WO2006068170A1 - Procede ameliorant la stabilite thermique d'une deshydrogenase de glucose - Google Patents

Procede ameliorant la stabilite thermique d'une deshydrogenase de glucose Download PDF

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WO2006068170A1
WO2006068170A1 PCT/JP2005/023468 JP2005023468W WO2006068170A1 WO 2006068170 A1 WO2006068170 A1 WO 2006068170A1 JP 2005023468 W JP2005023468 W JP 2005023468W WO 2006068170 A1 WO2006068170 A1 WO 2006068170A1
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Prior art keywords
enzyme
gdh
glucose
activity
pqqgdh
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PCT/JP2005/023468
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English (en)
Japanese (ja)
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WO2006068170A9 (fr
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Tadanobu Matsumura
Masao Kitabayashi
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Toyo Boseki Kabushiki Kaisha
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Priority claimed from JP2004371277A external-priority patent/JP4591074B2/ja
Priority claimed from JP2005327281A external-priority patent/JP2007129966A/ja
Application filed by Toyo Boseki Kabushiki Kaisha filed Critical Toyo Boseki Kabushiki Kaisha
Publication of WO2006068170A1 publication Critical patent/WO2006068170A1/fr
Publication of WO2006068170A9 publication Critical patent/WO2006068170A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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

Definitions

  • the present invention relates to a method for improving the stability of a composition comprising a soluble coenzyme-bound type of gnolecose dehydrogenase (hereinafter referred to as GDH).
  • GDH soluble coenzyme-bound type of gnolecose dehydrogenase
  • the present invention relates to a glucose measurement method using GDH with improved stability and a genolecose sensor.
  • the present invention also relates to a method for improving the holoformation rate or stability in a dry state of a composition containing soluble coenzyme-linked gnolecose dehydrogenase (hereinafter, dalcose dehydrogenase is also referred to as GDH).
  • GDH soluble coenzyme-linked gnolecose dehydrogenase
  • the present invention relates to a glucose measuring method using a GDH with improved holo-formation rate or stability, and a genolecose sensor.
  • the PQQGDH of the present invention is useful for the determination of glucose in clinical examinations and food analysis.
  • Blood glucose self-measurement is important for diabetics to grasp their normal blood glucose level and use it for treatment.
  • an enzyme using gnolecose as a substrate is used for sensors used for blood glucose self-measurement.
  • An example of such an enzyme is gnorecoxidase (EC 1. 1. 3. 4).
  • Glucose oxidase has been used for a long time as an enzyme for blood glucose sensors because of its high specificity to glucose and excellent heat stability, and its first announcement was made about 40 years ago. Go back.
  • measurement is performed by passing electrons generated in the process of oxidizing glucose and converting it to D dalcono ⁇ rataton to the electrode through a mediator.
  • dissolved oxygen affects the measured value because the protons generated in the above are easily passed to oxygen.
  • Dependent glucose dehydrogenase (EC1. 1. 5. 2 (BEC1. 1. 99. 17)) is used as an enzyme for blood glucose sensors.
  • NAD (P) -dependent glucose dehydrogenase has poor stability and requires the addition of a coenzyme.
  • PQQ-dependent glucose dehydrogenase PQQ-dependent glucose dehydrogenase is
  • QQGDH has the disadvantage that it affects the accuracy of measured values because it also acts on sugars other than glucose, such as maltose and ratatoose, which have poor substrate specificity.
  • Patent Document 1 discloses a flavin-binding glucose dehydrogenase derived from the genus Aspergillus. This enzyme is superior in that it has excellent substrate specificity and is not affected by dissolved oxygen. With regard to thermal stability, the activity remaining rate is about 89% after 15 minutes of treatment at 50 ° C, and it is said that the stability is also excellent. However, considering the fact that heat treatment may be required in the process of manufacturing the sensor chip, it is by no means sufficient stability.
  • Patent Literature l WO 2004/058958
  • Fig. 1 shows the residual rate (%) of PQQGDH activity after treatment for 16 hours at 50 ° C in a PQQGDH composition based on PIPES buffer (pH 6.5) in the presence of various compounds. Show.
  • Figure 2 shows the results after treatment of PQQGDH composition in the presence of various compounds based on phthalate buffer (pH 7.0) and potassium phosphate buffer (pH 7.0) at 50 ° C for 16 hours. P Indicates the residual rate (%) of QQGDH activity.
  • FIG. 3 shows the residual rate (%) of NADGDH activity after 1 hour treatment of NADGDH composition coexisting with various compounds at 50 ° C.
  • FIG. 4 shows the residual rate (%) of FADGDH activity after treatment for 30 minutes at 50 ° C. with a FADGDH composition in the presence of a proteinaceous stabilizer.
  • FIG. 5 shows the residual ratio (%) of FADGDH activity after treatment of a FADGDH composition coexisting with a dicarboxylic acid compound at 50 ° C. for 30 minutes.
  • FIG. 6 shows the FADGDH activity remaining rate (%) after a 15 minute treatment at 50 ° C. with a FADGDH composition in the presence of a salt compound.
  • Fig. 7 shows the optimum temperature for PQQ-dependent GDH.
  • Fig. 8 shows the thermal stability of PQQ-dependent GDH.
  • FIG. 9 shows the results of studying the thermal stability effect of heating treatment. 4 after heating
  • Residual activity was calculated with the activity value of Sampu Nore stored at ° C as 100%.
  • FIG. 10 shows the results of the examination of the heat treatment conditions (temperature X time). The residual activity was calculated with the activity value of the sample stored at 4 ° C after the heating treatment as 100%.
  • FIG. 11 shows the results of an examination of the effect of re-cooking treatment at 50 ° C for 16 hours after the heating treatment.
  • the residual activity after heat treatment at 55 ° C for 1 hour was calculated with the activity value of the sampnore stored at 4 ° C as 100%.
  • FIG. 12 Percentage of expression level of holo-type PQQGDH calculated by dividing holo-type PQQGDH activity (U / ml) expressed per unit liquid volume by total PQQGDH activity (U / ml).
  • FIG. 13 Percentage of expression level of holo-type PQQGDH calculated by dividing holo-type PQQGDH activity (U / ml) expressed per unit volume by total PQQGDH activity (U / ml).
  • An object of the present invention is to provide a reagent for measuring a blood glucose level that overcomes the above-mentioned drawbacks related to the stability of known enzymes for blood glucose sensors and is more practically advantageous.
  • Patent Document 2 has reported on measures to improve the stability of PQQGDH, among which there are reports of studies using PQQGDH modification means at the gene level. The possibility of increasing means It was not touched.
  • Patent Document 2 WO02 / 072839
  • the present invention has the following configuration.
  • a method for improving the thermal stability of a composition comprising a soluble coenzyme-linked glucose dehydrogenase comprising a step of heating the composition.
  • Item 2 The method for improving thermal stability according to Item 1, wherein the coenzyme is pyrroloquinone quinoline or a flavin compound.
  • a glucose assembly kit comprising the composition according to Item 5.
  • the improvement in thermal stability according to the present invention reduces the heat inactivation of the enzyme during preparation of the glucose measurement reagent, the gnolecose assay kit, and the glucose sensor, thereby reducing the amount of the enzyme used and the measurement accuracy. Enable improvement. It also makes it possible to provide a blood glucose measurement reagent using GDH, which has excellent storage stability.
  • the improvement of the holo-type ratio or storage stability according to the present invention also improves the storage stability of glucose measuring reagents, glucose assembly kits, and glucose sensors, thereby reducing the amount of the enzyme used and improving the measurement accuracy. To. It also makes it possible to provide a blood glucose level measurement reagent using GDH, which has excellent storage stability.
  • GDH is an enzyme that catalyzes the following reaction.
