WO2010053161A1 - Glucose déshydrogénase flavine-adénine-dinucléotide-dépendante modifiée - Google Patents

Glucose déshydrogénase flavine-adénine-dinucléotide-dépendante modifiée Download PDF

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WO2010053161A1
WO2010053161A1 PCT/JP2009/069006 JP2009069006W WO2010053161A1 WO 2010053161 A1 WO2010053161 A1 WO 2010053161A1 JP 2009069006 W JP2009069006 W JP 2009069006W WO 2010053161 A1 WO2010053161 A1 WO 2010053161A1
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amino acid
fadgdh
acid sequence
seq
mutation
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Japanese (ja)
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理文 八尾
仁司 近藤
伸一 横堀
明彦 山岸
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ユニチカ株式会社
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)

Definitions

  • the present invention relates to an active FADGDH gene obtained by inserting a specific amino acid sequence into a flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter referred to as FADGDH) -like protein derived from Aspergillus oryzae RIB40 strain, and
  • the present invention relates to an active FADGDH consisting of an amino acid sequence encoded by the gene.
  • the present invention comprises a modified FADGDH gene whose substrate affinity and / or specific activity is improved by changing a specific amino acid of the active FADGDH to another amino acid, and an amino acid sequence encoded by the gene It relates to modified FADGDH.
  • a typical example is a glucose sensor mainly used in the medical field.
  • the glucose sensor is for constructing a reaction system including an enzyme and an electron transfer substance.
  • glucose is quantified using, for example, an amperometric technique.
  • enzymes glucose oxidase (GOD) and glucose dehydrogenase (GDH) have been used.
  • GOD has high substrate specificity for glucose and excellent thermal stability, and has the advantage that the production cost is low compared to other enzymes because mass production of the enzyme is possible. Since the used system is easily affected by dissolved oxygen in the measurement sample, there is a problem that the dissolved oxygen affects the measurement result.
  • the system using GDH is not easily affected by dissolved oxygen in the measurement sample. For this reason, the system using GDH can measure the glucose concentration accurately even when measuring in an environment where the oxygen partial pressure is low or measuring a high concentration sample that requires a large amount of oxygen. Can do.
  • GDH is superior to GOD in that it is not affected by dissolved oxygen.
  • NADGDH in which addition of NAD is essential as a coenzyme is poor in stability
  • PQQGDH having pyrrolopinolinequinone (PQQ) as a coenzyme is a substrate.
  • an enzyme used as a glucose sensor is not affected by dissolved oxygen, and an enzyme having low reactivity to sugars other than glucose is desired.
  • FADGDH having flavin adenine dinucleotide (FAD) as a coenzyme can be cited as an enzyme that satisfies the above specifications.
  • FAD flavin adenine dinucleotide
  • the purified enzyme was shown to have high substrate specificity and high specific activity. (See Non-Patent Document 1).
  • the activity per culture broth of Aspergillus oryzae was low and was insufficient for industrial production.
  • the gene sequence of the enzyme is unknown, industrial mass production by genetic recombination was impossible.
  • there have been many problems such as isolation and purification of the enzyme, decoding of a partial amino acid sequence, and decoding of a gene sequence encoding the enzyme.
  • the full-length FADGDH gene has been recently cloned from mRNA extracted from Aspergillus oryzae T1 strain using the whole genome sequence database of Aspergillus oryzae RIB40 strain (see Patent Document 3), and random mutation using PCR Heat-stabilized FADGDH and the like have been obtained by a method combining the introduction method and the screening of a heat-stabilized enzyme (Patent Document 4).
  • An object of the present invention is to develop and provide a more practical enzyme for blood glucose measurement sensor. More specifically, an active FADGDH gene is obtained and modified by a genetic engineering technique so as to have higher performance. More specifically, it is to obtain a modified FADGDH with improved substrate affinity and specific activity so that it can withstand widespread use in sensor applications.
  • the present inventors use a National Center forology Biotechnology Information (NCBI) database including partial amino acid sequence information of FADGDH obtained by culturing Aspergillus oryzae and whole genome information of Aspergillus oryzae.
  • NCBI National Center forology Biotechnology Information
  • a FADGDH-like gene was obtained, but the FADGDH-like protein encoded by the gene did not have GDH activity. Therefore, the present inventors have determined the cause of the absence of GDH activity in the FADGDH-like protein using the published amino acid sequence of FADGDH derived from Aspergillus terreus, NCBI database, and bioinformatics and genetic engineering techniques, An active FADGDH was obtained by inserting a specific amino acid sequence into the amino acid sequence of the protein.
  • the first aspect of the present invention is an amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3, between positions 202 and 203 of the amino acid sequence, GIPVT, GIPRT, GIPQT, GIPTT, An amino acid sequence in which a 5-amino acid sequence selected from the group consisting of GYPVT and GYPRT is inserted, and the amino acid sequence “MLFSLAFLSALSLATA” from the 1st position to the 16th position or the amino acid sequence “MLFSLAFLSALSLATASPAGRA” from the 1st position to the 22nd position is deleted
  • FDGDH active flavin adenine dinucleotide-dependent glucose dehydrogenase
  • the second aspect of the present invention is the amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3, wherein methionine (M) at position 202 of the amino acid sequence is substituted with alanine (A);
  • a 5-amino acid sequence selected from the group consisting of GIPVT, GIPRT, GYPVT and GYPRT is inserted between position 1 and position 203, and the amino acid sequence “MLFSLAFLSALSLATA” from position 1 to position 16 or from position 1 to position 22
  • FDGDH active flavin adenine dinucleotide-dependent glucose dehydrogenase
  • the third aspect of the present invention is the active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) described in the first and second aspects, wherein the following groups (1) to (33) are used:
  • the gist is a modified FADGDH in which at least one amino acid substitution selected is made and the Km value is reduced compared to the corresponding active FADGDH before the modification.
  • the fourth aspect of the present invention is the active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) described in the first and second aspects, comprising the following (1) to (27):
  • the gist of the present invention is a modified FADGDH in which at least one amino acid substitution selected from the group is made and the specific activity value is increased as compared with the corresponding active FADGDH before modification.
  • an amino acid sequence in which at least one modification selected from deletion, substitution and addition of one or more amino acids in the amino acid sequence of the active FADGDH or the modified FADGDH described above is provided.
  • the gist of the present invention is the active FADGDH or modified FADGDH.
  • the sixth aspect of the present invention is a gist of a DNA represented by a DNA sequence encoding the amino acid sequence of the active FADGDH or modified FADGDH described above.
  • the codon usage is Escherichia coli.
  • the gist is DNA which is a codon usage optimized for the above.
  • the seventh aspect of the present invention is summarized as a recombinant expression plasmid containing the above-described DNA.
  • the eighth aspect of the present invention is characterized by a transformant obtained by transforming the above-described recombinant expression plasmid into a host, preferably a transformant whose host is E. coli.
  • the ninth aspect of the present invention is a gist of a method for producing active FADGDH or modified FADGDH, which comprises culturing the above-described transformant.
  • the tenth aspect of the present invention is a glucose assay kit containing any of the above-mentioned active FADGDH or modified FADGDH.
  • the eleventh aspect of the present invention is summarized as a glucose sensor including any one of the active FADGDH or the modified FADGDH described above.
  • a twelfth aspect of the present invention is a gist of a method for measuring a glucose concentration containing any one of the active FADGDH or modified FADGDH described above.
  • active FADGDH has been obtained by inserting a specific amino acid sequence into the amino acid sequence of the FADGDH-like protein derived from Aspergillus oryzae. Furthermore, it has become possible to provide a modified FADGDH in which the Km value, which is an indicator of enzyme substrate affinity, is decreased and / or the specific activity is increased.
  • the purpose of identifying the cause of the absence of GDH activity in the flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) -like protein derived from Aspergillus oryzae RIB40 strain, and expressing the GDH activity in the FADGDH-like protein by site-directed mutagenesis based on the consensus method It is a figure which shows a part of alignment diagram of the amino acid sequence of the homologous protein derived from 15 species used in total. It is a figure which shows the electrophoresis photograph of the refine
  • the left lane (M) is a molecular weight marker.
  • the right lane (active GDH) is active FADGDH.
  • the number on the left is the molecular weight of each band of the molecular weight marker, and the unit is kDa.
  • the arrow on the right side shows a band of active FADGDH isolated and purified.
  • the result of the absorption spectrum from 250 nm to 600 nm is shown.
  • absorption maximums of about 370 nm and about 450 nm were observed.
  • the figure of FIG. 3A was expanded in the range of about 300 nm to 500 nm.
  • Flavin adenine dinucleotide-dependent glucose dehydrogenase is an enzyme having flavin adenine dinucleotide (FAD) as a coenzyme and having glucose dehydrogenase (GDH) activity.
  • the active flavin adenine dinucleotide-dependent glucose dehydrogenase of the present invention (hereinafter also referred to as active FADGDH) is an amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3 at positions 202 and 203 of the amino acid sequence.
