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    • 专利摘要:
    In water-soluble glucose dehydrogenase with pyrroloquinolinequinone as a coenzyme, it is thought that it exists on the surface of the enzyme molecule and the side chain is exposed on the surface of the molecule, so that it does not interact largely with other residues. A modified glucose dehydrogenase is disclosed in which one or more amino acid residues selected from the group consisting of glutamine, asparagine and threonine are substituted with arginine. This modified enzyme can be efficiently recovered after recombinant production.
    公开号:KR20040062945A
    申请号:KR10-2004-7004432
    申请日:2002-09-26
    公开日:2004-07-09
    发明作者:고지 소데
    申请人:고지 소데；
    IPC主号:
    专利说明:

    Glucose Dehydrogenase {GLUCOSE DEHYDROGENASE}
    [2] Blood glucose levels are important markers of diabetes and are very important indicators for clinical diagnosis. In addition, the determination of glucose concentration in fermentation production using microorganisms is an important item in process monitoring. Conventionally, glucose was quantified by an enzymatic method using glucose b oxidase (GOD) or glucose hexaphosphate dehydrogenase (G6PDH). In recent years, the application of glucose dehydrogenase (PQQGDH) which uses pyrroloquinolinequinone as a coenzyme as a new enzyme is attracting attention. Since PQQGDH has high oxidative activity against glucose and PQQGDH does not require oxygen as an electron acceptor because PQQGDH is a coenzyme-bound enzyme, it is expected to be applied to the field of assay including the recognition device of glucose sensor. have.
    [3] PQQGDH is a glucose dehydrogenase that uses pyrroloquinolinequinone as a coenzyme and catalyzes a reaction for oxidizing glucose to produce gluconolactone.
    [4] PQQGDH is known to contain membrane-bound enzymes and water-soluble enzymes. Membrane-bound PQQGDH is a single peptide protein having a molecular weight of about 87 kDa and is widely found in various Gram-negative bacteria. On the other hand, water-soluble PQQGDH has been identified in several states of Acinetobacter calcoaceticus (Biosci. Biotech. Biochem. (1995), 59 (8), 1548-1555), and its structural gene has been cloned to reveal the amino acid sequence. (Mol. Gen. Genet. (1989), 217: 430-436). A. calcoaceticus-derived water-soluble PQQGDH is a water-soluble enzyme that forms a homodimer composed of two pieces of the sub-unit molecular weight of about 50kDa, requires Ca 2+ and PQQ to indicate that the activity, such as 2200U / ㎎~7400U / ㎎ It shows high enzymatic activity. It is known that the isoelectric point is a basic protein having an apoenzyme not associated with PQQ of about 9.2 and a homoenzyme of about 10.2 (K. Matsushita, et al. (1995) Biosci. Biotech. Biochem., 59). , 1548-1555). The results of X-ray structural analysis of water-soluble PQQGDH have also been published (A. Oubrie, et al. (1999) J. Mol. Bio., 289, 319-333 and A. Oubrie, et al. (1999) The EMBO Journal, 18 (19), 5187-5194), the conformational structure of water-soluble PQQGDH and the estimated presence position of PQQ and Ca 2+ are known.
    [5] Regarding the purification of the water-soluble PQQGDH, Duine et al. Completely purified the water-soluble PQQGDH from A. calcoaceticus to obtain an inactivation of 640 U / mg at a recovery rate of 10% (P. Dokter. Et al. (1986) Biochem. J. , 239, 163-167). They were subjected to cation exchange chromatography, gel filtration chromatography, cation exchange chromatography, and gel filtration chromatography on water-soluble fractions prepared from A. calcoaceticus, and identified a single band of about 50 kDa by SDS-PAGE. It was. Subsequent studies yielded an inactivation of 2214U / mg with 44% recovery (as described above in K. Matsushita, et al.). In addition, they recombinantly produced the soluble PQQGDH structural gene into Escherichia coli, and performed two cation exchange chromatography and fractional chromatography to obtain an inactivation of 7400U / mg with a recovery rate of 41% (AJJ Olsthoorn, and JA Duine ( 1996) Archives of Biochem.Biophys., 336, 42-48).
