CN1161374A

CN1161374A - Fructosyl amino acid oxidase and process for producing same

Info

Publication number: CN1161374A
Application number: CN 96107304
Authority: CN
Inventors: 加藤畅夫; 阪井康能; 谷吉树; 酒井敏克; 石丸香
Original assignee: Kyoto Daiichi Kagaku KK
Current assignee: Arkray Inc
Priority date: 1996-04-03
Filing date: 1996-04-03
Publication date: 1997-10-08

Abstract

This invention provides a fructosyl amino acid oxidase derived from genus fusarium, which is active on both fructosyl lysine and fructosyl valine, and also the process for producing the enzyme, an assay of an amadori compound using the enzyme, a reagnet or a kit containign the enzyme.

Description

Fructosyl amino acid oxidase and process for producing the same

The present invention relates to a novel fructosamino oxidase. In particular, it relates to a novel fructosyl amino acid oxidase derived from fusarium, a method for producing the enzyme, and a method for assaying amadori (amadori) compounds using the enzyme and a reagent or kit containing the enzyme.

When active substances such as proteins, peptides and amino acids with one (or more) amino group(s) are present together with a reducing sugar such as aldose containing one (or more) aldehyde group(s), the two undergo non-enzymatically irreversible binding via the amino and aldehyde groups, followed by amadori rearrangement to form an amadori compound. The production rate of the amadori compound is a function of reactant concentration, contact time, temperature, etc., and a lot of useful information about the sample containing the active substance(s) can be obtained from the amount of the amadori compound. Examples of a substance containing a dammara sinus compound include foods such as soy sauce and body fluids such as blood.

In a living body, fructosamine is produced by the reaction of glucose with an amino acid, and the resulting glycated derivatives of hemoglobin, albumin and protein in blood are referred to as glycohemoglobin, glycoalbumin and fructosamine, respectively. Since the concentration of these glycated derivatives in blood reflects the average level of blood glucose over a specific period of time, it can be an important indicator for the diagnosis and control of diabetes. Therefore, it is very clinically useful to establish a method for measuring the amadori compound in blood.

In addition, the preservation conditions and time after production of the food product can be determined according to the amount of the amadori compound in the food product. Thus, the method for measuring the amadori compound also contributes to the quality control of foods.

In this regard, the assay for amadori compounds is very useful in many fields including pharmaceuticals and foods.

Known methods for assaying amadori compounds are: high performance liquid chromatography [ chromatogr.sci.10: 659(1979)]boric acid is applied to a chromatography column packed with solid matter [ clin. chem.28: 2088-2094(1982)], electrophoresis [ clin. chem.26: 1598-: 620(1993), j.clin.lab.inst.reading.16: 33-37(1993), a method for determining the amount of fructosamine [ clin. 87-95(1982), colorimetric determination after oxidation with thiobarbituric acid [ Clin. 197-204(1981), and so forth. However, these prior methods require expensive equipment, lack the necessary accuracy and are not rapid enough.

Currently, methods utilizing enzyme catalysis are widely used in the fields of clinical assays and food analysis because substances to be assayed can be accurately and rapidly selected for analysis based on the characteristics of the enzyme (specificity in terms of substrate, reaction, structure, active site, etc.).

There have been proposed measurement methods involving a reaction of an oxidoreductase with amadori compounds, measuring oxygen consumption or hydrogen peroxide production as an index of the amount of amadori compounds (e.g., Japanese patent specifications (kokoku) Nos. 5-33997 and 6-65300, and Japanese laid-open patent specifications No. 2-195900, 3-155780, 4-4874, 5-192193 and 6-46846). Further, the determination of glycated proteins for the diagnosis of diabetes has also been disclosed (Japanese laid-open patent publication Nos. 2-195899, 2-195900, 5-192193 and 6-46846).

The decomposition reaction of a damascene compound catalyzed by a redox enzyme can be represented by the following reaction formula: wherein R is¹Is an aldose residue, R²Is an amino acid, protein or peptide residue.

Examples of enzymes catalyzing the above reaction are as follows:

1. a fructosyl amino acid oxidase derived from Corynebacterium (Japanese patent specifications 5-33997 and 6-65300) or Aspergillus (Japanese laid-open patent specification 3-155780);

2. fructosamine deglycosidase derived from Candida (Japanese laid-open patent Specification No. 6-46846);

3. a fructosyl amino acid-desugarizing enzyme derived from Penicillium (Japanese laid-open patent publication No. 4-4874);

4. ketoamine oxidase derived from Corynebacterium, Fusarium, Acremonium or Dekkera (Japanese laid-open patent Specification No. 5-192193)]; and

5. an alkyllysine enzyme which can be prepared according to the method described in J.biol.chem., Vol.239, pp.3790-3796 (1964).

However, assays involving these prior enzymes have several drawbacks.

For example, glycated protein in blood, which serves as an index for diagnosis of diabetes, is glycated albumin, glycated hemoglobin and fructosamine glucose binds to a lysine residue at the epsilon-position of the protein molecule to produce glycated albumin [ J.biol.chem., 261: 13542 13545(1986)]. if glycated hemoglobin is produced, glucose also binds to the N-terminal valine of β -chain [ J.biol.chem.254: 3892-3898(1979)]. therefore, glycated protein must be assayed as an effective index for diagnosis of diseases using an enzyme highly specific for fructosyl valine and fructosyl lysine, but the existing enzyme derived from Corynebacterium does not act on fructosyl lysine, as for the enzyme derived from Aspergillus, it does not significantly act on glycated protein and its hydrolysate.although the ketoamine oxidase described in Japanese laid-open patent publication No. 5-192193 acts on fructosyl valine, it cannot accurately assay lysine residue because glycated protein binding to sugar is not reacted with fructosyl lysine, fructose, and fructose is not reacted with specific for fructosyl lysine residue, and fructose, it is not reliably determined because of fructose residue due to the fact that fructose-lysine residue is not reacted with fructose, fructose-lysine residue, fructose-lysine-dehydrogenase, fructose-lysine-and fructose-lysine-dehydrogenase cannot be determined accurately.

