, but cannot accurately measure a glycated protein having a sugar bound to a lysine residue. Since fructosylamine deglycase has a high activity for difructosyl lysine, it is not possible to specifically measure the glycosylated ε-position of lysine residue, and specifically the glycosylated valine residue. I can't do it either. 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. Japanese Patent Laid-Open No. 4-4
The enzyme derived from the genus Penicillium described in 874 is an enzyme that acts on fructosyl lysine and fructosyl alanine. As described above, conventional enzymes are not suitable for accurate quantification of glycosylated proteins, and development of enzymes with high specificity for fructosyl lysine and fructosyl valine has been awaited.

In general, in order for an analytical method using an enzyme to be accurate and useful, it is necessary to select an enzyme which is optimum for the purpose of the 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.

[0008]

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
It was found that when the bacterium ( m ) is cultured in the presence of fructosyl lysine and / or fructosyl N ^α -Z-lysine, an enzyme having a desired activity is induced, and the present invention has been completed. That is, the present invention relates to the genus Fusarium ( Fusa
The present invention provides a fructosyl amino acid oxidase produced by culturing a fungus of Rium ) in a medium containing fructosyl lysine and / or fructosyl N ^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.
It contains fructosyl lysine and / or fructosyl N ^α -Z-lysine (hereinafter sometimes abbreviated as FZL) obtained by autoclaving at ˜150 ° C. for 3 to 60 minutes. As will be described later, the fructosyl amino acid oxidase of the present invention is active in both fructosyl lysine and fructosyl valine, but its activity is characterized in that the activity for the former is equal to or higher than the activity for the latter. In addition, in this specification, the fructosyl amino acid oxidase of this invention may be called FAOD.

The enzyme of the present invention can be produced by culturing a Fusarium bacterium having a fructosyl amino acid oxidase-producing ability in a medium containing fructosyl lysine and / or fructosyl N ^α -Z-lysine. Such bacteria include Fusarium oxysporum fs
p. linear (IFO NO.5880) (Fusarium oxysporum f.sp. lin
i ), Fusarium oxysporum f.sp. batatas (IFO
NO.4468) (Fusarium oxysporum f.sp. batatas) , Fusarium oxysporum · f.sp. Nibeumu (IFO NO.4471) (F
usarium oxysporum f.sp. niveum), Fusarium oxysporum · f.sp. Kukumeriniumu (IFO NO.6384) (Fusariu
m oxysporum f.sp. cucumerinum), Fusarium oxysporum · f.sp. Merongenae (IFO NO.7706) (Fusarium
oxysporum f.sp. melongenae), Fusarium oxysporum · f.sp. Api (IFO NO.9964) (Fusarium oxysporum f .
sp. apii ), Fusarium oxysporum f.sp. pini (IF
O NO.9971) (Fusarium oxysporum f.sp. pini ) and Fusarium oxysporum · f.sp. Furagarie (IFO NO.3118
0) ( Fusarium oxysporum f.sp. fragariae ) and the like.

The FAODs of the present invention 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 4.0 to 13.0, optimum pH is 8 0.5; 3) Stable temperature is 20 to 50 ° C, optimum temperature is 30 to 35 ° C.
4) When measured by a gel filtration method using 200 pg of Superdex, the molecular weight is about 106,000 (106 kD).
a).

The fructosyl lysine and / or FZL used in the production of FAOD of the present invention contains glucose of 0.01 to
50% by weight and lysine and / or N ^α -Z-lysine 0.0
1 to 20% by weight in a solution is autoclaved at 100 to 150 ° C. for 3 to 60 minutes. Specifically, it can be produced by dissolving 200 g of glucose and 10 g of N ^α -Z-lysine in a solution having a total volume of 1000 ml and usually autoclaving at 120 ° C for 20 minutes. Further, the fructosyl lysine and / or FZL-containing medium used for the production of FAOD of the present invention (hereinafter referred to as FZL medium) is obtained by adding the fructosyl lysine and / or FZL obtained by the above method to a usual medium. Or, for example, 0.01 to 50% by weight of glucose, 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
_{Wt%, MgSO 4 · 7H 2 O} 0.05 wt%, CaCl
₂ · 2H ₂ O 0.01% by weight and yeast extract 0.2 wt%
1 containing a mixture (preferably pH 5.6-6.0)
It can be obtained by autoclaving at 00 to 150 ° C. for 3 to 60 minutes.