    [6] As a method for efficiently producing PQQGDH, a method for recombinant production in Escherichia coli and a method for recombinant production using a yeast or enterobacterial bacterial group as a host have been reported. When recombinantly expressing water-soluble PQQGDH in this manner, this enzyme is expressed as a water-soluble protein, and since its isoelectric point is very high and basic, cation exchange chromatography is mainly used for purification. However, it is difficult to remove other basic proteins from the host by cation exchange chromatography alone.
    [7] On the other hand, as a method for purifying a basic protein, a method of adding an arginine tag to the C terminal as an affinity tag is known for the purpose of improving affinity to a cation exchange column (HM Sassenfeld, and SJ Brewe (1984). ) Biotechnology, 2, 76-81). Application of this method to the water-soluble PQQGDH basic protein is expected to increase the surface charge of the water-soluble PQQGDH, thereby improving the affinity for the cation exchange column. However, the production of E. coli-added protein by E. coli results in the difficulty of obtaining a single enzyme by arranging the arginine residue from the C-terminal side by the outer membrane protease of E. coli.
    [8] Accordingly, an object of the present invention is to provide a modified water-soluble PQQGDH that enables efficient recovery of recombinantly produced PQQGDH.
    [1] The present invention relates to a modified glucose dehydrogenase in which specific amino acid residues of glucose dehydrogenase (GDH) containing pyrroloquinolinequinone (PQQ) as coenzymes are substituted with other amino acid residues. The modified enzyme of the present invention is useful for quantification of glucose in clinical trials, food analysis, and the like.
    [14] 1 shows a method for producing a mutant gene encoding a modified enzyme of the present invention.
    [15] Fig. 2 shows the structure of plasmid pGB2 used in the present invention.
    [16] Fig. 3 is a photograph showing SDS-PAGE of the modified enzyme of the present invention.
    [17] Figure 4 shows the enzyme activity for each fraction of the chromatography of the modified enzyme of the present invention.
    [18] 5 shows an assay for glucose using the modified PQQGDH of the present invention.
    [19] 6 shows a calibration curve of an enzyme sensor using the modified PQQGDH of the present invention.
    [20] Best Mode for Carrying Out the Invention
    [21] The contents of all patents and references expressly cited in the present specification are to be incorporated herein by reference in their entirety. In addition, all the content of the specification of the Japan patent application 2001-294846 which is an application of the priority application of this application is included in this specification by reference.
    [22] Design of the modified PQQGDH
    [23] In order to manufacture the modified PQQGDH of the present invention, based on the conformational information of the natural water-soluble PQQGDH, a site capable of increasing surface charge without changing various properties such as enzyme activity and stability by amino acid substitution is selected. do. The selection is carried out according to the following criteria: those present on the surface of the water-soluble PQQGDH protein, being neutral residues, being polar residues, and the side chains are exposed on the molecular surface so that large interactions with other residues Those not considered to be present, or those present in regions not believed to be enzyme-activating or substrate-binding sites. By replacing the selected amino acid residues with basic residues, especially arginine residues, the surface charge of the protein can be increased without significantly affecting the enzyme activity. Preferably, the amino acid residues to be mutated are selected from the group consisting of glutamine, asparagine and threonine.
    [24] Since the modified PQQGDH of the present invention thus obtained is firmly bound to cation exchange chromatography by increased surface charge, it can be easily separated and purified from other proteins derived from the host using cation exchange chromatography.
    [25] In the modified glucose dehydrogenase of the present invention, as long as it has the desired glucose dehydrogenase activity, part of another amino acid residue may be deleted or substituted, and another amino acid residue may be added.
    [26] Also, those skilled in the art can easily select PQQGDH having a higher surface charge by selecting neutral polar residues present on the molecular surface and replacing the amino acid residues with arginine, for water-soluble PQQGDH derived from other bacteria. You can get it.
    [27] Manufacturing method of modified PQQGDH
    [28] The sequence of genes encoding the natural water soluble PQQGDH from Acinetobacter calcoaceticus is defined by SEQ ID NO: 2.
    [29] The gene encoding the modified PQQGDH of the present invention can be constructed by replacing a nucleotide sequence encoding an amino acid residue to be substituted with a nucleotide sequence encoding arginine in a gene encoding natural water-soluble PQQGDH. Various methods for such site-specific nucleotide substitutions are known in the art and are described, for example, in Sambrook et al., "Molecular Cloning; A Laboratory Manual", 2nd Edition, 1989, Cold Spring Harbor Laboratory Press, New York It is described in.
