WO2018062103A1 - Glucose dehydrogenase - Google Patents

Glucose dehydrogenase Download PDF

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WO2018062103A1
WO2018062103A1 PCT/JP2017/034547 JP2017034547W WO2018062103A1 WO 2018062103 A1 WO2018062103 A1 WO 2018062103A1 JP 2017034547 W JP2017034547 W JP 2017034547W WO 2018062103 A1 WO2018062103 A1 WO 2018062103A1
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amino acid
enzyme
glucose
seq
acid sequence
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PCT/JP2017/034547
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French (fr)
Japanese (ja)
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享一 西尾
裕三 小嶋
庄太郎 山口
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天野エンザイム株式会社
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Priority to JP2018542558A priority Critical patent/JP7044709B2/en
Publication of WO2018062103A1 publication Critical patent/WO2018062103A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • the present invention relates to glucose dehydrogenase (glucose dehydrogenase). Specifically, the present invention relates to a flavin adenine dinucleotide (FAD) -dependent glucose dehydrogenase (E.C.1.1.99.10) having improved low-temperature reactivity, a gene thereof, and the like.
  • FAD flavin adenine dinucleotide
  • E.C.1.1.99.10 flavin adenine dinucleotide
  • 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).
  • FAD-GDH has a problem of reactivity with xylose, it has excellent substrate specificity and is regarded as a promising enzyme for glucose sensors. In putting FAD-GDH into practical use, the reactivity with xylose becomes a problem as described above. 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. In view of such circumstances, it is an object of the present invention to provide FAD-GDH that is highly practical, particularly for glucose sensors, and uses thereof. In addition, although FAD-GDH having low reactivity to xylose has been reported (Patent Document 6), characteristics such as pH stability which are particularly important when applied to a glucose sensor have not been clarified, and its practical use. Target value is unknown.
  • the present inventors conducted large-scale screening for a wide range of microorganisms.
  • the inventors succeeded in obtaining a novel FAD-GDH having a characteristic suitable for glucose sensor use that exhibits high activity in a wide pH range in addition to the characteristic of low reactivity to xylose (Japanese Patent Application No. 2015).
  • -Patent application as -218852.
  • the modification of the FAD-GDH was attempted with the aim of further reducing the xylose reactivity.
  • a plurality of mutant enzymes having improved activity at low temperatures ie, low temperature reactivity
  • mutations amino acid substitutions
  • the xylose reactivity was also decreased. That is, the above mutant enzyme not only improved the low-temperature reactivity, but also improved the substrate specificity to glucose, and was extremely practical.
  • Glucose dehydrogenase having the following amino acid sequence (a) or (b): (a) an amino acid sequence in which the lysine at position 354 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine, isoleucine, proline or glutamine; (b) an amino acid sequence having 80% or more identity with the amino acid sequence of (a), wherein the polypeptide comprising the amino acid sequence is a glucose dehydrogenase as compared to the polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • An amino acid sequence with improved low temperature reactivity of activity is provided.
  • Glucose dehydrogenase gene comprising any DNA selected from the group consisting of the following (A) to (C): (A) DNA encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24; (B) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 25 to 28; (C) DNA encoding a protein having a base sequence equivalent to any one of SEQ ID NOs: 25 to 28 and having glucose dehydrogenase activity. [6] A recombinant DNA comprising the glucose dehydrogenase gene according to [5]. [7] A microorganism having the recombinant DNA according to [6].
  • a method for preparing glucose dehydrogenase comprising the following steps (1) to (3): (1) A step of preparing the glucose dehydrogenase gene according to [5]; (2) expressing the gene, and (3) collecting the expression product.
  • a glucose measurement method comprising measuring glucose in a sample using the glucose dehydrogenase according to any one of [1] to [4].
  • a glucose measurement reagent comprising the glucose dehydrogenase according to any one of [1] to [4].
  • a glucose measurement kit comprising the glucose measurement reagent according to [10].
  • a glucose sensor comprising the glucose dehydrogenase according to any one of [1] to [4].
  • An enzyme agent comprising the glucose dehydrogenase according to any one of [1] to [4].
  • M represents a molecular weight marker (200, 116, 97.2, 66.4 KDa from the top), and the lane number is the fraction number when separated with Superdex®200.
  • isolated is used herein interchangeably with “purified”.
  • 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.
  • mutant enzyme is an enzyme obtained by mutating or modifying an existing enzyme. “Mutant enzyme”, “mutant enzyme” and “modified enzyme” are used interchangeably.
  • the existing enzyme to be mutated is typically a wild-type enzyme.
  • the 1st aspect of this invention is related with glucose dehydrogenase mutant enzyme (henceforth this enzyme).
  • One embodiment of this enzyme has an amino acid sequence in which lysine (K) at position 354 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V), isoleucine (I), proline (P), or glutamine (Q).
  • the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of glucose dehydrogenase produced by Aspergillus iizukae No.5453 strain. This strain is the same as the 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.
  • NBRC National Institute of Technology and Evaluation
  • the amino acid residue to be substituted that is, lysine (K) at position 354, was found as an amino acid residue important for temperature characteristics by the present inventors.
  • the low temperature reactivity is improved as compared with the wild-type enzyme by substituting the amino acid residue with valine (V), isoleucine (I), proline (P) or glutamine (Q).
  • the low temperature in this specification is “15 ° C. to 25 ° C.”.
  • Improvement of low temperature reactivity can be evaluated based on, for example, activity at 20 ° C. relative to activity at 37 ° C. (relative activity at 20 ° C. based on 37 ° C. activity). Since this enzyme has improved low-temperature reactivity compared to the wild-type enzyme (ie, the amino acid at position 354 is not substituted), the relative activity of the enzyme is higher than that of the wild-type enzyme. Get higher. The relative activity of this enzyme is, for example, 1.2 to 2.5 times that of the wild-type enzyme.
  • the amino acid residue (lysine at position 354) was important not only for low-temperature reactivity but also for xylose reactivity.
  • This enzyme in which the amino acid residue is substituted with valine, isoleucine, proline or glutamine has a further feature that xylose reactivity is low.
  • the reactivity to D-xylose when the reactivity to D-glucose is 100% is 10% or less.
  • the reactivity is 8% or less. More preferably, the reactivity is 7% or less.
  • the reactivity of the wild type enzyme is 14% (Examples described later).
  • the present enzyme having excellent substrate specificity as described above is preferable as an enzyme for accurately measuring the amount of glucose in a sample. That is, according to this enzyme, even when D-xylose is present in the sample, the target glucose amount can be measured more accurately. Therefore, it can be said that this enzyme is suitable for applications in which the presence of D-xylose is expected or concerned in the sample (typically measurement of glucose level in blood). It can be said that it is applicable to the above, that is, the 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.
  • the enzyme include an enzyme having the amino acid sequence of SEQ ID NO: 21 (K354V mutant enzyme), an enzyme having the amino acid sequence of SEQ ID NO: 22 (K354I mutant enzyme), and an enzyme having the amino acid sequence of SEQ ID NO: 23 (K354P mutant enzyme). And an enzyme having the amino acid sequence of SEQ ID NO: 24 (K354Q mutant enzyme).
  • 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.
  • the above-mentioned mutant enzyme that is, the enzyme having the amino acid sequence of SEQ ID NO: 21 (K354V mutant enzyme), the enzyme having the amino acid sequence of SEQ ID NO: 22 (K354I mutant enzyme), the amino acid of SEQ ID NO: 23
  • the enzyme having the sequence (K354P mutant enzyme) or the enzyme having the amino acid sequence of SEQ ID NO: 24 (K354Q mutant enzyme) although there is a slight difference in the amino acid sequence, there is a substantial difference in the characteristics.
  • “Slight difference in amino acid sequence” as used herein typically means deletion of one to several amino acids (upper limit is 3, 5, 7, 10) constituting an amino acid sequence, It means that a mutation (change) has occurred in the amino acid sequence by substitution or addition, insertion, or a combination of 1 to several amino acids (the upper limit is 3, 5, 7, 10).
  • the difference in amino acid sequence occurs at a position other than the position where the amino acid substitution (position of 354 lysine) is performed.
  • it is preferable that the histidine (H) at position 525 (H) and the histidine at position 568 (H) presumed to constitute an active center are not targeted for deletion or substitution.
  • the identity (%) between the amino acid sequence of “substantially identical enzyme” and the amino acid sequence of the reference mutant enzyme (sequence of any of SEQ ID NOs: 21 to 24) is, for example, 60% or more, preferably 70 % Or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, Most preferably, it is 99% or more.
  • the difference in amino acid sequence may occur at a plurality of positions. “Slight differences in amino acid sequence” 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.
  • 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 eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • ⁇ -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.
  • the identity (%) of two amino acid sequences or two nucleic acids can be determined by the following procedure, for example.
  • two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence).
  • a molecule amino acid residue or nucleotide
  • Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used.
  • gap weight 12, 10, 8, 6, or 4
  • the enzyme may be part of a larger protein (eg, a fusion protein).
  • a larger protein eg, a fusion protein
  • 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 present enzyme having the above amino acid sequence can be easily prepared by a genetic engineering technique. 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.
  • a suitable host cell for example, E. coli
  • modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur.
  • modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.
  • 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.
  • 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.
  • 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.
  • sequences of genes encoding this enzyme are SEQ ID NO: 25 (sequence encoding K354V mutant enzyme), SEQ ID NO: 26 (sequence encoding K354I mutant enzyme), SEQ ID NO: 27 (sequence encoding K354P mutant enzyme), This is shown in SEQ ID NO: 28 (sequence encoding K354Q mutant enzyme).
  • 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.
  • 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.
  • a base sequence defining an equivalent nucleic acid is also referred to as an “equivalent base sequence”.
  • 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.
  • plural refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it depends 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, 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 85% or more with respect to the base sequence serving as a reference (sequence of any of SEQ ID NOs: 25 to 28). More preferably about 90% or more, even more preferably 95% or more, 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. %, 99.9% nucleic acid having the same base sequence 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.
  • 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 ⁇ g / ml denaturation
  • 5 ⁇ Denhardt solution 1% SDS
  • 10% dextran sulfate 10 ⁇ g / ml denaturation
  • 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 ⁇ g / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).
  • nucleic acid having a base sequence of a gene encoding the present enzyme or a part of a base sequence complementary thereto.
  • 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.
  • a nucleic acid fragment can be labeled.
  • fluorescent substances, enzymes, and radioisotopes 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.
  • 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.
  • Examples of vectors using insect cells as hosts include pAc and pVL, and examples of vectors using mammalian cells as hosts include pCDM8 and pMT2PC.
  • 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).
  • 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.
  • E. coli include E. coli BL21 (DE3) pLysS when T7 promoter is used, and E. coli JM109 otherwise.
  • 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.
  • calcium chloride method Frnal 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.
  • microorganism of the present invention can be used for producing this enzyme. it can.
  • a further aspect of the present invention relates to a method for preparing the enzyme.
  • a mutant enzyme 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 any one of the amino acid sequences of SEQ ID NOs: 21 to 24 is prepared.
  • the “nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24” is a nucleic acid from which a polypeptide having the amino acid sequence is obtained when expressed, and a base corresponding to the amino acid sequence.
  • nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS: 21 to 24 refers to sequence information disclosed in this specification or the attached sequence listing, and uses standard genetic engineering techniques, molecular biological techniques, It can be prepared in an isolated state by using a biochemical method or the like.
  • amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 23, and the amino acid sequence of SEQ ID NO: 24 all mutated the amino acid sequence of GDH derived from Aspergillus iizukae No. 5453. It is a thing. Therefore, a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24 can be obtained by adding a necessary mutation to the gene encoding GDH derived from Aspergillus iizukae No.5453 (base sequence of SEQ ID NO: 2). (Gene) can be obtained.
  • position-specific base sequence substitution Many methods for position-specific base sequence substitution are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method is selected from them. Can be used.
  • a position-specific mutation introducing method a position-specific amino acid saturation mutation method can be employed.
  • the position-specific amino acid saturation mutation method is a “Semi-rational, semi-random” technique in which amino acid saturation mutation is introduced by estimating the position where the desired function is involved based on the three-dimensional structure of the protein (J. Mol. Biol. 331, 585-592 (2003)).
  • a site-specific amino acid saturation mutation can be introduced by using a kit such as Quick change (Stratagene) and overlap extention PCR (Nucleic Acid Res. 16,7351-7367 (1988)).
  • a DNA polymerase used for PCR Taq polymerase or the like can be used.
  • a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo), Pfu turbo (Stratagene).
  • 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.
  • 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.
  • the pH of the medium is about 6.0 to 9.0 (preferably around pH 7.0).
  • the expression product (mutant enzyme) is recovered (step (3)).
  • 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.
  • 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.
  • 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.
  • 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) Purify and obtain the purified product of mutant enzyme Door can be.
  • DEAE Sepharose CL-6B GE Healthcare Bioscience
  • Octyl Sepharose CL-6B GE Healthcare Bioscience
  • CM Sepharose CL-6B GE Healthcare Bioscience
  • the microbial cells are collected by filtering, centrifuging, etc., and then the microbial cells are subjected to mechanical methods such as pressure treatment, ultrasonic treatment, or enzymatic methods such as lysozyme. After destruction by the method, a purified product of the mutant enzyme 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.
  • the enzyme can be purified to have a specific activity of 10 to 1000 (U / mg), preferably 50 to 500 (U / mg).
  • the final form may be liquid or solid (including powder).
  • the purified enzyme obtained as described above by pulverizing it by, for example, freeze drying, vacuum drying or spray drying.
  • the purified enzyme may be dissolved in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer or GOOD buffer in advance.
  • a phosphate buffer or a triethanolamine buffer can be used.
  • PIPES, MES, or MOPS is mentioned as a GOOD buffer 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.
  • 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.
  • ribosomes necessary for protein synthesis
  • various factors such as initiation factors
  • various enzymes such as tRNA.
  • 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.
  • a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis.
  • 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.
  • RNA is used as a template to synthesize proteins.
  • 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.
  • conditions to which factors necessary for each reaction are added are set so that the transcription reaction and the translation reaction proceed continuously.
  • a further aspect of the invention relates to the use of the enzyme.
  • a glucose measurement method using the present enzyme is provided.
  • 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).
  • fermented foods for example, vinegar
  • 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.
  • 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.
  • 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.
  • this invention also provides the glucose sensor containing this enzyme.
  • 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).
  • 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
  • the enzyme agent of the present invention may contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline and the like.
  • excipient starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used.
  • Phosphate, citrate, acetate, etc. can be used as the buffer.
  • the stabilizer propylene glycol, ascorbic acid or the like can be used.
  • preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
  • preservatives ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • the reactivity to maltose and xylose is relative value when the reactivity to glucose is 100% (measured value when maltose (or xylose) is used as substrate / measured value when glucose is used as substrate x 100)
  • Glucose oxidase (GO) was expressed as a relative value to glucose dehydrogenase (measured value of glucose oxidase (GO) activity / measured value of glucose dehydrogenase activity ⁇ 100).
