JPH10313857A - Kinase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new kinase, having high substrate specificity for 1,5- anhydroglucitol (1,5-AG), capable of phosphorylating the 1,5-AG in the presence of nucleoside diphosphates and Mg ions and useful for the measurement, etc., of the 1,5-AG with a high accuracy. SOLUTION: This new ADP-dependent hexokinase has higher substrate specificity for 1,5-anhydroglucitol (1,5-AG) than that in the case of using glucose as a substrate, is capable of catalyzing a reaction for substituting hydroxyl group at the 6-position of the 1,5-AG with phosphate group in the presence of nucleoside diphosphates and magnesium ions and producing 1,5-anhydroglucitol phosphate and nucleoside monphosphates, is used for simply measuring the 1,5-AG with a high accuracy and is useful for diagnosis, etc., of diabetes by measuring the amount of the 1,5-AG in blood plasma. The enzyme is obtained by culturing a strain of Thermococcus litoralis ATCC51850 in a culture medium at 95 deg.C for 20 hr, then separating and crushing the microbial cell and extracting and purifying an active fraction from the resultant crushed liquid of the microbial cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for substituting 1,6-anhydroglucitol (hereinafter abbreviated as 1,5AG) at the 6-position hydroxyl group with a phosphate group in the presence of nucleoside diphosphates and magnesium ions. Catalyzes a reaction for producing 5-anhydroglucitol hexaphosphate (hereinafter abbreviated as 1,5AG6P) and nucleoside monophosphate, and comprises 1,5AG
Novel ADP-dependent hexokinase having high substrate specificity for the enzyme and the field of diagnostic enzymes.

[0002]

2. Description of the Related Art 1,5AG is a compound which is present in human cerebrospinal fluid, plasma, serum, urine and the like, and has been reported to decrease the amount in certain diseases, especially in diabetes (Japanese Internal Medicine). Journal of the Society of Japan 80, 1198-1204, 19
91), a substance that has recently been used as a diagnostic marker for diabetes.

Conventionally, as a method for measuring 1,5AG in a biological sample, gas chromatography (diabetes, 25
Vol. 11, 115-1118, 1982) and a method of measuring using pyranose oxidase (JP-A-63-1853).
No. 97), a method of measuring using glucokinase or hexokinase (JP-A-8-107796).
Etc. were known.

[0004] However, the gas chromatography method requires labeling of 1,5AG as a pretreatment of a test solution, and requires a long time for measurement, and it is difficult to measure a large number of samples simultaneously. In addition, there are drawbacks such as the need for advanced technology for maintenance and management of analytical instruments, and it is inconvenient to apply it to actual clinical practice. In addition, the method for measuring 1,5AG using pyranose oxidase is based on the fact that pyranose oxidase acts widely on pyranoses as a substrate.
5AG could not be measured accurately.

Further, ATP is used as a phosphate donor for 1,5
Hexokinase and glucokinase were known as enzymes that phosphorylate AG, but hexokinase and glucokinase show high specificity for glucose.
Has almost no effect on 5AG, and practically 1,5-A
Measurement of G was not possible.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel enzyme having a high specificity for 1,5AG and, at the same time, having a long-term storage stability as a liquid reagent.

[0007]

Means for Solving the Problems First, the present inventors have set forth 1,
When a wide variety of enzymes that specifically act on 5AG were screened from microorganisms, it was found that ADP, which is a kind of nucleoside diphosphates, was present in the cells of the ultra-high thermophilic bacterium Thermococcus litoralis ATCC 51850.
ADP-dependent hexokinase that catalyzes the reaction that phosphorylates 5AG to generate 1,5AG6P (hereinafter ADP-
HK) for the first time, and in particular, it has been found that 1,5AG has a higher substrate specificity than using glucose as a substrate.

The present invention has been completed on the basis of the above findings, and is 1,5 times smaller than when glucose is used as a substrate.
It has high substrate specificity for AG, and catalyzes the reaction of generating 1,5AG6P and nucleoside monophosphates by substituting the hydroxyl group at the 6-position of 1,5AG with a phosphate group in the presence of nucleoside diphosphates and magnesium ions. AD
It concerns P-HK.

