JP2000262281A - Crosslinked glucose dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme for glucose sensors, etc., comprising PQQ glucose dehydrogenase crosslinked by a bifunctional reagent, having high heat stability and capable of simply and rapidly measuring blood glucose level useful for diagnose, etc., of diabetes mellitus. SOLUTION: This enzyme is a new water-soluble PQQ glucose dehydrogenase (PQQGDH) crosslinked by a bifunctional reagent and is improved so as to increase heat stability and useful for glucose sensor elements, etc., capable of simply and rapidly measuring blood glucose level which is important for diagnosis of diabetes mellitus, home control, etc., of patients. The enzyme is obtained by incubating a glucose dehydrogenase derived from Acinetobacter calcoaceticus in the presence of 1 mM pyrroloquinolinequinone(PQQ) and 1 mM CaCl2 at room temperature for 30 min and adding a bifunctional reagent thereto and stirring these components at room temperature for 30 min to carry out crosslinking reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a glucose dehydrogenase improved to increase thermostability.

[0002]

2. Description of the Related Art Diabetes patients tend to increase year by year,
Since diabetes diagnosis and home care of patients are very important, glucose sensors capable of simply and quickly measuring a blood glucose level have been developed. Glucose oxidase (GOD) is most often used as a glucose sensor element. GOD has been frequently used because it is an enzyme that is stable to heat and is supplied in large quantities at low cost. G
As the principle of detecting OD glucose, an oxygen electrode type for detecting oxygen consumed in the oxidation reaction of GOD glucose or a hydrogen peroxide electrode type for detecting generated hydrogen peroxide has been developed. However, since this method is affected by other redox substances in the blood due to the high applied potential, mediator sensors that reduce the applied potential using various electron mediators have been developed since the 1980s. ing. However, GOD transfers electrons to oxygen instead of the mediator when the dissolved oxygen concentration is high, so that accurate measurement cannot be performed. Thus, glucose dehydrogenase (GDH) has been attracting attention as an ideal mediator-type sensor element that is not affected by the concentration of dissolved oxygen. Among GDH, coenzyme-linked PQQ glucose dehydrogenase (PQQGDH)
Has a high catalytic activity and a high turnover number, and therefore has a high response current value and no longer a response time. That is, accurate and quick measurement is possible. Further, since it is a coenzyme-bound type, it is not necessary to add an expensive coenzyme to the reaction solution. Furthermore, if the enzyme is water-soluble, a surfactant is not required in the buffer solution, and there is an advantage that the enzyme is easy to handle.
icus-derived water-soluble PQQGDH (PQQGDH-
B) is very ideal as an element of a glucose sensor.

[0003]

However, PQQ
GDH has a narrower dynamic range than GOD,
PQQ that is practical because of low substrate specificity and stability
In order to develop a GDH sensor, it is necessary to improve the enzyme in these respects.

Accordingly, the present invention provides a PQ having improved thermal stability.
It is intended to provide QGDH-B.

[0005]

SUMMARY OF THE INVENTION The present invention provides a water soluble PQQGDH crosslinked with a bifunctional reagent. The crosslinked PQQGDH of the present invention has higher thermal stability than natural water-soluble PQQGDH. Preferably, the PQ of the present invention
QGDH is Acinetobacter calco
aceticus-derived water-soluble PQQGDH. Also, preferably, the bifunctional reagent is glutaraldehyde.

[0006] The present invention also provides a glucose sensor using the crosslinked water-soluble PQQGDH according to the present invention.

[0007]

DETAILED DESCRIPTION OF THE INVENTION Crosslinked The present invention relates to a water-soluble PQQ crosslinked with a bifunctional reagent.
It features GDH. As used herein,
"Bifunctional reagent" refers to a chemical species having two reactive functional groups. Bifunctional reagents that can be used in the present invention include, for example, glutaraldehyde and glyoxal.

It is well known to use a bifunctional reagent to immobilize an enzyme on a carrier such as a polymer. In this case, it has been generally believed that intramolecular cross-linking of the enzyme itself should be avoided in order to maintain the conformation of the enzyme and retain its activity. Therefore, it was a surprising finding that the stability of the enzyme was improved by intramolecular crosslinking.