The medium used for the production of FAOD of the present invention is
It may be an ordinary synthetic or natural medium containing a carbon source, a nitrogen source, an inorganic substance, and other nutrient sources, examples of the carbon source include glucose, xylose, glycerin, etc., and examples of the nitrogen source include peptone and casein digestion. Thing, yeast extract, etc. can be used. Further, as the inorganic substance, those contained in a usual medium such as sodium, potassium, calcium, manganese, magnesium, cobalt and the like can be used. The FAOD of the present invention is best induced when cultured in a medium containing fructosyl lysine and / or FZL. As an example of a preferable medium, FZL medium obtained by the above method is used as a single nitrogen source and glucose is used as a carbon source (1.0% glucose, 0.5% FZL, 1.0% K ₂ HPO). ₄ , 0.1% N
_{aH 2 PO 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 a total volume of 1.0
Glucose 20g (2%), FZL 10g in 00ml
(1%), K ₂ HPO ₄ 1.0 g (0.1%), NaH ₂ P
_{O 4 1.0g (0.1%),} MgSO 4 · 7H 2 0 0.5g
(0.05%), CaCl ₂ .2H ₂ O 0.1 g (0.01
%) And yeast extract (2.0 g, 0.2%) (pH 5.6-6.0). The FZL medium can be prepared by adding FZL to an ordinary medium or autoclaving a medium containing glucose and N ^α -Z-lysine. The medium obtained by any of the methods has a brown color due to the presence of fructosyl lysine and / or FZL.
L (glycated lysine and / or glycated N)
^α- Z-lysine) browning 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 prepared according to the state of each bacterium and are not limited to the above. For example, Fusarium oxysporum f. sp. Lini under this condition 20 to 20
When cultured for 100 hours, preferably 80 hours, FAOD
Are accumulated in the culture medium (Fig. 1). 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. Since the enzyme activity of FAOD of the present invention is accumulated in the bacterial cells, the bacterial cells in the culture are crushed and used for enzyme production. The disruption of cells may be carried out by mechanical means or autolysis using a solvent, freezing, sonication, pressurization and the like. A method for separating and purifying an enzyme is also known, and purification is performed by combining salting out with ammonium sulfate, precipitation with an organic solvent such as ethanol, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography and the like. For example, the culture is centrifuged or suction filtered to collect mycelium, washed, and
It is suspended in 1M Tris-hydrochloric acid (pH 8.5) and the mycelium is disrupted by Dino-Mill. Then, the supernatant obtained by centrifugation is used as a cell-free extract and purified by treating with ammonium sulfate fractionation and phenyl-sepharose hydrophobic chromatography.

However, for the purposes of the present invention, FAO
D includes enzyme-containing substances and solutions at any purification stage, including culture broth, as long as they can catalyze the oxidation reaction of Amadori compounds regardless of their degree of purification. Further, 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 an Amadori compound oxidizing activity is also included. FAO obtained in this way
D is useful for the quantification of Amadori compounds, especially glycated proteins 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 free or protective group. The present invention provides a method for producing fructosyl amino acid oxidase.
Furthermore, the present invention cultivates a strain of the genus Fusarium capable of producing a fructosyl amino acid oxidase in a medium containing fructosyl lysine and / or fructosyl N ^α -Z-lysine, and recovering the fructosyl amino acid oxidase from the culture. The present invention provides a method for producing a fructosyl amino acid oxidase, which comprises: FAODs produced by strains of these genera are all useful for solving the technical problems to be solved by the present invention. In the present specification, if necessary, Fusarium oxysporum f. sp. FAOD derived from Lini may also be referred to as FAOD-L.

The characteristics of the FAOD of the present invention will be described in detail below. 1. General Inducing Properties The FAOD of the present invention comprises fructosyl lysine and / or FZ.
L-inducible enzyme, fructosyl lysine and / or FZL as a nitrogen source, glucose as a carbon source fructosyl lysine and / or FZL medium,
It is produced by culturing a fructosyl amino acid oxidase-producing bacterium of the genus Fusarium. FAOD is both glucose and lysine and / or N ^alpha -Z- lysine be induced in a GL brown-colored medium obtained by autoclaving glucose and lysine and / or N ^alpha -Z- lysine separately autoclave treatment The enzyme specifically acts on the Amadori compound because it is not induced in the medium prepared by.

2. Reaction specificity and substrate specificity The FAOD of 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 ₂ + H ₂ O ₂ (wherein R ¹ represents an aldose residue, R ² represents an amino acid, protein or peptide residue) has catalytic activity in the reaction . In the above reaction formula, R ¹ is —O.
_{H, - (CH 2) n} - or _{- [CH (OH)] n} -CH 2 O
H (in the formula, n is an integer of 0-6) and R ² is —CHR ³
- [CONHR ^3] (wherein, R ³ is α- amino acid side chain residue, m represents an integer of 1-480) _m COOH preferred Amadori compound substrate represented by. Among them, R ³ is a side chain residue of an amino acid selected from lysine, polylysine, valine, asparagine, etc., and n is 5 to 6,
Compounds in which m is 55 or less are preferred.

