JP3229430B2 - Determination of 1,5-anhydroglucitol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying 1,5-anhydroglucitol, and more particularly, to 1,5-anhydroglucitol (hereinafter referred to as "1 -anhydroglucitol") which is practically used as a diagnostic marker for diabetes. 1,5-AG ").

[0002]

2. Description of the Related Art A method for quantifying 1,5-AG using an enzyme is as follows.
It has already been proposed by one of the present applicants (Japanese Unexamined Patent Publication No.
2-79780, JP-A-63-185397).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel method for determining 1,5-anhydroglucitol.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a method for measuring the presence of 1,
In quantifying 5-anhydroglucitol by enzymatic method, it is obtained from microorganisms other than the genus Flavobacterium.
A method for quantifying 1,5-anhydroglucitol, which comprises using an enzyme capable of oxidizing the hydroxyl group at the 3-position of 1,5-anhydroglucitol.

Hereinafter, the present invention will be described in detail. The enzyme having the ability to oxidize the hydroxyl group at the 3-position of 1,5-AG used in the present invention is a microorganism other than the genus Flavobacterium.
Obtained from things. Specifically, for example, a reference by Hayano et al. (The Journal of Biological Chemistry vol.24
2, No. 16, pp. 3665-3672, 1967).
Agrobacterium rhizogenes (Ag), an enzyme that oxidizes the 3-position of glucose obtained from "cterium tumefacience"
robacterium rhizogenes) IFO15188, Agrobacterium sp. IFO1371
4. Agrobacterium radiobacter
ium radiobacter) IFO13532, Agrobacterium tumefacience
Enzyme obtained from a microorganism belonging to the genus Agrobacterium such as IFO3058, Shi Tofaga-Heparina (Cytophag
a heparina) IFO12017, and enzymes obtained from microorganisms belonging to the genus Cytophaga, such as Cytophaga keratolytica IFO14087.

[0006] In order to obtain the enzyme used in the present invention from the above microorganisms, since this enzyme is present in the cytoplasmic fraction of the cells, the cells are first separated from the culture and cultured in an appropriate buffer. It is necessary to destroy the body and then obtain the cytoplasmic fraction from the treatment. Then, as a method for destroying the cells, a French press or an ultrasonic crusher can be used. The cytoplasmic fraction is obtained by centrifuging the cell disruption liquid and separating it as a precipitate from the membrane fraction, cell wall components and the like.

In the present invention, a target enzyme can be isolated from the supernatant obtained by centrifugation. As an isolation method, a polyethylene glycol fraction or an ammonium sulfate fraction, which is generally known as a method for purifying an enzyme, can be used. And if you need more pure enzyme,
It can be purified by conventional ion exchange chromatography and gel filtration column chromatography over like. However, in the present invention, not only the purified enzyme but also the centrifugal supernatant obtained after the disruption of the cells can be used. Further, these can be used by fixing them to a carrier such as a resin or a film.

[0008] In order to carry out the method of the present invention, for example, the above enzyme is allowed to act on 1,5-AG in the presence of an electron acceptor. Then, from the amount of the reduced form of the electron acceptor, 1,5-AG
Is quantified.

The electron acceptor is not particularly limited as long as it is involved in the oxidation reaction of 1,5-AG, and examples thereof include oxygen, phenazine methosulfate, and dichlorophenolindophenol. Further, ferricyanide compounds such as potassium ferricyanide, sodium ferricyanide, and ammonium ferricyanide, and coenzymes such as cytochrome C, NAD ⁺ , NADP ⁺ , and FMN can also be used.

In the present invention, any sample requiring 1,5-AG concentration measurement can be used. For example, typical examples include cerebrospinal fluid, plasma, serum, urine, and the like. Further, in order to facilitate the measurement of the 1,5-AG concentration, a treatment solution from which proteins, saccharides, and the like have been removed may be used.

In the present invention, the determination of the reduced form of the electron acceptor can be carried out according to the method described in JP-A-62-79780. As a typical method, a method utilizing a change in the degree of coloring of the electron acceptor will be described below.

First, a Tris-HCl buffer (0.05 Mp
H7) 0.7 ml, 0.1 M potassium ferricyanide solution 0.1 ml, enzyme and 1,5-AG solution 0.1 ml
Is put in a container and reacted at 34 ° C. for 10 minutes. Then
0.5 ml of ferric sulfate-Dubanol reagent (ferric sulfate 5 g, sodium lauryl sulfate 3 g, 85% phosphoric acid 95 ml, distilled water 900 ml) and distilled water 3.5 ml were added, and the mixture was allowed to stand for 10 minutes at 660 nm. Measure the absorbance. The concentration of 1,5-AG is calculated from the absorbance of the sample using a calibration curve prepared with a 1,5-AG solution having a known concentration.