  • a 5-amino acid sequence selected from the group consisting of GIPVT, GIPRT, GIPQT, GIPTT, GYPVT and GYPRT is inserted between the positions, and the amino acid sequence “MLFSLAFLSALSLATA” from position 1 to position 16 or positions 1 to 22
  • It is a polypeptide consisting of an amino acid sequence from which the amino acid sequence “MLFSLAFLSALSLATASPAGRA” is deleted.
  • the active FADGDH of the present invention has an amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3, wherein methionine (M) at position 202 of the amino acid sequence is substituted with alanine (A), and 202
  • a 5-amino acid sequence selected from the group consisting of GIPVT, GIPRT, GYPVT and GYPRT is inserted between position 1 and position 203, and the amino acid sequence “MLFSLAFLSALSLATA” from position 1 to position 16 or from position 1 to position 22 Is a polypeptide consisting of an amino acid sequence from which the amino acid sequence “MLFSLAFLSALSLATASPAGRA” is deleted.
  • glycine (Gly) is G
  • alanine (Ala) is A
  • valine (Val) is V
  • leucine (Leu) is L
  • isoleucine (Ile) is I
  • phenylalanine (Phe) is F
  • tyrosine (Tyr) is Y
  • Tryptophan (Trp) is W
  • serine (Ser) is S
  • threonine (Thr) is T
  • cysteine (Cys) is C
  • methionine (Met) is M
  • aspartic acid (Asp) is D
  • glutamic acid (Glu) is E
  • Asparagine (Asn) is N
  • glutamine (Gln) is Q
  • lysine (Lys) is K
  • arginine (Arg) is R
  • histidine (His) is H
  • proline (Pro) is P.
  • N49D means that the 49th amino acid N from the N-terminal side in a specific amino acid sequence is substituted with amino acid D.
  • T111S + T113S means that amino acid substitutions of T111S and T113S are introduced simultaneously.
  • the modified FADGDH of the present invention is a polypeptide having an improved substrate affinity or / and specific activity when converted to a polypeptide having GDH activity as compared to the corresponding active FADGDH before modification described above. is there.
  • the modified FADGDH with improved substrate affinity of the present invention preferably has a reduced Km value, which is an index of substrate affinity, compared to the corresponding active FADGDH before modification, and the Km value is 30 or less.
  • the modified FADGDH having improved substrate affinity according to the present invention is at least one selected from the group consisting of the following (1) to (33) in the above-mentioned active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH): A polypeptide consisting of an amino acid sequence in which amino acid substitutions are made is preferred.
  • the position of the following amino acid substitution is the amino acid position of the amino acid sequence in which 5 amino acid sequences are inserted between positions 202 and 203 of the amino acid sequence of SEQ ID NO: 3, ie, the amino acid sequence of the amino acid sequence of SEQ ID NO: 53 The position corresponding to the residue.
  • the active FADGDH of the present invention at least one amino acid substitution selected from the group consisting of G35A, N49D, A82K, H288Y, T330G, A118I + T120I, A118I + T120I + H288Y, A118I + T120I + T330G, and H288Y + T330G is preferable.
  • the position of the amino acid substitution is the amino acid position of the amino acid sequence in which 5 amino acid sequences are inserted between positions 202 and 203 of the amino acid sequence of SEQ ID NO: 3, that is, the amino acid residue of the amino acid sequence of SEQ ID NO: 53 This is the position corresponding to the group.
  • the Km value is also referred to as Michaelis constant, which is a constant included with the maximum velocity Vmax in the Michaelis-Menten-type enzyme reaction rate equation, and in the case of glucose dehydrogenase, is a numerical value representing affinity for glucose. In the case of enzymes having different Km values for the same substrate, the effect is stronger as the Km is smaller.
  • the Km value in the present invention means a value measured and calculated under the conditions described later.
  • the modified FADGDH with improved specific activity of the present invention preferably has an increased specific activity value compared to the corresponding active FADGDH before modification, and the specific activity value is 700 U / mg-protein or more. And more preferably 1000 U / mg protein or more.
  • FADGDH active flavin adenine dinucleotide-dependent glucose dehydrogenase
  • FADGDH consisting of an amino acid sequence having at least one amino acid substitution selected from the group consisting of the following (1) to (27) is preferable.
  • the position of the following amino acid substitution is the amino acid position of the amino acid sequence in which 5 amino acid sequences are inserted between positions 202 and 203 of the amino acid sequence of SEQ ID NO: 3, ie, the amino acid sequence of the amino acid sequence of SEQ ID NO: 53 The position corresponding to the residue.
  • the active FADGDH of the present invention at least one of S53K, A82K, A107V, I295L, V311I, T330G, A82K + I295L, H288Y + I295L, H288Y + T330G, H288Y + S583A + L585M, T330G + S583 + preferable.
  • the position of the amino acid substitution is the amino acid position of the amino acid sequence in which 5 amino acid sequences are inserted between positions 202 and 203 of the amino acid sequence of SEQ ID NO: 3, that is, the amino acid residue of the amino acid sequence of SEQ ID NO: 53 This is the position corresponding to the group.
  • the specific activity value represents an activity value per unit weight of the enzyme.
  • the specific activity value in the present invention means a value measured and calculated under the conditions described later.
  • N49D amino acid substitution notations.
  • N49D means that the 49th amino acid N from the N-terminal side in a specific amino acid sequence is substituted with amino acid D.
  • T111S + T113S means that amino acid substitutions of T111S and T113S are introduced simultaneously.
  • the active FADGDH and modified FADGDH of the present invention are the above-mentioned active FADGDH or modified FADGDH, wherein part or all of the amino acid sequence corresponding to positions 1 to 22 of the amino acid sequence of SEQ ID NO: 3 is N-terminal.
  • polypeptides removed from Preferably, a polypeptide from which amino acid sequences corresponding to positions 1 to 16 of the amino acid sequence of SEQ ID NO: 3 have been removed, or an amino acid sequence corresponding to positions 1 to 22 of the amino acid sequence of SEQ ID NO: 3 A polypeptide from which is removed.
  • a polypeptide obtained by removing the amino acid sequence “MLFSLAFLSALSLATA” from the 1st position to the 16th position of the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence “MLFSLAFLSALSLATASPAGRA” from the 1st position to the 22nd position of the amino acid sequence of SEQ ID NO: 3 Can be mentioned.
  • an amino acid sequence in which at least one modification selected from deletion, addition, insertion and substitution of one or a plurality of amino acid residues in the amino acid sequences of the active FADGDH and the modified FADGDH described above is provided.
  • a polypeptide having an amino acid sequence in which at least one modification selected from deletion, addition, insertion and substitution of 1 to 20 amino acid residues is preferable, and preferably 1 to 10 amino acids. More preferred is a polypeptide having an amino acid sequence in which at least one modification in which residues are selected from deletion, addition, insertion and substitution is made.
  • polypeptide having an amino acid sequence in which 1 to 6 amino acid residues are at least one modified selected from deletion, addition, insertion and substitution is preferred, and several (1 to 2 or 3) polypeptides are preferred. More preferred is a polypeptide having an amino acid sequence in which at least one modification of amino acid residues selected from deletion, addition, insertion and substitution is made. More preferred is a polypeptide having an amino acid sequence in which one amino acid residue is deleted, added, inserted or substituted. Furthermore, a polypeptide having GDH activity is preferred. Furthermore, the present invention includes a polypeptide having homology to the above-mentioned active FADGDH or modified FADGDH.
  • “homology” refers to the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol) of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. Lett., Vol.174, 247-250, 1999). Examples of parameters include Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • the DNA represented by the DNA sequence encoding the amino acid sequence of the active FADGDH or modified FADGDH of the present invention includes, for example, DNA that can be obtained by a method for synthesizing genes described later or a method for introducing a mutation of DNA.
  • the above DNA or a cell having the same is subjected to mutation treatment, and DNA that hybridizes under stringent conditions with the DNA having the sequence of SEQ ID NO: 3, for example, is selected from these DNAs or cells.
  • the DNA obtained by “Stringent conditions” as used herein refers to conditions under which so-called specific hybrids are formed, but non-specific hybrids are not formed.
  • nucleic acids with high homology for example, DNAs having homology of 70 to 90% or more hybridize, and nucleic acids with lower homology hybridize.
  • under stringent conditions refers to the following conditions, for example.
  • 6 ⁇ SSC (1 ⁇ 1) containing 0.5% SDS, 5 ⁇ Denhartz [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 ⁇ g / ml salmon sperm DNA X SSC refers to the conditions of incubation in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at 50 ° C. to 65 ° C. for 4 hours to overnight. Hybridization can be performed under the stringent conditions described above.
  • a nylon membrane on which a DNA library or cDNA library encoding the active FADGDH or modified FADGDH of the present invention is immobilized is prepared, and 6 ⁇ SSC, 0.5% SDS, 5 ⁇ Denharz, 100 ⁇ g / ml salmon sperm Block nylon membrane at 65 ° C in prehybridization solution containing DNA. Thereafter, each probe labeled with 32P is added and incubated overnight at 65 ° C.
  • This nylon membrane is placed in 6 x SSC for 10 minutes at room temperature, in 2 x SSC containing 0.1% SDS, for 10 minutes at room temperature, in 0.2 x SSC containing 0.1% SDS for 30 minutes at 45 ° C. After washing, autoradiography can be taken to detect DNA that specifically hybridizes with the probe.
