WO2019230614A1 - Improvement of glucose dehydrogenase - Google Patents

Abstract

The present invention addresses the problem of providing a flavin adenine dinucleotide-dependent glucose dehydrogenase having further improved substrate specificity and/or low-temperature reactivity and having higher practically value. Disclosed is a flavin adenine dinucleotide-dependent glucose dehydrogenase having reduced xylose reactivity, which is produced utilizing a circular permutation (CP) mutation.

Description

Improvement of glucose dehydrogenase

The present invention relates to glucose dehydrogenase (glucose dehydrogenase). More specifically, the present invention relates to a flavin adenine dinucleotide (FAD) -dependent glucose dehydrogenase (E.C.1.1.5.9) having improved properties (substrate specificity, low temperature reactivity) and its gene. This application claims priority based on Japanese Patent Application No. 2018-102829 filed on May 29, 2018, the entire contents of which are incorporated by reference.

Diabetes patients are increasing year by year, and diabetic patients, particularly insulin dependent patients, need to monitor blood glucose levels on a daily basis to control blood sugar levels. In recent years, it has become possible to check the blood glucose level of diabetic patients with an auto-blood glucose meter that can easily and accurately measure in real time using an enzyme. Glucose oxidase (EC1.1.3.4) and PQQ-dependent glucose dehydrogenase (EC1.1.5.2) (see, for example, Patent Documents 1 to 3) have been developed for glucose sensors (for example, sensors used in self blood glucose meters). However, oxygen reactivity, maltose and galactose reactivity became problems. In order to solve this problem, FAD-dependent glucose dehydrogenase (hereinafter abbreviated as “FAD-GDH”) has been developed (see, for example, Patent Documents 4 and 5 and Non-Patent Documents 1 to 4).

In general, not only oral glucose tolerance test but also oral xylose tolerance test and intravenous xylose tolerance test are performed at the time of diabetes determination test. FAD-GDH is generally known to react with xylose, and when FAD-GDH is used, there is a problem in that it affects blood glucose levels during the above-mentioned load test. On the other hand, it is known that the measurement accuracy of a blood glucose meter using FAD-GDH is affected by environmental temperature. In addition, it has been pointed out that when measured in a low-temperature environment where the reactivity of FAD-GDH used in the sensor decreases, the measured value is lower than the actual blood glucose level in the low blood glucose region. Yes. As a solution to these problems, the present applicant has reported FAD-GDH having low reactivity to xylose, that is, excellent substrate specificity, and its improved enzyme (Patent Documents 6 and 7). Patent Document 8 reports FAD-GDH having low reactivity to xylose, but characteristics such as pH stability that are particularly important when applied to a glucose sensor have not been clarified. The practical value is unknown.

JP 2000-350588 A JP 2001-197888 A Japanese Patent Laid-Open No. 2001-346587 International Publication No. 2004/058958 Pamphlet International Publication No. 2007/139013 Pamphlet International Publication No. 2017/077924 Pamphlet International Publication No. 2018/062103 Pamphlet International Publication No. 2015/060150 Pamphlet

As described above, FAD-GDH, which has low reactivity with xylose, has been developed, but there is still room for improvement in substrate specificity. The same applies to the low temperature reactivity, and further improvement is desired. Therefore, an object of the present invention is to provide FAD-GDH having further improved substrate specificity and / or low-temperature reactivity and higher practical value.

In the course of studying to solve the above-mentioned problems, the present inventors focused on Circular Permutation (CP) mutation and attempted to improve FAD-GDH reported in the previous patent applications (Patent Documents 6 and 7). CP mutation is a technique for changing the N-terminus and C-terminus of a target protein while maintaining the protein structure (Non-patent Document 5). It has been reported that this method may be able to modify enzyme activity and substrate specificity without significantly changing the structure of the active site (Non-Patent Documents 6 and 7).

After comprehensive screening and evaluation of the designed mutant enzymes, multiple mutant enzymes with improved substrate specificity were identified. When the low-temperature reactivity of these mutant enzymes was examined, the low-temperature reactivity was also improved. As described above, the present inventors have succeeded in creating FAD-GDH having high practicality suitable for use in a glucose sensor for blood glucose measurement. The following invention is based on the results.
[1] The first sequence consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having an identity of 70% or more with the amino acid sequence and the amino acid sequence of SEQ ID NO: 5 or the identity of the amino acid sequence is 70% or more A primary structure in which a second sequence comprising an amino acid sequence is linked via a peptide linker, or a third sequence comprising the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having an identity with said amino acid sequence of 70% or more, and SEQ ID NO: 7 Or a fourth sequence consisting of an amino acid sequence having an identity of 70% or more with the amino acid sequence has a primary structure linked through a peptide linker, and is compared with the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1. Thus, glucose dehydrogenase having low reactivity to D-xylose and / or improved low-temperature reactivity of glucose dehydrogenase activity.
[2] The glucose dehydrogenase according to [1], wherein the identity defining the first sequence, the second sequence, the third sequence, and the fourth sequence is 80% or more.
[3] The glucose dehydrogenase according to [1], wherein the identity defining the first sequence, the second sequence, the third sequence, and the fourth sequence is 85% or more.
[4] The glucose dehydrogenase according to [1], wherein the identity defining the first sequence, the second sequence, the third sequence, and the fourth sequence is 90% or more.
[5] A primary structure in which the amino acid sequence of SEQ ID NO: 4 in which 173 lysine is substituted with valine, isoleucine, proline or glutamine and the amino acid sequence of SEQ ID NO: 5 linked via a peptide linker, or SEQ ID NO: The amino acid sequence according to [1], wherein the amino acid sequence of No. 128 of 6 amino acid sequence is substituted with valine, isoleucine, proline or glutamine and the amino acid sequence of SEQ ID NO: 7 is linked via a peptide linker. Glucose dehydrogenase.
[6] The amino acid sequence in which the 125th serine in the amino acid sequence of SEQ ID NO: 6 was replaced with cysteine and the amino acid sequence in which the 160th serine of the amino acid sequence in SEQ ID NO: 7 was replaced with cysteine were linked via a peptide linker. The primary structure or the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence in which amino acid sequence 144 of tyrosine No. 7 was replaced with cysteine and amino acid sequence 184 of glycine was replaced with cysteine were linked via a peptide linker. The glucose dehydrogenase according to [1], which has a primary structure.
[7] The glucose dehydrogenase according to any one of [1] to [6], wherein the number of amino acids constituting the peptide linker is 5 to 30.
[8] The glucose dehydrogenase according to [7], wherein the peptide linker is ESSSGTSSSSGTSESSS (SEQ ID NO: 23) or GTSSS (SEQ ID NO: 25).
[9] (a) the amino acid sequence of SEQ ID NO: 8,
(b) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 8,
(c) the amino acid sequence of SEQ ID NO: 9,
(d) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 9,
(e) the amino acid sequence of SEQ ID NO: 32, or (f) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 32,
A glucose dehydrogenase having a low reactivity to D-xylose and / or improved low-temperature stability of glucose dehydrogenase activity as compared to the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1.
[10] The glucose dehydrogenase according to [9], wherein the identity defining the amino acid sequence of (b), the amino acid sequence of (d), and the amino acid sequence of (f) is 80% or more.
[11] The glucose dehydrogenase according to [9], wherein the identity defining the amino acid sequence of (b), the amino acid sequence of (d) and the amino acid sequence of (f) is 85% or more.
[12] The glucose dehydrogenase according to [9], wherein the identity defining the amino acid sequence of (b), the amino acid sequence of (d) and the amino acid sequence of (f) is 90% or more.
[13] An amino acid sequence in which the lysine 173 in the amino acid sequence of SEQ ID NO: 8 is substituted with valine, isoleucine, proline or glutamine, or a lysine 128 in the amino acid sequence of SEQ ID NO: 9 is substituted with valine, isoleucine, proline or glutamine The glucose dehydrogenase according to [9], comprising the amino acid sequence.
[14] The glucose dehydrogenase according to [9], comprising the amino acid sequence of SEQ ID NO: 36 or 37.
[15] The glucose dehydrogenase according to any one of [1] to [14], wherein the reactivity to D-xylose is 8% or less when the reactivity to D-glucose is 100%.
[16] A glucose dehydrogenase gene encoding the glucose dehydrogenase of any one of [1] to [15].
[17] The glucose dehydrogenase gene according to [14], comprising any one of DNAs selected from the group consisting of the following (A) to (C):
(A) DNA encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36 or 37;
(B) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 10 to 13 and 38 to 43;
(C) DNA encoding a protein having a nucleotide sequence equivalent to any one of SEQ ID NOs: 10 to 13 and 38 to 43 and having glucose dehydrogenase activity.
[18] A recombinant DNA comprising the glucose dehydrogenase gene according to [16] or [17].
[19] A microorganism having the recombinant DNA according to [18].
[20] A method for preparing glucose dehydrogenase, comprising the following steps (1) to (3):
(1) A step of preparing a glucose dehydrogenase gene according to [16] or [17];
(2) expressing the gene; and (3) collecting the expression product.
[21] A glucose measurement method comprising measuring glucose in a sample using the glucose dehydrogenase according to any one of [1] to [15].
[22] A glucose measurement reagent comprising the glucose dehydrogenase according to any one of [1] to [15].
[23] A glucose measurement kit comprising the glucose measurement reagent according to [22].
[24] A glucose sensor comprising the glucose dehydrogenase according to any one of [1] to [15].
[25] An enzyme agent comprising the glucose dehydrogenase according to any one of [1] to [15].