[0013]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the present invention.

Enzyme Production Example 1 (Production of glucose-3-dehydrogenase by Agrobacterium rhizogenes (IFO15188)) 100 ml of a medium having the composition shown in the following Table 1 was dispensed into a 500 ml Sakaguchi flask, and then 120 ° C for 15 minutes. After sterilization, one loopful of Agrobacterium rhizogenes (IFO15188) was inoculated and cultured by shaking at 27 ° C. for 24 hours.
In addition, the numerical value of the medium composition in Table 1 represents weight%.

[0015]

(Table 1) (NH ₄ ) ₂ SO ₄ : 0.1 Urea: 0.05 NaCl: 0.02 MgSO ₄ .7H ₂ O: 0.02 CaCl ₂ .H ₂ O: 0.01 FeSO ₄ .7H ₂ O: 0.0025 1 M phosphate buffer (pH 7): 5 Sucrose: 2

Next, the obtained culture solution was subjected to 15000 G ×
The cells were obtained by centrifugation under the conditions of 20 minutes. The cells were washed with 50 mM phosphate buffer (pH 7) and suspended in the same buffer. The suspension was treated with an ultrasonic crusher under cooling to crush the cells. 15000G × 2 crushed liquid
Centrifugation was performed under 0 minute conditions to remove cell residue, and a crude enzyme solution was obtained. Ammonium sulfate powder was added to this crude enzyme solution and dissolved by stirring. At this time, 15
Centrifugation was performed under the condition of 000 G × 20 minutes, and the glucose-3-dehydrogenase activity of each ammonium sulfate fraction was measured. The activity was mainly present in the 40% -80% saturated fraction.

Further, the above ammonium sulfate fraction was added to a phosphate buffer (10
mM pH 7, containing 10 mM sucrose) and desalted using a dialysis membrane. Ammonium sulfate powder was added to the desalted liquid and dissolved by stirring. The protein precipitated in the 40% to 80% saturated fraction was centrifuged under the condition of 15000 G × 20 minutes to obtain a fraction. This fraction was dissolved in phosphate buffer (containing 5 mM pH7 and 10 mM glycerol) and desalted using a dialysis membrane.

After the desalted solution was passed through a DEAE-cellulofine column to adsorb the enzyme, the enzyme was eluted with a phosphate buffer containing 0 to 0.1 M KCl (containing 10 mM pH7 and 10 mM glycerol). The activity fraction was obtained by measuring the glucose-3-dehydrogenase activity of each eluted fraction. The active fraction was concentrated using an Advantech UF membrane to obtain an enzyme solution. The activity of this enzyme solution was 9.2 units / ml, and 2 units of enzyme were obtained per 1 g of wet cells.

Enzyme Production Example 2 (Production of Glucose-3-dehydrogenase by Cytophaga heparina (IFO12017)) Polypeptone: 1% by weight, Yeast extract: 0.2% by weight,
MgSO ₄ · 7H ₂ O: containing 0.1 wt%, pH 7
Was dispensed into a 500 ml Sakaguchi flask, sterilized at 120 ° C. for 15 minutes, and inoculated with a platinum loop of Sitofaga heparina (IFO12017).
For 24 hours. 15,000 G of culture solution
The cells were centrifuged under conditions of × 20 minutes to obtain bacterial cells. The cells were washed with 50 mM phosphate buffer (pH 7) and suspended in the same buffer. The suspension was treated with an ultrasonic crusher under cooling to crush the cells. 15000G of crushed liquid
Centrifugation was performed for 20 minutes to remove cell residue, and a crude enzyme solution was obtained.

[0020]

Next, typical enzymes used in the present invention, for example, Agrobacterium rhizogenes (IFO151)
88), Agrobacterium sp (IFO1371)
4), Agrobacterium radiobacter (IFO1)
3532), Agrobacterium Tsumefuashiensu (IFO3058), Shi Tofuaga-Heparina (IFO
12017), Sitophaga keratricha (IFO1
4087) has a substrate specificity of oxidizing glucose, 2-deoxyglucose, 1,5-AG, and sucrose. Therefore, these enzymes are similar to the enzymes described in the aforementioned Hayano et al.
It seems to be a dehydrogenase that oxidizes the 3-position hydroxyl group of -AG.