  • GDH that can be applied to the method of the present invention is not particularly limited as long as it is a soluble coenzyme-bound dalcos dehydrogenase (GDH).
  • coenzyme for example, pyrroloquinone quinoline or flavin compound or nicotinamide adenine dinucleotide (NAD) can be used.
  • NAD nicotinamide adenine dinucleotide
  • the GD H (PQQGDH) taking pyrroloquinone quinoline as a coenzyme that can be applied to the method of the present invention is not particularly limited, but for example, acinetopacter • force no coreaceticus (Acinetobacter calcoaceticus) from LMD79.41 (A. M. Cleton—Jansen et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Genet., 217, 430 (1989)), Escherichia E. coli) (A. M. Cleton—Jansen et al., J.
  • Acinetobacter baumannii (CIcinetobacter baumannii) NCIMB1151 7 or more, ij, Acinetobacter calcoaceticus (this was classified).
  • 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 GLD-321 manufactured by Toyobo Co., Ltd. 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) Cultivated transformants transformed into suitable plasmids (many are known in the art, eg, E. coli), and culture fluid force centrifugation 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.
  • a water-soluble fraction containing PQQGDH can be obtained. Or expressed P by using an appropriate host vector system.
  • QQGDH can be secreted directly into the culture medium.
  • 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 should preferably be 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.
  • GDH taking FAD as a coenzyme (FAD-dependent GDH) that can be applied to the method of the present invention is not particularly limited.
  • filamentous fungi belonging to the category of eukaryotes And those derived from microorganisms such as Penicillium and Aspergillus. These microbial strains can be easily obtained by requesting distribution from each strain storage organization.
  • Penicillium Penicillium, Lirashino Echinulatam is registered with the Product Evaluation Technology Foundation's “Biological Resources Department” under the deposit number NBRC6092.
  • the medium for culturing the microorganism is not particularly limited as long as the microorganism grows and can produce the GDH shown in the present invention. More preferably, the medium is a carbon source or inorganic necessary for the growth of the microorganism.
  • a liquid medium containing a nitrogen source and Z or an organic nitrogen source is more preferable, and a liquid medium suitable for aeration stirring is preferred.
  • liquid medium for example, glucose, dextran, soluble starch, sucrose, etc. as the carbon source.
  • the nitrogen source for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, defatted soybeans, A potato extract etc. are illustrated.
  • other nutrients for example, inorganic salts such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride, Vitamins and the like).
  • the culture method follows a method known in the art. For example, spore of microorganisms or growing cells are inoculated in a liquid medium containing the above nutrients, and the cells are allowed to grow by standing or aeration stirring, but preferably cultured by aeration stirring.
  • 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.
  • 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.
  • the change is monitored by sampling the culture medium and measuring the GDH activity in the culture liquid, and the point when the increase in GDH activity over time is eliminated is regarded as the peak. To stop the culture.
  • a method of extracting GDH from the above 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 as a solvent, Preferably resuspended in water or buffer.
  • the resuspended cells can be disrupted by a known method to extract GDH in the cells into a solvent.
  • a crushing method a lytic enzyme can be used, or a physical crushing method can be used.
  • the lytic enzyme is not particularly limited as long as it has the ability to digest the fungal cell wall. Examples of applicable enzymes include “Lyticase” manufactured by Sigma.
  • Examples of the physical crushing method include ultrasonic crushing, glass bead crushing, and French press. After the crushing treatment, the residue can be removed by centrifugation or filtration to obtain a GDH crude solution.
  • the culture method in the present invention may be solid culture.
  • the eukaryotic microorganism having the ability to produce GDH of the present invention is grown on wheat bran or the like while appropriately controlling temperature, humidity and the like.
  • 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 solid matter such as bacterial cell bran by centrifugation or filtration.
  • GDH GDH
  • Purification of GDH can be performed by appropriately combining various separation techniques that are usually used depending on the fraction in which GDH activity is present. From the GDH extract above, for example Salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing PAGE, SDS_PAGE, ion exchange chromatography, hydroxyapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric A known separation method such as point electrophoresis can be selected appropriately. Various stabilizers and the like can also be added to the extracted GDH or the purified GDH solution.
  • Such substances include sugars and sugar alcohols such as mannitol, trehalose, sucrose, sonorebitonore, erythritole, glyceronole, etc., amino acids such as bovine serum albumin, egg white albumin and various Proteins such as chaperones and peptides.
  • sugars and sugar alcohols such as mannitol, trehalose, sucrose, sonorebitonore, erythritole, glyceronole, etc.
  • amino acids such as bovine serum albumin, egg white albumin and various Proteins such as chaperones and peptides.
  • the GDH of the present invention can also be pulverized by force S, lyophilization, vacuum drying, spray drying or the like which can be provided in a liquid state.
  • GDH dissolved in a buffer solution or the like can be used, and it is preferable to add sugar / sugar alcohols, amino acids, proteins, peptides, etc. as excipients or stabilizers. It can also be granulated after pulverization.
  • the composition of the buffer used for the GDH extraction, purification, pulverization, and stability test described above is not particularly limited, but preferably has a buffer capacity in the range of pH 5-8.
  • buffers such as boric acid, Tris hydrochloric acid, potassium phosphate, BES, Bicine, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, M ⁇ PS ⁇ , PIPES, P ⁇ Good buffering agents such as PS0, TAPS, TAPS0, TES, Tricine are listed.
  • 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 buffering agent 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, or may be contained in advance when a glucose measuring reagent, a glucose assay kit, or a glucose sensor described later is prepared. In this case, the liquid state, the dry state, etc. are not limited, and it is sufficient to function during measurement.
  • the improvement in stability as used in the present invention means that the residual rate (%) of the maintained GDH enzyme increases after heat treatment of the composition containing the GDH enzyme at a certain temperature for a certain period of time.
  • the sampnore stored at 4 ° C is regarded as 100%, and the activity value of the GDH solution after heat treatment at 55 ° C for 1 hour is compared with that of the enzyme. Survival rate is calculated. If this residual rate was increased compared to that without heating treatment, it was judged that the thermal stability of GDH was improved.
  • the PQQGDH activity value (a) after 4 hours at 16 ° C and the GDH activity value (b) after heat treatment were measured.
  • the relative value ((b) / (a) X 100) was calculated when a) was 100. This relative value was defined as the residual rate (%). Then, the presence or absence of the heating treatment was compared, and it was determined that the thermal stability was improved when the residual rate increased due to the execution.
  • heat treatment in the above description refers to a treatment for confirming the stability of the enzyme and is different from the “warming treatment” of the present invention. The same applies to the explanations other than in this paragraph.
  • the heating treatment requires that at least 50% or more of the activity be maintained before and after the heating treatment, preferably 80% or more of the activity is maintained, and more preferably 90% or more. It is desirable that the activity of is maintained.
  • the temperature at which large heat inactivation of the enzyme occurs is the absolute value of the slope of the approximate expression obtained from three or more consecutive data when the residual activity of the enzyme is examined at several different processing temperatures. This means the point where the temperature changes more than twice (processing temperature).
  • processing temperature For example, in Fig. 1, the absolute expression of the approximate expression at 0, 30, and 40 ° C is 0.23, and the inclination of the approximate expression at 40, 50, and 60 ° C is 0.233. The absolute value is 3.63. Therefore, under this heat treatment condition, 40 ° C is a temperature at which large thermal deactivation occurs.
  • the temperature at which large heat inactivation of the enzyme in this patent occurs is the temperature at which the residual activity of the enzyme is 80% or less when heat-treated at several different temperatures.
  • the lower temperature is referred to as “temperature at which large heat inactivation of the enzyme occurs”. If it can only be determined by one method, the temperature determined by that method is defined as “the temperature at which a large heat inactivation of the enzyme occurs”.
  • the temperature at which this large heat inactivation occurs varies depending on the type of enzyme, and even for the same enzyme, it varies depending on the enzyme concentration used in the study.