    [30] The mutant gene thus obtained is inserted into a gene expression vector (e.g., plasmid) and transformed into a suitable host (e.g., E. coli). Many vectors and host systems for expressing foreign proteins are known in the art, and various types of bacteria, yeasts, cultured cells and the like can be used as hosts, for example.
    [31] After culturing the transformant expressing the modified PQQGDH obtained as described above, and recovering the cells from the culture by centrifugation or the like, the cells are crushed by a French press or the like, or periplasmic shock by osmotic shock. The enzyme is released into the medium. This can be ultracentrifuged to obtain an aqueous fraction containing PQQGDH. Alternatively, by using an appropriate host vector system, the expressed PQQGDH can be secreted into the culture solution.
    [32] The water-soluble fraction thus obtained is purified by cation exchange chromatography. Purification can be carried out according to the textbooks generally known in the art for chromatographic purification of proteins. Various cation exchange chromatography columns that can be used for purification of proteins are known in the art, and any of these may be used. For example, CM-5PW, CM-Toyopearl 650M, SP-5PW (above, Toso Corporation), S-Sepharose, Mono-S, and S-Resorce (above Pharmacia) can be used. Equilibrate the column with a suitable buffer and load the sample onto the column to wash off the unadsorbed components. As a buffer, a phosphate buffer, a MOPS buffer, etc. can be used, for example.
    [33] Next, a higher salt buffer is used to elute the adsorbed components to the column. Salt concentration can be varied by linear gradient, or by a combination thereof, using a plurality of buffers having different salt concentrations. Elution of the sample is monitored by absorbance measurement or the like and aliquoted in an appropriate amount. The modified enzyme of the present invention can be obtained as a purified product by measuring the enzyme activity for each fraction and recovering the desired fraction.
    [34] Further, before or after the cation chromatography, purification by other techniques known in the art may be carried out for purification of proteins, such as filtration, dialysis, gel filtration chromatography, affinity chromatography, and the like, if necessary.
    [35] The purity of the protein can be readily ascertained using methods known in the art, such as SDS-PAGE, HPLC and the like.
    [36] Method of measuring enzyme activity
    [37] PQQGDH of the present invention has a function of catalyzing the reaction of oxidizing glucose to produce gluconolactone using PQQ as a coenzyme. The enzyme activity can be quantified by the color reaction of the redox dye to reduce the amount of PQQ reduced by the oxidation of glucose by PQQGDH. As the color reagent, for example, PMS (phenazinemethsulfate) -DCIP (2,6-dichlorophenol indophenol), potassium ferricyanide, ferrocene and the like can be used.
    [38] Glucose Assay Kit
    [39] The invention also features a glucose assay kit containing the modified PQQGDH according to the invention. The glucose assay kit of the present invention contains the modified PQQGDH according to the present invention in an amount sufficient for at least one assay. The kit typically includes, in addition to the modified PQQGDH of the present invention, the buffer required for the assay, the mediator, the glucose standard for the preparation of the calibration curve, and instructions for use. The modified PQQGDH according to the present invention may be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable preservation solution. Preferably, the modified PQQGDH of the present invention is provided in a complete form. However, it may be provided in the form of an apoenzyme and may be perfected at the time of use.
    [40] Glucose sensor
    [41] The invention also features a glucose sensor using the modified PQQGDH according to the invention. As an electrode, a carbon electrode, a gold electrode, a platinum electrode, etc. are used, and the enzyme of this invention is immobilized on this electrode. The immobilization method includes 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, a redox polymer, or the like, or with an electronic mediator represented by ferrocene or a derivative thereof. The polymer may be fixed or adsorbed on the electrode, or may be used in combination. Preferably, the modified PQQGDH of the present invention is immobilized on an electrode in a complete form, which may be immobilized in the form of an apoenzyme, and PQQ may be provided as another layer or in solution. Typically, the modified PQQGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent having an amine group to block the free functional group of glutaraldehyde.
    [42] Measurement of glucose concentration can be performed as follows. Buffer is added to the incubation cell and PQQ and CaCl 2 and mediator are added to maintain a constant temperature. Potassium ferricyanide, phenazine metsulfate, etc. can be used as a mediaier. As the working electrode, an electrode on which the modified PQQGDH of the present invention is immobilized is used, and a counter electrode (for example, platinum electrode) and a reference electrode (for example, Ag / AgCl electrode) are used. After a constant voltage was applied to the carbon electrode and the current became normal, an increase in the current was measured by adding a sample containing glucose. The glucose concentration in the sample can be calculated according to the calibration curve produced by the glucose solution of the standard concentration.
