WO2006095758A1 - Methode pour eviter l'inhibition d'un substrat par pqqgdh - Google Patents

Methode pour eviter l'inhibition d'un substrat par pqqgdh Download PDF

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Publication number
WO2006095758A1
WO2006095758A1 PCT/JP2006/304440 JP2006304440W WO2006095758A1 WO 2006095758 A1 WO2006095758 A1 WO 2006095758A1 JP 2006304440 W JP2006304440 W JP 2006304440W WO 2006095758 A1 WO2006095758 A1 WO 2006095758A1
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Prior art keywords
glucose
pqqgdh
gnolecose
measurement
substrate inhibition
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PCT/JP2006/304440
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English (en)
Japanese (ja)
Inventor
Tadanobu Matsumura
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Toyo Boseki Kabushiki Kaisha
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Publication of WO2006095758A1 publication Critical patent/WO2006095758A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Definitions

  • the present invention relates to a method for avoiding substrate inhibition of pyroguchi quinoline quinone-dependent glucose dehydrogenase.
  • Pyroguchi quinoline quinone-dependent gnolecose dehydrogenase (also referred to as PQQGDH in this application) is gnorecose dehydrogenase (GDH) using pyroguchi quinoline quinone (PQQ) as a coenzyme. Since it catalyzes the reaction that oxidizes gnolecose to produce dalconolatatone, it can be used for blood glucose measurement. Blood glucose concentration is an extremely important index for clinical diagnosis as an important marker of diabetes.
  • Patent Document 1 Japanese Patent Laid-Open No. 11-243949
  • Pyromouth quinoline quinone-dependent glucose dehydrogenase catalyzes the reaction that oxidizes D-glucose to produce D-darcono 1,5-latataton, and is not affected by dissolved oxygen in the reaction system. Because it has enzyme properties that do not require the addition of coenzymes, it is expected to have a wide range of uses such as blood glucose biochemical diagnostic agents as well as blood glucose sensors.
  • Patent Document 2 has reported studies using PQQGDH modification means at the gene level, but the mechanism of substrate inhibition has also been disclosed. There was no suggestion, and it was not even mentioned as a means to resolve substrate inhibition from the viewpoint of measurement reaction conditions.
  • Patent Document 2 W003Zl06668
  • Item 1 A method for reducing substrate inhibition in dalcose measurement, characterized in that the pH during measurement reaction is acidic in a method for measuring gno-record in a system containing pyroguchi quinoline quinone-dependent glucose dehydrogenase Item 2.
  • Glucose measuring reagent with reduced substrate inhibition characterized by containing pyroguchi quinoline quinone-dependent glucose dehydrogenase and acidic ⁇ during the measurement reaction Item 4.
  • Ferricyanide ion as a mediator The glucose measurement reagent according to Item 3, comprising
  • Glucose assay kit with reduced substrate inhibition characterized by containing pyroguchi quinoline quinone-dependent glucose dehydrogenase and ⁇ at the time of measurement reaction being acidic Item 6.
  • Ferricyanide as a mediator The glucose assembly kit according to Item 5, comprising an ion
  • Glucose sensor with reduced substrate inhibition characterized by containing pyroguchi quinoline quinone-dependent glucose dehydrogenase and acidic ⁇ during the measurement reaction Item 8. Felicyanide ion as a mediator
  • Avoidance of substrate inhibition according to the present invention enables high-concentration glucose measurement using a gnolecose measuring reagent, a gnolecose assembly kit, and a gnolecose sensor.
  • PQQGDH which can be applied to the method of the present invention, coordinates Pyroguchi quinoline quinone as a coenzyme and oxidizes D-glucose to produce D-darcono 1,5-latataton. It is an enzyme that catalyzes (ECl.
  • PQQGDH that can be applied to the method of the present invention is derived from, for example, Acinetobacter calcoaceticus LMD79.41 (A. M. Cleton- Jansen et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Gen et., 217, 430 (1989)), derived from Escherichia coli (A. M Cleton—Jansen et al., J. Bacteriol., 172, 6308 (1990)), derived from Gluconobacter oxydans (Mol. Gen. Genet., 229, 20 6 (1991) And those derived from Acine tobacter baumanni NCIMB11517 reported in Patent Document 1.
  • Acinetobacter calcoaceticus LMD79.41 A. M. Cleton- Jansen et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Gen et., 217, 430 (1989)
  • Acinetobacter baumannii (Acinetobacter baumannii) NCIMB 1151 7fe3 ⁇ 4 ⁇ 11, Acinetobacter calcoaceticus.
  • PQQGDH that can be applied to the method of the present invention is not limited to those exemplified above, as long as it has glucose dehydrogenase activity. Other amino acid residues may be added.
  • PQQGDH for example, commercially available products such as Toyobo GLD-321 can be used. Alternatively, it can be easily produced by those skilled in the art using known techniques in the technical field.
  • the above-mentioned natural microorganisms that produce PQQGDH, or the gene encoding natural PQQGDH as it is or after mutation expression vectors (many are known in the art)
