JP2005270082A - Glucose dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified water-soluble pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) having high selectivity toward glucose. <P>SOLUTION: This modified glucose dehydrogenase comprises a water-soluble glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme and is formed by substituting a residue on the 193 position of a water-soluble PQQGDH derived from Acinetobacter calcoaceticus, or an amino acid residue of another strain on a position equivalent to the above, with an amino acid residue having a neutral or acidic side chain. Further, a gene encoding the modified water-soluble glucose dehydrogenase, a vector containing the gene, a transformant containing the gene, a glucose assay kit containing the modified water-soluble glucose dehydrogenase, and a glucose sensor are provided, respectively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

Detailed Description of the Invention

  The present invention relates to the production of glucose dehydrogenase (PQQGDH) using pyrroloquinoline quinone as a coenzyme and its use in the determination of glucose.

  Blood glucose concentration is an important marker of diabetes. In fermentation production using microorganisms, the glucose concentration is quantified to monitor the process. Conventionally, glucose has been quantified by an enzymatic method using glucose oxidase (GOD) or glucose 6-phosphate dehydrogenase (G6PDH). However, in the method using GOD, it is necessary to add catalase or peroxidase to the assay system in order to quantify hydrogen peroxide generated during the glucose oxidation reaction. G6PDH has been used for glucose determination based on spectroscopic techniques, but NAD (P), a coenzyme, must be added to the reaction system.

  Therefore, the application of PQQGDH has attracted attention as a new enzyme that replaces the enzyme that has been used in conventional glucose enzyme determination methods. PQQGDH is a glucose dehydrogenase that uses pyrroloquinoline quinone as a coenzyme, and catalyzes a reaction in which glucose is oxidized to produce gluconolactone.

  PQQGDH is known to have a membrane-bound enzyme and a water-soluble enzyme. 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. For example, AM. Cleton-Jansen et al. , J .; Bacteriol. (1990) 172, 6308-6315. On the other hand, water-soluble PQQGDH has been confirmed in several strains of Acinetobacter calcaceticus (Biosci. Biotech. Biochem. (1995), 59 (8), 1548-1555), and its amino acid sequence has been cloned. (Mol. Gen. Genet. (1989), 217: 430-436). A. Calcoaceticus-derived water-soluble PQQGDH is a homodimer having a molecular weight of about 50 kDa. There is almost no homology on the primary structure of the protein with other PQQ enzymes.

  Recently, the results of X-ray crystal structure analysis of water-soluble PQQGDH derived from Acinetobacter calcoaceticus have been reported, and the higher-order structure of the enzyme including the active center has been clarified. (A. Oubrie et al., J. Mol. Biol., 289, 319-333 (1999); A. Oubrie et al., The EMBO Journal, 18 (19) 5187-5194 (1999); al., PNAS, 96 (21), 11787-11791 (1999)). These papers revealed that water-soluble PQQGDH is a β-propeller protein composed of six W-motifs.

  Since PQQGDH has high oxidative activity on glucose, and PQQGDH is a coenzyme-linked enzyme, it does not require oxygen as an electron acceptor. Application to the field is expected. However, PQQGDH has a problem of low selectivity for glucose.

Problems to be solved by the invention

  Therefore, an object of the present invention is to provide a modified water-soluble PQQGDH having high selectivity for glucose.

Means for solving the problem

  As a result of earnest research to develop modified PQQGDH that can be applied to clinical tests and food analysis by improving genetic engineering of water-soluble PQQGDH, the present inventor has conducted research on water-soluble PQQGDH. By introducing amino acid mutations in specific regions, we succeeded in obtaining enzymes with high selectivity for glucose.

  That is, the present invention provides a water-soluble glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme, wherein one or more amino acid residues of a natural water-soluble glucose dehydrogenase are substituted with other amino acid residues. And a modified glucose dehydrogenase having high selectivity for glucose compared to the natural water-soluble glucose dehydrogenase. The modified glucose dehydrogenase of the present invention has a high selectivity for glucose compared to natural water-soluble glucose dehydrogenase. Preferably, the modified PQQGDH of the present invention is less reactive to lactose or maltose than the wild type compared to the reactivity to glucose. More preferably, when the reactivity to glucose is 100%, the activity against maltose is 40% or less, more preferably 30% or less, and further preferably 20% or less.