As described above, the existing enzymes do not provide a necessary accurate measurement method for glycated proteins, and therefore, there is a need for the development of an enzyme having high specificity for fructosyl lysine and fructosyl valine.

In general, in order to improve the accuracy and practicality of an assay method comprising an enzymatic step, it is necessary to use an enzyme having catalytic activity suitable for the assay purpose. Therefore, it is necessary to select an appropriate enzyme for accurate measurement and reproducible production in consideration of many factors such as the substance to be measured, the enzyme substrate, the condition of the sample, the measurement conditions, and the like. In order to select a suitable enzyme, a number of enzymes must first be obtained and analyzed for their activity, substrate specificity, temperature stability, pH stability, etc. Therefore, it is necessary to develop more fructosyl amino acid oxidases and to perform the same analysis of their properties.

The present inventors have conducted extensive studies to provide a novel fructosyl amino acid oxidase specific to amadori compounds, particularly glycated proteins, and have found that fructosyl lysine and/or fructose N can be produced by reacting^αThe desired enzyme is obtained by culturing the strain of Fusarium in the presence of-Z-lysine.

Thus, the present invention provides a novel fructosyl amino acid oxidase active on both fructosyl lysine and fructosyl valine by incorporating fructosyl lysine and/or fructose N^α-Z-lysine in a medium, wherein the Fusarium strain produces a fructosyl amino acid oxidase.

In the present invention, a culture medium containing fructosyl lysine may contain fructosyl lysine and/or fructose N^α-Z-lysine for use in the cultivation of a microorganism capable of producing the fructosyl amino acid oxidase of the invention. Fructosyl lysine and/or fructose N^α-Z-lysine(hereinafter abbreviated as "FZL") by glucose with lysine and/or N^α-Z-lysine is obtained by treatment in an autoclave at 100-150 ℃ for 3-60 minutes. As described below, the fructosaminyl group of the present inventionThe enzyme oxidases have unexpected specificity for fructosyllysines including FZL and fructosylvaline, wherein the activity for the former is equal to or greater than the activity for the latter. In the present specification, the term "fructosyl amino acid oxidase" is simply referred to as "FAOD".

The enzyme of the present invention can be produced by culturing a strain of fusarium capable of producing FAOD in a medium containing fructosyl lysine and/or FZL.

Examples of fusarium strains include: fusarium oxysporum Linne (IFONO.5880), Fusarium oxysporum Kuehne (IFO NO.4668), Fusarium oxysporum Kuehne (IFO NO.4471), Fusarium oxysporum Cucumidis (IFO NO.6384), Fusarium oxysporum Mali (IFO NO.7706), Fusarium oxysporum Queensis (9964), Fusarium oxysporum Franch (IFO NO.9971) and Fusarium oxysporum Kuntze (IFONO.31180).

The FAOD of the present invention generally has the following physicochemical properties:

1) capable of catalyzing the oxidation of amadori compounds in the presence of oxygen to produce α -ketoaldehyde, amine derivatives and hydrogen peroxide;

2) is stable at a pH of about 4.0-13.0, preferably at a pH of about 8.5;

3) can be kept stable at a temperature of about 20-50 deg.C, preferably 30-35 deg.C; and

4) the molecular weight was estimated to be about 106,000(106kDa) by gel filtration over Superdex 200 pg.

Mixing 0.01-50% (W/W) glucose with 0.01-20% (W/W) lysine and/or N^αThe solution of-Z-lysine can be obtained by treating in an autoclave at 100-150 ℃ for 3-60 minutesThe fructosyl lysine and/or FZL of the FAOD of the invention are prepared. In particular, the total volume of the solution is 1000ml, and the solution contains 200g of glucose and N^αA solution of 10g of-Z-lysine was treated in an autoclave at 120 ℃ for 20 minutes to prepare FZL.

Although a fructosyl lysine and/or FZL-containing medium (which may be referred to as FZL-medium) can be obtained by adding the fructosyl lysine and/or FZL prepared by the above-mentioned method to any one of the conventional media, it is more convenient to prepare the fructosyl lysine and/or FZL-containing medium by adding the fructosyl lysine and/or FZL-containing medium to a medium containing 0.01 to 50% (W/W) of glucose and 0.01 to 20% (W/W) of lysine and/or N^α-Z-lysine, 0.1% (W/W) K₂HPO₄，0.1％(W/W)NaH₂PO₄，0.05％(W/W)MgSO₄·7H₂O，0.01％(W/W)CaCl₂·2H₂The mixture of O and 0.2% (W/W) yeast extract (preferably pH 5.6-6.0) was treated in an autoclave at 100-150 ℃ for 3-60 minutes.

The medium used for preparing the FAOD of the present invention may be a synthetic or natural medium commonly used in the art, which contains a carbon source, a nitrogen source, inorganic substances, and other nutrients. Examples of the carbon source include glucose, xylose, glycerol and the like, and examples of the nitrogen source include peptone, casein hydrolysate, yeast extract and the like; examples of the inorganic substances include sodium, potassium, calcium, manganese, magnesium, cobalt and the like which are generally contained in a medium.

When a microorganism is cultured in a medium containing fructosyl lysine and/or FZL, the FAOD of the present invention is induced to a large extent. Preferred media are fructosyl-lysine-and/or FZL-containing media (1.0% glucose, 0.5% fructosyl-lysine and/or FZL,1.0％K₂HPO₄，0.1％NaH₂PO₄，0.05％MgSO₄·7H₂O，0.01％CaCl₂·2H₂a mixture of O and 0.01% vitamins) wherein fructosyl lysine and/or FZL is the sole source of nitrogen and glucose is the source of carbon.

A particularly preferred medium is a medium containing 20g (2%) glucose, 10g (1%) fructosyllysine and/or FZL, 1.0g (0.1%) K in a total volume of 1000ml₂HPO₄，1.0(0.1％)NaH₂PO₄，0.5g(0.05％)MgSO₄·7H₂O，0.1g(0.01％)CaCl₂·2H₂O and 2.0g (0.2%) of yeast extract (pH 5.6-6.0).

