WO2010053161A1 - Modified flavin-adenine-dinucleotide-dependent glucose dehydrogenase - Google Patents

Modified flavin-adenine-dinucleotide-dependent glucose dehydrogenase 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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PCT/JP2009/069006
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French (fr)
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.

Abstract

Disclosed are: a gene for an activated flavin-adenine-dinucleotide-dependent glucose dehydrogenase (GDH) (FADGDH), which is produced by inserting a specific amino acid sequence into a protein derived from Aspergillus oryzae, and an activated FADGDH comprising an amino acid sequence encoded by the gene; and a gene for a modified FADGDH having improved affinity for a substrate and/or an improved specific activity, which is produced by replacing a specific amino acid residue in the activated FADGDH by another amino acid residue, and an activated FADGDH comprising an amino acid sequence encoded by the gene.  It becomes possible to provide a GDH which has excellent affinity for a substrate and an excellent specific activity and is unaffected by dissolved oxygen.

Description

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Modified flavin adenine dinucleotide-dependent glucose dehydrogenase
　本発明は、アスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）ＲＩＢ４０株由来の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（以下、ＦＡＤＧＤＨという。）様タンパク質に特定のアミノ酸配列を挿入することにより得られる活性型ＦＡＤＧＤＨの遺伝子ならびに該遺伝子にコードされるアミノ酸配列から成る活性型ＦＡＤＧＤＨに関する。さらに本発明は、該活性型ＦＡＤＧＤＨの特定のアミノ酸を他のアミノ酸に変更することにより基質親和性および／または比活性を向上させた改変型ＦＡＤＧＤＨの遺伝子ならびに該遺伝子にコードされるアミノ酸配列から成る改変型ＦＡＤＧＤＨに関する。 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. Furthermore, 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.
　特定の基質に対して特異的に反応する酵素を用いたバイオセンサの開発は、産業の分野を問わず盛んに行われてきた。その代表的なものとして、主に医療分野で使用されるグルコースセンサが挙げられる。グルコースセンサは、酵素と電子伝達物質を含む反応系を構築するためのものであり、このグルコースセンサを利用する場合には、たとえばアンペロメトリックな手法を用いてグルコースが定量される。酵素としては、グルコースオキシダーゼ（ＧＯＤ） やグルコースデヒドロゲナーゼ（ＧＤＨ） が利用されてきた。 Development of biosensors using enzymes that react specifically with specific substrates has been actively conducted regardless of the industrial field. 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. When this glucose sensor is used, glucose is quantified using, for example, an amperometric technique. As enzymes, glucose oxidase (GOD) and glucose dehydrogenase (GDH) have been used.
　ＧＯＤはグルコースに対する基質特異性が高く熱安定性に優れており、酵素の量産化が可能であるために生産コストが他の酵素と比べて安価であるといった利点があるが、その反面、ＧＯＤ を使用した系は測定サンプル中の溶存酸素の影響を受けやすいため、溶存酸素が測定結果に影響を及ぼすといった問題があった。 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.
　一方、ＧＤＨを使用した系は、測定サンプル中の溶存酸素の影響を受けにくい。このため、ＧＤＨを使用した系は、酸素分圧が低い環境下で測定を行ったり、酸素量が多く要求される高濃度サンプルを測定する場合であっても、精度よくグルコース濃度を測定することができる。しかしながら、ＧＤＨは溶存酸素の影響を受けない点においてＧＯＤに勝るが、例えば補酵素としてＮＡＤの添加が必須のＮＡＤＧＤＨは安定性に乏しく、ピロロピノリンキノン（ＰＱＱ）を補酵素としてもつＰＱＱＧＤＨは基質特異性が低くマルトース等への糖類にも作用するため、グルコースセンサとして利用した場合、測定値の正確性が損なわれるといった問題があった。特に、マルトースに対する反応性に関しては、インスリン製剤とマルトース療法を併用する糖尿病患者に対し測定血糖値が偽高値となって、インスリン過剰投与の結果、低血糖症を引き起こす危険性が指摘されてきた。 On the other hand, 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. However, GDH is superior to GOD in that it is not affected by dissolved oxygen. For example, NADGDH in which addition of NAD is essential as a coenzyme is poor in stability, and PQQGDH having pyrrolopinolinequinone (PQQ) as a coenzyme is a substrate. Since it has a low specificity and acts on saccharides such as maltose, there is a problem that the accuracy of the measured value is impaired when used as a glucose sensor. In particular, regarding the reactivity to maltose, it has been pointed out that there is a risk of causing hypoglycemia as a result of insulin overdose as a result of a measured high blood glucose level in a diabetic patient using an insulin preparation and maltose therapy in combination.
　したがって、グルコースセンサとして利用する酵素は溶存酸素の影響を受けず、かつグルコース以外の糖への反応性の低い酵素が望まれる。上記のスペックを満たす酵素としてフラビンアデニンジヌクレオチド（ＦＡＤ）を補酵素としてもつＦＡＤＧＤＨが挙げられる。該酵素は約４０年前にアスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）の培養外液および菌体内に存在することが明らかにされ、精製された酵素は基質特異性が高く、比活性も高いことが示された（非特許文献１参照）。しかしながら、アスペルギルス・オリゼの培養液当たりの活性は低く、工業的に生産するには不十分であった。さらに該酵素の遺伝子配列が不明であるため、遺伝子組み換えによる工業的な大量生産も不可能であった。また、該酵素の遺伝子を特定して工業的に利用するには、酵素の単離精製、部分アミノ酸配列の解読、該酵素をコードする遺伝子配列の解読といった課題が山積していた。 Therefore, 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. About 40 years ago, it was revealed that the enzyme was present in the outer culture and cells of Aspergillus oryzae, and the purified enzyme was shown to have high substrate specificity and high specific activity. (See Non-Patent Document 1). However, the activity per culture broth of Aspergillus oryzae was low and was insufficient for industrial production. Furthermore, since the gene sequence of the enzyme is unknown, industrial mass production by genetic recombination was impossible. Moreover, in order to specify the gene of the enzyme and use it industrially, 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.
　一方、近年、アスペルギルス・オリゼＲＩＢ４０株の全ゲノム配列の解読が完了し、一般に公開されて利用することができるようになった。さらに、アスペルギルス・テレウス（Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ）由来ＦＡＤＧＤＨが単離同定され（特許文献１参照）、遺伝子配列およびアミノ酸配列も同定されたことから（特許文献２参照）、バイオインフォマティクスを利用してアスペルギルス・オリゼ由来ＦＡＤＧＤＨ遺伝子の探索と開発を進める素地が整いつつあった。実際に、ごく最近、アスペルギルス・オリゼＲＩＢ４０株の全ゲノム配列データベースを利用してアスペルギルス・オリゼＴ１株から抽出したｍＲＮＡより完全長ＦＡＤＧＤＨ遺伝子がクローニングされ（特許文献３参照）、ＰＣＲを利用したランダム変異導入法と熱安定化酵素のスクリーニングを組み合わせた手法により、熱安定化したＦＡＤＧＤＨ等が得られている（特許文献４）。 On the other hand, in recent years, the complete genome sequence of Aspergillus oryzae RIB40 strain has been completed, and it has become publicly available. Furthermore, since FADGDH derived from Aspergillus terreus was isolated and identified (see Patent Document 1), and the gene sequence and amino acid sequence were also identified (see Patent Document 2), Aspergillus oryzae was utilized using bioinformatics. The basis for the search and development of the origin FADGDH gene was being prepared. Actually, 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).
　また、センサ用途の酵素全般に求められることではあるが、センサの性能を評価する際、測定値の正確さ以外に、測定時間の短さも重要な評価項目となる。上述したように、測定値の正確さはセンサに使用している酵素の基質特異性に依存するが、測定時間の短さは酵素の基質親和性と比活性の両方に依存する。したがって、センサ用途の酵素を開発する際には、基質親和性と比活性の両方にも注目する必要がある。 In addition, as is required for all enzymes for sensor applications, shortness of measurement time is an important evaluation item in addition to the accuracy of measured values when evaluating the performance of sensors. As described above, the accuracy of the measurement value depends on the substrate specificity of the enzyme used in the sensor, but the short measurement time depends on both the substrate affinity and specific activity of the enzyme. Therefore, when developing enzymes for sensor applications, it is necessary to pay attention to both substrate affinity and specific activity.
国際公開第２００４／０５８９５８号International Publication No. 2004/058958 国際公開第２００６／１０１２３９号International Publication No. 2006/101239 国際公開第２００７／１１６７１０号International Publication No. 2007/116710 国際公開第２００８／０５９７７７号International Publication No. 2008/059777
　本発明の目的は、より実用的な血糖測定センサ用酵素を開発し、広く提供することである。より具体的には、活性型ＦＡＤＧＤＨの遺伝子を取得し、より高い性能を有するよう遺伝子工学的手法により改変することにある。さらに詳しく述べれば、センサ用途での広範な利用に耐えうるよう、基質親和性と比活性を向上させた改変型ＦＡＤＧＤＨを取得することにある。 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.
　本発明者らは上記目的を達成するために、アスペルギルス・オリゼを培養して得たＦＡＤＧＤＨの部分アミノ酸配列情報とアスペルギルス・オリゼの全ゲノム情報を含むＮａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）データベースを利用してＦＡＤＧＤＨ様遺伝子を取得したが、該遺伝子にコードされるＦＡＤＧＤＨ様タンパク質はＧＤＨ活性を有していなかった。そこで本発明者らは、公開されていたアスペルギルス・テレウス由来ＦＡＤＧＤＨのアミノ酸配列とＮＣＢＩデータベース、およびバイオインフォマティクスと遺伝子工学的手法を利用して該ＦＡＤＧＤＨ様タンパク質にＧＤＨ活性が発現しない原因を突き止め、該タンパク質のアミノ酸配列に特定のアミノ酸配列を挿入することにより活性型ＦＡＤＧＤＨを得るに至った。さらに、該活性型ＦＡＤＧＤＨ遺伝子を鋳型にバイオインフォマティクスと遺伝子工学的手法および進化工学的手法を利用して複数の変異を導入することにより、もとの活性型ＦＡＤＧＤＨに比べ基質親和性と比活性を向上させうることを見出し、本発明に達した。 In order to achieve the above-mentioned object, 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. Thus, 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. Furthermore, by introducing a plurality of mutations using the active FADGDH gene as a template using bioinformatics, genetic engineering techniques and evolutionary engineering techniques, substrate affinity and specific activity can be improved compared to the original active FADGDH. The inventors have found that it can be improved and have reached the present invention.
　すなわち、本発明の第一の態様は、配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＩＰＱＴ、ＧＩＰＴＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列を有する活性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（ＦＡＤＧＤＨ）を要旨とするものである。 That is, 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 An active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) having the followings.
　また本発明の第二の態様は、配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位のメチオニン（Ｍ）がアラニン（Ａ）に置換され、かつ２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列を有する活性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（ＦＡＤＧＤＨ）を要旨とするものである。 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 The active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) having an amino acid sequence in which the amino acid sequence “MLFSLAFLSALSLATASPAGRA” is deleted is summarized.
　また本発明の第三の態様は、上記の第一と第二の態様に記載される活性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（ＦＡＤＧＤＨ）において、以下の（１）～（３３）からなる群より選択される少なくとも一種のアミノ酸置換がなされ、改変前の対応する該活性型ＦＡＤＧＤＨに比べＫｍ値が低下した改変型ＦＡＤＧＤＨを要旨とするものである。 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.
　（１）Ｎ４９Ｄ
　（２）Ｓ５３Ｋ
　（３）Ｓ５３Ｎ
　（４）Ａ１１８Ｉ＋Ｔ１２０Ｉ
　（５）Ａ１１８Ｉ＋Ｔ１２０Ａ
　（６）Ａ１１８Ｍ＋Ｔ１２０Ｉ
　（７）Ａ１１８Ｍ＋Ｔ１２０Ａ
　（８）Ｙ１４７Ｆ＋Ｌ１４８Ｋ
　（９）Ｉ２９５Ｖ
　（１０）Ｌ３０４Ｑ＋Ｉ３０５Ｌ
　（１１）Ｌ３０４Ｋ＋Ｉ３０５Ｌ
　（１２）Ｖ３１１Ｉ
　（１３）Ｔ３３０Ｇ
　（１４）Ｇ３５Ａ
　（１５）Ｖ４２Ｌ
　（１６）Ａ８２Ｋ
　（１７）Ａ８２Ｅ
　（１８）Ａ１０７Ｖ
　（１９）Ｈ２８８Ｙ
　（２０）Ｎ４４６Ｓ＋Ｉ４４７Ｖ
　（２１）Ｎ４４６Ｓ＋Ｉ４４７Ｌ
　（２２）Ａ５３２Ｃ＋Ａ５３３Ｒ
　（２３）Ｎ５４５Ｐ
　（２４）Ｖ５６２Ｉ＋Ｌ５６３Ｍ
　（２５）Ｓ５８３Ａ＋Ｌ５８５Ｍ
　（２６）Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｈ２８８Ｙ
　（２７）Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｔ３３０Ｇ
　（２８）Ｈ２８８Ｙ＋Ｔ３３０Ｇ
　（２９）Ａ８２Ｋ＋Ｈ２８８Ｙ
　（３０）Ｉ２９５Ｌ＋Ｔ３３０Ｇ
　（３１）Ｉ２９５Ｌ＋Ｈ２８８Ｙ
　（３２）Ｌ３０４Ｑ＋Ｉ３０５Ｌ＋Ｖ３１１Ｉ
　（３３）Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
　ここで、該アミノ酸置換の位置は、配列番号３に記載されるアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入された配列のアミノ酸残基の位置に相当する位置を示す。すなわち、該アミノ酸置換の位置は、配列番号５３のアミノ酸配列のアミノ酸残基に相当する位置である。
　また、本発明の第四の態様は、上記の第一と第二の態様に記載される活性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（ＦＡＤＧＤＨ）において、以下の（１）～（２７）からなる群より選択される少なくとも一種のアミノ酸置換がなされ、改変前の対応する該活性型ＦＡＤＧＤＨに比べ比活性値が上昇した改変型ＦＡＤＧＤＨを要旨とするものである。 (1) N49D
(2) S53K
(3) S53N
(4) A118I + T120I
(5) A118I + T120A
(6) A118M + T120I
(7) A118M + T120A
(8) Y147F + L148K
(9) I295V
(10) L304Q + I305L
(11) L304K + I305L
(12) V311I
(13) T330G
(14) G35A
(15) V42L
(16) A82K
(17) A82E
(18) A107V
(19) H288Y
(20) N446S + I447V
(21) N446S + I447L
(22) A532C + A533R
(23) N545P
(24) V562I + L563M
(25) S583A + L585M
(26) A118I + T120I + H288Y
(27) A118I + T120I + T330G
(28) H288Y + T330G
(29) A82K + H288Y
(30) I295L + T330G
(31) I295L + H288Y
(32) L304Q + I305L + V311I
(33) T330G + S583A + L585M
Here, the position of the amino acid substitution corresponds to the position of the amino acid residue in the sequence in which a 5-amino acid sequence is inserted between positions 202 and 203 in the amino acid sequence shown in SEQ ID NO: 3. Indicates the position. That is, the position of the amino acid substitution is a position corresponding to the amino acid residue of the amino acid sequence of SEQ ID NO: 53.
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.
　（１）Ｎ４９Ｄ
　（２）Ｓ５３Ｋ
　（３）Ｓ５３Ｎ
　（４）Ａ１１８Ｍ＋Ｔ１２０Ａ
　（５）Ｙ１４７Ｆ＋Ｌ１４８Ｋ
　（６）Ｉ２９５Ｌ
　（７）Ｉ２９５Ｖ
　（８）Ｌ３０４Ｑ＋Ｉ３０５Ｌ
　（９）Ｌ３０４Ｋ＋Ｉ３０５Ｌ
　（１０）Ｖ３１１Ｉ
　（１１）Ｔ３３０Ｇ
　（１２）Ｇ３５Ａ
　（１３）Ａ８２Ｋ
　（１４）Ａ８２Ｅ
　（１５）Ａ１０７Ｖ
　（１６）Ｈ２８８Ｙ
　（１７）Ｎ４４６Ｓ＋Ｉ４４７Ｖ
　（１８）Ｎ４４６Ｓ＋Ｉ４４７Ｌ
　（１９）Ａ５３２Ｃ＋Ａ５３３Ｒ
　（２０）Ｓ５８３Ａ＋Ｌ５８５Ｍ
　（２１）Ｔ３３０Ｇ＋Ｈ２８８Ｙ
　（２２）Ｉ２９５Ｌ＋Ｔ３３０Ｇ
　（２３）Ｉ２９５Ｌ＋Ａ８２Ｋ
　（２４）Ｉ２９５Ｌ＋Ｈ２８８Ｙ
　（２５）Ｌ３０４Ｑ＋Ｉ３０５Ｌ＋Ｖ３１１Ｉ
　（２６）Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
　（２７）Ｈ２８８Ｙ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
　ここで、該アミノ酸置換の位置は、配列番号３に記載されるアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入された配列のアミノ酸残基の位置に相当する位置を示す。すなわち、該アミノ酸置換の位置は、配列番号５３のアミノ酸配列のアミノ酸残基に相当する位置である。 (1) N49D
(2) S53K
(3) S53N
(4) A118M + T120A
(5) Y147F + L148K
(6) I295L
(7) I295V
(8) L304Q + I305L
(9) L304K + I305L
(10) V311I
(11) T330G
(12) G35A
(13) A82K
(14) A82E
(15) A107V
(16) H288Y
(17) N446S + I447V
(18) N446S + I447L
(19) A532C + A533R
(20) S583A + L585M
(21) T330G + H288Y
(22) I295L + T330G
(23) I295L + A82K
(24) I295L + H288Y
(25) L304Q + I305L + V311I
(26) T330G + S583A + L585M
(27) H288Y + S583A + L585M
Here, the position of the amino acid substitution corresponds to the position of the amino acid residue in the sequence in which a 5-amino acid sequence is inserted between positions 202 and 203 in the amino acid sequence shown in SEQ ID NO: 3. Indicates the position. That is, the position of the amino acid substitution is a position corresponding to the amino acid residue of the amino acid sequence of SEQ ID NO: 53.
　また本発明の第五の態様は、前記した活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨのアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換及び付加から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有する活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを要旨とするものである。 According to a fifth aspect of the present invention, there is provided 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.
　また本発明の第六の態様は、前記した活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨのアミノ酸配列をコードするＤＮＡ配列で表されるＤＮＡを要旨とするものであり、好ましくは、該ＤＮＡにおいて、コドンユーゼージが大腸菌に最適化したコドンユーゼージであるＤＮＡを要旨とするものである。 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. Preferably, in the DNA, the codon usage is Escherichia coli. The gist is DNA which is a codon usage optimized for the above.
　また本発明の第七の態様は、前記したＤＮＡを含む組み換え発現プラスミドを要旨とするものである。 The seventh aspect of the present invention is summarized as a recombinant expression plasmid containing the above-described DNA.
　また本発明の第八の態様は、前記した組み換え発現プラスミドを宿主に形質転換した形質転換体を要旨とするものであり、好ましくは、宿主が大腸菌である形質転換体である。 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.
　また本発明の第九の態様は、前記した形質転換体を培養することを含む活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨの製造方法を要旨とするものである。
　また本発明の第十の態様は、前記したいずれかの活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを含むグルコースアッセイキットを要旨とするものである。
　また本発明の第十一の態様は、前記したいずれかの活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを含むグルコースセンサを要旨とするものである。
　また本発明の第十二の態様は、前記したいずれかの活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを含むグルコース濃度の測定法を要旨とするものである。 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.
　本発明により、アスペルギルス・オリゼ由来のＦＡＤＧＤＨ様タンパク質のアミノ酸配列に特定のアミノ酸配列を挿入することにより、活性型ＦＡＤＧＤＨを得るに至った。さらに、酵素の基質親和性の指標であるＫｍ値が低下し、および／または比活性が上昇した改変型ＦＡＤＧＤＨを提供することが可能となった。 According to the present invention, 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.
アスペルギルス・オリゼＲＩＢ４０株由来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（ＦＡＤＧＤＨ）様タンパク質にＧＤＨ活性がない原因を特定し、コンセンサス法に基づく部位特異的変異導入により該ＦＡＤＧＤＨ様タンパク質にＧＤＨ活性を発現せしめる目的で使用した、計１５生物種由来の相同タンパク質のアミノ酸配列のアライメント図の一部を示す図である。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. 精製した活性型ＦＡＤＧＤＨの電気泳動写真を示す図である。左側のレーン（Ｍ）は分子量マーカー。右側のレーン（活性型ＧＤＨ）が活性型ＦＡＤＧＤＨ。左側の数字は分子量マーカーの各バンドの分子量で単位はｋＤａ。右側の矢印は、単離精製された活性型ＦＡＤＧＤＨのバンドを示す。It is a figure which shows the electrophoresis photograph of the refine | purified active type FADGDH. 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. 精製した活性型ＦＡＤＧＤＨのスペクトル分析結果。２５０ｎｍから６００ｎｍまでの吸収スペクトルの結果を示している。約２８０ｎｍの吸収極大以外に、約３７０ｎｍと約４５０ｎｍの吸収極大が観察された。The spectrum analysis result of refine | purified active form FADGDH. The result of the absorption spectrum from 250 nm to 600 nm is shown. In addition to the absorption maximum of about 280 nm, absorption maximums of about 370 nm and about 450 nm were observed. 図３Ａの図を約３００ｎｍから５００ｎｍの範囲で拡大した図。The figure of FIG. 3A was expanded in the range of about 300 nm to 500 nm. コンセンサス法に基づく部位特異的変異導入および、推定祖先型アミノ酸配列に基づく部位特異的変異導入に使用したアライメント図を、サイズの問題から３分割した図のうち、Ｎ末端側の図である。It is a figure of the N terminal side among the diagrams which divided the alignment figure used for the site-directed mutagenesis based on the consensus method and the site-directed mutagenesis based on the deduced ancestral amino acid sequence from the size problem. コンセンサス法に基づく部位特異的変異導入および、推定祖先型アミノ酸配列に基づく部位特異的変異導入に使用したアライメント図を、サイズの問題から３分割した図のうち、真ん中の図である。It is a middle figure among the figures which divided the alignment figure used for the site-directed mutagenesis based on the consensus method and the site-directed mutagenesis based on the deduced ancestral amino acid sequence into 3 parts due to the size problem. コンセンサス法に基づく部位特異的変異導入および、推定祖先型アミノ酸配列に基づく部位特異的変異導入に使用したアライメント図を、サイズの問題から３分割した図のうち、Ｃ末端側の図である。It is a figure of the C terminal side among the diagrams which divided the alignment figure used for the site-specific mutagenesis based on the consensus method and the site-specific mutagenesis based on the presumed ancestral amino acid sequence from the size problem. 図４から図６のアライメント図のアミノ酸配列情報と最尤法に基づく系統樹作成プログラムにより作成した分子系統樹を示す図である。It is a figure which shows the molecular phylogenetic tree created with the phylogenetic tree creation program based on the amino acid sequence information and the maximum likelihood method of the alignment diagrams of FIGS.
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
　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（ＦＡＤＧＤＨ）は、フラビンアデニンジヌクレオチド（ＦＡＤ）を補酵素として持ち、グルコースデヒドロゲナーゼ（ＧＤＨ）活性を持つ酵素である。 Flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) is an enzyme having flavin adenine dinucleotide (FAD) as a coenzyme and having glucose dehydrogenase (GDH) activity.
