WO1997013872A1 - Method and assaying amodori compounds - Google Patents

Abstract

A method for enzymatically assaying Amadori compounds in biological components or foods, which comprises measuring the saccharification rate of a sample, determining the amount of fructosylamine, or measuring the concentration of saccharified matters.

Description

 Specification

 Method for measuring Amadori compounds

Technical field

 The present invention relates to a method for measuring an Amadori compound using an enzyme, and more particularly to a novel method for measuring an Amadori compound using fructosylamino acid oxidase, and a reagent and a kit used in the method.

Background art

 Amadori compounds are non-enzymatic and irreversible when amino and aldehyde groups coexist with a substance having an amino group such as protein, peptide and amino acid and a reducing sugar such as aldose. It is produced by binding and Amadori rearrangement. The rate of Amadori compound formation is a function of the concentration of the reactive substance, contact time, temperature, and so on. Therefore, it is thought that various information on the substance containing the reactive substance can be obtained from the generation s. Substances containing Amadori compounds include 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 amino acid are combined, is produced. For example, a fructosylvanine 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 blood protein is glycated is called fructosamine. These blood levels reflect the average blood glucose over a period of time in the past, and the measured values can be important indicators for diagnosing and managing the symptoms of diabetes. Is extremely useful clinically. In addition, by measuring the Amadori compound in food, it is possible to know the storage status and period of the food after its manufacture, which will be useful for quality control. As such, the analysis of Amadori compounds is in medicine and food. Useful in a wide range of fields, including goods.

 Conventional methods for measuring Amadori compounds include high-performance liquid chromatography [Chromatogr. Sci. 10: 659 (1979)] and a method using a column filled with solids mixed with boric acid [Clin. Chem. 28: 2088-2094 (1982)], electrophoresis [Clin. Chem. 26: 1598-1602 (1980)]> Method using antigen-antibody reaction [JJCLA 18: 620 (1993). 16: 33-37 (1993)]. Method for measuring fructosamine [Clin. Chim. Acta 127: 87-95 (1982) 3, Colorimetric determination after oxidation using thiobarbituric acid [Clin. Chim Acta 112: 197-204 (1981)] is known, but expensive equipment was required, and it was not always an accurate and quick method.

 In recent years, methods that utilize enzyme reactions have been clinically used because of the ability to rapidly and accurately analyze target substances due to the properties of enzymes (specificity of substrate, reaction, structure, position ff, etc.). It has become widespread in the field of analysis and food analysis. Analytical methods have already been proposed to measure Amadori compounds by reacting an Amadori compound with an oxidizing enzyme and measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated in the reaction (eg, JP-B 5-33997, JP-B 6-65300, JP-A-2-195900, JP-A-3-155780, JP-A-4-4874, JP-A-5-192193, JP-A-6 -46846). In addition, methods for measuring glycated proteins for the diagnosis of diabetes have been disclosed (Japanese Patent Publication No. 2-195899, Japanese Patent Publication No. 2-195900, and Japanese Patent Application No. 5-192193) (EP 0 526 150). A), JP-A-6-46846 (EP 0 576 838 A)).

 The decomposition reaction of the Amadori compound by the oxidizing enzyme can be represented by the following general formula.

R ¹ -CO-CH ₂ -NH-R ² + 0 ₂ + H ₂₀ →

R—CO—CHO + R—NH ₂ + H ₂ 0 ₂ (Where R is an aldose residue and R ² is an amino acid, protein or peptide residue)

 The following are known as enzymes that catalyze the above reaction.

1. Fructosyl amino acid oxidase: Corynebacterium (Japanese Patent Publication No. 5-33997, Japanese Patent Publication No. 6-65300), Aspergillus (Japanese Patent Publication No. 3-155780) ₀

 2. Fructosylamine deglycase: Candida (JP-A-6-46846)

 3. Fructosyl amino acid-decomposing enzyme: enicUliuin (JP-A-4-4874).

 4. Ketoamine okidase: Corynebacterium) S, Fusarium, Acremonium or Debryomyces (Japanese Patent Application Laid-Open No. 5-192193)

 5. Alkyl lysinase: Prepared by the method described in J. Biol. Chem. 239, pp. 3790-3796 (1964).

However, the conventional methods using these yeasts have the following problems. That is, glycated albumin, glycated hemoglobin, and fructosamine are indicators for the diagnosis of diabetes. Glycated albumin is produced by the coupling of glucose to the ε-position of a lysine residue in a protein molecule [J. Biol. Chem. 261: 13542-13545 (1986)]. In glycated hemoglobin, hemoglobin; glucose is also silicified to the 3-terminal N-terminal variant [J. Biol. Chem. 254: 3892-3 898 (1979)]. Therefore, it was necessary to accurately measure fructosyl lysine and Z or fructosyl valine in order to measure a glycated protein as an indicator of diabetes. However, fructosyl lysine cannot be measured accurately by the methods described in JP-B 5-33997 and JP-B 6-65300, and JP-A 3-155780 discloses that glycated protein or its hydrolyzate cannot be measured. The effect is not disclosed. On the other hand, the method described in JP-A-5-192193 cannot accurately measure a glycated protein in which a saccharide is bound to a lysine residue. According to the method described in JP-A-6-46846, a saccharified product at the ε-position of a lysine residue cannot be measured extraordinarily, and a saccharified product at a parinic residue cannot be specifically measured. Furthermore, the method described in Japanese Patent Publication No. 2-195900 using the enzyme described in J. Biol. Chen, Vol. 239, pp. 3790-3796 (1964) also measures substances in which residues other than saccharides are bonded to lysine. There was a question that the specificity for saccharides was low, and accurate measurements could not be expected. In the method described in JP-A-4-4874, only fructosyl lysine and fructosylalanine can be measured.

 According to these conventional methods, the Amadori compound (usually a protein) is decomposed into amino acids, and then hydrogen peroxide generated by the reaction of the liberated saccharification site amino acid residues with the enzyme is consumed or consumed. The amount of oxygen needed to be measured and could not be processed quickly. Furthermore, Amadori compounds having different glycation sites could not be specifically measured.

 As described above, the conventional method is not suitable for accurate measurement of glycated protein, and development of a method for rapidly and specifically measuring fructosyl lysine and / or fructosyl valine and a peptide containing at least one of them is awaited. Was.

Disclosure of the invention

 The present inventors have conducted intensive studies with the aim of providing a measurement method for accurately analyzing an Amadori compound, particularly a glycated protein, and as a result, have found that certain fungi have been identified as fructosyl lysine and / or fructosyl The present inventors have found that the purpose can be achieved by using a nitrogen atom obtained by culturing in the presence of N′—Z-lysine, and completed the present invention.

That is, the present invention is characterized in that the measurement is performed using an enzyme. It is intended to provide a method for measuring an Amadori compound in a substance-containing sample.

 The subject of the method of the present invention is arbitrary, but usually a biological component or food, as long as it can contain the Amadori compound.

 In the method of the present invention, the Amadori compound is preferably measured by measuring the saccharification rate of the sample, quantifying fructosylamine, or measuring the saccharified substance concentration. Above all, analysis of Amadori compounds by measuring the saccharification rate using yeast is novel. When measuring Amadori compounds, especially saccharified proteins, there are two methods: measuring the absolute amount (concentration) of the saccharified protein and measuring the percentage of saccharified protein (saccharification rate).

 When measuring only the saccharified amount of the protein component to be measured, whether the decrease is due to a change in the total amount of the target protein component or a change in the blood glucose level is determined. Unknown. When measuring the saccharification rate, it is expressed as the ratio of the amount of saccharified protein in the total amount of the target protein component, so only the change derived from the blood glucose level, that is, the change in the pathology of diabetes, is reflected independently of the change in the protein amount Value can be shown. In the conventional method of colorimetric determination using fructosamine method and thioparbituric acid, accurate information on the amount of saccharified substances cannot be obtained due to the influence of the coexisting substance because of the measurement of bioactivity. The amount of saccharified product obtained by the present method measures the absolute amount of the coupling between amino acid residues and glucose constituting the protein, so that accurate information on the number of saccharified amino acids can be obtained.

 In addition, when measuring the saccharification rate by this method, the total amount of the protein component to be measured and the saccharification amount are separately measured, and the saccharification rate is obtained by calculation. It has great clinical significance. High-performance liquid chromatography, a method using a column packed with solids mixed with boric acid, electrophoresis, and a method using an antigen-antibody reaction include non-glycated tans.

-D- Since the saccharification rate is determined from the relative ratio between the amount of protein and the amount of saccharified protein, there is a disadvantage that information on ft cannot be obtained. With this method, it is possible to measure the saccharification rate that is important for the diagnosis of diabetes mellitus without being affected by the metabolism and amount of protein, and to determine the nutritional status and the total protein amount for determining the degree of liver injury. Measurement can be performed simultaneously. This indicates that this method is also effective in diagnosing diabetes in patients with diseases involving fluctuations in protein levels.

 In addition, the measurement of glycated proteins using enzymes has high specificity and can be performed in the same manner as conventional biochemical measurements. There is an advantage that simultaneous measurement is possible. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing the relationship between the fructosamine value and the amount of hydrogen peroxide generated by the FAUD action.

 FIG. 2 is a graph showing the relationship between the concentration of glycated human serum albumin and the amount of hydrogen peroxide generated by the action of FAOD derived from Fusarium oxysvolum S-1F4.

 FIG. 3 is a graph showing the relationship between the concentration of saccharified human serum albumin and the amount of hydrogen peroxide generated by the action of FAOD derived from Giperella fujikuroi.

 FIG. 4 is a graph showing the relationship between the concentration of glycated human blood albumin and the amount of purple water peroxide generated by the action of F AOD from Fusarium oxasbum 'f.sp' lini.

FIG. 5 is a graph showing the relationship between the concentration of glycated human serum albumin and the amount of hydrogen peroxide generated by the action of FAOD derived from Aspergillus terreus GP1. 2 P

FIG. 6 is a graph showing the relationship between the saccharification rate of human serum albumin and the amount of hydrogen peroxide generated by the action of FAOD derived from Fusarium oxasvolum f.sp.lini.

 FIG. 7 is a graph showing the relationship between the saccharification rate of human serum albumin and the amount of hydrogen peroxide generated by the action of FAOD derived from Aspergillus terreus GP1.

