JP2886846B2 - 1,5-anhydroglucitol dehydrogenase and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel 1,5-anhydroglucitol dehydrogenase useful as a reagent for measuring 1,5-anhydroglucitol useful for the diagnosis of diabetes and the like, and a novel 1,5-anhydroglucitol dehydrogenase useful therefor. Related to manufacturing method.

[0002]

2. Description of the Related Art 1,5-Anhydroglucitol (hereinafter, referred to as "anhydroglucitol")
“1,5-AG”) is present in body fluids such as human serum, plasma, and urine, and its amount in certain kinds of diseases, particularly diabetes, greatly fluctuates. Therefore, 1,5-
The measured value of AG has become a useful diagnostic index and has recently become an important test item in clinical practice.

[0003] As a method for determining 1,5-AG, a method in which pyranose oxidase is allowed to act and the generated hydrogen peroxide is colorimetrically determined using a peroxidase color developing system (Japanese Patent Publication No. 5-41)
238) has become mainstream, and has recently been applied to general-purpose automatic analyzers.

As another method, 1,5-AG oxidase (see Japanese Patent Publication No. 3-24200) is acted to reduce the amount of oxygen consumed, the reduced form of the electron acceptor or the oxidized 1,5-AG. Methods for measuring reaction products and the like are known.

[0005]

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. In the case of the oxidase coloring system, it was difficult to say that the sensitivity was sufficient.

[0006] In the method of measuring the reduced form using an oxidoreductase utilizing an electron acceptor, the reduced form itself such as 2,6-dichlorophenolindophenol or a ferricyan compound which is an electron acceptor is also used. However, there are problems such as the instability of the sample, the sensitivity of the sample is not sufficient, and the application to a general-purpose automatic analyzer for processing a large number of samples is difficult.

[0007] Accordingly, an object of the present invention is to provide a novel 1,5 AG that specifically acts on 1,5-AG and has excellent storage stability and is applicable to a quantitative reagent for a general-purpose automatic analyzer. -AG
An object of the present invention is to provide a dehydrogenase and a method for producing the same.

[0008]

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
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 microorganisms belonging to the genus bacterium. When 1,5-AG is reacted with 1,5-AG dehydrogenase, it oxidizes the 2-position hydroxyl group of 1,5-AG and reduces the electron acceptors such as 2,6-dichlorophenolindophenol. When catalyzed and the reaction is carried out using a reducing color former, even in the absence of an electron carrier, the 2-position hydroxyl group of 1,5-AG is oxidized and the reducing color former is directly reduced to produce a color forming substance. Have already filed a patent application.
-323969).

However, in the above-mentioned application, 1,5-AG dehydrogenase is produced by culturing known strains such as Agrobacterium tumefaciens IF013532, Agrobacterium tumefaciens IF013533, and the like. 1,5-AG dehydrogenase was not yet satisfactory in terms of storage stability.

[0010] In order to obtain 1,5-AG dehydrogenase having excellent storage stability suitable as a measurement reagent, a broad search was conducted for microorganisms capable of producing 1,5-AG dehydrogenase. Have found that a novel strain belonging to Agrobacterium tumefaciens newly isolated from soil by S. et al. Produces 1,5-AG dehydrogenase with extremely excellent storage stability, and completes the present invention. Reached.

That is, one aspect of the present invention is to provide a 1,5-AG dehydrogenase having the following physicochemical properties.

(1) Action: In the absence of an electron carrier, it specifically catalyzes a reaction that specifically acts on 1,5-AG to oxidize the 2-position hydroxyl group and directly reduce the reducing color former.

(2) substrate specificity: strongly acting on 1,5-AG,
Acts weakly 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: 90% or more stable up to around 40 ° C.

(7) Method for measuring titer 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% nonionic surfactant, 50 mM A total of 3.0 ml of the enzyme-containing solution consisting of 0.3 ml of 5-anhydroglucitol aqueous solution, 0.45 ml of water, and 0.15 ml of the enzyme solution was reacted at 37 ° C.
The absorbance change (△ mOD / min) is measured. The molecular extinction coefficient of the formazan dye produced under these conditions is 16.7x
And 10 ^3, 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 (2).