　本発明の活性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（以下、活性型ＦＡＤＧＤＨともいう）は、配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＩＰＱＴ、ＧＩＰＴＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列からなるポリペプチドである。また、本発明の活性型ＦＡＤＧＤＨは、配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位のメチオニン（Ｍ）がアラニン（Ａ）に置換され、かつ２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列からなるポリペプチドである。 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. Further, 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.
　配列表を除いた本出願におけるアミノ配列中の２０種類のアミノ酸残基は、一文字略記で表現している。すなわち、グリシン（Ｇｌｙ）はＧ、アラニン（Ａｌａ）はＡ、バリン（Ｖａｌ）はＶ、ロイシン（Ｌｅｕ）はＬ、イソロイシン（Ｉｌｅ）はＩ、フェニルアラニン（Ｐｈｅ）はＦ、チロシン（Ｔｙｒ）はＹ、トリプトファン（Ｔｒｐ）はＷ、セリン（Ｓｅｒ）はＳ、スレオニン（Ｔｈｒ）はＴ、システイン（Ｃｙｓ）はＣ、メチオニン（Ｍｅｔ）はＭ、アスパラギン酸（Ａｓｐ）はＤ、グルタミン酸（Ｇｌｕ）はＥ、アスパラギン（Ａｓｎ）はＮ、グルタミン（Ｇｌｎ）はＱ、リジン（Ｌｙｓ）はＫ、アルギニン（Ａｒｇ）はＲ、ヒスチジン（Ｈｉｓ）はＨ、プロリン（Ｐｒｏ）はＰである。 The 20 amino acid residues in the amino sequence in the present application excluding the sequence table are expressed by single letter abbreviations. That is, 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, and proline (Pro) is P.
　本出願における「Ｎ４９Ｄ」等の表現は、アミノ酸置換の表記法である。例えば「Ｎ４９Ｄ」とは、ある特定のアミノ酸配列におけるN末端側から４９番目のアミノ酸Nを、アミノ酸Dに置換することを意味する。さらに、例えば「Ｔ１１１Ｓ＋Ｔ１１３Ｓ」とは、Ｔ１１１ＳとＴ１１３Ｓのアミノ酸置換を同時に導入することを意味する。 In the present application, expressions such as “N49D” are notation for amino acid substitution. For example, “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. Furthermore, for example, “T111S + T113S” means that amino acid substitutions of T111S and T113S are introduced simultaneously.
　以下、本発明の改変型ＦＡＤＧＤＨについて説明する。本発明の改変型ＦＡＤＧＤＨは、上述の改変前の対応する活性型ＦＡＤＧＤＨと比較して、ＧＤＨ活性を有するポリペプチドに変換された場合に、基質親和性または/および比活性が向上したポリペプチドである。 Hereinafter, the modified FADGDH of the present invention will be described. 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.
　すなわち、本発明の基質親和性が向上した改変型ＦＡＤＧＤＨは、改変前の対応する活性型ＦＡＤＧＤＨと比較して、基質親和性の指標であるＫｍ値が低下したものが好ましく、Ｋｍ値が３０以下、１以上であるものがさらに好ましい。
　さらに、本発明の基質親和性が向上した改変型ＦＡＤＧＤＨは上述の活性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（ＦＡＤＧＤＨ）において、以下の（１）～（３３）からなる群より選択される少なくとも一種のアミノ酸置換がなされたアミノ酸配列からなるポリペプチドが好ましい。ここで、以下のアミノ酸置換の位置は、配列番号３のアミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入されたアミノ酸配列のアミノ酸位置であり、すなわち配列番号５３のアミノ酸配列のアミノ酸残基に相当する位置である。 That is, 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. One or more is more preferable.
Furthermore, 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. Here, 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.
　（１）Ｎ４９Ｄ
　（２）Ｓ５３Ｋ
　（３）Ｓ５３Ｎ
　（４）Ａ１１８Ｉ＋Ｔ１２０Ｉ
　（５）Ａ１１８Ｉ＋Ｔ１２０Ａ
　（６）Ａ１１８Ｍ＋Ｔ１２０Ｉ
　（７）Ａ１１８Ｍ＋Ｔ１２０Ａ
　（８）Ｙ１４７Ｆ＋Ｌ１４８Ｋ
　（９）Ｉ２９５Ｖ
　（１０）Ｌ３０４Ｑ＋Ｉ３０５Ｌ
　（１１）Ｌ３０４Ｋ＋Ｉ３０５Ｌ
　（１２）Ｖ３１１Ｉ
　（１３）Ｔ３３０Ｇ
　（１４）Ｇ３５Ａ
　（１５）Ｖ４２Ｌ
　（１６）Ａ８２Ｋ
　（１７）Ａ８２Ｅ
　（１８）Ａ１０７Ｖ
　（１９）Ｈ２８８Ｙ
　（２０）Ｎ４４６Ｓ＋Ｉ４４７Ｖ
　（２１）Ｎ４４６Ｓ＋Ｉ４４７Ｌ
　（２２）Ａ５３２Ｃ＋Ａ５３３Ｒ
　（２３）Ｎ５４５Ｐ
　（２４）Ｖ５６２Ｉ＋Ｌ５６３Ｍ
　（２５）Ｓ５８３Ａ＋Ｌ５８５Ｍ
　（２６）Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｈ２８８Ｙ
　（２７）Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｔ３３０Ｇ
　（２８）Ｈ２８８Ｙ＋Ｔ３３０Ｇ
　（２９）Ａ８２Ｋ＋Ｈ２８８Ｙ
　（３０）Ｉ２９５Ｌ＋Ｔ３３０Ｇ
　（３１）Ｉ２９５Ｌ＋Ｈ２８８Ｙ
　（３２）Ｌ３０４Ｑ＋Ｉ３０５Ｌ＋Ｖ３１１Ｉ
　（３３）Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
　さらに、本発明の基質親和性が向上した改変型ＦＡＤＧＤＨは、Ｋｍ値が、改変前の対応する前記した活性型ＦＡＤＧＤＨに比較した９０％以下であり、好ましくは８０％以下であり、より好ましくは７０％以下であり、最も好ましくは６０％以下のものである。 (1) N49D
(2) S53K
(3) S53N
(4) A118I + T120I
(5) A118I + T120A
(6) A118M + T120I
(7) A118M + T120A
(8) Y147F + L148K
(9) I295V
(10) L304Q + I305L
(11) L304K + I305L
(12) V311I
(13) T330G
(14) G35A
(15) V42L
(16) A82K
(17) A82E
(18) A107V
(19) H288Y
(20) N446S + I447V
(21) N446S + I447L
(22) A532C + A533R
(23) N545P
(24) V562I + L563M
(25) S583A + L585M
(26) A118I + T120I + H288Y
(27) A118I + T120I + T330G
(28) H288Y + T330G
(29) A82K + H288Y
(30) I295L + T330G
(31) I295L + H288Y
(32) L304Q + I305L + V311I
(33) T330G + S583A + L585M
Furthermore, the modified FADGDH having improved substrate affinity according to the present invention has a Km value of 90% or less, preferably 80% or less, more preferably compared to the corresponding active FADGDH before modification. 70% or less, most preferably 60% or less.
　さらに、本発明の活性型ＦＡＤＧＤＨにおいて、Ｇ３５Ａ、Ｎ４９Ｄ、Ａ８２Ｋ、Ｈ２８８Ｙ、Ｔ３３０Ｇ、Ａ１１８Ｉ＋Ｔ１２０Ｉ、Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｈ２８８Ｙ、Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｔ３３０Ｇ、Ｈ２８８Ｙ＋Ｔ３３０Ｇからなる群より選択される少なくとも一種のアミノ酸置換がなされたものが好ましい。ここで、上記アミノ酸置換の位置は、配列番号３のアミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入されたアミノ酸配列のアミノ酸位置であり、すなわち配列番号５３のアミノ酸配列のアミノ酸残基に相当する位置である。 Further, in 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. Here, 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.
　ここで、Ｋｍ値とはミカエリス定数ともいい、ミカエリス・メンテン型の酵素反応速度式において最大速度Ｖmaxとともに含まれる定数で、グルコースデヒドロゲナーゼの場合はグルコースに対する親和性を表す数値である。同じ基質に対してＫｍ値が異なる酵素の場合はＫｍが小さいほど作用が強い。本発明におけるＫｍ値は、後述の条件で測定し、計算した値を意味する。 Here, 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.
　また、本発明の比活性が向上した改変型ＦＡＤＧＤＨは、改変前の対応する活性型ＦＡＤＧＤＨと比較して、比活性値が上昇したものが好ましく、比活性値が７００Ｕ／ｍｇ－タンパク質以上であるものが好ましく、１０００Ｕ／ｍｇ－タンパク質以上であるものがさらに好ましい。
　さらに、上記活性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（ＦＡＤＧＤＨ）において、以下の（１）～（２７）からなる群より選択される少なくとも一種のアミノ酸置換がなされたアミノ酸配列からなるＦＡＤＧＤＨが好ましい。ここで、以下のアミノ酸置換の位置は、配列番号３のアミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入されたアミノ酸配列のアミノ酸位置であり、すなわち配列番号５３のアミノ酸配列のアミノ酸残基に相当する位置である。 Further, 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.
Furthermore, in the above active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH), 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. Here, 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.
　（１）Ｎ４９Ｄ
　（２）Ｓ５３Ｋ
　（３）Ｓ５３Ｎ
　（４）Ａ１１８Ｍ＋Ｔ１２０Ａ
　（５）Ｙ１４７Ｆ＋Ｌ１４８Ｋ
　（６）Ｉ２９５Ｌ
　（７）Ｉ２９５Ｖ
　（８）Ｌ３０４Ｑ＋Ｉ３０５Ｌ
　（９）Ｌ３０４Ｋ＋Ｉ３０５Ｌ
　（１０）Ｖ３１１Ｉ
　（１１）Ｔ３３０Ｇ
　（１２）Ｇ３５Ａ
　（１３）Ａ８２Ｋ
　（１４）Ａ８２Ｅ
　（１５）Ａ１０７Ｖ
　（１６）Ｈ２８８Ｙ
　（１７）Ｎ４４６Ｓ＋Ｉ４４７Ｖ
　（１８）Ｎ４４６Ｓ＋Ｉ４４７Ｌ
　（１９）Ａ５３２Ｃ＋Ａ５３３Ｒ
　（２０）Ｓ５８３Ａ＋Ｌ５８５Ｍ
　（２１）Ｔ３３０Ｇ＋Ｈ２８８Ｙ
　（２２）Ｉ２９５Ｌ＋Ｔ３３０Ｇ
　（２３）Ｉ２９５Ｌ＋Ａ８２Ｋ
　（２４）Ｉ２９５Ｌ＋Ｈ２８８Ｙ
　（２５）Ｌ３０４Ｑ＋Ｉ３０５Ｌ＋Ｖ３１１Ｉ
　（２６）Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
　（２７）Ｈ２８８Ｙ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
　さらに、本発明の比活性が向上した改変型ＦＡＤＧＤＨの比活性値は、改変前の対応する前記した活性型ＦＡＤＧＤＨに比較して１２０％以上であり、好ましくは１５０％以上であり、より好ましくは１８０％以上であり、最も好ましくは２２０％以上のものである。 (1) N49D
(2) S53K
(3) S53N
(4) A118M + T120A
(5) Y147F + L148K
(6) I295L
(7) I295V
(8) L304Q + I305L
(9) L304K + I305L
(10) V311I
(11) T330G
(12) G35A
(13) A82K
(14) A82E
(15) A107V
(16) H288Y
(17) N446S + I447V
(18) N446S + I447L
(19) A532C + A533R
(20) S583A + L585M
(21) T330G + H288Y
(22) I295L + T330G
(23) I295L + A82K
(24) I295L + H288Y
(25) L304Q + I305L + V311I
(26) T330G + S583A + L585M
(27) H288Y + S583A + L585M
Furthermore, the specific activity value of the modified FADGDH with improved specific activity of the present invention is 120% or more, preferably 150% or more, more preferably compared to the corresponding active FADGDH before modification. 180% or more, most preferably 220% or more.
　さらに、本発明の活性型ＦＡＤＧＤＨにおいて、Ｓ５３Ｋ、Ａ８２Ｋ、Ａ１０７Ｖ、Ｉ２９５Ｌ、Ｖ３１１Ｉ、Ｔ３３０Ｇ、Ａ８２Ｋ＋Ｉ２９５Ｌ、Ｈ２８８Ｙ＋Ｉ２９５Ｌ、Ｈ２８８Ｙ＋Ｔ３３０Ｇ、Ｈ２８８Ｙ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ、Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ、Ｓ５８３Ａ＋Ｌ５８５Ｍからなる群より選択される少なくとも一種のアミノ酸置換がなされたものが好ましい。ここで、上記アミノ酸置換の位置は、配列番号３のアミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入されたアミノ酸配列のアミノ酸位置であり、すなわち配列番号５３のアミノ酸配列のアミノ酸残基に相当する位置である。 Furthermore, in 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. Here, 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.
　ここで、比活性値とは、酵素単位重量当たりの活性値を表す。本発明における比活性値は、後述の条件で測定し、計算した値を意味する。 Here, 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.
　ここで「Ｎ４９Ｄ」等の表現は、アミノ酸置換の表記法である。例えば「Ｎ４９Ｄ」とは、ある特定のアミノ酸配列におけるN末端側から４９番目のアミノ酸Nを、アミノ酸Dに置換することを意味する。さらに、例えば「Ｔ１１１Ｓ＋Ｔ１１３Ｓ」とは、Ｔ１１１ＳとＴ１１３Ｓのアミノ酸置換を同時に導入することを意味する。 Here, expressions such as “N49D” are amino acid substitution notations. For example, “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. Furthermore, for example, “T111S + T113S” means that amino acid substitutions of T111S and T113S are introduced simultaneously.
　本発明の活性型ＦＡＤＧＤＨおよび改変型ＦＡＤＧＤＨは、上記活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨにおいて、配列番号３のアミノ酸配列の1位から２２位までに相当する位置のアミノ酸配列の一部または全てをN末端から除去したポリペプチドも含む。好ましくは、配列番号３のアミノ酸配列の１位から１６位までに相当する位置のアミノ酸配列を除去したポリペプチド、又は配列番号３のアミノ酸配列の1位から２２位までに相当する位置のアミノ酸配列を除去したポリペプチドが挙げられる。さらに好ましくは、配列番号３のアミノ酸配列の１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」、又は配列番号３のアミノ酸配列の1位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」を除去したポリペプチドが挙げられる。 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. Also included are 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. More preferably, 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.
　本発明には、上述した活性型ＦＡＤＧＤＨおよび改変型ＦＡＤＧＤＨのアミノ酸配列において、1個又は複数個のアミノ酸残基が欠失、付加、挿入及び置換から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有するポリペプチドも含有される。上述したアミノ酸配列において、1個乃至２０個のアミノ酸残基が欠失、付加、挿入及び置換から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有するポリペプチドが好ましく、1個乃至１０個のアミノ酸残基が欠失、付加、挿入及び置換から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有するポリペプチドがさらに好ましい。さらに、1個乃至６個のアミノ酸残基が欠失、付加、挿入及び置換から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有するポリペプチドが好ましく、数個（１から２または３個）のアミノ酸残基が欠失、付加、挿入及び置換から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有するポリペプチドがさらに好ましい。さらに好ましくは、1個のアミノ酸残基が欠失、付加、挿入または置換されたアミノ酸配列を有するポリペプチドが挙げられる。さらに、ＧＤＨ活性を有するポリペプチドが好ましい。
　さらに、本発明は、上述した活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨに相同性を有するポリペプチドを含む。その例は、少なくとも８０%以上、好ましくは９０%以上、さらに好ましくは９５%以上、さらに好ましくは９９％以上の相同性を有するポリペプチドをあげることができる。さらに、ＧＤＨ活性を有するポリペプチドが好ましい。
　ここで、「相同性」とは、BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]のbl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol.174, 247-250, 1999)により得られる同一性の値を示す。パラメーターとしては、Gap insertion Cost value: 11、Gap extension Cost value: 1が例示される。 In the present invention, there is provided 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. Polypeptides having are also included. In the amino acid sequence described above, 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. Furthermore, a 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. Examples thereof include polypeptides having homology of at least 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 99% or more. Furthermore, a polypeptide having GDH activity is preferred.
Here, “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.
　以下、本発明の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨをコードするＤＮＡ配列で表されるＤＮＡ、該ＤＮＡを含む組み換え発現プラスミド、該プラスミドを導入した形質転換体、およびそれらの製造方法、また、該形質転換体を用いた活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨの製造方法について説明する。 Hereinafter, a DNA represented by a DNA sequence encoding the active FADGDH or modified FADGDH of the present invention, a recombinant expression plasmid containing the DNA, a transformant introduced with the plasmid, a production method thereof, A method for producing active FADGDH or modified FADGDH using the transformant will be described.
　本発明の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨのアミノ酸配列をコードするＤＮＡ配列で表されるＤＮＡは、例えば後述の遺伝子の全合成法やＤＮＡの変異導入法により得ることができるＤＮＡが挙げられる。
　本発明のＤＮＡは、上記ＤＮＡ又はこれを有する細胞に変異処理を行い、これらのＤＮＡ若しくは細胞から、例えば配列番号３の配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡを選択することによって得られるＤＮＡも含む。
　ここでいう「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成されるが、非特異的なハイブリッドは形成されない条件をいう。この条件を明確に数値化することは困難であるが、相同性が高い核酸同士、例えば70～90％以上の相同性を有するＤＮＡ同士がハイブリダイズし、それより相同性が低い核酸同士がハイブリダイズしない条件等が挙げられる。
　ここでいう「ストリンジェントな条件下」とは、例えば以下の条件をいう。すなわち 0.5％ＳＤＳ、５×デンハルツ〔Denhartz's、0.１％ウシ血清アルブミン（BSA ）、0.１％ポリビニルピロリドン、0.１％フィコール400 〕及び 100μg/mlサケ精子DNA を含む６×SSC （１×SSC は、0.15 M NaCl 、0.015 M クエン酸ナトリウム、pH7.0 ）中で、50℃～65℃で４時間～一晩保温する条件をいう。
　ハイブリダイゼーションは、上記に示したストリンジェントな条件下で行うことができる。例えば、本発明の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨをコードするDNA ライブラリーまたはcDNAライブラリーを固定化したナイロン膜を作成し、６×SSC 、 0.5％SDS 、５×デンハルツ、100 μg/mlサケ精子DNA を含むプレハイブリダイゼーション溶液中、65℃でナイロン膜をブロッキングする。その後、32Ｐでラベルした各プローブを加えて、65℃で一晩保温する。このナイロン膜を６×SSC 中、室温で10分間、0.１％ SDSを含む２×SSC 中、室温で10分間、0.１％ SDSを含む0.２×SSC 中、45℃で30分間洗浄した後、オートラジオグラフィーをとり、プローブと特異的にハイブリダイズするDNA を検出することができる。また、洗いなどの条件を変えることによって様々な相同性を示す遺伝子を得ることができる。 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.
For the DNA of the present invention, 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. Although it is difficult to quantify this condition clearly, nucleic acids with high homology, for example, DNAs having homology of 70 to 90% or more hybridize, and nucleic acids with lower homology hybridize. The conditions etc. which do not soy are mentioned.
Here, “under stringent conditions” refers to the following conditions, for example. That is, 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 μg / 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. For example, 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 μg / 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. In addition, genes having various homologies can be obtained by changing conditions such as washing.
　また、上記ＤＮＡに相同性を有するＤＮＡも含まれる。相同性としては、少なくとも８０%以上、好ましくは９０%以上の相同性を有する遺伝子、さらに好ましくは９５%以上、さらに好ましくは９８%以上である。
　ここで、ＤＮＡの「相同性」とは、BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]のbl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol.174, 247-250, 1999)により得られる同一性の値を示す。パラメーターとしては、Gap insertion Cost value: 11、Gap extension Cost value: 1が例示される。
　さらに、該ＤＮＡは、コドンユーゼージが宿主に最適化したものが好ましく、コドンユーゼージが大腸菌に最適化したＤＮＡがさらに好ましい。コドンユーゼージを表す指標として、各コドンの宿主最適コドン使用頻度の総計を採択する。最適コドンとは、同じアミノ酸に対応するコドンのうち出現頻度が最も高いコドンと定義する。コドンユーゼージは、宿主に最適化したものであれば特に限定されないが、例えば、大腸菌のコドンユーゼージの一例として以下のものが挙げられる。
　Ｆ：フェニルアラニン（ｔｔｃ）、Ｌ：ロイシン（ｃｔｇ）、Ｉ：イソロイシン（ａｔｃ）、Ｍ：メチオニン（ａｔｇ）、Ｖ：バリン（ｇｔａ）、Ｙ：チロシン（ｔａｃ）、終止コドン（ｔａａ）、Ｈ：ヒスチジン（ｃａｃ）、Ｑ：グルタミン（ｃａｇ）、Ｎ：アスパラギン（ａａｃ）、Ｋ：リジン（ａａｇ）、Ｄ：アスパラギン酸（ｇａｃ）、Ｅ：グルタミン酸（ｇａａ）、Ｓ：セリン（ｔｃｔ）、Ｐ：プロリン（ｃｃｇ）、Ｔ：スレオニン（ａｃｃ）、Ａ：アラニン（ｇｃｔ）、Ｃ：システイン（ｔｇｃ）、Ｗ：トリプトファン（ｔｇｇ）、Ｒ：アルギニン（ｃｇｔ）、Ｇ：グリシン（ｇｇｔ） Moreover, 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.
Here, “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.
Further, 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. As an index indicating codon usage, 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)
　上記ＤＮＡを、発現ベクターに連結させることにより、組み換え発現プラスミド（以下、組み換えベクターともいう）が得られる。
　ここで発現ベクターとしては、宿主内で自律的に増殖し得るファージまたはプラスミドから遺伝子組み換え用として構築されたものが適している。ファージとしては、たとえば後述する大腸菌（エシェリヒア・コリ）を宿主とする場合には、Ｌａｍｂｄａ ｇｔ１０、Ｌａｍｂｄａ ｇｔ１１などが例示される。一方、プラスミドとしては、たとえば、大腸菌を宿主とする場合には、ｐＢＲ３２２、ｐＵＣ１８、ｐＵＣ１１８、ｐＵＣ１９、ｐＵＣ１１９、ｐＴｒｃ９９Ａ、ｐＢｌｕｅｓｃｒｉｐｔおよびコスミドであるＳｕｐｅｒ Ｃｏｓ Ｉなどが例示される。シュードモナスを用いる場合には、グラム陰性菌用広宿主域ベクターであるＲＳＦ１０１０、ｐＢＢＲ１２２、ｐＣＮ５１などが例示される。 By ligating the above DNA to an expression vector, a recombinant expression plasmid (hereinafter also referred to as a recombinant vector) can be obtained.
Here, 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. On the other hand, 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. When Pseudomonas is used, RSF1010, pBBR122, pCN51, etc., which are broad host range vectors for Gram-negative bacteria, are exemplified.
　作製された活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨのＤＮＡは、発現ベクターと結合した状態で宿主中に安定に保存される。 The produced active FADGDH or modified FADGDH DNA is stably stored in the host in a state of being bound to the expression vector.
　宿主としては、組み換えベクターが安定であり、かつ自律増殖可能で外来性遺伝子の形質を発現できるのであれば特に制限されない。宿主としては、真核生物でも原核生物でもよいが、微生物が好ましく、好ましくは大腸菌、さらに好ましくは大腸菌ＤＨ５α、ＸＬ－１ Ｂｌｕｅ ＭＲなどを用いることができる。 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 DH5α, XL-1 Blue MR, or the like.