Figure 8 is Ru graph der showing the relationship between the amount of hydrogen peroxide generated by the action of FAOD derived from glycation rate and Giperera-fujikuroi of human serum albumin ₀

 FIG. 9 is a graph showing the relationship between the saccharification rate of human serum albumin and the amount of hydrogen peroxide generated by the action of FAOD derived from Fusarium oxysporum S-1F4.

 FIG. 10 is a graph showing the relationship between the concentration of saccharified hemoglobin and the amount of hydrogen peroxide generated by the action of FAOD derived from Fusarium, oxysporum, f. Sp., And lini.

 FIG. 11 is a graph showing the relationship between the concentration of saccharified hemoglobin and the amount of hydrogen peroxide generated by the action of FAOD derived from Aspergillus terreus GP1.

 FIG. 12 is a graph showing the relationship between the amount of saccharified hemoglobin and the amount of hydrogen peroxide generated by the action of FAOD derived from Penicillium jansinerum S-313.

 FIG. 13 is a graph showing the relationship between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated by the action of FAOD derived from Penicillium jansinerum S-3413.

Figure 14 shows valine as a function of total hemoglobin content (absorbance at 415 nm). 4 is a graph showing the relationship between the ratio of saccharification amount (absorbance at 727 nm) and hemoglobin A 1c value.

 FIG. 15 is a graph showing the relationship between hemoglobin A 1 c value and hydrogen peroxide ft generated by the action of F AOD derived from Penicillium ′ Jansinerum S—34 13.

 No. 160 shows the relationship between the concentration of fructosylvalin and the amount of hydrogen peroxide produced by the use of FAOD derived from Penicillium 'Jansinerum S-314, detected by a chemical method. It is a graph shown.

 FIG. 17 is a graph showing the relationship between the concentration of fructosyl lysine and the amount of hydrogen peroxide generated by the action of immobilized FAOD derived from Fusarium 'oxysporum S-1F4.

Good form for carrying out the invention ¾

 An enzyme that acts on the saccharification site of the substrate includes fructosylamino acid oxidase. When fructosylamino acid oxidase is used, the measurement of the Amadori compound is performed by measuring the amount of oxygen consumed or the amount of a product in the reaction mixture. Products include hydrogen peroxide and glucosone. Quantification of hydrogen peroxide is usually measured using a chromogen that produces a dye upon decomposition of hydrogen peroxide by a catalyst having peroxidase or peroxidase-like activity, or by an electrochemical method. Can be. Alternatively, the amount of hydrogen peroxide generated can be quantified by measuring the amount of aldehyde generated in the presence of catalase and alcohol.

In the method of the present invention, an enzyme produced by culturing a fungus in a medium containing fructosyl lysine and Z or fructosyl N "-Z-pyridine can be used. Fructoshi used in the invention method The amino acid oxidase is called FAOD.

The FAOD used in the method of the present invention is obtained by culturing a fungus having fructosylamino acid oxidase-producing ability in a medium containing fructosyl lysine and / or fructosyl N'-I-Z-lysine (hereinafter sometimes abbreviated as FZL). Can be produced. Such fungi include Fusarium 厲 usariuo), Gibberella fe (Gibberella), Penicillium, Almiraria Ar (Armillaria), Caldarionivces 、, Ganoderma As and Aspergillus As. ) And the like. Examples include Fusarium oxysporum S-1F4 (Fu_sariuni oxysporum S-1F4) (1-3-1-3 Tsukuba-Higashi, Ibaraki Pref.) Deposited under BP-5010 (Original Deposit: February 24, 1994: Transfer to International Deposit: February 22, 1995), Fusarium Oxysporum f. Sp. Rini (IFO NO 5880) (Fusarium oxysporum f. Sp. Lini), Fusarium oxysporum f. Sp. Batatas (Fusarium oxysporum f. Sp. Batatas) Fusarium oxysporum f. Sp. (IFO NO. 4471) (Fusarium oxysporum f. Sp. Niveuni). Fusarium oxysporum f. Sp. Kucumulinium (IFO NO. 63 84) Fusarium oxysporum f. Sp. Cucumerinuin, Fusarium xysporum f. sp. melongenae (IFO NO. 7706) (Fusarium oxysporum f. sp. melonge nae), Fusarium oxy Borm · f. Sp. Avi (IFO NO. 9964) (Fusarium oxysporum f. Sp. Apii), Fusarium 'Oxisborg · f. Sp. Vinyl (IFO NO. 9971) (Fusarium oxysporum f. Sp. Pini) and Fusarium · Foxy sporum f. Sp. Fragarie (IFO NO. 31180) (Fusarium oxysporum f. Sp. Fragariae); Giperera fujikuroi (IFO NO. 6356. 6605) (Gibberella fuj ikuroi); iuta janthinellum S-3413) Depositary institution: Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry of the Ministry of International Trade and Industry; Deposit No .: FERM BP-5475, Original Deposit Date: March 28, 1995: Transfer to International Deposit: March 14, 1996 Sun), Penicillium 'Jansinerum (IFO NO.4651, 6581, 7905) (Peni cillium janthinellum), Penicillium' Oxalicum (IFO NO.5748) (Penicillium oxalicum), Penicillium 'Jabanicum (IFO NO.4639) Penicillium j3vanicum), Penicillium 'chrysogenum (IFO NO.4897) (Penicilli um chrysogenuni). Penicillium cyanium (IFO NO.5337) (Penicillium cyaneum); , Aspergillus' Teleus GP-1 (Contractor: Ministry of International Trade and Industry at the above address; Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology; Deposit No .: FERil BP-5684; Deposited by Hara: May 31, 1996; Transfer: September 30, 1996) (Aspergillus terr eus GP-1) Aspergillus oryzae (IFO 4242) (Aspergillus oryzae), Aspergillus oryzae (IF05710) (Aspergillus oryzae), and the like, but are not limited thereto.

 The FAODs produced by the above method generally catalyze the reaction of oxidizing the Amadori compound in the presence of an acid to produce α-ketoaldehyde, an amide derivative and hydrogen peroxide.

Fourques Toshirurijin及beauty / or FZL used for the production of FAOD to be used in the method of the present invention, the glucose 01-50% by weight and lysine and / or New ^beta one Ζ- lysine 0.01 to 20 wt% in solution, 100 Manufactured by autoclaving at ~ 150 to 3-60 minutes. Specifically, glucose 200 g in a solution of total volume 100 Om 1, New ^beta one Zeta - dissolved lysine emissions 10 g, usually 120. C, can be manufactured by autoclaving for 20 minutes.

Further, fructosyl sulfate for production of FAOD used in the method of the present invention. Gin and / or FZL-containing medium (hereinafter referred to as FZL medium) may be prepared by adding fructosyl lysine and FZL or FZL obtained by the above method to an ordinary medium, for example, by adding 0.01 to 50% by weight of glucose, / or New ^beta - zeta-lysine 0.01,20 weight ¾, K ₂ HP0 ₄ 0.1 wt%, NaH ₂ P0 ₄ 0.1 _{wt%, MgS0 4 · 7H 2 0} 0.05 _{wt%, CaC 1 2 · 2Η 2} 0 0.01 weight % And yeast extract 0.2% by weight (preferably ΡΗ5.6-6.0) can be obtained by subjecting the mixture to autoclaving at 100 to 50 L for 3 to 60 minutes.

 The medium used for the production of FAOD used in the method of the present invention 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. As a nitrogen source, peptone, casein digest, yeast extract, etc. can be used. Further, as inorganic substances, those contained in ordinary culture media such as sodium, potassium, calcium, manganese, magnesium, and cobalt can be used.

FAOD used in the method of the present invention is most induced when cultured in a medium containing fructosyl lysine and F or FZL. Examples of preferred media, the FZL obtained by the method described above as a single nitrogen source, FZ L medium (1.0% glucose using glucose as the carbon crowd source, 0.5% FZL, 0.1% K Z HPO "0 _{. l% NaH 2 P0 4,} 0.05% MgS0 4 · 7H 2 0, 0.01% C a C 12 · 2H 2 0 and 0 ... 0.01% vitamin mixture) can Rukoto cited. particularly preferred medium, the total amount 1, 00 Oinl glucose 20 g (2, FZL 10 g (1% _{in), K 2 HP0 4 1.0 g} (0.1%), N a H 2 P 041.0 g (0.1%), MgSO 7H 2 0 0.5 g ( _{0.0 5%), CaC 1 2} · 2H 2 0 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 a normal medium or by autoclaving a medium containing glucose and! ^ '-Z-lysine. Medium be obtained by any process has exhibited a brown color by the presence of Fourques Toshirurijin and Roh or FZL, FZL browning medium or GL (Guriketetsu Dorijin and / or Gurike ferrous ^ΚΝ β - Ζ- lysine) ¾ change medium Called.

 Culturing is usually performed at about 25 to 37, preferably at 28. The ρΗ of the medium is in the range of 4.0 to 8.0, preferably 5.5 to 6.0. However, these conditions are those which are appropriately polished in accordance with the condition of each bacterium, and are not limited to the above.

 The tie obtained in this manner can remove nucleic acids, cell wall fragments, and the like according to a conventional method to obtain an enzyme product.

 Since the enzyme activity of F AOD used in the method of the present invention is accumulated in the cells, the cells in the culture are disrupted and used for enzyme production.

 The disruption of the cells may be any of autolysis, freezing, sonication, and pressurization using mechanical means or a solvent.

 Methods for separating and purifying yeast cord are also known, and include a combination of salting out using ammonium sulfate or the like, precipitation with an organic solvent such as ethanol, ion exchange chromatography, hydrophobic chromatography, gel filtration, and affinity chromatography. Make some.

For example, the culture tie is collected by centrifugation or suction filtration to collect mycelium, washed, made 0.1 M Tris-HCl buffer (ρΗ8.δ), and crushed by Dyno-llill. Next, the supernatant obtained by centrifugation is treated as a cell-free extract by ammonium sulfate fractionation and fluorosepharose monofiltration. To purify.