Construction method of CP mutant enzyme library. Low temperature reactivity of CP mutant enzyme. The low-temperature reactivity of two CP mutant enzymes (CP-L184, CP-V229) with reduced xylose reactivity was compared with the wild-type enzyme. The relative activity was evaluated with the activity at 50 ° C. as 100%.

1. Terminology The term “isolated” is used herein interchangeably with “purified”. The term “isolated” is used to distinguish a product that is produced without human intervention from its natural state, ie, a state that exists in nature. In the case of a product produced through intervening, it is used to distinguish it from those that have not undergone an isolation step or a purification step. In the former case, an artificial operation of isolation results in an “isolated state” that is different from the natural state, and the isolated is clearly and decisively different from the natural product itself. On the other hand, in the latter case, impurities are typically removed or reduced in quantity by the isolation process or purification process, and the purity is increased. The purity of the isolated enzyme is not particularly limited. However, if application to a use requiring high purity is planned, it is preferable that the purity of the isolated enzyme is high.

The term “modified glucose dehydrogenase” is an enzyme in which an existing glucose dehydrogenase (typically a wild-type enzyme) is mutated or modified. The term “modified glucose dehydrogenase” is used interchangeably with the term “mutant enzyme”. In the present specification, glucose dehydrogenase may be abbreviated as “GDH”.

2. Modified glucose dehydrogenase (GDH)
The first aspect of the present invention relates to modified GDH (hereinafter also referred to as “the present enzyme”). This enzyme has a characteristic structure that was found by examination using CP mutation. Specifically, this enzyme has a sequence corresponding to the N-terminal region of the wild-type enzyme (referred to as “N-terminal sequence”) and a sequence corresponding to the same C-terminal region (referred to as “C-terminal sequence”). ) Have a primary structure linked via a peptide linker. In this enzyme, the N-terminal sequence, the peptide linker, and the C-terminal sequence are arranged in this order from the N-terminus toward the C-terminus.

This enzyme exhibits superior properties than the wild type enzyme. The property here is substrate specificity and / or low temperature reactivity. Typically, the enzyme has an improvement in these two properties. When the substrate specificity is improved, it acts selectively on D-glucose, and the reactivity to D-xylose is lower than that of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1. In the present enzyme having improved substrate specificity, the reactivity to D-xylose when the reactivity to D-glucose is 100% is preferably 8% or less, more preferably 7% or less, even more. Preferably it is 6% or less. It should be noted that the present enzyme having improved substrate specificity typically has lower reactivity to maltose, D-galactose, and the like than the wild-type enzyme.

This enzyme having excellent substrate specificity is preferable as an enzyme for accurately measuring the amount of glucose in a sample. That is, according to this enzyme, the target glucose level can be measured more accurately even when impurities such as D-xylose, maltose or D-galactose are present in the sample. Therefore, it can be said that this enzyme is suitable for applications in which the presence of such contaminants is expected or concerned (typically, measurement of the amount of glucose in blood), and there are various applications including such applications. It can be said that it is applicable to various uses, that is, versatility is high. In addition, the reactivity and substrate specificity of this enzyme can be measured and evaluated by the method shown in the below-mentioned Example.

On the other hand, the improvement in low-temperature reactivity can be evaluated based on, for example, the activity at 20 ° C. relative to the activity at 50 ° C. (relative activity at 20 ° C. based on the activity at 50 ° C.). When the low-temperature reactivity is improved, the relative activity is higher for the present enzyme than for the wild-type enzyme. The relative activity of this enzyme is, for example, 2 to 5 times that of the wild-type enzyme. This enzyme with improved low-temperature reactivity is suitable for applications that are expected to be used in low-temperature environments (typically glucose sensors for blood glucose measuring devices). In order to facilitate understanding of the characteristics of the present invention and comparison with the wild-type enzyme, the low temperature in this specification is “15 ° C. to 25 ° C.”.

In the first embodiment of the present enzyme, the N-terminal sequence consists of the amino acid sequence of SEQ ID NO: 4, and the C-terminal sequence consists of the amino acid sequence of SEQ ID NO: 5. This embodiment corresponds to the mutant enzyme CP-L184 (see Examples below). On the other hand, in the second embodiment of the present invention, the N-terminal sequence consists of the amino acid sequence of SEQ ID NO: 6, and the C-terminal sequence consists of the amino acid sequence of SEQ ID NO: 7. This embodiment corresponds to the mutant enzyme CP-V229 (see Examples below). For convenience of explanation, the N-terminal sequence and C-terminal sequence of the first aspect are referred to as the first sequence and the second sequence, respectively, and the N-terminal sequence and C-terminal sequence of the second aspect are respectively the third sequence and Called the fourth array.

By the way, in general, when a part of the amino acid sequence of a certain protein is mutated, the protein after the mutation may have the same function as the protein before the mutation. That is, the amino acid sequence mutation does not substantially affect the protein function, and the protein function may be maintained before and after the mutation. In consideration of this technical common sense, a slight difference in the amino acid sequence is recognized but no substantial difference is observed in the characteristics when compared with the above enzyme (first aspect or second aspect). Can be regarded as substantially the same enzyme as the present enzyme. “Slight difference in amino acid sequence” used herein typically means 1 to several tens of amino acid sequences (upper limit is 20, 30, 50, 90), preferably 1 to Several (upper limit is 3, 5, 7, 10) amino acid deletion, substitution, or 1 to several tens (upper limit is 20, 30, 50, 90), preferably Means that the amino acid sequence has been altered (changed) by addition, insertion, or a combination of 1 to several amino acids (the upper limit is 3, 5, 7, 10).

The substantially identical enzyme in the case of the first embodiment is a sequence corresponding to the first sequence, that is, a sequence consisting of an amino acid sequence having an identity of 70% or more with the amino acid sequence of SEQ ID NO: 4 (“first homologous sequence”) And a sequence corresponding to the second sequence, that is, a sequence consisting of an amino acid sequence having an identity of 70% or more with the amino acid sequence of SEQ ID NO: 5 (referred to as “second homologous sequence”) via a peptide linker. Primary structure connected together. Note that either one of the first homologous sequence and the second homologous sequence may be 100% identical to the sequence serving as a reference for identity (that is, the first sequence or the second sequence).

Similarly, in the case of the second embodiment, the substantially identical enzyme is a sequence corresponding to the third sequence, that is, a sequence consisting of an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 6 (“third homology” A sequence corresponding to the fourth sequence, that is, a sequence consisting of an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 7 (referred to as a “fourth homologous sequence”) is a peptide It has a primary structure linked through a linker. Note that one of the third homologous sequence and the fourth homologous sequence may be 100% identical to the sequence serving as a reference for identity (that is, the third sequence or the fourth sequence).

Homologous sequences in the “substantially identical enzyme” (first homologous sequence, second homologous sequence, third homologous sequence, fourth homologous sequence) and reference mutant enzyme sequence (first sequence relative to the first homologous sequence, first The identity (%) to the second sequence for the second homologous sequence, the third sequence for the third homologous sequence, the fourth sequence for the fourth homologous sequence) is preferably 80% or more, more preferably 85% or more, Still more preferably, it is 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more. The difference in amino acid sequence may occur at a plurality of positions.

“Slight amino acid sequence differences” are preferably caused by conservative amino acid substitutions. “Conservative amino acid substitution” refers to substitution of an amino acid residue with an amino acid residue having a side chain of similar properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). A conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.

