Mutant glucose dehydrogenase with improved thermostability and use thereof

Abstract

To provide glucose dehydrogenases with improved thermostability.SOLUTION: The present invention is a mutant FAD-dependent glucose dehydrogenase, comprising a catalytic subunit, an electron transport subunit, and a hitchhiker subunit, each of said catalytic subunit amino acid sequence and said electron transport subunit amino acid sequence comprising a cysteine residue introduced therein, and the catalytic subunit and the electron transport subunit being linked to each other through disulfide bonds between the cysteine residues, resulting in improved thermal stability.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to biotechnology and the electrochemical measurement of glucose using mutant glucose dehydrogenases. [Background technology]

[0002] Numerous methods have been developed to improve the thermal stability of proteins. Examples of known artificial approaches for improvement include glycosylation, chemical cross-linking, and conjugation to polymers. These methods are broadly applicable to any target protein because they stabilize these target proteins by comprehensively protecting them. However, in the case of enzymes, these methods have a significant impact on enzyme activity, reducing the reproducibility of results and posing practical problems, such as the need for additional purification and modification steps.

[0003] U.S. Patent No. 9,074,239 (Patent Document 1) and U.S. Patent No. 8,945,359 (Patent Document 2) disclose techniques for improving the thermostability of FAD-dependent glucose dehydrogenase (FAD-GDH) by site-directed mutagenesis. However, these documents do not suggest stabilizing the intersubunit bond in connection with the introduction of a mutation within the catalytic subunit. U.S. Patent No. 8,945,359 (Patent Document 2) describes that the specific activity of the mutant at 50°C is only 50% of the specific activity at 30°C, and that the mutant is inactivated at a high temperature of 60°C. Biotechnol Lett, pp. 1774-8 (2015) (Non-Patent Document 1) reports that a mutant enzyme containing cysteine ​​residues introduced into two specific sites in the catalytic subunit exhibited improved thermostability while maintaining activity and specificity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 9,074,239 [Patent Document 2] U.S. Patent No. 8,945,359 [Non-patent literature]

[0005] [Non-Patent Document 1] Biotechnol Lett magazine, pages 1774-8 (2015) Summary of the Invention [Problem to be solved by the invention]

[0006] Glucose dehydrogenase is an enzyme that is often used in glucose sensors. Glucose sensors routinely used by diabetic patients for self-monitoring of blood glucose are required to provide accurate measurement values ​​under any circumstances or environments, and therefore the enzyme, which is an important factor for performance, must have high stability to avoid heat inactivation. Therefore, an object of the present invention is to provide a glucose dehydrogenase with improved thermostability. [Means for solving the problem]

[0007] Based on the information on the crystal structure of FAD-GDH, a heterotrimer containing electron transfer subunits, the present inventors have proposed a method for introducing cysteine ​​residues into specific sites on the intersubunit interface to form disulfide bonds on the interface, thereby altering the higher-order structure of the heterotrimer. Furthermore, the electron transfer activity of this mutant enzyme may be significantly enhanced by the introduction of cysteine ​​residues at positions believed to be within the intramolecular electron transfer pathway of the enzyme, and the inventors have found that these cysteine ​​residues play an important role not only in bridging the intersubunit gap but also in promoting electron transfer.

[0008] One aspect of the present invention provides a mutant glucose dehydrogenase that has stable thermostability without reducing enzymatic activity.

[0009] According to one aspect of the present invention, catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit wherein the amino acid sequence of the catalytic subunit and the amino acid sequence of the electron transfer subunit each contain a cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bonded to each other via a disulfide bond between the cysteine ​​residues.

[0010] According to another aspect of the present invention, there is provided the mutant FAD-dependent glucose dehydrogenase, wherein the amino acid sequence of the electron transfer subunit contains another cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues.

[0011] According to another aspect of the present invention, there is provided the mutant FAD-dependent glucose dehydrogenase, wherein the amino acid sequence of the catalytic subunit and the amino acid sequence of the electron transfer subunit contain cysteine ​​residues introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues, and at the same time, the amino acid sequence of the electron transfer subunit contains another cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues.

[0012] According to another aspect of the present invention, there is provided an enzyme electrode comprising the mutant FAD-dependent glucose dehydrogenase.

[0013] According to another aspect of the present invention, there is provided a biosensor comprising the enzyme electrode. [Effects of the Invention]

[0014] According to the present invention, there is provided an FAD-dependent glucose dehydrogenase with improved thermostability. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the enzyme activity of each GDH (wild-type containing a full-length β subunit and mutants 1 to 3) derived from Burkholderia cepacia (Bc) at 25°C to 65°C. [Figure 2] 1 is a graph showing the enzyme activity of each GDH (wild-type and mutants 1 to 3 containing truncated β subunits) derived from Burkholderia cepacia at 25°C to 65°C. [Figure 3] 1 is a graph showing the enzyme activity of each GDH (mutant 4 and mutant 5 containing a full-length or truncated β subunit) derived from Burkholderia cepacia at 25°C to 75°C. [Figure 4] 1 is a graph showing the enzyme activity at 75°C of each GDH derived from Burkholderia cepacia (wild-type and mutants 1 to 5 containing truncated β subunits). [Figure 5] FIG. 1 shows the results of measuring glucose-dependent current using enzyme electrodes containing each GDH derived from Burkholderia cepacia (wild-type containing a full-length or truncated β subunit, mutant 3, and mutant 5). [Figure 6] FIG. 1 shows the results of continuous measurement of current response using an enzyme electrode containing a mutant GDH derived from Burkholderia cepacia (mutant 5: QYY α205Cβ383C-γ155Cβ349C) or QYY GDH (control). [Figure 7]FIG. 1 shows the results of daily measurements of glucose-dependent current responses using an enzyme electrode containing a mutant GDH derived from Burkholderia cepacia (mutant 5: QYY α205Cβ383C-γ155Cβ349C) or QYY GDH (control). [Figure 8] 1 is a graph showing the enzyme activity of GDH (disulfide-forming mutant or wild-type) derived from Ewingella americana (Ea) at 65°C. [Figure 9] Figure 1 shows a sequence alignment of the hitchhiker subunit between Burkholderia cepacia and Ewingella americana. [Figure 10] Figure 1 shows a sequence alignment of catalytic subunits between Burkholderia cepacia and Ewingella americana. [Figure 11] FIG. 1 shows a sequence alignment of electron transfer subunits between Burkholderia cepacia and Ewingella americana. [Figure 12] 1 is a graph showing the enzyme activity of GDH (wild-type, αP204CβH382C, or αP204CβH382C-γA168CβA347C) derived from Silvimonas terrae (St) at 30 to 70°C. [Figure 13] 1 is a graph showing the time course of the enzyme activity of GDH (wild type, αP204CβH382C, or αP204CβH382C-γA168CβA347C) derived from Silvimonas terrae (St) at 60°C. [Figure 14] 1 is a graph showing the enzyme activity of GDH (wild-type, αP200CβD389C, or αP200CβD389C-γR182CβY355C) derived from Zymobacter palmae (Zp) at 30 to 70°C. [Figure 15] 1 is a graph showing the time course of enzyme activity of GDH (wild type, αP200CβD389C, or αP200CβD389C-γR182CβY355C) derived from Zymobacter palmae (Zp) at 60°C. [Figure 16]1 is a graph showing the enzyme activity of GDH (wild-type or αP204CβS467C-γQ198CβY433C) derived from Covetia sp. (Cb) L2A1 strain at 30 to 70°C. [Figure 17] 1 is a graph showing the time course of enzyme activity of GDH (wild-type or αP204CβS467C-γQ198CβY433C) derived from Covetia sp. (Cb) L2A1 strain at 60°C. [Figure 18] Figure 1 shows sequence alignment of catalytic subunits between Burkholderia cepacia, Ewingella americana, Silvimonas terrae, Zymobacter palmae, and Covetia sp. [Figure 19] Figure 1 shows sequence alignment of electron transport subunits between Burkholderia cepacia, Ewingella americana, Silvimonas terrae, Zymobacter palmae, and Covetia sp. [Figure 20] Figure 1 shows sequence alignment of the hitchhiker subunit between Burkholderia cepacia, Ewingella americana, Silvimonas terrae, Zymobacter palmae, and Covetia sp. DETAILED DESCRIPTION OF THE INVENTION

[0016] According to one embodiment of the present invention, the glucose dehydrogenase is catalytic subunit (α subunit), electron transfer subunit (β subunit), and Hitchhiker subunit (γ subunit) wherein at least the catalytic subunit and the electron transfer subunit each contain a cysteine ​​residue introduced therein, and these cysteine ​​residues form a disulfide bond between them. Said glucose dehydrogenase will hereinafter be referred to as mutant glucose dehydrogenase (GDH) or mutant FAD-GDH. As used herein, an enzyme is transformed into a mutant by introducing a cysteine ​​residue, and its catalytic activity depends on flavin adenine dinucleotide (FAD). The introduction of a cysteine ​​residue herein refers to the introduction of a cysteine ​​residue by substituting it for an existing residue in the sequence (e.g., at the residue shown below), or the introduction of a cysteine ​​residue into the sequence (e.g., immediately before or after the residue shown below). Therefore, the mutants of the present invention are The atypical GDH may contain cysteine ​​residues at the residue positions shown below, or may contain cysteine ​​residues at the residue positions shown below. The residue may contain a cysteine ​​residue immediately before or immediately after the adjacent residue.

[0017] Thus, the mutant glucose dehydrogenase is a trimer composed of three subunits, which may be linked to each other via linkers and / or the like. Known examples of FAD-GDH, which is a trimer composed of the three subunits, include Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia thailandensis, Ralstonia pickettii, Ralstonia sojae, and others. Ralstonia solanacearum, Burkholderia phytofirmans, Ralstonia sp., Moraxella Moraxellaceae bacterium, Pseudomonas knaackmussii knackmussii), Yersinia nurmii, Edagophila cremaea Examples of FAD-GDHs that can be used include FAD-GDHs from Edaphovirga cremea, Silvimonas terrae, Zymobacter palmae, Cobetia sp., Halomonas sp., and Ewingella americana. The mutant FAD-GDH according to one embodiment of the present invention can be obtained by introducing a mutation into the FAD-GDH.