 However, for the purpose of the present invention, the FAOD used in the method of the present invention, regardless of the degree of purification, is not limited to enzyme-containing substances in all purification stages, including culture medium, as long as it can catalyze the oxidation reaction of Amadori compounds. Solution. In addition, the present invention can achieve the object of the present invention only at the site of the fermented purple molecule that is involved in the activity of the enzyme. Therefore, the present invention also encompasses any fragment having the oxidation activity of the Amadori compound. The FAOD thus obtained is useful not only for the measurement of Amadori compounds, especially for the measurement of glycated proteins for the diagnosis of diabetes, but also for solving the technical problems to be solved by the present invention. It is.

 Furthermore, an enzyme obtained by culturing a living organism transformed by an expression vector containing a DNA encoding FAOD can also be used in the method of the present invention. The FAOD thus obtained is useful not only for the measurement of Amadori compounds, particularly for the measurement of glycated proteins for the diagnosis of diabetes, but also for the technical problems to be solved by the present invention. Useful. Hereinafter, the characteristics of the FAOD used in the method of the present invention will be described in detail. 1. General induction characteristics

 FAOD used in the method of the present invention is an inducible enzyme induced by fructonyl lysine and / or FZL.Fructosyl lysine and / or FZL is used as a nitrogen source and glucose is used as a carbon source. It is produced by culturing fungal fructosylamino acid oxidase-producing bacteria.

FAOD is induced in a GL browning medium obtained by autoclaving both glucose and lysine and / or N'-Z-lysine. Prepared by toraping ¾enzyme acts specifically on the Amadori compound, since it is not induced in the cultivated medium.

 2. Properties and substrate specificity

 FAOD used in the method of the present invention has the formula:

^{R'-CO-CHz- HR 2 +} 0 2 tens of H ₂ 0 →

R ¹ -CO-CHO + R with NH ₂ + H ₂ 0 ₂

(Wherein, R ¹ represents an aldose residue, R ² represents an amino acid, protein or peptide residue)

Has a catalytic activity in the reaction represented by In the above reaction formula, R ^{1 is} —OH, one (CH ₂ ) „— or one [CH (OH)] n-CHzOH (where n is an integer of 0-6), and R ^{2 is} —CHR Amadori compounds represented by ³ — [CONHR ³ ] _n COOH (wherein, R ³ is a residue on one amino acid side and m is an integer of 1 to 480) are preferred as the substrate, among which R ^{3 is} lysine And a side residue of an amino acid selected from boryridine, parin, asparagine, etc., and a compound wherein n is 5 to 6 and m is 55 or less. The results are shown in Table 1.

Table 1

 Various properties of funolecto cinnorea mino acid cyst ^ ^ Producing bacterium Gibberella Fusarium F. oxysporua penicillun Aspergillus fujikuroi oxysporuB S-14F f. Sp. Lini janthinellum S-3413 terreus GP1

<IF06356> Ku PERM BP-5010> <IF05880> <FER BP-5475> <Box BP-5684> Molecular weight

 (Gel filtration) 47,000 45,000 106.000 38.700 94,000 (SDS-PAGE) 52.000 50.000 51.000 48.700 48.000 Coenzyme FAD and covalent FAD and covalent FAD and covalent FAD and covalent FAD and covalent FAD and covalent

 (U / rag

 (Fructosyl lysine) 16.7 48.9 32.0 4.18 20.6

 (Fructosi) V-palin) N.D. * 1 N.D. 9.62 18.6 10.4 Michaelis constant

(aFZL) * ² 0.45mM 1.37m 0.51IDN 0.62πιΜ

(ε FZL) * ³ 0.13ιαΝ 0.22πιϊ 0.37 IDM 0.192mN

specificity contant

 (Kn / Vmax) (aFZL) 86 94 131 240

 (£ FZ L) 176 555 107 26.8

Optimum pH 8.0 8.0 8.5 7.5 8.0 Optimum temperature C) 35 45 35 25 25-30 Isoelectric point 4.8 6.8

Effect of SH reagent Yes Yes Yes Yes Yes

* 1: Not detected

* 2: N ^β —fructosyl Ν '— Ζ—resin

* 3: N'- Fourques Toshiru ^Ν β - Ζ- lysine

Table 1 shows that F AOD used in the method of the present invention has high specificity for fructosyl lysine and / or fructosyl valine. Further, the activity of FAOD for each substrate is shown in Table 2 below.

Table 2

 Substrate specificity of F A O D Substrate concentration Relative activity (%)

 Cibberella Fusariun oxysporum F. oxysporum Penicillium Aspergillus fujikuroi S-1F4 f.sp. lini janthinellum terreus GPl IFO NO.6356> <FER «BP-5010> <IF0 NO.5880> S-3413 <FER BP-5475) <FERM BP- 5684

N'-Fructosyl N "-Z-lysine 1.67mM 100 100 100 100 100 (FZL)

Fructosyl valine 1.67 ND ¹ ND 30.1 446 32.1

N'-Methyl-L-lysine 1.67 N.D.N.D.N.D.N.D.N.D.

 Fructosyl poly-L-lysine 0.02% 1.0 2.3 0.24 N.D.0.30 (FPL)

 Poly-L-lysine 0.02 N.D.N.D.N.D.N.D.N.D.

FBSA * ² 0.17 NDNDNDNDND

FHSA * ³ 0.17 NDNDNDNDND

 Tryptic FBSA 0.17 0.19 4.6 0.62 N.D.0.58

Tryptic FHSA 0.17 N.D.2.3 N.D.N.D.N.D.

Tryptic FPL 0.17 59.7

* 1: Not detected

 * 2: Funorectosyl bovine serum albumin

 * 3: Fructosyl human serum albumin

 As shown in Table 2, FAOD used in the method of the present invention has activity against fructosyl lysine and Z or fructosyl boryl lysine, which indicates that the FAOD is useful for measuring glycated albumin. It indicates that there is. Further, FAODs derived from Fusarium oxysvolum.f.sp.'lini, Aspergillus'Teleus GP1 and Penicillium jansinerum S-3413 used in the method of the present invention have activity against fructosylvalin. This indicates that the FAOD is also useful for measuring glycated hemoglobin. Furthermore, FAOD used in the method of the present invention also has an activity on protease digests of glycated proteins.

3. Measurement of titer

 The power grinding measurement of the bran was performed by the following method.

 (1) A method of measuring the generated hydrogen peroxide by a colorimetric method.

 A. Speed method

The 10 OmM FZL solution was prepared by dissolving FZL obtained in advance with distilled water. 45 mM 4-Aminoantipyrine, 60 units / m 1 peroxidase solution, 100/1 each of 6 OmM Fuunol solution, 0.1 M Tris-HCl 锾 mouth solution (pH 8.0) Im and »¾ solution 51 Mix and make up to 3.0 ml with distilled water. After incubating for 2 minutes at 30 ° C., 501 OmM solution was added, and the absorbance at 505 nm was measured over time. From the molecular extinction coefficient (5.16 x 1 O ^-'cm- ¹ ) of the quinone dye produced, calculate the micromoles of purple water per minute produced per minute, and use this figure as the enzyme activity unit (unit: U ) When I do.

 B. terminal law

 Treat in the same manner as in Method A above, add the substrate, incubate at 30 ° C for 30 minutes, measure the absorbance at 505 nm, and measure the absorbance generated from the calibration curve prepared in advance using a standard aqueous peroxide peroxide solution. Enzyme activity is measured by calculating hydrogen oxide S.

 (2) Method of measuring oxygen absorption by enzyme reaction

 Mix lm 1 of 0.1 M Tris-HCl buffer (pH 8.0) with 501 of enzyme solution, make up to 3.0 ml with distilled water, and put into a cell of Rank Brothers' public electrode. After mixing with 3 CTC and equilibrating the temperature with the dissolved acid purple, 50 mM FZL 1001 is added, and the oxygen absorption is continuously measured with a recorder to obtain the initial velocity. Calculate the amount of oxygen absorbed in one minute from the standard curve, and use this as the enzyme unit.

 As described above, FAOD is useful for measuring an Amadori compound, and therefore, the present invention provides a method for measuring oxygen consumption or a reaction product by bringing a sample containing an Amadori compound into contact with FAOD. Another object of the present invention is to provide a method for measuring an Amadori compound in a sample. The method of the present invention is carried out based on the measurement of the amount and / or saccharification rate of a glycated protein in a biological component or food, or the determination of fructosylamine.

 The enzyme activity of FAOD is measured based on the following reaction.

R ¹ -CO-CH ₂ -NH-R ² + 0 ₂ + H ₂₀ →

R'-C O-CHO + R with NH ₂ + H ₂ 0 ₂

(Wherein, R ¹ represents an aldose residue, R ² represents an amino acid, protein or peptide residue)

As the test liquid, use any sample solution containing the Amadori compound. Examples thereof include foods such as blood (whole blood, blood serum or serum), urine and other biological samples, as well as oils.

 In the analysis method of the present invention, any of the following methods for measuring an Amadori compound is used.

 (1) Method based on the amount of reaction product

 The action of FAOD produces hydrogen peroxide and glucosone. Hydrogen peroxide is measured by a method known in the art, for example, a coloring method, a method using a hydrogen oxide electrode, etc., and a standard curve prepared for the amounts of water peroxide purple and the Amadori compound. The Amadori compound in the sample is measured by comparing with. Specifically, it is based on the measurement of the titer described in 3 above. However, the amount of FAOD shall be 1 unit Zm 1, an appropriately diluted sample shall be added, and the amount of hydrogen peroxide generated shall be measured. The color development system for the colorimetric method of hydrogen peroxide is as follows: カ ッ プ Couplers such as 4-aminoantivirin (AAA) and 3-methyl-12-benzothiazolinone hydrazone (MBTH) in the presence of oxidase. It is possible to use a system that produces a dye by oxidative condensation of phenol with a chromogen such as phenol.

Examples of chromogens include fuynol derivatives, aniline derivatives, and toluidine derivatives. For example, N-ethyl-N- (2-hydroquinone-3-sulfopropyl) 1 m-toluidine, N, N-dimethylaniline, N.N-dimethylaniline N-Jetyla diphosphine, 2,4-dichroic phenol, N-ethyl-N- (2-hydroxy-13-sulfoprovir) -1,3,5-Dimethoxyanilinine, N-ethyl-N- (3-sulfo Propyl) -1-3.5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -1,5-dimethylaniline (MAOS) and the like. Also, a leuco-type coloring reagent which is oxidized in the presence of peroxidase to give a color can be used. Various leuco-type coloring reagents are known to those skilled in the art, and include 0-dian ginseng, 0-trizine, 3.3-diaminobenzidine, 3,3.5.5-tetramethylbenzidine, and N- (carboxymethylaminocarbonyl). And 1,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboquinmethylaminocarbonyl) -1,3,7-bis (dimethylamino) phenothiazine (DA A67), and the like.