Incidentally, the identity (%) of two amino acid sequences or two nucleic acids (hereinafter, “two sequences” is used as a term including them) can be determined by the following procedure, for example. First, two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is the same. The identity of two sequences is a function of the number of identical positions common to the two sequences (ie identity (%) = number of identical positions / total number of positions × 100), preferably the optimal alignment Take into account the number and size of gaps required for conversion.

比較 Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. Specific examples of mathematical algorithms that can be used for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 226 87: 2264-68; Karlin and Altschul (1993) Proc. Natl. There is a modified algorithm in Acad. Sci. USA 90: 5873-77, but it is not limited to this. Such an algorithm is incorporated in the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search may be carried out with the score = s100 and wordlength = 12 in the NBLAST program. In order to obtain an amino acid sequence equivalent to this enzyme, for example, a BLAST polypeptide search may be performed using the XBLAST program with score = 50 and wordlength = 3. In order to obtain a gap alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. Please refer to http://www.ncbi.nlm.nih.gov for details. Examples of other mathematical algorithms that can be used for sequence comparison include those described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is incorporated in the ALIGN program available on, for example, the GENESTREAM network server (IGH （Montpellier, France) or the ISREC server. When using the ALIGN program for comparison of amino acid sequences, for example, a PAM120 residue mass table can be used, with a gap length penalty = 12 and a gap penalty = 4.

The identity of two amino acid sequences, using the Gloss program in the GCG software package, using a Blossom 62 matrix or PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3 Or 4 can be determined. In addition, the degree of homology between two nucleic acid sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com) with gap weight = 50 and gap length weight = 3. it can.

The enzyme may be part of a larger protein (eg, a fusion protein). Examples of sequences added in the fusion protein include sequences useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

The “peptide linker” that is a constituent element of the enzyme is a linker composed of a peptide in which amino acids are linked in a linear form. The amino acid which comprises a peptide linker is not specifically limited. Preferably, amino acids having a small size (for example, glycine (G), serine (S)) are used, but the peptide linker may be formed using threonine (T), glutamic acid (E), or the like. When a peptide linker is constituted by using a small-sized amino acid such as glycine or serine, the movement of the peptide linker becomes difficult to be restricted, and the influence on the three-dimensional structure formation is reduced.

One of the preferred peptide linker design strategies is to use or mimic a part of the amino acid sequence of the wild type enzyme, that is, to change a part of the amino acid sequence of the wild type enzyme as it is or partially (change of constituent amino acids, The addition of amino acid residues on one or both sides or in the middle) is used for the peptide linker. A specific example of the peptide linker designed by this policy is ESSSGTSSSSGTSESSS (SEQ ID NO: 23), and it has been confirmed that this peptide linker exhibits a desired function (see Examples described later). In addition to this peptide linker, specific examples of peptide linkers include GTSSSS (SEQ ID NO: 16), GTEESSS (SEQ ID NO: 17), SSSGTSSSS (SEQ ID NO: 18), GTSQPESSS (SEQ ID NO: 19), GTSWAQPESSS (SEQ ID NO: 20), GTSAPWAQPESSS (sequence) No. 21), YLTDTSPTEFESSS (SEQ ID NO: 22), ESSSGTSSSSGTSESSS (SEQ ID NO: 23), YVKGSDKNVKPVKGESSS (SEQ ID NO: 24), GTSSS (SEQ ID NO: 25), GTSSSSGTS (SEQ ID NO: 26) and GTSSESSSGTSSSSGTSESSS (SEQ ID NO: 27) can be mentioned. . As these examples show, examples of preferred linker design strategies include configuring the linker to include one or more of G, S, and T, and only G, S, and T. To construct a linker.

The length of the peptide linker, that is, the number of constituent amino acid residues is, for example, 5 to 30, preferably 5 to 25, and more preferably 5 to 21. Considering an important design strategy for CP mutation (a method to change the N-terminus and C-terminus of the target protein (enzyme) while maintaining the structure), maintaining the positional relationship (configuration) between the N-terminus and the C-terminus The length of the peptide linker may be determined.

Examples of the amino acid sequence of this enzyme, that is, an amino acid sequence in which an N-terminal side sequence, a peptide linker, and a C-terminal side sequence are arranged in this order from the N-terminus to the C-terminus, include the following (a) to (d ).
(a) amino acid sequence of SEQ ID NO: 8 (b) amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 8 (c) amino acid sequence of SEQ ID NO: 9 (d) identical to amino acid sequence of SEQ ID NO: 9 (E) amino acid sequence of SEQ ID NO: 32 (f) amino acid sequence having identity of 70% or more

The identity defining the amino acid sequence of (b), the amino acid sequence of (d) and the amino acid sequence of (f) is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more. More preferably 92% or more, particularly preferably 95% or more, and most preferably 99% or more. The difference in amino acid sequence may occur at a plurality of positions.

By the way, a mutation (amino acid substitution) effective for improving low-temperature reactivity has been found for GDH (GDH produced by Aspergillus iizukae No.5453) which is the origin of this enzyme ( (See WO 2018/062103). Based on this fact, a modified GDH to which the mutation is applied is provided. In this modified GDH, an amino acid sequence in which the 173 lysine of the amino acid sequence of SEQ ID NO: 4 is substituted with valine, isoleucine, proline or glutamine or a homologous sequence thereof as the first sequence (N-terminal sequence of the first embodiment) As the third sequence (N-terminal sequence of the second embodiment), an amino acid sequence in which the 128th lysine of the amino acid sequence of SEQ ID NO: 6 is replaced with valine, isoleucine, proline or glutamine or a homologous sequence thereof is used. For example, the amino acid sequence in which the 173 lysine of the amino acid sequence of SEQ ID NO: 8 is substituted with valine, isoleucine, proline or glutamine, or the lysine 128 of the amino acid sequence of SEQ ID NO: 9 is substituted with valine, isoleucine, proline or glutamine. It will have a substituted amino acid sequence. The amino acid residue to be substituted (SEQ ID NO: 8 for amino acid 173, SEQ ID NO: 9 for amino acid 128) corresponds to amino acid 354 of the wild-type enzyme (including the signal peptide and shown in SEQ ID NO: 2).

In addition, Aspergillus iizukae (Aspergillus iizukae) No.5453 strain is the same strain as NBRC-8869 strain. NBRC 8869 shares are stored in the National Institute of Technology and Evaluation (NBRC) (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture 292-0818), and will be sold in accordance with the prescribed procedures. be able to.

On the other hand, the inventors succeeded in finding an amino acid substitution that brings about an improvement in heat resistance when used in combination with a CP mutation (for details, see Examples below). Based on this result, a modified GDH to which the amino acid substitution is applied is provided. In an example of this modified GDH, an amino acid sequence in which the 125th serine of the amino acid sequence of SEQ ID NO: 6 is substituted with cysteine or a homologous sequence thereof is used as the third sequence (N-terminal sequence of the second embodiment). As the 4 sequences (the C-terminal side sequence of the second embodiment), an amino acid sequence in which the 160th serine in the amino acid sequence of SEQ ID NO: 7 is substituted with cysteine or a homologous sequence thereof is used. The amino acid sequence of a specific example (CP-V229-5aa- (S186C / S351C)) of the modified GDH is shown in SEQ ID NO: 36. In another example, as the fourth sequence (C-terminal sequence of the second embodiment), an amino acid sequence in which the 144th tyrosine of the amino acid sequence of SEQ ID NO: 7 is replaced with cysteine and the 184th glycine is replaced with cysteine, or The homologous sequence is used (the third sequence is the amino acid sequence of SEQ ID NO: 6 or a homologous sequence thereof). The amino acid sequence of a specific example (CP-V229-5aa- (Y170C / G210C)) of the modified GDH is shown in SEQ ID NO: 37. In addition, the 125th amino acid (125th amino acid in SEQ ID NO: 36) of the amino acid sequence of SEQ ID NO: 6 is the same as the 351st amino acid of the wild-type enzyme (including the signal peptide and shown in SEQ ID NO: 2, and the same). In the amino acid sequence of No. 7, the 160th amino acid (530th amino acid in SEQ ID NO: 36) is the 186th amino acid of the wild type enzyme, and the 144th amino acid of the amino acid sequence of SEQ ID NO: 7 (the 514th amino acid in the SEQ ID NO: 37) is the wild type enzyme. Amino acid No. 170 in the amino acid sequence of SEQ ID NO: 7 (amino acid 570 in SEQ ID NO: 37) corresponds to amino acid 210 of the wild-type enzyme. In addition, as shown in the below-mentioned Example, it was shown that said 2 sets of amino acid substitution is effective for heat resistance improvement also with respect to the wild type enzyme (sequence number 1) which has not added CP mutation. Based on this finding, in the present application, serine 170 of the wild type enzyme (SEQ ID NO: 1) is substituted with cysteine (corresponding to S186C above), and serine 335 is substituted with cysteine (corresponding to S351C above), The modified GDH improved in heat resistance by the substitution of the two amino acids, the 154th tyrosine of the wild-type enzyme (SEQ ID NO: 1) is replaced with cysteine (corresponding to Y170C above), and the 194th glycine is replaced with cysteine. (Corresponding to the above-mentioned G210C), a modified GDH having improved heat resistance by the substitution of the two amino acids is also provided.