First, a method for obtaining ADP-HK of the present invention will be described. 1,5AG compared to using the glucose of the present invention
ADP-HK having high substrate specificity is not limited as long as it is an enzyme having high substrate specificity to 1,5AG as a substrate. For example, ADP-HK is a thermophilic bacterium Thermococcus litoralis. ADP-HK derived from ATCC 51850 strain. The hyperthermophilic bacterium Thermococcus litoralis ATCC 51850 is from the American Type Culture Collection (ATC).
C) and the ATCC catalog (19th, 199th)
6 years) and is available to many.

ADP-HK-producing bacteria can be cultured, for example, by using the same medium as in the examples described later without aeration, and the culture temperature may be within the temperature range in which the bacteria grow. ~ 110 ° C, preferably 85 ~ 10
5 ° C. The culture time may be the culture time at which the target enzyme has the highest titer, for example, 1 to 3 days, and the target enzyme may be collected.

ADP-HK is mainly contained and accumulated in the cells, and may be extracted from the cells.
For example, the extraction method of ADP-HK is to separate cells from the culture solution by centrifugation or the like, suspend the cells in a buffer such as a phosphate buffer or a Tris-HCl buffer, and then add lysozyme,
The mixture is crushed by ultrasonic waves, glass beads, etc., and centrifuged, and the soluble fraction is recovered as a crude enzyme solution.

The crude ADP-H thus obtained
The purified ADP-HK can be obtained by treating the K-containing solution using known protein and enzyme isolation and purification means. For example, a general enzyme purification method such as a fractional precipitation method using an organic solvent such as acetone or ethanol, a salting out method using ammonium sulfate, an ion exchange chromatography method, a hydrophobic chromatography method, an affinity chromatography method, and a gel filtration method is appropriately selected. ,
Purified ADP-HK can be obtained in combination with 5 to 50% of a suitable stabilizer such as sucrose, glycerol or the like.
Alternatively, amino acids, coenzymes, and the like may be added in an amount of about 0.01 to 0.1%, and may be cryopreserved or lyophilized.

Next, the reaction mechanism when 1,5AG of ADP-HK is used as a substrate is Escherichia coli DH
1,5 present in the cells of strain 1 (ATCC 33849)
It examined using anhydroglucitol 6-phosphate dehydrogenase (1,5AG6PDH). These 1,5
The reaction of ADP-HK using AG as a substrate is represented by the following reaction formula.

[0014]

Embedded image

## EQU1 ## Also, 1,5AG6PDH
Is an enzyme that catalyzes at least the following reaction formula.

[0016]

Embedded image

## EQU1 ## 1,5AG6PDH uses 1,5AG6P as a substrate, and consumes oxidized nicotinamide adenine dinucleotide (phosphate) [oxidized NAD (P): NAD (P) ⁺ ] as a coenzyme to reduce nicotinamide adenine. There is no particular limitation as long as it is 1,5AG6PDH that produces dinucleotide (phosphate) [reduced NAD (P): NAD (P) H + H ⁺ ].

The 1,5AG6PDH producing bacterium is not limited as long as it is a 1,5AG6PDH producing bacterium belonging to the genus Escherichia.
The mutant having the ability to produce H may be used.
Preferably, a production bacterium belonging to the genus Escherichia coli is considered, and more preferably, Escherichia coli D.
H1 (ATCC 33849) is stored in the ATCC,
It is described in the catalog of TCC (18th, 1992), and is available to many people.
AG6PDH is preferred.

Next, in obtaining 1,5AG6PDH, preferably Escherichia coli DH1 (ATCC3
3849), and the culture means may be solid culture or liquid culture, but is preferably aeration culture using a flask, a jar or the like. As the medium, those commonly used for culturing microorganisms are widely used. As a carbon source, glucose, glycerol, sorbitol, lactose, etc., as a nitrogen source, yeast extract, meat extract, tryptone, peptone, etc., and as an inorganic salt, sodium chloride, magnesium chloride, magnesium sulfate, calcium chloride, etc. may be used. Culture conditions include, for example, pH
The target enzyme may be collected at 6.5 to 7.5 at a culture temperature of 25 to 37 ° C. and a culture time at which the target enzyme has the highest titer, for example, 18 to 30 hours.