    [9] The present inventors earnestly researched to develop a modified PQQGDH that can facilitate purification by improving the conventional water-soluble PQQGDH, and as a result, by replacing arginine with a specific residue present on the surface of the water-soluble PQQGDH, It was successful to obtain a mutant enzyme that can be easily purified by.
    [10] That is, in the present invention, in the water-soluble glucose dehydrogenase containing co-enzyme of pyrroloquinolinequinone, one or more amino acid residues selected from the group consisting of glutamine, asparagine and threonine on the surface of the enzyme molecule are substituted with arginine. It provides modified modified glucose dehydrogenase.
    [11] Preferably, the water-soluble glucose dehydrogenase which uses pyrroloquinolinequinone as a coenzyme is water-soluble PQQGDH derived from Acinetobacter calcoaceticus.
    [12] The present invention also provides a water-soluble glucose dehydrogenase derived from Pyrroloquinolinequinone derived from Acinetobacter calcoaceticus as a coenzyme, wherein the 209th glutamine residue is selected from the group consisting of the 240th asparagine residue and the 389th threonine residue. Provided is a modified glucose dehydrogenase wherein more amino acid residues are substituted with arginine. More preferably, the 209th glutamine residue, the 240th asparagine residue, and the 389th threonine residue are each substituted with arginine.
    [13] The present invention also provides a glucose assay kit and a glucose containing the above-described modified glucose dehydrogenase gene, a vector containing the gene and a transformant containing the gene, and the modified glucose dehydrogenase of the present invention. Provide a sensor.
    [43] The present invention will be described in detail with reference to the following Examples, but the present invention is not limited to these embodiments.
    [44] Example 1
    [45] Construction of the modified enzyme PQQGDH gene
    [46] The mutation was introduced based on the structural gene of Acinetobacter calcoaceticus- derived PQQGDH shown in SEQ ID NO: 2. Plasmid pGB2 inserts the structural gene which codes PQQGDH derived from Acinetobacter calcoaceticus into the multicloning site | part of vector pTrc99A (made by Pharmacia) (FIG. 1). By site-specific mutagenesis according to a conventional method, the base sequences encoding glutamine 209, asparagine 240 and threonine 389 were replaced with base sequences encoding arginine, respectively. Site-specific mutations were performed by the method shown in FIG. 2 using plasmid pGB2. The arrangement of the synthetic oligonucleotide target primer specifically used is shown below.
    [47] Q209R
    [48] 5'-gCCAACTCAACgTgAACTgAATg-3 '(SEQ ID NO: 3)
    [49] D229R
    [50] 5'-CTTAAATCTTCgTggAAgTATTC-3 '(SEQ ID NO: 4)
    [51] N240R
    [52] 5'-CCAAgTTTTCgCggggTggTTAg-3 '(SEQ ID NO: 5)
    [53] A Kpn I-HindIII fragment containing a portion of a gene encoding PQQGDH derived from Acinetobacter calcoaceticus was inserted into the vector plasmid pKF18k ( Takarashujo Co., Ltd.). 50 fmol of the template and 5 pmol of the selection primer attached to the Mutan® Express Km kit manufactured by Takarashu Co., Ltd. and 50 pmol of the phosphorylated target primer were used in a total amount of 1/10 of the copper kit. The mixture was mixed with the annealing buffer, and the plasmid was denatured by heat treatment at 100 ° C. for 3 minutes to form one chain. The selection primer is for reversing double amber mutations on the kanamycin resistance gene of pKF18k. This was placed on ice for 5 minutes and the primers annealed. The complementary chain was synthesize | combined by adding 3 microliters of this copper kit extension buffer, 1 microliter of T4 DNA ligase, 1 microliter of T4 DNA polymerase, and 5 microliter of sterile water.
    [54] This was transformed with E. coli BMH 71-18 mutS, a mismatch repair defect defect of DNA, and shaken culture was performed overnight to amplify the plasmid.