This enzyme can be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (for example, E. coli) with DNA encoding the present enzyme and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, if this enzyme is obtained as a recombinant protein, various modifications are possible. For example, if a DNA encoding this enzyme and another appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. This enzyme can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

3. Nucleic acid encoding modified GDH, etc. The second aspect of the present invention provides a nucleic acid related to the enzyme. That is, a gene encoding the enzyme, a nucleic acid that can be used as a probe for identifying the nucleic acid encoding the enzyme, and a nucleic acid that can be used as a primer for amplifying or mutating the nucleic acid encoding the enzyme Is provided.

The gene encoding this enzyme is typically used for the preparation of this enzyme. According to a genetic engineering preparation method using a gene encoding this enzyme, it is possible to obtain the enzyme in a more homogeneous state. This method can also be said to be a suitable method when preparing a large amount of the present enzyme. The use of the gene encoding this enzyme is not limited to the preparation of this enzyme. For example, the nucleic acid can also be used as an experimental tool for elucidating the mechanism of action of the present enzyme, or as a tool for designing or creating a further mutant of the enzyme.

In the present specification, the “gene encoding the enzyme” refers to a nucleic acid from which the enzyme is obtained when it is expressed, not to mention a nucleic acid having a base sequence corresponding to the amino acid sequence of the enzyme. Also included are nucleic acids obtained by adding sequences that do not encode amino acid sequences to such nucleic acids. Codon degeneracy is also considered.

Examples of sequences of genes encoding this enzyme are SEQ ID NO: 10 (sequence encoding mutant enzyme CP-L184, not including signal sequence), SEQ ID NO: 11 (sequence encoding mutant enzyme CP-L184, including signal sequence) ), SEQ ID NO: 12 (sequence encoding the mutant enzyme CP-V229, not including the signal sequence), SEQ ID NO: 13 (sequence encoding the mutant enzyme CP-V229, including the signal sequence), SEQ ID NO: 38 (mutant enzyme CP -Sequence encoding V229-5aa (not including signal sequence), sequence encoding SEQ ID NO: 39 (mutant enzyme CP-V229-5aa). Including a signal sequence), SEQ ID NO: 40 (sequence encoding a mutant enzyme CP-V229-5aa (S186C / S351C); not including a signal sequence), SEQ ID NO: 41 (mutant enzyme CP-V229-5aa (S186C / S351C) A sequence encoding a signal sequence), SEQ ID NO: 42 (a sequence encoding a mutant enzyme CP-V229-5aa (Y170C / G210C); no signal sequence), SEQ ID NO: 43 (a mutant enzyme CP-V229-5aa (Sequence encoding Y170C / G210C) including signal sequence).

The nucleic acid of the present invention is isolated by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. Can be prepared.

In another embodiment of the present invention, a nucleic acid (hereinafter referred to as an “equivalent nucleic acid”) having a base sequence different from that of a protein encoded by the enzyme that is equivalent in function to the base sequence of the gene encoding the enzyme. In addition, a base sequence defining an equivalent nucleic acid is also referred to as an “equivalent base sequence”). As an example of an equivalent nucleic acid, an enzyme characteristic of this enzyme comprising a base sequence including substitution, deletion, insertion, addition, or inversion of one or more bases based on the base sequence of the nucleic acid encoding this enzyme Mention may be made of DNA encoding a protein having activity (ie GDH activity). Base substitution or deletion may occur at a plurality of sites. The term “plurality” as used herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid. It is.

The equivalent nucleic acid is, for example, 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably, with respect to the base sequence serving as a reference (sequence of any of SEQ ID NOs: 10 to 13, 38 to 43) Has an identity of 90% or more, even more preferably 92% or more, particularly preferably 95% or more, and most preferably 99% or more.

Such equivalent nucleic acids include, for example, restriction enzyme treatment, treatment with exonuclease and DNA ligase, position-directed mutagenesis (MolecularMCloning, lonThird Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) It can be obtained by introducing mutations by mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). The equivalent nucleic acid can also be obtained by other methods such as ultraviolet irradiation.

Another aspect of the present invention relates to a nucleic acid having a base sequence complementary to the base sequence of the gene encoding this enzyme. Still another embodiment of the present invention is at least about 60%, 70%, 80%, 90%, 95%, 99% of the base sequence of the gene encoding the enzyme of the present invention, or a base sequence complementary thereto. A nucleic acid having a nucleotide sequence that is% or 99.9% identical is provided.

Still another embodiment of the present invention relates to a nucleic acid having a base sequence that hybridizes under stringent conditions to a base sequence of a gene encoding the enzyme or a base sequence complementary to the equivalent base sequence. The “stringent conditions” here are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987) Can be set with reference to. As stringent conditions, for example, hybridization solution (50% formamide, 10 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denaturation Conditions of incubation at about 42 ° C to about 50 ° C using salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), followed by washing at about 65 ° C to about 70 ° C using 0.1 x SSC, 0.1% SDS Can be mentioned. Further preferable stringent conditions include, for example, 50% formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

Still another embodiment of the present invention provides a nucleic acid (nucleic acid fragment) having a base sequence of a gene encoding the present enzyme or a part of a base sequence complementary thereto. Such a nucleic acid fragment can be used for detecting, identifying, and / or amplifying a nucleic acid having a base sequence of a gene encoding this enzyme. The nucleic acid fragment is, for example, a nucleotide portion continuous in the base sequence of the gene encoding the present enzyme (eg, about 10 to about 100 bases in length, preferably about 20 to about 100 bases in length, more preferably about 30 to about 100 bases in length). It is designed to include at least a portion that hybridizes to the. When used as a probe, a nucleic acid fragment can be labeled. For labeling, for example, a fluorescent substance, an enzyme, or a radioactive element can be used.

Still another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (gene encoding the enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein into a target such as a cell.

An appropriate vector is selected according to the purpose of use (cloning, protein expression) and in consideration of the type of host cell. M13 phage or a modified form thereof, λ phage or a modified form thereof, pBR322 or a modified form thereof (pB325, pAT153, pUC8, etc.), etc., as a vector using E. coli as a host, pYepSec1, pMFa, pYES2 Examples of vectors that use insect cells as a host include pAc and pVL, examples of vectors that use mammalian cells as a host include pCDM8 and pMT2PC, and examples of vectors that use filamentous fungi as a host include pUC19.

The vector of the present invention is preferably an expression vector. “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell. Expression vectors usually contain a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence / absence (and extent) of introduction of the expression vector can be confirmed using a selection marker.

Insertion of the nucleic acid of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. are performed using standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press and New York, which can be referred to, are known methods using restriction enzymes and DNA ligases).

As host cells, microorganisms such as Escherichia coli (Escherichia coli), budding yeast (Saccharomyces cerevisiae), and filamentous fungi (Aspergillus oryzae) are preferably used from the viewpoint of easy handling, but the recombinant DNA replicates. Any host cell capable of expressing the gene of the present enzyme can be used. Examples of E. coli include E. coli BL21 (DE3) pLysS when T7 promoter is used, and E. coli JM109 otherwise. Examples of filamentous fungi include Aspergillus oryzae RIB40. Examples of budding yeast include budding yeast SHY2, budding yeast AH22, or budding yeast INVSc1 (Invitrogen).

Still another aspect of the present invention relates to a microorganism (that is, a transformant) having the recombinant DNA of the present invention. The microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention. For example, calcium chloride method (Journal of Molecular Biology (J. Mol. Biol.), Volume 53, pp. 159 (1970)), Hanahan Method (Journal of Molecular Biology, Volume 166, 557) (1983), SEM (Gene, 96, 23 (1990)), Chung et al. (Proceedings of the National Academy of Sciences of the USA, 86 Vol., P. 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84,7413-7417 (1984)) etc. The microorganism of the present invention can be used for producing this enzyme. it can.