　宿主に組み換えベクターを移入する方法としては、たとえば宿主が大腸菌の場合には、カルシウム処理によるコンピテントセル法やエレクトロポーレーション法などを用いることができる。 As a method for transferring the recombinant vector into the host, for example, when the host is Escherichia coli, a competent cell method using calcium treatment, an electroporation method, or the like can be used.
　組み換え発現プラスミドＤＮＡの分離・精製は、微生物を溶菌して得られる溶菌物に基づいて行われる。溶菌の方法としては、たとえばリゾチームなどの溶菌酵素により処理が施され、必要に応じてプロテアーゼや他の酵素やラウリル硫酸ナトリウム（ＳＤＳ）などの界面活性剤が併用される。さらに、凍結融解やフレンチプレス処理のような物理的破砕方法を組み合わせてもよい。溶菌物からのＤＮＡの分離・精製は、たとえばフェノール処理やプロテアーゼ処理による除蛋白処理、リボヌクレアーゼ処理、アルコール沈殿処理および市販のキットを適宜組み合わせることにより行うことができる。 Separation and purification of recombinant expression plasmid DNA is performed based on a lysate obtained by lysing a microorganism. As 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.
　ＤＮＡの切断は、常法にしたがい、たとえば制限酵素処理を用いて行うことができる。制限酵素としては、たとえば特定のヌクレオチド配列に作用するII 型制限酵素を用いられる。ポリヌクレオチドと発現ベクターとの結合は、たとえばＤＮＡリガーゼを用いて行われる。 The DNA can be cleaved according to a conventional method, for example, using a restriction enzyme treatment. As 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.
　次いで、組み換えベクターにマーカーを施して、この組み換えベクターを宿主に移入して形質転換体を形成する。この形質転換体から、組み換えベクターのマーカーと酵素活性の発現を指標としてスクリーニングして、ＦＡＤＧＤＨ をコードする遺伝子を含有する組み換えベクターを保持する遺伝子供与微生物を得る。 Next, 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.
　ＦＡＤＧＤＨをコードする遺伝子の塩基配列は、公知のジデオキシ法により解読できる。ＦＡＤＧＤＨのアミノ酸配列は、上記のように決定された塩基配列より推定できる。 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.
　形質転換体の培養形態は、宿主の栄養生理的性質を考慮して培養条件を選択すればよく、好ましくは液体培養で行う。工業的には通気攪拌培養を行うのが有利である。 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.
　培地の栄養源としては、微生物の培養に通常用いられるものが使用され得る。炭素源としては、資化可能な炭素化合物であればよく、たとえばグルコース、シュークロース、ラクトース、マルトース、糖蜜、ピルビン酸などが使用される。窒素源としては、資化可能な窒素化合物であればよく、たとえばペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物などが使用される。その他に、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガン、亜鉛などの塩類、特定のアミノ酸、特定のビタミンなどが必要に応じて使用される。 As the nutrient source of the medium, those commonly used for culturing microorganisms can be used. As the carbon source, any carbon compound that can be assimilated may be used. For example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used. As the nitrogen source, any assimilable nitrogen compound may be used. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, 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.
　培養温度は、宿主が生育し、宿主がＦＡＤＧＤＨを産生する範囲で適宜変更し得るが、好ましくは２０～３７℃程度である。培養は、ＦＡＤＧＤＨが最高収量に達する時期を見計らって適当時期に完了すればよく、通常は培養時間が１２～４８時間程度である。培地のｐＨ は、宿主が発育し、宿主がＦＡＤＧＤＨを産生する範囲で適宜変更し得るが、好ましくはｐＨ５．０～９．０程度の範囲である。 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.
　形質転換体を培養し、培養液を遠心分離などの方法により培養上清または菌体を回収し、菌体は超音波またはフレンチプレスといった機械的方法またはリゾチームなどの溶菌酵素により処理を施し、必要に応じてプロテアーゼや他の酵素やラウリル硫酸ナトリウム（ＳＤＳ）などの界面活性剤を併用することにより可溶化し、ＦＡＤＧＤＨを含む水溶性画分を得ることができる。または適当な発現ベクターと宿主を選択することにより、発現したＦＡＤＧＤＨを培養液中に分泌させることができる。 Culture the transformant, collect the culture supernatant or cells by centrifugation, etc., and treat the cells with a mechanical method such as ultrasonic or French press or a lytic enzyme such as lysozyme. Accordingly, 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. Alternatively, the expressed FADGDH can be secreted into the culture medium by selecting an appropriate expression vector and host.
　上記のようにして得られたＦＡＤＧＤＨを含む水溶性画分から該酵素を精製する方法としては、この溶液から直ちに行うこともできるが、この溶液中のＦＡＤＧＤＨを濃縮した後に行うこともできる。濃縮は、たとえば、減圧濃縮、膜濃縮、塩析処理、または親水性有機溶媒（たとえば、メタノール、エタノール、アセトン）による分別沈殿法により行うことができる。ＦＡＤＧＤＨの濃縮には、加熱処理や等電点処理も有効な精製手段である。濃縮液の精製は、たとえば、ゲルろ過、吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティクロマトグラフィーを適宜組み合わせることによって行うことができる。これらの方法はすでに公知であり、適当な文献、雑誌、教科書等を参照することで進めることができる。このようにして得られた精製酵素は、たとえば凍結乾燥、真空乾燥、スプレードライにより粉末化して市場に流通させることができる。 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.
　本発明はまた、本発明における活性型ＦＡＤＧＤＨ及び/又は改変型ＦＡＤＧＤＨを含むグルコースアッセイキットを含む。本発明のグルコースアッセイキットは、本発明に従う活性型ＦＡＤＧＤＨ及び/又は改変型ＦＡＤＧＤＨを少なくとも１回のアッセイに十分な量で含む。キットは、本発明の活性型ＦＡＤＧＤＨ及び/又は改変型ＦＡＤＧＤＨに加えて、アッセイに必要な緩衝液、メディエータ、キャリブレーションカーブ作製のためのグルコース標準溶液、および使用にあたってのプロトコルを含んでもよい。本出願における活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨは種々の形態で、例えば、凍結乾燥された試薬として、または適切な保存溶液中の溶液として提供することができる。 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. In addition to the active FADGDH and / or modified FADGDH of the present invention, 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.
　本発明はまた、本発明における活性型ＦＡＤＧＤＨ及び/又は改変型ＦＡＤＧＤＨを用いるグルコースセンサを含む。電極としては、カーボン電極、金電極、白金電極などを用いることができ、この電極上に本発明の酵素を固定化する。固定化方法としては、架橋試薬を用いる方法、高分子マトリックス中に封入する方法、透析膜で被覆する方法、光架橋性ポリマー、導電性ポリマー、酸化還元ポリマーなどが挙げられる。または、フェロセンおよびその誘導体に代表される電子メディエータとともにポリマー中に固定または電極上に吸着固定してもよく、またこれらを組み合わせて用いてもよい。例えば、グルタルアルデヒドを用いて本出願における活性型ＦＡＤＧＤＨ及び/又は改変型ＦＡＤＧＤＨをカーボン電極上に固定化し、アミン基を有する試薬で処理してグルタルアルデヒドをブロッキングすることができる。 The present invention also includes a glucose sensor using the active FADGDH and / or the modified FADGDH in the present invention. As an electrode, 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. Examples of 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. Alternatively, 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. For example, 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.
　グルコース濃度の測定は、次のようにして行うことができる。恒温セルに緩衝液を入れ、一定温度に維持する。メディエータとしては、フェリシアン化カリウム、フェナジンメトサルフェート等を使用できる。作用電極として本出願における活性型ＦＡＤＧＤＨ及び/又は改変型ＦＡＤＧＤＨを固定化した電極を用い、対極および参照電極を用いることができる。カーボン電極に一定の電圧を印加して、電流が定常になった後、グルコースを含む試料を加えて電流の増加を測定する。標準濃度のグルコース溶液により作製したキャリブレーションカーブに従い、試料中のグルコース濃度を計算することができる。 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.
　本発明において、ＧＤＨ活性の測定は以下の条件で行う。
（試薬）
１００ ｍＭ リン酸バッファーｐＨ６．５
４ ｍＭ ２,６－ジクロロフェノールインドフェノール(ＤＣＩＰ)
４ ｍＭ ＰＭＳ
１ Ｍ Ｄ－グルコース
１０％ ＴｒｉｔｉｏｎＸ－１００
上記リン酸バッファーを１２．３ｍＬ、ＤＣＩＰ溶液を０．５ｍＬ、ＰＭＳ溶液を１．０ｍＬ、グルコース溶液を６ｍＬ、ＴｒｉｔｏｎＸ－１００溶液０．２ｍＬを混合して酵素反応測定試薬とする。
（測定条件）
　酵素反応測定試薬０．９ｍＬを分光光度計用セルに入れ、３７℃で３分間以上プレインキュベートする。ＧＤＨ溶液０．００５ｍＬを添加してよく混合し、３７℃で予めインキュベートされた分光光度計で、６００ｎｍの吸光度変化を９０秒間記録し、直線部分から１分間あたりの吸光度変化（ΔＯＤ）を測定する。ブランクは、ＧＤＨ溶液の代わりにＧＤＨの溶媒を酵素反応測定試薬に混合して上記のように１分間あたりの吸光度変化（ΔＯＤｂｌａｎｋ）を測定する。これらの値から、下記の計算式にしたがって活性値を求める。 In the present invention, 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. Add 0.005 mL of GDH solution, mix well, record absorbance change at 600 nm for 90 seconds with a spectrophotometer pre-incubated at 37 ° C., and measure the absorbance change per minute (ΔOD) from the linear portion. . In the blank, the change in absorbance per minute (ΔODblank) is measured by mixing GDH solvent in the enzyme reaction measurement reagent instead of the GDH solution as described above. From these values, the activity value is determined according to the following formula.
　ここでＧＤＨ活性における１単位（Ｕ）とは、濃度３００ｍＭのグルコース存在下において、１分間に１マイクロモルのＤＣＩＰを還元する酵素量として定義される。 Here, 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.
　活性（Ｕ／ｍＬ）＝　｛－（ΔＯＤ－ΔＯＤｂｌａｎｋ）×９０５×希釈倍率｝／（１８．５×５×１．０）
　なお、上記計算式の９０５は酵素反応測定試薬と酵素溶液の液量、１８．５は本測定条件におけるＤＣＩＰの分子吸光係数（ｃｍ２／マイクロモル）、５は酵素溶液の液量、１．０は酵素活性測定に使用するセルの光路長（ｃｍ）を示す。 Activity (U / mL) = {− (ΔOD−ΔODblank) × 905 × dilution factor} / (18.5 × 5 × 1.0)
In the above calculation formula, 905 is the amount of the enzyme reaction measurement reagent and the enzyme solution, 18.5 is the molecular extinction coefficient of DCIP (cm 2 / micromol) under the present measurement conditions, and 5 is the amount of the enzyme solution. 0 shows the optical path length (cm) of the cell used for enzyme activity measurement.
　本発明において、ＦＡＤＧＤＨの比活性の測定は以下の条件で行う。
（試薬）
酵素希釈液　２０ｍＭ 酢酸アンモニウムバッファー ｐＨ５．２、０．１％ ＢＳＡ、０．１％ ＴｒｉｔｏｎＸ－１００
酵素反応測定試薬　上記のＧＤＨ活性の測定に使用した試薬と同じ組成のもの
ブラッドフォード試薬　例えば市販品（ＢｉｏＲａｄ社製）を使用する
（測定条件）
　必要であれば酵素原液を酵素希釈液で希釈し、上記活性測定法により活性を求める。酵素原液のタンパク質濃度を、ブラッドフォード試薬により定法に従って測定する。検量線はＢＳＡでとり、ブランクは酵素希釈液でとる。 In the present invention, 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 For example, a commercially available product (manufactured by BioRad) is used (measurement conditions)
If necessary, 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.
　上記計算式により、酵素原液の活性（Ｕ／ｍＬ）を求め、同時にＢＳＡを対照として求めた酵素原液のタンパク質濃度を求める。これらの値から、以下の計算式にしたがって、比活性を求める。 Using the above formula, 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.
　比活性（Ｕ／ｍｇ）＝　（活性）／（タンパク質濃度）
　本発明において、ＦＡＤＧＤＨのＫｍの測定は以下の条件で行う。
（試薬）
１００ ｍＭ リン酸バッファーｐＨ６．５
４ ｍＭ ２,６－ジクロロフェノールインドフェノール(ＤＣＩＰ)
４ ｍＭ ＰＭＳ
Ｄ－グルコース
１０％ ＴｒｉｔｉｏｎＸ－１００
　Ｄ－グルコースは、１M、０．５M、０．２５M、０．１２５M、０．０９４M、０．０６３M、０．０３１Mの計７種類の溶液を準備する。 Specific activity (U / mg) = (activity) / (protein concentration)
In the present invention, 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
For 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.
　上記リン酸バッファーを１２．３ｍＬ、ＤＣＩＰ溶液を０．５ｍＬ、ＰＭＳ溶液を１．０ｍＬ、グルコース溶液を６ｍＬ、ＴｒｉｔｏｎＸ－１００溶液を０．２ｍＬを混合して計７種類のグルコース濃度の異なるＫｍ測定用試薬を調整する。Ｋｍ測定用試薬のグルコース終濃度は、３００ｍＭ、１５０ｍＭ、７５ｍＭ、３７．５ｍＭ、２８ｍＭ、１８．８ｍＭ、９．４ｍＭとなる。
（測定条件）
　必要であれば酵素原液を酵素希釈液で希釈する。酵素反応測定試薬０．９ｍＬを３７℃で３分間以上プレインキュベートする。ＧＤＨ溶液０．００５ｍＬを添加してよく混合し、３７℃で予めインキュベートされた分光光度計で、６００ｎｍの吸光度変化を９０秒間記録し、直線部分から１分間あたりの吸光度変化（ΔＯＤ）を測定する。ブランクは、ＧＤＨ溶液の代わりにＧＤＨの溶媒を酵素反応測定試薬に混合して上記のように１分間あたりの吸光度変化（ΔＯＤｂｌａｎｋ）を測定する。これらの値から、下記の計算式にしたがって反応速度を求める。 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. Add 0.005 mL of GDH solution, mix well, record absorbance change at 600 nm for 90 seconds with a spectrophotometer pre-incubated at 37 ° C., and measure the absorbance change per minute (ΔOD) from the linear portion. . In the blank, the change in absorbance per minute (ΔODblank) is measured by mixing GDH solvent in the enzyme reaction measurement reagent instead of the GDH solution as described above. From these values, the reaction rate is determined according to the following formula.
　ここで反応速度とは、計７通りのグルコース濃度において、１分間の吸光度変化として定義される。 Here, the reaction rate is defined as a change in absorbance per minute at a total of seven glucose concentrations.
　反応速度(ΔＡ)　＝　－（ΔＯＤ－ΔＯＤｂｌａｎｋ）
　横軸にグルコース濃度、縦軸にΔＡをプロットすると、ミカエリス・メンテンプロットが描ける。Ｋｍ値は、公知のコンピュータープログラムを利用してラインフィッティング法により引いた曲線から算出させる。 Reaction rate (ΔA) = − (ΔOD−ΔODblank)
Plotting the glucose concentration on the horizontal axis and ΔA 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.
　以下、本発明の一態様を詳細に説明する。 Hereinafter, one embodiment of the present invention will be described in detail.
　真菌由来ＦＡＤＧＤＨをコードする遺伝子の入手方法については、次の（１）～（４）のような様々な方法が考えられた。 Various methods such as the following (1) to (4) have been considered for obtaining a gene encoding the fungal-derived FADGDH.
　（１）真菌の菌体からｍＲＮＡを抽出し、それらを鋳型にＲＮＡ依存性ＤＮＡポリメラーゼによる逆転写反応によってｃＤＮＡを合成する。次にｃＤＮＡを鋳型としてＦＡＤＧＤＨ特異的なオリゴヌクレオチドでＰＣＲすることにより、ＦＡＤＧＤＨタンパク質をコードする遺伝子を入手する。 (1) mRNA is extracted from fungal cells, and cDNA is synthesized by reverse transcription reaction using RNA-dependent DNA polymerase using them as a template. Next, PCR is performed with a FADGDH-specific oligonucleotide using cDNA as a template to obtain a gene encoding the FADGDH protein.
　（２）真菌の菌体からゲノムＤＮＡを抽出し、それらを鋳型にＦＡＤＧＤＨ特異的なオリゴヌクレオチドでＰＣＲすることにより、ＦＡＤＧＤＨタンパク質をコードするイントロンを含んだ遺伝子を入手する。 (2) 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.
　（３）所望の真菌由来のＦＡＤＧＤＨのアミノ酸配列または遺伝子配列が明らかになっており公知のデータベースに登録されている複数の遺伝子の中からＦＡＤＧＤＨ遺伝子を検索可能である場合、データベース上で公開されている遺伝子の塩基配列に基づいて、後述するＰＣＲによる遺伝子の全合成法（Ｄｉｌｌｏｎ，Ｐ．Ｊ．，Ｒｏｓｅｎ，Ｃ．Ａ．，Ｈｕｍａｎａ Ｐｒｅｓｓ，Ｔｏｔｏｗａ，Ｎｅｗ Ｊｅｒｓｅｙ，Ｖｏｌ．１５，ｐ２６３，１９９３を参照）に従って、所望の遺伝子の塩基配列と全く同じ遺伝子を増幅し、入手する。 (3) If the 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.
　（４）所望の真菌由来のＦＡＤＧＤＨのアミノ酸配列または遺伝子配列が明らかになっておらず、公知のデータベースに登録されている遺伝子群の中からＦＡＤＧＤＨをコードする遺伝子を特定することが不可能である場合、ＦＡＤＧＤＨを生産する真菌から該酵素を単離し、部分アミノ酸配列分析により部分アミノ酸配列情報を得、使用した真菌の全ゲノム配列情報に対して検索することで、上記部分アミノ酸配列を有したタンパク質のアミノ酸配列情報および遺伝子の塩基配列情報を得る。得られた配列情報を元に、後述する遺伝子の全合成法によって目的の遺伝子を得る。 (4) 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 In this case, 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.
　ここでの遺伝子の全合成法としては、簡便に利用できる以下のような方法が挙げられるが限定はされない。 Here, examples of the total synthesis method of the gene include the following methods that can be easily used, but are not limited thereto.
　ＰＣＲを使用した遺伝子の全合成法には、材料として好ましくは１００ｍｅｒ程度の合成オリゴヌクレオチドを使用する。合成オリゴヌクレオチドは、所望の遺伝子配列に基づいて設計することもできるし、所望の遺伝子を異種発現させる際には宿主のコドンユーゼージを使用して設計することもできる。例えば、真菌の遺伝子を大腸菌で発現させる際は、合成する遺伝子配列のコドンユーゼージを大腸菌に最適化するようにオリゴヌクレオチドを合成する。所望の遺伝子が２００ｂｐを超える長鎖である場合には、数本から数十本に分割した合成オリゴヌクレオチドを互いにアニーリング部位をもつように設計する。これらの合成オリゴヌクレオチドを鋳型にＰＣＲを行うことにより、所望の遺伝子を増幅させ、利用することができる。 In the total gene synthesis method using PCR, 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. When the desired gene has a long chain exceeding 200 bp, 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.
　アスペルギルス・オリゼの全ゲノム配列、遺伝子配列とコードされている推定アミノ酸配列は公開されており、例えばNCBIのホームページ（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／）等から閲覧可能であり、アスペルギルス・オリゼＴ１株由来ＦＡＤＧＤＨ遺伝子配列は同定されている（特許文献３参照）。 The entire genome sequence of Aspergillus oryzae and the deduced amino acid sequence encoded by the gene sequence are publicly available, and can be viewed from the NCBI website (http://www.ncbi.nlm.nih.gov/), for example. Aspergillus oryzae T1 strain-derived FADGDH gene sequence has been identified (see Patent Document 3).
　真菌由来ＦＡＤＧＤＨをコードする遺伝子の入手方法として、上記（１）～（４）のように様々な方法が考えられたが、必ずしも成功するとは限らず、本発明者らは様々な困難に対処しつつ鋭意研究を行った結果、上記（４）の方法を利用して、アスペルギルス・オリゼ由来のＦＡＤＧＤＨをコードすると思われる遺伝子を得ることができた。具体的な方法を以下に説明する。 As a method for obtaining a gene encoding FADGDH derived from a fungus, various methods as described in the above (1) to (4) have been considered, but it is not always successful, and the present inventors have dealt with various difficulties. As a result of extensive research, it was possible to obtain a gene that seems to encode FADGDH derived from Aspergillus oryzae using the method of (4) above. A specific method will be described below.
　アスペルギルス・オリゼＩＦＯ４２２０株の培養上清から、後述する各種の精製方法によってＦＡＤＧＤＨを精製することができた。該精製酵素を電気泳動することにより、目的のＦＡＤＧＤＨと予想されるタンパク質のバンドを得ることができた。該タンパク質を極めて解像度の高いＬＣ－ＭＳ／ＭＳ分析に供することによって該タンパク質の部分アミノ酸配列分析を行い、部分アミノ酸配列情報を得た。得られた部分アミノ酸配列情報をアスペルギルス・オリゼＲＩＢ４０株のゲノムデータベースに照合することにより、配列番号１のＦＡＤＧＤＨ候補遺伝子情報の取得に至った。 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.
　しかしながら、該ＦＡＤＧＤＨ候補遺伝子を遺伝子の全合成法によりコドンユーゼージを大腸菌へ最適化せしめ、N末端シグナル配列の除去も実施したが、当該遺伝子を大腸菌で発現させてもＧＤＨ活性を有したタンパク質が発現しないことが明らかになった。したがって、データベースに登録されていた該候補遺伝子にはＧＤＨ活性を有するタンパク質のアミノ酸配列がコードされていないことが初めて明らかになった（なお、同様の知見は後日、上述した特許文献３において公開された）。 However, although the FADGDH candidate gene was optimized for E. coli by codon usage by the gene total synthesis method and the N-terminal signal sequence was removed, a protein having GDH activity was not expressed even when the gene was expressed in E. coli. It became clear. Therefore, it became clear for the first time that the candidate gene registered in the database does not encode the amino acid sequence of a protein having GDH activity (the same knowledge was later disclosed in Patent Document 3 described above). )
　上記ＦＡＤＧＤＨ候補タンパク質がＧＤＨ活性を有さない原因を調べる手法については様々な方法が考えられたが、必ずしも原因が特定できるわけではない。本発明者らは鋭意研究の結果、以下のインフォマティクスを使用した方法を用いることにより、上記ＦＡＤＧＤＨ候補タンパク質がＧＤＨ活性を有さない原因を特定するに至った。 Although various methods have been considered for investigating the cause of the above FADGDH candidate protein not having GDH activity, the cause cannot always be identified. As a result of intensive studies, the present inventors have identified the cause of the fact that the FADGDH candidate protein does not have GDH activity by using a method using the following informatics.
