JP2872983B2 - Method for quantifying 1,5-anhydroglucitol and reagent for quantification - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and a reagent for quantifying 1,5-anhydroglucitol, which are useful for diagnosing diabetes and the like.

[0002]

2. Description of the Related Art 1,5-Anhydroglucitol (hereinafter referred to as "1,
5-AG ”) is present in body fluids such as human serum, plasma, and urine, and its amount in body fluids varies greatly in certain diseases, especially in diabetes. Is becoming an important inspection item.

[0003] As a method for quantifying 1,5-AG, there is a method in which pyranose oxidase or sorbose oxidase is acted on and the generated hydrogen peroxide is colorimetrically quantified using a peroxidase coloring system (Japanese Patent Publication No. 5-41238). ) Has become mainstream, and has recently been applied to general-purpose automatic analyzers.
However, since these enzymes have low substrate specificity and react strongly with saccharides such as glucose, it was necessary to completely remove or eliminate the saccharides in the sample in advance.

Japanese Patent Publication No. 3-24200 discloses a Pseudomonas sp. 1,5-AG produced from NK-85001
A method for quantifying the amount of oxygen consumed after reaction with 1,5-AG and the reaction products such as reduced forms of electron acceptors using 1,5-AG oxidase, which has high specificity, has been proposed. I have. However, the method of quantifying the amount of consumed oxygen is not suitable for multi-analyte processing, and the method of quantifying the reduced form of the electron acceptor also includes a ferricyanide compound used in the examples of the publication. When dichlorophenol indophenol or the like is used as an electron acceptor, the reductant itself is reversible and unstable, and cannot be said to be sufficient in terms of sensitivity, and is not suitable for practical applications.

When quantification is carried out using an enzyme such as 1,5-AG dehydrogenase using NAD as a coenzyme (see Japanese Patent Application Laid-Open No. 2-268679), a biological sample using NAD as a coenzyme is used. Accuracy is problematic because it may be affected by enzymes involved, such as lactate dehydrogenase.

Further, a soluble fraction of the genus Agrobacterium (The Journal of Biological Chemistry vol. 242, No. 1)
6, pp. 3665-3672, 1967) and glucoside 3-dehydrogenase [EC 1.1.99.13], which is confirmed to be present in the membrane fraction of Flavobacterium, can also use 1,5-AG as a substrate. Known (Journal of Biochemistry vol.100, No
4, pp. 1049-1055, 1986). However, these enzymes also
Since the substrate specificity is weak, it is necessary to completely remove or eliminate the saccharides in the sample in advance, and since a ferricyan compound or dichlorophenolindophenol is used as the electron acceptor, safety and sensitivity are reduced. There's a problem.

On the other hand, as a clinical significance of 1,5-AG, in diabetic patients, the concentration of 1,5-AG in serum or plasma is specifically reduced to about several μg / ml. Conversely, while the glucose concentration is about 100 mg / dl in a healthy person, it can reach 1,000 mg / dl in a diabetic patient, and the concentration difference from 1,5-AG is several thousand times. A completely specific enzyme capable of directly quantifying 1,5-AG in a sample in which high-concentration glucose coexists has not been found yet.

The specificity for 1,5-AG is poor,
Rather, when quantifying using pyranose oxidase or sorbose oxidase that reacts strongly with glucose, it is necessary to completely remove or eliminate glucose. For that purpose, various methods have been proposed. For example, Japanese Patent Publication No. 5-41238 proposes a method for combining saccharides such as glucose with a pretreatment operation using an ion exchange resin. Further, as a glucose elimination method, JP-A-1-320998, JP-B-7-71514,
JP-A-6-237795, JP-A-3-27299, JP-A-Hei.
No. 6,245,796 discloses that glucose 6-position kinase (glucokinase or hexokinase) is used, and in order to complete this reaction, the equilibrium of glucose phosphorylation is completely adjusted to glucose-6-phosphate. There is disclosed a method of making a contrivance (a conjugate system combining several types of enzymes and substrates). This also serves as a device for reducing the inhibitory effect of ATP on pyranose oxidase.However, as in JP-B-7-102154 and JP-B-7-108236, it is necessary to define pH conditions under which pyranose oxidase acts. Also, a method of eliminating the ATP inhibitory effect by devising pyranose oxidase of different origin has been proposed.

However, of the above methods, the method using an ion exchange resin is complicated and unsuitable for multi-sample processing. Further, in the method using a conjugated system in which a glucose 6-phosphorylating enzyme is combined with several types of enzymes, the reaction system is complicated, and it is necessary to consider the stability of the enzyme and substrate used. In addition, the method of treating with the glucose 6-phosphorylating enzyme alone also has limitations such as a large excess of ATP required for complete conversion and elimination of glucose, and limitations on the origin and reaction conditions of pyranose oxidase. In many cases, versatility is poor when combined as a 1,5-AG measurement reagent.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned drawbacks of the conventional method for determining 1,5-AG, and to provide a method for detecting 1,5-AG in a sample.
An object of the present invention is to provide a method for quantifying 1,5-AG and a reagent for quantification, which can measure G with high accuracy and high sensitivity by a simple operation and can be applied to an automatic analyzer.