 The measurement of hydrogen peroxide using a chromogen includes a colorimetric method, a luminescence method, and a chemical luminescence method. For the fluorescence method, a compound that emits fluorescence by oxidation, for example, homovanillic acid, 4-hydroquinphenyl sulfonic acid, tyramine, nociclecresol, diacetylfluorescin derivative and the like can be used. In the chemiluminescence method, veroxidase, lithium furocyanide, hemin, or the like can be used as a catalyst, and luminol, noresigenin, isorminol, pyrogallol, or the like can be used as a substrate.

 In addition, for the measurement of hydrogen peroxide, a system in which catalase is allowed to act in the presence of an alcohol (eg, methanol) and the resulting aldehyde is colored by a Hunch reaction or a condensation reaction with MBTH can be used. This aldehyde can also be conjugated to aldehyde dehydrogenase to measure changes in NAD (NADH).

 For measurement of glucosone, an aldose reagent such as diphenylamine can be used.

When measuring hydrogen peroxide using an electrode, the anode can be any material that can transfer electrons to and from hydrogen peroxide, but platinum, gold, silver, etc. are preferred. . The measurement can be performed by a method known to those skilled in the art, such as ambiometry, potentiometry, and coulometry. In addition, the reaction between the FAOD or the substrate and the positive electrode is mediated by an electron carrier, The resulting oxidation or reduction current or the amount of electricity can also be measured. The electron conductor may be a substance known to those skilled in the art, such as a fluorinated conductor, a quinone derivative, or any substance having an electron transfer function that can be generally considered by those skilled in the art.

 Furthermore, a proton conductor can be interposed between the hydrogen peroxide generated by the FAD reaction and the electrode, and the resulting oxidation, a-source current, or its air volume can be measured.

 C 2) Oxygen consumption

 The value obtained by subtracting the amount of oxygen at the end of the reaction from the amount of oxygen at the start of the reaction (oxygen quenching β *) is measured and compared with the quasi-curve created for the amounts of oxygen quenched Si and Amadori compounds in the sample. Is measured. Specifically, the measurement is performed in accordance with the above-mentioned titration measurement. However, the amount of F A O D to be used shall be 1 unit / ml, and an appropriately diluted sample shall be added to determine the amount of oxygen eliminated. Although the method of the present invention can be carried out using the sample solution as it is, depending on the target Amadori compound, it is preferable to carry out the sample while or after the sample is treated so that the FAOD reacts.

Such purposes include the use of protease (enzymatic method), the use of chemicals such as trifluorosulfuric acid (chemical method), and the use of physical methods such as heat (physical method). . For the enzymatic method, endo- and exo-type proteases known to those skilled in the art can be used alone or in combination. Endo-type proteases are abundant substances that are degraded from the inside of proteins.For example, trypsin, chymotrypsin, subtilisin, brotinase K, papain, cathepsin Β, pepsin, thermolysin, protease X〗 V, protease XVII, protease XXI, lysylende butide, pro-leather, promelain F, etc. On the other hand, the exo-type Oral protease is an enzyme that decomposes sequentially from the end of the peptide chain, and examples thereof include aminopeptidase and carboxypeptidase. Enzyme treatment methods are also known and can be performed, for example, by the method described in the following Examples.

These endo-type and exo-type proteases are preferably used depending on the saccharification site of the Amadori compound to be measured by utilizing its properties. For example, glycated albumin is an endo-type protease because the internal lysine residue is glycated, and hemoglobin Ale is an exo-type protease because the / 9 chain N-terminal valine residue is glycated. Processing can be performed more efficiently. These can be read from Tables 3 and 4 below.

Table 3 Treatment method for glycated human albumin Protease F AOD activity Relative activity

 (U / ml)

End-type protease

 Specific

 Tribcine 2.5 100 Residual peptidase 6.3 250

^ Specific

 Proteinase A 5.0 200 Subtilisin δ.7 230 Human cathepsin 4.2 170 Protease XIV 7.7 310 Bronase 5.8 230 Bronase E 8.4 340 Proteinase K 3.1 120 Blotase P 3.0 120 Blotase N 4.4 180 Proleather 3.0 120 Papain 5.0 200 Protease A 5.9 240 Bromelain F 4.0 160 exo-type protease

Carboxypeptidase Υ 0.82 32 aminopeptidase Τ 0.73 30 aminopeptidase I 0.86 34 aminopeptidase Μ 0.89 36 aminopeptidase C ― 0.54 22 Table 4 Treatment method for hemoglobin A1c Protease FAOD activity Relative activity

 (U / ml)

End-type proteases

 Specific

 Tribcine 0.22 5.8 Non-specific

 Pepsin 0.20 5.3 Brotinase A 0.13 3.5 Subtilisin 0.04 1.1 Dicatecatesin 0.07 1.9 Human cathepsin 0.10 2.6 Bronase 0.05 1.Protease N 0.06 1.6 Protease A 0.14 3.7 Papain 0.06 1.6 Exo-type protease

Carboxypeptidase B 1.27 33.3 Aminopeptidase 3.82 100.0 Aminobutidase M 0.43 11.2 Furthermore, according to the method of the present invention, unlike the conventional method, it is not necessary to completely decompose the Amadori compound into amino acids and release saccharified amino acid residues in the treatment by the thixotropy method. The processing time can be shortened because the saccharified peptide may be in a state in which FAOD can easily react.

 In the chemical method, an acid, an alkali, a surfactant, a protein denaturant and the like can be used alone or in combination. In the physical method, heat, microwave, pressure, etc. can be used alone or in combination.

 The enzymatic method, the chemical method, and the physical method can be used alone, but may be used in an appropriate combination. Further, these processes can be performed before or at the time of the F AOD reaction. In the conventional method, it was necessary to measure after processing the sample, but in the method of the present invention, the processing of the sample and the FAOD reaction can be performed simultaneously, so the operation is simpler and more labor-intensive than the conventional method. Instead, it can be measured in a short time.

 As described above, since FAOD used in the method of the present invention has high substrate specificity for fructosyl lysine contained in glycated proteins, it includes the measurement of glycated albumin in a blood sample, diagnosis of diabetes, etc. Useful for In addition, since fructosylvaline also has specificity, it is useful for measuring hemoglobin A1c.

 When a blood sample (whole blood, plasma or serum) is used as the specimen, the collected blood sample is used as it is or after being subjected to a treatment such as folding.

If the sample is a whole blood or hemolyzed sample, the sample will show a unique absorption spectrum due to the presence of hemoglobin. When measuring by the colorimetric method, the wavelength to be detected overlaps with the absorption of hemoglobin, and accurate measurement cannot be performed. Therefore, it is necessary to subtract the previously measured hemoglobin spectrum from the spectrum after the FAOD reaction and measure the difference. However Moreover, this method is not always accurate, and is cumbersome. According to the present invention, when the measurement is performed by the colorimetric method, the absorption of hemoglobin itself can be avoided, and the measurement can be performed in a long wavelength region (600 to 800 nm), whereby the shadow of hemoglobin in the sample can be obtained. And more accurate measurement is possible. For example, hydrogen peroxide produced by the action of FAOD was produced by oxidative coloring of DA64, DA67, 4AA / MAOS, 4AAZM APS, etc. in the presence of peroxidase, and by the action of lipase. It is detected by color development due to the condensation reaction between aldehyde and MBTH. By this method, glycated albumin as well as Amadori compounds containing hemoglobin Ale in whole blood and hemolyzed samples can be measured. However, the detection wavelength is not limited to this, and even if it is in the wavelength range that overlaps with hemoglobin absorption, as described above, the previously measured hemoglobin spectrum is measured after the FAOD reaction. The method of subtracting from the spectrum and measuring the difference is also included in the method of the present invention.

 Furthermore, the enzymes such as FAOD, peroxidase, lipase, and protease used in the method of the present invention may be used in the form of a solution, or may be immobilized on a suitable solid support. For example, by packing an enzyme immobilized on beads into a column and incorporating it into an automated device, routine analysis of a large number of samples such as clinical tests can be performed efficiently. In addition, the immobilized yeast purple can be reused, which is preferable in terms of economic efficiency.

 Furthermore, by appropriately combining an enzyme, a coloring dye, and a processing reagent, it is possible to obtain a kit for analyzing Amadori compounds useful not only for clinical analysis but also for food analysis.

Immobilization of the enzyme can be performed by a method known in the art. For example, the method is carried out by a carrier binding method, a cross-linking method, an inclusive method, a complex method, or the like. Carriers include polymer gels, microcapsules, agarose, alginic acid, and carrageen One Nan, and so on. Coupling is performed by a method known to those skilled in the art using covalent bonding, ionic bonding, physical adsorption, and biochemical affinity.

 When using an immobilized enzyme, the analysis may be either flow or batch. As noted above, immobilized enzymes are particularly useful for routine analysis (glycos) of glycated proteins in blood samples.

 If the laboratory test is aimed at diagnosing diabetes, the result should be expressed as glycated protein concentration, the ratio of glycated protein concentration to the total protein K concentration in the sample (glycation rate), or fructosylamine 4. . The total protein concentration can be determined by a conventional method known to those skilled in the art (eg, absorbance at 280 ηιη, Bradford method, Lowly method, Buret method, etc.). If the Amadori compound (glycated protein) to be measured is glycated albumin, phthalein dyes such as bromcresol green (BCG), bromcresol monopurple (BCP), and bromphenol blue (BPB) are used for the total albumin S level. Method, methyl orange, a method using azo dyes such as 2- (4'-hydroquinbenzenazo) benzoic acid (HAB (C) A), nephrometry, a method using the natural fluorescence of albumin, etc. Can be determined. When the measurement target is glycated hemoglobin, the total hemoglobin concentration can be measured by a gas method, a cyanmethemoglobin method, an azamethemoglobin method, a method using the absorbance of hemoglobin itself, etc. However, the present invention is not limited to this.

 The present invention also provides a method for measuring an Amadori compound in a sample, comprising: FAOD; and a processing reagent for treating the sample such that a saccharification site of the Amadori compound in the sample reacts with FA0D to form a sieve. It provides a reagent or kit.