  • genes having various homologies can be obtained by changing conditions such as washing.
  • DNA having homology to the above DNA is also included.
  • the homology is at least 80% or more, preferably 90% or more of genes having homology, more preferably 95% or more, more preferably 98% or more.
  • “homology” of DNA refers to the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. FEMS Microbiol. Lett., Vol. 174, 247-250, 1999). Examples of parameters include Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • the DNA is preferably a DNA whose codon usage is optimized for the host, and more preferably a DNA whose codon usage is optimized for E. coli.
  • the total of the optimal host codon usage for each codon is adopted.
  • the optimal codon is defined as the codon having the highest appearance frequency among codons corresponding to the same amino acid.
  • the codon usage is not particularly limited as long as it is optimized for the host. Examples of codon usage of E. coli include the following.
  • F phenylalanine (ttc), L: leucine (ctg), I: isoleucine (atc), M: methionine (atg), V: valine (gta), Y: tyrosine (tac), stop codon (taa), H: Histidine (cac), Q: glutamine (cag), N: asparagine (aac), K: lysine (aag), D: aspartic acid (gac), E: glutamic acid (gaa), S: serine (tct), P: Proline (ccg), T: Threonine (acc), A: Alanine (gct), C: Cysteine (tgg), W: Tryptophan (tgg), R: Arginine (cgt), G: Glycine (ggt)
  • a recombinant expression plasmid (hereinafter also referred to as a recombinant vector) can be obtained.
  • suitable expression vectors are those constructed for gene recombination from phages or plasmids that can autonomously grow in the host. Examples of the phage include Lambda gt10 and Lambda gt11 when Escherichia coli (Escherichia coli) described later is used as a host.
  • examples of plasmids include pBR322, pUC18, pUC118, pUC19, pUC119, pTrc99A, pBluescript, and Super Cos I which is a cosmid when E. coli is used as a host.
  • Pseudomonas RSF1010, pBBR122, pCN51, etc., which are broad host range vectors for Gram-negative bacteria, are exemplified.
  • the produced active FADGDH or modified FADGDH DNA is stably stored in the host in a state of being bound to the expression vector.
  • the host is not particularly limited as long as the recombinant vector is stable, can autonomously propagate, and can express a foreign gene trait.
  • the host may be eukaryotic or prokaryotic, but is preferably a microorganism, preferably Escherichia coli, more preferably Escherichia coli DH5 ⁇ , XL-1 Blue MR, or the like.
  • a competent cell method using calcium treatment, an electroporation method, or the like can be used.
  • Separation and purification of recombinant expression plasmid DNA is performed based on a lysate obtained by lysing a microorganism.
  • a method for lysis for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease, other enzyme, or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as necessary. Further, physical crushing methods such as freeze-thawing and French press treatment may be combined.
  • Separation and purification of DNA from the lysate can be performed by appropriately combining, for example, deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment, and a commercially available kit.
  • the DNA can be cleaved according to a conventional method, for example, using a restriction enzyme treatment.
  • a restriction enzyme for example, a type II restriction enzyme acting on a specific nucleotide sequence is used.
  • the polynucleotide and the expression vector are bound using, for example, DNA ligase.
  • a marker is applied to the recombinant vector, and this recombinant vector is transferred to a host to form a transformant. From this transformant, screening is performed using the expression of the recombinant vector marker and the enzyme activity as an index to obtain a gene-donating microorganism holding the recombinant vector containing the gene encoding FADGDH.
  • the base sequence of the gene encoding FADGDH can be decoded by a known dideoxy method.
  • the amino acid sequence of FADGDH can be deduced from the base sequence determined as described above.
  • the culture form of the transformant may be selected in consideration of the nutritional physiological properties of the host, and is preferably a liquid culture. Industrially, aeration and agitation culture is advantageous.
  • the nutrient source of the medium those commonly used for culturing microorganisms can be used.
  • the carbon source any carbon compound that can be assimilated may be used.
  • the nitrogen source any assimilable nitrogen compound may be used.
  • peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used.
  • salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.
  • the culture temperature can be appropriately changed within the range in which the host grows and the host produces FADGDH, but is preferably about 20 to 37 ° C.
  • the culture may be completed at an appropriate time in anticipation of the time when FADGDH reaches the maximum yield, and the culture time is usually about 12 to 48 hours.
  • the pH of the medium can be appropriately changed within the range where the host grows and the host produces FADGDH, but is preferably in the range of about pH 5.0 to 9.0.
  • a water-soluble fraction containing FADGDH can be obtained by solubilization by using a protease, another enzyme, or a surfactant such as sodium lauryl sulfate (SDS) in combination.
  • the expressed FADGDH can be secreted into the culture medium by selecting an appropriate expression vector and host.
  • the method for purifying the enzyme from the water-soluble fraction containing FADGDH obtained as described above can be performed immediately from this solution, but can also be performed after concentrating FADGDH in this solution.
  • Concentration can be performed by, for example, vacuum concentration, membrane concentration, salting-out treatment, or fractional precipitation with a hydrophilic organic solvent (for example, methanol, ethanol, acetone).
  • Heat treatment and isoelectric point treatment are also effective purification means for the concentration of FADGDH.
  • Purification of the concentrated solution can be performed by appropriately combining, for example, gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography. These methods are already known and can be carried out by referring to appropriate documents, magazines, textbooks and the like.
  • the purified enzyme thus obtained can be pulverized, for example, by freeze drying, vacuum drying, or spray drying, and distributed to the market.
  • the present invention also includes a glucose assay kit containing active FADGDH and / or modified FADGDH in the present invention.
  • the glucose assay kit of the present invention contains active FADGDH and / or modified FADGDH according to the present invention in an amount sufficient for at least one assay.
  • the kit may contain a buffer necessary for the assay, a mediator, a glucose standard solution for preparing a calibration curve, and a protocol for use.
  • the active FADGDH or modified FADGDH in the present application can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.
  • the present invention also includes a glucose sensor using the active FADGDH and / or the modified FADGDH in the present invention.
  • a carbon electrode, a gold electrode, a platinum electrode, or the like can be used, and the enzyme of the present invention is immobilized on this electrode.
  • the immobilization method include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, and a redox polymer.
  • it may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator typified by ferrocene and derivatives thereof, or a combination thereof.
  • the active FADGDH and / or modified FADGDH in the present application can be immobilized on a carbon electrode using glutaraldehyde, and glutaraldehyde can be blocked by treatment with a reagent having an amine group.
  • the glucose concentration can be measured as follows. Put buffer in constant temperature cell and maintain at constant temperature.
  • As the mediator potassium ferricyanide, phenazine methosulfate, or the like can be used.
  • As the working electrode an electrode in which the active FADGDH and / or modified FADGDH in the present application is immobilized can be used, and a counter electrode and a reference electrode can be used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.
  • GDH activity is measured under the following conditions.
  • (reagent) 100 mM phosphate buffer pH 6.5 4 mM 2,6-dichlorophenolindophenol (DCIP) 4 mM PMS 1 M D-glucose 10% TritionX-100
  • the above phosphate buffer (12.3 mL), DCIP solution (0.5 mL), PMS solution (1.0 mL), glucose solution (6 mL), and Triton X-100 solution (0.2 mL) are mixed to prepare an enzyme reaction measurement reagent.
  • (Measurement condition) Place 0.9 mL of the enzyme reaction measurement reagent in the spectrophotometer cell and preincubate at 37 ° C. for 3 minutes or more.
  • 1 unit (U) in GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCIP per minute in the presence of glucose at a concentration of 300 mM.
  • the specific activity of FADGDH is measured under the following conditions.
  • (reagent) Enzyme dilution 20 mM ammonium acetate buffer pH 5.2, 0.1% BSA, 0.1% Triton X-100
  • Enzyme reaction measurement reagent Bradford reagent having the same composition as the reagent used for the above-mentioned measurement of GDH activity
  • a commercially available product manufactured by BioRad
  • the enzyme stock solution is diluted with an enzyme diluent, and the activity is determined by the above activity measurement method.
  • the protein concentration of the enzyme stock solution is measured according to a standard method using Bradford reagent.
  • the calibration curve is taken with BSA, and the blank is taken with enzyme diluent.
  • the activity (U / mL) of the enzyme stock solution is obtained, and at the same time, the protein concentration of the enzyme stock solution obtained using BSA as a control is obtained. From these values, the specific activity is determined according to the following formula.
  • KAD of FADGDH is measured under the following conditions.
  • (reagent) 100 mM phosphate buffer pH 6.5 4 mM 2,6-dichlorophenolindophenol (DCIP) 4 mM PMS D-glucose 10% TritionX-100
  • D-glucose a total of seven types of solutions of 1M, 0.5M, 0.25M, 0.125M, 0.094M, 0.063M, and 0.031M are prepared.
  • the above phosphate buffer (12.3 mL), DCIP solution (0.5 mL), PMS solution (1.0 mL), glucose solution (6 mL), Triton X-100 solution (0.2 mL) were mixed, and a total of 7 different Km measurements with different glucose concentrations Prepare reagents for use.