  • glucose oxidase derived from Aspergillus niger
  • PQQ-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus
  • FAD-dependent glucose dehydrogenase derived from Aspergillus oryzae
  • Aspergillus iizukae No. 5543 has a significantly lower reactivity to maltose and xylose than the existing PQQ-dependent glucose dehydrogenase and FAD-dependent glucose dehydrogenase.
  • Aspergillus iizukae No.5453 is the same as Aspergillus iizukae Sugiyama NBRC 8869, which is stored in the National Institute of Technology and Evaluation (NBRC) (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818). Is a stock.
  • the crude enzyme solution was purified (salting out, hydrophobic bond chromatography, ion exchange chromatography, gel filtration chromatography) to obtain a purified enzyme.
  • the purified enzyme was analyzed by gel filtration (using GE Healthcare Superdex®200) and SDS-PAGE. The results of SDS-PAGE are shown in FIG. The fraction with the highest glucose dehydrogenase activity (No. 34) was used in subsequent experiments.
  • HPLC separation conditions High-performance liquid chromatograph (HPLC): LC-20A system (Shimadzu Corporation) Column: Cadenza CD-C18 (2.0mmI.D. ⁇ 150mm) (Intact Corporation) Column temperature: 50 ° C Detection wavelength: 214mm Injection volume: 70 ⁇ L Mobile phase flow rate: 0.2mL / min Mobile phase A: Water / trifluoroacetic acid (1000/1) Mobile phase B: acetonitrile / water / trifluoroacetic acid (800/200/1)
  • FIG. 3 shows the amino acid sequence identified by analysis of the peaks obtained by HPLC separation (13 peaks were identified and numbered in order from the shorter retention time).
  • PCR was performed using the designed primer and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) using the genomic DNA of Aspergillus iizukae No.5453 as a template to obtain an amplified DNA fragment.
  • PCR conditions were as follows. (Reaction solution) PrimeSTAR Max Premix (2 ⁇ ) 25 ⁇ L GDH5453-F 15 pmol GDH5453-5-1-R 15 pmol Genomic DNA (1/1000 dilution) 1 ⁇ L Adjust to 50 ⁇ L with sterile distilled water (cycle conditions) 35 cycles at 98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 2 minutes
  • the obtained DNA fragment was subcloned using Mighty Cloning Reagent Set (Blunt End) (Takara Bio Inc.), and the base sequence of the DNA fragment was confirmed according to a conventional method.
  • Primer FS51R07F AACCGTCTGTCTGAAGACCC (SEQ ID NO: 7)
  • Primer FS51R07R TACTTCCTTTTGCTCG (SEQ ID NO: 8)
  • PCR was performed using the designed primers and PCR® DIG® Probe® Synthesis® Kit (Roche Diagnostics) to obtain a DNA probe labeled with digoxigenin. Southern hybridization was performed using this probe. Chromosomal DNA was completely digested with restriction enzymes BamHI, KpnI, PstI, SacI, SphI, and XbaI, and those digested with a combination of these restriction enzymes and restriction enzyme SalI were separated by 0.8% agarose electrophoresis. Subsequently, the membrane was transferred to a zeta probe membrane (Bio-Rad Inc.) to obtain a membrane for Southern hybridization.
  • zeta probe membrane Bio-Rad Inc.
  • Southern hybridization was performed according to a conventional method using DIG Easy Hyb. (Roche Diagnostics). Detection was performed using a digoxigenin antibody labeled with alkaline phosphatase, and a restriction enzyme map (FIG. 4) around the target gene was prepared from the detection results.
  • the restriction enzyme map around the target DNA revealed that the target gene was contained in a fragment of about 5.7 Kbp that was completely digested with the restriction enzyme SphI, so 0.8% of the chromosomal DNA completely digested with the restriction enzyme SphI
  • a fragment of about 5.7 Kbp was recovered from agarose and inserted into the restriction enzyme SphI site of pUC18 (Takara Bio Inc.) plasmid.
  • 1,000 strains of E. coli JM109 (Takara Bio Inc.) transformed with the recombinant plasmid were prepared.
  • 5453K354_FW cctgtctcctaccccaac (SEQ ID NO: 16)
  • 5453K354V_R1 ggggtaggagacaggaactccaccggagagagt (SEQ ID NO: 17)
  • 5453K354I_R2 ggggtaggagacagggattccaccggagagagt (SEQ ID NO: 18)
  • 5453K354P_R3 ggggtaggagacaggaggtccaccggagagagt (SEQ ID NO: 19)
  • 5453K354Q_R4 ggggtaggagacaggttgtccaccggagagagt (SEQ ID NO: 20)
  • An Aspergillus iizukae No.5453 GDH gene sequence comprising an expression cassette connected between the Takaamylase-modified CS3 promoter and an Aspergillus oryzae-derived FAD-dependent glucose dehydrogenase terminator gene, and an Aspergillus oryzae-derived orotidine 5'-phosphate de
  • PCR was performed using the designed primer and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) using the principle of inverse PCR. And an amplified DNA fragment was obtained.
  • PCR conditions were as follows.
  • the obtained DNA fragment was phosphorylated and then ligated, followed by transformation into E. coli to obtain an expression plasmid into which the mutation was introduced.
  • a pyrG gene-deficient strain of Aspergillus oryzae RIB40 was transformed, and a transformant was obtained using uridine requirement.
  • liquid culture was carried out using soluble starch as a C source under takaamylase induction conditions to obtain a culture solution containing a mutant enzyme.
  • Various purifications were performed from the obtained culture broth to obtain a partially purified mutant enzyme.
  • the optimum temperature was evaluated by the following method.
  • the present GDH catalyzes a reaction in which a hydroxyl group of glucose is oxidized to produce glucono- ⁇ -lactone in the presence of an electron acceptor. GDH activity was detected by the following reaction system.
  • PMS represents Phenazine methosulfate
  • DCIP represents 2,6-Dichlorophenol-indophenol solution.
  • reduced PMS is generated with the oxidation of glucose
  • the reduced DCIP generated by the reduction of DCIP by the reduced PMS in the reaction (2) is measured at a wavelength of 600 nm.
  • the enzyme activity (unit) is calculated by the following formula.
  • Vt is the total liquid volume
  • Vs is the sample volume
  • 16.3 is the extinction coefficient (cm 2 / ⁇ mol) per 1 mol of reduced DCIP
  • 1.0 is the optical path length (cm)
  • df is the dilution factor.
  • Mutant enzyme (K354V mutant enzyme, K354I mutant enzyme, K354P mutant enzyme, K354Q mutant enzyme) in which lysine at position 354 is replaced with valine, isoleucine, proline, or glutamine (site-specific mutation is introduced) is compared with the wild-type enzyme Thus, the activity at 20 ° C. relative to 37 ° C. (relative activity) was improved.
  • the amino acid sequence of each mutant enzyme and the gene sequence encoding it are as follows.
  • K354V mutant enzyme SEQ ID NO: 21 (amino acid sequence); SEQ ID NO: 25 (gene sequence) K354I mutant enzyme: SEQ ID NO: 22 (amino acid sequence); SEQ ID NO: 26 (gene sequence) K354P mutant enzyme: SEQ ID NO: 23 (amino acid sequence); SEQ ID NO: 27 (gene sequence) K354Q mutant enzyme: SEQ ID NO: 24 (amino acid sequence); SEQ ID NO: 28 (gene sequence)
  • GD41F ccacagaaggcatttatgttgggcaaactcacgttctt (SEQ ID NO: 29)
  • GD42R gctttatctaccaaactacacagcagcagcatcgg (SEQ ID NO: 30)
  • PCR was carried out using the designed primer and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) using the mutant enzyme expression vector constructed in the above experiment as a template to obtain an amplified DNA fragment.
  • PCR conditions were as follows. (Reaction solution) PrimeSTAR Max Premix (2 ⁇ ) 25 ⁇ L GD41F 15 pmol GD42R 15 pmol Genomic DNA (1/1000 dilution) 1 ⁇ L Adjust to 50 ⁇ L with sterile distilled water (cycle conditions) 35 cycles at 98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 2 minutes
  • the amplified product after PCR was inserted into Saccharomyces cerevisiae expression system pYES2 plasmid to construct a plasmid after mutagenesis.
  • the constructed plasmid after mutation introduction was transformed into Escherichia coli DH5 ⁇ , followed by plasmid extraction to prepare a mutation library.
  • the obtained library was transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen).
  • the obtained transformant was subjected to liquid culture, and GDH activity and substrate specificity when xylose was used as a substrate were examined.
  • culture experiment operation referred to the manual of pYES2.
  • Substrate specificity evaluation method The present GDH catalyzes a reaction in which the hydroxyl group of glucose is oxidized to produce glucono- ⁇ -lactone in the presence of an electron acceptor. GDH activity was detected by the following reaction system.
  • 1-Methoxy PMS represents 1-Methoxy phenazine methosulfate
  • NTB represents Nitrotetrazorium blue.
  • 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 reactivity to xylose was expressed as a relative value when the reactivity to glucose was 100% (measured value when xylose was used as a substrate / measured value when glucose was used as a substrate x 100). Moreover, the relative value when the reactivity value of the wild-type enzyme to xylose was defined as 100% was also determined.
  • the mutant enzyme in which lysine at position 354 was substituted with valine, isoleucine, proline, or glutamine showed a marked decrease in reactivity to xylose, and showed a substrate specificity that was markedly superior to that of the wild-type enzyme.
  • the glucose dehydrogenase of the present invention is excellent in low temperature stability. Therefore, it is suitable for applications that are expected to be used in a low temperature environment (typically a glucose sensor for a blood glucose meter).
  • the glucose dehydrogenase of the present invention has improved substrate specificity and is particularly suitable for use in a glucose sensor.
  • Sequence number 4 Description of artificial sequence: Primer GDH5453-F Sequence number 5: Description of artificial sequence: Primer GDH5453-5-1-R SEQ ID NO: 6: description of artificial sequence: PCR product SEQ ID NO: 7: description of artificial sequence: primer FS51R07F SEQ ID NO: 8: Description of artificial sequence: Primer FS51R07R SEQ ID NO: 16: Description of artificial sequence: Primer 5453K354_FW SEQ ID NO: 17: Description of artificial sequence: Primer 5453K354V_R1 SEQ ID NO: 18: Description of artificial sequence: Primer 5453K354I_R2 SEQ ID NO: 19: Description of artificial sequence: Primer 5453K354P_R3 SEQ ID NO: 20: Description of artificial sequence: Primer 5453K354Q_R4 SEQ ID NO: 21: description of artificial sequence: K354V mutant enzyme SEQ ID NO: 22: description of artificial sequence: K354I mutant enzyme SEQ ID NO: 23: description of artificial sequence: K35

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Abstract

The present invention addresses the problem of providing novel FAD-GDH having high practicality. Provided is glucose dehydrogenase having the amino acid sequence of (a) or (b) as follows. (a) The amino acid sequence of SEQ ID NO. 1 in which the lysine at position 354 has been substituted with a valine, isoleucine, proline, or glutamine, or (b) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), wherein a polypeptide having said amino acid sequence has improved low-temperature reactivity in terms of glucose dehydrogenase activity compared to a polypeptide having the amino acid sequence of SEQ ID NO. 1.

Description

グルコースデヒドロゲナーゼGlucose dehydrogenase
　本発明はグルコースデヒドロゲナーゼ（グルコース脱水素酵素）に関する。詳しくは、低温反応性が改善されたフラビンアデニンジヌクレオチド（FAD）依存性グルコースデヒドロゲナーゼ（E.C.1.1.99.10）及びその遺伝子等に関する。本出願は、２０１６年９月２８日に出願された日本国特許出願第２０１６－１９０３９８号に基づく優先権を主張するものであり、当該特許出願の全内容は参照により援用される。 The present invention relates to glucose dehydrogenase (glucose dehydrogenase). Specifically, the present invention relates to a flavin adenine dinucleotide (FAD) -dependent glucose dehydrogenase (E.C.1.1.99.10) having improved low-temperature reactivity, a gene thereof, and the like. This application claims priority based on Japanese Patent Application No. 2016-190398 filed on Sep. 28, 2016, the entire contents of which are incorporated by reference.
　糖尿病患者は年々増加しており、糖尿病患者、特にインスリン依存性の患者は血糖値を日常的に監視し血糖をコントロールする必要がある。近年、酵素を用いてリアルタイムで簡便にかつ正確に測定できる自己血糖測定器で糖尿病患者の血糖値をチェック出来るようになった。グルコースセンサ（例えば、自己血糖測定器に使用されるセンサ）用として、グルコースオキシダーゼ（E.C.1.1.3.4）、PQQ依存性グルコースデヒドロゲナーゼ（E.C.1.1.5.2）（例えば特許文献１～３を参照）が開発されたが、酸素反応性、マルトース、ガラクトースへの反応性が問題となった。この問題を解決すべく、FAD依存性グルコースデヒドロゲナーゼ（以下、「FAD-GDH」と略称する）が開発された（例えば特許文献４、５、非特許文献１～４を参照）。 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, reactivity to maltose and galactose 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).
　一般に、糖尿病判定検査時には、経口グルコース負荷試験だけでなく、経口キシロース負荷試験、経静脈キシロース負荷試験が実施される。FAD-GDHは概してキシロースへ反応することが知られており、FAD-GDHを用いた場合、上記負荷試験時に血糖値へ影響することが問題となる。 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.
特開２０００－３５０５８８号公報JP 2000-350588 A 特開２００１－１９７８８８号公報JP 2001-197888 A 特開２００１－３４６５８７号公報Japanese Patent Laid-Open No. 2001-346587 国際公開第２００４／０５８９５８号パンフレットInternational Publication No. 2004/058958 Pamphlet 国際公開第２００７／１３９０１３号パンフレットInternational Publication No. 2007/139013 Pamphlet 国際公開第２０１５／０６０１５０号パンフレットInternational Publication No. 2015/060150 Pamphlet
　FAD-GDHはキシロースに対する反応性の問題はあるものの、基質特異性に優れ、グルコースセンサ用の酵素として有望視されている。FAD-GDHを実用化するにあたっては、上記の通り、キシロースに対する反応性が問題となる。一方、FAD-GDHを利用した血糖測定器の測定精度は環境温度に影響を受けることが知られている。また、特に重要な問題として、センサに使用するFAD-GDHの反応性が低下する低温環境で測定した場合、血糖値が低い領域において実際の血糖値よりも測定値が低くなることが指摘されている。本発明は、このような状況に鑑み、特にグルコースセンサ用として実用性の高いFAD-GDH及びその用途等を提供することを課題とする。尚、キシロースに対する反応性が低いFAD-GDHも報告されているが（特許文献６）、グルコースセンサに応用した場合に特に重要となるpH安定性等の特性は明らかにされておらず、その実用的価値は不明である。 Although FAD-GDH has a problem of reactivity with xylose, it has excellent substrate specificity and is regarded as a promising enzyme for glucose sensors. In putting FAD-GDH into practical use, the reactivity with xylose becomes a problem as described above. 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. In view of such circumstances, it is an object of the present invention to provide FAD-GDH that is highly practical, particularly for glucose sensors, and uses thereof. In addition, although FAD-GDH having low reactivity to xylose has been reported (Patent Document 6), characteristics such as pH stability which are particularly important when applied to a glucose sensor have not been clarified, and its practical use. Target value is unknown.
