JP2008035748A - New glucose dehydrogenase derived from bacterium of genus aspergillus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a glucose dehydrogenase more suitable for a glucose sensor, etc. <P>SOLUTION: It is found out that various bacteria of the genus Aspergillus produce the new glucose dehydrogenase having a low property of acting on maltose. Thereby, the glucose dehydrogenase suitable for the glucose sensor, etc., can be produced. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel glucose dehydrogenase derived from Aspergillus.

  Blood glucose self-measurement is important for diabetics to grasp their normal blood glucose level and use it for treatment. An enzyme using glucose as a substrate is used for a sensor used for blood glucose self-measurement. An example of such an enzyme is glucose oxidase (EC 1.1.3.4). Glucose oxidase has been used as an enzyme for blood glucose sensors for a long time because it has the advantage of high specificity for glucose and excellent thermal stability, and its first announcement dates back to about 40 years ago. . In a blood glucose sensor using glucose oxidase, measurement is performed by passing electrons generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone to the electrode through a mediator. There is a problem that dissolved oxygen affects the measured value because the protons generated in the above are easily passed to oxygen.

  In order to avoid such problems, for example, NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.47) or pyrrolopinoline quinone-dependent glucose dehydrogenase (EC 1.1.5.2 (formerly EC1 .1.99.17)) is used as an enzyme for blood glucose sensors. These are superior in that they are not affected by dissolved oxygen, but the former NAD (P) -dependent glucose dehydrogenase has poor stability and the complexity of requiring the addition of a coenzyme. On the other hand, the latter pyrrolopinoline quinone-dependent glucose dehydrogenase has poor substrate specificity and acts on saccharides other than glucose, such as maltose and lactose, and thus has the disadvantage of impairing the accuracy of measured values.

References 1 to 4 report glucose dehydrogenase derived from Aspergillus oryzae, but do not report glucose dehydrogenase derived from other Aspergillus species.
Biochim Biophys Acta. 1967 Jul 11; 139 (2): 265-76. Biochim Biophys Acta. 1967 Jul 11; 139 (2): 277-93. Biochim Biophys Acta. 146 (2): 317-27 Biochim Biophys Acta. 146 (2): 328-35

Further, as a flavin-binding glucose dehydrogenase, Patent Document 1 discloses an Aspergillus terreus-derived flavin-binding glucose dehydrogenase. This enzyme is advantageous in that it does not require the addition of a coenzyme, is excellent in substrate specificity, and is not affected by dissolved oxygen. Regarding thermal stability, the residual activity rate is about 89% after treatment at 50 ° C. for 15 minutes, and the stability (hereinafter also referred to as heat resistance) is said to be excellent.
WO 2004/058958

  An object of the present invention is to provide an enzyme for a blood glucose sensor that is more practically advantageous. More specifically, a novel glucose dehydrogenase excellent in heat stability and maltose activity is obtained and used from a microorganism that has not been known to produce glucose dehydrogenase (hereinafter also referred to as GDH). is there.

In order to achieve the above object, the present inventors screened 75 types of Aspergillus spp. From among microorganisms that have not been known to produce glucose dehydrogenase until now, using glucose dehydrogenase activity as an index. It was. As a result, 13 types of Aspergillus were found to have GDH activity, and 10 types of glucose dehydrogenase produced by them were found to have excellent heat stability and maltose activity, leading to the present application. It was.
ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to acquire the novel glucose dehydrogenase excellent in heat stability and maltose action property from Aspergillus genus bacteria.