    [55] Next, the plasmid extracted here was transformed with E. coli MV1184, and the plasmid was extracted from the colony. Then, sequencing was performed on these plasmids to confirm the introduction of the target mutations. This fragment was replaced with a Kpn I-Hind III fragment of a gene encoding wild type PQQGDH on plasmid pGB2, to construct a modified PQQGDH gene (hereinafter modified PQQGDH) having three mutations of Q209R, D229R and N240R. It was.
    [56] Example 2
    [57] Preparation of modified enzyme
    [58] The gene encoding wild-type or modified PQQGDH was inserted into the multicloning site of pTrc99A (Pharmacia), an expression vector for E. coli, and the constructed plasmid was transformed with E. coli DH5α strain. This was cultured by shaking overnight at 37 ° C using a Sakaguchi flask in 450 ml of L medium (containing 50 µg / ml of ampicillin), and incubated in 7 L of L medium containing 1 mM CaCl 2 , 500 µM PQQ. About 3 hours after the start of the culture, isopropylthiogalactoside was added to a final concentration of 0.3 mM, and then incubated for 1.5 hours. The cells were collected by centrifugation (5,000 × g, 10 minutes, 4 ° C.), and then washed twice with 0.85% NaCl solution. The cells were suspended in 10 mM phosphate buffer (pH 7.0), crushed with a French press (110 MPa), and centrifuged (15,000 x g, 15 minutes, 4 ° C) twice. Removed as precipitation. This supernatant was ultracentrifuged (40,000 rpm, 90 minutes, 4 ° C.), and the supernatant was obtained as an aqueous solution fraction. This was dialyzed overnight at 4 ° C. with A buffer (10 mM MOPS-NaOH buffer (pH 7.0)) to obtain crude fractions.
    [59] Example 3
    [60] Purification by Cation Chromatography
    [61] The crude fraction prepared in Example 1 was filtered through a 0.2 μm filter before adsorbing the column. The column used CM-5PW (Toso Corporation), 10 mM MOPS-NaOH buffer (pH7.0) as A buffer, 0.8 M NaCl + 10 mM MOPS-NaOH buffer (pH 7.0) as B buffer.
    [62] The column was first equilibrated with A buffer, the sample was adsorbed and then washed with 5 times the A buffer of column capacity. Thereafter, a linear gradient of OM-0.64 M NaCl (120 minutes) was added using B buffer to elute the target enzyme. In addition, the flow rate was 0.5 ml / min, and the eluted protein was detected by the light absorption wavelength of 280 nm. The eluate was fractionated for 2 minutes. Wild type water-soluble PQQGDH showed a peak eluting at a salt concentration of about 80 mM in a cation exchange chromatography for about 20 minutes, and a modified water-soluble PQQGDH showed a peak eluting at a salt concentration of about 190 mM for about 38 minutes.
    [63] The fraction of an eluting peak was analyzed by SDS-PAGE, and the result is shown in FIG. About the modified water-soluble PQQGDH, a single band of 50 kDa, which is the desired size, was obtained, and this chromatography was able to purify almost completely. In contrast, wild-type water-soluble PQQGDH showed a band of contaminants.
    [64] Example 4
    [65] Determination of Enzyme Activity
    [66] Determination of enzymatic activity was carried out using PMS (phenadinemethsulfate) -DCIP (2,6-dichlorophenol indophenol) in 10 mM MOPS-NaOH buffer (pH7.0) to spectrophotometric changes in absorbance at 600 nm of DCIP. Tracking was carried out using a photometer, and the rate of decrease in absorbance was defined as the reaction rate of enzyme. At this time, 1 unit of enzymatic activity in which 1 mol of DCIP was reduced in 1 minute was used. In addition, the molar extinction coefficient at the pH 7.0 of DCIP was 16.3 mM -1 .
    [67] Enzyme activity for each fraction of chromatography is shown in FIG. 4. The abscissa is the elution time and the ordinate is the GDH activity.