[0018] According to one embodiment of the present invention, in the mutant FAD-GDH, each of the amino acid sequences of the catalytic subunit and the electron transfer subunit contains an introduced cysteine ​​residue therein, and the catalytic subunit and the electron transfer subunit are bonded to each other via a disulfide bond between the cysteine ​​residues. The introduction of the cysteine ​​residue can be by amino acid substitution or insertion.

[0019] Each subunit will be described below using FAD-GDH from Burkholderia cepacia as an example, although the mutant GDH is not limited to the following embodiments. The Burkholderia cepacia strain is not limited. For example, the Burkholderia cepacia KS1 strain may be used. The Burkholderia cepacia KS1 strain was deposited at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (currently the National Institute of Technology and Evaluation (NITE) IPOD) on September 25, 2000, under the accession number FERM BP-7306.

[0020] <Catalytic subunit> The catalytic subunit, also called the α subunit, is FAD (flavin adenine dinucleotide). It is a polypeptide that has catalytic activity for glucose dehydrogenation dependent on a catalytic subunit (e.g., a phosphodiesterase). The catalytic subunit preferably contains an Fe-S cluster. SEQ ID NO: 3 is the amino acid sequence of the FAD-GDH catalytic subunit of Burkholderia cepacia strain KS1. The following explanation will be made with reference to this sequence.

[0021] The site for cysteine ​​introduction in the amino acid sequence of the catalytic subunit is preferably located in a region that forms a contact surface with the electron transfer subunit in the spatial structure of the FAD-GDH. For example, cysteine ​​is introduced at a position in the region of amino acid positions 200 to 240 of SEQ ID NO: 3. Thus, amino acid residues in the region of amino acid positions 200 to 240 of SEQ ID NO: 3 are substituted with cysteine. This region in the catalytic subunit is located on the contact surface with the electron transfer subunit in the spatial structure of GDH. Specific examples of the amino acids in this region include the proline residue at position 205, the glycine residue at position 208, the asparagine residue at position 215, the isoleucine residue at position 224, and the glutamic acid residue at position 235 in the amino acid sequence of SEQ ID NO: 3. Introduction of a cysteine ​​residue includes substitution with cysteine ​​and insertion of cysteine. For example, any of the above amino acid residues may be substituted with a cysteine ​​residue. Alternatively, when a cysteine ​​residue is inserted, the cysteine ​​residue may be inserted immediately before or after these residues.

[0022] The above amino acid substitution positions are positions in SEQ ID NO: 3. However, the amino acid sequence of the catalytic subunit into which the mutation is introduced may have a length different from that of SEQ ID NO: 3. For example, if the amino acid sequence of the catalytic subunit into which the mutation is introduced is shorter by three amino acids on the N-terminal side compared to SEQ ID NO: 3, the proline residue at position 205 of SEQ ID NO: 3 corresponds to the proline at position 202 of the amino acid sequence. Such a case is also considered to be a substitution of a proline residue corresponding to the "proline residue at position 205" and is included in the scope of the present invention. The same applies to other residues.

[0023] Of the above amino acid residues, the proline residue at position 205 is preferably substituted with a cysteine ​​residue.P In one embodiment, a cysteine ​​residue is introduced to replace a proline residue in this motif. The catalytic subunit of the mutant glucose dehydrogenase then has an amino acid sequence "ARNSRCYD" (SEQ ID NO: 24) that has at least 60, 70, 80, 90, or 95% identity to the amino acid sequence of SEQ ID NO: 3, 7, 13, 17, or 21.

[0024] FAD-GDHs are also known from other microorganisms. In the amino acid sequences of their catalytic subunits, e.g., amino acid sequences having at least 60%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO: 3, cysteines may be introduced into the above regions or at amino acid residue positions corresponding to the above specific amino acid residues. More specifically, the amino acid sequence of the catalytic subunit into which the mutation is to be introduced may be aligned with the amino acid sequence of SEQ ID NO: 3, and amino acid residues in the region of amino acid positions 200 to 240 of SEQ ID NO: 3, for example, amino acids at positions corresponding to the proline residue at position 205, the glycine residue at position 208, the asparagine residue at position 215, the isoleucine residue at position 224, or the glutamic acid residue at position 235, may be substituted with cysteine.

[0025] Any polypeptide having a certain degree of sequence identity with SEQ ID NO: 3 or other sequences of the catalytic subunit shown below can be used to prepare a mutant glucose dehydrogenase catalytic subunit, as long as it maintains the function of the catalytic subunit, for example, to form a complex with an electron transfer subunit and a hitchhiker subunit and exert glucose dehydrogenase activity. Sequence identity is the percentage of identity between two amino acid sequences, and can be determined by visual inspection or calculation. The identity of the amino acid sequence here can be obtained by comparing two amino acids, if necessary. It can be defined by aligning the sequences while inserting gaps as needed to maximize the number of matching amino acids, and then calculating the ratio of the number of matching amino acids to the total number of amino acids in the aligned portion (same below).

[0026] Examples of GDH α-subunit amino acid sequences having 60% or more identity to SEQ ID NO: 3 include Burkholderia cenocepacia strain J2315 (GenBank accession number WP_006482972), Burkholderia thailandensis strain TXDOH (WP_009900298), Ralstonia pickettii strain 12D (WP_012761508), Ralstonia solanacearum strain IPO1609 (WP_003265143), Burkholderia phytofirmans strain PsJN (WP_012428610), Ralstonia sp. (WP_048931828), Moraxellaceae bacterium (WP_1148 99537), Pseudomonas knaackmussii (WP_043248521), Yersinia nurumii (WP_049596850), Edahobirga cremea (WP_114193339), Silvimonas terrae (WP_184102398), Zymobacter palmae (WP_027705356), Covetia sp. (WP_158773851), Halomonas sp. (WP_107334716) and Ewingella americana ( WP_034788281) are listed.

[0027] As described above, the region and residue into which the cysteine ​​residue is introduced in these catalytic subunits can be determined by aligning the sequence of each catalytic subunit with the sequence of the catalytic subunit of Burkholderia cepacia (SEQ ID NO: 3). For example, in the case of the catalytic subunit of Ewingella americana GDH (SEQ ID NO: 7), as shown in Figure 10, regions 200-240 in SEQ ID NO: 3 correspond to regions 197-237 in SEQ ID NO: 7, and the proline residue at position 205, the glycine residue at position 208, the asparagine residue at position 215, the isoleucine residue at position 224, and the glutamic acid residue at position 235 correspond to the proline residue at position 202, the glycine residue at position 205, the asparagine residue at position 212, the isoleucine residue at position 221, and the glutamic acid residue at position 232 in SEQ ID NO: 7, respectively.

[0028] Other embodiments of the catalytic subunit include a catalytic subunit comprising an amino acid sequence having at least 60%, 80%, 90%, or 95% identity to SEQ ID NO:7, wherein a cysteine ​​has been introduced at a position corresponding to the proline residue at position 202, the glycine residue at position 205, the asparagine residue at position 212, the isoleucine residue at position 221, or the glutamic acid residue at position 232 in SEQ ID NO:7.

[0029] For example, in the catalytic subunit of Silvimonas terrae GDH (SEQ ID NO: 13), As shown in FIG. 18, the proline residue at position 205 of SEQ ID NO:3 corresponds to the proline residue at position 204 of SEQ ID NO:13. Another embodiment of the catalytic subunit has at least 60%, 80%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO:13 and has a cysteine ​​residue introduced at the position corresponding to the proline residue at position 204 of SEQ ID NO:13.

[0030] For example, in the catalytic subunit of Zymobacter palmae GDH (SEQ ID NO: 17) As shown in FIG. 18, the proline residue at position 205 of SEQ ID NO:3 corresponds to the proline residue at position 200 of SEQ ID NO:17. Another embodiment of the catalytic subunit has at least 60%, 80%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO:17 and has a cysteine ​​residue introduced at the position corresponding to the proline residue at position 200 of SEQ ID NO:17.

[0031] For example, in the catalytic subunit of Covetia sp. GDH (SEQ ID NO: 21), as shown in FIG. 18, the proline residue at position 205 of SEQ ID NO: 3 corresponds to the proline residue at position 204 of SEQ ID NO: 21. Another embodiment of the catalytic subunit has at least 60%, 80%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO:21 and has a cysteine ​​residue introduced at the position corresponding to the proline residue at position 204 of SEQ ID NO:21.

[0032] The amino acid sequence of the catalytic subunit (α subunit) may contain not only the cysteine-introducing mutation but also other mutations. Known examples of mutants containing mutations in the catalytic subunit of FAD-dependent GDH derived from Burkholderia cepacia include a mutant with substitutions at positions 472 and 475 (WO 2005 / 103248), a mutant with substitutions at positions 326, 365, and 472 (QYY mutant, JP 2012-090563 A), and a mutant with substitutions at positions 365, 326, 472, 475, 529, etc. (WO 2006 / 137283). The catalytic subunit of the mutant GDH according to one embodiment of the present invention may contain these mutations.

[0033] <Electron transfer subunit> The electron transfer subunit, also called the β subunit, contains at least one heme-binding domain and has the function of transferring electrons generated in a reaction using glucose as a substrate to an electrode (electron transfer function). The electron transfer subunit includes a cysteine ​​residue introduced therein that forms a disulfide bond with the cysteine ​​residue introduced into the catalytic subunit.

[0034] The electron transfer subunit is not limited as long as it has the function of transferring electrons generated by glucose dehydrogenation to the electrode. Electron transfer subunits derived from various organisms, including known electron transfer subunits, may be used. The electron transfer subunit may be derived from the same microorganism as the catalytic subunit. An example of the electron transfer subunit is the electron transfer subunit of GDH from Burkholderia cepacia. An example of the GDH electron transfer subunit from Burkholderia cepacia strain KS1 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 4 is the amino acid sequence of the GDH β subunit from Burkholderia cepacia strain KS1. The following description will be made with reference to this sequence.