　アスペルギルス・オリゼ由来ＦＡＤＧＤＨと一次構造が似ている遺伝子配列または該遺伝子にコードされているアミノ酸配列を、適当なデータベースに対して相同性検索することにより、アスペルギルス・オリゼ由来ＦＡＤＧＤＨと相同な複数の遺伝子配列またはアミノ酸配列を入手することができる。これらの配列を、後述の公知のプログラムなどを利用してマルチプルアライメント（多重整列）させ、ＦＡＤＧＤＨ候補のアミノ酸配列も同時に並べることにより、アミノ酸配列の欠失等、候補タンパク質を発現させても活性が発現しない原因となっている部位が明らかになる場合がある。すなわち、マルチプルアライメント図のすべての座位は、コンピュータープログラムにより挿入、欠失、置換等が最小となるように並べられる。したがって、アミノ酸配列の欠失等により候補タンパク質に活性がない場合には、候補タンパク質のアミノ酸配列の特定の座位が欠失し、候補遺伝子以外のアミノ酸配列には何らかのアミノ酸が配置されている状況が観察される可能性がある。 A plurality of genes that are homologous to Aspergillus oryzae-derived FADGDH by homologous searching for a gene sequence similar in primary structure to Aspergillus oryzae-derived FADGDH or an amino acid sequence encoded by the gene against an appropriate database Sequences or amino acid sequences can be obtained. These sequences are subjected to multiple alignment (multiple alignment) using a known program to be described later, and the amino acid sequence of FADGDH candidate is also arranged at the same time, so that the activity can be obtained even if the candidate protein is expressed such as deletion of amino acid sequence. The site causing the non-expression may be clarified. That is, 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.
　本発明者らはアスペルギルス・オリゼ由来ＦＡＤＧＤＨ候補タンパク質のアミノ酸配列と相同性の高い特許文献２に記載のアスペルギルス・テレウス（Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ）由来ＦＡＤＧＤＨのアミノ酸配列を用いてＮＣＢＩデータベースに対して相同性検索を行い、複数の相同なアミノ酸配列情報を収集し、それらをマルチプルアライメントソフトによりマルチプルアライメントし、ＦＡＤＧＤＨ候補タンパク質のアミノ酸配列も同時にアライメントさせた。結果を図１に示した。図１において、Ｓｅｑ０１からＳｅｑ１５はぞれぞれ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 ｐｕｎｃｔｉｆｏｒｍｅ）、ｇｌｕｃｏｓｅ－ｍｅｔｈａｎｏｌ－ｃｈｏｌｉｎｅ ｏｘｉｄｏｒｅｄｕｃｔａｓｅ（Ｓｅｑ０７、Ａｎａｂａｅｎａ ｖａｒｉａｂｉｌｉｓ）、Ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ ａｎｄ ｒｅｌａｔｅｄ ｆｌａｖｏｐｒｏｔｅｉｎｓ（Ｓｅｑ０８、ＺＰ＿００１１０５３８、Ｎｏｓｔｏｃ ｐｕｎｃｔｉｆｏｒｍｅ）、ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ０９、ＺＰ＿０１１６２８３６、Ｐｈｏｔｏｂａｃｔｅｒｉｕｍ ｓｐ）、ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１０、ＮＰ＿４１４８４５、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）、ｈｙｐｏｔｈｅｔｉｃａｌ ａｌｃｏｈｏｌ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１１、ＺＰ＿０１１０９７８４、Ａｌｔｅｒｏｍｏｎａｓ ｍａｃｌｅｏｄｉｉ）、ａｌｃｏｈｏｌ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１２、ＹＰ＿６９４４３０、Ａｌｃａｎｉｖｏｒａｘ ｂｏｒｋｕｍｅｎｓｉｓ）、Ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ ａｎｄ ｒｅｌａｔｅｄ ｆｌａｖｏｐｒｏｔｅｉｎｓ（Ｓｅｑ１３、ＺＰ＿００８９１６９２、Ｂｕｒｋｈｏｌｄｅｒｉａ ｐｓｅｕｄｏｍａｌｌｅｉ）、ＦＡＤ－ｏｘｉｄｏｒｅｄｕｃｔａｓｅ ｐｒｏｔｅｉｎ（Ｓｅｑ１４、ＹＰ＿４７１８３９、Ｒｈｉｚｏｂｉｕｍ ｅｔｌｉ）、アスペルギルス・オリゼＲＩＢ４０株由来ＦＡＤＧＤＨ様タンパク質のアミノ酸配列（Ｓｅｑ１５）である。また、Ｓｅｑ１から１４は、それぞれ配列番号１３２から１４５に記載の配列であり、Ｓｅｑ１５は配列番号３に記載の配列である。右端の数字はＮ末端を１位としたときのアミノ酸の位置番号で、例えばＳｅｑ１５の右端のアミノ酸Ｅは、２４８位のアミノ酸であることを示す。（－）はアライメントのギャップを示す。アスペルギルス・オリゼＲＩＢ４０株由来ＦＡＤＧＤＨ様タンパク質のアミノ酸配列に観察された５アミノ酸欠失をアンダーバーで示し、５アミノ酸配列欠失領域と表記している。 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. In Figure 1, 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, Nostoc punctiforme), choline dehydrogenase (Seq09, ZP_01162836, Photobacterium sp), choline dehydrogenase (Seq10, NP_414845 Escherichia coli), hypothetical alcohol dehydrogenase (Seq11 , ZP_01109784, Alteromonas macleodii), alcohol dehydrogenase (Seq12, YP_694430, Alcanivorax borkumensis), Choline dehydrogenase and related flavoproteins (Seq13, ZP_00891692, Burkholderia pseudomallei), FAD-oxidoreductase protein (Seq14, YP_471839, Rhizobium etli), Aspergillus oryzae RIB40 shares It is an amino acid sequence (Seq15) of origin FADGDH-like protein. 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. For example, 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.
　図１に示すようにＦＡＤＧＤＨ候補タンパク質のアミノ酸配列において、２０２位と２０３位のアミノ酸残基の間に５アミノ酸配列の欠失が観察され、本発明者らはこのアミノ酸配列の欠失が該ＦＡＤＧＤＨ候補タンパク質にＧＤＨ活性が発現しない原因である可能性があると考えた。 As shown in FIG. 1, in the amino acid sequence of the FADGDH candidate protein, a deletion of 5 amino acid sequences was observed between the amino acid residues at positions 202 and 203, and the present inventors confirmed that the deletion of this amino acid sequence was the FADGDH. It was thought that this may be the cause of the GDH activity not expressing in the candidate protein.
　例えば、特許文献３に記載されている方法のように、アスペルギルス・オリゼのｍＲＮＡを抽出し、それらを鋳型にＲＮＡ依存性ＤＮＡポリメラーゼによる逆転写反応によってｃＤＮＡを合成し、次にｃＤＮＡを鋳型としてＦＡＤＧＤＨ特異的なオリゴヌクレオチドでＰＣＲすることにより、上記のような問題を回避して微生物からＦＡＤＧＤＨタンパク質をコードする遺伝子を直接入手することができることが知られている。 For example, as in the method described in Patent Document 3, mRNAs 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. It is known that 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.
　しかしながら本出願においては、以下に示す（１）コンセンサス法や（２）系統学的手法に基づいた祖先型アミノ酸導入法といった確率論的な手法を駆使することにより、活性型ＦＡＤＧＤＨの取得に至った。
（１）コンセンサス法
　マルチプルアライメント図に基づくコンセンサス法とは、本来は抗体の機能改変を目的として利用され始め、酵素の熱安定性の向上を目的として利用された実績もあるＤＮＡ配列またはアミノ酸配列における部位特異的変異導入法（配列上のどの位置にどの変異を導入するか部位特異的に決定する方法）であり、本発明のように酵素の活性がない原因となっている部位の改変に利用された実績もある（詳細については、Ｂ．Ｓｔｅｉｐｅ，ｅｔ ａｌ．，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２４０，１８８－１９２，１９９４を参照）。しかし、必ずしも成功するとは限らない。 However, in the present application, 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.
　マルチプルアライメント図に基づくコンセンサス法の材料としては、既知のアミノ酸配列を公知のデータベースに対して相同性検索することで得られる複数のアミノ酸配列を、公知のアライメントプログラムなどを利用してマルチプルアライメントさせた図を使用する。マルチプルアライメント図のすべての座位は、コンピュータープログラムにより挿入、欠失、置換等が最小となるように並べられる。したがって、アミノ酸配列の欠失等により候補タンパク質に活性がない場合には、候補タンパク質のアミノ酸配列の特定の座位が欠失し、候補遺伝子以外のアミノ酸配列には何らかのアミノ酸が配置されている状況が観察されることがある。その座位において候補タンパク質以外のアミノ酸残基に例えばメチオニン（Ｍ）が多く配列していれば、該欠失部位にＭを挿入する。同様にセリン（Ｓ）が多く配置していれば、該欠失部位にＳを挿入する。このような多数決的な決定による変異導入法をコンセンサス法と呼ぶ。
（２）系統学的手法に基づいた祖先型アミノ酸導入法
　系統学的手法に基づいた祖先型アミノ酸導入法は、ある特定の酵素についての複数の生物種における共通祖先のアミノ酸配列を推定し、該アミノ酸配列をもとの酵素に変異として導入することにより共通祖先の酵素の機能を推察する目的で開発された手法である。一般的に共通祖先型の酵素はもとの酵素に対して耐熱化することが示されており、全生物の共通祖先が超好熱菌であるとの仮説を支持するものであるが、本発明における利用のように酵素活性が発現しない原因となっている部位の改変にも使用した実績もある（詳細については、「Ｈｉｓａｋｏ，Ｉ．，ｅｔ ａｌ．，ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌｅｔｔｅｒｓ，２４３，３９３－３９８，２００５」、「Ｋｅｉｋｏ， Ｗ．，ｅｔ　ａｌ．，ＦＥＢＳ Ｌｅｔｔｅｒｓ，５８０，３８６７－３８７１，２００６」、「ＪＰＡ２００２－２４７９９１」を参照）。しかし、必ずしも成功するとは限らない。 As a material for consensus method based on multiple alignment diagrams, multiple amino acid sequences obtained by homology search of known amino acid sequences against a known database were subjected to multiple alignment using a known alignment program etc. Use the figure. All loci in the multiple alignment diagram are aligned 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. If, for example, methionine (M) is abundantly arranged at amino acid residues other than the candidate protein at that locus, M is inserted into the deletion site. Similarly, if serine (S) is arranged in a large amount, S is inserted at the deletion site. Such a method for introducing a mutation by majority decision is called a consensus method.
(2) An ancestral amino acid introduction method based on a phylogenetic method An ancestral amino acid introduction method based on a phylogenetic method estimates an amino acid sequence of a common ancestor in a plurality of species for a specific enzyme, This technique was developed for the purpose of inferring the functions of common ancestor enzymes by introducing amino acid sequences into the original enzyme as mutations. In general, 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.
　材料としては、候補遺伝子のアミノ酸配列をデータベースに対して相同性検索することで得られた複数の相同アミノ酸配列、それらを基に作成した分子系統樹（以下、系統樹とも表記する）および系統樹作成のためのアルゴリズム、マルチプルアライメント図を利用する。 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.
　系統樹作成のための様々なアルゴリズム、例えば、最大節約原理に基づくアルゴリズム等が知られており、それを実現するコンピュータープログラムも利用または入手することができる。例えば、ＣＬＵＳＴＡＬ　Ｗ、ＰＵＺＺＬＥ、ＭＯＬＰＨＹ、ＰＨＹＬＩＰ等の種々の系統樹推定プログラムが利用できる。または、最尤原理に基づくアルゴリズム等も知られており、それを実現するコンピュータープログラムも利用または入手することができる。例えば、ＭｏｄｅｌＴｅｓｔ、ＰＨＹＭＬ、ＰＨＹＬＩＰ、ＴｒｅｅＦｉｎｄｅｒ等の種々の系統樹推定プログラムが利用できる。それらを用いて系統樹を作製することができるが、より簡便には、既に公表されている系統樹を利用することもできる。 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. For example, various phylogenetic tree estimation programs such as CLUSTAL W, PUZZLE, MOLPHY, and PHYLIP can be used. Alternatively, 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. For example, 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.
　このような系統樹においては、分子進化的に近い位置の生物種は、系統樹中で近い位置に現れる。また、系統樹中で根元に近い位置にある生物種はより祖先に近いと考えられる。 In such a phylogenetic tree, species close to molecular evolution appear in close positions in the phylogenetic tree. In addition, the species near the root in the phylogenetic tree are considered to be closer to the ancestors.
　候補遺伝子のアミノ酸配列をデータベースに対して相同性検索することで得られた複数の相同アミノ酸配列のデータを基に適当なプログラムを使用してマルチプルアラインメントの結果が得られたならば、特定の系統樹における祖先型タンパク質のアミノ酸配列を推定することができる。 If multiple alignment results are obtained using an appropriate program based on the data of multiple homologous amino acid sequences obtained by homology search of the amino acid sequences of candidate genes against a database, a specific strain The amino acid sequence of the ancestral protein in the tree can be deduced.
　本発明においては、特に最大節約法または最尤法（「Young, Z., Kumar,S., Nei.M, Genetics 141, 1641-16510, 1995」、「Stewart, C.-B.Active ancestral molecules, Nature 374, 12-13,1995」、「根井正利『分子進化遺伝学』培風館、「根井正利、S. クマー『分子進化と分子系統学』培風館」）を用いた。 In the present invention, 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 ””.
　本発明に使用し得る最大節約法とは、祖先型を仮定したときにその後生じると予想される変異の事象の数が最も少ない祖先型の過程を真の祖先型と推定する方法である。また、最大節約法に基づいてアミノ酸配列から直接に祖先型推定を行なうためのプログラムＰＲＯＴＰＡＲＳも利用可能である。この方法では原理的には系統樹の推定と祖先型アミノ酸の推定が同時に行われるため、必ずしも系統樹を作成することは必要ではないが、特に手動計算で祖先型アミノ酸を推定する場合には系統樹を作成することが好ましい。 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. In principle, 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.
　本発明に使用し得る最尤法とは、予め決定した系統樹の樹形とアミノ酸置換モデルに基づいて、樹形の特定の位置に（主に系統樹の根にあたる部分）おけるあらゆる祖先型アミノ酸配列を推定し、最も尤度の高い配列を最も有望な祖先型アミノ配列として選択する方法である。また、最尤法に基づいて、系統樹およびアミノ酸配列のマルチプルアライメントから祖先型推定を行うためのプログラムＰＡＭＬ等も利用可能である。 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.
　いずれかの方法で得られた系統樹を利用してマルチプルアラインメントしたアミノ酸残基のそれぞれの部位に関して祖先型アミノ酸を決定することができる。このようにして、マルチプルアラインメントした配列の各々の残基に関して祖先型アミノ酸残基を推定し、その結果、対応する領域の祖先型アミノ酸配列を推定することができる。 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.
　この場合、祖先型アミノ酸配列を推定するために用いる生物種を変えると、系統樹の樹形が変化し、それと関連して異なる祖先型アミノ残基が得られる場合もあり、その位置と種類は比較に用いるタンパク質にも依存する。 In this case, 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.
　従って、そのような変動が比較的少ない位置のアミノ酸残基を改変の対象とすることが考えられる。そのようなアミノ酸残基は、系統樹の作成に用いる生物種を変える、または、生物種は変えずに系統樹作成に使用するアミノ酸配列情報の一部のみを用いるなど、系統樹の作成に使用するアミノ酸配列情報を変化させた場合の樹形変化の程度を見積り、樹形への影響の少ない残基を選択することによって決定することが考えられる。 Therefore, it is conceivable that 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.
　上述のようにして祖先型アミノ酸残基が決定されたならば、解析対象としたタンパク質について非祖先型であるアミノ酸残基の少なくとも１つを祖先型アミノ酸残基に置換してそのタンパク質を改変することができる。本発明者らは、候補遺伝子の欠失部分において推定された祖先型アミノ酸配列を、欠失部分に挿入した。 Once the ancestral amino acid residue is determined as described above, 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.
　特定の部位に特定の変異を導入する方法として、キットなどが広く販売され当業者が容易に利用可能なＤＮＡの部位特異的変異導入法などを利用すればよい。そのようにして変異を導入した候補遺伝子の活性を調べるには、該遺伝子を適当な発現ベクターに連結して適当な宿主に導入し、酵素を発現させて活性の有無を確認すればよい。すなわち、タンパク質の遺伝情報を有するＤＮＡの特定の塩基を変換することにより、或いは特定の塩基を挿入または欠失させることにより、改変ＤＮＡ が作成される。ＤＮＡ中の塩基を変換する具体的な方法としては、例えば市販のキット（ＴｒａｎｓｆｏｒｍｅｒＭｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ：Ｃｌｏｎｅｔｅｃｈ製、ＥＸＯＩＩＩ／Ｍｕｎｇ　Ｂｅａｎ　Ｄｅｌｅｔｉｏｎ　Ｋｉｔ：Ｓｔｒａｔａｇｅｎｅ製、 ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ　Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ：Ｓｔｒａｔａｇｅｎｅ製等）の使用等が挙げられる。また、ＤＮＡを幾つかの断片に分割して化学合成し、ＤＮＡリガーゼで結合するという手法を採ることも可能である。 As a method for introducing a specific mutation into a specific site, 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. In order to examine the activity of a candidate gene into which mutations have been introduced in this manner, 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.
　本発明者らは、ＧＤＨ活性を発現しない配列番号３のアミノ酸配列から成るタンパク質を改変してＧＤＨ活性を発現させるべく鋭意検討した結果、具体的には実施例３に示すようにアミノ酸配列のマルチプルアライメント図とコンセンサス法および祖先型アミノ酸導入法を参考とし、鋭意研究を行い、配列番号３のアミノ酸配列に特定の部位特異的変異導入を行うことにより、活性型ＦＡＤＧＤＨを得ることができることを初めて見出した。 As a result of intensive studies to modify the protein consisting of the amino acid sequence of SEQ ID NO: 3 that does not express GDH activity to express GDH activity, the present inventors specifically showed that multiple amino acid sequences as shown in Example 3 For the first time, it has been found that active FADGDH can be obtained by conducting extensive research with reference to alignment diagrams, consensus methods, and ancestral amino acid introduction methods, and introducing specific site-specific mutations into the amino acid sequence of SEQ ID NO: 3. It was.
　すなわち、本発明の活性型ＦＡＤＧＤＨは、配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＩＰＱＴ、ＧＩＰＴＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列からなるポリペプチド及び配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位のメチオニン（Ｍ）がアラニン（Ａ）に置換され、かつ２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列からなるポリペプチドである。 That is, 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. In which 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.
　さらに本発明者らは、該活性型ＦＡＤＧＤＨの機能改変に取り組んだ結果、基質親和性と比活性値を向上させる機能改変方法をも見出した。 Furthermore, as a result of working on functional modification of the active FADGDH, the present inventors have also found a functional modification method that improves the substrate affinity and specific activity value.
　活性型ＦＡＤＧＤＨの機能改変に関する方法としては、以下の（１）～（３）に示すように、マルチプルアライメント図に基づくコンセンサス法や系統学的手法に基づいた祖先型アミノ酸導入法および進化工学的手法が考えられた。 As a method for functional modification of active FADGDH, as shown in the following (1) to (3), an ancestral amino acid introduction method and evolutionary engineering method based on a consensus method based on multiple alignment diagrams and a phylogenetic method Was considered.
　（１）コンセンサス法
　例えば、マルチプルアライメント図に基づくコンセンサス法とは、上述したような方法により実施することができる。 (1) Consensus method For example, the consensus method based on multiple alignment diagrams can be implemented by the method as described above.
　（２）系統学的手法に基づいた祖先型アミノ酸導入法
例えば、系統学的手法に基づいた祖先型アミノ酸導入法とは、上述したような方法により実施することができる。 (2) An ancestral amino acid introduction method based on a phylogenetic technique For example, an ancestral amino acid introduction method based on a phylogenetic technique can be performed by the method described above.
　（３）進化工学的手法
　例えば、進化工学的手法を利用した酵素の機能改変は、まず目的のタンパク質の遺伝子配列にランダム変異を導入するところから始める。ランダム変異導入とは、主としてＰＣＲを利用した手法であり、ＰＣＲの正確性をわざと低く設定することで遺伝子上の様々な部位に様々な変異を導入する手法であり、例えば、Ｄｉｖｅｒｓｉｔｙ ＰＣＲ Ｒａｎｄｏｍ Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ（Ｃｌｏｎｔｅｃｈ製）、ＧｅｎｅＭｏｒｐｈ　ＩＩ　ＲａｎｄｏｍＭｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ製）等のキットを使用して簡便に実施することができる。そのようにして変異を導入した遺伝子の変異体を後述するような方法により適当な発現プラスミドに連結して適当な宿主に形質転換し、プレート培地上に撒いてコロニーを形成させることにより、変異体ライブラリを作成することができる。 (3) Evolutionary engineering method For example, functional modification of an enzyme using an evolutionary engineering method is started by first introducing a random mutation into the gene sequence of the target protein. 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. For example, Diversity PCR Random Mutagenesis Kit (Manufactured by Clontech), GeneMorph II Randommutagenesis Kit (manufactured by Stratagene), etc. 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.
　上記の変異体ライブラリは単コロニーずつ、９６ウェルプレート等において液体培養し、変異体酵素を発現させることが可能である。発現させた変異体酵素は９６ウェルフォーマット等で後の実験に使用することができる。酵素の機能改変ターゲットが基質親和性であれば、上記９６ウェルフォーマットの変異体酵素のライブラリを、同じく９６ウェルフォーマットで調製した通常よりも基質濃度の低い活性測定試薬へ入れて酵素反応をモニターし、他の変異体に比べ酵素反応の反応速度が速い変異体をピックアップすることにより、基質親和性の高い変異体酵素をスクリーニングすることが可能である。変異体遺伝子のどの部位にどのような変異が導入されたかは、後述するようにシーケンスによりDNA配列を読むことにより確認できる。 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.
　本発明者らは、上述の方法を用いて鋭意検討した結果、基質親和性および／または比活性を向上させた改変型ＦＡＤＧＤＨの取得に至り、本発明に至った。 As a result of intensive studies using the above-described method, the present inventors have obtained modified FADGDH with improved substrate affinity and / or specific activity, leading to the present invention.
　次に、本発明を実施例に具体的に説明するが、本発明は以下に限定されるものではない。
＜実施例１＞
アスペルギルス・オリゼ由来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遺伝子（以下、ＦＡＤＧＤＨとも表記する）の取得
　本研究を開始した当初、アスペルギルス属においてＦＡＤＧＤＨの遺伝子配列は公開されていなかった。したがって、ＦＡＤＧＤＨを単離、精製し、アミノ酸分析を行って部分アミノ酸配列を得、公知のゲノム情報データベースとの照合によって該遺伝子配列の取得を試みた。
（１－１）アスペルギルス・オリゼの培養
　まず、自社保有のアスペルギルス・オリゼＩＦＯ４２２０株のグリセロールストックをポテトデキストロース寒天培地（日水製薬製）に植菌して２５℃で４８時間培養して該糸状菌の菌糸体を得た。培養液（１％グルコース、２％ポリペプトンNS（日本製薬製）、０．５％コーンスティープリカー、０．１％硫酸マグネシウム７水和物、ｐＨ６．０）１Lを調整し、５Lフラスコに入れて１２０℃で２０分オートクレーブ滅菌し、上記寒天培地を植菌して３０℃で２４時間培養し、０．１ｍＭ　ＥＤＴＡ、０．１ｍＭ　ヒドロキノンを添加してさらに２４時間培養した。培養終了後、菌糸体をガーゼで濾過して培養上清を得た。培養上清のＧＤＨ活性を測定した結果、活性が見出されたので、以下の精製の手順へ進んだ。
（１－２）ＧＤＨの精製
　濾過した培養上清を６５％飽和硫酸アンモニウムにより塩析し、遠心分離によりＧＤＨ活性を有する上清画分を得た。さらに上清画分を９５％飽和硫安となるように硫酸アンモニウムを添加して塩析し、遠心分離によりＧＤＨ活性を有する沈殿画分を得た。沈殿を２０ｍＭ酢酸アンモニウムバッファー（pＨ５．２）に懸濁し、さらに同バッファーで透析した。上記バッファーで平衡化したＤＥＡＥ－Ｓｅｐｈａｒｏｓｅカラムに、透析したＧＤＨ溶液を素通りさせて共雑物を除いた。次に、２０ｍＭ酢酸アンモニウムバッファー（pＨ５．２）で平衡化したＲＥＳＯＵＲＣＥ　Ｓカラム（ＧＥヘルスケア製）にＤＥＡＥ－Ｓｅｐｈａｒｏｓｅカラムを素通りさせたＧＤＨ溶液を吸着させ、０Ｍから１ＭのＮａＣｌの濃度勾配でＧＤＨを溶出させた。 Next, although an Example demonstrates this invention concretely, this invention is not limited to the following.