1,5AG6PDH is mainly contained and accumulated in the cells, and may be extracted from the cells. If the extraction method of 1,5AG6PDH is exemplified, the cells are separated from the culture solution by centrifugation, and the cells are suspended in a buffer such as a phosphate buffer or a Tris-HCl buffer, and then lysozyme, ultrasound, Crushed by glass beads or the like and centrifuged, and the soluble fraction is collected as a crude enzyme solution.

The thus obtained crude 1,5AG
By treating the 6PDH-containing solution using known protein and enzyme isolation and purification means, the purified 1,5AG6
PDH can be obtained. For example, a general enzyme purification method such as a fractional precipitation method using an organic solvent such as acetone or ethanol, a salting out method using ammonium sulfate, an ion exchange chromatography method, a hydrophobic chromatography method, an affinity chromatography method, and a gel filtration method is appropriately selected. Can be combined with each other to obtain purified 1,5AG6PDH, and about 5 to 50% of an appropriate stabilizer such as sucrose, glycerol, etc., and 0.1% of an amino acid, a coenzyme or the like.
It may be cryopreserved or lyophilized by adding about 01 to 0.1%.

When 1,5AG of ADP-HK was used as a substrate, the reaction product was found to be 1,5 anhydroglucitol hexaphosphate because of the action of 1,5AG6PDH.
The reaction formula when 1,5AG is used as a substrate is

[0023]

Embedded image

## EQU1 ## Next, the ADP-HK of the present invention
The method for measuring the physicochemical properties and enzyme activity of is described. ADP-HK Enzyme Activity Assay Measuring Reagent 50 mM Tris-HCl buffer (pH 7.5) 20 mM Glucose 2 mM ADP 2 mM MgCl ₂ 5 U / ml glucose 6-phosphate dehydrogenase (G6PDH, manufactured by Toyobo) 0.025% NBT 1 mM NADP 1% Triton X-100 5 U / ml diaphorase (NADPH) After preliminarily heating 1 ml of the measurement reagent at 37 ° C. for 1 minute, 0.
Add 02 ml of enzyme solution and react for 10 minutes. After the reaction, the reaction was stopped by adding 2 ml of 0.1N hydrochloric acid, and
Using a cell with a layer length of 1.0 cm within a
The absorbance at m is measured (As). As a blind test, the same operation is performed using 0.02 ml of distilled water instead of the enzyme solution, and the absorbance is measured (Ab). The absorbance difference between the absorbance of the enzyme (As) and the absorbance of the blind test (Ab) (A
The enzyme activity is determined from s-Ab). One unit of enzyme activity is 3
The amount of the enzyme that generates 1 μmol of reduced NADP per minute at 7 ° C. is defined as the amount of the enzyme, and the calculation formula is as follows. Enzyme activity (U / ml) = (As-Ab) × 0.795 ×
Enzyme dilution ratio Physicochemical properties of ADP-HK (1) Enzymatic action Enzymatic action when glucose is used as a substrate is shown below.

[0025]

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(2) Substrate specificity Substrate specificity is determined by using each substrate in the presence of ADP and MgCl _2.
After reacting with DP-HK, the amount of change in ADP was measured using Showdex Asahippak.
ak) It was measured by an HPLC method using a GS-320HQ column (manufactured by Showa Denki Kogyo KK). Table 1 shows the results. From Table 1, the ADP-HK of the present invention showed the highest substrate specificity for 1,5AG.