  • a plasmid is inserted into a suitable host (many are known in the art, such as E. coli), and the transformant is cultured and centrifuged from the culture. After harvesting the cells, etc., destroy the cells by mechanical methods or enzymatic methods such as lysozyme, and if necessary, solubilize by adding a chelating agent such as EDT A or a surfactant. Water soluble, including PQQGDH A fraction can be obtained.
  • the expressed PQQGDH can be secreted directly into the culture medium by using an appropriate host vector system.
  • the PQQGDH-containing solution obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or hydrophilic organic solvents such as methanol, ethanol, acetone and the like. Precipitate by the fractional precipitation method. Heat treatment and isoelectric point treatment are also effective purification means.
  • purified PQQGDH can be obtained by performing gel filtration using an adsorbent or gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography.
  • the purified enzyme preparation is preferably purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).
  • a heat treatment of preferably 25 to 50 ° C, more preferably 30 to 45 ° C may be performed.
  • concentration of PQQGDH in the present invention is not particularly limited.
  • the enzyme activity of PQQGDH can be measured by the following method.
  • NTB nitrotetrazolium blue
  • PMS phenazine methosulfate
  • One unit refers to the amount of PQQGDH enzyme that forms 0.5 mmol of diformazan per minute under the conditions described below.
  • D glucose solution: 0.5M (0.9 g D—glucose (molecular weight 180 ⁇ 16) / 10ml H20)
  • PIPES NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302.36) suspended in 60 mL of water, dissolved in 5 N NaOH, 2. 2 ml of 10% Triton X_100 (Use 5N NaOH for 25. Adjust the ptl to 6.5 ⁇ 0.05 with C and add 100 ml with water)
  • E. Enzyme Diluent ImM CaC12, 0.1% Triton X-100, 0.1 mM BSA-containing 50 mM PIPES _Na 0H buffer (pH 6.5)
  • the enzyme powder was dissolved in ice-cold enzyme diluent (E) immediately before the assembly and diluted to 0.1 -0.8 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).
  • Vt Total volume (3. lml)
  • the pH at the time of measurement is acidic.
  • the pH is less than 7.0 and is not particularly limited, but the upper limit is preferably 6.5 or less, more preferably 6.0 or less, and even more preferably 5.5 or less in the present invention.
  • the lower limit is preferably pH 3.0 or higher, more preferably pH 3.5 or higher.
  • the preferred range in the present invention is pH 3.5 to 5.5.
  • buffers to make the pH during measurement acidic.
  • a buffer is not particularly limited as long as it has a buffer capacity capable of keeping the pH acidic.
  • Commonly used buffers include tris hydrochloric acid, boric acid, phosphoric acid, acetic acid, succinic acid, succinic acid, phthalic acid, maleic acid, glycine and their salts, MES, Bis_Tris, ADA, PIPES , ACES, MOPS 0, DES buffer such as BES, MOPS, TES, HEPES, etc. Do not form insoluble salts with calcium, buffer is preferred.
  • the concentration of these additives is not particularly limited as long as it has a buffering capacity, but the preferable upper limit is 10 mM or less, more preferably 50 mM or less. A preferred lower limit is 5 mM or more.
  • the content of the buffer in the lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.:! To 30% (weight ratio). Used in the range of.
  • These buffers may be added at the time of measurement, and may be preliminarily contained when preparing a reagent for measuring gnolecose, a gnolecose assembly kit or a glucose sensor described later. .
  • the liquid state, the dry state, etc. are not limited, and it is sufficient to function during measurement.
  • Substrate inhibition as used in the present invention refers to a phenomenon in which the reaction rate decreases at a constant concentration when the glucose (substrate) concentration of the sample to be measured is increased. When samples with various darose concentrations are measured, the glucose concentration immediately before the reaction rate decreases is measured.
  • reducing substrate inhibition means increasing the "maximum concentration at which substrate inhibition does not occur”.
  • normoglycemia is about 5 mM (90 mg / dl), and hyperglycemia is present. Value is 10mM
  • the effect of the present invention becomes more remarkable in a system including a mediator.
  • the mediator that can be applied to the method of the present invention is not particularly limited. (PMS) and 2,6-dichlorophenol indophenol (DCPIP), PMS and nitroblue tetrazolium (NBT), DCPIP alone, ferricyanide ion (as a compound) Potassium ferricyanide, etc.) alone, and Huekousen alone. Of these, ferricyanide ions (such as ferricyanium potassium as the compound) are preferred.
  • mediators may be added at the time of measurement, or may be included in advance when a glucose measurement reagent, a glucose assay kit, or a glucose sensor described later is prepared. In that case, the liquid state, the dry state, etc. are not asked and the measurement is not performed.
  • various components can coexist as necessary.
  • surfactants for example, surfactants, stabilizers, excipients and the like may be added.
  • PQQGDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as glutamic acid, glutamine, and lysine, and serum albumin.
  • PQQGDH can be stabilized by containing calcium ions or calcium salts.