<Example 1>
Acquisition of Aspergillus oryzae-derived flavin adenine dinucleotide-dependent glucose dehydrogenase gene (hereinafter also referred to as FADGDH) At the beginning of this study, the gene sequence of FADGDH was not disclosed in the genus Aspergillus. Therefore, FADGDH was isolated and purified, amino acid analysis was performed to obtain a partial amino acid sequence, and acquisition of the gene sequence was attempted by collation with a known genome information database.
(1-1) Cultivation of Aspergillus oryzae First, the glycerol stock of Aspergillus oryzae IFO4220 strain owned by the company was inoculated into potato dextrose agar medium (manufactured by Nissui Pharmaceutical) and cultured at 25 ° C. for 48 hours, and then the filamentous fungus Obtained mycelium. Prepare 1 L of culture solution (1% glucose, 2% polypeptone NS (Nippon Pharmaceutical Co., Ltd.), 0.5% corn steep liquor, 0.1% magnesium sulfate heptahydrate, pH 6.0) and place in a 5 L flask. The mixture was sterilized by autoclaving at 120 ° C. for 20 minutes, inoculated with the agar medium, cultured at 30 ° C. for 24 hours, added with 0.1 mM EDTA and 0.1 mM hydroquinone, and further cultured for 24 hours. After completion of the culture, the mycelium was filtered with gauze to obtain a culture supernatant. As a result of measuring the GDH activity of the culture supernatant, the activity was found, so the procedure proceeded to the following purification procedure.
(1-2) Purification of GDH The filtered culture supernatant was salted out with 65% saturated ammonium sulfate, and a supernatant fraction having GDH activity was obtained by centrifugation. Furthermore, 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. Next, 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.
　アスペルギルス・オリゼの分泌型ＦＡＤＧＤＨは糖鎖修飾を受けていることが示されており（非特許文献１、Ｔｃｈａｎ－ｇｉ Ｂａｋ，Ｂｉｏｃｈｉｍ．Ｂｉｏｐｈｙｓ．Ａｃｔａ，１３９，２７７－２９３，１９６７）、定法で使用されるタンパク質染色剤のクマシーブリリアントブルー（ＣＢＢ）では十分に染色されない可能性が示唆されたので、ＣＢＢ染色と、糖タンパク質染色用のＰＡＳ染色（ＳＩＧＭＡ製）の両方の染色法を使用して、GDH画分をＳＤＳ－ＰＡＧＥした。結果、いずれの染色法でも７５ｋＤａと１００ｋＤａの分子量マーカーの間にバンドが観察された。
（１－３）部分アミノ酸配列分析
　ＣＢＢ染色により得られた上記の単一バンドをゲルから切り出してＬＣ－ＭＳ／ＭＳ分析（島津製作所）を実施したところ、部分アミノ酸配列データを得ることができた。該部分アミノ酸配列情報をアスペルギルス・オリゼＲＩＢ４０株の全ゲノムデータベースに対して照合したところ、配列番号１の遺伝子であると推定された。以下、該遺伝子をＦＡＤＧＤＨ候補遺伝子と表記する。
＜実施例２＞
ＦＡＤＧＤＨ候補遺伝子の合成と大腸菌における発現
　実施例１より、ＦＡＤＧＤＨ候補遺伝子として配列番号１の塩基配列情報を得ることができたので、該遺伝子を大腸菌で発現させるべく合成した。その具体的な実施例を以下に示す。
（２－１）コドンユーゼージを変更したＦＡＤＧＤＨ候補遺伝子の設計
　配列番号１に示したＤＮＡ配列のコドンユーゼージは真菌のものであるため、該遺伝子を大腸菌で効率よく発現させるためには、遺伝子の全配列のコドンを大腸菌に適したものに変更する必要がある。まず、配列番号１に記載のＤＮＡ配列のコドンを変更して設計し直すところから始めた。 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.
(1-3) Partial amino acid sequence analysis The above single band obtained by CBB staining was cut out from the gel and subjected to LC-MS / MS analysis (Shimadzu Corporation). As a result, partial amino acid sequence data could be obtained. . When the partial amino acid sequence information was collated against the whole genome database of Aspergillus oryzae RIB40 strain, it was estimated to be the gene of SEQ ID NO: 1. Hereinafter, this gene is referred to as a FADGDH candidate gene.
<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.
　変更後のコドンは以下のように設定した。一文字目のアルファベットから、アミノ酸の一文字表記、アミノ酸の名称（設計に使用したコドン）の順に表記する。 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：フェニルアラニン（ｔｔｃ）、Ｌ：ロイシン（ｃｔｇ）、Ｉ：イソロイシン（ａｔｃ）、Ｍ：メチオニン（ａｔｇ）、Ｖ：バリン（ｇｔａ）、Ｙ：チロシン（ｔａｃ）、終止コドン（ｔａａ）、Ｈ：ヒスチジン（ｃａｃ）、Ｑ：グルタミン（ｃａｇ）、Ｎ：アスパラギン（ａａｃ）、Ｋ：リジン（ａａｇ）、Ｄ：アスパラギン酸（ｇａｃ）、Ｅ：グルタミン酸（ｇａａ）、Ｓ：セリン（ｔｃｔ）、Ｐ：プロリン（ｃｃｇ）、Ｔ：スレオニン（ａｃｃ）、Ａ：アラニン（ｇｃｔ）、Ｃ：システイン（ｔｇｃ）、Ｗ：トリプトファン（ｔｇｇ）、Ｒ：アルギニン（ｃｇｔ）、Ｇ：グリシン（ｇｇｔ）。 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).
　配列番号１の１番目のコドン「ａｔｇ」から順に、上記コドンに変更して大腸菌に適したＤＮＡ配列の設計を行った。ただし、ロングプライマーＰＣＲ時のスリッピングによる望まない変異の導入を防ぐため、可能な限り３つ以上同じ塩基が連続しない（例えば「ｔｔｔ」や「ｇｇｇ」）ように工夫して設計し、配列番号２のＤＮＡ配列情報を得た。すなわち、例えばアミノ酸がアスパラギン酸－プロリン（ＤＰ）と連続する場合、上述したコドンでＤＮＡを設計すると「ｇａｃｃｃｇ」となりｃが３つ連続してしまうので、Ｄのコドンを、ｇａｃよりコドン使用頻度の低いｇａｔとするようにした。なお、配列番号１の遺伝子と配列番号２の遺伝子は、コドンユーゼージが異なるだけで、ともに配列情報３のアミノ酸配列をコードしている。
（２－２）全合成に使用するロングプライマー
　参考にした遺伝子の全合成方法（非特許文献２）は、１００ｍｅｒ程度の長い合成オリゴヌクレオチド（ロングプライマー）を使用する。設計した配列番号２に記載のＤＮＡ配列をもとにして、配列番号４から配列番号２５に記載のロングプライマーを合成し、以下のＰＣＲによる遺伝子の全合成に使用した。ロングプライマーのアニーリング領域は、１８～２２ｍｅｒとした。
（２－３）ＰＣＲによる遺伝子の全合成
　参考にしたＰＣＲによる遺伝子の全合成法は、２回のＰＣＲステップ、すなわち、ＰＣＲ１とＰＣＲ２を必要とする。まずＰＣＲ１で上記の一部アニーリングするロングプライマーどうしをＰＣＲにより伸長させ、微量の全長遺伝子を得る。次に、ＰＣＲ２によって、ＰＣＲ１で生じた微量の全長遺伝子を鋳型に、遺伝子全体を増幅するように設計した特異的プライマーによりＰＣＲを行い、所望の遺伝子を増幅させることができる。 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. However, in order to prevent the introduction of undesired mutations due to slipping during long primer PCR, 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. Note that the gene of SEQ ID NO: 1 and the gene of SEQ ID NO: 2 both encode the amino acid sequence of sequence information 3 with only the difference in codon usage.
(2-2) Long Primer Used for Total Synthesis The reference total gene synthesis method (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.
(2-3) Total Synthesis of Genes by PCR The method of total synthesis of genes by reference PCR requires two PCR steps, that is, PCR1 and PCR2. First, in PCR1, the above-mentioned long primers that partially anneal are extended by PCR to obtain a trace amount of a full-length gene. Next, 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.
　特異的プライマーの塩基配列は配列番号２６と配列番号２７に示す。配列番号２６の特異的プライマーは、配列番号２のＤＮＡ配列５’端にＮｄｅＩ制限酵素認識配列を追加し、配列番号２７の特異的プライマーは配列番号２のＤＮＡ配列３’に終止コドンを２回連続して配置し、さらにＢａｍＨＩ制限酵素認識配列を追加するように設計した。 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反応溶液（１×ＫＯＤ～ｐｌｕｓ～緩衝液（東洋紡製）、０．２ｍＭ　ｄＮＴＰｓ、１ｍＭ　ＭｇＳＯ４、０．２μＭ　ロングプライマー、１Ｕ　ＫＯＤポリメラーゼ（東洋紡製））を５０μＬ調整し、以下のサイクル様式でＰＣＲを行った。 50 μL of PCR1 reaction solution (1 × KOD-plus-buffer (Toyobo), 0.2 mM dNTPs, 1 mM MgSO4, 0.2 μM long primer, 1 U KOD polymerase (Toyobo)) was prepared, and PCR was performed in the following cycle mode. Went.
　初期変性 ９４℃、１分間
　１２サイクル ９４℃、３０秒間
５５℃、１分間
６８℃、２分間
　最終伸長 ６８℃、５分間
　ＰＣＲ１完了後、ＰＣＲ２反応溶液（１×ＫＯＤ～ｐｌｕｓ～緩衝液（東洋紡製）、０．２ｍＭ　ｄＮＴＰｓ、１ｍＭ　ＭｇＳＯ４、０．３μＭ　特異的プライマー、１μＬ　ＰＣＲ１反応溶液、１Ｕ　ＫＯＤポリメラーゼ（東洋紡製））を５０μＬ調整し、以下のサイクル様式でＰＣＲを行った。 Initial denaturation 94 ° C, 1 minute 12 cycles 94 ° C, 30 seconds 55 ° C, 1 minute 68 ° C, 2 minutes Final extension 68 ° C, 5 minutes After completion of PCR1, PCR2 reaction solution (1 × KOD-plus-buffer (Toyobo) ), 0.2 mM dNTPs, 1 mM MgSO 4, 0.3 μM specific primer, 1 μL PCR1 reaction solution, 1U KOD polymerase (manufactured by Toyobo)) was adjusted to 50 μL, and PCR was performed in the following cycle mode.
　初期変性 ９４℃、１分間
　１２サイクル ９４℃、３０秒間
５５℃、３０分間
６８℃、２分間
　最終伸長 ６８℃、５分間
　ＰＣＲ２完了後、反応溶液の一部をとって定法によりアガロースゲル電気泳動したところ、約１８００ｂｐの単一なバンドが観察された。
（２－４）Ｎ末端側シグナル配列の除去
　本発明者らは、該酵素は本来、菌体外へ分泌される酵素であるため、タンパク質のN末端側に分泌シグナルを有している可能性があると考えた。そこで公知の推定プログラムを使用してシグナル配列の領域とその切断部位を推定したところ、本発明者らは、配列番号３のアミノ酸配列の１６番目のAと１７番目のSの間、または２２番目のAと２３番目のKの間で切断される可能性を推測した。そこで、１５番目までのアミノ酸を除去し、１６番目のアミノ酸を開始コドンのメチオニン（M）に変更して配列番号２８のDNA配列が増幅できるように設計した配列番号２９と配列番号３０のオリゴヌクレオチドで（２－３）のＰＣＲ産物をさらにＰＣＲした。同様に、２１番目までのアミノ酸を除去し、２２番目のアミノ酸を開始コドンのメチオニン（M）に変更して配列番号３１のＤＮＡ配列が増幅できるように設計した配列番号３２と配列番号３０のオリゴヌクレオチドで（２－３）のＰＣＲ産物をさらにＰＣＲした。（２－３）のＰＣＲ産物および上記のＮ末シグナル配列を除去するように増幅させたＰＣＲ産物をそれぞれ定法により精製した。
（２－５）合成遺伝子の大腸菌における発現
　上記遺伝子を発現させるプラスミドにはｐＢｌｕｅｓｃｒｉｐｔを使用した。該プラスミドはｌａｃプロモーターによりＬａｃＺ遺伝子を発現するが、ｌａｃＺ開始コドン近傍配列「ｃｔａｔｇ」をＰＣＲによる部位特異的変異導入法により「ｃａｔａｔｇ」とすることにより、ＮｄｅＩ認識配列とすることができ、ＮｄｅＩで切断した遺伝子をクローニングし、ｌａｃプロモーターにより遺伝子を比較的ゆるやかに発現させることができる。以下、ＮｄｅＩ認識配列をｌａｃＺ開始コドンに挿入したｐＢｌｕｅｓｃｒｉｐｔをｐＢＳＮと表記する。 Initial denaturation 94 ° C, 1 minute 12 cycles 94 ° C, 30 seconds 55 ° C, 30 minutes 68 ° C, 2 minutes Final extension 68 ° C, 5 minutes After completion of PCR2, a part of the reaction solution was taken and subjected to agarose gel electrophoresis by a conventional method However, a single band of about 1800 bp was observed.
(2-4) Removal of N-terminal signal sequence Since the enzyme is originally an enzyme that is secreted out of the cell body, it may have a secretion signal on the N-terminal side of the protein. I thought there was. Thus, when a signal sequence region and its cleavage site were estimated using a known estimation program, 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. The PCR product (2-3) was further subjected to PCR. Similarly, 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. Hereinafter, pBluescript in which the NdeI recognition sequence is inserted into the lacZ start codon is denoted as pBSN.
　ＦＡＤＧＤＨ候補遺伝子を大腸菌で発現させるために、（２－３）および（２－４）に記載のＤＮＡとｐＢＳＮをＮｄｅＩおよびＢａｍＨＩで切断してそれぞれを精製し、リガーゼで両遺伝子断片どうしを結合して発現プラスミドを作製した。 In order to express the FADGDH candidate gene in E. coli, the DNA and pBSN described in (2-3) and (2-4) were cleaved with NdeI and BamHI, purified, and the two gene fragments were ligated with each other. Thus, an expression plasmid was prepared.
　作製した発現プラスミドにより大腸菌ＤＨ５αを形質転換し、寒天プレート培地上で培養してコロニーを形成させ、各発現プラスミドで形質転換した形質転換体を得た。 Escherichia coli DH5α 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.
　得られた形質転換体を２ｍＬのＬＢ培地で培養し、定法に従いプラスミドを精製した。合成した遺伝子に特異的なプライマーを使用してシーケンスを行い、所望の配列番号２、配列番号２８、配列番号３１のＤＮＡが組み込まれた発現プラスミドであることを確認した。以下、配列番号２、配列番号２８、配列番号３１の遺伝子が組み込まれた発現プラスミドを、それぞれｐＢＳＮＧＤＨ、ｐＢＳＮＧＤＨＭ１７、ｐＢＳＮＧＤＨＭ２３と表記する。 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. Hereinafter, 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.
　ｐＢＳＮＧＤＨ、ｐＢＳＮＧＤＨＭ１７、ｐＢＳＮＧＤＨＭ２３で形質転換された形質転換体の単コロニーを２ｍＬのＬＢ培地に植菌して３７℃で１６時間培養し、１００μｇ／mＬのアンピシリンを含むＴｅｒｒｉｆｉｃ培養液２０ｍＬに植菌して２７℃で４８時間培養した。 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 μg / mL ampicillin. The cells were cultured at 27 ° C. for 48 hours.
　培養終了後、菌体を破砕して活性の有無を確認したが、ｐＢＳＮＧＤＨ、ｐＢＳＮＧＤＨＭ１７、ｐＢＳＮＧＤＨＭ２３で形質転換した大腸菌でともにＧＤＨ活性は観察されなかった。以上の結果より、アスペルギルス・オリゼＲＩＢ４０株のゲノムデータベースより得られたＦＡＤＧＤＨ候補遺伝子から発現させたタンパク質およびそのシグナル配列除去タンパク質は活性を有していないことが示された。
＜実施例３＞
活性を有したＦＡＤＧＤＨ（以降、活性型ＦＡＤＧＤＨとも表記する）遺伝子の合成、大腸菌における発現解析
　実施例２より、データベースに登録されていた配列番号３のアミノ酸配列から成るタンパク質はＧＤＨ活性を有していないことが明らかになった。そこで配列番号３のアミノ酸配列と、既に公開されているアスペルギルス・テレウス由来ＦＡＤＧＤＨ（以下ＡＴＧＤＨとする）のアミノ酸配列およびデータベースに登録されているＡＴＧＤＨに相同なアミノ酸配列を比較することにより、配列番号３のアミノ酸配列から成るＦＡＤＧＤＨ候補タンパク質にＧＤＨ活性が認められない原因を調べた。
（３－１）ＧＤＨ活性が認められない原因の究明
　この時点で、アスペルギルス・テレウス由来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（以下ＡＴＧＤＨとする）のＤＮＡ配列およびアミノ酸配列は公開されており（特許文献２、ＷＯ２００６／１０１２３９）、ＡＴＧＤＨと相同なアミノ酸配列群をＮＣＢＩのデータベース上（ｈｔｔｐ：／／ｗｗｗ.ｎｃｂｉ.ｎｌｍ.ｎｉｈ.ｇｏｖ／）で検索することが可能であった。さらに、配列番号３のアミノ酸配列とＡＴＧＤＨのアミノ酸配列は比較的相同性が高かったことから、配列番号３のアミノ酸配列に変異（アミノ酸の欠失等）が存在するかどうか、それぞれのアミノ酸配列をアライメントさせることにより検討した。 After completion of the culture, the cells were disrupted to confirm the presence or absence of activity. However, no GDH activity was observed in E. coli transformed with pBSNGDH, pBSNGDH17, and pBSNGDHHM23. From the above results, it was shown that the protein expressed from the FADGDH candidate gene obtained from the genome database of Aspergillus oryzae RIB40 strain and its signal sequence-removed protein have no activity.
<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. Therefore, by comparing the amino acid sequence of SEQ ID NO: 3 with the previously published amino acid sequence of FADGDH (hereinafter referred to as ATGDH) derived from Aspergillus terreus and the amino acid sequence homologous to ATGDH registered in the database, 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.
(3-1) Investigation of the cause of the absence of GDH activity At this time, the DNA sequence and amino acid sequence of flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter referred to as ATGDH) derived from Aspergillus tereus have been published (patent document) 2, WO2006 / 101239) and amino acid sequence groups homologous to ATGDH could be searched on the NCBI database (http://www.ncbi.nlm.nih.gov/). Furthermore, since the 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.
　公開されていたＡＴＧＤＨのアミノ酸配列を相同性検索ソフトのＢｌａｓｔｐにより検索し、該配列と相同性の高いアミノ酸配列群のアミノ酸配列情報を得た。具体的には、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 ｐｕｎｃｔｉｆｏｒｍｅ）、ｇｌｕｃｏｓｅ－ｍｅｔｈａｎｏｌ－ｃｈｏｌｉｎｅ ｏｘｉｄｏｒｅｄｕｃｔａｓｅ（Ｓｅｑ０７、Ａｎａｂａｅｎａ ｖａｒｉａｂｉｌｉｓ）、Ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ ａｎｄ ｒｅｌａｔｅｄ ｆｌａｖｏｐｒｏｔｅｉｎｓ（Ｓｅｑ０８、ＺＰ＿００１１０５３８、Ｎｏｓｔｏｃ ｐｕｎｃｔｉｆｏｒｍｅ）、ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ０９、ＺＰ＿０１１６２８３６、Ｐｈｏｔｏｂａｃｔｅｒｉｕｍ ｓｐ）、ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１０、ＮＰ＿４１４８４５、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）、ｈｙｐｏｔｈｅｔｉｃａｌ ａｌｃｏｈｏｌ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１１、ＺＰ＿０１１０９７８４、Ａｌｔｅｒｏｍｏｎａｓ ｍａｃｌｅｏｄｉｉ）、ａｌｃｏｈｏｌ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１２、ＹＰ＿６９４４３０、Ａｌｃａｎｉｖｏｒａｘ ｂｏｒｋｕｍｅｎｓｉｓ）、Ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ ａｎｄ ｒｅｌａｔｅｄ ｆｌａｖｏｐｒｏｔｅｉｎｓ（Ｓｅｑ１３、ＺＰ＿００８９１６９２、Ｂｕｒｋｈｏｌｄｅｒｉａ ｐｓｅｕｄｏｍａｌｌｅｉ）、ＦＡＤ－ｏｘｉｄｏｒｅｄｕｃｔａｓｅ ｐｒｏｔｅｉｎ（Ｓｅｑ１４、ＹＰ＿４７１８３９、Ｒｈｉｚｏｂｉｕｍ ｅｔｌｉ）、のアミノ酸配列情報を得た。さらにこれらの配列情報に、配列番号３のアミノ酸配列（Ｓｅｑ１５）を追加した。ここで、Ｓｅｑ～とは図１に示した配列番号を指し、間の数値はＮＣＢＩデータベースに登録されているそれぞれのアミノ酸配列のアクセッションナンバーを指す。さらにその後に生物種の名称を示した。また、Ｓｅｑ１から１４は、それぞれ配列番号１３２から１４５に記載の配列であり、Ｓｅｑ１５は配列番号３に記載の配列である。 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. Specifically, 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 variabilis), Choline dehydrogenase and related flavoproteins (Seq08, ZP_00110538, Nostoc punctiforme), choline dehydrogenase (Seq09, ZP_01162836, Photobacterium sp), choline dehydrogenase (Seq10, NP_414845, Escherichia coli ), Hypothetical alcohol dehydrogenase (Seq11, ZP_01109784, Alter omonas macleodii), alcohol dehydrogenase (Seq12, YP_694430, Alcanivorax borkumensis), Choline dehydrogenase and related flavoproteins (Seq13, ZP_00891692, Burkholderia pseudomallei), FAD-oxidoreductase protein (Seq14, YP_471839, to obtain a Rhizobium etli), amino acid sequence information. Furthermore, the amino acid sequence of SEQ ID NO: 3 (Seq15) was added to these sequence information. Here, 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.
　上記１５種類のアミノ酸配列データを公知のプログラムによりマルチプルアライメントした結果、配列番号３のアミノ酸配列の２０２番目のMと２０３番目のEの間に明らかな５アミノ酸の欠失が見出された（図１）。本発明者らは、この５アミノ酸の欠失が、配列番号３のアミノ酸配列から成るタンパク質にＧＤＨ活性が認められなかった原因と考えた。そこで、この５アミノ酸の欠失を埋め、かつＧＤＨ活性を回復させるために、（３－２）以下に記載するコンセンサス概念に基づくアミノ酸配列の挿入実験を行った。 As a result of multiple alignment of the above 15 types of amino acid sequence data using a known program, a clear 5-amino acid deletion was found between the 202nd M and the 203rd E of the amino acid sequence of SEQ ID NO: 3 (Fig. 1). The present inventors considered that this 5 amino acid deletion was the cause of the absence of GDH activity in the protein consisting of the amino acid sequence of SEQ ID NO: 3. Therefore, in order to fill in this deletion of 5 amino acids and restore GDH activity, an insertion experiment of an amino acid sequence based on the consensus concept described in (3-2) below was performed.