4). Method for Preparing Modified GDH A further aspect of the present invention relates to a method for preparing the enzyme. In the preparation method of the present invention, modified GDH successfully obtained by the present inventors is prepared by a genetic engineering technique. Specifically, first, a gene encoding this enzyme is prepared (step (1)). Specifically, for example, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36, or 37 is prepared. Here, the “nucleic acid encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36 or 37” is a nucleic acid from which a polypeptide having the amino acid sequence can be obtained when expressed, Not only nucleic acids consisting of the corresponding base sequences, but also extra sequences (either amino acid sequence encoding sequences or non-amino acid sequence encoding sequences) may be added to such nucleic acids. Good. Codon degeneracy is also considered. “Nucleic acid encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36, or 37” refers to standard genetic engineering techniques, molecular biology, with reference to the sequence information disclosed in this specification or the attached sequence listing. It can be prepared in an isolated state by using a conventional method, a biochemical method or the like. Here, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 36, and the amino acid sequence of SEQ ID NO: 37 are amino acids of GDH derived from Aspergillus iizukae No.5453 strain The sequence is modified. Therefore, a nucleic acid (gene) encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36 or 37 by using a gene encoding GDH derived from Aspergillus iizukae No. 5453 (base sequence of SEQ ID NO: 3). Can be obtained. Specific examples of DNA encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36, or 37 are the nucleotide sequences of SEQ ID NOs: 10-13 and 38-43.

Following step (1), the prepared gene is expressed (step (2)). For example, first, an expression vector into which the above gene is inserted is prepared, and a host cell is transformed using the expression vector. Next, the transformant is cultured under conditions where a mutant enzyme that is an expression product is produced. The transformant may be cultured according to a conventional method. The carbon source used in the medium may be any assimitable carbon compound. For example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used. The nitrogen source may be any nitrogen compound that can be used. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

The culture temperature can be set in consideration of the growth characteristics of the transformant to be cultured and the production characteristics of the mutant enzyme. Preferably, it can be set within the range of 30 ° C. to 40 ° C. (more preferably around 37 ° C.). The culture time can be set in consideration of the growth characteristics of the transformant to be cultured and the production characteristics of the mutant enzyme. The pH of the medium is adjusted so that the transformant grows and the enzyme is produced. Preferably, the pH of the medium is about 6.0 to 9.0 (preferably around pH 7.0).

Subsequently, the expression product (modified GDH) is recovered (step (3)). Although the culture solution containing the cultured microbial cells can be used as it is or after concentration, removal of impurities, etc., it can be used as an enzyme solution. In general, the expression product is once recovered from the culture solution or microbial cells. If the expression product is a secreted protein, it can be recovered from the culture solution, and if not, it can be recovered from the fungus body. When recovering from the culture solution, for example, the culture supernatant is filtered and centrifuged to remove insolubles, followed by concentration under reduced pressure, membrane concentration, salting out using ammonium sulfate or sodium sulfate, methanol, ethanol, acetone, etc. Various chromatographic methods such as fractional precipitation, dialysis, heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography (eg, Sephadex gel (GE Healthcare Bioscience)) Separation using a combination of gel filtration, DEAE Sepharose CL-6B (GE Healthcare Bioscience), Octyl Sepharose CL-6B (GE Healthcare Bioscience), CM Sepharose CL-6B (GE Healthcare Bioscience) Purified to obtain a refined modified GDH product Door can be. On the other hand, when recovering from the bacterial cells, the bacterial cells are collected by filtering, centrifuging, and the like, and then the bacterial cells are mechanically treated by pressure treatment, ultrasonic treatment, or enzyme d by lysozyme or the like. After disruption by a conventional method, a purified product of modified GDH can be obtained by separation and purification in the same manner as described above.

The degree of purification of the enzyme is not particularly limited. For example, the enzyme can be purified to a specific activity of 0.1 to 1000 (U / mg), preferably 1 to 500 (U / mg). The final form may be liquid or solid (including powder).

It is also possible to provide the purified enzyme obtained as described above by pulverizing it by, for example, freeze drying, vacuum drying or spray drying. At that time, the purified enzyme may be dissolved in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer or GOOD buffer in advance. Preferably, a phosphate buffer or a triethanolamine buffer can be used. In addition, PIPES, MES, or MOPS is mentioned as a GOOD buffer here.

Usually, gene expression and expression product (modified GDH) are collected using an appropriate host-vector system as described above, but a cell-free synthesis system may be used. Here, “cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)” refers to a ribosome derived from a live cell (or obtained by a genetic engineering technique), not a live cell. This refers to the in vitro synthesis of mRNA and protein encoded by a template nucleic acid (DNA or mRNA) using transcription / translation factors. In a cell-free synthesis system, a cell extract obtained by purifying a cell disruption solution as needed is generally used. Cell extracts generally contain ribosomes necessary for protein synthesis, various factors such as initiation factors, and various enzymes such as tRNA. When protein is synthesized, other substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, and creatine phosphate are added to the cell extract. Of course, a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis.

Development of a transcription / translation system that reconstitutes each molecule (factor) necessary for protein synthesis has also been reported (Shimizu, Y. et al .: Nature Biotech., 19, 751-755, 2001). In this synthesis system, three types of initiation factors constituting bacterial protein synthesis system, three types of elongation factors, four types of factors involved in termination, 20 types of aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and Genes of 31 kinds of factors consisting of methionyl tRNA formyltransferase are amplified from the Escherichia coli genome, and the protein synthesis system is reconstructed in vitro using these genes. In the present invention, such a reconstructed synthesis system may be used.

The term “cell-free transcription / translation system” is used interchangeably with a cell-free protein synthesis system, in-vitro translation system or in-vitro transcription / translation system. In an in vitro translation system, RNA is used as a template to synthesize proteins. As the template RNA, total RNA, mRNA, in vitro transcript and the like are used. The other in vitro transcription / translation system uses DNA as a template. The template DNA should contain a ribosome binding region and preferably contain an appropriate terminator sequence. In the in vitro transcription / translation system, conditions to which factors necessary for each reaction are added are set so that the transcription reaction and the translation reaction proceed continuously.

5. Uses of Modified GDH A further aspect of the invention relates to the use of the enzyme. In this aspect, first, a glucose measurement method using the present enzyme is provided. In the glucose measuring method of the present invention, the amount of glucose in a sample is measured using an oxidation-reduction reaction by this enzyme. The present invention can be applied to various uses in which changes due to this reaction can be used.

The present invention is used, for example, for measurement of blood glucose level, measurement of glucose concentration in foods (such as seasonings and beverages), and the like. Moreover, you may utilize this invention in order to investigate a fermentation degree in the manufacturing process of fermented foods (for example, vinegar) or fermented drinks (for example, beer and liquor).

The present invention also provides a glucose measuring reagent containing the present enzyme. The reagent is used in the glucose measurement method of the present invention described above. Serum albumin, proteins, surfactants, saccharides, sugar alcohols, inorganic salts, and the like may be added for the purpose of stabilizing the glucose measuring reagent and activating it during use.

A reagent for measuring glucose can also be used as a component of the measurement kit. In other words, the present invention also provides a kit (glucose measurement kit) containing the glucose measurement reagent. The kit of the present invention contains the above-mentioned reagent for glucose measurement as an essential component. In addition, a reaction reagent, a buffer solution, a glucose standard solution, a container and the like are included as optional elements. The glucose measurement kit of the present invention usually includes an instruction manual.

It is possible to construct a glucose sensor using this enzyme. That is, this invention also provides the glucose sensor containing this enzyme. In a typical structure of the glucose sensor of the present invention, an electrode system including a working electrode and a counter electrode is formed on an insulating substrate, and a reagent layer containing the present enzyme and mediator is formed thereon. A measurement system that also includes a reference electrode may be used. If such a so-called three-electrode measurement system is used, the potential of the working electrode can be expressed based on the potential of the reference electrode. The material of each electrode is not particularly limited. Examples of the electrode material for the working electrode and the counter electrode are gold (Au), carbon (C), platinum (Pt), and titanium (Ti). As the mediator, a ferricyan compound (such as potassium ferricyanide), a metal complex (such as a ruthenium complex, an osmium complex, or a vanadium complex), a quinone compound (such as pyrroloquinoline quinone), or the like is used. The structure of the glucose sensor and the electrochemical measurement method using the glucose sensor are detailed in, for example, the actual bioelectrochemistry-practical development of biosensors and biobatteries (issued in March 2007, CMC Publishing). .