When performing end-point analysis, FAOD jt in reagents is usually 1 to 1 per sample. 100 nits / ml, and the buffer solution is preferably Tris-HCl (pH 8.0). When the Amadori compound is measured based on the amount of hydrogen peroxide generated, the color-forming system may be a system that forms a color by oxidative condensation described in the above-mentioned “(1) Method based on the amount of reaction product”, or a leuco-type. A coloring reagent or the like can be used. A kit can also be obtained by combining the reagent for measuring the Amadori compound of the present invention, an appropriate color former, and a color standard or standard substance for ratio. Such a kit would be useful for preliminary diagnosis and testing. The above measuring reagents and kits are used for measuring the amount and / or saccharification rate of glycated protein in biological components or foods, or for quantifying fructosylamine.

 Hereinafter, the present invention will be described in more detail with reference to Examples.

Production example 1 Production of FAOD derived from Giperella

Gibberella fujikuroi (IFO NO.6356) (Gibberella fujikuroi) from FZL 0.5%, glucose 1.0%, diphosphoric acid 0.1%, phosphoric acid-sodium 0.1%, magnesium sulfate 0.05%, calcium chloride 0.01% The cells were inoculated into 10 L of a medium (pH 6.0) containing 0.2% yeast extract, and cultured with stirring using a jar fermenter with aeration of 2 LZ for 28 hours at a stirring speed of 400 rpm. Cultures were collected over time. 170 s (wet weight) of mycelium was suspended in 1 L of 0.1 M Tris-monohydrochloride bite solution (pH 8.5) containing 2 mM DTT, and the mycelium was disrupted with Dyno-Mill. The crushed liquid was centrifuged at 10,000 Orpin for 15 minutes, and the obtained liquid was used as a crude enzyme liquid (non-fine extract). Ammonia sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40% saturated, stirred, and centrifuged at 12.00 Orpm for 10 minutes. Ammonium sulfate was added to the obtained supernatant to 75% saturation, stirred, and centrifuged at 12.00 Orpm for 10 minutes. Precipitate contains 2 mM DTT Was dissolved in 50 mM Tris-HCl buffer (pH 8.5) (buffer A) and dialyzed against buffer A overnight. The dialysate was adsorbed on a DEAE-Sephacel column equilibrated with buffer A. After washing with buffer A, elution was performed with a linear gradient of 0-0.5M potassium chloride. The active fraction was collected, subjected to a 55% to 75% ammonium sulfate fraction, and dialyzed against buffer A. Ammonium sulfate was added to the dialysate so as to be 25% saturated, and the dialysate was adsorbed to a full-length Yopearl column equilibrated with buffer A containing 25% saturated ammonium sulfate. After washing with the same buffer, elution was carried out with a linear gradient of ammonium sulfate concentration of 25-0% saturation. The active fractions were collected, ammonium sulfate was added to achieve 40% saturation, and the mixture was adsorbed on a Petit Root Yopal column equilibrated with buffer A containing 40% saturated ammonium sulfate. After washing with the same buffer solution, elution was carried out with a linear concentration gradient of ammonium sulfate concentration of 40-0% saturation. The active fractions were collected, ammonium sulfate was added to 80% saturation, stirred, centrifuged at 12.000 rpB for 10 minutes, and the resulting precipitate was dissolved in 0.1 M buffer A. The enzyme solution was subjected to cefacryl s-200 gel a¾chromatography containing 0.1M buffer chloride A and containing 0.1M buffer A and made into a flat mouth. The active fraction was collected and concentrated by ultrafiltration. By treating the concentrate with a Pharmacia FPLC system using a Mono Q column (elution with 0-0.5M linear concentration gradient of potassium chloride in buffer A), 30-60 nits of purified enzyme can be obtained. Production Example 2 Production of FAOD derived from Fusarium II

Fusarium oxisbolum S-1 F4 (FERM BP-5010) (Fusarium ox ysporum S-1F4) was prepared from FZL 0.5%, glucose 1.0%, dicalcium phosphate 0.1%, monosodium phosphate 0.1%, magnesium sulfate Inoculate 10 L of a medium (pH 6.0) containing 0.05%, 0.01% calcium chloride and 0.2% yeast extract, and use a jar armor for 2 minutes with a stirring speed of 40 Orpm. The culture was agitated for hours. Cultures are collected by filtration. I did. A portion (200 g) of the mycelium was suspended in 1 L of 0.1 M Tris-monohydrochloride buffer (pH 8.5) containing 0-chome, and the mycelium was disrupted by Dyno-Mill. The crushed liquid was centrifuged at 10,000 Orpn for 15 minutes, and the obtained liquid was used as a crude enzyme liquid (cell-free extract). Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40% saturated, and the mixture was stirred and separated at 12.00 Orpin for 10 minutes. Ammonium sulfate was added to the obtained supernatant to 75% saturation, stirred, and eccentrically separated at 12.00 Orpin for 10 minutes. The precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.5) containing 2 mM DTT (hereinafter abbreviated as buffer A), and dialyzed against mouth liquid A overnight. The dialysate was adsorbed on a DEAE-Sephacel column equilibrated with buffer A. After washing with the same buffer A, elution was carried out with a linear concentration gradient of 0 to 0.5 M potassium chloride. The active fraction was collected, subjected to a 55% to 75% ammonium sulfate fraction, and dialyzed against buffer A overnight. Ammonium sulfate was added to the dialysate so as to be 25% saturated, and the dialysate was adsorbed to a Fuyroot yopal column equilibrated with buffer A containing 25% saturated ammonium sulfate. After washing with the same buffer solution, elution was performed with a linear gradient of 25-0% saturation. The active fractions were collected, ammonium sulfate was added to 40% saturation, and the mixture was adsorbed on a Petil-Toyopearl column equilibrated with Buffer A containing 40% saturated ammonium sulfate. After washing with the same buffer, elution was carried out with a linear concentration gradient of ammonium sulfate S degree 40-0% saturation. The active fractions were collected, ammonium sulfate was added to 80% saturation, and the mixture was stirred, centrifuged at 12.000 rpm for 10 minutes, and the obtained precipitate was dissolved in 0.1 M buffer A. The enzyme solution was applied to a Cefacryl S-200 gel filtration chromatograph which had been equilibrated with 0.1 M buffer A and contained 0.1 M of chloride. Active fractions were collected and concentrated by ultrafiltration. Treating the concentrate with a Pharmacia FPLC system using a Mono Q column (0-0.5 M linear chlorinated chloride using buffer A, elution with a gradient), 30-60 units To obtain a purified mellow purple.

Production example 3 Production of FAOD from Fusarium or Aspergillus JR

 Fusarium 'oxysporum f.sp.lini (IFO .5880) CFusarium oxysporum f. Sp. Lini) or Aspergillus terreus GP 1 (FERM BP-5684) (Aspergillus terreus GP1) 0.5% FZL, glucose 1. Medium containing 0%, 0.1% dipotassium phosphate, 0.1% monosodium phosphate, 0.05% magnesium sulfate, 0.01% calcium chloride, and 0.2% yeast extract (pH 6. 0) 10 L was inoculated and cultivated with a jar arm for 28 hours under the conditions of an aeration amount of 2 LZ and a stirring speed of 40 Orpa for 28 hours. The tie stuff was collected after a.

 Next, 270 g of mycelium (wet weight S) was mixed with 800 ml of 0.1 M Tris-hanic acid buffer (pH 8.5) containing 2 mM DTT, and the mycelium was disrupted by Dyno-Hill. . The disrupted solution was centrifuged at 9,50 Orpin for 20 minutes, and the resulting supernatant (cell-free extract) was purified as a crude enzyme solution by the following method.

Step 1: Ammonium sulfate fractionation

Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so that it became 40% saturated, and the protein was removed by centrifugation (4 ^EC . 12.00ΟΓΡΠ). 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 dissolved in 5 OmM Tris-monohydrochloride buffer (pH 8.5) containing 2 mM DTT (hereinafter abbreviated as buffer A) and contains an equal amount of 40% ammonium sulfate Quench A was added. 200 ml of butyl-TOYOP EARL resin was added to the crude enzyme solution, and adsorption was performed by the batch method. Elution was also performed by the batch method using buffer A, and the active fraction was sulfuric acid. The solution was concentrated by precipitation.

Step 3: Hydrophobic chromatography

 The concentrated active fraction was adsorbed to a phenyl-TOYOPEARL column equilibrated with buffer A containing 25% ammonium sulfate, washed with the same buffer, and eluted with a linear gradient of 25 to 0% ammonium sulfate. . The collected active fraction was concentrated by ammonium sulfate precipitation and used for the next step.

Step 4: Hydrophobic chromatography (column method)

 The collected active fraction was used for a butyl topopearl column (equilibrated with mouth liquid A containing 40% ammonium sulfate). The concentrate was adsorbed and washed with the same buffer. The active fraction was obtained with a linear gradient of 40 to 0% ammonium sulfate.

Step 5: ion exchange mouth chromatography

 Next, DEAE-TOYOPEARL column chromatography was performed (equilibrated with buffer A). Since FAOD activity was observed in the washed fraction, this was collected, concentrated with ammonium sulfate, and used in the next step.

Step 6: Gel filtration

 After that, gel migration was performed with Sephacryl® 300 (equilibrated with 0.1 M Tris-HCl buffer (ρΗ8.δ) including 0.1 M Na CI. As a result, 70 to 100 units of the enzyme preparation were obtained.

Production Example 4 Manufacture of {Nisylium} -derived FAOD

Benzinlium Jansinerum S-3413 (FERM BP-5475) (Penicill iuB janthinellum S-3413) was prepared from 0.5% FZL, 1.0% glucose, 0.1% dibasic phosphate, 0.1% sodium phosphate, 0.1% magnesium sulfate, magnesium sulfate Inoculated in 10 L of a medium (pH 6.0) containing 0.05% calcium chloride, 0.01% calcium chloride, and yeast extract 0.2%. The mixture was cultured for 28 to 36 hours under conditions of a flow rate of 2 LZ and a stirring speed of 50 Orpm. Cultures were collected over time.