That is, the present invention has the following configuration.
[Section 1]
Aspergillus carbonarius, Aspergillus foetidus, Aspergillus ustus, Aspergillus foetidus var.pallidus, Aspergillus flavus, Aspergillus niger var. Intermedius, Aspergillus flavus var.columnaris, Aspergillus aculeatus, Aspergillus oryzae sper It is obtained by culturing at least one microorganism of Aspergillus thomii and Aspergillus kambarensis in a nutrient medium and extracting and purifying it from the culture solution using glucose dehydrogenase activity, maltose activity and / or heat stability as indicators. Glucose dehydrogenase having excellent maltose activity and / or heat stability.
[Section 2]
Aspergillus carbonarius NBRC4030, Aspergillus foetidus NBRC4031, Aspergillus ustus NBRC4104, Aspergillus foetidus var. Pallidus NBRC4118, Aspergillus flavus NBRC4186, Aspergillus niger var. IntermedusNBRC4281, Aspergillus flavus var. Columnaris NBRC5324, Aspergillus aculeatus NBRC5330, Aspergillus oryzae var. OryzaeNBRC6215, Aspergillus phoenicis NBRC6648, At least one microorganism of Aspergillus brunneo-uniseriatus NBRC6993, Aspergillus avenaceus NBRC8129, Aspergillus thomii NBRC8135, Aspergillus kambarensis NBRC8870 is cultured in a nutrient medium, and glucose dehydrogenase activity and maltose activity and / or thermal stability are indicated. And glucose dehydrogenase excellent in maltose activity and / or heat stability, which can be obtained by extraction and purification from a culture solution.
[Section 3]
At least one microorganism among the microorganisms described in claim 1 or 2 is cultured in a nutrient medium, and extracted from the culture solution using glucose dehydrogenase activity, maltose activity and / or heat stability as indicators. -Method for producing glucose dehydrogenase including a purification step
[Section 4]
A method for measuring glucose concentration using the glucose dehydrogenase according to claim 1 or 2.
[Section 5]
A glucose assay kit comprising the glucose dehydrogenase according to claim 1 or 2.
[Section 6]
A glucose sensor comprising the glucose dehydrogenase according to claim 1 or 2.

  The glucose dehydrogenase of the present invention is excellent in thermostability and maltose activity, and can provide a blood glucose sensor enzyme that is more advantageous in practical use in medical applications such as in vitro diagnostic drugs and sensors, and other glucose measurement applications. it can.

One embodiment of the present invention is aspergillus carbonarius, Aspergillus foetidus, Aspergillus ustus, Aspergillus foetidus var.pallidus, Aspergillus flavus, Aspergillus niger var.intermedius, Aspergillus flavus var. At least one microorganism of Aspergillus brunneo-uniseriatus, Aspergillus avenaceus, Aspergillus thomii, Aspergillus kambarensis is cultivated in a nutrient medium, and glucose bromide activity and / or maltose activity and / or thermostability are used as indicators in the culture solution Glucose dehydrogenase excellent in maltose activity and / or heat stability, which can be obtained by extraction and purification from
More preferably, Aspergillus carbonarius NBRC4030, Aspergillus foetidus NBRC4031, Aspergillus ustus NBRC4104, Aspergillus foetidus var. Pallidus NBRC4118, Aspergillus flavus NBRC4186, Aspergillus niger var. Aspergillus phoenicis NBRC6648, Aspergillus brunneo-uniseriatus NBRC6993, Aspergillus avenaceus NBRC8129, Aspergillus thomii NBRC8135, Aspergillus kambarensis NBRC8870, cultured in nutrient medium, glucose dehydrogenase activity, and / or maltose activity A glucose dehydrogenase excellent in maltose activity and / or heat stability, which can be obtained by extraction and purification from a culture solution using stability as an index.

  In another embodiment of the present invention, at least one of the microorganisms exemplified above is cultured in a nutrient medium, and glucose dehydrogenase activity, maltose activity and / or heat stability are used as indicators. And a method for producing glucose dehydrogenase, comprising a step of extracting and purifying glucose dehydrogenase from the culture solution.

The measurement method of glucose dehydrogenase activity follows the following method.
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
14 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution 15.8 ml of the above PIPES buffer, 0.2 ml of DCPIP solution, and 4 ml of D-glucose solution are mixed to obtain a reaction reagent.
<Measurement conditions>
Prewarm 2.9 ml of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 ml of GDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute (ΔOD _TEST) ). In the blind test, a change in absorbance per minute (ΔOD _BLANK ) is similarly measured by adding a solvent that dissolves GDH to the reagent mixture instead of the GDH solution. From these values, the GDH activity is determined according to the following formula 1. Here, 1 unit (U) in GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of 200 mM D-glucose.

(Formula 1)
Activity (U / ml) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution factor} ÷ (16.3 × 0.1 × 1.0)

In the formula, 3.0 is the amount of the reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm ² / micromole) under the conditions for this activity measurement, and 0.1 is the solution of the enzyme solution. Amount (ml), 1.0 indicates the optical path length (cm) of the cell.