This enzyme can also be provided in the form of an enzyme agent. In addition to the active ingredient (the present enzyme), the enzyme agent of the present invention may contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline and the like. As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used. As the buffering agent, phosphate, citrate, acetate and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The following studies were conducted with the aim of developing FAD-GDH with high practical value. Specifically, we aimed to improve (especially, decrease xylose reactivity) Aspergillus iizukae-derived GDH (AiGDH) using CP mutation.

(1) Examination of linker sequence of CP mutant enzyme A gene cloned by PCR method based on GDH wild-type gene sequence (SEQ ID NO: 3) derived from Aspergillus iizukae No.5453 was inserted into pYES2 plasmid of Saccharomyces cerevisiae expression system A plasmid for yeast expression was constructed. Next, homology modeling was performed from the amino acid sequence of GDH derived from Aspergillus iizukae No.5453 (SEQ ID NO: 1) to predict the three-dimensional structure. At this time, it was based on the known three-dimensional structure of FAD-GDH derived from Aspergillus flavus (Yoshida, H., et al. (2015). Sci Rep 5: 13498). In order to create a CP mutant enzyme, it is necessary to connect the N-terminus (or vicinity thereof) and the C-terminus (or vicinity thereof) of the wild-type enzyme with an artificial linker sequence. Therefore, the distance between the N-terminal region and the C-terminal region was predicted from the modeled three-dimensional structure. The 27th amino acid (Y) and 591 amino acid (V) were the shortest distance (about 13.5 mm). Therefore, it was decided to remove the peptide of amino acid numbers 19 to 26, leaving the signal peptide (MLGKLTFFSALSLAVA: SEQ ID NO: 14) and 2 amino acids (AP) (amino acid numbers 1 to 18) as they were.

Next, a linker sequence connecting the N-terminal region and the C-terminal region was examined. A region where a new N-terminus could be created was predicted from the modeled three-dimensional structure. From the three-dimensional structure analysis, it is known that the FAD-GDH region around FAD is an important region for enzyme activity (Yoshida, H., et al. (2015). Sci Rep 5: 13498). Therefore, attention was paid to amino acid sequences that are far from the amino acids around FAD and that do not form an α-helix or β-sheet structure. Specifically, a plurality of candidate points were selected from amino acids in a three-dimensional loop structure. In addition, 3 amino acids (62 valine (V), 184 leucine (L), 502 proline (P)) are selected from the candidate points, and 4 different linker sequences (3 amino acids) Long, 4 amino acids, 6 amino acids, 7 amino acids). For example, when the amino acid at position 184 is the new N-terminal, the resulting enzyme is a signal peptide-AiGDH (amino acid numbers 184-591)-linker sequence-AiGDH (amino acid numbers 27-183). After transforming Saccharomyces cerevisiae INVsc1 with the prepared plasmid for expressing the mutant enzyme, the transformant was cultured in a liquid medium containing galactose for inducing expression to obtain a culture solution containing the mutant enzyme.

Screening was performed using enzyme activity against glucose as an index. GDH catalyzes a reaction that oxidizes the hydroxyl group of glucose to produce glucono-δ-lactone in the presence of an electron acceptor. GDH activity was detected by the following reaction system.

In the formula, 1-Methoxy PMS represents 1-Methoxy phenazine methosulfate, and NTB represents Nitrotetrazorium blue.

In reaction (1), reduced 1-Methoxy PMS is generated with the oxidation of glucose, and further, Diformazan generated by reduction of NTB by reduced 1-Methoxy PMS in reaction (2) is measured at a wavelength of 570 nm.

The enzyme activity is calculated by the following calculation formula.

In the formula, Vt is the total liquid volume, Vs is the sample volume, 20.1 is the absorbance coefficient (cm ² /0.5 μmol) per 0.5 μmol of Diformazan, 1.0 is the optical path length (cm), and df is Each dilution factor is indicated.

100% mmol / L PIPES-NaOH containing 0.1% Triton X-100 pH 7.0 to 24 mL, 1 mol / L substrate solution 3 mL, 3 mmol / L 1-Methoxy PMS solution 2 mL, 6.6 mmol / L NTB solution 1 mL Mixed to prepare assay reagents. A culture sample was added to 1 mL of the assay reagent containing each substrate, reacted at 37 ° C., and then the absorbance (570 nm) was measured.

Each mutant enzyme was evaluated by the above enzyme activity measurement method. As a result of evaluating a novel N-terminal mutant enzyme (CP-L184) of No. 184 leucine (L), a CP mutant enzyme having activity was obtained when a linker having a length of 6 amino acids or more was introduced (Table 1). ). On the other hand, in the CP mutant enzyme having 62 valine (V) or 502 proline (P) as the new N-terminus, the activity was below the detection limit even when any of the four types of linkers was used.

Next, the activity of CP-L184 when the linker was lengthened was measured. As a result, enzyme activity could be detected with a linker having 6 to 18 amino acids (Table 1). In the subsequent examination, a 17 amino acid long linker (ESSSGTSSSSGTSESSS: SEQ ID NO: 23) was used.

Examination of linker sequence (CP-L184)

(2) Screening of CP mutant enzyme Signal peptide-AiGDH (amino acid numbers 27 to 591)-Linker sequence-Template DNA encoding the structure (amino acid sequence) of AiGDH (amino acid numbers 27 to 591) was prepared. PCR primers were designed to correspond to newly created N-terminal and C-terminal positions. First, for the primary screening, a total of 278 primers capable of comprehensively covering AiGDH were prepared while shifting the positions of the N-terminal and C-terminal by 4 amino acids. PCR was performed using a combination of two primers, and the amplified DNA fragment was incorporated into a plasmid containing a signal peptide sequence to create a total of 139 CP mutant enzyme expression plasmids with different N-terminal and C-terminal positions (Fig. 1). Saccharomyces cerevisiae INVsc1 was transformed with a plasmid library for expression of these mutant enzymes, and an exhaustive mutant enzyme library was prepared. Each transformant was cultured in a liquid medium containing galactose for inducing expression to obtain a culture solution containing the mutant enzyme. Regarding the reaction for oxidizing the hydroxyl group of glucose, Diformazan produced by reduction of NTB was measured at a wavelength of 570 nm using reduced 1-Methoxy PMS as an electron accepting mediator. In the primary screening, effective mutation points were identified using the enzyme activity for glucose as an index. For the mutation points that were active in the primary screening, a sub-library was created by shifting the positions of the N-terminal and C-terminal one amino acid before and after that. As a result of evaluating the sub-library by the above enzyme activity measurement method, 16 types of CP mutant enzymes showing activity against glucose were obtained (Table 2).

CP mutant enzyme obtained by screening

(3) Recombinant expression of the CP mutant enzyme and purification of the enzyme In the Saccharomyces cerevisiae expression system, the enzyme activity of the culture medium used for activity evaluation was low, and detailed evaluation of the CP mutant enzyme was difficult. Therefore, we decided to insert the CP mutant enzyme gene into the expression cassette connected between the Takaamylase-modified CS3 promoter and the terminator gene of Aspergillus oryzae-derived FAD-GDH. In this plasmid, the Aspergillus oryzae-derived orotidine 5′-phosphate decarboxylase gene (pyrG gene) is inserted into pUC19. A pyrG gene-deficient strain of Aspergillus oryzae RIB40 strain was transformed, and a transformant was obtained using uridine requirement. The obtained transformed strain was subjected to liquid culture using soluble starch as a C source under takaamylase induction conditions to obtain a culture solution containing CP mutant enzyme.

The culture solution was collected, filtered and sterilized, and further purified with a hydrophobic column. The purification conditions are shown below.
Purification column: HiTrap Phenyl FF (1 ml)
Binding buffer: 50 mM phosphate buffer, 2 M ammonium sulfate (pH 6.0)
Elution buffer: 50 mM phosphate buffer (pH 6.0)

AKTA prime was used to increase the concentration of the elution buffer with a gradient, and the protein adsorbed on the column was eluted. As a result, it was found that there was no enzyme activity in the flow-through fraction, and the enzyme activity was concentrated in the first peak. All active peaks were collected and concentrated by ultrafiltration. The following evaluation was performed using each CP mutant enzyme purified and concentrated by the above method and a wild-type enzyme prepared by the same method.