Example 1 Method Using Dichlorophenol Indophenol (DCIP) as Electron Acceptor (Preparation of Calibration Curve) In 2.0 ml of 0.5 M phosphate buffer (pH 7.0), 1.
0.7 ml of a 6 mM DCIP solution and 0.2 ml of the enzyme solution prepared in Enzyme Production Example 1 were added and stirred to prepare a reaction solution. In a quartz cell having an optical path length of 1 cm, a standard solution obtained by diluting a 1,5-AG solution having a concentration of 50 mg / l twice with distilled water.
ml and 1.0 ml of distilled water were added, and the mixture was set in a cell holder of a spectrometer thermostatized at 30 ° C. After allowing to stand for 3 minutes to allow the temperature to reach a constant temperature, 0.1 ml of the above reaction solution was added to the quartz cell, and the mixture was inverted and stirred, and the absorbance at 600 nm was measured. One minute after the start of the reaction, the relationship between the rate of decrease in absorbance for one minute and the concentration of 1,5-AG was measured.
It was shown to.

Example 2 Method of using potassium ferricyanide as an electron acceptor (preparation of calibration curve) 1.0 ml of a 0.1 M potassium ferricyanide solution in 5.0 ml of 20 mM Tris / hydrochloric acid buffer, and the agropreparation prepared in Enzyme Production Example 1 Bacterium rhizogenes (IFO15
188) of the enzyme (6.4 units / ml) 0.5 ml,
1.5 ml of distilled water was added to prepare a coloring reagent. 50μ
0.4 ml of a coloring reagent was added to 0.1 ml of a solution obtained by diluting a 1,5-AG standard solution having a concentration of g / ml twice with distilled water.
The reaction was performed at 5 ° C. for 1 hour. After the reaction, the reaction solution was added to a ferric-Dupanol reagent (1.00 g of ferric sulfate, 0.604 g of sodium dodecyl sulfate, 19 m of 85% phosphoric acid).
1 was dissolved in water and adjusted to 200 ml) 0.25
The reaction was stopped by adding 1.75 ml of distilled water, and the mixture was stirred and the absorbance at 660 nm was measured. The result is shown in FIG.

Example 3 Measurement of Human Serum 1,5-AG Concentration A commercially available kit for measuring 1,5-AG, Rana AG (anhydroglucitol) (manufactured by Nippon Kayaku Co., Ltd.) , Discard the packing solution in the pretreatment column, and
Conditioning was performed by washing with ml of distilled water. After adding 0.1 ml of a human serum specimen to the column, the column was eluted twice with 0.5 ml of distilled water to obtain 1.1 ml of an eluate from which protein and glucose were removed. Similarly, the 1,5-AG standard solution was treated with the pretreatment column. 1.0 ml of the thus-obtained sample and the column treatment solution of the 1,5-AG standard solution were placed in a quartz cell having an optical path length of 1 cm, and reacted according to the method of Example 1.
The AG concentration was measured. Table 2 shows the results.

[0025]

< Table 2 ><Measurement measurement of human serum sample> (numerical value is mg / l) Sample Result of Example 2 Result by Rana AG A 32.3 33.0 B 42.1 42.4 C 33.5 33.2

As shown in Table 2, 1,5-A according to the present invention
The measured value of G almost agrees with the measured value of commercially available rana AG (anhydroglucitol) (using pyranos oxidase which oxidizes the 2-position of 1,5-AG as an enzyme).

[0027]

According to the present invention described above, 1,5-
1,5-AG by an enzyme that oxidizes the hydroxyl group at the 3-position of AG
Can be quantified. Therefore, the present invention greatly contributes to, for example, the field of diabetes diagnosis.

[Brief description of the drawings]

FIG. 1 is a calibration curve when dichlorophenol indophenol is used as an electron acceptor, the vertical axis represents the rate of decrease in absorbance (ABS: 600 nm / min), and the horizontal axis represents 1,5-A
G concentration (mg / L).

FIG. 2 is a calibration curve when potassium ferricyanide is used as an electron acceptor, and the vertical axis represents absorbance (660 n
m), and the horizontal axis represents 1,5-AG concentration (mg / ml).

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Toshikatsu Hirahara 95-7 Minoki-cho, Fukuyama City, Hiroshima Prefecture (56) References JP-A-62-79780 (JP, A) JP-A-63-185397 (JP, A) J. Biochem. , 100 (4), 1049-1055 (1986) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-3/00 CA (STN)

Claims (1)

(57) [Claims] 1. Agrobacterium for determining 1,5-anhydroglucitol in a sample by an enzymatic method.
Obtained from a microorganism belonging to the genus or the genus Cytophaga ,
1, an enzyme having an ability to oxidize the hydroxyl group at the 3-position of 1,5-anhydroglucitol is used.
A method for determining 5-anhydroglucitol.