The glucose dehydrogenase of the present invention is excellent in heat stability and / or maltose activity.
The action of maltose means an action of dehydrogenating maltose, and an activity measurement value when maltose is used as a substrate (in the above glucose activity measurement method, 1M maltose solution is used instead of 1M D-glucose solution) is glucose. Is defined as the activity divided by the substrate.
From the viewpoint of glucose measurement, it is preferable that the action on maltose is low. The effect on maltose is preferably 10% or less, more preferably 5% or less, and still more preferably 2% or less with respect to glucose.
Thermal stability can be evaluated by the method described in Example 2 described later.

  The microorganism used in the present invention can be obtained from the National Institute for Product Evaluation Technology.

  The nutrient medium for culturing the microorganism is not particularly limited as long as the microorganism can grow and produce the glucose dehydrogenase shown in the present invention. More preferably, the carbon source, inorganic nitrogen source and One containing an organic nitrogen source is preferable, and a liquid medium suitable for aeration stirring is more preferable. In the case of a liquid medium, for example, glucose, dextran, soluble starch, and sucrose are used as the carbon source, and examples of the nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, defatted soybeans, potato An extract etc. are illustrated. Moreover, other nutrients (for example, inorganic salts such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride, vitamins, etc.) may be contained as desired.

  The culture method follows methods known in the art. For example, spore of microorganisms or cells in a growing state are inoculated in a liquid medium containing the above nutrients, and the cells are allowed to grow by standing or stirring with aeration. Preferably, the cells are cultured by stirring with aeration. The pH of the culture solution is preferably 5-9, more preferably 6-8. The temperature is usually 14 to 42 ° C, more preferably 20 to 40 ° C. Usually, the culture is continued for 14 to 144 hours, but the culture is preferably terminated when the expression level of GDH is maximized under each culture condition. As a measure to identify such a time point, the change is monitored by sampling the culture solution and measuring the GDH activity in the culture solution, and the time point when the increase in GDH activity with time is stopped is regarded as the peak What is necessary is just to stop culture | cultivation.

  As a method for extracting GDH from the culture solution, when collecting GDH accumulated in the microbial cells, only the microbial cells are collected by an operation such as centrifugation or filtration, and the microbial cells are collected in a solvent, preferably water. Alternatively, resuspend in buffer. The resuspended cells can be crushed by a known method to extract GDH in the cells into a solvent. As a crushing method, a lytic enzyme can be used, or a physical crushing method may be used. The lytic enzyme is not particularly limited as long as it has the ability to digest fungal cell walls. Examples of applicable enzymes include “Lyticase” manufactured by Sigma. Examples of the physical crushing method include ultrasonic crushing, glass bead crushing, and French press. The residue after the crushing treatment can be removed by centrifugation or filtration to obtain a GDH crude extraction solution. When collecting GDH secreted outside the cells, the culture supernatant obtained by removing the cells by centrifugation or filtration may be used as the crudely extracted GDH.

  In addition, the culture method in the present invention may be solid culture. Preferably, the eukaryotic microorganism having the ability to produce the GDH of the present invention is grown on bran such as wheat while appropriately controlling temperature, humidity and the like. At this time, the culture may be performed by standing or may be mixed by stirring the culture. GDH extraction is performed by adding a solvent, preferably water or a buffer, to the culture to dissolve GDH, and removing solids such as bacterial cells and bran by centrifugation or filtration.

  As a method for purifying GDH from the GDH-containing solution obtained as described above, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate and sodium sulfate, or hydrophilic organic solvents such as methanol, ethanol, What is necessary is just to precipitate by the fractional precipitation method with acetone etc. Heat treatment and isoelectric point treatment are also effective purification means. Further, purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

These can proceed according to the following literature, for example.
(A) Protein Experiment Protocol Volume 1 Functional Analysis, Volume 2 Structural Analysis (Syujunsha) Supervised by Yoshifumi Nishimura and Shigeo Ohno (b) Revised Protein Experiment Notes Extraction and separation and purification (Yodosha) Masato Okada, Kaori Miyazaki
Editing (c) How to proceed with protein experiments (Yodosha) Edited by Masato Okada and Kaori Miyazaki or by the method exemplified below.
For example, gel filtration using Sephadex gel (manufactured by GE Healthcare Bioscience), DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience), octyl Sepharose CL-6B (manufactured by GE Healthcare Bioscience) And purified by column chromatography such as) to obtain a purified enzyme preparation. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