　上記課題を解決すべく本発明者らは、広範な微生物を対象として大規模なスクリーニングを実施した。その結果、キシロースに対する反応性が低いという特性に加え、広範囲のpH域で高い活性を示すという、グルコースセンサ用途に適した特性を備えた新規FAD-GDHを取得することに成功した（特願２０１５－２１８８５２号として特許出願した）。次に、キシロース反応性の更なる低下を目指し、当該FAD-GDHの改変を試みた。その結果、驚くべきことに、低温での活性（即ち、低温反応性）が改善した複数の変異酵素が得られ、低温反応性の改善に有効な変異（アミノ酸置換）が特定された。 In order to solve the above problems, the present inventors conducted large-scale screening for a wide range of microorganisms. As a result, the inventors succeeded in obtaining a novel FAD-GDH having a characteristic suitable for glucose sensor use that exhibits high activity in a wide pH range in addition to the characteristic of low reactivity to xylose (Japanese Patent Application No. 2015). -Patent application as -218852). Next, the modification of the FAD-GDH was attempted with the aim of further reducing the xylose reactivity. As a result, surprisingly, a plurality of mutant enzymes having improved activity at low temperatures (ie, low temperature reactivity) were obtained, and mutations (amino acid substitutions) effective for improving low temperature reactivity were identified.
　一方、得られた変異酵素の特性を調べたところ、キシロース反応性も低下していた。即ち、上記の変異酵素は低温反応性が改善しているだけでなく、グルコースに対する基質特異性も改善しており、実用性が極めて高いものであった。 On the other hand, when the characteristics of the obtained mutant enzyme were examined, the xylose reactivity was also decreased. That is, the above mutant enzyme not only improved the low-temperature reactivity, but also improved the substrate specificity to glucose, and was extremely practical.
　以下の発明は、以上の成果及び考察に基づく。
　［１］以下の(a)又は(b)のアミノ酸配列を有する、グルコースデヒドロゲナーゼ：
　(a)配列番号１のアミノ酸配列の354位リジンがバリン、イソロイシン、プロリン又はグルタミンに置換されたアミノ酸配列；
　(b)(a)のアミノ酸配列との同一性が80%以上のアミノ酸配列であって、該アミノ酸配列からなるポリペプチドは、配列番号１のアミノ酸配列からなるポリペプチドに比較して、グルコースデヒドロゲナーゼ活性の低温反応性が向上している、アミノ酸配列。
　［２］前記同一性が85%以上である、［１］に記載のグルコースデヒドロゲナーゼ。
　［３］前記同一性が90%以上である、［１］に記載のグルコースデヒドロゲナーゼ。
　［４］配列番号２１～２４のいずれかのアミノ酸配列からなる、［１］に記載のグルコースデヒドロゲナーゼ。
　［５］以下の(A)～(C)からなる群より選択されるいずれかのDNAからなるグルコースデヒドロゲナーゼ遺伝子：
　(A)配列番号２１～２４のいずれかのアミノ酸配列をコードするDNA；
　(B)配列番号２５～２８のいずれかの塩基配列からなるDNA；
　(C)配列番号２５～２８のいずれかの塩基配列と等価な塩基配列を有し、且つグルコースデヒドロゲナーゼ活性を有するタンパク質をコードするDNA。
　［６］［５］に記載のグルコースデヒドロゲナーゼ遺伝子を含む組換えDNA。
　［７］［６］に記載の組換えDNAを保有する微生物。
　［８］以下のステップ(1)～(3)を含む、グルコースデヒドロゲナーゼの調製法：
　(1)［５］に記載のグルコースデヒドロゲナーゼ遺伝子を用意するステップ；
　(2)前記遺伝子を発現させるステップ、及び
　(3)発現産物を回収するステップ。
　［９］［１］～［４］のいずれか一項に記載のグルコースデヒドロゲナーゼを用いて試料中のグルコースを測定することを特徴とする、グルコース測定法。
　［１０］［１］～［４］のいずれか一項に記載のグルコースデヒドロゲナーゼを含む、グルコース測定用試薬。
　［１１］［１０］に記載のグルコース測定用試薬を含む、グルコース測定用キット。
　［１２］［１］～［４］のいずれか一項に記載のグルコースデヒドロゲナーゼを含む、グルコースセンサ。
　［１３］［１］～［４］のいずれか一項に記載のグルコースデヒドロゲナーゼを含有する酵素剤。 The following invention is based on the above results and considerations.
[1] Glucose dehydrogenase having the following amino acid sequence (a) or (b):
(a) an amino acid sequence in which the lysine at position 354 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine, isoleucine, proline or glutamine;
(b) an amino acid sequence having 80% or more identity with the amino acid sequence of (a), wherein the polypeptide comprising the amino acid sequence is a glucose dehydrogenase as compared to the polypeptide comprising the amino acid sequence of SEQ ID NO: 1. An amino acid sequence with improved low temperature reactivity of activity.
[2] The glucose dehydrogenase according to [1], wherein the identity is 85% or more.
[3] The glucose dehydrogenase according to [1], wherein the identity is 90% or more.
[4] The glucose dehydrogenase according to [1], comprising the amino acid sequence of any one of SEQ ID NOs: 21 to 24.
[5] Glucose dehydrogenase gene comprising any DNA selected from the group consisting of the following (A) to (C):
(A) DNA encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24;
(B) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 25 to 28;
(C) DNA encoding a protein having a base sequence equivalent to any one of SEQ ID NOs: 25 to 28 and having glucose dehydrogenase activity.
[6] A recombinant DNA comprising the glucose dehydrogenase gene according to [5].
[7] A microorganism having the recombinant DNA according to [6].
[8] A method for preparing glucose dehydrogenase, comprising the following steps (1) to (3):
(1) A step of preparing the glucose dehydrogenase gene according to [5];
(2) expressing the gene, and (3) collecting the expression product.
[9] A glucose measurement method comprising measuring glucose in a sample using the glucose dehydrogenase according to any one of [1] to [4].
[10] A glucose measurement reagent comprising the glucose dehydrogenase according to any one of [1] to [4].
[11] A glucose measurement kit comprising the glucose measurement reagent according to [10].
[12] A glucose sensor comprising the glucose dehydrogenase according to any one of [1] to [4].
[13] An enzyme agent comprising the glucose dehydrogenase according to any one of [1] to [4].
Aspergillus iizukae No.5453株の培養液から精製した酵素（精製酵素）SDS-PAGEによる分析の結果。Mは分子量マーカー（上から200、116、97.2、66.4KDa）を表し、レーン番号はSuperdex 200で分離した際のフラクション番号である。Results of analysis by SDS-PAGE of enzyme (purified enzyme) purified from the culture solution of Aspergillus iizukae No.5453 strain. M represents a molecular weight marker (200, 116, 97.2, 66.4 KDa from the top), and the lane number is the fraction number when separated with Superdex®200. N末端アミノ酸配列を問い合わせ配列としたBLAST解析の結果。Results of BLAST analysis using the N-terminal amino acid sequence as a query sequence. 内部アミノ酸配列の解析結果。HPLC分離で得られた各ピークのアミノ酸配列を示す。Analysis results of internal amino acid sequence. The amino acid sequence of each peak obtained by HPLC separation is shown. 目的遺伝子（グルコースデヒドロゲナーゼ遺伝子）周辺の制限酵素マップ。Restriction enzyme map around the target gene (glucose dehydrogenase gene). 変異酵素の温度反応性。Temperature reactivity of the mutant enzyme.
１．用語
　本明細書において用語「単離された」は「精製された」と交換可能に使用される。用語「単離された」は、人為的操作が介在することなく産生される物の場合、天然の状態、即ち、自然界において存在している状態のものと区別するために使用され、人為的操作が介在して生産される物の場合、単離工程又は精製工程を経ていないものと区別するために使用される。前者の場合、単離するという人為的操作によって、天然の状態とは異なる状態である「単離された状態」となり、単離されたものは天然物自体と明確且つ決定的に相違する。一方、後者の場合、典型的には、単離工程又は精製工程によって不純物が除去され又はその量が低減され、純度が高まる。 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.
　単離された酵素の純度は特に限定されない。但し、純度の高いことが要求される用途への適用が予定されるのであれば、単離された酵素の純度は高いことが好ましい。 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.
　用語「変異酵素」とは、既存の酵素を変異ないし改変して得られる酵素である。「変異酵素」、「変異型酵素」及び「改変型酵素」は置換可能に用いられる。変異対象となる既存の酵素は、典型的には野生型酵素である。 The term “mutant enzyme” is an enzyme obtained by mutating or modifying an existing enzyme. “Mutant enzyme”, “mutant enzyme” and “modified enzyme” are used interchangeably. The existing enzyme to be mutated is typically a wild-type enzyme.
２．変異酵素
　本発明の第１の局面はグルコースデヒドロゲナーゼ変異酵素（以下、本酵素とも呼ぶ）に関する。本酵素の一態様は、配列番号１のアミノ酸配列の354位リジン（K）がバリン（V）、イソロイシン（I）、プロリン（P）又はグルタミン（Q）に置換されたアミノ酸配列を有する。 2. Mutant enzyme The 1st aspect of this invention is related with glucose dehydrogenase mutant enzyme (henceforth this enzyme). One embodiment of this enzyme has an amino acid sequence in which lysine (K) at position 354 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V), isoleucine (I), proline (P), or glutamine (Q).
　配列番号１のアミノ酸配列はアスペルギルス・イイズカエ（Aspergillus iizukae）No.5453株の産生するグルコースデヒドロゲナーゼのアミノ酸配列である。当該菌株はNBRC 8869株と同一株である。NBRC 8869株は独立行政法人製品評価技術基盤機構（NBRC）（〒292-0818　千葉県木更津市かずさ鎌足２－５－８）に保存されており、所定の手続きを経ることによってその分譲を受けることができる。 The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of glucose dehydrogenase produced by Aspergillus iizukae No.5453 strain. This strain is the same as the 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.
　本発明において置換対象となるアミノ酸残基、即ち354位のリジン（K）は、本発明者らの検討によって温度特性に重要なアミノ酸残基として見出されたものである。本酵素では当該アミノ酸残基がバリン（V）、イソロイシン（I）、プロリン（P）又はグルタミン（Q）に置換されることにより、野生型酵素に比較して低温反応性が改善されている。尚、本発明の特性の理解及び野生型酵素との比較を容易にするため、本明細書における低温は「15℃～25℃」とする。 In the present invention, the amino acid residue to be substituted, that is, lysine (K) at position 354, was found as an amino acid residue important for temperature characteristics by the present inventors. In this enzyme, the low temperature reactivity is improved as compared with the wild-type enzyme by substituting the amino acid residue with valine (V), isoleucine (I), proline (P) or glutamine (Q). 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.”.
　低温反応性の改善は、例えば、37℃での活性に対する20℃での活性（37℃の活性を基準とした20℃での相対活性）に基づき評価できる。本酵素は野生型酵素（即ち354位のアミノ酸置換が行われていないもの）に比較して低温反応性が改善されていることから、当該相対活性は、本酵素の方が野生型酵素よりも高くなる。本酵素の当該相対活性は、例えば、野生型酵素の1.2倍～2.5倍である。 Improvement of low temperature reactivity can be evaluated based on, for example, activity at 20 ° C. relative to activity at 37 ° C. (relative activity at 20 ° C. based on 37 ° C. activity). Since this enzyme has improved low-temperature reactivity compared to the wild-type enzyme (ie, the amino acid at position 354 is not substituted), the relative activity of the enzyme is higher than that of the wild-type enzyme. Get higher. The relative activity of this enzyme is, for example, 1.2 to 2.5 times that of the wild-type enzyme.
　後述の実施例に示す通り、上記アミノ酸残基（354位のリジン）は、低温反応性のみならず、キシロース反応性の低下にも重要であった。当該アミノ酸残基をバリン、イソロイシン、プロリン又はグルタミンに置換した本酵素は、キシロース反応性が低いという、更なる特徴を備える。具体的には、D-グルコースに対する反応性を100％としたときのD-キシロースに対する反応性が10％以下である。好ましくは当該反応性が8％以下である。更に好ましくは当該反応性が7％以下である。尚、野生型酵素の当該反応性は14%である（後述の実施例）。 As shown in Examples below, the amino acid residue (lysine at position 354) was important not only for low-temperature reactivity but also for xylose reactivity. This enzyme in which the amino acid residue is substituted with valine, isoleucine, proline or glutamine has a further feature that xylose reactivity is low. Specifically, the reactivity to D-xylose when the reactivity to D-glucose is 100% is 10% or less. Preferably, the reactivity is 8% or less. More preferably, the reactivity is 7% or less. The reactivity of the wild type enzyme is 14% (Examples described later).
　以上のような優れた基質特異性を有する本酵素は、試料中のグルコース量を正確に測定するための酵素として好ましい。即ち、本酵素によれば試料中にD-キシロースが存在していた場合であっても目的のグルコース量をより正確に測定することが可能である。従って本酵素は、試料中にD-キシロースの存在が予想又は懸念される用途（典型的には血液中のグルコース量の測定）に適したものであるといえ、しかも当該用途も含め様々な用途に適用可能であること、即ち汎用性が高いともいえる。尚、本酵素の反応性及び基質特異性は、後述の実施例に示す方法で測定・評価することができる。 The present enzyme having excellent substrate specificity as described above is preferable as an enzyme for accurately measuring the amount of glucose in a sample. That is, according to this enzyme, even when D-xylose is present in the sample, the target glucose amount can be measured more accurately. Therefore, it can be said that this enzyme is suitable for applications in which the presence of D-xylose is expected or concerned in the sample (typically measurement of glucose level in blood). It can be said that it is applicable to the above, that is, the 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.
　本酵素の具体例として、配列番号２１のアミノ酸配列を有する酵素（K354V変異酵素）、配列番号２２のアミノ酸配列を有する酵素（K354I変異酵素）、配列番号２３のアミノ酸配列を有する酵素（K354P変異酵素）、及び配列番号２４のアミノ酸配列を有する酵素（K354Q変異酵素）を挙げることができる。 Specific examples of the enzyme include an enzyme having the amino acid sequence of SEQ ID NO: 21 (K354V mutant enzyme), an enzyme having the amino acid sequence of SEQ ID NO: 22 (K354I mutant enzyme), and an enzyme having the amino acid sequence of SEQ ID NO: 23 (K354P mutant enzyme). And an enzyme having the amino acid sequence of SEQ ID NO: 24 (K354Q mutant enzyme).
