JPH08336386A - Fructosylamino acid oxidase and its production - Google Patents

Abstract

PURPOSE: To obtain the subject enzyme useful for diagnosing diabetic symptoms, administering such symptoms, and understanding the food preservation status and/or period after producing foods, through the quantitative analyses of Amadori compounds. CONSTITUTION: This enzyme is obtained by culturing in a fructosyllysine-contg. medium Penicillium bacteria capable of producing fructosylamino acid oxidase [e.g. Penicillium janthinellums-3413 (FERM BP-5475)] and has the following physicochemical properties: (1) catalyzing the reaction forming an α-ketaldehyde, amine derivative and H2 O2 by oxidizing an Amadori compound in the presence of oxygen; (2) optimum pH : 7.5, and s table pH: 4.0-11.0; (3) optimum temperature: 25 deg.C, and stable temperature: 15-50 deg.C; and (4) molecular weight: about 38700 (gel filtration method).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel fructosyl amino acid oxidase, more specifically, a fructosyl amino acid oxidase derived from a fungus of the genus Penicillium , a method for producing the enzyme, and an Amadori compound using the enzyme. The present invention relates to an analytical method, and 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.
For example, in a living body, a fructosylamine derivative which is an Amadori compound in which glucose and an amino acid are bound to each other 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 reducing ability of a fructosylamine derivative in an alkaline solution is called fructosamine. These blood concentrations reflect the average blood glucose level for a certain period in the past, and the measured value is
The establishment of measuring means is clinically extremely useful because it can be an important index for diagnosis of diabetic condition and management of symptoms. 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, JP-A-6-46846, and JP-A-7-289.
No. 253). Furthermore, a method for quantifying glycated protein for the diagnosis of diabetes has also been disclosed (JP-A-2-1958).
99, JP-A-2-195900, JP-A-5
-192193, JP-A-6-46846,
JP-A-7-289253).

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 is an aldose residue, R ² Represents an amino acid, protein or peptide residue) The following are known as enzymes that catalyze the above reaction. 1. Fructosyl amino acid oxidase: genus Corynebacterium (Japanese Patent Publication No. 5-33997)
JP, Kokoku 6-65300 discloses), Aspergillus (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 (JP-A-5-192193) Alkyl lysinase: J. Biol. Chem. 239, 379
Prepared by the method described on pages 0 to 3996 (1964).

[0006]

However, the methods using these enzymes have the following problems. That is, the indicators in the diagnosis of diabetes are glycated albumin, glycated hemoglobin and fructosamine. Glycated albumin is
Glucose is bound to the ε-position of a lysine residue in a protein molecule to form glucose [J. Biol. Chem. 261: 13542-13545 (198
6)]. In glycated hemoglobin, glucose is also bound to the N-terminal valine of the hemoglobin β chain [J. Biol. Chem. 25.
4: 3892-3898 (1979)]. 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, the ketoamine oxidase described in JP-A-5-192193 is an enzyme capable of decomposing fructosyl valine and cannot accurately measure a glycated protein having a sugar bound to a lysine residue. Since fructosylamine deglycase has high activity for difructosyl lysine, it is not possible to specifically measure the glycosylated ε-position of the lysine residue, and also the glycosylated valine residue specifically. Nor can it be measured. Furthermore, the method using alkyl lysinase also acts on substances other than saccharides bound to lysine,
There was a problem that the specificity for saccharified products was low, and accurate measurement could not be expected. The enzyme derived from the genus Penicillium described in JP-A-4-4874 is an enzyme that acts on fructosyl lysine and fructosyl alanine. in this way,
Conventional enzymes are not suitable for accurate quantification of glycated proteins, and the 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 inventors of the present invention have conducted extensive studies for the purpose of providing a novel fructosyl amino acid oxidase that specifically acts on an Amadori compound, especially a glycated protein, and as a result, the genus Penicilliu
m ) to fructosyl lysine and / or fructosyl
It was found that an enzyme having a desired activity was induced by culturing in the presence of N ^α -Z-lysine, and completed the present invention. That is, the present invention relates to the genus Peni
The present invention provides a fructosyl amino acid oxidase produced by culturing a fungus of Cillium ) 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, 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 described below, the fructosyl amino acid oxidase of the present invention is active on both fructosyl valine and fructosyl lysine,
Its activity is characterized by a higher activity on the former than on the latter. In the present specification, the fructosyl amino acid oxidase of the present invention may be referred to as FAOD.