The partial amino acid sequence of the purified GDH preparation is determined, and DNA encoding GDH derived from the microorganism or other microorganisms is cloned using a probe or primer prepared based on the partial amino acid sequence. It is easy for a person skilled in the art to introduce the cloned DNA into various microorganisms, determine culture and purification conditions as appropriate, and collect GDH.
The base sequence of the GDH gene obtained by the above method can be decoded by the dideoxy method described in Science, Vol. 214, 1205 (1981). The amino acid sequence of GDH can be deduced from the base sequence determined as described above.

Furthermore, as long as the GDH obtained above has glucose dehydrogenase activity, some of the other amino acid residues may be deleted or substituted with those exemplified above, and other amino acid residues may It may be added.
In addition, the gene encoding GDH can be similarly modified.
Such modifications can be easily carried out by those skilled in the art using known techniques in the art. For example, various methods for substituting or inserting a base sequence of a gene encoding a protein in order to introduce a site-specific mutation into the protein are described in Sambrook et al., Molecular Cloning; A Laboratory Manual 2nd edition (1989). Cold Spring Harbor Laboratory Press, New York.
In addition, the GDH gene obtained above may further include a modified codon usage according to the purpose of improving the expression of GDH.

  The purified enzyme obtained as described above can be pulverized and distributed, for example, by freeze drying, vacuum drying, spray drying, or the like.

  In that case, you may add another stabilizer etc. to the enzyme of this invention, a kit, or a sensor in the range which does not have a bad influence especially on reaction of GDH. The blending method of the stabilizer of the present invention is not particularly limited. For example, a method of blending a stabilizer in a buffer solution containing GDH, a method of blending GDH in a buffer solution containing a stabilizer, or a method of blending GDH and a stabilizer in a buffer solution at the same time can be mentioned.

  The buffer component that can coexist is not particularly limited, and examples thereof include Tris buffer, phosphate buffer, borate buffer, and GOOD buffer. The pH of the buffer is adjusted in the range of about 5.0 to 9.0 according to the purpose of use. The content of the buffering agent in the lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.1 to 30% (weight ratio). used.

  If necessary, other components such as surfactants, stabilizers, excipients, salts, amino acids, sugars, preservatives and the like may be added.

  The GDH of the present invention can take various forms such as liquid (aqueous solution, suspension, etc.), powder, and lyophilization. The lyophilization method is not particularly limited and may be performed according to a conventional method. The composition containing the enzyme of the present invention is not limited to a lyophilized product, and may be in a solution state in which the lyophilized product is redissolved. Moreover, when measuring glucose, various forms, such as a glucose assay kit and a glucose sensor, can be taken.

  In the lyophilized composition (including on the sensor), the GDH content varies depending on the origin of the enzyme, but is usually suitably used in the range of about 5 to 70% (weight ratio). When converted into enzyme activity, it is preferably used in the range of 10 to 3000 U / mg.

  In the GDH of the present invention, it is possible to reduce the concentration of additives / mixtures that may affect the reaction to such an extent that they are not affected, or not to coexist substantially. For example, albumin such as bovine serum albumin (BSA) and ovalbumin (OVA) can be substantially not coexisted.

  The glucose dehydrogenase that can be produced as described above can be used for measurement of glucose concentration.

Glucose Assay Kit The present invention is also a glucose assay kit containing the GDH of the present invention. The glucose assay kit of the present invention contains the GDH of the present invention in an amount sufficient for at least one assay. Typically, the kit includes the GDH of the present invention, plus buffers necessary for the assay, mediators, glucose standard solution for creating a calibration curve, and directions for use. The GDH of the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.

Glucose Sensor The present invention also features a glucose sensor that uses the GDH of the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. Examples of the immobilization method include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, a redox polymer, or the like, or ferrocene or It may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator typified by a derivative thereof, or may be used in combination.