The culture medium containing fructose lysine and/or FZL can be prepared by adding fructose lysine and/or FZL to any of the conventional culture media, or by adding glucose and lysine and/or N^αThe medium of-Z-lysine is prepared by treatment in an autoclave. The medium prepared by either method is brown due to the presence of fructosyllysine and/or FZL and is therefore referred to as "FZL brown medium or GL (glycated lysine and/or glycated N)^α-Z-lysine) brown medium ".

The culture is usually carried out at a temperature of 25 to 37 ℃ and preferably 28 ℃ in a medium having a pH of 4.0 to 8.0 (preferably pH of 5.5 to 6.0), but the conditions of the culture may vary depending on various factors such as the condition of the microorganism and the like, and are not limited to the conditions as described above. For example, Fusarium flax strain, when cultured under these conditions for 20-100 hours, preferably 80 hours, has FAOD accumulated in the medium (see FIG. 1).

The resulting culture medium is then treated by a conventional method to remove nucleic acids, cell wall fragments and the like, to obtain an enzyme preparation.

Since the enzyme activity of the FAOD of the present invention is usually accumulated in the cells of bacteria/molds, the cells in the culture medium are collected and ground to extract the enzyme.

The cells may be milled using conventional methods, such as mechanical milling, autohydrolysis with a solvent, freezing, sonication, autoclaving and the like.

A method for isolating and purifying an enzyme is known in the art and can be carried out in combination with known methods such as salting out with ammonium sulfate, precipitation with an organic solvent such as ethanol or the like, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography or the like.

For example, the mycelia are collected by centrifugation or suction filtration of the resulting culture, washed and suspended in 0.1M Tris-HCl buffer (pH8.5), ground with Dino-Mill and centrifuged. The supernatant as extracellular extract was then fractionated with ammonium sulfate and purified by benzene-agarose hydrophobic column chromatography.

In the present invention, the FAOD includes any enzyme-containing substance and solution obtained from all the purification steps, regardless of the purity of the enzyme, and it also includes the medium as long as these substances and solution have the effect of catalyzing the oxidation of the amadori compound as described above.

Furthermore, any FAOD fragment having FAOD activity or associated with catalytic activity of FAOD enzyme is within the scope of the present invention, as such fragments may also be used in the present invention.

The FAOD thus obtained can be used for the assay of a mesangial compound, particularly a glycated protein in the diagnosis of diabetes.

Thus, the present invention provides a method for producing a FAOD, which comprises culturing a mold strain in a medium containing a glycated amino acid and/or a glycated protein which are selectively protected.

The invention also provides a method for producing FAOD, which comprises adding fructose-containing lysine and/or fructose N^αCulturing a strain of Fusarium capable of producing FAOD in a medium containing Z-lysine, and recovering the FAOD from the culture medium.

All FAODs produced by Fusarium strains in the present invention can be used to solve the problems addressed by the present invention. For temporary needs, the FAOD produced by a fusarium oxysporum strain in the specification is referred to as FAOD-L.

The FAOD of the present invention has the following characteristics:

1. general Induction Properties

The FAOD of the present invention is an inducible enzyme induced by fructosyl lysine and/or FZL, and can be produced by culturing a strain of Fusarium capable of producing FAOD in a medium containing fructosyl lysine and/or FZL as a nitrogen source and glucose as a carbon source. FAOD can be induced in a GL brown medium by mixing glucose with lysine and/or N^α-Z-lysine is obtained by treatment in an autoclave, but not comprising glucose and lysine and/or N, which have been autoclaved separately^α-Z-lysine medium. This indicates that the enzyme is specific for amadori compounds.

2. Reaction specificity and enzyme substrate specificity

The FAOD of the present invention has a catalytic action on the reaction represented by the following formulaThe following steps are used: wherein R is¹Is an aldose residue, R²Is an amino acid, protein or peptide residue.

In the above reaction scheme, the general formula R¹-CO-CH₂-NH-R²Amarum sinus of the formula (I), wherein R¹is-OH, - (CH)₂)_n-or- [ CH (OH)]_n-CH₂OH (n is an integer of 0 to 6), R²is-CHR³-[CONHR³]_mCOOH(R³Is a side chain residue of α -amino acid, m is an integer of 1-480) is a preferred substrate³For an amino acid side chain residue selected from lysine, polylysine, valine, aspartic acid, etc., a compound in which n is 5 to 6 and m.ltoreq.55 is a more preferred substrate.

The substrate specificity of the FAOD of the present invention is shown in table 1.

TABLE 1

Specificity of enzyme substrates for purified FAOD-L

Enzyme substrate concentration specific Activity (%)

N^e-fructose N^α-Z-lysine 1.67mM 100

Fructose valine 1.6730.1

N^e-methyl-l-lysine 1.67 n.d.^*)

N^e-fructose poly-l-lysine 0.02% 0.24

Poly-l-lysine 0.02 n.d.

FBSA^*20.17 N.D.

FHSA^*30.17 N.D.

FBSA 0.170.62 with trypsin

FHSA 0.17 N.D with trypsin.

1): not detected

2): fructose bovine serum albumin

3): fructose human serum albumin

As is evident from table 1: the FAOD of the invention has high specificity to both FZL and fructosyl valine. FAOD has activity on the protease hydrolysates of fructosyl polylysine and glycated proteins.

Examples of FAOD producing fusarium strains are shown in table 2.

TABLE 2

Enzyme substrate pairs were purified from fusarium strains grown on FZL brown medium

Specificity of the degraded FAOD

Specific Activity (10)^-2Mu/mg protein) IFONO, Fusarium oxysporum strain

Fructose N^α-Z-lysineFructose valine L/V¹⁾4468 sweet potato Strain 3.760.22816.54471 melon Strain 3.370.3409.95880 flax Strain 48.613.53.66384 cucumber Strain 2.260.2349.77706 apple Strain 4.860.27617.69964 celery Strain 2.650.16915.79971 pine Strain 1.920.13813.931180 strawberry Strain 25.22.2711.1

1): activity on FZL/Activity on fructosyl valine

As is evident from table 2: the FAOD of the present invention has activity on both fructosyl lysine and fructosyl valine, which indicates that the FAOD can also be used for analysis of glycated hemoglobin.

pH and temperature conditions

Measurement of pH conditions:

the optimum pH value is determined by performing the enzyme reaction in a conventional reaction system for determining the FAOD activity (as shown in "4-titer evaluation" below) by replacing the conventional buffer with various buffers having pH values of 4 to 13 such as 0.1M potassium phosphate buffer (KPB), tris hydrochloride buffer, glycine (Gly) -NaOH buffer, etc.