  • the temperature at which large heat inactivation occurred was 40 ° C (Fig. 1)
  • the heating treatment is examined at 50 ° C. or less.
  • the improvement of the holo-type ratio of the present invention is thought to lead to an increase in stability.
  • the improvement of the holo-type ratio in the present invention means that the residual rate (%) of GDH enzyme is maintained after the composition containing GDH enzyme is heat-treated at a certain temperature for a certain time. Means. In the present invention, 100 sampnole stored at 4 ° C, in which activity is almost completely maintained, is 100. / 0 and to, by comparing the activity value of GDH solution after heat treatment certain time and this at a constant temperature, and calculates the residual rate of that enzyme. When this residual ratio was increased compared to that without the compound, it was judged that the holo ratio of GDH was improved.
  • the improvement in stability as used in the present invention means that the residual rate (%) of the maintained GDH enzyme increases after heat treatment of the composition containing the GDH enzyme at a certain temperature for a fixed time. Means. In the present invention, 100 sampnole stored at 4 ° C, in which activity is almost completely maintained, is 100. / 0, by comparing the activity value of GDH solution after heat treatment predetermined time which is constant temperature, and calculates the residual rate of the enzyme. When this residual ratio was increased as compared with that without the compound, it was judged that the storage stability of GDH was improved.
  • the GDH activity value (a) of the solution stored at 4 ° C and the GDH activity value after heat treatment at a constant temperature for a certain time (b) ) was measured, and the relative value ((b) / (a) X 100) with respect to the measured value (a) as 100 was determined. This relative value was defined as the residual rate (%). Then, by comparing the presence or absence of the additive of the compound, it was determined that the stability was improved when the residual ratio increased by the addition.
  • Each GDH using different coenzymes is considering improvement of the holo-type ratio or stability under different conditions.
  • PQQGDH the enzyme solution prepared to 5 U / ml with a pH 6.5 buffer solution is heat-treated at 50 ° C for 16 hours, and then the remaining PQQGDH activity is compared to improve the holo ratio or stability. I have confirmed.
  • NADGDH was heat-treated at 50 ° C for 1 hour with a pH 8.0 buffer solution at 85 U / ml
  • the remaining N ADGDH activity was compared to improve the holo ratio or stability. I have confirmed.
  • the enzyme solution prepared at 5 U / ml in a buffer solution of ⁇ 6.5 was heat-treated at 50 ° C for 15-30 minutes, and then the remaining FADGDH activity was compared to determine the holo ratio or stability. The improvement is confirmed.
  • the determination as to whether the holo-type ratio is improved can also be made as follows.
  • the GDH activity value (a) measured by adding a sufficient amount of coenzyme to the activity measurement method described in the measurement method for GDH enzyme activity described below, and measured without adding any coenzyme.
  • the GDH activity value (b) was measured, and the relative value ((b) Z (a) X 100) with respect to the measured value (a) as 100 was determined. This relative value was defined as a holo-formation rate (%). Then, by comparing the presence or absence of the addition of the compound, it was determined that the stability was improved when the holoformation rate was increased by the additive.
  • the effect of the present invention becomes more remarkable in a system including a mediator.
  • the mediator applicable to the method of the present invention is not particularly limited, but a combination of phenazine methosulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP), PMS and nitro blue tetra Combinations with Zorium (NBT), DCPIP alone, ferricyanide ions (such as ferricyanium potassium as a compound) alone, and furcene alone. Of these, ferricyanide ions (such as potassium ferricyanide as the compound) are preferable.
  • Each of these mediators has various differences in sensitivity, so it is not necessary to uniformly define the concentration of addition, but generally an additive of ImM or higher is desirable.
  • mediators may be added at the time of measurement, or may be contained in advance when a glucose measuring reagent, a glucose assay kit, or a gnolecose sensor described later is prepared.
  • the liquid state, the dry state, etc. may be used, and it may be dissociated during the reaction at the time of measurement so as to be in an ionic state.
  • various components can be allowed to coexist if necessary.
  • surfactants, stabilizers, excipients and the like may be added.
  • PQQGDH it is possible to further stabilize PQQGDH by adding calcium ions or salts thereof, amino acids such as glutamic acid, gnoretamine, 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 10-4-1 X 10-2M.
  • the stabilizing effect of PQQGDH by containing calcium ions or calcium salts can be further improved by containing an amino acid selected from the group consisting of gnoretamic acid, gnoletamine 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.
  • egg white albumin (OVA) may be added.
  • one or more compounds selected from the group consisting of aspartic acid, glutamic acid, monoketoglutaric acid, malic acid, monoketognoreconic acid, a- cyclodextrin and their salts, and (2 ) PQQGDH can be stabilized by coexisting albumin.
  • PQQGDH as an enzyme is a glucose dehydrogenase having pyroguchi quinoline quinone (PQQ) as a coenzyme. Since it catalyzes the reaction that oxidizes glucose to produce darconoratone, it can be used for blood glucose measurement. Blood glucose level is diabetes It is an extremely important index for clinical diagnosis as an important marker of disease. Currently, blood glucose levels are measured mainly using a biosensor that uses glucose oxidase, but the reaction is affected by the dissolved oxygen concentration, which may cause errors in the measured values. was there. PQQ-dependent glucose dehydrogenase is attracting attention as a new enzyme that replaces this glucose oxidase.
  • the inventors of the present invention have made extensive studies in order to solve the above-mentioned problems. As a result, the aqueous solution containing the PQQ-dependent dalcoyl dehydrogenase is subjected to heat treatment, so that the holo in the total GDH enzyme protein during production can be obtained. The proportion of type PQQGDH could be improved and the present invention was finally completed.
  • Improvement in productivity as a holo-type PQQGDH according to the present invention results in a reduction in manufacturing cost. Furthermore, the increase in the proportion of holo-type PQQGDH eliminates the need to add a PQQ to form holo to obtain active PQQGDH, resulting in a reduction in manufacturing costs. These make it possible to manufacture PQQGDH inexpensively. Furthermore, it is also possible to provide a dull course assembly kit and a gnore course sensor at a low price.
  • PQQGDH used in the present invention is an enzyme that catalyzes the reaction when P-Q quinoline quinone is coordinated as a coenzyme to oxidize D-darcose to produce D-darcono 1,5 ratatones. (ECl. 1. 5. 2 (formerly ECl. 1. 99. 17)), and the origin and structure are not particularly limited.
  • Seticus Acinetobacter calcoaceticus LMD79. 41 (AM Cleton—Jans en et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Genet., 217, 430 (1989)), Escherichia coli ) (AM Cleton—Jansen et al., J. Bacteriol., 172, 6308 (1990)), Gnoreconactor oxydans (Mol. Gen. Genet., 229, 206 (1991)) And Acinetobacter baumanni NCIMB11517 reported in Patent Document 1.
  • Acinetobacter baumannii NCIMB11517 strain was categorized as ij, Acinetobacter calcoaceticus.
  • the amino acid sequence of PQQGDH derived from the genus Acinetobacter is preferably the AQ / DH of PQQu ⁇ DH derived from Acinetob acter calcoaceticus or AcinetoDacter baumanmi.
  • SEQ ID NO: 1 is preferable.
  • the wild-type PQ QGDH protein represented by SEQ ID NO: 1 and its base sequence represented by SEQ ID NO: 2 originated from Acinetobacter baumannii strain NCIMB 11517, and disclosed in JP-A-11-243949 Is disclosed.
  • the amino acid notation is numbered with 1 for aspartic acid from which the signal sequence has been removed.
  • PQQGDH used in the present invention has glucose dehydrogenase activity
  • a part of another amino acid residue may be deleted or substituted. Residues may be added.
  • the produced DNA having the genetic information of the modified protein is transferred into a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the modified protein.