　ＦＡＤＧＤＨの遺伝子情報を得る方法としては、実施例１で使用したアスペルギルス・オリゼＩＦＯ４２２０株から抽出したｍＲＮＡを鋳型に完全長ｃＤＮＡを合成し、特異的プライマーを使用したＰＣＲによりＦＡＤＧＤＨのＤＮＡを増幅し、シーケンスによりその塩基配列を得る方法がある。しかしながらこの方法は、きわめて分解しやすいｍＲＮＡを材料に使用すること、完全長ｃＤＮＡを得るのが容易でないなど、技術的難易度の高いプロセスがいくつか存在する。さらに、最も重大な問題は、アスペルギルス・オリゼＩＦＯ４２２０株が再現良くＦＡＤＧＤＨを発現しないことであった。本発明者らは、ＦＡＤＧＤＨの発現はすべて当該菌のフラスコ培養で実施しており、培養条件の十分なコントロールができていなかったことが原因として考えたが、再現良くＦＡＤＧＤＨを発現させる方策を見出せなかった。上記のような理由により、配列情報さえ揃えば比較的簡単に実施できるコンセンサス概念に基づいた変異導入法を選択した。
（３－２）コンセンサス概念に基づいたアミノ酸配列の挿入実験
　得られたアライメント図からコンセンサス法により、５アミノ酸欠失領域のどの部位にどのアミノ酸を導入するべきかを推定した。すなわち、図１のＳｅｑ１５が配列番号３に記載のアミノ酸配列であり、２０２番目のMと２０３番目のEの間に（－）で示された５アミノ酸の欠失が観察され（５アミノ酸配列欠失領域と示している）、他のＳｅｑ０１からＳｅｑ１４に関しては何らかのアミノ酸残基が埋まっている。 As a method for obtaining FADGDH gene information, 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. There is a method for obtaining the base sequence by sequencing. However, in 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. Furthermore, the most serious problem was that the Aspergillus oryzae IFO4220 strain did not reproducibly express FADGDH. The present inventors considered that all FADGDH expression was carried out in flask culture of the bacterium, and that the culture conditions were not sufficiently controlled. However, the inventors could find a method for expressing FADGDH with good reproducibility. There wasn't. For the reasons described above, a mutagenesis method was selected based on the consensus concept that can be implemented relatively easily if all the sequence information is available.
(3-2) Insertion experiment of amino acid sequence based on the consensus concept It was estimated from which alignment figure the amino acid to be introduced into which part of the 5-amino acid deletion region by the consensus method. That is, Seq15 in FIG. 1 is the amino acid sequence set forth in SEQ ID NO: 3, and a 5-amino acid deletion indicated by (−) is observed between the 202nd M and the 203rd E (5 amino acid sequence missing). In other Seq01 to Seq14, some amino acid residues are filled.
　Ｎ末端側から数えて１番目の欠失に関しては、Ｓｅｑ０１からＳｅｑ１４の配列においてＧが１３個、Ｄが１個であり、Ｇが優勢である。したがって、本発明者らは、挿入する１番目のアミノ酸はＧと推定した。２番目の欠失に関しては、Ｓｅｑ０１からＳｅｑ１４の配列においてＹが６個、Ｌが４個、Ｉが３個であった。詳細は実施例５に示すが、図１で得たアライメント図から系統学的手法により推定した祖先型アミノ酸配列において、２番目の欠失に相当するアミノ酸残基はＩであった。したがって、本発明者らは、挿入する２番目のアミノ酸をＩまたはＹを推定した。同様の手法により、３番目から５番目までの挿入アミノ酸を推定した。 Regarding the first deletion counted from the N-terminal side, there are 13 G and 1 D in the sequence from Seq01 to Seq14, and G is dominant. Therefore, the inventors estimated that the first amino acid to be inserted was G. Regarding the second deletion, there were 6 Y, 4 L, and 3 I in the sequence of Seq01 to Seq14. Although details are shown in Example 5, 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. By the same method, the 3rd to 5th inserted amino acids were estimated.
　結果、挿入する５アミノ酸の配列をＧＩＰＶＴ、ＧＩＰＲＴ、ＧＩＰＱＴ、ＧＩＰＴＴ、ＧＹＰＶＴ、ＧＹＰＲＴと設定した。さらに祖先型アミノ酸の検討から２０２位のメチオニン（Ｍ）をアラニン（Ａ）に変更する組み合わせも追加した。これらのアミノ酸配列は、上記ｐＢＳＮＧＤＨＭ１７またはｐＢＳＮＧＤＨＭ２３を鋳型に特異的オリゴヌクレオチドを使用して部位特異的変異導入することにより、配列番号３のアミノ酸配列の２０２位のMと２０３位のEの間に対応する位置に挿入した。 As a result, 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.
　すなわち、以下の（１）～（１８）の組み合わせについて変異体酵素の遺伝子を作製した。
（１）Ｍ１７；ＭＧＩＰＶＴ
（２）Ｍ１７；ＭＧＩＰＲＴ
（３）Ｍ１７；ＭＧＩＰＱＴ
（４）Ｍ１７；ＭＧＩＰＴＴ
（５）Ｍ１７；ＭＧＹＰＶＴ
（６）Ｍ１７；ＭＧＹＰＲＴ
（７）Ｍ１７；ＡＧＩＰＶＴ
（８）Ｍ１７；ＡＧＩＰＲＴ
（９）Ｍ１７；ＡＧＹＰＶＴ
（１０）Ｍ１７；ＡＧＹＰＲＴ
（１１）Ｍ２３；ＭＧＩＰＶＴ
（１２）Ｍ２３；ＭＧＩＰＲＴ
（１３）Ｍ２３；ＭＧＹＰＶＴ
（１４）Ｍ２３；ＭＧＹＰＲＴ
（１５）Ｍ２３；ＡＧＩＰＶＴ
（１６）Ｍ２３；ＡＧＩＰＲＴ
（１７）Ｍ２３；ＡＧＹＰＶＴ
（１８）Ｍ２３；ＡＧＹＰＲＴ
　ここで、Ｍ１７はｐＢＳＮＧＤＨＭ１７を鋳型にした変異体、Ｍ２３は、ｐＢＳＮＧＤＨＭ２３を鋳型にした変異体を示し、コロン（；）の後の文字列は、左から１番目が配列番号３の２０２位のメチオニン（Ｍ）か、アミノ酸置換として導入する祖先型アミノ酸のアラニン（Ａ）を示し、後に続く５アミノ酸は２０２位と２０３位の間に挿入したアミノ酸配列を示す。 That is, mutant enzyme genes were prepared for the following combinations (1) to (18).
(1) M17; MGIPVT
(2) M17; MGIPRT
(3) M17; MGIPQT
(4) M17; MGIPTT
(5) M17; MGYPVT
(6) M17; MGYPRT
(7) M17; AGIPVT
(8) M17; AGIPRT
(9) M17; AGYPVT
(10) M17; AGYPRT
(11) M23; MGIPVT
(12) M23; MGIPRT
(13) M23; MGYPVT
(14) M23; MGYPRT
(15) M23; AGIPVT
(16) M23; AGIPRT
(17) M23; AGYPVT
(18) M23; AGYPRT
Here, M17 is a mutant using pBSNGDHM17 as a template, M23 is a mutant using pBSNGDH23 as a template, and 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 alanine (A) to be introduced as an amino acid substitution, and the subsequent 5 amino acids represent an amino acid sequence inserted between positions 202 and 203.
　ＰＣＲによる部位特異的変異導入に使用した鋳型ＤＮＡと上記（１）から（１８）のアミノ酸を導入するのに使用したオリゴヌクレオチドの組み合わせ、および作製した発現プラスミドの名称を以下に順に示す。
（１）ｐＢＳＮＧＤＨＭ１７、配列番号３３と３４、ｐＢＳＮＵ１
（２）ｐＢＳＮＧＤＨＭ１７、配列番号３５と３６、ｐＢＳＮＵ２
（３）ｐＢＳＮＧＤＨＭ１７、配列番号３７と３８、ｐＢＳＮＵ３
（４）ｐＢＳＮＧＤＨＭ１７、配列番号３９と４０、ｐＢＳＮＵ４
（５）ｐＢＳＮＧＤＨＭ１７、配列番号４１と４２、ｐＢＳＮＵ５
（６）ｐＢＳＮＧＤＨＭ１７、配列番号４３と４４、ｐＢＳＮＵ６
（７）ｐＢＳＮＧＤＨＭ１７、配列番号４５と４６、ｐＢＳＮＵ７
（８）ｐＢＳＮＧＤＨＭ１７、配列番号４７と４８、ｐＢＳＮＵ８
（９）ｐＢＳＮＧＤＨＭ１７、配列番号４９と５０、ｐＢＳＮＵ９
（１０）ｐＢＳＮＧＤＨＭ１７、配列番号５１と５２、ｐＢＳＮＵ１０
（１１）ｐＢＳＮＧＤＨＭ２３、配列番号３３と３４、ｐＢＳＮＵ１１
（１２）ｐＢＳＮＧＤＨＭ２３、配列番号３５と３６、ｐＢＳＮＵ１２
（１３）ｐＢＳＮＧＤＨＭ２３、配列番号４１と４２、ｐＢＳＮＵ１３
（１４）ｐＢＳＮＧＤＨＭ２３、配列番号４３と４４、ｐＢＳＮＵ１４
（１５）ｐＢＳＮＧＤＨＭ２３、配列番号４５と４６、ｐＢＳＮＵ１５
（１６）ｐＢＳＮＧＤＨＭ２３、配列番号４７と４８、ｐＢＳＮＵ１６
（１７）ｐＢＳＮＧＤＨＭ２３、配列番号４９と５０、ｐＢＳＮＵ１７
（１８）ｐＢＳＮＧＤＨＭ２３、配列番号５１と５２、ｐＢＳＮＵ１８
　これらのオリゴヌクレオチドの組み合わせにより、ｐＢＳＮＧＤＨＭ１７とｐＢＳＮＧＤＨＭ２３を鋳型に、ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ　Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ製）を使用してＰＣＲによる部位特異的変異導入を行った。方法はキット付属のプロトコルに従い、変異導入した発現プラスミドを大腸菌ＤＨ５αに形質転換し、寒天プレート上で培養してコロニーを形成させた。 The combinations of the template DNA used for site-directed mutagenesis by PCR and the oligonucleotides used for introducing the amino acids (1) to (18) above, and the names of the prepared expression plasmids are shown below in order.
(1) pBSNGDH17, SEQ ID NOs: 33 and 34, pBSNU1
(2) pBSNGDH17, SEQ ID NOs: 35 and 36, pBSNU2
(3) pBSNGDH17, SEQ ID NOs: 37 and 38, pBSNU3
(4) pBSNGDH17, SEQ ID NOs: 39 and 40, pBSNU4
(5) pBSNGDH17, SEQ ID NOs: 41 and 42, pBSNU5
(6) pBSNGDH17, SEQ ID NOs: 43 and 44, pBSNU6
(7) pBSNGDH17, SEQ ID NOs: 45 and 46, pBSNU7
(8) pBSNGDH17, SEQ ID NOs: 47 and 48, pBSNU8
(9) pBSNGDH17, SEQ ID NOs: 49 and 50, pBSNU9
(10) pBSNGDH17, SEQ ID NOs: 51 and 52, pBSNU10
(11) pBSNGDH23, SEQ ID NOs: 33 and 34, pBSNU11
(12) pBSNGDH23, SEQ ID NOs: 35 and 36, pBSNU12
(13) pBSNGDH23, SEQ ID NOs: 41 and 42, pBSNU13
(14) pBSNGDH23, SEQ ID NOs: 43 and 44, pBSNU14
(15) pBSNGDH23, SEQ ID NOs: 45 and 46, pBSNU15
(16) pBSNGDH23, SEQ ID NOs: 47 and 48, pBSNU16
(17) pBSNGDH23, SEQ ID NOs: 49 and 50, pBSNU17
(18) pBSNGDH23, SEQ ID NOs: 51 and 52, pBSNU18
By combining these oligonucleotides, site-directed mutagenesis by PCR was performed using QuickChange Site Directed Mutagenesis Kit (manufactured by Stratagene) using pBSNGDH17 and pBSNGDH23 as templates. In accordance with the protocol attached to the kit, the mutated expression plasmid was transformed into E. coli DH5α and cultured on an agar plate to form colonies.
　得られた形質転換体を２ｍＬのＬＢ培地で培養し、定法に従いプラスミドを精製した。合成した遺伝子に特異的なプライマーを使用してシーケンスを行い、所望の変異が導入されていることを確認した。 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.
　ｐＢＳＮＵ１からｐＢＳＮＵ１８までを大腸菌ＤＨ５αに導入した形質転換体を、１００μｇ／mＬのアンピシリンを含む液体培地Ｔｂｒｏｔｈ中で２７℃、４８時間培養した。培養終了後、菌体を超音波破砕してＧＤＨ活性を確認した。 A transformant obtained by introducing pBSNU1 to pBSNU18 into E. coli DH5α was cultured in a liquid medium Tbroth containing 100 μg / mL ampicillin at 27 ° C. for 48 hours. After completion of the culture, the bacterial cells were sonicated to confirm GDH activity.
　なお、N末端側シグナル配列を除去しないｐＢＳＮＧＤＨを鋳型にして、５アミノ酸配列ＧＩＰＶＴを挿入するように部位特異的変異導入を行った変異体を、上記の方法により発現させたところ、ほとんどＧＤＨ活性を示さなかった。したがって、十分なＧＤＨ活性を有した該タンパク質の発現には、シグナル配列の除去が必須であることが明らかになった。
＜実施例４＞
活性型ＦＡＤＧＤＨの発現、精製、性能評価
　実施例３で得られた活性型ＦＡＤＧＤＨの発現プラスミドを形質転換した大腸菌を使用して、活性型ＦＡＤＧＤＨの発現と精製を具体的には以下のように実施した。
（４－１）活性型ＦＡＤＧＤＨの発現
　活性型ＦＡＤＧＤＨの発現プラスミドをそれぞれ大腸菌ＤＨ５αに形質転換し、寒天プレート培地上で培養してコロニーを形成させた。単一のコロニーをLB液体培地２ｍＬに植菌し、３７℃で１８時間、振とう培養した。LB液体培地を２０ｍＬ調整して５００mＬフラスコに入れ、１２０℃で２０分オートクレーブ滅菌して上記培養液を植菌し、３７℃、６時間、振とう培養した。オートクレーブした１００μｇ／mＬのアンピシリンを含むＴｅｒｒｉｆｉｃ培養液１Ｌに上記培養液２０ｍＬを植菌し、５Ｌフラスコにおいて、２７℃で48時間、振とう培養した。培養完了後、培養液を遠心分離（８０００ｒｐｍ、１０分）して菌体を回収し、精製に使用するまで－８０℃で保存した。
（４－２）活性型ＦＡＤＧＤＨの精製
　－８０℃保存した菌体ペレットに３０ｍＬの２０ｍＭ　酢酸アンモニウムバッファー（ｐＨ５．２）に懸濁して超音波破砕した。菌体破砕液を遠心分離（８０００ｒｐｍ、４０分、４℃）して上清を回収した。得られた上清を、２０ｍＭ　酢酸アンモニウムバッファー（ｐＨ５．２）で平衡化した２０ｍＬのＤＥＡＥ－Ｓｅｐｈａｒｏｓｅカラムに素通りさせた。続けて、同じく２０ｍＭ　酢酸アンモニウムバッファー（pＨ５．２）で平衡化した２０ｍＬのＣＭ－Ｓｅｐｈａｒｏｓｅカラムに吸着させて、０Ｍから０．５ＭのＮａＣｌグラジエントで溶出し、ＧＤＨ活性を有する画分を得た。得られたＧＤＨ画分に、氷冷下で３０％飽和硫安となるように硫酸アンモニウムを添加して硫安沈殿し、遠心分離（１００００ｒｐｍ、２０分、４℃）によりＧＤＨ画分を有する上清を得た。 In addition, when pBSNGDH that does not remove the N-terminal signal sequence was used as a template, a mutant in which site-directed mutagenesis was introduced so as to insert the 5-amino acid sequence GIPVT was expressed by the above method. Not shown. Therefore, it was revealed that removal of the signal sequence is essential for the expression of the protein having sufficient 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 DH5α 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 μg / mL ampicillin autoclaved, and cultured with shaking at 27 ° C. for 48 hours in a 5 L flask. After completion of the culture, 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). Subsequently, 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.
　ＦＰＬＣ装置（ＧＥヘルスケア製）に６ｍＬのＲＥＳＯＵＲＣＥ　ＰＨＥカラム（ＧＥヘルスケア製）をセットし、３０％飽和硫安＋２０ｍＭ酢酸アンモニウムバッファー（ｐＨ５．２）で装置全体を平衡化した。硫安沈殿の上清をアプライしてＧＤＨ画分をカラムに吸着させ、３０％から０％の硫酸アンモニウムのグラジエントで溶出し、ＧＤＨ画分を得た。得られたＧＤＨ画分を０．１％ ＴｒｉｔｏｎＸ－１００＋２０ｍＭ　酢酸アンモニウムバッファー（ｐＨ５．２）で透析し、ＧＤＨ溶液を得た。 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.
　上記ＧＤＨ溶液をＳＤＳ－ＰＡＧＥにより電気泳動して精製度を確認したところ、単一バンドになるまで精製されていた（図２）。観察された分子量は約６０ｋＤａであり、アミノ酸配列から計算された分子量６３．７ｋＤａに比べてほとんど違いがなかった。一方、得られた活性型ＦＡＤＧＤＨは、補酵素にＦＡＤを有するＡＴＧＤＨと相同性が高いタンパク質に５アミノ酸を挿入して得られた酵素であるが、補酵素にＦＡＤを有しているか断定できなかった。そこで、以下の実験を行った。
（４－３）補酵素の確認
　精製された活性型ＦＡＤＧＤＨの吸収スペクトルを測定した結果、約２８０ｎｍの吸収極大以外に、約３７０ｎｍと約４５０ｎｍの吸収極大が観察された（図３Ａ、図３Ｂ）。これらの吸収極大は、活性型ＦＡＤＧＤＨがフラビンタンパク質であることを示しており、ＦＡＤまたはＦＭＮを補酵素としていることが示唆された。 When 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. On the other hand, 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.
(4-3) Confirmation of coenzyme As a result of measuring the absorption spectrum of purified active FADGDH, absorption maxima of about 370 nm and about 450 nm were observed in addition to the absorption maxima of about 280 nm (FIGS. 3A and 3B). . These absorption maxima indicate that active FADGDH is a flavin protein, suggesting that FAD or FMN is a coenzyme.
　次に、得られたＧＤＨ溶液を酸－硫酸アンモニウム処理して透析することにより、該酵素の補酵素を除いてアポ化し、ＧＤＨ活性をなくした上で、ＦＡＤ溶液またはＦＭＮ溶液もしくはリボフラビン溶液を添加して室温で２０分間静置した。 Next, 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.
　上記処理を行ったタンパク質溶液を使用してＧＤＨ活性を測定したところ、ＦＡＤを添加した溶液にのみ、ＧＤＨ活性が観察された。したがって、活性型ＦＡＤＧＤＨはＦＡＤを補酵素とするホロ酵素であり、ＦＡＤＧＤＨであることが確認された。
（４－４）活性型ＦＡＤＧＤＨのＫｍと比活性の測定
　精製された活性型ＦＡＤＧＤＨのＫｍならびに比活性を測定したところ、表１に示す結果であった。Ｋｍ値についてはいずれの変異体についても約３０程度であることが明らかになったが、比活性値に関しては、変異体酵素によってかなりばらつきがあり、結果として２０２位のアミノ酸がメチオニン（Ｍ）で２０２位と２０３位の間にＧＩＰＶＴを挿入した変異体が最も比活性が高いことが示された。 When the GDH activity was measured using the protein solution subjected to the above treatment, the GDH activity was observed only in the solution to which FAD was added. Therefore, it was confirmed that active FADGDH is a holoenzyme using FAD as a coenzyme and is FADGDH.
(4-4) Measurement of Km and specific activity of active FADGDH The Km and specific activity of purified active FADGDH were measured and the results shown in Table 1 were obtained. The Km value was found to be about 30 for all mutants, but the specific activity value varied considerably depending on the mutant enzyme. As a result, the amino acid at position 202 was methionine (M). It was shown that the mutant in which GIPVT was inserted between positions 202 and 203 had the highest specific activity.
　上記のように、大腸菌で発現させた活性型ＦＡＤＧＤＨは、本出願に記載の測定方法において比較的低いＫｍ値、および比較的高い比活性を示していることが明らかになった。しかしながら、グルコースセンサ用途としてはまだ満足のいくものではなく、この酵素をもとに、Ｋｍ値の低下、比活性値の上昇を目標に、実施例５以下に示す一連の変異導入実験を行った。なお、上記実施例で使用した、配列番号３の２０２番目のMと２０３番目のEの間にＧＩＰＶＴを挿入したアミノ酸配列を配列番号５３に、該アミノ酸配列をコードするＤＮＡ配列を配列番号５４に示す。 As described above, it was revealed that the active FADGDH expressed in Escherichia coli showed a relatively low Km value and a relatively high specific activity in the measurement method described in the present application. However, it is not yet satisfactory as a glucose sensor application, and based on this enzyme, a series of mutagenesis experiments shown in Example 5 and below were conducted with the aim of decreasing the Km value and increasing the specific activity value. . In addition, the amino acid sequence used in the above Example in which GIPVT is inserted between the 202nd M and the 203rd E of SEQ ID NO: 3 is represented by SEQ ID NO: 53, and the DNA sequence encoding the amino acid sequence is represented by SEQ ID NO: 54. Show.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜実施例５＞
活性型ＦＡＤＧＤＨへの部位特異的変異導入
　実施例４より、大腸菌により発現させた活性型ＦＡＤＧＤＨのスペックはセンサ用途としてはまだ不十分であることが示された。この酵素のＫｍの減少と比活性の上昇を目的として、以下に示す推定祖先型アミノ酸配列に基づく変異導入およびコンセンサス概念に基づく変異導入実験を行った。
（５－１）活性型ＦＡＤＧＤＨ相同タンパク質情報の取得
　配列番号５３に示した活性型ＦＡＤＧＤＨのアミノ酸配列とＡＴＧＤＨのアミノ酸配列を使用して、Ｂｌａｓｔｐ（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｂｌａｓｔ．ｃｇｉ）により相同性検索を行い、活性型ＦＡＤＧＤＨと相同な様々な生物種由来のアミノ酸配列情報をランダムに得た。 <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. For the purpose of reducing the Km and increasing the specific activity of this enzyme, mutagenesis experiments based on the following deduced ancestral amino acid sequences and the consensus concept were performed.
(5-1) Acquisition of Active FADGDH Homologous Protein Information Using the active FADGDH amino acid sequence and ATGDH amino acid sequence shown in SEQ ID NO: 53, Blastp (http://blast.ncbi.nlm.nih.gov) /Blast.cgi), and amino acid sequence information derived from various species homologous to active FADGDH was randomly obtained.