[0035] The site for cysteine ​​introduction in the amino acid sequence of the electron transfer subunit is preferably located in a region that forms a contact surface with the catalytic subunit. For example, cysteine ​​is introduced at a position in the region of amino acid positions 330 to 400 of the amino acid sequence of SEQ ID NO: 4. Thus, amino acid residues in the region of amino acid positions 330 to 400 of SEQ ID NO: 4 are substituted with cysteine. This region in the electron transfer subunit is located on the contact surface with the catalytic subunit in the spatial structure of GDH. More specific examples of such amino acids in this region include the threonine residue at position 336, the glycine residue at position 385, the aspartic acid residue at position 383, and the tyrosine residue at position 391 in the amino acid sequence of SEQ ID NO: 4. Any of these residues may be substituted with a cysteine ​​residue. When a cysteine ​​residue is inserted, it may be inserted immediately before or after these residues.

[0036] Of the above amino acid residues, it is preferred that the aspartic acid residue at position 383 be substituted with a cysteine ​​residue. M P(A / G)F" (array In one embodiment, a cysteine ​​residue is introduced to replace the amino acid residue three amino acids before this motif. The electron transfer subunit of the mutant glucose dehydrogenase then has an amino acid sequence having at least 60, 70, 80, 90, or 95% identity with the amino acid sequence of SEQ ID NO: 4, 8, 14, 18, or 22, which is "CXX M P(A / G)F" (SEQ ID NO: 26).

[0037] Preferred examples of combinations of the cysteine ​​introduction site within the region in the catalytic subunit (the region of amino acid positions 200 to 240 of SEQ ID NO: 3) and the cysteine ​​introduction site within the region in the electron transfer subunit (the region of amino acid positions 330 to 400 of SEQ ID NO: 4) include a combination of proline at position 205 of the catalytic subunit and aspartic acid at position 383 of the electron transfer subunit, and a combination of asparagine at position 215 of the catalytic subunit and threonine at position 336 of the electron transfer subunit. These combinations of residues are spaced approximately 5 Å apart from each other in the spatial structure of FAD-GDH and are capable of forming disulfide bonds more efficiently.

[0038] The mutant glucose dehydrogenase may be a mutant glucose dehydrogenase in which an additional introduced cysteine ​​residue is contained in the amino acid sequence of the electron transfer subunit, and the cysteine ​​residue forms a disulfide bond with the cysteine ​​residue introduced into the amino acid sequence of the hitchhiker subunit, thereby bonding the hitchhiker subunit and the electron transfer subunit to each other.

[0039] The additional cysteine ​​residue is preferably introduced into a region of the electron transfer subunit that is on the interface with the hitchhiker subunit. An example of such a region is the region of amino acid positions 341 to 350 in the amino acid sequence of SEQ ID NO: 4. Thus, amino acid residues in the region of amino acid positions 341 to 350 of SEQ ID NO: 4 may be substituted with cysteine. This region in the electron transfer subunit is located on the interface with the hitchhiker subunit in the spatial structure of GDH. More specific examples of the amino acids in this region include the threonine residue at position 345, the proline residue at position 346, and the tyrosine residue at position 349 in the amino acid sequence of the region of SEQ ID NO: 4. Introduction of a cysteine ​​residue includes substitution with cysteine ​​and insertion of cysteine. For example, any of the above amino acid residues may be substituted with a cysteine ​​residue. Alternatively, when a cysteine ​​residue is inserted, the cysteine ​​residue may be inserted immediately before or after these residues.

[0040] Of the above amino acid residues, it is preferred that the tyrosine residue at position 349 be substituted with a cysteine ​​residue. YPS(L / M) " (SEQ ID NO: 27). In one embodiment, a cysteine ​​residue is introduced to replace the amino acid residue immediately preceding this motif. The electron transfer subunit of the mutant glucose dehydrogenase then has "CYPS(L / M)" (SEQ ID NO: 28) in an amino acid sequence that is at least 60, 70, 80, 90, or 95% identical to the amino acid sequence of SEQ ID NO: 4, 8, 14, 18, or 22.

[0041] The amino acid substitution mutation positions described above are positions in SEQ ID NO: 4. However, the amino acid sequence of the electron transfer subunit into which the mutation is introduced may have a length different from that of SEQ ID NO: 4. For example, if the amino acid sequence of the electron transfer subunit into which the mutation is introduced is shorter by 5 amino acids at the N-terminus compared to SEQ ID NO: 4, the threonine residue at position 336 of SEQ ID NO: 4 corresponds to the threonine at position 331 of the amino acid sequence. Such a case is also considered to be a substitution of the threonine residue corresponding to the "threonine residue at position 336" and is included in the scope of the present invention. The same applies to other residues.

[0042] The electron transfer subunit may contain additional mutations in addition to the mutation that introduces a cysteine.

[0043] The electron transfer subunit contains three heme-binding domains. The electron transfer subunit may be a mutant (truncated) electron transfer subunit in which the first and second heme-binding domains, i.e., the first and second domains counting from the N-terminus, are deleted. Such mutant electron transfer subunits are disclosed in U.S. Patent Application Publication No. 2019-0010215.

[0044] Therefore, in the electron transfer subunit having three heme-binding domains (CXXCH motif (SEQ ID NO: 31)), the first and second heme-binding domains counting from the N-terminus or the region containing these domains may be deleted. The following explanation uses the electron transfer subunit of Burkholderia cepacia as a representative example. SEQ ID NO: 4 contains the first heme-binding domain (amino acid positions 43-47), the second heme-binding domain (amino acid positions 191-195), and the third heme-binding domain (amino acid positions 334-338). Of these, the first and second heme-binding domains may be deleted, or the region containing the first and second heme-binding domains (amino acid positions 43-195) may be deleted. Examples of mutant electron transfer subunits lacking the region containing the first and second heme-binding domains include mutant electron transfer subunits consisting of amino acid positions 314 to 425 or positions 330 to 425 of SEQ ID NO:4.

[0045] GDHs are also known in other microorganisms. In the amino acid sequences of their electron transfer subunits, such as those of SEQ ID NO: 4, or amino acid sequences having at least 60%, at least 80%, at least 90%, or at least 95% identity to the truncated sequence consisting of amino acid positions 314 to 425 or amino acid positions 330 to 425 of SEQ ID NO: 4, cysteines may be introduced at positions corresponding to amino acid residues such as the threonine residue at position 336, the glycine residue at position 385, the aspartic acid residue at position 383, or the tyrosine residue at position 391, and / or the threonine residue at position 345, the proline residue at position 346, or the tyrosine residue at position 349. Specifically, the amino acid sequence of the electron transfer subunit into which the mutation is to be introduced may be aligned with the amino acid sequence of SEQ ID NO: 4, and the amino acid at a position corresponding to the threonine residue at position 336, the glycine residue at position 385, the aspartic acid residue at position 383, or the tyrosine residue at position 391 of SEQ ID NO: 4; or the threonine residue at position 345, the proline residue at position 346, or the tyrosine residue at position 349 may be substituted with cysteine.

[0046] Any polypeptide having a certain degree of sequence identity with SEQ ID NO: 4 or other sequences of the electron transfer subunit shown below can be used to prepare the electron transfer subunit of a mutant glucose dehydrogenase, as long as it maintains the function of the electron transfer subunit. The function of the electron transfer subunit means, for example, the function of forming a complex with the catalytic subunit and the hitchhiker subunit and donating and receiving electrons. The electron transfer subunit preferably has three heme-binding regions, and in the case of a truncated form, has a third heme-binding region. Examples of the amino acid sequence of the GDH electron transport subunit having at least 60% identity to SEQ ID NO: 4 include Burkholderia cenocepacia strain J2315 (WP_006482958), Burkholderia thailandensis strain TXDOH (WP_009900297), Ralstonia pickettii strain 12D (WP_012761509), Ralstonia solanacearum strain IPO1609 (WP_049281214), Burkholderia phytofirmans strain PsJN (WP_012428609), Ralstonia Sp. (WP_048931829), Moraxellaceae bacteria (WP_11489953 6), Pseudomonas knaackii (WP_043248520), Yersinia nurumii (WP_049596851), Edagophila cremaea (WP_114193 338), Silvimonas terrae (WP_184102397), Zymobacter pallidum Mae (WP_211245188), Cobetia sp. (W P_158773850), Halomonas sp. (WP_107336529) and E. The sequences of each electron transfer subunit of Wingella americana (WP_034788317) are shown.

[0047] As described above, the region and residue into which the cysteine ​​residue is introduced in these electron transfer subunits can be determined by aligning the sequence of each electron transfer subunit with the sequence of the electron transfer subunit of Burkholderia cepacia (SEQ ID NO: 4). For example, in the case of the electron transfer subunit of Ewingella americana (SEQ ID NO: 8), as shown in Figure 11, the region 330-400 in SEQ ID NO: 4 corresponds to the region 322-392 in SEQ ID NO: 8, and the threonine residue at position 336, the aspartic acid residue at position 383, the glycine residue at position 385, and the tyrosine residue at position 391 correspond to the serine residue at position 328, the glutamic acid residue at position 375, the phenylalanine residue at position 377, and the aspartic acid residue at position 383 in SEQ ID NO: 8, respectively.

[0048] Furthermore, in the case of the electron transfer subunit of Ewingella americana (SEQ ID NO: 8), as shown in Figure 11, regions 341 to 350 in SEQ ID NO: 4 are located in regions 333 to 342 in SEQ ID NO: 8, and the threonine residue at position 345, the proline residue at position 346, and the tyrosine residue at position 349 correspond to the serine residue at position 337, the glutamine residue at position 338, and the tyrosine residue at position 341 in SEQ ID NO: 8, respectively.

[0049] SEQ ID NO: 8 contains a first heme-binding domain (amino acid positions 41 to 45), a second heme-binding domain (amino acid positions 187 to 191), and a third heme-binding domain (amino acid positions 326 to 330). Of these, the first and second heme-binding domains may be deleted, or the region containing the first and second heme-binding domains (amino acid positions 41 to 191) may be deleted. Examples of mutant electron transfer subunits lacking the region containing the first and second heme-binding domains include mutant electron transfer subunits consisting of amino acid positions 306 to 420 or amino acid positions 322 to 420 of SEQ ID NO: 8.