  • the final glucose concentration of the reagent for measuring Km is 300 mM, 150 mM, 75 mM, 37.5 mM, 28 mM, 18.8 mM, 9.4 mM. (Measurement condition) If necessary, dilute the enzyme stock solution with enzyme diluent. Preincubate 0.9 mL of enzyme reaction measurement reagent at 37 ° C. for 3 minutes or more.
  • reaction rate is defined as a change in absorbance per minute at a total of seven glucose concentrations.
  • Reaction rate ( ⁇ A) ⁇ ( ⁇ OD ⁇ ODblank) Plotting the glucose concentration on the horizontal axis and ⁇ A on the vertical axis gives a Michaelis-Menten plot.
  • the Km value is calculated from a curve drawn by a line fitting method using a known computer program.
  • mRNA is extracted from fungal cells, and cDNA is synthesized by reverse transcription reaction using RNA-dependent DNA polymerase using them as a template.
  • PCR is performed with a FADGDH-specific oligonucleotide using cDNA as a template to obtain a gene encoding the FADGDH protein.
  • Genomic DNA is extracted from fungal cells, and PCR is performed with FADGDH-specific oligonucleotides using them as templates, thereby obtaining a gene containing an intron encoding the FADGDH protein.
  • FADGDH amino acid sequence or gene sequence of the desired fungal origin is known and the FADGDH gene can be searched from among a plurality of genes registered in a known database, Based on the base sequence of the gene, see the method for total synthesis of genes by PCR (Dillon, PJ, Rosen, CA, HumanaanPress, Totowa, New Jersey, Vol. 15, p263, 1993). ) Amplify and obtain the same gene as the base sequence of the desired gene.
  • the amino acid sequence or gene sequence of FADGDH derived from the desired fungus has not been clarified, and it is impossible to specify a gene encoding FADGDH from among a group of genes registered in a known database
  • the protein having the partial amino acid sequence is obtained by isolating the enzyme from the fungus producing FADGDH, obtaining partial amino acid sequence information by partial amino acid sequence analysis, and searching the entire genome sequence information of the used fungus. Amino acid sequence information and gene base sequence information are obtained. Based on the obtained sequence information, a target gene is obtained by a total synthesis method of genes described later.
  • examples of the total synthesis method of the gene include the following methods that can be easily used, but are not limited thereto.
  • a synthetic oligonucleotide of about 100 mer is preferably used as a material.
  • Synthetic oligonucleotides can be designed based on the desired gene sequence, or can be designed using the codon usage of the host when heterologously expressing the desired gene. For example, when a fungal gene is expressed in E. coli, an oligonucleotide is synthesized so that the codon usage of the gene sequence to be synthesized is optimized in E. coli.
  • synthetic oligonucleotides divided into several to several tens are designed so as to have an annealing site for each other. By performing PCR using these synthetic oligonucleotides as templates, a desired gene can be amplified and used.
  • FADGDH could be purified from the culture supernatant of Aspergillus oryzae IFO 4220 strain by various purification methods described below. By subjecting the purified enzyme to electrophoresis, a protein band expected to be the target FADGDH could be obtained. By subjecting the protein to LC-MS / MS analysis with extremely high resolution, partial amino acid sequence analysis of the protein was performed to obtain partial amino acid sequence information. By collating the obtained partial amino acid sequence information with the genome database of Aspergillus oryzae RIB40 strain, the FADGDH candidate gene information of SEQ ID NO: 1 was obtained.
  • all loci in the multiple alignment diagram are arranged by a computer program so that insertions, deletions, substitutions, etc. are minimized. Therefore, when the candidate protein is not active due to deletion of the amino acid sequence or the like, there is a situation where a specific locus of the amino acid sequence of the candidate protein is deleted and some amino acid is arranged in the amino acid sequence other than the candidate gene. May be observed.
  • the present inventors performed homology search against NCBI database using the amino acid sequence of Aspergillus terreus-derived FADGDH described in Patent Document 2 having high homology with the amino acid sequence of the FADGDH candidate protein derived from Aspergillus oryzae. A plurality of homologous amino acid sequence information was collected, they were subjected to multiple alignment with multiple alignment software, and the amino acid sequences of FADGDH candidate proteins were also aligned at the same time. The results are shown in FIG.
  • Seq01 from Seq15 Wazorezore ATGDH (Seq01), Glucose oxidase (Seq02, CAC12802, Aspergillus niger), choline dehydrogenase (Seq03, YP_363880, Xanthomonas campestris), putative choline dehydrogenase (Seq04, CAK12001, Rhizobium leguminosarum) , GMC oxidoreductase (Seq05, ZP_00629293, Paracoccus denitrificans), Choline dehydrogenase and l related flavoproteins (Seq06, ZP _00105746, Nostoc punctiforme), glucose-methanol-choline oxidoreductase (Seq07, Anabaena variabilis), Choline dehydrogenase and related flavoproteins (Seq08, ZP_00110538, Nos
  • Seq1 to 14 are the sequences described in SEQ ID NOs: 132 to 145, respectively, and Seq15 is the sequence described in SEQ ID NO: 3.
  • the number on the right end is the position number of the amino acid when the N-terminus is the 1st position.
  • the rightmost amino acid E of Seq15 indicates that it is the 248th amino acid.
  • (-) Indicates an alignment gap.
  • the 5-amino acid deletion observed in the amino acid sequence of the FADGDH-like protein derived from Aspergillus oryzae RIB40 strain is indicated by an underbar and is denoted as a 5-amino acid sequence deletion region.
  • RNAs of Aspergillus oryzae are extracted, cDNA is synthesized by reverse transcription reaction using RNA-dependent DNA polymerase using them as templates, and then FADGDH using cDNA as a template.
  • the gene encoding the FADGDH protein can be directly obtained from a microorganism by performing PCR with a specific oligonucleotide while avoiding the above problems.
  • the active FADGDH has been obtained by making full use of probabilistic methods such as (1) consensus method and (2) ancestral amino acid introduction method based on phylogenetic methods shown below. .
  • (1) Consensus method A consensus method based on multiple alignment diagrams is a DNA sequence or amino acid sequence that has been used for the purpose of modifying the function of an antibody and has been used for the purpose of improving the thermal stability of an enzyme. This is a site-specific mutagenesis method (a method for site-specific determination of which mutation is to be introduced at which position on the sequence) and is used to modify the site causing the absence of enzyme activity as in the present invention. (See B. Steipe, et al., J. Mol. Biol., 240, 188-192, 1994 for details). However, it is not always successful.
  • common ancestor-type enzymes have been shown to be thermostable to the original enzyme, and support the hypothesis that the common ancestors of all organisms are hyperthermophilic bacteria. It has also been used for modification of a site causing no enzyme activity as used in the invention (for details, see “Hisako, I., et al., FEMS Microbiology Letters, 243, 393-398. 2005, “Keiko, W., et al., FEBS Letters, 580, 3867-3871, 2006”, “JPA 2002-247991”). However, it is not always successful.
  • the materials include a plurality of homologous amino acid sequences obtained by homology search of the amino acid sequences of candidate genes against a database, a molecular phylogenetic tree (hereinafter also referred to as a phylogenetic tree) and a phylogenetic tree created based on them. Use the algorithm for creation, multiple alignment diagram.
  • Various algorithms for creating a phylogenetic tree such as an algorithm based on the principle of maximum saving, are known, and a computer program for realizing it can also be used or obtained.
  • various phylogenetic tree estimation programs such as CLUSTAL W, PUZZLE, MOLPHY, and PHYLIP can be used.
  • an algorithm based on the maximum likelihood principle is also known, and a computer program that realizes the algorithm can also be used or obtained.
  • various phylogenetic tree estimation programs such as ModelTest, PHYML, PHYLIP, and TreeFinder can be used.
  • a phylogenetic tree can be prepared using them, but a phylogenetic tree that has already been published can also be used more conveniently.
  • the maximum saving method or the maximum likelihood method (“Young, Z., Kumar, S., Nei.M, Genetics 141, 1641-16510, 1995”, “Stewart, C.-B.Active ancestral molecules , Nature 374, 12-13, 1995 ”,“ Makoto Toshii “Molecular Evolution Genetics”, “Masato Nei, S. Kukuma“ Molecular Evolution and Molecular Phylogenetics ””.
  • the maximum saving method that can be used in the present invention is a method of estimating an ancestor type process having the smallest number of mutation events that are expected to occur after assuming an ancestor type as a true ancestor type.
  • a program PROTPARS for estimating ancestral types directly from amino acid sequences based on the maximum saving method is also available.
  • phylogenetic tree estimation and ancestral amino acid estimation are performed simultaneously in this method, so it is not always necessary to create a phylogenetic tree. It is preferable to create a tree.
  • the maximum likelihood method that can be used in the present invention is any ancestral amino acid at a specific position in the tree shape (mainly the root of the tree) based on the tree tree and amino acid substitution model determined in advance. This is a method of estimating the sequence and selecting the most likely sequence as the most promising ancestral amino sequence. Further, based on the maximum likelihood method, a program PAML for performing ancestor type estimation from a phylogenetic tree and multiple alignments of amino acid sequences can be used.