The enzyme of the present invention can be produced by culturing a fungus of the genus Penicillium having the ability to produce fructosyl amino acid oxidase in a medium containing fructosyl lysine and / or fructosyl N ^α -Z-lysine.
As such a fungus, Penicillium yancinerm S-3413
( Penicillium janthinellum S-3413) (FERM BP-5475),
Penicillium yansinerum (IFO NO.4651,6581,7905)
( Penicillium janthinellum ), Penicillium oxalicum (IFO NO.5748) ( Penicillium oxalicum ), Penicillium yavanicum (IFO NO.7994) ( Penicillium javanicu
m ), Penicillium chrysogenum (IFO NO.4897) ( Penici
llium chrysogenum ), Penicillium cyaneum (IFO N
O.5337) ( Penicillium cyaneum ) and the like.

The FAODs of the present invention generally have the following physicochemical properties: 1) Oxidizes Amadori compounds in the presence of oxygen to catalyze the reaction to form α-ketoaldehydes, amine derivatives and hydrogen peroxide; 2) Stable pH is 4.0 to 11.0, optimum pH is 7 .3; stable temperature is 15 to 50 ° C., optimum temperature is 25 ° C .; 4) molecular weight is about 38,700 (38.7 kD) when measured by gel filtration method using 200 pg of Superdex.
a).

The fructosyl lysine and / or FZL used in the production of the 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 (hereinafter referred to as FZL medium) used for the production of FAOD of the present invention 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% by weight, MgSO ₄ .7H ₂ O 0.05% by weight, CaCl ₂
・ 0.01% by weight of 2H ₂ O and 0.2% by weight of yeast extract
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 producing 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 using FZL as a single nitrogen source and glucose 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)
Can be mentioned. Particularly preferred medium is 1
Glucose 20g (2%) in 000ml, FZL 10
g (1%), K ₂ HPO ₄ 1.0 g (0.1%), NaH _{2
PO 4 1.0g (0.1%),} MgSO 4 · 7H 2 0 0.5
g (0.05%), CaCl ₂ .2H ₂ O 0.1 g (0.0
It is a medium (pH 5.6-6.0) containing 1%) and 2.0 g (0.2%) of yeast extract. The FZL medium is prepared by adding FZL to a normal medium or by adding glucose and N ^α -Z-
It can be prepared by autoclaving a medium containing lysine. The medium obtained by either method is fructosyl lysine and / or FZL.
It exhibits a brown color due to the presence of C. and is referred to as FZL browning medium or GL (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 adjusted according to the state of each bacterium, and are not limited to the above. For example, Penicillium yancinerm S-3413 strain was
When cultured for 0 to 48 hours, preferably 36 hours, FAO
D accumulates 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. FAOD of the present invention
Since the enzyme activity of is accumulated in the bacterial cells, the bacteria in the culture are crushed and the enzyme is extracted. Crushing of cells may be carried out by any of 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, and after washing, 5
Suspend in 0 mM potassium phosphate buffer (pH 7.5),
Crush the mycelium with Dynomill. Then, the supernatant obtained by centrifugation was used as a cell-free extract, and ammonium sulfate fractionation and DEAE were performed.
-Purify by treatment with Sephacel ion exchange 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 cultivates a strain belonging to the genus Penicillium and capable of producing fructosyl amino acid oxidase in a fructosyl lysine and / or fructosyl N ^α -Z-lysine-containing medium, and removing fructosyl amino acid oxidase from the culture. The present invention provides a method for producing a fructosyl amino acid oxidase, which comprises recovering.