The pH stability was determined by measuring the activity of FAOD after adding FAOD to the above-mentioned various buffers and incubating at 30 ℃ for 10 minutes under usual conditions (30 ℃, pH 8.0).

Measurement of temperature conditions

The optimum temperature was determined by conducting the reaction at various temperatures of 20-60 ℃ and measuring the enzyme activity. The temperature stability was measured on the basis of the maintenance of activity under ordinary conditions in an enzyme solution prepared by dissolving FAOD in 0.1M Tris-HCl buffer (pH8.0) and incubating at various temperatures ranging from 20 to 60 ℃ for 10 minutes.

The FAOD of the present invention was evaluated according to these methods and it was stable in the range of pH 4.0 to 13.0, preferably pH 7.5 to 9.0, more preferably pH8.5 (see FIG. 2).

The enzymatic reaction can be carried out efficiently at 20-50 deg.C, preferably at 25-40 deg.C, more preferably at 35 deg.C (see figure 3). The FAOD of the present invention is stable at a temperature range of 20 to 50 ℃.

4. Evaluation of Titers

The titration was carried out as follows:

(1) method for the colorimetric determination of hydrogen peroxide produced

A. Measuring the rate of production

The FZL that had been obtained was dissolved in distilled water to prepare a 100mMFZL solution. To 100. mu.l of 45mM 4-aminoantipyrine, 100. mu.l of peroxidase (60U/ml), 100. mu.l of 60mM phenol, 1ml ofA mixture of 0.1M Tris-HCl buffer (pH8.0) and 50. mu.l of an enzyme solution was added with distilled water to give a total volume of 3.0 ml. The solution was incubated at 30 ℃ for 2 minutes. The length of time of absorbance at 505nm was measured after the addition of 50. mu.l of 100mM FZL solution. According to the molar absorptivity (5.16X 10) of the quinone pigment produced³m^-1cm^-1) The amount of hydrogen peroxide produced per minute (. mu. mol) was calculated and the resulting value was taken as one unit (U) of the enzyme activity.

B. Endpoint method

A solution was prepared and an enzyme substrate solution was added thereto in the same manner as in the above-mentioned method A. The absorbance at 505nm was measured after incubation at 30 ℃ for 30 minutes. The enzyme activity was evaluated based on the amount of hydrogen peroxide produced, with reference to a calibration curve obtained with a standard hydrogen peroxide solution.

(2) Method for determining oxygen uptake of enzymatic reactions

Distilled water was added to a mixture of 1ml of 0.1M Tris-HCl buffer (pH8.0) and 50. mu.l of an enzyme solution to give a total volume of 3.0 ml. The resulting solution was energized in an oxygen cell manufactured by Lank brother. The solution was stirred at 30 ℃ to equilibrate the oxygen dissolved therein at this temperature and 100. mu.500 mM FZL was added to the solution. The oxygen absorbance was then measured continuously with a recorder to obtain the initial rate. The oxygen uptake in one minute was determined according to a standard curve and was regarded as one enzyme unit.

5. Inhibition, activation and stabilization of enzymes

(1) Influence of Metal

To an enzyme solution, which is a 0.1M Tris-HCl buffer (pH8.0) containing a metal ion to be measured at a final concentration of 1mM, was added a solution, and after incubation at 30 ℃ for 5 minutes, the enzyme activity was evaluated, and the results are shown in Table 3 below.

TABLE 3

Effect of Metal ions on FAOD-L Activity Metal (1mM) -specific Activity

(％) (％)

No 100 FeSO₄93

LiCl 99 CoSO₄110

KCl 104 CuCl₂35

NaCl 109 ZnSO₄11

RbCl 109 AgNO ₃0

CsCl 108 BaCl₂116

MgCl₂103 HgCl ₂0

CaCl₂98 FeCl₃77

MnCl₂143

As is evident from table 3: the activity of FAOD of the present invention is slightly inhibited by copper and zinc ions, and is completely inhibited by silver and mercury ions.

(2) Effect of multiple inhibitors

The inhibitory effects of various substances were determined in a manner substantially similar to that in (1) above. The final concentration of PCMB (Mercury p-chlorobenzoate) was 0.1mM, and the other final concentrations were 1 mM. The results are shown in table 4. The stabilization was determined by dialyzing the purified FAOD against 50mM Tris-HCl buffer (pH8.5) containing 2mM Dithiothreitol (DTT) overnight and measuring the activity of the enzyme.

TABLE 4

Effect of multiple inhibitors on FAOD Activity reagent (1mM) specific reagent (1mM)

Activity (%)

100-free semicarbazide 96

PCMB

^*10 phenylhydrazine 49

DTNB

^*20 hydrazine 10

Iodoacetic acid 90 hydroxylamine 17

NaN₃102 Clorgyline N.D^*3

α' -O-bipyridine 103 n, α -dimethyl-n-2-propynyl phenethylamine 104

o-phenanthroline 114 aminoguanidine 73

*1: PCMB, Mercury p-chlorobenzoate

*2: DTNB, 5, 5' -dithiobis (2-nitrobenzoic acid)

*3: not detected

As is evident from table 4: the activity of FAOD was strongly inhibited by PCMB, DTNB, hydrazine and phenylhydrazine, indicating that SH and/or carbonyl groups play an important role in the enzymatic reaction.

The enzyme is stabilized with dithiothreitol DTT, and 50mM Tris-HCl buffer (pH8.5) containing 2mM DTT is a preferred solvent in the present invention.