The activity of FAOD of the present invention for each substrate is shown in Table 1 below. Table 1 Substrate specificity of purified FAOD-L

[Table 1] * 1: not detected * 2: fructosyl bovine serum albumin * 3: fructosyl human serum albumin From Table 1, it has high specificity for fructosyl N ^α -Z-lysine and fructosyl valine. Further, the FAOD of the present invention has an activity against fructosyl polylysine and further has an activity against a protease digestion product of a glycated protein. Strains of the genus Fusarium having FAOD production capacity are exemplified in Table 2 below. Table 2 Substrate specificity of FAOD purified from Fusarium sp. Cultured in FZL browning medium

[Table 2] 1): (activity against fructosyl N ^α -Z-lysine) /
(Activity against fructosyl valine)

As shown in Table 2, the FA of the present invention
OD is active against both fructosyl lysine and fructosyl valine, which indicates that the FAOD
Is also useful for the measurement of glycated hemoglobin.

3. pH and temperature conditions Examination of pH conditions The optimum pH is the buffer solution used in the usual FAOD activity measuring method (see 4. Measuring method of titer below) of various buffer solutions of pH 4 to 13 (0.1M phosphoric acid). Potassium buffer (KPB),
Tris-hydrochloric acid buffer solution and glycine (Gly) -NaOH buffer solution) were replaced and the enzymatic reaction was examined. In addition, after adding FAOD to the above-mentioned various buffers and incubating at 30 ° C for 10 minutes, the normal conditions (30 ° C, pH 8.
The stability by pH was examined by measuring the activity in 0). Examination of temperature conditions In order to examine the optimum temperature, the reaction temperature was changed from 20 to 60 ° C. to measure the enzyme activity. Regarding temperature stability, FAOD dissolved in 0.1 M Tris-hydrochloric acid buffer (pH 8.0) was incubated at each temperature of 20 to 60 ° C for 10 minutes, and the residual activity was used under normal conditions using the enzyme solution. It was measured. As a result of measurement by the above method, FAOD of the present invention
The optimum pH was 7.5 to 9.0, more preferably 8.5 (Fig. 2), and the stable pH range was 4.0 to 13.0. The FAOD enzyme reaction proceeded efficiently at 20 to 50 ° C, preferably 25 to 40 ° C, more preferably 35 ° C (Fig. 3). The stable temperature range was 20 to 50 ° C.

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 each 10
0 μl and 0.1 M Tris-HCl buffer (pH 8.0)
1 ml and 50 μl of enzyme solution are mixed, and the total amount is made up to 3.0 ml with distilled water. After incubating at 30 ° C. for 2 minutes, 50 μl of 100 mM FZL solution was added, and 50
Absorbance at 5 nm was measured over time. From the molecular extinction coefficient of the quinone dye produced (5.16 × 10 ³ M ^-1 cm ^-1 ), calculate the number of micromoles of hydrogen peroxide produced per minute, and use this number as the enzyme activity unit (unit: U). And

B. Terminal method After the same treatment as in the above method A and adding the substrate, 30 minutes at 30 ° C.
The enzyme activity is measured by measuring the absorbance at 505 nm after incubating at 1, and calculating the amount of hydrogen peroxide generated from a calibration curve prepared in advance using a standard hydrogen peroxide solution. (2) Method of measuring oxygen absorption by enzyme reaction 1 ml of 0.1 M Tris-hydrochloric acid buffer solution (pH 8.0) and 50 μl of enzyme solution were mixed, and the total amount was adjusted to 3.0 ml with distilled water,
Place in a cell with an oxygen electrode of Rank Brothers. 30
After stirring at ℃ to equilibrate the dissolved oxygen and temperature, 50 mM
100 μl of FZL is added, and oxygen absorption is continuously measured with a recorder to obtain an initial velocity. The amount of oxygen absorbed per minute is calculated from the standard curve, and this is used as the enzyme unit.