    [68] Example 5
    [69] Evaluation of Enzyme Activity and Substrate Specificity
    [70] The active fraction, the nonadsorbed fraction, and the crude fraction obtained by chromatography were dialyzed overnight at 4 ° C. with 100 times of 10 mM MOPS-NaOH buffer solution (pH7.0), and were completed for 1 hour or more in the presence of 1 μMPQQ and 1 mM CaCL 2 , respectively. It was done. Pour this in 187 μl portions, 3 μl of active reagent (48 μl of 6 mM DCIP, 8 μl of 600 mM PMS, 16 μl of 10 mM phosphate buffer pH7.0) and substrate, 20 mM of glucose, 2-deoxy-D 10 µl of a solution of -glucose, mannose, arose, 3-o-methyl-D-glucose, galactose, xylose, lactose or maltose was added, and the enzyme activity was measured at room temperature by the method shown in Example 4. Km and Vmax were obtained from a plot of substrate concentration versus enzymatic activity.
    [71] The activity against glucose was about 7100 U / mg wild type water-soluble PQQGDH and about 7800 U / mg modified water-soluble PQQGDH, showing approximately the same activity. In addition, the Km value and the Vmax value for each substrate other than glucose showed approximately the same values for both wild type and modified water soluble PQQGDH, and no change in substrate specificity due to mutagenesis was observed.
    [72] Example 6
    [73] Assay of Glucose
    [74] Glucose was assayed using modified PQQGDH. The modified enzyme was perfected for 1 hour or more in the presence of 1 μM PQQ, 1 mM CaCL 2 , and the enzyme activity was measured in the presence of various concentrations of glucose and 5 μM PQQ, 10 mM CaCL 2 . The method used the change of the absorbance of 600 nm of DCIP as an index according to the measuring method of the enzyme activity described in Example 4. As shown in Fig. 5, the modified PQQGDH can be used to quantify glucose in the range of 5 mM to 50 mM.
    [75] Example 7
    [76] Fabrication and Evaluation of Enzyme Sensors
    [77] 20 mg of carbon paste was added to 5 U of the modified enzyme and lyophilized. After mixing this well, only the surface of the carbon paste electrode which already filled about 40 mg of carbon paste was filled, and it grind | polished on the filter paper. This electrode was treated for 30 minutes at room temperature in 10 mM MOPS buffer (pH7.0) containing 1% glutaraldehyde, followed by room temperature in 10 mM MOPS buffer (pH7.0) containing 20 mM Lysine (Lysin). The glutaraldehyde was blocked by treatment for 20 minutes at. This electrode was equilibrated in 10 mM MOPS buffer (pH 7.0) at room temperature for at least 1 hour. The electrode was stored at 4 ° C.
    [78] Glucose concentration was measured using the produced enzyme sensor. The obtained calibration curve is shown in FIG. That is, glucose determination was possible in the range of 1 mM-12 mM using the enzyme sensor to which the modified PQQGDH of the present invention was immobilized.
    [79] A modified water soluble PQQGDH is provided which allows for efficient recovery of PQQGDH recombinantly produced by the present invention. The modified enzyme of the present invention is useful for quantifying glucose in clinical tests, food analysis, and the like.