[0016] Among the bacteria that produce FAOD of the present invention, Penicillium Yanshinerumu S-3413 (Penicillium janthinel
lum S-3413) (hereinafter referred to as S-3413 strain) is a strain newly isolated by the present inventors from the soil. The strain was examined for its mycological characteristics with reference to "Fungal Encyclopedia" by Shunichi Udagawa et al. (Kodansha Scientific 1993). As a result, it was identified as Penicillium janthinellum based on the following grounds. This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology under the deposit number FERM BP-5475. (1) Do not form an ascidian generation. (2) Penicillus formation was observed. (3) The phialide is a sharp type, and it suddenly becomes narrow and has a long tip. (4) Penicilli branch randomly and are spread type. (5) Does not form sclerotia. (6) Conidia linkage is remarkably open. Furthermore, (7) Growth status in medium Rapidly grows on Czapek agar medium. When it is cultured for 10 days in a thermostat at 25 ° C, it spreads like wool and exhibits a pale yellow or gray green color. Also, deep wrinkles are formed radially.

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 is an inducible enzyme induced by fructosyl lysine and / or fructosyl N ^α- Z-lysine (FZL), fructosyl lysine and / or FZL.
It is produced by culturing a fructosyl amino acid oxidase-producing strain of the genus Penicillium in a fructosyl lysine and / or FZL medium containing nitrogen as a nitrogen source and glucose as a carbon source. FAOD is induced in a GL browning medium obtained by autoclaving glucose and lysine and / or N ^α- Z-lysine together, but glucose and lysine and / or N ^α- Z-lysine were autoclaved separately. Since it is not induced in the medium prepared by the above method, the enzyme specifically acts on the Amadori compound.

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 and R ² represents an amino acid, protein or peptide residue) has a catalytic activity in the reaction. In the above reaction formula, R ¹ is —O.
_{H, - (CH 2) n} -, or _{- [CH (OH)] n} -C
H ₂ OH (where n is an integer of 0-6) and R ² is -C.
An Amadori compound represented by HR ^3- [CONHR ³ ] _m COOH (in the formula, R ³ represents an α-amino acid side chain residue, m represents an integer of 1 to 480) is preferable as a substrate. Above all, R
³ is a side chain residue of an amino acid selected from lysine, polylysine, valine, asparagine, etc., and n is 5 to 5.
A compound having 6, m of 55 or less is preferable.

The activity of FAOD of the present invention for each substrate is shown in Table 1 below. Table 1 Purified Penicillium Jansinerum S-34
Substrate specificity of FAOD derived from 13

[Table 1] * 1: not detected * 2: fructosyl human serum albumin From Table 1, FAOD of the present invention is fructosyl N ^α -Z-
It has a high specificity for lysine and fructosyl valine. Strains of the genus Penicillium having FAOD production ability are exemplified in Table 2 below. Table 2 Substrate specificity of FAOD extracted from Penicillium sp. Cultured in FZL browning medium

[Table 2] 1): Not detected

As shown in Table 2, the FA of the present invention
OD had a higher activity on fructosyl valine compared to fructosyl lysine, which indicates that the FA
This suggests that OD is useful for measuring glycated hemoglobin.