5. Inhibition, activation and stabilization of enzymes (1) Effect of metal Add various metal ions to 0.1M Tris-HCl buffer (pH 8.0) to a final concentration of 1 mM, and add 5 at 30 ° C.
After incubating for minutes, the activity was measured. The results are shown in Table 3 below. Table 3 Effect of metal ions on FAOD-L activity

[Table 3] As is clear from Table 3, copper ions and zinc ions are inhibitory to the activity of FAOD of the present invention, and silver ions and mercury ions are 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. However, the final concentration of mercuric parachlorobenzoate was 0.1 mM, and the other concentrations were 1 mM. Table 4 shows the results
Shown in In addition, for stabilization, 2 mM dithiothreitol (DTT) was added to 50 mM Tris-HCl buffer (pH 8.5).
The purified enzyme was dialyzed overnight against the enzyme to which was added, and the activity was measured. Table 4 Effect of various substances on FAOD activity

[Table 4] * 1: PCMB, mercuric parachlorobenzoate * 2: DTNB, 5,5'-dithiobis (2-nitrobenzoic acid) * 3: Not detectable. As apparent from Table 4, FAOD activity is PCMB, DTN.
It was strongly inhibited by B, hydrazine and phenylhydrazine. From this, it is expected that the SH group and the carbonyl group play an important role in the enzymatic reaction. On the other hand, a solvent that is stabilized by dithiothreitol and is suitable for storage is 50 mM Tris-hydrochloric acid buffer (pH 8.5) supplemented with 2 mM dithiothreitol.

6. Molecular weight SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis) was performed according to the method of Davis.
Electrophoresis was performed at 40 mA for 3 hours using a 10% gel, and protein staining was performed with Coomassie Brilliant Blue G-250. Phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase and soybean trypsin inhibitor were similarly electrophoresed as standard proteins, and a calibration curve was prepared to determine the molecular weight. As a result, the molecular weight of the subunit was about 51,000 (51 kDa) (Fig. 4). Gel filtration with 200 pg of Superdex determined the molecular weight to be about 106,000 (106 kDa) (Fig. 5), suggesting that the FAOD of the present invention is a dimer.

7. FAO as a result of measurement by isoelectric focusing method
D had a pI of 6.8.

8. Comparison with a known enzyme The existing fructosyl amino acid oxidase derived from bacteria was compared with the FAOD of the present invention. Table 5 Comparison of fructosyl amino acid oxidases from various microorganisms

[Table 5] 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 for fructosyl N ^α- Z-lysine 4): Specific activity for N ^ε -D-fructosyl N ^α -formyllysine From Table 5, the present invention The following differences are observed between the FAOD of S. cerevisiae and the fructosyl amino acid oxidases derived from the other two strains. (1) Difference in molecular weight: FAOD of the present invention has a clearly higher molecular weight than the other two enzymes. (2) Coenzyme: The FAOD of the present invention has covalently bound FAD as a coenzyme, while all other enzymes have non-covalently bound FAD. (3) Substrate specificity: The FAOD of the present invention has higher specificity for fructosyl lysine than fructosyl valine, but the enzyme derived from Corynebacterium does not act on fructosyl lysine and is derived from Aspergillus. Although the enzyme acts on fructosyl lysine, its activity is lower than that on fructosyl valine. (4) Michaelis constant: Shows that the affinity of FAOD of the present invention for the substrate fructosyl lysine is higher than that of other enzymes. (5) Optimum pH, optimum temperature, and inhibition by SH reagent:
The difference between FAOD of the present invention and the other two enzymes is shown.

As mentioned above, the enzyme FAOD of the present invention is
It is useful for quantifying Amadori compounds. Therefore, the present invention provides a sample containing the Amadori compound and the FAO of the present invention.
The present invention provides an analytical method for an Amadori compound in a sample, which comprises contacting with D and measuring the consumption of oxygen or the generation of hydrogen peroxide. The analysis method of the present invention comprises measuring the amount and / or saccharification rate of glycated proteins in biological components,
Alternatively, it is performed based on the quantification of fructosamine. F
The enzyme activity of AOD 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 2 + H 2 O 2 ( wherein, R ¹ is an aldose residue, R ² represents amino, a protein or peptide residue) as the test solution, Amadori compound Any sample solution containing a can be used, and examples include biological samples such as blood (whole blood, plasma or serum) and urine, and food products such as soy sauce.

The FAOD of the present invention is allowed to act on the solution containing the Amadori compound in a suitable buffer. PH of reaction solution,
The temperature is in the range satisfying the above conditions, that is, pH 4.0 to 4.0.
The temperature is 13.0, preferably 8.5, and the temperature is 20 to 50 ° C, preferably 35 ° C. Tris-hydrochloric acid or the like is used as the buffer solution. The amount of FAOD used is usually 0.1 unit / ml or more, preferably 1 to 100 unit / ml in the end point analysis method.