6. Molecular weight

The determination of the molecular weight was determined by SDS-PAGE (sodium dodecyl sulphate Polyacrylamide gel electrophoresis) and gel filtration on Superdex 200 pg.

SDS-PAGE was performed at 40mA for three hours using a 10% gel according to the Davis method, and the protein was stained with Coomassie Brilliant blue G-250. A calibration curve is obtained by performing electrophoresis on several standard known proteins such as phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase and soybean trypsin inhibitor in the same manner, and the molecular weight is determined based on the calibration curve. As a result, the molecular weight of one subunit was about 51,000(51 kDa). (see attached FIG. 4)

Gel filtration over Suprerdex 200pg showed a molecular weight of about 106,000(106KDa) (see fig. 5), indicating that the FAOD of the present invention is a dimer.

7. Isoelectric point

The isoelectric Point (PI) of the FAOD was 6.8 as measured by disc-focus electrophoresis.

8. Control with known enzymes

The FaOD of the present invention was compared with known fructosyl amino acid oxidases derived from various microorganisms.

TABLE 5

Control with various fructosyl amino acid oxidases derived from microorganisms

Microorganism fusarium flax corynebacterium¹⁾Aspergillus sp²⁾

Sporotrichum (IF05880)

Molecular weight (gel filtration 106,00088,00083,000 (SDS-PAGE) 51,00044,00043,000

Specificity of coenzyme covalently bound FAD non-covalently bound FAD enzyme substrate (U/mg protein) (fructosyl lysine) 18.5³⁾N.D.⁴⁾11.28⁴⁾(fructose valine) 6.837.0959.8 Melten's constant 0.37mM (FZL) 0.74mM (Gly) 2.2mM (Gly)

Optimum pH 8.58.37.7 and optimum temperature (DEG C) of 30-354040

Inactivation of isoelectric point 6.84.66.8 with SH reagent inactivation non-inactivated 1): t.horiuchi et al, Agric.biol.chem., 53(1), 103-110(1989)

2)：T.Horiuchi et al.，Agric.Biol.Chem.，53(2)333-338(1991)

3): for fructose N^αSpecific Activity of-Z-lysine

4): to N^e-D-fructose N^αSpecific Activity of formyl lysine

As is apparent from Table 5, the FAOD of the present invention has the following differences from other enzymes derived from two strains.

(1) Molecular weight: the molecular weight of the FAOD of the present invention is significantly greater than the molecular weight of the other two enzymes.

(2) Coenzyme: the coenzymes of the FAODs of the present invention are covalently bound FADs, while coenzymes of other enzymes are non-covalently bound FADs.

(3) Specificity of the substrate: the FAOD of the present invention has a higher specificity for fructosyl lysine than for fructosyl valine, whereas the enzyme derived from Corynebacterium has no effect on fructosyl lysine, and the enzyme derived from Aspergillus has less effect on fructosyl lysine than for fructosyl valine.

(4) The Michaelis constant: the difference in the Michaelis constant indicates that the FAOD of the present invention has a greater affinity for the fructosyl lysine substrate than other enzymes.

(5) Optimal pH, optimal temperature, isoelectric point and inhibition thereof by SH reagent: the data show that the FAOD of the present invention is significantly different from other enzymes.

As described above, the FAOD of the present invention can be used in assays for amadori compounds.

Accordingly, the present invention provides a method for determining the amount of a damascene compound in a sample, the method comprising contacting a sample containing a damascene compound with a FAOD of the present invention and determining the amount of oxygen consumed or the amount of hydrogen perchlorate produced. The present invention is based on the measurement of the glycated protein amount and/or the glycation degree, or based on the measurement of fructosamine in a sample derived from a living body.

The enzyme activity of FAOD is represented by the following reaction formula: wherein Ris¹Is an aldose residue, R²Is an amino acid, protein or peptide residue.

As the sample solution to be tested, any solution containing one (or more) amadori compounds may be used, for example, a solution derived from food such as soy sauce. And derived from living organisms such as blood (e.g., whole blood, plasma or serum), urine, and the like.

The FAOD of the present invention is reacted with a sample containing a damascene compound in a suitable buffer. Although the appropriate pH and temperature of the reaction mixture vary depending on the enzyme used and the sample to be tested, they have a range as described above, that is, pH of 4.0 to 13.0, preferably 8.5, and temperature of 20 to 50 ℃, preferably 35 ℃. As the buffer, Tris-HCl buffer may be used.

The amount of FAOD to be used for the measurement is usually 0.1 unit/ml or more, and if the end-point method is used, an amount of 1 to 100 units/ml is a preferable amount.

The present invention may be used to assay for amadori compounds by any of the known methods as shown below.

(1) Determination of the amount of hydrogen peroxide produced

The amount of the amadori compound in the sample can be estimated from the amount of hydrogen peroxide generated according to a hydrogen peroxide measuring method such as a colorimetric method or a method using a hydrogen peroxide electrode. The amount of amadori compound in the sample is calculated from a calibration curve relating the amount of hydrogen peroxide to the amount of amadori compound. Specifically, the calculation was carried out by a method similar to that described in "evaluation of titration in Table 4", which is similar except that the amount of FAOD was 1 unit/ml and the sample to be tested was diluted before the amount of hydrogen peroxide produced was measured.

As the system for developing hydrogen peroxide, any system can be used which develops color by oxidative condensation of a chromogen such as phenol with a coupler such as 4-antipyrine, 3-methyl-2-benzothiazolinone in the presence of peroxidase. The chromogens are phenol derivatives, aniline derivatives and toluidine derivatives, such as 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 and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline. Also employable is a colorless type color developing agent which develops color by oxidation in the presence of peroxidase, which are known in the art, and examples thereof include: o-dianisidine, o-tolidine, 3, 3-diaminobenzidine, 3, 3, 5, 5-tetramethylbenzidine, N- (carboxymethylaminocarbonyl) -4, 4-bis (dimethylamino) -dianiline, 10- (carboxymethylaminocarbonyl) -3, 7-bis (dimethylamino) phenothiazine, and the like.