  • a host microorganism for example, pBluescript, pUC18 and the like can be used when Escherichia coli is used as a host microorganism.
  • host microorganisms that can be used include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5.
  • a method for transferring the recombinant vector into the host microorganism for example, when the host microorganism belongs to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. Further, an elect mouth position method may be used. Furthermore, a commercially available combi- tive cell (for example, combitent high JM109; manufactured by Toyobo) may be used.
  • Such a gene can be extracted from these strains or chemically synthesized. Furthermore, a DNA fragment containing the PQQGDH gene can be obtained by using the PCR method.
  • methods for obtaining a gene encoding PQQGDH include the following methods. For example, after isolating and purifying the chromosome of Acinetobacter calcoaceticus NCIMB11517, DNA was cleaved using sonication, restriction enzyme treatment, etc., linear expression vector and blunt ends or attachment of both DNAs A recombinant vector is constructed by closing and ligating DNA ends with DNA ligase. Recombinant vector containing a gene encoding GDH having PQQ as a prosthetic group after transferring the recombinant vector into a replicable host microorganism and screening using the expression of the vector marker and enzyme activity as an indicator Get the microorganisms that hold.
  • the microorganism carrying the recombinant vector is cultured, the recombinant vector is isolated and purified from the cells of the cultured microorganism, and a gene encoding GDH is obtained from the expression vector.
  • a gene encoding GDH is obtained from the expression vector.
  • the chromosomal DNA of the gene donor Acinetopacter 'calcoaceticus NCIMB11517 is specifically collected as follows.
  • the gene-donating microorganism is centrifuged, for example:! ⁇ For 3 days with stirring.
  • the lysate containing GDH gene having PQQ as a prosthetic group can be prepared by collecting the bacterium and then lysing it.
  • a method for lysis for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease or other enzyme or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as necessary.
  • SDS sodium lauryl sulfate
  • it may be combined with a physical crushing method such as freeze-thawing or French press treatment.
  • a method such as deproteinization by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment or the like is appropriately performed according to a conventional method. It can be done by combining.
  • a method for cleaving DNA separated and purified from a microorganism can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Type II restriction enzymes that act on specific nucleotide sequences are suitable.
  • a vector constructed for gene recombination from a phage or plasmid capable of autonomously growing in a host microorganism is suitable.
  • the fage include Lambda gtlO and Lambda gtl l when Escherichia coli is used as a host microorganism.
  • plasmids include pBR322, pUC19, and pBluescript when Escherichia coli is used as a host microorganism.
  • a vector fragment can be obtained by cleaving the above-described vector with the restriction enzyme used for cleaving the microbial DNA that is the gene donor encoding GDH described above. It is not necessary to use the same restriction enzyme as that used to cleave the microbial DNA.
  • the microbial DNA fragment and the vector DNA fragment may be combined by any known DNA ligase method. For example, after annealing the attachment end of the microbial DNA fragment and the attachment end of the vector fragment, an appropriate DNA ligase may be used. Use to create a recombinant vector of microbial and vector DNA fragments. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.
  • the host microorganism used for cloning is not particularly limited as long as the recombinant vector is stable, can autonomously proliferate, and can express a foreign gene.
  • Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5, etc. can be used.
  • a combi- tive cell method using calcium treatment or an electo-baudation method can be used as a method for transferring the recombinant vector into the host microorganism.
  • the microorganism which is a transformant obtained as described above, can stably produce a large amount of GDH by being cultured in a nutrient medium.
  • the selection of whether or not the target recombinant vector is transferred to the host microorganism may be performed by searching for a microorganism that simultaneously expresses GDH activity by adding PQQ and the drug resistance marker of the vector holding the target DNA. For example, a microorganism that grows on a selective medium based on a drug resistance marker and produces GDH may be selected.
  • transfer from a recombinant vector carrying a GDH gene having PQQ selected once as a prosthetic group to a recombinant vector capable of replicating in a microorganism capable of producing PQQ is as follows. It can be easily carried out by collecting DNA, which is a GDH gene, from a recombinant vector carrying the GDH gene by restriction enzymes or PCR and linking it to other vector fragments. In addition, the transformation of microorganisms capable of producing PQQ with these vectors can be carried out by using the calcium treatment, the electoral cell method, the electopore method, or the like.
  • microorganisms capable of producing PQQ include methanol-utilizing bacteria such as Methylobacterium, acetic acid bacteria belonging to the genus Acetobacter and Gluconobacter, and Flavobacterium (Flavobacterium). And bacteria of the genus Genus, Syudomonas, and Acinetopacter. Among them, a host-vector system that can use a genus Pseudomonas and a bacterium belonging to the genus Acinetopacter has been established and is preferable because it is easy to use.
  • Pseudomonas In the genus Pseudomonas, Pseudomonas' Elginosa, Pseudomonas' fluorescens, Pseudomonas' Putida and the like can be used.
  • Acinetobacter 1 'Calcoaceticus For bacteria belonging to the genus Acinetopacter, Acinetobacter 1 'Calcoaceticus, Acinetobacter 1' Baumannii and the like can be used.
  • a vector derived from RSF1010 or a vector having a similar replicon can be used for bacteria belonging to the genus Pseudomonas.
  • ⁇ 240, ⁇ 24, etc. MM Bagdasarian et al., Gene, 26, 273 (1983)
  • pCN40, pCN60 etc. CC Nieto et al., Gene, 87, 145 (1990)
  • pTS 1137 gene recombination practical technology
  • pME290 etc. Y. Itoh et al., Gene, 36, 27 (1985)
  • pNIll, pNI20C N. Itoh et al., J. Biochem., 110, 614 (1991)
  • pNIll pNI20C
  • Microorganisms which are transformants thus obtained, can be produced as a whole P QQGDH protein by cultivating them in a nutrient medium, ⁇ the ability to produce QQGDH protein S, and adding an organic solvent to the medium. Both productivity improvement and productivity improvement as holo-type PQQGDH protein are possible.
  • the culture conditions should be selected in consideration of the nutritional physiological properties of the host. Industrially, aeration and agitation culture is advantageous.
  • the holo-type PQQGDH refers to the PQQGDH enzyme itself in which the coenzyme PQQ is bound to the GDH protein and the state thereof, and the enzyme having the GDH activity without the addition of PQQ and the state thereof. It is.
  • all PQQGDH means (1) holo-type PQQGDH, (2) apo-type PQQGDH in which coenzyme PQQ is not bound to GDH protein, and (3) the binding state of PQQGDH is not bound. This refers to the total GDH enzyme protein combined with PQ QGDH, which does not have GDH activity because it is complete, and its state.
  • PQQ-dependent gnolecose dehydrogenase or “PQQGDH” is a single protein, but as an enzyme co-enzyme complex, it is a mixture containing at least one of the above (1) to (3) when viewed from the binding state of PQ Q. means.
  • a nutrient source of the medium those commonly used for culturing microorganisms can be widely used.
  • Any carbon compound that can be assimilated may be used as the carbon source.
  • the nitrogen source may be any available nitrogen compound.
  • peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used.
  • phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc and other salts, specific amino acids, specific vitamins and the like are used as necessary.
  • the culture temperature is a force that can be appropriately changed within the range in which the bacteria grow and produce PQQGDH.
  • the culture time is slightly different depending on the conditions. It is usually about 6 to 48 hours, preferably about 16 to 36 hours if the culture is completed at an appropriate time in consideration of the time when the maximum yield of PQQGDH is reached.
  • the pH of the medium can be appropriately changed within the range in which the bacteria grow and produce PQQGDH, but is preferably in the range of about ⁇ 6.0 to 9.0, more preferably in the range of ⁇ 6.5 to 8.0.
