JP6520106B2 - Mutant pyrroloquinoline quinone-dependent glucose dehydrogenase, isolated nucleic acid molecule encoding mutant pyrroloquinoline quinone-dependent glucose dehydrogenase, enzyme electrode fixed with mutant pyrroloquinoline quinone-dependent glucose dehydrogenase, enzyme electrode Battery and biosensor equipped with - Google Patents

Description

  The present invention provides a mutant pyrroloquinoline quinone-dependent glucose dehydrogenase, an isolated nucleic acid molecule encoding the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase, and immobilizing the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase The present invention relates to an enzyme electrode, a biobattery provided with the enzyme electrode, and a biosensor. More specifically, the present invention is a mutant pyrroloquinoline capable of stably exhibiting its catalytic activity and achieving high densification of the catalyst current density when it is adsorbed and immobilized on an electrode substrate as an electrode catalyst to form an enzyme electrode. A quinone-dependent glucose dehydrogenase, an isolated nucleic acid molecule encoding the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase, an enzyme electrode on which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase is immobilized, the enzyme electrode The present invention relates to a provided biobattery and a biosensor.

  BACKGROUND ART In recent years, bio batteries utilizing bio resources have been proposed as the next generation energy because of their high energy efficiency and low environmental load. Organisms including microorganisms produce high-energy substances such as ATP in the in vivo metabolic process that oxidizes and degrades carbohydrates, proteins, lipids, etc. by biocatalysts such as enzymes, and acquires energy necessary for life activities doing. A bio battery is a power generation device that takes out energy generated in such in vivo metabolic process as electrical energy to an electrode.

  In the practical application of a biobattery utilizing the catalytic function of an enzyme, the choice of the enzyme determines the success or failure. Above all, construction of a bio-cell fueled with glucose, which is abundant in nature and has high energy conversion efficiency, has been attracting attention as a highly practical technology. Here, as an enzyme that oxidizes glucose, glucose oxidase, glucose dehydrogenase and the like are known. However, since glucose oxidase reacts with oxygen as an electron donor, in order to convert the glucose oxidation energy catalyzed by glucose oxidase into electrical energy, it is necessary to remove dissolved oxygen in the reaction solution in advance. On the other hand, since glucose dehydrogenase has no reactivity with oxygen unlike glucose oxidase, it has the advantage of not being affected by the dissolved oxygen in the reaction solution during electrical energy conversion, and also has a reaction rate. Very early. Due to such advantageous properties, glucose dehydrogenase is used for detection of saccharides, electrocatalysis of bio batteries, etc., and is an enzyme having high utility value in various fields such as food chemistry, clinical chemistry, biochemistry and the like.

  As glucose dehydrogenase, nicotinamide adenine dinucleotide (hereinafter sometimes abbreviated as "NAD +") Nicotinamide adenine dinucleotide phosphate (hereinafter sometimes abbreviated as "NADP +") is a coenzyme The one that functions as is widely known. Such NAD (P) + -dependent glucose dehydrogenases have been isolated from a wide variety of organisms. However, in the case of NAD (P) +-dependent glucose dehydrogenase, if it is attempted to couple NAD (P) + reduced by oxidation of glucose to the electrode reaction, additional components such as diaphorase are required, and the electrode structure is There was also the problem of becoming complicated.

  In recent years, pyrroloquinoline quinone (hereinafter sometimes abbreviated as "PQQ") has been found as a new coenzyme of glucose dehydrogenase. As such PQQ-dependent enzymes, an aldose sugar hydrogenase derived from Escherichia coli YliI strain (Escherichia coli YliI) and a glucose dehydrogenase derived from Acinetobacter calcoaceticus have been known. PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) is present in the periplasmic fraction of bacterial cells and is involved in energy production by transferring electrons obtained by oxidation to the respiratory chain. It was known to be.

  Studies such as analysis of sequence information of such PQQ-dependent glucose dehydrogenase (hereinafter sometimes referred to as "PQQGDH") and conformational analysis of proteins have been conducted (Non-patent document 1 and Non-patent document 2 etc. See for reference). Non-Patent Document 1 reports that the entire base sequence of the structural gene of PQQGDH derived from Acinetobacter calcoaceticus was identified and the primary structure of the protein encoded by them was clarified. Non-Patent Document 2 performs X-ray crystallographic analysis of the PQQGDH, and reports its three-dimensional structure. The three-dimensional structure of PQQGDH is a structure in which six antiparallel β sheets consisting of four β chains are assembled (6 propeller structure), and many of the other quinoproteins have an 8 propeller structure. It is known to be a unique enzyme.

  As mentioned above, it has the advantage that the reaction rate is very fast and insensitive to dissolved oxygen as compared to other enzymes that oxidize glucose. Taking advantage of this advantage, PQQGDH is being investigated for use as an electrode catalyst for biosensors and bio batteries. For example, Patent Document 1 discloses production of a glucose sensor for blood glucose measurement using PQQGDH.

  However, many enzymes, including PQQGDH, have lower thermal stability and durability than general metal catalysts etc., and their steric structure changes with the change of external environment and their catalytic activity decreases. There was a problem of that. Therefore, it has been a major obstacle in the practical application of biobattery and biosensor as an electrode catalyst. Therefore, in order to produce a more stable enzyme electrode, research on the construction of a thermostable enzyme is in progress. For example, Patent Document 2 and Non-patent Document 3 focus on the fact that PQQGDH forms a homodimer composed of identical subunits and expresses activity, and predetermined amino acids located in the 5CD loop region of PQQGDH. It has been reported that heat resistance was greatly improved by substituting a residue with cysteine. This is intended to stabilize the active homodimer by introducing a disulfide bond between dimerized PQQGDH monomers.

  The techniques of Patent Document 2 and Non-Patent Document 3 focus on the heat resistance of the enzyme, but when immobilizing the enzyme on the electrode substrate etc., the steric structure of the enzyme by contact with a hydrophobic electrode substrate It is necessary to avoid various problems including the reduction of the enzyme catalytic activity accompanying the change of structure etc. For example, when PQQGDH is adsorbed and fixed to an electrode substrate, it may occur that the homodimer of PQQGDH is dissociated in the inactive monomer. As a result, since it is known that the catalytic current density of the constructed enzyme electrode is reduced, it has been desired to provide PQQGDH which can be suitably used as an enzyme electrode or the like.

Unexamined-Japanese-Patent No. 2005-233976 (Japanese Patent Application No. 2005-109100) International Publication No. 2002/072839 (Japanese Patent Application No. 2002-571892)

Cleton-Janseb AM, Goosen N., Vink K., van de Putte P. et al., "Cloning, characterization and DNA sequencing of the gene encoding the Mr 50000 quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus" Mol. Gene. Genet. Volume 2-3, pp. 430-436, 1989 Oubrie A., Rozeboom HJ., Kalk KH., Olsthoorn AJ., DUINE JA., Dijkstra BW. Et al., "Structure and mechanism of soluble quinoprotein glucose dehydrogenase" EMBO J., Vol. 18, No. 19, No. 5187 -5194 pages, 1999 Igarashi S., Sode K. et al., "Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase." Mol. Biotechnol., Vol. 24, No. 2, pp. 97-104, 2003.

  Therefore, in order to solve the above problems, the present invention is able to stably exhibit its catalytic activity as an electrode catalyst of an enzyme electrode, and can achieve higher catalytic current density compared to that of a wild-type PQQ-dependent glucose dehydrogenase The purpose is to provide. Furthermore, the present invention aims to provide an enzyme electrode, that is, a biobattery and a biosensor utilizing the characteristics of the enzyme.

  As a result of repeated researches to solve the above-mentioned problems, the present inventors substituted cysteine with at least two specific amino acid residues of PQQ-dependent glucose dehydrogenase from wild type Acinetobacter calcoaceticus. When the immobilized enzyme is adsorbed and immobilized on the electrode substrate as an electrode catalyst to form an enzyme electrode, the mutant enzyme can stably exhibit its catalytic activity, and the catalyst current density of the enzyme electrode can be increased. I found it. It has been found that by utilizing the physicochemical properties of such a mutant-type enzyme, it is possible to construct an enzyme electrode that is more advantageous for practical use, and a biobattery and a biosensor using the enzyme electrode. The present inventors have completed the present invention based on these findings.

  That is, in order to achieve the above-mentioned object, the invention shown in the following [1] to [8] is provided.