(2) According to oxygen consumption determination

The amount of the amadori compound in the sample was estimated by calculating the oxygen consumption amount by subtracting the oxygen amount at the completion of the reaction from the oxygen amount at the start of the reaction using a calibration curve regarding the relationship between the oxygen consumption amount and the amount of the amadori compound. In particular, it can be determined by a titration method similar to that described in "4. titer evaluation" above, except that the amount of FAOD is 1 unit/ml and that the sample to be added is appropriately diluted before the oxygen consumption is determined.

According to the assay method of the present invention, the method can also be applied to a sample solution, but it is preferable to subject the sampleto pretreatment so as to dilute the sugar-linked lysine and/or valine residues in the glycated protein.

For such purposes, the sample is treated with a protease (enzymatic method) or a chemical (chemical method) such as hydrochloric acid, enzymatic method being the preferred method. In this case, both endo-and exo-proteases known in the art can be used in the method of the invention. Examples of endo-proteases include: trypsin, 2-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B, pepsin, thermolysin, protease XIV, lysine endopeptidase, proleser and bromelain F. Examples of exo proteases include: aminopeptidases and carboxypeptidases. Methods of enzyme treatment are also known, for example, trypsin treatment as described in the examples below can be performed.

As described above, the FAOD of the present invention has high specificity for fructosyl lysine in a glycoprotein. Thus, it finds application in the diagnosis and control of diabetic conditions, which involves the determination of glycoproteins in blood samples. On the other hand, the FAOD of the present invention has specificity for fructosyl valine. Therefore, the method can be used for measuring the glycosylated hemoglobin.

When blood is measured (e.g., whole blood, plasma or serum), a blood sample derived from a living body can be used in this manner or after pretreatment such as dialysis.

In addition, the enzyme (e.g., FAOD, peroxidase, etc.) used in the present invention may be used in a liquid form or may be used after being immobilized on a suitable solid support. For example, an automated apparatus for assaying glycated proteins or the like can be obtained using a column packed with an enzyme immobilized on a particle, which can improve the efficiency of conventional assays such as clinical tests in which many samples must be assayed quickly and accurately. Since thisimmobilized enzyme can be used repeatedly, it has another advantage in economic efficiency.

Furthermore, it is possible to provide a kit by combining the enzyme(s) with the chromogenic agent(s) in a suitable manner. The kit can be used for clinical detection and food analysis of the amadori compound.

Immobilization of the enzyme may be performed by methods known in the art. For example, by a carrier binding method, a crosslinking method, an inclusion method, a coordination method, and the like. Examples of carriers include polymer gels, microcapsules, agarose, alginic acid, carrageenan, and the like. The enzyme may be bound to the support by covalent bonds, ionic bonds, physical adsorption, biochemical affinity, or the like, according to methods known in the art.

Where immobilized enzymes are used, the assay may be performed in a flow or batch system. As mentioned above, this immobilized enzyme is particularly useful for routine detection (clinical testing) of glycoproteins in blood samples.

When this clinical test is used to guide the diagnosis of diabetes, the standard result for the diagnosis of diabetes is expressed by a fructosamine value, a glycoprotein concentration or a glycation degree, which is the ratio of the glycoprotein concentration to the total protein concentration in a blood sample. The total protein concentration can be determined by conventional methods, for example, by absorbance measurement at 280nm, the Bradford method, the Lory method, the ballistic method, albumin native fluorescence, or hemoglobin absorbance.

The present invention also provides a reagent or a kit for assaying a dammardraining compound, which consists of the FAOD of the present invention and a buffer solution, preferably having a pH of 7.5 to 8.5, and an optimal pH of 8.0. For immobilizing the FAOD, the solid support may be selected from polymer gels and the like, and alginic acid is preferable.

In the end-point assay, the reagent for one sample usually contains 1-100 units/ml of FAOD, and Tris-HCl (pH8.0) is used as a buffer.

When the amadori compound is measured based on the generated hydrogen peroxide, any color developing system that develops color by oxidative condensation reaction, colorless type color developer, and the like as described above "(1) measurement based on the amount of generated hydrogen peroxide" can be used.

The reagent for assaying a mesangial compound of the present invention can be combined with a suitable color-developing agent to prepare a kit for preliminary diagnosis and examination, together with a color-developing standard or standard substance.

The reagent or the kit as described above can be used for the measurement of the amount of glycoprotein and/or glycation degree or the measurement of fructosamine in a sample derived from a living body.

As is evident from the above: the FAOD of the present invention has specificity to both fructosyl lysine and fructosyl valine, wherein the specificity to the former is greater, and is different from the existing fructosyl amino acid oxidase in terms of substrate specificity and the like. Therefore, the FAOD of the present invention can be used for developing a new clinical detection method and a food analysis method. Therefore, it is very helpful to the diagnosis of diabetes and the quality control of food. In particular, it is useful for the diagnosis of diabetes, in which the amount of glycated protein and/or the glycation degree or the fructosamine value in blood can be used as an index for the diagnosis or control of the diabetic state. Now, by using a method for measuring a reagent for a mesangin compound of the present invention, it becomes possible to accuratelyand efficiently determine glycoproteins, which can facilitate diagnosis or control of a diabetic state.

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

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

FIG. 3 is a graph illustrating the relationship between temperature in a solvent and FAOD activity.

FIG. 4 shows a migration method obtained by SDS-PAGE of purified FAOD.

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

FIG. 6 shows the absorption spectrum of purified FAOD-L

FIG. 7 is a graph showing the relationship between the concentration of glycated human serum albumin as a substrate and the amount of hydrogen peroxide generated due to the FAOD effect.

FIG. 8 is a graph showing the relationship between the glycation degree of human serum albumin and the amount of hydrogen peroxide produced by the FAOD effect.

FIG. 9 is a graph showing the relationship between the concentration of glycated hemoglobin and the amount of hydrogen peroxide generated due to the FAOD effect.

The following examples further illustrate the present invention in detail, but do not limit the scope of the present invention.