(4) Evaluation of substrate specificity of CP mutant enzyme The substrate specificity of CP mutant enzyme expressed in Aspergillus oryzae RIB40 strain was evaluated. In the same manner as in (1), Diformazan produced by reduction of NTB was measured at a wavelength of 570 nm using reduced 1-Methoxy PMS as an electron accepting mediator for the reaction of oxidizing the hydroxyl group of glucose or other saccharides. As the substrate solution, glucose, maltose, galactose, mannose, xylose and 2-deoxy-D-glucose having a final concentration of 0.1M were used.

Each CP mutant enzyme was evaluated for specific activity against glucose and specific activity against xylose. CP-L184 (the amino acid sequence and gene sequence of the mature form are shown in SEQ ID NO: 8 and SEQ ID NO: 10, respectively) and CP-V229 (mature) The amino acid sequence and gene sequence of the body are shown in SEQ ID NO: 9 and SEQ ID NO: 12, respectively, and showed a decrease in xylose reactivity.

The enzyme activity against glucose was taken as 100%, and the activity against other saccharides was evaluated as a relative value. As a result, CP-L184 and CP-V229 showed reduced mannose reactivity in addition to xylose (Table 4). In addition, the reactivity of maltose, galactose and 2-deoxy-D-glucose was equal to or lower than that of the wild type (Table 3). This result indicates that the substrate specificity of these two CP mutant enzymes is superior to the wild type.

Substrate specificity of CP mutant enzyme

(5) Evaluation of low-temperature reactivity of CP mutant enzymes Low-temperature reactivity of CP mutant enzymes (CP-L184 and CP-V229) that showed excellent substrate specificity was verified. Regarding the enzyme activity against glucose, the activity at 50 ° C. was set to 100%, and the relative activity was evaluated while changing the reaction temperature. As a result, it was suggested that the relative activities of CP mutant enzymes CP-L184 and CP-V229 were improved from 15 ° C. to 35 ° C. compared to the wild type enzyme (FIG. 2). This result indicates that CP-L184 and CP-V229 have improved low-temperature reactivity and have superior properties as glucose sensors for blood glucose measurement.

(6) Examination of linkers Expression vectors of CP-L184 and CP-V229 in which a decrease in xylose reactivity was observed were modified to construct expression vectors with different linker lengths. First, a CP mutant enzyme in which a 5-amino acid linker was introduced as the shortest linker was prepared. In order to facilitate evaluation, the amino acid composition was limited to glycine (G), serine (S), and threonine (T), and 6 amino acids and 9 amino acids were prepared. Finally, a 21 amino acid linker was designed by adding 4 amino acids to the 17 amino acids used in the above study with a longer linker (Table 4). CP-L184 and CP-V229 plasmids with different linkers were constructed for expression in budding yeast. After transformation into budding yeast INVsc1, transformants were obtained, cultured in a medium containing galactose, and the culture broth was collected to evaluate enzyme activity.

Linker sequence

The xylose reactivity of wild-type enzyme and CP-L184 and CP-V229 was compared. Comparing CO mutant enzymes with linker lengths changed to 5, 6, 9, 17, and 21 amino acids, the xylose reactivity was 5.1-5.7% in CP-L184 vs. 11.4% in wild-type, CP-V229 It was 3.5-4.4% (Table 5). That is, regardless of the number of amino acids in the linker and the amino acid composition, the xylose reactivity was lower than that of the wild type regardless of which linker was used. From this, it was concluded that the linker length and composition basically had no effect on substrate specificity.

Substrate specificity of CP mutant enzyme with different number of amino acids in the linker

(7) Examination of heat resistance As a feature of CP mutation design, it can be combined with amino acid substitution mutation. Thus, amino acid substitution mutations were introduced into CP mutant enzymes to try to improve heat resistance. First, mutation points were designed based on the three-dimensional structure information of the wild-type enzyme. A plurality of patents have been filed and published for improving the heat resistance of FAD-GDH by introducing amino acid mutations (for example, Patent No. 5873379, Patent No. 4348563, WO 2015/099112 A1).

A mutation was designed to replace serine (S), which is close in structure, with cysteine (C). By forming a disulfide bond between the introduced cysteines, protein structure stabilization can be expected. The following 8 types of mutant enzymes were prepared.
(i) S46C / S243C, (ii) S165C / S205C, (iii) S186C / S351C, (iv) S188C / S351C, (v) S308C / S322C, (vi) S436C / S471C, (vii) S482C / S489C, viii) P417C / C566

Next, with reference to the structure of Aspergillus niger-derived glucose oxidase (GO) or eringi-derived alcohol oxidase (AAO) whose three-dimensional structure is disclosed, a mutant enzyme in which an amino acid was substituted was designed. (Ix) Y170C / G210C was designed to mimic the disulfide bond of GO. In addition, (x) G273C / G287C was designed by mimicking the disulfide bond of AAO.

The above 10 types of mutation points were designed, and amino acid mutations were introduced into the wild type gene by PCR and in-fusion reaction. Plasmid construction, transformation into budding yeast, and recombinant expression were performed. An enzyme sample was prepared and heated at 30 to 80 ° C. for 30 minutes using a thermal cycler. The activity of the sample after heating was measured at 37 ° C., and the 50% inactivation temperature (T50 value) was measured. As a result, compared to the wild-type enzyme, (iii) S186C / S351C and (ix) Y170C / G210C showed improved heat resistance (Table 6).

Temperature stability evaluation of amino acid substitution enzyme (mutation introduced into wild type enzyme)

* Indicated based on wild-type T50

Next, S186C / S351C and Y170C / G210C, mutation points with improved heat resistance, were introduced into CP-V229. Amino acid mutations were introduced into CP-V229-5aa (amino acid sequence and gene sequence are shown in SEQ ID NO: 32 and SEQ ID NO: 38, respectively) by PCR and in-fusion reaction. Plasmid construction, transformation into budding yeast, and recombinant expression were performed. An enzyme sample was prepared and heated at 30 to 80 ° C. for 30 minutes using a thermal cycler. By measuring the activity of the heated sample at 37 ° C., the 50% inactivation temperature (T50 value) was measured. As a result, for all mutations, after mutation introduction (CP-V229-5aaaa (S186C / S351C), CP-V229-5aa (Y170C / G210C)) compared to before mutation introduction (CP-V229-5aa) The heat resistance was greatly improved (Table 7). The amino acid sequence and gene sequence of CP-V229-5aa229 (S186C / S351C) are shown in SEQ ID NO: 36 and SEQ ID NO: 40, respectively, and the amino acid sequence and gene sequence of CP-V229-5aa (Y170C / G210C) are shown in SEQ ID NO: 37 and SEQ ID NO: 42.

Temperature stability evaluation of amino acid substitution enzyme (mutation introduced into CP-V229)

* Represented based on T50 value of CP-V229

Furthermore, xylose reactivity was evaluated. When xylose reactivity was compared between wild-type enzyme, CP mutant enzyme before mutagenesis, and CP mutant enzyme after mutagenesis, it was found that CP mutant enzyme (CP-V229-5aa after mutagenesis) (S186C / S351C) and CP-V229-5aa (Y170C / G210C)) were 5.4% (CP-V229-5aa (S186C / S351C)) and 5.8% (CP-V229-5aa (Y170C / G210C)) (Table 8). That is, compared to the wild type, the xylose reactivity of the CP mutant enzyme after mutagenesis was low, indicating the same level of xylose reactivity as the CP mutant enzyme (CP-V229-5aa) before mutagenesis. Thus, the property of low xylose reactivity was maintained even after the introduction of a mutation effective for improving heat resistance.

Substrate specificity of amino acid substitution CP mutant enzyme

As described above, we succeeded in improving the heat resistance of CP mutant enzyme. CP mutant enzyme with improved heat resistance has low xylose reactivity and is extremely useful as an enzyme for blood glucose measurement.

The GDH of the present invention has improved substrate specificity (particularly reactivity with xylose) and is particularly suitable for use in a glucose sensor. According to the present invention, the low temperature reactivity can also be improved. GDH with improved low-temperature reactivity is suitable for applications that are expected to be used in low-temperature environments (typically glucose sensors for blood glucose measuring devices).