    <110> SODE, KOJI<120> Glucose Dehydrogenase<130> psg9008WO<150> JP 2001-294846<151> 2001-09-26<160> 5<170> KopatentIn 1.71<210> 1<211> 454<212> PRT<213> Acinetobacter calcoaceticus<400> 1Asp Val Pro Leu Thr Pro Ser Gln Phe Ala Lys Ala Lys Ser Glu Asn  1 5 10 15Phe Asp Lys Lys Val Ile Leu Ser Asn Leu Asn Lys Pro His Ala Leu             20 25 30Leu Trp Gly Pro Asp Asn Gln Ile Trp Leu Thr Glu Arg Ala Thr Gly         35 40 45Lys Ile Leu Arg Val Asn Pro Glu Ser Gly Ser Val Lys Thr Val Phe     50 55 60Gln Val Pro Glu Ile Val Asn Asp Ala Asp Gly Gln Asn Gly Leu Leu 65 70 75 80Gly Phe Ala Phe His Pro Asp Phe Lys Asn Asn Pro Tyr Ile Tyr Ile                 85 90 95Ser Gly Thr Phe Lys Asn Pro Lys Ser Thr Asp Lys Glu Leu Pro Asn            100 105 110Gln Thr Ile Ile Arg Arg Tyr Thr Tyr Asn Lys Ser Thr Asp Thr Leu        115 120 125Glu Lys Pro Val Asp Leu Leu Ala Gly Leu Pro Ser Ser Lys Asp His    130 135 140Gln Ser Gly Arg Leu Val Ile Gly Pro Asp Gln Lys Ile Tyr Tyr Thr145 150 155 160Ile Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn                165 170 175Gln Ala Gln His Thr Pro Thr Gln Gln Glu Leu Asn Gly Lys Asp Tyr            180 185 190His Thr Tyr Met Gly Lys Val Leu Arg Leu Asn Leu Asp Gly Ser Ile        195 200 205Pro Lys Asp Asn Pro Ser Phe Asn Gly Val Val Ser His Ile Tyr Thr    210 215 220Leu Gly His Arg Asn Pro Gln Gly Leu Ala Phe Thr Pro Asn Gly Lys225 230 235 240Leu Leu Gln Ser Glu Gln Gly Pro Asn Ser Asp Asp Glu Ile Asn Leu                245 250 255Ile Val Lys Gly Gly Asn Tyr Gly Trp Pro Asn Val Ala Gly Tyr Lys            260 265 270Asp Asp Ser Gly Tyr Ala Tyr Ala Asn Tyr Ser Ala Ala Ala Asn Lys        275 280 285Ser Ile Lys Asp Leu Ala Gln Asn Gly Val Lys Val Ala Ala Gly Val    290 295 300Pro Val Thr Lys Glu Ser Glu Trp Thr Gly Lys Asn Phe Val Pro Pro305 310 315 320Leu Lys Thr Leu Tyr Thr Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro                325 330 335Thr Cys Gly Glu Met Thr Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser            340 345 350Ser Ala Tyr Val Tyr Lys Gly Gly Lys Lys Ala Ile Thr Gly Trp Glu        355 360 365Asn Thr Leu Leu Val Pro Ser Leu Lys Arg Gly Val Ile Phe Arg Ile    370 375 380Lys Leu Asp Pro Thr Tyr Ser Thr Thr Tyr Asp Asp Ala Val Pro Met385 390 395 400Phe Lys Ser Asn Asn Arg Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly                405 410 415Asn Val Leu Tyr Val Leu Thr Asp Thr Ala Gly Asn Val Gln Lys Asp            420 425 430Asp Gly Ser Val Thr Asn Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys        435 440 445Phe Thr Tyr Lys Ala Lys    450<210> 2<211> 1612<212> DNA<213> Acinetobacter calcoaceticus<400> 2agctactttt atgcaacaga gcctttcaga aatttagatt ttaatagatt cgttattcat 60cataatacaa atcatataga gaactcgtac aaacccttta ttagaggttt aaaaattctc 120ggaaaatttt gacaatttat aaggtggaca catgaataaa catttattgg ctaaaattgc 180tttattaagc gctgttcagc tagttacact ctcagcattt gctgatgttc ctctaactcc 240atctcaattt gctaaagcga aatcagagaa ctttgacaag aaagttattc tatctaatct 300aaataagccg catgctttgt tatggggacc agataatcaa atttggttaa ctgagcgagc 360aacaggtaag attctaagag ttaatccaga gtcgggtagt gtaaaaacag tttttcaggt 420accagagatt gtcaatgatg ctgatgggca gaatggttta ttaggttttg ccttccatcc 480tgattttaaa aataatcctt atatctatat ttcaggtaca tttaaaaatc cgaaatctac 540agataaagaa ttaccgaacc aaacgattat tcgtcgttat acctataata aatcaacaga 600tacgctcgag aagccagtcg atttattagc aggattacct tcatcaaaag accatcagtc 660aggtcgtctt gtcattgggc cagatcaaaa gatttattat acgattggtg accaagggcg 720taaccagctt gcttatttgt tcttgccaaa tcaagcacaa catacgccaa ctcaacaaga 780actgaatggt aaagactatc acacctatat gggtaaagta ctacgcttaa atcttgatgg 840aagtattcca aaggataatc caagttttaa cggggtggtt agccatattt atacacttgg 900acatcgtaat ccgcagggct tagcattcac tccaaatggt aaattattgc agtctgaaca 960aggcccaaac tctgacgatg aaattaacct cattgtcaaa ggtggcaatt atggttggcc 1020gaatgtagca ggttataaag atgatagtgg ctatgcttat gcaaattatt cagcagcagc 1080caataagtca attaaggatt tagctcaaaa tggagtaaaa gtagccgcag gggtccctgt 1140gacgaaagaa tctgaatgga ctggtaaaaa ctttgtccca ccattaaaaa ctttatatac 1200cgttcaagat acctacaact ataacgatcc aacttgtgga gagatgacct acatttgctg 1260gccaacagtt gcaccgtcat ctgcctatgt ctataagggc ggtaaaaaag caattactgg 1320ttgggaaaat