　具体的には、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 ｐｕｎｃｔｉｆｏｒｍｅ）、ｇｌｕｃｏｓｅ－ｍｅｔｈａｎｏｌ－ｃｈｏｌｉｎｅ ｏｘｉｄｏｒｅｄｕｃｔａｓｅ（Ｓｅｑ０７、Ａｎａｂａｅｎａ ｖａｒｉａｂｉｌｉｓ）、Ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ ａｎｄ ｒｅｌａｔｅｄ ｆｌａｖｏｐｒｏｔｅｉｎｓ（Ｓｅｑ０８、ＺＰ＿００１１０５３８、Ｎｏｓｔｏｃ ｐｕｎｃｔｉｆｏｒｍｅ）、ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ０９、ＺＰ＿０１１６２８３６、Ｐｈｏｔｏｂａｃｔｅｒｉｕｍ ｓｐ）、ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１０、ＮＰ＿４１４８４５、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）、ｈｙｐｏｔｈｅｔｉｃａｌ ａｌｃｏｈｏｌ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１１、ＺＰ＿０１１０９７８４、Ａｌｔｅｒｏｍｏｎａｓ ｍａｃｌｅｏｄｉｉ）、ａｌｃｏｈｏｌ ｄｅｈｙｄｒｏｇｅｎａｓｅ（Ｓｅｑ１２、ＹＰ＿６９４４３０、Ａｌｃａｎｉｖｏｒａｘ ｂｏｒｋｕｍｅｎｓｉｓ）、Ｃｈｏｌｉｎｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ ａｎｄ ｒｅｌａｔｅｄ ｆｌａｖｏｐｒｏｔｅｉｎｓ（Ｓｅｑ１３、ＺＰ＿００８９１６９２、Ｂｕｒｋｈｏｌｄｅｒｉａ ｐｓｅｕｄｏｍａｌｌｅｉ）、ＦＡＤ－ｏｘｉｄｏｒｅｄｕｃｔａｓｅ ｐｒｏｔｅｉｎ（Ｓｅｑ１４、ＹＰ＿４７１８３９、Ｒｈｉｚｏｂｉｕｍ ｅｔｌｉ）のアミノ酸配列情報を得た。さらにこれらの配列情報に、配列番号５３のアミノ酸配列からＮ末端側の１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」を除去して開始コドンのメチオニン（Ｍ）を付加した配列（Ｓｅｑ１５）も付け加えた。ここで、Ｓｅｑ～とは図４～図６に示した配列番号を指し、間の数値はデータベースに登録されているそれぞれのアミノ酸配列のアクセッションナンバーを指す。さらにその後に生物種の名称を示した。また、Ｓｅｑ１から１５は、それぞれ配列番号１３２から１４６に記載の配列である。
（５－２）活性型ＦＡＤＧＤＨを含むマルチプルアライメント図の作成
　上記のデータベースに登録されたアミノ酸配列データを、定法にしたがって公知のマルチプルアライメントソフトによりアライメントし、アライメント図を得た（図４～図６）。得られたアライメント図は、以降の祖先型アミノ酸配列の推定と、コンセンサス法による部位特異的変異導入に使用した。
（５－３）分子系統樹の作成と祖先型アミノ酸配列の推定
　上記アミノ酸配列のアライメント図に基づき、公知のコンピュータープログラムを使用して最尤法による系統樹を作成した（図７）。得られた系統樹に基づいて、１５種の生物由来の共通祖先（系統樹の根の位置）のアミノ酸配列を、公知のコンピュータープログラムを使用して最尤法により推定した。配列番号５５の共通祖先のアミノ酸配列（祖先型アミノ酸配列）を、該アミノ酸配列のすべての座位が活性型ＦＡＤＧＤＨの座位と対応するように、図４～図６のアライメント図と同時に並べた。
（５－４）活性型ＦＡＤＧＤＨに対するアミノ酸置換
　活性型ＦＡＤＧＤＨに対し、どの部位に祖先型アミノ酸を導入するか決定した。同時に、該アライメント図においてコンセンサス法により、どの部位にどのアミノ酸配列を導入するか決定した。 Specifically, 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 (Seq09, ZP_01162836, Photobacterium sp), choline dehydrogenase (Seq10, NP_414845, Escherichia coli ), Hypothetical alcohol dehydrogenase (Seq11, ZP_01109784, Alteramonas ma leodii), alcohol dehydrogenase (Seq12, YP_694430, Alcanivorax borkumensis), Choline dehydrogenase and related flavoproteins (Seq13, ZP_00891692, Burkholderia pseudomallei), FAD-oxidoreductase protein (Seq14, YP_471839, to obtain amino acid sequence information of Rhizobium etli). Furthermore, 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. It was. Here, 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.
(5-2) Creation of Multiple Alignment Diagrams Containing Active FADGDH The amino acid sequence data registered in the above database was aligned by known multiple alignment software according to a conventional method, and alignment diagrams were obtained (FIGS. 4 to 6). ). The obtained alignment diagram was used for the subsequent ancestral amino acid sequence estimation and site-specific mutagenesis by consensus method.
(5-3) Creation of molecular phylogenetic tree and estimation of ancestral amino acid sequence Based on the alignment diagram of the amino acid sequence, a phylogenetic tree was created by a maximum likelihood method using a known computer program (FIG. 7). Based on the obtained phylogenetic tree, 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.
　すなわち、配列番号５３の活性型ＦＡＤＧＤＨアミノ酸配列に対し、変異１．Ｖ３２Ｉ＋Ｇ３５Ａ＋Ｔ３７Ｓ＋Ｓ３８Ａ＋Ｌ４０Ｃ＋Ｖ４２Ｌ、変異２．Ｎ４９Ｄ、変異３．Ｓ５３Ｋ、変異４．Ｓ５３Ｎ、変異５．Ｔ１１１Ｓ＋Ｔ１１３Ｓ、変異６．Ａ１１８Ｉ＋Ｔ１２０Ｉ、変異７．Ａ１１８Ｉ＋Ｔ１２０Ａ、変異８．Ａ１１８Ｍ＋Ｔ１２０Ｉ、変異９．Ａ１１８Ｍ＋Ｔ１２０Ａ、変異１０．Ｋ１４１Ｄ＋Ｌ１４３Ｖ、変異１１．Ｋ１４１Ｑ＋Ｌ１４３Ｖ、変異１２．Ｙ１４７Ｆ＋Ｌ１４８Ｒ、変異１３．Ｙ１４７Ｆ＋Ｌ１４８Ｋ、変異１４．Ｉ２９５Ｌ、変異１５．Ｉ２９５Ｖ、変異１６．Ｌ３００Ｉ＋Ｒ３０１Ｎ、変異１７．Ｌ３０４Ｑ＋Ｉ３０５Ｌ、変異１８．Ｌ３０４Ｋ＋Ｉ３０５Ｌ、変異１９．Ｖ３１１Ｉ、変異２０．Ｔ３３０Ｇ、変異２１．Ｖ３２Ｉ、変異２２．Ｇ３５Ａ、変異２３．Ｔ３７Ｓ＋Ｓ３８Ａ、変異２４．Ｌ４０Ｃ、変異２５．Ｖ４２Ｌ、変異２６．Ａ８２Ｋ、変異２７．Ａ８２Ｅ、変異２８．Ａ１０７Ｖ、変異２９．Ｈ２８８Ｙ、変異３０．Ｎ４４６Ｔ＋Ｉ４４７Ｖ、変異３１．Ｎ４４６Ｓ＋Ｉ４４７Ｖ、変異３２．Ｎ４４６Ｔ＋Ｉ４４７Ｌ、変異３３．Ｎ４４６Ｓ＋Ｉ４４７Ｌ、変異３４．Ａ５３２Ｃ＋Ａ５３３Ｋ、変異３５．Ａ５３２Ｃ＋Ａ５３３Ｒ、変異３６．Ｎ５４５Ｐ、変異３７．Ｖ５６２Ｉ＋Ｌ５６３Ｍ、変異３８．Ｓ５８３Ａ＋Ｌ５８５Ｍの変異を導入することを決定した。 That is, mutation 1 with respect to the active FADGDH amino acid sequence of SEQ ID NO: 53. V32I + G35A + T37S + S38A + L40C + V42L, mutation2. 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. 10. K141D + L143V, mutation 11. K141Q + L143V, mutation 12. Y147F + L148R, mutation 13. Y147F + L148K, mutation 14. I295L, mutation 15. I295V, mutation 16. L300I + R301N, mutation17. L304Q + I305L, mutation 18. L304K + I305L, mutation 19. V311I, mutation 20. T330G, mutation 21. V32I, mutation 22. G35A, mutation 23. T37S + S38A, mutation 24. L40C, mutation 25. V42L, mutation 26. A82K, mutation27. A82E, mutation 28. A107V, mutation 29. H288Y, mutation 30. N446T + I447V, mutation 31. N446S + I447V, mutation 32. N446T + I447L, mutation 33. N446S + I447L, mutation34. A532C + A533K, mutation 35. A532C + A533R, mutation 36. N545P, mutation37. V562I + L563M, mutation38. It was decided to introduce a mutation of S583A + L585M.
　ここで「Ｎ４９Ｄ」とは、配列番号５３のN末端側から４９番目のアミノ酸Nを、アミノ酸Dに置換することを意味する。さらに「Ｔ１１１Ｓ／Ｔ１１３Ｓ」とは、Ｔ１１ＳとＴ１１３Ｓのアミノ酸置換を、同時に導入することを意味する。 Here, “N49D” means that the 49th amino acid N from the N-terminal side of SEQ ID NO: 53 is substituted with amino acid D. Furthermore, “T111S / T113S” means that amino acid substitutions of T11S and T113S are introduced simultaneously.
　活性型ＦＡＤＧＤＨに対する上記部位特異的へに導入は以下のように行った。上記変異１から変異３８の変異導入を実施するため、ＰＣＲによる部位特異的変異導入に使用するオリゴヌクレオチドを設計した。変異１については配列番号５６と配列番号５７、変異２については配列番号５８と配列番号５９、変異３については配列番号６０と配列番号６１、変異４については配列番号６２と６３、変異５については配列番号６４と配列番号６５、変異６については配列番号６６と配列番号６７、変異７については配列番号６８と配列番号６９、変異８については配列番号７０と配列番号７１、変異９については配列番号７２と配列番号７３、変異１０については配列番号７４と配列番号７５、変異１１については配列番号７６と配列番号７７、変異１２については配列番号７８と配列番号７９、変異１３については配列番号８０と配列番号８１、変異１４については配列番号８２と配列番号８３、変異１５については配列番号８４と配列番号８５、変異１６については配列番号８６と配列番号８７、変異１７については配列番号８８と配列番号８９、変異１８については配列番号９０と配列番号９１、変異１９については配列番号９２と配列番号９３、変異２０については配列番号９４と配列番号９５、変異２１については配列番号９６と配列番号９７、変異２２については配列番号９８と配列番号９９、変異２３については配列番号１００と配列番号１０１、変異２４については配列番号１０２と配列番号１０３、変異２５については配列番号１０４と配列番号１０５、変異２６については配列番号１０６と配列番号１０７、変異２７については配列番号１０８と配列番号１０９、変異２８については配列番号１１０と配列番号１１１、変異２９については配列番号１１２と配列番号１１３、変異３０については配列番号１１４と配列番号１１５、変異３１については配列番号１１６と配列番号１１７、変異３２については配列番号１１８と配列番号１１９、変異３３については配列番号１２０と配列番号１２１、変異３４については配列番号１２２と配列番号１２３、変異３５については配列番号１２４と配列番号１２５、変異３６については配列番号１２６と配列番号１２７、変異３７については配列番号１２８と配列番号１２９、変異３８については配列番号１３０と配列番号１３１のオリゴヌクレオチドを設定した。 The introduction into the site-specific manner with respect to the active FADGDH was carried out as follows. In order to introduce mutations from mutation 1 to mutation 38, an oligonucleotide used for site-directed mutagenesis by PCR was designed. SEQ ID NO: 56 and SEQ ID NO: 57 for mutation 1, SEQ ID NO: 58 and SEQ ID NO: 59 for mutation 2, SEQ ID NO: 60 and SEQ ID NO: 61 for mutation 3, SEQ ID NO: 62 and 63 for mutation 4, and SEQ ID NO: 62 and 63 SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67 for mutation 6, SEQ ID NO: 68 and SEQ ID NO: 69 for mutation 7, SEQ ID NO: 70 and SEQ ID NO: 71 for mutation 8, SEQ ID NO: for mutation 9 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 for Mutant 10, SEQ ID NO: 76 and SEQ ID NO: 77 for Mutant 11, SEQ ID NO: 78 and SEQ ID NO: 79 for Mutant 12, and SEQ ID NO: 80 for Mutant 13 SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83 for mutation 14, SEQ ID NO: 84 and SEQ ID NO: 85 for mutation 15, 16 for SEQ ID NO: 86 and 87, SEQ ID NO: 88 and 89 for variant 17, SEQ ID NO: 90 and 91 for variant 18, SEQ ID NO: 92 and 93 for variant 19 Are SEQ ID NO: 94 and SEQ ID NO: 95, mutation 21 is SEQ ID NO: 96 and SEQ ID NO: 97, mutation 22 is SEQ ID NO: 98 and SEQ ID NO: 99, mutation 23 is SEQ ID NO: 100 and SEQ ID NO: 101, and mutation 24 is the sequence. 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. Set oligonucleotides of SEQ ID NO: 130 and SEQ ID NO: 131.
　上記の相補的なオリゴヌクレオチドを利用して、ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ　Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ製）を使用し、ｐＢＳＮＵ１を鋳型としてＰＣＲによる部位特異的変異導入を行った。変異導入したそれぞれのプラスミドＤＮＡにより、大腸菌ＤＨ５αを形質転換してクローニングし、所望の変異が導入できたかどうか、シーケンスにより確認した。 Using the above complementary oligonucleotide, site-directed mutagenesis by PCR was performed using QuickChange Site Directed Mutagenesis Kit (manufactured by Stratagene) using pBSNU1 as a template. Escherichia coli DH5α was transformed with each of the mutated plasmid DNAs and cloned, and whether or not the desired mutation was introduced was confirmed by sequencing.
　得られた変異導入プラスミドＤＮＡを、変異１から変異３８の順に、ｐＢＳＮＵ１－１、ｐＢＳＮＵ１－２、ｐＢＳＮＵ１－３、ｐＢＳＮＵ１－４、ｐＢＳＮＵ１－５、ｐＢＳＮＵ１－６、ｐＢＳＮＵ１－７、ｐＢＳＮＵ１－８、ｐＢＳＮＵ１－９、ｐＢＳＮＵ１－１０、ｐＢＳＮＵ１－１１、ｐＢＳＮＵ１－１２、ｐＢＳＮＵ１－１３、ｐＢＳＮＵ１－１４、ｐＢＳＮＵ１－１５、ｐＢＳＮＵ１－１６、ｐＢＳＮＵ１－１７、ｐＢＳＮＵ１－１８、ｐＢＳＮＵ１－１９、ｐＢＳＮＵ１－２０、ｐＢＳＮＵ１－２１、ｐＢＳＮＵ１－２２、ｐＢＳＮＵ１－２３、ｐＢＳＮＵ１－２４、ｐＢＳＮＵ１－２５、ｐＢＳＮＵ１－２６、ｐＢＳＮＵ１－２７、ｐＢＳＮＵ１－２８、ｐＢＳＮＵ１－２９、ｐＢＳＮＵ１－３０、ｐＢＳＮＵ１－３１、ｐＢＳＮＵ１－３２、ｐＢＳＮＵ１－３３、ｐＢＳＮＵ１－３４、ｐＢＳＮＵ１－３５、ｐＢＳＮＵ１－３６、ｐＢＳＮＵ１－３７、ｐＢＳＮＵ１－３８と命名し、実施例７の大腸菌における発現解析に使用した。
＜実施例６＞
活性型ＡｓｐＧＤＨへのランダム変異導入
　実施例５では確率論的変異導入推定理論に基づき部位特異的変異導入を行ったが、それと同時に、基質親和性の向上した変異体を得るべく構築したスクリーニング系を使用して、ランダム変異導入を行った活性型ＦＡＤＧＤＨに対し、以下に具体的に示す有望変異体のスクリーニングを行い、有望変異株の取得を目指した。
（６－１）活性型ＦＡＤＧＤＨへのランダム変異導入
　配列番号５４に記載のＤＮＡを鋳型にして、Ｄｉｖｅｒｓｉｔｙ ＰＣＲ Ｒａｎｄｏｍ　Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ（Ｃｌｏｎｔｅｃｈ製）により、ランダム変異を導入した変異ＤＮＡを作製した。 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. -9, pBSNU1-10, pBSNU1-11, pBSNU1-12, pBSNU1-13, pBSNU1-14, pBSNU1-15, pBSNU1-16, pBSNU1-17, pBSNU1-18, pBSNU1-19, pBSNU1-20, pBSNU1-21 PBSNU1-22, pBSNU1-23, pBSNU1-24, pBSNU1-25, pBSNU1-26, pBSNU1-27, pBSNU1-28, pBSNU1-29, pBSNU1-30, pBSNU1-31, p SNU1-32, pBSNU1-33, pBSNU1-34, pBSNU1-35, pBSNU1-36, pBSNU1-37, named PBSNU1-38, it was used for expression analysis in E. coli in Example 7.
<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).
　該変異ＤＮＡとｐＢＳＮを、制限酵素ＮｄｅＩとＢａｍＨＩで処理した。処理方法は制限酵素のマニュアルに従い、処理条件は３７℃で２時間とした。 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.
　制限酵素処理した変異ＤＮＡとベクターをそれぞれアガロースゲル電気泳動してゲルから切り出し、定法によりＤＮＡ断片を精製した。精製したＤＮＡ断片どうしをリガーゼで結合してランダム変異導入ＦＡＤＧＤＨ発現プラスミドを構築した。 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.
　該発現プラスミドにより大腸菌ＤＨ５αを定法により形質転換し、寒天プレート培地上で３７℃、１８時間培養してシングルコロニーを形成させ、ランダム変異体ライブラリとした。
（６－２）ランダム変異導入活性型ＦＡＤＧＤＨの発現
　まず、上記（６－１）で得られたシングルコロニーをそれぞれ９６ウェルプレートの１００μｇ／ｍＬのアンピシリンを添加したＬＢ液体培地（５０μＬ）に植菌した。２ウェルを、ランダム変異を導入していない活性型ＦＡＤＧＤＨを発現する大腸菌株に当てた。ウェルプレート上面をガスパーマブル粘着シート（ＡＢｇｅｎｅ製）でシールし、さらに付属のふたをして３７℃で１６時間、培養した。 Escherichia coli DH5α 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.
(6-2) Expression of random mutagenesis activation type FADGDH First, the single colonies obtained in (6-1) above were inoculated into LB liquid medium (50 μL) supplemented with 100 μg / 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.
　この培養液２５μＬずつを新しいウェルプレートで、予め分注した２５μＬの０．２Ｎ　ＮａＯＨとよく混合した後、プレートにふたをして３７℃で１０分間、インキュベートすることにより溶菌した。 25 μL each of this culture solution was mixed well with 25 μL of 0.2 N NaOH previously dispensed in a new well plate, and then the plate was covered and incubated at 37 ° C. for 10 minutes for lysis.
　次に室温で１００μＬの０．１Ｍ リン酸バッファー（ｐＨ６．８）を加え、液を中和した。このとき非加熱のコントロールサンプルとしてランダム変異導入を行っていない活性型ＡｓｐＧＤＨサンプル２つのうち１つを取り分けて氷上で保存した。
（６－３）基質親和性向上変異体のスクリーニング
　上述した９６ウェルフォーマットで発現させた変異体酵素を使用して以下の実験を行った。プレートリーダー用９６ウェルプレートにグルコース濃度を減じた活性測定用試薬を特別に調整して１ウェルずつに２００μＬずつ分注し、３７℃で５分間、プレインキュベートした。プレインキュベートした活性測定試薬に氷上保存していた酵素サンプルを１μＬずつ８連ピペットで添加してミキサーで５秒間攪拌し、予め３７℃にインキュベートしておいたプレートリーダーに挿入した。 Next, 100 μL of 0.1 M phosphate buffer (pH 6.8) was added at room temperature to neutralize the solution. At this time, one of the two active AspGDH samples not subjected to random mutagenesis was separated as an unheated control sample and stored on ice.
(6-3) Screening for mutants with improved substrate affinity The following experiment was carried out using the mutant enzymes expressed in the 96-well format described above. A reagent for activity measurement with a reduced glucose concentration was prepared in a 96-well plate for a plate reader, dispensed 200 μL per well, and pre-incubated at 37 ° C. for 5 minutes. 1 μL of the enzyme sample stored on ice was added to the pre-incubated activity measurement reagent with an 8-unit pipette, stirred for 5 seconds with a mixer, and inserted into a plate reader that had been incubated at 37 ° C. in advance.
　６００nmにおける吸光度減少を５分間測定し、吸光度減少が直線的である時間内で、１分間当たりの吸光度変化量を求め、活性値を測定し、ランダム変異を導入していないｖＧＤＨと残存活性を比較した。ランダム変異を導入していない活性型ＦＡＤＧＤＨに比べて１分間当たりの吸光度変化量が高く測定された形質転換体について、基質親和性向上型の変異体候補として保存した。
（６－４）熱安定性変異体の精製
　６－２で保存したランダム変異体クローンを実施例４に示した培養方法、精製方法により精製した。精製した変異体酵素は実施例７の性能評価に供した。
＜実施例７＞
活性型ＦＡＤＧＤＨ変異体の発現解析と酵素の精製、性能評価
（７－１）変異体酵素の発現と精製
　実施例５で得られたpBSNU1－１からpBSNU1－３８でそれぞれ形質転換した大腸菌DH5αを使用して、実施例4で示した方法により変異体酵素を発現、精製した。
（７－２）Ｋｍと比活性の測定
　実施例４と同様に、変異体１から変異体３８の変異体酵素のＫｍと比活性を測定した。測定結果を表２に示す。計３８種類の変異体酵素のうち、変異１、変異１２、変異１６、変異２１、変異２３、変異２４、変異３０、変異３２、変異３４には活性が見出せなかった。Ｋｍ値に関しては、変異を導入していない活性型ＦＡＤＧＤＨに比べて数値上減少したのは、変異体２、変異体３、変異体４、変異体６、変異体７、変異体８、変異体９、変異体１１、変異体１３、変異体１５、変異体１７、変異体１８、変異体１９、変異体２０、変異体２２、変異体２５、変異体２６、変異体２７、変異体２８、変異体２９、変異体３１、変異体３３、変異体３５、変異体３６、変異体３７、変異体３８であり、かなりの効率で改変型ＦＡＤＧＤＨを得ることができた。特に、Ｋｍ値に関しては、変異体２、変異体６、変異体７、変異体８、変異体９、変異体１３、変異体２０、変異体２２、変異体２６、変異体２９で明らかに減少していることが観察された。比活性値に関しては、変異を導入していない活性型ＦＡＤＧＤＨに比べて数値上上昇したのは、変異体２、変異体３、変異体４、変異体９、変異体１３、変異体１４、変異体１５、変異体１７、変異体１８、変異体１９、変異体２０、変異体２２、変異体２６、変異体２７、変異体２８、変異体２９、変異体３１、変異体３３、変異体３５、変異体３８であり、かなりの効率で改変型ＦＡＤＧＤＨを得ることができた。特に、比活性値に関しては、変異２、変異３、変異４、変異１３、変異１４、変異１５、変異１９、変異２０、変異２６、変異２７、変異２８、変異２９、変異３１、変異３３、変異３５、変異３８で明らかに上昇した。ランダム変異導入により得られた変異体は、シーケンスの結果、変異２２と同等の変異が入っていることが明らかになった（発現プラスミド名を表２において、ｐＳｃ０１と表記した）。 Measure the decrease in absorbance at 600 nm for 5 minutes, determine the amount of change in absorbance per minute within the time period in which the decrease in absorbance is linear, measure the activity value, and compare the remaining activity with vGDH into which no random mutation has been introduced. did. Transformants whose absorbance change per minute was measured higher than that of active FADGDH into which random mutations were not introduced were stored as mutant candidates with improved substrate affinity.