In the analysis method of the present invention, any 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, it is based on the above-mentioned measurement of titer 4. However, the FAOD amount is set to 1 unit / ml, an appropriately diluted sample is added, and the amount of hydrogen peroxide produced is measured. As a coloring system for hydrogen peroxide, a system that develops color by the oxidative condensation of a coupler such as 4-aminoantipyrine, 3-methyl-2-benzothiazolinonehydrazone and a chromogen such as phenol in the presence of peroxidase is used. Can be used. Chromogens include phenol derivatives, aniline derivatives, toluidine derivatives and the like. For example, N-ethyl-
N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N, N-dimethylaniline, N, N-diethylaniline, 2,4-dichlorophenol, N-ethyl-
N- (2-hydroxy-3-sulfopropyl) -3,5
-Dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-
N- (2-hydroxy-3-sulfopropyl) -3,5
-Dimethylaniline and the like can be mentioned. Also, a leuco-type coloring reagent that exhibits oxidative coloring in the presence of peroxidase can be used, and such a leuco-type coloring reagent is known to those skilled in the art, and o-dianisidine, o-trizine, 3,3
-Diaminobenzidine, 3,3,5,5-tetramethylbenzidine, N- (carboxymethylaminocarbonyl)
-4,4-bis (dimethylamino) biphenylamine,
10- (carboxymethylaminocarbonyl) -3,7
-Bis (dimethylamino) phenothiazine and the like. (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 above-mentioned titer measurement. However, FAOD used
The amount is 1 unit / ml, and an appropriately diluted sample is added to determine the amount of oxygen consumed.

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 preferred that the lysine and / or valine residue to which the sugar has been bound be liberated beforehand. When using a proteolytic enzyme for such purpose (enzymatic method)
In some cases, a chemical substance such as hydrochloric acid is used (chemical method), but the former is preferable. In that case, endo-type and exo-type proteolytic enzymes (proteases) known to those skilled in the art can be used. The endo-type protease includes, for example, trypsin, α-chymotrypsin, subtilisin, proteinase K, papain,
Cathepsin B, pepsin, thermolysin, protease XIV, lysyl endopeptidase, proleather, bromelain F and the like. On the other hand, exo-type proteases include aminopeptidase, carboxypeptidase and the like. The method of enzyme treatment is also known, and for example, the method described in the following examples can be used.

As described above, since the FAOD of the present invention has a high substrate specificity for fructosyl lysine contained in glycated protein, it is possible to diagnose diabetes mellitus including the measurement of glycated protein in a blood sample. It is useful for Further, since it has specificity to fructosyl valine, it is also useful for measuring glycated hemoglobin. When a blood sample (whole blood, plasma or serum) is used as the sample, the sampled blood is used as it is or after being subjected to a treatment such as folding. Further, FAO used in the method of the present invention
The enzyme such as D and peroxidase may be used in a solution state, or 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 methods known in the art. For example, it is carried out by a carrier binding method, a cross-linking method, an entrapping 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 a clinical test is aimed at diagnosing diabetes, the result is expressed as a glycated protein concentration, or a ratio of the glycated protein concentration to the total protein concentration in a sample (glycation rate) or a fructosamine value. Total protein concentration was determined by standard methods (absorbance at 280 nm, Bradford method, Lowry
Method, Burette method, natural fluorescence of albumin, absorbance of hemoglobin, etc.).

The present invention also provides an analytical reagent or kit for an Amadori compound containing the FAOD of the present invention. The reagent for quantifying the Amadori compound of the present invention is FAOD of the present invention, preferably pH 7.5 to 8.5,
More preferably, it comprises a buffer solution having a pH of 8.0. The FAO
When D is immobilized, the solid support is selected from polymeric gels and the like, preferably alginic acid. The amount of FAOD in the reagent is
Usually, 1 to 100 units / ml, and the buffer is preferably Tris-hydrochloric acid (pH 8.0). When quantifying the Amadori compound based on the amount of hydrogen peroxide produced, the coloring system is
The system that develops color by oxidative condensation described in the above-mentioned “(1) Method based on the amount of generated hydrogen peroxide”, a leuco-type color reagent, and the like can be used. It is also possible to prepare a kit by combining the analytical reagent for the Amadori compound of the present invention, an appropriate color former and a color reference or standard substance for comparison. Such a kit is considered to be useful for preliminary diagnosis and examination. The above analytical reagents and kits are used for measuring the amount and / or glycation rate of glycated proteins in biological components, or for quantifying fructosamine. Hereinafter, the present invention will be described in more detail with reference to examples.