Crosslinking with a bifunctional reagent can be carried out by dissolving the enzyme in a suitable solvent, adding the bifunctional reagent, reacting at room temperature or lower, and then removing the unreacted reagent. it can. The reaction was performed at an enzyme concentration of 1 μg / m
It is preferable to carry out in the range of 1-100 μg / ml. When the concentration is lower than 1 μg / ml, the reaction rate is extremely low and is not practical. On the other hand, if the enzyme concentration is higher than 100 μg / ml, the proper conformation of the enzyme is not maintained, so that the enzyme activity decreases. If the concentration is further increased, it is expected that intermolecular cross-linking will occur, and that the enzyme protein will aggregate and the enzyme activity will be significantly reduced. Most preferably, the enzyme concentration is 50 μg / ml or less. The final concentration of the bifunctional reagent is preferably 0.01-5%, more preferably 0.1-2.5%. Furthermore, if the unreacted bifunctional reagent remains in the solution, the enzyme activity may be reduced.Therefore, it is necessary to remove the unreacted bifunctional reagent by a method such as dialysis or column chromatography. Is preferred. Glucose sensor The present invention also provides a crosslinked PQQGDH according to the invention on an electrode.
Is characterized by a glucose sensor in which is immobilized. As the electrode, a gold electrode, a platinum electrode, a carbon paste electrode, a glassy carbon electrode, a graphite electrode, or the like can be used. The enzyme of the present invention is immobilized on this electrode. As the immobilization method, a method using a crosslinking reagent,
There is a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of adsorbing to a conductive polymer,
These may be used in combination. Preferably, the crosslinked PQQGDH of the present invention is immobilized on the electrode in a holated form, but it can also be immobilized in the form of an apoenzyme and PQQ provided as a separate layer or in solution. Typically, the crosslinked PQQGDH of the present invention is immobilized on a carbon electrode by mixing with a carrier protein and treating with glutaraldehyde. Preferably, glutaraldehyde is then blocked by treatment with a reagent having an amine group. This is because the number of free radicals required for immobilization becomes insufficient by the crosslinking treatment of the enzyme, and it is considered that leakage of the enzyme can be prevented by forming a network of the carrier protein. As a carrier protein, PQQG
Any protein, such as bovine serum albumin, can be used as long as it does not adversely affect the enzyme activity of DH.

[0010] The measurement of the glucose concentration can be performed as follows. PQQ and CaCl in constant temperature cell
₂ and the buffer containing the mediator are added and maintained at a constant temperature. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate, and the like can be used. A working electrode is a carbon paste electrode on which the modified PQQGDH of the present invention is immobilized, and a counter electrode (for example, a platinum electrode)
And a reference electrode (eg, an Ag / AgCl electrode). After a constant voltage is applied to the carbon paste electrode and the current becomes steady, a sample containing glucose is added and the increase in the current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

[0011]

EXAMPLES In the following examples, the following reagents and equipment were used. The substrate pyrroloquinoline quinone (PQQ) was purchased from Mitsubishi Gas Chemical Company. Glutaraldehyde 25% aqueous solution (primary) for crosslinking treatment (Kishida Chemical Co., Ltd.)
Was used. For the production of the electrodes, a carbon paste electrode (inner diameter 3.0 mm, outer diameter 6.0 mm) and a carbon paste oil base (both from BAS) were used. Example 1 Preparation of Enzyme For production of PQQGDH-B, the plasmid vector pT was used.
NcoI-HindI of rc99A (Pharmacia)
Gene 1.5 encoding PQQGDH-B at the II site
Plasmid pGB2 constructed by inserting kb (FIG. 1)
Was introduced into Escherichia coli PP2418 which lacks the ability to produce PQQGDH. Escherichia coli was cultured by a conventional method using a 2 liter fermenter and induced with IPTG. The collected and washed cells are suspended in 10 mM phosphate buffer (pH 7.0), crushed by a French press, and centrifuged (10000 g, 20 minutes, 4 ° C.) twice to remove unbroken cells. did. The supernatant was subjected to ultracentrifugation (50,000 rpm, 60 minutes, 4 ° C.), and the supernatant was obtained as a water-soluble fraction. This is 100 times the amount of 1
It was dialyzed overnight against 0 mM phosphate buffer (pH 7.0).