[0011]

Means for Solving the Problems In order to solve the above problems, the present inventors studied reaction systems that can be used for the analysis of 1,5-AG and searched for enzymes that can be used for them. As a result, Agrobacterium (Ag
1,5-A in the membrane fraction of microorganisms belonging to the genus robacterium)
The existence of a novel enzyme that specifically oxidizes G (hereinafter referred to as "1,5-AG dehydrogenase"). When this 1,5-AG dehydrogenase acts on 1,5-AG, Catalyzing the reaction of oxidizing the 2-position hydroxyl group of 5,5-AG and reducing an electron acceptor such as 2,6-dichlorophenolindophenol, and performing the reaction using a reducing color former It has been found that, even in the absence of a carrier, the 2-position hydroxyl group of 1,5-AG is oxidized and the reducing color former is directly reduced to form a color-forming substance. Based on these facts, the present invention was completed. Reached.

That is, one aspect of the present invention is to use a body fluid as a sample, act on 1,5-AG, and catalyze a reaction for directly reducing a reducing color former in the absence of an electron carrier.
Using 1,5-AG dehydrogenase, after changing the glucose in the sample to a structure that does not react with the 1,5-AG dehydrogenase with a glucose scavenger, or while changing the glucose in the sample, In the presence of a reducing color former,
An object of the present invention is to provide a method for quantifying 1,5-AG, which comprises measuring a reduced color-forming substance produced by the action of an AG dehydrogenase.

Another object of the present invention is to provide a 1,5-AG dehydrogenase which acts on 1,5-AG and catalyzes a reaction for directly reducing a reducing color former in the absence of an electron carrier. A glucose eliminator that changes glucose into a structure that does not react with the 1,5-AG dehydrogenase, and a reagent for quantifying 1,5-AG, comprising a reducing color former. is there.

According to the present invention, a body fluid is used as a sample, and a glucose erasing agent and a reducing color former are added to the sample.
By causing the AG dehydrogenase to act, the glucose in the sample is changed to a structure that does not react with 1,5-AG dehydrogenase by the glucose scavenger, and It acts specifically on 5-AG to oxidize 1,5-AG and directly reduces the reducing color former to form color. The reduction reaction of the reducing coloring agent is an irreversible reaction, and is not affected by enzymes contained in a biological sample or the like, so that highly sensitive and accurate quantification can be performed. Further, since the reaction system is simple and the number of constituent reagents is small, workability is improved and application to an automatic analyzer is easy. In addition, since the body fluid of a diabetic patient or the like contains glucose at a concentration several thousand times higher than that of 1,5-AG, 1,5-AG having a high specificity to 1,5-AG
Even with 5-AG dehydrogenase, the measurement accuracy is reduced by the influence of glucose to a considerable extent. The glucose concentration of the enzyme has almost no problem, and high measurement accuracy can be obtained.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a specimen
For example, body fluids such as serum, plasma or urine are used. However, in the present invention, the body fluid includes not only the body fluid itself but also those prepared from the body fluid by various methods.

The 1,5-AG dehydrogenase used in the present invention specifically acts on 1,5-AG and catalyzes a reaction for directly reducing a reducing color former in the absence of an electron carrier. Is not particularly limited. However, preferably, microorganisms belonging to the genus Agrobacterium and capable of producing 1,5-AG dehydrogenase, particularly Agrobacterium tumefaciens IFO13532, Agrobacterium tumefaciens IFO13533, and Agrobacterium tumefaciens NT1130 strain ( The product produced from the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry, deposit number FERM BP-5997) is used. Among them, Agrobacterium tumefaciens NT1130 strain is a strain isolated from the soil by the present inventors, and has excellent storage stability compared to 1,5-AG dehydrogenase derived from existing strains. Is particularly preferred. Of course, not only to select from the stock strains,
The strain may be further isolated from the natural world such as soil, a mutant strain may be used, or a gene involved in enzyme production may be incorporated into a host from these strains to produce the enzyme.

For reference, the mycological properties of the Agrobacterium tumefaciens NT1130 strain are described as follows:
It is as follows.