  • the calcium salt include calcium chloride, calcium acetate, calcium salt of inorganic acid such as calcium citrate, and calcium salt of organic acid.
  • the calcium ion content is preferably 1 X 10-4 to 1 X 10-2M.
  • the stabilizing effect of containing calcium ions or calcium salts is further improved by containing an amino acid selected from the group consisting of gnoretamic acid, glutamine and lysine.
  • the amino acid selected from the group consisting of glutamic acid, glutamine and lysine may be one type or two or more types.
  • BSA bovine serum albumin
  • OVA ovalbumin
  • (1) aspartic acid, gnoretamic acid, a-ketoglutaric acid, malic acid, a-ke PQQGDH can be stabilized by the coexistence of one or more compounds selected from the group consisting of togluconic acid, a- cyclodextrin and their salts, and (2) albumin.
  • glucose can be measured by the following various methods.
  • the reagent for measuring glucose, the glucose assay kit, and the glucose sensor of the present invention can take various forms such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying or spray drying, freeze drying, etc. .
  • the freeze-drying method is not particularly limited, but should be performed according to conventional methods.
  • the composition containing the enzyme of the present invention is not limited to a lyophilized product, but may be a solution in which the lyophilized product is redissolved.
  • the reagent for measuring glucose of the present invention typically includes a reagent necessary for measurement such as PQQGDH, a buffer solution, a mediator, a glucose standard solution for preparing a calibration curve, and a usage guideline.
  • the kit of the present invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution.
  • the PQQGDH of the present invention can be provided in the form of a force apoenzyme provided in a holified form and can be holoed into a used B temple.
  • the glucose assay kit of the present invention typically includes reagents necessary for measurement such as PQQGDH, buffer, and mediator, a glucose standard solution for preparing a calibration curve, and usage guidelines.
  • the kit of the present invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution.
  • the PQQGDH of the present invention is provided in the form of a holo, but it can also be provided in the form of an apoenzyme and holoed at the time of use.
  • a carbon electrode, a gold electrode, a platinum electrode, or the like is used as an electrode, and PQQGLD is immobilized on this electrode.
  • 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 photocrosslinking polymer, a conductive polymer, a redox polymer, or the like. It can be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator represented by Huekousen or its derivatives, or a combination thereof.
  • the PQQGDH of the present invention may be immobilized in the form of a force apoenzyme that is immobilized on the electrode in a holo form, and PQQ may be supplied as a separate layer or in solution.
  • PQQGDH of the present invention is immobilized on a carbon electrode using dartalaldehyde, and then treated with a reagent having an amine group to block the gnoretaraldehyde.
  • Measurement of the gnolecose concentration can be performed as follows. Put the buffer in the thermostatic cell, and keep CaC12 and mediator at a constant temperature.
  • As the mediator potassium ferricyanide, phenazine methosulfate, or the like can be used.
  • As the working electrode an electrode on which the PQQGDH of the present invention is immobilized is used, 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 the calibration curve prepared with the standard concentration of gnolecose solution.
  • D-Dalcono 1,5-Lataton + ferrocyanide ion The presence of ferrocyanide ion produced by reduction of ferricyanide ion was confirmed by measuring the decrease in absorbance at a wavelength of 420 nm by spectrophotometry.
  • PIPES NaOH buffer, ⁇ 6 ⁇ 5: 50 mM (1.51 g PIPES (molecular weight 302. 36) suspended in 60 mL of water was dissolved in 5 N NaOH, and 2 ml of 10% Triton X— 1 Calorie 00. Adjust the pH to 6 ⁇ 5 ⁇ 0 ⁇ 05 at 25 ° C using 5N NaOH, and add water to make 100 ml.
  • D-Gnolecose solution Based on 1500mM glucose solution prepared at 150, 300, 450, 600, 750, 900, 1050, 1200, 1350 and 1500mM respectively 1/10, 2/10, 3/10, 4 /
  • the same method was performed except that distilled water was added instead of the glucose solution, and a blank ( ⁇ OD blank) was measured.
  • the above operation was performed using a glucose solution having a concentration of 150 mM to 1500 mM.
  • FIG. 19 shows the concentration of each component in the reaction solution.
  • the change in absorbance per unit time was determined by calculating A ODZmin (A0D test 1 ⁇ OD blank).
  • the horizontal axis of the graph plots the glucose concentration in the reaction solution, and the vertical axis plots ⁇ OD / min corresponding to each glucose concentration.
  • Example 1 Based on the procedure of Example 1, the pH of the buffer was changed from 6.5 to 5.5. All other conditions were in accordance with Example 1.
  • the avoidance of substrate inhibition according to the present invention enables high-concentration glucose measurement with a gnolecose measurement reagent, a glucose assembly kit and a gnolecose sensor.