acattattgg ttccatcttt aaaacgtggt gtcattttcc gtattaagtt 1380agatccaact tatagcacta cttatgatga cgctgtaccg atgtttaaga gcaacaaccg 1440ttatcgtgat gtgattgcaa gtccagatgg gaatgtctta tatgtattaa ctgatactgc 1500cggaaatgtc caaaaagatg atggctcagt aacaaataca ttagaaaacc caggatctct 1560cattaagttc acctataagg ctaagtaata cagtcgcatt aaaaaaccga tc 1612<210> 3<211> 23<212> DNA<213> Artificial Sequence<220><223> primer for point mutation<400> 3gccaactcaa cgtgaactga atg 23<210> 4<211> 23<212> DNA<213> Artificial Sequence<220><223> primer for point mutation<400> 4cttaaatctt cgtggaagta ttc 23<210> 5<211> 23<212> DNA<213> Artificial Sequence<220><223> primer for point mutation<400> 5ccaagttttc gcggggtggt tag 23
    权利要求:
    Claims (11)
    [1" claim-type="Currently amended] In a water-soluble glucose dehydrogenase with pyrroloquinolinequinone as a coenzyme, a modified glucose in which one or more amino acid residues selected from the group consisting of glutamine, asparagine, and threonine present on the surface of an enzyme molecule is substituted with arginine Dehydrogenase.
    [2" claim-type="Currently amended] The modified glucose dehydrogenase according to claim 1, wherein the water-soluble glucose dehydrogenase comprising pyrroloquinolinequinone as a coenzyme is water-soluble PQQGDH derived from Acinetobacter calcoaceticus .
    [3" claim-type="Currently amended] In a water-soluble glucose dehydrogenase derived from acinetobacter calcoaceticus pyrroloquinolinequinone as a coenzyme, in the group consisting of the 209th glutamine residue, the 240th asparagine residue, and the 389th threonine residue A modified glucose dehydrogenase wherein one or more selected amino acid residues are substituted with arginine.
    [4" claim-type="Currently amended] 4. The modified glucose dehydrogenase of claim 3, wherein the 209th glutamine residue, the 240th asparagine residue, and the 389th threonine residue are each substituted with arginine.
    [5" claim-type="Currently amended] A gene encoding the modified glucose dehydrogenase according to any one of claims 1 to 4.
    [6" claim-type="Currently amended] The vector containing the gene of Claim 5.
    [7" claim-type="Currently amended] The transformant containing the gene of Claim 5.
    [8" claim-type="Currently amended] The organism in which the gene according to claim 5 is inserted into a main chromosome.
    [9" claim-type="Currently amended] The method for producing a modified glucose dehydrogenase according to any one of claims 1 to 4, wherein the organism according to claim 8 is used.
    [10" claim-type="Currently amended] A glucose assay kit containing the modified glucose dehydrogenase according to any one of claims 1 to 4.
    [11" claim-type="Currently amended] A glucose sensor containing the modified glucose dehydrogenase according to any one of claims 1 to 4.
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    同族专利:
    公开号 | 公开日
    EP1437411A4|2006-02-08|
    IL161060D0|2004-08-31|
    CN1571841A|2005-01-26|
    US20040265828A1|2004-12-30|
    CA2461755A1|2003-04-03|
    EP1437411A1|2004-07-14|
    WO2003027294A1|2003-04-03|
    JP2003093071A|2003-04-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-09-26|Priority to JPJP-P-2001-00294846
    2001-09-26|Priority to JP2001294846A
    2002-09-26|Application filed by 고지 소데
    2002-09-26|Priority to PCT/JP2002/009943
    2004-07-09|Publication of KR20040062945A
    优先权:
    申请号 | 申请日 | 专利标题
    JPJP-P-2001-00294846|2001-09-26|
    JP2001294846A|JP2003093071A|2001-09-26|2001-09-26|Glucose dehydrogenase|
    PCT/JP2002/009943|WO2003027294A1|2001-09-26|2002-09-26|Glucose dehydrogenase|
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