  In one embodiment of the present invention, in the modified glucose dehydrogenase of the present invention, 1 or more in the region from residue 186 to residue 206 of water-soluble PQQGDH derived from Acinetobacter calcoaceticus or an equivalent region in other species These amino acid residues are substituted with other amino acid residues (that is, amino acid residues different from the corresponding amino acid residues in the naturally occurring PQQ glucose dehydrogenase). In the present specification, amino acid positions are numbered with 1 as the starting methionine.

  As used herein with respect to amino acid residue positions or regions, the term “equivalent” refers to biologically equivalent amino acid residues or regions in two or more proteins that are structurally similar but not identical. Or it represents having a biochemical function. For example, in water-soluble PQQGDH derived from organisms other than Acinetobacter calcaceticus, residues 186 to 206 in an enzyme having 80% or more homology with Acinetobacter calcaceticus-derived water-soluble PQQGDH and glucose dehydrogenase activity And a region having high amino acid sequence similarity, and it is reasonably considered that the region plays the same role in the protein as seen from the secondary structure of the protein, the region is expressed as “Acinetobacter calcoaceticus-derived water The region equivalent to the region from residue 186 to residue 206 of sex PQQGDH ”. Further, the eighth amino acid residue in the region is said to be “an amino acid residue at a position equivalent to the 193 residue of water-soluble PQQGDH derived from Acinetobacter calcoaceticus”.

  Preferably, the modified glucose dehydrogenase of the present invention has a neutral or acidic side chain at the 193th leucine residue of water-soluble PQQGDH derived from Acinetobacter calcoaceticus or an amino acid residue at an equivalent position in other species Substituted with an amino acid residue.

  More preferably, in the modified glucose dehydrogenase of the present invention, the 193rd leucine residue of the amino acid sequence represented by SEQ ID NO: 1 is replaced with glutamic acid, aspartic acid, histidine, tyrosine, threonine, glutamine, or asparagine residue. Has been replaced.

In another aspect, the modified glucose dehydrogenase of the present invention has the sequence:
Gly-Arg-Asn-Xaa1-Xaa2-Ala-Tyr-Leu
(Where Xaa1, Xaa2 are any natural amino acid residue, provided that when Xaa1 is Gln, Xaa2 is Glu, Asp, His, Tyr, Thr, Gln or Asn).

  The present invention also provides a gene encoding the above-mentioned modified glucose dehydrogenase, a vector containing the gene and a transformant containing the gene, and a glucose assay kit and glucose sensor containing the modified glucose dehydrogenase of the present invention. I will provide a.

  Since the enzyme protein of the modified glucose dehydrogenase of the present invention exhibits high selectivity for glucose and has high oxidation activity for glucose, it can be used for highly selective and sensitive measurement of glucose. Can be applied.

Structure of modified PQQGDH

  In the preferred modified glucose dehydrogenase of the present invention, one or more amino acid residues in the region of residues 186 to 206 of water-soluble PQQGDH derived from Acinetobacter calcoaceticus or the equivalent region in other species Of amino acid residues. Preferably, in the modified PQQGDH of the present invention, the 193rd leucine residue of the amino acid sequence represented by SEQ ID NO: 1 is substituted with glutamic acid, aspartic acid, histidine, tyrosine, threonine, glutamine, or asparagine residue. .

In another aspect, the modified PQQGDH of the present invention has the sequence:
Gly-Arg-Asn-Xaa1-Xaa2-Ala-Tyr-Leu
(Where Xaa1, Xaa2 are any natural amino acid residue, provided that when Xaa1 is Gln, Xaa2 is Glu, Asp, His, Tyr, Thr, Gln or Asn).