3. pH and temperature conditions Measurement of pH conditions 0.1 M acetic acid (Ac), potassium phosphate (K-P) buffer, Tris-HCl buffer and glycine (Gly) -N
FAOD was added to an aOH buffer (pH 4.0 to 11.0) and incubated at 25 ° C for 10 minutes, and then the activity was measured under normal conditions (25 ° C, pH 8.0). When measured by the above method, the stable pH range of the FAOD of the present invention is about pH 4.0 to 11.0, preferably pH 6.0 to 9.
The optimum pH was about 7.5 (see FIG. 2). Measurement of temperature conditions 15 in 50 mM potassium phosphate buffer (pH 7.5)
After adding FAOD to a temperature condition of -60 ° C and incubating for 10 minutes, the activity was measured under normal conditions. The stable temperature range is 15 to 50 ° C, preferably 15 to 45 ° C, more preferably 15 ° C, and the enzyme reaction is 15 to 45 ° C.
It efficiently proceeds at preferably 15 to 40 ° C, more preferably 25 ° C. (See Figure 3)

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 fructosyl valine (FV) solution was prepared by dissolving FV obtained in advance with distilled water. 45 mM 4-aminoantipyrine, 60 units / ml peroxidase solution, and 60 mM phenol solution (100 μl each), 0.1 M Tris-hydrochloric acid buffer solution (pH 8.0) 1 ml, and enzyme solution 50 μl were mixed, and the whole amount was distilled water. Make up to 2.95 ml. After equilibration at 25 ° C., 50 μl of 100 mM FV solution was added, and the absorbance at 505 nm was measured with time. Molecular extinction coefficient of the quinone dye (5.16 × 10 ³ M ^-1
cm ^-1 ), calculate the micromoles of hydrogen peroxide produced per minute, and use this number as the enzyme activity unit (unit: U)
And

B. Terminal method The same treatment as in the above method A was carried out, and after the addition of the substrate, 30 minutes at 25 ° C.
The enzyme activity is measured by measuring the absorbance at 505 nm after the incubation at 1, and separately calculating the amount of hydrogen peroxide generated from a calibration curve previously prepared using a standard hydrogen peroxide solution. (2) Method for measuring oxygen absorption by enzyme reaction Mix 1 ml of 0.1 M Tris-hydrochloric acid buffer (pH 8.0) and 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 25 ° C to equilibrate the temperature with dissolved oxygen, 50
100 μl of mM FV is added, and oxygen absorption is continuously measured by 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 enzyme (1) Effect of metal Various metal ions with a final concentration of 1 mM were added under the condition of 0.1 M Tris-hydrochloric acid buffer solution (pH 8.0) and incubated at 30 ° C for 5 minutes. After that, the activity was measured. The results are shown in Table 3 below. Table 3 Penicillium yansinerum S-3 of metal ion
Effect on 413-derived FAOD activity

[Table 3] It is clear from Table 3 that copper ion and barium ion are inhibitory to the activity of FAOD of the present invention, and cobalt ion, zinc ion, silver ion and mercury ion are strongly 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 (PCMB) was 0.1 mM, and other concentrations were 1 mM. The results are shown in Table 4. Stabilization was examined by dialysis overnight against 50 mM potassium phosphate buffer (pH 7.5) to which 0.1 mM dithiosuitol (DTT) was added, and then measuring the activity. It was Table 4 Effect of various substances on FAOD activity

[Table 4] * 1: PCMB, mercuric parachlorobenzoate * 2: DNTB, 5,5'-dithiobis (2-nitrobenzoic acid) * 3: EDTA, ethylenediaminetetraacetic acid Table 4 clearly shows that FAOD activity is PCMB and DNT.
It is strongly inhibited by B, hydrazine, phenylhydrazine and hydroxylamine, and 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 potassium phosphate buffer (pH 7.5) supplemented with 0.1 mM dithiothreitol.

6. The molecular weight was about 38,700 (38.7 kDa) as a result of gel filtration with 200 pg of Superdex (Fig. 4). SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis) was run according to the method of Davis using a 10% gel at 40 mA for 3 hours, and protein staining was Coomassie Brilliant Blue G.
Performed at -250. Also, in SDS-PAGE,
Phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor were similarly electrophoresed as standard proteins, and the molecular weight was measured using a calibration curve. As a result, the molecular weight of the subunit was about 48,700 (48 It was shown to be 0.7 kDa) (Fig. 5). Therefore, it is clear that the FAOD of the present invention is a monomer.