  • An ancestral amino acid can be determined for each site of multiple aligned amino acid residues using the phylogenetic tree obtained by either method. In this way, an ancestral amino acid residue can be estimated for each residue of a multiple aligned sequence, and as a result, an ancestral amino acid sequence of the corresponding region can be estimated.
  • changing the species used to estimate the ancestral amino acid sequence may change the tree shape of the phylogenetic tree, resulting in different ancestral amino residues. It also depends on the protein used for comparison.
  • amino acid residues at positions where such fluctuations are relatively small are targeted for modification.
  • Such amino acid residues are used to create a phylogenetic tree, such as changing the species used to create a phylogenetic tree, or using only part of the amino acid sequence information used to create a phylogenetic tree without changing the species. It can be determined by estimating the degree of dendritic change when the amino acid sequence information to be changed is selected, and selecting residues that have little influence on the dendritic shape.
  • the protein to be analyzed is modified by substituting at least one of the non-ancestral amino acid residues for the protein to be analyzed with the ancestral amino acid residue. be able to.
  • the present inventors inserted the ancestral amino acid sequence estimated in the deletion part of the candidate gene into the deletion part.
  • a DNA site-specific mutagenesis method or the like that is widely sold in kits and easily available to those skilled in the art may be used.
  • the gene may be linked to an appropriate expression vector, introduced into an appropriate host, and an enzyme may be expressed to confirm the presence or absence of activity. That is, a modified DNA cage is created by converting a specific base of DNA having genetic information of a protein, or by inserting or deleting a specific base.
  • Specific methods for converting bases in DNA include, for example, commercially available kits (Transformer Mutagenesis Kit: manufactured by Clonetech, EXOIII / Mung Bean Selection Kit: manufactured by Stratagene, etc., used by QuickChange SiteDirectedMutageSensedKitageStrainedKitageSensitatedKitageStrain It is done. It is also possible to divide DNA into several fragments, chemically synthesize them, and join them with DNA ligase.
  • the active FADGDH of the present invention is a GIPVT, GIPRT, GIPQT, GIPTT, GYPVT between amino acids 202 and 203 in the amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3. And an amino acid sequence selected from the group consisting of GYPRT and an amino acid sequence “MLFSLAFLSALSLATA” from the 1st position to the 16th position or an amino acid sequence “MLFSLAFLSALSLATASPAGRA” from the 1st position to the 22nd position is deleted.
  • the methionine (M) at position 202 of the amino acid sequence is substituted with alanine (A) in the amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3 and A 5-amino acid sequence selected from the group consisting of GIPVT, GIPRT, GYPVT and GYPRT is inserted between positions 202 and 203, and the amino acid sequence “MLFSLAFLSALSLATA” from positions 1 to 16 or positions 1 to 22 It is a polypeptide consisting of an amino acid sequence from which the amino acid sequence “MLFSLAFLSALSLATASPAGRA” is deleted.
  • the present inventors have also found a functional modification method that improves the substrate affinity and specific activity value.
  • Consensus method For example, the consensus method based on multiple alignment diagrams can be implemented by the method as described above.
  • an ancestral amino acid introduction method based on a phylogenetic technique can be performed by the method described above.
  • Random mutagenesis is a technique mainly using PCR, which is a technique for introducing various mutations at various sites on a gene by intentionally setting the accuracy of PCR low.
  • Diversity PCR Random Mutagenesis Kit Manufactured by Clontech
  • GeneMorph II Randommutagenesis Kit manufactured by Stratagene
  • a mutant of the gene thus introduced with a mutation is ligated to an appropriate expression plasmid by a method as described below, transformed into an appropriate host, and spread on a plate medium to form a colony. You can create a library.
  • the above mutant library can be liquid-cultured in a 96-well plate or the like for each single colony to express the mutant enzyme.
  • the expressed mutant enzyme can be used in subsequent experiments in a 96-well format or the like. If the enzyme function-modified target is substrate affinity, the enzyme reaction is monitored by placing the above-mentioned 96-well format mutant enzyme library in an activity measurement reagent having a lower substrate concentration than that prepared in the same 96-well format. It is possible to screen for mutant enzymes having a high substrate affinity by picking up mutants having a higher enzyme reaction rate than other mutants. It can be confirmed by reading the DNA sequence by sequence as described later which mutation has been introduced at which site of the mutant gene.
  • Example 1 Acquisition of Aspergillus oryzae-derived flavin adenine dinucleotide-dependent glucose dehydrogenase gene (hereinafter also referred to as FADGDH)
  • FADGDH Aspergillus oryzae-derived flavin adenine dinucleotide-dependent glucose dehydrogenase gene
  • the supernatant fraction was salted out by adding ammonium sulfate to 95% saturated ammonium sulfate, and a precipitate fraction having GDH activity was obtained by centrifugation.
  • the precipitate was suspended in 20 mM ammonium acetate buffer (pH 5.2) and dialyzed against the same buffer.
  • the dialyzed GDH solution was passed through a DEAE-Sepharose column equilibrated with the above buffer to remove contaminants.
  • a GDH solution in which a DEAE-Sepharose column is passed through is adsorbed on a RESOURCE S column (manufactured by GE Healthcare) equilibrated with 20 mM ammonium acetate buffer (pH 5.2), and GDH is added with a concentration gradient from 0 M to 1 M NaCl. Was eluted.
  • Aspergillus oryzae secreted FADGDH has been shown to be glycosylated (Non-patent Document 1, Tchan-gi Bak, Biochim. Biophys. Acta, 139, 277-293, 1967) and used in a conventional manner. It was suggested that the protein stain Coomassie Brilliant Blue (CBB) may not be sufficiently stained, so using both staining methods of CBB staining and PAS staining for glycoprotein staining (manufactured by SIGMA) The GDH fraction was subjected to SDS-PAGE. As a result, a band was observed between the molecular weight markers of 75 kDa and 100 kDa in any staining method.
  • CBB Coomassie Brilliant Blue
  • Example 2 Synthesis of FADGDH candidate gene and expression in Escherichia coli From Example 1, since the base sequence information of SEQ ID NO: 1 was obtained as the FADGDH candidate gene, the gene was synthesized to be expressed in Escherichia coli. Specific examples thereof are shown below. (2-1) Design of FADGDH candidate gene with modified codon usage Since the codon usage of the DNA sequence shown in SEQ ID NO: 1 is that of a fungus, in order to efficiently express the gene in E. coli, the entire sequence of the gene It is necessary to change the codon to be suitable for E. coli. First, it started from redesigning by changing the codon of the DNA sequence described in SEQ ID NO: 1.
  • the changed codons were set as follows. From the first letter of the alphabet, it is written in the order of one letter of amino acid and the name of amino acid (codon used for design).
  • F phenylalanine (ttc), L: leucine (ctg), I: isoleucine (atc), M: methionine (atg), V: valine (gta), Y: tyrosine (tac), stop codon (taa), H: Histidine (cac), Q: glutamine (cag), N: asparagine (aac), K: lysine (aag), D: aspartic acid (gac), E: glutamic acid (gaa), S: serine (tct), P: Proline (ccg), T: threonine (acc), A: alanine (gct), C: cysteine (tgc), W: tryptophan (tgg), R: arginine (cgt), G: glycine (ggt).
  • a DNA sequence suitable for Escherichia coli was designed by changing to the above-mentioned codon in order from the first codon “atg” of SEQ ID NO: 1.
  • it is designed by devising so that three or more identical bases do not continue as much as possible (for example, “ttt” or “ggg”).
  • Two DNA sequence information was obtained. That is, for example, when an amino acid is continuous with aspartic acid-proline (DP), if DNA is designed with the above-mentioned codons, it becomes “gacccc” and three c are consecutive. Therefore, the codon of D is more frequently used than gac. A low ga was set.
  • Non-patent Document 2 uses a long synthetic oligonucleotide (long primer) of about 100 mer. Based on the designed DNA sequence shown in SEQ ID NO: 2, the long primers shown in SEQ ID NO: 4 to SEQ ID NO: 25 were synthesized and used for the following total synthesis of genes by PCR. The annealing region of the long primer was 18-22mer.
  • PCR1 and PCR2 Two PCR steps, that is, PCR1 and PCR2.
  • PCR1 the above-mentioned long primers that partially anneal are extended by PCR to obtain a trace amount of a full-length gene.
  • PCR 2 can be performed by PCR using specific primers designed to amplify the entire gene using a small amount of the full-length gene generated in PCR 1 as a template, and a desired gene can be amplified.
  • the base sequences of specific primers are shown in SEQ ID NO: 26 and SEQ ID NO: 27.
  • the specific primer of SEQ ID NO: 26 adds an NdeI restriction enzyme recognition sequence to the DNA sequence 5 ′ end of SEQ ID NO: 2, and the specific primer of SEQ ID NO: 27 has two stop codons in the DNA sequence 3 ′ of SEQ ID NO: 2. It was designed to be arranged sequentially and to add a BamHI restriction enzyme recognition sequence.