　ところで、一般に、あるタンパク質のアミノ酸配列の一部を変異させた場合において変異後のタンパク質が変異前のタンパク質と同等の機能を有することがある。即ちアミノ酸配列の変異がタンパク質の機能に対して実質的な影響を与えず、タンパク質の機能が変異前後において維持されることがある。この技術常識を考慮すれば、上記の変異酵素、即ち、配列番号２１のアミノ酸配列を有する酵素（K354V変異酵素）、配列番号２２のアミノ酸配列を有する酵素（K354I変異酵素）、配列番号２３のアミノ酸配列を有する酵素（K354P変異酵素）、又は配列番号２４のアミノ酸配列を有する酵素（K354Q変異酵素）と比較した場合に、アミノ酸配列の僅かな相違が認められるものの、特性に実質的な差が認められないものは、上記変更変異と実質同一の酵素とみなすことができる。ここでの「アミノ酸配列の僅かな相違」とは、典型的には、アミノ酸配列を構成する１～数個（上限は例えば３個、５個、７個、１０個）のアミノ酸の欠失、置換、若しくは１～数個（上限は例えば３個、５個、７個、１０個）のアミノ酸の付加、挿入、又はこれらの組合せによりアミノ酸配列に変異（変化）が生じていることをいう。アミノ酸配列の相違は上記アミノ酸置換（354位リジンの置換）が施された位置以外の位置で生ずることとする。また、活性中心を構成すると推定される525位ヒスチジン（H）及び568位ヒスチジン（H）は欠失又は置換の対象にしないことが好ましい。 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, the above-mentioned mutant enzyme, that is, the enzyme having the amino acid sequence of SEQ ID NO: 21 (K354V mutant enzyme), the enzyme having the amino acid sequence of SEQ ID NO: 22 (K354I mutant enzyme), the amino acid of SEQ ID NO: 23 When compared with the enzyme having the sequence (K354P mutant enzyme) or the enzyme having the amino acid sequence of SEQ ID NO: 24 (K354Q mutant enzyme), although there is a slight difference in the amino acid sequence, there is a substantial difference in the characteristics. Those that are not can be regarded as enzymes that are substantially identical to the modified mutation. “Slight difference in amino acid sequence” as used herein typically means deletion of one to several amino acids (upper limit is 3, 5, 7, 10) constituting an amino acid sequence, It means that a mutation (change) has occurred in the amino acid sequence by substitution or addition, insertion, or a combination of 1 to several amino acids (the upper limit is 3, 5, 7, 10). The difference in amino acid sequence occurs at a position other than the position where the amino acid substitution (position of 354 lysine) is performed. Moreover, it is preferable that the histidine (H) at position 525 (H) and the histidine at position 568 (H) presumed to constitute an active center are not targeted for deletion or substitution.
　「実質同一の酵素」のアミノ酸配列と、基準となる変異酵素のアミノ酸配列（配列番号２１～２４のいずれかの配列）との同一性（％）は、例えば60％以上であり、好ましくは70％以上、より好ましくは80%以上であり、より一層好ましくは85%以上であり、更に好ましくは90%以上であり、更に一層好ましくは95%以上であり、特に好ましくは98%以上であり、最も好ましくは99%以上である。尚、アミノ酸配列の相違は複数の位置で生じていてもよい。「アミノ酸配列の僅かな相違」は、好ましくは保存的アミノ酸置換により生じている。「保存的アミノ酸置換」とは、あるアミノ酸残基を、同様の性質の側鎖を有するアミノ酸残基に置換することをいう。アミノ酸残基はその側鎖によって塩基性側鎖（例えばリジン、アルギニン、ヒスチジン）、酸性側鎖（例えばアスパラギン酸、グルタミン酸）、非荷電極性側鎖（例えばグリシン、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システイン）、非極性側鎖（例えばアラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン）、β分岐側鎖（例えばスレオニン、バリン、イソロイシン）、芳香族側鎖（例えばチロシン、フェニルアラニン、トリプトファン、ヒスチジン）のように、いくつかのファミリーに分類されている。保存的アミノ酸置換は好ましくは、同一のファミリー内のアミノ酸残基間の置換である。 The identity (%) between the amino acid sequence of “substantially identical enzyme” and the amino acid sequence of the reference mutant enzyme (sequence of any of SEQ ID NOs: 21 to 24) is, for example, 60% or more, preferably 70 % Or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, Most preferably, it is 99% or more. The difference in amino acid sequence may occur at a plurality of positions. “Slight differences in amino acid sequence” 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.
　ところで、二つのアミノ酸配列又は二つの核酸（以下、これらを含む用語として「二つの配列」を使用する）の同一性（％）は例えば以下の手順で決定することができる。まず、最適な比較ができるよう二つの配列を並べる（例えば、第一の配列にギャップを導入して第二の配列とのアライメントを最適化してもよい）。第一の配列の特定位置の分子（アミノ酸残基又はヌクレオチド）が、第二の配列における対応する位置の分子と同じであるとき、その位置の分子が同一であるといえる。二つの配列の同一性は、その二つの配列に共通する同一位置の数の関数であり（すなわち、同一性（％）＝同一位置の数／位置の総数 × 100）、好ましくは、アライメントの最適化に要したギャップの数およびサイズも考慮に入れる。 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.
　二つの配列の比較及び同一性の決定は数学的アルゴリズムを用いて実現可能である。配列の比較に利用可能な数学的アルゴリズムの具体例としては、KarlinおよびAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68に記載され、KarlinおよびAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77において改変されたアルゴリズムがあるが、これに限定されることはない。このようなアルゴリズムは、Altschulら (1990) J. Mol. Biol. 215:403-10に記載のNBLASTプログラムおよびXBLASTプログラム（バージョン2.0）に組み込まれている。本発明の核酸分子に等価なヌクレオチド配列を得るには例えば、NBLASTプログラムでscore = 100、wordlength = 12としてBLASTヌクレオチド検索を行えばよい。本酵素に等価なアミノ酸配列を得るには例えば、XBLASTプログラムでscore = 50、wordlength = 3としてBLASTポリペプチド検索を行えばよい。比較のためのギャップアライメントを得るためには、Altschulら (1997) Amino Acids Research 25(17):3389-3402に記載のGapped BLASTが利用可能である。BLASTおよびGapped BLASTを利用する場合は、対応するプログラム（例えばXBLASTおよびNBLAST）のデフォルトパラメータを使用することができる。詳しくはhttp://www.ncbi.nlm.nih.govを参照されたい。配列の比較に利用可能な他の数学的アルゴリズムの例としては、MyersおよびMiller (1988) Comput Appl Biosci. 4:11-17に記載のアルゴリズムがある。このようなアルゴリズムは、例えばGENESTREAMネットワークサーバー（IGH Montpellier、フランス）またはISRECサーバーで利用可能なALIGNプログラムに組み込まれている。アミノ酸配列の比較にALIGNプログラムを利用する場合は例えば、PAM120残基質量表を使用し、ギャップ長ペナルティ＝12、ギャップペナルティ＝4とすることができる。 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.
　二つのアミノ酸配列の同一性を、GCGソフトウェアパッケージのGAPプログラムを用いて、Blossom 62マトリックスまたはPAM250マトリックスを使用し、ギャップ加重＝12、10、8、6、又は4、ギャップ長加重＝2、3、又は4として決定することができる。また、二つの核酸配列の相同度を、GCGソフトウェアパッケージ（http://www.gcg.comで利用可能）のGAPプログラムを用いて、ギャップ加重＝50、ギャップ長加重＝3として決定することができる。 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.
　本酵素が、より大きいタンパク質（例えば融合タンパク質）の一部であってもよい。融合タンパク質において付加される配列としては、例えば、多重ヒスチジン残基のような精製に役立つ配列、組み換え生産の際の安定性を確保する付加配列等が挙げられる。 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.
　上記アミノ酸配列を有する本酵素は、遺伝子工学的手法によって容易に調製することができる。例えば、本酵素をコードするDNAで適当な宿主細胞（例えば大腸菌）を形質転換し、形質転換体内で発現されたタンパク質を回収することにより調製することができる。回収されたタンパク質は目的に応じて適宜精製される。このように組換えタンパク質として本酵素を得ることにすれば種々の修飾が可能である。例えば、本酵素をコードするDNAと他の適当なDNAとを同じベクターに挿入し、当該ベクターを用いて組換えタンパク質の生産を行えば、任意のペプチドないしタンパク質が連結された組換えタンパク質からなる本酵素を得ることができる。また、糖鎖及び／又は脂質の付加や、あるいはN末端若しくはC末端のプロセッシングが生ずるような修飾を施してもよい。以上のような修飾により、組換えタンパク質の抽出、精製の簡便化、又は生物学的機能の付加等が可能である。 The present enzyme having the above amino acid sequence can be easily prepared by a genetic engineering technique. 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．変異酵素をコードする核酸等
　本発明の第２の局面は本酵素に関連する核酸を提供する。即ち、本酵素をコードする遺伝子、本酵素をコードする核酸を同定するためのプローブとして用いることができる核酸、本酵素をコードする核酸を増幅又は突然変異等させるためのプライマーとして用いることができる核酸が提供される。 3. Nucleic acid encoding mutant enzyme, 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.
　本酵素をコードする遺伝子は典型的には本酵素の調製に利用される。本酵素をコードする遺伝子を用いた遺伝子工学的調製法によれば、より均質な状態の本酵素を得ることが可能である。また、当該方法は大量の本酵素を調製する場合にも好適な方法といえる。尚、本酵素をコードする遺伝子の用途は本酵素の調製に限られない。例えば、本酵素の作用機構の解明などを目的とした実験用のツールとして、或いは酵素の更なる変異体をデザイン又は作製するためのツールとして、当該核酸を利用することもできる。 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.
　本明細書において「本酵素をコードする遺伝子」とは、それを発現させた場合に本酵素が得られる核酸のことをいい、本酵素のアミノ酸配列に対応する塩基配列を有する核酸は勿論のこと、そのような核酸にアミノ酸配列をコードしない配列が付加されてなる核酸をも含む。また、コドンの縮重も考慮される。 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.
　本酵素をコードする遺伝子の配列の例を配列番号２５（K354V変異酵素をコードする配列）、配列番号２６（K354I変異酵素をコードする配列）、配列番号２７（K354P変異酵素をコードする配列）、配列番号２８（K354Q変異酵素をコードする配列）に示す。 Examples of sequences of genes encoding this enzyme are SEQ ID NO: 25 (sequence encoding K354V mutant enzyme), SEQ ID NO: 26 (sequence encoding K354I mutant enzyme), SEQ ID NO: 27 (sequence encoding K354P mutant enzyme), This is shown in SEQ ID NO: 28 (sequence encoding K354Q mutant enzyme).
　本発明の核酸は、本明細書又は添付の配列表が開示する配列情報を参考にし、標準的な遺伝子工学的手法、分子生物学的手法、生化学的手法などを用いることによって、単離された状態に調製することができる。 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.
　本発明の他の態様では、本酵素をコードする遺伝子の塩基配列と比較した場合にそれがコードするタンパク質の機能は同等であるものの一部において塩基配列が相違する核酸（以下、「等価核酸」ともいう。また、等価核酸を規定する塩基配列を「等価塩基配列」ともいう）が提供される。等価核酸の例として、本酵素をコードする核酸の塩基配列を基準として１若しくは複数の塩基の置換、欠失、挿入、付加、又は逆位を含む塩基配列からなり、本酵素に特徴的な酵素活性（即ちGDH活性）を有するタンパク質をコードするDNAを挙げることができる。塩基の置換や欠失などは複数の部位に生じていてもよい。ここでの「複数」とは、当該核酸がコードするタンパク質の立体構造におけるアミノ酸残基の位置や種類によっても異なるが例えば2～40塩基、好ましくは2～20塩基、より好ましくは2～10塩基である。 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 depends on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid It is.
　等価核酸は、基準となる塩基配列（配列番号２５～２８のいずれかの配列）に対して、例えば60％以上、好ましくは70%以上、より好ましくは80％以上、より一層好ましくは85％以上、さらに好ましくは約90％以上、さらに一層好ましくは95％以上、最も好ましくは99%以上の同一性を有する。 The equivalent nucleic acid is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 85% or more with respect to the base sequence serving as a reference (sequence of any of SEQ ID NOs: 25 to 28). More preferably about 90% or more, even more preferably 95% or more, most preferably 99% or more.
　以上のような等価核酸は例えば、制限酵素処理、エキソヌクレアーゼやDNAリガーゼ等による処理、位置指定突然変異導入法（Molecular Cloning, Third Edition, Chapter 13 ,Cold Spring Harbor Laboratory Press, New York）やランダム突然変異導入法（Molecular Cloning, Third Edition, Chapter 13 ,Cold Spring Harbor Laboratory Press, New York）による変異の導入などによって得られる。また、紫外線照射など他の方法によっても等価核酸を得ることができる。 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.
　本発明の他の態様は、本酵素をコードする遺伝子の塩基配列に対して相補的な塩基配列を有する核酸に関する。本発明の更に他の態様は、本発明の本酵素をコードする遺伝子の塩基配列、或いはそれに相補的な塩基配列に対して少なくとも約60%、70%、80%、90%、95%、99%、99.9%同一な塩基配列を有する核酸を提供する。 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. %, 99.9% nucleic acid having the same base sequence is provided.
　本発明の更に別の態様は、本酵素をコードする遺伝子の塩基配列又はその等価塩基配列に相補的な塩基配列に対してストリンジェントな条件下でハイブリダイズする塩基配列を有する核酸に関する。ここでの「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。このようなストリンジェントな条件は当業者に公知であって例えばMolecular Cloning（Third Edition, Cold Spring Harbor Laboratory Press, New York)やCurrent protocols in molecular biology（edited by Frederick M. Ausubel et al., 1987）を参照して設定することができる。ストリンジェントな条件として例えば、ハイブリダイゼーション液（50％ホルムアミド、10×SSC(0.15M NaCl, 15mM sodium citrate, pH 7.0)、5×Denhardt溶液、1％ SDS、10％ デキストラン硫酸、10μg/mlの変性サケ***DNA、50mMリン酸バッファー(pH7.5)）を用いて約42℃～約50℃でインキュベーションし、その後0.1×SSC、0.1％ SDSを用いて約65℃～約70℃で洗浄する条件を挙げることができる。更に好ましいストリンジェントな条件として例えば、ハイブリダイゼーション液として50％ホルムアミド、5×SSC(0.15M NaCl, 15mM sodium citrate, pH 7.0)、1×Denhardt溶液、1％SDS、10％デキストラン硫酸、10μg/mlの変性サケ***DNA、50mMリン酸バッファー(pH7.5)）を用いる条件を挙げることができる。 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)).
　本発明の更に他の態様は、本酵素をコードする遺伝子の塩基配列、或いはそれに相補的な塩基配列の一部を有する核酸（核酸断片）を提供する。このような核酸断片は、本酵素をコードする遺伝子の塩基配列を有する核酸などを検出、同定、及び／又は増幅することなどに用いることができる。核酸断片は例えば、本酵素をコードする遺伝子の塩基配列において連続するヌクレオチド部分（例えば約10～約100塩基長、好ましくは約20～約100塩基長、更に好ましくは約30～約100塩基長）にハイブリダイズする部分を少なくとも含むように設計される。プローブとして利用される場合には核酸断片を標識化することができる。標識化には例えば、蛍光物質、酵素、放射性同位元素を用いることができる。 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, fluorescent substances, enzymes, and radioisotopes can be used.
　本発明のさらに他の局面は、本発明の遺伝子（本酵素をコードする遺伝子）を含む組換えDNAに関する。本発明の組換えDNAは例えばベクターの形態で提供される。本明細書において用語「ベクター」は、それに挿入された核酸を細胞等のターゲット内へと輸送することができる核酸性分子をいう。 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.