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Abstract

L'invention concerne une méthode pour éviter toute inhibition de substrat par une déshydrogénase de glucose pyrroloquinoline quinone dépendante. L'invention concerne une méthode de réduction d'inhibition de substrat, lors d'une mesure de glucose dans un système contenant une déshydrogénase de glucose pyrroloquinoline quinone dépendante. Cette méthode est caractérisée en ce que la valeur du pH, lors de la mesure de la réaction, est comprise à l'intérieur d'une zone acide.
PCT/JP2006/304440 2005-03-11 2006-03-08 Methode pour eviter l'inhibition d'un substrat par pqqgdh WO2006095758A1 (fr)

Applications Claiming Priority (4)

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JP2005069000 2005-03-11
JP2005-069000 2005-03-11
JP2005-175453 2005-06-15
JP2005175453A JP2006280360A (ja) 2005-03-11 2005-06-15 Ｐｑｑｇｄｈの基質阻害を回避する方法

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002073181A1 (fr) * 2001-03-13 2002-09-19 Koji Sode Electrode enzymatique
EP1367120A2 (fr) * 2002-05-27 2003-12-03 Toyo Boseki Kabushiki Kaisha Glucose Déhydrogénase dépendante de la Pyrroloquinoline Quinone avec une specifité de substrat et une stabilité supérieure
WO2003106668A1 (fr) * 2002-06-13 2003-12-24 Sode Koji Glucose déshydrogénase
JP2004173538A (ja) * 2002-11-25 2004-06-24 Amano Enzyme Inc ピロロキノリンキノン依存性グルコース脱水素酵素
WO2005026340A1 (fr) * 2003-09-08 2005-03-24 Toyo Boseki Kabushiki Kaisha Forme modifiée de la glucose déshydrogénase (gdh) pyrroloquinoline quinone (pqq) présentant une excellente spécificité de substrat
JP2005073630A (ja) * 2003-09-02 2005-03-24 Toyobo Co Ltd 酵素の反応性を改変する方法および反応性を改変した修飾酵素
JP2005073631A (ja) * 2003-09-02 2005-03-24 Toyobo Co Ltd 酵素の基質特異性を改変する方法および基質特異性を改変した修飾酵素
JP2006034165A (ja) * 2004-07-27 2006-02-09 Toyobo Co Ltd Ｐｑｑｇｄｈ製造法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002073181A1 (fr) * 2001-03-13 2002-09-19 Koji Sode Electrode enzymatique
EP1367120A2 (fr) * 2002-05-27 2003-12-03 Toyo Boseki Kabushiki Kaisha Glucose Déhydrogénase dépendante de la Pyrroloquinoline Quinone avec une specifité de substrat et une stabilité supérieure
WO2003106668A1 (fr) * 2002-06-13 2003-12-24 Sode Koji Glucose déshydrogénase
JP2004173538A (ja) * 2002-11-25 2004-06-24 Amano Enzyme Inc ピロロキノリンキノン依存性グルコース脱水素酵素
JP2005073630A (ja) * 2003-09-02 2005-03-24 Toyobo Co Ltd 酵素の反応性を改変する方法および反応性を改変した修飾酵素
JP2005073631A (ja) * 2003-09-02 2005-03-24 Toyobo Co Ltd 酵素の基質特異性を改変する方法および基質特異性を改変した修飾酵素
WO2005026340A1 (fr) * 2003-09-08 2005-03-24 Toyo Boseki Kabushiki Kaisha Forme modifiée de la glucose déshydrogénase (gdh) pyrroloquinoline quinone (pqq) présentant une excellente spécificité de substrat
JP2006034165A (ja) * 2004-07-27 2006-02-09 Toyobo Co Ltd Ｐｑｑｇｄｈ製造法

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