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WO2018062103A1 - Glucose déshydrogénase - Google Patents

Glucose déshydrogénase Download PDF

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Publication number
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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Prior art keywords
amino acid
enzyme
glucose
seq
acid sequence
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English (en)
Japanese (ja)
Inventor
享一 西尾
裕三 小嶋
庄太郎 山口
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Amano Enzyme Inc
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Amano Enzyme Inc
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Priority to JP2018542558A priority Critical patent/JP7044709B2/ja
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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

La présente invention vise à fournir une nouvelle FAD-GDH très pratique. L'invention concerne par conséquent une glucose déshydrogénase ayant la séquence d'acides aminés de (a) ou (b) comme suit. (a) La séquence d'acides aminés de SEQ ID NO : 1 dans laquelle la lysine en position 354 a été substituée par une valine, une isoleucine, une proline ou une glutamine, ou (b) une séquence d'acides aminés qui est identique à au moins 80 % à la séquence d'acides aminés de (a), un polypeptide qui possède ladite séquence d'acides aminés ayant une réactivité à basse température améliorée en termes d'activité de la glucose déshydrogénase comparé à un polypeptide ayant la séquence d'acides aminés de SEQ ID NO : 1.
PCT/JP2017/034547 2016-09-28 2017-09-25 Glucose déshydrogénase Ceased WO2018062103A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2019230614A1 (fr) * 2018-05-29 2019-12-05 天野エンザイム株式会社 Perfectionnement apporté à la glucose déshydrogénase

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WO2004058958A1 (fr) * 2002-12-24 2004-07-15 Ikeda Food Research Co., Ltd. Glucose dehydrogenase de liaison coenzymatique
WO2007139013A1 (fr) * 2006-05-29 2007-12-06 Amano Enzyme Inc. Glucose déshydrogénase de liaison au flavine-adénine-dinucléotide
JP2011217731A (ja) * 2010-03-26 2011-11-04 Toyobo Co Ltd 改変型フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼ
WO2015060150A1 (fr) * 2013-10-21 2015-04-30 東洋紡株式会社 Glucose déshydrogénase inédite
JP2015146773A (ja) * 2014-02-06 2015-08-20 東洋紡株式会社 新規なグルコースデヒドロゲナーゼ
JP2016116488A (ja) * 2014-12-22 2016-06-30 東洋紡株式会社 新規なグルコースデヒドロゲナーゼ
WO2017077924A1 (fr) * 2015-11-06 2017-05-11 天野エンザイム株式会社 Nouvelle glucose déshydrogénase

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Publication number Priority date Publication date Assignee Title
WO2004058958A1 (fr) * 2002-12-24 2004-07-15 Ikeda Food Research Co., Ltd. Glucose dehydrogenase de liaison coenzymatique
WO2007139013A1 (fr) * 2006-05-29 2007-12-06 Amano Enzyme Inc. Glucose déshydrogénase de liaison au flavine-adénine-dinucléotide
JP2011217731A (ja) * 2010-03-26 2011-11-04 Toyobo Co Ltd 改変型フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼ
WO2015060150A1 (fr) * 2013-10-21 2015-04-30 東洋紡株式会社 Glucose déshydrogénase inédite
JP2015146773A (ja) * 2014-02-06 2015-08-20 東洋紡株式会社 新規なグルコースデヒドロゲナーゼ
JP2016116488A (ja) * 2014-12-22 2016-06-30 東洋紡株式会社 新規なグルコースデヒドロゲナーゼ
WO2017077924A1 (fr) * 2015-11-06 2017-05-11 天野エンザイム株式会社 Nouvelle glucose déshydrogénase

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019230614A1 (fr) * 2018-05-29 2019-12-05 天野エンザイム株式会社 Perfectionnement apporté à la glucose déshydrogénase

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