　使用目的（クローニング、タンパク質の発現）に応じて、また宿主細胞の種類を考慮して適当なベクターが選択される。大腸菌を宿主とするベクターとしてはM13ファージ又はその改変体、λファージ又はその改変体、pBR322又はその改変体（pB325、pAT153、pUC8など）等、酵母を宿主とするベクターとしてはpYepSec1、pMFa、pYES2等、昆虫細胞を宿主とするベクターとしてはpAc、pVL等、哺乳類細胞を宿主とするベクターとしてはpCDM8、pMT2PC等を例示することができる。 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 using insect cells as hosts include pAc and pVL, and examples of vectors using mammalian cells as hosts include pCDM8 and pMT2PC.
　本発明のベクターは好ましくは発現ベクターである。「発現ベクター」とは、それに挿入された核酸を目的の細胞（宿主細胞）内に導入することができ、且つ当該細胞内において発現させることが可能なベクターをいう。発現ベクターは通常、挿入された核酸の発現に必要なプロモーター配列や、発現を促進させるエンハンサー配列等を含む。選択マーカーを含む発現ベクターを使用することもできる。かかる発現ベクターを用いた場合には、選択マーカーを利用して発現ベクターの導入の有無（及びその程度）を確認することができる。 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.
　本発明の核酸のベクターへの挿入、選択マーカー遺伝子の挿入（必要な場合）、プロモーターの挿入（必要な場合）等は標準的な組換えDNA技術（例えば、Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New Yorkを参照することができる、制限酵素及びDNAリガーゼを用いた周知の方法）を用いて行うことができる。 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).
　宿主細胞としては、取り扱いの容易さの点から、大腸菌（エシェリヒア・コリ）、出芽酵母（サッカロマイセス・セレビシエ）、糸状菌（アスペルギルス・オリゼ）などの微生物を用いることが好ましいが、組換えDNAが複製可能で且つ本酵素の遺伝子が発現可能な宿主細胞であれば利用可能である。大腸菌の例としてT7系プロモーターを利用する場合は大腸菌BL21(DE3)pLysS、そうでない場合は大腸菌JM109を挙げることができる。また、出芽酵母の例として出芽酵母SHY2、出芽酵母AH22あるいは出芽酵母INVSc1（インビトロジェン社）を挙げることができる。 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 budding yeast include budding yeast SHY2, budding yeast AH22, or budding yeast INVSc1 (Invitrogen).
　本発明の更に他の局面は、本発明の組換えDNAを保有する微生物（即ち形質転換体）に関する。本発明の微生物は、上記本発明のベクターを用いたトランスフェクション乃至はトランスフォーメーションによって得ることができる。例えば、塩化カルシウム法（ジャーナル オブ モレキュラー バイオロジー(J.Mol. Biol.)、第53巻、第159頁 (1970)）、ハナハン（Hanahan）法（ジャーナル オブ モレキュラー バイオロジー、第166巻、第557頁 (1983)）、SEM法（ジーン（Gene)、第96巻、第23頁(1990)〕、チャング（Chung）らの方法（プロシーディングズ オブ ザ ナショナル アカデミー オブ サイエンシーズ オブ ザ USA、第86巻、第2172頁(1989)）、リン酸カルシウム共沈降法、エレクトロポーレーション(Potter,H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165(1984))、リポフェクション(Felgner, P.L. et al.,  Proc. Natl. Acad. Sci. U.S.A. 84,7413-7417(1984))等によって実施することができる。尚、本発明の微生物は、本酵素を生産することに利用することができる。 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．変異酵素の調製法
　本発明の更なる局面は本酵素の調製法に関する。本発明の調製法では、本発明者らが取得に成功した変異酵素を遺伝子工学的手法で調製する。具体的には、まず本酵素をコードする遺伝子を用意する（ステップ(1)）。具体的には、例えば、配列番号２１～２４のいずれかのアミノ酸配列をコードする核酸を用意する。ここで、「配列番号２１～２４のいずれかのアミノ酸配列をコードする核酸」は、それを発現させた場合に当該アミノ酸配列を有するポリペプチドが得られる核酸であり、当該アミノ酸配列に対応する塩基配列からなる核酸は勿論のこと、そのような核酸に余分な配列（アミノ酸配列をコードする配列であっても、アミノ酸配列をコードしない配列であってもよい）が付加されていてもよい。また、コドンの縮重も考慮される。「配列番号２１～２４のいずれかのアミノ酸配列をコードする核酸」は、本明細書又は添付の配列表が開示する配列情報を参考にし、標準的な遺伝子工学的手法、分子生物学的手法、生化学的手法などを用いることによって、単離された状態に調製することができる。ここで、配列番号２１のアミノ酸配列、配列番号２２のアミノ酸配列、配列番号２３のアミノ酸配列、配列番号２４のアミノ酸配列はいずれも、アスペルギルス・イイズカエNo.5453株由来GDHのアミノ酸配列に変異を施したものである。従って、アスペルギルス・イイズカエNo.5453株由来GDHをコードする遺伝子（配列番号２の塩基配列）に対して必要な変異を加えることによっても、配列番号２１～２４のいずれかのアミノ酸配列をコードする核酸（遺伝子）を得ることができる。位置特異的塩基配列置換のための方法は当該技術分野において数多く知られており（例えば、Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New Yorkを参照）、その中から適切な方法を選択して用いることができる。位置特異的変異導入法として、位置特異的アミノ酸飽和変異法を採用することができる。位置特異的アミノ酸飽和変異法は、タンパクの立体構造を基に、求める機能の関与する位置を推定し、アミノ酸飽和変異を導入する「Semi-rational,semi-random」手法である（J.Mol.Biol.331,585-592(2003)）。例えば、Quick change（ストラタジーン社）等のキット、Overlap extention PCR(Nucleic Acid Res. 16,7351-7367(1988)）を用いて位置特異的アミノ酸飽和変異を導入することが可能である。PCRに用いるDNAポリメラーゼはTaqポリメラーゼ等を用いることができる。但し、KOD-PLUS-（東洋紡社）、Pfu turbo（ストラタジーン社）などの精度の高いDNAポリメラーゼを用いることが好ましい。 4). Method for Preparing Mutant Enzyme A further aspect of the present invention relates to a method for preparing the enzyme. In the preparation method of the present invention, a mutant enzyme 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 any one of the amino acid sequences of SEQ ID NOs: 21 to 24 is prepared. Here, the “nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24” is a nucleic acid from which a polypeptide having the amino acid sequence is obtained when expressed, and a base corresponding to the amino acid sequence. An extra sequence (which may be a sequence encoding an amino acid sequence or a sequence not encoding an amino acid sequence) may be added to such a nucleic acid as well as a nucleic acid comprising a sequence. Codon degeneracy is also considered. “Nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS: 21 to 24” refers to sequence information disclosed in this specification or the attached sequence listing, and uses standard genetic engineering techniques, molecular biological techniques, It can be prepared in an isolated state by using a biochemical method or the like. Here, the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 23, and the amino acid sequence of SEQ ID NO: 24 all mutated the amino acid sequence of GDH derived from Aspergillus iizukae No. 5453. It is a thing. Therefore, a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24 can be obtained by adding a necessary mutation to the gene encoding GDH derived from Aspergillus iizukae No.5453 (base sequence of SEQ ID NO: 2). (Gene) can be obtained. Many methods for position-specific base sequence substitution are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method is selected from them. Can be used. As a position-specific mutation introducing method, a position-specific amino acid saturation mutation method can be employed. The position-specific amino acid saturation mutation method is a “Semi-rational, semi-random” technique in which amino acid saturation mutation is introduced by estimating the position where the desired function is involved based on the three-dimensional structure of the protein (J. Mol. Biol. 331, 585-592 (2003)). For example, a site-specific amino acid saturation mutation can be introduced by using a kit such as Quick change (Stratagene) and overlap extention PCR (Nucleic Acid Res. 16,7351-7367 (1988)). As a DNA polymerase used for PCR, Taq polymerase or the like can be used. However, it is preferable to use a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo), Pfu turbo (Stratagene).
　ステップ（1)に続いて、用意した遺伝子を発現させる（ステップ(2)）。例えば、まず上記遺伝子を挿入した発現ベクターを用意し、これを用いて宿主細胞を形質転換する。次に、発現産物である変異酵素が産生される条件下で形質転換体を培養する。形質転換体の培養は常法に従えばよい。培地に使用する炭素源としては資化可能な炭素化合物であればよく、例えばグルコース、シュークロース、ラクトース、マルトース、糖蜜、ピルビン酸などが使用される。また、窒素源としては利用可能な窒素化合物であればよく、例えばペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物などが使用される。その他、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガン、亜鉛などの塩類、特定のアミノ酸、特定のビタミンなどが必要に応じて使用される。 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.
　培養温度は培養対象の形質転換体の生育特性や変異型酵素の産生特性などを考慮して設定することができる。好ましくは30℃～40℃の範囲内（より好ましくは37℃付近）で設定することができる。培養時間は、培養対象の形質転換体の生育特性や変異型酵素の産生特性などを考慮して設定することができる。培地のpHは、形質転換体が生育し且つ酵素が産生される範囲内に調製される。好ましくは培地のpHを6.0～9.0程度（好ましくはpH7.0付近）とする。 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).
　続いて、発現産物（変異酵素）を回収する（ステップ(3)）。培養後の菌体を含む培養液をそのまま、或いは濃縮、不純物の除去などを経た後に酵素溶液として利用することもできるが、一般的には培養液又は菌体より発現産物を一旦回収する。発現産物が分泌型タンパク質であれば培養液より、それ以外であれば菌体内より回収することができる。培養液から回収する場合には、例えば培養上清をろ過、遠心処理して不溶物を除去した後、減圧濃縮、膜濃縮、硫酸アンモニウムや硫酸ナトリウムを利用した塩析、メタノールやエタノール又はアセトンなどによる分別沈殿法、透析、加熱処理、等電点処理、ゲルろ過や吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー等の各種クロマトグラフィー（例えば、セファデックス（Sephadex）ゲル（GEヘルスケアバイオサイエンス）などによるゲルろ過、DEAEセファロースCL-6B （GEヘルスケアバイオサイエンス）、オクチルセファロースCL-6B （GEヘルスケアバイオサイエンス）、CMセファロースCL-6B（GEヘルスケアバイオサイエンス））などを組み合わせて分離、精製を行ことにより変異酵素の精製品を得ることができる。他方、菌体内から回収する場合には、培養液をろ過、遠心処理等することによって菌体を採取し、次いで菌体を加圧処理、超音波処理などの機械的方法またはリゾチームなどによる酵素的方法で破壊した後、上記と同様に分離、精製を行うことにより変異酵素の精製品を得ることができる。 Subsequently, the expression product (mutant enzyme) 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) Purify and obtain the purified product of mutant enzyme Door can be. On the other hand, when recovering from the microbial cells, the microbial cells are collected by filtering, centrifuging, etc., and then the microbial cells are subjected to mechanical methods such as pressure treatment, ultrasonic treatment, or enzymatic methods such as lysozyme. After destruction by the method, a purified product of the mutant enzyme can be obtained by separation and purification in the same manner as described above.
　酵素の精製度は特に限定されないが、例えば比活性が10～1000（U/mg）、好ましくは比活性が50～500（U/mg）の状態に精製することができる。また、最終的な形態は液体状であっても固体状（粉体状を含む）であってもよい。 The degree of purification of the enzyme is not particularly limited. For example, the enzyme can be purified to have a specific activity of 10 to 1000 (U / mg), preferably 50 to 500 (U / mg). The final form may be liquid or solid (including powder).
　上記のようにして得られた精製酵素を、例えば凍結乾燥や真空乾燥或いはスプレードライなどにより粉末化して提供することも可能である。その際、精製酵素を予めリン酸緩衝液、トリエタノールアミン緩衝液、トリス塩酸緩衝液やGOODの緩衝液に溶解させておいてもよい。好ましくは、リン酸緩衝液、トリエタノールアミン緩衝液を使用することができる。尚、ここでGOODの緩衝液としてはPIPES、MES又はMOPSが挙げられる。 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.
　通常は、以上のように適当な宿主－ベクター系を利用して遺伝子の発現～発現産物（変異酵素）の回収を行うが、無細胞合成系を利用することにしてもよい。ここで、「無細胞合成系（無細胞転写系、無細胞転写／翻訳系）」とは、生細胞を用いるのではく、生細胞由来の（或いは遺伝子工学的手法で得られた）リボソームや転写・翻訳因子などを用いて、鋳型である核酸（DNAやmRNA）からそれがコードするmRNAやタンパク質をin vitroで合成することをいう。無細胞合成系では一般に、細胞破砕液を必要に応じて精製して得られる細胞抽出液が使用される。細胞抽出液には一般に、タンパク質合成に必要なリボソーム、開始因子などの各種因子、tRNAなどの各種酵素が含まれる。タンパク質の合成を行う際には、この細胞抽出液に各種アミノ酸、ATP、GTPなどのエネルギー源、クレアチンリン酸など、タンパク質の合成に必要なその他の物質を添加する。勿論、タンパク質合成の際に、別途用意したリボソームや各種因子、及び／又は各種酵素などを必要に応じて補充してもよい。 Usually, gene expression and expression product (mutant enzyme) 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.
　タンパク質合成に必要な各分子（因子）を再構成した転写／翻訳系の開発も報告されている（Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001）。この合成系では、バクテリアのタンパク質合成系を構成する３種類の開始因子、３種類の伸長因子、終結に関与する４種類の因子、各アミノ酸をtRNAに結合させる２０種類のアミノアシルtRNA合成酵素、及びメチオニルtRNAホルミル転移酵素からなる３１種類の因子の遺伝子を大腸菌ゲノムから増幅し、これらを用いてタンパク質合成系をin vitroで再構成している。本発明ではこのような再構成した合成系を利用してもよい。 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.
　用語「無細胞転写／翻訳系」は、無細胞タンパク質合成系、in vitro翻訳系又はin vitro転写／翻訳系と交換可能に使用される。in vitro翻訳系ではRNAが鋳型として用いられてタンパク質が合成される。鋳型RNAとしては全RNA、mRNA、in vitro転写産物などが使用される。他方のin vitro転写／翻訳系ではDNAが鋳型として用いられる。鋳型DNAはリボソーム結合領域を含むべきであって、また適切なターミネータ配列を含むことが好ましい。尚、in vitro転写／翻訳系では、転写反応及び翻訳反応が連続して進行するように各反応に必要な因子が添加された条件が設定される。 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．変異酵素の用途
　本発明の更なる局面は本酵素の用途に関する。この局面ではまず、本酵素を用いたグルコース測定法が提供される。本発明のグルコース測定法では本酵素による酸化還元反応を利用して試料中のグルコース量を測定する。この反応による変化が利用できる各種用途に本発明を適用可能である。 5). Uses of the Mutant Enzyme 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.
　本発明は例えば血糖値の測定、食品（調味料や飲料など）中のグルコース濃度の測定などに利用される。また、発酵食品（例えば食酢）又は発酵飲料（例えばビールや酒）の製造工程において発酵度を調べるために本発明を利用してもよい。 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).