[0035]

EXAMPLES Example 1 Cultivation of Fusarium oxysporum f.sp. lini and purification of FAOD-L 1) Culture Fusarium oxysporum f.sp. lini (IFO NO.58
80; Fusarium oxysporum f.sp. lini ) to FZL 0.5
%, Glucose 1.0%, dipotassium phosphate 0.1%,
Monosodium phosphate 0.1%, magnesium sulfate 0.0
5%, calcium chloride 0.01%, yeast extract 0.0.
The medium was inoculated into 10 L of medium (pH 6.0) containing 2%, aeration rate was 2 L / min and stirring speed was 4 using a jar fermenter.
Culture was carried out with stirring at 28 rpm at 80 rpm for 80 hours. The culture was filtered and collected. 2) Preparation of crude enzyme solution 270 g (wet weight) of mycelium containing 2 mM DTT,
The cells were suspended in 800 ml of 0.1 M Tris-hydrochloric acid buffer (pH 8.5) and the mycelium was disrupted with Dino-Mill. The disrupted solution was centrifuged at 9,500 rpm for 20 minutes, and the resulting supernatant (cell-free extract) was used as a crude enzyme solution and purified by the following method. 3) Purification Step 1 : Ammonium Sulfate Fractionation Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40% saturated, and the mixture was centrifuged (4 ° C, 12,
(000 rpm) to remove excess protein. Further, ammonium sulfate was added to the supernatant so as to be 75% saturated, and the precipitate was recovered. Step 2 : Hydrophobic chromatography (batch method) The precipitate obtained in Step 1 is treated with 50 mM Tris-hydrochloric acid buffer (pH 8.5) containing 2 mM DTT (hereinafter, referred to as
Buffer A, which was dissolved in Buffer A) and contained an equal amount of ammonium sulfate 40%, was added. Butyl toyopearl (bu
200 ml of tyl-TOYOPEARL resin was added and adsorption was performed by the batch method. Elution was also performed by the batch method using buffer solution A, and the active fraction was concentrated by ammonium sulfate precipitation. Step 3 : Hydrophobic Chromatography The concentrated active fraction was adsorbed on a phenyl-TOYOPEARL column equilibrated with buffer A containing 25% ammonium sulfate, washed with the same buffer and washed with 25-0% ammonium sulfate. Elution with a linear gradient. The collected active fraction was concentrated by ammonium sulfate precipitation and used in the next step. Step 4 : Hydrophobic chromatography (column method) The recovered active fraction was applied to a butyl Toyopearl column (equilibrated with buffer solution A containing 40% ammonium sulfate). The concentrated solution was adsorbed and washed with the same buffer. The active fraction was obtained with a linear gradient of 40-0% ammonium sulfate. Step 5 : Ion exchange chromatography Next, DEAE-TOYOPEARL (DEAE-TOYOPE)
ARL) column chromatography was performed (buffer solution A).
Equilibrated with). Since FAOD activity was observed in the washed fraction, it was collected, concentrated with ammonium sulfate, and used in the next step. Step 6 : Gel filtration Finally, gel filtration with Sephacryl-300 was carried out (equilibration with 0.1 M Tris-hydrochloric acid buffer solution (pH 8.5) containing 0.1 M NaCl and 2 mM DTT). As a result, 70 to 100 units of enzyme preparation was obtained.

The UV absorption spectrum of the purified enzyme is shown in FIG. FIG. 6 shows that this enzyme is a flavin enzyme. The molecular weight of the purified enzyme preparation was determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and gel filtration using Superdex 200 pg. SDS-PAGE was performed at 40 mA using a 10% gel according to the method of Davis.
After time migration, protein staining was performed with Coomassie Brilliant Blue G-250. Phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase and soybean trypsin inhibitor were similarly electrophoresed as standard proteins to prepare a calibration curve. As a result, the subunit molecular weight of the purified enzyme was about 51,000 (51 kD
It was a). On the other hand, gel filtration was performed using 0.1M Tris-hydrochloric acid buffer containing 0.1M NaCl (pH 8.5). As a result, as shown in FIG.
kDa). Furthermore, FAO purified in this example
The enzyme activity of DL, optimum pH and temperature, pH and temperature stability, influence of metals and inhibitors, etc. are as described above.