[1] Compared to the wild-type pyrroloquinoline quinone-dependent glucose dehydrogenase consisting of the amino acid sequence of the following (a), (b) or (c) and shown in the amino acid sequence of SEQ ID NO: 2, as an electrocatalyst A mutant pyrroloquinoline quinone-dependent glucose dehydrogenase with improved catalytic current density obtained when it is adsorbed and immobilized on an electrode substrate to form an enzyme electrode.
(A) Oite the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 2 26th valine and the 334 amino acid sequence in which amino acid residues are substituted into the cysteine at position serine of the amino acid sequence shown in (b 1) In the amino acid sequence of (a), one or several amino acids are substituted, deleted, or added at positions other than the amino acid residues at the positions corresponding to the 26th and 334th positions of the amino acid sequence shown in Or an amino acid sequence inserted in (c) an amino acid sequence having at least 90 % or more identity with the amino acid sequence shown in SEQ ID NO: 2, and the 26th valine and the 334th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 Amino acid sequence [2] in which the amino acid residue at the position corresponding to serine is substituted with cysteine [2] In the amino acid sequence shown in SEQ ID NO: 2, the 26th valine and the 334rd serine are cis Mutant pyrroloquinoline quinone-dependent glucose dehydrogenase according to [1] comprising the amino acid sequence replaced to the in.
[3] The mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the above-mentioned [1] or [2] consisting of the amino acid sequence shown in SEQ ID NO: 3.

  According to the constitutions of the above [1] to [3], provision of a novel mutant pyrroloquinoline quinone dependent glucose dehydrogenase becomes possible. The mutant pyrroloquinoline quinone-dependent glucose dehydrogenase provided by the present invention is a catalyst obtained when adsorbed and immobilized on an electrode substrate to form an enzyme electrode, as compared to wild-type pyrroloquinoline quinone-dependent glucose dehydrogenase The current density is improving. The pyrroloquinoline quinone-dependent glucose dehydrogenase needs to form a homodimer for its catalytic activity, but in the process of preparation of the enzyme electrode, etc., the enzyme is adsorbed and immobilized on the surface of the electrode substrate etc. In this case, it may occur that this homodimer adsorbs in the state of being dissociated into monomers. As a result, it has been known that the catalytic activity of the enzyme is lost and the catalytic current density of the constructed enzyme electrode is reduced. The mutant pyrroloquinoline quinone-dependent glucose dehydrogenases of the present invention form stable dimers and ameliorate the above-mentioned undesirable phenomena. That is, the enzyme electrode on which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention is immobilized can smoothly exhibit its catalytic function to achieve high density of the enzyme catalytic current. The mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention having such properties can be applied to techniques requiring glucose dehydrogenation in various industrial fields such as medical, food and environmental fields. In particular, it can be used as a biobattery and a biosensor electrode catalyst, and can contribute to the increase in output of the biobattery and the increase in sensitivity of the biosensor.

[4] An isolated nucleic acid molecule encoding the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the above [1] to [3].

  According to the configuration of the above [4], a nucleic acid molecule encoding the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention can be provided. In addition, since the nucleic acid molecule encoding the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention can be obtained, the enzyme can be mass-produced inexpensively and industrially by genetic engineering methods. This can further improve the industrial utility value of the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention.

[5] An enzyme electrode in which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the above [1] to [3] is adsorbed and immobilized on an electrode substrate.
[6] The enzyme electrode according to the above [4], wherein the electrode substrate is a carbon substrate.

  According to the constitutions of the above [5] and [6], it is possible to provide an enzyme electrode in which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention is adsorbed and immobilized as an electrocatalyst. Since the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention forms a stable dimer, it does not dissociate into an inactive monomer even when immobilized on an electrode substrate. Although a hydrophobic carbon substrate is generally used as an electrode substrate of an enzyme electrode, it is also stably fixed to such a carbon substrate. Therefore, the enzyme electrode on which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention is adsorbed and immobilized can smoothly exhibit its catalytic function to achieve high densification of the enzyme catalytic current, which is a bio battery and bio It can be used as a sensor electrode catalyst, and can contribute to the increase in output of a bio battery and the increase in sensitivity of a biosensor.

[7] A biobattery comprising the enzyme electrode of the above [5] or [6].

  According to the configuration of the above [7], it is possible to provide a biobattery comprising an enzyme electrode on which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention is immobilized as an electrocatalyst. The enzyme electrode of the present invention utilizes the catalytic function of the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention, and can smoothly exhibit its catalytic function to achieve high density of the enzyme catalytic current. it can. Therefore, a biocell provided with such an enzyme electrode can efficiently oxidize fuel such as glucose to extract electric energy, and can improve the cell output. The bio battery of the present invention can be used as a safe and clean energy source in various industrial fields such as medical care, welfare, information communication, mobile and portable electronic devices.

[8] A biosensor provided with the enzyme electrode of the above [5] or [6].

  According to the configuration of the above [8], it is possible to provide a biosensor provided with an enzyme electrode on which a mutant pyrroloquinoline quinone-dependent glucose dehydrogenase is immobilized as an electrocatalyst. The enzyme electrode of the present invention utilizes the catalytic function of the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention, and can smoothly exhibit its catalytic function to achieve high density of the enzyme catalytic current. it can. Therefore, a biosensor provided with such an enzyme electrode can detect and measure a target substance such as glucose with high sensitivity and speed. The biosensor of the present invention can be used in all fields requiring detection and measurement of substances that can be substrates of pyrroloquinoline quinone-dependent glucose dehydrogenase such as glucose, and in particular, medical, food, environmental fields, etc. It is available in various industrial fields.

FIG. 6 shows the amino acid sequences of wild-type PQQGDH and mutant PQQGDH (mutant V26C_S334C and mutant Q352C) obtained in Example 1. It is an electrophoresis chart which shows the result of Example 1 which examined the confirmation of the dimerization of mutant type PQQGDH (mutant type V26C_S334C and mutant type Q352C). It is a graph which shows the result of Example 4 which examined the electrochemical characterization (CV) of a mutant type PQQGDH (mutant type V26C_S334C and a mutant type Q352C) fixed electrode. It is a graph which shows the result of Example 5 which examined electrochemical characterization (CA) of a mutant type PQQGDH (mutant type V26C_S334C) fixed electrode.

  Hereinafter, the present invention will be described in detail.

(Mutated pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention)
The mutant pyrroloquinoline quinone-dependent glucose dehydrogenase (hereinafter abbreviated as “PQQGDH”) of the present invention, an amino acid at a specific position in the amino acid sequence of a naturally occurring wild-type PQQGDH is a specific other amino acid It is substituted, and compared with wild-type PQQGDH, the catalyst current density obtained when it is adsorbed and immobilized on an electrode substrate as an electrode catalyst to form an enzyme electrode is improved. PQQGDH needs to form a homodimer in order to exert its catalytic activity, but it is necessary to adsorb and immobilize PQQGDH on the surface of an electrode substrate or the like in the process of enzyme electrode preparation etc. It may occur that dissociates into monomers in the dissociated state. As a result, it has been known that the catalytic activity of the enzyme is lost and the catalytic current density of the constructed enzyme electrode is reduced. The mutant PQQGDH of the present invention forms a stable dimer and ameliorates the above-mentioned undesirable phenomenon.

Here, PQQGDH has the ability to catalyze the dehydrogenation reaction of glucose by using glucose as a substrate, and the coenzyme pyrroloquinoline quinone (hereinafter sometimes abbreviated as "PQQ") is present as follows. Below, it catalyzes a reaction that acts on glucose to form gluconolactone.
D-glucose + electron acceptor → D-gluconolactone + reduced electron acceptor

  Wild-type PQQGDH means PQQGDH which is the basis of the mutant PQQGDH of the present invention, and in which the amino acid sequence of the standard PQQGDH isolated from nature is not intentionally or unintentionally mutated Means Therefore, as long as it does not have a mutation site, it includes not only naturally derived ones but also those produced by artificial means such as recombinants.