Example 1 fermentation of Fusarium Linearum Strain and purification of FAOD-L

1) Fermentation of

Fusarium limanii of FusariumThe strain (IFO 5880) was cultivated in a medium containing O.5% FZL, 1.0% glucose, 0.1% K₂HPO₄，0.1％NaH₂PO₄，0.05％MgSO₄·7H₂O，0.01％CaCl₂·2H₂O and 0.2% yeast extract, and incubated at 28 ℃ for 80 hours while aeration (2 liters/min) and agitation (400 rpm) are performed with a fermenter. The culture was filtered to obtain hyphae.

2) Preparation of crude extract

A part of the mycelia (270g wet weight) was suspended in 0.1M Tris-HCl buffer (pH8.5, 800ml) containing 2mM DTT and ground with Dino-Mill, and the ground mixture was centrifuged at 9,500 rpm for 20 minutes to obtain a supernatant (cell-free extract) as a crude extract, which was then purified.

3) Purification of

The first step is as follows: ammonium sulfate fractionation

The crude extract was added to ammonium sulfate to 40% saturation, and the mixture was centrifuged at 12,000 rpm, and maintained at 4 ℃ to remove precipitates. Ammonium sulfate was added to the supernatant to 75% saturation and the precipitate was isolated.

The second step is that: hydrophobic chromatography (batch method)

The precipitate obtained from the above first step was dissolved in 50mM Tris-HCl (pH8.5) (hereinafter referred to as "buffer A") containing 2mM DTT. After the same volume of buffer A containing 40% ammonium sulfate was added, butyl-Toyopearl resin (200ml) was added to the solution of the crude extract for adsorption. Elution was carried out by the batch method with buffer A. The active fraction was concentrated with ammonium sulfate.

The third step: hydrophobic column chromatography

The concentrate obtained from the second step above was adsorbed on a phenyl-Toyopearl column equilibrated with buffer a containing 25% saturated ammonium sulfate. After washing with the same buffer, elution was carried out under a linear gradient of 25-0% saturated ammonium sulfate. The active fraction was collected and concentrated with ammonium sulfate, and the resulting concentrate was used in the next step.

The fourth step: hydrophobic chromatography (column method)

The concentrate obtained in the above third step was adsorbed on a butyl-Toyopearl column equilibrated with buffer A containing 40% saturated ammonium sulfate, and the column was washed with the same buffer. Elution was performed under a linear gradient of 40-0% saturation ammonium sulfate to obtain the active fraction.

The fifth step: ion exchange column chromatography

Then, the active fraction obtained in the fourth step above was applied to a DEAE-Toyopearl column equilibrated with buffer A. FAOD activity was detected in the wash fractions collected and concentrated with ammonium sulfate. The resulting concentrate was used in the next step.

And a sixth step: gel filtration

As a final step, gel filtration was performed using sephacryl 300 equilibrated with 0.1M Tris-HCl buffer containing 0.1M NaCl and 2mM DTT to obtain 70-100 units of an enzyme preparation.

The UV absorption spectrum of the purified enzyme is shown in FIG. 6, which shows that the enzyme is a flavoenzyme.

The molecular weight of the purified enzyme was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and gel filtration on Superdex 200 pg.

SDS-PAGE was performed according to the Davis method using 10% gels at 40mA for three hours. Proteins were stained with Coomassie Brilliant blue G-250. The molecular weight is calculated using a calibration curve obtained by electrophoresis of standard proteins such as phosphorylase B, Bovine Serum Albumin (BSA), ovalbumin, phosphoanhydrase and soybean trypsin inhibitor in the same manner. SDS-PAGE showed: the molecular weight of the subunit of the purified enzyme was about 51,000(51kDa) (see FIG. 4).

Gel filtration with 0.1M Tris-HCl buffer (pH8.5) containing 0.1M NaCl showed a molecular weight of about 106,000(106kDa), as shown in FIG. 5.

In addition, the FAOD-L prepared in example 1 showed the same numerical values and physicochemical characteristics as those related to the enzyme activity, optimum pH and temperature, stability of pH and temperature, influence of metal and inhibitor, and the like, as described above.

Example 2 measurement of glycated human Albumin amount

A set of glycated human albumin solutions with varying concentrations between 0-10% was prepared by dissolving glycated human serum albumin (Sigma) in 0.9% NaCl solution. The measurement was carried out using these solutions in the following manner.

1) Protease treatment

A mixture of glycated albumin solution (60. mu.l) and 12.5mg/ml protease XIV (Sigma) solution (60. mu.l) was incubated at 37 ℃ for 30 minutes. The mixture was then heated at about 90 ℃ for 5 minutes to terminate the reaction.

2) Activity assay

The FAOD reaction mixture was prepared from the following reagents:

30. mu.l of 45mM 4-aminoantipyrine solution

30. mu.l of a 60mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine solution

Peroxidase solution (60 units/ml) 30. mu.l

300. mu.l of 0.1M tris-HCl buffer (pH8.0)

FAOD-L solution (6 units/ml) 50. mu.l

After mixing the reagents, the total volume was adjusted to 1ml with distilled water. A FAOD-L solution (6 units/ml) was prepared by diluting the purified FAOD-L obtained in example 1 with 0.1M Tris-HCl buffer (pH 8.0). After the FAOD reaction mixture was incubated at 30 ℃ for 2 minutes, each protease-treated solution (100. mu.l) was added thereto. Then, the absorbance at 555nm after 30 minutes was measured to evaluate the relationship between the glycated albumin concentration and the absorbance. The results are shown in FIG. 7, in which the ordinate represents the absorbance at 555nm, which corresponds to the amount of hydrogen peroxide produced, and the abscissa represents the glycated albumin concentration. FIG. 7 shows: the concentration of glycated albumin is correlated with the amount of hydrogen peroxide.

Example 3 measurement of human serum Albumin glycation Rate

Glycated human serum albumin (sigmaCo.) (150mg) and human serum albumin (Sigma Co.) (150mg) were dissolved in 0.9% NaCl solution (3ml), respectively. These solutions were combined to prepare solutions of different glycation degrees, and the glycation degrees thereof were measured with a glycated albumin automatic measuring apparatus (kyoto Daiichi kagakuco. ltd.). The measurement shows that: the saccharification rate is 24.6-61.1%. The measurement was carried out using these solutions in the following manner.