  • a culture solution containing cells that produce PQQGDH in the culture can be collected and used as is, but generally, if PQQGDH is present in the culture solution, it is filtered and centrifuged according to conventional methods. It is used after separating the PQQGDH-containing solution and microbial cells by the above.
  • PQQGDH is present in the microbial cells
  • the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are collected by a mechanical method or an enzymatic method such as lysozyme. Destroy it, and if necessary, add a chelating agent such as EDTA and a surfactant to solubilize GDH and separate and collect it as an aqueous solution.
  • 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, etc. It may be precipitated by the fractional precipitation method. Heat treatment and isoelectric point treatment are also effective purification means. Also, adsorbent or gel Purified PQQGDH can be obtained by gel filtration using a filter medium, adsorption chromatography, ion exchange chromatography, and affinity chromatography.
  • the purified enzyme preparation can be obtained by separation and purification by column chromatography.
  • the purified enzyme preparation is preferably purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).
  • the heat treatment described above plays a role of improving the ratio of holo-type PQQGDH to the total GDH enzyme protein.
  • This treatment that improves PQQGDH activity without adding PQQ from the outside is extremely useful for industrial applications. This phenomenon is presumed to occur when the GDH enzyme protein is expressed, and because it binds to PQQ but is in an inactive state due to its incomplete binding state, GDH enzyme protein strength S, and heat treatment conformation. Yong's change is considered to have improved the binding state with PQQ and became active. Thus, it was surprising and surprising that there may be GDH enzyme proteins that bind to PQQ but are inactive due to imperfect binding.
  • the heat treatment conditions are preferably 25 ° C to 50 ° C, more preferably 30 ° C to 45 ° C.
  • This heat treatment is not only useful for production using a host producing PQQ as described above. It is also useful when preparing a holo-type PQQGDH by holizing apo-type GDH protein. For example, after cloning the GDH gene, the apo-type GDH protein is expressed directly using Escherichia coli DH5, purified in the same manner as PQQGDH, and then the purified apo-type GDH protein is holoed to prepare the holo-type PQQGDH And so on.
  • the purified enzyme obtained as described above can be pulverized and distributed, for example, by freeze drying, vacuum drying, spray drying, or the like.
  • the purified enzyme can be dissolved in phosphate buffer, Tris-HCl buffer or GOOD buffer.
  • Preferred are GOOD buffers, with PIPES, MES or MOPS buffers being particularly preferred.
  • PQQGDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as gnoretamine, gnoretamine, and lysine, and serum albumin.
  • the PQQGDH protein can take various forms such as liquid (aqueous solution, suspension, etc.), powder, and lyophilized.
  • the freeze-drying 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. Further, when measuring the genole course, various forms such as a glucose assembly kit and a glucose sensor can be taken.
  • the purified modified protein thus obtained can be stabilized by the following method.
  • the PQQGDH content varies depending on the origin of the enzyme, but is usually suitably used in the range of about 5 to 50% (weight ratio). In terms of enzyme activity, it is preferably used in the range of 100 to 2000 U / mg.
  • Salts of aspartic acid, gnoretamic acid, ⁇ -ketoglutaric acid, malic acid, and hyketogluconic acid include salts such as sodium, potassium, ammonium, calcium, and magnesium, but are not particularly limited. .
  • the addition amount of the above compound, its salt and dicyclodextrin is preferably 1 to 90% (weight ratio). These substances can be used alone or in combination.
  • the buffer solution to be contained is not particularly limited, but Tris buffer solution, phosphate buffer Examples include impulse solution, borate buffer solution, and GOOD buffer solution.
  • the pH of the buffer solution is adjusted in accordance with the intended use within a range of about 5.0.90.
  • 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. 30% (weight ratio). Used in range.
  • albumin examples include bovine serum albumin (BSA) and ovalbumin (OVA). BSA is particularly preferable.
  • the albumin content is preferably in the range of 180% (weight ratio), more preferably 570% (weight ratio).
  • Further stabilizers and the like may be added to the composition in a range that does not particularly adversely affect the reaction of PQQGDH.
  • the blending method of the stabilizer of the present invention is not particularly limited. For example, a method in which a stabilizer is added to a buffer solution containing PQQGDH, a method in which PQQGDH is added to a buffer solution containing a stabilizer, or a method in which PQQGDH and a stabilizer are simultaneously added to a buffer solution may be mentioned.
  • a stabilizing effect can also be obtained by adding calcium ions. That is, the modified protein 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 content of calcium ions is preferably 1 X 10 1 X 10_ 2 M .
  • the stabilizing effect by containing calcium ions or calcium salts is further improved by containing an amino acid selected from the group consisting of glutamic acid, glutamine and lysine.
  • the amino acid selected from the group consisting of gnoretamic acid, glutamine and lysine may be one kind or two or more kinds.
  • the content of an amino acid selected from the group consisting of gnoretamic acid, gnoletamine and lysine is preferably 0.01 to 0.2% by weight.
  • serum albumin may be added.
  • the content is preferably 0.05 to 0.5% by weight.
  • the buffering agent a normal one is used, and the buffer having a pH of 5 to 10 is usually used. I like it. Specifically, Tris-HCl, boric acid, and the power for which Good's buffer is used Any buffer that does not form an insoluble salt with calcium can be used.
  • a surfactant such as sodium EDTA, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite
  • the present invention includes a step of heating the composition in a composition containing a soluble coenzyme-bound type of gnolecose dehydrogenase.
  • This is a method for producing a composition containing soluble coenzyme-linked gnolecose dehydrogenase with improved thermostability.
  • the present invention provides a composition comprising a soluble coenzyme-bound glucose dehydrogenase produced by the method described above, wherein a soluble coenzyme-bound glucose dehydrogenase having improved thermal stability is obtained. It is a composition to contain.
  • gnole course can be measured by the following various methods.
  • the reagent for measuring gnolecose, the gnolecose assembly kit, and the gnolecose sensor of the present invention are various forms such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying, spray drying, etc. Can be taken.
  • the drying 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, but may be a solution in which the dried product is redissolved.
  • gnole course can be measured by the following various methods.
  • the reagent for measuring gnolecose of the present invention typically includes reagents necessary for measurement such as GDH, buffer solution, mediator, 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 GDH 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 holo- ed at the time of use.
  • the invention also features a glucose assembly kit comprising GDH according to the invention.
  • the glucose assay kit of the present invention is a GDH according to the present invention is converted into at least one assay. Contains enough.
  • the kit contains the GDH of the present invention plus the buffers necessary for assembly, mediators, glucose standard solutions for creating calibration curves, and usage guidelines.
  • the GDH according to the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.
  • the GDH 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 hololated at the time of use.
  • the invention also features a glucose sensor using GDH according to the invention.
  • 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.
  • Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, an oxidation-reduction polymer, or the like in the polymer together with a mediator. These can be adsorbed and fixed on the electrode, or a combination of these may be used.
  • the GDH of the present invention may be immobilized in the form of apoenzyme in the form of a holoform and immobilized on the electrode, and the coenzyme may be supplied as a separate layer or in solution.
  • the GDH of the present invention is immobilized on a carbon electrode using dartalaldehyde, and then treated with a reagent having an amine group to block dartalaldehyde.
  • the glucose concentration can be measured as follows. Put the buffer in the thermostatic cell and add the mediator to maintain a constant temperature.
  • a working electrode an electrode on which GDH of the present invention is immobilized is used, and a counter electrode (for example, platinum electrode) and a reference electrode (for example, Ag / Ag C1 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. It is possible to calculate the glucose concentration in the sample according to the calibration curve prepared with the standard concentration glucose solution.
  • a method for improving the stability of the enzyme comprising a step of subjecting the composition to a heating treatment, wherein the composition comprises a soluble coenzyme-linked glucose dehydrogenase.
  • a soluble complement with improved thermal stability produced by a method comprising a step of subjecting a composition comprising a soluble coenzyme-linked glucose dehydrogenase to a heating treatment.