A. Morphological features 1) Cell shape and size: Bacillus, size (0.3-0.
5) × (1.0 to 3.0) μm. 2) Presence or absence of spores: no 3) Motility: Motile, with periflagellate. 4) Gram staining: negative

B. Physiological properties 1) Catalase: Positive 2) Oxidase: Positive 3) Urease: Positive 4) Gelatin liquefaction: Negative 5) Indole production: Negative 6) Hydrogen sulfide production: Negative 7) Use of citric acid: Negative 8) Esculin Degradability: positive 9) β-galactosidase: positive 10) Reduction of nitrate: positive (±) 11) Attitude to oxygen: aerobic 12) Growth temperature: 25-35 ° C 13) Attitude to sugars: glucose, xylose, mannose, Arabinose, fructose, maltose,
It produces acid from rhamnose, mannitol and saccharose without generating gas.

C. Quinone composition analysis Ubiquinone-10 (98%)

From the above results, this strain is considered to be classified into the genus Agrobacterium. Usually, when identifying microorganisms, the microorganisms are identified in comparison with the `` Birgies Manual of Deterministic Bacteology, '' which summarizes the taxonomic properties of each microorganism. Regarding the genus Agrobacterium which is thought to belong to, recently, 16Sr
Systematic classification based on RNA gene sequence was attempted (In
ternational Journal of Bacteriology vol.43, 1993,
p.694-702 and vol.44, No.2, 1994, p.373-374), and the classification criteria were changed and reorganized based on the results. As a result, species consolidation and strain classification changes have been made, and the consistency with the "Barges Manual of Deterministic Bacteriology" has been lost.

Therefore, the 16S rRNA is also
Systematic classification based on the gene sequence of As a method, the entire gene sequence of the 16S rRNA of this bacterium was determined, and the homology with the gene sequence of a microorganism registered and published in a gene database (GenBank, EMBL and DDBJ) was searched. As a result, Agrobacterium tumefaciens ( Agroba
cterium tumefaciens) was significantly consistent with 99.4%. From this result, the strain was identified as a bacterium belonging to the species Agrobacterium tumefaciens, and the strain was identified as Agrobacterium tumefaciens NT.
The strain was named 1130 strain (Agrobacterium tumefaciens NT1130). This fungus was deposited on June 27, 1997 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 1-3, Higashi, Tsukuba, Ibaraki, Japan), and the deposit number was as described above. FERM BP-5997.

The 1,5-AG dehydrogenase used in the present invention is, for example, a method in which the above strain is cultured in a nutrient medium to produce and accumulate 1,5-AG dehydrogenase in a membrane fraction and to collect it. Can be manufactured. As the nutrient medium, a carbon source,
A medium containing an inorganic nitrogen source and an inorganic salt is used. As the carbon source, any one can be used as long as it basically utilizes a saccharide and induces L-sorbose, glucose and the like with an enzyme. As the inorganic nitrogen source, ammonium sulfate, ammonium chloride, ammonium phosphate, etc., as the inorganic salt,
Salts such as sodium, potassium, magnesium, iron, manganese and the like can be used.

The cultivation is preferably carried out under aerobic conditions such as shaking and aeration and stirring, and is preferably carried out at pH 6 to 8 and 25 to 35 ° C. The culture period is 1 to 7 days, usually 2 to 4 days.
Complete in days. By culturing by the above method, 1,5-AG dehydrogenase is mainly produced and accumulated in the membrane fraction of bacterial cells.

In order to obtain the present enzyme from the microorganisms after culturing, the cells are disrupted in a suitable buffer solution by dynomill, ultrasonic treatment or the like, and the membrane fraction is separated from the treated solution by ultracentrifugation or the like. . Next, the enzyme is released from the membrane fraction with a nonionic surfactant such as Triton X-100, and the insoluble matter is removed by centrifugation, whereby an extract containing 1,5-AG dehydrogenase can be obtained. The purified enzyme can be obtained by purifying the extract by appropriately combining techniques generally used for enzyme purification such as hydrophobic chromatography, ion exchange chromatography, hydroxyapatite, and gel filtration.

The 1,5-AG dehydrogenase collected from the Agrobacterium tumefaciens NT1130 strain by the method described above has the following physicochemical properties. In addition,
The titer of 1,5-AG dehydrogenase was measured by the following method. That is, 0.2 M potassium phosphate buffer (pH 8.0) 1.5
ml, 0.25 wt% nitrotetrazolium blue 0.3 ml, 2
0.3% by weight of nonionic surfactant, 0.3ml of 50mM 1,5-AG aqueous solution, 0.45ml of water, and 0.15ml of enzyme solution were reacted at 37 ° C with a total of 3.0ml of enzyme-containing solution, and absorbance change at 540nm (△ mOD / min) is measured. The molecular extinction coefficient of the formazan dye generated under these conditions is set to 16.7 × 10 ^3, and the activity of 1,5-AG dehydrogenase is defined as Determined according to Equation 2.

(Equation 2)

(1) Action: 100 mM containing 1 mM 2,6-dichlorophenol indophenol and 2.5 mM 1,5-AG
Add the enzyme of the present invention to a Tris / HCl buffer (pH 9.0), and add
The reaction was carried out at a temperature of ° C, and the molar balance between the product in the reaction solution and the reaction was analyzed by high performance liquid chromatography (HPLC) over time under the conditions shown in Table 1.