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

SEQ ID NO: 4: Description of artificial sequence: N-terminal sequence of mutant enzyme CP-L184 SEQ ID NO: 5: Description of artificial sequence: C-terminal sequence of mutant enzyme CP-L184 SEQ ID NO: 6: Description of artificial sequence: Mutant enzyme CP -V229 N-terminal sequence SEQ ID NO: 7: description of artificial sequence: C-terminal sequence of mutant enzyme CP-V229 SEQ ID NO: 8: description of artificial sequence: mutant enzyme CP-L184
SEQ ID NO: 9 Description of artificial sequence: Mutant enzyme CP-V229
SEQ ID NO: 10: Description of artificial sequence: Mutant enzyme CP-L184 gene SEQ ID NO: 11: Description of artificial sequence: Mutant enzyme CP-L184 gene SEQ ID NO: 12: Description of artificial sequence: Mutant enzyme CP-V229 gene SEQ ID NO: 13: Artificial Sequence Description: Mutant Enzyme CP-V229 Gene SEQ ID NO: 14: Artificial Sequence Description: Signal Peptide SEQ ID NO: 15: Description of Artificial Sequence: Linker SEQ ID NO: 16: Description of Artificial Sequence: Linker SEQ ID NO: 17: Description of Artificial Sequence: Linker SEQ ID NO: 18: description of artificial sequence: linker SEQ ID NO: 19: description of artificial sequence: linker SEQ ID NO: 20: description of artificial sequence: linker SEQ ID NO: 21: description of artificial sequence: linker SEQ ID NO: 22: description of artificial sequence: Linker SEQ ID NO: 23: description of artificial sequence: linker SEQ ID NO: 24: description of artificial sequence: linker SEQ ID NO: 25: description of artificial sequence: linker -SEQ ID NO: 26: description of artificial sequence: linker SEQ ID NO: 27: description of artificial sequence: linker SEQ ID NO: 28: description of artificial sequence: mutant enzyme CP-L184-5aa
SEQ ID NO: 29: Description of artificial sequence: Mutant enzyme CP-L184-6aa
SEQ ID NO: 30: Description of artificial sequence: Mutant enzyme CP-L184-9aa
SEQ ID NO: 31 Description of artificial sequence: Mutant enzyme CP-L184-21aa
SEQ ID NO: 32: Description of artificial sequence: Mutant enzyme CP-V229-5aa
SEQ ID NO: 33: Description of artificial sequence: Mutant enzyme CP-V229-6aa
SEQ ID NO: 34: Description of artificial sequence: Mutant enzyme CP-V229-9aa
SEQ ID NO: 35: Description of artificial sequence: Mutant enzyme CP-V229-21aa
SEQ ID NO: 36: Description of artificial sequence: Mutant enzyme CP-V229-5aa (S186C / S351C)
SEQ ID NO: 37: Description of artificial sequence: Mutant enzyme CP-V229-5aa (Y170C / G210C)
SEQ ID NO: 38: Description of artificial sequence: Mutant enzyme CP-V229-5aa gene SEQ ID NO: 39: Description of artificial sequence: Mutant enzyme CP-V229-5aa gene SEQ ID NO: 40: Description of artificial sequence: Mutant enzyme CP-V229-5aa (S186C / S351C) gene SEQ ID NO: 41: Description of artificial sequence: Mutant enzyme CP-V229-5aa (S186C / S351C) gene SEQ ID NO: 42: Description of artificial sequence: Mutant enzyme CP-V229-5aa (Y170C / G210C) gene SEQ ID NO: 43: Description of artificial sequence: Mutant enzyme CP-V229-5aa (Y170C / G210C) gene

Claims (25)

1. A first sequence consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having an identity of 70% or more with the amino acid sequence, and an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having an identity of 70% or more of the amino acid sequence A primary structure in which the second sequence is linked via a peptide linker, or a third sequence consisting of the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 70% or more identity with the amino acid sequence, and the amino acid sequence of SEQ ID NO: 7 Or a fourth sequence consisting of an amino acid sequence having 70% or more identity with the amino acid sequence has a primary structure linked via a peptide linker, and compared to a wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1, Glucose dehydrogenase with low reactivity to D-xylose and / or improved low temperature reactivity of glucose dehydrogenase activity.
2. The glucose dehydrogenase according to claim 1, wherein the identity defining the first sequence, the second sequence, the third sequence and the fourth sequence is 80% or more.
3. The glucose dehydrogenase according to claim 1, wherein the identity defining the first sequence, the second sequence, the third sequence and the fourth sequence is 85% or more.
4. The glucose dehydrogenase according to claim 1, wherein the identity defining the first sequence, the second sequence, the third sequence and the fourth sequence is 90% or more.
5. A primary structure in which the 173 lysine of the amino acid sequence of SEQ ID NO: 4 is substituted with valine, isoleucine, proline or glutamine and the amino acid sequence of SEQ ID NO: 5 linked via a peptide linker, or the amino acid of SEQ ID NO: 6 The glucose dehydrogenase according to claim 1, which has a primary structure in which the amino acid sequence in which the 128th lysine in the sequence is substituted with valine, isoleucine, proline or glutamine and the amino acid sequence of SEQ ID NO: 7 are linked via a peptide linker.
6. A primary structure in which an amino acid sequence in which the 125th serine of the amino acid sequence of SEQ ID NO: 6 is substituted with cysteine and an amino acid sequence in which the 160th serine of the amino acid sequence of SEQ ID NO: 7 is substituted with cysteine are linked via a peptide linker; Or a primary structure in which the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence in which the 144th tyrosine of the amino acid sequence of SEQ ID NO: 7 is replaced with cysteine and the 184th glycine is replaced with cysteine are linked via a peptide linker. The glucose dehydrogenase according to claim 1, comprising:
7. The glucose dehydrogenase according to any one of claims 1 to 6, wherein the number of amino acids constituting the peptide linker is 5 to 30.
8. The glucose dehydrogenase according to claim 7, wherein the peptide linker is ESSSGTSSSSGTSESSS (SEQ ID NO: 23) or GTSSS (SEQ ID NO: 25).
9. (a) the amino acid sequence of SEQ ID NO: 8,
   (b) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 8,
   (c) the amino acid sequence of SEQ ID NO: 9,
   (d) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 9,
   (e) the amino acid sequence of SEQ ID NO: 32, or (f) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 32,
   A glucose dehydrogenase having a low reactivity to D-xylose and / or improved low-temperature stability of glucose dehydrogenase activity as compared to the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1.
10. The glucose dehydrogenase according to claim 9, wherein the identity defining the amino acid sequence (b), the amino acid sequence (d) and the amino acid sequence (f) is 80% or more.
11. The glucose dehydrogenase according to claim 9, wherein the identity defining the amino acid sequence (b), the amino acid sequence (d) and the amino acid sequence (f) is 85% or more.
12. The glucose dehydrogenase according to claim 9, wherein the identity defining the amino acid sequence (b), the amino acid sequence (d), and the amino acid sequence (f) is 90% or more.
13. An amino acid sequence in which the lysine 173 in the amino acid sequence of SEQ ID NO: 8 is substituted with valine, isoleucine, proline or glutamine, or an amino acid sequence in which the lysine No. 128 in the amino acid sequence of SEQ ID NO: 9 is substituted with valine, isoleucine, proline or glutamine The glucose dehydrogenase according to claim 9, comprising:
14. The glucose dehydrogenase according to claim 9, consisting of the amino acid sequence of SEQ ID NO: 36 or 37.
15. The glucose dehydrogenase according to any one of claims 1 to 14, wherein the reactivity to D-xylose is 8% or less when the reactivity to D-glucose is 100%.
16. A glucose dehydrogenase gene encoding the glucose dehydrogenase according to any one of claims 1 to 15.
17. The glucose dehydrogenase gene according to claim 14, comprising any DNA selected from the group consisting of the following (A) to (C):
    (A) DNA encoding the amino acid sequence of SEQ ID NO: 8, 9, 32, 36 or 37;
    (B) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 10 to 13 and 38 to 43;
    (C) DNA encoding a protein having a nucleotide sequence equivalent to any one of SEQ ID NOs: 10 to 13 and 38 to 43 and having glucose dehydrogenase activity.
18. A recombinant DNA comprising the glucose dehydrogenase gene according to claim 16 or 17.
19. A microorganism having the recombinant DNA according to claim 18.
20. A method for preparing glucose dehydrogenase comprising the following steps (1) to (3):
    (1) providing the glucose dehydrogenase gene according to claim 16 or 17;
    (2) expressing the gene; and (3) collecting the expression product.
21. A glucose measuring method comprising measuring glucose in a sample using the glucose dehydrogenase according to any one of claims 1 to 15.
22. A glucose measurement reagent comprising the glucose dehydrogenase according to any one of claims 1 to 15.
23. A glucose measurement kit comprising the glucose measurement reagent according to claim 22.
24. A glucose sensor comprising the glucose dehydrogenase according to any one of claims 1 to 15.
25. An enzyme agent comprising the glucose dehydrogenase according to any one of claims 1 to 15.

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