　本発明はまた、本酵素を含むグルコース測定用試薬を提供する。当該試薬は上記の本発明のグルコース測定法に使用される。グルコース測定用試薬の安定化や使用時の活性化等を目的として、血清アルブミン、タンパク質、界面活性剤、糖類、糖アルコール、無機塩類等を添加してもよい。 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.
　グルコース測定用試薬を測定キットの構成要素にすることもできる。換言すれば、本発明は、上記グルコース測定用試薬を含むキット（グルコース測定用キット）も提供する。本発明のキットは必須の構成要素として上記グルコース測定用試薬を含む。また、反応用試薬、緩衝液、グルコース標準液、容器などを任意の要素として含む。尚、本発明のグルコース測定キットには通常、使用説明書が添付される。 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.
　本酵素を利用してグルコースセンサを構成することが可能である。即ち、本発明は、本酵素を含むグルコースセンサも提供する。本発明のグルコースセンサの典型的な構造では、絶縁性基板上に作用電極及び対極を備えた電極系が形成され、その上に本酵素とメディエータを含む試薬層が形成される。参照電極も備えた測定系を用いることにしてもよい。このような、いわゆる３電極系の測定系を用いれば、参照電極の電位を基準として作用電極の電位を表すことが可能となる。各電極の材料は特に限定されない。作用電極及び対極の電極材料の例を示せば、金（Au）、カーボン（C）、白金（Pt）、チタン（Ti）である。メディエータとしてはフェリシアン化合物（フェリシアン化カリウムなど）、金属錯体（ルテニウム錯体、オスミウム錯体、バナジウム錯体など）、キノン化合物（ピロロキノリンキノンなど）などが使用される。尚、グルコースセンサの構成、グルコースセンサを利用した電気化学的測定法については、例えば、バイオ電気化学の実際－バイオセンサ・バイオ電池の実用展開－（２００７年３月発行、シーエムシー出版）に詳しい。 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). .
　本酵素を酵素剤の形態で提供することもできる。本発明の酵素剤は有効成分（本酵素）の他、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水などを含有していてもよい。賦形剤としてはデンプン、デキストリン、マルトース、トレハロース、乳糖、D-グルコース、ソルビトール、D-マンニトール、白糖、グリセロール等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としてはエタノール、塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等を用いることができる。 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. Phosphate, citrate, acetate, etc. can be used as the buffer. 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.
１．微生物からのスクリーニング
　公的機関から入手した保存菌株や自然界から入手した菌株を含む13,000株を培養して得られた培養液を試料として、以下の条件、即ち、グルコースデヒドロゲナーゼ活性が高いこと、マルトースに反応しないこと、キシロースへの反応性が低いこと、及びグルコースオキシダーゼ活性を示さないこと、を満たすものを以下の方法で選出した。 1. Screening from microorganisms Using as a sample a culture solution obtained by culturing 13,000 strains including stocks obtained from public institutions and strains obtained from the natural world, the following conditions, namely, high glucose dehydrogenase activity, maltose Those satisfying that no reaction, low reactivity to xylose, and no glucose oxidase activity were selected by the following method.
グルコースデヒドロゲナーゼ活性の測定方法
（測定試液）
　100 mmol/L PIPES cont. 0.1％(w/v) Triton X-100 pH 7.0：　24mL
　3 mmol/L 1-Methoxy PMS（Phenazine methanesulfate）：　2mL
　6.6 mmol/L NTB（Nitrotetrazorium blue）：　1mL
　1 mol/L グルコース：　3mL Method for measuring glucose dehydrogenase activity (measuring solution)
100 mmol / L PIPES cont. 0.1% (w / v) Triton X-100 pH 7.0: 24mL
3 mmol / L 1-Methoxy PMS (Phenazine methanesulfate): 2mL
6.6 mmol / L NTB (Nitrotetrazorium blue): 1mL
1 mol / L glucose: 3mL
（測定手順）
　サンプルを20μLずつ96wellプレートに分注後、測定試液を１ウェルあたり200μLずつ添加し、37℃、60分後、570nmの吸光度をプレートリーダーで測定した。上記測定試液のうち、グルコースをマルトース又はキシロースへ変更して同様に測定し、マルトース、キシロースへの反応性も確認した。 (Measurement procedure)
After dispensing 20 μL of each sample into a 96-well plate, 200 μL of measurement reagent was added per well, and after 60 minutes at 37 ° C., the absorbance at 570 nm was measured with a plate reader. Among the above measuring solutions, glucose was changed to maltose or xylose and the same measurement was performed, and the reactivity to maltose and xylose was also confirmed.
グルコースオキシダーゼ活性の測定方法
（測定試液）
　100 mmol/L PIPES cont. 0.1％(w/v) Triton X-100 pH 7.0：　23mL
　5g/dL フェノール試液：　0.5mL
　25u/mL PO“Amano”3（天野エンザイム株式会社）溶液：　3mL
　0.5g/dL 4-アミノアンチピリン試液：　0.5mL
　1 mol/L グルコース：　3mL Method for measuring glucose oxidase activity (measuring solution)
100 mmol / L PIPES cont. 0.1% (w / v) Triton X-100 pH 7.0: 23mL
5g / dL phenol reagent: 0.5mL
25u / mL PO “Amano” 3 (Amano Enzyme Inc.) solution: 3mL
0.5g / dL 4-aminoantipyrine test solution: 0.5mL
1 mol / L glucose: 3mL
（測定手順）
　サンプルを20μLずつ96wellプレートに分注後、測定試液を１ウェルあたり200μLずつ添加し、37℃、60分後、500nmの吸光度をプレートリーダーで測定した。 (Measurement procedure)
After dispensing 20 μL of each sample into a 96-well plate, 200 μL of the measurement reagent was added per well, and after 60 minutes at 37 ° C., the absorbance at 500 nm was measured with a plate reader.
　検討の結果、マルトースに反応せず、キシロースへの反応性が低く、しかもグルコースオキシダーゼではない、Aspergillus iizukae No.5453株の生産するグルコースデヒドロゲナーゼが見出された。Aspergillus iizukae No.5453株について、マルトース及びキシロースへの反応性とグルコースオキシダーゼ（GO）活性を以下の表に示す。マルトース及びキシロースへの反応性は、グルコースへの反応性を100％としたときの相対値（マルトース（又はキシロース）を基質とした場合の測定値／グルコースを基質とした場合の測定値　×　１００）で表した。また、グルコースオキシダーゼ（GO）はグルコースデヒドロゲナーゼに対する相対値（グルコースオキシダーゼ（GO）活性の測定値／グルコースデヒドロゲナーゼ活性の測定値　×　１００）で表した。比較のため、グルコースオキシダーゼ（Aspergillus niger由来）、PQQ依存性グルコースデヒドロゲナーゼ（Acinetobacter calcoaceticus由来）、FAD依存性グルコースデヒドロゲナーゼ（Aspergillus oryzae由来）の結果も示した。
Figure JPOXMLDOC01-appb-T000001
 As a result of the examination, a glucose dehydrogenase produced by Aspergillus iizukae No. 5453, which does not react with maltose, has low reactivity with xylose and is not glucose oxidase, was found. The following table shows the reactivity to maltose and xylose and glucose oxidase (GO) activity of Aspergillus iizukae No.5453. The reactivity to maltose and xylose is relative value when the reactivity to glucose is 100% (measured value when maltose (or xylose) is used as substrate / measured value when glucose is used as substrate x 100) Expressed in Glucose oxidase (GO) was expressed as a relative value to glucose dehydrogenase (measured value of glucose oxidase (GO) activity / measured value of glucose dehydrogenase activity × 100). For comparison, results of glucose oxidase (derived from Aspergillus niger), PQQ-dependent glucose dehydrogenase (derived from Acinetobacter calcoaceticus), and FAD-dependent glucose dehydrogenase (derived from Aspergillus oryzae) are also shown.
Figure JPOXMLDOC01-appb-T000001
　Aspergillus iizukae No.5453株の生産するグルコースデヒドロゲナーゼは、既存のPQQ依存性グルコースデヒドロゲナーゼ及びFAD依存性グルコースデヒドロゲナーゼと比較して優位にマルトース及びキシロースへの反応性が低いことがわかる。尚、Aspergillus iizukae No.5453株は独立行政法人製品評価技術基盤機構（NBRC）（〒292-0818　千葉県木更津市かずさ鎌足２－５－８）に保存されているAspergillus iizukae Sugiyama NBRC 8869と同一株である。 It can be seen that the glucose dehydrogenase produced by Aspergillus iizukae No. 5543 has a significantly lower reactivity to maltose and xylose than the existing PQQ-dependent glucose dehydrogenase and FAD-dependent glucose dehydrogenase. Aspergillus iizukae No.5453 is the same as Aspergillus iizukae Sugiyama NBRC 8869, which is stored in the National Institute of Technology and Evaluation (NBRC) (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818). Is a stock.
２．精製酵素の調製
　以上の検討によって見出された、Aspergillus iizukae No.5453株の生産するグルコースデヒドロゲナーゼについて、その精製酵素を取得すべく、Aspergillus iizukae No.5453株を以下の培地で30℃、5日間培養した。得られた培養液から菌体を除去し、粗酵素液とした。
（培地）
　グルコース：　15.0％(w/v)
　酵母エキス：　3.0％(w/v)
　大豆ペプトン：　6.0％(w/v)
　KH2PO4：　0.3％(w/v)
　K2HPO4：　0.2％(w/v)
　ヒドロキノン(pH6.0)：　4mM 2. Preparation of purified enzyme Regarding glucose dehydrogenase produced by Aspergillus iizukae No.5453 found by the above examination, in order to obtain the purified enzyme, Aspergillus iizukae No.5453 was cultured in the following medium at 30 ° C for 5 days. Cultured. The cells were removed from the obtained culture solution to obtain a crude enzyme solution.
(Culture medium)
Glucose: 15.0% (w / v)
Yeast extract: 3.0% (w / v)
Soybean peptone: 6.0% (w / v)
KH 2 PO 4 : 0.3% (w / v)
K 2 HPO 4 : 0.2% (w / v)
Hydroquinone (pH6.0): 4mM
　粗酵素液を精製（塩析、疎水結合クロマト、イオン交換クロマト、ゲル濾過クロマトグラフィー）し、精製酵素を得た。精製酵素をゲル濾過（GEヘルスケア社製Superdex 200を使用）及びSDS-PAGEで分析した。SDS-PAGEの結果を図１に示す。グルコースデヒドロゲナーゼ活性が最も高いフラクション（No.34）を以降の実験に使用した。 The crude enzyme solution was purified (salting out, hydrophobic bond chromatography, ion exchange chromatography, gel filtration chromatography) to obtain a purified enzyme. The purified enzyme was analyzed by gel filtration (using GE Healthcare Superdex®200) and SDS-PAGE. The results of SDS-PAGE are shown in FIG. The fraction with the highest glucose dehydrogenase activity (No. 34) was used in subsequent experiments.
３．N末端アミノ酸及び内部アミノ酸配列の決定
　目的の酵素の活性ピークであるフラクションNo.34をSDS-PAGEで分離して得られたバンドについて、常法に従い、PVDF膜へブロッティングした後、N末アミノ酸解析を実施したところ、約60KDaのタンパク質（図１、矢印）で「SSSYDYIVIGGGTSGLTVAN（配列番号３）」の配列情報が得られた。同配列を問い合わせ配列として、NCBIが提供するBLAST解析を実施したところ、グルコースデヒドロゲナーゼである可能性が高いことが判明した。BLAST解析結果を図２に示す。 3. Determination of N-terminal amino acid and internal amino acid sequence The band obtained by separating fraction No. 34, which is the activity peak of the target enzyme, by SDS-PAGE was blotted to a PVDF membrane according to a conventional method, followed by N-terminal amino acid analysis. As a result, the sequence information of “SSSYDYIVIGGGTSGLTVAN (SEQ ID NO: 3)” was obtained with a protein of about 60 KDa (FIG. 1, arrow). When BLAST analysis provided by NCBI was performed using the same sequence as a query sequence, it was found that there was a high possibility of glucose dehydrogenase. The BLAST analysis results are shown in FIG.
　次に、本タンパク質の全長配列を入手すべく、内部アミノ酸解析を実施した。まず、以下の方法でトリプシン処理し（ゲル内消化）、得られたペプチド断片を高速液体クロマトグラフ（HPLC）で分離した。その後、プロテインシークエンサーPPSQ-33A（株式会社島津製作所）を用いてアミノ酸解析をした。 Next, internal amino acid analysis was performed to obtain the full-length sequence of this protein. First, trypsinization was performed by the following method (in-gel digestion), and the obtained peptide fragments were separated by high performance liquid chromatography (HPLC). Thereafter, amino acid analysis was performed using a protein sequencer PPSQ-33A (Shimadzu Corporation).
（トリプシン処理）
　(i)電気泳動後のゲル試料を適当な大きさに切断する。
　(ii)2-メルカプトエタノール及び４－ビニルピリジンを用いて還元アルキル化処理。
　(iii)緩衝液（0.1mol/L炭酸水素アンモニウム）中でプロメガ社製Sequencing Grade Modified Trypsinを用いて消化後、ゲルからペプチド断片を抽出する。 (Trypsin treatment)
(i) The gel sample after electrophoresis is cut into an appropriate size.
(ii) Reductive alkylation treatment using 2-mercaptoethanol and 4-vinylpyridine.
(iii) After digestion in a buffer solution (0.1 mol / L ammonium bicarbonate) using Sequencing Grade Modified Trypsin manufactured by Promega, peptide fragments are extracted from the gel.
（HPLC分離条件）
　高速液体クロマトグラフ（HPLC）：　LC-20Aシステム（株式会社島津製作所）
　カラム：　Cadenza CD-C18(2.0mmI.D.×150mm)（インタクト株式会社）
　カラム温度：　50℃
　検出波長：　214mm
　注入量：　70μL
　移動相流速：　0.2mL/min
　移動相A：　水/トリフルオロ酢酸（1000/1）
　移動相B：　アセトニトリル/水/トリフルオロ酢酸（800/200/1） (HPLC separation conditions)
High-performance liquid chromatograph (HPLC): LC-20A system (Shimadzu Corporation)
Column: Cadenza CD-C18 (2.0mmI.D. × 150mm) (Intact Corporation)
Column temperature: 50 ° C
Detection wavelength: 214mm
Injection volume: 70μL
Mobile phase flow rate: 0.2mL / min
Mobile phase A: Water / trifluoroacetic acid (1000/1)
Mobile phase B: acetonitrile / water / trifluoroacetic acid (800/200/1)
　HPLC分離で得られたピーク（１３個のピークを特定し、保持時間の短い方から順に番号を付した）の解析によって同定したアミノ酸配列を図３に示す。 FIG. 3 shows the amino acid sequence identified by analysis of the peaks obtained by HPLC separation (13 peaks were identified and numbered in order from the shorter retention time).
４．遺伝子配列の決定
　上記３．で得られたN末アミノ酸解析結果とピークNo.5のアミノ酸解析結果に基づき、以下のプライマーを設計した。
　プライマーGDH5453-F：TAYGAYTAYATHGTNATHGGNGGNGGNACNWSNGG（配列番号４）
　プライマーGDH5453-5-1-R：NSWNGCDATRTGNACRTTNCC（配列番号５） 4). Determination of gene sequence 3. The following primers were designed based on the N-terminal amino acid analysis results obtained in step 1 and the amino acid analysis results of peak No. 5.