[0028]

[Table 1]

As a result, the peak of 1,5-AG having a retention time of 10.0 min. Decreased with the progress of the reaction, and a new peak of the reactant appeared at a position of 9.6 min. The molar balance of this reaction was consistent. Also, 1,
Glucoside that oxidizes the 3-position hydroxyl group of 5-AG
-3- Dehydrogenase (EC1.1.99.13) was reacted under the same conditions and analyzed by HPLC.As a result, a peak of the reactant appeared at a retention time of 10.8 min with a decrease in the peak of 1,5-AG. Was. On the other hand, pyranose oxidase (EC 1.1.3.1) has the action of oxidizing the 2-hydroxyl group of 1,5-AG.
0) in 40 mM borate buffer containing 2.5 mM 1,5-AG (pH
As a result of adding at 7.5 ° C and reacting at 37 ° C, a peak of the reaction product appeared at a new retention time of 9.6 minutes with a decrease in the peak of 1,5-AG. In these results,
Since the retention time of the reaction product obtained when the enzyme was allowed to act on 1,5-AG and the reaction product obtained when the pyranose oxidase was acted on was the same, the oxidation of 1,5-AG by the enzyme was observed. It can be seen that the site is a 2-position hydroxyl group.

(2) Substrate specificity: In the aforementioned enzyme activity measurement method, the relative reactivity (substrate specificity) when using various sugar solutions (all at a final concentration of 5 mM) instead of 1,5-AG was determined. It is shown in Table 2. This enzyme had a strong effect on 1,5-AG, a weak effect on L-sorbose, and a slight effect on D-glucose.

[0031]

[Table 2]

(3) Optimum pH: Using this enzyme, citrate buffer, potassium phosphate buffer, and Tris-HCl buffer having various pHs are used instead of the buffer in the aforementioned enzyme activity measurement method. And the enzyme activity when a glycine buffer was used. The result is shown in FIG. FIG. 1 shows that the optimum pH of the present enzyme is around 8.0 to 9.0.

(4) pH stability: The enzyme was added to citrate buffer, potassium phosphate buffer, Tris-HCl buffer and glycine buffer (each buffer was 200 mM) having various pH, and the mixture was added at 37 ° C. , And the residual enzyme activity was measured. The result is shown in FIG. Figure 2 shows that the stable pH of the enzyme
It can be seen that the range is from 6.0 to 9.0.

(5) Optimum temperature: This enzyme was reacted for 10 minutes at various temperatures with the composition in the above-mentioned enzyme activity measurement method. The result is shown in FIG. FIG. 3 shows that the optimum temperature of the present enzyme is around 35 to 45 ° C.

(6) Temperature stability: The enzyme was added to 20 mM potassium phosphate buffer, treated at various temperatures for 10 minutes, and the residual enzyme activity was measured. The result is shown in FIG. FIG. 4 shows that the enzyme is stable at 90% or more up to around 40 ° C.

(7) Electron acceptors: When various electron acceptors coexist with this enzyme in a 100 mM potassium phosphate buffer (pH 8.0), the activity at each electron acceptor is determined by relative activity. And shown in Table 3. From Table 3, it was found that 2,6-dichlorophenolindophenol and ferricyanide compounds can be used as electron acceptors in addition to nitrotetrazolium blue, which is a reducing color former.

[0037]

[Table 3]

(8) Molecular weight: The molecular weight of this enzyme determined by dodecyl sulfate-polyacrylamide electrophoresis is about
55,000.

(9) Km value: Line weaver bark (Line
The Km value of this enzyme with respect to 1,5-AG was determined by a weaver-Burk) plot, and was found to be about 0.5 mM.

(10) Storage stability: dissolved in 100 mM Tris-HCl buffer (pH 7.0) at a concentration of 0.1 u / ml,
FIG. 5 shows the results of measuring the residual activity after storage for 7 days and 14 days under the conditions described above.

On the other hand, for comparison, Agrobacterium tumefaciens IF013532 and Agrobacterium.
FIG. 5 also shows the results of measuring the storage stability of 1,5-AG dehydrogenase produced by the same method as described above using Tumefaciens IF013533 in the same manner as described above.

In FIG. 5, ■-■ indicate Agrobacterium.
The results of the enzyme derived from Tumefaciens NT1130 strain are shown.
− ◆ indicates Agrobacterium tumefaciens IF0135
The results of enzymes derived from Agrobacterium tumefaciens IF013533 are shown, and the results of enzymes derived from Agrobacterium tumefaciens IF013533 are shown.

