JPH10155479A - 1,5-anhydroglucitol dehydrogenase, its production and determination of 1,5-anhydroglucitol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a 1,5-anhydroglucitol (1,5-AG) dehydrogenase capable of simply measuring 1,5-AG in a specimen in high accuracy, extremely useful for diagnosing especially diabetes, etc. SOLUTION: This 1,5-AG dehydrogenase has the following physical and chemical properties. (1) Action: specifically acting on 1,5-AG in the absence of an electron carrier to oxidize a hydroxyl group at the 2-position and to catalyze a reaction to reduce a direct reducing coloring agent. (2) Substrate specificity: strong in 1.5-AG, weak in L-sorbose and somewhat acting on D- glucose. (3) Optimum pH: pH about 8.0-9.0. (4) pH Stability: stable at pH 7.0-9.0. (5) Optimum temperature: at about 35-40 deg.C. (6) Temperature stability: >=90% stable at up to about 40 deg.C. (7) Electron acceptor: capable of using a reducing coloring agent besides 2,6-dichlorophenolindophenol, ferricyanide compound, etc., but incapable of using a coenzyme such as NAD, NADP, etc., and a dissolved enzyme. (8) Molecular weight: about 55,000 (SDS-PACE method).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel 1,5-anhydroglucitol dehydrogenase and a method for producing the same, and a simple and highly accurate diagnosis of diabetes and the like using the enzyme. The present invention relates to a method for determining 1,5-anhydroglucitol.

[0002]

2. Description of the Related Art 1,5-Anhydroglucitol (hereinafter, referred to as "1")
"1,5-AG") is a clinically important test item in recent years as a useful diagnostic index because the amount of serum, plasma, urine, etc. in body fluids fluctuates greatly in certain diseases, especially diabetes. It is becoming. As a method for quantifying 1,5-AG, a method of performing colorimetric quantification of generated hydrogen peroxide by a pyranose oxidase using a peroxidase coloring system (Japanese Patent Publication No. 5-41238) has become mainstream. , Is applied to general-purpose automatic analyzers. Alternatively,
There is a method in which an oxidase having 1,5-AG oxidizing ability is acted on to measure the amount of consumed oxygen, a reduced form of an electron acceptor, or an oxidized 1,5-AG reaction product.

[0003]

However, the concentration of 1,5-AG in a human-derived subject, for example, serum, is low, and in a diabetic patient, the level is further lowered. The oxidase coloring system is not sufficiently satisfactory in terms of sensitivity. Also, in the method of measuring the reductant using an oxidoreductase utilizing an electron acceptor, the reductant itself, such as 2,6-dichlorophenolindophenol or a ferricyan compound, which is an electron acceptor, is unstable. And the sensitivity is not sufficient, and it is difficult to apply it to a general-purpose automatic analyzer for processing a large number of samples.

[0004] Therefore, there has been a demand for the development of a method for quantitatively determining 1,5-AG which can easily and accurately measure 1,5-AG and can be easily applied to a general-purpose automatic analyzer.

[0005]

Under such circumstances, the present inventors have studied the 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
It has been found that a novel enzyme that specifically oxidizes 1,5-AG (hereinafter referred to as "1,5-AG dehydrogenase") is present in the membrane fraction of a microorganism belonging to the genus ium). When this 1,5-AG dehydrogenase acts on 1,5-AG, it oxidizes the 2-position hydroxyl group of 1,5-AG and simultaneously activates an electron acceptor such as 2,6-dichlorophenolindophenol. Catalyzing the reduction reaction, and performing the reaction using a reducing color former, oxidizes the 2-position hydroxyl group of 1,5-AG and directly reduces the reducing color former even in the absence of an electron carrier. The inventors have found that a color-forming substance is produced, and have completed the present invention.

That is, the present invention provides a 1,5-AG dehydrogenase having the following physicochemical properties.

(1) Action: In the absence of an electron carrier, it acts specifically on 1,5-anhydroglucitol to give 2
It has the effect of catalyzing the reaction of oxidizing the hydroxyl group at the position and directly reducing 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 7.0 to 9.0.

(5) Optimum temperature: The optimum temperature is around 35 to 40 ° C.

(6) Temperature stability: stable to 90% or more up to around 40 ° C.

(7) Electron acceptor: In addition to 2,6-dichlorophenolindophenol and ferricyan compound, a reducing color former can be used. In addition, coenzymes such as NAD and NADP and dissolved enzymes cannot be used.

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

[0015] The present invention also relates to a method for culturing a microorganism belonging to the genus Agrobacterium and having 1,5-AG dehydrogenase-producing ability in a nutrient medium, and adding 1,5-AG dehydrogenase in a membrane fraction. It is intended to provide a method for producing 1,5-AG dehydrogenase, which comprises producing, accumulating, and collecting the resulting product.