[0027]

[Table 1]

A: Bacillus stearothermophilus-derived glucokinase (manufactured by Pharmacia) B: Yeast-derived hexokinase (manufactured by Oriental Yeast Co., Ltd.) C: Pyrococcus furiosus-derived ADP-HK (Japanese Patent Application No. 7-340482) (3) Isoelectric point 4.1 ± 0.5 (by electrophoresis using carrier ampholine at pH 3.5 to 10) (4) Molecular weight 50,000 ± 5000 (gel filtration using TSK-G3000SW _XL ) (5) Optimum pH pH 4.0 to 5.0 is acetate buffer (○-○), pH 5.0.
0-6.5 is citrate buffer (△-△), pH 6.5-
7.5 is a phosphate buffer solution (□-□), pH 7.5 to 8.5
Is Tris-HCl buffer (●-●), pH 8.5-10.
5 used a glycine-NaOH buffer (▲-▲). FIG. 1 shows the examination results of the optimum pH. From Fig. 1, the optimum pH is 7
~ 7.5. (6) pH stability pH 4.0 to 5.0 is acetate buffer (液-○), pH 5.0.
0-6.5 is citrate buffer (△-△), pH 6.5-
7.5 is a phosphate buffer solution (□-□), pH 7.5 to 8.5
Is Tris-HCl buffer (●-●), pH 8.5-10.
5 used a glycine-NaOH buffer (▲-▲). 3
FIG. 2 shows the examination results of the pH stability after the treatment at 7 ° C. for 60 minutes. From FIG. 2, the best stability was exhibited in the pH range of 6.5 to 10.5. (7) Optimum temperature FIG. 3 shows the results of the examination of the optimum temperature. 3, the optimum temperature was 80 to 100 ° C. (8) Thermal stability 100 mM Tris-HCl buffer (pH 7.5) (ADP
-HK 0.5 U / ml) after heat treatment at each temperature for 10 minutes. As a result, the residual activity was stable from 80% or more to at least 95 ° C. (FIG. 4). (9) Influence of divalent metal ions In place of magnesium ions, various divalent metal ions were added, and the enzymatic activities were measured. The results are shown in Table 2. From Table 2, the ADP-HK of the present invention was most activated by magnesium ions and inhibited by copper ions.

[0029]

[Table 2]

(10) Influence of various additives Table 3 shows the influence of various additives such as other metal ions and surfactants based on the activity measurement method (containing ₂ mM MgCl ₂ ). According to Table 3, the ADP-HK of the present invention is ED
Inhibited by TA.

[0031]

[Table 3]

(11) Action on nucleoside diphosphates ADP, GDP, CD as nucleoside diphosphates
Table 4 shows the results of activity measurement using P, UDP and IDP. From Table 4, the ADP-HK of the present invention showed high specificity for ADP and CDP.

[0033]

[Table 4]

[0034]

Next, the present invention will be described in detail with reference to examples and reference examples, but the present invention is not limited to these examples.

[0035]

[Reference Example 1] Culture of 1,5AG6PDH-producing bacteria Culture of Escherichia coli / DH1 (ATCC33849) Medium composition 0.5% Meat extract 1.5% Sorbitol 1.0% Tryptone 1.5% Beer yeast extract 50 ml of a liquid medium (pH 7.5) containing the mixture is dispensed into two 300 ml Erlenmeyer flasks.
After heat sterilization for 0 minutes, Escherichia. Coli D
1 ml of a cell suspension of H1 (ATCC 33849) was transplanted and cultured at 30 ° C. for 15 hours while stirring to obtain a seed culture solution. The above medium components and antifoam FS Antifoam 0
Liquid medium 5 containing 0.3% 28 (manufactured by Dow Corning)
After sterilizing the mixture in two 10-liter jars, the seed culture was transplanted, and the pH of the culture was controlled at 37 ° C. with 5N NaOH to be 6.5 or more while aerobically stirring.
After culturing for 4 hours, 10 l of a culture solution having a culture titer of 15 mU / ml
I got

Purification of 1,5AG6PDH 10 l of the obtained culture was centrifuged, and the obtained cells were washed once with 50 mM Tris-HCl buffer (pH 8.5). The washed cells were washed with 50 mM Tris-HCl buffer (p.
H8.5), adjusted to 1.2 l, and DYNO-M
The cells were disrupted using ILL (manufactured by Willy A. Bachofen) to obtain a disrupted cell suspension.