  • PCR1 reaction solution (1 ⁇ KOD-plus-buffer (Toyobo), 0.2 mM dNTPs, 1 mM MgSO4, 0.2 ⁇ M long primer, 1 U KOD polymerase (Toyobo)
  • PCR1 reaction solution 1 ⁇ KOD-plus-buffer (Toyobo), 0.2 mM dNTPs, 1 mM MgSO4, 0.2 ⁇ M long primer, 1 U KOD polymerase (Toyobo)
  • PCR2 reaction solution (1 ⁇ KOD-plus-buffer (Toyobo) ), 0.2 mM dNTPs, 1 mM MgSO 4, 0.3 ⁇ M specific primer, 1 ⁇ L PCR1 reaction solution, 1U KOD polymerase (manufactured by Toyobo)) was adjusted to 50 ⁇ L, and PCR was performed in the following cycle mode.
  • the present inventors found that between the 16th A and 17th S of the amino acid sequence of SEQ ID NO: 3, or the 22nd The possibility of cleavage between A and the 23rd K was estimated. Therefore, the oligonucleotides of SEQ ID NO: 29 and SEQ ID NO: 30 designed so that the DNA sequence of SEQ ID NO: 28 can be amplified by removing the amino acid up to 15th and changing the 16th amino acid to methionine (M) of the start codon.
  • M methionine
  • oligos of SEQ ID NO: 32 and SEQ ID NO: 30 designed to remove the amino acid up to the 21st and change the 22nd amino acid to the methionine (M) of the start codon to amplify the DNA sequence of SEQ ID NO: 31.
  • the PCR product of (2-3) was further PCRed with nucleotides.
  • the PCR product (2-3) and the PCR product amplified so as to remove the N-terminal signal sequence were purified by conventional methods.
  • (2-5) Expression of synthetic gene in E. coli pBluescript was used as a plasmid for expressing the above gene.
  • the plasmid expresses the LacZ gene by the lac promoter, but the lacZ initiation codon neighboring sequence “ctatg” can be converted to “catatg” by site-directed mutagenesis by PCR, whereby an NdeI recognition sequence can be obtained.
  • the cleaved gene can be cloned, and the gene can be expressed relatively slowly by the lac promoter.
  • pBluescript in which the NdeI recognition sequence is inserted into the lacZ start codon is denoted as pBSN.
  • Escherichia coli DH5 ⁇ was transformed with the prepared expression plasmid, cultured on an agar plate medium to form colonies, and transformants transformed with each expression plasmid were obtained.
  • the obtained transformant was cultured in 2 mL of LB medium, and the plasmid was purified according to a conventional method. Sequencing was performed using primers specific to the synthesized gene, and it was confirmed that the expression plasmid had the desired DNAs of SEQ ID NO: 2, SEQ ID NO: 28, and SEQ ID NO: 31 incorporated therein.
  • expression plasmids in which the genes of SEQ ID NO: 2, SEQ ID NO: 28, and SEQ ID NO: 31 are incorporated are denoted as pBSNGDH, pBSNGDH17, and pBSNGDH23, respectively.
  • a single colony of a transformant transformed with pBSNGDH, pBSNGDHM17, and pBSNGDHHM23 is inoculated into 2 mL of LB medium, cultured at 37 ° C. for 16 hours, and inoculated into 20 mL of a Terific culture solution containing 100 ⁇ g / mL ampicillin. The cells were cultured at 27 ° C. for 48 hours.
  • Example 3 Synthesis of active FADGDH (hereinafter also referred to as active FADGDH) gene, expression analysis in E. coli From Example 2, the protein consisting of the amino acid sequence of SEQ ID NO: 3 registered in the database has GDH activity It became clear that there was no.
  • SEQ ID NO: 3 The cause of the absence of GDH activity in the FADGDH candidate protein consisting of the amino acid sequence was investigated.
  • ATGDH flavin adenine dinucleotide-dependent glucose dehydrogenase
  • amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of ATGDH were relatively highly homologous, whether or not there was a mutation (such as an amino acid deletion) in the amino acid sequence of SEQ ID NO: 3 It examined by making it align.
  • the amino acid sequence of the publicly disclosed ATGDH was searched using the homology search software Blastp, and amino acid sequence information of amino acid sequences having high homology with the sequence was obtained.
  • ATGDH (Seq01), Glucose oxidase (Seq02, CAC12802, Aspergillus niger), choline dehydrogenase (Seq03, YP_363880, Xanthomonas campestris), putative choline dehydrogenase (Seq04, CAK12001, Rhizobium leguminosarum), GMC oxidoreductase (Seq05, ZP_00629293 , Paracoccus denitrificans), Choline dehydrogenase and related flavoproteins (Seq06, ZP_00105746, Nostoc p nctiforme), glucose-methanol-choline oxidoreductase (Seq07, Anabaena vari
  • Seq15 amino acid sequence of SEQ ID NO: 3 (Seq15) was added to these sequence information.
  • Seq ⁇ indicates the sequence number shown in FIG. 1, and the numerical value between them indicates the accession number of each amino acid sequence registered in the NCBI database. After that, the name of the species was shown.
  • Seq1 to 14 are the sequences described in SEQ ID NOs: 132 to 145, respectively, and Seq15 is the sequence described in SEQ ID NO: 3.
  • full-length cDNA was synthesized using the mRNA extracted from Aspergillus oryzae IFO 4220 strain used in Example 1 as a template, and FADGDH DNA was amplified by PCR using specific primers.
  • this method there are several processes with high technical difficulty, such as using mRNA that is very easily degraded as a material, and obtaining a full-length cDNA.
  • the most serious problem was that the Aspergillus oryzae IFO4220 strain did not reproducibly express FADGDH.
  • the second deletion there were 6 Y, 4 L, and 3 I in the sequence of Seq01 to Seq14.
  • the amino acid residue corresponding to the second deletion was I in the ancestral amino acid sequence estimated by the phylogenetic technique from the alignment diagram obtained in FIG. Therefore, we estimated I or Y as the second amino acid to be inserted.
  • the 3rd to 5th inserted amino acids were estimated.
  • the sequence of 5 amino acids to be inserted was set as GIPVT, GIPRT, GIPQT, GIPTT, GYPVT, GYPRT. Furthermore, the combination which changes methionine (M) of the 202nd position into alanine (A) was also added from examination of the ancestral type amino acid. These amino acid sequences correspond between M at position 202 and E at position 203 of the amino acid sequence of SEQ ID NO: 3 by site-directed mutagenesis using the above pBSNGDH17 or pBSNGDH23 as a template and a specific oligonucleotide. Inserted at the position to be.
  • mutant enzyme genes were prepared for the following combinations (1) to (18).
  • M17 is a mutant using pBSNGDHM17 as a template
  • M23 is a mutant using pBSNGDH23 as a template
  • the character string after the colon (;) is the first methionine at position 202 of SEQ ID NO: 3 from the left. (M) or an ancestral amino acid
  • the obtained transformant was cultured in 2 mL of LB medium, and the plasmid was purified according to a conventional method. Sequencing was performed using primers specific to the synthesized gene, and it was confirmed that the desired mutation was introduced.
  • a transformant obtained by introducing pBSNU1 to pBSNU18 into E. coli DH5 ⁇ was cultured in a liquid medium Tbroth containing 100 ⁇ g / mL ampicillin at 27 ° C. for 48 hours. After completion of the culture, the bacterial cells were sonicated to confirm GDH activity.
  • Example 4 Expression, purification, and performance evaluation of active FADGDH Using Escherichia coli transformed with the expression plasmid of active FADGDH obtained in Example 3, the expression and purification of active FADGDH were specifically performed as follows. did. (4-1) Expression of active FADGDH Expression plasmids of active FADGDH were each transformed into E.
  • coli DH5 ⁇ and cultured on an agar plate medium to form colonies.
  • a single colony was inoculated into 2 mL of LB liquid medium and cultured with shaking at 37 ° C. for 18 hours.
  • 20 mL of LB liquid medium was prepared, put into a 500 mL flask, autoclaved at 120 ° C. for 20 minutes, inoculated with the above culture solution, and cultured with shaking at 37 ° C. for 6 hours.
  • 20 mL of the above culture solution was inoculated into 1 L of Terific culture solution containing 100 ⁇ g / mL ampicillin autoclaved, and cultured with shaking at 27 ° C. for 48 hours in a 5 L flask.
  • the culture solution was centrifuged (8000 rpm, 10 minutes) to recover the cells and stored at ⁇ 80 ° C. until used for purification.
  • (4-2) Purification of active FADGDH The cell pellet stored at ⁇ 80 ° C. was suspended in 30 mL of 20 mM ammonium acetate buffer (pH 5.2) and sonicated. The cell disruption solution was centrifuged (8000 rpm, 40 minutes, 4 ° C.), and the supernatant was collected. The resulting supernatant was passed through a 20 mL DEAE-Sepharose column equilibrated with 20 mM ammonium acetate buffer (pH 5.2).
  • the resultant was adsorbed on a 20 mL CM-Sepharose column equilibrated with 20 mM ammonium acetate buffer (pH 5.2) and eluted with a NaCl gradient from 0 M to 0.5 M to obtain a fraction having GDH activity.