Primer GDH5453-F: TAYGAYTAYATHGTNATHGGNGGNGGNACNWSNGG (SEQ ID NO: 4)
Primer GDH5453-5-1-R: NSWNGCDATRTGNACRTTNCC (SEQ ID NO: 5)
　Aspergillus iizukae No.5453株のゲノムDNAをテンプレートにして、設計したプライマーとPrimeSTAR（登録商標） Max DNA Polymerase（タカラバイオ株式会社）を用いてPCRを行い、増幅されたDNA断片を得た。PCR条件は以下の通りとした。
（反応液）
　PrimeSTAR Max Premix（2×）　25μL
　GDH5453-F　15 pmol
　GDH5453-5-1-R　15 pmol
　ゲノムDNA(1/1000希釈)　1μL
　滅菌蒸留水で50μLに調整
（サイクル条件）
　98℃で10秒、55℃で15秒、72℃で2分の条件で３５サイクル PCR was performed using the designed primer and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) using the genomic DNA of Aspergillus iizukae No.5453 as a template to obtain an amplified DNA fragment. PCR conditions were as follows.
(Reaction solution)
PrimeSTAR Max Premix (2 ×) 25μL
GDH5453-F 15 pmol
GDH5453-5-1-R 15 pmol
Genomic DNA (1/1000 dilution) 1μL
Adjust to 50 μL with sterile distilled water (cycle conditions)
35 cycles at 98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 2 minutes
　得られたDNA断片をMighty Cloning Reagent Set (Blunt End)（タカラバイオ株式会社）を用いてサブクローニングし、常法に従いDNA断片の塩基配列を確認した。 The obtained DNA fragment was subcloned using Mighty Cloning Reagent Set (Blunt End) (Takara Bio Inc.), and the base sequence of the DNA fragment was confirmed according to a conventional method.
　得られた塩基配列（配列番号６）に基づき、以下のプライマーを設計した。
　プライマーFS51R07F：AACCGTCTGTCTGAAGACCC（配列番号７）
　プライマーFS51R07R：TACTTCCTTTTGCTCG（配列番号８） Based on the obtained base sequence (SEQ ID NO: 6), the following primers were designed.
Primer FS51R07F: AACCGTCTGTCTGAAGACCC (SEQ ID NO: 7)
Primer FS51R07R: TACTTCCTTTTGCTCG (SEQ ID NO: 8)
　上記DNA断片をテンプレートにして、設計したプライマーとPCR DIG Probe Synthesis Kit（ロシュ・ダイアグノスティックス株式会社）を用いてPCRを行い、ジゴキシゲニン標識されたDNAプローブを得た。このプローブを用いてサザンハイブリダイゼーションを行った。染色体DNAを制限酵素BamHI、KpnI、PstI、SacI、SphI、XbaIで完全消化したものと、これら制限酵素と制限酵素SalIを組み合わせて完全消化したものを0.8％アガロース電気泳動で分離した。続いて、ゼータープローブメンブレン（Bio-Rad株式会社）に転写し、サザンハイブリダイゼーション用の膜を得た。サザンハイブリダイゼーションは、DIG Easy Hyb. (ロシュ・ダイアグノスティックス株式会社)を用い、常法に従って行った。アルカリホスファターゼで標識したジゴキシゲニン抗体を用いて検出し、検出結果から目的遺伝子周辺の制限酵素マップ（図４）を作製した。 Using the above DNA fragment as a template, PCR was performed using the designed primers and PCR® DIG® Probe® Synthesis® Kit (Roche Diagnostics) to obtain a DNA probe labeled with digoxigenin. Southern hybridization was performed using this probe. Chromosomal DNA was completely digested with restriction enzymes BamHI, KpnI, PstI, SacI, SphI, and XbaI, and those digested with a combination of these restriction enzymes and restriction enzyme SalI were separated by 0.8% agarose electrophoresis. Subsequently, the membrane was transferred to a zeta probe membrane (Bio-Rad Inc.) to obtain a membrane for Southern hybridization. Southern hybridization was performed according to a conventional method using DIG Easy Hyb. (Roche Diagnostics). Detection was performed using a digoxigenin antibody labeled with alkaline phosphatase, and a restriction enzyme map (FIG. 4) around the target gene was prepared from the detection results.
　目的DNA周辺の制限酵素マップより、制限酵素SphIで完全消化した約5.7Kbpの断片中に目的の遺伝子が含まれることが明らかとなったため、染色体DNAを制限酵素SphIで完全消化したものを0.8％アガロース電気泳動後、約5.7Kbp付近の断片をアガロースより回収し、pUC18(タカラバイオ株式会社)プラスミドの制限酵素SphIサイトに挿入した。当該組換えプラスミドで形質転換した大腸菌（E.coli）JM109(タカラバイオ株式会社)を1000株作製した。 The restriction enzyme map around the target DNA revealed that the target gene was contained in a fragment of about 5.7 Kbp that was completely digested with the restriction enzyme SphI, so 0.8% of the chromosomal DNA completely digested with the restriction enzyme SphI After agarose electrophoresis, a fragment of about 5.7 Kbp was recovered from agarose and inserted into the restriction enzyme SphI site of pUC18 (Takara Bio Inc.) plasmid. 1,000 strains of E. coli JM109 (Takara Bio Inc.) transformed with the recombinant plasmid were prepared.
　得られた形質転換体のコロニーをナイロンメンブレン(ロシュ・ダイアグノスティックス株式会社)に転写した後、DIG Easy Hyb. (ロシュ・ダイアグノスティックス株式会社)を用いてコロニーハイブリダイゼーションを常法で行ったところ、ポジティブクローンが数株得られた。当該クローンからプラスミドを回収し、常法に従い塩基配列を決定した。当該塩基配列（配列番号２）から予想されるアミノ酸配列（配列番号１）は上記３．で決定した精製酵素のN末端及び内部アミノ酸配列を含んでいた。このことから、得られた組換えプラスミドにはグルコースデヒドロゲナーゼをコードする遺伝子が含まれることが確認された。 After transferring the colonies of the obtained transformant to a nylon membrane (Roche Diagnostics Inc.), colony hybridization is performed in a conventional manner using DIG Easy Hyb. (Roche Diagnostics Inc.). As a result, several positive clones were obtained. A plasmid was recovered from the clone and the nucleotide sequence was determined according to a conventional method. The amino acid sequence (SEQ ID NO: 1) deduced from the base sequence (SEQ ID NO: 2) is the above 3. The N-terminal and internal amino acid sequences of the purified enzyme determined in (1) were included. From this, it was confirmed that the obtained recombinant plasmid contained a gene encoding glucose dehydrogenase.
５．アスペルギルス・オリゼ発現系での変異導入酵素の発現および評価
　Aspergillus iizukae No.5453株由来GDHの遺伝子配列（配列番号２）をもとに以下のプライマーを設計した。
　5453K354_FW: cctgtctcctaccccaac（配列番号１６）
　5453K354V_R1: ggggtaggagacaggaactccaccggagagagt（配列番号１７）
　5453K354I_R2: ggggtaggagacagggattccaccggagagagt（配列番号１８）
　5453K354P_R3: ggggtaggagacaggaggtccaccggagagagt（配列番号１９）
　5453K354Q_R4: ggggtaggagacaggttgtccaccggagagagt（配列番号２０） 5). Expression and evaluation of mutation-inducing enzyme in Aspergillus oryzae expression system The following primers were designed based on the gene sequence of GDH derived from Aspergillus iizukae No.5453 (SEQ ID NO: 2).
5453K354_FW: cctgtctcctaccccaac (SEQ ID NO: 16)
5453K354V_R1: ggggtaggagacaggaactccaccggagagagt (SEQ ID NO: 17)
5453K354I_R2: ggggtaggagacagggattccaccggagagagt (SEQ ID NO: 18)
5453K354P_R3: ggggtaggagacaggaggtccaccggagagagt (SEQ ID NO: 19)
5453K354Q_R4: ggggtaggagacaggttgtccaccggagagagt (SEQ ID NO: 20)
　Aspergillus iizukae No.5453株由来GDHの遺伝子配列がタカアミラーゼ改変CS3プロモーターとアスペルギルス・オリゼ由来FAD依存性グルコースデヒドロゲナーゼのターミネータ遺伝子の間につながれた発現カセットと、アスペルギルス・オリゼ由来オロチジン5'-リン酸デカルボキシラーゼ遺伝子(pyrG遺伝子)がpUC19に挿入された発現プラスミドpUCPGDH5453をテンプレートにして、設計したプライマーとPrimeSTAR（登録商標）Max DNA Polymerase（タカラバイオ株式会社）を用いてインバースPCRの原理を用いてPCRを行い、増幅されたDNA断片を得た。PCR条件は以下の通りとした。
（反応液）
　PrimeSTAR Max Premix（2×）　25μL
　FWプライマー　15 pmol
　R1～4プライマー　15 pmol
　プラスミドDNA　1μL
　滅菌蒸留水で50μLに調整
（サイクル条件）
　98℃で10秒、55℃で15秒、72℃で2分の条件で35サイクル An Aspergillus iizukae No.5453 GDH gene sequence comprising an expression cassette connected between the Takaamylase-modified CS3 promoter and an Aspergillus oryzae-derived FAD-dependent glucose dehydrogenase terminator gene, and an Aspergillus oryzae-derived orotidine 5'-phosphate de Using the expression plasmid pUCPGDH5453 with the carboxylase gene (pyrG gene) inserted into pUC19 as a template, PCR was performed using the designed primer and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) using the principle of inverse PCR. And an amplified DNA fragment was obtained. PCR conditions were as follows.
(Reaction solution)
PrimeSTAR Max Premix (2 ×) 25μL
FW primer 15 pmol
R1-4 primer 15 pmol
Plasmid DNA 1μL
Adjust to 50 μL with sterile distilled water (cycle conditions)
35 cycles at 98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 2 minutes
　得られたDNA断片をリン酸化後、ライゲーションを行い、続いて大腸菌に形質転換を行い、変異が導入された発現プラスミドを取得した。構築された発現プラスミドを用いて、アスペルギルス・オリゼRIB40のpyrG遺伝子欠損株を形質転換し、ウリジン要求性を利用して形質転換株を取得した。 The obtained DNA fragment was phosphorylated and then ligated, followed by transformation into E. coli to obtain an expression plasmid into which the mutation was introduced. Using the constructed expression plasmid, a pyrG gene-deficient strain of Aspergillus oryzae RIB40 was transformed, and a transformant was obtained using uridine requirement.
　得られた形質転換株を用いて、可溶性でんぷんをC源にタカアミラーゼ誘導条件で液体培養し、変異酵素を含む培養液を取得した。得られた培養液から各種精製を行い、部分精製された変異酵素を取得した。部分精製した変異酵素用いて、以下の方法で至適温度を評価した。 Using the obtained transformant, liquid culture was carried out using soluble starch as a C source under takaamylase induction conditions to obtain a culture solution containing a mutant enzyme. Various purifications were performed from the obtained culture broth to obtain a partially purified mutant enzyme. Using the partially purified mutant enzyme, the optimum temperature was evaluated by the following method.
至適温度評価方法
　本GDHは、電子受容体存在下でグルコースの水酸基を酸化してグルコノ－δ－ラクトンを生成する反応を触媒する。GDH活性の検出は、下記の反応系で行った。
Figure JPOXMLDOC01-appb-M000002
 　尚、式中のPMSはPhenazine methosulfateを表し、DCIPは2,6-Dichlorophenol-indophenol solutionを表す。
　反応（１）において、グルコースの酸化に伴って還元型PMSが生成し、更に反応（２）において還元型PMSによるDCIPの還元により生成した還元型DCIPを600nmの波長で測定する。 Optimal Temperature Evaluation Method The present GDH catalyzes a reaction in which a hydroxyl group of glucose is oxidized to produce glucono-δ-lactone in the presence of an electron acceptor. GDH activity was detected by the following reaction system.
Figure JPOXMLDOC01-appb-M000002
 In the formula, PMS represents Phenazine methosulfate, and DCIP represents 2,6-Dichlorophenol-indophenol solution.
In the reaction (1), reduced PMS is generated with the oxidation of glucose, and the reduced DCIP generated by the reduction of DCIP by the reduced PMS in the reaction (2) is measured at a wavelength of 600 nm.
　酵素活性（ユニット）は以下の計算式によって算出される。
Figure JPOXMLDOC01-appb-M000003
 　尚、式中のVtは総液量を、Vsはサンプル量を、16.3は還元型DCIPの1μmol当たりの吸光係数（cm2/μmol）を、1.0は光路長（cm）を、dfは希釈倍数をそれぞれ表す。 The enzyme activity (unit) is calculated by the following formula.
Figure JPOXMLDOC01-appb-M000003
 In the formula, Vt is the total liquid volume, Vs is the sample volume, 16.3 is the extinction coefficient (cm 2 / μmol) per 1 mol of reduced DCIP, 1.0 is the optical path length (cm), and df is the dilution factor. Respectively.
　50mmol/L リン酸緩衝液pH 6.5 2.05mL、1mol/L グルコース溶液0.60mL、15mmol/L PMS溶液 0.10mL、2mmol/L DCIP溶液 0.15mLを混合し、各種測定温度で5分間保温後、0.1%BSAを含む50mmol/L リン酸緩衝液pH 6.5で希釈した酵素溶液を0.1mL添加し、反応を開始した。酵素反応の進行と共に600nmに吸収を持つ還元型DCIPが生成される。１分間あたりの600nmにおける吸光度の増加を測定することにより、GDH活性を測定した。 50 mmol / L phosphate buffer solution pH 6.5 to 2.05 mL, 1 mol / L glucose solution 0.60 mL, 15 mmol / L PMS solution 0.10 mL, 2 mmol / L DCIP solution 0.15 mL, mixed at various measurement temperatures for 5 minutes, 0.1% 0.1 mL of an enzyme solution diluted with 50 mmol / L phosphoric acid buffer solution pH 6.5 containing BSA was added to initiate the reaction. Reduced DCIP having absorption at 600 nm is generated as the enzymatic reaction proceeds. GDH activity was measured by measuring the increase in absorbance at 600 nm per minute.
　測定結果を図５に示す。354位のリジンをバリン、イソロイシン、プロリン、又はグルタミンに置換（部位特異的変異を導入）した変異酵素（K354V変異酵素、K354I変異酵素、K354P変異酵素、K354Q変異酵素）は、野生型酵素と比較して、37℃に対する20℃での活性（相対活性）が向上していた。尚、各変異酵素のアミノ酸配列及びそれをコードする遺伝子配列は以下の通りである。
　K354V変異酵素：配列番号２１（アミノ酸配列）；配列番号２５（遺伝子配列）
　K354I変異酵素：配列番号２２（アミノ酸配列）；配列番号２６（遺伝子配列）
　K354P変異酵素：配列番号２３（アミノ酸配列）；配列番号２７（遺伝子配列）
　K354Q変異酵素：配列番号２４（アミノ酸配列）；配列番号２８（遺伝子配列） The measurement results are shown in FIG. Mutant enzyme (K354V mutant enzyme, K354I mutant enzyme, K354P mutant enzyme, K354Q mutant enzyme) in which lysine at position 354 is replaced with valine, isoleucine, proline, or glutamine (site-specific mutation is introduced) is compared with the wild-type enzyme Thus, the activity at 20 ° C. relative to 37 ° C. (relative activity) was improved. The amino acid sequence of each mutant enzyme and the gene sequence encoding it are as follows.