Further, the present invention provides a method for preparing 1,1,
Provided is a method for quantifying 1,5-AG, which comprises reacting the above-mentioned 1,5-AG dehydrogenase on a sample containing 5-anhydroglucitol and measuring the resulting reduced color forming substance. Things.

[0017]

DETAILED DESCRIPTION OF THE INVENTION Conventionally, from microorganisms belonging to the genus Agrobacterium, Hayano et al. (Journal of Biological Chemistry, Vol. 242, 3665-3672, 1967)
Year) confirmed the presence of glucoside-3-dehydrogenase in the cytoplasmic fraction, separately from Takeuchi et al. (Journal of Biochemistry, 100, 1049-1055, 19
(1986) reported that 1,5-AG could be a substrate for glucoside-3-dehydrogenase. From the strains belonging to the genus Pseudomonas, NAKAMURA et al. (Journal of Biochemistry, Vol. 99, 607-613, 198)
6 years), an enzyme that converts 1,5-AG to 1,5-anhydro-D-fructose and consumes oxygen only in the presence of an electron acceptor in the membrane fraction has been reported. I have.

However, no metabolic enzyme that is specific to 1,5-AG and oxidizes 1,5-AG in the absence of an electron carrier to directly reduce a reductive coloring agent has not yet been reported. That is, the action of the enzyme of the present invention is shown in the following formula. The enzyme showing such action is not yet known and is novel.

[0019]

(Equation 1)

The microorganism used for the production of the enzyme of the present invention may be any microorganism that belongs to the genus Agrobacterium and produces 1,5-AG dehydrogenase. And Agrobacterium tumefaciens IFO13532, Agrobacterium tumefaciens IFO13533, which are depositable and segregable and known to the Fermentation Research Institute.

As a medium for culturing the above cells, a medium containing a carbon source, an inorganic nitrogen source and an inorganic salt is used. Basically, a saccharide is used as a carbon source, and any one can be used as long as it can induce L-sorbose, glucose, and the like. As the inorganic nitrogen source, ammonium sulfate, ammonium chloride, ammonium phosphate and the like can be used, and as the inorganic salt, salts such as sodium, potassium, magnesium, iron and manganese 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
Complete in 4 days. By culturing according to the above method, 1,5-AG dehydrogenase is mainly produced and accumulated in the membrane fraction of bacterial cells.

After culturing, to obtain the enzyme of the present invention from the microorganisms, 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. I do. Next, the enzyme is released from the membrane fraction using 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.

Next, Agrobacterium.
1, of the present invention obtained using Tumefaciens IFO13533
5 shows the properties of 5-AG dehydrogenase. Note that 1,5-
The activity of AG dehydrogenase was measured by the following method.

<Method for measuring enzyme activity> 1.5 ml of 0.2 M potassium phosphate buffer (pH 8.0), 0.3 ml of 0.25 wt% nitrotetrazolium blue, 0.3 ml of 2 wt% Triton X-100, 50 ml of 50 mM
A total of 3.0 ml of the enzyme-containing solution consisting of 0.3 ml of 5-AG aqueous solution, 0.45 ml of water and 0.15 ml of the enzyme 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), was determined activity of 1,5-AG dehydrogenase according to the following equation.

[0026]

(Equation 2)

<Physicochemical Properties> (1) Action: Add the enzyme of the present invention to a 100 mM Tris / hydrochloric acid buffer (pH 9.0) containing 1 mM 2,6-dichlorophenol indophenol and 2.5 mM 1,5-AG. The reaction was carried out at 37 ° C., and the molar balance between the product in the reaction solution and the reaction with time was shown in Table 1.
Was analyzed by HPLC under the conditions shown in (1).

[0028]

[Table 1]

As a result, the peak of 1,5-AG having a retention time of 10.0 minutes decreased with the progress of the reaction, and a new peak of the reactant appeared at a position of 9.6 minutes of the retention time. The molar balance of this reaction was consistent. Also, 1,
Glucoside-3, which has the effect of oxidizing the 3-position hydroxyl group of 5-AG
-Dehydrogenase (EC 1.1.99.13) was reacted under the same conditions, and analyzed by HPLC. As a result, a peak of the reaction product appeared at a new retention time of 10.8 minutes with a decrease in the peak of 1,5-AG. On the other hand, pyranose oxidase (EC 1.1.3.1), which has the action of oxidizing the 2-position hydroxyl group of 1,5-AG
0) in 40 mM borate buffer containing 2.5 mM 1,5-AG (pH
The reaction was carried out at 37 ° C. As a result, the peak of the reaction product appeared at a new retention time of 9.6 minutes, along with the decrease of the 1,5-AG peak. In these results,
Since the retention time of the reaction product obtained when the enzyme of the present invention is acted on 1,5-AG and the reaction product obtained when pyranose oxidase is acted on the 1,5-AG by the enzyme of the present invention, It can be seen that the oxidation site of is a hydroxyl group at the 2-position.