As shown in FIG. 5, 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens NT1130 strain has a residual activity of 90% or more after storage for 14 days, and has excellent storage stability. Shows sex. In contrast, 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IF013532 and Agrobacterium tumefaciens IF
[0135] 1,5-AG dehydrogenase derived from 1,5-AG dehydrogenase has a residual activity of 40% or less after storage for 14 days, and thus has poor storage stability.

The 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens NT1130 strain found by the present inventors is Agrobacterium tumefaciens IF01.
Compared to 1,5-AG dehydrogenase derived from 3532 and 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IF013533, the storage stability is significantly different, but other physicochemical There were no significant differences in properties.

The method for quantifying 1,5-AG of the present invention can be performed, for example, by the following method. That is, a glucose erasing agent and a reducing color former are added to a body fluid or a specimen composed of the one prepared therefrom, and 1,5-AG dehydrogenase or an enzyme preparation containing the same is added thereto and incubated, By producing 2-keto-1,5-AG and a reduced color-forming substance and measuring the amount of the generated color-forming substance, the amount of 1,5-AG in the specimen can be quantified. The addition of the glucose eliminator, the reducing chromogenic agent and the 1,5-AG dehydrogenase to the sample may all be performed simultaneously, but preferably, the glucose erasing agent and the reducing chromogenic agent are added first. ,
After changing the glucose in the sample to a form in which 1,5-AG dehydrogenase does not act, it is preferable to add 1,5-AG dehydrogenase to cause a reaction.

Buffers that can be used in the above-mentioned quantification method include:
Any of the commonly used ones such as a phosphate buffer having a pH of 6 to 10, a Tris-HCl buffer, a Good buffer, a borate buffer and the like can be used.

As the reducing color former, tetrazolium or a salt thereof can be used. Specifically, nitrotetrazolium blue (hereinafter abbreviated as NTB), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H tetrazolium chloride, 3- (4,5-dimethyl- 2-thiazolyl) -2,
5-diphenyl-2H tetrazolium bromide, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H tetrazolium salt (hereinafter abbreviated as WST-1), etc. It can be used and its use concentration is 50-2000 μmol / l, especially 1
The range of from 00 to 1000 μmol / l is preferred.

In the quantification method of the present invention, 1,5-AG dehydrogenase is preferably used in the range of 20 to 10,000 units / liter, particularly preferably in the range of 200 to 2,000 units / liter.

In the measurement of the reduced color-forming substance in the reaction solution, the reaction is carried out as described above, and the absorbance after a certain time or the change in the absorbance within a certain time is measured at an absorption wavelength specific to the color-forming substance. It can be done by doing.

In the quantification method of the present invention, the glucose in the sample is changed to a form in which 1,5-AG dehydrogenase does not act, or while the glucose is changed, the reducing activity of 1,5-AG in the sample is reduced. 1,5-AG dehydrogenase is allowed to act in the presence of a color former. For this reason, the reagent contains a glucose scavenger. As the glucose scavenger, those containing glucose 6-position kinase and adenosine triphosphate are preferably used.

The 1,5-AG dehydrogenase used in the present invention is 1,5-AG dehydrogenase.
Although highly specific for 5-AG, it is not completely specific and has some effect on glucose. For this reason, when measuring 1,5-AG in the serum or plasma of a diabetic patient whose concentration difference between 1,5-AG and glucose is several thousand times, the influence of glucose is not small. Become. Therefore, by using a glucose scavenger in combination, it is possible to further improve the measurement accuracy when using serum, plasma or the like of a diabetic patient as a sample.

In this case, according to the present invention, if the coexisting glucose in the sample is reduced to a certain extent, the effect of glucose becomes almost negligible due to the high specificity for 1,5-AG, and the glucose is completely eliminated. There is no need to delete. Therefore, as a glucose scavenger, 1,
A simple substance that can reduce glucose to a concentration level at which it does not react with 5-AG dehydrogenase is sufficient.

Glucose 6-phosphorylating enzyme used as a glucose scavenger includes glucokinase, hexokinase and the like, and is not particularly limited as long as it is classified into EC 2.7.1.2 and EC 2.7.1.1. Commercially available ones can be used, but glucokinase having high specificity for glucose is preferably used. The amount of their use varies depending on the amount of glucose in the sample, but is generally 0.1 to 50 u / ml.
It is. The amount of adenosine triphosphate (ATP) required for glucose phosphorylation varies depending on the amount of glucose in the sample, but is generally 1 to 20 mM. Further, magnesium ions are usually contained in an amount of about 5 to 50 mM as inorganic or organic salts to promote the glucose phosphorylation reaction. The glucose phosphorylation reaction for eliminating glucose is carried out in a buffer solution having a pH of 6 to 10 at 20 to 50 ° C, preferably 25 to 37 ° C, for about 10 minutes immediately after the addition.
As a buffer that can be used, any commonly used buffer such as a glycine buffer, a Tris-HCl buffer, and a Good buffer can be used.