Method for Producing Modified PQQGDH The sequence of the gene encoding natural water-soluble PQQGDH derived from Acinetobacter calcaceticus is defined by SEQ ID NO: 2. In the gene encoding the modified PQQGDH of the present invention, the base sequence encoding the amino acid residue to be substituted in the gene encoding natural water-soluble PQQGDH is replaced with the base sequence encoding the desired amino acid residue. Can be constructed. Various methods for such site-specific base sequence substitution are known in the art, for example, Sambrook et al., “Molecular Cloning; Laboratory Manual”, 2nd edition, 1989, Cold Spring Harbor Laboratory Press. , New York.

  The mutant gene thus obtained is inserted into a gene expression vector (for example, a plasmid) and transformed into a suitable host (for example, E. coli). Many vectors and host systems for expressing foreign proteins are known in the art, and various hosts such as bacteria, yeast, and cultured cells can be used as the host.

  In the modified PQQGDH of the present invention, as long as it has a desired glucose dehydrogenase activity, a part of another amino acid residue may be deleted or substituted, and another amino acid residue may be added. Good. Various methods for such site-specific base sequence substitution are well known in the art.

  Furthermore, the person skilled in the art also compares the primary structure of proteins in water-soluble PQQGDH derived from other bacteria in parallel or the secondary structure predicted based on the primary structure of the enzyme. Can easily recognize a region equivalent to the region from residue 186 to residue 206 of water-soluble PQQGDH derived from Acinetobacter calcoaceticus, and according to the present invention, amino acid residues in this region can be By substituting with an amino acid residue, a modified glucose dehydrogenase with improved glucose selectivity can be obtained. These modified glucose dehydrogenases are also within the scope of the present invention.

  The transformant expressing the modified PQQGDH obtained as described above is cultured, and the cells are collected from the culture solution by centrifugation or the like, and then the cells are crushed with a French press or the like, or osmotic The periplasmic enzyme is released into the medium by shock. This can be ultracentrifuged to obtain a water-soluble fraction containing PQQGDH. Alternatively, the expressed PQQGDH can be secreted into the culture medium by using an appropriate host vector system. The modified PQQGDH of the present invention is prepared by purifying the obtained water-soluble fraction by ion exchange chromatography, affinity chromatography, HPLC or the like.

Method for Measuring Enzyme Activity PQQGDH of the present invention has an action of catalyzing a reaction in which glucose is oxidized to produce gluconolactone using PQQ as a coenzyme. In the measurement of enzyme activity, the amount of PQQ that is reduced with the oxidation of glucose by PQQGDH can be quantified by a color reaction of the redox dye. Examples of the color reagent include PMS (phenazine methosulfate) -DCIP (2,6-dichlorophenolindophenol), potassium ferricyanide, ferrocene, and the like.

Selectivity for glucose The selectivity of PQQGDH of the present invention for glucose is determined by measuring the enzyme activity as described above using various sugars such as galactose and maltose as substrates, and the relative activity to the activity when glucose is used as a substrate. It can be evaluated by examining.

  The modified PQQGDH of the present invention has improved selectivity for glucose compared to the wild-type enzyme, and in particular has higher reactivity to glucose compared to reactivity to maltose. Therefore, an assay kit or enzyme sensor prepared using this modified enzyme has the advantage of high selectivity for glucose measurement, and can detect glucose with high sensitivity even in samples containing various sugars.

Glucose Assay Kit The present invention also features a glucose assay kit comprising a modified PQQGDH according to the present invention. The glucose assay kit of the present invention comprises a modified PQQGDH according to the present invention in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the modified PQQGDH of the present invention, buffers necessary for the assay, mediators, a glucose standard solution for creating a calibration curve, and directions for use. The modified PQQGDH 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. Preferably, the modified PQQGDH of the present invention is provided in the form of a holo, but can be provided in the form of an apoenzyme and can be holoed at the time of use.

Glucose Sensor The present invention also features a glucose sensor that uses a modified PQQGDH according to the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. 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, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination thereof may be used. Preferably, the modified PQQGDH of the present invention is immobilized on the electrode in a holo form, but may be immobilized in the form of an apoenzyme and the PQQ provided as a separate 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 glutaraldehyde.