  The glucose concentration can be measured as follows. A buffer solution is put into a thermostatic cell, and a coenzyme (such as flavin) and a mediator are added to maintain a constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the GDH of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is limited to the following examples.

<Example 1>
Acquisition of GDH derived from Aspergillus sp. 75 Aspergillus spp. Were purchased from National Institute of Technology and Evaluation, each L dry specimen was inoculated into potato dextrose agar medium (Difco) at 25 ° C. Restored by incubating. The mycelium on the restored plate was recovered by scraping off the agar and inoculated into 50 ml of 1% malt extract, 1.5% soybean peptide, 2% glucose, pH 6.5 placed in a 500 ml flask. After culturing at 30 ° C. for 3 days, the culture broth was centrifuged to collect the culture supernatant, and the GDH activity was measured according to the activity measurement method described above.
As a result, GDH activity was confirmed in 14 of 75 strains.

<Example 2>
Temperature stability Each crude enzyme solution in which GDH activity was confirmed in Example 1 was subjected to a heating treatment at 50C for 15 minutes, and the activities after the treatment before the treatment were compared. The activity measurement method followed the above-described test example. The table shows the residual activity after the heating treatment relative to the activity before the heating treatment. Aspergillus foetidus var. Pallidus, Aspergillus flavus, Aspergillus niger var.intermedius, Aspergillus flavus var. Columnaris, Aspergillus oryzae var. It was thought that high stability was maintained after heat treatment.

<Example 3>
Substrate specificity Among the crude enzyme solutions obtained in Example 1, maltose activity was examined except for those with extremely poor stability. The activity measurement method is based on the above test example, and the ratio of activity to maltose / activity to glucose is defined as maltose activity. It was shown to. In all strains, the maltose activity was 5% or less, and it was confirmed that the maltose activity was extremely low.

In the present invention, it has been found that a novel glucose dehydrogenase having superior thermal stability and lower maltose activity than Aspergillus strains can be produced. These enzymes are considered to be more suitable for glucose sensors and the like.

The glucose dehydrogenase excellent in thermostability and maltose activity of the present invention can be used for medical applications such as in vitro diagnostic drugs and sensors, and other glucose measurement applications.

Claims (6)

Aspergillus carbonarius, Aspergillus foetidus, Aspergillus ustus, Aspergillus foetidus var.pallidus, Aspergillus flavus, Aspergillus niger var. Intermedius, Aspergillus flavus var.columnaris, Aspergillus aculeatus, Aspergillus oryzae sper It is obtained by culturing at least one microorganism among Aspergillus thomii and Aspergillus kambarensis in a nutrient medium, and extracting and purifying it from the culture solution using glucose dehydrogenase activity, maltose activity and / or heat stability as indicators. Glucose dehydrogenase having excellent maltose activity and / or heat stability. Aspergillus carbonarius NBRC4030, Aspergillus foetidus NBRC4031, Aspergillus ustus NBRC4104, Aspergillus foetidus var. Pallidus NBRC4118, Aspergillus flavus NBRC4186, Aspergillus niger var. IntermedusNBRC4281, Aspergillus flavus var. Columnaris NBRC5324, Aspergillus aculeatus NBRC5330, Aspergillus oryzae var. OryzaeNBRC6215, Aspergillus phoenicis NBRC6648, At least one microorganism of Aspergillus brunneo-uniseriatus NBRC6993, Aspergillus avenaceus NBRC8129, Aspergillus thomii NBRC8135, Aspergillus kambarensis NBRC8870 is cultured in a nutrient medium, and glucose dehydrogenase activity and maltose activity and / or thermal stability are indicated. And glucose dehydrogenase excellent in maltose activity and / or heat stability, which can be obtained by extraction and purification from a culture solution. At least one microorganism among the microorganisms according to claim 1 or 2 is cultured in a nutrient medium, and extracted from the culture solution using glucose dehydrogenase activity, maltose activity and / or heat stability as indicators. -Method for producing glucose dehydrogenase including a purification step A method for measuring glucose concentration using the glucose dehydrogenase according to claim 1 or 2. A glucose assay kit comprising the glucose dehydrogenase according to claim 1 or 2. A glucose sensor comprising the glucose dehydrogenase according to claim 1 or 2.