[0050] Another embodiment of the electron transfer subunit includes an electron transfer subunit comprising an amino acid sequence having at least 60%, 80%, 90%, or 95% identity to SEQ ID NO: 8 or to amino acids 306 to 420 of SEQ ID NO: 8, wherein a first cysteine ​​has been introduced at a position corresponding to the serine residue at position 328, the glutamic acid residue at position 375, the phenylalanine residue at position 377, or the aspartic acid residue at position 383 in SEQ ID NO: 8, and a second cysteine ​​has been introduced at a position corresponding to the serine residue at position 337, the glutamine residue at position 338, or the tyrosine residue at position 341 in SEQ ID NO: 8.

[0051] Furthermore, for example, the electron transfer subunit of Silvimonas terrae GDH (SEQ ID NO: 14) 19, the aspartic acid residue at position 383 of SEQ ID NO: 4 corresponds to the histidine residue at position 382 of SEQ ID NO: 14. The tyrosine residue at position 349 of SEQ ID NO: 4 corresponds to the alanine residue at position 347 of SEQ ID NO: 14. SEQ ID NO: 14 contains the first heme-binding region (amino acid numbers 43 to 47), the second heme-binding region (amino acid numbers 191 to 195), and the third heme-binding region (amino acid numbers 332 to 336). Of these, the first and second heme-binding regions may be deleted, or the region containing the first and second heme-binding regions (amino acid numbers 43 to 195) may be deleted. An example of a mutant electron transfer subunit lacking the region containing the first and second heme-binding regions has the amino acid sequence consisting of amino acid numbers 332 to 481 of SEQ ID NO: 14. Another embodiment of the electron transfer subunit has at least 60%, 80%, 90%, or 95% sequence identity with SEQ ID NO: 14 or the amino acid sequence of amino acids 332 to 481 of SEQ ID NO: 14, and has a first cysteine ​​residue introduced at a position corresponding to the histidine residue at position 382 of SEQ ID NO: 14, and / or a second cysteine ​​residue introduced at a position corresponding to the alanine residue at position 347 of SEQ ID NO: 14.

[0052] Furthermore, for example, the electron transfer subunit of Zymobacter palmae GDH (SEQ ID NO: 1 In 8), as shown in Figure 19, the aspartic acid residue at position 383 of SEQ ID NO: 4 corresponds to the aspartic acid residue at position 389 of SEQ ID NO: 18. The tyrosine residue at position 349 of SEQ ID NO: 4 corresponds to the tyrosine residue at position 355 of SEQ ID NO: 18. SEQ ID NO: 18 contains the first heme-binding region (amino acid numbers 49 to 53), the second heme-binding region (amino acid numbers 196 to 200), and the third heme-binding region (amino acid numbers 340 to 344). Of these, the first and second heme-binding regions may be deleted, or the region containing the first and second heme-binding regions (amino acid numbers 49 to 200) may be deleted. An example of a mutant electron transfer subunit lacking the region containing the first and second heme-binding regions has the amino acid sequence of amino acids 340 to 433 of SEQ ID NO: 18. Another embodiment of the electron transfer subunit has at least 60%, 80%, 90%, or 95% sequence identity with SEQ ID NO: 18 or the amino acid sequence of amino acids 340 to 433 of SEQ ID NO: 18, and has a first cysteine ​​residue introduced at a position corresponding to the aspartic acid residue at position 389 of SEQ ID NO: 18, and / or a second cysteine ​​residue introduced at a position corresponding to the tyrosine residue at position 355 of SEQ ID NO: 18.

[0053] Furthermore, for example, in the electron transfer subunit of Covetia sp. GDH (SEQ ID NO: 22), as shown in Figure 19, the aspartic acid residue at position 383 of SEQ ID NO: 4 corresponds to the serine residue at position 467 of SEQ ID NO: 22. The tyrosine residue at position 349 of SEQ ID NO: 4 corresponds to the tyrosine residue at position 433 of SEQ ID NO: 22. SEQ ID NO: 22 contains the first heme-binding region (amino acid numbers 84 to 88), the second heme-binding region (amino acid numbers 231 to 235), and the third heme-binding region (amino acid numbers 418 to 422). Of these, the first and second heme-binding regions may be deleted, or the region containing the first and second heme-binding regions (amino acid numbers 84 to 235) may be deleted. An example of a mutant electron transfer subunit lacking the region containing the first and second heme-binding regions has the amino acid sequence of amino acid numbers 418 to 522 of SEQ ID NO: 22. Another embodiment of the electron transfer subunit has at least 60%, 80%, 90%, or 95% sequence identity with SEQ ID NO:22 or the amino acid sequence of amino acids 418 to 522 of SEQ ID NO:22, and has a first cysteine ​​residue introduced at a position corresponding to the serine residue at position 467 of SEQ ID NO:22, and / or a second cysteine ​​residue introduced at a position corresponding to the tyrosine residue at position 433 of SEQ ID NO:22.

[0054] <Hitchhiker Subunit> The glucose dehydrogenase also contains a hitchhiker subunit. The hitchhiker subunit, also called a γ subunit, forms a complex with the α subunit and the β subunit and functions to be secreted into the periplasm. The hitchhiker subunit is not limited as long as it has this function. γ subunits derived from various organisms, including known γ subunits, may be used. The hitchhiker subunit may be derived from the same microorganism as the catalytic subunit or electron transfer subunit. An example of the hitchhiker subunit is the hitchhiker subunit of GDH from Burkholderia cepacia. SEQ ID NO: 2 is the GDH hitchhiker subunit of Burkholderia cepacia strain KS1. The following explanation will be made with reference to this sequence.

[0055] As noted above, the hitchhiker subunit may include a cysteine ​​residue introduced therein to form a disulfide bond with the electron transfer subunit. The site for cysteine ​​introduction in the amino acid sequence of the hitchhiker subunit is preferably located in a region that forms a contact surface with the electron transfer subunit. For example, cysteine ​​is introduced at a position in the region of amino acid positions 140 to 160 in the amino acid sequence of SEQ ID NO: 2. Thus, amino acid residues in the region of amino acid positions 140 to 160 of SEQ ID NO: 2 are substituted with cysteine. This region in the hitchhiker subunit is located on the contact surface with the electron transfer subunit in the spatial structure of GDH. Specific examples of such amino acids in this region include the threonine residue at position 145, the asparagine residue at position 154, and the lysine residue at position 155 in the amino acid sequence of SEQ ID NO: 2. Introduction of a cysteine ​​residue includes substitution with and insertion of a cysteine. For example, any of the above amino acid residues may be substituted with a cysteine ​​residue. Alternatively, when a cysteine ​​residue is inserted, the cysteine ​​residue may be inserted immediately before or after these residues.

[0056] Of the above amino acid residues, the lysine residue at position 155 is preferably substituted with a cysteine ​​residue. PXXWXXXP " (SEQ ID NO: 29). In one embodiment, a cysteine ​​residue is introduced to replace the amino acid residue one amino acid before this motif. The hitchhiker subunit of the mutant glucose dehydrogenase then has an amino acid sequence having at least 60, 70, 80, 90, or 95% identity with the amino acid sequence of SEQ ID NO: 2, 6, 12, 16, or 20, which contains the amino acid sequence "C". PXX WXXXP" (SEQ ID NO: 30).

[0057] The amino acid substitution mutation positions described above are positions in SEQ ID NO: 2. However, the amino acid sequence of the hitchhiker subunit into which the mutation is introduced may have a length different from that of SEQ ID NO: 2. For example, if the amino acid sequence of the hitchhiker subunit into which the mutation is introduced is longer by two amino acids at the N-terminus compared to SEQ ID NO: 2, the threonine residue at position 145 of SEQ ID NO: 2 corresponds to the threonine at position 147 of the amino acid sequence. Such a case is also considered to be a substitution of the threonine residue corresponding to the "threonine residue at position 145" and is included in the scope of the present invention. The same applies to other residues.

[0058] Preferred examples of combinations of the cysteine ​​introduction site for disulfide formation with the hitchhiker subunit in the electron transfer subunit (the region of amino acid positions 341 to 350 of SEQ ID NO: 4) and the cysteine ​​introduction site in the hitchhiker subunit (the region of amino acid positions 140 to 160 of SEQ ID NO: 2) include a combination of threonine at position 345 of the electron transfer subunit and asparagine at position 154 of the hitchhiker subunit, and a combination of tyrosine at position 349 of the electron transfer subunit and lysine at position 155 of the hitchhiker subunit. These combinations of residues are spaced approximately 5 Å apart in the spatial structure of FAD-GDH and are capable of more efficient disulfide bond formation.

[0059] The hitchhiker subunit may contain additional mutations in addition to the mutation introducing a cysteine.

[0060] GDHs are also known in other microorganisms. Cysteines may be introduced into the above regions or at positions corresponding to the above specific amino acid residues in the amino acid sequences of the hitchhiker subunits thereof, for example, amino acid sequences having at least 60%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO: 2. . Specifically, the amino acid sequence of the hitchhiker subunit into which the mutation is to be introduced may be aligned with the amino acid sequence of SEQ ID NO: 2, and the amino acid at the position corresponding to the threonine residue at position 145, the asparagine residue at position 154, or the lysine residue at position 155 of SEQ ID NO: 2 may be substituted with a cysteine.

[0061] Any polypeptide having a certain degree of sequence identity with SEQ ID NO: 2 or other sequences of the hitchhiker subunit shown below can be used to prepare a mutant glucose dehydrogenase hitchhiker subunit, as long as it maintains the function of the hitchhiker subunit, such as forming a complex with the catalytic subunit and the electron transfer subunit and being secreted into the periplasm. Examples of the GDH hitchhiker subunit amino acid sequence having at least 60% identity to SEQ ID NO: 2 include Burkholderia cenocepacia strain J2315 (WP_006482974), Burkholderia thailandensis strain TXDOH (WP_009900299), Ralstonia pickettii strain 12D (WP_012761507), Ralstonia solanacearum strain IPO1609 (WP_003265142), Burkholderia phytofirmans strain PsJN (WP_012428611), Ralstonia sp. (WP_048931990), Moraxellaceae bacterium (WP_11489 9538), Pseudomonas knaackmussii (WP_043248523), Yersinia nurumii (WP_049596849), Edahobirga cremea (WP_114193340), Silvimonas terrae (WP_184102399), Zymobacter palmae (BBG29837), Covetia sp. (WP_158773852), Halomonas sp. (WP_107334715) and Ewingella americana (W The sequences of the γ subunits of each of the following genes are listed:

[0062] As described above, the region and residue into which the cysteine ​​residue is introduced in these hitchhiker subunits can be determined by aligning the sequence of each hitchhiker subunit with the sequence of the Burkholderia cepacia hitchhiker subunit (SEQ ID NO: 2). For example, in the case of the Ewingella americana hitchhiker subunit (SEQ ID NO: 6), as shown in Figure 9, regions 140-160 in SEQ ID NO: 2 correspond to regions 142-162 in SEQ ID NO: 6, and the threonine residue at position 145, the asparagine residue at position 154, and the lysine residue at position 155 correspond to the valine residue at position 147, the asparagine residue at position 156, and the arginine residue at position 157 in SEQ ID NO: 6, respectively.