The glucose elimination treatment in the sample using each of the above reagents is preferably carried out in the form of a pretreatment for the reaction of 1,5-AG dehydrogenase. Since the enzyme has a high substrate specificity, it is sufficient that glucose can be reduced to a concentration level that does not react with 1,5-AG dehydrogenase. By combining under the conditions, it is also possible to perform the glucose treatment and the 1,5-AG quantification in each of the above reagents in one liquid.

Therefore, the reagent for quantifying 1,5-AG of the present invention may be a combination of a glucose eliminator and an enzyme for quantification, or a mixture of the glucose eliminator and the enzyme for quantification to form one reagent. Can also be quantified. More specifically, it is preferable to use two reagents, a first reagent containing a reducing color former and a glucose scavenger, and a second reagent containing 1,5-AG dehydrogenase. It may be a single reagent containing all of the color former, glucose scavenger, and 1,5-AG dehydrogenase.

[0056]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Production Example 1 [Acquisition of 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens NT1130 strain] Dipotassium phosphate 2%, monopotassium phosphate 0.5%, potassium chloride 0.75%, sodium chloride 2.5%, Ammonium chloride 1.
25%, sodium sulfate 0.025%, magnesium sulfate 0.005%
And 50 ml of a medium (pH 7.0) containing 0.1% L-sorbose and 20%
Add to a 0 ml culture flask, sterilize at 120 ° C for 15 minutes, cool, and mix with Agrobacterium tumefaciens NT1130.
One loopful of the strain (FERM BP-5997) was inoculated and shake-cultured at 30 ° C. for 3 days to obtain a seed culture solution. 10 ml of the resulting seed culture
Was inoculated into 1000 ml of a liquid medium having the same composition as above in a 2 liter culture flask, and cultured with shaking at 30 ° C. for 3 days.

After the completion of the culture, the cells were collected by centrifugation. The amount of the obtained cells was about 2 g per liter of the culture solution. The obtained cells were suspended in 2.5 ml / g of 20 mM potassium phosphate buffer (pH 7.2), and the cells were cut into pieces with an ultrasonic homogenizer. The obtained crushed liquid was centrifuged at 10,000 g, and the supernatant was further centrifuged at 100,000 g to obtain a membrane fraction. 10 ml of 1% Triton X-10 per gram of membrane fraction
0 in 20 mM potassium phosphate buffer (pH 7.2)
The enzyme was solubilized from the membrane fraction overnight with stirring. The obtained lysate was centrifuged again at 100,000 g to remove membrane residues, and a crude enzyme solution was obtained. When the enzymatic activity of this crude enzyme solution was measured based on the above-mentioned assay method, 2 units of activity were obtained per 1 g of bacterial cells.

Production Example 2 [Purification of 1,5-AG dehydrogenase] The solubilized solution obtained in Production Example 1 was previously purified with 1% Triton X-10.
The enzyme is adsorbed through a hydroxyapatite column equilibrated with 20 mM potassium phosphate buffer (pH 7.2) containing 0%, washed with a buffer of the same composition, and containing 1% Triton X-100.
Elution was performed with a linear gradient of 20 mM potassium phosphate buffer (pH 7.2), and the active fraction of the enzyme was collected. In order to further increase the specific activity, the same operation as above was performed again through a hydroxyapatite column. The active fraction was collected and concentrated by ultrafiltration to obtain a purified enzyme having a specific activity of 14 units / mg protein. The purified preparation showed a single band in polyacrylamide electrophoresis.

Reference Example [Quantification of 1,5-AG in Reagent System without Glucose Elimination Agent] WST-1 was used as a reducing color former and 1,5-AG dehydrogenase obtained in Production Example 2 was used. And the first shown in Table 4 below.
A quantification reagent consisting of the reagent and the second reagent was prepared. Using this reagent, 1,5-AG solutions of various concentrations and glucose solutions of various concentrations were measured.

That is, to each 10 μl of a solution containing 1,5-AG of each concentration or a solution containing a fixed concentration of 1,5-AG with a varied glucose concentration, After adding 320 μl of one reagent and reacting at 37 ° C. for 5 minutes, and then adding 70 μl of the second reagent and reacting at 37 ° C. for 5 minutes, a Hitachi 7070 type automatic analyzer was used to obtain a main wavelength of 450 nm and a sub wavelength of 700 nm. The absorbance was measured by a two-point assay.

The results are shown in FIGS. FIG. 6 shows the results of measurements for solutions containing 1,5-AG at each concentration, and FIG. 7 shows the results of measurements for solutions containing a constant concentration of 1,5-AG and glucose at each concentration.