[0019]

(Equation 2)

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

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

(10) Storage stability: dissolved in 100 mM Tris-HCl buffer (pH 7.0) at a concentration of 0.1 u / ml,
Is 90% or more after storage for 14 days under the above conditions.

The 1,5-AG dehydrogenase belongs to the genus Agrobacterium, and is preferably produced by a microorganism capable of producing 1,5-AG dehydrogenase.

Another aspect of the present invention is to culture a microorganism belonging to the genus Agrobacterium and capable of producing 1,5-AG dehydrogenase having the above-mentioned physicochemical properties in a nutrient medium, and adding the microorganism to a membrane fraction. It is intended to provide a method for producing 1,5-AG dehydrogenase, which comprises producing, accumulating 1,5-AG dehydrogenase, and collecting this.

In this method, the microorganism having an ability to produce 1,5-AG dehydrogenase includes Agrobacterium tumefaciens NT1130 (Agrobacterium tumefaciens NT1 strain).
1130, FERM BP-5997 strain).

According to the present invention, in the absence of an electron carrier, it specifically acts on 1,5-AG to oxidize the hydroxyl group at the 2-position and catalyze the reaction of directly reducing the reducing color former. , 5-AG dehydrogenase can be provided. Then, by measuring the reduced coloring substance directly generated by the action of the enzyme, 1,5-AG in the sample can be measured simply and accurately. In addition, the 1,5-AG dehydrogenase has excellent storage stability, and is extremely advantageous for commercialization as a quantitative reagent.

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Enzymes conventionally produced by microorganisms belonging to the genus Agrobacterium include Hayano et al. (Journal of Biological Chemistry, 242, 3).
665-3692, 1967) confirmed the presence of glucoside-3-dehydrogenase in the cytoplasmic fraction. Meanwhile, Takeuchi et al. (Journal of Biochemistry, Vol. 100, 1049-1055, 1986)
It has been reported that AG can be a substrate for glucoside-3-dehydrogenase.

On the other hand, as described above, the present inventors have found that in the membrane fraction of a microorganism belonging to the genus Agrobacterium, 1,1 in the absence of an electron carrier.
A metabolic enzyme that oxidizes 5-AG and directly reduces a reducing color former has been found (Japanese Patent Application No. 8-323969). The action of this enzyme is as shown in the following chemical formula 1.

[0029]

Embedded image

The enzyme of the present invention also comprises Agrobacterium (Ag
robacterium), which is found in the membrane fraction of a microorganism belonging to the genus robacterium, and has the action shown in the above chemical formula 1.
However, the enzyme of the present invention is disclosed in Japanese Patent Application No. 8-323969.
The difference is that the storage stability is remarkably superior to that of the enzyme described in the above publication.

The microorganism used for producing the enzyme of the present invention belongs to the genus Agrobacterium and produces 1,5-AG dehydrogenase having the above-mentioned physicochemical properties and excellent storage stability. Any strain may be used as long as it is present, but a preferable example is Agrobacterium tumefaciens NT1130 (Agrobacterium tumefaciens NT1130, FERM BP-5997) newly isolated from soil by the present inventors.
Co., Ltd.). The mycological properties of this microorganism are 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 against this bacterium
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 bacterium was deposited on June 27, 1997 with the Research Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry, and its deposit number was
FERM BP-5997.

The 1,5-AG dehydrogenase of the present invention can be obtained, for example, by culturing the above strain in a nutrient medium, producing and accumulating 1,5-AG dehydrogenase in a membrane fraction, and collecting the resulting product. Can be manufactured. As the nutrient medium, 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 inorganic nitrogen sources, ammonium sulfate, ammonium chloride,
Ammonium phosphate and the like, sodium as an inorganic salt,
Salts such as potassium, magnesium, iron and manganese can be used.

The cultivation is preferably performed under aerobic conditions such as shaking and aeration and stirring, and is preferably performed 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 microorganism, 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. Then, 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.

Next, Agrobacterium.
1, of the present invention obtained using Tumefaciens NT1130 strain
5 shows the properties of 5-AG dehydrogenase. Note that 1,5-
The activity of the AG dehydrogenase was measured in accordance with the above-described titer measurement method.