1) Protease treatment

2) Activity assay

The FAOD reaction mixture was prepared from the following reagents:

30. mu.l of 45mM 4-aminoantipyrine solution

30. mu.l of a 60mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine solution

Peroxidase solution (60 units/ml) 30. mu.l

0.1M Tris-HCl buffer (pH8.0) 300. mu.l

FAOD-L solution (6 units/ml) 50. mu.l

After mixing the reagents, the total volume was adjusted to 1ml with distilled water. A FAOD-L solution (6 units/ml) was prepared by diluting the purified FAOD-L obtained in example 1 with 0.1M Tris-HCl buffer (pH 8.0).

After the FAOD reaction mixture was incubated at 30 ℃ for 2 minutes, each protease-treated solution (100. mu.l) was added thereto, and then, the absorbance thereof at 555nm after 30 minutes was measured to evaluate the relationship between the albumin glycation degree and the absorbance. The results are shown in FIG. 8, in which the ordinate represents the absorbance at 555nm, which corresponds to the amount of hydrogen peroxide produced, and the abscissa represents the glycation degree of albumin. FIG. 8 shows: the glycation degree of albumin and the amount of hydrogen peroxide are correlated with each other.

Example 4 measurement of glycated hemoglobin level

A set of 0-35% glycated hemoglobin solutions was prepared by dissolving glycated hemoglobin control (Sigma) in distilled water. These solutions were used for the measurement in the following manner.

1) Protease treatment

A mixture of a glycated hemoglobin solution (25. mu.l), a 500 unit/ml aminopeptidase solution (5. mu.l) and 0.1M Tris-HCl buffer (pH8.0) was incubated at 30 ℃ for 30 minutes, towhich 10% trichloroacetic acid (50. mu.l) was added and stirred. After the mixture was allowed to stand at 0 ℃ for 30 minutes, it was centrifuged at 12000 rpm for 10 minutes. The supernatant was neutralized with about 50. mu.l of 2M NaOH.

2) Activity assay

The FAOD reaction mixture was prepared from the following reagents:

30 μ l of 3mM N- (carboxymethylaminocarbonyl) -4, 4-bis (dimethylamino) diphenylamine solution

Peroxidase solution (60 units/ml) 30. mu.l

0.1M Tris-HCl buffer (pH8.0) 300. mu.l

FAOD-L solution (4 units/ml) 10. mu.l

After mixing the reagents, the total volume was adjusted to 1ml with distilled water. A FAOD-L solution (4 units/ml) was prepared by diluting the purified FAOD-L obtained in example 1 with 0.1M Tris-HCl buffer (pH 8.0). After the FAOD reaction mixture was incubated at 30 ℃ for 2 minutes, each protease-treated solution (80. mu.l) was added thereto. Then, the absorbance at 727nm after 30 minutes was measured to evaluate the relationship between the glycated hemoglobin concentration and the absorbance. The results are shown in FIG. 9, in which the ordinate represents the absorbance at 727nm, which corresponds to the amount of hydrogen peroxide produced, and the abscissa represents the glycated hemoglobin concentration. FIG. 9 shows that the concentration of glycated hemoglobin is correlated with the amount of hydrogen peroxide.

Claims

1. A fructosyl amino acid oxidase which is active on both fructosyl lysine and fructosyl valine and is produced by culturing a strain of Fusarium which produces the fructosyl amino acid oxidase in a medium containing the fructosyl lysine.

2. Fructosyl amino acid oxidase according to claim 1, which has an activity on fructosyl lysine which is higher than or equal to the activity on fructosyl valine.

3. The fructosyl amino acid oxidase as claimed in claim 1, wherein the fructosyl lysine-containing medium contains glucose and lysine and/or N at 100-150 ℃^αFructosyllysine and/or fructose N obtained by co-treatment of-Z-lysine in an autoclave for 3 to 60 minutes^α-Z-lysine.

4. The fructosyl amino acid oxidase of claim 1, wherein the strain of fusarium is selected from the group consisting of fusarium oxysporum linn (IFO No.5880), fusarium oxysporum carrots (IFO No.4468), fusarium cucumerinum (IFO No.4471), fusarium cucumerinum (IFO No.6384), fusarium oxysporum mali (IFO No.7706), fusarium oxysporum crispum (9964), fusarium oxysporum (IFO No.9971) and fusarium graminearum (IFO No. 31180).

5. Fructosyl amino acid oxidase according to any of claims 1 to 4, characterized by the following physicochemical properties:

1) capable of catalyzing the oxidation of amadori (amadori) compounds in the presence of oxygen to produce α -ketoaldehyde, an amine derivative, and hydrogen peroxide;

2) can be stable at a pH of about 4.0-13.0, and has an optimum pH of 8.5;

3) can be kept stable at a temperature of about 20-50 deg.C, and the optimal temperature is 30-35 deg.C; and

4) the molecular weight was estimated to be about 106,000(106kDa) by Superdex 200pg gel filtration.

6. A process for producing a fructosyl amino acid oxidase of claim 1, which comprises culturing a mold in a medium containing an optionally protected fructosyl amino acid and/or glycated protein.

7. The method of claim 6, wherein a compound capable of producing a fructosyl amino acid oxygen is addedFusarium strain containing chemozyme, fructosyl lysine and/or fructose N^α-Z-lysine, and recovering the fructosyl amino acid oxidase from the resulting culture.

8. A method for assaying an amadori compound in a sample containing the same, comprising contacting the sample with the fructosyl amino acid oxidase of claim 1 and measuring the amount of oxygen consumed or hydrogen peroxide produced.

9. The assay of claim 8 wherein the sample is derived from a living body and the assaying of the amadori compound is carried out as an assay of the amount of glycated protein and/or the glycation degree in the sample or an assay of fructosamine.

10. A reagent or kit for assaying a mesangial compound, which comprises the fructosyl amino acid oxidase of claim 1.

11. The reagent or kit according to claim 10, which is used for measuring the glycated protein amount and/or the glycation degree, or fructosamine, in a sample derived from a living body.