Example 2 Measurement of Glycated Human Serum Albumin Concentration Glycated human serum albumin (Sigma) was dissolved in a 0.9% aqueous sodium chloride solution to prepare glycated human serum albumin solutions having different concentrations in the range of 0 to 10%. did. The following operations were performed using these solutions. 1) Protease treatment Saccharified albumin solution 60 μl 12.5 mg / ml Protease XIV (Sigma) solution 60 μl This mixed solution was incubated at 37 ° C. for 30 minutes and then heated at about 90 ° C. for 5 minutes to stop the reaction. . 2) Activity measurement The FAOD reaction solution was prepared as follows. 45 mM 4-aminoantipyrine solution 30 μl 60 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine solution 30 μl 60 units / ml peroxidase solution 30 μl 0.1 M Tris-HCl buffer (pH 8.0) ) 300 μl 6 units / ml FAOD-L solution 50 μl Distilled water was added to make the total volume 1 ml. 6 units / ml FAOD
The -L solution contained 0.1 M Tris-hydrochloric acid buffer solution (p of FAOD-L obtained by the method of Example 1 so as to be 6 units / ml).
It was prepared by diluting with H8.0). The FAOD reaction solution is 30
After incubating at 0 ° C. for 2 minutes, 100 μl of each protease treatment solution was added, and the absorbance at 555 nm after 30 minutes was measured. FIG. 7 shows the relationship between the concentration of glycated albumin and the absorbance obtained by this method. 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 concentration of glycated albumin. The figure shows that there is a correlation between the concentration of glycated albumin and the amount of hydrogen peroxide generated.

Example 3 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. 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 24.6% to 6%.
It was 1.1%. The following operations were performed using these solutions. 1) Protease treatment Saccharified albumin solution 60 μl 12.5 mg / ml Protease XIV (Sigma) solution 60 μl This solution was incubated at 37 ° C. for 30 minutes and then heated at about 90 ° C. for 5 minutes to stop the reaction. 2) Activity measurement The FAOD reaction solution was prepared as follows. 45 mM 4-aminoantipyrine solution 30 μl 60 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine solution 30 μl 60 units / ml peroxidase solution 30 μl 0.1 M Tris-HCl buffer (pH 8.0) ) 300 μl 6 units / ml FAOD-L solution 50 μl Distilled water was added to make the total volume 1 ml. 6 units / ml FAOD
The -L solution contained 0.1 M Tris-hydrochloric acid buffer solution (p of FAOD-L obtained by the method of Example 1 so as to be 6 units / ml).
It was prepared by diluting with H8.0). The FAOD reaction solution is 30
After incubating at 0 ° C. for 2 minutes, 100 μl of each protease treatment solution was added, and the absorbance at 555 nm after 30 minutes was measured. The relationship between the glycation ratio of albumin and the absorbance obtained by this method is 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 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 Measurement of glycated hemoglobin concentration Glycohemoglobin control (Sigma) was dissolved in distilled water to prepare glycated hemoglobin solutions having different concentrations in the range of 0 to 30%. The following operations were performed using these solutions. 1) Protease treatment Glycated hemoglobin solution 25 μl 500 units / ml aminopeptidase solution 5 μl 0.1 M Tris-HCl buffer (pH 8.0) 20 μl This mixture was incubated at 30 ° C. for 30 minutes. Then, add 50 μl of 10% trichloroacetic acid and stir,
After standing still at 0 ° C. for 30 minutes, centrifugation was performed at 12000 rpm for 10 minutes. About 5M of 2M NaOH was added to the obtained supernatant.
0 μl was added to make a neutral solution. 2) Activity measurement The FAOD reaction solution was prepared as follows. 3 mM N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine solution 30 μl 60 units / ml peroxidase solution 30 μl 0.1 M Tris-hydrochloric acid buffer solution (pH 8.0) 300 μl 4 units / ml FAOD -L solution 10 μl Distilled water was added to make the total volume 1 ml. 4 units / ml FAOD
The -L solution contained 0.1 M Tris-hydrochloric acid buffer solution (p) so that FAOD-L obtained by the method of Example 1 became 4 units / ml.
It was prepared by diluting with H8.0). The FAOD reaction solution is 30
After incubating at 0 ° C. for 2 minutes, 80 μl of each protease treatment solution was added, and the absorbance at 727 nm after 30 minutes was measured. The relationship between the concentration of glycated hemoglobin and the absorbance obtained by this method is shown in FIG. In the figure, the vertical axis represents the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the concentration of glycated hemoglobin. The figure shows that there is a correlation between the concentration of glycated hemoglobin and the amount of hydrogen peroxide generated.

[0040]

INDUSTRIAL APPLICABILITY The FAOD of the present invention, unlike the conventional homologous enzyme fructosyl amino acid oxidase, specifically acts on both fructosyl lysine and fructosyl valine and has higher specificity to the former. . 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 of glycated protein and / or glycation rate in blood or the amount of fructosamine is used as an index to help diagnose the pathology of diabetes. Further, the FA of the present invention
Glycated proteins can be accurately quantified by an Amadori compound analysis reagent and analysis method using OD, which can contribute to diabetes diagnosis and symptom management.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between FAOD production in a culture medium and culture time.