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

[0042]

[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-3, which has the effect of oxidizing the 3-hydroxyl group of 5-AG
-Dehydrogenase (EC1.1.99.13) was reacted under the same conditions, and analyzed by HPLC. As a result, the peak of the reaction product appeared at a new retention time of 10.8 minutes along with a decrease in the 1,5-AG peak. On the other hand, pyranose oxidase (EC1.1.3.10), which has the action of oxidizing the 2-position hydroxyl group of 1,5-AG,
The solution was added to 40 mM borate buffer (pH 7.5) containing 2.5 mM 1,5-AG and reacted at 37 ° C. As a result, the peak of 1,5-AG decreased, and a new retention time of 9.6 minutes was reached. A peak of the reaction product appeared. In these results, the retention time of the reaction product obtained when the enzyme of the present invention was reacted with 1,5-AG and the reaction product obtained when the enzyme of the present invention was reacted with pyranose oxidase were the same. 1,
It can be seen that the oxidation site of 5-AG is a 2-position hydroxyl group.

(2) Substrate specificity: In the aforementioned enzyme activity measurement method, the relative reactivity (substrate specificity) in the case where various sugar solutions (all at a final concentration of 5 mM) were used instead of 1,5-AG was determined. It is shown in Table 2. The enzyme of the present invention acts strongly on 1,5-AG, weakly on L-sorbose, and slightly on D-glucose.

[0045]

[Table 2]

(3) Optimum pH: Using the enzyme of the present invention, instead of the buffer in the above-mentioned enzyme activity measurement method,
Enzyme activities were measured using a citrate buffer, a potassium phosphate buffer, a Tris-HCl buffer, and a glycine buffer having a pH. The result is shown in FIG. From FIG.
It can be seen 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 was added to citrate buffer, potassium phosphate buffer, Tris-HCl buffer, and glycine buffer (200 mM for each buffer) having various pH values. After treatment at 60 ° 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 6.0 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 45 ° C.

(6) Temperature stability: The enzyme of the present invention was added to a 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 of the present invention is 90% or more stable up to around 40 ° C.

(7) Electron acceptor: 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. 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.

[0051]

[Table 3]

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

(9) Km value: Line weaver bark (Line
The Km value of the enzyme of the present invention with respect to 1,5-AG was determined by a weaver-Burk) plot 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, known strains Agrobacterium tumefaciens IF013532 and Agrobacterium.
1,5-AG dehydrogenase was produced in the same manner as in Examples 1 and 2, using Tumefaciens IF013533. The results of measuring the storage stability of these enzymes in the same manner as described above are also shown in FIG.

In FIG. 5, ■-■ indicate Agrobacterium.
The results of the enzyme of the present invention derived from T. faecalis NT1130 strain are shown, ◆-◆ indicates the result of the enzyme derived from Agrobacterium tumefaciens IF013532, and ▲-▲ indicates the result of the enzyme derived from Agrobacterium tumefaciens IF013533. Show.

As shown in FIG. 5, the 1,5-AG dehydrogenase of the present invention derived from Agrobacterium tumefaciens NT1130 strain has a residual activity of 90% or more after storage for 14 days, and is excellent. Storage stability. In contrast, 1,5-AG derived from Agrobacterium tumefaciens IF013532
Dehydrogenase and Agrobacterium tumefaciens
All the 1,5-AG dehydrogenases derived from IF013533 have a residual activity of 40% or less after storage for 14 days, and thus have extremely poor storage stability.

The Agrobacterium tumefaciens NT1130 is different from the known Agrobacterium tumefaciens in that it produces 1,5-AG dehydrogenase having excellent storage stability. It was determined to be a new strain belonging to B. tumefaciens.

The 1,5-AG dehydrogenase of the present invention is a 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IF013532.
AG dehydrogenase, and 1,5-AG dehydrogenase from Agrobacterium tumefaciens IF013533, there was no significant difference in other physicochemical properties, but the storage stability was significantly different In this respect, it can be clearly distinguished from the 1,5-AG dehydrogenase derived from the above-mentioned known strain.

The method for quantifying 1,5-AG using the enzyme of the present invention can be performed, for example, by the following method. That is, a test sample 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.