7. 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.Ch
em., 53 (1), 103-110 (1989) 2): T. Horiuchi et al. Agric.Biol.Ch
em., 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 is a monomer, whereas the other two enzymes are dimers, which are clearly different. (2) Coenzyme: FAOD has covalently bound FAD as a coenzyme, while all other enzymes have non-covalently bound FAD. (3) Differences such as optimum pH, optimum temperature, and inhibition by SH reagent indicate the difference between FAOD and the other two enzymes.

Further, a fructosyl amino acid degrading enzyme derived from the genus Penicillium described in JP-A-4-4874,
The following differences are observed when comparing with the FAOD of the present invention. (1) Substrate specificity: The fructosyl amino acid degrading enzyme described in JP-A-4-4874 has activity on fructosyl lysine and fructosyl alanine, whereas FAOD of the present invention has fructosyl lysine and fructosyl valine. , And the activity is stronger for fructosyl valine. (2) Inducing conditions: The fructosyl amino acid degrading enzyme described in JP-A-4-4874 is also produced in a medium containing no fructosyl amino acid, but the FAOD of the present invention should be cultured in a medium containing fructosyl lysine. Induced by. (3) Production method: The FAOD of the present invention is produced by culturing using a browning medium.

As mentioned above, the enzyme FAOD of the present invention is
It is useful for quantifying Amadori compounds. Therefore, the present invention also relates to a sample containing the Amadori compound and the FA of the present invention.
The present invention provides an analytical method for an Amadori compound in a sample, which comprises contacting with OD and measuring the consumption of oxygen or the generation of hydrogen peroxide. The analysis method of the present invention is carried out based on the measurement of the amount and / or the saccharification rate of glycated protein in biological components, or the quantification of fructosylamine. The enzyme activity of FAOD 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 an appropriate buffer. PH of reaction solution,
The temperature is in the range satisfying the above conditions, that is, pH 6.0 to 6.0.
The temperature is 9.0 to preferably 7.5 and the temperature is 15 to 45 ° C, preferably 15 to 40 ° C. Potassium phosphate 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 1 in the end point analysis method.
00 units / ml.

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 Hydrogen Peroxide Generation Amount Regarding the amount of hydrogen peroxide and Amadori compound, which is measured by a quantification 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 the hydrogen peroxide coloring system, a system which develops a color by 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. be able to. 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 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 that the valine and / or lysine residue to which the sugar has been bound be released in advance. 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, the FAOD of the present invention has a high specificity for fructosyl valine and is therefore useful for the measurement of glycated hemoglobin. In addition, since fructosyl lysine contained in glycated protein also has high substrate specificity, it is useful for diagnosing diabetes, including measuring glycated protein in a blood sample. 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 treatment such as folding. Further, FA used in the method of the present invention
Enzymes such as OD and peroxidase 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 carried out 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 clinical tests are aimed at diagnosing diabetes, the results are expressed as a glycated protein concentration, or as a ratio of the glycated protein concentration to the total protein concentration in the sample (glycation rate) or the amount of fructosylamine. To be done. The total protein concentration is
It can be measured by an ordinary method (absorbance at 280 nm, Bradford method, Burette method, Lowry method, or 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 6.0-9.0,
More preferably, it comprises a buffer solution having a pH of 7.5. 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 potassium phosphate (pH 7.5) is preferable as the buffer solution. 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 reagent and kit are used for measuring the amount and / or glycation rate of glycated protein in biological components, or for quantifying fructosylamine. Hereinafter, the present invention will be described in more detail with reference to examples.