  The wild-type PQQGDH may be derived from any organism as long as it has the above-mentioned properties. However, PQQGDH from Acinetobacter calcoaceticus is preferred. In particular, Acinetobacter calcoaceticus described in Non-Patent Document 1 (JOURNAL Mol. Gen. Genet., Vol. 217, Vol. A glucose dehydrogenase (GENBANK ACCESSION No: X15871) derived from Acinetobacter calcoaceticus is preferably exemplified. An example of the nucleotide sequence and the amino acid sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. This enzyme is present in the periplasmic fraction of Acinetobacter bacteria, and is involved in energy production by transferring electrons obtained by oxidation to the respiratory chain. Compared with other glucose oxidases, it is characterized by its reaction speed being very fast, and being insusceptible to the influence of dissolved oxygen, so it is an enzyme that is extremely useful as an enzyme electrode. Therefore, it is widely used for a self-blood glucose meter, and application as an enzyme catalyst of the enzyme cell which used glucose as fuel is expected.

  In the mutant PQQGDH of the present invention, the amino acid residue at the position corresponding to the 26th and 334th amino acids in the amino acid sequence shown in the above-mentioned SEQ ID NO: 2 showing the preferred example of the amino acid sequence of wild type PQQGDH is substituted with a cysteine residue It is done. PQQGDH is an enzyme that needs to form a homodimer to exert glucose dehydrogenation activity, and can not express its catalytic activity in monomers. Then, at the time of homodimer formation, the 26th valine in the amino acid sequence shown in SEQ ID NO: 2 of one of the monomers is the other 334th serine, and the 334th serine is the other 26th Conformation to valine has been revealed by three-dimensional structural analysis. By substituting the adjacent 26th and 334rd with cysteine, the 26th and 334th of one monomer form a disulfide bond with the other 334th and 26th, respectively, and a stable amount of 2 is formed. Become to form a body. Since the 26th and 334rd positions in the monomer are at spaced positions, the mutant PQQGDH of the present invention forms a very stable homodimer. Such a disulfide bond does not become steric hindrance at the time of activity expression, and the PQQGDH of the present invention is “a catalyst current density obtained when it is adsorbed and fixed to an electrode substrate to form an enzyme electrode”.

  In the conformational analysis of wild-type PQQGDH, between the 26th valine and the other 334rd serine in the amino acid sequence shown in SEQ ID NO: 2 of one monomer, conversely the 334th serine and the other 26th It is known that the Cβ distance between valine (A) is as close as 4.2, and the distance between the 352nd glutamine is also as close as 4.6 (Table 1). However, even if glutamine at position 352 is substituted with cysteine to form a disulfide bond, “the catalyst current density obtained when adsorbed and immobilized on an electrode substrate to form an enzyme electrode like the mutant PQQGDH of the present invention is improved. The effect is not obtained. From this, it is considered that the single disulfide bond can not suppress the dissociation of the homodimer when the electrode substrate is adsorbed and fixed. As a result, substitution of the 415th amino acid residue with cysteine disclosed in Patent Document 2 (Japanese Patent Application No. 2002-571892) in the section of the prior art document mentioned above and cysteine with the 414th amino acid residue are disclosed. It is considered that homodimer dissociation can not be effectively suppressed similarly for the mutant PQQGDH (Table 1) obtained by substitution. The above prior art documents describe that two disulfide bonds are formed by substitution to cysteines 340 and 418 shown in Table 1 to stabilize a dimer, but As compared with the distance between the 26th and 334th amino acid residues, the distance between the 340th and 418th amino acid residues is separated, and the homodimer at the time of adsorption fixation of the electrode substrate is It is considered that the dissociation can not be effectively suppressed. In addition, when introducing such a disulfide bond between dimers, it is also necessary that the position of the enzyme does not cause steric hindrance in expressing the activity of the enzyme. Therefore, even if a cysteine is introduced to form a disulfide bond at an amino acid residue at a close position during homodimer formation, the enzyme catalytic activity is reduced or the enzyme electrode can not exhibit its function as an electrode catalyst. There is a case.

  Here, “the catalyst current density obtained when adsorbed and immobilized on the electrode substrate to form an enzyme electrode is improved” is an enzyme electrode constructed by adsorbing and modifying the modified PQQGDH of the present invention on the electrode substrate, It means that the catalyst current density is improved by exhibiting the catalytic function smoothly. When the catalyst current density is increased by preferably 10% or more, for example, about 5 to 10%, as compared with the enzyme electrode on which wild-type PQQGDH is immobilized, It can be determined that the current density is improved.

  The mutant PQQGDH of the present invention can be obtained by known methods. For example, the gene encoding wild-type PQQGDH which is the basis of modification is modified, and the resulting mutant gene is used to transform host cells, and culture of such transformants is carried out from the above-mentioned present invention. Physicochemical properties of mutant PQQGDH, that is, the ability to catalyze the dehydrogenation reaction of glucose, and the enzyme electrode constructed by adsorbing and fixing the enzyme as an electrode catalyst to the electrode substrate facilitates its catalytic function It can be obtained by collecting a protein having the characteristic that the catalyst current density is enhanced compared to wild-type PQQGDH. The confirmation of such physicochemical properties can be performed, for example, based on the description of Examples 3 to 5.

  The gene encoding wild-type PQQGDH, which is the basis of modification, can be obtained using a known gene cloning technology. For example, genomic DNA derived from an organism by a hybridization method using as a probe a DNA prepared based on the desired wild-type PQQGDH gene base sequence which can be obtained by searching a known database such as GenBank. A nucleic acid molecule encoding a desired enzyme can be prepared from cDNA or the like synthesized by reverse transcription from total RNA. The probe used here is an oligonucleotide containing a sequence complementary to a desired enzyme, and can be prepared based on a conventional method. For example, a chemical synthesis method based on the phosphoamidite method etc., and if a target nucleic acid has already been obtained, its restriction enzyme fragment etc. can be used. As such a probe, an oligonucleotide consisting of 10 or more, preferably 15 or more, more preferably about 20 to 50 consecutive bases of this base sequence based on the base sequence of the nucleic acid molecule encoding the desired enzyme is exemplified. Be done. The probe may be appropriately labeled if necessary, and examples of such a label include radioisotopes, fluorescent dyes and the like.

  Also, by using PCR as a primer prepared based on the desired wild-type PQQGDH gene base sequence, a nucleic acid molecule encoding a desired enzyme can be similarly prepared using genomic DNA derived from an organism and cDNA as a template. it can. The primers used when using PCR are oligonucleotides containing a sequence complementary to the desired enzyme, and can be prepared based on a conventional method. For example, a chemical synthesis method based on the phosphoamidite method etc., and if a target nucleic acid has already been obtained, its restriction enzyme fragment etc. can be used. When preparing a primer based on a chemical synthesis method, the primer is designed based on the sequence information of the target nucleic acid prior to the synthesis. Primer design can be designed to amplify a desired region, for example, using primer design support software. After synthesis, the primers are purified by means such as HPLC. Moreover, when performing chemical synthesis, it is also possible to use a commercially available automatic synthesizer. As such a primer, an oligonucleotide having 10 or more, preferably 15 or more, more preferably about 20 to 50 bases, which is designed to sandwich a desired amplification region, is exemplified.

  Here, complementary means that the probe or primer and the target nucleic acid molecule can specifically bind to each other according to the base pairing rule to form a stable duplex structure. Here, not only perfect complementarity, but only some of the nucleobases are in line with the base pairing rules, as long as the probe or primer and the target nucleic acid molecule are sufficient to be able to form stable duplex structures with each other. Even partial complementarity that is compatible is acceptable. The number of bases must be long enough to specifically recognize the target nucleic acid molecule, but if it is too long, nonspecific reactions are induced, which is not preferable. Therefore, an appropriate length is determined depending on many factors such as sequence information of the target nucleic acid such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution.

  It can also be obtained by synthesis based on known genetic information by a nucleic acid synthesis method such as the conventional phosphoramidite (pHos pHoramidite) method.

  Here, as the sequence information of PQQGDH suitable as a basis of the variant of the present invention, the amino acid sequence and nucleotide sequence of the above-mentioned glucose dehydrogenase from Acinetobacter calcoaceticus (GENBANK ACCESSION No: X15871), respectively, in the sequence listing It is shown in SEQ ID NO: 2 and SEQ ID NO: 1.

  There is no particular limitation on the method for inserting a mutation site into the gene encoding wild-type PQQGDH, and mutagenesis techniques for production of mutant proteins known to those skilled in the art can be used. For example, known mutagenesis techniques such as PCR mutation which introduces mutations using site-directed mutagenesis, PCR or the like, or transposon insertion mutagenesis can be used. Alternatively, a commercially available mutation introduction kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene)) may be used.