FIG. 6 shows that linear color absorbance was obtained from 1,5-AG concentration of 0 to 50 μg / ml, and that 1,5-AG could be quantified. From FIG. 7, it can be seen that the coexistence of glucose causes an increase in absorbance in proportion to the increase in glucose concentration, but the coexistence of about 100 μg / ml shows a small change in absorbance, which is 2 μg / 1.5% in terms of 1,5-AG. less than ml. From this result, when 1,5-AG dehydrogenase having a high substrate specificity obtained from Production Example 2 was used, 1,5-AG was almost accurate even when glucose at the 1,5-AG concentration level remained. It can be seen that it can be measured. This means that it is not necessary to completely eliminate glucose.

[0064]

[Table 4]

Example 1 [Quantification of 1,5-AG in Reagent System Combined with Glucose Elimination Agent] A first reagent containing a glucose elimination agent having the composition shown in Table 5 and obtained in Production Example 2 A second reagent containing 1,5-AG dehydrogenase was used, and a solution obtained by changing the glucose concentration to a solution containing 1,5-AG at a constant concentration was used as a sample. 1,5-AG was measured by the method. The result is shown in FIG.

As shown in FIG.
Even in the sample containing 00 mg / dl, the same absorbance as that of the sample containing no glucose was obtained.
It turns out that 1,5-AG can be accurately quantified.

[0067]

[Table 5]

Example 2 [Quantitative determination of 1,5-AG in a reagent system containing a glucose scavenger] A glucose scavenger, a reducing color former, and a composition shown in Table 6
And using a reagent consisting of one solution containing 1,5-AG dehydrogenase obtained in Production Example 2, and adding a solution obtained by changing the glucose concentration to a solution containing a certain concentration of 1,5-AG. 1,5-AG was measured as a sample.

That is, 312 μl of the reagent shown in Table 6 was added to 8 μl of the sample, and the mixture was reacted for 15 minutes.
Main wavelength 450nm, subwavelength 700 by type 0 automatic analyzer
The absorbance was measured by a one-point assay at a measurement temperature of 37 ° C. The result is shown in FIG.

As can be seen from FIG.
In the sample containing mg / dl, almost the same absorbance as that of the sample containing no glucose was obtained. Therefore, even in the sample containing high-concentration glucose, the 1,5-AG It can be seen that can be accurately determined.

[0071]

[Table 6]

[0072]

As described above, according to the present invention,
A reducing color former was added to a sample containing 1,5-AG, and
By causing 5-AG dehydrogenase to act, 1,5-AG dehydrogenase specifically acts on 1,5-AG to oxidize 1,5-AG, and at the same time, directly reduces the color former. Since the color is developed by reduction, 1,5-AG in the sample can be quantified from the absorbance. The above reaction is an irreversible reaction and is not affected by an enzyme contained in a biological sample or the like, so that highly sensitive and accurate quantification can be performed. Further, since the reaction system is simple and the number of constituent reagents is small, workability is improved and application to an automatic analyzer is easy.

Further, by reducing the coexistence of glucose in the specimen to a certain extent by using a glucose scavenger in combination, the high specificity of 1,5-AG dehydrogenase used in the present invention for 1,5-AG Because the effect of glucose is almost negligible, high measurement accuracy can be obtained even when using serum or plasma from diabetic patients whose concentration difference between 1,5-AG and glucose is several thousand times. Can be obtained.

[Brief description of the drawings]

Fig. 1 Agrobacterium tumefaciens NT1130
4 is a table showing the optimum pH of 1,5-AG dehydrogenase obtained by culturing the strain.

FIG. 2 is a table showing the pH stability of the 1,5-AG dehydrogenase.

FIG. 3 is a chart showing an optimum temperature of the 1,5-AG dehydrogenase.

FIG. 4 is a table showing the temperature stability of the 1,5-AG dehydrogenase.

FIG. 5. The 1,5-AG dehydrogenase, Agrobacterium
1 is a table showing a comparison of the storage stability of 1,5-AG dehydrogenase derived from Tumefaciens IF013532 and 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IF013533.

FIG. 6 shows a first reagent containing a reducing color former and 1,5-AG
Using a second reagent containing dehydrogenase, various concentrations of 1,
5 is a table showing the results of measuring a 5-AG solution.

FIG. 7 shows a first reagent containing a reducing color former and 1,5-AG
Using a second reagent containing dehydrogenase, a fixed concentration of 1,
5 is a chart showing the results of measuring a solution added to a solution containing 5-AG while changing the glucose concentration.

FIG. 8 shows a solution containing a fixed concentration of 1,5-AG, using a first reagent containing a reducing color former and a glucose scavenger and a second reagent containing 1,5-AG dehydrogenase. 4 is a table showing the results of measuring solutions added with varying glucose concentrations.