K354V mutant enzyme: SEQ ID NO: 21 (amino acid sequence); SEQ ID NO: 25 (gene sequence)
K354I mutant enzyme: SEQ ID NO: 22 (amino acid sequence); SEQ ID NO: 26 (gene sequence)
K354P mutant enzyme: SEQ ID NO: 23 (amino acid sequence); SEQ ID NO: 27 (gene sequence)
K354Q mutant enzyme: SEQ ID NO: 24 (amino acid sequence); SEQ ID NO: 28 (gene sequence)
６．サッカロマイセス・セレビシエ発現系での変異導入酵素の発現、及び基質特異性の評価
　Aspergillus iizukae No.5453株由来GDHの遺伝子配列をもとに、以下のプライマーを設計した。
　GD41F: ccacagaaggcatttatgttgggcaaactcacgttctt（配列番号２９）
　GD42R: gctttatctaccaaactacacagcagcagcatcgg（配列番号３０） 6). Expression of mutation-inducing enzyme in Saccharomyces cerevisiae expression system and evaluation of substrate specificity Based on the gene sequence of GDH derived from Aspergillus iizukae No.5453, the following primers were designed.
GD41F: ccacagaaggcatttatgttgggcaaactcacgttctt (SEQ ID NO: 29)
GD42R: gctttatctaccaaactacacagcagcagcatcgg (SEQ ID NO: 30)
　５．の実験で構築した変異酵素発現用ベクターをテンプレートにして、設計したプライマーとPrimeSTAR（登録商標） Max DNA Polymerase（タカラバイオ株式会社）を用いてPCRを行い、増幅されたDNA断片を得た。PCR条件は以下の通りとした。
（反応液）
　PrimeSTAR Max Premix（2×）　25μL
　GD41F　15 pmol
　GD42R　15 pmol
　ゲノムDNA(1/1000希釈)　1μL
　滅菌蒸留水で50μLに調整
（サイクル条件）
　98℃で10秒、55℃で15秒、72℃で2分の条件で35サイクル 5). PCR was carried out using the designed primer and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) using the mutant enzyme expression vector constructed in the above experiment as a template to obtain an amplified DNA fragment. PCR conditions were as follows.
(Reaction solution)
PrimeSTAR Max Premix (2 ×) 25μL
GD41F 15 pmol
GD42R 15 pmol
Genomic DNA (1/1000 dilution) 1μL
Adjust to 50 μL with sterile distilled water (cycle conditions)
35 cycles at 98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 2 minutes
　PCR後の増幅産物をサッカロマイセス・セレビシエ発現系pYES2プラスミドに挿入して、変異導入後のプラスミドを構築した。構築された変異導入後のプラスミドを大腸菌DH5αに形質転換後、プラスミド抽出を行い、変異ライブラリーを作製した。得られたライブラリーをサッカロマイセス・セレビシエINVSc1（インビトロジェン社）に形質転換した。得られた形質転換株について液体培養を行い、GDH活性、及びキシロースを基質にした際の基質特異性を調べた。尚、培養実験操作はpYES2のマニュアルを参考にした。 The amplified product after PCR was inserted into Saccharomyces cerevisiae expression system pYES2 plasmid to construct a plasmid after mutagenesis. The constructed plasmid after mutation introduction was transformed into Escherichia coli DH5α, followed by plasmid extraction to prepare a mutation library. The obtained library was transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen). The obtained transformant was subjected to liquid culture, and GDH activity and substrate specificity when xylose was used as a substrate were examined. In addition, culture experiment operation referred to the manual of pYES2.
基質特異性評価方法
　本GDHは、電子受容体存在下でグルコースの水酸基を酸化してグルコノ－δ－ラクトンを生成する反応を触媒する。GDH活性の検出は、下記の反応系で行った。
Figure JPOXMLDOC01-appb-M000004
 　尚、式中の1-Methoxy PMSは1-Methoxy phenazine methosulfateを表し、NTBはNitrotetrazorium blueを表す。 Substrate specificity evaluation method The present GDH catalyzes a reaction in which the hydroxyl group of glucose is oxidized to produce glucono-δ-lactone in the presence of an electron acceptor. GDH activity was detected by the following reaction system.
Figure JPOXMLDOC01-appb-M000004
 In the formula, 1-Methoxy PMS represents 1-Methoxy phenazine methosulfate, and NTB represents Nitrotetrazorium blue.
　反応（１）において、グルコースの酸化に伴って還元型1-Methoxy PMSが生成し、更に反応（２）において還元型1-Methoxy PMSによるNTBの還元により生成したDiformazanを570nmの波長で測定する。 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.
　0.1% Triton X-100を含む50mmol/L PIPES-NaOH pH 7.0 24mL、1mol/L 基質溶液（グルコース又はキシロース） 3mL、3mmol/L 1-Methoxy PMS溶液 1mL、6.6mmol/L NTB溶液 3mLを混合し、アッセイ用試薬を調製した。各基質を含むアッセイ用試薬200μL対して20μLの培養上清を添加し、37℃で１時間反応させた後の570nmの吸光度をプレートリーダーにて測定した。キシロースへの反応性は、グルコースへの反応性を100％としたときの相対値（キシロースを基質とした場合の測定値／グルコースを基質とした場合の測定値　×　１００）で表した。また、野性型酵素のキシロースへの反応性の値を100％とした場合の相対値も求めた。 Mix 50 mmol / L PIPES-NaOH pH 0.1 to 24 mL, 1 mol / L substrate solution (glucose or xylose) 3 mL, 3 mmol / L 1-Methoxy PMS solution 1 mL, 6.6 mmol / L NTB solution 3 mL containing 0.1% Triton X-100. An assay reagent was prepared. 20 μL of the culture supernatant was added to 200 μL of the assay reagent containing each substrate, and after reacting at 37 ° C. for 1 hour, the absorbance at 570 nm was measured with a plate reader. The reactivity to xylose was expressed as a relative value when the reactivity to glucose was 100% (measured value when xylose was used as a substrate / measured value when glucose was used as a substrate x 100). Moreover, the relative value when the reactivity value of the wild-type enzyme to xylose was defined as 100% was also determined.
　結果を以下の表に示す。354位のリジンをバリン、イソロイシン、プロリン、又はグルタミンに置換した変異酵素はキシロースへの反応性低下が顕著であり、野生型酵素に比較して格段に優れた基質特異性を示した。
Figure JPOXMLDOC01-appb-T000005
 The results are shown in the table below. The mutant enzyme in which lysine at position 354 was substituted with valine, isoleucine, proline, or glutamine showed a marked decrease in reactivity to xylose, and showed a substrate specificity that was markedly superior to that of the wild-type enzyme.
Figure JPOXMLDOC01-appb-T000005
　本発明のグルコースデヒドロゲナーゼは低温安定性に優れる。従って、低温環境下での利用が想定される用途（典型的には血糖測定器用のグルコースセンサ）に適する。本発明のグルコースデヒドロゲナーゼは基質特異性も改善されており、特にグルコースセンサへの利用に適したものである。 The glucose dehydrogenase of the present invention is excellent in low temperature stability. Therefore, it is suitable for applications that are expected to be used in a low temperature environment (typically a glucose sensor for a blood glucose meter). The glucose dehydrogenase of the present invention has improved substrate specificity and is particularly suitable for use in a glucose sensor.
　この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 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.
　この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 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.
　配列番号４：人工配列の説明：プライマーGDH5453-F
　配列番号５：人工配列の説明：プライマーGDH5453-5-1-R
　配列番号６：人工配列の説明：PCR産物
　配列番号７：人工配列の説明：プライマーFS51R07F
　配列番号８：人工配列の説明：プライマーFS51R07R
　配列番号１６：人工配列の説明：プライマー5453K354_FW
　配列番号１７：人工配列の説明：プライマー5453K354V_R1
　配列番号１８：人工配列の説明：プライマー5453K354I_R2
　配列番号１９：人工配列の説明：プライマー5453K354P_R3
　配列番号２０：人工配列の説明：プライマー5453K354Q_R4
　配列番号２１：人工配列の説明：K354V変異酵素
　配列番号２２：人工配列の説明：K354I変異酵素
　配列番号２３：人工配列の説明：K354P変異酵素
　配列番号２４：人工配列の説明：K354Q変異酵素
　配列番号２５：人工配列の説明：K354V変異酵素
　配列番号２６：人工配列の説明：K354I変異酵素
　配列番号２７：人工配列の説明：K354P変異酵素
　配列番号２８：人工配列の説明：K354Q変異酵素
　配列番号２９：人工配列の説明：プライマーGD41F
　配列番号３０：人工配列の説明：プライマーGD42R Sequence number 4: Description of artificial sequence: Primer GDH5453-F
Sequence number 5: Description of artificial sequence: Primer GDH5453-5-1-R
SEQ ID NO: 6: description of artificial sequence: PCR product SEQ ID NO: 7: description of artificial sequence: primer FS51R07F
SEQ ID NO: 8: Description of artificial sequence: Primer FS51R07R
SEQ ID NO: 16: Description of artificial sequence: Primer 5453K354_FW
SEQ ID NO: 17: Description of artificial sequence: Primer 5453K354V_R1
SEQ ID NO: 18: Description of artificial sequence: Primer 5453K354I_R2
SEQ ID NO: 19: Description of artificial sequence: Primer 5453K354P_R3
SEQ ID NO: 20: Description of artificial sequence: Primer 5453K354Q_R4
SEQ ID NO: 21: description of artificial sequence: K354V mutant enzyme SEQ ID NO: 22: description of artificial sequence: K354I mutant enzyme SEQ ID NO: 23: description of artificial sequence: K354P mutant enzyme SEQ ID NO: 24: description of artificial sequence: K354Q mutant enzyme SEQ ID NO: 25: Description of artificial sequence: K354V mutant enzyme SEQ ID NO: 26: Description of artificial sequence: K354I mutant enzyme SEQ ID NO: 27: Description of artificial sequence: K354P mutant enzyme SEQ ID NO: 28: Description of artificial sequence: K354Q mutant enzyme SEQ ID NO: 29: Description of artificial sequence: Primer GD41F
SEQ ID NO: 30: Description of artificial sequence: Primer GD42R

Claims (13)

  1. 　以下の(a)又は(b)のアミノ酸配列を有する、グルコースデヒドロゲナーゼ：
    　(a)配列番号１のアミノ酸配列の354位リジンがバリン、イソロイシン、プロリン又はグルタミンに置換されたアミノ酸配列；
    　(b)(a)のアミノ酸配列との同一性が80%以上のアミノ酸配列であって、該アミノ酸配列からなるポリペプチドは、配列番号１のアミノ酸配列からなるポリペプチドに比較して、グルコースデヒドロゲナーゼ活性の低温反応性が向上している、アミノ酸配列。     Glucose dehydrogenase having the following amino acid sequence (a) or (b):
    (a) an amino acid sequence in which the lysine at position 354 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine, isoleucine, proline or glutamine;
    (b) an amino acid sequence having 80% or more identity with the amino acid sequence of (a), wherein the polypeptide comprising the amino acid sequence is a glucose dehydrogenase as compared to the polypeptide comprising the amino acid sequence of SEQ ID NO: 1. An amino acid sequence with improved low temperature reactivity of activity.
  2. 　前記同一性が85%以上である、請求項１に記載のグルコースデヒドロゲナーゼ。 The glucose dehydrogenase according to claim 1, wherein the identity is 85% or more.
  3. 　前記同一性が90%以上である、請求項１に記載のグルコースデヒドロゲナーゼ。 The glucose dehydrogenase according to claim 1, wherein the identity is 90% or more.
  4. 　配列番号２１～２４のいずれかのアミノ酸配列からなる、請求項１に記載のグルコースデヒドロゲナーゼ。 The glucose dehydrogenase according to claim 1, consisting of the amino acid sequence of any one of SEQ ID NOs: 21 to 24.
  5. 　以下の(A)～(C)からなる群より選択されるいずれかのDNAからなるグルコースデヒドロゲナーゼ遺伝子：
    　(A)配列番号２１～２４のいずれかのアミノ酸配列をコードするDNA；
    　(B)配列番号２５～２８のいずれかの塩基配列からなるDNA；
    　(C)配列番号２５～２８のいずれかの塩基配列と等価な塩基配列を有し、且つグルコースデヒドロゲナーゼ活性を有するタンパク質をコードするDNA。     Glucose dehydrogenase gene consisting of any DNA selected from the group consisting of (A) to (C) below:
    (A) DNA encoding any one of the amino acid sequences of SEQ ID NOs: 21 to 24;
    (B) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 25 to 28;
    (C) DNA encoding a protein having a base sequence equivalent to any one of SEQ ID NOs: 25 to 28 and having glucose dehydrogenase activity.
  6. 　請求項５に記載のグルコースデヒドロゲナーゼ遺伝子を含む組換えDNA。 Recombinant DNA containing the glucose dehydrogenase gene according to claim 5.
  7. 　請求項６に記載の組換えDNAを保有する微生物。 A microorganism having the recombinant DNA according to claim 6.
  8. 　以下のステップ(1)～(3)を含む、グルコースデヒドロゲナーゼの調製法：
    　(1)請求項５に記載のグルコースデヒドロゲナーゼ遺伝子を用意するステップ；
    　(2)前記遺伝子を発現させるステップ、及び
    　(3)発現産物を回収するステップ。     A method for preparing glucose dehydrogenase comprising the following steps (1) to (3):
    (1) A step of preparing the glucose dehydrogenase gene according to claim 5;
    (2) expressing the gene, and (3) collecting the expression product.
  9. 　請求項１～４のいずれか一項に記載のグルコースデヒドロゲナーゼを用いて試料中のグルコースを測定することを特徴とする、グルコース測定法。 A glucose measuring method, wherein glucose in a sample is measured using the glucose dehydrogenase according to any one of claims 1 to 4.
  10. 　請求項１～４のいずれか一項に記載のグルコースデヒドロゲナーゼを含む、グルコース測定用試薬。 A glucose measurement reagent comprising the glucose dehydrogenase according to any one of claims 1 to 4.
  11. 　請求項１０に記載のグルコース測定用試薬を含む、グルコース測定用キット。 A glucose measurement kit comprising the glucose measurement reagent according to claim 10.
  12. 　請求項１～４のいずれか一項に記載のグルコースデヒドロゲナーゼを含む、グルコースセンサ。 A glucose sensor comprising the glucose dehydrogenase according to any one of claims 1 to 4.
  13. 　請求項１～４のいずれか一項に記載のグルコースデヒドロゲナーゼを含有する酵素剤。 An enzyme agent comprising the glucose dehydrogenase according to any one of claims 1 to 4.
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