  • Composition comprising enzyme-linked glucose dehydrogenase
  • a glucose sensor comprising the composition according to [5].
  • Soluble coenzyme-bound glucose dehydrogenase with improved stability comprising a step of subjecting the composition to soluble coenzyme-bound glucose dehydrogenase to a heating treatment.
  • a method for producing a composition comprising:
  • the present invention has the following configuration from another viewpoint.
  • a method comprising a composition containing a soluble coenzyme-bound glucose dehydrogenase, wherein a compound having a carbocinole group or an amino group is added to the composition to improve the mouth ratio of the enzyme.
  • composition comprising a soluble coenzyme-bound dalcoose dehydrogenase having an improved holo-type ratio by the method of [1] to [3].
  • a glucose sensor comprising the composition according to [ 9 ].
  • a method for improving the ratio of holo-type PQQGDH to total GDH enzyme protein characterized by heat-treating an aqueous solution containing PQQ-dependent glucose dehydrogenase
  • a method for producing a PQQ-dependent glucose dehydrogenase comprising heat-treating an aqueous solution containing the PQQ-dependent glucose dehydrogenase
  • a glucose sensor comprising a PQQ-dependent glucose dehydrogenase produced by the method according to [2].
  • Example 1 Construction of an expression plasmid for the PQQ-dependent gnolecose dehydrogenase gene
  • Wild-type PQQ-dependent glucose dehydrogenase expression plasmid PNPG5 is expressed in the multiple cloning site of the vector pBluescript SK (-).
  • Acinetobacter baumannii A structural gene encoding PQQ-dependent glucose dehydrogenase derived from NCIMB 1 1517 strain.
  • the base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of PQQ-dependent glucose dehydrogenase deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.
  • PNPG5 DNA (5 ⁇ g) was cleaved with restriction enzymes BamHI and Xhol (Toyobo Co., Ltd.), and the structural gene part of mutant PQQ-dependent glucose dehydrogenase was isolated.
  • the isolated DNA and pTM33 (1 ⁇ g) cleaved with BamHI and Xhol were reacted with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA.
  • the ligated DNA was transformed using a competent cell of Escherichia coli DH5a.
  • the resulting expression plasmid was named pNPG6.
  • Pseudomonas' Putida TE3493 (Microeken No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C for 16 hours, and the cells were collected by centrifugation (12,000 rpm, 10 minutes) Then, 8 ml of 5 mM K-phosphate buffer (PH7.0) containing 300 mM sucrose cooled on ice was added to the cells to suspend the cells. The cells are collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer ( ⁇ 7.0) containing 300 mM sucrose ice-cooled is added to the cells. The body was suspended.
  • LBG medium LB medium + 0.3% glycerol
  • the suspension was transformed with the expression plasmid PNPG6 obtained in Example 1 by the electrovolution method.
  • the desired transformant was obtained from a colony grown on LB agar medium containing 100 ⁇ g Zml of streptomycin.
  • the PQQ-dependent glucose dehydrogenase activity at the end of the culture is about 30 U / ml per 1 ml of the culture solution, as measured by the above activity measurement.
  • the cells are collected by centrifugation, suspended in 20 mM phosphate buffer ( PH 7.0), disrupted by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. It was.
  • the obtained crude enzyme solution was separated and purified by HiTrap_SP (Amersham-Falmasia) ion exchange column chromatography. Next, after dialyzing with 10 mM PIPES_Na 0 H buffer (pH 6.5), calcium chloride was added to a final concentration of mM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Falmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band on SDS-PAGE.
  • Test Example 1 Method for measuring PQQ-dependent GDH activity
  • the activity of PQQ-dependent GDH is measured under the following conditions.
  • DCPIP 2,6-dichlorophenol-indophenol
  • PMS phenazine methosulfate
  • One unit refers to the amount of PQQGDH enzyme that forms 1.0 mmol of DCPIP (red) per minute under the conditions described below.
  • D-glucose solution 1. OM (l. 8g D-glucose (molecular weight 180. 16) / l0ml H20)
  • PIPES_NaOH buffer solution pH 6.5: 50 mM (1.51 g PIPES (molecular weight 302. 36) suspended in 60 mL water was dissolved in 5 N NaOH, and 2 ml 10% Triton X— 1 Get 00 power Q. The pH was adjusted to 6.5 ⁇ 0.05 at 25 ° C using 5N NaOH, and water was added to make 100 ml. )
  • the enzyme powder was dissolved in ice-cooled enzyme diluent (E) immediately before the assembly and diluted to 0.05-0.10 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).
  • the above activity measurement procedure was carried out using another type of sugar solution as a substrate instead of the glucose solution.
  • Vt Total volume (3. lml)
  • Vs Sample volume (0 ⁇ lml)
  • Test example 2 NAD-dependent GDH activity measurement method
  • NAD-dependent GDH activity is measured under the following conditions.
  • Toyobo's glucose dehydrogenase (GLD311) was used as the NAD-dependent GDH enzyme preparation.
  • the amount of D-gnolecose + NAD + ⁇ D-gnolecono_1,5-latataton + NADH + H + NADH was measured by the change in absorbance at 340 nm.
  • One unit is the amount of NADGDH enzyme that forms 1.0 micromolar NADH per minute under the conditions described below.
  • D-glucose solution 1.5M (2.7g D-glucose (molecular weight 180.16) / l0ml H20)
  • NAD solution 8% (80mg NAD (molecular weight 717.48) / lmlH20)
  • the enzyme powder was dissolved in ice-cold enzyme diluent (D) immediately before the assembly and diluted to 0.10-0.70 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).
  • each L dry specimen was inoculated into potato dextrose agar medium (manufactured by Difco) and incubated at 25 ° C.
  • the mycelium on the restored plate was collected together with the agar and suspended in filter sterilized water.
  • Production medium 1% malt extract, 1.5% soy peptide, 0.1% MgS04'7 hydrate, 2./.Dalcos, pH6.5
  • the above mycelial suspensions were respectively added to start the culture.
  • the culture conditions were a temperature of 30 ° C, an aeration rate of 2 L / min, and a stirring rate of 3 80 i "pm. After 64 hours from the start of the culture, the culture was stopped and a Nutsche filter was used. The cells of each strain were collected on the filter paper by suction filtration. Concentrate 5 L of the culture solution to 1/10 volume with a hollow fiber module for ultrafiltration with a molecular weight of 10,000,000 cut, and add ammonium sulfate to the concentrate to a final concentration of 60% saturation (456 g / U).
  • the mediator used in the glucose measurement composition, glucose assay kit, glucose sensor, or glucose measurement method of the present invention is not particularly limited, but preferably 2, 6-dichlorophenol- indophenol (abbreviation). DCPIP), ferrocene or their derivatives (eg potassium ferricyanide, phenazine methosulfate, etc.). These mediators are commercially available.
  • Test Example 3 Method for measuring FAD-dependent GDH activity
  • the activity of FAD-dependent GDH is measured under the following conditions.
  • 1 unit (G) 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-darcose.
  • TEST BLANK In the formula, 3.0 is the volume of the reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm2 / micromole) under the conditions for this activity measurement, 0.1 is the enzyme solution The amount of liquid (ml), 1.0 indicates the optical path length (cm) of the cell.
  • Example 5 Confirmation of holo ratio or storage stability using glucose measurement system The examination was performed according to the method for measuring PQQGDH activity in Test Example 1 described above. In addition, in order to measure the enzyme activity of PQQGDH including the apo-type, the activity was also measured in a reaction mixture containing a final concentration of 860 nM PQQ.
  • each sample was diluted 10-fold with an enzyme diluent, and then PQQGDH activity was measured.
  • the enzyme activity of each sample stored at 4 ° C for 16 hours was defined as 100, and the activity values after treatment at 50 ° C for 16 hours were compared and calculated as a relative value (%).