FIG. 2 is a graph showing the relationship between the activity of FAOD in a solvent and the optimum pH.

FIG. 3 is a graph showing the relationship between the activity of FAOD in a solvent and the optimum temperature.

FIG. 4 FAOD in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
-A copy of a photograph showing the migration pattern of L.

FIG. 5 is a graph showing the results of molecular weight measurement of FAOD-L by gel filtration using Superdex 200 pg.

FIG. 6 is an absorption spectrum of FAOD-L.

FIG. 7 is a graph showing the relationship between the concentration of glycated human serum albumin and the amount of hydrogen peroxide produced by the FAOD action.

FIG. 8 is a graph showing the relationship between the saccharification rate of human serum albumin and the amount of hydrogen peroxide produced by the FAOD action.

FIG. 9 is a graph showing the relationship between the concentration of glycated hemoglobin and the amount of hydrogen peroxide produced by the FAOD action.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:77) (72) Inventor Toshikatsu Sakai 28 Sannocho, Shogoin, Sakyo-ku, Kyoto-shi, Kyoto (72) ) Inventor, Kaoru Ishimaru, No. 302, Saint Amu No. 2, 23, Kitabata-cho, Fushimi-ku, Kyoto-shi, Kyoto

Claims (11)

[Claims] 1. A fructosyl amino acid oxidase produced by culturing a Fusarium bacterium capable of producing fructosyl amino acid oxidase in a fructosyl lysine-containing medium, which comprises fructosyl lysine and fructosyl valine. A fructosyl amino acid oxidase having activity against. 2. The fructosyl amino acid oxidase according to claim 1, wherein the activity against fructosyl lysine is equal to or higher than the activity against fructosyl valine. 3. The fructosyl lysine-containing medium contains glucose and lysine and / or N ^α -Z-lysine at a temperature of 100.
The fructosyl amino acid oxidase according to claim 1, which contains fructosyl lysine and / or fructosyl N ^α- Z-lysine, which is obtained by autoclaving at −150 ° C. for 3 to 60 minutes. 4. Fusarium genus fungi, Fusarium oxysporum, f.sp. linear (IFO NO.5880) (Fusarium oxyspo
rum f.sp. lini ), Fusarium oxysporum f.sp.
Batatasu (IFO NO.4468) (Fusarium oxysporum f.sp. bata
tas ), Fusarium oxysporum f.sp. niveum (IF
O NO.4471) ( Fusarium oxysporum f.sp. niveum ), Fusarium oxysporum f.sp.
384) (Fusarium oxysporum f.sp. cucumerinum) , Fusarium oxysporum · f.sp. Merongenae (IFO NO.770
6) (Fusarium oxysporum f.sp. melongenae) , Fusarium oxysporum · f.sp. Api (IFO NO.9964) (Fusarium
oxysporum f.sp.apii ), Fusarium oxysporum
f.sp. Pini (IFO NO.9971) (Fusarium oxysporum f.sp. pin
i ) and Fusarium oxysporum f.sp.
(IFO NO.31180) ( Fusarium oxysporum f.sp.fragariae )
The fructosyl amino acid oxidase according to claim 1, which is selected from the group consisting of: 5. A fructosyl amino acid oxidase according to any one of claims 1 to 4, which has the following physicochemical properties: 1) Oxidation of an Amadori compound in the presence of oxygen to form α-ketoaldehyde, amine Catalyzes the reaction to form a derivative and hydrogen peroxide; 2) stable pH is 4.0 to 13.0, optimum pH is 8.5; 3) stable temperature is 20 to 50 ° C, optimum temperature is 30-35 ° C
4) When measured by a gel filtration method using 200 pg of Superdex, the molecular weight is about 106,000 (106 kD).
a). 6. 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 allowing the fungus to produce fructosyl amino acid oxidase. The method for producing the fructosyl amino acid oxidase according to claim 1. 7. A strain belonging to the genus Fusarium and capable of producing fructosyl amino acid oxidase is cultured in a fructosyl lysine and / or fructosyl N ^α -Z-lysine-containing medium, and the fructosyl amino acid oxidase is recovered from the culture. 7. The method according to claim 6, wherein 8. 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 to measure the consumption of oxygen or the generation of hydrogen peroxide. Analytical method of compounds. 9. 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. 10. An Amadori compound analysis reagent or kit containing the fructosyl amino acid oxidase according to claim 1. 11. Amount and / or amount of glycated protein in biological components
11. The analytical reagent or kit according to claim 10, which is used for measuring the saccharification rate or quantifying fructosamine.