The crude purified enzyme after dialysis was filtered through a 0.2 μm filter, and then filtered through a 10 m filter.
M phosphate buffer (pH 7.0) with A buffer, 0.8
Packing column for cation exchange column chromatography equilibrated with A buffer using MNaCl 10 mM phosphate buffer (pH 7.0) as B buffer, CMTOY
Using an OPEARL 650M column, B buffer 4
PQQGDH-B was eluted with a gradient of 0% / 60 minutes. The flow rate was 3 ml / min. For the detection of the protein, the absorbance at 280 nm was used, and the peak portion was collected.

[0013] Next, the fractionated sample was subjected to 0.2 M N
aCl, loaded on a hydroxyapatite column equilibrated with 10 mM phosphate buffer (pH 7.0), and 0.2 M-
0.5 M NaCl, 10 mM phosphate buffer (pH 7.
PQQGDH-B was eluted with a gradient of 0) / 60 min. The elution was performed at 1 ml / min, and the eluate was collected every 2 minutes. The obtained active fraction is 100 times the amount of 1
It was dialyzed against 0 mM MOPS buffer (pH 7.0) and desalted. Example 2 Method for measuring the enzyme activity of PQQGDH-B The enzyme activity of PQQGDH-B was measured using PMS (phenazine methosulfate) -DCIP (2,6-dichlorophenolindophenol) in 10 mM phosphate buffer pH 7.0. I did it. DCIP 60
The change in the absorbance at 0 nm was followed, and the rate of decrease in the absorbance was defined as the reaction rate of the enzyme. At this time, 1 μmol per minute
The enzyme activity by which DCIP was reduced was defined as 1 unit.
The molar extinction coefficient of DCIP at pH 7.0 is 1
It was 6.3 mM ^-1 . Spectrophotometer UV-120 for measurement
0 (manufactured by Shimadzu Corporation) was used. Example 3 Preparation of Cross-Linked Enzyme The enzyme prepared in Example 1 was added to 1 mM PQQ, 1 mM
Incubated for 30 minutes at room temperature in the presence of CaCl ₂ and holated. The final concentration of 0.1, 1.
GA was added so that the concentration became 0 and 2.5%, and the mixture was stirred at room temperature for 30 minutes. As an uncrosslinked control, the same treatment as the crosslinking treatment was performed using water instead of GA. This was dialyzed against 1000 volumes of a 10 mM MOPS buffer (pH 7.0), or unreacted GA was removed using a NAP-10 column. The activity of these enzymes was measured according to the method described above.

FIG. 2 shows the results. Assuming that the activity of the enzyme before the crosslinking treatment was 100%, when the crosslinking treatment was performed using 0.1% GA, about 28% of the activity was maintained. When treated with higher concentrations of GA, the activity was more reduced. Activity was reduced to about 12% when treated with water as a control. Example 4 Evaluation of thermostability The protein prepared at the time of crosslinking was 99.09, 49.55, 39.64, and 33.03, respectively, for the enzyme prepared in Example 1.
0.1 μg / ml according to Example 3 as μg / ml
A cross-linking treatment was performed. Next, the cross-linked enzyme was adjusted to a protein concentration of 1.0 μg / ml, heat-treated by incubating at 55 ° C. for 0 to 40 minutes, and the residual activity of the enzyme was measured. As an uncrosslinked control, the same treatment as the crosslinking treatment was performed using water instead of GA.