  In particular, it is preferably exemplified that PCR is performed using, as a primer, a DNA containing wild type PQQGDH as a template and an oligonucleotide having a desired modification (deletion or substitution) as a template. Here, in the preparation of mutant PQQGDH, a primer used when using PCR comprises a sequence complementary to a nucleic acid molecule encoding wild type PQQGDH, and is designed so that a desired mutation can be inserted. It can be prepared based on a conventional method. For example, chemical synthesis methods based on the phosphoramidite method etc. can be used. When preparing a primer based on a chemical synthesis method, it is designed based on the sequence information of the target nucleic acid prior to the synthesis. Primer design can be designed to amplify a desired region, for example, using primer design support software. After synthesis, the primers are purified by means such as HPLC. Moreover, when performing chemical synthesis, it is also possible to use a commercially available automatic synthesizer. As such a primer, an oligonucleotide consisting of 10 or more, preferably 15 or more, more preferably about 20 to 50 bases is exemplified.

  In addition, once the amino acid sequence of the desired mutant PQQGDH is determined, the appropriate base sequence encoding it can be determined, and the mutant PQQGDH of the present invention can be prepared using nucleic acid synthesis techniques such as conventional phosphoramidite method. A nucleic acid molecule that encodes can be chemically synthesized.

  In addition, such mutant PQQGDH of the present invention is obtained from among mutants produced by natural or artificial mutation, by screening whether it has physicochemical properties of the above-mentioned mutant PQQGDH of the present invention. it can. Furthermore, the mutant PQQGDH of the present invention can also be produced by chemical synthesis techniques. For example, it can be prepared by synthesizing using a peptide synthesizer and reconstituting the resulting polypeptide under appropriate conditions.

  As the mutant PQQGDH of the present invention, in the amino acid sequence shown in SEQ ID NO: 2 which is an example of the amino acid sequence of wild type PQQGDH (GENBANK ACCESSION: X15871) derived from Acinetobacter calcoaceticus, Preferred are those having an amino acid sequence in which the 26th valine and the 334rd serine are substituted with cysteine. Particularly preferably, the mutant PQQGDH of the present invention has the amino acid sequence shown in SEQ ID NO: 3.

  Furthermore, among amino acid sequences of wild-type PQQGDH derived from other strains of Acinetobacter calcoaceticus than strains that produce wild-type PQQGDH (GENBANK ACCESSION No: X15871), and cells of other species and organisms. Those in which the amino acid residue at the position corresponding to the 26th valine of the amino acid sequence shown in 2 and the serine at the 334rd position are substituted with cysteine also have the physicochemical properties of the above-mentioned mutant PQQGDH of the present invention As long as it is present, it is included in the mutant PQQGDH of the present invention. Therefore, also in the amino acid sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2 such as the homologue of PQQGDH shown in GENBANK ACCESSION No: X15871, the 26th valine and 334th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 Those in which the amino acid residue at the position corresponding to serine is replaced with cysteine are also included in the mutant PQQGDH of the present invention. Such substitution positions correspond to the 26th valine and the 334th serine of the amino acid sequence shown in SEQ ID NO: 2 by aligning the amino acid sequence of the target wild-type PQQGDH with the amino acid sequence shown in SEQ ID NO: 2 It can be determined by searching the position to be

  In addition to the substitution of the amino acid residue at the position corresponding to the 26th valine and the 334rd serine of the amino acid sequence shown in SEQ ID NO: 2, as long as the physicochemical properties of the above-mentioned mutant PQQGDH are retained. The amino acid residue may have a modification site in which modification has occurred in a specific amino acid residue other than the above amino acid residue. The term "modification" means that one or more amino acids in the amino acid sequence of the protein on which the modification is based have a modification comprising at least one of deletion, substitution, insertion and addition. Further, “modification in which one or more amino acids are composed of at least one of deletion, substitution, insertion and addition” means known DNA recombination technology for a gene encoding a protein which is the basis of modification, a point mutation introducing method It means that as many amino acids as can be deleted, substituted, inserted or added are deleted, substituted, inserted or added, and the like, including combinations thereof. For example, such a mutant has a homology of 70% or more, preferably 80% or more, more preferably 90%, 95% or more at the amino acid level to the amino acid sequence shown in SEQ ID NO: 2 can do.

  A protein having such a modification can be obtained by screening a protein having the activity among mutants produced by natural or artificial mutation. Alternatively, it can also be obtained by modifying the nucleic acid molecule encoding mutant PQQGDH by a known method. About this method, it can carry out according to the method mentioned above.

  Those skilled in the art can easily predict the modification that retains the enzyme activity of mutant PQQGDH upon modification of the amino acid sequence. Specifically, for example, in the case of amino acid substitution, it can be substituted with an amino acid having similar properties to the amino acid before substitution in terms of polarity, charge, hydrophilicity or hydrophobicity from the viewpoint of protein structure retention . Such substitutions are well known to those skilled in the art as conservative substitutions. As a specific example, for example, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan are classified into nonpolar amino acids, and thus have similar properties. In addition, non-charged amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Acidic amino acids also include aspartic acid and glutamic acid. In addition, examples of basic amino acids include lysine, arginine and histidine. Amino acid substitutions within each of these groups are tolerated as the function of the protein is maintained. In addition, for convenience of subsequent purification, immobilization to a solid phase, etc., one having a histidine tag (His-tag), FLAG tag (FLAG-tag) or the like added to the N or C terminal of the amino acid sequence is also preferable. It is illustrated. The introduction of such a tag peptide can be carried out by a conventional method. In addition, it may be a truncated form in which the amino acid residue at the C-terminal side or the N-terminal side is cleaved within a range that does not cause the disappearance of the enzyme activity. Furthermore, chemical modifications such as glucosylation may be added.

  In the mutant PQQGDH of the present invention, compared with wild-type PQQGDH, the catalytic current density obtained when it is adsorbed and immobilized on an electrode substrate as an electrode catalyst to form an enzyme electrode is improved. This is because the mutant PQQGDH of the present invention substitutes the adjacent 26th and 334th amino acids for cysteine formation with a cysteine so that the 26th and 334th amino acids of one monomer are the other, the 334th amino acid. The 26th and 26th, respectively, form a disulfide bond to form a stable dimer, and are able to stably maintain the dimer structure even when adsorbed and fixed to the electrode substrate. Moreover, such a disulfide bond does not become steric hindrance at the time of activity expression. As described above, the enzyme electrode on which the mutant PQQGDH of the present invention is immobilized can smoothly exhibit its catalytic function to achieve high density of enzyme catalytic current. The mutant PQQGDH of the present invention having such properties can be applied to techniques requiring glucose dehydrogenation in various industrial fields such as medical, food and environmental fields. In particular, it can be used as a biobattery and a biosensor electrode catalyst, and can contribute to the increase in output of the biobattery and the increase in sensitivity of the biosensor.

(Nucleic acid molecule encoding mutant PQQGDH)
The mutant PQQGDH genes of the present invention include those encoding all mutant PQQGDHs having the physicochemical properties of the mutant PQQGDHs of the present invention described above. Preferably, all the polynucleotides encoding a protein having an amino acid sequence in which the 26th valine and the 334rd serine are substituted with cysteine in the amino acid sequence shown in SEQ ID NO: 2. In particular, all polynucleotides encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3. Here, the polynucleotide in the present invention includes both DNA and RNA, and in the case of DNA, it does not matter whether it is single-stranded or double-stranded.

  Since the nucleic acid molecule encoding the mutant PQQGDH of the present invention has a base sequence clarified based on its amino acid sequence in the present specification, DNA such as conventional phosphoramidite method is used based on such sequence information. It can be chemically synthesized using synthetic methods. It can also be produced by modifying DNA encoding wild type PQQGDH which is the basis of modification. The details are described above.