[0036]

Example 1 Production and purification of FAOD derived from Penicillium yansinerum S-3413 Penicillium yancinerm S-3413 (FERM BP-54
75; Penicillium janthinellum S-3413) to FZL 0.
5%, glucose 1.0%, dipotassium phosphate 0.1
%, Monosodium phosphate 0.1%, magnesium sulfate
The medium was inoculated into 10 L of a medium (pH 6.0) containing 0.05%, calcium chloride 0.01%, and yeast extract 0.2%, and an aeration rate was 2 L / min and a stirring speed was 500 rpm using a jar fermenter. The cells were cultured at 28 ° C. for 36 hours with stirring. The culture was filtered and collected. 410 g (wet weight) of mycelium was suspended in 800 ml of 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 mM DTT, and the mycelium was crushed with a Dyno mill. Crush liquid at 9,500 rpm
After centrifugation for 20 minutes, the resulting solution was used as a crude enzyme solution (cell-free extract) and purified by the following method. 40 for crude enzyme solution
Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added so that the solution became 100% 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. 50m of precipitate containing 0.1 mM DTT
M potassium phosphate buffer (pH 7.5) (hereinafter buffer A
Abbreviated). The resulting enzyme solution was dialyzed against buffer A overnight. The external liquid was exchanged twice. The dialyzed enzyme solution was applied to a DEAE-Sephacel column (4.2 x 26 cm) equilibrated with buffer solution A. Since the active fraction was found in the fraction washed with the same buffer, it was collected and subjected to the ammonium sulfate fraction at 0-55% saturation. Next, a phenyl-Sepharose 6FF (Low Substitute) column (HR10 / 1) equilibrated with buffer A containing 25% saturated ammonium sulfate was used.
Adsorbed on 0). After washing with the same buffer, ammonium sulfate concentration 2
Elution was with a linear gradient of 5-0% saturation. Collect the active fractions,
After concentration with ammonium sulfate, the resulting enzyme solution was subjected to gel filtration using a Superdex 200 pg column equilibrated with 0.2 M potassium phosphate buffer (pH 7.5) containing 0.1 mM DTT to give 70 to 100 units. A purified enzyme 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 obtained purified enzyme preparation was determined by SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis). SDS-PAGE was carried out according to the method of Davis, using a 10% gel, running at 40 mA for 3 hours, and then Coomassie Brilliant Blue G-250.
Protein staining was performed at. Phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor were similarly electrophoresed as standard proteins, and the molecular weight was determined from the calibration curve. As a result, the molecular weight of the subunit was about 48,700 (48.7 kDa). Was shown (FIG. 5). Also, Superdex 2
In the molecular weight measurement by gel filtration with 00 pg, as is clear from the calibration curve diagram of FIG. 4, about 38,700 (38.7
kDa). Regarding the enzyme activity, pH and temperature stability of FAOD prepared in this example, the influence of metals and inhibitors, etc., the above-mentioned values or properties were shown.

Example 2 Measurement of Glycated Hemoglobin Amount 1) Sample Treatment 0 to 15 mg of glycohemoglobin control E (Sigma) was dissolved in 100 μl of distilled water. Acetone hydrochloride (1N hydrochloric acid / acetone: 1/100) (1 ml) was added to these samples and the mixture was centrifuged at 12,000 rpm for 10 minutes. The precipitate was washed with 500 μl of diethyl ether and dried under reduced pressure. Further, 100 μl of 8M urea was added, and the mixture was heated in boiling water for 20 minutes, cooled, and then added to 5.4 units / ml trypsin 3
Mix with 00 μl and incubate at 37 ° C. for 3 hours.
Then, it heated in boiling water for 5 minutes, and prepared the sample. 2) Activity measurement A 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-HCl buffer (pH 8.0) 300 μl 25 units / ml FAOD Solution 5 μl Distilled water up to 1 ml. 25 units / ml FAO
The D solution was adjusted to 25 units / ml of FAOD obtained by the method of Example 1 with 0.1 M potassium phosphate buffer solution (p
It was prepared by diluting with H7.5). To the FAOD reaction solution, 150 μl of each of the above-mentioned treated substrates was added, incubated at 30 ° C., and the absorbance at 727 nm after 30 minutes was measured. The relationship between the amount of glycated hemoglobin obtained by this method and the absorbance is shown in FIG. The vertical axis in the figure is the absorbance at 727 nm
(Corresponding to the amount of hydrogen peroxide), the horizontal axis represents the amount of glycated hemoglobin. The figure shows that there is a correlation between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated.