  • Ammonium sulfate is added to the obtained GDH fraction so as to be 30% saturated ammonium sulfate under ice-cooling, and ammonium sulfate is precipitated.
  • Centrifugation (10000 rpm, 20 minutes, 4 ° C.) provides a supernatant having the GDH fraction. It was.
  • a 6 mL RESOURCE PHE column (manufactured by GE Healthcare) was set in an FPLC apparatus (manufactured by GE Healthcare), and the entire apparatus was equilibrated with 30% saturated ammonium sulfate + 20 mM ammonium acetate buffer (pH 5.2). The supernatant of ammonium sulfate precipitation was applied, and the GDH fraction was adsorbed onto the column, and eluted with a gradient of 30% to 0% ammonium sulfate to obtain a GDH fraction. The obtained GDH fraction was dialyzed against 0.1% Triton X-100 + 20 mM ammonium acetate buffer (pH 5.2) to obtain a GDH solution.
  • the GDH solution was electrophoresed by SDS-PAGE to confirm the degree of purification, it was purified to a single band (FIG. 2).
  • the observed molecular weight was about 60 kDa, which was almost the same as the molecular weight of 63.7 kDa calculated from the amino acid sequence.
  • the obtained active FADGDH is an enzyme obtained by inserting 5 amino acids into a protein having high homology with ATGDH having FAD as a coenzyme, but it cannot be determined whether the coenzyme has FAD. It was. Therefore, the following experiment was conducted.
  • the resulting GDH solution is dialyzed by treating with acid-ammonium sulfate to remove the coenzyme of the enzyme to apotheize it, and after eliminating the GDH activity, add the FAD solution, FMN solution or riboflavin solution. And left at room temperature for 20 minutes.
  • Example 5 Site-directed mutagenesis into active FADGDH
  • Example 4 shows that the specifications of active FADGDH expressed by E. coli are still insufficient for sensor applications.
  • mutagenesis experiments based on the following deduced ancestral amino acid sequences and the consensus concept were performed.
  • ATGDH (Seq01), Glucose oxidase (Seq02, CAC12802, Aspergillus niger), choline dehydrogenase (Seq03, YP_363880, Xanthomonas campestris), putative choline dehydrogenase (Seq04, CAK12001, Rhizobium leguminosarum), GMC oxidoreductase (Seq05, ZP_00629293 , Paracoccus denitrificans), Choline dehydrogenase and related flavoproteins (Seq06, ZP_00105746, Nostoc puncti forme), glucose-methanol-choline oxidoreductase (Seq07, Anabaena variabilis), Choline dehydrogenase and related flavoproteins (Seq08, ZP_00110538, Nostoc punctiforme), choline dehydrogenase (Se
  • Seq15 a sequence (Seq15) in which the amino acid sequence “MLFSLAFLSALSLATA” from the 1st to 16th positions on the N-terminal side is removed from the amino acid sequence of SEQ ID NO: 53 and the start codon methionine (M) is added is added to these sequence information.
  • Seq ⁇ refers to the sequence numbers shown in FIG. 4 to FIG. 6, and the numerical value in between refers to the accession number of each amino acid sequence registered in the database. After that, the name of the species was shown.
  • Seq 1 to 15 are the sequences described in SEQ ID NOs: 132 to 146, respectively.
  • the amino acid sequence of the common ancestor (the root position of the phylogenetic tree) derived from 15 organisms was estimated by the maximum likelihood method using a known computer program.
  • the amino acid sequence of the common ancestor of SEQ ID NO: 55 (ancestral amino acid sequence) was aligned with the alignment diagrams of FIGS. 4 to 6 so that all the loci of the amino acid sequence corresponded to the loci of active FADGDH.
  • (5-4) Amino Acid Substitution for Active FADGDH It was determined at which site the ancestral amino acid was introduced into active FADGDH. At the same time, it was determined which amino acid sequence was introduced into which site by the consensus method in the alignment diagram.
  • mutation 1 with respect to the active FADGDH amino acid sequence of SEQ ID NO: 53.
  • N49D mutation 3.
  • S53K mutation 4.
  • S53N mutation 5.
  • T111S + T113S mutation 6.
  • A118I + T120I mutation 7.
  • A118I + T120A mutation 8.
  • A118M + T120I mutation 9.
  • A118M + T120A mutation 10.
  • K141D + L143V mutation 11.
  • I295L mutation 15. I295V, mutation 16.
  • L300I + R301N mutation17.
  • N49D means that the 49th amino acid N from the N-terminal side of SEQ ID NO: 53 is substituted with amino acid D.
  • T111S / T113S means that amino acid substitutions of T11S and T113S are introduced simultaneously.
  • SEQ ID NO: 104 and SEQ ID NO: 105 for mutation 25 SEQ ID NO: 106 and SEQ ID NO: 107 for mutation 26, SEQ ID NO: 108 and SEQ ID NO: 109 for mutation 27, SEQ ID NO: 110 for mutation 28 And SEQ ID NO: 111, and for mutation 29, SEQ ID NO: 112 and SEQ ID NO: 11 SEQ ID NO: 114 and SEQ ID NO: 115 for mutation 30, SEQ ID NO: 116 and SEQ ID NO: 117 for mutation 31, SEQ ID NO: 118 and SEQ ID NO: 119 for mutation 32, SEQ ID NO: 120 and SEQ ID NO: 121 for mutation 33, mutation SEQ ID NO: 122 and SEQ ID NO: 123 for mutation 34, SEQ ID NO: 124 and SEQ ID NO: 125 for mutation 35, SEQ ID NO: 126 and SEQ ID NO: 127 for mutation 36, SEQ ID NO: 128, SEQ ID NO: 129, and mutation 38 for mutation 37.
  • site-directed mutagenesis by PCR was performed using QuickChange Site Directed Mutagenesis Kit (manufactured by Stratagene) using pBSNU1 as a template.
  • Escherichia coli DH5 ⁇ was transformed with each of the mutated plasmid DNAs and cloned, and whether or not the desired mutation was introduced was confirmed by sequencing.
  • the obtained mutation-introduced plasmid DNA was transformed into pBSNU1-1, pBSNU1-2, pBSNU1-3, pBSNU1-4, pBSNU1-5, pBSNU1-6, pBSNU1-7, pBSNU1-8, pBSNU1 in the order of mutation 1 to mutation 38.
  • Example 6 Random mutagenesis into active AspGDH In Example 5, site-specific mutagenesis was performed based on the probabilistic mutagenesis estimation theory. At the same time, a screening system constructed to obtain mutants with improved substrate affinity was prepared. Using the active FADGDH subjected to random mutagenesis, screening of promising mutants specifically shown below was aimed at obtaining promising mutants. (6-1) Random mutation introduction into active FADGDH Using the DNA described in SEQ ID NO: 54 as a template, a mutant DNA into which random mutation was introduced was prepared by Diversity PCR Random Mutagenesis Kit (Clontech).
  • the mutant DNA and pBSN were treated with restriction enzymes NdeI and BamHI.
  • the treatment method followed the restriction enzyme manual, and the treatment conditions were 37 ° C. for 2 hours.
  • the restriction enzyme-treated mutant DNA and vector were each excised from the gel by agarose gel electrophoresis, and the DNA fragment was purified by a conventional method.
  • the purified DNA fragments were joined with ligase to construct a random mutagenesis FADGDH expression plasmid.
  • Escherichia coli DH5 ⁇ was transformed with the expression plasmid by a conventional method, and cultured on an agar plate medium at 37 ° C. for 18 hours to form a single colony to obtain a random mutant library.
  • (6-1) Expression of random mutagenesis activation type FADGDH
  • the single colonies obtained in (6-1) above were inoculated into LB liquid medium (50 ⁇ L) supplemented with 100 ⁇ g / mL ampicillin in 96-well plates. did.
  • Two wells were applied to an E. coli strain expressing active FADGDH without any random mutation introduced.
  • the upper surface of the well plate was sealed with a gas-permable adhesive sheet (manufactured by ABgene), further covered with an attached lid, and cultured at 37 ° C. for 16 hours.
  • thermostable mutant The random mutant clone stored in 6-2 was purified by the culture method and purification method described in Example 4. The purified mutant enzyme was subjected to the performance evaluation of Example 7.
  • Example 7 Expression analysis of active FADGDH mutant, enzyme purification, performance evaluation (7-1) Expression and purification of mutant enzyme Using Escherichia coli DH5 ⁇ transformed with pBSNU1-1 to pBSNU1-38 obtained in Example 5 respectively Then, the mutant enzyme was expressed and purified by the method shown in Example 4. (7-2) Measurement of Km and specific activity In the same manner as in Example 4, the Km and specific activity of mutant enzymes from Mutant 1 to Mutant 38 were measured. The measurement results are shown in Table 2. Of the 38 types of mutant enzymes, no activity was found in mutation 1, mutation 12, mutation 16, mutation 21, mutation 23, mutation 24, mutation 30, mutation 32, and mutation 34. Regarding the Km value, the numerical values decreased compared to the active FADGDH in which no mutation was introduced.