  Furthermore, in the amino acid sequence shown in SEQ ID NO: 2, a protein having an amino acid sequence in which valine at position 26 and serine at position 334 are substituted with cysteine in the amino acid sequence shown in SEQ ID NO: 2 The present invention also includes those comprising a polynucleotide that hybridizes under stringent conditions with a polynucleotide complementary to the polynucleotide consisting of the encoding base sequence. Such a polynucleotide can be prepared by using known mutagenesis techniques or DNA synthesis methods such as conventional phosphoramidite method. Alternatively, exonuclease is allowed to act on a polynucleotide consisting of a nucleotide sequence encoding a protein having an amino acid sequence in which the 26th valine and the 334rd serine are substituted with cysteine in the amino acid sequence shown in SEQ ID NO: 2 Can be obtained by As such a polynucleotide, in the polynucleotide consisting of a nucleotide sequence encoding a protein having an amino acid sequence in which the 26th valine and the 334rd serine are substituted with cysteine in the amino acid sequence shown in SEQ ID NO: 2 Also included are those in which multiple bases are deleted, substituted, added or inserted. Therefore, one having a base sequence encoding a tag peptide added to the 3 'or 5' end of the base sequence is also preferably exemplified.

  Here, under stringent conditions, DNAs having an identity of 60% or more, preferably 70%, more preferably 80% or more, particularly preferably 90%, 95% or more in the base sequence are preferentially used. It refers to conditions that can be hybridized. Stringency can be prepared by appropriately changing the salt concentration and temperature during the hybridization reaction and washing. For example, conditions for Southern hybridization described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York et al.

  More specifically, hybridization in 50% (v / v) formamide, 5 × SSC at 42 ° C. for 16 hours is exemplified. Here, 1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. Also, it may contain 0.1 to 1.0% (v / v) of sodium dodecyl sulfate (SDS) and 0 to 200 μl of denatured nonspecific DNA. And, as washing conditions, washing for 5 minutes at 5 ° C. in 2 × SSC, 0.1% SDS, and washing for 30 minutes to 4 hours at 65 ° C. in 0.1 × SSC, 0.1% SDS are exemplified. . In addition, conditions equivalent to these will be easily understood by those skilled in the art.

(Recombinant vector of the present invention)
The recombinant vector of the present invention can be constructed by incorporating the nucleic acid molecule encoding the mutant PQQGDH of the present invention into a suitable vector. There are no particular limitations on the available vectors, as long as they can incorporate foreign DNA and can be autonomously replicated in host cells. Thus, the vector comprises a sequence of at least one restriction site into which a nucleic acid molecule encoding a mutant PQQGDH can be inserted. For example, plasmid vectors (pEX system, pUC system, pBR system etc.), phage vectors (λgt10, λgt11, λZAP etc.), cosmid vectors, virus vectors (vaccinia virus, baculovirus etc.) etc. are included.

  The recombinant vector of the present invention is incorporated such that the nucleic acid molecule encoding mutant PQQGDH can express its function. Therefore, other known base sequences necessary for functional expression of the nucleic acid molecule may be included. For example, a promoter sequence, a leader sequence, a signal sequence, a ribosome binding sequence and the like can be mentioned. As a promoter sequence, for example, when the host is E. coli, lac promoter, trp promoter and the like are suitably exemplified. However, known promoter sequences can be used without being limited thereto. Furthermore, the recombinant vector of the present invention can also contain a marking sequence or the like that can confer phenotypic selection in a host. Examples of such marking sequences include sequences encoding genes such as drug resistance and auxotrophy. Specifically, kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene and the like are exemplified.

  Insertion of a nucleic acid molecule or the like encoding a mutant PQQGDH into a vector is carried out, for example, by cleaving the gene of the present invention with an appropriate restriction enzyme and inserting it into a restriction enzyme site or multicloning site of an appropriate vector for ligation. Etc. can be used, but it is not limited thereto. For ligation, known methods such as a method using DNA ligase can be used. In addition, commercially available ligation kits such as DNA Ligation Kit (Takara-bio) can also be used.

(Transformant of the present invention)
The transformant of the present invention can be constructed by transforming appropriate cells with a recombinant vector containing the nucleic acid molecule encoding the mutant PQQGDH of the present invention. Here, the host cell is not particularly limited as long as it is a host cell capable of efficiently expressing the nucleic acid molecule encoding the mutant PQQGDH of the present invention. Prokaryotes can be suitably used, in particular E. coli. Besides, Bacillus subtilis, Bacillus bacteria, Pseudomonas bacteria and the like can also be used. As E. coli, for example, E. coli DH5α, E. coli BL21, E. coli JM109 and the like can be used. Furthermore, it is possible to use eukaryotic cells without being limited to prokaryotes. For example, yeasts such as Saccharomyces cerevisiae, insect cells such as Sf9 cells, animal cells such as CHO cells, and COS-7 cells can be used. As transformation methods, known methods such as calcium chloride method, electroporation method, liposome transfection method, microinjection method and the like can be used.

(Method for producing mutant PQQGDH of the present invention)
The method for producing mutant PQQGDH of the present invention has the ability to catalyze the dehydrogenation reaction of glucose from the culture obtained by culturing the above-mentioned transformant of the present invention, and using the enzyme as an electrode catalyst It is carried out by collecting a protein having physicochemical properties that the enzyme electrode constructed by adsorbing and fixing to the electrode substrate smoothly exhibits its catalytic function and the catalytic current density is improved compared to wild-type PQQGDH. . That is, it comprises a culture step of culturing the above-mentioned transformant of the present invention, and a recovery step of recovering the protein expressed in the culture step. Thus, expression in a suitable host enables mass production of mutant PQQGDH at low cost.

  The culture step is carried out by inoculating the transformant of the present invention in an appropriate medium and culturing it according to a conventional method. The culture conditions of the transformant of the present invention may be selected in consideration of the nutritional and physiological characteristics of the host cell. The medium to be used is not particularly limited as long as it contains nutrients which can be assimilated by the host cell and can efficiently express a protein in a transformant. Therefore, a medium containing a carbon source, a nitrogen source and other essential nutrients necessary for the growth of host cells is preferable, and either a natural medium or a synthetic medium may be used. For example, as a carbon source, glucose, dextran, starch and the like can be mentioned, and as a nitrogen source, ammonium salts, nitrates, amino acids, peptone, casein and the like can be mentioned. As other nutrients, if desired, inorganic salts, vitamins, antibiotics and the like can be included. When the host cell is E. coli, LB medium, M9 medium and the like can be suitably used. Also, the culture form is not particularly limited, but a liquid medium can be suitably used from the viewpoint of mass culture.

  Selection of host cells carrying the recombinant vector of the present invention can be performed, for example, by the presence or absence of expression of the marking sequence. For example, when using a drug resistance gene as a marking sequence, it can be carried out by culturing in a drug-containing medium corresponding to the drug resistance gene.

  The purification step may be performed by recovering, ie, isolating and purifying mutant PQQGDH from the culture of the transformant obtained in the above-mentioned culture step. Depending on the fraction in which the enzyme of the present invention is present, a procedure according to a general protein isolation and purification method may be applied. Specifically, when the mutant PQQGDH is produced outside the host cell, the culture solution is used as it is, or the host cell is removed by means of centrifugation, filtration or the like to obtain a culture supernatant. Subsequently, the enzyme of the present invention can be isolated and purified by appropriately selecting a known protein purification method for the culture supernatant. For example, known isolation and purification techniques such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, hydrophobic, anionic, cationic, various chromatography such as affinity chromatography can be applied singly or in combination as appropriate. . In particular, when using affinity chromatography, it is preferable to express the enzyme of the present invention as a fusion protein with a tag peptide such as His Tag to utilize the affinity to such tag peptide. When the mutant PQQGDH is produced in a host cell, the culture is recovered by centrifugation, filtration or the like. Subsequently, host cells are disrupted by an enzymatic disruption method such as lysozyme treatment or a physical disruption method such as ultrasonication, freeze-thaw, osmotic shock, or the like. After crushing, the solubilized fraction is collected by means of centrifugation, filtration and the like. The obtained solubilized fraction can be isolated and purified by the same treatment as in the case where it can be produced extracellularly as described above.