Example 3 Measurement of Glycated Hemoglobin Amount 1) Sample Treatment 30 mg Glycohemoglobin Control E (Sigma)
Was dissolved in 200 μl of distilled water, 8M urea, 0.2% ED
1 ml of 570 mM Tris-HCl buffer (pH 8.8) containing TA / sodium and 2-mercaptoethanol 40
μl was added, and the mixture was left standing for 2 hours under nitrogen. afterwards,
400 μl of 1M sodium iodoacetate was added, 30
After standing for a minute, 40 μl of 2-mercaptoethanol was added. After dialysis against 0.1 M ammonium bicarbonate, it was mixed with 10 μl of 10 mg / ml TPCK-trypsin and incubated at 37 ° C. for 3 hours. Then, the sample was prepared by heating for 5 minutes in boiling water. 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 0.1M Tris-HCl buffer (pH 8.0) 300 μl 25 units / ml FAOD Solution 10 μl Treated sample 0 to 13.2 mg Distilled water was added to bring the total volume to 900 μl. 25 units / ml
The FAOD solution was prepared by using the FAOD obtained by the method of Example 1 at 25
It was prepared by diluting it with 0.1 M potassium phosphate buffer (pH 7.5) so that the unit / ml was obtained. This FAOD reaction solution was incubated at 30 ° C., and after 30 minutes, 727 nm
The absorbance at was measured. The relationship between the amount of glycated hemoglobin obtained by this method and the absorbance 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 amount of glycated hemoglobin. The figure shows that there is a correlation between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated.

Example 4 Measurement of hemoglobin A1c value Hemoglobin A0 reagent (Sigma Co.) was diluted to 2.3 mM with distilled water.
Melted to become. This solution was fractionated using an automatic glycohemoglobin measuring device (Kyoto Daiichi Kagaku), and the hemoglobin A1c fraction and hemoglobin A0 fraction were fractionated and purified. By mixing both fractions in a ratio, hemoglobin A
A substrate sample having a 1c value of 0% to 52.0% was obtained. 1) Sample treatment Substrate sample 250 μg 500 units / ml aminopeptidase solution 5 μl 1.0 M Tris-hydrochloric acid buffer solution (pH 8.0) 15 μl These were mixed to make a total volume of 200 μl. This mixture was incubated at 30 ° C. for 30 minutes. Then, add 200 μl of 10% trichloroacetic acid and stir,
After standing still at 0 ° C. for 20 minutes, centrifugation was performed at 12,000 rpm for 10 minutes. About 4 N of 5N NaOH was added to the obtained supernatant.
0 μl was added to make a neutral solution. 2) Activity measurement A FAOD reaction solution was prepared as follows. 3 mM N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine solution 100 μl 60 units / ml peroxidase solution 100 μl 0.1 M Tris-hydrochloric acid buffer (pH 8.0) 1000 μl 16 units / ml FAOD Solution 15 μl Distilled water to a total volume of 2.6 ml. 16 units / ml F
An AOD solution was prepared by diluting FAOD obtained by the method of Example 1 with 0.1 M potassium phosphate buffer (pH 7.5) so that the FAOD was 16 units / ml. The FAOD reaction solution was incubated at 30 ° C. for 2 minutes, 400 μl of each of the above-mentioned treated substrates was added, and the absorbance at 727 nm was measured after further incubation for 30 minutes. The relationship between the hemoglobin A1c value and the absorbance of the substrate 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 hemoglobin A1c value. The figure shows that there is a correlation between the hemoglobin A1c value and the hydrogen peroxide generation amount.