410 g of mycelium (wet weight)

The cells were suspended in 800 ml of a 0.1 M potassium phosphate buffer (pH 7.5) containing 0-chome, and the mycelium was disrupted with Dyno-Mill. The crushed liquid was eccentrically separated at 9.50 Orpm for 20 minutes, and the obtained liquid was used as a crude enzyme liquid (cell-free extract) and purified by the following method.

 Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40¾ saturated, stirred, and centrifuged at 12.00 Orp for 10 minutes. Ammonium sulfate was added to the obtained supernatant to 75% saturation, stirred, and eccentrically separated at 12.ΟΟΟΓΡΒ for 10 minutes. The precipitate was dissolved in a 5 OmM calcium phosphate buffer (pH 7.5) containing ImM DTT (hereinafter referred to as buffer A). The obtained solution was dialyzed against buffer A. The external solution was exchanged twice. The enzyme solution after the analysis was ablated on a DEAE-Sephacel column (4.226 cm) equilibrated with buffer A. The active fraction was found in the fraction washed with the same buffer, and was collected and subjected to ammonium sulfate fraction of 0-55% saturation. Next, the enzyme was equilibrated with buffer A containing 25% saturated ammonium sulfate.

(Low Substitute) Column (HR 10Z10). After washing with the same contacting solution, elution was performed with a linear gradient of ammonium sulfate concentration of 25-0% saturation. The active fractions were collected, concentrated with ammonium sulfate, and the resulting enzyme solution was subjected to gel chromatography using a Superdex 200 pg column equilibrated with 0.2 M potassium phosphate buffer (pH 7.5) containing 0.1 ImM. After filtration, 70-100 units of purified nitrogen were obtained.

Example 1

 In this example, FAO D derived from Giperella JR obtained in Production Example 1 was used.

0.1% fructosyl polylysine solution was analyzed using the BMY / NBT assay. Test showed a fructosamine value of 750 ol / 1.

 By diluting this solution with distilled water, a series of samples varying in the range of 0 to 750 μαοΐ / 1 was prepared. The FAOD reaction solution was prepared as follows.

 4 δπιΜ 4 Aminoantipyrine solution 50 1 6 ΟπιΜ Fuyunol solution 50 1

 3 5

 60 units Zml peroxidase solution 50 10.1 Tris-HCl buffer (pH 8.0) 500 il

7.6 units / ml FAOD solution 30 1 0.05% Tribcine (endo-type protease) solution 100 1 The total volume was adjusted to 1400 l with distilled water.

 The 7.6 unit ZmlF AOD solution was prepared using 0.1 M Tris-hydrochloric acid buffer (pH 8) so that the FAOD derived from Giperella fujikuroi (IFO NO. .0) to make silk.

 30 of this reaction solution. C, and each of the prepared fructosyl boridine resin solutions was added, and the absorbance at 505ππ was measured 30 minutes later. Figure 1 shows the relationship between the fructosamine value and the absorbance obtained by this method. In the figure, the vertical axis represents the absorbance at 505 mn (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the fructosamine value. The figure shows that the fructosamine value and the amount of generated hydrogen peroxide are correlated.

Example 2 Measurement of glycated human serum albumin concentration

In this example, FAOD derived from Fusarium obtained in Production Example 2 was used. Glycated human serum albumin (Sigma) is dissolved in 0.9% sodium chloride aqueous solution. Then, saccharified human serum albumin solutions having different concentrations in the range of 0 to 10% were prepared.

 The following operations were performed using these solutions.

 1) Sample processing

 Saccharified albumin solution 60/1

12.5 ig / ml protease XIV (Sigma)

 (Endo-type protease) Solution 60 1 Incubate this mixture for 30 minutes at 37 ° C. Heat denaturation by heating at C for 5 minutes.

 2) Activity measurement

 The F A 0 D reaction solution was prepared as follows.

 45mM 4-aminotinvirine solution 30 16 OoM N-ethyl-N- (2-hydroquinone

 3-sulfopropyl) 1 m—toluidine solution 30 1

60 units per ml peroxidase solution 30 ^ 1

0.1M Tris-hydrochloric acid bite liquid (pH 8.0) 300 ^ 1

10.3 units / ml FAOD solution 20 1 Distilled water was used to bring the total volume to 1 ml.

 10.3 Unit No. ml FAOD solution was prepared by adjusting the FAOD derived from Fusarium oxysbolum S-1 F4 (FERM BP-5010) (Fusarium oxysporum S-IF4) obtained in Production Example 2 to 0.13 units / ml so as to obtain 10.3 units / ml It was diluted with Tris-HCl buffer (PH8.0) and made into a net.

After 2 minutes Q Inkyupeto the FAOD reaction mixture at 30 ^e C, each processing solution described above 100 1 added, the absorbance was measured at 555nn after 30 minutes. Fig. 2 shows the relationship between the concentration of saccharified albumin obtained by this method and the absorbance. You. The vertical bar in the figure indicates the absorbance of 555ππ (corresponding to the amount of hydrogen peroxide), and the horizontal axis indicates the concentration of glycated albumin. The figure shows that the concentration of saccharified albumin and the amount of hydrogen peroxide generated are correlated.

Example 3 Measurement of glycated human serum albumin concentration

 In this example, the same operation as in Example 2 was repeated, except that the FAOD derived from Giperella II obtained in Production Example 1 was used. The FAOD solution was prepared by mixing FAOD derived from Gibberella fujikuroi obtained in Production Example 1 (IFO No. 6356) (Gibberella fujikuroi) with 0.1 M Tris-hydrochloric acid buffer (pH 8. 0).

 Figure 3 shows the relationship between the concentration of saccharified albumin (horizontal axis in the figure) and absorbance (corresponding to hydrogen peroxide i: vertical axis) obtained by this method. The figure shows that the concentration of saccharified albumin and the amount of generated hydrogen peroxide are correlated.

Example 4 Glycated human blood? f Measurement of albumin concentration

 In this example, the same operation as in Example 2 was repeated, except that the FAOD derived from Fusarium 厲 obtained in Production Example 3 was used 50 // 1. The FAOD solution was prepared so that the FAOD derived from Fusarium 'oxysbolum · f.sp. · lini (IFO NO.5880) (Fusariura oxys orum f.sp. lini) obtained in Production Example 3 was 6.0 units Zml. It was prepared by diluting with 0.1 M Tris-HCl buffer (pH 8.0).

 Figure 4 shows the relationship between the concentration of saccharified albumin (horizontal axis in the figure) and the absorbance (corresponding to the amount of hydrogen peroxide: vertical bow) obtained by this method. The figure shows that the concentration of saccharified albumin and the amount of generated hydrogen peroxide are correlated.

Example 5 Measurement of saccharified human serum albumin concentration

In this example, the same operation as in Example 2 was repeated, except that FAOD derived from the genus Albergillus obtained in Production Example 3 was used. That is, FA0 96 2964

The D solution was prepared such that the FA D derived from Aspergillus terreus GP1 CFEB1I BP-5684 (Aspergillus terreus GP1) was adjusted to 0.1 units / ml.

The product was diluted with M Tris-hydrochloride mouth liquid (pH 8.0) and manufactured by IS.

 Figure 5 shows the relationship between the concentration of saccharified albumin obtained by this method (horizontal axis in the figure) and the absorbance (corresponding to the amount of hydrogen peroxide: vertical axis). The figure shows that the concentration of saccharified albumin and the amount of generated hydrogen peroxide are correlated.

Example 6 Measurement of saccharification rate of human blood albumin

 In this example, the FASARD derived from Fusarium 得 obtained in Production Example 3 was used.

In 3 ml of 0.9% sodium chloride aqueous solution, 150 mg of glycated human serum albumin (Sigma) and 150 mg of human blood serum albumin (Sigma) were dissolved. By mixing these solutions to prepare solutions with different saccharification rates, and testing using an automatic glycoalbumin measuring device (Kyoto Daiichi Kagaku), the saccharification rate was 24.6% to 61.1%. %Met.

 The following operations were performed using these solutions.

 1) Sample processing

 Saccharified albumin solution 60 / a

12.5 mg / ol brothase) (IV (Sigma)

 (Endo-protease) solution 60 μΐ 37 The mixture was incubated at C for 30 minutes, and then heat denatured by heating at about 90 minutes for 5 minutes.

 2) Activity measurement

 The F A 0D reaction solution was subjected to ISSi as follows.

 45πιΜ 4 Aminoantipyrine solution 30 ^ 1

6 Om N—Ethyru N— (2-Hydroxy

3—Sulfoprovir) 1 πι— Toluidine solution 30 1 60 units Zml peroxidase solution 30 1

0.1 M Tris-HCl buffer (pH 8.0) 300 β \

6 units / ml FAOD solution The total volume was adjusted to 1 ml with 50 fi I distilled water.

 The 6 unit / ml FAOD solution was prepared by converting FAOD from Fusarium oxysporum f.sp. lini (Fusarium oxysporum f. Sp. Lini) obtained in Production Example 3 to 6 unit Znl. It was prepared by diluting with 0.1 M Tris-HCl buffer (pH 8.0).

 After incubating the FAOD reaction solution at 30 ° C for 2 minutes, each of the above-mentioned treatment solutions was added to lO Oiil, and the absorbance at 555ππι was measured 30 minutes later. FIG. 6 shows the relationship between the saccharification rate of albumin obtained by this method and the absorbance. The vertical axis in the figure indicates the absorbance of 555na (corresponding to the amount of hydrogen peroxide), and the horizontal axis indicates the saccharification rate of albumin. The figure shows that the saccharification rate of albumin and the hydrogen peroxide generator are in a relationship.

Example 7 Measurement of saccharification rate of human serum albumin

 In this example, the same operation as in Example 6 was repeated, except that the FAO D derived from Alpergillus 厲 obtained in Production Example 3 was used. In other words, the FAOD solution is prepared by diluting FAOD derived from Aspergillus' Teleus GP1 (FERM BP-5684) (Aspergillus terreus GP1) with 0.1 Tris-HCl buffer (pH 8.0) to 6.0 nit / ml. IS made.

 Figure 7 shows the relationship between the saccharification rate of albumin (horizontal axis in the figure) and absorbance (corresponding to the amount of hydrogen peroxide: vertical axis) obtained by this method. The figure shows that the saccharification rate of albumin and the amount of generated hydrogen peroxide are correlated.