FIG. 9 shows a reducing color former, a glucose scavenger, and 1,5-A
4 is a table showing the results of measuring a solution obtained by changing the glucose concentration to a solution containing a fixed concentration of 1,5-AG using a reagent containing G dehydrogenase in one solution.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification code FI (C12N 9/04 C12R 1:01) (58) Investigated field (Int.Cl. ⁶ , DB name) C12Q 1/32 C12Q 1 / 54 G01N 33/49 G01N 33/493 C12N 9/04 BIOSIS (DIALOG) WPI (DIALOG)

Claims (10)

(57) [Claims] 1. A 1,5-anhydroglucitol which acts on 1,5-anhydroglucitol using a body fluid as a sample and catalyzes a reaction for directly reducing a reducing color former in the absence of an electron carrier. Using toll dehydrogenase, after or while changing the glucose in the sample to a structure that does not react with the 1,5-anhydroglucitol dehydrogenase with a glucose scavenger, Wherein the 1,5-anhydroglucitol dehydrogenase is allowed to act on 1,5-anhydroglucitol in the presence of a reducing color former, and the resulting reduced color former is measured. For the determination of 1,5-anhydroglucitol. 2. The method according to claim 1, wherein the glucose scavenger is glucose 6
The method for quantifying 1,5-anhydroglucitol according to claim 1, wherein the method comprises a phosphatase and adenosine triphosphate. 3. The method for quantifying 1,5-anhydroglucitol according to claim 1, wherein the reducing color former is tetrazolium or a salt thereof. 4. The method according to claim 1, wherein the 1,5-anhydroglucitol dehydrogenase belongs to the genus Agrobacterium and is produced by a microorganism capable of producing 1,5-anhydroglucitol dehydrogenase. The method for quantifying 1,5-anhydroglucitol according to any one of claims 1 to 3. 5. The method according to claim 1, wherein the 1,5-anhydroglucitol dehydrogenase has the following physicochemical properties.
5. The method for quantifying 1,5-anhydroglucitol according to any one of Items 1 to 4. (1) Action: In the absence of an electron carrier, it specifically catalyzes a reaction that specifically acts on 1,5-anhydroglucitol to oxidize the hydroxyl group at the 2-position and directly reduce the reducing color former. (2) Substrate specificity: Strongly acts on 1,5-anhydroglucitol and weakly acts on L-sorbose. It also slightly acts on D-glucose. (3) Optimum pH: The optimum pH is around 8.0 to 9.0. (4) pH stability: stable at pH 6.0 to 9.0. (5) Optimum temperature: The optimum temperature is around 35 to 45 ° C. (6) Temperature stability: stable at least 90% up to around 40 ° C. (7) Method for measuring titer 0.2 M potassium phosphate buffer (pH 8.0) 1.5 ml, 0.25% by weight
Nitrotetrazolium blue 0.3 ml, 0.3 wt% nonionic surfactant 0.3 ml, 50 mM 1,5-anhydroglucitol aqueous solution 0.3 ml, water 0.45 ml, and enzyme solution 0.15 ml, total 3.0 ml enzyme-containing solution Is reacted at 37 ° C., and the change in absorbance at 540 nm (ΔmOD / min) is measured. The molecular extinction coefficient of the formazan dye produced under these conditions is 16.7 × 10
³ and then, the amount of enzyme that produces the formazan 1μmol per minute as one unit (Unit), obtaining the 1,5-anhydroglucitol dehydrogenase activity according to the following equation (1). (Equation 1) (8) Electron acceptor: In addition to 2,6-dichlorophenolindophenol, ferricyan compound, etc., a reducing color former can also be used. Further, coenzymes such as NAD and NADP and dissolved oxygen cannot be used. (9) Molecular weight: The molecular weight by dodecyl sulfate-polyacrylamide electrophoresis is about 55,000. 6. A 1,5-anhydroglucitol dehydrogenase which acts on 1,5-anhydroglucitol and catalyzes a reaction for directly reducing a reducing color former in the absence of an electron carrier. A glucose eliminator that changes glucose into a structure that does not react with the 1,5-anhydroglucitol dehydrogenase, and a reducing color former, Reagent for quantification. 7. The method according to claim 7, wherein the glucose scavenger is glucose 6
7. The reagent for quantifying 1,5-anhydroglucitol according to claim 6, which comprises a phosphatase and adenosine triphosphate. 8. The reagent for quantifying 1,5-anhydroglucitol according to claim 6, wherein the reducing color former is tetrazolium or a salt thereof. 9. The method according to claim 1, wherein the 1,5-anhydroglucitol dehydrogenase belongs to the genus Agrobacterium and is produced by a microorganism capable of producing 1,5-anhydroglucitol dehydrogenase. The reagent for quantifying 1,5-anhydroglucitol according to any one of claims 6 to 8. 10. The method according to claim 6, wherein the 1,5-anhydroglucitol dehydrogenase is an enzyme having the physicochemical properties described in claim 5. Five-
Reagent for quantification of anhydroglucitol.