The buffer which can be used in the above-mentioned quantitative method is pH
Any commonly used phosphate buffer, Tris-HCl buffer, Good buffer, borate buffer, and the like can be used.

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), 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 this assay, 1,5-AG dehydrogenase is used in an amount of 20 to 10,000 units / liter, particularly 200 to 2,000 units / liter.
It is preferred to use in the liter range.

The samples to which the above quantification method can be applied include:
Examples include biological samples such as serum, plasma, and urine containing 5-AG.

In the measurement of the reduced color-forming substance in the reaction solution, the reaction is carried out as described above, and then 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.

[0066]

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 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 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.

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 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.

Test Example 1 [Quantification of 1,5-AG Using NTB] Various concentrations of 1,5-AG were added to 2.25 ml of 350 mM potassium phosphate buffer (pH 8.0) containing 0.35 mM NTB and 1% Triton X-100. , 5-AG solution and 0.1 ml of 1,5-AG dehydrogenase solution obtained in Example 2, 0.3 ml (0.54 units) were added, and a total of 3.0 ml of the reaction solution was added.
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. 6 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.

Test Example 2 [Quantification 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 Test Example 1 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. 7 shows the result. The calibration curve was linear from 0 to 50 μg / ml, indicating that 1,5-AG could be quantified using WST-1 as a reducing color former.

[0072]

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, by measuring the reduced chromogenic substance directly generated by the action of the above specific enzyme, 1,5-
AG can be measured easily and accurately and is extremely useful especially for diagnosis of diabetes and the like. In addition, the 1,5-AG dehydrogenase of the present invention has excellent storage stability, and is therefore extremely advantageous in commercializing it as a quantitative reagent.

[Brief description of the drawings]

Fig. 1 Agrobacterium tumefaciens NT1130
Of the 1,5-AG dehydrogenase of the present invention obtained by culturing the strain
4 is a chart showing pH.

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

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

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

FIG. 5 shows the 1,5-AG dehydrogenase of the present invention, 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IF013532, and Agrobacterium tumefaciens IF01.
1 is a table showing the storage stability of 1,533-AG dehydrogenase derived from 3533 in comparison.

FIG. 6 is a calibration curve for the measurement of 1,5-AG using the 1,5-AG dehydrogenase of the present invention and NTB as a reducing color former.

FIG. 7 is a calibration curve for the measurement of 1,5-AG using the 1,5-AG dehydrogenase of the present invention and WST-1 as a reducing color former.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12N 9/04 C12Q 1/32 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A 1,5-anhydroglucitol dehydrogenase having the following physicochemical properties: (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: 90% or more stable up to around 40 ° C. (7) Method of measuring titer 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% nonionic surfactant, 50 mM 1,5-anne A total of 3.0 ml of an enzyme-containing solution consisting of 0.3 ml of an aqueous hydroglucitol solution, 0.45 ml of water, and 0.15 ml of an enzyme solution was reacted at 37 ° C.
The absorbance change (△ mOD / min) is measured. The molecular extinction coefficient of the formazan dye produced under these conditions is 16.7x
And 10 ^3, 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 and ferricyan compound, a reducing color former can be used. Also,
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. (10) Storage stability: 100 mM Tris-HCl buffer (pH 7.
0) is dissolved at a concentration of 0.1 u / ml and stored at 4 ° C. for 14 days, and the residual activity is 90% or more. 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. The method according to claim 1, which belongs to the genus Agrobacterium.
The microorganism having the ability to produce 1,5-anhydroglucitol dehydrogenase described is cultured in a nutrient medium to produce and accumulate 1,5-anhydroglucitol dehydrogenase in the membrane fraction. A method for producing 1,5-anhydroglucitol dehydrogenase, which comprises collecting. 4. The method according to claim 1, which belongs to the genus Agrobacterium.
The microorganism having the ability to produce 1,5-anhydroglucitol dehydrogenase according to the present invention is Agrobacterium tumefaciens NT1130 strain (Agrobacterium tumefaciens NT1130, FERM
4. The method for producing 1,5-anhydroglucitol dehydrogenase according to claim 3, which is BP-5997 strain).