(Method of detecting glucose)
The mutant PQQGDH of the present invention can be used for the detection of glucose in a sample. The detection of glucose can be performed by measuring the activity of the enzyme that catalyzes a dehydrogenation reaction in a PQQ-dependent manner on glucose. The activity of the enzyme can be carried out using any method known as a method for measuring the activity of an enzyme that catalyzes the dehydrogenation of saccharides in a PQQ-dependent manner. For example, it can be quantified by a color reaction of a redox reagent. For example, dichloroindophenol (hereinafter abbreviated as "DCIP"), nitrotetrazolium blue (hereinafter abbreviated as "NTB"), etc. can be used. Specifically, the mutant PQQGDH of the present invention is reacted with a sample to be measured in the coexistence of an electron mediator such as phenazine methosulfate (hereinafter abbreviated as "PMS"). Oxidation of glucose produces reduced PMS, followed by reduction of DCIP by reduced PMS at a wavelength of 600 nm, or diformazan formed by reduction of NTB by reduced PMS at a wavelength of 570 nm It can carry out by measuring by photometry. The change in absorbance can be the activity of the enzyme, and thus the presence of glucose can be detected. For measurement of absorbance, a known microplate reader (manufactured by Molecular Device) can be used. Also, if the appropriate test conditions are selected, the changes in electrode oxidation current and absorbance described above are linearly proportional to the enzyme activity to be measured. At this time, the glucose concentration can be determined based on the change value obtained by the measurement, based on a standard curve prepared in advance using a glucose solution of standard concentration within the target concentration range. In addition, the mutant PQQGDH-immobilized electrode of the present invention can be reacted with a sample to be measured in the coexistence of an electron mediator to measure the electrode oxidation current. In this case, the electrode oxidation current can be used as the activity of the enzyme, and thus the presence of glucose can be detected.

(Enzyme electrode of the present invention)
The mutant PQQGDH of the present invention can be used as an electrode catalyst of an enzyme electrode, and such an enzyme electrode also constitutes a part of the present invention. The enzyme electrode of the present invention can be constructed by adsorbing and fixing the mutant PQQGDH of the present invention to an electrode substrate.

As the electrode substrate, carbon substrates such as graphite and glassy carbon which can be connected to an external circuit, metals or alloys such as aluminum, copper, gold, platinum, silver, nickel and palladium, SnO ₂ , In ₂ O ₃ , WO _3, TiO ₂ conductive oxides such like, can be used a conductive substrate of the conventional known materials. Preferably, it is a carbon base material. Moreover, you may comprise these electroconductive substrates by single layer or 2 or more types of laminated structure. The size, shape, and the like of the electrode are not particularly limited, and can be appropriately adjusted according to the purpose of use.

  Immobilization of the enzyme on the electrode substrate can be carried out by known methods such as physical adsorption, ionic bond, carrier binding method of immobilization via covalent bond, and the like. In addition, a crosslinking method in which crosslinking and immobilization are performed with a crosslinking reagent having a divalent functional group such as glutaraldehyde can also be used. Furthermore, entrapment methods etc. which are enclosed and fixed in semipermeable membranes, such as polysaccharides, such as alginic acid and carrageenan, conductive polymers, redox polymers, polymers having a network structure such as photocrosslinkable polymers, dialysis membranes, etc. Can also be used. Moreover, you may use combining these. Preferably, the enzyme is immobilized on the electrode substrate by physical adsorption.

  Physical adsorption is to fix the modified PQQGDH of the present invention on an electrode substrate by physical force. And, in the case of fixation by adsorption bonding, the electrode substrate can be brought into contact with a solution containing the modified PQQGDH of the present invention and dried, and then it can be fixed easily. At the time of fixation, it is preferable to fix the mutant PQQGDH in the state of holoenzyme containing coenzyme PQQ. However, it may be immobilized in the form of the apoenzyme and supplied as a separate layer or in a form in which the PQQ is dissolved in a suitable buffer. In addition, other substances necessary for expression of the catalytic activity of the enzyme, such as calcium ion, may be supplied as a separate layer or in a form dissolved in an appropriate buffer.

  Since the enzyme electrode of the present invention forms a stable dimer, the mutant PQQGDH of the present invention, which is an electrocatalyst, dissociates into a monomer that is inactive even when immobilized on the electrode substrate Absent. Although a hydrophobic carbon substrate is generally used as an electrode substrate of an enzyme electrode, it is also stably fixed to such a carbon substrate. Therefore, the enzyme electrode on which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention is immobilized can smoothly exhibit its catalytic function to achieve high densification of the enzyme catalytic current, a bio battery and a biosensor It can be used as an electrode catalyst, and can contribute to high power output of a bio battery and high sensitivity of a biosensor.

(Bio battery of the present invention)
The mutant PQQGDH of the present invention can be used to construct a biobattery, which also constitutes a part of the present invention. The bio battery of the present invention is composed of, for example, an anode electrode performing oxidation reaction, and a cathode electrode performing reduction reaction, and is composed of an electrolyte layer separating the anode and the cathode as necessary. And the enzyme electrode of the present invention immobilized on the electrode substrate connected to the external circuit. Preferably, the enzyme electrode in which the mutant PQQGDH of the present invention is immobilized on an electrode substrate connected to an external circuit is constructed as an anode electrode. By this configuration, on the anode electrode side, the mutant PQQGDH extracts electrons generated by oxidizing the substrate to the electrode and generates protons. On the other hand, the cathode side is preferably constructed by fixing a catalyst capable of transferring electrons by reducing an oxidant such as oxygen or hydrogen peroxide, and protons generated on the anode side react with oxygen. Configured to produce water. Moreover, as a cathode side electrode, the electrode to which enzymes, such as multi copper enzymes, such as pyruvate oxidase and laccase, were fixed can also be used, for example.

  As the electrolyte layer, any substance can be used as long as it has no electron transfer ability and can transport protons.

  If necessary, an electron mediator that mediates electron transfer between the enzyme reaction and the electrode can be used. The mediator is not particularly limited, and examples thereof include quinones, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferricyanide, ferredoxins, ferrocene and derivatives thereof and the like.

  By this configuration, the enzyme on the anode electrode side oxidizes the fuel substrate and receives electrons. Then, this electron is delivered to the anode electrode directly or through a mediator for mediating electron transfer between the enzyme reaction and the electrode. Then, a current is generated by passing electrons from the anode electrode to the cathode electrode through the external circuit. On the other hand, protons generated on the anode electrode side move to the cathode electrode side through the electrolyte layer, and react with electrons moving from the anode side through the external circuit to generate water. In the reaction with glucose, the following current response occurs to generate a current.

Glucose + glucose dehydrogenase (oxidized form)
→ gluconolactone + glucose dehydrogenase (reduced form);
Glucose dehydrogenase (reduced type) → glucose dehydrogenase (oxidized type) + e-

  The biobattery of the present invention utilizes the catalytic function of the mutant PQQGDH of the present invention, and is provided with an enzyme electrode on which PQQGDH is immobilized as an electrocatalyst. Since such an enzyme electrode can smoothly exhibit its catalytic function to achieve high densification of the enzyme catalytic current, the biocell of the present invention efficiently oxidizes fuel such as glucose to produce electricity. Energy can be taken out, and battery output can be improved. The bio battery of the present invention can be used as a safe and clean energy source in various industrial fields such as medical care, welfare, information communication, mobile and portable electronic devices.

(Biosensor)
The mutant PQQGDH of the present invention can be used to construct a biosensor such as a glucose sensor for detecting glucose, and such a biosensor also constitutes a part of the present invention. The glucose sensor of the present invention is configured by providing a working electrode configured as an enzyme electrode having a mutant PQQGDH immobilized on an electrode substrate, and a counter electrode thereof. If necessary, in order to increase the reliability of measurement accuracy, a three-electrode system provided with a reference electrode such as silver silver halide may be used. Here, an enzyme electrode in which a mutant PQQGDH is immobilized on an electrode substrate serving as a working electrode can be constructed according to an electrode of a bio battery.

  The measurement by the glucose sensor of the present invention is performed by bringing a measurement sample into contact with the glucose sensor. The contact causes an oxidation-reduction reaction to occur between the enzyme immobilized on the electrode and the substance to be measured which is a substrate of the enzyme, which is performed by detecting the current generated thereby. Such response current value can be used to measure the presence or absence or concentration of the substrate in the sample. Specifically, for example, when a sample to be measured is brought into contact, glucose contained in the sample reacts with the mutant PQQGDH immobilized on the working electrode, and then the electron mediator is reduced. Then, a voltage is applied to the electrode system to oxidize the reductant of the electron acceptor, and glucose in the sample can be detected from the change in the oxidation current value obtained. At this time, the glucose concentration can be determined based on the obtained oxidation current value by preparing a standard curve in advance using a glucose solution of standard concentration within the target concentration range. Here, as samples, all samples in which the presence of saccharides is expected can be targeted. For example, biological samples derived from organisms such as blood, urine, and saliva, food samples, environmental samples and the like are exemplified, but not limited thereto. In addition, it may also include samples obtained by appropriately treating these samples as necessary.