  • FIG. 1 shows the residual rate (%) of PQQGDH activity after treatment of PQQ GDH composition coexisting with various compounds based on PIPES buffer (pH 6.5) at 50 ° C. for 16 hours.
  • Figure 2 shows the residual rate of PQQG DH activity after treatment of PQQGDH composition in the presence of phthalate buffer ( ⁇ 7.0) and potassium phosphate buffer ( ⁇ 7.0) in the presence of various compounds at 50 ° C for 16 hours. (%).
  • Example 6 Confirmation of holo-type ratio or storage stability using glucose measurement system 1 The examination was performed according to the method for measuring GDH activity described above. First, NADGDH (Toyobo GLD-311) was diluted to 80-90 U / ml with enzyme dilution (ImM CaC12, 0.1%
  • the holo-type ratio or the storage stability was improved by adding the compounds to the NADGDH composition.
  • the highest effect was observed when succinic acid or maleic acid was added.
  • Fig. 3 shows the residual ratio (%) of NA DGDH activity after 1 hour treatment of NADGDH composition coexisting with various compounds at 50 ° C.
  • Example 7 Confirmation of holo ratio or storage stability using glucose measurement system 2 The study was conducted according to the NADGDH activity measurement method of Test Example 2 described above.
  • Example 8 Confirmation of holo ratio or storage stability using glucose measurement system 3
  • the examination was performed according to the method for measuring FADGDH activity in Test Example 3 described above.
  • the enzyme dilution solution (ImM) was adjusted so that the FADGLD obtained in Example 4 was about 10 U / ml.
  • FIG. 4 shows the residual ratio (%) of FADGDH activity after treatment at 50 ° C. for 30 minutes with a FADGDH composition in the presence of a proteinaceous stabilizer.
  • FIG. 5 shows the residual ratio (%) of FADGDH activity after treatment of a FADGDH composition coexisting with a dicarboxylic acid compound at 50 ° C. for 30 minutes.
  • FIG. 6 shows the residual rate (%) of the FAD GDH activity after treatment with a salt compound in the presence of a FADGDH composition at 50 ° C. for 15 minutes.
  • Example 7 Construction of PQQ-dependent Gnorecose Dehydrogenase Gene Expression Plasmid Wild-type PQQ-dependent glucose dehydrogenase expression plasmid PNPG5 was added to the multiple cloning site of the beta pBluescript SK (—). Acinetobacter baumannii) A structural gene encoding a PQQ-dependent darcos dehydrogenase derived from NCIMB11517 strain. The base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of PQQ-dependent glucose dehydrogenase deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.
  • QuickChangeTM Site -Directed Mutagenes is Kit (manufactured by STRATAGENE) based on a recombinant plasmid pN PG5 containing a wild-type PQQ-dependent glucose dehydrogenase gene and a synthetic oligonucleotide of about 40mer containing a triplet encoding the amino acid at the site of mutation introduction
  • Mutation treatment operation according to the protocol using J, J168, Q168A, 169Y, L169P, A170L, E245D, M342I, N429D, 430P mutations introduced, mutant PQQ-dependent type with improved substrate specificity Glucose dehydro
  • a recombinant plasmid (pNPG5_Q168A + 169Y + L169P + Al70L + E245D + M342I + N429D + 430P) encoding a genease was obtained.
  • the nucleotide sequence of the obtained candidate strain was determined, and the 168th glutamine of the amino acid sequence described in SEQ ID NO: 1 was alanine, the 169th leucine was proline, the 170th alanine was leucine, and the 245th gnore.
  • Mutant PQQ with thamic acid replaced with aspartic acid, 342th methionine replaced with isoleucine, 429th asparagine replaced with aspartic acid, 168th followed by tyrosine and 429th after proline It was confirmed that it encodes type glucose dehydrogenase.
  • E. coli competent cells (Escherichia coli JM 109; manufactured by Toyobo Co., Ltd.) were transformed with the recombinant plasmid to obtain transformants.
  • Example 9 Construction of an expression vector that can replicate in Pseudomonas bacteria
  • Recombinant plasmid obtained in Example 8 pNPG5-Q 168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P DNA 5 ⁇ g cleaved with restriction enzymes BamHI and Xhol (manufactured by Toyobo) and dependent on mutant PQQ The structural gene portion of type glucose dehydrogenase was isolated. The isolated DNA was reacted with pTM33 (1 ig) cleaved with BamHI and Xhol and 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA.
  • the ligated DNA was transformed using a competent cell of Escherichia coli DH5a.
  • the obtained expression plasmid was designated as pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P.
  • Pseudomonas.Putida TE3493 (Microeken No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C for 16 hours, and the cells were collected by centrifugation (12,000 rpm, 10 minutes) Then, 8 ml of 5 mM K_phosphate buffer solution (PH7.0) containing 300 mM sucrose cooled with ice was added to the cells to suspend the cells. The cells are collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer ( ⁇ 7.0) containing 300 mM sucrose cooled on ice is added to the cells. The body was suspended.
  • LBG medium LB medium + 0.3% glycerol
  • Example 9 the expression plasmid pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P obtained in Example 9 was added to 0.5 / ig calecto, electopores. Transformation was performed by the Yong method. A target transformant was obtained from a colony grown on an LB agar medium containing 100 ⁇ g / ml streptomycin.
  • the PQQ-dependent glucose dehydrogenase activity at the end of the culture was about 30 U / ml per 1 ml of the culture solution after the measurement of the activity, and the cells were collected by centrifugation, and the 20 mM phosphate buffer solution ( After suspending in pH 7.0), the mixture was crushed by sonication and further centrifuged to obtain a supernatant as a crude enzyme solution.
  • the resulting crude enzyme solution was separated and purified by HiTrap-SP (Amersham-Falmasia) ion exchange column chromatography. Then, after dialyzing with 10 mM PIPES-NaOH buffer ( ⁇ 6 ⁇ 5), calcium chloride was added so that the final concentration force was SlmM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Falmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band on SDS-PAGE.
  • the purified enzyme thus obtained was used as a PQQ-dependent GLD evaluation sample.
  • each L dry specimen was inoculated into potato dextrose agar medium (manufactured by Difco) and incubated at 25 ° C.
  • the mycelium on the restored plate was collected together with the agar and suspended in filter sterilized water.
  • Production medium 1% malt extract, 1.5% soy peptide, 0.1% MgS04'7 hydrate, 2./.Dalcos, pH6.5
  • 10L jar mentors Prepare the above mycelial suspension after autoclaving at 120 ° C for 15 minutes. Each was charged and culture was started.
  • the culture conditions were a temperature of 30 ° C., an aeration rate of 2 L / min, and a stirring rate of 3 80 ⁇ ⁇ ⁇ .
  • the culture was stopped 64 hours after the start of the culture, and the cells of each strain were collected on the filter paper by suction filtration using a Nutsche filter.
  • the mediator used in the composition for measuring glucose, the glucose assay kit, the glucose sensor, or the glucose measuring method of the present invention is not particularly limited, but preferably 2, 6-dichlorophenol- indophenol (abbreviation). DCPIP), ferrocene or their derivatives (eg ferricyanium potassium, phenazine methosulfate, etc.). These mediators are commercially available.
  • Example 13 Optimal temperature and thermal stability of PQQ-dependent GDH
  • Thermal stability is 50, 40, 50, 60, 70 with 50 mM PIPES-NaOH (pH 6.5), ImU CaC12 composition and 5.0 U / ml PQQ-dependent GDH. Compare the GDH activity remaining after heat treatment at C for 30 minutes with the activity value of the sample stored at 4 ° C. Residual activity (%) was calculated (FIG. 8). Until the heat treatment temperature was 40 ° C, there was no significant decrease in activity, and there was a tendency to be largely deactivated by the 50 ° C treatment. Thermal stability is likely to vary depending on the solution composition and enzyme concentration. For example, the higher the enzyme concentration, the higher the temperature at which large heat inactivation is observed (it will be 50 ° C or higher).