This crushed liquid was subjected to 50 m
After dialyzing overnight at 5 ° C. against 20 l of M Tris-HCl buffer (pH 8.5), the mixture was centrifuged at 8000 rpm for 60 minutes to obtain 1.0 l (enzyme activity: 120 U) of supernatant.
This supernatant was treated with 50 mM Tris-HCl buffer (pH 8.
DEAE-Sepharose FF buffered in 5)
(Pharmacia) 1200ml (2.6 x 38c
The column was eluted with a linear gradient of 0 to 0.25 M NaCl. As a result, about 0.15
An active fraction (75 U) was eluted at an M NaCl concentration.
NaCl was dissolved in the obtained active fraction to a concentration of 3 M, and a 50 mM Tris-HCl buffer (pH 8.5),
Phenyl-Seph equilibrated with 3M NaCl
arose CL4B (Pharmacia) 500ml
It was passed through a (2.6 × 38 cm) column and eluted with a linear gradient of 2.5 to 0 M NaCl. As a result, the active fraction (50
U) was obtained. The obtained active fraction was dialyzed overnight against 20 mM Tris-HCl buffer (pH 8.5),
After passing through a 50 ml (2.6 × 19 cm) column of Blue-sepharose (Pharmacia) buffered with 20 mM Tris-HCl buffer (pH 8.5),
Elution was performed with a linear gradient of １1 M NaCl, 20% ethylene glycol. As a result, about 0.6
An active fraction (30 U) was eluted at a concentration of M NaCl and 12% ethylene glycol. The obtained active fraction was dialyzed overnight against 50 mM Tris-HCl buffer (pH 8.5), and then buffered with 50 mM Tris-HCl buffer (pH 8.5) Resource Q (Pharmacia).
Elution was performed with a linear gradient of 0-0.3 M NaCl through a 6 ml column. As a result, about 0.
An active fraction (28 U) was obtained at a NaCl concentration of 15 M,
A homogeneous enzyme preparation was obtained.

Physicochemical properties of 1,5AG6PDH (1) Action 1,5AG6PDH is dehydrogenated in the presence of a coenzyme to give a compound of the formula (C ₆ H ₁₁ O ₈ P ₁ )

[0039]

Embedded image

Catalyze the reaction represented by (2) Measuring method of titer Measuring method of enzyme activity of 1,5AG6PDH Measuring reagent 50 mM CAPS-NaOH buffer (pH 10.0) 1 mM NADP 5 mM 1,5AG6P (CAPS: N-Cyclohexyl-3-aminopropanesu-sulfonic acid) Measurement 1 ml of the reagent is placed in a cell having an optical path length of 1 cm,
After preliminarily heating for 0.01 minute, add 0.01 ml of enzyme solution and
The absorbance (Aa) at a wavelength of 340 nm after 5 minutes and the absorbance (Ab) 1.5 minutes after addition of the enzyme solution are measured. The enzyme activity is determined from the absorbance difference (Ab-Aa) between the absorbances (Aa) and (Ab). When the absorbance (Ab) becomes 0.2 or more, the enzyme solution is diluted with 50 mM CAPS-NaOH buffer (pH 10.0) for measurement.
One unit of enzyme activity is 1 μmol of reduced N per minute at 37 ° C.
The amount of the enzyme that generates ADP is calculated by the following formula. Enzyme activity (U / ml) = (Ab-Aa) x 13.5 x dilution ratio of enzyme (3) Substrate specificity Using the above titration method, 1,5AG in the reaction solution was used.
Instead of 6P, using various substrates, 1,5AG6P
Table 5 shows the results of the determination of the relative activity to As a result, this enzyme showed substrate specificity for at least 1,5AG6P.

[0041]

[Table 5]

(3) Optimum pH pH 7.0 to 9.0 is determined using Tris-HCl buffer (□-□), p
H9.5 to 10.5 is a CAPS-NaOH buffer (■-
(2) was used. The optimum pH was around 9 to 10, and the results are shown in FIG. (4) pH stability pH 4.0 to 5.5 is citrate buffer (液-○), pH
5.5-7.0 are bistris-HCl buffers (●-●), p
H7.0-9.0 are Tris-HCl buffer (□-□), pH
9.5 to 11.0 is a CAPS-NaOH buffer solution (■-
(2) was used. It was stable at pH 6 to 9 after treatment at 50 ° C. for 30 minutes, and the results are shown in FIG.