  • Mutant 2 Mutant 3, Mutant 4, Mutant 6, Mutant 7, Mutant 8, Mutant 9, Mutant 11, Mutant 13, Mutant 15, Mutant 17, Mutant 18, Mutant 19, Mutant 20, Mutant 22, Mutant 25, Mutant 26, Mutant 27, Mutant 28, Mutant 29, Mutant 31, Mutant 33, Mutant 35, Mutant 36, Mutant 37, Mutant 38, and modified FADGDH could be obtained with considerable efficiency.
  • the Km value is clearly decreased in Mutant 2, Mutant 6, Mutant 7, Mutant 8, Mutant 9, Mutant 13, Mutant 20, Mutant 22, Mutant 26, and Mutant 29.
  • the mutant FADGDH could be obtained with considerable efficiency.
  • mutation 2, mutation 3, mutation 4, mutation 13, mutation 14, mutation 15, mutation 19, mutation 20, mutation 26, mutation 27, mutation 28, mutation 29, mutation 31, mutation 33 It was clearly increased by mutation 35 and mutation 38.
  • the mutant obtained by random mutagenesis was found to contain a mutation equivalent to mutation 22 (the expression plasmid name was expressed as pSc01 in Table 2).
  • Example 8 Combinations of Promising Mutations From the series of experiments conducted in Example 7, many mutants with improved substrate affinity and / or specific activity were obtained compared to the active FADGDH before mutation introduction. Combining the mutations introduced into these promising mutations might yield mutants with improved substrate affinity and specific activity, so we first examined the promising mutation combinations.
  • the present inventors made combinations of (1) a combination of mutations with decreased Km, (2) a combination of mutations with increased specific activity, and (3) a mutation with decreased Km and a mutation with increased specific activity. The combination of thought.
  • the combination of mutations used in the specific experiment of Example 9 is A combination of mutation 6 (A118I / T120I) and mutation 20 (T330G), A combination of mutation 6 (A118I / T120I) and mutation 26 (A82K), A combination of mutation 6 (A118I / T120I) and mutation 29 (H288Y), A combination of mutation 20 (T330G) and mutation 26 (A82K), A combination of mutation 20 (T330G) and mutation 29 (H288Y), A combination of mutation 26 (A82K) and mutation 29 (H288Y), A combination of mutation 14 (I295L) and mutation 20 (T330G), A combination of mutation 14 (I295L) and mutation 26 (A82K), A combination of mutation 14 (I295L) and mutation 28 (A107V), A combination of mutation 20 (T330G) and mutation 28 (A107V), A combination of mutation 26 (A82K) and mutation 28 (A107V), A combination of mutation 2 (N49D) and mutation 3 (S53K), A
  • mutations are in order of mutation 39 (A118I / T120I / T330G), mutation 40 (A82K / A118I / T120I), mutation 41 (A118I / T120I / H288Y), mutation 42 (A82K / T330G), mutation 43 (H288Y / T330G), Mutant 44 (A82K / H288Y), Mutant 45 (I295L / T330G), Mutant 46 (A82K / I295L), Mutant 47 (A107V / I295L), Mutant 48 (A107V / T330G), Mutant 49 (A82K / A107V), Mutant 50 (N49D / S53K), mutation 51 (H288Y / I295L), mutation 52 (L304Q / I305L / V311I), mutation 53 (T330G / S583A / L585M), mutation 54 (H288Y / S583A / L585M), mutation 55 (A
  • Example 9 Expression analysis of combination mutants, enzyme purification, and performance evaluation
  • the combination mutant genes shown in Example 8 were introduced by PCR using site-directed mutagenesis using the complementary oligonucleotides shown in Example 5. did.
  • mutation 39 is introduced using pBSNU1-6, which is an expression plasmid introduced with mutation 6, as a template, and complementary oligonucleotides of SEQ ID NO: 94 and SEQ ID NO: 95 designed to introduce mutation 20. Then, site-directed mutagenesis by PCR was performed using QuickChange Site Directed Mutagenesis Kit (manufactured by Stratagene).
  • Escherichia coli DH5 ⁇ was transformed by a conventional method using each of the plasmid DNAs mutated, and cultured on an agar plate medium at 37 ° C. for 18 hours to obtain a single colony of the transformant.
  • the obtained colonies were inoculated into an LB liquid medium supplemented with 100 ⁇ g / mL ampicillin, cultured with shaking at 37 ° C. for 18 hours, and plasmid DNA was purified by a conventional method. These plasmid DNAs were sequenced to confirm that the desired mutation was introduced.
  • the obtained mutation-introduced plasmid DNAs were obtained in the order of mutation 39 to mutation 55 in the order of pBSNU1-39, pBSNU1-40, pBSNU1-1-41, pBSNU1-42, pBSNU1-43, pBSNU1-44, pBSNU1-45, pBSNU1-46, pBSNU1 -47, pBSNU1-48, pBSNU1-49, pBSNU1-50, pBSNU1-51, pBSNU1-52, pBSNU1-53, pBSNU1-54, pBSNU1-55.
  • the mutant enzyme was expressed and purified using E. coli DH5 ⁇ transformed with pBSNU1-39 to pBSNU1-55, respectively. Expression, purification, and performance evaluation of the mutant enzyme were carried out in the same manner as in Example 4.
  • Table 3 shows the results of the performance evaluation. No activity was found in mutation 40, mutation 42, mutation 47, mutation 48, mutation 49, mutation 50, and mutation 55 among the total 17 types of combination mutant enzymes. Regarding the Km value, the mutation 39, the mutation 41, the mutation 43, the mutation 45, and the mutation 51 clearly decreased as compared with the active FADGDH in which no mutation was introduced. The specific activity was clearly increased in mutation 43, mutation 45, mutation 46, mutation 51, mutation 52, mutation 53, and mutation 54, compared to active FADGDH in which no mutation was introduced.
  • Glucose sensor An enzyme-immobilized glucose sensor using the modified FADGDH of the variant 20 used in Example 7 above was produced as a prototype, and a known method (Non-Patent Document 10, S. Tsujimura, et al., Biosci. Biotechnol. Biochem., 70, 654-659, 2006), D-glucose was measured.
  • the present invention it has become possible to express GDH activity in an FADGDH-like protein derived from Aspergillus oryzae that has no GDH activity registered in the database.
  • the experimental method used in the present invention is that gene data that is not industrially useful can be modified into a useful gene by utilizing other gene databases that are publicly available. It is very significant from the viewpoint of effective use of the database, and is expected to be widely used in general.
  • SEQ ID NOs: 2, 4-32 The sequences described in Example 2.
  • SEQ ID NOs: 33-52 are the sequences described in Example 3.
  • SEQ ID NOs: 53 and 54 The sequences described in Example 4.
  • SEQ ID NOs: 55-131 The sequences described in Example 5.
  • SEQ ID NO: 146 Sequence shown in FIGS. 4-6.

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Abstract

La présente invention concerne : un gène pour une glucose déshydrogénase flavine-adénine-dinucléotide-dépendante (GDH) (FADGDH) activée, qui est produite par insertion d’une séquence d’acides aminés spécifique dans une protéine dérivée d’Aspergillus oryzae, et une FADGDH activée comprenant une séquence d’acides aminés codée par le gène ; et un gène pour une FADGDH modifiée ayant une affinité améliorée pour un substrat et/ou une activité spécifique améliorée, qui est produite par remplacement d’un résidu d’acide aminé spécifique dans la FADGDH activée par un autre résidu d’acide aminé, et une FADGDH activée comprenant une séquence d’acides aminés codée par le gène. Il devient possible de produire une GDH qui a une excellente affinité pour un substrat et une excellente activité spécifique et n’est pas affectée par l’oxygène dissous.
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JP2012055229A (ja) * 2010-09-09 2012-03-22 Toyobo Co Ltd 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の比活性を向上するための方法
JPWO2012169512A1 (ja) * 2011-06-07 2015-02-23 キッコーマン株式会社 フラビン結合型グルコースデヒドロゲナーゼ、フラビン結合型グルコースデヒドロゲナーゼの製造方法、およびそれを用いたグルコース測定方法
WO2013022074A1 (fr) * 2011-08-11 2013-02-14 東洋紡株式会社 Nouvelle glucose déshydrogénase
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US9260699B2 (en) 2011-10-31 2016-02-16 Toyobo Co., Ltd. Glucose dehydrogenase
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WO2013118799A1 (fr) * 2012-02-09 2013-08-15 東洋紡株式会社 Nouvelle glucose déshydrogénase
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US11072809B2 (en) 2015-01-16 2021-07-27 Toyobo Co., Ltd. FAD-dependent glucose dehydrogenase
US10913971B2 (en) 2015-04-09 2021-02-09 Toyobo Co., Ltd. Enzyme preparation for use in measurement of glucose
JP2017104130A (ja) * 2017-03-06 2017-06-15 東洋紡株式会社 新規なグルコース脱水素酵素
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CN112481227B (zh) * 2020-12-05 2022-10-28 河北省微生物研究所有限公司 利用维生素b2提高fad依赖的葡萄糖脱氢酶产量的方法
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