(2) Substrate specificity: In the enzyme activity measurement method described above, 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. Table 2
Shown in The enzyme of the present invention acts strongly on 1,5-AG, weakly on L-sorbose, and slightly on D-glucose.

[0031]

[Table 2]

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

(4) pH stability: The enzyme of the present invention is added to citrate buffer, potassium phosphate buffer, Tris-HCl buffer and glycine buffer (each buffer is 200 mM) having various pH, After treatment at 37 ° C for 60 minutes, the residual enzyme activity was measured. The result is shown in FIG. FIG. 2 shows that the stable pH range of the enzyme of the present invention is 7.0 to 9.0.

(5) Optimum temperature: The enzyme of the present invention 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 enzyme of the present invention is around 35 to 40 ° C.

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

(7) Electron acceptors: When various electron acceptors are present together with the enzyme of the present invention in a 100 mM potassium phosphate buffer (pH 8.0), the activity at each electron acceptor is determined by the relative activity. It was expressed by. Table 3 shows that 2,6-dichlorophenol indophenol and ferricyan compounds have high activity as electron acceptors, but nitrotetrazolium blue can be an electron acceptor even in the absence of an electron carrier.

[0037]

[Table 3]

(8) Molecular weight: The molecular weight of the enzyme of the present invention determined by dodecyl sulfate-polyacrylamide electrophoresis was
It was about 55,000.

(9) Isoelectric point: The isoelectric point of the enzyme of the present invention using a chromatofocusing column (manufactured by Pharmacia) was about 4.0.

(10) Km value: Lineweaverk (Linew
eaver-Burk) plot, the Km value of the enzyme of the present invention with respect to 1,5-AG was found to be about 1.0 mM.

The method for determining 1,5-AG of the present invention
It is performed as follows using 1,5-AG dehydrogenase. That is, the subject is incubated with 1,5-AG dehydrogenase or an enzyme preparation containing the same in the presence of a reducing color former to produce 2-keto-1,5-AG and a reduced color former. By measuring the amount of the generated coloring substance, the amount of 1,5-AG in the subject can be quantified.

As the buffer which can be used in the quantification method of the present invention, any commonly used buffer 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. It is possible.

Examples of the reducing color former include tetrazolium and salts thereof. 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) and the like can be used at a concentration of 50 to 2000 μmol / l, particularly 100 to 100 μmol / l.
A range of 00 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.

Examples of the sample to which the quantification method of the present invention can be applied include biological samples containing 1,5-anhydroglucitol, such as serum, plasma, and urine.

In the measurement of the reduced chromogenic substance in the reaction solution, after the reaction as described above, 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 chromogenic substance. It can be done by doing.

[0047]

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.

Example 1 [Acquisition of 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IFO13533] 2% dipotassium phosphate, 0.5% monopotassium phosphate, 0.75% potassium chloride, 2.5% sodium chloride, 2.5% sodium chloride Medium consisting of 1.25% ammonium, 0.025% sodium sulfate, 0.005% magnesium sulfate and 0.1% L-sorbose (pH 7.
0) Add 50 ml to a 200 ml culture flask,
After sterilization for 1 minute, the mixture was cooled, a loopful of Agrobacterium tumefaciens IFO13533 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 placed in a 2 liter culture flask in a liquid medium 1000 having the same composition as above.
The mixture was inoculated into 30 ml and shake-cultured at 30 ° C. for 3 days. After 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. 2.5 g / g of 20 mM potassium phosphate buffer (pH 7.2)
And the cells were chopped 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. The membrane fraction was suspended in 10 ml of 20 mM potassium phosphate buffer (pH 7.2) containing 1% Triton X-100 per 1 g of the membrane fraction, and the enzyme was solubilized from the membrane fraction overnight with stirring. The obtained lysate was centrifuged again at 100,000 g to remove a membrane residue, thereby obtaining a crude enzyme solution. When the enzymatic activity of this crude enzyme solution was measured based on the above-mentioned assay method, an activity of 0.5 unit was obtained per 1 g of bacterial cells.

Example 2 [Purification of 1,5-AG dehydrogenase] The solubilized solution obtained in Example 1 was previously purified with 1% Triton X-10.
The enzyme is adsorbed through a hydroxyapatite column equilibrated with a 20 mM potassium phosphate buffer (pH 7.2) containing 0%, washed with a buffer of the same composition, and then washed with a buffer containing 1% Triton X-100.
Elution was performed with a linear gradient of 0 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 12 units / mg protein. The purified preparation showed a single band in polyacrylamide electrophoresis.