(5) Optimum temperature The results of enzymatic reaction performed by changing the temperature conditions using the measuring method in the above titration method are as shown in FIG. 37-50 ° C. (6) Thermal stability 50 m comprising 1 U / ml of 1,5AG6PDH
The MCAPS-NaOH buffer (pH 9.5) was left at each temperature for 30 minutes and then according to the titration method described above.
As a result of measuring the residual activity of 1,5AG6PDH, as shown in FIG. 8, the enzyme was stable up to around 45 ° C.

(7) Influence by various substances In the above titration method, various additives were added.
Table 6 shows the results of measurement of the enzyme activity of 1,5AG6PDH against 1,5AG6P.

[0045]

[Table 6]

(8) Molecular weight 78,000 ± 6000 (TSKgel.G3000SW)
(By gel filtration method using XL (manufactured by Tosoh Corporation)) (9) Isoelectric point 4.7 ± 0.5 (carrier ampholite pH 3.5-
(Measured by electrophoresis using pH 10 (manufactured by Pharmacia)) (10) Km value: Km value for 1,5AG6P: 25 mM Km value for NADP: 0.09 mM (11) Mechanism of action confirmation test 1,5AG6P, NADP, triethylamine weight A reaction solution containing a carbonate buffer (pH 10.0) and 1,5AG6PDH is reacted at 37 ° C. for 8 hours. The carbonyl group generated in 1,5AG6P was reacted with 2,4-dinitrophenylhydrazine and HCl and separated as a hydrazone, and the mass spectrum was measured to find that the product had the composition formula (C ₆ H ₁₁ O ₈ P ₁ ) Was confirmed.

[0047]

Example 1 Thermococcus litoralis (Therm
ococcus litoralis) ATCC518
50 0.5% culture medium composition of 0.1% yeast extract strains tryptone 0.72% maltose _{2.39% NaCl 0.4% Na 2 SO} 4 0.07% KCl 0.02% NaHCO 3 0.01% KBr 0.03% H ₃ BO ₄ 1.08% MgCl ₂ 0.15% CaCl ₂ 0.0025% SrCl ₂ 0.025% NH ₄ Cl 0.014% K ₂ HPO ₄ 0.1% CH ₃ COONa 0 0.0015% N (COOH) ₃ 0.0005% MnSO ₄ 0.0014% FeSO ₄ 0.0002% NiCl ₂ 0.0001% CoSO ₄ 0.0001% ZnSO ₄ 0.00001% CuSO ₄ 0.000001% Na ₂ Wo ₄ 0.000001% Na ₂ Mo ₄ 0.1% cysteine hydrochloride 500 ml of a liquid medium (pH 7) containing the above medium components
Dispense into a single 00 ml Erlenmeyer flask,
After heat sterilization for 10 minutes, 10 ml of a cell suspension of Thermococcus litoralis ATCC 51850 strain was transplanted into this, and cultured at 95 ° C. for 20 hours with stirring to obtain a seed culture solution. 20 l / 30 liquid medium containing the above medium components
After sterilizing the jar, the seed culture is transplanted and cultured at 95 ° C. for 15 hours while gently stirring, and 5 mU / ml
Of the culture was obtained.

[0048]

Example 2 Purification of ADP-HK Twenty liters of the obtained culture was centrifuged, and the obtained cells were treated with 20 mM Tris-HCl buffer (pH 7.5) containing 0.9% NaCl. Washed twice. 20 mM washed cells
Suspended in Tris-HCl buffer (pH 7.5).
ml, and an ultrasonic crusher manufactured by Kubota (INSON
(ATOR 201M) at 180 W for 30 minutes to obtain a disrupted cell suspension.