[0063] Another embodiment of the hitchhiker subunit includes a hitchhiker subunit comprising an amino acid sequence having at least 60%, 80%, 90%, or 95% identity to SEQ ID NO:6, wherein a cysteine ​​has been introduced at a position corresponding to the valine residue at position 147, the asparagine residue at position 156, or the arginine residue at position 157 in SEQ ID NO:6.

[0064] For example, the hitchhiker subunit of Silvimonas terrae GDH (SEQ ID NO: 12) 20, the lysine residue at position 155 of SEQ ID NO:2 corresponds to the alanine residue at position 168 of SEQ ID NO:12. Another embodiment of the hitchhiker subunit has at least 60%, 80%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO:12 and has a cysteine ​​residue introduced at a position corresponding to the alanine residue at position 168 of SEQ ID NO:12.

[0065] For example, the hitchhiker subunit of Zymobacter palmae GDH (SEQ ID NO: 16 ), as shown in FIG. 20, the lysine residue at position 155 of SEQ ID NO: 2 is This corresponds to the arginine residue at position 182 of No. 16. Another embodiment of the hitchhiker subunit has at least 60%, 80%, 90%, or 95% sequence identity to SEQ ID NO:16 or the amino acid sequence of amino acids 19 to 195 of SEQ ID NO:16, and has a cysteine ​​residue introduced at a position corresponding to the arginine residue at position 182 of SEQ ID NO:16.

[0066] For example, in the hitchhiker subunit of Covetia sp. GDH (SEQ ID NO: 20), as shown in FIG. 20, the lysine residue at position 155 of SEQ ID NO: 2 corresponds to the glutamine residue at position 198 of SEQ ID NO: 20. Another embodiment of the hitchhiker subunit has at least 60%, 80%, 90%, or 95% sequence identity to SEQ ID NO:20 or the amino acid sequence of amino acids 14 to 207 of SEQ ID NO:20, and has a cysteine ​​residue introduced at a position corresponding to the glutamine residue at position 198 of SEQ ID NO:20.

[0067] In one embodiment of the mutant FAD-dependent glucose dehydrogenase, the catalytic subunit is SEQ ID NO:3 the electron transfer subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4, and has a first cysteine ​​introduced at a position corresponding to aspartic acid at position 383 of SEQ ID NO:4 and a second cysteine ​​introduced at a position corresponding to threonine at position 345 of SEQ ID NO:4; the hitchhiker subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:2, and has a cysteine ​​introduced at a position corresponding to asparagine at position 154 of SEQ ID NO:2.

[0068] In one embodiment of the mutant FAD-dependent glucose dehydrogenase, the catalytic subunit is SEQ ID NO:3 the electron transfer subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4, and has a first cysteine ​​introduced at a position corresponding to aspartic acid at position 383 of SEQ ID NO:4 and a second cysteine ​​introduced at a position corresponding to tyrosine at position 349 of SEQ ID NO:4; the hitchhiker subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:2, and has a cysteine ​​introduced at a position corresponding to lysine at position 155 of SEQ ID NO:2.

[0069] In one embodiment of the mutant FAD-dependent glucose dehydrogenase, the catalytic subunit is SEQ ID NO:7 the electron transfer subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of amino acids 326 to 420 of SEQ ID NO: 8, and has a first cysteine ​​introduced at a position corresponding to glutamic acid at position 375 of SEQ ID NO: 8 and a second cysteine ​​introduced at a position corresponding to tyrosine at position 341 of SEQ ID NO: 8; the hitchhiker subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 6, and has a cysteine ​​introduced at a position corresponding to arginine at position 157 of SEQ ID NO: 6.

[0070] In one embodiment of the mutant FAD-dependent glucose dehydrogenase, the catalytic subunit is SEQ ID NO: 1 the electron transfer subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of amino acids 332 to 481 of SEQ ID NO: 14, and has a first cysteine ​​introduced at a position corresponding to histidine at position 382 of SEQ ID NO: 14 and a second cysteine ​​introduced at a position corresponding to alanine at position 347 of SEQ ID NO: 14; the hitchhiker subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 12, and has a cysteine ​​introduced at a position corresponding to alanine at position 168 of SEQ ID NO: 12.

[0071] In one embodiment of the mutant FAD-dependent glucose dehydrogenase, the catalytic subunit is SEQ ID NO: 1 the electron transfer subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence of amino acids 340 to 465 of SEQ ID NO: 18, and has a first cysteine ​​introduced at a position corresponding to aspartic acid at position 389 of SEQ ID NO: 18 and a second cysteine ​​introduced at a position corresponding to tyrosine at position 355 of SEQ ID NO: 18; the hitchhiker subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of amino acids 19 to 195 of SEQ ID NO: 16, and has a cysteine ​​introduced at a position corresponding to arginine at position 182 of SEQ ID NO: 16.

[0072] In one embodiment of the mutant FAD-dependent glucose dehydrogenase, the catalytic subunit is SEQ ID NO:2 the electron transfer subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:22 or the amino acid sequence of amino acids 418 to 522 of SEQ ID NO:22, and has a first cysteine ​​introduced at a position corresponding to serine at position 467 of SEQ ID NO:22 and a second cysteine ​​introduced at a position corresponding to tyrosine at position 433 of SEQ ID NO:22; the hitchhiker subunit has an amino acid sequence that is at least 60%, 70%, 80%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:20 or the amino acid sequence of amino acids 14 to 207 of SEQ ID NO:20, and has a cysteine ​​introduced at a position corresponding to glutamine at position 198 of SEQ ID NO:20.

[0073] According to one embodiment of the present invention, the mutant GDH can be obtained by genetic engineering. More specifically, the mutant GDH can be obtained by introducing a desired mutation into a gene encoding GDH by site-directed mutagenesis or the like, and then expressing the resulting mutant GDH gene in any host cell or cell-free translation system. Therefore, the present invention also encompasses a method for producing a mutant FAD-dependent GDH, which comprises introducing the mutations described herein into a nucleic acid molecule that expresses an FAD-dependent GDH and expressing the mutant FAD-dependent GDH. The GDH is preferably isolated or purified.

[0074] The gene encoding the GDH catalytic (α) subunit is not limited as long as it has a nucleotide sequence corresponding to the amino acid sequence of the GDH α subunit. A specific example of this gene is a DNA consisting of nucleotide positions 764 to 2380 of SEQ ID NO: 1. The α subunit gene is a DNA consisting of the nucleotide positions 764 to 2380 of the nucleotide sequence of SEQ ID NO: 1. It may be DNA having a nucleotide sequence consisting of positions 764 to 2380, or DNA encoding a protein with GDH activity that hybridizes under stringent conditions with a probe that can be prepared from this sequence (for example, DNA having a base sequence that is at least 80%, 90%, or 95% identical to DNA consisting of nucleotide positions 764 to 2380 of SEQ ID NO: 1).

[0075] A specific example of a gene encoding the GDH electron transfer (β) subunit is DNA comprising a nucleotide sequence consisting of nucleotide positions 2386 to 3660 of SEQ ID NO: 1. The β subunit gene may be DNA having a nucleotide sequence consisting of nucleotide positions 2386 to 3660 of SEQ ID NO: 1, or DNA encoding a protein that can function as a β subunit, which hybridizes under stringent conditions with a probe that can be prepared from this sequence (for example, DNA having a nucleotide sequence that is at least 80%, 90%, or 95% identical to DNA consisting of nucleotide positions 2386 to 3660 of SEQ ID NO: 1).

[0076] A specific example of a gene encoding the GDH hitchhiker subunit (γ) is DNA comprising a nucleotide sequence consisting of nucleotide positions 258 to 761 of SEQ ID NO: 1. The γ subunit gene may be DNA having a nucleotide sequence consisting of nucleotide positions 258 to 761 of SEQ ID NO: 1, or DNA encoding a protein that can function as a γ subunit and that hybridizes under stringent conditions with a probe that can be prepared from this sequence (for example, DNA having a nucleotide sequence that is at least 80%, 90%, or 95% identical to DNA consisting of nucleotide positions 258 to 761 of SEQ ID NO: 1).

[0077] Examples of the above-mentioned stringent conditions include conditions that allow hybridization between DNAs that preferably have at least 80%, more preferably at least 90%, and particularly preferably at least 95% identity, and do not allow hybridization between DNAs that have less than 80%, less than 90%, or less than 95% identity. Specific examples of such stringent conditions include conditions in which washing is performed using 0.1×SSC and 0.1% SDS at 60°C, 65°C, or 68°C.

[0078] The α subunit gene, β subunit gene, and γ subunit gene can be obtained, for example, by PCR using the chromosomal DNA of the Burkholderia cepacia strain KS1 as a template. PCR primers for this purpose can be prepared by chemical synthesis based on the above nucleotide sequence. Alternatively, the genes can be obtained from the chromosomal DNA of the Burkholderia cepacia strain KS1 by hybridization using oligonucleotides prepared based on the above sequence as probes. Strains other than the KS1 strain, such as the Burkholderia cenocepacia J2315 strain, Burkholderia thailandensis TXDOH strain, Ralstonia pickettii 12D strain, Ralstonia solanacearum IPO1609 strain, and Burkholderia phytofirmans PsJN strain, may also be used.