The glucose concentration can be measured as follows. The buffer is placed in a thermostatic cell, and PQQ and CaCl ₂ and mediator are added to maintain a constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. As the working electrode, an electrode on which the 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 is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

Example 1
Construction of Modified Enzyme PQQGDH Gene Mutation was introduced based on the structural gene of Acinetobacter calcoaceticus-derived PQQGDH shown in SEQ ID NO: 2. The plasmid pGB2 is obtained by inserting a structural gene encoding PQQGDH derived from Acinetobacter calcaceticus into the multiple cloning site of the vector pTrc99A (Pharmacia). The base sequence encoding the 193rd leucine residue was replaced with the base sequence encoding a glutamic acid residue by site-directed mutagenesis according to a conventional method. Site-directed mutagenesis was performed using plasmid pGB2. Table 1 shows the sequences of the synthetic oligonucleotide target primers used for the mutation.

  A vector plasmid pKF18k (Takara Shuzo Co., Ltd.) was incorporated with a Kpn I-Hind III fragment containing a part of the gene encoding PQQGDH derived from Acinetobacter calcoaceticus and used as a template. 50 fmol of this template was mixed with 5 pmol of the selection primer attached to Mutan (registered trademark) -Express Km kit manufactured by Takara Shuzo Co., Ltd. and 50 pmol of the phosphorylated target primer together with an annealing buffer of 1/10 of the total (20 μl) of the same kit. The plasmid was denatured by heat treatment at 100 ° C. for 3 minutes to form a single strand. The selection primer is for reverting the double amber mutation on the kanamycin resistance gene of pKF18k. This was placed on ice for 5 minutes to anneal the primer. To this was added 3 μl of the same kit extension buffer, 1 μl of T4 DNA ligase, 1 μl of T4 DNA polymerase and 5 μl of sterile water to synthesize complementary strands. This is the strain E. deficient in mismatch repair ability of DNA. E. coli BMH71-18mutS was transformed with shaking culture overnight to amplify the plasmid.

  Next, the plasmid extracted from this was transformed into E. coli. E. coli MV1184 was transformed and a plasmid was extracted from the colony. These plasmids were sequenced to confirm the introduction of the intended mutation. This fragment was replaced with the KpnI-HindIII fragment of the gene encoding wild type PQQGDH on the plasmid pGB2 to construct a modified PQQGDH gene.

Example 2
Preparation of Modified Enzyme The gene encoding wild type or modified PQQGDH was transformed into E. coli. The plasmid constructed by inserting it into the multicloning site of pTrc99A (Pharmacia), an expression vector for E. coli, was E. coli. E. coli DH5α strain was transformed. This was cultured in 450 ml of L medium (containing 50 μg / ml of ampicillin and 30 μg / ml of chloramphenicol) with shaking overnight at 37 ° C., and planted in 71 L medium containing 1 mM CaCl ₂ and 500 μMPQQ. Fungus. About 3 hours after the start of the culture, isopropylthiogalactoside was added to a final concentration of 0.3 mM, and then cultured for 1.5 hours. The cells were collected from the culture solution by centrifugation (5000 × g, 10 minutes, 4 ° C.), and the cells were washed twice with a 0.85% NaCl solution. The collected cells were crushed with a French press, and unbroken cells were removed by centrifugation (10000 × g, 15 minutes, 4 ° C.). The supernatant was ultracentrifuged (160500 × g (40000 rpm), 90 minutes, 4 ° C.) to obtain a water-soluble fraction. This was used in the following examples as a crudely purified enzyme preparation.

Example 3
Measurement of enzyme activity The crude enzyme preparations of wild type and each modified PQQGDH obtained in Example 2 were holoed for 1 hour or more in the presence of 1 μMPQQ and 1 mM CaCl ₂ , respectively. 187 μl of this was dispensed, 3 μl of active reagent (6 mM DCIP 48 μl, 600 mM PMS 8 μl, 10 mM phosphate buffer pH 7.0 16 μl) and 10 μl of D-glucose solution of each concentration were added, and the enzyme activity was measured.