This crushed liquid was centrifuged at 8000 rpm for 30 minutes to obtain 210 ml (enzyme activity: 122 U) of a supernatant. The supernatant was dialyzed overnight at 5 ° C. against 8 liters of 10 mM Tris-HCl buffer (pH 7.5) using a dialysis tube, and then 10 mM Tris-HCl buffer (pH 7.0).
DEAE-Sepharose FF buffered in 5)
(Pharmacia) 200ml (2.6 × 38cm)
And elution was performed with a linear gradient of 0 to 1 mol of NaCl. As a result, an active fraction (96 U) was eluted at a NaCl concentration of 0.65 to 0.7 mol.
NaCl was dissolved to 4 M in the obtained active fraction, and Phenyl-S buffered with 4 M NaCl was used.
epharose FF (Pharmacia) 200m
1 (2.6 × 38 cm) column, 4 to 0 M N
Elution was performed with a linear gradient of aCl. As a result, an active fraction (70) was obtained at a NaCl concentration of 0.02 to 0.07 mol. The obtained active fraction is 10 m
After dialysis overnight at 5 ° C. against 8 l of M Tris-hydrochloric acid (pH 7.5), hydroxyapatite (Pentax) 1 buffered with 10 mM Tris-hydrochloric acid buffer (pH 7.5) 1
Pass through a 00 ml (2.6 × 19 cm) column,
Elution was performed with a linear gradient of 0.5 M phosphate buffer (pH 7.5). As a result, 0.02-0.
The active fraction (60 U) was eluted at a phosphate buffer concentration of 03 M to obtain a homogeneous enzyme preparation.

[0050]

According to the present invention, a novel ADP-HK having a high substrate specificity to 1,5AG and capable of being used for simple and accurate measurement of 1,5AG can be provided.

[Brief description of the drawings]

FIG. 1 shows an optimal pH curve of ADP-HK of the present invention.

FIG. 2 shows a pH stability curve of ADP-HK of the present invention.

FIG. 3 shows an optimal temperature curve of ADP-HK of the present invention.

FIG. 4 shows a thermal stability curve of ADP-HK of the present invention.

FIG. 5 shows an optimum pH curve of 1,5AG6PDH.

FIG. 6 shows a pH stability curve of 1,5AG6PDH.

FIG. 7 shows an optimum temperature curve of 1,5AG6PDH.

FIG. 8 shows a thermal stability curve of 1,5AG6PDH.

[Procedure amendment]

[Submission date] July 24, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0023

[Correction method] Change

[Correction contents]

[0023]

Embedded image

Claims (3)

[Claims] The present invention has a high substrate specificity for 1,5 anhydroglucitol as compared with the case where glucose is used as a substrate, and has a high specificity for 1,5 anhydroglucitol in the presence of nucleoside diphosphates and magnesium ions. ADP-dependent hexokinase that catalyzes the reaction of substituting a hydroxyl group at the 2-position with a phosphate group to generate 1,5 anhydroglucitol hexaphosphate and nucleoside monophosphates. 2. The nucleoside diphosphate is ADP, and the nucleoside monophosphate is AMP.
The hexokinase as described. 3. The ADP-dependent hexokinase according to claim 1 has at least the following physicochemical properties. (1) Enzyme action Glucose as substrate, A as nucleoside diphosphate
The enzymatic action when DP is used is shown below. Embedded image (2) Substrate specificity Higher specificity for 1,5 anhydroglucitol compared to using glucose as a substrate. (3) Isoelectric point 4.1 ± 0.5 (by electrophoresis using carrier ampholine of pH 3.5 to 10) (4) Molecular weight 50,000 ± 5000 (for gel filtration using TSK-G3000SW _XL ) (5) (5) Optimum pH The optimum pH was in the range of 7 to 7.5. (6) pH stability The best stability was exhibited in the pH range of 6.5 to 10.5. (7) Optimum temperature The optimum temperature was 80 to 100 ° C. (8) Thermal stability 100 mM Tris-HCl buffer (pH 7.5) (ADP
Dependent hexokinase (0.5 U / ml) at each temperature
As a result of measuring the residual activity after heat treatment for 5 minutes, it was stable at least up to 95 ° C.