  • Example 14 Effect of heating treatment on the stability of PQQ-dependent GDH
  • Figure 9 shows the results of studying the thermal stability effect of heating treatment.
  • the residual activity was calculated with the activity value of the sample stored at 4 ° C after the heating treatment as 100%.
  • Figure 10 shows the results of the examination of temperature treatment conditions (temperature X time). The residual activity was calculated by setting the activity value of the sample stored at 4 ° C after heating to 100%.
  • Fig. 11 shows the results of studying the effects of rewarming after warming.
  • the residual activity was calculated with the activity value of the sample stored at 4 ° C after the heating treatment as 100%.
  • the composition of the present invention is a composition comprising a soluble coenzyme-linked glucose dehydrogenase, which is produced by a method comprising a step of subjecting the composition to a heating treatment, and having improved heat stability.
  • Example 15 Construction of expression plasmid for PQQ-dependent gnolecose dehydrogenase gene
  • Wild-type PQQ-dependent gnolecose dehydrogenase expression plasmid PNPG5 is a cinetopactor ⁇ ⁇ ⁇ at the multiple cloning site of the vector pBluescript SK (-).
  • a structural gene encoding a PQQ-dependent glucose dehydrogenase derived from Acinetobacter baumannii NCIMB11517 strain was inserted.
  • the base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of PQQ-dependent glucose dehydrogenase deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.
  • Example 16 Construction of an expression vector capable of replicating in Pseudomonas bacteria
  • DNA 5 ⁇ g of the recombinant plasmid pNPG5 obtained in Example 15 was cleaved with restriction enzymes BamHI and XHoI (Toyobo Co., Ltd.) to depend on PQQ.
  • the structural gene portion of gnolecose dehydrogenase was isolated.
  • the isolated DNA and pTS 1 137 (1 ⁇ g) cleaved with BamHI and XHoI were reacted with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA.
  • the ligated DNA was transformed using Escherichia coli DH5a competent cells.
  • the resulting expression plasmid was named PNPG6.
  • Pseudomonas' Putida TE3493 (Microeken No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C for 16 hours, and the cells were collected by centrifugation (12,000 rpm, 10 minutes) Then, 8 ml of 5 mM K-phosphate buffer (PH7.0) containing 300 mM sucrose cooled on ice was added to the cells to suspend the cells. The cells are collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer ( ⁇ 7.0) containing 300 mM sucrose ice-cooled is added to the cells. The body was suspended.
  • LBG medium LB medium + 0.3% glycerol
  • the expression plasmid pNPG6 obtained in Example 16 was transformed to 0. 0, and transformed by the electrovolution method.
  • the desired transformant was obtained from a colony grown on LB agar medium containing 100 ⁇ g / ml streptomycin.
  • NTB nitrotetrazolium blue
  • PMS phenazine methosulfate
  • One unit forms 0.5 mmol of diformazan per minute under the conditions described below Check the amount of PQQGDH enzyme.
  • D glucose solution: 0.5M (0.9 g D—glucose (molecular weight 180. 16) / l0ml H 2 O)
  • 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 5N NaOH, 2. 2 ml of 10% Triton X Calorie free 100. Use 5N NaOH 25. Adjust the pH to 6.5 ⁇ 0.05 with C and add 100 ml with water.
  • reaction mixture into a test tube (plastic) and preheat at 37 ° C for 5 minutes 0.1 ml of enzyme solution was added and gently inverted to mix.
  • 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 measurement was carried out in the same manner after the enzyme was diluted with 50 mM PIPES NaOH buffer (pH 6.5) containing 0.1% Triton X—100, 0.1% BSA and ⁇ ⁇ PQQ.
  • GDH production medium (1.5% glycerol, 4% yeast extract, 1.25% K2HP04, 0.23% KH2P04, pH 6.8) into a 500 ml Sakaguchi flask. C, 20 minutes A one-clave was performed and the medium was sterilized. After standing to cool, separately sterile filtered streptomycin was added to 100 ⁇ g / ml, and ethanol was further added to 1% (V / V). The transformant obtained in Example 17 was cultured in this medium at 33 ° C for 24 hours, and the cells were collected by centrifugation (12000 rpm, 5 minutes). The cells are suspended in 20 mM phosphate buffer (pH 7.0), crushed by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. According to Test Examples 4 and 5, PQQGDH Activity was measured.
  • the ratio of the expression level of holo PQQGDH was calculated by dividing the holo PQQGD H activity (U / ml) expressed per unit volume by the total PQQGDH activity (U / ml).
  • Figure 12 shows the measurement results.
  • Example 19 Purification of PQQGDH and confirmation of improvement in proportion of holo-type PQQGDH by heat treatment HiTrap- SP (Amersham-Farmasia) ion buffered with 20 mM phosphate buffer (pH 7.0)
  • the exchange column chromatography was charged and the GDH protein was adsorbed on the resin.
  • 20 mM phosphate buffer (pH 7.0) which is twice the amount of column resin
  • 20 mM phosphate buffer (pH 7.0) containing twice the same amount of 0.3 M NaCl GDH protein was eluted from the resin, and the eluted fraction was collected.
  • calcium chloride was added to a final concentration of SlmM.
  • the dialysate is divided into 2 minutes, (A) a method for obtaining a purified enzyme preparation after heat treatment, and (B) a method for obtaining a purified enzyme preparation without heat treatment, respectively. Carried out.
  • the stability of GDH itself is increased by heating the GDH composition. It is possible to provide a reagent with high storage stability by performing a heating process at the time of production of the glucose measuring reagent.
  • the improvement of the holo-type ratio or the storage stability according to the present invention can improve the measurement accuracy with a glucose measuring reagent, a glucose assay kit and a glucose sensor.
  • productivity as a holo type PQQGDH can be improved, and PQQGDH can be manufactured at low cost.
  • the activity value per unit protein weight of PQQGDH is improved by increasing the proportion of holo-type PQQG DH.
  • PQQGDH is preferable for use because it allows a reduction in the amount of protein added to the glucose assembly kit and glucose sensor.

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Abstract

Problème à résoudre: trouver un procédé améliorant la stabilité d'une composition comprenant une déshydrogenase de glucose soluble se liant par coenzyme. Solution: un procédé améliorant la stabilité thermique d'une déshydrogenase de glucose soluble se liant par coenzyme dans une composition comprenant cette enzyme, ce procédé consistant à traiter cette composition avec de la chaleur, un processus de production d'une composition comprenant cette enzyme possédant cette stabilité thermique améliorée, une composition produite par ce procédé, un kit de dosage de glucose et un capteur de glucose comprenant cette composition et un procédé permettant de déterminer une concentration de glucose au moyen de cette composition.
PCT/JP2005/023468 2004-12-22 2005-12-21 Procede ameliorant la stabilite thermique d'une deshydrogenase de glucose WO2006068170A1 (fr)

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JP2005327281A JP2007129966A (ja) 2005-11-11 2005-11-11 グルコースデヒドロゲナーゼの熱安定性を向上する方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2083074A1 (fr) * 2006-11-14 2009-07-29 Toyo Boseki Kabushiki Kaisha Glucose deshydrogenase dependante du flavine-adenine dinucleotide modifiee
EP2083074A4 (fr) * 2006-11-14 2010-01-27 Toyo Boseki Glucose deshydrogenase dependante du flavine-adenine dinucleotide modifiee
US8039248B2 (en) 2006-11-14 2011-10-18 Toyo Boseki Kabushiki Kaisha Modified flavin adenine dinucleotide dependent glucose dehydrogenase

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