FIG. 3 shows the results. It was found that when the enzyme concentration at the time of crosslinking was about 50 μg / ml or less, the thermal stability was significantly improved by GA crosslinking. When the enzyme concentration is high, the activity is considered to be reduced due to intermolecular crosslinking. Example 5 Blocking The free aldehyde group after the GA treatment was blocked, and the effect was examined. A glutaraldehyde (GA) solution was added to the enzyme holated according to Example 1 to a concentration of 0.1%, followed by stirring at room temperature for 30 minutes (the protein concentration at the time of crosslinking was about 27 μg / ml). Purified water was added in place of GA, and the same treatment was carried out to obtain an uncrosslinked control.
This was dialyzed against (1) 1000-fold volume of 10 mM MOPS buffer (pH 7.0), and (2) 10 mM Tris-
Dialysis against HCl buffer (pH 7.0), (3) final concentration 50
After stirring for 20 minutes at room temperature as mM lysine, 10m
Three treatments of dialysis with MMOPS buffer (pH 7.0) were performed, freeze-dried, and redissolved in water. These crosslinked enzymes were heat-treated at 55 ° C. for 0 to 40 minutes, and the residual activities were measured.

FIG. 4 shows the results. The thermal stability is further improved by blocking. Example 6 Characterization of Cross-Linked Enzyme For the enzyme cross-linked under the optimal conditions obtained in Example 2,
The activity was measured in a glucose concentration range of 0 to 100 mM. FIG. 5 shows the results. Vmax of the crosslinked enzyme of the present invention with respect to glucose was 388 U / mg, and Km was 20 mM. Example 7 Preparation of Enzyme Sensor Using Crosslinked PQQGDH The enzyme sample prepared in Example 1 was prepared using 1 mM PQQ,
Incubation was carried out at room temperature for 30 minutes in the presence of 1 mM CaCl _{2 for} holiation. The protein concentration is 30 μg / ml.
And add GA so that the final concentration is 0.1%.
After stirring at room temperature for 1 minute, 1000 times the volume of 10 mM MO
Dialyzed with PS. 5 U of this enzyme is added to carbon paste 20
mg and lyophilized overnight. After this was mixed well, only the surface of the carbon paste electrode already filled with about 40 mg of carbon paste was filled and polished on filter paper. 1 mg / ml bovine serum albumin 5 on the electrode surface
μl was added dropwise and dried at room temperature. This electrode is 1% G
A in a 10 mM MOPS buffer (pH 7.0) containing 10 mM MOPS buffer (pH 7.0) for 30 minutes at room temperature.
Stirred at room temperature for minutes for blocking GA.

Next, the electrode was stirred in a 10 mM MOPS buffer (pH 7.0) for 1 hour or more at room temperature to equilibrate. 1 mM PQQ, 1 mM CaCl except during measurement
₂ was stored at 4 ° C. in a 10 mM MOPS buffer (pH 7.0).

1 mM PQQ, 1 mM Ca
10 mM MOPS buffer containing Cl ₂ (pH 7.0)
And a final concentration of 1 mM m-P as a mediator
MS was added to bring the total volume to 10 ml. There, a carbon paste electrode prepared as a working electrode was used, a platinum electrode was used as a counter electrode, and an Ag / AgCl electrode was used as a reference electrode.
All measurements were performed at 25 ° C. A potential of +100 mV was applied, and when the current became steady, an appropriate concentration of glucose was added and the increased current value was measured. The current value when glucose was not added was set to 0A.

FIG. 6 shows a calibration curve.
The glucose sensor using the crosslinked enzyme of the present invention was able to measure glucose in the range of 1 mM to 12 mM.

[Brief description of the drawings]

FIG. 1 shows the structure of plasmid pGB2.

FIG. 2 shows the residual activity after treatment of the enzyme with glutaraldehyde.

FIG. 3 shows the thermostability of the crosslinked enzyme of the present invention.

FIG. 4 shows the effect of blocking after glutaraldehyde treatment.

FIG. 5 shows the activity of the crosslinked enzyme of the present invention at various glucose concentrations.

FIG. 6 shows a calibration curve of an enzyme sensor using the crosslinked enzyme of the present invention.

Claims (4)

[Claims] 1. A water-soluble P cross-linked by a bifunctional reagent.
QQGDH. 2. The method according to claim 1, wherein the PQQGDH is Acinetob.
from actor calcoaceticus,
The water-soluble PQQGDH according to claim 1. 3. The water-soluble PQQGDH according to claim 1, wherein the bifunctional reagent is glutaraldehyde. 4. A glucose sensor comprising the water-soluble PQQGDH according to claim 1 immobilized on an electrode.