Example 3 [Quantification of 1,5-AG using NTB] Various concentrations of 1-AG were added to 2.55 ml of 350 mM potassium phosphate buffer (pH 8.0) containing 0.35 mM NTB and 1% Triton X-100. 0.15 ml of a 1,5-AG solution and 0.3 ml (0.54 units) of a 1,5-AG dehydrogenase solution obtained in Example 2 were added to give a total of 3.0 ml of a reaction solution.
The reaction was carried out at 37 ° C. for 10 minutes, and the absorbance at 540 nm was measured to prepare a calibration curve. FIG. 5 shows the result. The calibration curve is 0
It becomes a straight line up to μ50 μg / ml, indicating that 1,5-AG can be quantified using NTB as a reducing color former.

Example 4 [Quantitative determination of 1,5-AG using WST-1] The above procedure was repeated except that WST-1 was used in place of the reducing color former of Example 3 and the absorbance measurement wavelength was 450 nm. Measurement was carried out in the same manner as in the above to prepare a calibration curve. FIG. 6 shows the result. The calibration curve was a straight line from 0 to 50 μg / ml, indicating that 1,5-AG can be quantified using WST-1 as a reducing color former.

[0052]

As described above, the 1,5-AG dehydrogenase of the present invention acts specifically on 1,5-AG in the absence of an electron carrier to directly reduce a reducing color former. Have. Therefore, the method for quantifying 1,5-AG of the present invention using this method measures a reduced color-forming substance produced directly by the action of a specific enzyme. It can be measured simply and accurately and is extremely useful especially for diagnosis of diabetes and the like.

[Brief description of the drawings]

Figure 1: Agrobacterium tumefaciens IFO135
Optimal of the 1,5-AG dehydrogenase of the present invention obtained by culturing 33
It is a graph which shows pH.

[Figure 2] Agrobacterium tumefaciens IFO135
3 is a graph showing the pH stability of 1,5-AG dehydrogenase of the present invention obtained by culturing No. 33.

Fig. 3 Agrobacterium tumefaciens IFO135
1 is a graph showing the optimum temperature of 1,5-AG dehydrogenase of the present invention obtained by culturing No. 33.

FIG. 4 Agrobacterium tumefaciens IFO135
3 is a graph showing the temperature stability of 1,5-AG dehydrogenase of the present invention obtained by culturing No. 33.

FIG. 5: Agrobacterium tumefaciens IFO135
33 is a calibration curve of 1,5-AG measurement using 1,5-AG dehydrogenase of the present invention obtained by culturing No. 33 and NTB as a reducing color former.

FIG. 6: Agrobacterium tumefaciens IFO135
3 is a calibration curve of 1,5-AG measurement using 1,5-AG dehydrogenase of the present invention obtained by culturing No. 33 and WST-1 as a reducing color former.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:01)

Claims (6)

[Claims] 1. A 1,5-anhydroglucitol dehydrogenase having the following physicochemical properties: (1) Action: In the absence of an electron carrier, specifically acts on 1,5-anhydroglucitol to oxidize the hydroxyl group at the 2-position and catalyze the reaction of directly reducing the reducing color former. Have. (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 8.0-9.0. (4) pH stability: stable at pH 7.0 to 9.0. (5) Optimum temperature: The optimum temperature is around 35-40 ° C. (6) Temperature stability: Stable at least 90% up to around 40 ° C. (7) Electron acceptor: In addition to 2,6-dichlorophenolindophenol, ferricyan compound, etc., a reducing color former can also be used. In addition, coenzymes such as NAD and NADP and dissolved enzymes cannot be used. (8) Molecular weight: The molecular weight determined by dodecyl sulfate-polyacrylamide electrophoresis is about 55,000. 2. The 1,5-anhydroglucitol according to claim 1, which is produced by a microorganism belonging to the genus Agrobacterium and capable of producing 1,5-anhydroglucitol dehydrogenase. Dehydrogenase. 3. A microorganism belonging to the genus Agrobacterium and capable of producing 1,5-anhydroglucitol dehydrogenase is cultured in a nutrient medium, and 1,5-anhydroglucitol is contained in the membrane fraction. A method for producing 1,5-anhydroglucitol dehydrogenase, which comprises producing and accumulating dehydrogenase, and collecting the resulting dehydrogenase. 4. The method according to claim 3, wherein the microorganism belonging to the genus Agrobacterium and capable of producing 1,5-anhydroglucitol dehydrogenase is Agrobacterium tumefaciens. 5. A sample containing 1,5-anhydroglucitol, which is reacted with the 1,5-anhydroglucitol dehydrogenase according to claim 1 in the presence of a reducing color former to form a sample. Measuring the reduced color forming substance
Determination of anhydroglucitol. 6. The method according to claim 5, wherein the reducing color former is tetrazolium or a salt thereof.