The enzyme activity was measured by using PMS (phenazine methosulfate) -DCIP (2,6-dichlorophenolindophenol) in 10 mM MOPS-NaOH buffer (pH 7.0) at room temperature, and measuring the absorbance change of DCIP at 600 nm. Tracking was performed using a spectrophotometer, and the rate of decrease in absorbance was defined as the enzyme reaction rate. At this time, the enzyme activity that reduces 1 μmol of DCIP per minute was defined as 1 unit. The molar extinction coefficient of DCIP at pH 7.0 was 16.3 mM- ¹ .

  Km was determined from a plot of substrate concentration versus enzyme activity. As a result, it was 48 mM for glucose, 18 mM for galactose, and 20 mM for maltose.

Example 4
Evaluation of substrate specificity Substrate specificity was examined for crude enzyme preparations of each modified enzyme. The wild-type and modified PQQGDH crude purified enzyme preparations obtained in Example 2 were holoed for 1 hour or more in the presence of 1 μMPQQ and 1 mM CaCl ₂ , respectively. This was dispensed in 187 μl aliquots, and 3 μl of active reagent (containing 6 mM DCIP, 600 mM PMS, 10 mM phosphate buffer pH 7.0) and substrate were added. Various concentrations of glucose or other sugars were added as substrates, and the respective enzyme activities were examined. The value was expressed as a relative activity with respect to 100% of the activity when glucose was used as a substrate. As a result, when glucose was set to 100% as a value obtained from FIG. 1, it was 5% for maltose, and the enzyme of the present invention showed higher reactivity to glucose compared to reactivity to maltose.

Example 6
Preparation and Evaluation of Enzyme Sensor 20 mg of carbon paste was added to 5 units of Leu193Glu modified enzyme and freeze-dried. After this was mixed well, it was filled only on the surface of the carbon paste electrode already filled with about 40 mg of carbon paste, and was polished on a filter paper. This electrode was treated in 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde for 30 minutes at room temperature, and then treated in 10 mM MOPS buffer (pH 7.0) containing 20 mM lysine for 20 minutes at room temperature. To block glutaraldehyde. This electrode was equilibrated in a 10 mM MOPS buffer (pH 7.0) at room temperature for 1 hour or longer. The electrode was stored at 4 ° C.

  The glucose concentration was measured using the prepared enzyme sensor. Glucose could be selectively quantified in the range of 0.1 mM to 5 mM using the enzyme sensor to which the modified PQQGDH of the present invention was immobilized.

  It is a graph which shows the substrate concentration dependence of the activity of modified PQQGDH of this invention.

Claims (10)

  In the water-soluble glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme, one or more amino acid residues of the natural water-soluble glucose dehydrogenase are substituted with other amino acid residues, and the natural water-soluble glucose Modified glucose dehydrogenase having a high selectivity for glucose compared to neutral glucose dehydrogenase.   In glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme, the 193rd leucine residue of water-soluble PQQGDH derived from Acinetobacter calcoaceticus or the amino acid residue at the equivalent position in other species has a neutral or acidic side chain The modified glucose dehydrogenase according to claim 1, which is substituted with a residue.   The modified glucose dehydrogenation according to claim 7, wherein the 193rd leucine residue of the amino acid sequence represented by SEQ ID NO: 1 is substituted with a glutamic acid, aspartic acid, histidine, tyrosine, threonine, glutamine, or asparagine residue. enzyme. In glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme, the sequence Gly-Arg-Asn-Xaa1-Xaa2-Ala-Tyr-Leu
(In the formula, Xaa1 and Xaa2 are any natural amino acid residues, provided that when Xaa1 is Gln, Xaa2 is Glu, Asp, His, Tyr, Thr, Gln or Asn)
A modified glucose dehydrogenase comprising: A gene encoding the modified glucose dehydrogenase according to any one of claims 1 to 4. A vector comprising the gene according to claim 5. A transformant comprising the gene according to claim 5. The transformant of Claim 7 in which the gene of Claim 5 is integrated in the main chromosome. A glucose assay kit comprising the modified glucose dehydrogenase according to claim 1. A glucose sensor comprising the modified glucose dehydrogenase according to claim 1.

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