(6-4) Purification of 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 DH5α 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. In particular, 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. It was observed that Regarding the specific activity value, the numerical value increased compared to the active FADGDH in which no mutation was introduced, that is, the mutant 2, the mutant 3, the mutant 4, the mutant 9, the mutant 13, the mutant 14, and the mutation Body 15, Mutant 17, Mutant 18, Mutant 19, Mutant 20, Mutant 22, Mutant 26, Mutant 27, Mutant 28, Mutant 29, Mutant 31, Mutant 33, Mutant 35 The mutant FADGDH could be obtained with considerable efficiency. In particular, with respect to specific activity values, 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. As a result of sequencing, 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).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
＜実施例８＞
有望変異の組み合わせ
　実施例７で行った一連の実験から、変異導入前の活性型ＦＡＤＧＤＨに比べて基質親和性および／または比活性が向上した変異体が多数得られた。これらの有望変異に導入した変異を組み合わせることにより、さらに幅広く基質親和性と比活性の向上した変異体が得られる可能性が考えられたので、まず有望変異の組み合わせパターンについて検討した。本発明者らは、組み合わせのパターンとして、（１）Ｋｍの低下した変異どうしの組み合わせ、（２）比活性の上昇した変異どうしの組み合わせ（３）Ｋｍの低下した変異と比活性が上昇した変異の組み合わせが考えた。 <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.
　つまり、実施例９の具体的な実験に使用した変異の組み合わせは、
変異６（Ａ１１８Ｉ／Ｔ１２０Ｉ）と変異２０（Ｔ３３０Ｇ）の組み合わせ、
変異６（Ａ１１８Ｉ／Ｔ１２０Ｉ）と変異２６（Ａ８２Ｋ）の組み合わせ、
変異６（Ａ１１８Ｉ／Ｔ１２０Ｉ）と変異２９（Ｈ２８８Ｙ）の組み合わせ、
変異２０（Ｔ３３０Ｇ）と変異２６（Ａ８２Ｋ）の組み合わせ、
変異２０（Ｔ３３０Ｇ）と変異２９（Ｈ２８８Ｙ）の組み合わせ、
変異２６（Ａ８２Ｋ）と変異２９（Ｈ２８８Ｙ）の組み合わせ、
変異１４（Ｉ２９５Ｌ）と変異２０（Ｔ３３０Ｇ）の組み合わせ、
変異１４（Ｉ２９５Ｌ）と変異２６（Ａ８２Ｋ）の組み合わせ、
変異１４（Ｉ２９５Ｌ）と変異２８（Ａ１０７Ｖ）の組み合わせ、
変異２０（Ｔ３３０Ｇ）と変異２８（Ａ１０７Ｖ）の組み合わせ、
変異２６（Ａ８２Ｋ）と変異２８（Ａ１０７Ｖ）の組み合わせ、
変異２（Ｎ４９Ｄ）と変異３（Ｓ５３Ｋ）の組み合わせ、
変異１４（Ｉ２９５Ｌ）と変異２９（Ｈ２８８Ｙ）の組み合わせ、
変異１７（Ｌ３０４Ｑ／Ｉ３０５Ｌ）と変異１９（Ｖ３１１Ｉ）の組み合わせ、
変異２０（Ｔ３３０Ｇ）と変異３８（Ｓ５８３Ａ／Ｌ５８５Ｍ）の組み合わせ、
変異２９（Ｈ２８８Ｙ）と変異３８（Ｓ５８３Ａ／Ｌ５８５Ｍ）の組み合わせ、
変異２６（Ａ８２Ｋ）と変異３８（Ｓ５８３Ａ／Ｌ５８５Ｍ）の組み合わせである。 That is, 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 combination of mutation 14 (I295L) and mutation 29 (H288Y),
A combination of mutation 17 (L304Q / I305L) and mutation 19 (V311I),
A combination of mutation 20 (T330G) and mutation 38 (S583A / L585M),
A combination of mutation 29 (H288Y) and mutation 38 (S583A / L585M),
It is a combination of mutation 26 (A82K) and mutation 38 (S583A / L585M).
　上記変異を順に変異３９（Ａ１１８Ｉ／Ｔ１２０Ｉ／Ｔ３３０Ｇ）、変異４０（Ａ８２Ｋ／Ａ１１８Ｉ／Ｔ１２０Ｉ）、変異４１（Ａ１１８Ｉ／Ｔ１２０Ｉ／Ｈ２８８Ｙ）、変異４２（Ａ８２Ｋ／Ｔ３３０Ｇ）、変異４３（Ｈ２８８Ｙ／Ｔ３３０Ｇ）、変異４４（Ａ８２Ｋ／Ｈ２８８Ｙ）、変異４５（Ｉ２９５Ｌ／Ｔ３３０Ｇ）、変異４６（Ａ８２Ｋ／Ｉ２９５Ｌ）、変異４７（Ａ１０７Ｖ／Ｉ２９５Ｌ）、変異４８（Ａ１０７Ｖ／Ｔ３３０Ｇ）、変異４９（Ａ８２Ｋ／Ａ１０７Ｖ）、変異５０（Ｎ４９Ｄ／Ｓ５３Ｋ）、変異５１（Ｈ２８８Ｙ／Ｉ２９５Ｌ）、変異５２（Ｌ３０４Ｑ／Ｉ３０５Ｌ／Ｖ３１１Ｉ）、変異５３（Ｔ３３０Ｇ／Ｓ５８３Ａ／Ｌ５８５Ｍ）、変異５４（Ｈ２８８Ｙ／Ｓ５８３Ａ／Ｌ５８５Ｍ）、変異５５（Ａ８２Ｋ／Ｓ５８３Ａ／Ｌ５８５Ｍ）と命名した。
＜実施例９＞
組み合わせ変異体の発現解析と酵素の精製、性能評価
　実施例８に示した組み合わせ変異体遺伝子は、実施例５に示した相補的なオリゴヌクレオチドを用いて、ＰＣＲによる部位特異的変異導入法により導入した。例えば、変異３９の導入には、変異６を導入した発現プラスミドであるｐＢＳＮＵ１－６を鋳型とし、変異２０を導入するために設計した配列番号９４と配列番号９５の相補的なオリゴヌクレオチドを利用して、ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ　Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ製）によりＰＣＲによる部位特異的変異導入を行った。 The above 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 (A82K) S583A / L585M) and was named.
<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. For example, 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).
　変異導入されたそれぞれのプラスミドＤＮＡを使用して大腸菌ＤＨ５αを定法により形質転換し、寒天プレート培地上で３７℃、１８時間培養して該形質転換体の単一コロニーを得た。得られたコロニーを１００μｇ／ｍＬのアンピシリンを添加したＬＢ液体培地に植菌して３７℃で１８時間振とう培養し、定法によりプラスミドＤＮＡを精製した。これらのプラスミドＤＮＡを対象にシーケンスを行い、所望の変異が導入できたことを確認した。 Escherichia coli DH5α 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 μg / 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.
　得られた変異導入プラスミドDNAを、変異３９から変異５５の順に、ｐＢＳＮＵ１－３９、ｐＢＳＮＵ１－４０、ｐＢＳＮＵ１－４１、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と命名した。ｐＢＳＮＵ１－３９からｐＢＳＮＵ１－５５でそれぞれ形質転換した大腸菌ＤＨ５αを使用して、変異体酵素を発現、精製した。変異体酵素の発現と精製、性能評価は実施例４と同様に行った。 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 DH5α 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.
　性能評価の結果を表３に示す。計１７種類の組み合わせ変異体酵素のうち、変異４０、変異４２、変異４７、変異４８、変異４９、変異５０、変異５５には活性が見出されなかった。Km値に関しては、変異を導入していない活性型ＦＡＤＧＤＨに比べ、変異３９、変異４１、変異４３、変異４５および変異５１で明らかに低下した。比活性に関しては、変異を導入していない活性型ＦＡＤＧＤＨに比べ、変異４３、変異４５、変異４６、変異５１、変異５２、変異５３、変異５４で明らかに上昇した。 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.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
＜実施例１０＞
グルコースセンサ
　上記の実施例７で使用した変異体２０の改変型ＦＡＤＧＤＨを使用した酵素固定化グルコースセンサを試作し、公知の方法（非特許文献１０、Ｓ．Ｔｓｕｊｉｍｕｒａ，ｅｔ　ａｌ．，Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｃｈｅｍ．，７０，６５４－６５９，２００６）を参考にＤ－グルコースの測定を行った。 <Example 10>
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.
　変異体２０の改変型ＦＡＤＧＤＨを３Ｕ固定化させたグラッシーカーボン電極を使用し、Ｄ－グルコースに対する応答電流を測定した。電解セル中に０．１Ｍのリン酸カリウムバッファー（ｐＨ６．５）と、１Ｍのフェリシアンカリウム溶液を充填し、空気飽和条件化で既知の濃度のＤ－グルコースの測定を行った。既知のグルコース濃度に対して電流値をプロットすることにより、グルコース濃度の上限が１０００ｍｇ／ｄＬまでの検量線を描くことができた。これにより本発明の改変型ＦＡＤＧＤＨを使用した酵素固定化グルコースセンサによりグルコースの定量が可能であることが示された。 Using a glassy carbon electrode in which 3 U of the modified FADGDH of the variant 20 was immobilized, the response current to D-glucose was measured. An electrolysis cell was filled with 0.1 M potassium phosphate buffer (pH 6.5) and 1 M ferricyanide potassium solution, and D-glucose at a known concentration was measured under air saturation conditions. By plotting the current value against the known glucose concentration, it was possible to draw a calibration curve up to an upper limit of the glucose concentration of 1000 mg / dL. Thus, it was shown that glucose can be quantified by the enzyme-immobilized glucose sensor using the modified FADGDH of the present invention.
　本発明を詳細にまた特定の実施態様を参照して説明したが、本発明の精神と範囲を逸脱することなく様々な変更や修正を加えることができることは当業者にとって明らかである。
　本出願は、２００８年１１月６日出願の日本特許出願（特願２００８－２８５４６１）に基づくものであり、その内容はここに参照として取り込まれる。 Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.
This application is based on a Japanese patent application (Japanese Patent Application No. 2008-285461) filed on Nov. 6, 2008, the contents of which are incorporated herein by reference.
　本発明により、データベースに登録されていたＧＤＨ活性を有さないアスペルギルス・オリゼ由来ＦＡＤＧＤＨ様タンパク質にＧＤＨ活性を発現せしめることが可能になった。本発明において使用した実験手法は、産業上有用でない遺伝子データであっても、その他の大量に公開されている遺伝子データベース等を利用することにより有用な遺伝子に改変させることができるという点で、遺伝子データベースの有効利用という観点からも大変意義深く、広く一般に利用されることが期待される。 According to 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.
　また、本発明により創作された基質親和性および／または比活性の向上した変異体酵素をグルコースセンサ等に利用することにより、測定時間のさらなる短縮や測定データの正確さの向上といったスペック上のメリットが得られ、医療関連分野等の産業に幅広く貢献することが可能である。 In addition, by using a mutant enzyme with improved substrate affinity and / or specific activity created by the present invention for a glucose sensor, etc., it is possible to further reduce measurement time and improve measurement data accuracy. It is possible to contribute widely to industries such as medical-related fields.
配列番号２、４－３２：実施例２に記載の配列である。
配列番号３３－５２：実施例３に記載の配列である。
配列番号５３、５４：実施例４に記載の配列である。
配列番号５５－１３１：実施例５に記載の配列である。
配列番号１４６：図４－６に記載の配列である。 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.

Claims (14)

  1. 　配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＩＰＱＴ、ＧＩＰＴＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列を有する活性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（ＦＡＤＧＤＨ）。 5 selected from the group consisting of GIPVT, GIPRT, GIPQT, GIPTT, GYPVT and GYPRT between positions 202 and 203 in the amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3. Active flavin adenine dinucleotide-dependent glucose having an amino acid sequence inserted and having an amino acid sequence “MLFSLAFLSALSLATA” from position 1 to position 16 or an amino acid sequence “MLFSLAFLSALSLATASPAGRA” from position 1 to position 22 deleted Dehydrogenase (FADGDH).
  2. 　配列番号３に記載されるアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属由来のアミノ酸配列において、該アミノ酸配列の２０２位のメチオニン（Ｍ）がアラニン（Ａ）に置換され、かつ２０２位と２０３位の間に、ＧＩＰＶＴ、ＧＩＰＲＴ、ＧＹＰＶＴ及びＧＹＰＲＴからなる群より選択される５アミノ酸配列が挿入され、かつ、１位から１６位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡ」又は１位から２２位までのアミノ酸配列「ＭＬＦＳＬＡＦＬＳＡＬＳＬＡＴＡＳＰＡＧＲＡ」が欠失したアミノ酸配列を有する活性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（ＦＡＤＧＤＨ）。 In the amino acid sequence derived from the genus Aspergillus described in SEQ ID NO: 3, methionine (M) at position 202 of the amino acid sequence is substituted with alanine (A), and between positions 202 and 203, GIPVT, A 5-amino acid sequence selected from the group consisting of GIPRT, GYPVT and GYPRT was inserted, and the amino acid sequence “MLFSLAFLSALSLATA” from position 1 to position 16 or the amino acid sequence “MLFSLAFLSALSLATASPAGRA” from position 1 to position 22 was deleted. An active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) having an amino acid sequence.
  3. 　請求項1又は２に記載される活性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（ＦＡＤＧＤＨ）において、以下の（１）～（３３）からなる群より選択される少なくとも一種のアミノ酸置換がなされ、改変前の対応する該活性型ＦＡＤＧＤＨに比べＫｍ値が低下した改変型ＦＡＤＧＤＨ。
    　（１）Ｎ４９Ｄ
    　（２）Ｓ５３Ｋ
    　（３）Ｓ５３Ｎ
    　（４）Ａ１１８Ｉ＋Ｔ１２０Ｉ
    　（５）Ａ１１８Ｉ＋Ｔ１２０Ａ
    　（６）Ａ１１８Ｍ＋Ｔ１２０Ｉ
    　（７）Ａ１１８Ｍ＋Ｔ１２０Ａ
    　（８）Ｙ１４７Ｆ＋Ｌ１４８Ｋ
    　（９）Ｉ２９５Ｖ
    　（１０）Ｌ３０４Ｑ＋Ｉ３０５Ｌ
    　（１１）Ｌ３０４Ｋ＋Ｉ３０５Ｌ
    　（１２）Ｖ３１１Ｉ
    　（１３）Ｔ３３０Ｇ
    　（１４）Ｇ３５Ａ
    　（１５）Ｖ４２Ｌ
    　（１６）Ａ８２Ｋ
    　（１７）Ａ８２Ｅ
    　（１８）Ａ１０７Ｖ
    　（１９）Ｈ２８８Ｙ
    　（２０）Ｎ４４６Ｓ＋Ｉ４４７Ｖ
    　（２１）Ｎ４４６Ｓ＋Ｉ４４７Ｌ
    　（２２）Ａ５３２Ｃ＋Ａ５３３Ｒ
    　（２３）Ｎ５４５Ｐ
    　（２４）Ｖ５６２Ｉ＋Ｌ５６３Ｍ
    　（２５）Ｓ５８３Ａ＋Ｌ５８５Ｍ
    　（２６）Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｈ２８８Ｙ
    　（２７）Ａ１１８Ｉ＋Ｔ１２０Ｉ＋Ｔ３３０Ｇ
    　（２８）Ｈ２８８Ｙ＋Ｔ３３０Ｇ
    　（２９）Ａ８２Ｋ＋Ｈ２８８Ｙ
    　（３０）Ｉ２９５Ｌ＋Ｔ３３０Ｇ
    　（３１）Ｉ２９５Ｌ＋Ｈ２８８Ｙ
    　（３２）Ｌ３０４Ｑ＋Ｉ３０５Ｌ＋Ｖ３１１Ｉ
    　（３３）Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
    　ここで、該アミノ酸置換の位置は、配列番号３に記載されるアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入された配列のアミノ酸残基の位置に相当する位置を示す。     3. The activated flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) according to claim 1 or 2, wherein at least one amino acid substitution selected from the group consisting of the following (1) to (33) is made, The modified FADGDH having a decreased Km value compared to the corresponding active FADGDH.
    (1) N49D
    (2) S53K
    (3) S53N
    (4) A118I + T120I
    (5) A118I + T120A
    (6) A118M + T120I
    (7) A118M + T120A
    (8) Y147F + L148K
    (9) I295V
    (10) L304Q + I305L
    (11) L304K + I305L
    (12) V311I
    (13) T330G
    (14) G35A
    (15) V42L
    (16) A82K
    (17) A82E
    (18) A107V
    (19) H288Y
    (20) N446S + I447V
    (21) N446S + I447L
    (22) A532C + A533R
    (23) N545P
    (24) V562I + L563M
    (25) S583A + L585M
    (26) A118I + T120I + H288Y
    (27) A118I + T120I + T330G
    (28) H288Y + T330G
    (29) A82K + H288Y
    (30) I295L + T330G
    (31) I295L + H288Y
    (32) L304Q + I305L + V311I
    (33) T330G + S583A + L585M
    Here, the position of the amino acid substitution corresponds to the position of the amino acid residue in the sequence in which a 5-amino acid sequence is inserted between positions 202 and 203 in the amino acid sequence shown in SEQ ID NO: 3. Indicates the position.
  4. 　請求項１又は２に記載される活性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（ＦＡＤＧＤＨ）において、以下の（１）～（２７）からなる群より選択される少なくとも一種のアミノ酸置換がなされ、改変前の対応する該活性型ＦＡＤＧＤＨに比べ比活性値が上昇したことを特徴とする改変型ＦＡＤＧＤＨ。
    　（１）Ｎ４９Ｄ
    　（２）Ｓ５３Ｋ
    　（３）Ｓ５３Ｎ
    　（４）Ａ１１８Ｍ＋Ｔ１２０Ａ
    　（５）Ｙ１４７Ｆ＋Ｌ１４８Ｋ
    　（６）Ｉ２９５Ｌ
    　（７）Ｉ２９５Ｖ
    　（８）Ｌ３０４Ｑ＋Ｉ３０５Ｌ
    　（９）Ｌ３０４Ｋ＋Ｉ３０５Ｌ
    　（１０）Ｖ３１１Ｉ
    　（１１）Ｔ３３０Ｇ
    　（１２）Ｇ３５Ａ
    　（１３）Ａ８２Ｋ
    　（１４）Ａ８２Ｅ
    　（１５）Ａ１０７Ｖ
    　（１６）Ｈ２８８Ｙ
    　（１７）Ｎ４４６Ｓ＋Ｉ４４７Ｖ
    　（１８）Ｎ４４６Ｓ＋Ｉ４４７Ｌ
    　（１９）Ａ５３２Ｃ＋Ａ５３３Ｒ
    　（２０）Ｓ５８３Ａ＋Ｌ５８５Ｍ
    　（２１）Ｔ３３０Ｇ＋Ｈ２８８Ｙ
    　（２２）Ｉ２９５Ｌ＋Ｔ３３０Ｇ
    　（２３）Ｉ２９５Ｌ＋Ａ８２Ｋ
    　（２４）Ｉ２９５Ｌ＋Ｈ２８８Ｙ
    　（２５）Ｌ３０４Ｑ＋Ｉ３０５Ｌ＋Ｖ３１１Ｉ
    　（２６）Ｔ３３０Ｇ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
    　（２７）Ｈ２８８Ｙ＋Ｓ５８３Ａ＋Ｌ５８５Ｍ
    　ここで、該アミノ酸置換の位置は、配列番号３に記載されるアミノ酸配列において、該アミノ酸配列の２０２位と２０３位の間に５アミノ酸配列が挿入された配列のアミノ酸残基の位置に相当する位置を示す。     3. The active flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) according to claim 1 or 2, wherein at least one amino acid substitution selected from the group consisting of the following (1) to (27) is made, A modified FADGDH characterized by having an increased specific activity value as compared with the corresponding active FADGDH.
    (1) N49D
    (2) S53K
    (3) S53N
    (4) A118M + T120A
    (5) Y147F + L148K
    (6) I295L
    (7) I295V
    (8) L304Q + I305L
    (9) L304K + I305L
    (10) V311I
    (11) T330G
    (12) G35A
    (13) A82K
    (14) A82E
    (15) A107V
    (16) H288Y
    (17) N446S + I447V
    (18) N446S + I447L
    (19) A532C + A533R
    (20) S583A + L585M
    (21) T330G + H288Y
    (22) I295L + T330G
    (23) I295L + A82K
    (24) I295L + H288Y
    (25) L304Q + I305L + V311I
    (26) T330G + S583A + L585M
    (27) H288Y + S583A + L585M
    Here, the position of the amino acid substitution corresponds to the position of the amino acid residue in the sequence in which a 5-amino acid sequence is inserted between positions 202 and 203 in the amino acid sequence shown in SEQ ID NO: 3. Indicates the position.
  5. 　請求項１～４のいずれか１項に記載の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨのアミノ酸配列において、１若しくは複数個のアミノ酸が欠失、置換及び付加から選ばれる少なくとも1つの改変がなされたアミノ酸配列を有する活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨ。 The amino acid sequence of the active FADGDH or modified FADGDH according to any one of claims 1 to 4, wherein one or more amino acids have at least one modification selected from deletion, substitution and addition Active FADGDH or modified FADGDH having
  6. 　請求項１～５のいずれか１項に記載の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨのアミノ酸配列をコードするＤＮＡ配列で表されるＤＮＡ。 A DNA represented by a DNA sequence encoding the amino acid sequence of the active FADGDH or modified FADGDH according to any one of claims 1 to 5.
  7. 　請求項６に記載のＤＮＡにおいて、コドンユーゼージが大腸菌に最適化したコドンユーゼージであるＤＮＡ。 7. The DNA according to claim 6, wherein the codon usage is a codon usage optimized for E. coli.
  8. 　請求項７に記載のＤＮＡを含む組み換え発現プラスミド。 A recombinant expression plasmid comprising the DNA according to claim 7.
  9. 　請求項８に記載の組み換え発現プラスミドで形質転換された宿主を含む形質転換体。 A transformant comprising a host transformed with the recombinant expression plasmid according to claim 8.
  10. 　宿主が大腸菌である請求項９に記載の形質転換体。 The transformant according to claim 9, wherein the host is Escherichia coli.
  11. 　請求項１０に記載の形質転換体を培養することを含む活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨの製造方法。 A method for producing active FADGDH or modified FADGDH, comprising culturing the transformant according to claim 10.
  12. 　請求項１～５のいずれか１項に記載の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを含むグルコースアッセイキット。 A glucose assay kit comprising the active FADGDH or the modified FADGDH according to any one of claims 1 to 5.
  13. 　請求項１～５のいずれか１項に記載の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを含むグルコースセンサ。 A glucose sensor comprising the active FADGDH or the modified FADGDH according to any one of claims 1 to 5.
  14. 　請求項１～５のいずれか１項に記載の活性型ＦＡＤＧＤＨ又は改変型ＦＡＤＧＤＨを含むグルコース濃度の測定法。 A method for measuring a glucose concentration containing the active FADGDH or the modified FADGDH according to any one of claims 1 to 5.
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