[0041]

The FAOD of the present invention specifically acts on both fructosyl valine and 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 of glycated protein and / or the glycation rate or the amount of fructosylamine in blood is used as an index to help diagnose the pathological condition of diabetes. Also,
Glycated proteins can be accurately quantified by the Amadori compound analysis reagent and analysis method using FAOD of the present invention, which can contribute to diagnosis and management of symptoms of diabetes.

[Brief description of drawings]

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

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

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

FIG. 4 is a graph showing the results of molecular weight measurement by gel filtration using Superdex 200 pg.

FIG. 5 is a photograph showing a migration pattern obtained by subjecting purified FAOD derived from Penicillium yancinerm S-3413 to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

FIG. 6 is an absorption spectrum of FAOD derived from purified S-3413.

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

FIG. 8 is a graph showing the relationship between the amount of glycated hemoglobin and the amount of hydrogen peroxide produced by the FAOD action.

FIG. 9 is a graph showing the relationship between the hemoglobin A1c value and the amount of hydrogen peroxide produced by the FAOD action.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location (C12N 9/06 C12R 1:82) (72) Inventor Masayuki Yagi Santanda Nishikyogoku, Kyoto City Kyoto Prefecture Town 1 West Kyogoku housing complex 4 Room 302 (72) Inventor Fumiyo Funatsu 2-40-40, Eggplant made in Hirakata City, Osaka Prefecture

Claims (12)

[Claims] 1. A fructosyl amino acid oxidase produced by culturing a fungus of the genus Penicillium capable of producing fructosyl amino acid oxidase in a fructosyl lysine-containing medium. 2. The fructosyl lysine-containing medium contains glucose, lysine and / or N ^α -Z-lysine at a temperature of 10
The fructosyl amino acid oxidase according to claim 1, which contains fructosyl lysine and is obtained by autoclaving at 0 to 150 ° C for 3 to 60 minutes. 3. The fructosyl amino acid oxidase according to claim 1, which has an activity against fructosyl valine and / or fructosyl lysine. 4. The fungus of the genus Penicillium is Penicillium.
Penicillium janthinellum S-341
3) (FERM BP-5475), Penicillium Jansinerum (IFO N
O.4651,6581,7905) ( Penicillium janthinellum ), Penicillium oxalicum (IFO NO.5748) ( Penicillium ox
alicum ), Penicillium yavanicum (IFO NO.7994) ( Pen
icillium javanicum ), Penicillium chrysogenum (IF
The fructosyl amino acid oxidase according to claim 1, which is selected from the group consisting of O NO.4897) ( Penicillium chrysogenum ) and Penicillium cyaneum (IFO NO.5337) ( Penicillium cyaneum ). 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 11.0, optimum pH is 7.5; 3) stable temperature is 15 to 50 ° C, optimum temperature is 25 ° C .; 4) When measured by gel filtration method using 200 pg of Superdex, the molecular weight is about 38,700 (38.7 kD).
a). 6. A penicillium yansinerum S-3413 ( Pen
icillium janthinellum S-3413) (FERM BP-5475). 7. A method for producing a fructosyl amino acid oxidase in a fungus of the genus Penicillium by culturing the fungus of the genus Penicillium in a medium containing a glycosylated amino acid and / or a glycosylated protein having a free or protective group. And a method for producing a fructosyl amino acid oxidase. 8. A strain belonging to the genus Penicillium and capable of producing fructosyl amino acid oxidase is cultured in a medium containing fructosyl lysine and / or fructosyl N ^α -Z-lysine, and the fructosyl amino acid oxidase is recovered from the culture. The method of claim 7, wherein the method comprises: 9. 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. 10. 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 fructosylamine. Item 9. The method according to Item 9. 11. An Amadori compound analysis reagent or kit containing the fructosyl amino acid oxidase according to claim 1. 12. The amount of glycated protein in a biological component and / or
Alternatively, the analytical reagent or kit according to claim 11, which is used for measuring a saccharification rate or quantifying fructosylamine.