[0079] Each of the GDH α-subunit, β-subunit, and γ-subunit having a desired mutation can be obtained by introducing a nucleotide mutation corresponding to the desired amino acid mutation (e.g., substitution with a cysteine ​​residue) into the DNA encoding that GDH subunit by site-directed mutagenesis, and then expressing the resulting mutant DNA using an appropriate expression system. Furthermore, mutant GDH can be obtained by expressing it from the DNA encoding the mutant GDH α-subunit, and also from the DNA encoding the mutant β-subunit and the DNA encoding the mutant γ-subunit. Introduction of a mutation into the DNA encoding each subunit can be performed by the same method as for the γ-subunit, α-subunit, and This may be done using a polycistronic DNA fragment encoding the β subunit and the β subunit in that order.

[0080] The polycistronic DNA fragment encoding the γ subunit, α subunit, and β subunit in this order can be obtained, for example, by PCR using the chromosomal DNA of the Burkholderia cepacia KS1 strain as a template and oligonucleotides having the nucleotide sequences of SEQ ID NOs: 9 and 10 as primers.

[0081] Vectors used to obtain the GDH subunit genes, introduce the mutations, express the genes, etc. include vectors that function in bacteria belonging to the genus Escherichia, such as pTrc99A, pBR322, pUC18, pUC118, pUC19, pUC119, pACYC184, and pBBR122. Examples of promoters used for gene expression include lac, trp, tac, trc, PL, tet, and PhoA. By introducing the γ subunit gene, α subunit gene, and β subunit gene into appropriate sites in an expression vector containing a promoter, insertion of the genes into the vector and binding of the promoter can be carried out in the same process. Examples of such expression vectors include pTrc99A, pBluescript, and pKK223-3.

[0082] The γ subunit gene, α subunit gene, and β subunit gene may be integrated into the chromosomal DNA of a host microorganism in a manner that allows expression. Examples of methods for transforming microorganisms using recombinant vectors include the calcium-treated competent cell method, the protoplast method, and electroporation.

[0083] Examples of the host microorganism include, but are not limited to, bacteria belonging to the genus Bacillus such as Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus niger. Any host microorganism suitable for producing foreign proteins can be used. Transformants such as microorganisms transformed with the mutant GDH gene can be cultured by an appropriate culture method depending on the type of transformant, such as at 30 to 37°C for 12 to 24 hours. The mutant GDH may be used after purification. The mutant GDH may contain an additional sequence such as a tag sequence for purification.

[0084] According to one embodiment of the present invention, the mutant GDH or the microorganism expressing the mutant GDH may be used as a component of an enzyme electrode in a glucose sensor. A specific example of this glucose sensor is a glucose sensor that uses an enzyme electrode formed by immobilizing the mutant GDH on the surface of an electrode such as a gold electrode, a platinum electrode, or a carbon electrode as the working electrode. This sensor refers to a measurement system for electrochemically measuring the concentration of a target analyte. The sensor typically includes three electrodes: a working electrode (enzyme electrode), a counter electrode (e.g., platinum), and a reference electrode (e.g., Ag / AgCl). The sensor may also be a two-electrode system consisting of a working electrode and a counter electrode, e.g., an electrode used in a conventional simple blood glucose level system. The sensor preferably further includes a thermostatic cell for containing a buffer solution and a test sample, a power supply for applying a voltage to the working electrode, an ammeter, a recorder, and the like. The sensor may be either a batch-type sensor or a flow-type sensor. Specifically, the flow-type sensor may be capable of continuously measuring blood glucose levels. More specifically, the sensor may be a sensor having a two-electrode or three-electrode system on which the enzyme of the present invention is immobilized, and the measurement is performed by placing this electrode system in a continuously supplied blood sample or dialysis sample, or in blood or interstitial fluid. The structure of such an enzyme sensor is well known in the art and is described in Biosensors - Fundamental and Applicable ons, Anthony P.F. Turner, Isao Karube, and George S. Wilson, Oxford University Press, 1987. The sensor according to one embodiment of the present invention may be a direct electron transfer sensor that does not contain an electron transfer medium.

[0085] The method for immobilizing the enzyme (mutant GDH) on the electrode is not limited, and examples of this method include a method of chemically immobilizing the enzyme molecules on the electrode using a crosslinking agent or the like, a method of indirectly immobilizing the enzyme molecules on the electrode using a binder or the like, and a method of physically adsorbing the enzyme molecules to the electrode.

[0086] The method for chemically immobilizing the enzyme on the electrode using a crosslinker or the like may be a method for directly immobilizing the enzyme on the electrode. Alternatively, a method such as the method disclosed in U.S. Patent No. 10,563,242 (B2) can be used. More specifically, this method involves immobilizing a monolayer (SAM)-forming molecule on the electrode and then immobilizing a molecular recognition element containing the enzyme via the SAM-forming molecule. The monolayer-forming molecule is a compound that can bind to the electrode and bind the enzyme molecule to the electrode. A monolayer film can be formed by binding multiple molecules of the compound in the same direction on the electrode surface. The distance between the electrode and the enzyme molecule can be controlled by using the monolayer-forming molecule. The monolayer-forming molecules preferably include a first functional group having affinity for the electrode, a spacer region, and a second functional group reactive with the functional group contained in the enzyme molecule. More preferably, the monolayer-forming molecules have a structure in which the first functional group having affinity for the electrode is attached to a first end of the spacer region, and the second functional group reactive with the functional group contained in the enzyme molecule is attached to a second end of the spacer region. Examples of the first functional group having affinity for the electrode include thiol and dithiol groups when the electrode is metal, and pyrene and porphyrin when the electrode is carbon. Examples of the second functional group reactive with the functional group contained in the enzyme molecule include succinimide groups, which react with amino groups (including terminal amino groups and side-chain amino groups) contained in the enzyme molecule, and oxazoline groups, which react with carboxyl groups (including terminal carboxyl groups and side-chain carboxyl groups) contained in the enzyme molecule. An example of a monolayer-forming molecule containing a thiol or dithiol group and a succinimide group is DSH, which will be explained later in the Examples.

[0087] The glucose concentration can be measured using the glucose sensor of the present invention as follows: A buffer solution is placed in the thermostatic cell of the sensor, and the temperature of the cell is maintained constant. An enzyme electrode on which the mutant GDH is immobilized is used as the working electrode. A platinum electrode, for example, is used as the counter electrode. An Ag / AgCl electrode, for example, is used as the reference electrode. A constant voltage is applied to the working electrode. After the current becomes constant, a sample containing glucose is placed in the thermostatic cell, and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared using glucose solutions with standard concentrations.

[0088] The mutant GDH according to one embodiment of the present invention can also be used as a component of a glucose assay kit, which may include, in addition to the mutant GDH, a colorimetric or luminescent reagent, a dilution buffer, a standard substance, manufacturer's instructions, etc.

[0089] For example, a glucose sensor and a glucose assay kit using wild-type GDH from Burkholderia cepacia are described in U.S. Patent Application Publication No. 2004 / 0023330(A1). The mutant GDH can be applied in a similar manner. [Example]

[0090] The present invention will now be further illustrated by the following non-limiting examples.

[0091] Example 1 Expression of mutant GDH We used Burkholderia cepacia CyGDH as a GDH (CyGDH) containing a cytochrome C-containing subunit. This Burkholderia cepacia CyGDH is an oligomeric enzyme composed of a γ subunit, an α subunit, and a β subunit. These three subunits are encoded by a gene having the nucleotide sequence of SEQ ID NO: 1. Based on this sequence, we used pTrc99Aγα(QYY)β, which contains a polynucleotide modified so that the encoded α subunit has a QYY mutation, and pTrc99Aγα(QYY)β330-His, which contains a polynucleotide modified so that the α subunit has a QYY mutation and the encoded β subunit is a truncated β subunit consisting of amino acid positions 330 to 425 (U.S. Patent Application Publication No. 2019-0010215).

[0092] Using these vectors, site-directed mutagenesis was performed to introduce the following amino acid substitutions into the subunits: Mutant 1 is a mutant in which a cysteine ​​is substituted at position 154 of the γ subunit and at position 345 of the β subunit, forming a disulfide bond between these cysteines; Mutant 2 is a mutant in which a cysteine ​​is substituted at position 155 of the γ subunit and at position 349 of the β subunit, forming a disulfide bond between these cysteines; Mutant 3 is a mutant in which a cysteine ​​is substituted at position 205 of the α subunit and at position 383 of the β subunit, forming a disulfide bond between these cysteines. Mutant 4 is a mutant in which a cysteine ​​is substituted at position 205 of the α subunit and at position 383 of the β subunit, forming a disulfide bond (α-β disulfide bond) between these cysteines, a cysteine ​​is substituted at position 345 of the β subunit and at position 154 of the γ subunit, forming a disulfide bond (β-γ disulfide bond) between these cysteines, Mutant 5 is a mutant in which a cysteine ​​is substituted at position 205 of the α subunit and at position 383 of the β subunit, forming a disulfide bond (α-β disulfide bond) between these cysteines, a cysteine ​​is substituted at position 349 of the β subunit and at position 155 of the γ subunit, forming a disulfide bond (β-γ disulfide bond) between these cysteines.

[0093] [Table 1]

[0094] Each of the obtained vectors was used to transform Escherichia coli, and the resulting transformants were cultured to express each mutant GDH. Cultivation and purification of glucose dehydrogenase were carried out by the method described in U.S. Patent Application Publication No. 2019-0010215.

[0095] Measurement of enzyme activity Each mutant GDH was heat-treated at each temperature for 15 minutes, and then the enzyme activity was measured using a system using PMS / DCIP according to the method described in U.S. Patent Application Publication No. 2019-0010215.

[0096] The results are shown in Figure 1. QYY, which lacks the cysteine ​​residue, showed a dramatic decrease in activity from 55°C onwards, whereas mutants 1, 2, and 3 maintained their activity even at 55°C and 65°C.

[0097] As shown in FIG. 2, when the truncated β subunit was used and a cysteine ​​was introduced into the subunit, little decrease in activity was observed.