Example 8 Measurement of saccharification rate of human serum albumin

In this example, the FAOD derived from Giperella た obtained in Production Example 1 was used for 201 The same operation as in Example 6 was repeated, except that it was performed. The FAOD solution was prepared using 0.1 M Tris-HCl buffer (pH 8) such that the FAOD from Giperella fujikuroi (IFO No. 6356) (Gibberella fu jikuroi) obtained in Production Example 1 was adjusted to 6.6 units / m1. .0) and prepared.

 Fig. 8 shows the relationship between the saccharification rate of albumin obtained by this method (jux axis in the figure) and absorbance (corresponding to the appropriate amount of oxidized water purple: vertical axis). The figure shows that the saccharification rate of albumin and the amount of generated hydrogen peroxide are correlated.

Example 9 Measurement of saccharification rate of human serum albumin

 In the present example, the same operation as in Example 6 was repeated, except that 201 of the Fusarium-derived FA0D obtained in Production Example 2 was used. The FAOD solution was prepared so that the FAOD derived from Fusarium oxisbolum S-1F4 (FERH BP-5010) (Fusarium o ysporum S-1F4) obtained in Production Example 2 was 10.3 units. The mixture was diluted with 1M Tris-HCl buffer (pH 8.0) and made into an air-tight.

 Figure 9 shows the relationship between the saccharification rate of albumin obtained by this method (figure 铀 in Fig. Φ) and absorbance (corresponding to the amount of peroxidized water: vertical axis). The figure shows that there is a correlation between the concentration of saccharified albumin and the amount of hydrogen peroxide generated.

Example 10 Measurement of glycated hemoglobin concentration

 In this example, Fusarium-derived FAOD obtained in Production Example 3 was used. 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) Sample processing

Glycated hemoglobin solution 2 500 units ^/ 101 aminopeptidase (Exo-type mouth opening) Solution 5 1

0.1 M Tris-HCl buffer (pH 8.0) 20 を 30 Incubated at C for 30 minutes. Thereafter, 50% acetic acid at 10% trichloro mouth was added and the mixture was stirred. The mixture was kept at 0 ° C for 30 minutes and centrifuged at 1200 Orpn for 10 minutes. About 50 l of 2M NaOH was added to the obtained supernatant to make a neutral solution.

2) Activity measurement

 The F A 0 D reaction solution was prepared as follows.

 3 mM N— (carboxymethylaminocarbonyl)

 4,4 bis (dimethylamino) biphenylamine solution (DA64) 30 l 60 units / ml perokinidase solution 30 1 0.1 M Tris-HCl buffer (pH 8.0) 300 ^ 1

4 units / ml FAOD solution 10 // 1 The total volume was adjusted to 1 ml with distilled water.

 The 4-unit / ml FAOD solution was prepared by combining the FAOD derived from Fusarium oxyspornolem, f.sp., lini (IFO NO.5880) (Fusarium oxysporum f.sp. lini) obtained in Production Example 3 with 4 units / ml. It was prepared by diluting with a 0.1 M Tris-monohydrochloride buffer solution (pH 8.0).

 After the FAOD reaction solution was incubated at 30 ° C for 2 minutes, each of the above treatment solutions was added at 80/1, and the absorbance at 727 nm was measured 30 minutes later. Figure 10 shows the relationship between the concentration of saccharified hemoglobin and the absorbance obtained by this method. The vertical axis in the figure 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 the degree of glycated hemoglobin and the amount of generated hydrogen peroxide are correlated.

Example 11 Measurement of saccharified hemoglobin concentration In this example, the same operation as in Example 10 was repeated, except that FA 0D derived from Arlbergills JR obtained in Production Example 3 was used. That is, the FA OD solution was prepared by dissolving FA 0D derived from Aspergillus' Teleus GP1 (FERli BP-5684) (Aspergillus terreus GPl) in a 0.1 M Tris-HCl buffer (4.0 nit / ml). pH 8.0).

 FIG. 11 shows the relationship between the * degree and the absorbance of saccharified hemoglobin obtained by this method. The vertical axis in the figure indicates the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the vertical axis indicates the concentration of glycated hemoglobin. The figure shows that there is a correlation between the concentration of glycated hemoglobin and the amount of generated hydrogen peroxide.

Example 12 Measurement of glycated hemoglobin S

 1) Sample processing

 0 to 15 mg of glycohemoglobin control E (Sigma) was dissolved in 100 1 of distilled water. Acetone hydrochloride (1 N acetone hydrochloride: 1/100) inl was added to these samples, and centrifuged at 12,000 rpm for 10 minutes. The precipitate was washed with getyl ether 500 and dried under reduced pressure. Add 8Μ urea 100 1, heat in boiling water for 20 minutes, cool and mix with 5.2 units / ml trypsin 300 1 37. I incubated at C for 3 hours. Thereafter, the sample was heated in boiling water for 5 minutes to prepare a sample.

 2) Activity measurement

 The F A 0 D reaction solution was prepared as follows.

 3 mM N— (carboxymethylaminocarbonyl)

 4.4 Bis (dimethylamino) biphenylamine solution 30 iil

60 units / ml peroxidase solution 30 1

0.1 M Tris-HCl buffer (pH 8.0) 300 "1

25 units / ml FAOD solution 10 ^ 1 The total volume was adjusted to 1 ml with distilled water.

 The 25-unit Zml FAOD solution was prepared by diluting the FAOD obtained by the method of Production Example 4 with 0.1 M potassium phosphate / mouth liquid (pH 7.5) to 25 units / ml.

This FA OD reaction the processing substrate above 150 1 added, then 30 ^e C di Nkyupeto, the absorbance was measured at 727nm after 30 minutes. Fig. 12 shows the relationship between the amount of saccharified hemoglobin obtained by this method and the absorbance. The vertical axis in the figure indicates the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis indicates the concentration of saccharified hemoglobin. The figure shows that there is a correlation between the amount of glycated hemoglobin and the hydrogen peroxide generation S.

Example 13 Measurement of glycated hemoglobin amount

 1) Sample processing

 3 Omg of Glycohemoglobin Control E (Sigma) was dissolved in 200 ml of distilled water, and 1 ml of 570 mM Tris-hydrochloric acid buffer (pH8.8) containing 8 M urea and 0.2% EDTA. Beet ethanol 401 was added, and the mixture was allowed to stand for 2 hours under a nitrogen atmosphere. Thereafter, 1M sodium eodovine acetate 4001 was added, and the mixture was allowed to stand for 30 minutes, and then 40 il of 2-mercaptoethanol was added. After dialysis against 0.1 M ammonium bicarbonate, it was mixed with 1 OmgZinl TPCK-tribine 10 il. Incubated at C for 3 hours. Then, the sample was heated in boiling water for 5 minutes to prepare a sample.

 2) Activity measurement

 The F A OD reaction solution was prepared as follows.

 3mM N— (carboxymethylaminocarbonyl)

 4.4—Bis (dimethylamino) biphenylamine solution 3

60 units per ml peroxidase solution 30 1 0.1 M Tris monohydrochloride buffer ( _P H8.0) 300 // 1

25 units / ml F A0D solution 10 ^ 1 treated sample 0 to 13.2 n The total volume was adjusted to 900 / il with distilled water.

25 Yuni' DOO Znil FA0D solution, so that the FA 0 D obtained in Production Example 4 The method in 25 Yuni' concert ml, and prepared by dilution with 0. 1M potassium phosphate buffer _(P H 7. 5).

 The F AOD reaction solution was incubated with a pipette for 30 minutes, and the absorbance at 727 nm was measured 30 minutes later. FIG. 13 shows the relationship between the amount of glycated hemoglobin obtained by this method and the absorbance. In the figure, the vertical axis represents the absorbance at 727 mn (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the concentration of saccharified hemogbin. The figure shows that there is a correlation between the amount of saccharified hemoglobin and the amount of hydrogen peroxide generated.

Example 14 Determination of hemoglobin A 1 c value

 In the present Example, {Nisylium} -derived FAΔD obtained by the method described in Production Example 4 was used. Glycohemoglobin controls N and E (Sigma) were dissolved in distilled water. By mixing these solutions, solutions with different hemoglobin A lc values were prepared and assayed using an automatic glycohemoglobin analyzer (Kyoto Daiichi Kagaku). The values were between 5.1% and 9.2%. Met. The following operations were performed using these solutions.

 1) Sample processing

 Glycohemoglobin solution 25 il

500 Units 01 Amino-Butidase

(Exo-type open mouth thease) solution 5 0.1 M Tris-HCl buffer (pH 8.0) 20 βΐ The mixture was incubated for 30 minutes at 30 ^e C. Thereafter, 50 l of 10% trichloroacetic acid was added and the mixture was stirred, allowed to stand at 0 ° C for 30 minutes, and then centrifuged at 1200 rpm for 10 minutes. About 501 of 2M NaOH was added to the obtained supernatant to make a neutral solution.

 2) Activity measurement

 The FAOD reaction solution was prepared as follows.

 3mi N— (carboxymethylaminocarbonyl) one

 4,4-bis (dimethylamino) biphenylamine solution (DA64) 30 Hi 60 units / ml peroxidase solution 30 ^ 1 0.1 M Tris-HCl buffer (pH 8.0) 300 1

12 units / ml FAOD solution 10 1 The total volume was adjusted to 1 ml with distilled water.

 The 12 unit / ml FAOD solution was prepared by dissolving the FAOD derived from Penicillium. Chancinerum S—3413 (FESM BP-5475) CPenicillium janthinellum S-3413) obtained in Production Example 4 with 0.1 M phosphoric acid so as to obtain 12 units. It was prepared by diluting with a potassium hydroxide solution (PH7.5).

After the FAOD reaction solution was incubated at 30 ° C for 2 minutes, each of the above treatment solutions was added at 80 / zl, and the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide) was measured 30 minutes later. The amount of total hemoglobin was measured in advance by the absorbance at 415 nm, and the ratio of the amount of saccharified variin (absorbance at 727 nm) to the amount of total hemoglobin (absorbance at 415 nm) was calculated. Fig. 14 shows the relationship between the ratio of saccharified amount of palin to the total hemoglobin (vertical axis) and hemoglobin A1c value (horizontal axis) obtained by this method. The figure shows that there is a correlation between the hemoglobin A 1 c value and the glycation ratio of valine in hemoglobin measured using FAOD. Example 15 Measurement of hemoglobin A 1 c value

 Hemoglobin AO reagent (Sigma) was dissolved in distilled water to 2.3 M. This solution was fractionated using an automatic hemoglobin measuring device (Kyoto Daiichi Kagaku), and the hemoglobin A 1c fraction and the hemoglobin A 0 fraction were separated and purified. Substrate samples with hemoglobin Alc values between 0% and 52.0% were obtained.