  As a measurement method using the biosensor of the present invention, known methods such as chronoamperometry or coulometry measuring cyclic oxidation current or reduction current, cyclic voltammetry can be used.

  The biosensor of the present invention utilizes the catalytic function of mutant PQQGDH, and includes an enzyme electrode on which PQQGDH is immobilized as an electrocatalyst. Since such an enzyme electrode can smoothly exhibit its catalytic function to achieve high densification of the enzyme catalytic current, a biosensor provided with such an enzyme electrode has high sensitivity to a substance to be measured such as glucose. And it can be detected and measured quickly. Therefore, the biosensor of the present invention is applicable to all fields requiring detection and measurement of substances that can be substrates of pyrroloquinoline quinone-dependent glucose dehydrogenase such as glucose, and in particular, medical, food, environmental fields Etc. are available in various industrial fields.

  EXAMPLES The present invention will be described more specifically by the following examples, but the present invention is not limited to these examples.

  Wild type pyrroloquinoline quinone dependent glucose dehydrogenase (hereinafter referred to as "PQQGDH") needs to form a homodimer for expression of its enzyme activity. However, in the preparation of the enzyme electrode, etc., when the enzyme is adsorbed and fixed on the surface of the electrode substrate etc., it may occur that the dimer is dissociated in the dissociated state, and as a result, the enzyme activity is lost. The catalyst current density could not be obtained. Therefore, in the following examples, even when the treatment such as adsorption fixation is carried out, in order to obtain the mutant PQQGDH which can maintain the homodimer and can express the enzyme activity well, intensive study on the mutation introduction site did.

Example 1: Construction of mutant PQQGDH Even when an electrode substrate or the like is subjected to treatment such as adsorption and fixation, examination is performed to obtain mutant PQQGDH which can maintain good homodimer and express enzyme activity well. Did.

1. Construction of Expression Vector Encoding Mutant PQQGDH Based on the wild type PQQGDH gene (GENBANK ACCESSION No: X15871) derived from Acinetobacter calcoaceticus as described above, the 26th valine and the 334th serine by site-directed mutagenesis. A gene (mutant V26C_S334C) in which the codon encoding R.sup.1 was converted to a codon encoding cysteine, and a gene (mutant Q352C) in which the codon encoding glutamine at position 352 was converted to a codon encoding cysteine. The prepared gene was inserted into a pET-22b (+) (Novagen) vector using an NdeI and BamHI site so that a histidine tag was added to the C-terminus, to obtain a mutant PQQGDH expression vector. In the same manner, an expression vector containing a gene encoding wild type PQQGDH was constructed. The entire amino acid sequences expressed by the expression vectors of mutant V26C_S334C, mutant Q352C and wild type PQQGDH are shown in FIG. 1 and as SEQ ID NOs: 3, 4 and 5 in the sequence listing.

The sequence information of the primers used for mutagenesis is as follows.
For V26C
ACGDH_V26-5: CATTTGCTGATTGCCTCCTAACTCC (SEQ ID NO: 6)
ACGDH_V26-3: GGAGTTAGAGGGCAATCAGCAATG (SEQ ID NO: 7)
For S334C
ACGDH_S334-5: GACGAAAGAATGCGAATGGACTGG (SEQ ID NO: 8)
ACGDH_S334-3: CCAGTCCCATTCGCATTCTTTCGTC (SEQ ID NO: 9)
For Q352
ACGDH_Q352C-5: ATATACGGTTTGCGATACCTACA (SEQ ID NO: 10)
ACGDH_Q352C-3: TGTAGGTATCGCAAC AACGGTATAT (SEQ ID NO: 11)

2. Expression of mutant PQQGDH The BL21 (DE3) strain was transformed using the expression vectors of mutant V26C_S334C, mutant Q352C and wild type PQQGDH constructed in 1 above. This is cultured with shaking at 37 ° C. in LB medium, and when the culture solution becomes approximately OD ₆₀₀ = 0.6, isopropyl-β-D-thiogalactopyranoside (isopropyl-sD-thiogalactopyranoside) at a final concentration of 0.01 mM. : IPTG was added to induce expression. After induction of expression, the cells were cultured overnight at 28 ° C. to recover the cells. The collected cells were suspended in PBS, and the cells were disrupted by ultrasound. Subsequently, centrifugation was performed, and the supernatant was recovered as a water soluble fraction. From the collected water-soluble fraction, purification of mutant V26C_S334C, mutant Q352C and wild type PQQGDH having a histidine tag was performed by metal ion affinity chromatography using TALON resin (manufactured by CLONTECH). For purification, add the water-soluble fraction to PBS equilibrated TALON resin, wash the resin with PBS containing 1 mM imidazole, and finally, with mutant PBS containing 100 mM imidazole, mutant V26C_S334C, mutant Q352C, and wild type PQQGDH was eluted. The obtained eluted fraction was subjected to buffer exchange with 50 mM MOPS (pH 7.0) buffer containing 1 mM CaCl ₂ while removing imidazole using a Hitrap desalting column (manufactured by GE Healthcare).

3. Enhancement of holization and dimerization of mutant PQQGDH In the mutant V26C_S334C and mutant Q352C obtained in the above 2, PQQ for holization at a final concentration of 30 μM, and for promotion of dimerization Potassium ferricyanide was added to a final concentration of 1 μM and left overnight at 4 ° C. in the dark. After standing, excess PQQ and potassium ferricyanide were removed using Hitrap desalting column (manufactured by GE Healthcare). In addition, in order to remove PQQGDH which did not be dimerized, it was kept at 60 ° C. for 30 minutes and centrifuged to recover a supernatant.

4. Holization of Wild-type PQQGDH To the wild-type PQQGDH obtained in 2 above, 1 μM of PQQ for holization was added at a final concentration of 1 μM and left overnight at 4 ° C. in the dark. After standing, excess PQQ was removed using Hitrap desalting column (manufactured by GE Healthcare). Also, in order to increase the degree of purification, the mixture was incubated at 50 ° C. for 30 minutes and centrifuged to recover the supernatant.

5. Confirmation of Dimerization of Mutant PQQGDH The mutant V26C_S334C, the mutant Q352C and the wild type PQQGDH obtained in the above 3 were analyzed by SDS-PAGE to confirm whether or not a dimer was formed.

  The results of SDS-PAGE are shown in FIG. The left side of FIG. 2 shows the reduction treated sample, the right side shows the electrophoresis of the unreduced sample, lane 1 shows the result of wild type PQQGDH, lane 2 shows the result of mutant Q352C, and lane 3 the result of mutant V26C_S334C. From the results in FIG. 2, in the reduced state, a band was confirmed around 50 kDa for all of the mutant V26C_S334C, the mutant Q352C, and the wild-type PQQGDH. On the other hand, in the non-reduced state, a band was observed around 50 kDa in the wild-type PQQGDH as in the reduced state, but in the mutant V26C_S334C and the mutant Q352C, a band could be confirmed around 100 kDa. From this, it is considered that the mutant V26C_S334C and the mutant Q352C form a stable dimer by disulfide bond.

Example 2 Evaluation of Enzymatic Activity of Mutant PQQGDH In this example, the enzyme activity of the mutant PQQGDH obtained in Example 1 was evaluated.

  The enzyme activities of the mutant V26C_S334C and the mutant Q352C obtained in 3 of Example 1 and the wild type PQQGDH obtained in 4 of Example 1 were measured. The enzyme activity was determined by measuring the reduction reaction of 2,6-dichlorophenolindophenol (DCIP) using phenazine mesosulfate (PMS) as a mediator. Specifically, 50 mM glucose, 200 μM PMS, 50 μM DCIP, 1 nM enzyme were mixed in 0.5 M potassium phosphate buffer (pH 8.0), and the disappearance rate of absorbance at 600 nm was measured.

Here, to briefly explain the measurement principle, PQQGDH catalyzes a reaction of oxidizing a hydroxyl group of glucose to form D-gluconolactone. With the oxidation of glucose, reduced PQQ is generated, and reduced PQQ generates reduced PMS. The reductive decolorization reaction of DCIP with this reduced PMS was measured by spectrophotometry at a wavelength of 600 nm.

  The results are shown in Table 2.