[0098] We then evaluated the thermal stability of mutants 4 and 5, in which cysteines were introduced into not only the α subunit but also the γ subunit to form α-β disulfide bonds and β-γ disulfide bonds. As a result, mutant 4 exhibited a decrease in activity of approximately one-third of the original activity at 75°C, as shown in Figure 3. In contrast, mutant 5 exhibited approximately 70% of the original activity at 75°C for both the normal β subunit and the truncated subunit, indicating that the enzyme's performance is unlikely to deteriorate even at high temperatures.

[0099] The activity was then investigated after heat treatment at 75°C for various lengths of time. As a result, mutants 4 and 5 showed that their activity was maintained even after 60 minutes of heat treatment. In particular, mutant 5 was found to be a highly thermostable enzyme, retaining more than 60% of its original activity even after 60 minutes of heat treatment.

[0100] Preparation of enzyme electrode and measurement of glucose Mutant 5 was then used to prepare an enzyme electrode, which was then used to measure glucose concentration. First, an enzyme electrode was prepared by immobilizing the mutant on a gold surface via a monolayer-forming molecule, DSH shown below. [ka]

[0101] Specifically, a gold wire (diameter, 0.5 mm; length, 6-7 cm) was immersed in piranha solution (200 μl) at room temperature for 2 hours and then washed with acetone. The wire was immersed in a DSH solution (concentration: 20 μM) in acetone and incubated at 25° C. for 24 hours to bind the thiol group of DSH to the gold surface. The wire was then washed with acetone and immersed in a phosphate buffer solution (300 μl) containing the mutant GDH (concentration: 0.03 mg / ml) and incubated at 4° C. overnight to bind the mutant GDH via the functional group of DSH, thereby obtaining an enzyme electrode on whose surface the mutant GDH was immobilized.

[0102] Using the enzyme electrode prepared as described above, the response current to 0 mM (background), 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, or 50 mM aqueous glucose solutions was measured by chronoamperometry. Glucose measurements were performed at 37°C using a platinum (Pt) wire counter electrode and a silver / silver chloride reference electrode, applying a potential of +0.4 V (relative to silver / silver chloride) to the working electrode.

[0103] As a result, the glucose concentration dependency of the response current was found as shown in FIG. Thus, we demonstrated that glucose concentrations can be measured using sensors that utilize GDH in which cysteines have been introduced to link the subunits together via disulfide bonds, and surprisingly, we found that the introduction of these cysteines improved the electrode properties.

[0104] Continuous measurement of response current The enzyme electrode carrying mutant 5 prepared as described above was immersed in a 20 mM glucose solution at room temperature together with a counter electrode (platinum (Pt) wire) and a reference electrode (silver / silver chloride). A potential of +0.2 V (relative to silver / silver chloride) was applied to the working electrode to measure the current response continuously over a period of 4 weeks, and the results were compared with those of the enzyme electrode carrying QYY GDH without cysteine ​​residues. As shown in Figure 6, the current response of the enzyme electrode carrying mutant 5 was maintained at a high level over a period of 4 weeks, while the current response of the enzyme electrode carrying QYY GDH decreased by approximately 40%.

[0105] Next, the enzyme electrode carrying Mutant 5 was immersed in glucose solutions (0 mM, 1 mM, 3 mM, 5 mM, 10 mM, 15 mM, and 20 mM) at room temperature together with a counter electrode (platinum (Pt) wire) and a reference electrode (silver / silver chloride). A potential of +0.2 V (relative to silver / silver chloride) was continuously applied to the working electrode, and the response current was measured and plotted. The results were compared with those of the enzyme electrode carrying QYY GDH without cysteine ​​residues. As shown in Figure 7, the glucose concentration dependence of the current response of the enzyme electrode carrying Mutant 5 was maintained for approximately 5 weeks, while the glucose concentration dependence of the current response of the enzyme electrode carrying QYY GDH was almost lost on days 26 and 34. These data clearly demonstrate that the enzyme with the introduced cysteine ​​residue is highly stable and can be suitably used for long-term glucose measurement.

[0106] Example 2 Preparation of mutant GDH from Ewingella americana and evaluation of its heat resistance CyGDH derived from Ewingella americana was used as a GDH containing a cytochrome C-containing subunit. This CyGDH derived from Ewingella americana is an oligomeric enzyme composed of a γ subunit, an α subunit, and a β subunit. These three subunits are encoded by a gene having the nucleotide sequence of SEQ ID NO: 5. This CyGDH gene was inserted into pTrc99A.

[0107] This vector was used to perform site-directed mutagenesis to introduce the following amino acid substitutions into the subunits: Mutant E, in which position 202 of the α subunit was substituted with a cysteine ​​and position 375 of the β subunit was substituted with a cysteine; This mutant has a disulfide bond (α-β disulfide bond) formed between the cysteines, a cysteine ​​at position 341 in the β subunit, a cysteine ​​at position 157 in the γ subunit, and a disulfide bond (β-γ disulfide bond) formed between these cysteines.

[0108] Each of the obtained vectors was used to transform Escherichia coli, and the resulting transformants were cultured to express the mutant GDH. Cultivation and purification of glucose dehydrogenase were carried out by the method described in U.S. Patent Application Publication No. 2019-0010215.

[0109] Measurement of enzyme activity The mutant GDH was heat-treated at 65°C, and the enzyme activity was measured using a system using PMS / DCIP according to the method described in U.S. Patent Application Publication No. 2019-0010215, and the time-dependent changes in enzyme activity were plotted.

[0110] The results are shown in Figure 8. Wild-type GDH without cysteine ​​residues showed a drastic decrease in activity at 65°C, whereas the activity of the mutant GDH with α-β disulfide bonds and β-γ disulfide bonds was maintained for 30 minutes at 65°C.

[0111] Example 3 Preparation of mutant GDH from Silvimonas terrae and evaluation of its heat resistance CyGDH from Silvimonas terrae was used as a GDH containing a cytochrome C-containing subunit. This CyGDH is an oligomeric enzyme composed of a γ subunit, an α subunit, and a β subunit. These three subunits are encoded by a gene having the nucleotide sequence of SEQ ID NO: 11. This CyGDH gene was inserted into pTrc99A.

[0112] Using this vector, site-directed mutagenesis was performed to introduce the following amino acid substitutions into the subunits. Two mutant CyGDHs were constructed. One mutant (αP204CβH382C-γA168CβA347C) had a proline at position 204 in the α subunit substituted with a cysteine, a histidine at position 382 in the β subunit substituted with a cysteine, forming a disulfide bond (α-β disulfide bond) between these cysteines, and an alanine at position 347 in the β subunit substituted with a cysteine, and an alanine at position 168 in the γ subunit substituted with a cysteine, forming a disulfide bond (β-γ disulfide bond) between these cysteines. In this mutant, the proline at position 204 of the α subunit is replaced with a cysteine, and the histidine at position 382 of the β subunit is replaced with a cysteine, forming a disulfide bond (α-β disulfide bond) between these cysteines (αP204CβH382C).

[0113] Each of the obtained vectors was used to transform Escherichia coli, and the resulting transformants were cultured to express the mutant GDH. Cultivation and purification of glucose dehydrogenase were carried out by the method described in U.S. Patent Application Publication No. 2019-0010215.

[0114] Measurement of enzyme activity The mutant GDH was heat-treated at 60°C, and the enzyme activity was measured using a system using Ru / MTT according to the method described in U.S. Patent Application Publication No. 2019-0010215, and the time-dependent change in enzyme activity was plotted.

[0115] The results are shown in Figure 12. The activity of wild-type GDH without cysteine ​​residues decreased dramatically with increasing temperature, but the mutant GDH with α-β disulfide bonds showed improved thermostability, and the activity of the mutant GDH with α-β disulfide bonds and β-γ disulfide bonds was maintained up to 60°C. Also, as shown in Figure 13, the wild-type GDH without cysteine ​​residues showed a dramatic decrease in activity at 60°C, whereas the mutant GDH with α-β disulfide bonds showed improved thermostability, and the activity of the mutant GDH with α-β disulfide bonds and β-γ disulfide bonds was maintained for 30 minutes at 60°C.

[0116] Example 4 Preparation of mutant GDH from Zymobacter palmae and evaluation of its heat resistance CyGDH from Zymobacter palmae was used as a GDH containing a cytochrome C-containing subunit. This CyGDH is an oligomeric enzyme composed of a γ subunit, an α subunit, and a β subunit. These three subunits are encoded by a gene having the nucleotide sequence of SEQ ID NO: 15. A modified CyGDH gene was inserted into pTrc99A, in which g at position 55 in the nucleotide sequence of SEQ ID NO: 15 was replaced with a, and valine at position 19 in the amino acid sequence of SEQ ID NO: 16 was replaced with methionine, so that the γ subunit is translated from this position.

[0117] Using this vector, site-directed mutagenesis was performed to introduce the following amino acid substitutions into the subunits. Two mutant CyGDHs were constructed. One was a mutant (αP200CβD389C-γR182CβY355C) in which the proline at position 200 in the α subunit was substituted with cysteine, the aspartic acid at position 389 in the β subunit was substituted with cysteine, forming a disulfide bond (α-β disulfide bond) between these cysteines, the tyrosine at position 355 in the β subunit was substituted with cysteine, and the arginine at position 164 in the γ subunit (corresponding to the arginine at position 182 in SEQ ID NO: 16) was substituted with cysteine, forming a disulfide bond (β-γ disulfide bond) between these cysteines. The mutant (αP200CβD389C) has a proline substituted with a cysteine ​​in the αP200CβD389C subunit, and an aspartic acid at position 389 in the β subunit is substituted with a cysteine, forming a disulfide bond (α-β disulfide bond) between these cysteines.

[0118] Each of the obtained vectors was used to transform Escherichia coli, and the resulting transformants were cultured to express the mutant GDH. Cultivation and purification of glucose dehydrogenase were carried out by the method described in U.S. Patent Application Publication No. 2019-0010215.

[0119] Measurement of enzyme activity The mutant GDH was heat-treated at 60°C, and the enzyme activity was measured using a system using Ru / MTT according to the method described in U.S. Patent Application Publication No. 2019-0010215, and the time-dependent change in enzyme activity was plotted.