 1) Sample processing

 250 g substrate sample

500 units 0> 1 Aminobeptidase solution 5 1 1.0M Tris-HCl buffer (pH 8.0) 15 ^ 1 These were mixed, and the total amount was adjusted to 200 with distilled water. Then, 200 1 of 10% trichloro ^ acid was added, and the mixture was stirred, left at 0 ° C for 20 minutes, and then centrifuged at 12,000 卬 for 10 minutes. About 401 of 5 M NaOH was added to the obtained supernatant to make a neutral solution.

 2) Activity measurement

 The FAOD reaction solution was manufactured by IS as follows.

 3mM-(carboxymethylaminocarbonyl) 1

 4.4-Bis (dimethylamino) biphenylamine solution 100 1

60 units / nil peroxidase solution 30 il 0.1 M Tris-monohydrochloride buffer (pH 8.0) 1000 / ^ 1 16 units / ml FAOD solution 151 The total volume was made up to 2.6 ml with distilled water.

The 16-unit Zral FAOD solution was prepared by diluting the FAOD obtained in Production Example 4 with 0.1 M potassium phosphate buffer (pH 7.5) so as to become 16 units-Znl. After 2 minutes Inkyube Bok The FAOD reaction mixture at 30 ^e C, above the processing solution 400/1 was added, it was measured absorbance at 727nm after incubation for an additional 30 minutes. The relationship between the hemoglobin A 1 c value of the substrate obtained by this method and the substrate is shown in FIG. The vertical axis in the figure represents the absorbance at 727πιη (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the hemoglobin A 1c value. The figure shows that there is a correlation between hemoglobin A 1 c value and the amount of generated hydrogen peroxide.

Example 16 Electrode measurement of fructosyl valine concentration

 A platinum working electrode and a silver / silver chloride electrode were used as reference electrodes. These compresses were immersed in a reaction solution obtained by adding 12 units of FADII solution to 5 ml of 0.1 M potassium phosphate buffer solution (pH 7.5). The 12-unit ZoilF AOD solution was prepared by diluting the FAOD derived from Benicillium.Yannnerm S-3413 obtained in Production Example 4 with a 0.1 M potassium phosphate buffer solution (pH 7.5) to 12 units / ml. Prepared. Fructosyl valine was added to the reaction solution, and the current value was measured at 30 ° C. and a constant voltage of 60 OmV. Figure 16 shows the relationship between the concentration of fructosyl valine obtained by this method and the value of the compress. The vertical axis in the figure indicates the current value (corresponding to the amount of hydrogen peroxide) at a constant voltage of 60 OmV, and the horizontal axis indicates fructosylvaline Ban degree. The figure shows that fructosyl valine and hydrogen peroxide generation are correlated.

Example 17 Fructosyl lysine measurement using FAOD-immobilized enzyme

20 ml of a 3% sodium alginate aqueous solution was added to 10 ml of the 10.3 unit / ml FAOD solution. This mixture was placed in a syringe and dropped at a constant rate into a 0.05 M calcium chloride solution (pH 6 to 8) kept at 37 to obtain a bead. The beads were cured to obtain an immobilized enzyme. 10.3 Unitol mlFAOD solution was obtained from Fusarium oxasporum S- The 800 derived from 14 was diluted with 0.1 M Tris-hydrochloric acid buffer (PH8.0) so as to have a volume of 10.3 nit Zml, and made into a net.

 Using this immobilized yeast cord, the fructosyl lysine concentration was measured by the following method and the reaction solution

 0.1M Tris-HCl dimouth solution (pH 8.0) 300 1

45nM 4-aminoantipyrine solution 30〃16 OmM N-ethyl-N- (2-hydroxy

 3-sulfobrovir) 1-in-toluidine solution 30/1 160 units Zml peroxidase solution 30〃 1

FAOD immobilized enzyme 4 Omg All in distilled water :! To 1 ml.

 Fructosyl lysine was added to the reaction solution, and the absorbance at 555 nm was measured 30 and 3 minutes later. Fig. 17 shows the relationship between fructosyl lysine concentration and absorbance obtained by this method. The vertical axis in the figure indicates the absorbance at 555 nro (corresponding to the amount of hydrogen peroxide), and the horizontal axis indicates the fructosyl lysine concentration. The figure shows that there is a correlation between the concentration of fructosyl lysine and the hydrogen peroxide generating children.

Example 18 Kit for measuring saccharified albumin

Coloring reagent

 A: 135 mol 4 -amino antivirin / 30 ml

 B: 180 ^ mol N—Ethyru N— (2-Hydroquine 1-3—Sulfobu mouth building) 1 m—Toluidine / 40 ml

 C: 50 units FAOD + 180 units Peroxidase 3

Oral 0.1 M Tris monohydrochloride buffer (pH 8.0) Processing reagent

 D: 75 mg protease X I VZSml

 For measurement, use 1 ml of a mixed solution of 501 D solution and ABC for 50 X 1 of human blood sample.

Example 19 Kit for measuring hemoglobin A1c

Coloring reagent

 A: 9 mol N— (carboxymethylaminocarbonyl) -1,4,4-bis (dimethylamino) biphenylamine Z 7 Oml

 B: 30 units FAOD + 180 units Peroxidaseno 30 ml 0.1 M Tris-HCl buffer (pH 8.0)

Processing reagent

 C: 250 units aminopeptidase 2.5 ml 0.1 M Tris monohydrochloride buffer (pH 8.0)

 D: 500 og tricloacetic acid Z5nil

 Ε: 1 Ommo 1 NaOH / 5ml

 For the measurement, use 1 ml of a mixed solution of 25 Ai D.E solution 50 / ^ 1 and AB for human serum sample 251.

Claims

The scope of the claims

 1. A method for measuring an Amadori compound in a Amadori compound-containing sample, wherein the method is performed by using an enzyme.

 2. The method according to claim 1, wherein the sample is a biological component or a food.

 3. The method according to claim 1, wherein the method is performed by measuring a saccharification rate of a sample.

 4. The method according to claim 1 or 2, wherein the determination is performed by determining fructosylamine in the sample.

 5. The method according to claim 1, wherein the method is performed by measuring a saccharified substance concentration in a sample.

 6. The method according to any one of claims 1 to 5, wherein the enzyme is fructosyl amino acid kindase.

 7. The method according to claim 6, wherein the fructosylamino acid oxidase is obtained by culturing the fungus in a medium containing fructosyl lysine and / or fructosyl 1 ^ '-Z-lysine.

 8. After or while treating the sample, fructosylamino acid oxidase acts on the sample so that the saccharification site of the Amadori compound in the sample reacts easily with fructosylamino acid kinase, and is then added to the reaction mixture. The method according to any one of claims 6 to 7, wherein the amount of oxygen consumed or the amount of a product is measured.

 9. The method according to claim 8, wherein the method of treating the sample is a brothase treatment and / or a chemical or physical treatment.

 10. The method of claim 9 wherein the protease is an exo-type protease and a no- or end-type protease.

1 1. Chemical and physical treatments include acids, alkalis, surfactants, denaturants, heat, 10. The method according to claim 9, wherein the treatment is performed by microwave and pressure.

 12. After treating the sample with an exo-type protease, or while treating the sample, the sample is treated with fructosyl amino acid oxidase to measure the amount of oxygen-depleted S or the amount of a reaction product. Method for analyzing an Amadori compound having a saccharification site at the terminal of a peptide chain.

 13. The method according to claim 12, wherein the Amadori compound is hemoglobin Ale.

14. After treating the sample with endo-type protease, or while treating the sample, fructosylamino acid oxidase is acted on to determine the oxygen consumption or S of the reaction product. Method for analyzing Amadori compounds having a saccharification site inside the peptide chain in the inside.

 15. The method according to claim 14, wherein the Amadori compound is saccharified albumin.

16. The method according to any one of claims 1 to 15, wherein, when hemoglobin is contained in the sample, the Amadori compound is measured by an absorbance at 600 to 800 nm.

 17. The method according to claim 16, wherein a reagent that is oxidized with perokinidase to generate a dye is acted on the reaction mixture, and the amount of hydrogen peroxide generated in the reaction mixture is measured.

18. The reagents are N- (carboxymethylaminocarbonyl) 1,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -13.7-bis (dimethylamino) phenothiazine ( DA67), 4-Aminoantipyrine (4AA) ZN-ethyl-N- (2-hydroxy-3-3-sulfopropyl) -1,3,5-dimethylaniline (MAO S), 4-aminoantipyrine (4AA) 18. The method according to claim 17, wherein the method is selected from the group consisting of: -ethyl-N-sulfoprovir-3.5-dimethylaniline (MAPS).

1 9. The method according to claim 16, wherein catalase is acted on the reaction mixture, and the amount of aldehyde generated is measured.

20. The method according to any one of claims 1 to 15, wherein the Amadori compound is measured electrochemically.

 21. The method according to claim 20, characterized in that the amount of acid purple consumed by the action of fructosyl amino acid oxidase is measured at the south pole.

 2 2. The method according to claim 20, wherein hydrogen peroxide a produced by the action of fructosyl amino acid oxidase is measured at the positive electrode.

23. The method according to claim 20, wherein at least one or more electron carriers are interposed between the fructosylamino acid oxidase and the electrode, and the resulting oxidation, primary current, or the amount of electricity is measured. the method of.

 2 4. With at least one or more electron carriers between hydrogen peroxide and the electrode generated by the action of fructosyl amino acid oxidase, measure the resulting oxidation, primary current, or its charge. Claims characterized by the following:

20. The method of claim 20.

 25. Measurement of the Amadori compound in the sample, which includes fructosylamino acid okinidase and a reagent for treating the sample so that the saccharification site of the Amadori compound in the sample is easily reacted with the fructosylamino acid okinidase. Reagent or kit for determination.

 26. The reagent or kit according to claim 25, which is used for measuring the amount and Z or saccharification rate of glycated protein in a biological component, or for determining fructosylamine.