  According to the results in Table 2, mutant V26C_S334C and wild-type PQQGDH showed equivalent activity, but mutant Q352C had reduced activity by 20%. As described above, the mutant V26C_S334C and the mutant Q352C are obtained by converting amino acid residues adjacent to each other at the time of dimer formation of wild-type PQQGDH into cysteine. However, the enzyme activity of mutant Q352C was reduced.

Example 3 Preparation of Enzyme Electrode Immobilized with Mutant PQQGDH In this example, an enzyme electrode was prepared in which the mutant PQQGDH obtained in Example 1 was immobilized on an electrode substrate. Similarly, preparation of an enzyme electrode on which wild type PQQGDH was immobilized was also performed.

A glassy carbon electrode (manufactured by BAS) having an electrode area of about 0.07 cm ² was used as the electrode substrate. On the electrode surface, add mutant V26C_S334C, mutant Q352C, and wild type PQQGDH prepared at each concentration of 0.625, 1.25, 2.5, 5.0, 10.0, 20.0 μg / ml, and allow to stand overnight at 4 ° C. PQQGDH was adsorbed. For the mutant V26C_S334C and the mutant Q352C, the one obtained in 3 of Example 1 above was used, and for the wild-type PQQGDH, the one obtained in 4 of Example 1 above was used. After adsorption, the 50 mM containing CaCl ₂ in 1 mM MOPS (pH 7.0) buffer for 10 min electrodes were washed in, 50 mM MOPS (pH 7.0) containing PQQ and 1 mM of CaCl ₂ in 10 [mu] M buffer It was immersed in an enzyme electrode (wild-type PQQGDH fixed electrode, mutant V26C_S334C fixed electrode, mutant Q352C fixed electrode).

Example 4 Electrochemical characterization (CV) of mutant PQQGDH fixed electrode
In the present example, the electrochemical characteristics of the enzyme electrode produced in Example 3 were evaluated. Evaluation of the electrochemical characteristics was performed by cyclic voltammetry (CV) of the enzyme electrode.

  CV is the enzyme electrode (wild-type PQQGDH fixed electrode, mutant V26C_S334C fixed electrode, and mutant Q352C fixed electrode) prepared in Example 3 as a working electrode using an electrochemical measurement apparatus made by BAS, and silver as a reference electrode Measurement was performed by a three-electrode system using a silver chloride electrode and platinum as a counter electrode. As a measurement solution, 0.5 M potassium phosphate buffer (pH 8.0) containing 100 mM glucose and 1 mM 2,6-dichlorophenolindophenol (DCIP) was used. Then, the enzyme electrode, the reference electrode and the counter electrode were immersed in the measurement solution, and the range of -0.2 V to 0.3 V was measured at a sweep rate of 50 mV / s. In addition, as a control, CV measurement was also performed on a glassy carbon electrode on which the enzyme is not adsorbed and fixed according to the same procedure as described above.

The results are shown in FIG. FIG. 3 shows CV measurement results of a wild type PQQGDH fixed electrode, a mutant V26C_S334C fixed electrode, and a mutant Q352C fixed electrode at a concentration of 20 μg / ml, with the horizontal axis representing the potential (V vs. Ag / AgCl), the vertical axis Indicates the current (mA / cm ^-2 ). From the results in FIG. 3, it is found that the mutant V26C_S334C fixed electrode has a current response value higher than that of the wild-type PQQGDH fixed electrode. This proved that stable current response values can be obtained by having the V26C and S334C mutations in wild-type PQQGDH. However, in the mutation to the cysteine of Q352 which is adjacent at the time of dimer formation, the improvement of the current response value as observed in the mutant V26C_S334C was not confirmed. Further, in Example 2, a decrease in enzyme activity was confirmed. From this, even if cysteine is introduced to form a disulfide bond in an amino acid residue at a close position, there are cases where the enzyme catalytic activity is lowered or the function as an electrode catalyst of the enzyme electrode can not be exhibited. found. Therefore, in order to improve the catalytic current density of the enzyme electrode, the mutation position is designed to form a disulfide bond at a position that can form a stable dimer and does not become steric hindrance during activity expression. It can be understood that it is necessary.

Example 5 Electrochemical characterization (CA) of mutant PQQGDH electrode
In the present example, evaluation of the electrochemical characteristics of the enzyme electrode produced in the above-mentioned Example 3 was performed following the above-mentioned Example 4. The electrochemical properties were evaluated by chronoamperometry (CA) of the enzyme electrode.

  CA was performed under the same conditions as in the CV of Example 3 above at a set potential of 0.2 V with the enzyme electrode (wild-type PQQGDH fixed electrode and mutant V26C_S334C fixed electrode) prepared in Example 3 as a working electrode. The current response value after 3 minutes from the start of the measurement was recorded as the limiting current density. In the present example, no examination was made on the mutant Q352C fixed electrode in which a decrease in current response value was recognized in Example 4.

The results are shown in FIG. FIG. 4 shows the results of CA measurement (average value of three measurements) of V26C_S334C fixed electrode and wild type PQQGDH fixed electrode at each concentration, the horizontal axis is the concentration of immobilized enzyme on the electrode (μg / ml), the vertical axis Indicates the limiting current density (mA / cm ² ). From the results of FIG. 4, the limiting current density was saturated at the wild type PQQGDH fixed electrode and the mutant V26C_S334C fixed electrode at an adsorption concentration of 5 μg / ml, and the current densities were 1.7 mA / cm ² and 1.9 mA / cm ² , respectively. From this, it was found that the presence of the V26C and S334C mutations in PQQGDH improves the current density by 10% or more compared to the wild type. It is understood that the improvement of the current density is attributable to the suppression of the dissociation of the homodimer by having the mutations of V26C and S334C, in consideration of the results confirmed in 4 of Example 1.

  From the results of the above Examples 1 to 5, it is understood that the mutant PQQGDH of the present invention forms a stable homodimer. And, since the enzyme electrode on which the mutant PQQGDH is immobilized can exhibit its catalytic function smoothly, it exhibits higher current response than that of the enzyme electrode on which wild-type PQQGDH is immobilized, and densification of the enzyme catalyst current can be achieved. It also turned out. An enzyme electrode on which a mutant PQQGDH that gives such a high catalytic current is immobilized can be used as an electrode of a bio battery or a double sensor, and is expected to contribute to the high output of the bio battery and the high sensitivity of the biosensor.

  The present invention relates to mutant PQQGDH and related to the use thereof, and can be used in all fields where utilization of glucose dehydrogenase is required, and in particular in various industrial fields such as medical, food, environmental fields, etc. It is available.

Claims (8)

Compared with the wild-type pyrroloquinoline quinone-dependent glucose dehydrogenase consisting of the amino acid sequence of the following (a), (b) or (c) and shown in the amino acid sequence of SEQ ID NO: 2, an electrode substrate as an electrode catalyst A mutant pyrroloquinoline quinone-dependent glucose dehydrogenase with improved catalytic current density obtained when it is adsorbed and immobilized on an enzyme electrode.
(A) Oite the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 2 26th valine and the 334 amino acid sequence in which amino acid residues are substituted into the cysteine at position serine of the amino acid sequence shown in (b 1) In the amino acid sequence of (a), one or several amino acids are substituted, deleted, or added at positions other than the amino acid residues at the positions corresponding to the 26th and 334th positions of the amino acid sequence shown in SEQ ID NO: 2 Or an amino acid sequence inserted in (c) an amino acid sequence having at least 90 % or more identity with the amino acid sequence shown in SEQ ID NO: 2, and the 26th valine and the 334th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 Amino acid sequence in which the amino acid residue at the position corresponding to serine is substituted for cysteine   The mutant pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 1, comprising an amino acid sequence in which the 26th valine and the 334rd serine are substituted by cysteine in the amino acid sequence shown in SEQ ID NO: 2.   The mutant pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 1 or 2, which comprises the amino acid sequence shown in SEQ ID NO: 3.   An isolated nucleic acid molecule encoding a mutant pyrroloquinoline quinone dependent glucose dehydrogenase according to any one of claims 1-3. An enzyme electrode in which the mutant pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 3 is adsorbed and immobilized on an electrode substrate.   The enzyme electrode according to claim 5, wherein the electrode substrate is a carbon substrate.   A biobattery comprising the enzyme electrode according to claim 5 as an anode electrode.   A biosensor provided with the enzyme electrode according to claim 5 or 6.