[0120] The results are shown in Figure 14. The activity of wild-type GDH without cysteine ​​residues decreased dramatically with increasing temperature, but the activity of mutant GDH with an α-β disulfide bond and mutant GDH with an α-β disulfide bond and a β-γ disulfide bond was maintained up to 65°C. Also, as shown in Figure 15, the activity of wild-type GDH without cysteine ​​residues decreased dramatically to zero at 60°C, while the activity of mutant GDH with an α-β disulfide bond and mutant GDH with an α-β disulfide bond and a β-γ disulfide bond was maintained for 30 minutes at 60°C.

[0121] Example 5 Preparation of mutant GDH from Covetia sp. and evaluation of its thermostability CyG from Covetia sp. as a GDH containing cytochrome C-containing subunit CyGDH was used. This CyGDH is an oligomeric enzyme composed of a γ subunit, an α subunit, and a β subunit. These three subunits are encoded by a gene having the nucleotide sequence of SEQ ID NO: 19. In the nucleotide sequence of SEQ ID NO: 19, the atg at positions 40 to 42 serves as the initiation codon, and the γ subunit is translated from the methionine at position 14 of the amino acid sequence of SEQ ID NO: 20. The CyGDH gene was inserted into pTrc99A.

[0122] Using this vector, site-directed mutagenesis was performed to introduce the following amino acid substitutions into the subunits: One mutant CyGDH was created in which the proline at position 204 in the α subunit was substituted with cysteine, the serine at position 467 in the β subunit was substituted with cysteine, forming a disulfide bond (α-β disulfide bond) between these cysteines, the tyrosine at position 433 in the β subunit was substituted with cysteine, and the glutamine at position 185 in the γ subunit (corresponding to the glutamine at position 198 in SEQ ID NO: 20) was substituted with cysteine, forming a disulfide bond (β-γ disulfide bond) between these cysteines (αP204CβS467C-γQ198CβY433C).

[0123] Each of the obtained vectors was used to transform Escherichia coli, and the resulting transformants were cultured to express the mutant GDH. Cultivation and purification of glucose dehydrogenase were carried out by the method described in U.S. Patent Application Publication No. 2019-0010215.

[0124] Measurement of enzyme activity The mutant GDH was heat-treated at 60°C, and the enzyme activity was measured using a system using Ru / MTT according to the method described in U.S. Patent Application Publication No. 2019-0010215, and the time-dependent change in enzyme activity was plotted.

[0125] The results are shown in Figure 16. The activity of wild-type GDH without cysteine ​​residues decreased with increasing temperature, but the activity of mutant GDHs with α-β disulfide bonds and β-γ disulfide bonds was maintained up to 60°C. Also, as shown in Figure 17, the activity of wild-type GDH without cysteine ​​residues decreased dramatically to zero at 60°C, while the activity of mutant GDHs with α-β disulfide bonds and β-γ disulfide bonds was maintained for 30 minutes at 60°C.

[0126] These data clearly demonstrate that disulfide bond formation is also effective in improving the stability of oligomeric GDH enzymes of various origins.

[0127] The contents of all publications, including patents and patent applications and non-patent documents, cited in this specification are hereby incorporated by reference to the same extent as if the entire contents were expressly set forth.

Claims

1. catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit A mutant FAD-dependent glucose dehydrogenase comprising: the amino acid sequence of the catalytic subunit includes a cysteine ​​residue introduced therein, the amino acid sequence of the electron transfer subunit includes a first cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the amino acid sequence of the electron transfer subunit contains a second cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the catalytic subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO:3, and a cysteine ​​residue is introduced at a position corresponding to the proline residue at position 205 in SEQ ID NO:3; the electron transfer subunit has SEQ ID NO:4 or an amino acid sequence having at least 90% identity to amino acids 330-425 of SEQ ID NO:4, wherein a first cysteine ​​residue is introduced at a position corresponding to the aspartic acid residue at position 383 in SEQ ID NO:4, and a second cysteine ​​residue is introduced at a position corresponding to the tyrosine residue at position 349 in SEQ ID NO:4; and A mutant FAD-dependent glucose dehydrogenase, wherein the hitchhiker subunit has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and a cysteine ​​residue is introduced at a position corresponding to the lysine residue at position 155 in SEQ ID NO:

2.

2. A catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit A mutant FAD-dependent glucose dehydrogenase comprising: the amino acid sequence of the catalytic subunit includes a cysteine ​​residue introduced therein, the amino acid sequence of the electron transfer subunit includes a first cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the amino acid sequence of the electron transfer subunit contains a second cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the catalytic subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO:3, and a cysteine ​​residue is introduced at a position corresponding to the proline residue at position 205 in SEQ ID NO:3; the electron transfer subunit has SEQ ID NO:4 or an amino acid sequence having at least 90% identity to amino acids 330-425 of SEQ ID NO:4, wherein a first cysteine ​​residue is introduced at a position corresponding to the aspartic acid residue at position 383 in SEQ ID NO:4, and a second cysteine ​​residue is introduced at a position corresponding to the threonine residue at position 345 in SEQ ID NO:4; and A mutant FAD-dependent glucose dehydrogenase, wherein the hitchhiker subunit has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and a cysteine ​​residue is introduced at a position corresponding to the asparagine residue at position 154 in SEQ ID NO:

2.

3. A catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit A mutant FAD-dependent glucose dehydrogenase comprising: the amino acid sequence of the catalytic subunit includes a cysteine ​​residue introduced therein, the amino acid sequence of the electron transfer subunit includes a first cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the amino acid sequence of the electron transfer subunit contains a second cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the catalytic subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO:7, and a cysteine ​​residue is introduced at a position corresponding to the proline residue at position 202 in SEQ ID NO:7; the electron transfer subunit has SEQ ID NO:8 or an amino acid sequence having at least 90% identity to amino acids 306-420 of SEQ ID NO:8, a first cysteine ​​residue introduced at a position corresponding to the glutamine residue at position 375 in SEQ ID NO:8, and a second cysteine ​​residue introduced at a position corresponding to the tyrosine residue at position 341 in SEQ ID NO:8; and A mutant FAD-dependent glucose dehydrogenase, wherein the hitchhiker subunit has an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, and a cysteine ​​residue is introduced at a position corresponding to the arginine residue at position 157 in SEQ ID NO:

6.

4. A catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit A mutant FAD-dependent glucose dehydrogenase comprising: the amino acid sequence of the catalytic subunit includes a cysteine ​​residue introduced therein, the amino acid sequence of the electron transfer subunit includes a first cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the amino acid sequence of the electron transfer subunit contains a second cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the catalytic subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 13, and a cysteine ​​residue is introduced at a position corresponding to the proline residue at position 204 in SEQ ID NO: 13; the electron transfer subunit has SEQ ID NO:14 or an amino acid sequence having at least 90% identity to amino acids 332-481 of SEQ ID NO:14, wherein a first cysteine ​​residue is introduced at a position corresponding to the histidine residue at position 382 in SEQ ID NO:14, and a second cysteine ​​residue is introduced at a position corresponding to the alanine residue at position 347 in SEQ ID NO:14; and A mutant FAD-dependent glucose dehydrogenase, wherein the hitchhiker subunit has an amino acid sequence that is at least 90% identical to SEQ ID NO: 12, and a cysteine ​​residue is introduced at a position corresponding to the alanine residue at position 168 in SEQ ID NO:

12.

5. A catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit A mutant FAD-dependent glucose dehydrogenase comprising: the amino acid sequence of the catalytic subunit includes a cysteine ​​residue introduced therein, the amino acid sequence of the electron transfer subunit includes a first cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the amino acid sequence of the electron transfer subunit contains a second cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the catalytic subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 17, and a cysteine ​​residue is introduced at a position corresponding to the proline residue at position 200 in SEQ ID NO: 17; the electron transfer subunit has an amino acid sequence having at least 90% identity to SEQ ID NO:18 or amino acids 340-465 of SEQ ID NO:18, wherein a first cysteine ​​residue is introduced at a position corresponding to the aspartic acid residue at position 389 in SEQ ID NO:18, and a second cysteine ​​residue is introduced at a position corresponding to the tyrosine residue at position 355 in SEQ ID NO:18; and A mutant FAD-dependent glucose dehydrogenase, wherein the hitchhiker subunit has an amino acid sequence having at least 90% identity to SEQ ID NO: 16 or amino acids 19 to 195 of SEQ ID NO: 16, and a cysteine ​​residue has been introduced into a position corresponding to the arginine residue at position 182 in SEQ ID NO:

16.

6. A catalytic subunit, an electron transfer subunit, and Hitchhiker Subunit A mutant FAD-dependent glucose dehydrogenase comprising: the amino acid sequence of the catalytic subunit includes a cysteine ​​residue introduced therein; the amino acid sequence of the electron transfer subunit includes a first cysteine ​​residue introduced therein, and the catalytic subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the amino acid sequence of the electron transfer subunit contains a second cysteine ​​residue introduced therein, and the amino acid sequence of the hitchhiker subunit contains a cysteine ​​residue introduced therein, and the hitchhiker subunit and the electron transfer subunit are bound to each other via a disulfide bond between the cysteine ​​residues; the catalytic subunit comprises an amino acid sequence having at least 90% identity to SEQ ID NO:21, and a cysteine ​​residue is introduced at a position corresponding to the proline residue at position 204 in SEQ ID NO:21; the electron transfer subunit has an amino acid sequence having at least 90% identity to SEQ ID NO:22 or amino acids 418-522 of SEQ ID NO:22, wherein a first cysteine ​​residue is introduced at a position corresponding to the serine residue at position 467 in SEQ ID NO:22, and a second cysteine ​​residue is introduced at a position corresponding to the tyrosine residue at position 433 in SEQ ID NO:22; and A mutant FAD-dependent glucose dehydrogenase, wherein the hitchhiker subunit has an amino acid sequence having at least 90% identity to SEQ ID NO:20 or amino acids 14 to 207 of SEQ ID NO:20, and a cysteine ​​residue has been introduced into a position corresponding to the glutamine residue at position 198 in SEQ ID NO:

20.

7. An enzyme electrode comprising the FAD-dependent glucose dehydrogenase according to any one of claims 1 to 6.

8. A biosensor comprising the enzyme electrode according to claim 7.

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